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Sample records for high-affinity monoclonal igg1

  1. A high affinity monoclonal antibody recognizing the light chain of human coagulating factor VII.

    Science.gov (United States)

    Sarial, Sheila; Asadi, Farzad; Jeddi-Tehrani, Mahmood; Hadavi, Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Taghizadeh-Jahed, Masoud; Shokri, Fazel; Rabbani, Hodjattallah

    2012-12-01

    Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII.

  2. Production and Identification of High Affinity Monoclonal Antibodies Against Pesticide Carbofuran

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    To produce high-affinity monoclonal antibodies against pesticide carbofuran, and the develop immunochemical assays for people's health and environmental protection, the hapten 4-[[(2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy) carbonyl]-amino]-butanoic acid (BFNB) of carbofuran was synthesized and Balb/c mice were immunized by the hapten-carrier (BFNB-bovine serum albumin, BFNB-BSA) conjugates. The splenocytes of immunized mice were fused with Sp2/0 cells and the cultural supernatants of hybridoma cells were screened by the indirect enzyme-linked immunoabsorbent assay (ELISA), based on BFNB-ovoalbumin conjugates (BFNB-OVA). Purified monoclonal antibody (McAb) was obtained from fluids of ascites, deposited by octanoic acid and ammonium sulfate. The affinity and the specificity of McAb were characterized by ELISA or indirect competitive ELISA. A hybridoma cell line (5D3) secreting anti-carbofuran McAb had been established. The titer of culture medium and ascites was up to 1:2.048 × 103 and 1:1.024 × 106, respectively, and the subtype of the McAb was IgG1. The affinity constant of the McAb was about 2.54 × 109 L mol-1, with an IC50 value of 1.18 ng mL-1 and a detection limit of 0.01 ng mL-1. Cross-reactivity studies showed that the McAb was quiet specific for carbofuran, as among the four analogous compounds, they were all hardly recognized (4.59 × 10-4% for 2,3-dihydro-2,2-dimethyl-7-benzofuranol and less than 3.0 × 10-4% for others). The prepared McAb had a very high affinity and specificity,and it could be used to develop ELISA for rapid determination of carbofuran.

  3. Isolation and characterization of a monoclonal anti CK-2 alpha subunit antibody of the IgG1 subclass

    DEFF Research Database (Denmark)

    Schmidt-Spaniol, I; Boldyreff, B; Issinger, O G

    1992-01-01

    A monoclonal antibody was produced against the recombinant human alpha subunit of CK-2. The antibody was of the IgG1 subclass and it was isolated from serum-free cell culture media and purified by affinity chromatography on Protein G Sepharose. The antibody can be used to detect specifically the CK......-2 alpha subunit in immunoblots from tissue extracts. An ELISA detection test was also established which also allows the identification of the CK-2 alpha subunit....

  4. High-affinity monoclonal antibodies specific for deoxynucleosides structurally modified by alkylating agents: Applications for immunoanalysis

    International Nuclear Information System (INIS)

    Adamkiewicz, J.; Ahrens, O.; Rajewsky, M.F.

    1984-01-01

    So far the results of attempts to use monoclonal antibodies for the demonstration of carcinogen-DNA adducts in cells by immunostaining have been promising. Thus the authors have established a standardized procedure for the quantitation of specific alkyl-deoxynucleosides in the nuclear DNA of individual cells by direct immunofluorescence, using tetramethylrhodamine isothiocyanate-labeled monoclonal antibodies and a computer-based image analysis of electronically intensified fluorescence signals. With a fluorescent anti-(O/sup 6/-EtdGuo) monoclonal antibody, the present detection limit for O/sup 6/-Etd-Guo in the nuclei of individual cells previously exposed to an ethylating N-nitroso compound (e.g., N-ethyl-N-nitrosourea) is -- 700 O/sup 6/-EtdGuo molecules per diploid genome, i.e., similar to the detection limit for the same ethylation product in a hydrolysate of (O/sup 6/-EtdGuo)-containing DNA analyzed by competitive RIA

  5. Specific recognition of the C-terminal end of A beta 42 by a high affinity monoclonal antibody

    DEFF Research Database (Denmark)

    Axelsen, Trine Veje; Holm, Arne; Birkelund, Svend

    2009-01-01

    The neurotoxic peptide A beta(42) is derived from the amyloid precursor protein by proteolytic cleavage and is deposited in the brain of patients suffering from Alzheimer's disease (AD). In this study we generate a high affinity monoclonal antibody that targets the C-terminal end of A beta(42......) with high specificity. By this is meant that the paratope of the antibody must enclose the C-terminal end of A beta(42) including the carboxy-group of amino acid 42, and not just recognize a linear epitope in the C-terminal part of A beta. This has been accomplished by using a unique antigen construct made...... by the Ligand Presenting Assembly technology (LPA technology). This strategy results in dimeric presentation of the free C-terminal end of A beta(42). The generated Mab A beta1.1 is indeed specific for the C-terminal end of A beta(42) to which it binds with high affinity. Mab A beta1.1 recognizes the epitope...

  6. The production of high affinity monoclonal antibodies to human growth hormone

    International Nuclear Information System (INIS)

    Stuart, M.C.; Walichnowski, C.M.; Hussain, S.; Underwood, P.A.; Harman, D.F.; Rathjen, D.A.; Sturmer, S.R. von

    1983-01-01

    The primary aim of this work was to produce specific monoclonal antibodies to human growth hormone (hGH) for use in a diagnostic RIA of hGH levels in serum. Three different schedules were used for immunization of BALB/c mice and the splenocytes from each mouse were fused with myeloma cells Sp 2/0 Ag 14. Each fusion resulted in the production of hundreds of hybridomas secreting hGH-directed antibodies. Six antibodies have been fully characterized and have been grouped into pairs which recognize 3 different epitopes on the hGH molecule. One pair exhibits no cross reaction with the structurally related placental hormone, human placental lactogen (hPL), a second pair has low cross reaction with hPL (1.6-3%) and a third pair reacts equally well with hGH and hPL indicating binding to a common epitope in the 2 molecules. The highest affinity antibody, 74/6, which has an affinity constant of 4.4x10 10 l/mol and 3% cross-reactivity with hPL, has been used to establish a RIA for serum hGH measurements. Evidence is provided that hGH levels measured in this assay correlate well with those obtained in a conventional rabbit antiserum assay. (Auth.)

  7. Analyses of the peripheral immunome following multiple administrations of avelumab, a human IgG1 anti-PD-L1 monoclonal antibody

    OpenAIRE

    Donahue, Renee N.; Lepone, Lauren M.; Grenga, Italia; Jochems, Caroline; Fantini, Massimo; Madan, Ravi A.; Heery, Christopher R.; Gulley, James L.; Schlom, Jeffrey

    2017-01-01

    Background Multiple anti-PD-L1/PD-1 checkpoint monoclonal antibodies (MAb) have shown clear evidence of clinical benefit. All except one have been designed or engineered to omit the possibility to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) as a second potential mode of anti-tumor activity; the reason for this is the concern of lysis of PD-L1 positive immune cells. Avelumab is a fully human IgG1 MAb which has been shown in prior in vitro studies to mediate ADCC versus a range...

  8. Generation of a novel high-affinity monoclonal antibody with conformational recognition epitope on human IgM.

    Science.gov (United States)

    Sarikhani, Sina; Mirshahi, Manouchehr; Gharaati, Mohammad Reza; Mirshahi, Tooran

    2010-11-01

    As IgM is the first isotype of antibody which appears in blood after initial exposure to a foreign antigen in the pattern of primary response, detection, and quantification of this molecule in blood seems invaluable. To approach these goals, generation, and characterization of a highly specific mAb (monoclonal antibody) against human IgM were investigated. Human IgM immunoglobulins were used to immunize Balb/c mice. Spleen cells taken from the immunized animals were fused with SP2/O myeloma cells using PEG (polyethylene glycol, MW 1450) as fusogen. The hybridomas were cultured in HAT containing medium and supernatants from the growing hybrids were screened by enzyme-linked immunosorbent assay (ELISA) using plates coated with pure human IgM and the positive wells were then cloned at limiting dilutions. The best clone designated as MAN-1, was injected intraperitoneally to some Pristane-injected mice. Anti-IgM mAb was purified from the animals' ascitic fluid by protein-G sepharose followed by DEAE-cellulose ion exchange chromatography. MAN-1 interacted with human IgM with a very high specificity and affinity. The purity of the sample was tested by SDS-PAGE and the affinity constant was measured (K(a) = 3.5 x 10(9)M(-1). Immunoblotting and competitive ELISA were done and the results showed that the harvested antibody recognizes a conformational epitope on the mu chain of human IgM and there was no cross-reactivity with other subclasses of immunoglobulins. Furthermore, isotyping test was done and the results showed the subclass of the obtained mAb which was IgG(1)kappa.

  9. High affinity anti-TIM-3 and anti-KIR monoclonal antibodies cloned from healthy human individuals.

    Directory of Open Access Journals (Sweden)

    Stefan Ryser

    Full Text Available We report here the cloning of native high affinity anti-TIM-3 and anti-KIR IgG monoclonal antibodies (mAbs from peripheral blood mononuclear cells (PBMC of healthy human donors. The cells that express these mAbs are rare, present at a frequency of less than one per 105 memory B-cells. Using our proprietary multiplexed screening and cloning technology CellSpot™ we assessed the presence of memory B-cells reactive to foreign and endogenous disease-associated antigens within the same individual. When comparing the frequencies of antigen-specific memory B-cells analyzed in over 20 screening campaigns, we found a strong correlation of the presence of anti-TIM-3 memory B-cells with memory B-cells expressing mAbs against three disease-associated antigens: (i bacterial DNABII proteins that are a marker for Gram negative and Gram positive bacterial infections, (ii hemagglutinin (HA of influenza virus and (iii the extracellular domain of anaplastic lymphoma kinase (ALK. One of the native anti-KIR mAbs has similar characteristics as lirilumab, an anti-KIR mAb derived from immunization of humanized transgenic mice that is in ongoing clinical trials. It is interesting to speculate that these native anti-TIM-3 and anti-KIR antibodies may function as natural regulatory antibodies, analogous to the pharmacological use in cancer treatment of engineered antibodies against the same targets. Further characterization studies are needed to define the mechanisms through which these native antibodies may function in healthy and disease conditions.

  10. Protein Adsorption and Layer Formation at the Stainless Steel-Solution Interface Mediates Shear-Induced Particle Formation for an IgG1 Monoclonal Antibody.

    Science.gov (United States)

    Kalonia, Cavan K; Heinrich, Frank; Curtis, Joseph E; Raman, Sid; Miller, Maria A; Hudson, Steven D

    2018-03-05

    Passage of specific protein solutions through certain pumps, tubing, and/or filling nozzles can result in the production of unwanted subvisible protein particles (SVPs). In this work, surface-mediated SVP formation was investigated. Specifically, the effects of different solid interface materials, interfacial shear rates, and protein concentrations on SVP formation were measured for the National Institute of Standards and Technology monoclonal antibody (NISTmAb), a reference IgG1 monoclonal antibody (mAb). A stainless steel rotary piston pump was used to identify formulation and process parameters that affect aggregation, and a flow cell (alumina or stainless steel interface) was used to further investigate the effect of different interface materials and/or interfacial shear rates. SVP particles produced were monitored using flow microscopy or flow cytometry. Neutron reflectometry and a quartz crystal microbalance with dissipation monitoring were used to characterize adsorption and properties of NISTmAb at the stainless steel interface. Pump/shear cell experiments showed that the NISTmAb concentration and interface material had a significant effect on SVP formation, while the effects of interfacial shear rate and passage number were less important. At the higher NISTmAb concentrations, the adsorbed protein became structurally altered at the stainless steel interface. The primary adsorbed layer remained largely undisturbed during flow, suggesting that SVP formation at high NISTmAb concentration was caused by the disruption of patches and/or secondary interactions.

  11. Analyses of the peripheral immunome following multiple administrations of avelumab, a human IgG1 anti-PD-L1 monoclonal antibody.

    Science.gov (United States)

    Donahue, Renee N; Lepone, Lauren M; Grenga, Italia; Jochems, Caroline; Fantini, Massimo; Madan, Ravi A; Heery, Christopher R; Gulley, James L; Schlom, Jeffrey

    2017-01-01

    Multiple anti-PD-L1/PD-1 checkpoint monoclonal antibodies (MAb) have shown clear evidence of clinical benefit. All except one have been designed or engineered to omit the possibility to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) as a second potential mode of anti-tumor activity; the reason for this is the concern of lysis of PD-L1 positive immune cells. Avelumab is a fully human IgG1 MAb which has been shown in prior in vitro studies to mediate ADCC versus a range of human tumor cells, and clinical studies have demonstrated anti-tumor activity versus a range of human cancers. This study was designed to investigate the effect on immune cell subsets in the peripheral blood of cancer patients prior to and following multiple administrations of avelumab. One hundred twenty-three distinct immune cell subsets in the peripheral blood of cancer patients ( n  = 28) in a phase I trial were analyzed by flow cytometry prior to and following one, three, and nine cycles of avelumab. Changes in soluble (s) CD27 and sCD40L in plasma were also evaluated. In vitro studies were also performed to determine if avelumab would mediate ADCC of PBMC. No statistically significant changes in any of the 123 immune cell subsets analyzed were observed at any dose level, or number of doses, of avelumab. Increases in the ratio of sCD27:sCD40L were observed, suggesting potential immune activation. Controlled in vitro studies also showed lysis of tumor cells by avelumab versus no lysis of PBMC from five donors. These studies demonstrate the lack of any significant effect on multiple immune cell subsets, even those expressing PD-L1, following multiple cycles of avelumab. These results complement prior studies showing anti-tumor effects of avelumab and comparable levels of adverse events with avelumab versus other anti-PD-1/PD-L1 MAbs. These studies provide the rationale to further exploit the potential ADCC mechanism of action of avelumab as well as other human IgG1 checkpoint

  12. A high-affinity human monoclonal IgM antibody reacting with multiple strains of Mycoplasma hominis

    DEFF Research Database (Denmark)

    Moller, SA; Birkelund, Svend; Borrebaeck, CA

    1990-01-01

    Human monoclonal antibodies were produced against Mycoplasma hominis by in vitro immunization of peripheral blood lymphocytes from a healthy seropositive donor using low amounts of antigen (5 ng/ml). The immune B lymphocytes were subsequently immortalized by Epstein-Barr virus transformation...

  13. Analysis of Tn antigenicity with a panel of new IgM and IgG1 monoclonal antibodies raised against leukemic cells

    DEFF Research Database (Denmark)

    Blixt, Klas Ola; Lavrova, Olga I; Mazurov, Dmitriy V

    2012-01-01

    antigenicity remain poorly understood. As a consequence, a broad variety of anti-Tn monoclonal antibodies (mAbs) have been generated. To further investigate the nature and complexity of the Tn antigen, we generated seven different anti-Tn mAbs of IgM and IgG classes raised against human Jurkat T cells, which...... are Tn-positive due to the low activity of T-synthase and mutation in specific chaperone Cosmc. The binding analysis of anti-Tn mAbs with the array of synthetic saccharides, glycopeptides and O-glycoproteins revealed unexpected differences in specificities of anti-Tn mAbs. IgM mAbs bound the terminal Gal...

  14. Viscosity of high concentration protein formulations of monoclonal antibodies of the IgG1 and IgG4 subclass - Prediction of viscosity through protein-protein interaction measurements

    DEFF Research Database (Denmark)

    Neergaard, Martin S; Kalonia, Devendra S; Parshad, Henrik

    2013-01-01

    The purpose of this work was to explore the relation between protein-protein interactions (PPIs) and solution viscosity at high protein concentration using three monoclonal antibodies (mAbs), two of the IgG4 subclass and one of the IgG1 subclass. A range of methods was used to quantify the PPI...... low or high protein concentration determined using DLS. The PPI measurements were correlated with solution viscosity (measured by DLS using polystyrene nanospheres and ultrasonic shear rheology) as a function of pH (4-9) and ionic strength (10, 50 and 150mM). Our measurements showed that the highest...... solution viscosity was observed under conditions with the most negative kD, the highest apparent radius and the lowest net charge. An increase in ionic strength resulted in a change in the nature of the PPI at low pH from repulsive to attractive. In the neutral to alkaline pH region the mAbs behaved...

  15. Probing the Conformation of an IgG1 Monoclonal Antibody in Lyophilized Solids Using Solid-State Hydrogen-Deuterium Exchange with Mass Spectrometric Analysis (ssHDX-MS).

    Science.gov (United States)

    Moussa, Ehab M; Singh, Satish K; Kimmel, Michael; Nema, Sandeep; Topp, Elizabeth M

    2018-02-05

    Therapeutic proteins are often formulated as lyophilized products to improve their stability and prolong shelf life. The stability of proteins in the solid-state has been correlated with preservation of native higher order structure and/or molecular mobility in the solid matrix, with varying success. In the studies reported here, we used solid-state hydrogen-deuterium exchange with mass spectrometric analysis (ssHDX-MS) to study the conformation of an IgG1 monoclonal antibody (mAb) in lyophilized solids and related the extent of ssHDX to aggregation during storage in the solid phase. The results demonstrate that the extent of ssHDX correlated better with aggregation rate during storage than did solid-state Fourier-transform infrared (ssFTIR) spectroscopic measurements. Interestingly, adding histidine to sucrose at different formulation pH conditions decreased aggregation of the mAb, an effect that did not correlate with structural or conformational changes as measured by ssFTIR or ssHDX-MS. Moreover, peptide-level ssHDX-MS analysis in four selected formulations demonstrated global changes across the structure of the mAb when lyophilized with sucrose, trehalose, or mannitol, whereas site-specific changes were observed when lyophilized with histidine as the sole excipient.

  16. Variation in N-linked carbohydrate chains in different batches of two chimeric monoclonal IgG1 antibodies produced by different murine SP2/0 transfectoma cell subclones.

    Science.gov (United States)

    Bergwerff, A A; Stroop, C J; Murray, B; Holtorf, A P; Pluschke, G; Van Oostrum, J; Kamerling, J P; Vliegenthart, J F

    1995-06-01

    Two chimeric human/murine monoclonal antibodies were constructed by substitution of the murine constant regions with human gamma 1 and kappa constant regions for heavy and light chains, respectively. The chimeric human/murine molecules are anti-idiotypic antibodies, meaning that they were directed against the antigen binding site in the variable region of another antibody. Antibody batches were produced under identical production conditions, using two selected SP2/0 myeloma cell subclones, which produce chimeric antibodies with different variable regions, but identical constant regions. Several samples were collected during the production of the antibodies in hollow-fibre reactors. The heavy chain, but not the light chain, of the two different chimeric IgG1 antibodies is glycosylated. Structural analysis of the enzymically released N-linked carbohydrate chains by 1H-NMR spectroscopy, as well as by chromatographic profiling, demonstrated that the collection of N-glycans comprises a small amount of monoantennary, and for the greater part diantennary structures. The N-glycans are completely (alpha 1-->6)-fucosylated at the innermost GlcNAc residue. The antennae of the neutral diantennary N-glycans are built up from GlcNAc beta 1-->2, Gal beta 1-->4GlcNAc beta 1-->2 or Gal alpha 1-->3G alpha 1 beta 1-->4GlcNAc beta 1-->2 elements, whereas the antennae of the neutral monoantennary carbohydrate chains have only (beta 1-->2)-linked GlcNAc residues. Galactosylation of the GlcNAc beta 1-->2Man alpha 1-->6 branch occurs four times more frequently than that of the GlcNAc beta 1-->2Man alpha 1-->3 branch, independently of the production batch. A small amount of the diantennary N-glycans are mono- or disialylated, carrying N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc), exclusively (alpha 2-->6)-linked to beta Gal. Analysis of the different production batches demonstrates that the structures of the N-linked carbohydrate chains are identical in the two

  17. Generation and characterization of a human-mouse chimeric high-affinity antibody that detects the DYKDDDDK FLAG peptide.

    Science.gov (United States)

    Ikeda, Koki; Koga, Tomoaki; Sasaki, Fumiyuki; Ueno, Ayumi; Saeki, Kazuko; Okuno, Toshiaki; Yokomizo, Takehiko

    2017-05-13

    DYKDDDDK peptide (FLAG) is a useful tool for investigating the function and localization of proteins whose antibodies (Abs) are not available. We recently established a high-affinity monoclonal antibody (mAb) for FLAG (clone 2H8). The 2H8 Ab is highly sensitive for detecting FLAG-tagged proteins by flowcytometry and immunoprecipitation, but it can yield nonspecific signals in immunohistochemistry of mouse tissues because it is of mouse origin. In this study, we reduced nonspecific signals by generating a chimeric 2H8 Ab with Fc fragments derived from human immunoglobulin. We fused a 5' terminal cDNA fragments for the Fab region of 2H8 mAb with 3' terminal cDNA fragments for Fc region of human IgG1. We transfected both chimeric plasmids and purified the resulting human-mouse chimeric 2H8. The chimeric 2H8 Ab successfully detected FLAG-tagged proteins in flowcytometry with anti-human IgG secondary Ab with comparable sensitivity to 2H8 mAb. Importantly, chimeric 2H8 detected specific FLAG peptide signals without nonspecific signals in immunohistochemical analysis with mouse tissues. This human-mouse chimeric high-affinity anti-FLAG Ab will prove useful for future immunohistochemical analysis of mouse tissues. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Importance of neonatal FcR in regulating the serum half-life of therapeutic proteins containing the Fc domain of human IgG1: a comparative study of the affinity of monoclonal antibodies and Fc-fusion proteins to human neonatal FcR.

    Science.gov (United States)

    Suzuki, Takuo; Ishii-Watabe, Akiko; Tada, Minoru; Kobayashi, Tetsu; Kanayasu-Toyoda, Toshie; Kawanishi, Toru; Yamaguchi, Teruhide

    2010-02-15

    The neonatal FcR (FcRn) binds to the Fc domain of IgG at acidic pH in the endosome and protects IgG from degradation, thereby contributing to the long serum half-life of IgG. To date, more than 20 mAb products and 5 Fc-fusion protein products have received marketing authorization approval in the United States, the European Union, or Japan. Many of these therapeutic proteins have the Fc domain of human IgG1; however, the serum half-lives differ in each protein. To elucidate the role of FcRn in the pharmacokinetics of Fc domain-containing therapeutic proteins, we evaluated the affinity of the clinically used human, humanized, chimeric, or mouse mAbs and Fc-fusion proteins to recombinant human FcRn by surface plasmon resonance analysis. The affinities of these therapeutic proteins to FcRn were found to be closely correlated with the serum half-lives reported from clinical studies, suggesting the important role of FcRn in regulating their serum half-lives. The relatively short serum half-life of Fc-fusion proteins was thought to arise from the low affinity to FcRn. The existence of some mAbs having high affinity to FcRn and a short serum half-life, however, suggested the involvement of other critical factor(s) in determining the serum half-life of such Abs. We further investigated the reason for the relatively low affinity of Fc-fusion proteins to FcRn and suggested the possibility that the receptor domain of Fc-fusion protein influences the structural environment of the FcRn binding region but not of the FcgammaRI binding region of the Fc domain.

  19. Human IgG1 antibodies suppress angiogenesis in a target-independent manner

    NARCIS (Netherlands)

    Bogdanovich, Sasha; Kim, Younghee; Mizutani, Takeshi; Yasuma, Reo; Tudisco, Laura; Cicatiello, Valeria; Bastos-Carvalho, Ana; Kerur, Nagaraj; Hirano, Yoshio; Baffi, Judit Z; Tarallo, Valeria; Li, Shengjian; Yasuma, Tetsuhiro; Arpitha, Parthasarathy; Fowler, Benjamin J; Wright, Charles B; Apicella, Ivana; Greco, Adelaide; Brunetti, Arturo; Ruvo, Menotti; Sandomenico, Annamaria; Nozaki, Miho; Ijima, Ryo; Kaneko, Hiroki; Ogura, Yuichiro; Terasaki, Hiroko; Ambati, Balamurali K; Leusen, Jeanette HW; Langdon, Wallace Y; Clark, Michael R; Armour, Kathryn L; Bruhns, Pierre; Verbeek, J Sjef; Gelfand, Bradley D; De Falco, Sandro; Ambati, Jayakrishna

    2016-01-01

    Aberrant angiogenesis is implicated in diseases affecting nearly 10% of the world's population. The most widely used anti-angiogenic drug is bevacizumab, a humanized IgG1 monoclonal antibody that targets human VEGFA. Although bevacizumab does not recognize mouse Vegfa, it inhibits angiogenesis in

  20. Molecular basis of a high affinity murine interleukin-5 receptor.

    OpenAIRE

    Devos, R; Plaetinck, G; Van der Heyden, J; Cornelis, S; Vandekerckhove, J; Fiers, W; Tavernier, J

    1991-01-01

    The mouse interleukin-5 receptor (mIL-5R) consists of two components one of which, the mIL-5R alpha-chain, binds mIL-5 with low affinity. Recently we demonstrated that monoclonal antibodies (Mabs) recognizing the second mIL-5R beta-chain, immunoprecipitate a p130-140 protein doublet which corresponds to the mIL-3R and the mIL-3R-like protein, the latter chain for which so far no ligand has been identified. In this study we show that a high affinity mIL-5R can be reconstituted on COS1 cells by...

  1. Fc receptors for mouse IgG1 on human monocytes: polymorphism and role in antibody-induced T cell proliferation.

    Science.gov (United States)

    Tax, W J; Hermes, F F; Willems, R W; Capel, P J; Koene, R A

    1984-09-01

    In previous studies, it was shown that there is polymorphism in the mitogenic effect of mouse IgG1 monoclonal antibodies against the T3 antigen of human T cells. This polymorphism implies that IgG1 anti-T3 antibodies are not mitogenic for T cells from 30% of healthy individuals. The present results demonstrate that this polymorphism is caused by polymorphism of an Fc receptor for mouse IgG1, present on human monocytes. The Fc receptor for murine IgG1 could be detected by a newly developed rosetting assay on monocytes from all individuals responsive to the mitogenic effect of IgG1 anti-T3 antibodies. This Fc receptor was not detectable on monocytes from those individuals exhibiting no mitogenic responses to IgG1 anti-T3 monoclonal antibodies. Cross-linking of T3 antigens appears to be essential for antibody-induced mitosis of T cells, because mononuclear cells that did not proliferate in response to WT 31 (an IgG1 antibody against T3 antigen) showed a proliferative response to Sepharose beads coated with WT 31. The Fc receptor--if functionally present--may be involved in the cross-linking of T3 antigens through anti-T3 antibodies. Further evidence for the involvement of this Fc receptor in antibody-induced T cell proliferation was provided by inhibition studies. Immune complexes containing IgG1 antibodies were able to inhibit the proliferative response to IgG1 anti-T3 antibodies. This inhibition by immune complexes appears to be mediated through the monocyte Fc receptor for mouse IgG1. These findings are important for the interpretation of previously described inhibitory effects of anti-T cell monoclonal antibodies on T cell proliferation, and show that such inhibitory effects may be monocyte-mediated (via immune complexes) rather than caused by a direct involvement of the respective T cell antigens in T cell mitosis. The Fc receptor for mouse IgG1 plays a role in antibody-induced T cell proliferation. Its polymorphism may have important implications for the

  2. A potential therapy for chordoma via antibody-dependent cell-mediated cytotoxicity employing NK or high-affinity NK cells in combination with cetuximab.

    Science.gov (United States)

    Fujii, Rika; Schlom, Jeffrey; Hodge, James W

    2018-05-01

    OBJECTIVE Chordoma is a rare bone tumor derived from the notochord and is resistant to conventional therapies such as chemotherapy, radiotherapy, and targeting therapeutics. Expression of epidermal growth factor receptor (EGFR) in a large proportion of chordoma specimens indicates a potential target for therapeutic intervention. In this study the authors investigated the potential role of the anti-EGFR antibody cetuximab in immunotherapy for chordoma. METHODS Since cetuximab is a monoclonal antibody of the IgG1 isotype, it has the potential to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) employing natural killer (NK) cells as effectors. Polymorphisms in the CD16 allele expressed on NK cells have been shown to influence the degree of ADCC of tumor cells, with the high-affinity valine (V)/V allele being responsible for more lysis than the V/phenylalanine (F) or FF allele. Unfortunately, however, only approximately 10% of the population expresses the VV allele on NK cells. An NK cell line, NK-92, has now been engineered to endogenously express IL-2 and the high-affinity CD16 allele. These irradiated high-affinity (ha)NK cells were analyzed for lysis of chordoma cells with and without cetuximab, and the levels of lysis observed in ADCC were compared with those of NK cells from donors expressing the VV, VF, and FF alleles. RESULTS Here the authors demonstrate for the first time 1) that cetuximab in combination with NK cells can mediate ADCC of chordoma cells; 2) the influence of the NK CD16 polymorphism in cetuximab-mediated ADCC for chordoma cell lysis; 3) that engineered haNK cells-that is, cells transduced to express the CD16 V158 FcγRIIIa receptor-bind cetuximab with similar affinity to normal NK cells expressing the high-affinity VV allele; and 4) that irradiated haNK cells induce ADCC with cetuximab in chordoma cells. CONCLUSIONS These studies provide rationale for the use of cetuximab in combination with irradiated haNK cells for therapy for

  3. ADCC employing an NK cell line (haNK) expressing the high affinity CD16 allele with avelumab, an anti-PD-L1 antibody.

    Science.gov (United States)

    Jochems, Caroline; Hodge, James W; Fantini, Massimo; Tsang, Kwong Y; Vandeveer, Amanda J; Gulley, James L; Schlom, Jeffrey

    2017-08-01

    NK-92 cells, and their derivative, designated aNK, were obtained from a patient with non-Hodgkin lymphoma. Prior clinical studies employing adoptively transferred irradiated aNK cells have provided evidence of clinical benefit and an acceptable safety profile. aNK cells have now been engineered to express IL-2 and the high affinity (ha) CD16 allele (designated haNK). Avelumab is a human IgG1 anti-PD-L1 monoclonal antibody, which has shown evidence of clinical activity in a range of human tumors. Prior in vitro studies have shown that avelumab has the ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) of human tumor cells when combined with NK cells. In the studies reported here, the ability of avelumab to enhance the lysis of a range of human carcinoma cells by irradiated haNK cells via the ADCC mechanism is demonstrated; this ADCC is shown to be inhibited by anti-CD16 blocking antibody and by concanamycin A, indicating the use of the granzyme/perforin pathway in tumor cell lysis. Studies also show that while NK cells have the ability to lyse aNK or haNK cells, the addition of NK cells to irradiated haNK cells does not inhibit haNK-mediated lysis of human tumor cells, with or without the addition of avelumab. Avelumab-mediated lysis of tumor cells by irradiated haNK cells is also shown to be similar to that of NK cells bearing the V/V Fc receptor high affinity allele. These studies thus provide the rationale for the clinical evaluation of the combined use of avelumab with that of irradiated adoptively transferred haNK cells. © 2017 UICC.

  4. High affinity hemoglobin and Parkinson's disease.

    Science.gov (United States)

    Graham, Jeffrey; Hobson, Douglas; Ponnampalam, Arjuna

    2014-12-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra (SN) region of the midbrain. Oxidative damage in this region has been shown to play an important role in the pathogenesis of this disease. Human neurons have been discovered to contain hemoglobin, with an increased concentration seen in the neurons of the SN. High affinity hemoglobin is a clinical entity resulting from mutations that create a functional increase in the binding of hemoglobin to oxygen and an inability to efficiently unload it to tissues. This can result in a number of metabolic compensatory changes, including an elevation in circulating hemoglobin and an increase in the molecule 2,3-diphosphoglycerate (2,3-DPG). Population based studies have revealed that patients with PD have elevated hemoglobin as well as 2,3-DPG levels. Based on these observations, we hypothesize that the oxidative damage seen in PD is related to an underlying high affinity hemoglobin subtype. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. A fully human IgG1 anti-PD-L1 MAb in an in vitro assay enhances antigen-specific T-cell responses

    OpenAIRE

    Grenga, Italia; Donahue, Renee N; Lepone, Lauren M; Richards, Jacob; Schlom, Jeffrey

    2016-01-01

    Monoclonal antibodies (MAbs) that interfere with checkpoint molecules are being investigated for the treatment of infectious diseases and cancer, with the aim of enhancing the function of an impaired immune system. Avelumab (MSB0010718C) is a fully human IgG1 MAb targeting programmed death-ligand 1 (PD-L1), which differs from other checkpoint-blocking antibodies in its ability to mediate antibody-dependent cell-mediated cytotoxicity. These studies were conducted to define whether avelumab cou...

  6. Removal of a C-terminal serine residue proximal to the inter-chain disulfide bond of a human IgG1 lambda light chain mediates enhanced antibody stability and antibody dependent cell-mediated cytotoxicity

    Science.gov (United States)

    Shen, Yang; Zeng, Lin; Zhu, Aiping; Blanc, Tim; Patel, Dipa; Pennello, Anthony; Bari, Amtul; Ng, Stanley; Persaud, Kris; Kang, Yun (Kenneth); Balderes, Paul; Surguladze, David; Hindi, Sagit; Zhou, Qinwei; Ludwig, Dale L.; Snavely, Marshall

    2013-01-01

    Optimization of biophysical properties is a critical success factor for the developability of monoclonal antibodies with potential therapeutic applications. The inter-domain disulfide bond between light chain (Lc) and heavy chain (Hc) in human IgG1 lends structural support for antibody scaffold stability, optimal antigen binding, and normal Fc function. Recently, human IgG1λ has been suggested to exhibit significantly greater susceptibility to reduction of the inter Lc-Hc disulfide bond relative to the same disulfide bond in human IgG1κ. To understand the molecular basis for this observed difference in stability, the sequence and structure of human IgG1λ and human IgG1κ were compared. Based on this Lc comparison, three single mutations were made in the λ Lc proximal to the cysteine residue, which forms a disulfide bond with the Hc. We determined that deletion of S214 (dS) improved resistance of the association between Lc and Hc to thermal stress. In addition, deletion of this terminal serine from the Lc of IgG1λ provided further benefit, including an increase in stability at elevated pH, increased yield from transient transfection, and improved in vitro antibody dependent cell-mediated cytotoxicity (ADCC). These observations support the conclusion that the presence of the terminal serine of the λ Lc creates a weaker inter-chain disulfide bond between the Lc and Hc, leading to slightly reduced stability and a potential compromise in IgG1λ function. Our data from a human IgG1λ provide a basis for further investigation of the effects of deleting terminal serine from λLc on the stability and function of other human IgG1λ antibodies. PMID:23567210

  7. Comparative Diagnosis of Serum IgG1 and Coproantigen ELISA for Fasciolosis Detection of Goats in Mexico

    Science.gov (United States)

    Molina-Mendoza, Pedro; Hernández-Guzmán, Karina; Olivares-Pérez, Jaime; Sarracent-Pérez, Jorge; Zumaquero-Ríos, José

    2016-01-01

    The objective of present study was to determine the prevalence of natural caprine fasciolosis in the Mixteca region of Mexico using coproantigen and serum IgG1 ELISA tests for comparative purposes. A total of 1070 serum and faecal samples were analyzed for IgG1 antibodies and coproantigens, using ELISA with E/S products as antigen and a monoclonal antibody-based sandwich ELISA. Prevalence of 73.46% was found using the serological ELISA and a percentage of 77.20 was found for coproantigen ELISA. The diagnostic sensitivity and specificity for serum ELISA were 86.7% and 96.4%, and for the coproantigen ELISA they were 93.1% and 97.8%, respectively. The seropositive samples were further categorized as low, medium, or high positivity. Results show a great proportion of low and medium positive goats when the serum ELISA test was used. Correlation coefficients between coproantigens and seropositivity were statistically significant (P < 0.01) for low seropositivity (r = 0.93) and medium seropositivity (r = 0.84). The accuracy of faecal antigen ELISA was higher compared to indirect ELISA serological test. Two ELISAs were shown to be useful for demonstrating the current status of F. hepatica infection in the endemic areas and can be employed in studies on epidemiology as well as anthelmintics treatment for preventing economic loss and the risk of transmission to humans. PMID:27563665

  8. Generation of monoclonal antibodies against prostate specific antigen (PSA) for the detection of PSA and its purification

    International Nuclear Information System (INIS)

    Acevedo Castro, Boris Ernesto

    2012-01-01

    The prostate cancer in Cuba is a problem of health (2672 diagnosed cases and 2769 deaths in 2007). Various diagnostic methods have been implemented for the detection and management of this disease, emphasizing among them (PSA) prostate-specific antigen serological determination. At this work was generated and characterized a panel of 11 antibodies (AcMs) monoclonal IgG1 detected with high affinity described major epitopes of the PSA, both in solution and attached to the test plate. From the panel obtained AcMs was the standardization of an essay type ELISA for the detection of serum total PSA (associated and free) equimolar, based on antibody monoclonal CB-PSA.4 in the coating and the CB-PSA.9 coupled with biotin as liner, with a detection limit of 0.15 ng/mL. Similarly, standardized system for detection in serum free PSA, based on the AcMs CB-PSA.4 (coating) and CB-PSA.2 coupled with biotin (liner), with a detection limit of 0.5 ng/mL. Finally, with the purpose of using PSA as standard in trials type ELISA, developed a simple method of inmunopurificación based on the AcM, CB-PSA.2, which was obtained the PSA with a purity exceeding 90%. Immunoassay Centre on the basis of the AcMs panel and the results of this study, developed and recorded two diagnostic systems for the detection of PSA in human serum. (author)

  9. Structural insights into the interaction of human IgG1 with FcγRI: no direct role of glycans in binding

    Energy Technology Data Exchange (ETDEWEB)

    Oganesyan, Vaheh, E-mail: oganesyanv@medimmune.com; Mazor, Yariv; Yang, Chunning; Cook, Kimberly E.; Woods, Robert M. [MedImmune LLC, 1 MedImmune Way, Gaithersburg, MD 20878 (United States); Ferguson, Andrew [AstraZeneca Pharmaceuticals, 35 Gatehouse Drive, Mailstop E3, Waltham, MA 02451 (United States); Bowen, Michael A.; Martin, Tom; Zhu, Jie; Wu, Herren; Dall’Acqua, William F., E-mail: oganesyanv@medimmune.com [MedImmune LLC, 1 MedImmune Way, Gaithersburg, MD 20878 (United States)

    2015-10-31

    In an effort to identify the critical structural features responsible for the high-affinity interaction of IgG1 Fc with FcγRI, the structure of the corresponding complex was solved at a resolution of 2.4 Å. The three-dimensional structure of a human IgG1 Fc fragment bound to wild-type human FcγRI is reported. The structure of the corresponding complex was solved at a resolution of 2.4 Å using molecular replacement; this is the highest resolution achieved for an unmutated FcγRI molecule. This study highlights the critical structural and functional role played by the second extracellular subdomain of FcγRI. It also explains the long-known major energetic contribution of the Fc ‘LLGG’ motif at positions 234–237, and particularly of Leu235, via a ‘lock-and-key’ mechanism. Finally, a previously held belief is corrected and a differing view is offered on the recently proposed direct role of Fc carbohydrates in the corresponding interaction. Structural evidence is provided that such glycan-related effects are strictly indirect.

  10. [Preparation of monoclonal antibody against 4-amylphenol and homology modeling of its Fv fragment].

    Science.gov (United States)

    Cheng, Lei; Wu, Haizhen; Fei, Jing; Zhang, Lujia; Ye, Jiang; Zhang, Huizhan

    2017-03-01

    Objective To prepare and characterize a monoclonal antibody (mAb) against 4-amylphenol (4-AP), clone its cDNA sequence and make homology modeling for its Fv fragment. Methods A high-affinity anti-4-AP mAb was generated from a hybridoma cell line F10 using electrofusion between splenocytes from APA-BSA-immunized mouse and Sp2/0 myeloma cells. Then we extracted the mRNA of F10 cells and cloned the cDNA of mAb. The homology modeling and molecular docking of its Fv fragment was conducted with biological software. Results Under the optimum conditions, the ic-ELISA equation was y=A 2 +(A 1 -A 2 )/(1+(x/x 0 ) p ) (A 1 =1.28; A 2 =-0.066; x 0 =12560.75; p=0.74) with a correlation coefficient (R 2 ) of 0.997. The lowest detectable limit was 0.65 μg/mL. The heavy and light chains of mAb respectively belonged to IgG1 and Kappa. The homology modeling and molecular docking studies revealed that the binding of 4-Ap and mAb was attributed to the hydrogen bond and hydrophobic interactions. Conclusion The study successfully established a stable 4-AP mAb-secreting hybridoma cell line. The study on spatial structure of Fv fragment using homology modeling provided a reference for the development and design of single chain variable fragments.

  11. Development of a new high-affinity human antibody with antitumor activity against solid and blood malignancies.

    Science.gov (United States)

    Sioud, Mouldy; Westby, Phuong; Vasovic, Vlada; Fløisand, Yngvar; Peng, Qian

    2018-04-16

    mAbs have emerged as a promising strategy for the treatment of cancer. However, in several malignancies, no effective antitumor mAbs are yet available. Identifying therapeutic mAbs that recognize common tumor antigens could render the treatment widely applicable. Here, a human single-chain variable fragment (scFv) antibody library was sequentially affinity selected against a panel of human cancer cell lines and an antibody fragment (named MS5) that bound to solid and blood cancer cells was identified. The MS5 scFv was fused to the human IgG1 Fc domain to generate an antibody (MS5-Fc fusion) that induced antibody-dependent cellular cytotoxicity and phagocytosis of cancer cells by macrophages. In addition, the MS5-Fc antibody bound to primary leukemia cells and induced antibody-dependent cellular cytotoxicity. In the majority of analyzed cancer cells, the MS5-Fc antibody induced cell surface redistribution of the receptor complexes, but not internalization, thus maximizing the accessibility of the IgG1 Fc domain to immune effector cells. In vitro stability studies showed that the MS5-Fc antibody was stable after 6 d of incubation in human serum, retaining ∼60% of its initial intact form. After intravenous injections, the antibody localized into tumor tissues and inhibited the growth of 3 different human tumor xenografts (breast, lymphoma, and leukemia). These antitumor effects were associated with tumor infiltration by macrophages and NK cells. In the Ramos B-cell lymphoma xenograft model, the MS5-Fc antibody exhibited a comparable antitumor effect as rituximab, a chimeric anti-CD20 IgG1 mAb. These results indicate that human antibodies with pan-cancer abilities can be generated from phage display libraries, and that the engineered MS5-Fc antibody could be an attractive agent for further clinical investigation.-Sioud, M., Westby, P., Vasovic, V., Fløisand, Y., Peng, Q. Development of a new high-affinity human antibody with antitumor activity against solid and

  12. Monoclonal antibodies technology. Protocols

    International Nuclear Information System (INIS)

    Acevado Castro, B.E.

    1997-01-01

    reached at least 3/4 confluency, 2 ampules/cell culture. Save the supernatants for further studies. Regarding to separate the interested cells of irrelevant cells, you must been do a limited diluted procedures. Ascitis production. To produce ascitis the number of cells depend of the every particular clone. Usually we inoculated ip between 1 to 2 x10 6 cells/ml/mice in PBS buffer. The animals must be inoculated intraperitonealy with Pristan (Sigma) or mineral oil using 0.5 ml per mice. The inoculation could be occurred 7 to 10 days before to inoculated the clone. The weight of the animals must be controlled in order to obtain better yield. The bioreactors are an alternative method to produce AcMs without it have not used animals models. IgG purification process on protein A columns. Mouse antibodies of the IgG 1 subclass do not have a high affinity for protein A. Every subclass could be eluted at different peaks using a gradient pH. Usually we use 3M NaCl/1.5 M Gly pH 8.9 as a coupling buffer. For eluting IgGs we use 0.1M Citric Acid in a gradient pH (6 to IgG 1 , 5 to IgG 2a or IgG 2b , 4 IgG 3 ). 1. Adjust a Sepharose Protein A (Pharmacia) beads using NaCl/1.5 M Gly pH 8.9 (coupling buffer). 2. Diluted the ascitis 1:10 in coupling buffer using a filter paper for remove the aggregations. 3. Pass the solution trough a Protein A bead column at a standarized flow according your equipment. The capacity of the Protein A beads for IgGl is approximately 5 mg/ml of wet beads. Ascitis contain between 1-10 mg/ml. 4. Wash the colums with 10 volumes of coupling buffer. 5. Elute the column with 0.1 M Citric Acid in a gradient pH starting at 6 to 4. Collect the eluate in appropriate tube, and identify the immunoglobulin-containing fractions by absorbance a 280 nm (1 DO = 0.8 mg/ml)

  13. Detection of Waterborne Viruses Using High Affinity Molecularly Imprinted Polymers.

    Science.gov (United States)

    Altintas, Zeynep; Gittens, Micah; Guerreiro, Antonio; Thompson, Katy-Anne; Walker, Jimmy; Piletsky, Sergey; Tothill, Ibtisam E

    2015-07-07

    Molecularly imprinted polymers (MIPs) are artificial receptor ligands which can recognize and specifically bind to a target molecule. They are more resistant to chemical and biological damage and inactivation than antibodies. Therefore, target specific-MIP nanoparticles are aimed to develop and implemented to biosensors for the detection of biological toxic agents such as viruses, bacteria, and fungi toxins that cause many diseases and death due to the environmental contamination. For the first time, a molecularly imprinted polymer (MIP) targeting the bacteriophage MS2 as the template was investigated using a novel solid-phase synthesis method to obtain the artificial affinity ligand for the detection and removal of waterborne viruses through optical-based sensors. A high affinity between the artificial ligand and the target was found, and a regenerative MIP-based virus detection assay was successfully developed using a new surface plasmon resonance (SPR)-biosensor which provides an alternative technology for the specific detection and removal of waterborne viruses that lead to high disease and death rates all over the world.

  14. Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay.

    Science.gov (United States)

    Hall, Justin; Brault, Amy; Vincent, Fabien; Weng, Shawn; Wang, Hong; Dumlao, Darren; Aulabaugh, Ann; Aivazian, Dikran; Castro, Dana; Chen, Ming; Culp, Jeffrey; Dower, Ken; Gardner, Joseph; Hawrylik, Steven; Golenbock, Douglas; Hepworth, David; Horn, Mark; Jones, Lyn; Jones, Peter; Latz, Eicke; Li, Jing; Lin, Lih-Ling; Lin, Wen; Lin, David; Lovering, Frank; Niljanskul, Nootaree; Nistler, Ryan; Pierce, Betsy; Plotnikova, Olga; Schmitt, Daniel; Shanker, Suman; Smith, James; Snyder, William; Subashi, Timothy; Trujillo, John; Tyminski, Edyta; Wang, Guoxing; Wong, Jimson; Lefker, Bruce; Dakin, Leslie; Leach, Karen

    2017-01-01

    Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2', 3' -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2'-5' and 3'-5' phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (KD = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.

  15. Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay

    Energy Technology Data Exchange (ETDEWEB)

    Hall, Justin; Brault, Amy; Vincent, Fabien; Weng, Shawn; Wang, Hong; Dumlao, Darren; Aulabaugh, Ann; Aivazian, Dikran; Castro, Dana; Chen, Ming; Culp, Jeffrey; Dower, Ken; Gardner, Joseph; Hawrylik, Steven; Golenbock, Douglas; Hepworth, David; Horn, Mark; Jones, Lyn; Jones, Peter; Latz, Eicke; Li, Jing; Lin, Lih-Ling; Lin, Wen; Lin, David; Lovering, Frank; Niljanskul, Nootaree; Nistler, Ryan; Pierce, Betsy; Plotnikova, Olga; Schmitt, Daniel; Shanker, Suman; Smith, James; Snyder, William; Subashi, Timothy; Trujillo, John; Tyminski, Edyta; Wang, Guoxing; Wong, Jimson; Lefker, Bruce; Dakin, Leslie; Leach, Karen (UMASS, MED); (Pfizer)

    2017-09-21

    Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2', 3' -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2'-5' and 3'-5' phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (KD = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.

  16. A novel TNFα antagonizing peptide-Fc fusion protein designed based on CDRs of TNFα neutralizing monoclonal antibody

    International Nuclear Information System (INIS)

    Qin Weisong; Feng Jiannan; Zhang Wei; Li Yan; Shen, Beifen

    2004-01-01

    The variable regions of antibody molecules bind antigens with high affinity and specificity. The binding sites are imparted largely to the hypervariable portions (i.e., CDRs) of the variable region. Peptides derived from CDRs can bind antigen with similar specificity acting as mimic of antibody and become drug-designing core, although with markedly lower affinity. In order to increase the affinity and bioactivity, in this study, a novel peptide (PT) designed on CDRs of a TNFα neutralizing monoclonal antibody Z12 was linked with Fc fragment of human IgG1. The interaction mode of PT-linker-Fc (PLF) with TNFα was analyzed with computer-guided molecular modeling method. After expression in Escherichia coli and purification, recombinant PT-linker-Fc could bind directly with the TNFα coated on the ELISA plates. Furthermore, PLF could competitively inhibit the binding of Z12 to TNFα and also inhibit the TNFα-induced cytotoxicity on L929 cells. The TNFα antagonizing activity of PLF was significantly higher than that of the free peptide. This study highlights the potential of human Fc to enhance the potency of peptides designed on the CDRs of antibodies and could be useful in developing new TNFα antagonists

  17. Effect of pH, temperature, and salt on the stability of Escherichia coli- and Chinese hamster ovary cell-derived IgG1 Fc.

    Science.gov (United States)

    Li, Cynthia H; Narhi, Linda O; Wen, Jie; Dimitrova, Mariana; Wen, Zai-qing; Li, Jenny; Pollastrini, Joseph; Nguyen, Xichdao; Tsuruda, Trace; Jiang, Yijia

    2012-12-18

    The circulation half-life of a potential therapeutic can be increased by fusing the molecule of interest (an active peptide, the extracellular domain of a receptor, an enzyme, etc.) to the Fc fragment of a monoclonal antibody. For the fusion protein to be a successful therapeutic, it must be stable to process and long-term storage conditions, as well as to physiological conditions. The stability of the Fc used is critical for obtaining a successful therapeutic protein. The effects of pH, temperature, and salt on the stabilities of Escherichia coli- and Chinese hamster ovary cell (CHO)-derived IgG1 Fc high-order structure were probed using a variety of biophysical techniques. Fc molecules derived from both E. coli and CHO were compared. The IgG1 Fc molecules from both sources (glycosylated and aglycosylated) are folded at neutral pH and behave similarly upon heat- and low pH-induced unfolding. The unfolding of both IgG1 Fc molecules occurs via a multistep unfolding process, with the tertiary structure and C(H)2 domain unfolding first, followed by changes in the secondary structure and C(H)3 domain. The acid-induced unfolding of IgG1 Fc molecules is only partially reversible, with the formation of high-molecular weight species. The CHO-derived Fc protein (glycosylated) is more compact (smaller hydrodynamic radius) than the E. coli-derived protein (aglycosylated) at neutral pH. Unfolding is dependent on pH and salt concentration. The glycosylated C(H)2 domain melts at a temperature 4-5 °C higher than that of the aglycosylated domain, and the low-pH-induced unfolding of the glycosylated Fc molecule occurs at a pH ~0.5 pH unit lower than that of the aglycosylated protein. The difference observed between E. coli- and CHO-derived Fc molecules primarily involves the C(H)2 domain, where the glycosylation of the Fc resides.

  18. Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms.

    Science.gov (United States)

    Yang, Danlin; Singh, Ajit; Wu, Helen; Kroe-Barrett, Rachel

    2017-04-17

    Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the use of four routinely used biosensor platforms in our laboratory to evaluate the binding affinity and kinetics of ten high-affinity monoclonal antibodies (mAbs) against human proprotein convertase subtilisin kexin type 9 (PCSK9). While both Biacore T100 and ProteOn XPR36 are derived from the well-established Surface Plasmon Resonance (SPR) technology, the former has four flow cells connected by serial flow configuration, whereas the latter presents 36 reaction spots in parallel through an improvised 6 x 6 crisscross microfluidic channel configuration. The IBIS MX96 also operates based on the SPR sensor technology, with an additional imaging feature that provides detection in spatial orientation. This detection technique coupled with the Continuous Flow Microspotter (CFM) expands the throughput significantly by enabling multiplex array printing and detection of 96 reaction sports simultaneously. In contrast, the Octet RED384 is based on the BioLayer Interferometry (BLI) optical principle, with fiber-optic probes acting as the biosensor to detect interference pattern changes upon binding interactions at the tip surface. Unlike the SPR-based platforms, the BLI system does not rely on continuous flow fluidics; instead, the sensor tips collect readings while they are immersed in analyte solutions of a 384-well microplate during orbital agitation. Each of these biosensor platforms has its own advantages and disadvantages. To provide a direct comparison of these instruments' ability to provide quality kinetic data, the described protocols illustrate experiments that use the same assay format and the same high-quality reagents to characterize antibody-antigen kinetics that fit the simple 1:1 molecular interaction model.

  19. Engineering of bispecific affinity proteins with high affinity for ERBB2 and adaptable binding to albumin.

    Directory of Open Access Journals (Sweden)

    Johan Nilvebrant

    Full Text Available The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein.

  20. Monoclonal antibodies specific for the organophosphate pesticide azinphos-methyl

    NARCIS (Netherlands)

    Jones, WT; Harvey, D; Jones, SD; Ryan, GB; Wynberg, H; TenHoeve, W; Reynolds, PHS

    1995-01-01

    2-(2-Mercapto-5-methyl-1,3,2-dioxaphosphorinan-5-yl,2-sulphide) methoxyacetic acid has been synthesized and used to prepare an azinphos hapten and protein conjugates. Monoclonal antibodies of high affinity against the pesticide azinphos-methyl were prepared from mice immunized with the

  1. Fed-batch bioreactor performance and cell line stability evaluation of the artificial chromosome expression technology expressing an IgG1 in Chinese hamster ovary cells.

    Science.gov (United States)

    Combs, Rodney G; Yu, Erwin; Roe, Susanna; Piatchek, Michele Bailey; Jones, Heather L; Mott, John; Kennard, Malcolm L; Goosney, Danika L; Monteith, Diane

    2011-01-01

    The artificial chromosome expression (ACE) technology system uses an engineered artificial chromosome containing multiple site-specific recombination acceptor sites for the rapid and efficient construction of stable cell lines. The construction of Chinese hamster ovary(CHO) cell lines expressing an IgG1 monoclonal antibody (MAb) using the ACE system has been previously described (Kennard et al., Biotechnol Bioeng. 2009;104:540-553). To further demonstrate the manufacturing feasibility of the ACE system, four CHO cell lines expressing the human IgG1 MAb 4A1 were evaluated in batch and fed-batch shake flasks and in a 2-L fed-batch bioreactor. The batch shake flasks achieved titers between 0.7 and 1.1 g/L, whereas the fed-batch shake flask process improved titers to 2.5–3.0 g/L. The lead 4A1 ACE cell line achieved titers of 4.0 g/L with an average specific productivity of 40 pg/(cell day) when cultured in a non optimized 2-L fed-batch bioreactor using a completely chemically defined process. Generational stability characterization of the lead 4A1-expressing cell line demonstrated that the cell line was stable for up to 75 days in culture. Product quality attributes of the 4A1 MAb produced by the ACE system during the stability evaluation period were unchanged and also comparable to existing expression technologies such as the CHO-dhfr system. The results of this evaluation demonstrate that a clonal, stable MAb-expressing CHO cell line can be produced using ACE technology that performs competitively using a chemically defined fed-batch bioreactor process with comparable product quality attributes to cell lines generated by existing technologies.

  2. Structure-based engineering to restore high affinity binding of an isoform-selective anti-TGFβ1 antibody

    Science.gov (United States)

    Honey, Denise M.; Best, Annie; Qiu, Huawei

    2018-01-01

    ABSTRACT Metelimumab (CAT192) is a human IgG4 monoclonal antibody developed as a TGFβ1-specific antagonist. It was tested in clinical trials for the treatment of scleroderma but later terminated due to lack of efficacy. Subsequent characterization of CAT192 indicated that its TGFβ1 binding affinity was reduced by ∼50-fold upon conversion from the parental single-chain variable fragment (scFv) to IgG4. We hypothesized this result was due to decreased conformational flexibility of the IgG that could be altered via engineering. Therefore, we designed insertion mutants in the elbow region and screened for binding and potency. Our results indicated that increasing the elbow region linker length in each chain successfully restored the isoform-specific and high affinity binding of CAT192 to TGFβ1. The crystal structure of the high binding affinity mutant displays large conformational rearrangements of the variable domains compared to the wild-type antigen-binding fragment (Fab) and the low binding affinity mutants. Insertion of two glycines in both the heavy and light chain elbow regions provided sufficient flexibility for the variable domains to extend further apart than the wild-type Fab, and allow the CDR3s to make additional interactions not seen in the wild-type Fab structure. These interactions coupled with the dramatic conformational changes provide a possible explanation of how the scFv and elbow-engineered Fabs bind TGFβ1 with high affinity. This study demonstrates the benefits of re-examining both structure and function when converting scFv to IgG molecules, and highlights the potential of structure-based engineering to produce fully functional antibodies. PMID:29333938

  3. Antigen-binding properties of monoclonal antibodies reactive with EBNA1 and use in immunoaffinity chromatography.

    Directory of Open Access Journals (Sweden)

    Sarah J Duellman

    Full Text Available Epstein-Barr virus (EBV nuclear antigen 1 (EBNA1 was overexpressed and purified from Escherichia coli. Mouse monoclonal antibodies (mAbs were prepared that react with EBNA1. Eleven high affinity mAbs were recovered. Nine mAbs are isotype IgG (all subisotype IgG(1 and two mAbs are isotype IgM. All mAbs react strongly with EBNA1 in an ELISA assay while only one mAb (designated 1EB6 fails to react in a Western blot assay. The epitopes for these mAbs were mapped to seven different regions, providing good coverage of the entire EBNA1 protein. The mAbs had differing affinity for an EBNA1/DNA complex with four mAbs able to supershift the complex completely. All mAbs can immunoprecipitate EBNA1 from E. coli overexpressing EBNA1. A modified ELISA assay, termed ELISA-elution assay, was used to screen for mAbs that release EBNA1 in the presence of a low molecular weight polyhydroxylated compound (polyol and a nonchaotropic salt. MAbs with this property, termed polyol-responsive (PR-mAbs, allow gentle elution of labile proteins and protein complexes. Four mAbs are polyol-responsive with two showing usefulness in gentle immunoaffinity chromatography. Purification with these PR-mAbs may be useful in purifying EBNA1 complexes and elucidating EBNA1-associated proteins. This panel of anti-EBNA1 mAbs will advance the study of EBV by providing new tools to detect and purify EBNA1.

  4. Monoclonal antibody

    International Nuclear Information System (INIS)

    Oyamada, Hiyoshimaru

    1987-01-01

    Some aspects of monoclonal antibodies are described, centering on studies made by the author and those presented at the Second International Conference on Monoclonal Antibody Immunoconjugates for Cancer held in March this year (1987). The history of immuno-nuclear medicine and procedures for producing monoclonal antibodies are briefly outlined. Monoclonal antibodies are immunoglobulins. Here, the structure of IgG, which is used most frequently, is described. An IgG is composed of two antigen binding fragments (Fab) and one crystallizable fragment (Fc). The end portion of a Fab reacts with an antigen. One of the major applications of immuno-nuclear medicine is the diagnosis of cancer. As label nucleides, 131 I and 111 I were selected in most cases in the past while 123 I and 99m Tc are currently used more often. Advantages and disadvantages of this diagnosis method is discussed citing studies presented at the First (1986) and Second (1987) International Conference on Monoclonal Antibody Immunoconjugates for Cancer. The present status of the application of monoclonal antibodies to treatment of cancer is also described. (Nogami, K.)

  5. Generation of monoclonal antibodies against prostate specific antigen (PSA) for the detection of PSA and its purification; Generación de anticuerpos monoclonales contra el antígeno específico de próstata (PSA) para la detección del PSA y su purificación

    Energy Technology Data Exchange (ETDEWEB)

    Acevedo Castro, Boris Ernesto [Centro de Ingeniería Genética y Biotecnología, CIGB, La Habana (Cuba)

    2012-07-01

    The prostate cancer in Cuba is a problem of health (2672 diagnosed cases and 2769 deaths in 2007). Various diagnostic methods have been implemented for the detection and management of this disease, emphasizing among them (PSA) prostate-specific antigen serological determination. At this work was generated and characterized a panel of 11 antibodies (AcMs) monoclonal IgG1 detected with high affinity described major epitopes of the PSA, both in solution and attached to the test plate. From the panel obtained AcMs was the standardization of an essay type ELISA for the detection of serum total PSA (associated and free) equimolar, based on antibody monoclonal CB-PSA.4 in the coating and the CB-PSA.9 coupled with biotin as liner, with a detection limit of 0.15 ng/mL. Similarly, standardized system for detection in serum free PSA, based on the AcMs CB-PSA.4 (coating) and CB-PSA.2 coupled with biotin (liner), with a detection limit of 0.5 ng/mL. Finally, with the purpose of using PSA as standard in trials type ELISA, developed a simple method of inmunopurificación based on the AcM, CB-PSA.2, which was obtained the PSA with a purity exceeding 90%. Immunoassay Centre on the basis of the AcMs panel and the results of this study, developed and recorded two diagnostic systems for the detection of PSA in human serum. (author)

  6. A recombinant, fully human monoclonal antibody with antitumor activity constructed from phage-displayed antibody fragments

    NARCIS (Netherlands)

    Huls, GA; Heijnen, IAFM; Cuomo, ME; Koningsberger, JC; Boel, E; de Vries, ARV; Loyson, SAJ; Helfrich, W; Henegouwen, GPV; van Meijer, M; de Kruif, J; Logtenberg, T

    A single-chain Fv antibody fragment specific for the tumor-associated Ep-CAM molecule was isolated from a semisynthetic phage display library and converted into an intact, fully human IgG1 monoclonal antibody (huMab), The purified huMab had an affinity of 5 nM and effectively mediated tumor cell

  7. Human anti-rhesus D IgG1 antibody produced in transgenic plants

    DEFF Research Database (Denmark)

    Bouquin, Thomas; Thomsen, Mads; Nielsen, Leif Kofoed

    2002-01-01

    antigen, which is responsible for alloimmunization of RhD- mothers carrying an RhD+ fetus. Anti-RhD extracted from plants specifically reacted with RhD+ cells in antiglobulin technique, and elicited a respiratory burst in human peripheral blood mononuclear cells. Plant-derived antibody had equivalent......Transgenic plants represent an alternative to cell culture systems for producing cheap and safe antibodies for diagnostic and therapeutic use. To evaluate the functional properties of a 'plantibody', we generated transgenic Arabidopsis plants expressing full-length human IgG1 against the Rhesus D...... properties to CHO cell-produced anti-RhD antibody, indicating its potential usefulness in diagnostic and therapeutic programs....

  8. A novel lentiviral scFv display library for rapid optimization and selection of high affinity antibodies.

    Science.gov (United States)

    Qudsia, Sehar; Merugu, Siva B; Mangukiya, Hitesh B; Hema, Negi; Wu, Zhenghua; Li, Dawei

    2018-04-30

    Antibody display libraries have become a popular technique to screen monoclonal antibodies for therapeutic purposes. An important aspect of display technology is to generate an optimization library by changing antibody affinity to antigen through mutagenesis and screening the high affinity antibody. In this study, we report a novel lentivirus display based optimization library antibody in which Agtuzumab scFv is displayed on cell membrane of HEK-293T cells. To generate an optimization library, hotspot mutagenesis was performed to achieve diverse antibody library. Based on sequence analysis of randomly selected clones, library size was estimated approximately to be 1.6 × 10 6 . Lentivirus display vector was used to display scFv antibody on cell surface and flow cytometery was performed to check the antibody affinity to antigen. Membrane bound scFv antibodies were then converted to secreted antibody through cre/loxP recombination. One of the mutant clones, M8 showed higher affinity to antigen in flow cytometery analysis. Further characterization of cellular and secreted scFv through western blot showed that antibody affinity was increased by three fold after mutagenesis. This study shows successful construction of a novel antibody library and suggests that hotspot mutagenesis could prove a useful and rapid optimization tool to generate similar libraries with various degree of antigen affinity. Copyright © 2018 Elsevier Inc. All rights reserved.

  9. Detection of IgG1 and IgG4 subtypes reactive against potato apyrase in schistosomiasis patients

    Directory of Open Access Journals (Sweden)

    Priscila de Faria-Pinto

    2010-07-01

    Full Text Available In this paper, we showed for the first time that the conserved domains within Schistosoma mansoni ATP diphosphohydrolase isoforms, shared with potato apyrase, possess epitopes for the IgG1 and IgG4 subtypes, as 24 (80% of the 30 schistosomiasis patients were seropositive for this vegetable protein. The analyses for each patient cured (n = 14 after treatment (AT with praziquantel revealed variable IgG1 and IgG4 reactivity against potato apyrase. Different antigenic epitopes shared between the vegetable and parasite proteins could be involved in susceptibility or resistance to S. mansoni AT with praziquantel and these possibilities should be explored.

  10. Fcγ1 fragment of IgG1 as a powerful affinity tag in recombinant Fc-fusion proteins: immunological, biochemical and therapeutic properties.

    Science.gov (United States)

    Soleimanpour, Saman; Hassannia, Tahereh; Motiee, Mahdieh; Amini, Abbas Ali; Rezaee, S A R

    2017-05-01

    Affinity tags are vital tools for the production of high-throughput recombinant proteins. Several affinity tags, such as the hexahistidine tag, maltose-binding protein, streptavidin-binding peptide tag, calmodulin-binding peptide, c-Myc tag, glutathione S-transferase and FLAG tag, have been introduced for recombinant protein production. The fragment crystallizable (Fc) domain of the IgG1 antibody is one of the useful affinity tags that can facilitate detection, purification and localization of proteins and can improve the immunogenicity, modulatory effects, physicochemical and pharmaceutical properties of proteins. Fcγ recombinant forms a group of recombinant proteins called Fc-fusion proteins (FFPs). FFPs are widely used in drug discovery, drug delivery, vaccine design and experimental research on receptor-ligand interactions. These fusion proteins have become successful alternatives to monoclonal antibodies for drug developments. In this review, the physicochemical, biochemical, immunological, pharmaceutical and therapeutic properties of recombinant FFPs were discussed as a new generation of bioengineering strategies.

  11. Relative stabilities of IgG1 and IgG4 Fab domains: Influence of the light–heavy interchain disulfide bond architecture

    Science.gov (United States)

    Heads, James T; Adams, Ralph; D'Hooghe, Lena E; Page, Matt J T; Humphreys, David P; Popplewell, Andrew G; Lawson, Alastair D; Henry, Alistair J

    2012-01-01

    The stability of therapeutic antibodies is a prime pharmaceutical concern. In this work we examined thermal stability differences between human IgG1 and IgG4 Fab domains containing the same variable regions using the thermofluor assay. It was found that the IgG1 Fab domain is up to 11°C more stable than the IgG4 Fab domain containing the same variable region. We investigated the cause of this difference with the aim of developing a molecule with the enhanced stability of the IgG1 Fab and the biological properties of an IgG4 Fc. We found that replacing the seven residues, which differ between IgG1 CH1 and IgG4 CH1 domains, while retaining the native IgG1 light-heavy interchain disulfide (L–H) bond, did not affect thermal stability. Introducing the IgG1 type L–H interchain disulfide bond (DSB) into the IgG4 Fab resulted in an increase in thermal stability to levels observed in the IgG1 Fab with the same variable region. Conversely, replacement of the IgG1 L–H interchain DSB with the IgG4 type L–H interchain DSB reduced the thermal stability. We utilized the increased stability of the IgG1 Fab and designed a hybrid antibody with an IgG1 CH1 linked to an IgG4 Fc via an IgG1 hinge. This construct has the expected biophysical properties of both the IgG4 Fc and IgG1 Fab domains and may therefore be a pharmaceutically relevant format. PMID:22761163

  12. N-Oxide analogs of WAY-100635 : new high affinity 5-HT (1A) receptor antagonists

    NARCIS (Netherlands)

    Oberwinkler - Marchais, Sandrine; Nowicki, B; Pike, VW; Halldin, C; Sandell, J; Chou, YH; Gulyas, B; Brennum, LT; Farde, L; Wikstrom, H V

    2005-01-01

    WAY-100635 [N-(2-(1-(4-(2-methoxyphenyl)piperazinyl)ethyl))-N-(2-pyridinyl)cyclohexanecarboxamide] 1 and its O-des-methyl derivative DWAY 2 are well-known high affinity 5-HT1A receptor antagonists. which when labeled with carbon-II (beta(+): t(1/2) 20.4min) in the carbonyl group are effective

  13. ISOLATION AND CHARACTERIZATION OF THE HIGH-AFFINITY K+-TRANSLOCATING ATPASE FROM RHODOBACTER-SPHAEROIDES

    NARCIS (Netherlands)

    ABEE, T; SIEBERS, A; ALTENDORF, K; KONINGS, WN

    1992-01-01

    Cells of the purple nonsulfur bacterium Rhodobacter sphaeroides express a high-affinity K+ uptake system when grown in media with low K+ concentrations. A vanadate-sensitive, K+-stimulated and Mg2+-stimulated ATPase was purified from membranes of these cells by solubilization with

  14. High affinity calmodulin target sequence in the signalling molecule PI 3-kinase

    DEFF Research Database (Denmark)

    Fischer, R; Julsgart, J; Berchtold, M W

    1998-01-01

    M-binding peptide derived from the p110gamma isoform interacts with CaM in a calcium-dependent way. Using gel shift analysis and fluorescence spectrophotometry we discovered that the peptide forms a high affinity complex with CaM. Titration experiments using dansylated CaM gave an affinity constant of 5 n...

  15. The role of CH/π interactions in the high affinity binding of streptavidin and biotin.

    Science.gov (United States)

    Ozawa, Motoyasu; Ozawa, Tomonaga; Nishio, Motohiro; Ueda, Kazuyoshi

    2017-08-01

    The streptavidin-biotin complex has an extraordinarily high affinity (Ka: 10 15 mol -1 ) and contains one of the strongest non-covalent interactions known. This strong interaction is widely used in biological tools, including for affinity tags, detection, and immobilization of proteins. Although hydrogen bond networks and hydrophobic interactions have been proposed to explain this high affinity, the reasons for it remain poorly understood. Inspired by the deceased affinity of biotin observed for point mutations of streptavidin at tryptophan residues, we hypothesized that a CH/π interaction may also contribute to the strong interaction between streptavidin and biotin. CH/π interactions were explored and analyzed at the biotin-binding site and at the interface of the subunits by the fragment molecular orbital method (FMO) and extended applications: PIEDA and FMO4. The results show that CH/π interactions are involved in the high affinity for biotin at the binding site of streptavidin. We further suggest that the involvement of CH/π interactions at the subunit interfaces and an extended CH/π network play more critical roles in determining the high affinity, rather than involvement at the binding site. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Biotin-avidin sandwich elisa with specific human isotypes IgG1 and IgG4 for Culicidae mosquito blood meal identification from an epizootic yellow fever area in Brazil

    Directory of Open Access Journals (Sweden)

    AM Marassá

    2009-01-01

    Full Text Available With a view toward investigating the feeding behavior of Culicidae mosquitoes from an area of epizootic yellow fever transmission in the municipalities of Garruchos and Santo Antônio das Missões, Rio Grande do Sul State, Brazil, specimens were collected by aspiration from September 2005 to April 2007. The engorged females were submitted to blood meal identification by enzyme-linked immunosorbent assay (ELISA. A total of 142 blood-engorged samples were examined for human or monkey blood through species-specific IgG. Additional tests for specificity utilizing isotypes IgG1 and IgG4 of human monoclonal antibodies showed that only anti-human IgG1 was effective in recognizing blood meals of human origin. The results indicated a significant difference (p = 0.027 in detection patterns in samples of Haemagogus leucocelaenus recorded from human blood meals at Santo Antônio das Missões, which suggests some degree of exposure, since it was an area where epizootic outbreaks have been reported.

  17. High throughput production of mouse monoclonal antibodies using antigen microarrays

    DEFF Research Database (Denmark)

    De Masi, Federico; Chiarella, P.; Wilhelm, H.

    2005-01-01

    Recent advances in proteomics research underscore the increasing need for high-affinity monoclonal antibodies, which are still generated with lengthy, low-throughput antibody production techniques. Here we present a semi-automated, high-throughput method of hybridoma generation and identification....... Monoclonal antibodies were raised to different targets in single batch runs of 6-10 wk using multiplexed immunisations, automated fusion and cell-culture, and a novel antigen-coated microarray-screening assay. In a large-scale experiment, where eight mice were immunized with ten antigens each, we generated...

  18. A novel anti-GPC3 monoclonal antibody (YP7) | Center for Cancer Research

    Science.gov (United States)

    Glypican-3 (GPC3) is an emerging therapeutic target in hepatoma. A novel anti-GPC3 monoclonal antibody (YP7) has been generated through a combination of peptide immunization and high-throughput flow cytometry screening. YP7 binds cell-surface-associated GPC3 with high affinity and exhibits significant hepatoma xenograft growth inhibition in nude mice. The new antibody may have

  19. The fourth dimension in immunological space: how the struggle for nutrients selects high-affinity lymphocytes.

    Science.gov (United States)

    Wensveen, Felix M; van Gisbergen, Klaas P J M; Eldering, Eric

    2012-09-01

    Lymphocyte activation via the antigen receptor is associated with radical shifts in metabolism and changes in requirements for nutrients and cytokines. Concomitantly, drastic changes occur in the expression of pro-and anti-apoptotic proteins that alter the sensitivity of lymphocytes to limiting concentrations of key survival factors. Antigen affinity is a primary determinant for the capacity of activated lymphocytes to access these vital resources. The shift in metabolic needs and the variable access to key survival factors is used by the immune system to eliminate activated low-affinity cells and to generate an optimal high-affinity response. In this review, we focus on the control of apoptosis regulators in activated lymphocytes by nutrients, cytokines, and costimulation. We propose that the struggle among individual clones that leads to the formation of high-affinity effector cell populations is in effect an 'invisible' fourth signal required for effective immune responses. © 2012 John Wiley & Sons A/S.

  20. Selective high-affinity polydentate ligands and methods of making such

    Energy Technology Data Exchange (ETDEWEB)

    Denardo, Sally J.; Denardo, Gerald L.; Balhorn, Rodney L.

    2018-02-06

    This invention provides novel polydentate selective high affinity ligands (SHALs) that can be used in a variety of applications in a manner analogous to the use of antibodies. SHALs typically comprise a multiplicity of ligands that each bind different region son the target molecule. The ligands are joined directly or through a linker thereby forming a polydentate moiety that typically binds the target molecule with high selectivity and avidity.

  1. Quantifying high-affinity binding of hydrophobic ligands by isothermal titration calorimetry.

    Science.gov (United States)

    Krainer, Georg; Broecker, Jana; Vargas, Carolyn; Fanghänel, Jörg; Keller, Sandro

    2012-12-18

    A fast and reliable quantification of the binding thermodynamics of hydrophobic high-affinity ligands employing a new calorimetric competition experiment is described. Although isothermal titration calorimetry is the method of choice for a quantitative characterization of intermolecular interactions in solution, a reliable determination of a dissociation constant (K(D)) is typically limited to the range 100 μM > K(D) > 1 nM. Interactions displaying higher or lower K(D) values can be assessed indirectly, provided that a suitable competing ligand is available whose K(D) falls within the directly accessible affinity window. This established displacement assay, however, requires the high-affinity ligand to be soluble at high concentrations in aqueous buffer and, consequently, poses serious problems in the study of protein binding involving small-molecule ligands dissolved in organic solvents--a familiar case in many drug-discovery projects relying on compound libraries. The calorimetric competition assay introduced here overcomes this limitation, thus allowing for a detailed thermodynamic description of high-affinity receptor-ligand interactions involving poorly water-soluble compounds. Based on a single titration of receptor into a dilute mixture of the two competing ligands, this competition assay provides accurate and precise values for the dissociation constants and binding enthalpies of both high- and moderate-affinity ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation and high-affinity protein-inhibitor interactions, and explore its potential and limitations with the aid of simulations and statistical analyses.

  2. Amyloid-beta binds catalase with high affinity and inhibits hydrogen peroxide breakdown.

    OpenAIRE

    Milton, N G

    1999-01-01

    Amyloid-beta (Abeta) specifically bound purified catalase with high affinity and inhibited catalase breakdown of H(2)O(2). The Abeta-induced catalase inhibition involved formation of the inactive catalase Compound II and was reversible. CatalaseAbeta interactions provide rapid functional assays for the cytotoxic domain of Abeta and suggest a mechanism for some of the observed actions of Abeta plus catalase in vitro.

  3. Reconstitution of high-affinity opioid agonist binding in brain membranes

    Energy Technology Data Exchange (ETDEWEB)

    Remmers, A.E.; Medzihradsky, F. (Univ. of Michigan Medical School, Ann Arbor (United States))

    1991-03-15

    In synaptosomal membranes from rat brain cortex, the {mu} selective agonist ({sup 3}H)dihydromorphine in the absence of sodium, and the nonselective antagonist ({sup 3}H)naltrexone in the presence of sodium, bound to two populations of opioid receptor sites with K{sub d} values of 0.69 and 8.7 nM for dihydromorphine, and 0.34 and 5.5 nM for naltrexone. The addition of 5 {mu}M guanosine 5{prime}-({gamma}-thio)triphosphate (GTP({gamma}S)) strongly reduced high-affinity agonist but not antagonist binding. Exposure of the membranes to high pH reduced the number of GTP({gamma}-{sup 35}S) binding sites by 90% and low K{sub m}, opioid-sensitive GTPase activity by 95%. In these membranes, high-affinity agonist binding was abolished and modulation of residual binding by GTP({gamma}S) was diminished. Alkali treatment of the glioma cell membranes prior to fusion inhibited most of the low K{sub m} GTPase activity and prevented the reconstitution of agonist binding. The results show that high-affinity opioid agonist binding reflects the ligand-occupied receptor - guanine nucleotide binding protein complex.

  4. Single-experiment displacement assay for quantifying high-affinity binding by isothermal titration calorimetry.

    Science.gov (United States)

    Krainer, Georg; Keller, Sandro

    2015-04-01

    Isothermal titration calorimetry (ITC) is the gold standard for dissecting the thermodynamics of a biomolecular binding process within a single experiment. However, reliable determination of the dissociation constant (KD) from a single titration is typically limited to the range 100 μM>KD>1 nM. Interactions characterized by a lower KD can be assessed indirectly by so-called competition or displacement assays, provided that a suitable competitive ligand is available whose KD falls within the directly accessible window. However, this protocol is limited by the fact that it necessitates at least two titrations to characterize one high-affinity inhibitor, resulting in considerable consumption of both sample material and time. Here, we introduce a fast and efficient ITC displacement assay that allows for the simultaneous characterization of both a high-affinity ligand and a moderate-affinity ligand competing for the same binding site on a receptor within a single experiment. The protocol is based on a titration of the high-affinity ligand into a solution containing the moderate-affinity ligand bound to the receptor present in excess. The resulting biphasic binding isotherm enables accurate and precise determination of KD values and binding enthalpies (ΔH) of both ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation, explore its potential and limitations with the aid of simulations and statistical analyses, and elaborate on potential applications to protein-inhibitor interactions. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates

    Directory of Open Access Journals (Sweden)

    Tal Noy-Porat

    2016-03-01

    Full Text Available Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (koff < 1 × 10−7 s−1 that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication.

  6. Proadifen-sensitive high affinity binding of 3H-alaproclate to liver membranes

    International Nuclear Information System (INIS)

    Ross, S.B.

    1987-01-01

    3 H-alaproclate, a selective 5 h ydroxytryptamine uptake inhibitor, was found to bind to microsomal membranes from the rat liver with high affinity (K D -=3 nM) and large capacity (B max about 2 nmol/g liver). This binding was stereoselective since S-( - )-alaproclate was 30 times more potent than the R-( + )-enantiomer to displace the 3 H-labelled racemate. Proadifen (SKF 525A), an inhibitor of cytochrome P-450, displaced the 3 H-alaproclate binding with the same, high affinity (K i =3 nM) as alaproclate itself. Repeated treatment with phenobarbital sodium (5x75 mg/kg intraperitoneally) increased the number of alaproclate binding sites 7-8 times without changing the affinity. However, most of the phenobarbital induced 3 H-alaproclate binding was not displaceable by proadifen, showing the presence of at least two different high affinity binding sites. The possible involvement of cytochrome P-450 in the alaproclate binding is discussed. (author)

  7. Proadifen-sensitive high affinity binding of /sup 3/H-alaproclate to liver membranes

    Energy Technology Data Exchange (ETDEWEB)

    Ross, S.B.

    1987-01-01

    /sup 3/H-alaproclate, a selective 5/sub h/ydroxytryptamine uptake inhibitor, was found to bind to microsomal membranes from the rat liver with high affinity (K/sub D/-=3 nM) and large capacity (B/sub max/ about 2 nmol/g liver). This binding was stereoselective since S-( - )-alaproclate was 30 times more potent than the R-( + )-enantiomer to displace the /sup 3/H-labelled racemate. Proadifen (SKF 525A), an inhibitor of cytochrome P-450, displaced the /sup 3/H-alaproclate binding with the same, high affinity (K/sub i/=3 nM) as alaproclate itself. Repeated treatment with phenobarbital sodium (5x75 mg/kg intraperitoneally) increased the number of alaproclate binding sites 7-8 times without changing the affinity. However, most of the phenobarbital induced /sup 3/H-alaproclate binding was not displaceable by proadifen, showing the presence of at least two different high affinity binding sites. The possible involvement of cytochrome P-450 in the alaproclate binding is discussed.

  8. Charge-mediated Fab-Fc interactions in an IgG1 antibody induce reversible self-association, cluster formation, and elevated viscosity.

    Science.gov (United States)

    Arora, Jayant; Hu, Yue; Esfandiary, Reza; Sathish, Hasige A; Bishop, Steven M; Joshi, Sangeeta B; Middaugh, C Russell; Volkin, David B; Weis, David D

    Concentration-dependent reversible self-association (RSA) of monoclonal antibodies (mAbs) poses a challenge to their pharmaceutical development as viable candidates for subcutaneous delivery. While the role of the antigen-binding fragment (Fab) in initiating RSA is well-established, little evidence supports the involvement of the crystallizable fragment (Fc). In this report, a variety of biophysical tools, including hydrogen exchange mass spectrometry, are used to elucidate the protein interface of such non-covalent protein-protein interactions. Using dynamic and static light scattering combined with viscosity measurements, we find that an IgG1 mAb (mAb-J) undergoes RSA primarily through electrostatic interactions and forms a monomer-dimer-tetramer equilibrium. We provide the first direct experimental mapping of the interface formed between the Fab and Fc domains of an antibody at high protein concentrations. Charge distribution heterogeneity between the positively charged interface spanning complementarity-determining regions CDR3H and CDR2L in the Fab and a negatively charged region in C H 3/Fc domain mediates the RSA of mAb-J. When arginine and NaCl are added, they disrupt RSA of mAb-J and decrease the solution viscosity. Fab-Fc domain interactions between mAb monomers may promote the formation of large transient antibody complexes that ultimately cause increases in solution viscosity. Our findings illustrate how limited specific arrangements of amino-acid residues can cause mAbs to undergo RSA at high protein concentrations and how conserved regions in the Fc portion of the antibody can also play an important role in initiating weak and transient protein-protein interactions.

  9. Rationale for combination of therapeutic antibodies targeting tumor cells and immune checkpoint receptors: Harnessing innate and adaptive immunity through IgG1 isotype immune effector stimulation.

    Science.gov (United States)

    Ferris, Robert L; Lenz, Heinz-Josef; Trotta, Anna Maria; García-Foncillas, Jesús; Schulten, Jeltje; Audhuy, François; Merlano, Marco; Milano, Gerard

    2018-02-01

    Immunoglobulin (Ig) G1 antibodies stimulate antibody-dependent cell-mediated cytotoxicity (ADCC). Cetuximab, an IgG1 isotype monoclonal antibody, is a standard-of-care treatment for locally advanced and recurrent and/or metastatic squamous cell carcinoma of the head and neck (SCCHN) and metastatic colorectal cancer (CRC). Here we review evidence regarding the clinical relevance of cetuximab-mediated ADCC and other immune functions and provide a biological rationale concerning why this property positions cetuximab as an ideal partner for immune checkpoint inhibitors (ICIs) and other emerging immunotherapies. We performed a nonsystematic review of available preclinical and clinical data involving cetuximab-mediated immune activity and combination approaches of cetuximab with other immunotherapies, including ICIs, in SCCHN and CRC. Indeed, cetuximab mediates ADCC activity in the intratumoral space and primes adaptive and innate cellular immunity. However, counterregulatory mechanisms may lead to immunosuppressive feedback loops. Accordingly, there is a strong rationale for combining ICIs with cetuximab for the treatment of advanced tumors, as targeting CTLA-4, PD-1, and PD-L1 can ostensibly overcome these immunosuppressive counter-mechanisms in the tumor microenvironment. Moreover, combining ICIs (or other immunotherapies) with cetuximab is a promising strategy for boosting immune response and enhancing response rates and durability of response. Cetuximab immune activity-including, but not limited to, ADCC-provides a strong rationale for its combination with ICIs or other immunotherapies to synergistically and fully mobilize the adaptive and innate immunity against tumor cells. Ongoing prospective studies will evaluate the clinical effect of these combination regimens and their immune effect in CRC and SCCHN and in other indications. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  10. First-in-human phase 1 of YS110, a monoclonal antibody directed against CD26 in advanced CD26-expressing cancers.

    Science.gov (United States)

    Angevin, Eric; Isambert, Nicolas; Trillet-Lenoir, Véronique; You, Benoit; Alexandre, Jérôme; Zalcman, Gérard; Vielh, Philippe; Farace, Françoise; Valleix, Fanny; Podoll, Thomas; Kuramochi, Yu; Miyashita, Itaru; Hosono, Osamu; Dang, Nam H; Ohnuma, Kei; Yamada, Taketo; Kaneko, Yutaro; Morimoto, Chikao

    2017-04-25

    YS110 is a humanised IgG1 monoclonal antibody with high affinity to the CD26 antigen. YS110 demonstrated preclinical anti-tumour effects without significant side effects. This FIH study was designed to determine the maximal tolerated dose (MTD) and recommended phase 2 dose (RP2D) to assess the tolerance, pharmacokinetics (PK) and pharmacodynamics profiles of YS110 and preliminary efficacy. YS110 were initially administered intravenously once every 2 weeks (Q2W) for three doses and then, based on PK data, once every week (Q1W) for five doses in patients with CD26-expressing solid tumours. Thirty-three patients (22 mesothelioma) received a median of 3 (range 1-30) YS110 infusions across six dose levels (0.1-6 mg kg -1 ). MTD was not reached and two dose-limiting toxicities (infusion hypersensitivity reactions) led to the institution of a systemic premedication. Low-grade asthenia (30.3%), hypersensitivity (27.3%), nausea (15.2%), flushing (15.2%), chills (12.1%) and pyrexia (12.1%) were reported as ADRs. Pharmacokinetic parameters (AUC and C max ) increased in proportion with the dose. sCD26/DPPIV assays indicated CD26 modulation. Prolonged stable diseases were observed in 13 out of 26 evaluable patients. YS110 is well tolerated up to 6 mg kg -1 Q1W, which has been defined as the RP2D, with encouraging prolonged disease stabilisations observed in a number of patients with advanced/refractory mesothelioma.

  11. Passive immunization against Cryptococcus neoformans with an isotype-switch family of monoclonal antibodies reactive with cryptococcal polysaccharide.

    OpenAIRE

    Sanford, J E; Lupan, D M; Schlageter, A M; Kozel, T R

    1990-01-01

    The in vivo properties of an immunoglobulin isotype-switch family of monoclonal antibodies specific for the polysaccharide capsule of Cryptococcus neoformans were examined in a murine model of cryptococcosis. Subclass-switch variants were isolated by sequential sublining of an immunoglobulin G subclass 1 (IgG1)-secreting cell line. Antibodies of the IgG1, IgG2a, and IgG2b isotypes with identical reactivities with cryptococcal polysaccharide were prepared. The antibodies had the distinct biolo...

  12. Effect of quinolinic acid in the nucleus basalis magnocellularis on cortical high-affinity choline uptake

    Energy Technology Data Exchange (ETDEWEB)

    Metcalf, R.H.; Boegman, R.J.; Quirion, R.; Riopelle, R.J.; Ludwin, S.K.

    1987-08-01

    A transient 45% increase in cortical high-affinity choline uptake (HACU) was observed after an injection of quinolinic acid (QUIN) into the nucleus basalis magnocellularis (nbM) of the rat. This was followed by a steady decline in choline uptake, which resulted in a 46% decrease by day 7. Specific (/sup 3/H)hemicholinium-3 binding to coronal brain sections showed a similar pattern following injections of QUIN into the nbM. The increase in cortical HACU elicited by QUIN appeared to be dose dependent.

  13. Characteristics of high affinity and low affinity adenosine binding sites in human cerebral cortex

    International Nuclear Information System (INIS)

    John, D.; Fox, I.V.

    1986-01-01

    The binding characteristics of human brain cortical membrane fractions were evaluated to test the hypothesis that there are A 1 and A 2 adenosine binding sites. The ligands used were 2-chloro(8- 3 H) adenosine and N 6 -(adenine-2, 8- 3 H) cyclohexayladenosine. Binding of chloroadenosine to human brain cortical membranes was time dependent, reversible and concentration dependent. The kinetic constant determinations from binding studies of the adenosine receptor are presented. Utilizing tritium-cyclohexyladenosine as ligand the authors observed evidence for a high affinity binding site in human brain cortical membranes with a kd of 5 nM

  14. Novel and high affinity fluorescent ligands for the serotonin transporter based on (s)-citalopram

    DEFF Research Database (Denmark)

    Kumar, Vivek; Rahbek-Clemmensen, Troels; Billesbølle, Christian B

    2014-01-01

    Novel rhodamine-labeled ligands, based on (S)-citalopram, were synthesized and evaluated for uptake inhibition at the human serotonin, dopamine, and norepinephrine transporters (hSERT, hDAT, and hNET, respectively) and for binding at SERT, in transiently transfected COS7 cells. Compound 14 demons...... demonstrated high affinity binding and selectivity for SERT (K i = 3 nM). Visualization of SERT, using confocal laser scanning microscopy, validated compound 14 as a novel tool for studying SERT expression and distribution in living cells....

  15. Kinetics and tissue distribution of the radiolabeled chimeric monoclonal antibody MOv18 IgG and F(ab')2 fragments in ovarian carcinoma patients

    NARCIS (Netherlands)

    Buist, M. R.; Kenemans, P.; den Hollander, W.; Vermorken, J. B.; Molthoff, C. J.; Burger, C. W.; Helmerhorst, T. J.; Baak, J. P.; Roos, J. C.

    1993-01-01

    Twenty-four patients suspected of having ovarian carcinoma received i.v. injection with a combination of radiolabeled intact IgG (1 mg) and F(ab')2 fragments (1 mg) of the chimeric monoclonal antibody MOv18, each form labeled with 1.85 MBq 131I or 125I. Laparotomy was performed either 2 or 6 days

  16. A phase I monotherapy study of RG7212, a first-in-class monoclonal antibody targeting TWEAK signaling in patients with advanced cancers

    DEFF Research Database (Denmark)

    Lassen, Ulrik N; Meulendijks, Didier; Siu, Lilian L

    2015-01-01

    activation. A phase I study of RG7212, a humanized anti-TWEAK IgG1κ monoclonal antibody, was conducted in patients with advanced solid tumors expressing Fn14. EXPERIMENTAL DESIGN: Dose escalations, over a 200- to 7,200-mg range, were performed with patients enrolled in weekly (QW), bi-weekly (Q2W), or every...

  17. High affinity binding of [3H]cocaine to rat liver microsomes

    International Nuclear Information System (INIS)

    El-Maghrabi, E.A.; Calligaro, D.O.; Eldefrawi, M.E.

    1988-01-01

    ] 3 H]cocaine bound reversible, with high affinity and stereospecificity to rat liver microsomes. Little binding was detected in the lysosomal, mitochondrial and nuclear fractions. The binding kinetics were slow and the kinetically calculated K/sub D/ was 2 nM. Induction of mixed function oxidases by phenobarbital did not produce significant change in [ 3 H]cocaine binding. On the other hand, chronic administration of cocaine reduced [ 3 H]cocaine binding drastically. Neither treatment affected the affinity of the liver binding protein for cocaine. Microsomes from mouse and human livers had less cocaine-binding protein and lower affinity for cocaine than those from rat liver. Binding of [ 3 H]cocaine to rat liver microsomes was insensitive to monovalent cations and > 10 fold less sensitive to biogenic amines than the cocaine receptor in rat striatum. However, the liver protein had higher affinity for cocaine and metabolites except for norcocaine. Amine uptake inhibitors displaced [ 3 H]cocaine binding to liver with a different rank order of potency than their displacement of [ 3 H]cocaine binding to striatum. This high affinity [ 3 H]cocaine binding protein in liver is not likely to be monooxygenase, but may have a role in cocaine-induced hepatotoxicity

  18. Acylated heptapeptide binds albumin with high affinity and application as tag furnishes long-acting peptides.

    Science.gov (United States)

    Zorzi, Alessandro; Middendorp, Simon J; Wilbs, Jonas; Deyle, Kaycie; Heinis, Christian

    2017-07-17

    The rapid renal clearance of peptides in vivo limits this attractive platform for the treatment of a broad range of diseases that require prolonged drug half-lives. An intriguing approach for extending peptide circulation times works through a 'piggy-back' strategy in which peptides bind via a ligand to the long-lived serum protein albumin. In accordance with this strategy, we developed an easily synthesized albumin-binding ligand based on a peptide-fatty acid chimera that has a high affinity for human albumin (K d =39 nM). This ligand prolongs the elimination half-life of cyclic peptides in rats 25-fold to over seven hours. Conjugation to a peptide factor XII inhibitor developed for anti-thrombotic therapy extends the half-life from 13 minutes to over five hours, inhibiting coagulation for eight hours in rabbits. This high-affinity albumin ligand could potentially extend the half-life of peptides in human to several days, substantially broadening the application range of peptides as therapeutics.

  19. High affinity, ligand specific uptake of complexed copper-67 by brain tissue incubated in vitro

    International Nuclear Information System (INIS)

    Barnea, A.; Hartter, D.E.

    1987-01-01

    Copper is an essential metal that is highly concentrated in the brain. The blood, the sole source of tissue Cu, contains 16-20 μM Cu, of which >95% is complexed to proteins and 2 was 10 times greater than that of CuAlbumin or Cu(II). Within the range of 0.2-150μM Cu, multiple uptake sites for CuHis were apparent. Increasing the molar ratio of His:Cu had a differential effect on Cu uptake: enhancing uptake at [Cu] 1 μM. Thus, using a His:Cu ratio of 1000, they observed a high affinity process exhibiting saturating and half saturating values of 5 μM and 1.5 μM Cu, respectively; using a His:Cu ratio of 2, they observed a low affinity process exhibiting saturating and half-saturating values of 100 μM and 40 μM Cu, respectively. Both processes required thermic but not metabolic energy, suggestive of facilitated diffusion. Considering the blood brain barrier for proteins, CuHis appears to be the major substrate for Cu uptake by neuronal tissue. They demonstrate the existence of a ligand specific, high affinity (apparent Km about 1.5 μM Cu) uptake process for CuHis in the brain, operative at the physiological concentration range of CuHis and histidine

  20. High-Affinity Accumulation of Chloroquine by Mouse Erythrocytes Infected with Plasmodium berghei

    Science.gov (United States)

    Fitch, Coy D.; Yunis, Norman G.; Chevli, Rekha; Gonzalez, Yolanda

    1974-01-01

    Washed erythrocytes infected with chloroquine-susceptible (CS) or with chloroquine-resistant (CR) P. berghei were used in model systems in vitro to study the accumulation of chloroquine with high affinity. The CS model could achieve distribution ratios (chloroquine in cells: chloroquine in medium) of 100 in the absence of substrate. 200—300 in the presence of 10 mM pyruvate or lactate, and over 600 in the presence of 1 mM glucose or glycerol. In comparable studies of the CR model, the distribution ratios were 100 in the absence of substrate and 300 or less in the presence of glucose or glycerol. The presence of lactate stimulated chloroquine accumulation in the CR model, whereas the presence of pyruvate did not. Lactate production from glucose and glycerol was undiminished in the CR model, and ATP concentrations were higher than in the CS model. Cold, iodoacetate, 2,4-dinitrophenol, or decreasing pH inhibited chloroquine accumulation in both models. These findings demonstrate substrate involvement in the accumulation of chloroquine with high affinity. In studies of the CS model, certain compounds competitively inhibited chloroquine accumulation, while others did not. This finding is attributable to a specific receptor that imposes structural constraints on the process of accumulation. For chloroquine analogues, the position and length of the side chain, the terminal nitrogen atom of the side chain, and the nitrogen atom in the quinoline ring are important determinants of binding to this receptor. PMID:4600044

  1. High-affinity receptors for bombesin-like peptides in normal guinea pig lung membranes

    International Nuclear Information System (INIS)

    Lach, E.; Trifilieff, A.; Landry, Y.; Gies, J.P.

    1991-01-01

    The binding of the radiolabeled bombesin analogue [ 125 I-Tyr 4 ]bombesin to guinea-pig lung membranes was investigated. Binding of [ 125 I-Tyr 4 ]bombesin was specific, saturable, reversible and linearly related to the protein concentration. Scatchard analysis of equilibrium binding data at 25C indicated the presence of a single class of non-interacting binding sites for bombesin (B max = 7.7 fmol/mg protein). The value of the equilibrium dissociation constant (K D = 90 pM) agrees with a high-affinity binding site. Bombesin and structurally related peptides such as [ 125 I-Tyr 4 ]bombesin, neuromedin B and neuromedin C inhibited the binding of [ 125 I-Tyr 4 ]bombesin in an order of potencies as follows: [ 125 I-Tyr 4 ]bombesin > bombesin ≥ neuromedin C much-gt neuromedin B. These results indicate that guinea-pig lung membranes possess a single class of bombesin receptors with a high affinity for bombesin and a lower one for neuromedin B

  2. Reconstitution of high affinity α2 adrenergic agonist binding by fusion with a pertussis toxin substrate

    International Nuclear Information System (INIS)

    Kim, M.H.; Neubig, R.R.

    1986-01-01

    High affinity α 2 adrenergic agonist binding is thought to occur via a coupling of the α 2 receptor with N/sub i/, the inhibitory guanyl nucleotide binding protein. Human platelet membranes pretreated at pH 11.5 exhibit a selective inactivation of agonist binding and N/sub i/. To further study the mechanism of agonist binding, alkali treated membranes (ATM) were mixed with membranes pretreated with 10 μM phenoxybenzamine to block α 2 receptors (POB-M). The combined membrane pellet was incubated in 50% polyethylene glycol (PEG) to promote membrane-membrane fusion and assayed for binding to the α 2 agonist [ 3 H]UK 14,304 (UK) and the antagonist [ 3 H] yohimbine. PEG treatment resulted in a 2-4 fold enhancement of UK binding whereas yohimbine binding was unchanged. No enhancement of UK binding was observed in the absence of PEG treatment. The reconstitution was dependent on the addition of POB-M. They found that a 1:1 ratio of POB-M:ATM was optimal. Reconstituted binding was inhibited by GppNHp. Fusion of rat C6 glioma cell membranes, which do not contain α 2 receptors, also enhanced agonist binding to ATM. Fusion of C6 membranes from cells treated with pertussis toxin did not enhance [ 3 H] UK binding. These data show that a pertussis toxin sensitive membrane component, possibly N/sub i/, can reconstitute high affinity α 2 agonist binding

  3. Acylated heptapeptide binds albumin with high affinity and application as tag furnishes long-acting peptides

    Science.gov (United States)

    Zorzi, Alessandro; Middendorp, Simon J.; Wilbs, Jonas; Deyle, Kaycie; Heinis, Christian

    2017-07-01

    The rapid renal clearance of peptides in vivo limits this attractive platform for the treatment of a broad range of diseases that require prolonged drug half-lives. An intriguing approach for extending peptide circulation times works through a `piggy-back' strategy in which peptides bind via a ligand to the long-lived serum protein albumin. In accordance with this strategy, we developed an easily synthesized albumin-binding ligand based on a peptide-fatty acid chimera that has a high affinity for human albumin (Kd=39 nM). This ligand prolongs the elimination half-life of cyclic peptides in rats 25-fold to over seven hours. Conjugation to a peptide factor XII inhibitor developed for anti-thrombotic therapy extends the half-life from 13 minutes to over five hours, inhibiting coagulation for eight hours in rabbits. This high-affinity albumin ligand could potentially extend the half-life of peptides in human to several days, substantially broadening the application range of peptides as therapeutics.

  4. Human IgG1 Responses to Surface Localised Schistosoma mansoni Ly6 Family Members Drop following Praziquantel Treatment.

    Directory of Open Access Journals (Sweden)

    Iain W Chalmers

    Full Text Available The heptalaminate-covered, syncytial tegument is an important anatomical adaptation that enables schistosome parasites to maintain long-term, intravascular residence in definitive hosts. Investigation of the proteins present in this surface layer and the immune responses elicited by them during infection is crucial to our understanding of host/parasite interactions. Recent studies have revealed a number of novel tegumental surface proteins including three (SmCD59a, SmCD59b and Sm29 containing uPAR/Ly6 domains (renamed SmLy6A SmLy6B and SmLy6D in this study. While vaccination with SmLy6A (SmCD59a and SmLy6D (Sm29 induces protective immunity in experimental models, human immunoglobulin responses to representative SmLy6 family members have yet to be thoroughly explored.Using a PSI-BLAST-based search, we present a comprehensive reanalysis of the Schistosoma mansoni Ly6 family (SmLy6A-K. Our examination extends the number of members to eleven (including three novel proteins and provides strong evidence that the previously identified vaccine candidate Sm29 (renamed SmLy6D is a unique double uPAR/Ly6 domain-containing representative. Presence of canonical cysteine residues, signal peptides and GPI-anchor sites strongly suggest that all SmLy6 proteins are cell surface-bound. To provide evidence that SmLy6 members are immunogenic in human populations, we report IgG1 (as well as IgG4 and IgE responses against two surface-bound representatives (SmLy6A and SmLy6B within a cohort of S. mansoni-infected Ugandan males before and after praziquantel treatment. While pre-treatment IgG1 prevalence for SmLy6A and SmLy6B differs amongst the studied population (7.4% and 25.3% of the cohort, respectively, these values are both higher than IgG1 prevalence (2.7% for a sub-surface tegumental antigen, SmTAL1. Further, post-treatment IgG1 levels against surface-associated SmLy6A and SmLy6B significantly drop (p = 0.020 and p < 0.001, respectively when compared to rising IgG1 levels against sub-surface SmTAL1.Collectively, these results expand the number of SmLy6 proteins found within S. mansoni and specifically demonstrate that surface-associated SmLy6A and SmLy6B elicit immunological responses during infection in endemic communities.

  5. THE USE OF DEDICATED PEPTIDE LIBRARIES PERMITS THE DISCOVERY OF HIGH-AFFINITY BINDING PEPTIDES

    NARCIS (Netherlands)

    DEKOSTER, HS; AMONS, R; BENCKHUIJSEN, WE; FEIJLBRIEF, M; SCHELLEKENS, GA; DRIJFHOUT, JW

    1995-01-01

    The motif for peptide binding to monoclonal antibody mAb A16, which is known to be directed against glycoprotein D of Herpes simplex virus type 1, was determined using two dedicated peptide libraries. As a starting point for this study we used an A-16 binding lead sequence, which had previously been

  6. A rapid one-step radiometric assay for hepatitis B surface antigen utilising monoclonal antibodies

    International Nuclear Information System (INIS)

    Goodall, A.H.; Meek, F.L.; Waters, J.A.; Miescher, G.C.; Janossy, G.; Thomas, H.C.

    1982-01-01

    A two-site antigen assay for HBsAg has been developed that employs 3 monoclonal antibodies. The antibodies were selected for their high affinity and their particular epitope specificity to establish an assay with a sensitivity for the antigen comparable with that of a conventional assay with heterologous antisera. In addition, by selecting a monoclonal antibody for use as a tracer which does not compete for antigenic binding sites with the solid-phase monoclonal antibodies, it has been possible to perform a two-site assay in a single 1 h incubation step, achieving the same degree of sensitivity. This principle of using monoclonal antibodies in a one-step assay therefore gives advantages of speed and simplicity over assays using heterologous antisera and would be applicable to a variety of antigen assays for which appropriate monoclonal antibodies are available. (Auth.)

  7. Role of polymorphic Fc receptor Fc gammaRIIa in cytokine release and adverse effects of murine IgG1 anti-CD3/T cell receptor antibody (WT31).

    Science.gov (United States)

    Tax, W J; Tamboer, W P; Jacobs, C W; Frenken, L A; Koene, R A

    1997-01-15

    Anti-CD3 monoclonal antibody (mAb) OKT3 is immunosuppressive, but causes severe adverse effects during the first administration ("first-dose reaction"). These adverse effects are presumably caused by cytokine release that results from T-cell activation. In vitro, T-cell activation by anti-CD3 mAb requires interaction with monocyte Fc receptors. The Fc receptor for murine IgG1, Fc gammaRIIa, is polymorphic. In some individuals, murine IgG1 anti-CD3 mAb causes T-cell proliferation and cytokine release in vitro (high responders [HR]), whereas in individuals with the low-responder (LR) phenotype it does not. We have now investigated the role of this Fc gammaRIIa polymorphism in the release of cytokines in vivo and the occurrence of adverse effects after the administration of WT31, a murine IgG1 anti-CD3/T cell receptor mAb. WT31 caused an increase of plasma tumor necrosis factor-alpha in all four HR patients and none of the five LR patients. In all HR patients except one, plasma gamma-interferon and interleukin 6 also increased, and a first-dose response was observed, whereas no cytokine release or adverse effects occurred in any of the LR patients. WT31 caused lymphopenia in all HR and none of the LR patients. FACS analysis demonstrated that in HR patients, after the initial disappearance of CD3+ cells from peripheral blood, modulation of CD3 occurred, whereas in LR patients a high degree of coating of the lymphocytes was observed. Surprisingly, WT31 also induced a marked granulocytopenia, as well as a decrease of thrombocytes, in three of the four HR patients (and in none of the LR patients). These data provide direct clinical evidence that Fc receptor interaction determines the release of cytokines and the occurrence of adverse effects after administration of anti-CD3/T cell receptor mAb. Furthermore, these data suggest that tumor necrosis factor-alpha by itself is not sufficient to induce the first-dose reaction.

  8. Isolation and characterization of high affinity aptamers against DNA polymerase iota.

    Science.gov (United States)

    Lakhin, Andrei V; Kazakov, Andrei A; Makarova, Alena V; Pavlov, Yuri I; Efremova, Anna S; Shram, Stanislav I; Tarantul, Viacheslav Z; Gening, Leonid V

    2012-02-01

    Human DNA-polymerase iota (Pol ι) is an extremely error-prone enzyme and the fidelity depends on the sequence context of the template. Using the in vitro systematic evolution of ligands by exponential enrichment (SELEX) procedure, we obtained an oligoribonucleotide with a high affinity to human Pol ι, named aptamer IKL5. We determined its dissociation constant with homogenous preparation of Pol ι and predicted its putative secondary structure. The aptamer IKL5 specifically inhibits DNA-polymerase activity of the purified enzyme Pol ι, but did not inhibit the DNA-polymerase activities of human DNA polymerases beta and kappa. IKL5 suppressed the error-prone DNA-polymerase activity of Pol ι also in cellular extracts of the tumor cell line SKOV-3. The aptamer IKL5 is useful for studies of the biological role of Pol ι and as a potential drug to suppress the increase of the activity of this enzyme in malignant cells.

  9. Solution structure of the Grb2 SH2 domain complexed with a high-affinity inhibitor

    International Nuclear Information System (INIS)

    Ogura, Kenji; Shiga, Takanori; Yokochi, Masashi; Yuzawa, Satoru; Burke, Terrence R.; Inagaki, Fuyuhiko

    2008-01-01

    The solution structure of the growth factor receptor-bound protein 2 (Grb2) SH2 domain complexed with a high-affinity inhibitor containing a non-phosphorus phosphate mimetic within a macrocyclic platform was determined by nuclear magnetic resonance (NMR) spectroscopy. Unambiguous assignments of the bound inhibitor and intermolecular NOEs between the Grb2 SH2 domain and the inhibitor was accomplished using perdeuterated Grb2 SH2 protein. The well-defined solution structure of the complex was obtained and compared to those by X-ray crystallography. Since the crystal structure of the Grb2 SH2 domain formed a domain-swapped dimer and several inhibitors were bound to a hinge region, there were appreciable differences between the solution and crystal structures. Based on the binding interactions between the inhibitor and the Grb2 SH2 domain in solution, we proposed a design of second-generation inhibitors that could be expected to have higher affinity

  10. Haemoglobin Pierre-Benite--a high affinity variant associated with relative polycythaemia.

    Science.gov (United States)

    Beard, M E; Potter, H C; Spearing, R L; Brennan, S O

    2001-12-01

    This is the second reported example of Hb Pierre--Benite (beta90 Glu-->Asp). This mutation is associated with increased oxygen affinity and polycythaemia. No instability was found and there was no charge shift detected by cellulose acetate electrophoresis at pH 8.3. The mutation was however, clearly indicated by electrospray ionization mass spectrometry (ESI MS), which showed an abnormal beta chain with a 14 Da decrease in mass. Blood volume studies documented a relative rather than a true polycythaemia and this finding has been reported in at least two other high affinity haemoglobin variants--Hb Heathrow and Hb Rahere. This finding led to delay in diagnosis because high oxygen affinity variants are conventionally considered to cause a true polycythaemia.

  11. New immunogenic form for vasopressin: production of high-affinity antiserum and RIA for plasmatic AVP

    International Nuclear Information System (INIS)

    Rougon-Rappuzi, G.; Delaage, M.A.; Conte-Devolx, B.; Millet, Y.

    1977-01-01

    A highly sensitive and specific radioimmunoassay (RIA) for arginine-vasopressin (AVP) was developped and applied to the measurement of AVP in human plasma. High-affinity antivasopressin antibodies with limited association constant heterogeneity have been induced by immunizing rabbits with Lysine-vasopressine (LVP) coupled to a human immunoglobulin (IgA). Replacing air drying of acetone-petroleum ether extracts by lyophilisation increased significantly the yields of AVP. Equilibrium dialysis was used for separating bound and free antigen, thus reducing the total time required for the assay to 48 hours. Only 1 ml of plasma was required for routine determinations due to a sensitivity threshold better than 0.5 pg/ml. Plasma AVP levels of normal subjects and of patients with inappropriate ADH secretion (SIADH) were determined during different hydratation states and following nicotin of ethanol infusions. (orig.) [de

  12. Effects of anticonvulsants in vivo on high affinity choline uptake in vitro in mouse hippocampal synaptosomes.

    Science.gov (United States)

    Miller, J. A.; Richter, J. A.

    1985-01-01

    The effects of several anticonvulsant drugs on sodium-dependent high affinity choline uptake (HACU) in mouse hippocampal synaptosomes was investigated. HACU was measured in vitro after in vivo administration of the drug to mice. HACU was inhibited by drugs which have in common the ability to facilitate gamma-aminobutyric acid (GABA) transmission, pentobarbitone, phenobarbitone, barbitone, diazepam, chloridiazepoxide, and valproic acid. Dose-response relationships were determined for these drugs and the drugs' potencies at inhibiting HACU correlated well with their anticonvulsant potencies. Clonazepam, ethosuximide, carbamazepine, and barbituric acid had no effect on HACU in the doses used while phenytoin and trimethadione stimulated HACU. These results suggest that certain anticonvulsants may elicit a part of their anticonvulsant activity by modulating cholinergic neurones. This effect may be mediated through a GABA mechanism. PMID:3978310

  13. Exploiting the high-affinity phosphonate-hydroxyapatite nanoparticle interaction for delivery of radiation and drugs

    International Nuclear Information System (INIS)

    Ong, Hooi Tin; Loo, Joachim S. C.; Boey, Freddy Y. C.; Russell, Stephen J.; Ma Jan; Peng, Kah-Whye

    2008-01-01

    Hydroxyapatite is biocompatible and used in various biomedical applications. Here, we generated hydroxyapatite nanoparticles (HNPs) of various sizes (40-200 nm) and demonstrated that they can be stably loaded with drugs or radioisotopes by exploiting the high-affinity HA-(poly)phosphonate interaction. Clinically available phosphonates, clodronate, and Tc-99m-methylene-diphosphonate (Tc-99m-MDP), were efficiently loaded onto HNPs within 15 min. Biodistribution of radiolabeled HNP-MDP-Tc99m in mice was monitored non-invasively using microSPECT-CT. Imaging and dosimetry studies indicated that the HNPs, regardless of size, were quickly taken up by Kupffer cells in the liver after systemic administration into mice. Clodronate loaded onto HNPs remained biologically active and were able to result in selective depletion of Kupffer cells. This method of drug or isotope loading on HA is fast and easy as it eliminates the need for additional surface modifications of the nanoparticles

  14. High Affinity IgE-Fc Receptor alpha and gamma Subunit Interactions

    International Nuclear Information System (INIS)

    Rashid, A.; Housden, J. E. M.; Sabban, S.; Helm, B.

    2014-01-01

    Objective: To explore the relationships between the subunits (alpha, beta and gamma) of the high affinity IgE receptor (Fc and RI) and its ability to mediate transmembrane signaling. Study Design: Experimental study. Place and Duration of Study: Department of Molecular Biology and Biotechnology, University of Sheffield, UK, from 2008 to 2009. Methodology: The approach employed was to create a chimera (human alpha-gamma-gamma) using the extracellular (EC) domain of the human high affinity IgE receptor. The alpha subunit (huFc and RIalpha) of IgE receptor was spliced onto the rodent gamma TM and cytoplasmic domain (CD). This was transfected into the Rat Basophilic Leukemia cell line in order to assess the possibility of selectively activating cells transfected with this single pass construct for antigen induced mediator release. Results: The RBLs cell lines transfected with the huFc and RIalpha/gamma/gamma cDNA constructs were assessed for the cell surface expression of the huFc and RIalpha subunit and the response to the antigenic stimulus by looking for degranulation and intracellular Ca2+ mobilisation. The results obtained showed the absence of huFc and RIalpha subunit expression on the surface of transfected cells as seen by flowcytometric studies, beta-hexosaminidase assays and intracellular calcium mobilisation studies. Conclusion: In the present study the grounds for non-expression of huFc and RIalpha/gamma/gamma cDNA remains elusive but may be due to the fact that the human-rodent chimeric receptors are assembled differently than the endogenous rodent receptors as seen in study in which COS 7 cells were transfected with human/rat chimeric complexes. (author)

  15. Kinetics and autoradiography of high affinity uptake of serotonin by primary astrocyte cultures

    International Nuclear Information System (INIS)

    Katz, D.M.; Kimelberg, H.K.

    1985-01-01

    Primary astrocyte cultures prepared from the cerebral cortices of neonatal rats showed significant accumulation of serotonin (5-hydroxytryptamine; [ 3 H]-5-HT). At concentrations in the range of 0.01 to 0.7 microM [ 3 H]-5-HT, this uptake was 50 to 85% Na+ dependent and gave a Km of 0.40 +/- 0.11 microM [ 3 H]-5-HT and a Vmax of 6.42 +/- 0.85 (+/- SEM) pmol of [ 3 H]-5-HT/mg of protein/4 min for the Na+-dependent component. In the absence of Na+ the uptake was nonsaturable. Omission of the monoamine oxidase inhibitor pargyline markedly reduced the Na+-dependent component of [ 3 H]-5-HT uptake but had a negligible effect on the Na+-independent component. This suggest significant oxidative deamination of serotonin after it has been taken up by the high affinity system, followed by release of its metabolite. The authors estimated that this system enabled the cells to concentrate [ 3 H]-5-HT up to 44-fold at an external [ 3 H]-5-HT concentration of 10(-7) M. Inhibition of [ 3 H]-5-HT uptake by a number of clinically effective antidepressants was also consistent with a specific high affinity uptake mechanism for 5-HT, the order of effectiveness of inhibition being chlorimipramine greater than fluoxetine greater than imipramine = amitriptyline greater than desmethylimipramine greater than iprindole greater than mianserin. Uptake of [ 3 H]-5-HT was dependent on the presence of Cl- as well as Na+ in the medium, and the effect of omission of both ions was nonadditive. Varying the concentration of K+ in the media from 1 to 50 mM had a limited effect on [ 3 H]-5-HT uptake

  16. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

    International Nuclear Information System (INIS)

    Wang, Deng-Liang; Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan; Yang, Hai-Tao; Wang, Jiang-Jie; Yao, Pei-Sen; Pan, Ru-Jun; Yang, Chaoyong James; Kang, De-Zhi

    2014-01-01

    Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K d 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K d 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy

  17. Antibody Binding Selectivity: Alternative Sets of Antigen Residues Entail High-Affinity Recognition.

    Directory of Open Access Journals (Sweden)

    Yves Nominé

    Full Text Available Understanding the relationship between protein sequence and molecular recognition selectivity remains a major challenge. The antibody fragment scFv1F4 recognizes with sub nM affinity a decapeptide (sequence 6TAMFQDPQER15 derived from the N-terminal end of human papilloma virus E6 oncoprotein. Using this decapeptide as antigen, we had previously shown that only the wild type amino-acid or conservative replacements were allowed at positions 9 to 12 and 15 of the peptide, indicating a strong binding selectivity. Nevertheless phenylalanine (F was equally well tolerated as the wild type glutamine (Q at position 13, while all other amino acids led to weaker scFv binding. The interfaces of complexes involving either Q or F are expected to diverge, due to the different physico-chemistry of these residues. This would imply that high-affinity binding can be achieved through distinct interfacial geometries. In order to investigate this point, we disrupted the scFv-peptide interface by modifying one or several peptide positions. We then analyzed the effect on binding of amino acid changes at the remaining positions, an altered susceptibility being indicative of an altered role in complex formation. The 23 starting variants analyzed contained replacements whose effects on scFv1F4 binding ranged from minor to drastic. A permutation analysis (effect of replacing each peptide position by all other amino acids except cysteine was carried out on the 23 variants using the PEPperCHIP® Platform technology. A comparison of their permutation patterns with that of the wild type peptide indicated that starting replacements at position 11, 12 or 13 modified the tolerance to amino-acid changes at the other two positions. The interdependence between the three positions was confirmed by SPR (Biacore® technology. Our data demonstrate that binding selectivity does not preclude the existence of alternative high-affinity recognition modes.

  18. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Deng-Liang [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan [State Key Laboratory for Physical Chemistry of Solid Surfaces, Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Analytical Chemistry, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China); Yang, Hai-Tao; Wang, Jiang-Jie [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Yao, Pei-Sen [Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Pan, Ru-Jun [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Yang, Chaoyong James, E-mail: cyyang@xmu.edu.cn [State Key Laboratory for Physical Chemistry of Solid Surfaces, Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Analytical Chemistry, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China); Kang, De-Zhi, E-mail: kdzy99988@163.com [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China)

    2014-10-31

    Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K{sub d} 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K{sub d} 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.

  19. A rhodium(III) complex for high-affinity DNA base-pair mismatch recognition

    Science.gov (United States)

    Junicke, Henrik; Hart, Jonathan R.; Kisko, Jennifer; Glebov, Oleg; Kirsch, Ilan R.; Barton, Jacqueline K.

    2003-01-01

    A rhodium(III) complex, rac-[Rh(bpy)2phzi]3+ (bpy, 2,2′-bipyridine; phzi, benzo[a]phenazine-5,6-quinone diimine) has been designed as a sterically demanding intercalator targeted to destabilized mismatched sites in double-helical DNA. The complex is readily synthesized by condensation of the phenazine quinone with the corresponding diammine complex. Upon photoactivation, the complex promotes direct strand scission at single-base mismatch sites within the DNA duplex. As with the parent mismatch-specific reagent, [Rh(bpy)2(chrysi)]3+ [chrysene-5,6-quinone diimine (chrysi)], mismatch selectivity depends on the helix destabilization associated with mispairing. Unlike the parent chrysi complex, the phzi analogue binds and cleaves with high affinity and efficiency. The specific binding constants for CA, CC, and CT mismatches within a 31-mer oligonucleotide duplex are 0.3, 1, and 6 × 107 M−1, respectively; site-specific photocleavage is evident at nanomolar concentrations. Moreover, the specificity, defined as the ratio in binding affinities for mispaired vs. well paired sites, is maintained. The increase in affinity is attributed to greater stability in the mismatched site associated with stacking by the heterocyclic aromatic ligand. The high-affinity complex is also applied in the differential cleavage of DNA obtained from cell lines deficient in mismatch repair vs. those proficient in mismatch repair. Agreement is found between photocleavage by the mismatch-specific probes and deficiency in mismatch repair. This mismatch-specific targeting, therefore, offers a potential strategy for new chemotherapeutic design. PMID:12610209

  20. New Regulatory Roles of Galectin-3 in High-Affinity IgE Receptor Signaling

    Czech Academy of Sciences Publication Activity Database

    Bambousková, Monika; Polakovičová, Iva; Hálová, Ivana; Goel, G.; Dráberová, Lubica; Bugajev, Viktor; Doan, A.; Utekal, Pavol; Gardet, A.; Xavier, R.J.; Dráber, Petr

    2016-01-01

    Roč. 36, č. 9 (2016), s. 1366-1382 ISSN 0270-7306 R&D Projects: GA ČR(CZ) GBP302/12/G101 Institutional support: RVO:68378050 Keywords : fc-epsilon-ri * t-cell-activation * basophilic leukemia-cells * mast-cell * immunoglobulin-e * tyrosine kinase * lipid rafts * monoclonal-antibody * negative regulation * mediator release Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.398, year: 2016

  1. Correlating the Impact of Well-Defined Oligosaccharide Structures on Physical Stability Profiles of IgG1-Fc Glycoforms.

    Science.gov (United States)

    More, Apurva S; Toprani, Vishal M; Okbazghi, Solomon Z; Kim, Jae H; Joshi, Sangeeta B; Middaugh, C Russell; Tolbert, Thomas J; Volkin, David B

    2016-02-01

    As part of a series of articles in this special issue describing 4 well-defined IgG1-Fc glycoforms as a model system for biosimilarity analysis (high mannose-Fc, Man5-Fc, GlcNAc-Fc and N297Q-Fc aglycosylated), the focus of this work is comparisons of their physical properties. A trend of decreasing apparent solubility (thermodynamic activity) by polyethylene glycol precipitation (pH 4.5, 6.0) and lower conformational stability by differential scanning calorimetry (pH 4.5) was observed with reducing size of the N297-linked oligosaccharide structures. Using multiple high-throughput biophysical techniques, the physical stability of the Fc glycoproteins was then measured in 2 formulations (NaCl and sucrose) across a wide range of temperatures (10°C-90°C) and pH (4.0-7.5) conditions. The data sets were used to construct 3-index empirical phase diagrams and radar charts to visualize the regions of protein structural stability. Each glycoform showed improved stability in the sucrose (vs. salt) formulation. The HM-Fc and Man5-Fc displayed the highest relative stability, followed by GlcNAc-Fc, with N297Q-Fc being the least stable. Thus, the overall physical stability profiles of the 4 IgG1-Fc glycoforms also show a correlation with oligosaccharide structure. These data sets are used to develop a mathematical model for biosimilarity analysis (as described in a companion article by Kim et al. in this issue). Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  2. Opioid receptor subtypes mediating the noise-induced decreases in high-affinity choline uptake in the rat brain.

    Science.gov (United States)

    Lai, H; Carino, M A

    1992-07-01

    Acute (20 min) exposure to 100-dB white noise elicits a naltrexone-sensitive decrease in sodium-dependent high-affinity choline uptake in the frontal cortex and hippocampus of the rat. In the present study, the subtypes of opioid receptors involved were investigated by pretreating rats with microinjection of specific opioid-receptor antagonists into the lateral cerebroventricle before noise exposure. We found that the noise-induced decrease in high-affinity choline uptake in the hippocampus was blocked by pretreatment with either mu-, delta-, or kappa-opioid-receptor antagonists, whereas the effect of noise on frontal cortical high-affinity choline uptake was blocked by a mu- and delta- but not by a kappa-antagonist. These data further confirm the role of endogenous opioids in mediating the effects of noise on central cholinergic activity and indicate that different neural mechanisms are involved in the effects of noise on the frontal cortical and hippocampal cholinergic systems.

  3. Visual and Plasmon Resonance Absorption Sensor for Adenosine Triphosphate Based on the High Affinity between Phosphate and Zr(IV)

    OpenAIRE

    Qi, Wenjing; Liu, Zhongyuan; Zhang, Wei; Halawa, Mohamed Ibrahim; Xu, Guobao

    2016-01-01

    Zr(IV) can form phosphate and Zr(IV) (?PO3 2??Zr4+?) complex owing to the high affinity between Zr(IV) with phosphate. Zr(IV) can induce the aggregation of gold nanoparticles (AuNPs), while adenosine triphosphate(ATP) can prevent Zr(IV)-induced aggregation of AuNPs. Herein, a visual and plasmon resonance absorption (PRA)sensor for ATP have been developed using AuNPs based on the high affinity between Zr(IV)with ATP. AuNPs get aggregated in the presence of certain concentrations of Zr(IV). Aft...

  4. New Synthesis and Tritium Labeling of a Selective Ligand for Studying High-affinity γ-Hydroxybutyrate (GHB) Binding Sites

    Science.gov (United States)

    Vogensen, Stine B.; Marek, Aleš; Bay, Tina; Wellendorph, Petrine; Kehler, Jan; Bundgaard, Christoffer; Frølund, Bente; Pedersen, Martin H.F.; Clausen, Rasmus P.

    2013-01-01

    3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [3H]-1 is described. The radiosynthesis employs in situ generated lithium trimethoxyborotritide. Screening of 1 against different CNS targets establishes a high selectivity and we demonstrate in vivo brain penetration. In vitro characterization of [3H]-1 binding shows high specificity to the high-affinity GHB binding sites. PMID:24053696

  5. Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides

    Directory of Open Access Journals (Sweden)

    Weonu Choe

    2016-12-01

    Full Text Available The rapidly increasing application of antibodies has inspired the development of several novel methods to isolate and target antibodies using smart biomaterials that mimic the binding of Fc-receptors to antibodies. The Fc-binding domain of antibodies is the primary binding site for e.g., effector proteins and secondary antibodies, whereas antigens bind to the Fab region. Protein A, G, and L, surface proteins expressed by pathogenic bacteria, are well known to bind immunoglobulin and have been widely exploited in antibody purification strategies. Several difficulties are encountered when bacterial proteins are used in antibody research and application. One of the major obstacles hampering the use of bacterial proteins is sample contamination with trace amounts of these proteins, which can invoke an immune response in the host. Many research groups actively develop synthetic ligands that are able to selectively and strongly bind to antibodies. Among the reported ligands, peptides that bind to the Fc-domain of antibodies are attractive tools in antibody research. Besides their use as high affinity ligands in antibody purification chromatography, Fc-binding peptides are applied e.g., to localize antibodies on nanomaterials and to increase the half-life of proteins in serum. In this review, recent developments of Fc-binding peptides are presented and their binding characteristics and diverse applications are discussed.

  6. Structural insights into a high affinity nanobody:antigen complex by homology modelling.

    Science.gov (United States)

    Skottrup, Peter Durand

    2017-09-01

    Porphyromonas gingivalis is a major periodontitis-causing pathogens. P. gingivalis secrete a cysteine protease termed RgpB, which is specific for Arg-Xaa bonds in substrates. Recently, a nanobody-based assay was used to demonstrate that RgpB could represent a novel diagnostic target, thereby simplifying. P. gingivalis detection. The nanobody, VHH7, had a high binding affinity and was specific for RgpB, when tested towards the highly identical RgpA. In this study a homology model of VHH7 was build. The complementarity determining regions (CDR) comprising the paratope residues responsible for RgpB binding were identified and used as input to the docking. Furthermore, residues likely involved in the RgpB epitope was identified based upon RgpB:RgpA alignment and analysis of residue surface accessibility. CDR residues and putitative RgpB epitope residues were used as input to an information-driven flexible docking approach using the HADDOCK server. Analysis of the VHH7:RgpB model demonstrated that the epitope was found in the immunoglobulin-like domain and residue pairs located at the molecular paratope:epitope interface important for complex stability was identified. Collectively, the VHH7 homology model and VHH7:RgpB docking supplies knowledge of the residues involved in the high affinity interaction. This information could prove valuable in the design of an antibody-drug conjugate for specific RgpB targeting. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Characterization of a high affinity cocaine binding site in rat brain

    International Nuclear Information System (INIS)

    Calligaro, D.; Eldefrawi, M.

    1986-01-01

    Binding of [ 3 H]cocaine to synaptic membranes from whole rat brain was reversible and saturable. Nonlinear regression analysis of binding isotherms indicated two binding affinities: one with k/sub d/ = 16 nM, B/sub max/ = 0.65 pmoles/mg protein and the other with K/sub d/ = 660 nM, B/sub max/ = 5.1 pmoles/mg protein. The high-affinity binding of [ 3 H]cocaine was sensitive to the actions of trypsin and chymotrypsin but not carboxypeptidase, and was eliminated by exposure of the membranes to 95 0 C for 5 min. Specific binding at 2 nM was higher at pH 8.8 than at pH 7.0. Binding of [ 3 H]cocaine (15 nM) was inhibited by increasing concentrations of Na + ions. Several cocaine analogues, neurotransmitter uptake inhibitors and local anesthetics displaced specific [ 3 H]cocaine binding at 2 nM with various potencies. The cocaine analogue (-)-norcocaine was the most potent (IC 50 = 10 nM), while the local anesthetic tetracaine was the least potent in inhibiting [ 3 H]cocaine binding. Several biogenic amine uptake inhibitors, including tricyclic antidepressants and phencyclidine, had IC 50 values below μM concentrations

  8. Carbonate-sensitive phytotransferrin controls high-affinity iron uptake in diatoms

    Science.gov (United States)

    McQuaid, Jeffrey B.; Kustka, Adam B.; Oborník, Miroslav; Horák, Aleš; McCrow, John P.; Karas, Bogumil J.; Zheng, Hong; Kindeberg, Theodor; Andersson, Andreas J.; Barbeau, Katherine A.; Allen, Andrew E.

    2018-03-01

    In vast areas of the ocean, the scarcity of iron controls the growth and productivity of phytoplankton. Although most dissolved iron in the marine environment is complexed with organic molecules, picomolar amounts of labile inorganic iron species (labile iron) are maintained within the euphotic zone and serve as an important source of iron for eukaryotic phytoplankton and particularly for diatoms. Genome-enabled studies of labile iron utilization by diatoms have previously revealed novel iron-responsive transcripts, including the ferric iron-concentrating protein ISIP2A, but the mechanism behind the acquisition of picomolar labile iron remains unknown. Here we show that ISIP2A is a phytotransferrin that independently and convergently evolved carbonate ion-coordinated ferric iron binding. Deletion of ISIP2A disrupts high-affinity iron uptake in the diatom Phaeodactylum tricornutum, and uptake is restored by complementation with human transferrin. ISIP2A is internalized by endocytosis, and manipulation of the seawater carbonic acid system reveals a second-order dependence on the concentrations of labile iron and carbonate ions. In P. tricornutum, the synergistic interaction of labile iron and carbonate ions occurs at environmentally relevant concentrations, revealing that carbonate availability co-limits iron uptake. Phytotransferrin sequences have a broad taxonomic distribution and are abundant in marine environmental genomic datasets, suggesting that acidification-driven declines in the concentration of seawater carbonate ions will have a negative effect on this globally important eukaryotic iron acquisition mechanism.

  9. High-affinity binding of two molecules of cysteine proteinases to low-molecular-weight kininogen.

    Science.gov (United States)

    Turk, B.; Stoka, V.; Björk, I.; Boudier, C.; Johansson, G.; Dolenc, I.; Colic, A.; Bieth, J. G.; Turk, V.

    1995-01-01

    Human low-molecular-weight kininogen (LK) was shown by fluorescence titration to bind two molecules of cathepsins L and S and papain with high affinity. By contrast, binding of a second molecule of cathepsin H was much weaker. The 2:1 binding stoichiometry was confirmed by titration monitored by loss of enzyme activity and by sedimentation velocity experiments. The kinetics of binding of cathepsins L and S and papain showed the two proteinase binding sites to have association rate constants kass,1 = 10.7-24.5 x 10(6) M-1 s-1 and kass,2 = 0.83-1.4 x 10(6) M-1 s-1. Comparison of these kinetic constants with previous data for intact LK and its separated domains indicate that the faster-binding site is also the tighter-binding site and is present on domain 3, whereas the slower-binding, lower-affinity site is on domain 2. These results also indicate that there is no appreciable steric hindrance for the binding of proteinases between the two binding sites or from the kininogen light chain. PMID:8528085

  10. Cyclic GMP-AMP Containing Mixed Phosphodiester Linkages Is An Endogenous High Affinity Ligand for STING

    Science.gov (United States)

    Zhang, Xu; Shi, Heping; Wu, Jiaxi; Zhang, Xuewu; Sun, Lijun; Chen, Chuo; Chen, Zhijian J.

    2013-01-01

    The presence of microbial or self DNA in the cytoplasm of mammalian cells is a danger signal detected by the DNA sensor cyclic-GMP-AMP (cGAMP) synthase (cGAS), which catalyzes the production of cGAMP that in turn serves as a second messenger to activate innate immune responses. Here we show that endogenous cGAMP in mammalian cells contains two distinct phosphodiester linkages, one between 2′-OH of GMP and 5′-phosphate of AMP, and the other between 3′-OH of AMP and 5′-phosphate of GMP. This molecule, termed 2′3′-cGAMP, is unique in that it binds to the adaptor protein STING with a much greater affinity than cGAMP molecules containing other combinations of phosphodiester linkages. The crystal structure of STING bound to 2′3′-cGAMP revealed the structural basis of this high-affinity binding and a ligand-induced conformational change in STING that may underlie its activation. PMID:23747010

  11. Identification of a High Affinity Nucleocapsid Protein Binding Element from The Bovine Leukemia Virus Genome

    Science.gov (United States)

    Yildiz, F. Zehra; Babalola, Kathleen; Summers, Michael F.

    2012-01-01

    Retroviral genome recognition is mediated by interactions between the nucleocapsid (NC) domain of the virally encoded Gag polyprotein and cognate RNA packaging elements that, for most retroviruses, appear to reside primarily within the 5′-untranslated region (5′-UTR) of the genome. Recent studies suggest that a major packaging determinant of Bovine Leukemia Virus (BLV), a member of the human T-cell leukemia virus (HTLV)/BLV family and a non-primate animal model for HTLV-induced leukemogenesis, resides within the gag open reading frame. We have prepared and purified the recombinant BLV NC protein and conducted electrophoretic mobility shift and isothermal titration calorimetry studies with RNA fragments corresponding to these proposed packaging elements. The gag-derived RNAs did not exhibit significant affinity for NC, suggesting an alternate role in packaging. However, an 83-nucleotide fragment of the 5′-UTR that resides just upstream of the gag start codon binds NC stoichiometrically and with high affinity (Kd = 136 ± 21 nM). These nucleotides were predicted to form tandem hairpin structures, and studies with smaller fragments indicate that the NC binding site resides exclusively within the distal hairpin (residues G369- U399, Kd = 67 ± 8 nM at physiological ionic strength). Unlike all other structurally characterized retroviral NC binding RNAs, this fragment is not expected to contain exposed guanosines, suggesting that RNA binding may be mediated by a previously uncharacterized mechanism. PMID:22846919

  12. Identification of high-affinity calmodulin-binding proteins in rat liver

    International Nuclear Information System (INIS)

    Hanley, R.M.; Dedman, J.R.; Shenolikar, S.

    1987-01-01

    The Ca 2+ -dependent binding of [ 125 I] calmodulin (CaM) to hepatic proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was utilized to identify CaM binding or acceptor proteins or CAPs. Two proteins of apparent molecular weight of 60,000 (CAP-60) and 45,000 (CAP-45) comprised > 80% of the Ca 2+ -dependent CaM binding in rat liver cytosol. CAP-60 and CAP-45 were partially purified by a variety of chromatographic steps, including affinity chromatography on CaM Sepharose. CAP-60 possessed a native molecular size of 400,000, indicating it to be the CaM-binding subunit of a larger oligomeric complex. In contrast, CAP-45 was monomeric as judged by gel filtration. Neither CAP-60 nor CAP-45 possessed chromatographic properties consistent with known CaM-dependent enzymes reported in the literature. Two-dimensional peptide mapping provided convincing evidence that CAP-60 and CAP-45 were unrelated to other well-characterized CAPs, namely Ca 2+ (CaM)-dependent protein kinase II, calcineurin, or the CaM-dependent cyclic nucleotide phosphodiesterase. The relative abundance and high affinity for CaM could suggest that these novel target proteins, CAP-60 and CAP-45, represent a dominant pathway for CaM action in the mammalian liver

  13. Humic Acid Confers HIGH-AFFINITY K+ TRANSPORTER 1-Mediated Salinity Stress Tolerance in Arabidopsis.

    Science.gov (United States)

    Khaleda, Laila; Park, Hee Jin; Yun, Dae-Jin; Jeon, Jong-Rok; Kim, Min Gab; Cha, Joon-Yung; Kim, Woe-Yeon

    2017-12-31

    Excessive salt disrupts intracellular ion homeostasis and inhibits plant growth, which poses a serious threat to global food security. Plants have adapted various strategies to survive in unfavorable saline soil conditions. Here, we show that humic acid (HA) is a good soil amendment that can be used to help overcome salinity stress because it markedly reduces the adverse effects of salinity on Arabidopsis thaliana seedlings. To identify the molecular mechanisms of HA-induced salt stress tolerance in Arabidopsis, we examined possible roles of a sodium influx transporter HIGH-AFFINITY K+ TRANSPORTER 1 (HKT1). Salt-induced root growth inhibition in HKT1 overexpressor transgenic plants (HKT1-OX) was rescued by application of HA, but not in wild-type and other plants. Moreover, salt-induced degradation of HKT1 protein was blocked by HA treatment. In addition, the application of HA to HKT1-OX seedlings led to increased distribution of Na+ in roots up to the elongation zone and caused the reabsorption of Na+ by xylem and parenchyma cells. Both the influx of the secondary messenger calcium and its cytosolic release appear to function in the destabilization of HKT1 protein under salt stress. Taken together, these results suggest that HA could be applied to the field to enhance plant growth and salt stress tolerance via post-transcriptional control of the HKT1 transporter gene under saline conditions.

  14. Enhanced membrane pore formation through high-affinity targeted antimicrobial peptides.

    Directory of Open Access Journals (Sweden)

    Christopher J Arnusch

    Full Text Available Many cationic antimicrobial peptides (AMPs target the unique lipid composition of the prokaryotic cell membrane. However, the micromolar activities common for these peptides are considered weak in comparison to nisin, which follows a targeted, pore-forming mode of action. Here we show that AMPs can be modified with a high-affinity targeting module, which enables membrane permeabilization at low concentration. Magainin 2 and a truncated peptide analog were conjugated to vancomycin using click chemistry, and could be directed towards specific membrane embedded receptors both in model membrane systems and whole cells. Compared with untargeted vesicles, a gain in permeabilization efficacy of two orders of magnitude was reached with large unilamellar vesicles that included lipid II, the target of vancomycin. The truncated vancomycin-peptide conjugate showed an increased activity against vancomycin resistant Enterococci, whereas the full-length conjugate was more active against a targeted eukaryotic cell model: lipid II containing erythrocytes. This study highlights that AMPs can be made more selective and more potent against biological membranes that contain structures that can be targeted.

  15. Early signs of pathological cognitive aging in mice lacking high-affinity nicotinic receptors.

    Directory of Open Access Journals (Sweden)

    Eleni eKonsolaki

    2016-04-01

    Full Text Available In order to address pathological cognitive decline effectively, it is critical to adopt early preventive measures in individuals considered at risk. It is therefore essential to develop approaches that identify such individuals before the onset of irreversible dementia. Α deficient cholinergic system has been consistently implicated as one of the main factors associated with a heightened vulnerability to the aging process. In the present study we used mice lacking high affinity nicotinic receptors (β2-/-, which have been proposed as an animal model of accelerated/premature cognitive aging. Our aim was to identify behavioural signs that could serve as indicators or predictors of impending cognitive decline. We used test batteries in order to assess cognitive functions and additional tasks to investigate spontaneous behaviours, such as species-specific activities and exploration/locomotion in a novel environment. Our data confirm and extend the hypothesis that β2-/- animals exhibit age-related cognitive impairments, manifested in both spatial learning and recognition memory tasks. In addition, we reveal deficits in spontaneous behaviour and habituation processes earlier in life. To our knowledge, this is the first study to perform an extensive behavioural examination of an animal model of premature cognitive aging, and our results suggest that β2-nAChR dependent cognitive deterioration progressively evolves from initial subtle behavioural changes to global dementia due to the combined effect of the neuropathology and aging.

  16. High affinity soluble ILT2 receptor: a potent inhibitor of CD8(+) T cell activation.

    Science.gov (United States)

    Moysey, Ruth K; Li, Yi; Paston, Samantha J; Baston, Emma E; Sami, Malkit S; Cameron, Brian J; Gavarret, Jessie; Todorov, Penio; Vuidepot, Annelise; Dunn, Steven M; Pumphrey, Nicholas J; Adams, Katherine J; Yuan, Fang; Dennis, Rebecca E; Sutton, Deborah H; Johnson, Andy D; Brewer, Joanna E; Ashfield, Rebecca; Lissin, Nikolai M; Jakobsen, Bent K

    2010-12-01

    Using directed mutagenesis and phage display on a soluble fragment of the human immunoglobulin super-family receptor ILT2 (synonyms: LIR1, MIR7, CD85j), we have selected a range of mutants with binding affinities enhanced by up to 168,000-fold towards the conserved region of major histocompatibility complex (MHC) class I molecules. Produced in a dimeric form, either by chemical cross-linking with bivalent polyethylene glycol (PEG) derivatives or as a genetic fusion with human IgG Fc-fragment, the mutants exhibited a further increase in ligand-binding strength due to the avidity effect, with resident half-times (t(1/2)) on the surface of MHC I-positive cells of many hours. The novel compounds antagonized the interaction of CD8 co-receptor with MHC I in vitro without affecting the peptide-specific binding of T-cell receptors (TCRs). In both cytokine-release assays and cell-killing experiments the engineered receptors inhibited the activation of CD8(+) cytotoxic T lymphocytes (CTLs) in the presence of their target cells, with subnanomolar potency and in a dose-dependent manner. As a selective inhibitor of CD8(+) CTL responses, the engineered high affinity ILT2 receptor presents a new tool for studying the activation mechanism of different subsets of CTLs and could have potential for the development of novel autoimmunity therapies.

  17. Physiological epidermal growth factor concentrations activate high affinity receptors to elicit calcium oscillations.

    Directory of Open Access Journals (Sweden)

    Béatrice Marquèze-Pouey

    Full Text Available Signaling mediated by the epidermal growth factor (EGF is crucial in tissue development, homeostasis and tumorigenesis. EGF is mitogenic at picomolar concentrations and is known to bind its receptor on high affinity binding sites depending of the oligomerization state of the receptor (monomer or dimer. In spite of these observations, the cellular response induced by EGF has been mainly characterized for nanomolar concentrations of the growth factor, and a clear definition of the cellular response to circulating (picomolar concentrations is still lacking. We investigated Ca2+ signaling, an early event in EGF responses, in response to picomolar doses in COS-7 cells where the monomer/dimer equilibrium is unaltered by the synthesis of exogenous EGFR. Using the fluo5F Ca2+ indicator, we found that picomolar concentrations of EGF induced in 50% of the cells a robust oscillatory Ca2+ signal quantitatively similar to the Ca2+ signal induced by nanomolar concentrations. However, responses to nanomolar and picomolar concentrations differed in their underlying mechanisms as the picomolar EGF response involved essentially plasma membrane Ca2+ channels that are not activated by internal Ca2+ store depletion, while the nanomolar EGF response involved internal Ca2+ release. Moreover, while the picomolar EGF response was modulated by charybdotoxin-sensitive K+ channels, the nanomolar response was insensitive to the blockade of these ion channels.

  18. Physiological epidermal growth factor concentrations activate high affinity receptors to elicit calcium oscillations.

    Science.gov (United States)

    Marquèze-Pouey, Béatrice; Mailfert, Sébastien; Rouger, Vincent; Goaillard, Jean-Marc; Marguet, Didier

    2014-01-01

    Signaling mediated by the epidermal growth factor (EGF) is crucial in tissue development, homeostasis and tumorigenesis. EGF is mitogenic at picomolar concentrations and is known to bind its receptor on high affinity binding sites depending of the oligomerization state of the receptor (monomer or dimer). In spite of these observations, the cellular response induced by EGF has been mainly characterized for nanomolar concentrations of the growth factor, and a clear definition of the cellular response to circulating (picomolar) concentrations is still lacking. We investigated Ca2+ signaling, an early event in EGF responses, in response to picomolar doses in COS-7 cells where the monomer/dimer equilibrium is unaltered by the synthesis of exogenous EGFR. Using the fluo5F Ca2+ indicator, we found that picomolar concentrations of EGF induced in 50% of the cells a robust oscillatory Ca2+ signal quantitatively similar to the Ca2+ signal induced by nanomolar concentrations. However, responses to nanomolar and picomolar concentrations differed in their underlying mechanisms as the picomolar EGF response involved essentially plasma membrane Ca2+ channels that are not activated by internal Ca2+ store depletion, while the nanomolar EGF response involved internal Ca2+ release. Moreover, while the picomolar EGF response was modulated by charybdotoxin-sensitive K+ channels, the nanomolar response was insensitive to the blockade of these ion channels.

  19. Specific, high affinity receptors for insulin-like growth factor II in the rat kidney glomerulus

    International Nuclear Information System (INIS)

    Haskell, J.F.; Pillion, D.J.; Meezan, E.

    1988-01-01

    Rat renal glomeruli were isolated by a technique involving kidney perfusion with a solution containing magnetic iron oxide particles, followed by homogenization, sieving, and concentration over a strong magnet. Isolated glomeruli were treated with 1% Triton X-100 to solubilize plasma membrane components, while insoluble basement membrane components were removed by centrifugation. [ 125 I]Insulin-like growth factor II (IGF-II) binding to this preparation was competitively inhibited by increasing amounts of unlabeled IGF-II, with 50% inhibition at an IGF-II concentration of 1 ng/ml. [ 125 I]IGF-II was covalently cross-linked with disuccinimidyl suberate to its receptor in rat renal glomeruli and a specific high mol wt (255,000) band could be identified on autoradiograms of dodecyl sulfate-polyacrylamide gels. [ 125 I]IGF-II binding and cross-linking to this band was inhibited by a polyclonal antibody against the type II IGF receptor. These results demonstrate for the first time that the isolated rat renal glomerulus contains a high affinity receptor for IGF-II

  20. Affinity Crystallography: A New Approach to Extracting High-Affinity Enzyme Inhibitors from Natural Extracts.

    Science.gov (United States)

    Aguda, Adeleke H; Lavallee, Vincent; Cheng, Ping; Bott, Tina M; Meimetis, Labros G; Law, Simon; Nguyen, Nham T; Williams, David E; Kaleta, Jadwiga; Villanueva, Ivan; Davies, Julian; Andersen, Raymond J; Brayer, Gary D; Brömme, Dieter

    2016-08-26

    Natural products are an important source of novel drug scaffolds. The highly variable and unpredictable timelines associated with isolating novel compounds and elucidating their structures have led to the demise of exploring natural product extract libraries in drug discovery programs. Here we introduce affinity crystallography as a new methodology that significantly shortens the time of the hit to active structure cycle in bioactive natural product discovery research. This affinity crystallography approach is illustrated by using semipure fractions of an actinomycetes culture extract to isolate and identify a cathepsin K inhibitor and to compare the outcome with the traditional assay-guided purification/structural analysis approach. The traditional approach resulted in the identification of the known inhibitor antipain (1) and its new but lower potency dehydration product 2, while the affinity crystallography approach led to the identification of a new high-affinity inhibitor named lichostatinal (3). The structure and potency of lichostatinal (3) was verified by total synthesis and kinetic characterization. To the best of our knowledge, this is the first example of isolating and characterizing a potent enzyme inhibitor from a partially purified crude natural product extract using a protein crystallographic approach.

  1. High-affinity cannabinoid binding site in brain: A possible marijuana receptor

    International Nuclear Information System (INIS)

    Nye, J.S.

    1988-01-01

    The mechanism by which delta 9 tetrahydrocannabinol (delta 9 THC), the major psychoactive component of marijuana or hashish, produces its potent psychological and physiological effects is unknown. To find receptor binding sites for THC, we designed a water-soluble analog for use as a radioligand. 5'-Trimethylammonium-delta 8 THC (TMA) is a positively charged analog of delta- 8 THC modified on the 5' carbon, a portion of the molecule not important for its psychoactivity. We have studied the binding of [ 3 H]-5'-trimethylammonium-delta- 8 THC ([ 3 H]TMA) to rat neuronal membranes. [ 3 H]TMA binds saturably and reversibly to brain membranes with high affinity to apparently one class of sites. Highest binding site density occurs in brain, but several peripheral organs also display specific binding. Detergent solubilizes the sites without affecting their pharmacologial properties. Molecular sieve chromatography reveals a bimodal peak of [ 3 H]TMA binding activity of approximately 60,000 daltons apparent molecular weight

  2. A high affinity Ca2(+)-ATPase on the surface membrane of Leishmania donovani promastigote

    International Nuclear Information System (INIS)

    Ghosh, J.; Ray, M.; Sarkar, S.; Bhaduri, A.

    1990-01-01

    A Ca2(+)-dependent ATP-hydrolytic activity was detected in the crude membrane ghost of the promastigote or vector form of the protozoal parasite Leishmania donovani, the pathogen responsible for kala azar. The Ca2(+)-ATPase was purified to apparent homogeneity after solubilization with deoxycholate. The enzyme consists of two subunits of Mr = 51,000 and 57,000 and has an apparent molecular weight of 215,000 +/- 12,000. The enzyme activity is exclusively dependent on Ca2+, and the pure enzyme can hydrolyze 1.6 mumol of ATP/min/mg of protein. The apparent Km for Ca2+ is 35 nM, which is further reduced to 12 nM in the presence of heterologous calmodulin. The enzyme is sensitive to vanadate, but is insensitive to oligomycin and ouabain. The enzyme is strongly associated with the plasma membrane and has its catalytic site oriented toward the cytoplasmic face. The enzyme spans across the plasma membrane as surface labeling with radioiodine shows considerable radioactivity in the completely purified enzyme. The localization and orientation of this high affinity, calmodulin-sensitive Ca2(+)-ATPase suggest some role of this enzyme in Ca2+ movement in the life cycle of this protozoal parasite

  3. A high affinity Ca2(+)-ATPase on the surface membrane of Leishmania donovani promastigote

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh, J.; Ray, M.; Sarkar, S.; Bhaduri, A. (Indian Institute of Chemical Biology, Calcutta (India))

    1990-07-05

    A Ca2(+)-dependent ATP-hydrolytic activity was detected in the crude membrane ghost of the promastigote or vector form of the protozoal parasite Leishmania donovani, the pathogen responsible for kala azar. The Ca2(+)-ATPase was purified to apparent homogeneity after solubilization with deoxycholate. The enzyme consists of two subunits of Mr = 51,000 and 57,000 and has an apparent molecular weight of 215,000 +/- 12,000. The enzyme activity is exclusively dependent on Ca2+, and the pure enzyme can hydrolyze 1.6 mumol of ATP/min/mg of protein. The apparent Km for Ca2+ is 35 nM, which is further reduced to 12 nM in the presence of heterologous calmodulin. The enzyme is sensitive to vanadate, but is insensitive to oligomycin and ouabain. The enzyme is strongly associated with the plasma membrane and has its catalytic site oriented toward the cytoplasmic face. The enzyme spans across the plasma membrane as surface labeling with radioiodine shows considerable radioactivity in the completely purified enzyme. The localization and orientation of this high affinity, calmodulin-sensitive Ca2(+)-ATPase suggest some role of this enzyme in Ca2+ movement in the life cycle of this protozoal parasite.

  4. Inhibition of high affinity choline uptake by N-allyl-3-quinuclidinol

    International Nuclear Information System (INIS)

    Asermely, K.E.; O'Neill, J.J.

    1986-01-01

    The peripheral actions of N-allyl-3-quinuclidinol (N-Al-3-OHQ) on high affinity choline uptake (HAChU) on rat phrenic nerve diaphragm are described. Endplate regions (EPA) identified by the Koelle histochemical techniques for acetylcholinesterase, were dissected from adult rat hemidiaphragms and placed in cold Krebs solution (pH-7.35). All measurements of HAChU were at 37 0 C in buffers containing tritium choline (5 μM 0.124 μC/mmole) at intervals of 1, 2, 4, 8, 15 and 30 min. Tissues were washed 3x, digested in 1N NaOH and counted for tritium in Chaikoff's solution. All data are expressed as pmole Ch/g wet weight. Comparison between EPA and non-EPA tissue demonstrate HAChU and slow choline diffusion, respectively. Steady state is observed in 15 min. N-Al-3-OHQ produces 15% inhibition at 5 x 10 -5 M compared with 50% inhibition on brain synaptosomes. At 5 x 10 -4 M N-Al-3-OHQ, 30% inhibition is observed. Attempts to deplete ACh by pre-stimulation with high K + -ion (25 mM) were unsuccessful; tissue 3 H-choline uptake appeared to oscillate over a 30 min period

  5. Inhibition of high affinity choline uptake by N-allyl-3-quinuclidinol

    Energy Technology Data Exchange (ETDEWEB)

    Asermely, K.E.; O' Neill, J.J.

    1986-03-01

    The peripheral actions of N-allyl-3-quinuclidinol (N-Al-3-OHQ) on high affinity choline uptake (HAChU) on rat phrenic nerve diaphragm are described. Endplate regions (EPA) identified by the Koelle histochemical techniques for acetylcholinesterase, were dissected from adult rat hemidiaphragms and placed in cold Krebs solution (pH-7.35). All measurements of HAChU were at 37/sup 0/C in buffers containing tritium choline (5 ..mu..M 0.124 ..mu..C/mmole) at intervals of 1, 2, 4, 8, 15 and 30 min. Tissues were washed 3x, digested in 1N NaOH and counted for tritium in Chaikoff's solution. All data are expressed as pmole Ch/g wet weight. Comparison between EPA and non-EPA tissue demonstrate HAChU and slow choline diffusion, respectively. Steady state is observed in 15 min. N-Al-3-OHQ produces 15% inhibition at 5 x 10/sup -5/ M compared with 50% inhibition on brain synaptosomes. At 5 x 10/sup -4/ M N-Al-3-OHQ, 30% inhibition is observed. Attempts to deplete ACh by pre-stimulation with high K/sup +/-ion (25 mM) were unsuccessful; tissue /sup 3/H-choline uptake appeared to oscillate over a 30 min period.

  6. Synthesis of site-heterologous haptens for high-affinity anti-pyraclostrobin antibody generation.

    Science.gov (United States)

    Mercader, Josep V; Agulló, Consuelo; Abad-Somovilla, Antonio; Abad-Fuentes, Antonio

    2011-03-07

    The design and synthesis of functional chemical derivatives of small organic molecules is usually a key step for the intricate production of a variety of bioconjugates. In this respect, the derivatization site at which the spacer arm is introduced in immunizing conjugates constitutes a highly critical parameter for the generation of high-affinity and selective antibodies. However, due to the usual complexity of the required synthetic procedures, the appropriate comparison of alternative tethering positions has often been neglected. In the present study, meticulous strategies were followed to prepare synthetic derivatives of pyraclostrobin with the same linkers located at diverse rationally-chosen sites. Activity appraisal of antibodies and bioconjugates was carried out by bidimensional competitive direct and indirect immunoassays, and a superior performance of two of the three synthesized haptens was found. Finally, a detailed analysis of the conformations of the target molecule and the synthesized haptens in aqueous solution was done using computer assisted molecular modeling techniques. This study suggested that the lower titers and affinities of one set of antibodies are most probably due to conformational effects of the spacer arm in the immunizing bioconjugate.

  7. Comparison of the Fc fragment from a human IgG1 and its CH2, pFc', and tFc' subfragments. A study using reductive methylation and 13C NMR

    International Nuclear Information System (INIS)

    Jentoft, J.E.; Rayford, R.

    1989-01-01

    The Fc fragment of a human monoclonal IgG1 was compared with subfragments containing (a) the intact CH2 domain (CH2 fragment) or (b) the intact CH3 domain (pFc' and tFc' fragments). All fragments were reductively 13 C-methylated and their resulting dimethyllysyl resonances characterized in 0.1 M KCl as a function of pH by 13 C NMR spectroscopy. Seven resonances were characterized for the 18 lysine residues of the Fc fragment, eight for the 12 lysines of the CH2 fragment, and five each for the 9 lysines of the pFc' and the 6 lysines of the tFc' fragments, respectively. The multiplicity of resonances indicates that the lysine residues in each fragment exist in a variety of microenvironments and that the fragments are all highly structured. The correspondence between 6 of the 12 or 13 perturbed lysine residues in the Fc fragment and the smaller subfragments indicates that the conformation of the CH2 and CH3 domains is largely unchanged in the smaller fragments. However, in addition to three lysines at the CH2-CH3 domain interface, whose environments were known to be disrupted in the smaller fragments, three or four lysine residues have somewhat different properties in the Fc fragment and in the subfragments, indicating that some local perturbations are included in the domain structure in the subfragments

  8. Monoclonal antibodies to human factor VII: production of immunodepleted plasma for VII:C assays.

    OpenAIRE

    Takase, T; Tuddenham, E G; Chand, S; Goodall, A H

    1988-01-01

    A high affinity monoclonal antibody to factor VII (RFF-VII/1), coupled to sepharose, was used to immunodeplete factor VII from normal plasma. The plasma could be used as a substrate in a one stage coagulation assay and performed as well as, or better than, commercially available factor VII deficient plasma or plasma from congenitally deficient factor VII patients. Plasma from normal donors (n = 20), patients with liver disease (n = 20), and patients receiving warfarin (n = 20), or congenitall...

  9. Novel high-affinity and selective biaromatic 4-substituted ¿-hydroxybutyric acid (GHB) analogues as GHB ligands

    DEFF Research Database (Denmark)

    Høg, Signe; Wellendorph, Petrine; Nielsen, Birgitte

    2008-01-01

    Gamma-hydroxybutyrate (GHB) is a metabolite of gamma-aminobutyric acid (GABA) and has been proposed to function as a neurotransmitter or neuromodulator. GHB is used in the treatment of narcolepsy and is a drug of abuse. GHB binds to both GABA(B) receptors and specific high-affinity GHB sites...

  10. Are basophil histamine release and high affinity IgE receptor expression involved in asymptomatic skin sensitization?

    DEFF Research Database (Denmark)

    Jensen, Bettina Margrethe; Assing, K; Jensen, Lone Hummelshøj

    2006-01-01

    Immunoglobulin (Ig)E-sensitized persons with positive skin prick test, but no allergy symptoms, are classified as being asymptomatic skin sensitized (AS). The allergic type 1 disease is dependant on IgE binding to the high affinity IgE-receptor (FcepsilonRI) expressed on basophils and mast cells....

  11. New Synthesis and Tritium Labeling of a Selective Ligand for Studying High-Affinity γ-Hydroxybutyrate (GHB) Binding Sites

    DEFF Research Database (Denmark)

    Vogensen, Stine B.; Marek, Ales; Bay, Tina

    2013-01-01

    3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [3H]-1 is described. The radiosynthesis employs in situ generated lithium trimethoxyborotritide. Screen...

  12. Carbon-11-labelling of a novel, trishomocubane-derived, high affinity and selectivity DAT ligand

    International Nuclear Information System (INIS)

    Dolle, F.; Le Helleix, St.; Peyronneau, M.A.; Saba, W.; Tournier, N.; Valette, H.; Banister, S.; Kassiou, M.

    2011-01-01

    Complete text of publication follows: Objectives: Parkinson's disease, schizophrenia, attention deficit disorder and drug abuse are related to abnormalities within the brain's dopaminergic system. The neuronal dopamine transporter (DAT) plays a key role in regulating the synaptic concentration of dopamine and thus dopamine neurotransmission in the brain. Since the DAT can be considered as a marker of the integrity and number of the presynaptic striatal dopamine-producing neurons, considerable efforts have been spent in recent years on the design and development of DAT-selective radioligands for use in Positron Emission Tomography (PET) studies. Notably, the tropane PE2I and its fluorinated analogue LBT-999 were identified as having high affinity and selectivity for the DAT over the norepinephrine transporter (NET) and the serotonin transporter (SERT). Besides tropanes, only a few bicyclic frameworks, e.g. bicyclo[2.2.2]octanes, have delivered compounds with high affinity for the DAT. Recently, novel poly-carbocyclic DAT ligands with selectivity over the NET and the SERT were reported. The lead compound of this series (1, N-methyl-N-(3-fluoro) benzyl-pentacyclo[5.4.0.0 2, 6 .0 3, 10 .0 5, 9 ] undec-8-ylamine, Ki = 1.2 nM, ≥ 8300-fold selectivity over NET and SERT) was selected as a potential candidate for imaging the DAT with PET and isotopically labelled with carbon-11 using [ 11 C]methyl triflate. Methods: The trishomocubane derivatives 1 (reference) and 2 (precursor for labelling with carbon-11) were prepared from commercially available Cookson's diketone in 6 and 7 steps, respectively. Carbon-11 labelling of 1 was performed using a TRACERLab FX-C Pro synthesizer (GEMS) and comprises (1) trapping at -10 C of [ 11 C]MeOTf in acetone (0.4 mL) containing the nor-derivative 2 (0.6-0.9 mg, free base) and aq. 3N NaOH (8 μL); (2) heating at 110 C for 2 min; (3) concentration to dryness and taking up the residue in 1.0 mL of the HPLC mobile phase; (4) purification

  13. Blockage of High-Affinity Choline Transporter Increases Visceral Hypersensitivity in Rats with Chronic Stress

    Directory of Open Access Journals (Sweden)

    Chen Zhao

    2018-01-01

    Full Text Available Background. Visceral hypersensitivity is a common feature of irritable bowel syndrome. Cholinergic system involves in the development of visceral hypersensitivity, and high-affinity choline transporter (CHT1 is of crucial importance in choline uptake system. However, involvement of CHT1 in visceral hypersensitivity remains unknown. The research aimed to study the CHT1 expression in dorsal root ganglions (DRGs and the role of CHT1 in visceral hypersensitivity. Methods. Repetitive water avoidance stress (WAS was used to induce visceral hypersensitivity in rats. Colorectal distension (CRD was determined, and the abdominal withdrawal reflex (AWR and threshold intensity data were recorded to measure the visceral sensitivity. After intraperitoneal injection of hemicholinium-3 (HC-3, the specific inhibitor of CHT1, CRD data were also recorded. The CHT1 expression of DRGs was investigated by Western blotting, immunohistochemistry, and quantitative RT-PCR. Acetylcholine levels in the DRGs were detected by the assay kit. Results. Repetitive WAS increased the AWR score of CRD at high distension pressure and decreased the mean threshold of rats. The CHT1 expression and acetylcholine concentration of DRG were significantly increased in WAS rats. After the administration of HC-3, the AWR score in WAS group was significantly increased at higher distension pressure while the threshold intensity was significantly reduced compared to the normal saline group. Acetylcholine concentration was significantly lower than the normal saline rats. Conclusion. Our research firstly reports that CHT1 is overexpressed in noninflammatory visceral hypersensitivity, and blockage of CHT1 can enhance the visceral hypersensitivity. CHT1 may play an inhibitory role in visceral hypersensitivity.

  14. Blockage of High-Affinity Choline Transporter Increases Visceral Hypersensitivity in Rats with Chronic Stress

    Science.gov (United States)

    2018-01-01

    Background Visceral hypersensitivity is a common feature of irritable bowel syndrome. Cholinergic system involves in the development of visceral hypersensitivity, and high-affinity choline transporter (CHT1) is of crucial importance in choline uptake system. However, involvement of CHT1 in visceral hypersensitivity remains unknown. The research aimed to study the CHT1 expression in dorsal root ganglions (DRGs) and the role of CHT1 in visceral hypersensitivity. Methods Repetitive water avoidance stress (WAS) was used to induce visceral hypersensitivity in rats. Colorectal distension (CRD) was determined, and the abdominal withdrawal reflex (AWR) and threshold intensity data were recorded to measure the visceral sensitivity. After intraperitoneal injection of hemicholinium-3 (HC-3), the specific inhibitor of CHT1, CRD data were also recorded. The CHT1 expression of DRGs was investigated by Western blotting, immunohistochemistry, and quantitative RT-PCR. Acetylcholine levels in the DRGs were detected by the assay kit. Results Repetitive WAS increased the AWR score of CRD at high distension pressure and decreased the mean threshold of rats. The CHT1 expression and acetylcholine concentration of DRG were significantly increased in WAS rats. After the administration of HC-3, the AWR score in WAS group was significantly increased at higher distension pressure while the threshold intensity was significantly reduced compared to the normal saline group. Acetylcholine concentration was significantly lower than the normal saline rats. Conclusion Our research firstly reports that CHT1 is overexpressed in noninflammatory visceral hypersensitivity, and blockage of CHT1 can enhance the visceral hypersensitivity. CHT1 may play an inhibitory role in visceral hypersensitivity. PMID:29849603

  15. Endothelial targeting of high-affinity multivalent polymer nanocarriers directed to intercellular adhesion molecule 1.

    Science.gov (United States)

    Muro, Silvia; Dziubla, Thomas; Qiu, Weining; Leferovich, John; Cui, Xiumin; Berk, Erik; Muzykantov, Vladimir R

    2006-06-01

    Targeting of diagnostic and therapeutic agents to endothelial cells (ECs) provides an avenue to improve treatment of many maladies. For example, intercellular adhesion molecule 1 (ICAM-1), a constitutive endothelial cell adhesion molecule up-regulated in many diseases, is a good determinant for endothelial targeting of therapeutic enzymes and polymer nanocarriers (PNCs) conjugated with anti-ICAM (anti-ICAM/PNCs). However, intrinsic and extrinsic factors that control targeting of anti-ICAM/PNCs to ECs (e.g., anti-ICAM affinity and PNC valency and flow) have not been defined. In this study we tested in vitro and in vivo parameters of targeting to ECs of anti-ICAM/PNCs consisting of either prototype polystyrene or biodegradable poly(lactic-coglycolic) acid polymers (approximately 200 nm diameter spheres carrying approximately 200 anti-ICAM molecules). Anti-ICAM/PNCs, but not control IgG/PNCs 1) rapidly (t1/2 approximately 5 min) and specifically bound to tumor necrosis factor-activated ECs in a dose-dependent manner (Bmax approximately 350 PNC/cell) at both static and physiological shear stress conditions and 2) bound to ECs and accumulated in the pulmonary vasculature after i.v. injection in mice. Anti-ICAM/PNCs displayed markedly higher EC affinity versus naked anti-ICAM (Kd approximately 80 pM versus approximately 8 nM) in cell culture and, probably because of this factor, higher value (185.3 +/- 24.2 versus 50.5 +/- 1.5% injected dose/g) and selectivity (lung/blood ratio 81.0 +/- 10.9 versus 2.1 +/- 0.02, in part due to faster blood clearance) of pulmonary targeting. These results 1) show that reformatting monomolecular anti-ICAM into high-affinity multivalent PNCs boosts their vascular immuno-targeting, which withstands physiological hydrodynamics and 2) support potential anti-ICAM/PNCs utility for medical applications.

  16. 99mTc(CO)3-DTMA bombesin conjugates having high affinity for the GRP receptor

    International Nuclear Information System (INIS)

    Lane, Stephanie R.; Veerendra, Bhadrasetty; Rold, Tammy L.; Sieckman, Gary L.; Hoffman, Timothy J.; Jurisson, Silvia S.; Smith, Charles J.

    2008-01-01

    Introduction: Targeted diagnosis of specific human cancer types continues to be of significant interest in nuclear medicine. 99m Tc is ideally suited as a diagnostic radiometal for in vivo tumor targeting due to its ideal physical characteristics and diverse labeling chemistries in numerous oxidation states. Methods: In this study, we report a synthetic approach toward design of a new tridentate amine ligand for the organometallic aqua-ion [ 99m Tc(H 2 O) 3 (CO) 3 ] + . The new chelating ligand framework, 2-(N,N'-Bis(tert-butoxycarbonyl)diethylenetriamine) acetic acid (DTMA), was synthesized from a diethylenetriamine precursor and fully characterized by mass spectrometry and nuclear magnetic resonance spectroscopy ( 1 H and 13 C). DTMA was conjugated to H 2 N-(X)-BBN(7-14)NH 2 , where X=an amino acid or aliphatic pharmacokinetic modifier and BBN=bombesin peptide, by means of solid phase peptide synthesis. DTMA-(X)-BBN(7-14)NH 2 conjugates were purified by reversed-phase high-performance chromatography and characterized by electrospray-ionization mass spectrometry. Results: The new conjugates were radiolabeled with [ 99m Tc(H 2 O) 3 (CO) 3 ] + produced via Isolink radiolabeling kits to produce [ 99m Tc(CO) 3 -DTMA-(X)-BBN(7-14)NH 2 ]. Radiolabeled conjugates were purified by reversed-phase high-performance chromatography. Effective receptor binding behavior was evaluated in vitro and in vivo. Conclusions: [ 99m Tc(CO) 3 -DTMA-(X)-BBN(7-14)NH 2 ] conjugates displayed very high affinity for the gastrin releasing peptide receptor in vitro and in vivo. Therefore, these conjugates hold some propensity to be investigated as molecular imaging agents that specifically target human cancers uniquely expressing the gastrin releasing peptide receptor subtypes

  17. Characterization of high affinity [3H]triazolam binding in rat brain

    International Nuclear Information System (INIS)

    Earle, M.; Concas, A.; Yamamura, H.I.

    1986-01-01

    The hypnotic Triazolam (TZ), a triazolo (1,4)-benzodiazepine, displays a short physiological half life and has been used for the treatment of insomnia related to anxiety states. Specific binding properties of this recently tritiated TZ were characterized. The authors major objectives were the direct measurement of the temperature dependence and the GABA effect on [ 3 H]TZ binding. Saturation studies showed a shift to lower affinity at 37 0 C (K/sub d/ = 0.25 +/- 0.01 nM at O 0 C; K/sub d/ = 1.46 +/- 0.03 nM at 37 0 C) while the B/sub max/ values remained unchanged (1003 +/- 37 fmoles/mg prot. at 0 0 C and 1001 +/- 43 fmoles/mg prot. at 37 0 C). Inhibition studies showed that [ 3 H]TZ binding displayed no GABA shift at 0 0 C(K/sub i/ 0.37 +/- 0.03 nM/- GABA and K/sub i/ = 0.55 +/- 0.13 nM/+GABA) but a nearly two-fold shift was apparent at 37 0 C (K/sub i/ = 2.92 +/- 0.2 nM/-GABA; K/sub i/ = 1.37 +/- 0.11 mM/+GABA). These results were also confirmed by saturation studies in the presence or absence of GABA showing a shift to higher affinity in the presence of GABA only at 37 0 C. In Ro 15-1788/[ 3 H]TZ competition experiments the presence of GABA did not affect the inhibitory potency of Ro 15-1788 on [ 3 H]TZ binding at both temperatures. In conclusion [ 3 H]TZ binding showed an extremely high affinity for benzodiazepine receptors. In contrast to reported literature, the findings suggest that TZ interacts with benzodiazepine receptors similar to other benzodiazepine agonists

  18. Cartilage Acidic Protein 2 a hyperthermostable, high affinity calcium-binding protein.

    Science.gov (United States)

    Anjos, Liliana; Gomes, Ana S; Melo, Eduardo P; Canário, Adelino V; Power, Deborah M

    2013-03-01

    Cartilage Acidic Protein 2 (CRTAC2) is a novel protein present from prokaryotes to vertebrates with abundant expression in the teleost fish pituitary gland and an isoform of CRTAC1, a chondrocyte marker in humans. The two proteins are non-integrins containing N-terminal integrin-like Ca(2+)-binding motifs and their structure and function remain to be assigned. Structural studies of recombinant sea bream (sb)CRTAC2 revealed it is composed of 8.8% α-helix, 33.4% β-sheet and 57.8% unordered protein. sbCRTAC2 bound Ca(2+) with high affinity (K(d)=1.46nM) and favourable Gibbs free energy (∆G=-12.4kcal/mol). The stoichiometry for Ca(2+) bound to sbCRTAC2 at saturation indicated six Ca(2+) ligand-binding sites exist per protein molecule. No conformational change in sbCRTAC2 occurred in the presence of Ca(2+). Fluorescence emission revealed that the tertiary structure of the protein is hyperthermostable between 25°C and 95°C and the fully unfolded state is only induced by chemical denaturing (4M GndCl). sbCRTAC has a widespread tissue distribution and is present as high molecular weight aggregates, although strong reducing conditions promote formation of the monomer. sbCRTAC2 promotes epithelial cell outgrowth in vitro suggesting it may share functional homology with mammalian CRTAC1, recently implicated in cell-cell and cell-matrix interactions. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. Synthesis of protected peptides from the human IgG1 hinge region on PEG support using disulfide bond synthons and alkaline or enzymatic detachment

    Czech Academy of Sciences Publication Activity Database

    Niederhafner, Petr; Šafařík, Martin; Šebestík, Jaroslav; Gut, Vladimír; Maloň, Petr; Hlaváček, Jan

    2006-01-01

    Roč. 47, č. 6 (2006), s. 1023-1025 ISSN 0040-4039 R&D Projects: GA ČR(CZ) GA203/03/1362 Institutional research plan: CEZ:AV0Z40550506 Keywords : peptide synthesis * IgG1 hinge peptide * PEG carrier Subject RIV: CC - Organic Chemistry Impact factor: 2.509, year: 2006

  20. Physical stability comparisons of IgG1-Fc variants: effects of N-glycosylation site occupancy and Asp/Gln residues at site Asn 297.

    Science.gov (United States)

    Alsenaidy, Mohammad A; Okbazghi, Solomon Z; Kim, Jae Hyun; Joshi, Sangeeta B; Middaugh, C Russell; Tolbert, Thomas J; Volkin, David B

    2014-06-01

    The structural integrity and conformational stability of various IgG1-Fc proteins produced from the yeast Pichia pastoris with different glycosylation site occupancy (di-, mono-, and nonglycosylated) were determined. In addition, the physical stability profiles of three different forms of nonglycosylated Fc molecules (varying amino-acid residues at site 297 in the CH 2 domain due to the point mutations and enzymatic digestion of the Fc glycoforms) were also examined. The physical stability of these IgG1-Fc glycoproteins was examined as a function of pH and temperature by high-throughput biophysical analysis using multiple techniques combined with data visualization tools (three index empirical phase diagrams and radar charts). Across the pH range of 4.0-6.0, the di- and monoglycosylated forms of the IgG1-Fc showed the highest and lowest levels of physical stability, respectively, with the nonglycosylated forms showing intermediate stability depending on solution pH. In the aglycosylated Fc proteins, the introduction of Asp (D) residues at site 297 (QQ vs. DN vs. DD forms) resulted in more subtle changes in structural integrity and physical stability depending on solution pH. The utility of evaluating the conformational stability profile differences between the various IgG1-Fc glycoproteins is discussed in the context of analytical comparability studies. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

  1. Prognosis in monoclonal proteinaemia

    NARCIS (Netherlands)

    Schaar, Cornelis Gerardus

    2006-01-01

    Monoclonal proteinaemia (M-proteinemia) is associated with multiple myeloma (MM) or other hematological malignancies. In the absence of these diseases the term MGUS (Monoclonal Gammopathy of Undetermined Significance) is used. During 1991-1993 1464 patients with newly diagnosed M-proteinemia in the

  2. Therapeutic Recombinant Monoclonal Antibodies

    Science.gov (United States)

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  3. Nuclear medicine: Monoclonal antibodies

    International Nuclear Information System (INIS)

    Endo, K.; Sakahara, H.; Koizumi, M.; Kawamura, Y.; Torizuka, K.; Yokoyama, A.

    1986-01-01

    Antitumor monoclonal antibody was successfully labeled with Tc-99m by using dithiosemicarbazone (DTS) as a bifunctional chelating agent. In the first step, DTS was coupled to antibody without loss of immunoreactivity; the compound then efficiently formed a neutral 1:1 chelate with pentavalent or tetravalent Tc-99m. Imaging with Tc-99m-labeled monoclonal antibody to human osteosarcoma (OST-7) clearly displayed a small tumor in nude mice at 6 and 24 hours after intravenous administration. The tumor-to-blood ratio of the Tc-99m-labeled monoclonal antibody was higher than that of a radioiodinated antibody and similar to that of an In-111-labeled antibody. Thus, conjugation of DTS to monoclonal antibody followed by radiometalation is a simple and efficient method of preparing Tc-99m-labeled monoclonal antibody

  4. Neisseria meningitidis Group A IgG1 and IgG2 Subclass Immune Response in African Children Aged 12–23 Months Following Meningococcal Vaccination

    Science.gov (United States)

    Holme, Daniel; Findlow, Helen; Sow, Samba O.; Idoko, Olubukola T.; Preziosi, Marie-Pierre; Carlone, George; Plikaytis, Brian D.; Borrow, Ray

    2015-01-01

    Background. A group A meningococcal conjugate vaccine, PsA-TT, was licensed in 2010 and was previously studied in a phase 2 clinical trial to evaluate its safety and immunogenicity in African children 12–23 months of age. Methods. Subjects received either PsA-TT; meningococcal group A, C, W, Y polysaccharide vaccine (PsACWY); or Haemophilus influenzae type b conjugate vaccine (Hib-TT). Forty weeks following primary vaccination, the 3 groups were further randomized to receive either PsA-TT, one-fifth dose of PsACWY, or Hib-TT. Group A–specific immunoglobulin G (IgG) subclass response was characterized using an enzyme-linked immunosorbent assay. Results. The predominant IgG subclass response, regardless of vaccine, was IgG1. One month following primary vaccination, the geometric mean concentrations (GMCs) of IgG1 and IgG2 in the PsA-TT group were 21.73 µg/mL and 6.27 µg/mL, whereas in the PsACWY group the mean GMCs were 2.01 µg/mL and 0.97 µg/mL, respectively (P Group A–specific IgG1 and IgG2 GMCs remained greater in the PsA-TT group than in the PsACWY group 40 weeks following primary vaccination (P vaccines. Conclusions. Vaccination of African children aged 12–24 months with either PsA-TT or PsACWY elicited a predominantly IgG1 response. The IgG1:IgG2 mean ratio decreased following successive vaccination with PsACWY, indicating a shift toward IgG2, suggestive of the T-cell–independent immune response commonly associated with polysaccharide antigens. Clinical Trials Registration. SRCTN78147026. PMID:26553689

  5. Twins in spirit part II: DOTATATE and high-affinity DOTATATE - the clinical experience

    Energy Technology Data Exchange (ETDEWEB)

    Brogsitter, Claudia; Zoephel, Klaus; Hartmann, Holger; Kotzerke, Joerg [Technische Universitaet Dresden, Department of Nuclear Medicine, Dresden (Germany); Schottelius, Margret; Wester, Hans-Juergen [Technische Universitaet Muenchen, Pharmaceutical Radiochemistry and Department of Nuclear Medicine, Muenchen (Germany)

    2014-06-15

    Over recent decades interest in diagnosis and treatment of neuroendocrine tumours (NET) has steadily grown. The basis for diagnosis and therapy of NET with radiolabelled somatostatin (hsst) analogues is the variable overexpression of hsst receptors (hsst1-5 receptors). We hypothesized that radiometal derivatives of DOTA-iodo-Tyr{sup 3}-octreotide analogues might be excellent candidates for somatostatin receptor imaging. We therefore explored the diagnostic potential of {sup 68}Ga-DOTA-iodo-Tyr{sup 3}-octreotate [{sup 68}Ga-DOTA,3-iodo-Tyr{sup 3},Thr{sup 8}]octreotide ({sup 68}Ga-HA-DOTATATE; HA, high-affinity) compared to the established {sup 68}Ga-DOTA-Tyr{sup 3}-octreotate ({sup 68}Ga-DOTATATE) in vivo. The study included 23 patients with known somatostatin receptor-positive metastases from NETs, thyroid cancer or glomus tumours who were investigated with both {sup 68}Ga-HA-DOTATATE and {sup 68}Ga-DOTATATE. A patient-based and a lesion-based comparative analysis was carried out of normal tissue distribution and lesion detectability in a qualitative and a semiquantitative manner. {sup 68}Ga-HA-DOTATATE and {sup 68}Ga-DOTATATE showed comparable uptake in the liver (SUV{sub mean} 8.9 ± 2.2 vs. 9.3 ± 2.5, n.s.), renal cortex (SUV{sub mean} 13.3 ± 3.9 vs. 14.5 ± 3.7, n.s.) and spleen (SUV{sub mean} 24.0 ± 6.7 vs. 22.9 ± 7.3, n.s.). A somewhat higher pituitary uptake was found with {sup 68}Ga-HA-DOTATATE (SUV{sub mean} 6.3 ± 1.8 vs. 5.4 ± 2.1, p < 0.05). On a lesion-by-lesion basis a total of 344 lesions were detected. {sup 68}Ga-HA-DOTATATE demonstrated 328 lesions (95.3 % of total lesions seen), and {sup 68}Ga-DOTATATE demonstrated 332 lesions (96.4 %). The mean SUV{sub max} of all lesions was not significantly different between {sup 68}Ga-HA-DOTATATE and {sup 68}Ga-DOTATATE (17.8 ± 11.4 vs. 16.7 ± 10.7, n.s.). Our analysis demonstrated very good concordance between {sup 68}Ga-HA-DOTATATE and {sup 68}Ga-DOTATATE PET data. As the availability and use of {sup

  6. Dopamine inhibition of anterior pituitary adenylate cyclase is mediated through the high-affinity state of the D2 receptor

    International Nuclear Information System (INIS)

    Borgundvaag, B.; George, S.R.

    1985-01-01

    The diterpinoid forskolin stimulated adenylate cyclase activity (measured by conversion of [ 3 H]-ATP to [ 3 H]-cAMP) in anterior pituitary from male and female rats. Inhibition of stimulated adenylate cyclase activity by potent dopaminergic agonists was demonstrable only in female anterior pituitary. The inhibition of adenylate cyclase activity displayed a typically dopaminergic rank order of agonist potencies and could be completely reversed by a specific dopamine receptor antagonist. The IC 50 values of dopamine agonist inhibition of adenylate cyclase activity correlated with equal molarity with the dissociation constant of the high-affinity dopamine agonist-detected receptor binding site and with the IC 50 values for inhibition of prolactin secretion. These findings support the hypothesis that it is the high-affinity form of the D 2 dopamine receptor in anterior pituitary which is responsible for mediating the dopaminergic function of attenuating adenylate cyclase activity. 12 references, 4 figures, 1 table

  7. High affinity human antibody fragments to dengue virus non-structural protein 3.

    Directory of Open Access Journals (Sweden)

    Nicole J Moreland

    Full Text Available BACKGROUND: The enzyme activities catalysed by flavivirus non-structural protein 3 (NS3 are essential for virus replication. They are distributed between the N-terminal protease domain in the first one-third and the C-terminal ATPase/helicase and nucleoside 5' triphosphatase domain which forms the remainder of the 618-aa long protein. METHODOLOGY/PRINCIPAL FINDINGS: In this study, dengue full-length NS3 protein with residues 49 to 66 of NS2B covalently attached via a flexible linker, was used as bait in biopanning with a naïve human Fab phage-display library. Using a range of truncated constructs spanning the NS2B cofactor region and the full-length NS3, 10 unique Fab were identified and characterized. Of these, monoclonal Fab 3F8 was shown to bind α3″ (residues 526 through 531 within subdomain III of the helicase domain. The antibody inhibits the ATPase and helicase activites of NS3 in biochemical assays and reduces DENV replication in HEK293 cells that were previously transfected with Fab 3F8 compared with mock transfected cells. CONCLUSIONS/SIGNIFICANCE: Antibodies such as 3F8 are valuable tools for studying the molecular mechanisms of flaviviral replication and for the monospecific detection of replicating dengue virus in vivo.

  8. Characterization of high-affinity (/sup 3/H)ouabain binding in the rat central nervous system

    Energy Technology Data Exchange (ETDEWEB)

    Hauger, R.; Luu, H.M.; Meyer, D.K.; Goodwin, F.K.; Paul, S.M.

    1985-06-01

    The characteristics of (/sup 3/H)ouabain binding were examined in various areas of rat brain. In the striatum, Scatchard analysis revealed a single class of high-affinity binding sites with an apparent binding affinity (KD) of 10.4 +/- 0.9 nM and an estimated binding capacity (Bmax) of 7.6 +/- 1.9 pmol/mg protein. Similar monophasic Scatchard plots were found in the brainstem, cerebellum, hypothalamus, and frontal cerebral cortex. (/sup 3/H)Ouabain binding to rat brain was sodium- and ATP-dependent and strongly inhibited by potassium. Proscillariden A was the most potent cardiac glycoside tested in inhibiting specific (/sup 3/H)ouabain binding to brain membranes, and the rank order of inhibitory potencies for a series of cardiac glycosides was similar to that previously reported for inhibition of heart Na,K-ATPase. To assess whether the high-affinity binding sites for (/sup 3/H)ouabain were localized to neuronal or nonneuronal membranes, the effect of discrete kainic acid lesions on striatal (/sup 3/H)ouabain binding was examined. Kainic acid lesions of the striatum reduced (/sup 3/H)ouabain binding to striatal homogenates by 79.6 +/- 1.6%. This suggests that the high-affinity (/sup 3/H)ouabain binding sites measured in our experiments are localized to neuronal elements. Thus, the high-affinity binding of (/sup 3/H)ouabain to brain membranes may selectively label a neuronal form or conformation of Na,K-ATPase.

  9. Functional characterization of the high affinity IgG Receptor : making heads and tails of FcγRI

    NARCIS (Netherlands)

    van der Poel, C.E.

    2011-01-01

    This thesis focuses on human FcγRI, a high affinity receptor for antibodies of the IgG isotype. IgG is the most abundant antibody type in blood and all currently FDA approved therapeutic antibodies are of the IgG isotype. FcγRI, a member of the activating Fcγ receptors, exists as a complex of a

  10. Benzodiazepines have high-affinity binding sites and induce melanogenesis in B16/C3 melanoma cells.

    OpenAIRE

    Matthew, E; Laskin, J D; Zimmerman, E A; Weinstein, I B; Hsu, K C; Engelhardt, D L

    1981-01-01

    We found that two markers of differentiation, tyrosinase (monophenol, dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) activity and melanin synthesis, are induced by diazepam in B16/C3 mouse melanoma cells. We also demonstrated high-affinity binding sites for [3H]diazepam in these cells by radioreceptor assay, and we visualized binding to the cell surface by fluorescence microscopy with a benzodiazepine analog conjugated to a fluorescein-labeled protein. Our studies also showed tha...

  11. Photoaffinity labeling of mammalian α1-adrenergic receptors: identification of the ligand binding subunit with a high affinity radioiodinated probe

    International Nuclear Information System (INIS)

    Leeb-Lundberg, L.M.F.; Dickinson, K.E.J.; Heald, S.L.

    1984-01-01

    A description is given of the synthesised and characterization of a novel high affinity radioiodinated α 1 -adrenergic receptor photoaffinity probe, 4-amino-6,7-dimethoxy-2-[4-[5-(4-azido-3-[ 125 I]iodophenyl)pentanoyl]-1-piperazinyl] quinazoline. In the absence of light, this ligand binds with high affinity (K/sub d/ = 130 pm) in a reverisble and saturable manner to sites in rat hepatic plasma membranes. The binding is stereoselective and competitively inhibited by adrenergic agonists and antagonists with an α 1 -adrenergic specificity. Upon photolysis, this ligand incorporates irreversibly into plasma membranes prepared from several mammalian tissues including rat liver, rat, guinea pig, and rabbit spleen, rabbit lung, and rabbit aorta vascular smooth muscle cells, also with typical α 1 -adrenergic specificity. Autoradiograms of such membrane samples subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveal a major specifically labeled polypeptide at M/sub 4/ = 78,000-85,000, depending on the tissue used, in addition to some lower molecular weight peptides. Protease inhibitors, in particular EDTA, a metalloprotease inhibitor, dramatically increases the predominance of the M/sub r/ = 78,000-85,000 polypeptide while attenuating the labeling of the lower molecular weight bands. This new high affinity radioiodinated photoaffinity probe should be of great value for the molecular characterization of the α 1 -adrenergic receptor

  12. High-affinity RNA aptamers to C-reactive protein (CRP): newly developed pre-elution methods for aptamer selection

    International Nuclear Information System (INIS)

    Orito, N; Umekage, S; Sakai, E; Tanaka, T; Kikuchi, Y; Sato, K; Kawauchi, S; Tanaka, H

    2012-01-01

    We have developed a modified SELEX (systematic evolution of ligands by exponential enrichment) method to obtain RNA aptamers with high affinity to C-reactive protein (CRP). CRP is a clinical biomarker present in plasma, the level of which increases in response to infections and noninfectious inflammation. The CRP level is also an important prognostic indicator in patients with several syndromes. At present, CRP content in blood is measured immunochemically using antibodies. To develop a more sensitive method using RNA aptamers, we have attempted to obtain high-affinity RNA aptamers to CRP. We succeeded in obtaining an RNA aptamer with high affinity to CRP using a CRP-immobilized Sepharose column and pre-elution procedure. Pre-elution is a method that removes the weak binding portion from a selected RNA population by washing for a short time with buffer containing CRP. By surface plasmon-resonance (SPR) analysis, the affinity constant of this aptamer for CRP was calculated to be K D = 2.25x10 -9 (M). The secondary structure, contact sites with CRP protein, and application of this aptamer will be described.

  13. Differences between high-affinity forskolin binding sites in dopamine-riche and other regions of rat brain

    International Nuclear Information System (INIS)

    Poat, J.A.; Cripps, H.E.; Iversen, L.L.

    1988-01-01

    Forskolin labelled with [ 3 H] bound to high- and low-affinity sites in the rat brain. The high-affinity site was discretely located, with highest densities in the striatum, nucleus accumbens, olfactory tubercule, substantia nigra, hippocampus, and the molecular layers of the cerebellum. This site did not correlate well with the distribution of adenylate cyclase. The high-affinity striatal binding site may be associated with a stimulatory guanine nucleotide-binding protein. Thus, the number of sites was increased by the addition of Mg 2+ and guanylyl imidodiphosphate. Cholera toxin stereotaxically injected into rat striatum increased the number of binding sites, and no further increase was noted following the subsequent addition of guanyl nucleotide. High-affinity forskolin binding sites in non-dopamine-rich brain areas (hippocampus and cerebullum) were modulated in a qualitatively different manner by guanyl nucleotides. In these areas the number of binding sites was significantly reduced by the addition of guanyl nucleotide. These results suggest that forskolin may have a potential role in identifying different functional/structural guanine nucleotide-binding proteins

  14. Energy-dependent dissociation of ATP from high affinity catalytic sites of beef heart mitochondrial adenosine triphosphatase

    International Nuclear Information System (INIS)

    Penefsky, H.S.

    1985-01-01

    Incubation of [gamma- 32 P]ATP with a molar excess of the membrane-bound form of mitochondrial ATPase (F1) results in binding of the bulk of the radioactive nucleotide in high affinity catalytic sites (Ka = 10(12) M-1). Subsequent initiation of respiration by addition of succinate or NADH is accompanied by a profound decrease in the affinity for ATP. About one-third of the bound radioactive ATP appears to dissociate, that is, the [gamma- 32 P]ATP becomes accessible to hexokinase. The NADH-stimulated dissociation of [gamma- 32 P]ATP is energy-dependent since the stimulation is inhibited by uncouplers of oxidative phosphorylation and is prevented by respiratory chain inhibitors. The rate of the energy-dependent dissociation of ATP that occurs in the presence of NADH, ADP, and Pi is commensurate with the measured initial rate of ATP synthesis in NADH-supported oxidative phosphorylation catalyzed by the same submitochondrial particles. Thus, the rate of dissociation of ATP from the high affinity catalytic site of submitochondrial particles meets the criterion of kinetic competency under the conditions of oxidative phosphorylation. These experiments provide evidence in support of the argument that energy conserved during the oxidation of substrates by the respiratory chain can be utilized to reduce the very tight binding of product ATP in high affinity catalytic sites and to promote dissociation of the nucleotide

  15. Characterization of monoclonal antibodies directed against human thyroid stimulating hormone

    International Nuclear Information System (INIS)

    Soos, M.; Siddle, K.

    1982-01-01

    Monoclonal antibodies directed against human thyroid stimulating hormone (TSH) were obtained from hybrid myelomas, following fusion of mouse NSI myeloma cells with mouse spleen cells. Ten different antibodies were obtained from 4 separate fusions. Eight antibodies were of the IgG 1 subclass. Affinities of antibodies for TSH were in the range 2 x 10 8 -5 x 10 10 M -1 . Five of the antibodies were specific for TSH and did not react with LH, FSH or hCG. The remaining antibodies reacted with all these hormones and were assumed to recognise their common (α) subunit. The 5 specific antibodies fell into 3 subgroups recognising distinct antigenic determinants, whereas the 5 non-specific antibodies recognised a single determinant or closely related set of sites. It is concluded that these antibodies should be valuable reagents for use in sensitive and specific two-site immunoradiometric assays. (Auth.)

  16. High-affinity uranyl-specific antibodies suitable for cellular imaging

    Energy Technology Data Exchange (ETDEWEB)

    Reisser-Rubrecht, L.; Torne-Celer, C.; Renier, W.; Averseng, O.; Plantevin, S.; Quemeneur, E.; Bellanger, L.; Vidaud, C. [CEA Valrho, DSV, IBEB, Serv Biochim et Toxicol Nucl, F-30207 Bagnols Sur Ceze (France)

    2008-07-01

    Monoclonal antibodies (mAbs) have proved to be valuable models for the study of protein-metal interactions, and previous reports have described very specific antibodies to chelated metal ions, including uranyl. We raised specific mAbs against UO{sub 2}{sup 2+}-DCP-BSA (DCP, 1, 10-phenanthroline-2,9-dicarboxylic acid) to generate new sets of antibodies that might cross-react with various complexed forms of uranyl in different environments for further application in the field of toxicology. Using counter-screening with UO{sub 2}{sup 2+}-DCP-casein, we selected two highly specific mAbs against uranyl-DCP (K{sub D} = 10-100 pM): U04S and U08S. Competitive assays in the presence of different metal ions (UO{sub 2}{sup 2+}, Fe{sup 3+}, Zn{sup 2+}, Cu{sup 2+}, and Ca{sup 2+}) showed that uranyl in solution can act as a good competitor, suggesting some antibody ability to cross-react with chelating groups other than DCP in the UO{sub 2}{sup 2+} equatorial coordination plane. Interestingly, one of the antibodies could be used for revealing uranyl cations in cell samples. Fluorescence activated cell sorting analyses after immuno-labeling revealed the interaction of uranyl with human kidney cells HK2. The intracellular accumulation of uranyl could be directly visualized by metal-immunostaining using fluorescent-labeled mAb. Our results suggest that U04S mAb epitopes mostly include the uranyl fraction and its para-topes can accommodate a wide variety of chelating groups. (authors)

  17. High-affinity uranyl-specific antibodies suitable for cellular imaging

    International Nuclear Information System (INIS)

    Reisser-Rubrecht, L.; Torne-Celer, C.; Renier, W.; Averseng, O.; Plantevin, S.; Quemeneur, E.; Bellanger, L.; Vidaud, C.

    2008-01-01

    Monoclonal antibodies (mAbs) have proved to be valuable models for the study of protein-metal interactions, and previous reports have described very specific antibodies to chelated metal ions, including uranyl. We raised specific mAbs against UO 2 2+ -DCP-BSA (DCP, 1, 10-phenanthroline-2,9-dicarboxylic acid) to generate new sets of antibodies that might cross-react with various complexed forms of uranyl in different environments for further application in the field of toxicology. Using counter-screening with UO 2 2+ -DCP-casein, we selected two highly specific mAbs against uranyl-DCP (K D = 10-100 pM): U04S and U08S. Competitive assays in the presence of different metal ions (UO 2 2+ , Fe 3+ , Zn 2+ , Cu 2+ , and Ca 2+ ) showed that uranyl in solution can act as a good competitor, suggesting some antibody ability to cross-react with chelating groups other than DCP in the UO 2 2+ equatorial coordination plane. Interestingly, one of the antibodies could be used for revealing uranyl cations in cell samples. Fluorescence activated cell sorting analyses after immuno-labeling revealed the interaction of uranyl with human kidney cells HK2. The intracellular accumulation of uranyl could be directly visualized by metal-immunostaining using fluorescent-labeled mAb. Our results suggest that U04S mAb epitopes mostly include the uranyl fraction and its para-topes can accommodate a wide variety of chelating groups. (authors)

  18. Isolation of highly active monoclonal antibodies against multiresistant gram-positive bacteria.

    Directory of Open Access Journals (Sweden)

    Friederike S Rossmann

    Full Text Available Multiresistant nosocomial pathogens often cause life-threatening infections that are sometimes untreatable with currently available antibiotics. Staphylococci and enterococci are the predominant Gram-positive species associated with hospital-acquired infections. These infections often lead to extended hospital stay and excess mortality. In this study, a panel of fully human monoclonal antibodies was isolated from a healthy individual by selection of B-cells producing antibodies with high opsonic killing against E. faecalis 12030. Variable domains (VH and VL of these immunoglobulin genes were amplified by PCR and cloned into an eukaryotic expression vector containing the constant domains of a human IgG1 molecule and the human lambda constant domain. These constructs were transfected into CHO cells and culture supernatants were collected and tested by opsonophagocytic assay against E. faecalis and S. aureus strains (including MRSA. At concentrations of 600 pg/ml, opsonic killing was between 40% and 70% against all strains tested. Monoclonal antibodies were also evaluated in a mouse sepsis model (using S. aureus LAC and E. faecium, a mouse peritonitis model (using S. aureus Newman and LAC and a rat endocarditis model (using E. faecalis 12030 and were shown to provide protection in all models at a concentration of 4 μg/kg per animal. Here we present a method to produce fully human IgG1 monoclonal antibodies that are opsonic in vitro and protective in vivo against several multiresistant Gram-positive bacteria. The monoclonal antibodies presented in this study are significantly more effective compared to another monoclonal antibody currently in clinical trials.

  19. Computational modeling and molecular imprinting for the development of acrylic polymers with high affinity for bile salts.

    Science.gov (United States)

    Yañez, Fernando; Chianella, Iva; Piletsky, Sergey A; Concheiro, Angel; Alvarez-Lorenzo, Carmen

    2010-02-05

    This work has focused on the rational development of polymers capable of acting as traps of bile salts. Computational modeling was combined with molecular imprinting technology to obtain networks with high affinity for cholate salts in aqueous medium. The screening of a virtual library of 18 monomers, which are commonly used for imprinted networks, identified N-(3-aminopropyl)-methacrylate hydrochloride (APMA.HCl), N,N-diethylamino ethyl methacrylate (DEAEM) and ethyleneglycol methacrylate phosphate (EGMP) as suitable functional monomers with medium-to-high affinity for cholic acid. The polymers were prepared with a fix cholic acid:functional monomer mole ratio of 1:4, but with various cross-linking densities. Compared to polymers prepared without functional monomer, both imprinted and non-imprinted microparticles showed a high capability to remove sodium cholate from aqueous medium. High affinity APMA-based particles even resembled the performance of commercially available cholesterol-lowering granules. The imprinting effect was evident in most of the networks prepared, showing that computational modeling and molecular imprinting can act synergistically to improve the performance of certain polymers. Nevertheless, both the imprinted and non-imprinted networks prepared with the best monomer (APMA.HCl) identified by the modeling demonstrated such high affinity for the template that the imprinting effect was less important. The fitting of adsorption isotherms to the Freundlich model indicated that, in general, imprinting increases the population of high affinity binding sites, except when the affinity of the functional monomer for the target molecule is already very high. The cross-linking density was confirmed as a key parameter that determines the accessibility of the binding points to sodium cholate. Materials prepared with 9% mol APMA and 91% mol cross-linker showed enough affinity to achieve binding levels of up to 0.4 mmol g(-1) (i.e., 170 mg g(-1)) under flow

  20. Radioimmunoimaging of subacute infective endocarditis using a technetium-99m monoclonal granulocyte-specific antibody

    International Nuclear Information System (INIS)

    Munz, D.L.; Sandrock, D.; Emrich, D.; Morguet, A.J.; Heim, A.; Sold, G.; Figulla, H.R.; Kreuzer, H.

    1991-01-01

    Immunoscintigraphy with a technetium-99m murine monoclonal IgG 1 antibody directed against non-specific cross-reacting antigen (NCA-95) and carcinoembryonic antigen was performed with 20 patients with suspected subacute infective endocarditis (SIE) and 6 controls with suspected inflammatory/infectious disease elsewhere in the body. Immunoscintigraphy and echocardiography localised SIE in 11 of 15 patients in whom the disease could be confirmed. In 4 patients with validated SIE, the immunoscan was abnormal, and the echocardiogram was normal. In another 4 patients, the result was exactly the opposite. These findings suggest that the combination of immunoscintigraphy and echocardiography improves diagnostic efficacy in patients with suspected SIE. (orig.)

  1. Viral control in chronic HIV-1 subtype C infection is associated with enrichment of p24 IgG1 with Fc effector activity.

    Science.gov (United States)

    Chung, Amy; Makuba, Jenniffer M; Ndlovu, Bongiwe; Licht, Anna; Robinson, Hannah; Ramlakhan, Yathisha; Ghebremichael, Musie; Reddy, Tarylee; Goulder, Philip; Walker, Bruce; Ndung'u, Thumbi; Alter, Galit

    2018-04-03

    Postinfection HIV viral control and immune correlates analysis of the RV144 vaccine trial indicate a potentially critical role for Fc receptor-mediated antibody functions. However, the influence of functional antibodies in clade C infection is largely unknown. Plasma samples from 361 chronic subtype C-infected, antiretroviral therapy-naïve participants were tested for their HIV-specific isotype and subclass distributions, along with their Fc receptor-mediated functional potential. Total IgG, IgG subclasses and IgA binding to p24 clade B/C and gp120 consensus C proteins were assayed by multiplex. Antibody-dependent uptake of antigen-coated beads and Fc receptor-mediated natural killer cell degranulation were evaluated as surrogates for antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC), respectively. p24 IgG1 was the only subclass associated with viral control (P = 0.01), with higher p24-specific ADCP and ADCC responses detected in individuals with high p24 IgG1. Although p24 IgG1 levels were enriched in patients with elevated Gag-specific T-cell responses, these levels remained an independent predictor of low-viral loads (P = 0.04) and high CD4 counts (P = 0.004) after adjusting for Gag-specific T-cell responses and for protective HLA class I alleles. p24 IgG1 levels independently predict viral control in HIV-1 clade C infection. Whether these responses contribute to direct antiviral control via the recruited killing of infected cells via the innate immune system or simply mark a qualitatively superior immune response to HIV, is uncertain, but highlights the role of p24-specific antibodies in control of clade C HIV-1 infection.

  2. Monoclonal antibodies and cancer

    International Nuclear Information System (INIS)

    Haisma, H.J.

    1987-01-01

    The usefulness of radiolabeled monoclonal antibodies for imaging and treatment of human (ovarian) cancer was investigated. A review of tumor imaging with monoclonal antibodies is presented. Special attention is given to factors that influence the localization of the antibodies in tumors, isotope choice and methods of radiolabeling of the monoclonal antibodies. Two monoclonal antibodies, OC125 and OV-TL3, with high specificity for human epithelial ovarian cancer are characterized. A simple radio-iodination technique was developed for clinical application of the monoclonal antibodies. The behavior of monoclonal antibodies in human tumor xenograft systems and in man are described. Imaging of tumors is complicated because of high background levels of radioactivity in other sites than the tumor, especially in the bloodpool. A technique was developed to improve imaging of human tumor xenographs in nude mice, using subtraction of a specific and a non-specific antibody, radiolabeled with 111 In, 67 Ga and 131 I. To investigate the capability of the two monoclonal antibodies, to specifically localize in human ovarian carcinomas, distribution studies in mice bearing human ovarian carcinoma xenografts were performed. One of the antibodies, OC125, was used for distribution studies in ovarian cancer patients. OC125 was used because of availability and approval to use this antibody in patients. The same antibody was used to investigate the usefulness of radioimmunoimaging in ovarian cancer patients. The interaction of injected radiolabeled antibody OC125 with circulating antigen and an assay to measure the antibody response in ovarian cancer patients after injection of the antibody is described. 265 refs.; 30 figs.; 19 tabs

  3. A low redox potential affects monoclonal antibody assembly and glycosylation in cell culture.

    Science.gov (United States)

    Dionne, Benjamin; Mishra, Neha; Butler, Michael

    2017-03-20

    Glycosylation and intracellular assembly of monoclonal antibodies (MAbs) is important for glycan profile consistency. To better understand how these factors may be influenced by a lower redox potential, an IgG1-producing NS0 cell line was grown in the presence of varying concentrations of dithiothreitol (DTT). Cultures were monitored for growth and culture redox potential (CRP) with glycan heterogeneity determined using a HILIC-HPLC method. Macroheterogeneity was unchanged in all conditions whereas the Galactosylation Index (GI) decreased by as much as 50% in cultures with lower CRP or higher dithiothreitol levels. This shift in GI is reflected in more agalactosylated and asialylated species being produced. The MAb assembly pathway was determined using radioactive isotope 35 S incorporated into nascent IgG1 molecules. The assembly pathway for this IgG1 was shown to progress via HC→HC 2 →HC 2 LC→HC 2 LC 2 in all conditions tested and autoradiographs highlighted that the ratio of heavy chain dimer to heavy chain monomer increased over time with increasing DTT concentrations. This increase and correspondingly lower GI values may be due to disruption of the disulfide bonds at higher levels of assembly. A change in the assembly pathway may alter the final IgG glycan pattern and lead to control mechanisms that influence glycan profiles of MAbs. Copyright © 2017. Published by Elsevier B.V.

  4. Insights from the Fungus Fusarium oxysporum Point to High Affinity Glucose Transporters as Targets for Enhancing Ethanol Production from Lignocellulose

    Science.gov (United States)

    Ali, Shahin S.; Nugent, Brian; Mullins, Ewen; Doohan, Fiona M.

    2013-01-01

    Ethanol is the most-widely used biofuel in the world today. Lignocellulosic plant biomass derived from agricultural residue can be converted to ethanol via microbial bioprocessing. Fungi such as Fusarium oxysporum can simultaneously saccharify straw to sugars and ferment sugars to ethanol. But there are many bottlenecks that need to be overcome to increase the efficacy of microbial production of ethanol from straw, not least enhancement of the rate of fermentation of both hexose and pentose sugars. This research tested the hypothesis that the rate of sugar uptake by F. oxysporum would enhance the ethanol yields from lignocellulosic straw and that high affinity glucose transporters can enhance ethanol yields from this substrate. We characterized a novel hexose transporter (Hxt) from this fungus. The F. oxysporum Hxt represents a novel transporter with homology to yeast glucose signaling/transporter proteins Rgt2 and Snf3, but it lacks their C-terminal domain which is necessary for glucose signalling. Its expression level decreased with increasing glucose concentration in the medium and in a glucose uptake study the Km(glucose) was 0.9 mM, which indicated that the protein is a high affinity glucose transporter. Post-translational gene silencing or over expression of the Hxt in F. oxysporum directly affected the glucose and xylose transport capacity and ethanol yielded by F. oxysporum from straw, glucose and xylose. Thus we conclude that this Hxt has the capacity to transport both C5 and C6 sugars and to enhance ethanol yields from lignocellulosic material. This study has confirmed that high affinity glucose transporters are ideal candidates for improving ethanol yields from lignocellulose because their activity and level of expression is high in low glucose concentrations, which is very common during the process of consolidated processing. PMID:23382943

  5. Topography of the high-affinity lysine binding site of plasminogen as defined with a specific antibody probe

    International Nuclear Information System (INIS)

    Miles, L.A.; Plow, E.F.

    1986-01-01

    An antibody population that reacted with the high-affinity lysine binding site of human plasminogen was elicited by immunizing rabbits with an elastase degradation product containing kringles 1-3 (EDP I). This antibody was immunopurified by affinity chromatography on plasminogen-Sepharose and elution with 0.2 M 6-aminohexanoic acid. The eluted antibodies bound [ 125 I]EDP I, [ 125 I]Glu-plasminogen, and [ 125 I]Lys-plasminogen in radioimmunoassays, and binding of each ligand was at least 99% inhibited by 0.2 M 6-aminohexanoic acid. The concentrations for 50% inhibition of [ 125 I]EDP I binding by tranexamic acid, 6-aminohexanoic acid, and lysine were 2.6, 46, and l730 μM, respectively. Similar values were obtained with plasminogen and suggested that an unoccupied high-affinity lysine binding site was required for antibody recognition. The antiserum reacted exclusively with plasminogen derivatives containing the EDP I region and did not react with those lacking an EDP I region, or with tissue plasminogen activator or prothrombin, which also contains kringles. By immunoblotting analyses, a chymotryptic degradation product of M/sub r/ 20,000 was derived from EDP I that retained reactivity with the antibody. α 2 -Antiplasmin inhibited the binding of radiolabeled EDP I, Glu-plasminogen, or Lys-plasminogen by the antiserum, suggesting that the recognized site is involved in the noncovalent interaction of the inhibitor with plasminogen. The binding of [ 125 I]EDP I to fibrin was also inhibited by the antiserum. The observations provide independent evidence for the role of the high-affinity lysine binding site in the functional interactions of plasminogen with its primary substrate and inhibitor

  6. Structural implications of hERG K+ channel block by a high-affinity minimally structured blocker

    Science.gov (United States)

    Helliwell, Matthew V.; Zhang, Yihong; El Harchi, Aziza; Du, Chunyun; Hancox, Jules C.; Dempsey, Christopher E.

    2018-01-01

    Cardiac potassium channels encoded by human ether-à-go-go–related gene (hERG) are major targets for structurally diverse drugs associated with acquired long QT syndrome. This study characterized hERG channel inhibition by a minimally structured high-affinity hERG inhibitor, Cavalli-2, composed of three phenyl groups linked by polymethylene spacers around a central amino group, chosen to probe the spatial arrangement of side chain groups in the high-affinity drug-binding site of the hERG pore. hERG current (IhERG) recorded at physiological temperature from HEK293 cells was inhibited with an IC50 of 35.6 nm with time and voltage dependence characteristic of blockade contingent upon channel gating. Potency of Cavalli-2 action was markedly reduced for attenuated inactivation mutants located near (S620T; 54-fold) and remote from (N588K; 15-fold) the channel pore. The S6 Y652A and F656A mutations decreased inhibitory potency 17- and 75-fold, respectively, whereas T623A and S624A at the base of the selectivity filter also decreased potency (16- and 7-fold, respectively). The S5 helix F557L mutation decreased potency 10-fold, and both F557L and Y652A mutations eliminated voltage dependence of inhibition. Computational docking using the recent cryo-EM structure of an open channel hERG construct could only partially recapitulate experimental data, and the high dependence of Cavalli-2 block on Phe-656 is not readily explainable in that structure. A small clockwise rotation of the inner (S6) helix of the hERG pore from its configuration in the cryo-EM structure may be required to optimize Phe-656 side chain orientations compatible with high-affinity block. PMID:29545312

  7. Insights from the fungus Fusarium oxysporum point to high affinity glucose transporters as targets for enhancing ethanol production from lignocellulose.

    Directory of Open Access Journals (Sweden)

    Shahin S Ali

    Full Text Available Ethanol is the most-widely used biofuel in the world today. Lignocellulosic plant biomass derived from agricultural residue can be converted to ethanol via microbial bioprocessing. Fungi such as Fusarium oxysporum can simultaneously saccharify straw to sugars and ferment sugars to ethanol. But there are many bottlenecks that need to be overcome to increase the efficacy of microbial production of ethanol from straw, not least enhancement of the rate of fermentation of both hexose and pentose sugars. This research tested the hypothesis that the rate of sugar uptake by F. oxysporum would enhance the ethanol yields from lignocellulosic straw and that high affinity glucose transporters can enhance ethanol yields from this substrate. We characterized a novel hexose transporter (Hxt from this fungus. The F. oxysporum Hxt represents a novel transporter with homology to yeast glucose signaling/transporter proteins Rgt2 and Snf3, but it lacks their C-terminal domain which is necessary for glucose signalling. Its expression level decreased with increasing glucose concentration in the medium and in a glucose uptake study the Km((glucose was 0.9 mM, which indicated that the protein is a high affinity glucose transporter. Post-translational gene silencing or over expression of the Hxt in F. oxysporum directly affected the glucose and xylose transport capacity and ethanol yielded by F. oxysporum from straw, glucose and xylose. Thus we conclude that this Hxt has the capacity to transport both C5 and C6 sugars and to enhance ethanol yields from lignocellulosic material. This study has confirmed that high affinity glucose transporters are ideal candidates for improving ethanol yields from lignocellulose because their activity and level of expression is high in low glucose concentrations, which is very common during the process of consolidated processing.

  8. A rhodamine-labeled citalopram analogue as a high-affinity fluorescent probe for the serotonin transporter

    DEFF Research Database (Denmark)

    Zhang, Peng; Jørgensen, Trine Nygaard; Løland, Claus Juul

    2013-01-01

    A novel fluorescent ligand was synthesized as a high-affinity, high specificity probe for visualizing the serotonin transporter (SERT). The rhodamine fluorophore was extended from an aniline substitution on the 5-position of the dihydroisobenzofuran ring of citalopram (2, 1-(3-(dimethylamino......)propyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-carbonitrile), using an ethylamino linker. The resulting rhodamine-labeled ligand 8 inhibited [3H]5-HT uptake in COS-7 cells (Ki = 225 nM) with similar potency to the tropane-based JHC 1-064 (1), but with higher specificity towards the SERT relative...

  9. Autoradiographic imaging and quantification of the high-affinity GHB binding sites in rodent brain using (3)H-HOCPCA

    DEFF Research Database (Denmark)

    Klein, A B; Bay, T; Villumsen, I S

    2016-01-01

    analogue, 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) as a tritiated version ((3)H-HOCPCA) to radioactively label the specific GHB high-affinity binding site and gain further insight into the density, distribution and developmental profile of this protein. We show that, in low nanomolar concentrations...... brain development. Due to the high sensitivity of this radioligand, we were able to detect low levels of specific binding already at E15 in mouse brain, which increased progressively until adulthood. Collectively, we show that (3)H-HOCPCA is a highly sensitive radioligand, offering advantages over...

  10. Inhibition of Enterococcus faecium adherence to collagen by antibodies against high-affinity binding subdomains of Acm.

    Science.gov (United States)

    Nallapareddy, Sreedhar R; Sillanpää, Jouko; Ganesh, Vannakambadi K; Höök, Magnus; Murray, Barbara E

    2007-06-01

    Strains of Enterococcus faecium express a cell wall-anchored protein, Acm, which mediates adherence to collagen. Here, we (i) identify the minimal and high-affinity binding subsegments of Acm and (ii) show that anti-Acm immunoglobulin Gs (IgGs) purified against these subsegments reduced E. faecium TX2535 strain collagen adherence up to 73 and 50%, respectively, significantly more than the total IgGs against the full-length Acm A domain (28%) (P Acm adherence with functional subsegment-specific antibodies raises the possibility of their use as therapeutic or prophylactic agents.

  11. Monoclonal antibodies in oncology

    International Nuclear Information System (INIS)

    Chan, S.Y.T.; Sikora, K.

    1986-01-01

    Monoclonal antibodies (MCAs) can be used to differentiate between normal and neoplastic cells and thus exploited for diagnostic and, ultimately, therapeutic gain. The evidence for the existence of human tumour antigens is reviewed. Several areas of diagnosis are already benefiting from the application of the monoclonal technology. Immunohistology can help the pathologist with difficult diagnostic problems. New classifications of lymphoma and leukaemia can be based on specific surface molecules. Similarly, the detection of shed tumour antigens is already established as part of the routine assessment of many patients with common solid tumours. Isotopically labeled monoclonal antibodies have been used to localise primary and metastatic tumours. The use of antibodies in this way is not only a promising diagnostic tool but also the first step in studying the possibility of arming antibodies to provide therapeutic agents. Such trials are currently in progress. (Auth.)

  12. High-affinity DNA-binding Domains of Replication Protein A (RPA) Direct SMARCAL1-dependent Replication Fork Remodeling*

    Science.gov (United States)

    Bhat, Kamakoti P.; Bétous, Rémy; Cortez, David

    2015-01-01

    SMARCAL1 catalyzes replication fork remodeling to maintain genome stability. It is recruited to replication forks via an interaction with replication protein A (RPA), the major ssDNA-binding protein in eukaryotic cells. In addition to directing its localization, RPA also activates SMARCAL1 on some fork substrates but inhibits it on others, thereby conferring substrate specificity to SMARCAL1 fork-remodeling reactions. We investigated the mechanism by which RPA regulates SMARCAL1. Our results indicate that although an interaction between SMARCAL1 and RPA is essential for SMARCAL1 activation, the location of the interacting surface on RPA is not. Counterintuitively, high-affinity DNA binding of RPA DNA-binding domain (DBD) A and DBD-B near the fork junction makes it easier for SMARCAL1 to remodel the fork, which requires removing RPA. We also found that RPA DBD-C and DBD-D are not required for SMARCAL1 regulation. Thus, the orientation of the high-affinity RPA DBDs at forks dictates SMARCAL1 substrate specificity. PMID:25552480

  13. High-affinity DNA-binding domains of replication protein A (RPA) direct SMARCAL1-dependent replication fork remodeling.

    Science.gov (United States)

    Bhat, Kamakoti P; Bétous, Rémy; Cortez, David

    2015-02-13

    SMARCAL1 catalyzes replication fork remodeling to maintain genome stability. It is recruited to replication forks via an interaction with replication protein A (RPA), the major ssDNA-binding protein in eukaryotic cells. In addition to directing its localization, RPA also activates SMARCAL1 on some fork substrates but inhibits it on others, thereby conferring substrate specificity to SMARCAL1 fork-remodeling reactions. We investigated the mechanism by which RPA regulates SMARCAL1. Our results indicate that although an interaction between SMARCAL1 and RPA is essential for SMARCAL1 activation, the location of the interacting surface on RPA is not. Counterintuitively, high-affinity DNA binding of RPA DNA-binding domain (DBD) A and DBD-B near the fork junction makes it easier for SMARCAL1 to remodel the fork, which requires removing RPA. We also found that RPA DBD-C and DBD-D are not required for SMARCAL1 regulation. Thus, the orientation of the high-affinity RPA DBDs at forks dictates SMARCAL1 substrate specificity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Structure-guided development of a high-affinity human Programmed Cell Death-1: Implications for tumor immunotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Lázár-Molnár, Eszter; Scandiuzzi, Lisa; Basu, Indranil; Quinn, Thomas; Sylvestre, Eliezer; Palmieri, Edith; Ramagopal, Udupi A.; Nathenson, Stanley G.; Guha, Chandan; Almo, Steven C.

    2017-03-01

    Programmed Cell Death-1 (PD-1) is an inhibitory immune receptor, which plays critical roles in T cell co-inhibition and exhaustion upon binding to its ligands PD-L1 and PD-L2. We report the crystal structure of the human PD-1 ectodomain and the mapping of the PD-1 binding interface. Mutagenesis studies confirmed the crystallographic interface, and resulted in mutant PD-1 receptors with altered affinity and ligand-specificity. In particular, a high-affinity mutant PD-1 (HA PD-1) exhibited 45 and 30-fold increase in binding to PD-L1 and PD-L2, respectively, due to slower dissociation rates. This mutant (A132L) was used to engineer a soluble chimeric Ig fusion protein for cell-based and in vivo studies. HA PD-1 Ig showed enhanced binding to human dendritic cells, and increased T cell proliferation and cytokine production in a mixed lymphocyte reaction (MLR) assay. Moreover, in an experimental model of murine Lewis lung carcinoma, HA PD-1 Ig treatment synergized with radiation therapy to decrease local and metastatic tumor burden, as well as in the establishment of immunological memory responses. Our studies highlight the value of structural considerations in guiding the design of a high-affinity chimeric PD-1 Ig fusion protein with robust immune modulatory properties, and underscore the power of combination therapies to selectively manipulate the PD-1 pathway for tumor immunotherapy.

  15. Structure-guided development of a high-affinity human Programmed Cell Death-1: Implications for tumor immunotherapy

    Directory of Open Access Journals (Sweden)

    Eszter Lázár-Molnár

    2017-03-01

    Full Text Available Programmed Cell Death-1 (PD-1 is an inhibitory immune receptor, which plays critical roles in T cell co-inhibition and exhaustion upon binding to its ligands PD-L1 and PD-L2. We report the crystal structure of the human PD-1 ectodomain and the mapping of the PD-1 binding interface. Mutagenesis studies confirmed the crystallographic interface, and resulted in mutant PD-1 receptors with altered affinity and ligand-specificity. In particular, a high-affinity mutant PD-1 (HA PD-1 exhibited 45 and 30-fold increase in binding to PD-L1 and PD-L2, respectively, due to slower dissociation rates. This mutant (A132L was used to engineer a soluble chimeric Ig fusion protein for cell-based and in vivo studies. HA PD-1 Ig showed enhanced binding to human dendritic cells, and increased T cell proliferation and cytokine production in a mixed lymphocyte reaction (MLR assay. Moreover, in an experimental model of murine Lewis lung carcinoma, HA PD-1 Ig treatment synergized with radiation therapy to decrease local and metastatic tumor burden, as well as in the establishment of immunological memory responses. Our studies highlight the value of structural considerations in guiding the design of a high-affinity chimeric PD-1 Ig fusion protein with robust immune modulatory properties, and underscore the power of combination therapies to selectively manipulate the PD-1 pathway for tumor immunotherapy.

  16. Biphasic regulation of development of the high-affinity saxitoxin receptor by innervation in rat skeletal muscle

    International Nuclear Information System (INIS)

    Sherman, S.J.; Catterall, W.A.

    1982-01-01

    Specific binding of 3 H-saxitoxin (STX) was used to quantitate the density of voltage-sensitive sodium channels in developing rat skeletal muscle. In adult triceps surae, a single class of sites with a KD . 2.9 nM and a density of 21 fmol/mg wet wt was detected. The density of these high-affinity sites increased from 2.0 fmol/mg wet wt to the adult value in linear fashion during days 2-25 after birth. Denervation of the triceps surae at day 11 or 17 reduced final saxitoxin receptor site density to 10.4 or 9.2 fmol/mg wet wt, respectively, without changing KD. Denervation of the triceps surae at day 5 did not alter the subsequent development of saxitoxin receptor sites during days 5-9 and accelerated the increase of saxitoxin receptor sites during days 9-13. After day 13, saxitoxin receptor development abruptly ceased and the density of saxitoxin receptor sites declined to 11 fmol/wg wet wt. These results show that the regulation of high-affinity saxitoxin receptor site density by innervation is biphasic. During the first phase, which is independent of continuing innervation, the saxitoxin receptor density increases to 47-57% of the adult level. After day 11, the second phase of development, which is dependent on continuing innervation, gives rise to the adult density of saxitoxin receptors

  17. Preclinical evaluation of multistep targeting of diasialoganglioside GD2 using a IgG-scFv bispecific antibody with high affinity for GD2 and DOTA metal complex

    Science.gov (United States)

    Cheal, Sarah M.; Xu, Hong; Guo, Hong-fen; Zanzonico, Pat B.; Larson, Steven M.; Cheung, Nai-Kong

    2014-01-01

    Bispecific antibodies (BsAb) have proven to be useful targeting vectors for pretargeted radioimmunotherapy (PRIT). We sought to overcome key PRIT limitations such as high renal radiation exposure and immunogenicity (e.g. of streptavidin-antibody fusions), to advance clinical translation of this PRIT strategy for diasialoganglioside GD2-positive (GD2(+)) tumors. For this purpose, a IgG-scFv BsAb was engineered using the sequences for the anti-GD2 humanized monoclonal antibody hu3F8 (1) and C825, a murine scFv antibody with high affinity for the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) complexed with beta-particle emitting radiometals such as 177Lu and 90Y (2, 3). A three-step regimen including hu3F8-C825, a dextran-based clearing agent, and p-aminobenzyl-DOTA radiolabeled with 177Lu (as 177Lu-DOTA-Bn; t1/2 = 6.71 days (d)) was optimized in immunocompromised mice carrying subcutaneous (s.c.) human GD2(+) neuroblastoma (NB) xenografts. Absorbed doses for tumor and normal tissues were ∼85 cGy/MBq and ≤3.7 cGy/MBq, respectively, with therapeutic indicies (TI) of 142 for blood and 23 for kidney. A therapy study (n = 5 per group; tumor volume: 240 ± 160 mm3) with three successive PRIT cycles (total 177Lu: ∼33 MBq; tumor dose ∼3400 cGy), revealed complete tumor response in 5/5 animals, with no recurrence up to 28 d post-treatment. Tumor ablation was confirmed histologically in 4/5 mice, and normal organs showed minimal overall toxicities. All non-treated mice required sacrifice within 12 d (>1.0 cm3 tumor volume). We conclude that this novel anti-GD2 PRIT approach has sufficient TI to successfully ablate s.c. GD2(+)–NB in mice while sparing kidney and bone marrow. PMID:24944121

  18. Susceptibility to endotoxin induced uveitis is not reduced in mice deficient in BLT1, the high affinity leukotriene B4 receptor

    OpenAIRE

    Smith, J R; Subbarao, K; Franc, D T; Haribabu, B; Rosenbaum, J T

    2004-01-01

    Aim: To investigate the role of arachidonic acid derived chemotactic factor, LTB4, in the development of endotoxin induced uveitis (EIU), using mice deficient in the BLT1 gene which encodes the high affinity LTB4 receptor.

  19. Avaliação das subclasses IgG1 e IgG3 na doença hemolítica perinatal Assessment of IgG1 and IgG3 subclasses in perinatal hemolytic disease

    Directory of Open Access Journals (Sweden)

    Maria A. Araújo

    2003-01-01

    Full Text Available A doença hemolítica perinatal (DHPN ainda é um problema clínico. Nenhum teste isolado prediz, com segurança, a gravidade do quadro hemolítico. O objetivo do presente estudo foi determinar as subclasses de anticorpos IgG1 e IgG3 por citometria de fluxo no soro de 42 gestantes isoimunizadas e correlacionar os dados obtidos com a gravidade da DHPN. A distribuição dos fetos ou neonatos segundo a gravidade do quadro hemolítico evidenciou 13 casos com doença leve, 16 casos com doença moderada e 13 com doença grave. As subclasses foram detectadas em 33/42 (79% amostras. A subclasse IgG1, isoladamente, foi evidenciada em 14/33 (42,4% casos. Na relação entre gravidade da doença e subclasses de IgG, observou-se que IgG1 isolada foi encontrada em todos os grupos, e os valores da mediana de intensidade de fluorescência (MIF foram significativamente mais altos nas formas mais graves da DHPN (pThe hemolytic disease of the newborn (HDN continues to be a clinical problem in spite of prophylaxis. To date, none of the available tests, developed to predict the severity of HDN, has provided complete reliability. The objective of the present study was to determine the IgG1 and IgG3 subclasses in 42 isoimmunized pregnant women, and to correlate them with clinical severity of hemolytic disease. The IgG subclasses were determined employing flow cytometry. According to the clinical severity of HDN, fetuses and newborn babies were classified as 13 mild, 16 moderate and 13 severe cases. The IgG subclasses were detected in 33 of the 42 pregnant women. Of these, IgG1 was predominant in 72.7% of the cases; either isolated (42.4% or in association with IgG3 (30.3%. IgG1 was present in all the three clinical severity categories, however, its values were significantly higher in cases with greater clinical severity of HDN (p<0.01. On the other hand, the distribution of IgG3 values within each group was not statistically significant (p=0.11. IgG3 seems to be more associated with the mild hemolytic form of the disease, whereas the association of IgG1 and IgG3 suggested a clinically more severe form of HDN. It is possible, however, that the severity in these cases is related to the presence of IgG1. These results suggest that IgG1 and IgG3 isotypes should be included in multi-parametric protocols for the evaluation of clinical severity of HDN, as International literature does not give support to the use of IgG subclass determination alone as a reliable indicator to predict severity or prognosis of the disease.

  20. Monoclonal antibodies in myeloma

    DEFF Research Database (Denmark)

    Sondergeld, P.; van de Donk, N. W. C. J.; Richardson, P. G.

    2015-01-01

    The development of monoclonal antibodies (mAbs) for the treatment of disease goes back to the vision of Paul Ehrlich in the late 19th century; however, the first successful treatment with a mAb was not until 1982, in a lymphoma patient. In multiple myeloma, mAbs are a very recent and exciting add...

  1. Clearance of Human IgG1-Sensitised Red Blood Cells In Vivo in Humans Relates to the In Vitro Properties of Antibodies from Alternative Cell Lines

    Science.gov (United States)

    Armour, Kathryn L.; Smith, Cheryl S.; Ip, Natasha C. Y.; Ellison, Cara J.; Kirton, Christopher M.; Wilkes, Anthony M.; Williamson, Lorna M.; Clark, Michael R.

    2014-01-01

    We previously produced a recombinant version of the human anti-RhD antibody Fog-1 in the rat myeloma cell line, YB2/0. When human, autologous RhD-positive red blood cells (RBC) were sensitised with this IgG1 antibody and re-injected, they were cleared much more rapidly from the circulation than had been seen earlier with the original human-mouse heterohybridoma-produced Fog-1. Since the IgG have the same amino acid sequence, this disparity is likely to be due to alternative glycosylation that results from the rat and mouse cell lines. By comparing the in vitro properties of YB2/0-produced Fog-1 IgG1 and the same antibody produced in the mouse myeloma cell line NS0, we now have a unique opportunity to pinpoint the cause of the difference in ability to clear RBC in vivo. Using transfected cell lines that express single human FcγR, we showed that IgG1 made in YB2/0 and NS0 cell lines bound equally well to receptors of the FcγRI and FcγRII classes but that the YB2/0 antibody was superior in FcγRIII binding. When measuring complexed IgG binding, the difference was 45-fold for FcγRIIIa 158F, 20-fold for FcγRIIIa 158V and approximately 40-fold for FcγRIIIb. The dissimilarity was greater at 100-fold in monomeric IgG binding assays with FcγRIIIa. When used to sensitise RBC, the YB2/0 IgG1 generated 100-fold greater human NK cell antibody-dependent cell-mediated cytotoxicity and had a 103-fold advantage over the NS0 antibody in activating NK cells, as detected by CD54 levels. In assays of monocyte activation and macrophage adherence/phagocytosis, where FcγRI plays major roles, RBC sensitised with the two antibodies produced much more similar results. Thus, the alternative glycosylation profiles of the Fog-1 antibodies affect only FcγRIII binding and FcγRIII-mediated functions. Relating this to the in vivo studies confirms the importance of FcγRIII in RBC clearance. PMID:25302805

  2. Potential of Murine IgG1 and Human IgG4 to Inhibit the Classical Complement and Fcγ Receptor Activation Pathways

    Directory of Open Access Journals (Sweden)

    Gina-Maria Lilienthal

    2018-05-01

    Full Text Available IgG antibodies (Abs mediate their effector functions through the interaction with Fcγ receptors (FcγRs and the complement factors. The main IgG-mediated complement activation pathway is induced through the binding of complement C1q to IgG Abs. This interaction is dependent on antigen-dependent hexamer formation of human IgG1 and IgG3 to increase the affinity for the six-headed C1q molecule. By contrast, human IgG4 fails to bind to C1q. Instead, it has been suggested that human IgG4 can block IgG1 and IgG3 hexamerization required for their binding to C1q and activating the complement. Here, we show that murine IgG1, which functionally resembles human IgG4 by not interacting with C1q, inhibits the binding of IgG2a, IgG2b, and IgG3 to C1q in vitro, and suppresses IgG2a-mediated complement activation in a hemolytic assay in an antigen-dependent and IgG subclass-specific manner. From this perspective, we discuss the potential of murine IgG1 and human IgG4 to block the complement activation as well as suppressive effects of sialylated IgG subclass Abs on FcγR-mediated immune cell activation. Accumulating evidence suggests that both mechanisms seem to be responsible for preventing uncontrolled IgG (autoAb-induced inflammation in mice and humans. Distinct IgG subclass distributions and functionally opposite IgG Fc glycosylation patterns might explain different outcomes of IgG-mediated immune responses and provide new therapeutic options through the induction, enrichment, or application of antigen-specific sialylated human IgG4 to prevent complement and FcγR activation as well.

  3. Clearance of human IgG1-sensitised red blood cells in vivo in humans relates to the in vitro properties of antibodies from alternative cell lines.

    Directory of Open Access Journals (Sweden)

    Kathryn L Armour

    Full Text Available We previously produced a recombinant version of the human anti-RhD antibody Fog-1 in the rat myeloma cell line, YB2/0. When human, autologous RhD-positive red blood cells (RBC were sensitised with this IgG1 antibody and re-injected, they were cleared much more rapidly from the circulation than had been seen earlier with the original human-mouse heterohybridoma-produced Fog-1. Since the IgG have the same amino acid sequence, this disparity is likely to be due to alternative glycosylation that results from the rat and mouse cell lines. By comparing the in vitro properties of YB2/0-produced Fog-1 IgG1 and the same antibody produced in the mouse myeloma cell line NS0, we now have a unique opportunity to pinpoint the cause of the difference in ability to clear RBC in vivo. Using transfected cell lines that express single human FcγR, we showed that IgG1 made in YB2/0 and NS0 cell lines bound equally well to receptors of the FcγRI and FcγRII classes but that the YB2/0 antibody was superior in FcγRIII binding. When measuring complexed IgG binding, the difference was 45-fold for FcγRIIIa 158F, 20-fold for FcγRIIIa 158V and approximately 40-fold for FcγRIIIb. The dissimilarity was greater at 100-fold in monomeric IgG binding assays with FcγRIIIa. When used to sensitise RBC, the YB2/0 IgG1 generated 100-fold greater human NK cell antibody-dependent cell-mediated cytotoxicity and had a 103-fold advantage over the NS0 antibody in activating NK cells, as detected by CD54 levels. In assays of monocyte activation and macrophage adherence/phagocytosis, where FcγRI plays major roles, RBC sensitised with the two antibodies produced much more similar results. Thus, the alternative glycosylation profiles of the Fog-1 antibodies affect only FcγRIII binding and FcγRIII-mediated functions. Relating this to the in vivo studies confirms the importance of FcγRIII in RBC clearance.

  4. Large Scale Generation and Characterization of Anti-Human IgA Monoclonal Antibody in Ascitic Fluid of Balb/c Mice

    Science.gov (United States)

    Ezzatifar, Fatemeh; Majidi, Jafar; Baradaran, Behzad; Aghebati Maleki, Leili; Abdolalizadeh, Jalal; Yousefi, Mehdi

    2015-01-01

    Purpose: Monoclonal antibodies are potentially powerful tools used in biomedical research, diagnosis, and treatment of infectious diseases and cancers. The monoclonal antibody against Human IgA can be used as a diagnostic application to detect infectious diseases. The aim of this study was to improve an appropriate protocol for large-scale production of mAbs against IgA. Methods: For large-scale production of the monoclonal antibody, hybridoma cells that produce monoclonal antibodies against Human IgA were injected intraperitoneally into Balb/c mice that were previously primed with 0.5 ml Pristane. After ten days, ascitic fluid was harvested from the peritoneum of each mouse. The ELISA method was carried out for evaluation of the titration of produced mAbs. The ascitic fluid was investigated in terms of class and subclass by a mouse mAb isotyping kit. MAb was purified from the ascitic fluid by ion exchange chromatography. The purity of the monoclonal antibody was confirmed by SDS-PAGE, and the purified monoclonal antibody was conjugated with HRP. Results: Monoclonal antibodies with high specificity and sensitivity against Human IgA were prepared by hybridoma technology. The subclass of antibody was IgG1 and its light chain was the kappa type. Conclusion: This conjugated monoclonal antibody could have applications in designing ELISA kits in order to diagnose different infectious diseases such as toxoplasmosis and H. Pylori. PMID:25789225

  5. Large Scale Generation and Characterization of Anti-Human IgA Monoclonal Antibody in Ascitic Fluid of Balb/c Mice

    Directory of Open Access Journals (Sweden)

    Fatemeh Ezzatifar

    2015-03-01

    Full Text Available Purpose: Monoclonal antibodies are potentially powerful tools used in biomedical research, diagnosis, and treatment of infectious diseases and cancers. The monoclonal antibody against Human IgA can be used as a diagnostic application to detect infectious diseases. The aim of this study was to improve an appropriate protocol for large-scale production of mAbs against IgA. Methods: For large-scale production of the monoclonal antibody, hybridoma cells that produce monoclonal antibodies against Human IgA were injected intraperitoneally into Balb/c mice that were previously primed with 0.5 ml Pristane. After ten days, ascitic fluid was harvested from the peritoneum of each mouse. The ELISA method was carried out for evaluation of the titration of produced mAbs. The ascitic fluid was investigated in terms of class and subclass by a mouse mAb isotyping kit. MAb was purified from the ascitic fluid by ion exchange chromatography. The purity of the monoclonal antibody was confirmed by SDS-PAGE, and the purified monoclonal antibody was conjugated with HRP. Results: Monoclonal antibodies with high specificity and sensitivity against Human IgA were prepared by hybridoma technology. The subclass of antibody was IgG1 and its light chain was the kappa type. Conclusion: This conjugated monoclonal antibody could have applications in designing ELISA kits in order to diagnose different infectious diseases such as toxoplasmosis and H. Pylori.

  6. Large Scale Generation and Characterization of Anti-Human CD34 Monoclonal Antibody in Ascetic Fluid of Balb/c Mice

    Science.gov (United States)

    Aghebati Maleki, Leili; Majidi, Jafar; Baradaran, Behzad; Abdolalizadeh, Jalal; Kazemi, Tohid; Aghebati Maleki, Ali; Sineh sepehr, Koushan

    2013-01-01

    Purpose: Monoclonal antibodies or specific antibodies are now an essential tool of biomedical research and are of great commercial and medical value. The purpose of this study was to produce large scale of monoclonal antibody against CD34 in order to diagnostic application in leukemia and purification of human hematopoietic stem/progenitor cells. Methods: For large scale production of monoclonal antibody, hybridoma cells that produce monoclonal antibody against human CD34 were injected into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. 5 ml ascitic fluid was harvested from each mouse in two times. Evaluation of mAb titration was assessed by ELISA method. The ascitic fluid was examined for class and subclasses by ELISA mouse mAb isotyping Kit. mAb was purified from ascitic fluid by affinity chromatography on Protein A-Sepharose. Purity of monoclonal antibody was monitored by SDS -PAGE and the purified monoclonal antibody was conjugated with FITC. Results: Monoclonal antibodies with high specificity and sensitivity against human CD34 by hybridoma technology were prepared. The subclass of antibody was IgG1 and its light chain was kappa. Conclusion: The conjugated monoclonal antibody could be a useful tool for isolation, purification and characterization of human hematopoietic stem cells. PMID:24312838

  7. Large Scale Generation and Characterization of Anti-Human CD34 Monoclonal Antibody in Ascetic Fluid of Balb/c Mice

    Directory of Open Access Journals (Sweden)

    Koushan Sineh sepehr

    2013-02-01

    Full Text Available Purpose: Monoclonal antibodies or specific antibodies are now an essential tool of biomedical research and are of great commercial and medical value. The purpose of this study was to produce large scale of monoclonal antibody against CD34 in order to diagnostic application in leukemia and purification of human hematopoietic stem/progenitor cells. Methods: For large scale production of monoclonal antibody, hybridoma cells that produce monoclonal antibody against human CD34 were injected into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. 5 ml ascitic fluid was harvested from each mouse in two times. Evaluation of mAb titration was assessed by ELISA method. The ascitic fluid was examined for class and subclasses by ELISA mouse mAb isotyping Kit. mAb was purified from ascitic fluid by affinity chromatography on Protein A-Sepharose. Purity of monoclonal antibody was monitored by SDS -PAGE and the purified monoclonal antibody was conjugated with FITC. Results: Monoclonal antibodies with high specificity and sensitivity against human CD34 by hybridoma technology were prepared. The subclass of antibody was IgG1 and its light chain was kappa. Conclusion: The conjugated monoclonal antibody could be a useful tool for isolation, purification and characterization of human hematopoietic stem cells.

  8. Fc-Glycosylation in Human IgG1 and IgG3 Is Similar for Both Total and Anti-Red-Blood Cell Anti-K Antibodies

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    Myrthe E. Sonneveld

    2018-01-01

    Full Text Available After albumin, immunoglobulin G (IgG are the most abundant proteins in human serum, with IgG1 and IgG3 being the most abundant subclasses directed against protein antigens. The quality of the IgG-Fc-glycosylation has important functional consequences, which have been found to be skewed toward low fucosylation in some antigen-specific immune responses. This increases the affinity to IgG1-Fc-receptor (FcγRIIIa/b and thereby directly affects downstream effector functions and disease severity. To date, antigen-specific IgG-glycosylation have not been analyzed for IgG3. Here, we analyzed 30 pregnant women with anti-K alloantibodies from a prospective screening cohort and compared the type of Fc-tail glycosylation of total serum- and antigen-specific IgG1 and IgG3 using mass spectrometry. Total serum IgG1 and IgG3 Fc-glycoprofiles were highly similar. Fc glycosylation of antigen-specific IgG varied greatly between individuals, but correlated significantly with each other for IgG1 and IgG3, except for bisection. However, although the magnitude of changes in fucosylation and galactosylation were similar for both subclasses, this was not the case for sialylation levels, which were significantly higher for both total and anti-K IgG3. We found that the combination of relative IgG1 and IgG3 Fc-glycosylation levels did not improve the prediction of anti-K mediated disease over IgG1 alone. In conclusion, Fc-glycosylation profiles of serum- and antigen-specific IgG1 and IgG3 are highly similar.

  9. Studies of nontarget-mediated distribution of human full-length IgG1 antibody and its FAb fragment in cardiovascular and metabolic-related tissues.

    Science.gov (United States)

    Davidsson, Pia; Söderling, Ann-Sofi; Svensson, Lena; Ahnmark, Andrea; Flodin, Christine; Wanag, Ewa; Screpanti-Sundqvist, Valentina; Gennemark, Peter

    2015-05-01

    Tissue distribution and pharmacokinetics (PK) of full-length nontargeted antibody and its antigen-binding fragment (FAb) were evaluated for a range of tissues primarily of interest for cardiovascular and metabolic diseases. Mice were intravenously injected with a dose of 10 mg/kg of either human IgG1or its FAb fragment; perfused tissues were collected at a range of time points over 3 weeks for the human IgG1 antibody and 1 week for the human FAb antibody. Tissues were homogenized and antibody concentrations were measured by specific immunoassays on the Gyros system. Exposure in terms of maximum concentration (Cmax ) and area under the curve was assessed for all nine tissues. Tissue exposure of full-length antibody relative to plasma exposure was found to be between 1% and 10%, except for brain (0.2%). Relative concentrations of FAb antibody were the same, except for kidney tissue, where the antibody concentration was found to be ten times higher than in plasma. However, the absolute tissue uptake of full-length IgG was significantly higher than the absolute tissue uptake of the FAb antibody. This study provides a reference PK state for full-length whole and FAb antibodies in tissues related to cardiovascular and metabolic diseases that do not include antigen or antibody binding. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  10. Recombinant IgG1 Fc hexamers block cytotoxicity and pathological changes in experimental in vitro and rat models of neuromyelitis optica.

    Science.gov (United States)

    Tradtrantip, Lukmanee; Felix, Christian M; Spirig, Rolf; Morelli, Adriana Baz; Verkman, A S

    2018-05-01

    Intravenous human immunoglobulin G (IVIG) may have therapeutic benefit in neuromyelitis optica spectrum disorders (herein called NMO), in part because of the anti-inflammatory properties of the IgG Fc region. Here, we evaluated recombinant Fc hexamers consisting of the IgM μ-tailpiece fused with the Fc region of human IgG1. In vitro, the Fc hexamers prevented cytotoxicity in aquaporin-4 (AQP4) expressing cells and in rat spinal cord slice cultures exposed to NMO anti-AQP4 autoantibody (AQP4-IgG) and complement, with >500-fold greater potency than IVIG or monomeric Fc fragments. Fc hexamers at low concentration also prevented antibody-dependent cellular cytotoxicity produced by AQP4-IgG and natural killer cells. Serum from rats administered a single intravenous dose of Fc hexamers at 50 mg/kg taken at 8 h did not produce complement-dependent cytotoxicity when added to AQP4-IgG-treated AQP4-expressing cell cultures. In an experimental rat model of NMO produced by intracerebral injection of AQP4-IgG, Fc hexamers at 50 mg/kg administered before and at 12 h after AQP4-IgG fully prevented astrocyte injury, complement activation, inflammation and demyelination. These results support the potential therapeutic utility of recombinant IgG1 Fc hexamers in AQP4-IgG seropositive NMO. Copyright © 2018 Elsevier Ltd. All rights reserved.

  11. Tsetse salivary gland proteins 1 and 2 are high affinity nucleic acid binding proteins with residual nuclease activity.

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    Guy Caljon

    Full Text Available Analysis of the tsetse fly salivary gland EST database revealed the presence of a highly enriched cluster of putative endonuclease genes, including tsal1 and tsal2. Tsal proteins are the major components of tsetse fly (G. morsitans morsitans saliva where they are present as monomers as well as high molecular weight complexes with other saliva proteins. We demonstrate that the recombinant tsetse salivary gland proteins 1&2 (Tsal1&2 display DNA/RNA non-specific, high affinity nucleic acid binding with K(D values in the low nanomolar range and a non-exclusive preference for duplex. These Tsal proteins exert only a residual nuclease activity with a preference for dsDNA in a broad pH range. Knockdown of Tsal expression by in vivo RNA interference in the tsetse fly revealed a partially impaired blood digestion phenotype as evidenced by higher gut nucleic acid, hematin and protein contents.

  12. Characterization of glucagon-like peptide-1 receptor beta-arrestin 2 interaction: a high-affinity receptor phenotype

    DEFF Research Database (Denmark)

    Jorgensen, Rasmus; Martini, Lene; Schwartz, Thue W

    2005-01-01

    To dissect the interaction between beta-arrestin ((beta)arr) and family B G protein-coupled receptors, we constructed fusion proteins between the glucagon-like peptide 1 receptor and (beta)arr2. The fusion constructs had an increase in apparent affinity selectively for glucagon, suggesting...... that (beta)arr2 interaction locks the receptor in a high-affinity conformation, which can be explored by some, but not all, ligands. The fusion constructs adopted a signaling phenotype governed by the tethered (beta)arr2 with an attenuated G protein-mediated cAMP signal and a higher maximal internalization...... of that which has previously been characterized for family A G protein-coupled receptors, suggesting similarities in the effect of (beta)arr interaction between family A and B receptors also at the molecular level....

  13. High-affinity α4β2 nicotinic receptors mediate the impairing effects of acute nicotine on contextual fear extinction.

    Science.gov (United States)

    Kutlu, Munir Gunes; Holliday, Erica; Gould, Thomas J

    2016-02-01

    Previously, studies from our lab have shown that while acute nicotine administered prior to training and testing enhances contextual fear conditioning, acute nicotine injections prior to extinction sessions impair extinction of contextual fear. Although there is also strong evidence showing that the acute nicotine's enhancing effects on contextual fear conditioning require high-affinity α4β2 nicotinic acetylcholine receptors (nAChRs), it is unknown which nAChR subtypes are involved in the acute nicotine-induced impairment of contextual fear extinction. In this study, we investigated the effects of acute nicotine administration on contextual fear extinction in knock-out (KO) mice lacking α4, β2 or α7 subtypes of nAChRs and their wild-type (WT) littermates. Both KO and WT mice were first trained and tested for contextual fear conditioning and received a daily contextual extinction session for 4 days. Subjects received intraperitoneal injections of nicotine (0.18 mg/kg) or saline 2-4 min prior to each extinction session. Our results showed that the mice that lack α4 and β2 subtypes of nAChRs showed normal contextual fear extinction but not the acute nicotine-induced impairment while the mice that lack the α7 subtype showed both normal contextual extinction and nicotine-induced impairment of contextual extinction. In addition, control experiments showed that acute nicotine-induced impairment of contextual fear extinction persisted when nicotine administration was ceased and repeated acute nicotine administrations alone did not induce freezing behavior in the absence of context-shock learning. These results clearly demonstrate that high-affinity α4β2 nAChRs are necessary for the effects of acute nicotine on contextual fear extinction. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Visual and Plasmon Resonance Absorption Sensor for Adenosine Triphosphate Based on the High Affinity between Phosphate and Zr(IV).

    Science.gov (United States)

    Qi, Wenjing; Liu, Zhongyuan; Zhang, Wei; Halawa, Mohamed Ibrahim; Xu, Guobao

    2016-10-12

    Zr(IV) can form phosphate and Zr(IV) (-PO₃ 2- -Zr 4+ -) complex owing to the high affinity between Zr(IV) with phosphate. Zr(IV) can induce the aggregation of gold nanoparticles (AuNPs), while adenosine triphosphate(ATP) can prevent Zr(IV)-induced aggregation of AuNPs. Herein, a visual and plasmon resonance absorption (PRA)sensor for ATP have been developed using AuNPs based on the high affinity between Zr(IV)with ATP. AuNPs get aggregated in the presence of certain concentrations of Zr(IV). After the addition of ATP, ATP reacts with Zr(IV) and prevents AuNPs from aggregation, enabling the detection of ATP. Because of the fast interaction of ATP with Zr(IV), ATP can be detected with a detection limit of 0.5 μM within 2 min by the naked eye. Moreover, ATP can be detected by the PRA technique with higher sensitivity. The A 520nm / A 650nm values in PRA spectra increase linearly with the concentrations of ATP from 0.1 μM to 15 μM (r = 0.9945) with a detection limit of 28 nM. The proposed visual and PRA sensor exhibit good selectivity against adenosine, adenosine monophosphate, guanosine triphosphate, cytidine triphosphate and uridine triphosphate. The recoveries for the analysis of ATP in synthetic samples range from 95.3% to 102.0%. Therefore, the proposed novel sensor for ATP is promising for real-time or on-site detection of ATP.

  15. Visual and Plasmon Resonance Absorption Sensor for Adenosine Triphosphate Based on the High Affinity between Phosphate and Zr(IV

    Directory of Open Access Journals (Sweden)

    Wenjing Qi

    2016-10-01

    Full Text Available Zr(IV can form phosphate and Zr(IV (–PO32−–Zr4+– complex owing to the high affinity between Zr(IV with phosphate. Zr(IV can induce the aggregation of gold nanoparticles (AuNPs, while adenosine triphosphate(ATP can prevent Zr(IV-induced aggregation of AuNPs. Herein, a visual and plasmon resonance absorption (PRAsensor for ATP have been developed using AuNPs based on the high affinity between Zr(IVwith ATP. AuNPs get aggregated in the presence of certain concentrations of Zr(IV. After the addition of ATP, ATP reacts with Zr(IV and prevents AuNPs from aggregation, enabling the detection of ATP. Because of the fast interaction of ATP with Zr(IV, ATP can be detected with a detection limit of 0.5 μM within 2 min by the naked eye. Moreover, ATP can be detected by the PRA technique with higher sensitivity. The A520nm/A650nm values in PRA spectra increase linearly with the concentrations of ATP from 0.1 μM to 15 μM (r = 0.9945 with a detection limit of 28 nM. The proposed visual and PRA sensor exhibit good selectivity against adenosine, adenosine monophosphate, guanosine triphosphate, cytidine triphosphate and uridine triphosphate. The recoveries for the analysis of ATP in synthetic samples range from 95.3% to 102.0%. Therefore, the proposed novel sensor for ATP is promising for real-time or on-site detection of ATP.

  16. Two high-affinity ligand binding states of uterine estrogen receptor distinguished by modulation of hydrophobic environment

    International Nuclear Information System (INIS)

    Hutchens, T.W.; Li, C.M.; Zamah, N.M.; Besch, P.K.

    1987-01-01

    The steroid binding function of soluble (cytosolic) estrogen receptors from calf uteri was evaluated under conditions known to modify the extent of hydrophobic interaction with receptor-associated proteins. Receptor preparations were equilibrated into 6 M urea buffers and control buffers by chromatography through small columns of Sephadex G-25 or by dialysis at 0.6 0 C. Equilibrium dissociation constants (K/sub d/) and binding capacities (n) of experimental and control receptor preparations were determined by 13-point Scatchard analyses using concentrations of 17β-[ 3 H]estradiol from 0.05 to 10 nM. Nonspecific binding was determined at each concentration by parallel incubations with a 200-fold molar excess of the receptor-specific competitor diethylstilbestrol. The control receptor population was consistently found to be a single class of binding sites with a high affinity for estradiol which was unaffected by G-25 chromatography, by dialysis, by dilution, or by the presence of 0.4 M KCl. However, equilibration into 6 M urea induced a discrete (10-fold) reduction in receptor affinity to reveal a second, thermodynamically stable, high-affinity binding state. The presence of 0.4 M KCl did not significantly influence the discrete change in receptor affinity induced by urea. The effects of urea on both receptor affinity and binding capacity were reversible, suggesting a lack of covalent modification. These results demonstrate nonenzymatic means by which not only the binding capacity but also the affinity of receptor for estradiol can be reversibly controlled, suggesting that high concentrations of urea might be more effectively utilized during the physicochemical characterization and purification of steroid receptor proteins

  17. Cyr61/CCN1 displays high-affinity binding to the somatomedin B(1-44 domain of vitronectin.

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    Ivo M B Francischetti

    2010-02-01

    Full Text Available Cyr61 is a member of the CCN (Cyr61, connective tissue growth, NOV family of extracellular-associated (matricellular proteins that present four distinct functional modules, namely insulin-like growth factor binding protein (IGFBP, von Willebrand factor type C (vWF, thrombospondin type 1 (TSP, and C-terminal growth factor cysteine knot (CT domain. While heparin sulphate proteoglycans reportedly mediate the interaction of Cyr61 with the matrix and cell surface, the role of other extracellular associated proteins has not been revealed.In this report, surface plasmon resonance (SPR experiments and solid-phase binding assays demonstrate that recombinant Cyr61 interacts with immobilized monomeric or multimeric vitronectin (VTNC with K(D in the nanomolar range. Notably, the binding site for Cyr61 was identified as the somatomedin B domain (SMTB(1-44 of VTNC, which mediates its interaction with PAI-1, uPAR, and integrin alphav beta3. Accordingly, PAI-1 outcompetes Cyr61 for binding to immobilized SMTB(1-44, and Cyr61 attenuates uPAR-mediated U937 adhesion to VTNC. In contrast, isothermal titration calorimetry shows that Cyr61 does not display high-affinity binding for SMTB(1-44 in solution. Nevertheless, competitive ELISA revealed that multimeric VTNC, heat-modified monomeric VTNC, or SMTB(1-44 at high concentrations attenuate Cyr61 binding to immobilized VTNC, while monomeric VTNC was ineffective. Therefore, immobilization of VTNC exposes cryptic epitopes that recognize Cyr61 with high affinity, as reported for a number of antibodies, beta-endorphin, and other molecules.The finding that Cyr61 interacts with the SMTB(1-44 domain suggests that VTNC represent a point of anchorage for CCN family members to the matrix. Results are discussed in the context of the role of CCN and VTNC in matrix biology and angiogenesis.

  18. High affinity antigen recognition of the dual specific variants of herceptin is entropy-driven in spite of structural plasticity.

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    Jenny Bostrom

    Full Text Available The antigen-binding site of Herceptin, an anti-human Epidermal Growth Factor Receptor 2 (HER2 antibody, was engineered to add a second specificity toward Vascular Endothelial Growth Factor (VEGF to create a high affinity two-in-one antibody bH1. Crystal structures of bH1 in complex with either antigen showed that, in comparison to Herceptin, this antibody exhibited greater conformational variability, also called "structural plasticity". Here, we analyzed the biophysical and thermodynamic properties of the dual specific variants of Herceptin to understand how a single antibody binds two unrelated protein antigens. We showed that while bH1 and the affinity-improved bH1-44, in particular, maintained many properties of Herceptin including binding affinity, kinetics and the use of residues for antigen recognition, they differed in the binding thermodynamics. The interactions of bH1 and its variants with both antigens were characterized by large favorable entropy changes whereas the Herceptin/HER2 interaction involved a large favorable enthalpy change. By dissecting the total entropy change and the energy barrier for dual interaction, we determined that the significant structural plasticity of the bH1 antibodies demanded by the dual specificity did not translate into the expected increase of entropic penalty relative to Herceptin. Clearly, dual antigen recognition of the Herceptin variants involves divergent antibody conformations of nearly equivalent energetic states. Hence, increasing the structural plasticity of an antigen-binding site without increasing the entropic cost may play a role for antibodies to evolve multi-specificity. Our report represents the first comprehensive biophysical analysis of a high affinity dual specific antibody binding two unrelated protein antigens, furthering our understanding of the thermodynamics that drive the vast antigen recognition capacity of the antibody repertoire.

  19. Human monoclonal antibodies reactive with human myelomonocytic leukemia cells.

    Science.gov (United States)

    Posner, M R; Santos, D J; Elboim, H S; Tumber, M B; Frackelton, A R

    1989-04-01

    Peripheral blood mononuclear cells from a patient with chronic myelogenous leukemia (CML), in remission, were depleted of CD8-positive T-cells and cultured with Epstein-Barr virus. Four of 20 cultures (20%) secreted human IgG antibodies selectively reactive with the cell surfaces of certain human leukemia cell lines. Three polyclonal, Epstein-Barr virus-transformed, B-cell lines were expanded and fused with the human-mouse myeloma analogue HMMA2.11TG/O. Antibody from secreting clones HL 1.2 (IgG1), HL 2.1 (IgG3), and HL 3.1 (IgG1) have been characterized. All three react with HL-60 (promyelocytic), RWLeu4 (CML promyelocytic), and U937 (monocytic), but not with KG-1 (myeloblastic) or K562 (CML erythroid). There is no reactivity with T-cell lines, Burkitt's cell lines, pre-B-leukemia cell lines, or an undifferentiated CML cell line, BV173. Leukemic cells from two of seven patients with acute myelogenous leukemia and one of five with acute lymphocytic leukemia react with all three antibodies. Normal lymphocytes, monocytes, polymorphonuclear cells, red blood cells, bone marrow cells, and platelets do not react. Samples from patients with other diverse hematopoietic malignancies showed no reactivity. Immunoprecipitations suggest that the reactive antigen(s) is a lactoperoxidase iodinatable series of cell surface proteins with molecular weights of 42,000-54,000 and a noniodinatable protein with a molecular weight of 82,000. Based on these data these human monoclonal antibodies appear to react with myelomonocytic leukemic cells and may detect a leukemia-specific antigen or a highly restricted differentiation antigen.

  20. Histone H1(0) mapping using monoclonal antibodies.

    Science.gov (United States)

    Dousson, S; Gorka, C; Gilly, C; Lawrence, J J

    1989-06-01

    Monoclonal antibodies (mAb) to ox liver histone H1 degree were produced and characterized. Two sets of mice were immunized either with pure H1(0) or with an H1(0)-yeast tRNA complex. Eleven hybridomas of various clonal origin were selected. Typing of the antibodies indicated that all but three IgM belonged to the IgG1 class and contained kappa light chains. Immunoblotting experiments using peptides derived from H1(0) or H5 treated by various proteolytic agents (trypsin, N-bromosuccinimide, cyanogen bromide, acetic acid), revealed that nine of the mAb reacted with the globular part of H1(0). More advanced characterization of the antigenic determinants allowed us to determine distinct regions within this globular part which are involved in the antigenic recognition. The peptopes could be subdivided into two groups. Three mAb bound to residues 24-27 and were specific for H1(0). Six mAb bound to residues 27-30 and were specific for H1(0) except one of them which strongly cross-reacted with H5 and GH5. Two mAb reacted with the entire histone H1(0) but failed to react with any of the peptides, suggesting that the corresponding epitope is a conformational antigenic determinant. In order to confirm the localization of the two distinct regions which are involved in the antigenic recognition, a synthetic decapeptide corresponding to the beginning of human H1(0) globular part (from residue 19 to residue 28) was synthesized. Inhibition experiments of the reaction between H1(0) and the various IgG1 mAb by increasing amounts of peptide-bovine serum albumin conjugates were then performed.

  1. Characterization of monoclonal antibodies against human thyrotropin and use in an immunoradiometric assay and immunohistochemistry

    International Nuclear Information System (INIS)

    Benkirane, M.; Bon, D.; Bellot, F.; Prince, P.; Delori, P.; Hassoun, J.; Carayon, P.

    1987-01-01

    Monoclonal antibodies were prepared against human thyrotropin. 13 different antibodies were characterized. Ten antibodies were of the IgG1 subclass. The affinities of the antibodies were in the range 10 9 -10 11 mol -1 .l. Four of them were specific for hTSH and did not react with hLH, hFSH, hCG or αhCG. Four reacted with these hormones and recognized the α subunit of hCG. One cross-reacted only with HFSH. The remaining four antibodies recognized the holo-hTSH only, and thus were designated as anti-conformational determinants. Monoclonal antibodies reacting with different antigenic determinants on the hTSH molecule defined seven clusters. Two of them were used to develop a simplified two-site sandwich radioimmunoassay in which one monoclonal antibody was immobilized on tubes (anti-βTSH) and another (anti-α) labelled with 125 I. This assay was highly specific and demonstrated a sensitivity level of 0.1 μIU/ml. Two monoclonal antibodies were used in immunohistochemistry and their quality and specificity was assessed in the detection of hTSH immunoreactivity in human pituitary biological sections. 20 refs.; 6 figs.; 2 tabs

  2. Isolation and functional effects of monoclonal antibodies binding to thymidylate synthase.

    Science.gov (United States)

    Jastreboff, M M; Todd, M B; Malech, H L; Bertino, J R

    1985-01-29

    Monoclonal antibodies against electrophoretically pure thymidylate synthase from HeLa cells have been produced. Antibodies (M-TS-4 and M-TS-9) from hybridoma clones were shown by enzyme-linked immunoassay to recognize thymidylate synthase from a variety of human cell lines, but they did not bind to thymidylate synthase from mouse cell lines. The strongest binding of antibodies was observed to enzyme from HeLa cells. These two monoclonal antibodies bind simultaneously to different antigenic sites on thymidylate synthase purified from HeLa cells, as reflected by a high additivity index and results of cross-linked radioimmunoassay. Both monoclonal antibodies inhibit the activity of thymidylate synthase from human cell lines. The strongest inhibition was observed with thymidylate synthase from HeLa cells. Monoclonal antibody M-TS-9 (IgM subclass) decreased the rate of binding of [3H]FdUMP to thymidylate synthase in the presence of 5,10-methylenetetrahydrofolate while M-TS-4 (IgG1) did not change the rate of ternary complex formation. These data indicate that the antibodies recognize different epitopes on the enzyme molecule.

  3. Comparison of high affinity binding of 3H-proadifen and 3H-(-)-cocaine t rat liver membranes

    International Nuclear Information System (INIS)

    Ross, S.B.

    1995-01-01

    The characteristics of the binding of 3 H-proadifen to rat liver membranes were studied and compared to those of 3 H-cocaine. It was found that 3 H-proadifen was bound reversibly with high affinity (K D =1.8±0.5 nM) and large capacity (B max =2010±340 pmol/g wet tissue) to liver membranes. The corresponding values for the 3 H-cocaine binding were 3.5 nM and 1000 pmol/g wet tissue. The binding of 3 H-proadifen was mainly localised to the microsomal fraction. The number of binding sites was not increased by treatment of rats with phenobarbitone. With 1 μM CdCl 2 in the incubation buffer it was possible to differentiate between two 3 H-cocaine binding sites with K d values of 1.6 and 7.7 nM and B max values of 280 and 940 pmol/g wet liver tissue. S-(-)-Alaproclate inhibited the binding of 3 H-proadifen and 3 H-cocaine inhibited the binding of 3 H-proadifen (IC 50 =10 nM) and proadifen that of 3 H-cocaine (IC 50 =1 nM). There was a high correlation coefficient (r r =0.972; P 50 =100-500 nM): chloroquine, phenoxybenzamine, amitriptyline, ajmaline, remoxipride, imipramine and (-)-alaprenolol. CdCl 2 , ZnCl 2 and CuCl 2 inhibited the binding of both ligands with low Hill coefficients, indicating heterogeneous binding sites. The inhibition curve of Cd 2+ on the cocaine binding was biphasic with a high affinity part around 50 nM and a low affinity part at 15μM. The similarity of the characteristics of the binding of these ligands with that of 3 H-alaproclate is discussed. It is suggested that all three compounds bind to the same sites, although additional binding sites seem to exist for proadifen. (au) (9 refs.)

  4. Monoclonal antibodies against human angiotensinogen, their characterization and use in an angiotensinogen enzyme linked immunosorbent assay.

    Science.gov (United States)

    Rubin, I; Lykkegaard, S; Olsen, A A; Selmer, J; Ballegaard, M

    1988-01-01

    Monoclonal antibodies were produced against human angiotensinogen. An enzyme linked immunosorbent assay (ELISA) was developed using a high affinity monoclonal antibody as catching antibody and a polyclonal rabbit anti human angiotensinogen antibody as detecting antibody in a "sandwich" ELISA. Linear range of the ELISA was 15-450 pmol/l of human angiotensinogen. Intra- and inter- assay variation coefficients were in the range of 2% to 8%. A correlation coefficient, r = 0.97, (n = 20), with values obtained by radioimmunoassay. This correlation coefficient, obtained by using both normal and pregnant sera, confirmed that the ELISA fulfill the requirements for clinical useful assay. Characterization of the antibodies were performed with respect to affinity constant and epitopes.

  5. Usefulness of FC-TRIPLEX Chagas/Leish IgG1 as confirmatory assay for non-negative results in blood bank screening of Chagas disease.

    Science.gov (United States)

    Campos, Fernanda Magalhães Freire; Repoles, Laura Cotta; de Araújo, Fernanda Fortes; Peruhype-Magalhães, Vanessa; Xavier, Marcelo Antônio Pascoal; Sabino, Ester Cerdeira; de Freitas Carneiro Proietti, Anna Bárbara; Andrade, Mariléia Chaves; Teixeira-Carvalho, Andréa; Martins-Filho, Olindo Assis; Gontijo, Célia Maria Ferreira

    2018-04-01

    A relevant issue in Chagas disease serological diagnosis regards the requirement of using several confirmatory methods to elucidate the status of non-negative results from blood bank screening. The development of a single reliable method may potentially contribute to distinguish true and false positive results. Our aim was to evaluate the performance of the multiplexed flow-cytometry anti-T. cruzi/Leishmania IgG1 serology/(FC-TRIPLEX Chagas/Leish IgG1) with three conventional confirmatory criteria (ELISA-EIA, Immunofluorescence assay-IIF and EIA/IIF consensus criterion) to define the final status of samples with actual/previous non-negative results during anti-T. cruzi ELISA-screening in blood banks. Apart from inconclusive results, the FC-TRIPLEX presented a weak agreement index with EIA, while a strong agreement was observed when either IIF or EIA/IIF consensus criteria were applied. Discriminant analysis and Spearman's correlation further corroborates the agreement scores. ROC curve analysis showed that FC-TRIPLEX performance indexes were higher when IIF and EIA/IIF consensus were used as a confirmatory criterion. Logistic regression analysis further demonstrated that the probability of FC-TRIPLEX to yield positive results was higher for inconclusive results from IIF and EIA/IIF consensus. Machine learning tools illustrated the high level of categorical agreement between FC-TRIPLEX versus IIF or EIA/IIF consensus. Together, these findings demonstrated the usefulness of FC-TRIPLEX as a tool to elucidate the status of non-negative results in blood bank screening of Chagas disease. Copyright © 2018. Published by Elsevier B.V.

  6. Sudan ebolavirus long recovered survivors produce GP-specific Abs that are of the IgG1 subclass and preferentially bind FcγRI.

    Science.gov (United States)

    Radinsky, Olga; Edri, Avishay; Brusilovsky, Michael; Fedida-Metula, Shlomit; Sobarzo, Ariel; Gershoni-Yahalom, Orly; Lutwama, Julius; Dye, John; Lobel, Leslie; Porgador, Angel

    2017-07-20

    Ebolavirus is a highly lethal pathogen, causing a severe hemorrhagic disease with a high fatality rate. To better understand immune correlates of protection by virus specific IgG, we investigated the evolution of the Fcγ receptors (FcγRs)-activating capabilities of antiviral IgG in serum samples of long recovered survivors. To this end, longitudinal serum samples from survivors of Sudan ebolavirus (SUDV) infection, studied over years, were examined for the presence of Ebola-GP specific IgG subclasses, and for their binding to FcγRs. We developed a cell-based reporter system to quantitate pathogen-specific antibody binding to FcγRIIIA, FcγRIIA, FcγRIIB and FcγRI. With this system, we demonstrate that anti-GP-specific stimulation of the FcγRI reporter by survivors' sera was substantially high one year after acute infection, with a slight reduction in activity over a decade post infection. We further demonstrate that GP-specific IgG1 is by far the seroprevalent subclass that retained and even enhanced its presence in the sera, over ten years post infection; the prevalence of other GP-specific IgG subclasses was considerably reduced over time. In accordance, GP-specific FcγRI reporter response and GP-specific total IgG1 subclass correlated in the studied group of Ebola survivors. These observations are important for further informing Ebola vaccine and therapeutic development.

  7. The monoclonal S9.6 antibody exhibits highly variable binding affinities towards different R-loop sequences.

    Directory of Open Access Journals (Sweden)

    Fabian König

    Full Text Available The monoclonal antibody S9.6 is a widely-used tool to purify, analyse and quantify R-loop structures in cells. A previous study using the surface plasmon resonance technology and a single-chain variable fragment (scFv of S9.6 showed high affinity (0.6 nM for DNA-RNA and also a high affinity (2.7 nM for RNA-RNA hybrids. We used the microscale thermophoresis method allowing surface independent interaction studies and electromobility shift assays to evaluate additional RNA-DNA hybrid sequences and to quantify the binding affinities of the S9.6 antibody with respect to distinct sequences and their GC-content. Our results confirm high affinity binding to previously analysed sequences, but reveals that binding affinities are highly sequence specific. Our study presents R-loop sequences that independent of GC-content and in different sequence variations exhibit either no binding, binding affinities in the micromolar range and as well high affinity binding in the nanomolar range. Our study questions the usefulness of the S9.6 antibody in the quantitative analysis of R-loop sequences in vivo.

  8. Generation of monoclonal antibodies against highly conserved antigens.

    Directory of Open Access Journals (Sweden)

    Hongzhe Zhou

    Full Text Available BACKGROUND: Therapeutic antibody development is one of the fastest growing areas of the pharmaceutical industry. Generating high-quality monoclonal antibodies against a given therapeutic target is very crucial for the success of the drug development. However, due to immune tolerance, some proteins that are highly conserved between mice and humans are not very immunogenic in mice, making it difficult to generate antibodies using a conventional approach. METHODOLOGY/PRINCIPAL FINDINGS: In this report, the impaired immune tolerance of NZB/W mice was exploited to generate monoclonal antibodies against highly conserved or self-antigens. Using two highly conserved human antigens (MIF and HMGB1 and one mouse self-antigen (TNF-alpha as examples, we demonstrate here that multiple clones of high affinity, highly specific antibodies with desired biological activities can be generated, using the NZB/W mouse as the immunization host and a T cell-specific tag fused to a recombinant antigen to stimulate the immune system. CONCLUSIONS/SIGNIFICANCE: We developed an efficient and universal method for generating surrogate or therapeutic antibodies against "difficult antigens" to facilitate the development of therapeutic antibodies.

  9. Peptides in headlock – a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy

    Science.gov (United States)

    Braun, Michael B.; Traenkle, Bjoern; Koch, Philipp A.; Emele, Felix; Weiss, Frederik; Poetz, Oliver; Stehle, Thilo; Rothbauer, Ulrich

    2016-01-01

    Nanobodies are highly valuable tools for numerous bioanalytical and biotechnical applications. Here, we report the characterization of a nanobody that binds a short peptide epitope with extraordinary affinity. Structural analysis reveals an unusual binding mode where the extended peptide becomes part of a β-sheet structure in the nanobody. This interaction relies on sequence-independent backbone interactions augmented by a small number of specificity-determining side chain contacts. Once bound, the peptide is fastened by two nanobody side chains that clamp it in a headlock fashion. Exploiting this unusual binding mode, we generated a novel nanobody-derived capture and detection system. Matrix-coupled nanobody enables the fast and efficient isolation of epitope-tagged proteins from prokaryotic and eukaryotic expression systems. Additionally, the fluorescently labeled nanobody visualizes subcellular structures in different cellular compartments. The high-affinity-binding and modifiable peptide tag of this system renders it a versatile and robust tool to combine biochemical analysis with microscopic studies. PMID:26791954

  10. Peptides in headlock--a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy.

    Science.gov (United States)

    Braun, Michael B; Traenkle, Bjoern; Koch, Philipp A; Emele, Felix; Weiss, Frederik; Poetz, Oliver; Stehle, Thilo; Rothbauer, Ulrich

    2016-01-21

    Nanobodies are highly valuable tools for numerous bioanalytical and biotechnical applications. Here, we report the characterization of a nanobody that binds a short peptide epitope with extraordinary affinity. Structural analysis reveals an unusual binding mode where the extended peptide becomes part of a β-sheet structure in the nanobody. This interaction relies on sequence-independent backbone interactions augmented by a small number of specificity-determining side chain contacts. Once bound, the peptide is fastened by two nanobody side chains that clamp it in a headlock fashion. Exploiting this unusual binding mode, we generated a novel nanobody-derived capture and detection system. Matrix-coupled nanobody enables the fast and efficient isolation of epitope-tagged proteins from prokaryotic and eukaryotic expression systems. Additionally, the fluorescently labeled nanobody visualizes subcellular structures in different cellular compartments. The high-affinity-binding and modifiable peptide tag of this system renders it a versatile and robust tool to combine biochemical analysis with microscopic studies.

  11. High affinity [3H]glibenclamide binding sites in rat neuronal and cardiac tissue: Localization and developmental characteristics

    International Nuclear Information System (INIS)

    Miller, J.A.; Velayo, N.L.; Dage, R.C.; Rampe, D.

    1991-01-01

    We examined the binding of the antidiabetic sulfonylurea [3H] glibenclamide to rat brain and heart membranes. High affinity binding was observed in adult rat forebrain (Kd = 137.3 pM, maximal binding site density = 91.8 fmol/mg of protein) and ventricle (Kd = 77.1 pM, maximal binding site density = 65.1 fmol/mg of protein). Binding site density increased approximately 250% in forebrain membranes during postnatal development but was constant in ventricular membranes. Quantitative autoradiography was used to examine the regional distribution of [3H] glibenclamide binding sites in sections from rat brain, spinal cord and heart. The greatest density of binding in adult brain was found in the substantia nigra and globus pallidus, whereas the other areas displayed heterogenous binding. In agreement with the membrane binding studies, 1-day-old rat brain had significantly fewer [3H]glibenclamide binding sites than adult brain. Additionally, the pattern of distribution of these sites was qualitatively different from that of the adult. In adult rat spinal cord, moderate binding densities were observed in spinal cord gray and displayed a rostral to caudal gradient. In adult rat heart, moderate binding densities were observed and the sites were distributed homogeneously. In conclusion, significant development of [3H]glibenclamide binding sites was seen in the brain but not the heart during postnatal maturation. Furthermore, a heterogeneous distribution of binding sites was observed in both the brain and spinal cord of adult rats

  12. ZipA binds to FtsZ with high affinity and enhances the stability of FtsZ protofilaments.

    Directory of Open Access Journals (Sweden)

    Anuradha Kuchibhatla

    Full Text Available A bacterial membrane protein ZipA that tethers FtsZ to the membrane is known to promote FtsZ assembly. In this study, the binding of ZipA to FtsZ was monitored using fluorescence spectroscopy. ZipA was found to bind to FtsZ with high affinities at three different (6.0, 6.8 and 8.0 pHs, albeit the binding affinity decreased with increasing pH. Further, thick bundles of FtsZ protofilaments were observed in the presence of ZipA under the pH conditions used in this study indicating that ZipA can promote FtsZ assembly and stabilize FtsZ polymers under unfavorable conditions. Bis-ANS, a hydrophobic probe, decreased the interaction of FtsZ and ZipA indicating that the interaction between FtsZ and ZipA is hydrophobic in nature. ZipA prevented the dilution induced disassembly of FtsZ polymers suggesting that it stabilizes FtsZ protofilaments. Fluorescein isothiocyanate-labeled ZipA was found to be uniformly distributed along the length of the FtsZ protofilaments indicating that ZipA stabilizes FtsZ protofilaments by cross-linking them.

  13. Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING.

    Science.gov (United States)

    Zhang, Xu; Shi, Heping; Wu, Jiaxi; Zhang, Xuewu; Sun, Lijun; Chen, Chuo; Chen, Zhijian J

    2013-07-25

    The presence of microbial or self DNA in the cytoplasm of mammalian cells is a danger signal detected by the DNA sensor cyclic-GMP-AMP (cGAMP) synthase (cGAS), which catalyzes the production of cGAMP that in turn serves as a second messenger to activate innate immune responses. Here we show that endogenous cGAMP in mammalian cells contains two distinct phosphodiester linkages, one between 2'-OH of GMP and 5'-phosphate of AMP, and the other between 3'-OH of AMP and 5'-phosphate of GMP. This molecule, termed 2'3'-cGAMP, is unique in that it binds to the adaptor protein STING with a much greater affinity than cGAMP molecules containing other combinations of phosphodiester linkages. The crystal structure of STING bound to 2'3'-cGAMP revealed the structural basis of this high-affinity binding and a ligand-induced conformational change in STING that may underlie its activation. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. A soluble form of the high affinity IgE receptor, Fc-epsilon-RI, circulates in human serum.

    Directory of Open Access Journals (Sweden)

    Eleonora Dehlink

    Full Text Available Soluble IgE receptors are potential in vivo modulators of IgE-mediated immune responses and are thus important for our basic understanding of allergic responses. We here characterize a novel soluble version of the IgE-binding alpha-chain of Fc-epsilon-RI (sFcεRI, the high affinity receptor for IgE. sFcεRI immunoprecipitates as a protein of ∼40 kDa and contains an intact IgE-binding site. In human serum, sFcεRI is found as a soluble free IgE receptor as well as a complex with IgE. Using a newly established ELISA, we show that serum sFcεRI levels correlate with serum IgE in patients with elevated IgE. We also show that serum of individuals with normal IgE levels can be found to contain high levels of sFcεRI. After IgE-antigen-mediated crosslinking of surface FcεRI, we detect sFcεRI in the exosome-depleted, soluble fraction of cell culture supernatants. We further show that sFcεRI can block binding of IgE to FcεRI expressed at the cell surface. In summary, we here describe the alpha-chain of FcεRI as a circulating soluble IgE receptor isoform in human serum.

  15. Design, Synthesis, and Biological Evaluation of Small, High-Affinity Siglec-7 Ligands: Toward Novel Inhibitors of Cancer Immune Evasion.

    Science.gov (United States)

    Prescher, Horst; Frank, Martin; Gütgemann, Stephan; Kuhfeldt, Elena; Schweizer, Astrid; Nitschke, Lars; Watzl, Carsten; Brossmer, Reinhard

    2017-02-09

    Natural killer cells are able to directly lyse tumor cells, thereby participating in the immune surveillance against cancer. Unfortunately, many cancer cells use immune evasion strategies to avoid their eradication by the immune system. A prominent escape strategy of malignant cells is to camouflage themselves with Siglec-7 ligands, thereby recruiting the inhibitory receptor Siglec-7 expressed on the NK cell surface which subsequently inhibits NK-cell-mediated lysis. Here we describe the synthesis and evaluation of the first, high-affinity low molecular weight Siglec-7 ligands to interfere with cancer cell immune evasion. The compounds are Sialic acid derivatives and bind with low micromolar K d values to Siglec-7. They display up to a 5000-fold enhanced affinity over the unmodified sialic acid scaffold αMe Neu5Ac, the smallest known natural Siglec-7 ligand. Our results provide a novel immuno-oncology strategy employing natural immunity in the fight against cancers, in particular blocking Siglec-7 with low molecular weight compounds.

  16. High affinity γPNA sandwich hybridization assay for rapid detection of short nucleic acid targets with single mismatch discrimination.

    Science.gov (United States)

    Goldman, Johnathan M; Zhang, Li Ang; Manna, Arunava; Armitage, Bruce A; Ly, Danith H; Schneider, James W

    2013-07-08

    Hybridization analysis of short DNA and RNA targets presents many challenges for detection. The commonly employed sandwich hybridization approach cannot be implemented for these short targets due to insufficient probe-target binding strengths for unmodified DNA probes. Here, we present a method capable of rapid and stable sandwich hybridization detection for 22 nucleotide DNA and RNA targets. Stable hybridization is achieved using an n-alkylated, polyethylene glycol γ-carbon modified peptide nucleic acid (γPNA) amphiphile. The γPNA's exceptionally high affinity enables stable hybridization of a second DNA-based probe to the remaining bases of the short target. Upon hybridization of both probes, an electrophoretic mobility shift is measured via interaction of the n-alkane modification on the γPNA with capillary electrophoresis running buffer containing nonionic surfactant micelles. We find that sandwich hybridization of both probes is stable under multiple binding configurations and demonstrate single base mismatch discrimination. The binding strength of both probes is also stabilized via coaxial stacking on adjacent hybridization to targets. We conclude with a discussion on the implementation of the proposed sandwich hybridization assay as a high-throughput microRNA detection method.

  17. High-Affinity Interaction of the K-Ras4B Hypervariable Region with the Ras Active Site

    Science.gov (United States)

    Chavan, Tanmay S.; Jang, Hyunbum; Khavrutskii, Lyuba; Abraham, Sherwin J.; Banerjee, Avik; Freed, Benjamin C.; Johannessen, Liv; Tarasov, Sergey G.; Gaponenko, Vadim; Nussinov, Ruth; Tarasova, Nadya I.

    2015-01-01

    Ras proteins are small GTPases that act as signal transducers between cell surface receptors and several intracellular signaling cascades. They contain highly homologous catalytic domains and flexible C-terminal hypervariable regions (HVRs) that differ across Ras isoforms. KRAS is among the most frequently mutated oncogenes in human tumors. Surprisingly, we found that the C-terminal HVR of K-Ras4B, thought to minimally impact the catalytic domain, directly interacts with the active site of the protein. The interaction is almost 100-fold tighter with the GDP-bound than the GTP-bound protein. HVR binding interferes with Ras-Raf interaction, modulates binding to phospholipids, and slightly slows down nucleotide exchange. The data indicate that contrary to previously suggested models of K-Ras4B signaling, HVR plays essential roles in regulation of signaling. High affinity binding of short peptide analogs of HVR to K-Ras active site suggests that targeting this surface with inhibitory synthetic molecules for the therapy of KRAS-dependent tumors is feasible. PMID:26682817

  18. The RCK1 high-affinity Ca2+ sensor confers carbon monoxide sensitivity to Slo1 BK channels.

    Science.gov (United States)

    Hou, Shangwei; Xu, Rong; Heinemann, Stefan H; Hoshi, Toshinori

    2008-03-11

    Carbon monoxide (CO) is a lethal gas, but it is also increasingly recognized as a physiological signaling molecule capable of regulating a variety of proteins. Among them, large-conductance Ca(2+)- and voltage-gated K(+) (Slo1 BK) channels, important in vasodilation and neuronal firing, have been suggested to be directly stimulated by CO. However, the molecular mechanism of the stimulatory action of CO on the Slo1 BK channel has not been clearly elucidated. We report here that CO reliably and repeatedly activates Slo1 BK channels in excised membrane patches in the absence of Ca(2+) in a voltage-sensor-independent manner. The stimulatory action of CO on the Slo1 BK channel requires an aspartic acid and two histidine residues located in the cytoplasmic RCK1 domain, and the effect persists under the conditions known to inhibit the conventional interaction between CO and heme in other proteins. We propose that CO acts as a partial agonist for the high-affinity divalent cation sensor in the RCK1 domain of the Slo1 BK channel.

  19. ZrFsy1, a high-affinity fructose/H+ symporter from fructophilic yeast Zygosaccharomyces rouxii.

    Directory of Open Access Journals (Sweden)

    Maria José Leandro

    Full Text Available Zygosaccharomyces rouxii is a fructophilic yeast than can grow at very high sugar concentrations. We have identified an ORF encoding a putative fructose/H(+ symporter in the Z. rouxii CBS 732 genome database. Heterologous expression of this ORF in a S. cerevisiae strain lacking its own hexose transporters (hxt-null and subsequent kinetic characterization of its sugar transport activity showed it is a high-affinity low-capacity fructose/H(+ symporter, with Km 0.45 ± 0.07 mM and Vmax 0.57 ± 0.02 mmol h(-1 (gdw(-1. We named it ZrFsy1. This protein also weakly transports xylitol and sorbose, but not glucose or other hexoses. The expression of ZrFSY1 in Z. rouxii is higher when the cells are cultivated at extremely low fructose concentrations (<0.2% and on non-fermentable carbon sources such as mannitol and xylitol, where the cells have a prolonged lag phase, longer duplication times and change their microscopic morphology. A clear phenotype was determined for the first time for the deletion of a fructose/H(+ symporter in the genome where it occurs naturally. The effect of the deletion of ZrFSY1 in Z. rouxii cells is only evident when the cells are cultivated at very low fructose concentrations, when the ZrFsy1 fructose symporter is the main active fructose transporter system.

  20. VNARs: An Ancient and Unique Repertoire of Molecules That Deliver Small, Soluble, Stable and High Affinity Binders of Proteins

    Directory of Open Access Journals (Sweden)

    Caroline Barelle

    2015-09-01

    Full Text Available At 420 million years, the variable domain of New Antigen Receptors or VNARs are undoubtedly the oldest (and smallest antigen binding single domains identified in the vertebrate kingdom. Their role as an integral part of the adaptive immune system of sharks has been well established and has served to provide a greater understanding of the evolution of humoral immunity; their cellular components and processes as well as the underlying genetic organization and molecular control mechanisms. Intriguingly, unlike the variable domain of the camelid heavy chain antibodies or VHH, VNARs do not conform to all of the characteristic properties of classical antibodies with an ancestral origin that clearly distinguishes them from true immunoglobulin antibodies. However, this uniqueness of their origin only adds to their potential as next generation therapeutic biologics with their structural and functional attributes and commercial freedom all enhancing their profile and current success. In fact their small size, remarkable stability, molecular flexibility and solubility, together with their high affinity and selectivity for target, all reinforce the potential of these domains as drug candidates. The purpose of this review is to provide an overview of the existing basic biology of these unique domains, to highlight the drug-like properties of VNARs and describe current progress in their journey towards the clinic.

  1. Identification and properties of very high affinity brain membrane-binding sites for a neurotoxic phospholipase from the taipan venom

    Energy Technology Data Exchange (ETDEWEB)

    Lambeau, G.; Barhanin, J.; Schweitz, H.; Qar, J.; Lazdunski, M. (Centre de Biochimie, Nice (France))

    1989-07-05

    Four new monochain phospholipases were purified from the Oxyuranus scutellatus (taipan) venom. Three of them were highly toxic when injected into mice brain. One of these neurotoxic phospholipases, OS2, was iodinated and used in binding experiments to demonstrate the presence of two families of specific binding sites in rat brain synaptic membranes. The affinities were exceptionally high, Kd1 = 1.5 +/- 0.5 pM and Kd2 = 45 +/- 10 pM, and the maximal binding capacities were Bmax 1 = 1 +/- 0.4 and Bmax 2 = 3 +/- 0.5 pmol/mg of protein. Both binding sites were sensitive to proteolysis and demonstrated to be located on proteins of Mr 85,000-88,000 and 36,000-51,000 by cross-linking and photoaffinity labeling techniques. The binding of {sup 125}I-OS2 to synaptic membranes was dependent on Ca2+ ions and enhanced by Zn2+ ions which inhibit phospholipase activity. Competition experiments have shown that, except for beta-bungarotoxin, a number of known toxic snake or bee phospholipases have very high affinities for the newly identified binding sites. A good correlation (r = 0.80) was observed between toxicity and affinity but not between phospholipase activity and affinity.

  2. Identification and properties of very high affinity brain membrane-binding sites for a neurotoxic phospholipase from the taipan venom

    International Nuclear Information System (INIS)

    Lambeau, G.; Barhanin, J.; Schweitz, H.; Qar, J.; Lazdunski, M.

    1989-01-01

    Four new monochain phospholipases were purified from the Oxyuranus scutellatus (taipan) venom. Three of them were highly toxic when injected into mice brain. One of these neurotoxic phospholipases, OS2, was iodinated and used in binding experiments to demonstrate the presence of two families of specific binding sites in rat brain synaptic membranes. The affinities were exceptionally high, Kd1 = 1.5 +/- 0.5 pM and Kd2 = 45 +/- 10 pM, and the maximal binding capacities were Bmax 1 = 1 +/- 0.4 and Bmax 2 = 3 +/- 0.5 pmol/mg of protein. Both binding sites were sensitive to proteolysis and demonstrated to be located on proteins of Mr 85,000-88,000 and 36,000-51,000 by cross-linking and photoaffinity labeling techniques. The binding of 125 I-OS2 to synaptic membranes was dependent on Ca2+ ions and enhanced by Zn2+ ions which inhibit phospholipase activity. Competition experiments have shown that, except for beta-bungarotoxin, a number of known toxic snake or bee phospholipases have very high affinities for the newly identified binding sites. A good correlation (r = 0.80) was observed between toxicity and affinity but not between phospholipase activity and affinity

  3. A Dualistic Conformational Response to Substrate Binding in the Human Serotonin Transporter Reveals a High Affinity State for Serotonin*

    Science.gov (United States)

    Bjerregaard, Henriette; Severinsen, Kasper; Said, Saida; Wiborg, Ove; Sinning, Steffen

    2015-01-01

    Serotonergic neurotransmission is modulated by the membrane-embedded serotonin transporter (SERT). SERT mediates the reuptake of serotonin into the presynaptic neurons. Conformational changes in SERT occur upon binding of ions and substrate and are crucial for translocation of serotonin across the membrane. Our understanding of these conformational changes is mainly based on crystal structures of a bacterial homolog in various conformations, derived homology models of eukaryotic neurotransmitter transporters, and substituted cysteine accessibility method of SERT. However, the dynamic changes that occur in the human SERT upon binding of ions, the translocation of substrate, and the role of cholesterol in this interplay are not fully elucidated. Here we show that serotonin induces a dualistic conformational response in SERT. We exploited the substituted cysteine scanning method under conditions that were sensitized to detect a more outward-facing conformation of SERT. We found a novel high affinity outward-facing conformational state of the human SERT induced by serotonin. The ionic requirements for this new conformational response to serotonin mirror the ionic requirements for translocation. Furthermore, we found that membrane cholesterol plays a role in the dualistic conformational response in SERT induced by serotonin. Our results indicate the existence of a subpopulation of SERT responding differently to serotonin binding than hitherto believed and that membrane cholesterol plays a role in this subpopulation of SERT. PMID:25614630

  4. Monoclonal antibodies to Pneumocystis carinii

    DEFF Research Database (Denmark)

    Kovacs, J A; Halpern, J L; Lundgren, B

    1989-01-01

    To increase understanding of the antigenic structure of Pneumocystis carinii, we developed monoclonal antibodies to rat and human P. carinii. The specificity of the antibodies was demonstrated by immunofluorescence and immunoblot studies. Only one of five monoclonal antibodies to rat P. carinii r...

  5. Application of monoclonal antibodies in functional and comparative investigations of heavy-chain immunoglobulins in new world camelids.

    Science.gov (United States)

    Daley, L P; Gagliardo, L F; Duffy, M S; Smith, M C; Appleton, J A

    2005-03-01

    Of the three immunoglobulin G (IgG) isotypes described to occur in camelids, IgG2 and IgG3 are distinct in that they do not incorporate light chains. These heavy-chain antibodies (HCAbs) constitute approximately 50% of the IgG in llama serum and as much as 75% of the IgG in camel serum. We have produced isotype-specific mouse monoclonal antibodies (MAbs) in order to investigate the roles of HCAbs in camelid immunity. Seventeen stable hybridomas were cloned, and three MAbs that were specific for epitopes on the gamma chains of llama IgG1, IgG2, or IgG3 were characterized in detail. Affinity chromatography revealed that each MAb bound its isotype in solution in llama serum. The antibodies bound to the corresponding alpaca IgGs, to guanaco IgG1 and IgG2, and to camel IgG1. Interestingly, anti-IgG2 MAbs bound three heavy-chain species in llama serum, confirming the presence of three IgG2 subisotypes. Two IgG2 subisotypes were detected in alpaca and guanaco sera. The MAbs detected llama serum IgGs when they were bound to antigen in enzyme-linked immunosorbent assays and were used to discern among isotypes induced during infection with a parasitic nematode. Diseased animals, infected with Parelaphostrongylus tenuis, did not produce antigen-specific HCAbs; rather, they produced the conventional isotype, IgG1, exclusively. Our data document the utility of these MAbs in functional and physiologic investigations of the immune systems of New World camelids.

  6. High-affinity human leucocyte antigen class I binding variola-derived peptides induce CD4(+) T cell responses more than 30 years post-vaccinia virus vaccination

    DEFF Research Database (Denmark)

    Wang, M.; Tang, Sheila Tuyet; Lund, Ole

    2009-01-01

    Interferon-gamma secreting T lymphocytes against pox virus-derived synthetic 9-mer peptides were tested by enzyme-linked immunospot in peripheral blood of individuals vaccinated with vaccinia virus more than 30 years ago. The peptides were characterized biochemically as high-affinity human leucoc...

  7. High-Affinity Sites Form an Interaction Network to Facilitate Spreading of the MSL Complex across the X Chromosome in Drosophila

    NARCIS (Netherlands)

    Ramírez, Fidel; Lingg, Thomas; Toscano, Sarah; Lam, Kin Chung; Georgiev, Plamen; Chung, Ho-Ryun; Lajoie, Bryan R; de Wit, Elzo; Zhan, Ye; de Laat, Wouter; Dekker, Job; Manke, Thomas; Akhtar, Asifa

    2015-01-01

    Dosage compensation mechanisms provide a paradigm to study the contribution of chromosomal conformation toward targeting and spreading of epigenetic regulators over a specific chromosome. By using Hi-C and 4C analyses, we show that high-affinity sites (HAS), landing platforms of the male-specific

  8. Isolation and partial characterization of gypsy moth BTR-270, an anionic brush border membrane glycoconjugate that binds Bacillus thuringiensis Cry1A toxins with high affinity

    Science.gov (United States)

    Algimantas P. Valaitis; Jeremy L. Jenkins; Mi Kyong Lee; Donald H. Dean; Karen J. Garner

    2001-01-01

    BTR-270, a gypsy moth (Lymantria dispar) brush border membrane molecule that binds Bacillus thuringiensis (Bt) Cry1A toxins with high affinity, was purified by preparative gel electrophoresis. Rabbit antibodies specific for the Bt toxin-binding molecule were raised. Attempts to label BTR-270 by protein-directed techniques were...

  9. Contrast-enhanced CT with a High-Affinity Cationic Contrast Agent for Imaging ex Vivo Bovine, Intact ex Vivo Rabbit, and in Vivo Rabbit Cartilage

    OpenAIRE

    Stewart, Rachel C.; Bansal, Prashant N.; Entezari, Vahid; Lusic, Hrvoje; Nazarian, Rosalynn M.; Snyder, Brian D.; Grinstaff, Mark W.

    2013-01-01

    The high affinity of a cationic iodinated contrast agent for cartilage provides better tissue visualization, easier segmentation, higher contrast-to-noise ratios, and longer usable imaging windows and requires a lower dose of injected contrast agent compared with an anionic contrast agent.

  10. Soil carbon content and relative abundance of high affinity H2-oxidizing bacteria predict atmospheric H2 soil uptake activity better than soil microbial community composition

    NARCIS (Netherlands)

    Khdhiri, Mondher; Hesse, Laura; Popa, Maria Elena; Quiza, Liliana; Lalonde, Isabelle; Meredith, Laura K.; Röckmann, Thomas; Constant, Philippe

    2015-01-01

    Soil-atmosphere exchange of H2 is controlled by gas diffusion and the microbial production and oxidation activities in soil. Among these parameters, the H2 oxidation activity catalyzed by soil microorganisms harboring high affinity hydrogenase is the most difficult variable to parameterize because

  11. Amino propynyl benzoic acid building block in rigid spacers of divalent ligands binding to the Syk SH2 domains with equally high affinity as the natural ligand

    NARCIS (Netherlands)

    Dekker, Frank J; de Mol, Nico J; Fischer, Marcel J E; Liskamp, Rob M J; Dekker, Frank

    2003-01-01

    The construction of rigid spacers composed of amino propynyl benzoic acid building blocks is described. These spacers were used to link two phosphopeptide ligand sites towards obtaining divalent ligands with a high affinity for Syk tandem SH2 domains, which are important in signal transduction. The

  12. Mutational analysis of the high-affinity zinc binding site validates a refined human dopamine transporter homology model.

    Directory of Open Access Journals (Sweden)

    Thomas Stockner

    Full Text Available The high-resolution crystal structure of the leucine transporter (LeuT is frequently used as a template for homology models of the dopamine transporter (DAT. Although similar in structure, DAT differs considerably from LeuT in a number of ways: (i when compared to LeuT, DAT has very long intracellular amino and carboxyl termini; (ii LeuT and DAT share a rather low overall sequence identity (22% and (iii the extracellular loop 2 (EL2 of DAT is substantially longer than that of LeuT. Extracellular zinc binds to DAT and restricts the transporter's movement through the conformational cycle, thereby resulting in a decrease in substrate uptake. Residue H293 in EL2 praticipates in zinc binding and must be modelled correctly to allow for a full understanding of its effects. We exploited the high-affinity zinc binding site endogenously present in DAT to create a model of the complete transmemberane domain of DAT. The zinc binding site provided a DAT-specific molecular ruler for calibration of the model. Our DAT model places EL2 at the transporter lipid interface in the vicinity of the zinc binding site. Based on the model, D206 was predicted to represent a fourth co-ordinating residue, in addition to the three previously described zinc binding residues H193, H375 and E396. This prediction was confirmed by mutagenesis: substitution of D206 by lysine and cysteine affected the inhibitory potency of zinc and the maximum inhibition exerted by zinc, respectively. Conversely, the structural changes observed in the model allowed for rationalizing the zinc-dependent regulation of DAT: upon binding, zinc stabilizes the outward-facing state, because its first coordination shell can only be completed in this conformation. Thus, the model provides a validated solution to the long extracellular loop and may be useful to address other aspects of the transport cycle.

  13. Functional mapping and implications of substrate specificity of the yeast high-affinity leucine permease Bap2.

    Science.gov (United States)

    Usami, Yuki; Uemura, Satsohi; Mochizuki, Takahiro; Morita, Asami; Shishido, Fumi; Inokuchi, Jin-ichi; Abe, Fumiyoshi

    2014-07-01

    Leucine is a major amino acid in nutrients and proteins and is also an important precursor of higher alcohols during brewing. In Saccharomyces cerevisiae, leucine uptake is mediated by multiple amino acid permeases, including the high-affinity leucine permease Bap2. Although BAP2 transcription has been extensively analyzed, the mechanisms by which a substrate is recognized and moves through the permease remain unknown. Recently, we determined 15 amino acid residues required for Tat2-mediated tryptophan import. Here we introduced homologous mutations into Bap2 amino acid residues and showed that 7 residues played a role in leucine import. Residues I109/G110/T111 and E305 were located within the putative α-helix break in TMD1 and TMD6, respectively, according to the structurally homologous Escherichia coli arginine/agmatine antiporter AdiC. Upon leucine binding, these α-helix breaks were assumed to mediate a conformational transition in Bap2 from an outward-open to a substrate-binding occluded state. Residues Y336 (TMD7) and Y181 (TMD3) were located near I109 and E305, respectively. Bap2-mediated leucine import was inhibited by some amino acids according to the following order of severity: phenylalanine, leucine>isoleucine>methionine, tyrosine>valine>tryptophan; histidine and asparagine had no effect. Moreover, this order of severity clearly coincided with the logP values (octanol-water partition coefficients) of all amino acids except tryptophan. This result suggests that the substrate partition efficiency to the buried Bap2 binding pocket is the primary determinant of substrate specificity rather than structural amino acid side chain recognition. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Changes in medium radioactivity and composition accompany high-affinity uptake of glutamate and aspartate by mouse brain slices

    International Nuclear Information System (INIS)

    Latzkovits, L.; Neidle, A.; Lajtha, A.

    1984-01-01

    In measurements of high affinity transport in tissue slices, the incubation medium is often treated as an ''infinitely large pool''. External substrate concentrations, even at the micromolar level, are assumed to be constant and metabolic interactions between tissue and medium are neglected. In the present report we describe experiments in which glutamic and aspartic acid uptake by mouse brain slices were studied using techniques that could test these assumptions. Cerebral hemispheres were cut into 0.1 mm sections and about 90 mg of tissue incubated in 10 ml of oxygenated medium. After 45 minutes of equilibration, radioactive substrates were added and the concentrations and specific activities of the amino acids and their metabolites in the medium were determined. During the first 10 min following substrate addition, rapid decreases in glutamic and aspartic acid concentrations in the medium were accompanied by large decreases in specific activity caused by the continuous release of these amino acids from the tissue. In addition, extensive conversion of both substrates to glutamine and the preferential accumulation of this metabolite, in the medium, was found. These results demonstrate that metabolism and release occur simultaneously with uptake during transport experiments in vitro and that these processes can take place in specific tissue compartments. It is therefore necessary to measure the tissue and medium concentration levels of amino acids along with their radioactivity in such experiments, since all three processes (transport, metabolism, and compartmentation) are interrelated in the clearance of amino acids from the incubation medium and probably from the extracellular spaces in vivo as well

  15. NK1 receptor fused to beta-arrestin displays a single-component, high-affinity molecular phenotype.

    Science.gov (United States)

    Martini, Lene; Hastrup, Hanne; Holst, Birgitte; Fraile-Ramos, Alberto; Marsh, Mark; Schwartz, Thue W

    2002-07-01

    Arrestins are cytosolic proteins that, upon stimulation of seven transmembrane (7TM) receptors, terminate signaling by binding to the receptor, displacing the G protein and targeting the receptor to clathrin-coated pits. Fusion of beta-arrestin1 to the C-terminal end of the neurokinin NK1 receptor resulted in a chimeric protein that was expressed to some extent on the cell surface but also accumulated in transferrin-labeled recycling endosomes independently of agonist stimulation. As expected, the fusion protein was almost totally silenced with respect to agonist-induced signaling through the normal Gq/G11 and Gs pathways. The NK1-beta-arrestin1 fusion construct bound nonpeptide antagonists with increased affinity but surprisingly also bound two types of agonists, substance P and neurokinin A, with high, normal affinity. In the wild-type NK1 receptor, neurokinin A (NKA) competes for binding against substance P and especially against antagonists with up to 1000-fold lower apparent affinity than determined in functional assays and in homologous binding assays. When the NK1 receptor was closely fused to G proteins, this phenomenon was eliminated among agonists, but the agonists still competed with low affinity against antagonists. In contrast, in the NK1-beta-arrestin1 fusion protein, all ligands bound with similar affinity independent of the choice of radioligand and with Hill coefficients near unity. We conclude that the NK1 receptor in complex with arrestin is in a high-affinity, stable, agonist-binding form probably best suited to structural analysis and that the receptor can display binding properties that are nearly theoretically ideal when it is forced to complex with only a single intracellular protein partner.

  16. Specific capture and detection of Staphylococcus aureus with high-affinity modified aptamers to cell surface components.

    Science.gov (United States)

    Baumstummler, A; Lehmann, D; Janjic, N; Ochsner, U A

    2014-10-01

    Slow off-rate modified aptamer (SOMAmer) reagents were generated to several Staphylococcus aureus cell surface-associated proteins via SELEX with multiple modified DNA libraries using purified recombinant or native proteins. High-affinity binding agents with sub-nanomolar Kd 's were obtained for staphylococcal protein A (SpA), clumping factors (ClfA, ClfB), fibronectin-binding proteins (FnbA, FnbB) and iron-regulated surface determinants (Isd). Further screening revealed several SOMAmers that specifically bound to Staph. aureus cells from all strains that were tested, but not to other staphylococci or other bacteria. SpA and ClfA SOMAmers proved useful for the selective capture and enrichment of Staph. aureus cells, as shown by culture and PCR, leading to improved limits of detection and efficient removal of PCR inhibitors. Detection of Staph. aureus cells was enhanced by several orders of magnitude when the bacterial cell surface was coated with SOMAmers followed by qPCR of the SOMAmers. Furthermore, fluorescence-labelled SpA SOMAmers demonstrated their utility as direct detection agents in flow cytometry. Significance and impact of the study: Monitoring for microbial contamination of food, water, nonsterile products or the environment is typically based on culture, PCR or antibodies. Aptamers that bind with high specificity and affinity to well-conserved cell surface epitopes represent a promising novel type of reagents to detect bacterial cells without the need for culture or cell lysis, including for the capture and enrichment of bacteria present at low cell densities and for the direct detection via qPCR or fluorescent staining. © 2014 Soma Logic, Inc. published by John Wiley & Sons Ltd On behalf of the society for Applied Microbiology.

  17. Targeting the CXCR4 pathway using a novel anti-CXCR4 IgG1 antibody (PF-06747143 in chronic lymphocytic leukemia

    Directory of Open Access Journals (Sweden)

    Manoj K. Kashyap

    2017-05-01

    Full Text Available Abstract Background The CXCR4-CXCL12 axis plays an important role in the chronic lymphocytic leukemia (CLL-microenvironment interaction. Overexpression of CXCR4 has been reported in different hematological malignancies including CLL. Binding of the pro-survival chemokine CXCL12 with its cognate receptor CXCR4 induces cell migration. CXCL12/CXCR4 signaling axis promotes cell survival and proliferation and may contribute to the tropism of leukemia cells towards lymphoid tissues and bone marrow. Therefore, we hypothesized that targeting CXCR4 with an IgG1 antibody, PF-06747143, may constitute an effective therapeutic approach for CLL. Methods Patient-derived primary CLL-B cells were assessed for cytotoxicity in an in vitro model of CLL microenvironment. PF-06747143 was analyzed for cell death induction and for its potential to interfere with the chemokine CXCL12-induced mechanisms, including migration and F-actin polymerization. PF-06747143 in vivo efficacy was determined in a CLL murine xenograft tumor model. Results PF-06747143, a novel-humanized IgG1 CXCR4 antagonist antibody, induced cell death of patient-derived primary CLL-B cells, in presence or absence of stromal cells. Moreover, cell death induction by the antibody was independent of CLL high-risk prognostic markers. The cell death mechanism was dependent on CXCR4 expression, required antibody bivalency, involved reactive oxygen species production, and did not require caspase activation, all characteristics reminiscent of programmed cell death (PCD. PF-06747143 also induced potent B-CLL cytotoxicity via Fc-driven antibody-dependent cell-mediated cytotoxicity (ADCC and complement-dependent cytotoxicity activity (CDC. PF-06747143 had significant combinatorial effect with standard of care (SOC agents in B-CLL treatment, including rituximab, fludarabine (F-ara-A, ibrutinib, and bendamustine. In a CLL xenograft model, PF-06747143 decreased tumor burden and improved survival as a monotherapy, and in combination with bendamustine. Conclusions We show evidence that PF-06747143 has biological activity in CLL primary cells, supporting a rationale for evaluation of PF-06747143 for the treatment of CLL patients.

  18. Discovery of a Chemical Modification by Citric Acid in a Recombinant Monoclonal Antibody

    Science.gov (United States)

    2015-01-01

    Recombinant therapeutic monoclonal antibodies exhibit a high degree of heterogeneity that can arise from various post-translational modifications. The formulation for a protein product is to maintain a specific pH and to minimize further modifications. Generally Recognized as Safe (GRAS), citric acid is commonly used for formulation to maintain a pH at a range between 3 and 6 and is generally considered chemically inert. However, as we reported herein, citric acid covalently modified a recombinant monoclonal antibody (IgG1) in a phosphate/citrate-buffered formulation at pH 5.2 and led to the formation of so-called “acidic species” that showed mass increases of 174 and 156 Da, respectively. Peptide mapping revealed that the modification occurred at the N-terminus of the light chain. Three additional antibodies also showed the same modification but displayed different susceptibilities of the N-termini of the light chain, heavy chain, or both. Thus, ostensibly unreactive excipients under certain conditions may increase heterogeneity and acidic species in formulated recombinant monoclonal antibodies. By analogy, other molecules (e.g., succinic acid) with two or more carboxylic acid groups and capable of forming an anhydride may exhibit similar reactivities. Altogether, our findings again reminded us that it is prudent to consider formulations as a potential source for chemical modifications and product heterogeneity. PMID:25136741

  19. Application of monoclonal antibodies in diagnostics of the colorectal cancer

    International Nuclear Information System (INIS)

    Mladenov, B.; Milanov, S.; Peshev, N.; Tsanev, Ts.; Minchev, D.; Pencheva, V.

    1991-01-01

    Immunoscintigraphy with CEA monoclonal antibodies (MoA) in patients with colorectal cancer has been applied since 1987 by the authors. MoA from the hybridoma F023C5 are used (IgG 1 -class) and their fragments labelled with 131 I and 111 In. The labelled MoA are introduced intravenously in the course of 30 min, the total activity is 2.5 - 3.5 mCi. The scanning is made 48 and 96 hours on gamma camera. An additional activity on 99m Tc-sulfocolloid and 99m Tc-DTPA is applied for outlining the liver and kidney contours. Digital substraction technique is applied for image processing with contrast and background reduction. The thyroid is blocked with Lugol solution in a course of 5-6 days. Among all of the 18 investigated patients a positive result has been observed in 16. Metastases bigger than 1 cm have a positive scan. No initial invasion in the regional lymph nodes has been established. 3 figs., 4 refs

  20. Detection of hepatitis B virus infection in HBsAg-negative patients by monoclonal antibodies against HBsAg

    International Nuclear Information System (INIS)

    Fujita, Y.K.

    1986-01-01

    The technique of producing antibody secreting hybridomas has made available high-affinity antibodies of predefined specificity for use as diagnostic reagents. Recently, high-affinity monoclonal antibodies to hepatitis B surface antigens (HBsAg) were produced and characterized. Immunoassay was developed using these antibodies for the detection of HBsAg-associated determinants. The present study indicated the significance of the enhanced detection by monoclonal radioimmunoassay (M-RIA) of HBsAg in sera of patients with hepatitis B virus infection. The M-RIA detected HBsAg in sera of hemodialysis patients and blood donor defined as HBsAg-negative by polyclonal RIA (2.2 %, 0.14 %, respectively). Furthermore, individuals with chronic liver diseases were reactive only in the M-RIA (chronic hepatitis 4.8 %, liver cirrhosis 10.0 %, hepatocellular carcinoma 22.2 %). It is noteworthy that some of these patients were diagnosesed as so-called non-A non-B hepatitis because of no serological markers of hepatitis B virus infection such as HBsAb and HBcAb. The enhanced performance of the monoclonal RIA compared to conventional RIA was due to the increased sensitivity of the assay (55 pg vs 230 pg/ml). In immunohistochemical study, one of the monoclonal antibody named 5C3 was applied for detection of HBsAg in the formalin-fixed paraffin-embedded liver. HBsAg was detected in 6 out of 41 HBsAg-seronegative liver specimen. Thus, the studies showed the importance of the clinical application of monoclonal antibodies such as immunoassay and immunohistochemical study in the diagnosis of hepatitis B virus infection. (author)

  1. Detection of hepatitis B virus infection in HBsAg-negative patients by monoclonal antibodies against HBsAg

    Energy Technology Data Exchange (ETDEWEB)

    Fujita, Y K

    1986-11-01

    The technique of producing antibody secreting hybridomas has made available high-affinity antibodies of predefined specificity for use as diagnostic reagents. Recently, high-affinity monoclonal antibodies to hepatitis B surface antigens (HBsAg) were produced and characterized. Immunoassay was developed using these antibodies for the detection of HBsAg-associated determinants. The present study indicated the significance of the enhanced detection by monoclonal radioimmunoassay (M-RIA) of HBsAg in sera of patients with hepatitis B virus infection. The M-RIA detected HBsAg in sera of hemodialysis patients and blood donor defined as HBsAg-negative by polyclonal RIA (2.2 %, 0.14 %, respectively). Furthermore, individuals with chronic liver diseases were reactive only in the M-RIA (chronic hepatitis 4.8 %, liver cirrhosis 10.0 %, hepatocellular carcinoma 22.2 %). It is noteworthy that some of these patients were diagnosesed as so-called non-A non-B hepatitis because of no serological markers of hepatitis B virus infection such as HBsAb and HBcAb. The enhanced performance of the monoclonal RIA compared to conventional RIA was due to the increased sensitivity of the assay (55 pg vs 230 pg/ml). In immunohistochemical study, one of the monoclonal antibody named 5C3 was applied for detection of HBsAg in the formalin-fixed paraffin-embedded liver. HBsAg was detected in 6 out of 41 HBsAg-seronegative liver specimen. Thus, the studies showed the importance of the clinical application of monoclonal antibodies such as immunoassay and immunohistochemical study in the diagnosis of hepatitis B virus infection.

  2. Investigating high-concentration monoclonal antibody powder suspension in nonaqueous suspension vehicles for subcutaneous injection.

    Science.gov (United States)

    Bowen, Mayumi; Armstrong, Nick; Maa, Yuh-Fun

    2012-12-01

    Developing high-concentration monoclonal antibody (mAb) liquid formulations for subcutaneous (s.c.) administration is challenging because increased viscosity makes injection difficult. To overcome this obstacle, we investigated a nonaqueous powder suspension approach. Three IgG1 mAbs were spray dried and suspended at different concentrations in Miglyol® 840, benzyl benzoate, or ethyl lactate. Suspensions were characterized for viscosity, particle size, and syringeability; physical stability was visually inspected. Suspensions generally outperformed liquid solutions for injectability despite higher viscosity at the same mAb concentrations. Powder formulations and properties had little effect on viscosity or injectability. Ethyl lactate suspensions had lowest viscosity (Miglyol® 840 improved overall performance in high mAb concentration suspensions. This study demonstrated the viability of high mAb concentration (>300 mg/mL) in suspension formulations for s.c. administration. Copyright © 2012 Wiley Periodicals, Inc.

  3. Production of monoclonal antibodies for Avian Metapneumovirus (SHS-BR-121) isolated in Brazil

    OpenAIRE

    Coswig,LT; Stach-Machado,DR; Arns,CW

    2007-01-01

    Avian Metapneumovirus (aMPV), also called Turkey Rhinotracheitis Virus (TRTV), is an upper respiratory tract infection of turkeys, chickens and other avian species. Five monoclonal antibodies (MAbs) were created against the Brazilian isolate (SHS-BR-121) of aMPV, MAbs 1A5B8; 1C1C4; 2C2E9 and 2A4C3 of IgG1 and MAb 1C1F8 of IgG2a. Four Mabs (1A5B8; 1C1C4; 2C2E9 and 2A4C3) showed neutralizing activity and three (1A5B8; 1C1C4 and 2A4C3) inhibited cellular fusion in vitro. These MAbs were used to ...

  4. Pharmacokinetics and biodistribution of a radioiodine labeled peptidomimetic ligand for high-affinity nerve growth factor receptors

    Energy Technology Data Exchange (ETDEWEB)

    Jung, K. H.; Kim, D. H.; Paik, J. Y.; Koh, B. H.; Bae, J. S.; Choe, Y. S.; Lee, K. H.; Kim, B. T. [Samsung Medical Center, Seoul (Korea, Republic of)

    2005-07-01

    Some of the obstacles for the clinical application of whole nerve growth factor (NGF) may be overcome by utilizing small molecule mimetics. We thus investigated the in vivo pharmacokinetics and biodistribution of a small cyclic peptide derived from NGF-[C(92-96)] with high receptor binding affinity. I-125 C(92-96) was labeled with the Bolton-Hunter method, and binding to TrkA/IgG chimeric protein was confirmed on a polyacrylamide gel after cross-linking. Pharmacokinetic analysis was performed in normal ICR mice intravenously injected with 0.5 MBq I-125 C(92-96) containing varying doses of C(92-96). Biodistribution studies were done at 6 h after injection. Cross-linkage analysis confirmed binding of I-125 C(92-96) to the high affinity NGF receptor, TrkA. Intravenously injected I-125 C(92-96) was cleared from the blood in a biexponential manner with an early T1/2{alpha} of 5.2 min and late T1/2{beta} of 121.3 min. Log blood-concentration decreased over time with a k-slope of 0.0025, clearance of 11.8{+-}0.5 ml/min, T1/2 of 4.1{+-}0.4 hr, and volume of distribution of 69.7{+-}4.6 ml. The pattern of elimination from the blood remained essentially unchanged regardless of the dose of added C(92-96), with dose-proportionate increases in AUCs and peak concentrations consistent with linear pharmacokinetics. Biodistribution studies demonstrated high kidney activity suggesting renal excretion of I-125 C(92-96). There were moderate levels of accumulation in the spleen, lungs and liver, followed by the myocardium and skeletal muscle, whereas brain uptake was low (< 0.2 %ID/gm). Intravenously administered C(92-96) follows linear pharmacokinetics, and is cleared from the circulation at a rate comparable to whole NGF despite its substantially smaller size. Although intravenous C(92-96) does not adequately reach brain tissue, clinically relevant doses can achieve major organ accumulation levels that may be sufficient to elicit biologic responses through NGF receptors.

  5. Pharmacokinetics and biodistribution of a radioiodine labeled peptidomimetic ligand for high-affinity nerve growth factor receptors

    International Nuclear Information System (INIS)

    Jung, K. H.; Kim, D. H.; Paik, J. Y.; Koh, B. H.; Bae, J. S.; Choe, Y. S.; Lee, K. H.; Kim, B. T.

    2005-01-01

    Some of the obstacles for the clinical application of whole nerve growth factor (NGF) may be overcome by utilizing small molecule mimetics. We thus investigated the in vivo pharmacokinetics and biodistribution of a small cyclic peptide derived from NGF-[C(92-96)] with high receptor binding affinity. I-125 C(92-96) was labeled with the Bolton-Hunter method, and binding to TrkA/IgG chimeric protein was confirmed on a polyacrylamide gel after cross-linking. Pharmacokinetic analysis was performed in normal ICR mice intravenously injected with 0.5 MBq I-125 C(92-96) containing varying doses of C(92-96). Biodistribution studies were done at 6 h after injection. Cross-linkage analysis confirmed binding of I-125 C(92-96) to the high affinity NGF receptor, TrkA. Intravenously injected I-125 C(92-96) was cleared from the blood in a biexponential manner with an early T1/2α of 5.2 min and late T1/2β of 121.3 min. Log blood-concentration decreased over time with a k-slope of 0.0025, clearance of 11.8±0.5 ml/min, T1/2 of 4.1±0.4 hr, and volume of distribution of 69.7±4.6 ml. The pattern of elimination from the blood remained essentially unchanged regardless of the dose of added C(92-96), with dose-proportionate increases in AUCs and peak concentrations consistent with linear pharmacokinetics. Biodistribution studies demonstrated high kidney activity suggesting renal excretion of I-125 C(92-96). There were moderate levels of accumulation in the spleen, lungs and liver, followed by the myocardium and skeletal muscle, whereas brain uptake was low (< 0.2 %ID/gm). Intravenously administered C(92-96) follows linear pharmacokinetics, and is cleared from the circulation at a rate comparable to whole NGF despite its substantially smaller size. Although intravenous C(92-96) does not adequately reach brain tissue, clinically relevant doses can achieve major organ accumulation levels that may be sufficient to elicit biologic responses through NGF receptors

  6. Protoporphyrinogen oxidase: high affinity tetrahydrophthalimide radioligand for the inhibitor/herbicide-binding site in mouse liver mitochondria.

    Science.gov (United States)

    Birchfield, N B; Casida, J E

    1996-01-01

    Protoporphyrinogen oxidase (protox), the last common enzyme in heme and chlorophyll biosynthesis, is the target of several classes of herbicides acting as inhibitors in both plants and mammals. N-(4-Chloro-2-fluoro-5-(propargyloxy)phenyl)-3,4,5,6-tetrahydro phthalimide (a potent protox inhibitor referred to as THP) was synthesized as a candidate radioligand ([3H]-THP) by selective catalytic reduction of 3,6-dihydrophthalic anhydride (DHPA) with tritium gas followed by condensation in 45% yield with 4-chloro-2-fluoro-5-(propargyloxy)aniline. Insertion of tritium at the 3 and 6 carbons of DHPA as well as the expected 4 and 5 carbons resulted in high specific activity [3H]THP (92 Ci/mmol). This radioligand undergoes rapid, specific, saturable, and reversible binding to the inhibitor/herbicide binding site of the protox component of cholate-solubilized mouse liver mitochondria with an apparent Kd of 0.41 nM and Bmax of 0.40 pmol/mg of protein. In the standard assay, mouse preparation (150 micrograms of protein) and [3H]THP (0.5 nM) are incubated in 500 microL of phosphate buffer at pH 7.2 for 15 min at 25 degrees C followed by addition of ammonium sulfate and filtration with glass fiber filters. The potencies of five nitrodiphenyl ethers and two other herbicides as inhibitors of [3H]THP binding correlate well with those for inhibition of protox activity (r2 = 0.97, n = 7), thus validating the binding assay as relevant to enzyme inhibition. It is also suitable to determine in vivo block as illustrated by an approximately 50% decrease in [3H]THP binding in liver mitochondria from mice treated ip with oxyfluorfen at 4 mg/kg. This is the first report of a binding assay for protox in mammals. The high affinity and specific activity of [3H]THP facilitate quantitation of protox and therefore research on a sensitive inhibition site for porphyrin biosynthesis.

  7. Dissecting Immunogenicity of Monoclonal Antibodies

    National Research Council Canada - National Science Library

    Snyder, Christopher

    2003-01-01

    The potential of monoclonal antibodies, (mAbs), for use in therapeutic and diagnostic applications has not been fully realized in part due to counter-immune responses that often arise in patient recipients of mAb...

  8. Stability assessment on a library scale: a rapid method for the evaluation of the commutability and insertion of residues in C-terminal loops of the CH3 domains of IgG1-Fc.

    Science.gov (United States)

    Hasenhindl, Christoph; Traxlmayr, Michael W; Wozniak-Knopp, Gordana; Jones, Phil C; Stadlmayr, Gerhard; Rüker, Florian; Obinger, Christian

    2013-10-01

    Antigen-binding Fc fragments (Fcab) are generated by engineering the C-terminal loop regions in the CH3 domain of human immunoglobulin G class 1-crystallizable fragment (IgG1-Fc). For an optimum library design with high percentage of well-folded clones for efficient binder selection, information about the correlation between primary structure and stability is needed. Here, we present a rapid method that allows determination of the overall stability of whole libraries of IgG1-Fc on the surface of yeast by flow cytometry. Libraries of IgG1-Fc mutants with distinct regions in AB-, CD- and EF-loops of the CH3 domains randomized or carrying therein insertions of five additional residues were constructed, incubated at increasing temperatures and probed for residual binding of generic Fc ligands. Calculated temperatures of half-maximal irreversible denaturation of the libraries gave a clear hierarchy of tolerance to randomization of distinct loop positions. Experimental data were evaluated by a computational approach and are discussed with respect to the structure of IgG1-Fc and variation in sequence and length of these loops in homologous Fc proteins. Generally, the described method allows for quick assessment of the effects of randomization of distinct regions on the foldability and stability of a yeast-displayed protein library.

  9. Detection of FMD virus type specific IgG1, IgG2 and IgA antibodies in milk and serum of buffaloes vaccinated with oil adjuvanted polyvalent FMD vaccine

    Directory of Open Access Journals (Sweden)

    R. Sharma

    2010-02-01

    Full Text Available The present investigation was carried out on 15 randomly selected milch buffaloes divided into three groups on the basis of lactation at an organized farm, to study the foot and mouth disease virus type specific antibodies in milk and serum following FMD vaccination. Milk and serum samples collected before vaccination i.e. 0 day and on 7, 14, 28, 42 and 56 days post vaccination, were analyzed for the detection of FMD virus specific IgG1, IgG2 and IgA antibody response by indirect double antibody sandwich ELISA. Significant FMD virus type specific antibody titres (IgG1, IgG2 and IgA were detected in milk and serum of buffaloes on different days post vaccination, though the levels of antibodies were lower in milk as compared to serum. FMD virus type specific IgG1 was found to be the predominant subclass as compared to IgG2 and IgA both in milk and serum of vaccinated buffaloes. Milk and serum IgG1, IgG2 and IgA antibody titres were positively correlated with values of regression coefficient (R as 0.506, 0.434 and 0.396, respectively.

  10. Comparison of the C-mediating killing activity and C-activating properties of mouse monoclonal and polyclonal antibodies against Trypanosoma cruzi

    Directory of Open Access Journals (Sweden)

    T. L. Kipnis

    1992-01-01

    Full Text Available A Mouse polyclonal antiserum against Trypanosoma cruzi or its IgG and IgM fractions and five monoclonal antibodies (two IgM, two IgG1 and one IgG2a recognize and combine with membrane components of trypomastigote forms of the parasite as revealed by immunofluorescence. Although all these antibodies sensitize trypomastigotes and prepare them to activate the complement (C system, as measured by consumption of total C, C4, B and C3, only the polyclonal antiserum or its IgG, IgM and Fabμ fragments were able to induce trypanosome lysis by the alternative C pathway.

  11. Effect of producer cell line on functional activity of anti-D monoclonal antibodies destined for prevention of rhesus sensitization.

    Science.gov (United States)

    Olovnikova, N I; Ershler, M A; Belkina, E V; Nikolaeva, T L; Miterev, G Yu

    2009-04-01

    The ability of anti-D antibodies to cause antigen-specific immunosuppression depends on their interaction with low-affinity Fcgamma-receptors. Human monoclonal antibodies to D antigen of the rhesus system were investigated by antibody-dependent cytotoxicity assay in order to estimate their ability to induce hemolysis mediated by low-affinity Fcgamma receptors. We demonstrate that affinity of monoclonal antibodies to receptors of this type does not depend on primary structure of Fc-fragment, but depends on the producer cell line which expresses the antibodies. Monoclonal IgG1 antibodies interacting with FcgammaRIIa and FcgammaRIII lost this property, if they were secreted by human-mouse heterohybridoma, but not by human B-cell line. On the opposite, monoclonal antibodies that could not activate low-affinity Fcgamma receptors were highly active after human cells fusion with rat myeloma YB2/0. Hemolytic activity of IgG3 remained unchanged after fusion of human cells with rodent cells.

  12. Glucose uptake and growth of glucose-limited chemostat cultures of Aspergillus niger and a disruptant lacking MstA, a high-affinity glucose transporter

    DEFF Research Database (Denmark)

    Jørgensen, Thomas R; vanKuyk, Patricia A; Poulsen, Bjarne R

    2007-01-01

    This is a study of high-affinity glucose uptake in Aspergillus niger and the effect of disruption of a high-affinity monosaccharide-transporter gene, mstA. The substrate saturation constant (K(s)) of a reference strain was about 15 microM in glucose-limited chemostat culture. Disruption of mst......-affinity uptake system of A. niger. The mstA disruptant and a reference strain were cultivated in glucose-limited chemostat cultures at low, intermediate and high dilution rate (D=0.07 h(-1), 0.14 h(-1) and 0.20 h(-1)). Mycelium harvested from steady-state cultures was subjected to glucose uptake assays...

  13. In vitro and in vivo evaluation of new radiolabeled neurotensin(8-13) analogues with high affinity for NT1 receptors

    International Nuclear Information System (INIS)

    Garayoa, Elisa Garcia-; Allemann-Tannahill, Lesley; Blaeuenstein, Peter; Willmann, Martine; Carrel-Remy, Nathalie; Tourwe, Dirk; Iterbeke, Koen; Conrath, Peter; Schubiger, P. August

    2001-01-01

    The potential utility of neurotensin (NT) in cancer diagnosis and therapy is limited by its rapid degradation. New stabilized analogues were synthesized, labeled with [ 99m Tc] and screened in vitro and in vivo. High affinity and rapid internalization were obtained in binding assays. Despite their longer human plasma half-lives, a rapid degradation was observed with low concentrations as used in biodistribution tests. The tumor uptake rates were rather low but tumor/blood ratios increased according to the stability raise

  14. Na+-Dependent High-Affinity Nitrate, Phosphate and Amino Acids Transport in Leaf Cells of the Seagrass Posidonia oceanica (L. Delile

    Directory of Open Access Journals (Sweden)

    Lourdes Rubio

    2018-05-01

    Full Text Available Posidonia oceanica (L. Delile is a seagrass, the only group of vascular plants to colonize the marine environment. Seawater is an extreme yet stable environment characterized by high salinity, alkaline pH and low availability of essential nutrients, such as nitrate and phosphate. Classical depletion experiments, membrane potential and cytosolic sodium measurements were used to characterize the high-affinity NO3−, Pi and amino acids uptake mechanisms in this species. Net uptake rates of both NO3− and Pi were reduced by more than 70% in the absence of Na+. Micromolar concentrations of NO3− depolarized mesophyll leaf cells plasma membrane. Depolarizations showed saturation kinetics (Km = 8.7 ± 1 μM NO3−, which were not observed in the absence of Na+. NO3− induced depolarizations at increasing Na+ also showed saturation kinetics (Km = 7.2 ± 2 mM Na+. Cytosolic Na+ measured in P. oceanica leaf cells (17 ± 2 mM Na+ increased by 0.4 ± 0.2 mM Na+ upon the addition of 100 μM NO3−. Na+-dependence was also observed for high-affinity l-ala and l-cys uptake and high-affinity Pi transport. All together, these results strongly suggest that NO3−, amino acids and Pi uptake in P. oceanica leaf cells are mediated by high-affinity Na+-dependent transport systems. This mechanism seems to be a key step in the process of adaptation of seagrasses to the marine environment.

  15. [Study of anti-idiotype antibodies to human monoclonal antibody].

    Science.gov (United States)

    Harada, R; Takahashi, N; Owaki, I; Kannagi, R; Endo, N; Morita, N; Inoue, M

    1992-02-01

    A human monoclonal antibody, ll-50 (IgM, lambda), was generated, which reacted specifically with a major of glycolipid present in LS174T colon cancer cells. The glycolipid antigen which reacted with the ll-50 antibody was expected to four sugar residues from its TLC mobility, and it was ascertained that the glycolipid antigen which reacted with ll-50 antibody might be Lc4 antigen [Gal beta 1----3 GLcNAc beta 1----3 Gal beta 1----4 Glc beta 1----1 Cer] judging from TLC immunostaining and ELISA when the reactivity of ll-50 antibody was tested using various pure glycolipids in 3-5 sugar residues as an antigen. Sera in patients with malignant disorders and healthy individuals were analyzed by Sandwich assay of immobilized and biotinylated ll-50 antibody. The serum of the Lc4 antigen recognized by ll-50 antibody was significantly higher in patients with malignant disorders than that in healthy individuals (p less than 0.05). Three mouse monoclonal anti-idiotype antibodies, G3, B3 and C5 (all IgG1), were generated by the immunization of BALB/c mice with ll-50 antibody. These anti-idiotype antibodies specifically bound to to human monoclonal antibody, ll-50 and had a significant inhibitory activity towards the binding of ll-50 antibody to the Lc4 antigen. This indicated that these anti-idiotype antibodies, G3, B3, and C5, were paratope-related anti-idiotype antibodies. G3, B3, and C5 were expected to define the nearest idiotope because they could mutually inhibit ll-50 antibody. Sera in patients with malignant disorders and healthy individuals were analyzed by Sandwich assay of immobilized and biotinylated anti-idiotype antibodies, G3, B3, and C5. As to the ll-50 like antibodies defined by C5 (Id-C5+), the mean serum level in patients with malignant disorders was significantly higher than that in healthy individuals (p less than 0.05). As to the ll-50 like antibodies defined by B3 (Id-B3+), the mean serum level in patients with malignant disorders was significantly higher

  16. Humanised IgG1 antibody variants targeting membrane-bound carcinoembryonic antigen by antibody-dependent cellular cytotoxicity and phagocytosis.

    Science.gov (United States)

    Ashraf, S Q; Umana, P; Mössner, E; Ntouroupi, T; Brünker, P; Schmidt, C; Wilding, J L; Mortensen, N J; Bodmer, W F

    2009-11-17

    The effect of glycoengineering a membrane specific anti-carcinoembryonic antigen (CEA) (this paper uses the original term CEA for the formally designated CEACAM5) antibody (PR1A3) on its ability to enhance killing of colorectal cancer (CRC) cell lines by human immune effector cells was assessed. In vivo efficacy of the antibody was also tested. The antibody was modified using EBNA cells cotransfected with beta-1,4-N-acetylglucosaminyltransferase III and the humanised hPR1A3 antibody genes. The resulting alteration of the Fc segment glycosylation pattern enhances the antibody's binding affinity to the FcgammaRIIIa receptor on human immune effector cells but does not alter the antibody's binding capacity. Antibody-dependent cellular cytotoxicity (ADCC) is inhibited in the presence of anti-FcgammaRIII blocking antibodies. This glycovariant of hPR1A3 enhances ADCC 10-fold relative to the parent unmodified antibody using either unfractionated peripheral blood mononuclear or natural killer (NK) cells and CEA-positive CRC cells as targets. NK cells are far more potent in eliciting ADCC than either freshly isolated monocytes or granulocytes. Flow cytometry and automated fluorescent microscopy have been used to show that both versions of hPR1A3 can induce antibody-dependent cellular phagocytosis (ADCP) by monocyte-derived macrophages. However, the glycovariant antibody did not mediate enhanced ADCP. This may be explained by the relatively low expression of FcgammaRIIIa on cultured macrophages. In vivo studies show the efficacy of glycoengineered humanised IgG1 PR1A3 in significantly improving survival in a CRC metastatic murine model. The greatly enhanced in vitro ADCC activity of the glycoengineered version of hPR1A3 is likely to be clinically beneficial.

  17. High-affinity, noninhibitory pathogenic C1 domain antibodies are present in patients with hemophilia A and inhibitors

    Science.gov (United States)

    Batsuli, Glaivy; Deng, Wei; Healey, John F.; Parker, Ernest T.; Baldwin, W. Hunter; Cox, Courtney; Nguyen, Brenda; Kahle, Joerg; Königs, Christoph; Li, Renhao; Lollar, Pete

    2016-01-01

    Inhibitor formation in hemophilia A is the most feared treatment-related complication of factor VIII (fVIII) therapy. Most inhibitor patients with hemophilia A develop antibodies against the fVIII A2 and C2 domains. Recent evidence demonstrates that the C1 domain contributes to the inhibitor response. Inhibitory anti-C1 monoclonal antibodies (mAbs) have been identified that bind to putative phospholipid and von Willebrand factor (VWF) binding epitopes and block endocytosis of fVIII by antigen presenting cells. We now demonstrate by competitive enzyme-linked immunosorbent assay and hydrogen-deuterium exchange mass spectrometry that 7 of 9 anti-human C1 mAbs tested recognize an epitope distinct from the C1 phospholipid binding site. These mAbs, designated group A, display high binding affinities for fVIII, weakly inhibit fVIII procoagulant activity, poorly inhibit fVIII binding to phospholipid, and exhibit heterogeneity with respect to blocking fVIII binding to VWF. Another mAb, designated group B, inhibits fVIII procoagulant activity, fVIII binding to VWF and phospholipid, fVIIIa incorporation into the intrinsic Xase complex, thrombin generation in plasma, and fVIII uptake by dendritic cells. Group A and B epitopes are distinct from the epitope recognized by the canonical, human-derived inhibitory anti-C1 mAb, KM33, whose epitope overlaps both groups A and B. Antibodies recognizing group A and B epitopes are present in inhibitor plasmas from patients with hemophilia A. Additionally, group A and B mAbs increase fVIII clearance and are pathogenic in a hemophilia A mouse tail snip bleeding model. Group A anti-C1 mAbs represent the first identification of pathogenic, weakly inhibitory antibodies that increase fVIII clearance. PMID:27381905

  18. Cutting Edge: The murine high-affinity IgG receptor FcγRIV is sufficient for autoantibody-induced arthritis.

    Science.gov (United States)

    Mancardi, David A; Jönsson, Friederike; Iannascoli, Bruno; Khun, Huot; Van Rooijen, Nico; Huerre, Michel; Daëron, Marc; Bruhns, Pierre

    2011-02-15

    K/BxN serum-induced passive arthritis was reported to depend on the activation of mast cells, triggered by the activating IgG receptor FcγRIIIA, when engaged by IgG1 autoantibodies present in K/BxN serum. This view is challenged by the fact that FcγRIIIA-deficient mice still develop K/BxN arthritis and because FcγRIIIA is the only activating IgG receptor expressed by mast cells. We investigated the contribution of IgG receptors, IgG subclasses, and cells in K/BxN arthritis. We found that the activating IgG2 receptor FcγRIV, expressed only by monocytes/macrophages and neutrophils, was sufficient to induce disease. K/BxN arthritis occurred not only in mast cell-deficient W(sh) mice, but also in mice whose mast cells express no activating IgG receptors. We propose that at least two autoantibody isotypes, IgG1 and IgG2, and two activating IgG receptors, FcγRIIIA and FcγRIV, contribute to K/BxN arthritis, which requires at least two cell types other than mast cells, monocytes/macrophages, and neutrophils.

  19. Uses of monoclonal antibody 8H9

    Science.gov (United States)

    Cheung, Nai-Kong V.

    2013-04-09

    This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides different uses of the monoclonal antibody 8H9 or its derivative.

  20. Effectiveness of Alpha-toxin Fab Monoclonal Antibody Therapy in Limiting the Pathology of Staphylococcus aureus Keratitis.

    Science.gov (United States)

    Caballero, Armando R; Foletti, Davide L; Bierdeman, Michael A; Tang, Aihua; Arana, Angela M; Hasa-Moreno, Adela; Sangalang, Emma Ruth B; O'Callaghan, Richard J

    2015-08-01

    To investigate the effectiveness of a high-affinity human monoclonal antibody Fab fragment to Staphylococcus aureus alpha-toxin (LTM14 Fab) as therapy for S. aureus keratitis. A single topical drop of the LTM14 Fab antibody to alpha-toxin alone, or in 0.006% benzalkonium chloride (BAK), was applied every 30 min to S. aureus-infected rabbit corneas from 9 to 14 hours post-infection. Erosions and pathology were measured at 15 h post-infection. LTM14 Fab with BAK limited corneal erosions better than LTM14 Fab alone (p = 0.036), and both limited erosions compared to untreated eyes (p ≤ 0.0001). Overall pathology was similar in all groups (p ≥ 0.070), but iritis and chemosis were reduced by treatment (p ≤ 0.036). The high-affinity human monoclonal Fab fragment antibody (LTM14 Fab) to S. aureus alpha-toxin was effective in reducing corneal damage during S. aureus keratitis.

  1. Characterization of an AtCCX5 gene from Arabidopsis thaliana that involves in high-affinity K+ uptake and Na+ transport in yeast

    International Nuclear Information System (INIS)

    Zhang, Xinxin; Zhang, Min; Takano, Tetsuo; Liu, Shenkui

    2011-01-01

    Highlights: → The AtCCX5 protein coding a putative cation calcium exchanger was characterized. → AtCCX5 expressed in yeast was localized in the plasma membrane and nuclear periphery. → AtCCX5 protein did not show the same transport properties as the CAXs. → AtCCX5 protein involves in mediating high-affinity K + uptake in yeast. → AtCCX5 protein also involves in Na + transport in yeast. -- Abstract: The gene for a putative cation calcium exchanger (CCX) from Arabidopsis thaliana, AtCCX5, was cloned and its function was analyzed in yeast. Green fluorescent protein-tagged AtCCX5 expressed in yeast was localized in the plasma membrane and nuclear periphery. The yeast transformants expressing AtCCX5 were created and their growth in the presence of various cations (K + , Na + , Ca 2+ , Mg 2+ , Fe 2+ , Cu 2+ , Co 2+ , Cd 2+ , Mn 2+ , Ba 2+ , Ni 2+ , Zn 2+ , and Li + ) were analyzed. AtCCX5 expression was found to affect the response to K + and Na + in yeast. The AtCCX5 transformant also showed a little better growth to Zn 2+ . The yeast mutant 9.3 expressing AtCCX5 restored growth of the mutant on medium with low K + (0.5 mM), and also suppressed its Na + sensitivity. Ion uptake experiments showed that AtCCX5 mediated relatively high-affinity K + uptake and was also involved in Na + transport in yeast. Taken together, these findings suggest that the AtCCX5 is a novel transport protein involves in mediating high-affinity K + uptake and Na + transport in yeast.

  2. Tumor imaging with monoclonal antibodies

    International Nuclear Information System (INIS)

    Haisma, H.; Hilgers, J.

    1987-01-01

    Many monoclonal antibodies directed against tumor-associated antigens have been identified, but so far none of these are tumor specific. Polyclonal and monoclonal antibodies have been used for imaging of a wide variety of tumors with success. Radiolabeling of antibody is usually done with iodine isotopes of which 123 I is the best candidate for radioimmunodetection purposes. The labeling of antibodies through chelates makes it possible to use metal radioisotopes like 111 In, which is the best radioisotope for imaging with monoclonal antibodies due to its favorable half-life of 2.5 days. Usually imaging cannot be performed within 24 h after injection, but clearance of antibody can be increased by using F(ab) 2 of Fab. Another approach is to clear non-bound antibody by a second antibody, directed against the first. The detection limit of immunoimaging is about 2 cm, but will be improved by tomography or SPECT. There is still a high false positive and false negative rate, which makes it impossible to use radioimmunodetection as the only technique for diagnosis of tumors. In combination with other detection techniques, tumor imaging with monoclonal antibodies can improve diagnosis. 44 refs.; 3 tabs

  3. Radiolabeled monoclonal antibodies: a review

    International Nuclear Information System (INIS)

    Toledo e Souza, I.T. de; Okada, H.

    1990-05-01

    Since the description by Kohler and Milstein 1975 of their technique for producing monoclonal antibodies of predefined specificity, it has become a mainstay in most laboratories that utilize immunochemical techniques to study problems in basic, applied or clinical research. Paradoxically, the very success of monoclonal antibodies has generated a literature which is now so vast and scattered that it has become difficult to obtain a perspective. This brief review represents the distillation of many publications relating to the production and use of monoclonaal antibodies as radiopharmaceuticals. Significant advances were made possible in the last few years by combined developments in the fields of tumor-associated antigens and of monoclonal antibodies. In fact monoclonal antibodies against some well defined tumor-associated antigens, has led to significantly greater practical possibilities for producing highly specific radiolabeled antibodies as radiopharmaceuticals for diagnosis and therapy of human tumors. One of the main requirements of this methodology is the availability of stable radiopharmaceutical reagents which after labeling in vivo injection retain the capacity of specific interaction with the defined antigen and their molecular integrity. Since injection into human is the objetive of this kind of study all the specifications of radiopharmaceutical have to be fulfilled e.g. sterility, apirogenicity and absence of toxicity. (author) [pt

  4. In vitro and in vivo evaluation of new radiolabeled neurotensin(8-13) analogues with high affinity for NT1 receptors

    Energy Technology Data Exchange (ETDEWEB)

    Garayoa, Elisa Garcia-; Allemann-Tannahill, Lesley; Blaeuenstein, Peter; Willmann, Martine; Carrel-Remy, Nathalie; Tourwe, Dirk; Iterbeke, Koen; Conrath, Peter; Schubiger, P. August E-mail: schubiger@psi.ch

    2001-01-01

    The potential utility of neurotensin (NT) in cancer diagnosis and therapy is limited by its rapid degradation. New stabilized analogues were synthesized, labeled with [{sup 99m}Tc] and screened in vitro and in vivo. High affinity and rapid internalization were obtained in binding assays. Despite their longer human plasma half-lives, a rapid degradation was observed with low concentrations as used in biodistribution tests. The tumor uptake rates were rather low but tumor/blood ratios increased according to the stability raise.

  5. High-Affinity Methanotrophy Informed by Genome-Wide Analysis of Upland Soil Cluster Alpha (USCα) from Axel Heiberg Island, Canadian High Arctic

    Science.gov (United States)

    Rusley, C.; Onstott, T. C.; Lau, M.

    2017-12-01

    Methane (CH4) is a potent greenhouse gas whose proper budgeting is vital to climate predictions. Recent studies have identified upland Arctic mineral cryosols as consistent CH4 sinks, drawing CH4 from both the atmosphere and underlying anaerobic soil layers. Global atmospheric CH4 uptake is proposed to be mediated by high-affinity methanotrophs based on the detection of the marker gene pmoA (particulate methane monooxygenase beta subunit). However, a lack of pure cultures and scarcity of genomic information have hindered our understanding of their metabolic capabilities and versatility. Together with the missing genetic linkage between its pmoA and 16S ribosomal RNA (rRNA) gene, the factors that control the distribution and magnitude of high-affinity methanotrophy in the Arctic permafrost-affected region have remained elusive. Using 21 metagenomic datasets of surface soils obtained from long-term core incubation experiments,1 this bioinformatics study aimed to reconstruct the draft genome of the Upland Soil Cluster α-proteobacteria (USCα), the high-affinity methanotroph previously detected in the samples,2 and to determine its phylogeny and metabolic requirements. We obtained a genome bin containing the high-affinity form of the USCα-like pmoA gene. The 3.03 Mbp assembly is 91.6% complete with a unique set of single-copy marker genes. The 16S rRNA gene fragment of USCα belongs to the α-proteobacterial family Beijerinckiaceae. Genome annotation indicates possible formaldehyde oxidation via tetrahydromethanopterin-linked C1 transfer pathways, acetate utilization, carbon fixation via the Calvin-Benson-Bassham cycle, and glycogen production. Notably, the key enzymes for formaldehyde assimilation via the serine and ribulose monophosphate pathways are missing. The presence of genes encoding nitrate reductase and hemoglobin suggests adaptation to low O2 under water-logged conditions. Since USCα has versatile carbon metabolisms, it may not be an obligate methanotroph

  6. A high-affinity, dimeric inhibitor of PSD-95 bivalently interacts with PDZ1-2 and protects against ischemic brain damage

    DEFF Research Database (Denmark)

    Bach, Anders*; Clausen, Bettina H; Møller, Magda

    2012-01-01

    Inhibition of the ternary protein complex of the synaptic scaffolding protein postsynaptic density protein-95 (PSD-95), neuronal nitric oxide synthase (nNOS), and the N-methyl-d-aspartate (NMDA) receptor is a potential strategy for treating ischemic brain damage, but high-affinity inhibitors are ...... of Tat-N-dimer (3 nmol/g) to mice subjected to focal cerebral ischemia reduces infarct volume with 40% and restores motor functions. Thus, Tat-N-dimer is a highly efficacious neuroprotective agent with therapeutic potential in stroke....

  7. Production of monoclonal antibodies for Avian Metapneumovirus (SHS-BR-121 isolated in Brazil

    Directory of Open Access Journals (Sweden)

    LT Coswig

    2007-12-01

    Full Text Available Avian Metapneumovirus (aMPV, also called Turkey Rhinotracheitis Virus (TRTV, is an upper respiratory tract infection of turkeys, chickens and other avian species. Five monoclonal antibodies (MAbs were created against the Brazilian isolate (SHS-BR-121 of aMPV, MAbs 1A5B8; 1C1C4; 2C2E9 and 2A4C3 of IgG1 and MAb 1C1F8 of IgG2a. Four Mabs (1A5B8; 1C1C4; 2C2E9 and 2A4C3 showed neutralizing activity and three (1A5B8; 1C1C4 and 2A4C3 inhibited cellular fusion in vitro. These MAbs were used to investigate antigenic relationship among three strains (SHS-BR-121, STG 854/88 and TRT 1439/91 of aMPV subtypes A and B using cross-neutralization test. The results confirm that the monoclonal antibodies described can be used as a valuable tool in the epizootiological and serological studies, and also for the specific diagnosis of the subtypes in the infection for Avian Metapneumovirus.

  8. Passive immunization against Cryptococcus neoformans with an isotype-switch family of monoclonal antibodies reactive with cryptococcal polysaccharide.

    Science.gov (United States)

    Sanford, J E; Lupan, D M; Schlageter, A M; Kozel, T R

    1990-01-01

    The in vivo properties of an immunoglobulin isotype-switch family of monoclonal antibodies specific for the polysaccharide capsule of Cryptococcus neoformans were examined in a murine model of cryptococcosis. Subclass-switch variants were isolated by sequential sublining of an immunoglobulin G subclass 1 (IgG1)-secreting cell line. Antibodies of the IgG1, IgG2a, and IgG2b isotypes with identical reactivities with cryptococcal polysaccharide were prepared. The antibodies had the distinct biological properties associated with the heavy chains of each respective isotype. The antibodies were used prophylactically or therapeutically in an attempt to alter the course of cryptococcal infection in mice. Survival of mice and a tissue census of the numbers of viable cryptococci in the lung, spleen, and brain were used as indicators of efficacy. Passive immunization with the IgG2a and IgG2b antibodies effected a reduction in the numbers of cryptococci in lung and spleen. Passive immunization with the IgG1 antibody was markedly less effective. Passive immunization had little or no effect on the numbers of cryptococci in brain tissue, regardless of the immunoglobulin isotype. Despite apparent efficacy with regard to reduction in the numbers of yeast cells in the lung and spleen, the results showed no improvement in survival from murine cryptococcosis. Our results indicate that passive immunization produces a modest effect on the course of murine cryptococcosis in tissues other than brain. However, under the experimental conditions used, such treatment does not have a measurable impact on the ultimate outcome of the infection. PMID:2341184

  9. Growth inhibition of tumor cells in vitro by using monoclonal antibodies against gonadotropin-releasing hormone receptor.

    Science.gov (United States)

    Lee, Gregory; Ge, Bixia

    2010-07-01

    As the continuation of a previous study, synthetic peptides corresponding to the extracellular domains of human gonadotropin-releasing hormone (GnRH) receptor were used to generate additional monoclonal antibodies which were further characterized biochemically and immunologically. Among those identified to recognize GnRH receptor, monoclonal antibodies designated as GHR-103, GHR-106 and GHR-114 were found to exhibit high affinity (Kd L37), when cancer cells were incubated with GnRH or GHR-106. The widespread expressions of GnRH receptor in almost all of the studied human cancer cell lines were also demonstrated by RT-PCR and Western blot assay, as well as indirect immunofluorescence assay with either of these monoclonal antibodies as the primary antibody. In view of the longer half life of antibodies as compared to that of GnRH or its analogs, anti-GnRH receptor monoclonal antibodies in humanized forms could function as GnRH analogs and serve as an ideal candidate of anti-cancer drugs for therapeutic treatments of various cancers in humans as well as for fertility regulations.

  10. Monoclonal antibodies for the detection and quantitation of the endogenous plant growth regulator, abscisic acid

    Energy Technology Data Exchange (ETDEWEB)

    Mertens, R.; Weiler, E.W. (Bochum Univ. (Germany, F.R.). Lehrstuhl fuer Pflanzenphysiologie); Deus-Neumann, B. (Muenchen Univ. (Germany, F.R.). Inst. fuer pharmazeutische Biologie)

    1983-08-22

    Monoclonal antibodies (mAB) have been produced which recognize the physiologically active 2-cis-(S)-form of the endogenous plant growth regulator, abscisic acid (ABA). Cross-reaction with the ABA-catabolites, phaseic and dihydrophaseic acid, is negligible, and (R)-ABA, 2-trans-ABA, the ABA-conjugate, ABA-..beta..-D-glucopyranosyl ester, as well as the putative ABA precursor, xanthoxin, are totally unreactive. In addition to being very specific, the mAB exhibit high affinities for 2-cis-(S)-ABA; the K values were 7.9 x 10/sup 9/ l/mol and 3.7 x 10/sup 9/ l/mol for antibodies from two different clones. By mAB-radioimmunoassay (RIA), 4 pg of 2-cis-(S)-ABA (99.5% confidence level) can be detected. mAB-RIA can be used to quantitate ABA directly in unprocessed plant extracts.

  11. Monoclonal antibodies for the detection and quantitation of the endogenous plant growth regulator, abscisic acid

    International Nuclear Information System (INIS)

    Mertens, R.; Weiler, E.W.; Deus-Neumann, B.

    1983-01-01

    Monoclonal antibodies (mAB) have been produced which recognize the physiologically active 2-cis-(S)-form of the endogenous plant growth regulator, abscisic acid (ABA). Cross-reaction with the ABA-catabolites, phaseic and dihydrophaseic acid, is negligible, and (R)-ABA, 2-trans-ABA, the ABA-conjugate, ABA-β-D-glucopyranosyl ester, as well as the putative ABA precursor, xanthoxin, are totally unreactive. In addition to being very specific, the mAB exhibit high affinities for 2-cis-(S)-ABA; the K values were 7.9 x 10 9 l/mol and 3.7 x 10 9 l/mol for antibodies from two different clones. By mAB-radioimmunoassay (RIA), 4 pg of 2-cis-(S)-ABA (99.5% confidence level) can be detected. mAB-RIA can be used to quantitate ABA directly in unprocessed plant extracts. (Auth.)

  12. Monoclonal antibodies that bind the renal Na+/glucose symport system. 1. Identification

    International Nuclear Information System (INIS)

    Wu, J.S.R.; Lever, J.E.

    1987-01-01

    Phlorizin is a specific, high-affinity ligand that binds the active site of the Na + /glucose symporter by a Na + -dependent mechanism but is not itself transported across the membrane. The authors have isolated a panel of monoclonal antibodies that influence high-affinity, Na + -dependent phlorizin binding to pig renal brush border membranes. Antibodies were derived after immunization of mice either with highly purified renal brush border membranes or with apical membranes purified from LLC-PK 1 , a cell line of pig renal proximal tubule origin. Antibody 11A3D6, an IgG/sub 2b/, reproducibly stimulated Na + -dependent phlorizin binding whereas antibody 18H10B12, an IgM, strongly inhibited specific binding. These effects were maximal after 30-min incubation and exhibited saturation at increased antibody concentrations. Antibodies did not affect Na + -dependent sugar uptake in vesicles but significantly prevented transport inhibition by bound phlorizin. Antibodies recognized a 75-kDa antigen identified by Western blot analysis of brush border membranes, and a 75-kDa membrane protein could be immunoprecipitated by 18H10B12. These properties, provide compelling evidence that the 75-kDa antigen recognized by these antibodies is a component of the renal Na + /glucose symporter

  13. Monoclonal antibody fragment removal mediated by mixed mode resins.

    Science.gov (United States)

    O'Connor, Ellen; Aspelund, Matthew; Bartnik, Frank; Berge, Mark; Coughlin, Kelly; Kambarami, Mutsa; Spencer, David; Yan, Huiming; Wang, William

    2017-05-26

    Efforts to increase monoclonal antibody expression in cell culture can result in the presence of fragmented species requiring removal in downstream processing. Capto adhere, HEA Hypercel, and PPA Hypercel anion exchange/hydrophobic interaction mixed mode resins were evaluated for their fragment removal capabilities and found to separate large hinge IgG1 antibody fragment (LHF) from monomer. Removal of greater than 75% of LHF population occurred at pH 8 and low conductivity. The mechanism of fragment removal was investigated in two series of experiments. The first experimental series consisted of comparison to chromatographic behavior on corresponding single mode resins. Both single mode anion exchange and hydrophobic interaction resins failed to separate LHF. The second experimental series studied the impact of phase modifiers, ethylene glycol, urea, and arginine on the mixed mode mediated removal. The addition of ethylene glycol decreased LHF removal by half. Further decreases in LHF separation were seen upon incubation with urea and arginine. Therefore, it was discovered that the purification is the result of a mixed mode phenomena dominated by hydrophobic interaction and hydrogen bonding effects. The site of interaction between the LHF and mixed mode resin was determined by chemical labeling of lysine residues with sulfo-NHS acetate. The labeling identified the antibody hinge and light chain regions as mediating the fragment separation. Sequence analysis showed that under separation conditions, a hydrophobic proline patch and hydrogen bonding serine and threonine residues mediate the hinge interaction with the Capto adhere ligand. Additionally, a case study is presented detailing the optimization of fragment removal using Capto adhere resin to achieve purity and yield targets in a manufacturing facility. This study demonstrated that mixed mode resins can be readily integrated into commercial antibody platform processes when additional chromatographic abilities

  14. Production and characterization of a monoclonal antibody against enrofloxacin.

    Science.gov (United States)

    Chusri, Manaspong; Wongphanit, Pitikarn; Palaga, Tanapat; Puthong, Songchan; Sooksai, Sarintip; Komolpis, Kittinan

    2013-01-01

    Enrofloxacin is a fluoroquinolone antibiotic approved for the treatment of infections in animals. Because of the side effects to consumers of animal products, the maximum residue limits (MRLs) of enrofloxacin in animal tissues for consumption are regulated. In this study, a monoclonal antibody (mAb) against enrofloxacin was prepared and characterized for the development of a direct competitive enzyme-linked immunosorbent assay (ELISA). The obtained mAb, Enro44, was highly specific for enrofloxacin and had a 50% inhibition concentration (IC(50)) of 1.99 ng/ml in a competitive ELISA, and the limit of detection (LOD) was 0.50 ng/ml. The cross-reactivity of the mAb with other quinolones and fluoroquinolones was lower than 0.01%. The subclass of the mAb Enro44 was identified as IgG1. The antigen (Ag)-captured direct competitive ELISA using the mAb Enro44 was tested on different spiked samples, including chicken muscle, cattle milk, and cattle urine, and the assay demonstrated recoveries of 82-112%, 80-125%, and 78-124%, respectively. Furthermore, the quantitation of enrofloxacin obtained from the ELISA and from high-performance liquid chromatography (HPLC) was in good agreement, with the linear regression coefficient between 0.933 and 1.056. The cDNAs encoding a heavy-chain Fd fragment (VH and CH1) and a light chain of the mAb Enro44 were cloned and sequenced. Taken together, the results obtained reveal a potential use of this mAb in an ELISA for the detection of enrofloxacin in food samples. The information of amino acid sequence of this mAb will be useful for further modification and production of the mAb in a bioreactor.

  15. The ketamine analogue methoxetamine and 3- and 4-methoxy analogues of phencyclidine are high affinity and selective ligands for the glutamate NMDA receptor.

    Directory of Open Access Journals (Sweden)

    Bryan L Roth

    Full Text Available In this paper we determined the pharmacological profiles of novel ketamine and phencyclidine analogues currently used as 'designer drugs' and compared them to the parent substances via the resources of the National Institute of Mental Health Psychoactive Drug Screening Program. The ketamine analogues methoxetamine ((RS-2-(ethylamino-2-(3-methoxyphenylcyclohexanone and 3-MeO-PCE (N-ethyl-1-(3-methoxyphenylcyclohexanamine and the 3- and 4-methoxy analogues of phencyclidine, (1-[1-(3-methoxyphenylcyclohexyl]piperidine and 1-[1-(4-methoxyphenylcyclohexyl]piperidine, were all high affinity ligands for the PCP-site on the glutamate NMDA receptor. In addition methoxetamine and PCP and its analogues displayed appreciable affinities for the serotonin transporter, whilst the PCP analogues exhibited high affinities for sigma receptors. Antagonism of the NMDA receptor is thought to be the key pharmacological feature underlying the actions of dissociative anaesthetics. The novel ketamine and PCP analogues had significant affinities for the NMDA receptor in radioligand binding assays, which may explain their psychotomimetic effects in human users. Additional actions on other targets could be important for delineating side-effects.

  16. A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders

    Science.gov (United States)

    Cui, Naiwen; Zhang, Huidan; Schneider, Nils; Tao, Ye; Asahara, Haruichi; Sun, Zhiyi; Cai, Yamei; Koehler, Stephan A.; de Greef, Tom F. A.; Abbaspourrad, Alireza; Weitz, David A.; Chong, Shaorong

    2016-01-01

    Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-and-read drop-IVT2H method to screen a random DNA library. Drop-IVT2H was based on the correlation between the binding affinity of two interacting protein domains and transcriptional activation of a fluorescent reporter. A DNA library encoding potential peptide binders was encapsulated with IVT2H such that single DNA molecules were distributed in individual drops. We validated drop-IVT2H by screening a three-random-residue library derived from a high-affinity MDM2 inhibitor PMI. The current drop-IVT2H platform is ideally suited for affinity screening of small-to-medium-sized libraries (103–106). It can obtain hits within a single day while consuming minimal amounts of reagents. Drop-IVT2H simplifies and accelerates the drop-based microfluidics workflow for screening random DNA libraries, and represents a novel alternative method for protein engineering and in vitro directed protein evolution. PMID:26940078

  17. Specificity of Bacillus thuringiensis endotoxins is correlated with the presence of high-affinity binding sites in the brush border membrane of target insect midguts

    International Nuclear Information System (INIS)

    Hofmann, C.; Vanderbruggen, H.; Hoefte, H.; Van Rie, J.; Jansens, S.; Van Mellaert, H.

    1988-01-01

    Binding studies were performed with two 125 I-labeled Bacillus thuringiensis δ-endotoxins on brush border membrane vesicles prepared from the larval midgut of the tobacco hornworm Manduca sexta or the cabbage butterfly Pieris brassicae. One δ-endotoxin, Bt2-protoxin, is a 130-kDa recombinant crystalline protein from B. thuringiensis subsp. berliner. It kills larvae of both insect species. The active Bt2-toxin is a 60-kDa proteolytic fragment of the Bt2-protoxin. It binds saturably and with high affinity to brush border membrane vesicles from the midgut of both species. The other δ-endotoxin, Bt4412-protoxin, is a 136-kDa crystalline protein from B. thuringiensis subsp. thuringiensis, which is highly toxic for P. brassicae, but not for M. sexta larvae. Bt4412-toxin, obtained after proteolytic activation of Bt4412-protoxin, shows high-affinity saturable binding to P. brassicae vesicles but not to M. sexta vesicles. The correlation between toxicity and specific binding is further strengthened by competition studies. Other B. thuringiensis δ-endotoxins active against M. sexta compete for binding of 125 I-labeled Bt2-toxin to M. sexta vesicles, whereas toxins active against dipteran or coleopteran larvae do not compete. Bt2-toxin and Bt4412-toxin bind to different sites on P. brassicae vesicles

  18. Constitutive expression of a putative high-affinity nitrate transporter in Nicotiana plumbaginifolia: evidence for post-transcriptional regulation by a reduced nitrogen source.

    Science.gov (United States)

    Fraisier, V; Gojon, A; Tillard, P; Daniel-Vedele, F

    2000-08-01

    The NpNRT2.1 gene encodes a putative inducible component of the high-affinity nitrate (NO3-) uptake system in Nicotiana plumbaginifolia. Here we report functional and physiological analyses of transgenic plants expressing the NpNRT2.1 coding sequence fused to the CaMV 35S or rolD promoters. Irrespective of the level of NO3- supplied, NO3- contents were found to be remarkably similar in wild-type and transgenic plants. Under specific conditions (growth on 10 mM NO3-), the steady-state NpNRT2. 1 mRNA level resulting from the deregulated transgene expression was accompanied by an increase in 15NO3- influx measured in the low concentration range. This demonstrates for the first time that the NRT2.1 sequence codes a limiting element of the inducible high-affinity transport system. Both 15NO3- influx and mRNA levels decreased in the wild type after exposure to ammonium, in agreement with previous results from many species. Surprisingly, however, influx was also markedly decreased in transgenic plants, despite stable levels of transgene expression in independent transformants after ammonium addition. We conclude that the conditions associated with the supply of a reduced nitrogen source such as ammonium, or with the generation of a further downstream metabolite, probably exert a repressive effect on NO3- influx at both transcriptional and post-transcriptional levels.

  19. Septide and neurokinin A are high-affinity ligands on the NK-1 receptor: evidence from homologous versus heterologous binding analysis.

    Science.gov (United States)

    Hastrup, H; Schwartz, T W

    1996-12-16

    The three main tachykinins, substance P, neurokinin A (NKA), and neurokinin B, are believed to be selective ligands for respectively the NK-1, NK-2 and NK-3 receptors. However, NKA also has actions which cannot be mediated through its normal NK-2 receptor and the synthetic peptide [pGlu6,Pro9]-Substance P9-11--called septide--is known to have tachykinin-like actions despite its apparent lack of binding to any known tachykinin receptor. In the cloned NK-1 receptor expressed in COS-7 cells NKA and septide as expected were poor competitors for radiolabeled substance P. However, by using radiolabeled NKA and septide directly, it was found that both peptides in homologous binding assays as well as in competition against each other in fact bound to the NK-1 receptor with high affinity: Kd values of 0.51 +/- 0.15 nM (NKA) and 0.55 +/- 0.03 nM (septide). It is concluded that NKA and septide are high-affinity ligands for the NK-1 receptor but that they are poor competitors for substance P, which in contrast competes very well for binding with both NKA and septide.

  20. Customizing monoclonal antibodies for the treatment of methamphetamine abuse: current and future applications.

    Science.gov (United States)

    Peterson, Eric C; Gentry, W Brooks; Owens, S Michael

    2014-01-01

    Monoclonal antibody-based medications designed to bind (+)-methamphetamine (METH) with high affinity are among the newest approaches to the treatment of METH abuse and the associated medical complications. The potential clinical indications for these medications include treatment of overdose, reduction of drug dependence, and protection of vulnerable populations from METH-related complications. Research designed to discover and conduct preclinical and clinical testing of these antibodies suggests a scientific vision for how intact monoclonal antibody (mAb) (singular and plural) or small antigen-binding fragments of mAb could be engineered to optimize the proteins for specific therapeutic applications. In this review, we discuss keys to success in this development process including choosing predictors of specificity, efficacy, duration of action, and safety of the medications in disease models of acute and chronic drug abuse. We consider important aspects of METH-like hapten design and how hapten structural features influence specificity and affinity, with an example of a high-resolution X-ray crystal structure of a high-affinity antibody to demonstrate this structural relationship. Additionally, several prototype anti-METH mAb forms such as antigen-binding fragments and single-chain variable fragments are under development. Unique, customizable aspects of these fragments are presented with specific possible clinical indications. Finally, we discuss clinical trial progress of the first in kind anti-METH mAb, for which METH is the disease target instead of vulnerable central nervous system networks of receptors, binding sites, and neuronal connections. © 2014 Elsevier Inc. All rights reserved.

  1. Investigating the Interaction between the Neonatal Fc Receptor and Monoclonal Antibody Variants by Hydrogen/Deuterium Exchange Mass Spectrometry

    DEFF Research Database (Denmark)

    Jensen, Pernille Foged; Larraillet, Vincent; Schlothauer, Tilman

    2015-01-01

    The recycling of immunoglobulins by the neonatal Fc receptor (FcRn) is of crucial importance in the maintenance of antibody levels in plasma and is responsible for the long half-lives of endogenous and recombinant monoclonal antibodies. From a therapeutic point of view there is great interest...... in understanding and modulating the IgG-FcRn interaction to optimize antibody pharmacokinetics and ultimately improve efficacy and safety. Here we studied the interaction between a full-length human IgG1 and human FcRn via hydrogen/deuterium exchange mass spectrometry and targeted electron transfer dissociation...... to map sites perturbed by binding on both partners of the IgG-FcRn complex. Several regions in the antibody Fc region and the FcRn were protected from exchange upon complex formation, in good agreement with previous crystallographic studies of FcRn in complex with the Fc fragment. Interestingly, we found...

  2. Monoclonal antibodies for use in an immunoradiometric assay for α-foetoprotein

    International Nuclear Information System (INIS)

    Hunter, W.M.; Bennie, J.G.

    1982-01-01

    The advantages offered by a mouse IgG 1 monoclonal antibody to human α-foetoprotein (AFP) for the preparation of [ 125 I]antibody for use in an immunoradiometric assay (IRMA) have been investigated. The antibody was isolated from ascites fluid by sodium sulphate precipitation followed by gel filtration on Sephadex G-200. The freeze-dried powder and solutions thereof were stable and were used for iodination to 1 atom 125 I/molecule antibody by the chloramine-T procedure. At high antigen concentrations 70-80% of the added [ 125 ]Ab was present in the sandwich. Linear response curves in the range 1-100 μg antigen/l incubate were obtained when [ 125 I]Ab was in slight excess. In this region an Ag : Ab ratio 1.9 : 1 was obtained which is consistent with the saturation of a bifunctional antibody. Although non-specific binding (in the absence of antigen) was consistently 125 I]Ab, this was the main factor in determining assay detection limits. The serum AFP levels from both non-pregnant and pregnant subjects as measured by the IRMA using the [ 125 I]monoclonal Ab and by radioimmunoassay (RIA) using a sheep antiserum to AFP were in excellent agreement. The IRMA was manipulatively simple, employed a shorter incubation time (2h), required shorter counting times than the RIA and gave a much wider working range. The provision of a monoclonal antibody for labelling removes the one major practicability barrier which otherwise limits the development and use of the potentially superior IRMA system. (Auth.)

  3. Immunostimulatory CpG-oligonucleotides induce functional high affinity IL-2 receptors on B-CLL cells: costimulation with IL-2 results in a highly immunogenic phenotype.

    Science.gov (United States)

    Decker, T; Schneller, F; Kronschnabl, M; Dechow, T; Lipford, G B; Wagner, H; Peschel, C

    2000-05-01

    CpG-oligodeoxynucleotides (CpG-ODN) have been shown to induce proliferation, cytokine production, and surface molecule regulation in normal and malignant human B cells. In the present study, we investigated the potential of CpG-ODN to induce functional high-affinity receptors in leukemic and normal B cells and the effects of costimulation with IL-2 on proliferation, cytokine secretion, and surface molecule regulation. Highly purified B cells from B-CLL patients and normal controls were stimulated with CpG-ODN with or without IL-2. Expression of CD25 was determined using FACS, and the presence of high-affinity IL-2 receptors was determined by scatchard analysis. Costimulatory effects of IL-2 and CpG-ODN were investigated using proliferation assays, ELISA (IL-6, TNF-alpha), and FACS analysis (CD80, CD86 expression). Reactivity of autologous and allogeneic T cells toward activated B-CLL cells was determined in mixed lymphocyte reactions and Interferon-gamma Elispot assays. The CpG-ODN DSP30 caused a significantly stronger induction of the IL-2 receptor alpha chain in malignant as compared with normal B cells (p = 0.03). This resulted in the expression of functional high-affinity IL-2 receptors in B-CLL cells, but fewer numbers of receptors with less affinity were expressed in normal B cells. Although addition of IL-2 to CpG-ODN-stimulated cells augmented proliferation in both normal B cells and B-CLL cells, no costimulatory effect on cytokine production or surface molecule expression could be observed in normal B cells. In contrast, TNF-alpha and IL-6 production was increased in B-CLL cells, and the expression of CD80 and CD86 was further enhanced when IL-2 was used as a costimulus. Autologous and allogeneic immune recognition of B-CLL cells stimulated with CpG-ODN and IL-2 was increased compared with B-CLL cells stimulated with CpG-ODN alone. Stimulation of B-CLL cells with CpG-ODN and IL-2 might be an attractive strategy for potential immunotherapies for B

  4. Human metabolites of synthetic cannabinoids JWH-018 and JWH-073 bind with high affinity and act as potent agonists at cannabinoid type-2 receptors

    International Nuclear Information System (INIS)

    Rajasekaran, Maheswari; Brents, Lisa K.; Franks, Lirit N.; Moran, Jeffery H.; Prather, Paul L.

    2013-01-01

    K2 or Spice is an emerging drug of abuse that contains synthetic cannabinoids, including JWH-018 and JWH-073. Recent reports indicate that monohydroxylated metabolites of JWH-018 and JWH-073 retain high affinity and activity at cannabinoid type-1 receptors (CB 1 Rs), potentially contributing to the enhanced toxicity of K2 compared to marijuana. Since the parent compounds also bind to cannabinoid type-2 receptors (CB 2 Rs), this study investigated the affinity and intrinsic activity of JWH-018, JWH-073 and several monohydroxylated metabolites at human CB 2 Rs (hCB 2 Rs). The affinity of cannabinoids for hCB 2 Rs was determined by competition binding studies employing CHO-hCB 2 membranes. Intrinsic activity of compounds was assessed by G-protein activation and adenylyl cyclase (AC)-inhibition in CHO-hCB 2 cells. JWH-073, JWH-018 and several of their human metabolites exhibit nanomolar affinity and act as potent agonists at hCB 2 Rs. Furthermore, a major omega hydroxyl metabolite of JWH-073 (JWH-073-M5) binds to CB 2 Rs with 10-fold less affinity than the parent molecule, but unexpectedly, is equipotent in regulating AC-activity when compared to the parent molecule. Finally, when compared to CP-55,940 and Δ 9 -tetrahydrocannabinol (Δ 9 -THC), JWH-018, JWH-018-M5 and JWH-073-M5 require significantly less CB 2 R occupancy to produce similar levels of AC-inhibition, indicating that these compounds may more efficiently couple CB 2 Rs to AC than the well characterized cannabinoid agonists examined. These results indicate that JWH-018, JWH-073 and several major human metabolites of these compounds exhibit high affinity and demonstrate distinctive signaling properties at CB 2 Rs. Therefore, future studies examining pharmacological and toxicological properties of synthetic cannabinoids present in K2 products should consider potential actions of these drugs at both CB 1 and CB 2 Rs. - Highlights: • JWH-018 and JWH-073 are synthetic cannabinoids present in abused K2

  5. Human metabolites of synthetic cannabinoids JWH-018 and JWH-073 bind with high affinity and act as potent agonists at cannabinoid type-2 receptors

    Energy Technology Data Exchange (ETDEWEB)

    Rajasekaran, Maheswari; Brents, Lisa K.; Franks, Lirit N. [Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Moran, Jeffery H. [Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Arkansas Department of Public Health, Public Health Laboratory, Little Rock, AR 72205 (United States); Prather, Paul L., E-mail: pratherpaull@uams.edu [Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States)

    2013-06-01

    K2 or Spice is an emerging drug of abuse that contains synthetic cannabinoids, including JWH-018 and JWH-073. Recent reports indicate that monohydroxylated metabolites of JWH-018 and JWH-073 retain high affinity and activity at cannabinoid type-1 receptors (CB{sub 1}Rs), potentially contributing to the enhanced toxicity of K2 compared to marijuana. Since the parent compounds also bind to cannabinoid type-2 receptors (CB{sub 2}Rs), this study investigated the affinity and intrinsic activity of JWH-018, JWH-073 and several monohydroxylated metabolites at human CB{sub 2}Rs (hCB{sub 2}Rs). The affinity of cannabinoids for hCB{sub 2}Rs was determined by competition binding studies employing CHO-hCB{sub 2} membranes. Intrinsic activity of compounds was assessed by G-protein activation and adenylyl cyclase (AC)-inhibition in CHO-hCB{sub 2} cells. JWH-073, JWH-018 and several of their human metabolites exhibit nanomolar affinity and act as potent agonists at hCB{sub 2}Rs. Furthermore, a major omega hydroxyl metabolite of JWH-073 (JWH-073-M5) binds to CB{sub 2}Rs with 10-fold less affinity than the parent molecule, but unexpectedly, is equipotent in regulating AC-activity when compared to the parent molecule. Finally, when compared to CP-55,940 and Δ{sup 9}-tetrahydrocannabinol (Δ{sup 9}-THC), JWH-018, JWH-018-M5 and JWH-073-M5 require significantly less CB{sub 2}R occupancy to produce similar levels of AC-inhibition, indicating that these compounds may more efficiently couple CB{sub 2}Rs to AC than the well characterized cannabinoid agonists examined. These results indicate that JWH-018, JWH-073 and several major human metabolites of these compounds exhibit high affinity and demonstrate distinctive signaling properties at CB{sub 2}Rs. Therefore, future studies examining pharmacological and toxicological properties of synthetic cannabinoids present in K2 products should consider potential actions of these drugs at both CB{sub 1} and CB{sub 2}Rs. - Highlights: • JWH-018

  6. Comparison of high affinity binding of {sup 3}H-proadifen and {sup 3}H-(-)-cocaine t rat liver membranes

    Energy Technology Data Exchange (ETDEWEB)

    Ross, S.B. [Astra Arcus AB, Dept. of Neuropharmacology, Soedertaelje (Sweden)

    1995-06-01

    The characteristics of the binding of {sup 3}H-proadifen to rat liver membranes were studied and compared to those of {sup 3}H-cocaine. It was found that {sup 3}H-proadifen was bound reversibly with high affinity (K{sub D}=1.8{+-}0.5 nM) and large capacity (B{sub max}=2010{+-}340 pmol/g wet tissue) to liver membranes. The corresponding values for the {sup 3}H-cocaine binding were 3.5 nM and 1000 pmol/g wet tissue. The binding of {sup 3}H-proadifen was mainly localised to the microsomal fraction. The number of binding sites was not increased by treatment of rats with phenobarbitone. With 1 {mu}M CdCl{sub 2} in the incubation buffer it was possible to differentiate between two {sup 3}H-cocaine binding sites with K{sub d} values of 1.6 and 7.7 nM and B{sub max} values of 280 and 940 pmol/g wet liver tissue. S-(-)-Alaproclate inhibited the binding of {sup 3}H-proadifen and {sup 3}H-cocaine inhibited the binding of {sup 3}H-proadifen (IC{sub 50}=10 nM) and proadifen that of {sup 3}H-cocaine (IC{sub 50}=1 nM). There was a high correlation coefficient (r{sub r}=0.972; P<0.01; n=12) in the Spearman rank test between the inhibitory potencies of compounds examined in both systems. Beside some potent alaproclate analogues a couple of compounds had moderately high affinity (IC{sub 50}=100-500 nM): chloroquine, phenoxybenzamine, amitriptyline, ajmaline, remoxipride, imipramine and (-)-alaprenolol. CdCl{sub 2}, ZnCl{sub 2} and CuCl{sub 2} inhibited the binding of both ligands with low Hill coefficients, indicating heterogeneous binding sites. The inhibition curve of Cd{sup 2+} on the cocaine binding was biphasic with a high affinity part around 50 nM and a low affinity part at 15{mu}M. The similarity of the characteristics of the binding of these ligands with that of {sup 3}H-alaproclate is discussed. It is suggested that all three compounds bind to the same sites, although additional binding sites seem to exist for proadifen. (au) (9 refs.).

  7. Mapping of barley alpha-amylases and outer subsite mutants reveals dynamic high-affinity subsites and barriers in the long substrate binding cleft

    DEFF Research Database (Denmark)

    Kandra, L.; Abou Hachem, Maher; Gyemant, G.

    2006-01-01

    Subsite affinity maps of long substrate binding clefts in barley alpha-amylases, obtained using a series of maltooligosaccharides of degree of polymerization of 3-12, revealed unfavorable binding energies at the internal subsites -3 and -5 and at subsites -8 and +3/+4 defining these subsites...... as binding barriers. Barley a-amylase I mutants Y105A and T212Y at subsite -6 and +4 resulted in release or anchoring of bound substrate, thus modifying the affinities of other high-affinity subsites (-2 and +2) and barriers. The double mutant Y105A-T212Y displayed a hybrid subsite affinity profile......, converting barriers to binding areas. These findings highlight the dynamic binding energy distribution and the versatility of long maltooligosaccharide derivatives in mapping extended binding clefts in a-amylases....

  8. Two classes of astrocytes in the adult human and pig retina in terms of their expression of high affinity NGF receptor (TrkA).

    Science.gov (United States)

    Ruiz-Ederra, Javier; Hitchcock, Peter F; Vecino, Elena

    2003-02-13

    Astrocytes have been implicated in axon guidance and synaptic regeneration in the retina and these processes involve activation of the high affinity nerve growth factor receptor, known as the tyrosine kinase A (TrkA) receptor. The purpose of the present study was to characterize the expression of TrkA in astrocytes of the adult pig and human retina. To this end, sections of human and pig retinas were immunolabeled with a combination of antibodies to glial fibrillary acidic protein (GFAP) and TrkA. Our study revealed that most of the GFAP-positive cells express TrkA, whereas a rare, novel subpopulation of astrocytes was found to be devoid of TrkA. Our results support the idea that astrocytes play an important neurotrophic role in the retina.

  9. In Vivo Neutralization of α-Cobratoxin with High-Affinity Llama Single-Domain Antibodies (VHHs) and a VHH-Fc Antibody

    Science.gov (United States)

    Richard, Gabrielle; Meyers, Ashley J.; McLean, Michael D.; Arbabi-Ghahroudi, Mehdi; MacKenzie, Roger; Hall, J. Christopher

    2013-01-01

    Small recombinant antibody fragments (e.g. scFvs and VHHs), which are highly tissue permeable, are being investigated for antivenom production as conventional antivenoms consisting of IgG or F(ab’)2 antibody fragments do not effectively neutralize venom toxins located in deep tissues. However, antivenoms composed entirely of small antibody fragments may have poor therapeutic efficacy due to their short serum half-lives. To increase serum persistence and maintain tissue penetration, we prepared low and high molecular mass antivenom antibodies. Four llama VHHs were isolated from an immune VHH-displayed phage library and were shown to have high affinity, in the low nM range, for α-cobratoxin (α–Cbtx), the most lethal component of Naja kaouthia venom. Subsequently, our highest affinity VHH (C2) was fused to a human Fc fragment to create a VHH2-Fc antibody that would offer prolonged serum persistence. After in planta (Nicotiana benthamiana) expression and purification, we show that our VHH2-Fc antibody retained high affinity binding to α–Cbtx. Mouse α–Cbtx challenge studies showed that our highest affinity VHHs (C2 and C20) and the VHH2-Fc antibody effectively neutralized lethality induced by α–Cbtx at an antibody:toxin molar ratio as low as ca. 0.75×:1. Further research towards the development of an antivenom therapeutic involving these anti-α-Cbtx VHHs and VHH2-Fc antibody molecules should involve testing them as a combination, to determine whether they maintain tissue penetration capability and low immunogenicity, and whether they exhibit improved serum persistence and therapeutic efficacy. PMID:23894495

  10. Blockade of the high-affinity noradrenaline transporter (NET) by the selective 5-HT reuptake inhibitor escitalopram: an in vivo microdialysis study in mice

    Science.gov (United States)

    Nguyen, Hai T; Guiard, Bruno P; Bacq, Alexandre; David, Denis J; David, Indira; Quesseveur, Gaël; Gautron, Sophie; Sanchez, Connie; Gardier, Alain M

    2013-01-01

    BACKGROUND AND PURPOSE Escitalopram, the S(+)-enantiomer of citalopram is the most selective 5-HT reuptake inhibitor approved. Although all 5-HT selective reuptake inhibitors (SSRIs) increase extracellular levels of 5-HT ([5-HT]ext). some also enhance, to a lesser extent, extracellular levels of noradrenaline ([NA]ext). However, the mechanisms by which SSRIs activate noradrenergic transmission in the brain remain to be determined. EXPERIMENTAL APPROACH This study examined the effects of escitalopram, on both [5-HT]ext and [NA]ext in the frontal cortex (FCx) of freely moving wild-type (WT) and mutant mice lacking the 5-HT transporter (SERT−/−) by using intracerebral microdialysis. We explored the possibilities that escitalopram enhances [NA]ext, either by a direct mechanism involving the inhibition of the low- or high-affinity noradrenaline transporters, or by an indirect mechanism promoted by [5-HT]ext elevation. The forced swim test (FST) was used to investigate whether enhancing cortical [5-HT]ext and/or [NA]ext affected the antidepressant-like activity of escitalopram. KEY RESULTS In WT mice, a single systemic administration of escitalopram produced a significant increase in cortical [5-HT]ext and [NA]ext. As expected, escitalopram failed to increase cortical [5-HT]ext in SERT−/− mice, whereas its neurochemical effects on [NA]ext persisted in these mutants. In WT mice subjected to the FST, escitalopram increased swimming parameters without affecting climbing behaviour. Finally, escitalopram, at relevant concentrations, failed to inhibit cortical noradrenaline and 5-HT uptake mediated by low-affinity monoamine transporters. CONCLUSIONS AND IMPLICATIONS These experiments suggest that escitalopram enhances, although moderately, cortical [NA]extin vivo by a direct mechanism involving the inhibition of the high-affinity noradrenaline transporter (NET). PMID:22233336

  11. Distribution and ultrastructure of neurons in opossum piriform cortex displaying immunoreactivity to GABA and GAD and high-affinity tritiated GABA uptake

    International Nuclear Information System (INIS)

    Haberly, L.B.; Hansen, D.J.; Feig, S.L.; Presto, S.

    1987-01-01

    GABAergic neurons have been identified in the piriform cortex of the opossum at light and electron microscopic levels by immunocytochemical localization of GABA and the GABA-synthesizing enzyme glutamic acid decarboxylase and by autoradiographic visualization of high-affinity 3 H-GABA uptake. Four major neuron populations have been distinguished on the basis of soma size, shape, and segregation at specific depths and locations: large horizontal cells in layer Ia of the anterior piriform cortex, small globular cells with thin dendrites concentrated in layers Ib and II of the posterior piriform cortex, and multipolar and fusiform cells concentrated in the deep part of layer III in anterior and posterior parts of the piriform cortex and the subjacent endopiriform nucleus. All four populations were well visualized with both antisera, but the large layer Ia horizontal cells displayed only very light 3 H-GABA uptake, thus suggesting a lack of local axon collaterals or lack of high-affinity GABA uptake sites. The large, ultrastructurally distinctive somata of layer Ia horizontal cells receive a very small number of symmetrical synapses; the thin, axonlike dendrites of small globular cells are exclusively postsynaptic and receive large numbers of both symmetrical and asymmetrical synapses, in contrast to somata which receive a small number of both types; and the deep multipolar and fusiform cells receive a highly variable number of symmetrical and asymmetrical synapses on somata and proximal dendrites. Labeled puncta of axon terminal dimensions were found in large numbers in the neuropil surrounding pyramidal cell somata in layer II and in the endopiriform nucleus. Moderately large numbers of labeled puncta were found in layer I at the depth of pyramidal cell apical dendrites with greater numbers in layer Ia at the depth of distal apical segments than in layer Ib

  12. Production of Monoclonal Antibodies specific for Progesterone

    OpenAIRE

    YÜCEL, Fatıma

    2014-01-01

    Progesterone levels in milk and serum are indicators of pregnancy in cattle. The progesterone level reaches a peak on the 21 st and 22 nd days of pregnancy. Monoclonal antibodies specific to progesterone could be used for the immunodetection of milk and serum progesterone levels. We report here the development of hybrid cells prdoducing monoclonal antibodies specific for progesterone using hybridoma technology. Hybridoma cells secreting monoclonal antibodies against progesterone (MAM 2H1...

  13. Monoclonal antibodies for treating cancer

    International Nuclear Information System (INIS)

    Dillman, R.O.

    1989-01-01

    The purpose of this study is to assess the current status of in-vivo use of monoclonal antibodies for treating cancer. Publications appearing between 1980 and 1988 were identified by computer searches using MEDLINE and CANCERLIT, by reviewing the table of contents of recently published journals, and by searching bibliographies of identified books and articles. More than 700 articles, including peer-reviewed articles and book chapters, were identified and selected for analysis. The literature was reviewed and 235 articles were selected as relevant and representative of the current issues and future applications for in-vivo monoclonal antibodies for cancer therapy and of the toxicity and efficacy which has been associated with clinical trials. Approaches include using antibody alone (interacting with complement or effector cells or binding directly with certain cell receptors) and immunoconjugates (antibody coupled to radioisotopes, drugs, toxins, or other biologicals). Most experience has been with murine antibodies. Trials of antibody alone and radiolabeled antibodies have confirmed the feasibility of this approach and the in-vivo trafficking of antibodies to tumor cells. However, tumor cell heterogeneity, lack of cytotoxicity, and the development of human antimouse antibodies have limited clinical efficacy. Although the immunoconjugates are very promising, heterogeneity and the antimouse immune response have hampered this approach as has the additional challenge of chemically or genetically coupling antibody to cytotoxic agents. As a therapeutic modality, monoclonal antibodies are still promising but their general use will be delayed for several years. New approaches using human antibodies and reducing the human antiglobulin response should facilitate treatment. 235 references

  14. Llama VHH antibody fragments against GFAP: better diffusion in fixed tissues than classical monoclonal antibodies.

    Science.gov (United States)

    Perruchini, Claire; Pecorari, Frederic; Bourgeois, Jean-Pierre; Duyckaerts, Charles; Rougeon, François; Lafaye, Pierre

    2009-11-01

    Camelids produce antibodies made of homodimeric heavy chains, and the antigen-binding region being composed of a single domain called VHH. These VHHs are much smaller than complete IgG. They are also more thermostable and more soluble in water; they should, therefore, diffuse more readily in the tissues. VHHs, expressed in bacteria, are easier to produce than conventional monoclonal antibodies. Because of these special characteristics, these antibody fragments could have interesting developments in immunohistochemistry and in the development of biomarkers. To test the possibility of their use in immunohistochemistry (IHC), we selected the glial fibrillary acidic protein (GFAP), a well-known marker of astrocytes. One alpaca (Lama pacos) was immunized against GFAP. Lymphocytes were isolated; the DNA was extracted; the VHH-coding sequences were selectively amplified. Three VHHs with a high affinity for GFAP and their corresponding mRNA were selected by ribosome display. Large quantities of the recombinant VHHs coupled with different tags were harvested from transfected bacteria. One of them was shown to immunolabel strongly and specifically to GFAP of human astrocytes in tissue sections. The quality of the IHC was comparable or, in some aspects, superior to the quality obtained with conventional IgG. The VHH was shown to diffuse on a longer distance than conventional monoclonal antibodies in fixed cortical tissue: a property that may be useful in immunolabeling of thick sections.

  15. Production and characterization of monoclonal antibodies specific to the strobilurin pesticide pyraclostrobin.

    Science.gov (United States)

    Mercader, Josep V; Suárez-Pantaleón, Celia; Agulló, Consuelo; Abad-Somovilla, Antonio; Abad-Fuentes, Antonio

    2008-09-10

    Strobilurin fungicides are nowadays among the most important fungicides in the market of active agrochemicals. Pyraclostrobin, which belongs to the last generation of this family of molecules, shows a broader antifungal activity spectrum and higher efficiency and security profiles than previous fungicides. This paper describes the synthesis of functionalized haptens, the production of monoclonal antibodies, and the development of enzyme-linked immunosorbent assays (ELISA) for the detection of pyraclostrobin. A conformational analysis of hapten structure was performed, which provided relevant data concerning the length of the spacer arm. A very useful strategy has been followed for the screening of hybridomas, leading to the selection of a panel of high-affinity monoclonal antibodies to pyraclostrobin. Moreover, different immunoassays have been characterized using the conjugate-coated indirect ELISA format, and limits of detection below 0.1 microg/L have been obtained. Also, a simplified one-step procedure has been carried out with two indirect assays. Finally, these results have been compared with the performance of the same antibodies in the antibody-coated direct ELISA format.

  16. Monoclonal antibody-based immunoassays.

    Science.gov (United States)

    Appleby, P; Reischl, U

    1998-01-01

    An immunoassay may be defined as an assay that employs an immunological reagent, usually an antibody, to confer specificity for the ligand being measured. As a corollary to this, the discovery, and subsequent development, of monoclonal antibodies (MAbs) has greatly expanded the application and use of immunoassays. Polyclonal reagents, with their associated problems of specificity and quality control, have now been largely replaced by readily available MAbs of potential immortality and well-defined specificity and affinity. This has resulted, in the last two decades, in a great expansion in the range of immunoassays available and also a significant improvement in their reproducibility and reliability.

  17. Radiolabelled monoclonal antibodies against alpha-fetoprotein for in vivo localization of human hepatocellular carcinoma by immunotomoscintigraphy

    International Nuclear Information System (INIS)

    Bergmann, J.F.; Lumbroso, J.D.; Manil, L.; Saccavini, J.C.; Rougier, P.; Assicot, M.; Mathieu, A.; Bellet, D.; Bohuon, C.

    1987-01-01

    Two high affinity monoclonal antibodies, designated AF01 and AF04, directed against distinct epitopes of human alpha-fetoprotein (AFP) and the Fab fragments of one of them, were labelled with 131 I and injected into 18 patients with AFP producing hepatocellular carcinoma (HCC) in order to carry out imaging studies by tomoscintigraphy. Twelve patients were injected with whole antibody, only three of seven patients injected with AF01 and two of five patients injected with AF04 had a positive scan. In contrast, five out of six patients injected with labelled Fab fragments of AF04 had positive imaging. These results confirm that tumour imaging of HCC using 131 I labelled monoclonal antibody against AFP is feasible. Moreover, utilization of tomoscintigraphy in place of linear scintigraphy and Fab fragments instead of whole immunoglobulin may improve the sensitivity of radioimmunolocalization. This technique provides useful information on the in vivo distribution of monoclonal antibodies directed against AFP and on the practicability of the eventual therapeutic use of anti-AFP antibodies in HCC. (orig.)

  18. Monoclonal antibody hapten radiopharmaceutical delivery

    International Nuclear Information System (INIS)

    Goodwin, D.A.; McTigue, M.

    1986-01-01

    One hundred μg of monoclonal antibody (MoAb) CHA255 with a binding constant Kb of 4 x 10 9 was complexed with indium-111 labelled BLEDTA II, BLEDTA IV, benzyl EDTA, and an EDTA conjugate of Fab. The 24-h tumour and organ distribution of BALB/c mice bearing KHJJ tumours was studied for each compound alone, the antibody complex, and 3 h following a chelate chase of the antibody complex. Whole body biological half-life was measured for 7 days with and without a chelate chase for each antibody complex. The 24-h whole body counts dropped 20 to 60% and blood concentration fell over 89% within 3 h of administering the chelate chase. Theoretical equivalent human organ doses were calculated from the 24-h organ concentrations, effective half-life, and MIRD 11 S values (absorbed dose per cumulated activity). Liver and spleen were the target organs, with the dose ranging from 0.50 to 3.91 rads mCi -1 . The reduction in organ radiation dose varied up to 95% following the chelate chase. Rapid selective renal clearance of chelate labelled radiopharmaceuticals by competitive inhibition (chelate chase) of their reversible binding to monoclonal antibodies enhances tumour imaging and improves the radiation dosimetry. (author)

  19. Uses of monoclonal antibody 8H9

    Energy Technology Data Exchange (ETDEWEB)

    Cheung, Nai-Kong V.

    2018-04-10

    This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides a method of inhibiting the growth of tumor cells comprising contacting said tumor cells with an appropriate amount of monoclonal antibody 8H9 or a derivative thereof.

  20. Durvalumab: an investigational anti-PD-L1 monoclonal antibody for the treatment of urothelial carcinoma

    Directory of Open Access Journals (Sweden)

    Faiena I

    2018-01-01

    Full Text Available Izak Faiena,1,2 Amy L Cummings,3 Anna M Crosetti,3 Allan J Pantuck,1,2 Karim Chamie,1,2 Alexandra Drakaki1–3 1Department of Urology, 2Institute of Urologic Oncology, 3Department of Medicine, Division of Hematology and Oncology, David Geffen School of Medicine at University of California, Los Angeles, CA, USA Abstract: Our expanding knowledge of immunotherapy for solid tumors has led to an explosion of clinical trials aimed at urothelial carcinoma. The primary strategy is centered on unleashing the immune system by releasing the inhibitory signals propagated by programmed cell death-1 (PD-1 and its ligand programmed cell death ligand-1 (PD-L1. Many antibody constructs have been developed to block these interactions and are used in clinical trials. The Food and Drug Administration has already approved a number of checkpoint inhibitors such as anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4 monoclonal antibodies including ipilimumab; anti-PD-1 monoclonal antibodies including nivolumab and pembrolizumab; anti-PD-L1 antibodies including atezolizumab, avelumab, and durvalumab. One of the latest inhibitors is durvalumab, which is a high-affinity human immunoglobulin G1 kappa monoclonal antibody and blocks the interaction of PD-L1 with PD-1 and CD80. Currently, there are a number of ongoing trials in advanced urothelial carcinoma both using durvalumab monotherapy and in combination with other targeted therapies. In addition, durvalumab is being investigated in the non-muscle-invasive urothelial carcinoma, which is centered around intravenous formulations. These exciting developments have added a significant number of therapies in a previously limited treatment landscape. Keywords: durvalumab, checkpoint inhibitors, metastatic urothelial carcinoma

  1. An HIV-1 encoded peptide mimics the DNA binding loop of NF-κB and binds thioredoxin with high affinity

    International Nuclear Information System (INIS)

    Su Guoping; Wang Min; Taylor, Ethan Will

    2005-01-01

    Pro-fs is a human immunodeficiency virus type 1 (HIV-l)-encoded putative selenoprotein, predicted by a theoretical analysis of the viral genome; it is potentially expressed by a -1 frameshift from the protease coding region. Pro-fs has significant sequence similarity to the DNA binding loop of nuclear factor kappa B (NF-κB), which is known to bind thioredoxin (Trx). We hypothesize that the putative HIV-1 pro-fs gene product functions by mimicry of NF-κB via binding to Trx. The hypothesis was tested in vitro by co-immunoprecipitation and GST-pull down assays, using a purified mutant pro-fs protein, in which the two potential selenocysteine residues were mutated to cysteines, in order to permit expression in bacteria. Both experiments showed that pro-fs binds to human wild type Trx (Trx-wt) with high affinity. Mutation of the two conserved cysteine residues in the Trx active site redox center to serine (Ser) (Trx-CS) weakened but failed to abolish the interaction. In pro-fs-transfected 293T cells, using confocal microscopy and fluorescence resonance energy transfer (FRET), we have observed that pro-fs localizes in cell nuclei and forms oligomers. Upon stimulation by phorbol 12-myristate 13-acetate (PMA), Trx translocates into cell nuclei. Significant FRET efficiency was detected in the nuclei of PMA-stimulated 293T cells co-expressing fluorescence-tagged pro-fs and Trx-wt or Trx-CS. These results indicate that in living cells the double cysteine mutant of pro-fs binds to both Trx and Trx-CS with high affinity, suggesting that Trx-pro-fs binding is a structurally-specific interaction, involving more of the Trx molecule than just its active site cysteine residues. These results establish the capacity for functional mimicry of the Trx binding ability of the NF-κB/Rel family of transcription factors by the putative HIV-1 pro-fs protein

  2. Synthesis and characterization of [{sup 76}Br]-labeled high-affinity A{sub 3} adenosine receptor ligands for positron emission tomography

    Energy Technology Data Exchange (ETDEWEB)

    Kiesewetter, Dale O. [Positron Emission Tomography Radiochemistry Group, NIBIB, Clinical Center, National Institutes of Health, Bethesda, MD 20892 (United States)], E-mail: dk7k@nih.gov; Lang Lixin; Ma Ying; Bhattacharjee, Abesh Kumar [Positron Emission Tomography Radiochemistry Group, NIBIB, Clinical Center, National Institutes of Health, Bethesda, MD 20892 (United States); Gao, Zhan-Guo; Joshi, Bhalchandra V.; Melman, Artem; Castro, Sonia de; Jacobson, Kenneth A. [Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 (United States)

    2009-01-15

    Introduction: Bromine-76-radiolabeled analogues of previously reported high-affinity A{sub 3} adenosine receptor (A{sub 3}AR) nucleoside ligands have been prepared as potential radiotracers for positron emission tomography. Methods: The radiosyntheses were accomplished by oxidative radiobromination on the N{sup 6}-benzyl moiety of trimethyltin precursors. Biodistribution studies of the kinetics of uptake were conducted in awake rats. Results: We prepared an agonist ligand {l_brace}[{sup 76}Br](1'S,2'R,3'S,4'R,5'S)-4'-{l_brace}2-chloro-6-[(3-bromophenylmethyl)amino] purin-9-yl{r_brace}-1'-(methylaminocarbonyl)bicyclo[3.1.0]hexane-2',3'-diol (MRS3581){r_brace} in 59% radiochemical yield with a specific activity of 19.5 GBq/{mu}mol and an antagonist ligand {l_brace}[{sup 76}Br](1'R,2'R,3'S,4'R,5'S)-4'-(6-(3-bromobenzylamino) -2-chloro-9H-purin-9-yl)bicyclo[3.1.0]hexane-2',3'-diol (MRS5147){r_brace} in 65% radiochemical yield with a specific activity of 22 GBq/{mu}mol. The resultant products exhibited the expected high affinity (K{sub i}{approx}0.6 nM) and specific binding at the human A{sub 3}AR in vitro. Biodistribution studies in the rat showed uptake in the organs of excretion and metabolism. The antagonist MRS5147 exhibited increasing uptake in testes, an organ that contains significant quantities of A{sub 3}AR, over a 2-h time course, which suggests the presence of a specific A{sub 3}AR retention mechanism. Conclusion: We were able to compare uptake of the [{sup 76}Br]-labeled antagonist MRS5147 to [{sup 76}Br]agonist MRS3581. The antagonist MRS5147 shows increasing uptake in the testes, an A{sub 3}AR-rich tissue, suggesting that this ligand may have promise as a molecular imaging agent.

  3. Antilymphocytic antibodies and marrow transplantation. VIII. Recipient conditioning with Clq-affine monoclonal anti-pan T antibodies prevents GVHD in homozygous fully mismatched mice

    International Nuclear Information System (INIS)

    Thierfelder, S.; Kummer, U.; Schuh, R.; Mysliwietz, J.

    1986-01-01

    An approach to suppressing secondary disease with antibodies was studied that differed from conventional antibody treatment of donor marrow in vitro. It consisted of the selection of anti-Thy-1 antibodies with high affinity for Clq, the first subunit of the complement cascade, and a single injection of such antibodies into prospective irradiated marrow recipients. Monoclonal mouse IgM and rat IgG 2c antibodies of high titers in complement-dependent test systems but with low affinity for Clq caused little immunosuppression. Monoclonal rat IgG2b or mouse IgG2a anti-Thy-1 antibodies with high affinity for Clq prevented acute and chronic mortality of graft-v-host disease (GVHD), however, when injected in irradiated CBA or AKR mice prior to C57BL/6 spleen and/or bone marrow cell transfusion. This treatment simultaneously suppressed residual host-v-graft reactivity of the irradiated mice, so that permanent hematopoietic engraftment ensued even at 5 or 6 Gy. Full chimerism and specific tolerance were obtained. Primary immune response to SRBC was clearly depressed in the chimeras; secondary immune response was not. Clearance of T cell antibody activity (greater than 6 days), timing, and dose of injected antibody, as well as other modalities of the conditioning treatment that may have contributed to the remarkable immunosuppression, are discussed

  4. Neutralizing and non-neutralizing monoclonal antibodies against dengue virus E protein derived from a naturally infected patient

    Directory of Open Access Journals (Sweden)

    Isern Sharon

    2010-02-01

    Full Text Available Abstract Background Antibodies produced in response to infection with any of the four serotypes of dengue virus generally provide homotypic immunity. However, prior infection or circulating maternal antibodies can also mediate a non-protective antibody response that can enhance the course of disease in a subsequent heterotypic infection. Naturally occurring human monoclonal antibodies can help us understand the protective and pathogenic roles of the humoral immune system in dengue virus infection. Results Epstein-Barr Virus (EBV transformation of B cells isolated from the peripheral blood of a human subject with previous dengue infection was performed. B cell cultures were screened by ELISA for antibodies to dengue (DENV envelope (E protein. ELISA positive cultures were cloned by limiting dilution. Three IgG1 human monoclonal antibodies (HMAbs were purified and their binding specificity to E protein was verified by ELISA and biolayer interferometry. Neutralization and enhancement assays were conducted in epithelial and macrophage-like cell lines, respectively. All three HMAbs bound to E from at least two of the four DENV serotypes, one of the HMAbs was neutralizing, and all were able to enhance DENV infection. Conclusions HMAbs against DENV can be successfully generated by EBV transformation of B cells from patients at least two years after naturally acquired DENV infections. These antibodies show different patterns of cross-reactivity, neutralizing, and enhancement activity.

  5. Toll-like receptor 7 cooperates with IL-4 in activated B cells through antigen receptor or CD38 and induces class switch recombination and IgG1 production.

    Science.gov (United States)

    Tsukamoto, Yumiko; Nagai, Yoshinori; Kariyone, Ai; Shibata, Takuma; Kaisho, Tsuneyasu; Akira, Shizuo; Miyake, Kensuke; Takatsu, Kiyoshi

    2009-04-01

    IL-4 and 8-mercaptoguanosine (8-SGuo) stimulation of CD38-activated B cells induces mu to gamma1 class switch recombination (CSR) at the DNA level leading to a high level of IgG1 production. Although some of signaling events initiated by IL-4 in activated B cells have been characterized, the involvement of TLR/MyD88 and Btk pathway in IL-4-dependent mu to gamma1 CSR has not been thoroughly evaluated. In this study, we characterized receptors for 8-SGuo and differential roles of 8-SGuo and IL-4 in the induction and mu to gamma1 CSR and IgG1 production. The role of TLR7 and MyD88 in 8-SGuo-induced AID expression and mu to gamma1 CSR was documented, as 8-SGuo did not act on CD38-stimulated splenic B cells from Tlr7(-/-) and Myd88(-/-) mice. CD38-activated B cells from Btk-deficient mice failed to respond to TLR7 ligands for the AID expression and CSR, indicating that Btk is also indispensable for the system. Stimulation of CD38-activated B cells with 8-SGuo induced significant AID expression and DNA double strand breaks, but IL-4 stimulation by itself did not trigger mu to gamma1 CSR. Intriguingly, the mu to gamma1 CSR in the B cells stimulated with CD38 and 8-SGuo totally depends on IL-4 stimulation. Similar results were obtained in the activated B cells through BCR and loxoribine, a well-known TLR7 ligand, in place of 8-SGuo. In vivo administration of TLR7 ligand and anti-CD38 antibody induced the generation of CD138(+) IgG1(+) cells. These results indicate that TLR7 is a receptor for 8-SGuo and plays an essential role in the AID and Blimp-1 expression; however it is not enough to complete mu to gamma1 CSR in CD38-activated B cells. IL-4 may be required for the induction of DNA repair system together with AID for the completion of CSR.

  6. Human placenta: relative content of antibodies of different classes and subclasses (IgG1-IgG4) containing lambda- and kappa-light chains and chimeric lambda-kappa-immunoglobulins.

    Science.gov (United States)

    Lekchnov, Evgenii A; Sedykh, Sergey E; Dmitrenok, Pavel S; Buneva, Valentina N; Nevinsky, Georgy A

    2015-06-01

    The specific organ placenta is much more than a filter: it is an organ that protects, feeds and regulates the growth of the embryo. Affinity chromatography, ELISA, SDS-PAGE and matrix-assisted laser desorption ionization mass spectrometry were used. Using 10 intact human placentas deprived of blood, a quantitative analysis of average relative content [% of total immunoglobulins (Igs)] was carried out for the first time: (92.7), IgA (2.4), IgM (2.5), kappa-antibodies (51.4), lambda-antibodies (48.6), IgG1 (47.0), IgG2 (39.5), IgG3 (8.8) and IgG4 (4.3). It was shown for the first time that placenta contains sIgA (2.5%). In the classic paradigm, Igs represent products of clonal B-cell populations, each producing antibodies recognizing a single antigen. There is a common belief that IgGs in mammalian biological fluids are monovalent molecules having stable structures and two identical antigen-binding sites. However, similarly to human milk Igs, placenta antibodies undergo extensive half-molecule exchange and the IgG pool consists of 43.5 ± 15.0% kappa-kappa-IgGs and 41.6 ± 17.0% lambda-lambda-IgGs, while 15.0 ± 4.0% of the IgGs contained both kappa- and lambda-light chains. Kappa-kappa-IgGs and lambda-lambda-IgGs contained, respectively (%): IgG1 (47.7 and 34.4), IgG2 (36.3 and 44.5), IgG3 (7.4 and 11.8) and IgG4 (7.5 and 9.1), while chimeric kappa-lambda-IgGs consisted of (%): 43.5 IgG1, 41.0 IgG2, 5.6 IgG3 and 7.9 IgG4. Our data are indicative of the possibility of half-molecule exchange between placenta IgGs of various subclasses, raised against different antigens, which explains a very well-known polyspecificity and cross-reactivity of different human IgGs. © The Japanese Society for Immunology. 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. Monoclonal antibodies in pediatric allergy

    Directory of Open Access Journals (Sweden)

    Amelia Licari

    2015-10-01

    Full Text Available Production of monoclonal antibodies (mAbs involving human-mouse hybrid cells was first described in 1970s, but these biologics are now used for a variety of diseases including cancers, autoimmune disorders and allergic diseases. The aim of this article is to review current and future applications of mAbs, in particular focusing on anti-IgE therapy, in the field of pediatric allergy. Proceedings of the 11th International Workshop on Neonatology and Satellite Meetings · Cagliari (Italy · October 26th-31st, 2015 · From the womb to the adultGuest Editors: Vassilios Fanos (Cagliari, Italy, Michele Mussap (Genoa, Italy, Antonio Del Vecchio (Bari, Italy, Bo Sun (Shanghai, China, Dorret I. Boomsma (Amsterdam, the Netherlands, Gavino Faa (Cagliari, Italy, Antonio Giordano (Philadelphia, USA

  8. N- and C-terminally truncated forms of glucose-dependent insulinotropic polypeptide are high-affinity competitive antagonists of the human GIP receptor

    DEFF Research Database (Denmark)

    Hansen, L S; Sparre-Ulrich, A H; Christensen, M.

    2016-01-01

    functions and pharmacological potential. GIP(1-30)NH2 is a naturally occurring truncation of GIP(1-42). Here we characterize eight N-terminal trrncations of human GIP(1-30)NH2 : GIP(2- to 9-30)NH2 . EXPERIMENTAL APPROACH: COS-7 cells were transiently transfected with the human GIP receptor and assessed...... displayed lower affinities (Ki 2.3-347 nM) with highest affinities of GIP(3-30)NH2 and (5-30)NH2 . Agonism was only observed for GIP(1-30)NH2 with an Emax on 100% of GIP(1-42) and GIP(2-30)NH2 (Emax 20%). GIP(2- to 9-30)NH2 displayed antagonism (IC50 12-450 nM) and right-shifts of the GIP(1-42)-response......, but superior antagonist GIP(3-30)NH2 , that together with GIP(5-30)NH2 were high-affinity competitive antagonist and thus may be suitable tool compounds for basic GIP research and future pharmacological interventions....

  9. Premature Aging Phenotype in Mice Lacking High-Affinity Nicotinic Receptors: Region-Specific Changes in Layer V Pyramidal Cell Morphology.

    Science.gov (United States)

    Konsolaki, Eleni; Skaliora, Irini

    2015-08-01

    The mechanisms by which aging leads to alterations in brain structure and cognitive deficits are unclear. Α deficient cholinergic system has been implicated as one of the main factors that could confer a heightened vulnerability to the aging process, and mice lacking high-affinity nicotinic receptors (β2(-/-)) have been proposed as an animal model of accelerated cognitive aging. To date, however, age-related changes in neuronal microanatomy have not been studied in these mice. In the present study, we examine the neuronal structure of yellow fluorescent protein (YFP(+)) layer V neurons in 2 cytoarchitectonically distinct cortical regions in wild-type (WT) and β2(-/-) animals. We find that (1) substantial morphological differences exist between YFP(+) cells of the anterior cingulate cortex (ACC) and primary visual cortex (V1), in both genotypes; (2) in WT animals, ACC cells are more susceptible to aging compared with cells in V1; and (3) β2 deletion is associated with a regionally and temporally specific increase in vulnerability to aging. ACC cells exhibit a prematurely aged phenotype already at 4-6 months, whereas V1 cells are spared in adulthood but strongly affected in old animals. Collectively, our data reveal region-specific synergistic effects of aging and genotype and suggest distinct vulnerabilities in V1 and ACC neurons. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  10. Selective cortical decrease of high-affinity choline uptake carrier in Alzheimer's disease: an autoradiographic study using 3H-hemicholinium-3

    International Nuclear Information System (INIS)

    Rodriguez-Puertas, R.; Pazos, A.; Zarranz, J.J.; Pascual, J.

    1994-01-01

    H-hemicholinium-3 (H-HC-3) binding, a marker of the presynaptic high-affinity choline uptake carrier (HACU), was measured by autoradiography in several brain regions of 17 Alzheimer's disease (AD) patients and of 11 matched controls. A significant decrease in the density of H-HC-3 binding sites was found in entorhinal cortex, hippocampus and layers I-III of the frontal cortex. By contrast, in the caudate-putamen the number of H-HC-3 binding sites in AD cases was comparable to that of control striata. These data concur with previous results using classical presynaptic markers and reflect the loss in the activity of HACU, and, hence, in the synthesis of acetylcholine, that selectively occurs in cortical areas of AD brains due to the degeneration of presynaptic cholinergic terminals arising from the basal forebrain. However, the relatively low mean reduction in HACU in cortical areas (-40 %), together with the apparent indemnity of this marker in certain severely demented AD cases, suggest that AD dementia cannot be explained simply by the loss of presynaptic terminals originating in the basal forebrain. These data seem to be a good explanation for the poor response to cholinergic replacement in AD. (author)

  11. High Affinity vs. Native Fibronectin in the Modulation of αvβ3 Integrin Conformational Dynamics: Insights from Computational Analyses and Implications for Molecular Design.

    Directory of Open Access Journals (Sweden)

    Antonella Paladino

    2017-01-01

    Full Text Available Understanding how binding events modulate functional motions of multidomain proteins is a major issue in chemical biology. We address several aspects of this problem by analyzing the differential dynamics of αvβ3 integrin bound to wild type (wtFN10, agonist or high affinity (hFN10, antagonist mutants of fibronectin. We compare the dynamics of complexes from large-scale domain motions to inter-residue coordinated fluctuations to characterize the distinctive traits of conformational evolution and shed light on the determinants of differential αvβ3 activation induced by different FN sequences. We propose an allosteric model for ligand-based integrin modulation: the conserved integrin binding pocket anchors the ligand, while different residues on the two FN10's act as the drivers that reorganize relevant interaction networks, guiding the shift towards inactive (hFN10-bound or active states (wtFN10-bound. We discuss the implications of results for the design of integrin inhibitors.

  12. The Extracellular Domain of Human High Affinity Copper Transporter (hNdCTR1), Synthesized by E. coli Cells, Chelates Silver and Copper Ions In Vivo.

    Science.gov (United States)

    Sankova, Tatiana P; Orlov, Iurii A; Saveliev, Andrey N; Kirilenko, Demid A; Babich, Polina S; Brunkov, Pavel N; Puchkova, Ludmila V

    2017-11-03

    There is much interest in effective copper chelators to correct copper dyshomeostasis in neurodegenerative and oncological diseases. In this study, a recombinant fusion protein for expression in Escherichia coli cells was constructed from glutathione-S-transferase (GST) and the N-terminal domain (ectodomain) of human high affinity copper transporter CTR1 (hNdCTR1), which has three metal-bound motifs. Several biological properties of the GST-hNdCTR1 fusion protein were assessed. It was demonstrated that in cells, the protein was prone to oligomerization, formed inclusion bodies and displayed no toxicity. Treatment of E. coli cells with copper and silver ions reduced cell viability in a dose- and time-dependent manner. Cells expressing GST-hNdCTR1 protein demonstrated resistance to the metal treatments. These cells accumulated silver ions and formed nanoparticles that contained AgCl and metallic silver. In this bacterial population, filamentous bacteria with a length of about 10 µm were often observed. The possibility for the fusion protein carrying extracellular metal binding motifs to integrate into the cell's copper metabolism and its chelating properties are discussed.

  13. The Extracellular Domain of Human High Affinity Copper Transporter (hNdCTR1, Synthesized by E. coli Cells, Chelates Silver and Copper Ions In Vivo

    Directory of Open Access Journals (Sweden)

    Tatiana P. Sankova

    2017-11-01

    Full Text Available There is much interest in effective copper chelators to correct copper dyshomeostasis in neurodegenerative and oncological diseases. In this study, a recombinant fusion protein for expression in Escherichia coli cells was constructed from glutathione-S-transferase (GST and the N-terminal domain (ectodomain of human high affinity copper transporter CTR1 (hNdCTR1, which has three metal-bound motifs. Several biological properties of the GST-hNdCTR1 fusion protein were assessed. It was demonstrated that in cells, the protein was prone to oligomerization, formed inclusion bodies and displayed no toxicity. Treatment of E. coli cells with copper and silver ions reduced cell viability in a dose- and time-dependent manner. Cells expressing GST-hNdCTR1 protein demonstrated resistance to the metal treatments. These cells accumulated silver ions and formed nanoparticles that contained AgCl and metallic silver. In this bacterial population, filamentous bacteria with a length of about 10 µm were often observed. The possibility for the fusion protein carrying extracellular metal binding motifs to integrate into the cell’s copper metabolism and its chelating properties are discussed.

  14. A viral, transporter associated with antigen processing (TAP)-independent, high affinity ligand with alternative interactions endogenously presented by the nonclassical human leukocyte antigen E class I molecule.

    Science.gov (United States)

    Lorente, Elena; Infantes, Susana; Abia, David; Barnea, Eilon; Beer, Ilan; García, Ruth; Lasala, Fátima; Jiménez, Mercedes; Mir, Carmen; Morreale, Antonio; Admon, Arie; López, Daniel

    2012-10-12

    The transporter associated with antigen processing (TAP) enables the flow of viral peptides generated in the cytosol by the proteasome and other proteases to the endoplasmic reticulum, where they complex with nascent human leukocyte antigen (HLA) class I. Later, these peptide-HLA class I complexes can be recognized by CD8(+) lymphocytes. Cancerous cells and infected cells in which TAP is blocked, as well as individuals with unusable TAP complexes, are able to present peptides on HLA class I by generating them through TAP-independent processing pathways. Here, we identify a physiologically processed HLA-E ligand derived from the D8L protein in TAP-deficient vaccinia virus-infected cells. This natural high affinity HLA-E class I ligand uses alternative interactions to the anchor motifs previously described to be presented on nonclassical HLA class I molecules. This octameric peptide was also presented on HLA-Cw1 with similar binding affinity on both classical and nonclassical class I molecules. In addition, this viral peptide inhibits HLA-E-mediated cytolysis by natural killer cells. Comparison between the amino acid sequences of the presenting HLA-E and HLA-Cw1 alleles revealed a shared structural motif in both HLA class molecules, which could be related to their observed similar cross-reactivity affinities. This motif consists of several residues located on the floor of the peptide-binding site. These data expand the role of HLA-E as an antigen-presenting molecule.

  15. High affinity radiopharmaceuticals based upon lansoprazole for PET imaging of aggregated tau in Alzheimer's disease and progressive supranuclear palsy: synthesis, preclinical evaluation, and lead selection.

    Science.gov (United States)

    Fawaz, Maria V; Brooks, Allen F; Rodnick, Melissa E; Carpenter, Garrett M; Shao, Xia; Desmond, Timothy J; Sherman, Phillip; Quesada, Carole A; Hockley, Brian G; Kilbourn, Michael R; Albin, Roger L; Frey, Kirk A; Scott, Peter J H

    2014-08-20

    Abnormally aggregated tau is the hallmark pathology of tauopathy neurodegenerative disorders and is a target for development of both diagnostic tools and therapeutic strategies across the tauopathy disease spectrum. Development of carbon-11- or fluorine-18-labeled radiotracers with appropriate affinity and specificity for tau would allow noninvasive quantification of tau burden using positron emission tomography (PET) imaging. We have synthesized [(18)F]lansoprazole, [(11)C]N-methyl lansoprazole, and [(18)F]N-methyl lansoprazole and identified them as high affinity radiotracers for tau with low to subnanomolar binding affinities. Herein, we report radiosyntheses and extensive preclinical evaluation with the aim of selecting a lead radiotracer for translation into human PET imaging trials. We demonstrate that [(18)F]N-methyl lansoprazole, on account of the favorable half-life of fluorine-18 and its rapid brain entry in nonhuman primates, favorable kinetics, low white matter binding, and selectivity for binding to tau over amyloid, is the lead compound for progression into clinical trials.

  16. High Affinity Radiopharmaceuticals Based Upon Lansoprazole for PET Imaging of Aggregated Tau in Alzheimer’s Disease and Progressive Supranuclear Palsy: Synthesis, Preclinical Evaluation, and Lead Selection

    Science.gov (United States)

    2014-01-01

    Abnormally aggregated tau is the hallmark pathology of tauopathy neurodegenerative disorders and is a target for development of both diagnostic tools and therapeutic strategies across the tauopathy disease spectrum. Development of carbon-11- or fluorine-18-labeled radiotracers with appropriate affinity and specificity for tau would allow noninvasive quantification of tau burden using positron emission tomography (PET) imaging. We have synthesized [18F]lansoprazole, [11C]N-methyl lansoprazole, and [18F]N-methyl lansoprazole and identified them as high affinity radiotracers for tau with low to subnanomolar binding affinities. Herein, we report radiosyntheses and extensive preclinical evaluation with the aim of selecting a lead radiotracer for translation into human PET imaging trials. We demonstrate that [18F]N-methyl lansoprazole, on account of the favorable half-life of fluorine-18 and its rapid brain entry in nonhuman primates, favorable kinetics, low white matter binding, and selectivity for binding to tau over amyloid, is the lead compound for progression into clinical trials. PMID:24896980

  17. Carbon-11 labelling of eticlopride in two different positions - a selective high-affinity ligand for the study of dopamine D-2 receptors using PET

    International Nuclear Information System (INIS)

    Halldin, Christer; Hall, Haakan

    1990-01-01

    A new highly selective high-affinity dopamine D-2 receptor antagonist, eticlopride ((-)-(S)-5-chloro-3-ethyl-N-(1-ethyl-2-pyrrolidinyl)methyl)-6-methoxysalicylamide), was labelled with 11 C in two different positions ([N-ethyl- 11 C]eticlopride (I) and ([methyl- 11 C]eticlopride (II)). Product I was prepared by N-alkylation of the N-desethyl compound with [ 11 C]ethyl iodide. II was prepared by O-alkylation of the diphenolic precursor with [ 11 C]methyl iodide followed by separation of the two methylated products. The radiochemical yields were 15-20% (EOB) with an overall synthesis time of 45-60 min. Both compounds were isolated by semi-preparative HPLC and the radiochemical purity was in both cases > 99%. I was injected i.v. in a Cynomolgus monkey and brain radioactivity was measured by positron emission tomography (PET). The specific activity was 70 Ci/mmol at time of injection. There was a marked accumulation of radioactivity in the basal ganglia, regions known to have a high density of dopamine D-2 receptors. (author)

  18. Involvement of the VDE homing endonuclease and rapamycin in regulation of the Saccharomyces cerevisiae GSH11 gene encoding the high affinity glutathione transporter.

    Science.gov (United States)

    Miyake, Tsuyoshi; Hiraishi, Hiroyuki; Sammoto, Hiroyuki; Ono, Bun-Ichiro

    2003-10-10

    The Saccharomyces cerevisiae gene HGT1/GSH11 encodes the high affinity glutathione transporter and is repressed by cysteine added to the culture medium. It has been found previously that a 5'-upstream cis-element, CCGCCACAC, is responsible for regulating GSH11 expression and that several proteins bind to this element (Miyake, T., Kanayama, M., Sammoto, H., and Ono, B. (2002) Mol. Genet. Genomics 266, 1004-1011). In this report we present evidence that the most prominent of these proteins is VDE, known previously as the homing endonuclease encoded by VMA1. We show also that GSH11 is not expressed in a VDE-deleted strain and that inability to express the GSH11 of this strain is overcome by introduction of the coding region of VDE or the entire VMA1 gene. It is also found that VDE does not cut DNA in the vicinity of the GSH11 cis-element. Rapamycin, an inhibitor of the target of rapamycin (TOR) signal-transduction system, is found to enhance expression of GSH11 in a VDE-dependent manner under conditions of sulfur starvation. These results indicate that GSH11 is regulated by a system sensitive to sulfur starvation (presumably via cysteine depletion) and a more general system involving the nutritional starvation signal mediated by the TOR system. Both systems need to be operational (inhibition of TOR and sulfur starvation) for full expression of GSH11.

  19. Selective cortical decrease of high-affinity choline uptake carrier in Alzheimer`s disease: an autoradiographic study using {sup 3}H-hemicholinium-3

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez-Puertas, R; Pazos, A [Dept. of Physiology and Pharmacology, Unit of Pharmacology, Univ. of Cantabria, Santander (Spain); Zarranz, J J [Dept. of Neuroscience, Univ. of the Basque Country, Leioa (Spain); Pascual, J [Dept. of Medicine, Service of Neurology, Univ. Hospital ` Marques de Valdecilla` , Univ. of Cantabria, Santander (Spain)

    1994-12-31

    H-hemicholinium-3 (H-HC-3) binding, a marker of the presynaptic high-affinity choline uptake carrier (HACU), was measured by autoradiography in several brain regions of 17 Alzheimer`s disease (AD) patients and of 11 matched controls. A significant decrease in the density of H-HC-3 binding sites was found in entorhinal cortex, hippocampus and layers I-III of the frontal cortex. By contrast, in the caudate-putamen the number of H-HC-3 binding sites in AD cases was comparable to that of control striata. These data concur with previous results using classical presynaptic markers and reflect the loss in the activity of HACU, and, hence, in the synthesis of acetylcholine, that selectively occurs in cortical areas of AD brains due to the degeneration of presynaptic cholinergic terminals arising from the basal forebrain. However, the relatively low mean reduction in HACU in cortical areas (-40 %), together with the apparent indemnity of this marker in certain severely demented AD cases, suggest that AD dementia cannot be explained simply by the loss of presynaptic terminals originating in the basal forebrain. These data seem to be a good explanation for the poor response to cholinergic replacement in AD. (author).

  20. PCR-identification of a Nicotiana plumbaginifolia cDNA homologous to the high-affinity nitrate transporters of the crnA family.

    Science.gov (United States)

    Quesada, A; Krapp, A; Trueman, L J; Daniel-Vedele, F; Fernández, E; Forde, B G; Caboche, M

    1997-05-01

    A family of high-affinity nitrate transporters has been identified in Aspergillus nidulans and Chlamydomonas reinhardtii, and recently homologues of this family have been cloned from a higher plant (barley). Based on six of the peptide sequences most strongly conserved between the barley and C. reinhardtii polypeptides, a set of degenerate primers was designed to permit amplification of the corresponding genes from other plant species. The utility of these primers was demonstrated by RT-PCR with cDNA made from poly(A)+ RNA from barley, C. reinhardtii and Nicotiana plumbaginifolia. A PCR fragment amplified from N. plumbaginifolia was used as probe to isolate a full-length cDNA clone which encodes a protein, NRT2;1Np, that is closely related to the previously isolated crnA homologue from barley. Genomic Southern blots indicated that there are only 1 or 2 members of the Nrt2 gene family in N. plumbaginifolia. Northern blotting showed that the Nrt2 transcripts are most strongly expressed in roots. The effects of external treatments with different N sources showed that the regulation of the Nrt2 gene(s) is very similar to that reported for nitrate reductase and nitrite reductase genes: their expression was strongly induced by nitrate but was repressed when reduced forms of N were supplied to the roots.

  1. An in vitro-identified high-affinity nucleosome-positioning signal is capable of transiently positioning a nucleosome in vivo

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    Gracey Lia E

    2010-07-01

    Full Text Available Abstract Background The physiological function of eukaryotic DNA occurs in the context of nucleosomal arrays that can expose or obscure defined segments of the genome. Certain DNA sequences are capable of strongly positioning a nucleosome in vitro, suggesting the possibility that favorable intrinsic signals might reproducibly structure chromatin segments. As high-throughput sequencing analyses of nucleosome coverage in vitro and in vivo have become possible, a vigorous debate has arisen over the degree to which intrinsic DNA:nucleosome affinities orchestrate the in vivo positions of nucleosomes, thereby controlling physical accessibility of specific sequences in DNA. Results We describe here the in vivo consequences of placing a synthetic high-affinity nucleosome-positioning signal, the 601 sequence, into a DNA plasmid vector in mice. Strikingly, the 601 sequence was sufficient to position nucleosomes during an early phase after introduction of the DNA into the mice (when the plasmid vector transgene was active. This positioning capability was transient, with a loss of strong positioning at a later time point when the transgenes had become silent. Conclusions These results demonstrate an ability of DNA sequences selected solely for nucleosome affinity to organize chromatin in vivo, and the ability of other mechanisms to overcome these interactions in a dynamic nuclear environment.

  2. The Mitochondrial Metallochaperone SCO1 Is Required to Sustain Expression of the High-Affinity Copper Transporter CTR1 and Preserve Copper Homeostasis

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    Christopher J. Hlynialuk

    2015-02-01

    Full Text Available Human SCO1 fulfills essential roles in cytochrome c oxidase (COX assembly and the regulation of copper (Cu homeostasis, yet it remains unclear why pathogenic mutations in this gene cause such clinically heterogeneous forms of disease. Here, we establish a Sco1 mouse model of human disease and show that ablation of Sco1 expression in the liver is lethal owing to severe COX and Cu deficiencies. We further demonstrate that the Cu deficiency is explained by a functional connection between SCO1 and CTR1, the high-affinity transporter that imports Cu into the cell. CTR1 is rapidly degraded in the absence of SCO1 protein, and we show that its levels are restored in Sco1−/− mouse embryonic fibroblasts upon inhibition of the proteasome. These data suggest that mitochondrial signaling through SCO1 provides a post-translational mechanism to regulate CTR1-dependent Cu import into the cell, and they further underpin the importance of mitochondria in cellular Cu homeostasis.

  3. SKF 525-A and cytochrome P-450 ligands inhibit with high affinity the binding of [3H]dextromethorphan and σligands to guinea pig brain

    International Nuclear Information System (INIS)

    Klein, M.; Canoll, P.D.; Musacchio, J.M.

    1991-01-01

    The DM 1 /σ 1 site binds dextromethorphan (DM) and σ receptor ligands. The broad binding specificity of this site and its peculiar subcellular distribution prompted us to explore the possibility that this site is a member of the cytochrome P-450 superfamily of enzymes. We tested the effects of the liver microsomal monooxygenase inhibitor SKF 525-A (Proadifen), and other P-450 substrates on the binding of [ 3 H]dextromethorphan, [ 3 H]3-(3-Hydroxyphenyl)-N-(1-propyl)piperidine and (+)-[ 3 H]1,3-Di-o-tolyl-guanidine ([ 3 H]DTG) to the guinea pig brain. SKF 525-A, l-lobeline and GBR-12909 inhibited the binding of the three labeled ligands with nM affinity. Each drug has identical nM K i values for the high-affinity site labeled by the three ligands. This indicated that they displaced the labeled ligands from the common DM 1 σ 1 site. Debrisoquine and sparteine, prototypical substrates for liver debrisoquine 4-hydroxylase, displayed K i values of 9-13 and 3-4 μM respectively against the three labeled ligands. These results, the broad specificity of the DM 1 /σ 1 binding site, and its peculiar subcellular distribution, raises the possibility that this binding site is a member of the cytochrome P-450 superfamily of isozymes, rather than a neurotransmitter receptor

  4. [Structure-functional organization of eukaryotic high-affinity copper importer CTR1 determines its ability to transport copper, silver and cisplatin].

    Science.gov (United States)

    Skvortsov, A N; Zatulovskiĭ, E A; Puchkova, L V

    2012-01-01

    It was shown recently, that high affinity Cu(I) importer eukaryotic protein CTR1 can also transport in vitro abiogenic Ag(I) ions and anticancer drug cisplatin. At present there is no rational explanation how CTR1 can transfer platinum group, which is different by coordination properties from highly similar Cu(I) and Ag(I). To understand this phenomenon we analyzed 25 sequences of chordate CTR1 proteins, and found out conserved patterns of organization of N-terminal extracellular part of CTR1 which correspond to initial metal binding. Extracellular copper-binding motifs were qualified by their coordination properties. It was shown that relative position of Met- and His-rich copper-binding motifs in CTR1 predisposes the extracellular CTR1 part to binding of copper, silver and cisplatin. Relation between tissue-specific expression of CTR1 gene, steady-state copper concentration, and silver and platinum accumulation in organs of mice in vivo was analyzed. Significant positive but incomplete correlation exists between these variables. Basing on structural and functional peculiarities of N-terminal part of CTR1 a hypothesis of coupled transport of copper and cisplatin has been suggested, which avoids the disagreement between CTR1-mediated cisplatin transport in vitro, and irreversible binding of platinum to Met-rich peptides.

  5. Establishment of a novel high-affinity IgE receptor-positive canine mast cell line with wild-type c-kit receptors

    International Nuclear Information System (INIS)

    Amagai, Yosuke; Tanaka, Akane; Ohmori, Keitaro; Matsuda, Hiroshi

    2008-01-01

    Much is known regarding participations of mast cells with innate and acquired immunity by secreting various cytokines and chemical mediators. However, details of mast cell biology still remain unclear. In this study, we successfully established a novel growth factor-independent mast cell line (MPT-1) derived from canine mast cell tumor. MPT-1 cells manifested factor-independent proliferation as floating cells containing a large amount of histamine, as well as chymase-like dog mast cell protease 3, in cytosolic granules. Particularly, MPT-1 cells expressed high-affinity IgE receptors (FcεRI) and wild-type c-kit receptors. Degranulation of MPT-1 cells was induced not only by stimulation with calcium ionophore but also by cross-linkage of the surface IgE. Given that MPT-1 is the first mast cell line with FcεRI which has no c-kit mutations, MPT-1 cells may provide great contribution for investigation of IgE-mediated activation mechanisms of mast cells, leading to development of effective treatment for allergic disorders

  6. High affinity (3H) β-Alanine uptake by scar margins of ferric chloride-induced epileptogenic foci in rat isocortex

    International Nuclear Information System (INIS)

    Robitaille, Y.; Sherwin, A.

    1984-01-01

    Cortical astrocytes of normal mammalian brain are endowed with a high affinity uptake system for β-Alanine which is competitively inhibited by gamma aminobutyric acid (GABA), a neurotransmitter strongly implicated in epileptogenesis. The authors evaluated ( 3 H) β-Alanine uptake by reactive astrocytes proliferating within scar of epileptogenic foci induced in rat motor cortex by microinjections of 100 mM ferric chloride. Following in vitro incubation of scar tissue with ( 3 H) β-Alanine, ultrastructural morphometry of grain patterns at 5, 30 and 120 days post injection revealed early and significant grain count increases over astroglial processes, predominantly those related to perivascular glial end-feet. Astrocytic cell body and endothelial cell counts showed a more gradual and stepwise increase. Similar data were obtained by comparing visual and edited mean astrocytic grain counts. These results suggest that the enhanced uptake of reactive astrocytes may reflect a marked decrease of inhibitory GABAergic neurons within ferric chloride-induced scars. 7 figures, 1 table

  7. Mining Naïve Rabbit Antibody Repertoires by Phage Display for Monoclonal Antibodies of Therapeutic Utility.

    Science.gov (United States)

    Peng, Haiyong; Nerreter, Thomas; Chang, Jing; Qi, Junpeng; Li, Xiuling; Karunadharma, Pabalu; Martinez, Gustavo J; Fallahi, Mohammad; Soden, Jo; Freeth, Jim; Beerli, Roger R; Grawunder, Ulf; Hudecek, Michael; Rader, Christoph

    2017-09-15

    Owing to their high affinities and specificities, rabbit monoclonal antibodies (mAbs) have demonstrated value and potential primarily as basic research and diagnostic reagents, but, in some cases, also as therapeutics. To accelerate access to rabbit mAbs bypassing immunization, we generated a large naïve rabbit antibody repertoire represented by a phage display library encompassing >10 billion independent antibodies in chimeric rabbit/human Fab format and validated it by next-generation sequencing. Panels of rabbit mAbs selected from this library against two emerging cancer targets, ROR1 and ROR2, revealed high diversity, affinity, and specificity. Moreover, ROR1- and ROR2-targeting rabbit mAbs demonstrated therapeutic utility as components of chimeric antigen receptor-engineered T cells, further corroborating the value of the naïve rabbit antibody library as a rich and virtually unlimited source of rabbit mAbs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Two-monoclonal-antibody sandwich-type assay for thyrotropin, with use of an avidin-biotin separation technique

    International Nuclear Information System (INIS)

    Odell, W.D.; Griffin, J.; Zahradnik, R.

    1986-01-01

    We have developed a sensitive, specific, noncompetitive, sandwich-type radioimmunoassay for human thyrotropin (hTSH), which can be performed in 30 min. The assay involves two monoclonal antibodies, selected for high affinity and specificity and also for reaction against antigenic sites on hTSH that are distal from each other. One of these antibodies is labeled with 125 I; the other is conjugated covalently to biotin. Polystyrene beads were also conjugated covalently to biotin. After conjugation, the beads were incubated with avidin. These beads represent a rapid, simple method for separating hTSH-bound antibody from free antibody. The biotin-antibody-hTSH- 125 I-labeled antibody complexes bind to the beads and hTSH concentration is directly related to counts per minute. This assay can detect hTSH at a concentration of 0.06 milli-unit/L in serum

  9. Human anti-plague monoclonal antibodies protect mice from Yersinia pestis in a bubonic plague model.

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    Xiaodong Xiao

    2010-10-01

    Full Text Available Yersinia pestis is the etiologic agent of plague that has killed more than 200 million people throughout the recorded history of mankind. Antibiotics may provide little immediate relief to patients who have a high bacteremia or to patients infected with an antibiotic resistant strain of plague. Two virulent factors of Y. pestis are the capsid F1 protein and the low-calcium response (Lcr V-protein or V-antigen that have been proven to be the targets for both active and passive immunization. There are mouse monoclonal antibodies (mAbs against the F1- and V-antigens that can passively protect mice in a murine model of plague; however, there are no anti-Yersinia pestis monoclonal antibodies available for prophylactic or therapeutic treatment in humans. We identified one anti-F1-specific human mAb (m252 and two anti-V-specific human mAb (m253, m254 by panning a naïve phage-displayed Fab library against the F1- and V-antigens. The Fabs were converted to IgG1s and their binding and protective activities were evaluated. M252 bound weakly to peptides located at the F1 N-terminus where a protective mouse anti-F1 mAb also binds. M253 bound strongly to a V-antigen peptide indicating a linear epitope; m254 did not bind to any peptide from a panel of 53 peptides suggesting that its epitope may be conformational. M252 showed better protection than m253 and m254 against a Y, pestis challenge in a plague mouse model. A synergistic effect was observed when the three antibodies were combined. Incomplete to complete protection was achieved when m252 was given at different times post-challenge. These antibodies can be further studied to determine their potential as therapeutics or prophylactics in Y. pestis infection in humans.

  10. Development, characterization and application of monoclonal antibodies against Brazilian Dengue virus isolates.

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    Camila Zanluca

    Full Text Available Dengue is the most prevalent human arboviral disease. The morbidity related to dengue infection supports the need for an early, quick and effective diagnostic test. Brazil is a hotspot for dengue, but no serological diagnostic test has been produced using Brazilian dengue virus isolates. This study aims to improve the development of immunodiagnostic methods for dengue virus (DENV detection through the production and characterization of 22 monoclonal antibodies (mAbs against Brazilian isolates of DENV-1, -2 and -3. The mAbs include IgG2bκ, IgG2aκ and IgG1κ isotypes, and most were raised against the envelope or the pre-membrane proteins of DENV. When the antibodies were tested against the four DENV serotypes, different reactivity patterns were identified: group-specific, subcomplex specific (DENV-1, -3 and -4 and DENV-2 and -3 and dengue serotype-specific (DENV-2 or -3. Additionally, some mAbs cross-reacted with yellow fever virus (YFV, West Nile virus (WNV and Saint Louis encephalitis virus (SLEV. None of the mAbs recognized the alphavirus Venezuelan equine encephalitis virus (VEEV. Furthermore, mAbs D3 424/8G, D1 606/A12/B9 and D1 695/12C/2H were used to develop a capture enzyme-linked immunosorbent assay (ELISA for anti-dengue IgM detection in sera from patients with acute dengue. To our knowledge, these are the first monoclonal antibodies raised against Brazilian DENV isolates, and they may be of special interest in the development of diagnostic assays, as well as for basic research.

  11. Monoclonal antibody to serum immunoglobulins of Clarias batrachus and its application in immunoassays.

    Science.gov (United States)

    Sood, Neeraj; Chaudhary, Dharmendra K; Singh, Akhilesh; Rathore, Gaurav

    2012-12-15

    Serum immunoglobulins of Clarias batrachus (Cb-Ig) were purified by affinity chromatography using bovine serum albumin as capture ligand. Under reducing conditions in SDS-PAGE, Cb-Ig was composed of a heavy (H) chain (68.7 kDa) and two light (L) chains (27.4 and 26.3 kDa). Purified Cb-Ig was used to produce a monoclonal antibody (MAb) designated E4 MAb that belonged to IgG1 subclass. In Western blotting, this MAb showed binding to H chain of purified Cb-Ig and putative H chains in reduced sera of C. batrachus, Clarias gariepinus and Heteropneustes fossilis. However, no binding was observed with serum protein of Labeo rohita and Channa striata. Cross-reactivity of anti-Cb-Ig MAb was observed with serum of C. batrachus, C. gariepinus and H. fossilis in competitive ELISA. In immunoblotting of non-reduced Cb-Ig with E4 MAb, four bands assumed to be tetrameric, trimeric, dimeric and monomeric form were observed. In flow cytometric analysis of the gated lymphocytes, the number of surface Ig-positive (Ig+) cells in blood, spleen, kidney and thymus of C. batrachus was determined to be 50.1 ± 3.1, 55.1 ± 3.36, 42.4 ± 4.81 and 5.1 ± 0.89%, respectively, using E4 MAb. Ig+ cells were also demonstrated in formalin-fixed paraffin embedded tissue sections of spleen, kidney, thymus and smears of blood mononuclear cells in indirect immunoperoxidase test. The developed MAb was employed to detect pathogen-specific immunoglobulins in the sera of C. batrachus immunized with killed Edwardsiella tarda, by an indirect ELISA. This monoclonal antibody can be useful tool in immunological research and assays. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. A human/mouse chimeric monoclonal antibody against intercellular adhesion molecule-1 for tumor radioimmunoimaging

    International Nuclear Information System (INIS)

    Yamamura, Miyuki; Hinoda, Yuji; Sasaki, Shigeru; Tsujisaki, Masayuki; Imai, Kohzoh; Oriuchi, Noboru; Endo, Keigo.

    1996-01-01

    A mouse-human chimeric antibody for intercellular adhesion molecule-1 (ICAM-1) was established by using heavy chain loss mouse mutant hybridoma and human immunoglobulin expression vector. The HA58 hybridoma secreted anti-ICAM-1 monoclonal antibody (MoAb) (IgG1,κ). The gene of the mouse variable region of heavy chain was amplified and cloned by the polymerase chain reaction technique directly from the HA58 hybridoma RNA. The variable region of heavy chain was joined with an expression vector which contains human γ1 constant gene. The expression vector was transfected into heavy chain loss mutant cells HA58-7, which produced only murine immunoglobulin light chains. The resultant chimeric MoAb HA58, chHA58, retained full-binding reactivity to ICAM-1 compared with murine HA58 parental antibody. The chimeric MoAb chHA58 showed little antibody dependent cell-mediated cytotoxic activity against cultured tumor cells. Biodistribution studies with 99m Tc-labeled chHA58 in nude mice bearing human gastric carcinoma JRST cells, demonstrated that the tumor-blood ratio was 1.55 at 18 h after injection, when the tumors were clearly visible in gamma scintigraphy. These data suggest that chHA58 may be of practical use for radioimmunoimaging of a wide variety of tumors. (author)

  13. Monoclonal Antibody L1Mab-13 Detected Human PD-L1 in Lung Cancers.

    Science.gov (United States)

    Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Yanaka, Miyuki; Chang, Yao-Wen; Suzuki, Hiroyoshi; Kaneko, Mika K; Kato, Yukinari

    2018-04-01

    Programmed cell death ligand-1 (PD-L1) is a type I transmembrane glycoprotein expressed on antigen-presenting cells. It is also expressed in several tumor cells such as melanoma and lung cancer cells. A strong correlation has been reported between human PD-L1 (hPD-L1) expression in tumor cells and negative prognosis in cancer patients. Here, a novel anti-hPD-L1 monoclonal antibody (mAb) L 1 Mab-13 (IgG 1 , kappa) was produced using a cell-based immunization and screening (CBIS) method. We investigated hPD-L1 expression in lung cancer using flow cytometry, Western blot, and immunohistochemical analyses. L 1 Mab-13 specifically reacted hPD-L1 of hPD-L1-overexpressed Chinese hamster ovary (CHO)-K1 cells and endogenous hPD-L1 of KMST-6 (human fibroblast) in flow cytometry and Western blot. Furthermore, L 1 Mab-13 reacted with lung cancer cell lines (EBC-1, Lu65, and Lu99) in flow cytometry and stained lung cancer tissues in a membrane-staining pattern in immunohistochemical analysis. These results indicate that a novel anti-hPD-L1 mAb, L 1 Mab-13, is very useful for detecting hPD-L1 of lung cancers in flow cytometry, Western blot, and immunohistochemical analyses.

  14. Development of an Anti-HER2 Monoclonal Antibody H2Mab-139 Against Colon Cancer.

    Science.gov (United States)

    Kaneko, Mika K; Yamada, Shinji; Itai, Shunsuke; Kato, Yukinari

    2018-02-01

    Human epidermal growth factor receptor 2 (HER2) expression has been reported in several cancers, such as breast, gastric, lung, pancreatic, and colorectal cancers. HER2 is overexpressed in those cancers and is associated with poor clinical outcomes. Trastuzumab, a humanized anti-HER2 antibody, provides significant survival benefits for patients with HER2-overexpressing breast cancers and gastric cancers. In this study, we developed a novel anti-HER2 monoclonal antibody (mAb), H 2 Mab-139 (IgG 1 , kappa) and investigated it against colon cancers using flow cytometry, western blot, and immunohistochemical analyses. Flow cytometry analysis revealed that H 2 Mab-139 reacted with colon cancer cell lines, such as Caco-2, HCT-116, HCT-15, HT-29, LS 174T, COLO 201, COLO 205, HCT-8, SW1116, and DLD-1. Although H 2 Mab-139 strongly reacted with LN229/HER2 cells on the western blot, we did not observe a specific signal for HER2 in colon cancer cell lines. Immunohistochemical analyses revealed sensitive and specific reactions of H 2 Mab-139 against colon cancers, indicating that H 2 Mab-139 is useful in detecting HER2 overexpression in colon cancers using flow cytometry and immunohistochemical analyses.

  15. Anti-Podocalyxin Monoclonal Antibody 47-mG2a Detects Lung Cancers by Immunohistochemistry.

    Science.gov (United States)

    Yamada, Shinji; Itai, Shunsuke; Kaneko, Mika K; Kato, Yukinari

    2018-04-01

    Lung cancer is one of the leading causes of cancer-related deaths in the world. Regardless of the advances in lung cancer treatments, the prognosis is still poor. Podocalyxin (PODXL) is a highly glycosylated type I transmembrane protein that is expressed in normal tissues, including the heart, pancreas, and breast. It is also found and used as a diagnostic marker in many cancers, such as renal, brain, breast, oral, and lung cancers. We previously developed specific and sensitive anti-PODXL monoclonal antibodies, PcMab-47 (mouse IgG 1 , kappa) and its mouse IgG 2a -type (47-mG 2a ), both of which were suitable for immunohistochemical analyses of oral cancers. In this study, we investigated the utility of PcMab-47 and 47-mG 2a for the immunohistochemical analyses of lung cancers. PcMab-47 stained 51/70 (72.9%) cases of lung cancer, whereas 47-mG 2a stained 59/70 (84.3%) cases, indicating that the latter antibody is more sensitive and is useful for detecting PODXL in lung cancers.

  16. [Use of monoclonal antibodies against horse immunoglobulin in an enzyme immunoassay of bacterial toxins and anatoxins].

    Science.gov (United States)

    Burkin, M A; Gal'vidis, I A; Iakovleva, I V; Sviridov, V V

    2007-01-01

    Immunization of BALB/c mice by horse antiserum against diphtheria made it possible to obtain IgG1 monoclonal antibodies (MoAbs) 2B7E4 specific for light chains of horse immunoglobulin (Ig). Unlike commercial preparations of anti-horse immunoglobulin antibodies, which are specific for the whole Ig molecule or its Fc-fragment, the peroxidase (HRP) conjugate of the MoAb, 2B7E4-HRP did not interact with human, mouse, rabbit, and sheep Igs, or horse albumin. The conjugate obtained was used with MoAbs against bacterial toxins and commercial horse anatoxins, as a universal reagent in sandwich enzyme immunoassay (ELISA) for bacterial toxins and anatoxins. The detection sensitivity of diphtheria toxin/anatoxin equaled 0.0005 Lf/ml; tetanus toxin and anatoxin were detected with sensitivities of 20 LD50/ml and 0.005 UI/ml, respectively. A similar sandwich ELISA for botulinum anatoxins (group measurement) allowed types A, B, and E to be detected at 0.02, 0.002, and 0.001 UI/ml, respectively; selective measurement was only possible in the case of type E anatoxin (0.001 UI/ml).

  17. Preclinical Characterization of a Novel Monoclonal Antibody NEO-201 for the Treatment of Human Carcinomas

    Directory of Open Access Journals (Sweden)

    Massimo Fantini

    2018-01-01

    Full Text Available NEO-201 is a novel humanized IgG1 monoclonal antibody that was derived from an immunogenic preparation of tumor-associated antigens from pooled allogeneic colon tumor tissue extracts. It was found to react against a variety of cultured human carcinoma cell lines and was highly reactive against the majority of tumor tissues from many different carcinomas, including colon, pancreatic, stomach, lung, and breast cancers. NEO-201 also exhibited tumor specificity, as the majority of normal tissues were not recognized by this antibody. Functional assays revealed that treatment with NEO-201 is capable of mediating both antibody-dependent cellular cytotoxicity (ADCC and complement-dependent cytotoxicity (CDC against tumor cells. Furthermore, the growth of human pancreatic xenograft tumors in vivo was largely attenuated by treatment with NEO-201 both alone and in combination with human peripheral blood mononuclear cells as an effector cell source for ADCC. In vivo biodistribution studies in human tumor xenograft-bearing mice revealed that NEO-201 preferentially accumulates in the tumor but not organ tissue. Finally, a single-dose toxicity study in non-human primates demonstrated safety and tolerability of NEO-201, as a transient decrease in circulating neutrophils was the only related adverse effect observed. These findings indicate that NEO-201 warrants clinical testing as both a novel diagnostic and therapeutic agent for the treatment of a broad variety of carcinomas.

  18. Myeloma-Derived Light Chain Paired with a Diagnostic Monoclonal Antibody Hinders Immunoassay Performance.

    Science.gov (United States)

    Tu, Bailin; Tieman, Bryan; Moore, Jeffrey; Pan, You; Muerhoff, A Scott

    2017-06-01

    Monoclonal antibodies are widely used as the capture and detection reagents in diagnostic immunoassays. In the past, myeloma fusion partners expressing endogenous heavy and/or light chains were often used to generate hybridoma cell lines. As a result, mixed populations of antibodies were produced that can cause inaccurate test results, poor antibody stability, and significant lot-to-lot variability. We describe one such scenario where the P3U1 (P3X63Ag8U.1) myeloma fusion partner was used in the generation of a hybridoma producing protein induced vitamin K absence/antagonist-II (PIVKA II) antibody. The hybridoma produces three subpopulations of immunoglobulin as determined by ion exchange (IEx) chromatography that exhibit varying degrees of immunoreactivity (0%, 50%, or 100%) to the target antigen as determined by Surface Plasmon Resonance. To produce an antibody with the highest possible sensitivity and specificity, the antigen-specific heavy and light chain variable domains (VH and VL) were cloned from the hybridoma and tethered to murine IgG1 and kappa scaffolds. The resulting recombinant antibody was expressed in Chinese hamster ovary cells and is compatible for use in a diagnostic immunoassay.

  19. LpMab-23: A Cancer-Specific Monoclonal Antibody Against Human Podoplanin.

    Science.gov (United States)

    Yamada, Shinji; Ogasawara, Satoshi; Kaneko, Mika K; Kato, Yukinari

    2017-04-01

    Human podoplanin (hPDPN), the ligand of C-type lectin-like receptor-2, is involved in cancer metastasis. Until now, many monoclonal antibodies (mAbs) have been established against hPDPN. However, it is still difficult to develop a cancer-specific mAb (CasMab) against hPDPN because the protein sequence of hPDPN expressed in cancer cells is the same as that in normal cells. Herein, we report LpMab-23 of the mouse IgG 1 subclass, a novel CasMab against hPDPN. In an immunohistochemical analysis, LpMab-23 reacted with tumor cells of human oral cancer, but did not react with normal cells such as lymphatic endothelial cells (LECs). In contrast, LpMab-17, another anti-hPDPN mAb, reacted with both tumor cells and LECs. Furthermore, flow cytometric analysis revealed that LpMab-23 reacted with hPDPN-expressing cancer cell lines (LN319, RERF-LC-AI/hPDPN, Y-MESO-14/hPDPN, and HSC3/hPDPN) but showed little reaction with normal cells (LECs and HEK-293T), although another anti-hPDPN mAb, LpMab-7, reacted with both hPDPN-expressing cancer cells and normal cells, indicating that LpMab-23 is a CasMab against hPDPN.

  20. Monoclonal antibodies against peptidorhamnomannans of Scedosporium apiospermum enhance the pathogenicity of the fungus.

    Directory of Open Access Journals (Sweden)

    Livia C L Lopes

    2010-10-01

    Full Text Available Scedosporium apiospermum is part of the Pseudallescheria-Scedosporium complex. Peptidorhamnomannans (PRMs are cell wall glycopeptides present in some fungi, and their structures have been characterized in S. apiospermum, S. prolificans and Sporothrix schenckii. Prior work shows that PRMs can interact with host cells and that the glycopeptides are antigenic. In the present study, three monoclonal antibodies (mAbs, IgG1 to S. apiospermum derived PRM were generated and their effects on S. apiospermum were examined in vitro and in vivo. The mAbs recognized a carbohydrate epitope on PRM. In culture, addition of the PRM mAbs increased S. apiospermum conidia germination and reduced conidial phagocytosis by J774.16 macrophages. In a murine infection model, mice treated with antibodies to PRM died prior to control animals. Thus, PRM is involved in morphogenesis and the binding of this glycopeptide by mAbs enhanced the virulence of the fungus. Further insights into the effects of these glycopeptides on the pathobiology of S. apiospermum may lead to new avenues for preventing and treating scedosporiosis.

  1. Humanized versus murine anti-human epidermal growth factor receptor monoclonal antibodies for immunoscintigraphic studies

    International Nuclear Information System (INIS)

    Morales, Alejo A. Morales; Duconge, Jorge; Alvarez-Ruiz, Daniel; Becquer-Viart, Maria de Los Angeles; Nunez-Gandolff, Gilda; Fernandez, Eduardo; Caballero-Torres, Idania; Iznaga-Escobar, Normando

    2000-01-01

    The anti-human epidermal growth factor receptor (EGF-R) humanized antibody h-R3 (IgG 1 ), which binds to an extracellular domain of EGF-R, was used to evaluate the biodistribution on nude mice xenografted with A431 epidermoid carcinoma cell line. Results are compared with its murine version ior egf/r3 monoclonal antibody (mAb). Twenty-one athymic female 4NMRI nu/nu mice were injected intravenously with 10 μg/100 μCi of 99m Tc-labeled mAbs. The mAb ior C5 that recognizes an antigen expressed preferentially on the surface of malignant and cytoplasm of normal colorectal cells was used as negative control. Immunoreactivity of 99m Tc-labeled mAbs was measured by enzyme linked immunosorbent assay on A431 cell line and the immunoreactive fractions determined by Lindmo method. Among all organs significant accumulation was found in tumor (6.14±2.50 %ID/g, 5.06±2.61 %ID/g for murine and humanized mAbs, respectively) 4 h after injection. The immunoreactive fractions were found to be 0.88 and 0.81 for murine and humanized mAb, respectively. Thus, we expect better results using the humanized mAb h-R3 for diagnostic immunoscintigraphy

  2. Site-specific proteolytic degradation of IgG monoclonal antibodies expressed in tobacco plants.

    Science.gov (United States)

    Hehle, Verena K; Lombardi, Raffaele; van Dolleweerd, Craig J; Paul, Mathew J; Di Micco, Patrizio; Morea, Veronica; Benvenuto, Eugenio; Donini, Marcello; Ma, Julian K-C

    2015-02-01

    Plants are promising hosts for the production of monoclonal antibodies (mAbs). However, proteolytic degradation of antibodies produced both in stable transgenic plants and using transient expression systems is still a major issue for efficient high-yield recombinant protein accumulation. In this work, we have performed a detailed study of the degradation profiles of two human IgG1 mAbs produced in plants: an anti-HIV mAb 2G12 and a tumour-targeting mAb H10. Even though they use different light chains (κ and λ, respectively), the fragmentation pattern of both antibodies was similar. The majority of Ig fragments result from proteolytic degradation, but there are only a limited number of plant proteolytic cleavage events in the immunoglobulin light and heavy chains. All of the cleavage sites identified were in the proximity of interdomain regions and occurred at each interdomain site, with the exception of the VL /CL interface in mAb H10 λ light chain. Cleavage site sequences were analysed, and residue patterns characteristic of proteolytic enzymes substrates were identified. The results of this work help to define common degradation events in plant-produced mAbs and raise the possibility of predicting antibody degradation patterns 'a priori' and designing novel stabilization strategies by site-specific mutagenesis. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  3. Design, Synthesis, and in Vitro Pharmacology of New Radiolabeled γ-Hydroxybutyric Acid Analogues Including Photolabile Analogues with Irreversible Binding to the High-Affinity γ-Hydroxybutyric Acid Binding Sites

    DEFF Research Database (Denmark)

    Sabbatini, Paola; Wellendorph, Petrine; Høg, Signe

    2010-01-01

    γ-Hydroxybutyric acid (GHB) is a psychotropic compound endogenous to the brain. Despite its potential physiological significance, the complete molecular mechanisms of action remain unexplained. To facilitate the isolation and identification of the high-affinity GHB binding site, we herein report ...

  4. Aluminium fluoride and magnesium, activators of heterotrimeric GTP-binding proteins, affect high-affinity binding of the fungal toxin fusicoccin to the fusicoccin-binding protein in oat root plasma membranes.

    NARCIS (Netherlands)

    de Boer, A.H.; Van der Molen, G.W.; Prins, H.B.A.; Korthout, H.A.A.J.; van der Hoeven, P.C.J.

    1994-01-01

    The fusicoccin-binding protein was solubilised from purified oat root plasma membranes. The solubilised protein retained full binding activity, provided that protease inhibitors were included. Sodium fluoride reduced the high-affinity [H-3]fusicoccin binding to almost zero in a

  5. Comparative imaging and biodistribution studies with an anti-CEA monoclonal antibody and its F(ab)2 and Fab fragments in mice with colon carcinoma xenografts

    International Nuclear Information System (INIS)

    Andrew, S.M.; Pimm, M.V.; Baldwin, R.W.; Perkins, A.C.

    1986-01-01

    An IgG1 mouse monoclonal antibody directed against CEA has been digested with papain to yield F(ab) 2 and Fab fragments. Following radioiodination, intact antibody and fragments showed specific binding to cells of a CEA-producing tumour, although the immune reactivities of the fragments were lower than that of intact antibody. Gamma scintigraphy of nude mice bearing CEA producing human tumour xenografts and injected with 131 I-labelled fragments showed earlier and superior imaging of tumours than did 131 I-intact antibody, and this was most marked with the Fab fragment. Sequential dissection analyses showed that this was due to earlier and higher tumour-to-blood ratios with fragments than with intact antibody, but in absolute terms the degree of localization of both fragment types was significantly lower than that of intact antibody. (orig.)

  6. Monoclonal gammopathy in rheumatic diseases.

    Science.gov (United States)

    Yang, Yue; Chen, Long; Jia, Yuan; Liu, Yang; Wen, Lei; Liang, Yaoxian; An, Yuan; Chen, Shi; Su, Yin; Li, Zhanguo

    2018-07-01

    To analyze the clinical spectrum, laboratory characteristics, and outcomes of monoclonal gammopathy (MG) in patients with rheumatic diseases. Screening for the presence of MG was performed in 872 inpatients with rheumatic diseases from January 2010 to July 2017. A total of 41 patients were enrolled. Their clinical and biological features in addition to outcomes were described. For each patient with primary Sjögren syndrome (pSS), 2 age- and sex-matched pSS patients without MG were selected as controls. Risk factors for the presence of MG and malignant hematological neoplasias were assessed. MG was observed in patients with SS, rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, primary biliary cirrhosis, polymyositis, hypomyopathic dermatomyositis, psoriatic arthritis, ANCA-associated vasculitis, polyarteritis nodosa, and polymyalgia rheumatic, with SS the most frequent type. Serum M protein was detected in 37 patients. The monoclonal bands identified in serum were 16 IgG (5 κ, 11 λ), 11 IgA (6 κ, 5 λ), 6 IgM (5 κ, 1 λ), and 4 free λ chains. M components were observed in urine in the other 4 patients. High ESR, albumin/globulin inversion, rheumatoid factor positivity, hypergammaglobulinemia, and hypocomplementemia were common features, presented in more than half of the 41 patients. Patients with pSS, when complicated with MG, showed a higher rate of abnormal urine NAG (71.4 vs 15.8%, P = 0.025), higher levels of ESR [55.0 (53.5) mm/h vs 21.0 (31.8) mm/h, P = 0.001], ESSDAI [26.0 (25.0) vs 12.0 (9.0), P = 0.006], and ClinESSDAI scores [24.0 (25.0) vs 10.5 (10.0), P = 0.011]. Multivariate analysis revealed that the disease activity, assessed by either ESSDAI [adjusted OR 1.127 (95%CI 1.015-1.251), P = 0.025] or ClinESSDAI [adjusted OR 1.121 (95%CI 1.011-1.242), P = 0.030], was the only independent risk factor for the presence of MG. During the follow-up, 2 patients had transient serum M protein, 2 had isotype

  7. Relationship between hyperthyroidism and monoclonal gammapathy

    International Nuclear Information System (INIS)

    Canas, Carlos Alberto

    2007-01-01

    A 66-year-old man with primary hyperparathyroidism (PHPT) and monoclonal gammapathy associated to it of uncertain significance (MGUS). A possible pathogenic relationship between HPTP and MGUS is analyzed. Interleukin 6 could play a pivotal role.

  8. Monoclonal antibody therapy of inflammatory bowel disease

    NARCIS (Netherlands)

    van Deventer, S. J.; Camoglio, L.

    1997-01-01

    Animal models of inflammatory bowel disease have provided insight in the regulation of mucosal inflammation. This has resulted in novel therapeutic approaches that specifically target a single inflammatory mediator. Monoclonal antibody therapy has been used in steroid refractory Crohn's disease

  9. Pulmonary Administration of GW0742, a High-Affinity Peroxisome Proliferator-Activated Receptor Agonist, Repairs Collapsed Alveoli in an Elastase-Induced Mouse Model of Emphysema.

    Science.gov (United States)

    Ozawa, Chihiro; Horiguchi, Michiko; Akita, Tomomi; Oiso, Yuki; Abe, Kaori; Motomura, Tomoki; Yamashita, Chikamasa

    2016-01-01

    Pulmonary emphysema is a disease in which lung alveoli are irreversibly damaged, thus compromising lung function. Our previous study revealed that all-trans-retinoic acid (ATRA) induces the differentiation of human lung alveolar epithelial type 2 progenitor cells and repairs the alveoli of emphysema model mice. ATRA also reportedly has the ability to activate peroxisome proliferator-activated receptor (PPAR) β/δ. A selective PPARβ/δ ligand has been reported to induce the differentiation of human keratinocytes during wound repair. Here, we demonstrate that treatment using a high-affinity PPARβ/δ agonist, GW0742, reverses the lung tissue damage induced by elastase in emphysema-model mice and improves respiratory function. Mice treated with elastase, which collapsed their alveoli, were then treated with either 10% dimethyl sulfoxide (DMSO) in saline (control group) or GW0742 (1.0 mg/kg twice a week) by pulmonary administration. Treatment with GW0742 for 2 weeks increased the in vivo expression of surfactant proteins A and D, which are known alveolar type II epithelial cell markers. GW0742 treatment also shortened the average distance between alveolar walls in the lungs of emphysema model mice, compared with a control group treated with 10% DMSO in saline. Treatment with GW0742 for 3 weeks also improved tissue elastance (cm H2O/mL), as well as the ratio of the forced expiratory volume in the first 0.05 s to the forced vital capacity (FEV 0.05/FVC). In each of these experiments, GW0742 treatment reversed the damage caused by elastase. In conclusion, PPARβ/δ agonists are potential therapeutic agents for pulmonary emphysema.

  10. Synthesis of hapten and preparation of specific polyclonal antibody with high affinity for lenalidomide, the potent drug for treatment of multiple myeloma

    Directory of Open Access Journals (Sweden)

    Darwish Ibrahim A

    2012-10-01

    Full Text Available Abstract Background For therapeutic monitoring and pharmacokinetic studies of lenalidomide (LND, the potent drug for treatment of multiple myeloma (MM, a specific antibody was required for the development of a sensitive immunoassay system for the accurate determination of LND in plasma. Results In this study, a hapten of LND (N-glutaryl-LND was synthesized by introducing the glutaryl moiety, as a spacer, into the primary aromatic amine site of the LND molecular structure. The structure of the hapten (G-LND was confirmed by mass, 1H-NMR, and 13C spectrometric techniques. G-LND was coupled to each of bovine serum albumin (BSA and keyhole limpet hemocyanin (KLH proteins by ethyl-3-(3-dimethylaminopropyl carbodiimide as a coupling reagent. LND-KLH conjugate was used as an immunogen. Four female 2-3 months old New Zealand white rabbits were immunized with an emulsion of LND-KLH with Freund`s adjuvant. The immune response of the rabbits was monitored by direct enzyme-linked immunosorbent assay (ELISA using LND-BSA immobilized onto microwell plates as a solid phase. The rabbit that showed the highest antibody titer and affinity to LND was scarified and its sera were collected. The IgG fraction was isolated and purified by affinity chromatography on protein A column. The specificity of the purified antibody for LND was evaluated by indirect competitive ELISA using dexamethasone as a competitor as it is used with LND in a combination therapy. Conclusions The high affinity of the antibody (IC50 = 10 ng/mL will be useful in the development of an immunoassay system for the determination of plasma LND concentrations. Current research is going to optimize the assay conditions and validate the procedures for the routine application in clinical laboratories.

  11. Synthesis of hapten and preparation of specific polyclonal antibody with high affinity for lenalidomide, the potent drug for treatment of multiple myeloma.

    Science.gov (United States)

    Darwish, Ibrahim A; Alzoman, Nourh Z; Abuhejail, Reem M; El-Samani, Tilal E

    2012-10-26

    For therapeutic monitoring and pharmacokinetic studies of lenalidomide (LND), the potent drug for treatment of multiple myeloma (MM), a specific antibody was required for the development of a sensitive immunoassay system for the accurate determination of LND in plasma. In this study, a hapten of LND (N-glutaryl-LND) was synthesized by introducing the glutaryl moiety, as a spacer, into the primary aromatic amine site of the LND molecular structure. The structure of the hapten (G-LND) was confirmed by mass, 1H-NMR, and 13C spectrometric techniques. G-LND was coupled to each of bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) proteins by ethyl-3-(3-dimethylaminopropyl) carbodiimide as a coupling reagent. LND-KLH conjugate was used as an immunogen. Four female 2-3 months old New Zealand white rabbits were immunized with an emulsion of LND-KLH with Freund`s adjuvant. The immune response of the rabbits was monitored by direct enzyme-linked immunosorbent assay (ELISA) using LND-BSA immobilized onto microwell plates as a solid phase. The rabbit that showed the highest antibody titer and affinity to LND was scarified and its sera were collected. The IgG fraction was isolated and purified by affinity chromatography on protein A column. The specificity of the purified antibody for LND was evaluated by indirect competitive ELISA using dexamethasone as a competitor as it is used with LND in a combination therapy. The high affinity of the antibody (IC50 = 10 ng/mL) will be useful in the development of an immunoassay system for the determination of plasma LND concentrations. Current research is going to optimize the assay conditions and validate the procedures for the routine application in clinical laboratories.

  12. Spot 42 Small RNA Regulates Arabinose-Inducible araBAD Promoter Activity by Repressing Synthesis of the High-Affinity Low-Capacity Arabinose Transporter

    Science.gov (United States)

    Chen, Jiandong

    2016-01-01

    ABSTRACT The l-arabinose-inducible araBAD promoter (PBAD) enables tightly controlled and tunable expression of genes of interest in a broad range of bacterial species. It has been used successfully to study bacterial sRNA regulation, where PBAD drives expression of target mRNA translational fusions. Here we report that in Escherichia coli, Spot 42 sRNA regulates PBAD promoter activity by affecting arabinose uptake. We demonstrate that Spot 42 sRNA represses araF, a gene encoding the AraF subunit of the high-affinity low-capacity arabinose transporter AraFGH, through direct base-pairing interactions. We further show that endogenous Spot 42 sRNA is sufficient to repress araF expression under various growth conditions. Finally, we demonstrate this posttranscriptional repression has a biological consequence, decreasing the induction of PBAD at low levels of arabinose. This problem can be circumvented using strategies reported previously for avoiding all-or-none induction behavior, such as through constitutive expression of the low-affinity high-capacity arabinose transporter AraE or induction with a higher concentration of inducers. This work adds araF to the set of Spot 42-regulated genes, in agreement with previous studies suggesting that Spot 42, itself negatively regulated by the cyclic AMP (cAMP) receptor protein-cAMP complex, reinforces the catabolite repression network. IMPORTANCE The bacterial arabinose-inducible system is widely used for titratable control of gene expression. We demonstrate here that a posttranscriptional mechanism mediated by Spot 42 sRNA contributes to the functionality of the PBAD system at subsaturating inducer concentrations by affecting inducer uptake. Our finding extends the inputs into the known transcriptional control for the PBAD system and has implications for improving its usage for tunable gene expression. PMID:27849174

  13. Generation of high-affinity, internalizing anti-FGFR2 single-chain variable antibody fragment fused with Fc for targeting gastrointestinal cancers.

    Science.gov (United States)

    Borek, Aleksandra; Sokolowska-Wedzina, Aleksandra; Chodaczek, Grzegorz; Otlewski, Jacek

    2018-01-01

    Fibroblast growth factor receptors (FGFRs) are promising targets for antibody-based cancer therapies, as their substantial overexpression has been found in various tumor cells. Aberrant activation of FGF receptor 2 (FGFR2) signaling through overexpression of FGFR2 and/or its ligands, mutations, or receptor amplification has been reported in multiple cancer types, including gastric, colorectal, endometrial, ovarian, breast and lung cancer. In this paper, we describe application of the phage display technology to produce a panel of high affinity single chain variable antibody fragments (scFvs) against the extracellular ligand-binding domain of FGFR2 (ECD_FGFR2). The binders were selected from the human single chain variable fragment scFv phage display libraries Tomlinson I + J and showed high specificity and binding affinity towards human FGFR2 with nanomolar KD values. To improve the affinity of the best binder selected, scFvF7, we reformatted it to a bivalent diabody format, or fused it with the Fc region (scFvF7-Fc). The scFvF7-Fc antibody construct presented the highest affinity for FGFR2, with a KD of 0.76 nM, and was selectively internalized into cancer cells overexpressing FGFR2, Snu-16 and NCI-H716. Finally, we prepared a conjugate of scFvF7-Fc with the cytotoxic drug monomethyl-auristatin E (MMAE) and evaluated its cytotoxicity. The conjugate delivered MMAE selectively to FGFR2-positive tumor cells. These results indicate that scFvF7-Fc-vcMMAE is a highly potent molecule for the treatment of cancers with FGFR2 overexpression.

  14. High-affinity binding of [3H]estradiol-17 beta by an estrogen receptor in the liver of the turtle

    International Nuclear Information System (INIS)

    Ho, S.M.; Fehrer, S.; Yu, M.; Liang, L.C.; Press, D.

    1988-01-01

    Specific [3H]estradiol-17 beta ([3H]E2) binding activity (EBA) with characteristics of an estrogen receptor (ER) was demonstrated in cytosols and nuclear extracts of the female turtle, Chrysemys picta. Three different receptor assays (dextran-coated charcoal assay, hydroxylapatite batch procedure, and DNA-cellulose chromatography) were evaluated in terms of their applicability in analyzing large numbers of samples. For the measurement of cytosolic EBA, the hydroxylapatite batch procedure was found to be the most reliable assay. On the other hand, the dextran-coated charcoal assay was found to be the most appropriate method for the measurement of nuclear EBA. Turtle hepatic EBA binds [3H]E2 with high affinity (cytosolic, 17.4 +/- 2.8 X 10(9) M-1; nuclear, 17.7 +/- 1.9 X 10(9) M-1), limited capacity (cytosolic, 133.7 +/- 4.6 fmol/g tissue; nuclear, 81.1 +/- 9.0 fmol/g tissue), and strict steroid specificity. The EBA bound natural estrogens (E2, estrone, estriol) as well as the nonsteroidal estrogen, diethylstilbestrol, but exhibited little affinity for androgens, progesterone, or corticosterone. The turtle hepatic EBA resembled mammalian and avian ERs in terms of binding characteristics; however, unlike mammalian and avian ERs it was shown to be heat-labile. Incubation at 30 degrees caused rapid loss of [3H]E2 binding activity in both cytosolic and nuclear fractions. The exchange between [3H]E2 and the endogenously bound estrogen was slow at 4 and 15 degrees, but the exchange process was facilitated in the presence of the chaotropic salt, NaSCN. Establishment of quantitation methods for both cytosolic and nuclear forms of EBA will enable future investigation of the mechanism and regulation of estrogen action in the liver of this turtle species

  15. (3H)leukotriene B4 binding to the guinea pig spleen membranes: a rich tissue source for a high affinity leukotriene B4 receptor site

    International Nuclear Information System (INIS)

    Cheng, J.B.; Kohi, F.; Townley, R.G.

    1986-01-01

    To select a tissue rich for the high affinity leukotriene (LT)B 4 receptor site, they compared binding of 1 nM ( 3 H)LTB 4 (180 Ci/mmol) to the crude membrane preparations of guinea pig spleen, thymus, lung, uterus, bladder, brain, adrenal gland, small intestine, liver, kidney and heart. They found that the membrane preparations from spleen contained the highest binding activity per mg protein. They characterized the LTB 4 binding to the spleen preparation in detail. LTB 4 binding was rapid, reversible, stereoselective and saturable. The data from equilibrium experiments showed a linear Scatchard plot with a K/sub d/ of 1.6 nM and a binding site density of 259 fmol/mg prot. The rank order of agents competing for spleen ( 3 H)LTB 4 binding at 25 0 C was: LTB 4 (K/sub i/ = 2.8 nM) > 20-OH-LTB 4 (23 nM) > LTA 4 (48 nM) > LTA 4 methyl ester (0.13 μM) > 20-COOH-LTB 4 (> 6.6 μM) ≥ arachidonic acid (0.15 mM) similarly ordered FPL-55,712 (0.11 mM). At 4 0 C, LTB 4 (2.3 nM) competed at least 10x more effectively than 20-OH-LTB 4 (29 nM) and 20-COOH-LTB 4 (> 6.6 μM). HPLC analysis indicated that incubation of 84 ng LTB 4 with the spleen membrane at 25 0 C did not result in the formation of 20-OH-LTB 4 ( 3 H)LTB 4 receptor binding sites

  16. The high affinity K+ transporter AtHAK5 plays a physiological role in planta at very low K+ concentrations and provides a caesium uptake pathway in Arabidopsis.

    Science.gov (United States)

    Qi, Zhi; Hampton, Corrina R; Shin, Ryoung; Barkla, Bronwyn J; White, Philip J; Schachtman, Daniel P

    2008-01-01

    Caesium (Cs(+)) is a potentially toxic mineral element that is released into the environment and taken up by plants. Although Cs(+) is chemically similar to potassium (K(+)), and much is known about K(+) transport mechanisms, it is not clear through which K(+) transport mechanisms Cs(+) is taken up by plant roots. In this study, the role of AtHAK5 in high affinity K(+) and Cs(+) uptake was characterized. It is demonstrated that AtHAK5 is localized to the plasma membrane under conditions of K(+) deprivation, when it is expressed. Growth analysis showed that AtHAK5 plays a role during severe K(+) deprivation. Under K(+)-deficient conditions in the presence of Cs(+), Arabidopsis seedlings lacking AtHAK5 had increased inhibition of root growth and lower Cs(+) accumulation, and significantly higher leaf chlorophyll concentrations than wild type. These data indicate that, in addition to transporting K(+) in planta, AtHAK5 also transports Cs(+). Further experiments showed that AtHAK5 mediated Cs(+) uptake into yeast cells and that, although the K(+) deficiency-induced expression of AtHAK5 was inhibited by low concentrations of NH(4)(+) in planta, Cs(+) uptake by yeast was stimulated by low concentrations of NH(4)(+). Interestingly, the growth of the Arabidopsis atakt1-1 mutant was more sensitive to Cs(+) than the wild type. This may be explained, in part, by increased expression of AtHAK5 in the atakt1-1 mutant. It is concluded that AtHAK5 is a root plasma membrane uptake mechanism for K(+) and Cs(+) under conditions of low K(+) availability.

  17. Preliminary assessment of extrastriatal dopamine d-2 receptor binding in the rodent and nonhuman primate brains using the high affinity radioligand, {sup 18}F-fallypride

    Energy Technology Data Exchange (ETDEWEB)

    Mukherjee, Jogeshwar E-mail: jogeshwar-mukherjee@ketthealth.com; Yang, Z.-Y.; Brown, Terry; Lew, Robert; Wernick, Miles; Ouyang Xiaohu; Yasillo, Nicholas; Chen, C.-T.; Mintzer, Robert; Cooper, Malcolm

    1999-07-01

    We have identified the value of {sup 18}F-fallypride {l_brace}(S)-N-[(1-allyl-2-pyrrolidinyl)methyl]-5-(3-[{sup 18}F]fluoropropyl)-2,3-dim= ethoxybenzamide{r_brace}, as a dopamine D-2 receptor radiotracer for the study of striatal and extrastriatal receptors. Fallypride exhibits high affinities for D-2 and D-3 subtypes and low affinity for D-4 ({sup 3}H-spiperone IC{sub 50}s: D-2=0.05 nM [rat striata], D-3=0.30 nM [SF9 cell lines, rat recombinant], and D-4=240 nM [CHO cell lines, human recombinant]). Biodistribution in the rat brain showed localization of {sup 18}F-fallypride in striata and extrastriatal regions such as the frontal cortex, parietal cortex, amygdala, hippocampus, thalamus, and hypothalamus. In vitro autoradiographic studies in sagittal slices of the rat brain showed localization of {sup 18}F-fallypride in striatal and several extrastriatal regions, including the medulla. Positron emission tomography (PET) experiments with {sup 18}F-fallypride in male rhesus monkeys were carried out in a PET VI scanner. In several PET experiments, apart from the specific binding seen in the striatum, specific binding of {sup 18}F-fallypride was also identified in extracellular regions (in a lower brain slice, possibly the thalamus). Specific binding in the extrastriata was, however, significantly lower compared with that observed in the striata of the monkeys (extrastriata/cerebellum = 2, striata/cerebellum = 10). Postmortem analysis of the monkey brain revealed significant {sup 18}F-fallypride binding in the striata, whereas binding was also observed in extrastriatal regions such as the thalamus, cortical areas, and brain stem.

  18. SNF3 as high affinity glucose sensor and its function in supporting the viability of Candida glabrata under glucose-limited environment

    Directory of Open Access Journals (Sweden)

    Tzu Shan eNg

    2015-12-01

    Full Text Available Candida glabrata is an emerging human fungal pathogen that has efficacious nutrient sensing and responsiveness ability. It can be seen through its ability to thrive in diverse range of nutrient limited-human anatomical sites. Therefore, nutrient sensing particularly glucose sensing is thought to be crucial in contributing to the development and fitness of the pathogen. This study aimed to elucidate the role of SNF3 (Sucrose Non Fermenting 3 as a glucose sensor and its possible role in contributing to the fitness and survivability of C. glabrata in glucose-limited environment. The SNF3 knockout strain was constructed and subjected to different glucose concentrations to evaluate its growth, biofilm formation, amphotericin B susceptibility, ex vivo survivability and effects on the transcriptional profiling of the sugar receptor repressor (SRR pathway-related genes. The SNF3Δ strain showed a retarded growth in low glucose environments (0.01% and 0.1% in both fermentation and respiration-preferred conditions but grew well in high glucose concentration environments (1% and 2%. It was also found to be more susceptible to amphotericin B in low glucose environment (0.1% and macrophage engulfment but showed no difference in the biofilm formation capability. The deletion of SNF3 also resulted in the down-regulation of about half of hexose transporters genes (4 out of 9. Overall, the deletion of SNF3 causes significant reduction in the ability of C. glabrata to sense limited surrounding glucose and consequently disrupts its competency to transport and perform the uptake of this critical nutrient. This study highlighted the role of SNF3 as a high affinity glucose sensor and its role in aiding the survivability of C. glabrata particularly in glucose limited environment.

  19. A conserved motif in the linker domain of STAT1 transcription factor is required for both recognition and release from high-affinity DNA-binding sites.

    Science.gov (United States)

    Hüntelmann, Bettina; Staab, Julia; Herrmann-Lingen, Christoph; Meyer, Thomas

    2014-01-01

    Binding to specific palindromic sequences termed gamma-activated sites (GAS) is a hallmark of gene activation by members of the STAT (signal transducer and activator of transcription) family of cytokine-inducible transcription factors. However, the precise molecular mechanisms involved in the signal-dependent finding of target genes by STAT dimers have not yet been very well studied. In this study, we have characterized a sequence motif in the STAT1 linker domain which is highly conserved among the seven human STAT proteins and includes surface-exposed residues in close proximity to the bound DNA. Using site-directed mutagenesis, we have demonstrated that a lysine residue in position 567 of the full-length molecule is required for GAS recognition. The substitution of alanine for this residue completely abolished both binding to high-affinity GAS elements and transcriptional activation of endogenous target genes in cells stimulated with interferon-γ (IFNγ), while the time course of transient nuclear accumulation and tyrosine phosphorylation were virtually unchanged. In contrast, two glutamic acid residues (E559 and E563) on each monomer are important for the dissociation of dimeric STAT1 from DNA and, when mutated to alanine, result in elevated levels of tyrosine-phosphorylated STAT1 as well as prolonged IFNγ-stimulated nuclear accumulation. In conclusion, our data indicate that the kinetics of signal-dependent GAS binding is determined by an array of glutamic acid residues located at the interior surface of the STAT1 dimer. These negatively charged residues appear to align the long axis of the STAT1 dimer in a position perpendicular to the DNA, thereby facilitating the interaction between lysine 567 and the phosphodiester backbone of a bound GAS element, which is a prerequisite for transient gene induction.

  20. Dual regulation of root hydraulic conductivity and plasma membrane aquaporins by plant nitrate accumulation and high-affinity nitrate transporter NRT2.1.

    Science.gov (United States)

    Li, Guowei; Tillard, Pascal; Gojon, Alain; Maurel, Christophe

    2016-04-01

    The water status and mineral nutrition of plants critically determine their growth and development. Nitrate (NO3(-)), the primary nitrogen source of higher plants, is known to impact the water transport capacity of roots (root hydraulic conductivity, Lpr). To explore the effects and mode of action of NO3(-) on Lpr, we used an extended set of NO3(-) transport (nrt1.1, nrt1.2, nrt1.5 and nrt2.1), signaling (nrt1.1 and nrt2.1) and metabolism (nia) mutants in Arabidopsis, grown under various NO3(-) conditions. First, a strong positive relationship between Lpr and NO3(-) accumulation, in shoots rather than in roots, was revealed. Secondly, a specific 30% reduction of Lpr in nrt2.1 plants unraveled a major role for the high-affinity NO3(-) transporter NRT2.1 in increasing Lpr These results indicate that NO3(-)signaling rather than nitrogen assimilation products governs Lpr in Arabidopsis. Quantitative real-time reverse transcription-PCR and enzyme-linked immunosorbent assays (ELISAs) were used to investigate the effects of NO3(-) availability on plasma membrane aquaporin (plasma membrane intrinsic protein; PIP) expression. Whereas PIP regulation mostly occurs at the post-translational level in wild-type plants, a regulation of PIPs at both the transcriptional and translational levels was uncovered in nrt2.1 plants. In conclusion, this work reveals that control of Arabidopsis Lpr and PIP functions by NO3(-) involves novel shoot to root signaling and NRT2.1-dependent functions. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  1. Murine CMV Expressing the High Affinity NKG2D Ligand MULT-1: A Model for the Development of Cytomegalovirus-Based Vaccines

    Directory of Open Access Journals (Sweden)

    Lea Hiršl

    2018-05-01

    Full Text Available The development of a vaccine against human cytomegalovirus (CMV has been a subject of long-term medical interest. The research during recent years identified CMV as an attractive vaccine vector against infectious diseases and tumors. The immune response to CMV persists over a lifetime and its unique feature is the inflationary T cell response to certain viral epitopes. CMV encodes numerous genes involved in immunoevasion, which are non-essential for virus growth in vitro. The deletion of those genes results in virus attenuation in vivo, which enables us to dramatically manipulate its virulence and the immune response. We have previously shown that the murine CMV (MCMV expressing RAE-1γ, one of the cellular ligands for the NKG2D receptor, is highly attenuated in vivo but retains the ability to induce a strong CD8+ T cell response. Here, we demonstrate that recombinant MCMV expressing high affinity NKG2D ligand murine UL16 binding protein-like transcript (MULT-1 (MULT-1MCMV inserted in the place of its viral inhibitor is dramatically attenuated in vivo in a NK cell-dependent manner, both in immunocompetent adult mice and in immunologically immature newborns. MULT-1MCMV was more attenuated than the recombinant virus expressing RAE-1γ. Despite the drastic sensitivity to innate immune control, MULT-1MCMV induced an efficient CD8+ T cell response to viral and vectored antigens. By using in vitro assay, we showed that similar to RAE-1γMCMV, MULT-1 expressing virus provided strong priming of CD8+ T cells. Moreover, MULT-1MCMV was able to induce anti-viral antibodies, which after passing the transplacental barrier protect offspring of immunized mothers from challenge infection. Altogether, this study further supports the concept that CMV expressing NKG2D ligand possesses excellent characteristics to serve as a vaccine or vaccine vector.

  2. High-affinity interaction of hnRNP A1 with conserved RNA structural elements is required for translation and replication of enterovirus 71.

    Science.gov (United States)

    Levengood, Jeffrey D; Tolbert, Michele; Li, Mei-Ling; Tolbert, Blanton S

    2013-07-01

    Human Enterovirus 71 (EV71) is an emerging pathogen of infectious disease and a serious threat to public health. Currently, there are no antivirals or vaccines to slow down or prevent EV71 infections, thus underscoring the urgency to better understand mechanisms of host-enterovirus interactions. EV71 uses a type I internal ribosome entry site (IRES) to recruit the 40S ribosomal subunit via a pathway that requires the cytoplasmic localization of hnRNP A1, which acts as an IRES trans-activating factor. The mechanism of how hnRNP A1 trans activates EV71 RNA translation is unknown, however. Here, we report that the UP1 domain of hnRNP A1 interacts specifically with stem loop II (SLII) of the IRES, via a thermodynamically well-defined biphasic transition that involves conserved bulge 5'-AYAGY-3' and hairpin 5'-RY(U/A)CCA-3' loops. Calorimetric titrations of wild-type and mutant SLII constructs reveal these structural elements are essential to form a high-affinity UP1-SLII complex. Mutations that alter the bulge and hairpin primary or secondary structures abrogate the biphasic transition and destabilize the complex. Notably, mutations within the bulge that destabilize the complex correlate with a large reduction in IRES-dependent translational activity and impair EV71 replication. Taken together, this study shows that a conserved SLII structure is necessary to form a functional hnRNP A1-IRES complex, suggesting that small molecules that target this stem loop may have novel antiviral properties.

  3. Monoclonal for cancer detection and therapy

    International Nuclear Information System (INIS)

    Baldwin, R.W.; Byers, V.S.

    1985-01-01

    This book contains 18 chapters. Some of the chapter titles are: Monoclonal Antibodies to Breast Cancer and Their Application; Clinical Applications of Radioimmunolocalisation; Localisation of Cancer of the Ovary and Metastases Using 123 I-labelled Monoclonal Antibody HMFG-2 Compared to Surgical Findings; Interest of Globotriaosylceramide Membrane Antigen as Target for Immunotoxins; and Analysis, Results and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy

  4. Antibodies and Selection of Monoclonal Antibodies.

    Science.gov (United States)

    Hanack, Katja; Messerschmidt, Katrin; Listek, Martin

    Monoclonal antibodies are universal binding molecules with a high specificity for their target and are indispensable tools in research, diagnostics and therapy. The biotechnological generation of monoclonal antibodies was enabled by the hybridoma technology published in 1975 by Köhler and Milstein. Today monoclonal antibodies are used in a variety of applications as flow cytometry, magnetic cell sorting, immunoassays or therapeutic approaches. First step of the generation process is the immunization of the organism with appropriate antigen. After a positive immune response the spleen cells are isolated and fused with myeloma cells in order to generate stable, long-living antibody-producing cell lines - hybridoma cells. In the subsequent identification step the culture supernatants of all hybridoma cells are screened weekly for the production of the antibody of interest. Hybridoma cells producing the antibody of interest are cloned by limited dilution till a monoclonal hybridoma is found. This is a very time-consuming and laborious process and therefore different selection strategies were developed since 1975 in order to facilitate the generation of monoclonal antibodies. Apart from common automation of pipetting processes and ELISA testing there are some promising approaches to select the right monoclonal antibody very early in the process to reduce time and effort of the generation. In this chapter different selection strategies for antibody-producing hybridoma cells are presented and analysed regarding to their benefits compared to conventional limited dilution technology.

  5. Monoclonal antibodies protect from Staphylococcal Enterotoxin K (SEK) induced toxic shock and sepsis by USA300 Staphylococcus aureus.

    Science.gov (United States)

    Aguilar, Jorge L; Varshney, Avanish K; Pechuan, Ximo; Dutta, Kaushik; Nosanchuk, Joshua D; Fries, Bettina C

    2017-08-18

    Staphylococcus aureus is a leading infectious cause of life-threatening disease in humans, yet there is currently no vaccine to combat this bacterium. The pathogenesis of S. aureus is mediated by a diverse array of protein toxins including a large family of secreted pyrogenic superantigens. Neutralization of superantigens, including SEB and TSST-1, has proven to be protective in several animal models of toxic shock and sepsis. We demonstrate, for the first time, that a far more prevalent staphylococcal superantigen, SEK, can also induce lethal shock in mice. Additionally, we describe monoclonal antibodies (mAbs) that inhibit SEK-induced mitogenicity as well as protect against SEK-induced lethality, and enhance survival from S. aureus septicemia in murine models. MAb-4G3 (IgG2b), mAb-5G2 (IgG1), and mAb-9H2 (IgG1), all inhibit SEK-induced proliferation and cytokine production of human immune cells. We then demonstrate that passive immunization with a combination of mAb-4G3 and mAb-5G4, 2 mAbs that do not compete for epitope(s) on SEK, significantly enhance survival in a murine model of SEK-induced toxic shock (p = 0.006). In the setting of sepsis, passive immunization with this combination of mAbs also significantly enhances survival in mice after challenge with CA-MRSA strain USA300 (p = 0.03). Furthermore, septic mice that received mAb treatment in conjunction with vancomycin exhibit less morbidity than mice treated with vancomycin alone. Taken together, these findings suggest that the contribution of SEK to S. aureus pathogenesis may be greater than previously appreciated, and that adjunctive therapy with passive immunotherapy against SEs may be beneficial.

  6. Systemic radiotherapy with monoclonal antibodies

    International Nuclear Information System (INIS)

    Sautter-Bihl, M.L.; Matzku, S.; Bihl, H.

    1993-01-01

    In this experimental study, feasibility and efficiency of systematic radiotherapy with the I-131 labelled monoclonal antibody BW575/9 (radioimmunotherapy) are investigated using human SK-N-SH neuroblastoma transplated into nude mice. Series of six nude mice were treated with intravenous application of 400 μCi (group 1), 700 μCi (group 2) of the I-131 labelled and of the unlabelled MAb (group 3). An untreated group (group 4) served as control. Tumors of group (3) and (4) showed an identical growth. In group (1), tumor growth was arrested for seven days. In group (2), the tumor showed complete regression after eight days which lasted for 55 days. Thereafter, the tumor started to regrow. This growth characteristics are correlated with the doses achieved in the tumor using a medical radiation dose (MIRD) formulation. The biodistribution data necessary for MIRD calculation were obtained by previously performed experiments with the I-125 labelled MAb. The doses assessed in the tumor turned out to be five to ten times greater than those in normal tissues (liver, bone, etc.) These results confirm feasibility, selectivity and efficiency of radioimmunotherapy in the above described model. Moreover, this in vivo model seems suitable for further investigations concerning fundamental issues of radioimunotherapy. (orig.) [de

  7. PdeH, a high-affinity cAMP phosphodiesterase, is a key regulator of asexual and pathogenic differentiation in Magnaporthe oryzae.

    Directory of Open Access Journals (Sweden)

    Ravikrishna Ramanujam

    2010-05-01

    Full Text Available Cyclic AMP-dependent pathways mediate the communication between external stimuli and the intracellular signaling machinery, thereby influencing important aspects of cellular growth, morphogenesis and differentiation. Crucial to proper function and robustness of these signaling cascades is the strict regulation and maintenance of intracellular levels of cAMP through a fine balance between biosynthesis (by adenylate cyclases and hydrolysis (by cAMP phosphodiesterases. We functionally characterized gene-deletion mutants of a high-affinity (PdeH and a low-affinity (PdeL cAMP phosphodiesterase in order to gain insights into the spatial and temporal regulation of cAMP signaling in the rice-blast fungus Magnaporthe oryzae. In contrast to the expendable PdeL function, the PdeH activity was found to be a key regulator of asexual and pathogenic development in M. oryzae. Loss of PdeH led to increased accumulation of intracellular cAMP during vegetative and infectious growth. Furthermore, the pdeHDelta showed enhanced conidiation (2-3 fold, precocious appressorial development, loss of surface dependency during pathogenesis, and highly reduced in planta growth and host colonization. A pdeHDelta pdeLDelta mutant showed reduced conidiation, exhibited dramatically increased (approximately 10 fold cAMP levels relative to the wild type, and was completely defective in virulence. Exogenous addition of 8-Br-cAMP to the wild type simulated the pdeHDelta defects in conidiation as well as in planta growth and development. While a fully functional GFP-PdeH was cytosolic but associated dynamically with the plasma membrane and vesicular compartments, the GFP-PdeL localized predominantly to the nucleus. Based on data from cAMP measurements and Real-Time RTPCR, we uncover a PdeH-dependent biphasic regulation of cAMP levels during early and late stages of appressorial development in M. oryzae. We propose that PdeH-mediated sustenance and dynamic regulation of cAMP signaling

  8. 2,2'-Dithiobis(N-ethyl-spermine-5-carboxamide) is a high affinity, membrane-impermeant antagonist of the mammalian polyamine transport system.

    Science.gov (United States)

    Huber, M; Pelletier, J G; Torossian, K; Dionne, P; Gamache, I; Charest-Gaudreault, R; Audette, M; Poulin, R

    1996-11-01

    We have synthesized 2,2'-dithiobis(N-ethyl-spermine-5-carboxamide) (DESC), its thiol monomer (MESC), and the mixed MESC-cysteamine disulfide (DEASC) as potential inhibitors of polyamine transport in mammalian cells. DESC was the most potent antagonist of spermine transport in ZR-75-1 human breast cancer cells, with Ki values of 5. 0 +/- 0.7, 80 +/- 31, and 16 +/- 3 microM for DESC, MESC, and DEASC, respectively. DESC also strongly blocked putrescine and spermidine uptake in ZR-75-1 cells (Ki = 1.6 +/- 0.5 and 2.7 +/- 1.1 microM, respectively). While DESC and MESC were purely competitive inhibitors of putrescine transport, DEASC was a mixed competitive/noncompetitive antagonist. Remarkably, DESC was virtually impermeant in ZR-75-1 cells despite its low Ki toward polyamine transport. The marked difference in affinity between DESC and MESC was essentially due to the tail-to-tail juxtaposition of two spermine-like structures, suggesting that dimeric ligands of the polyamine transporter might simultaneously interact with more than one binding site. While DESC strongly decreased the initial rate of [3H]spermidine transport, even a 40-fold molar excess of antagonist could not completely abolish intracellular spermidine accumulation. Moreover, as little as 0.3 microM spermidine fully restored growth in ZR-75-1 cells treated with an inhibitor of polyamine biosynthesis in the presence of 50 microM DESC, thus emphasizing the importance of uptake of trace amounts of exogenous polyamines. Thus, reducing the exogenous supply of polyamines with a potent competitive inhibitor may be kinetically inadequate to block replenishment of the polyamine pool in polyamine-depleted tumor cells that display high transport capacity. These results demonstrate that polyamine analogues cross-linked into a dimeric structure such as DESC interact with high affinity with the mammalian polyamine carrier without being used as substrates. These novel properties provide a framework for the design of

  9. A vaccine of L2 epitope repeats fused with a modified IgG1 Fc induced cross-neutralizing antibodies and protective immunity against divergent human papillomavirus types.

    Science.gov (United States)

    Chen, Xue; Liu, Hongyang; Zhang, Ting; Liu, Yanchun; Xie, Xixiu; Wang, Zhirong; Xu, Xuemei

    2014-01-01

    Current human papillomavirus (HPV) major capsid protein L1 virus-like particles (VLPs)-based vaccines in clinic induce strong HPV type-specific neutralizing antibody responses. To develop pan-HPV vaccines, here, we show that the fusion protein E3R4 consisting of three repeats of HPV16 L2 aa 17-36 epitope (E3) and a modified human IgG1 Fc scaffold (R4) induces cross-neutralizing antibodies and protective immunity against divergent HPV types. E3R4 was expressed as a secreted protein in baculovirus expression system and could be simply purified by one step Protein A affinity chromatography with the purity above 90%. Vaccination of E3R4 formulated with Freunds adjuvant not only induced cross-neutralizing antibodies against HPV pseudovirus types 16, 18, 45, 52, 58, 6, 11 and 5 in mice, but also protected mice against vaginal challenges with HPV pseudovirus types 16, 45, 52, 58, 11 and 5 for at least eleven months after the first immunization. Moreover, vaccination of E3R4 formulated with FDA approved adjuvant alum plus monophosphoryl lipid A also induced cross-neutralizing antibodies against HPV types 16, 18 and 6 in rabbits. Thus, our results demonstrate that delivery of L2 antigen as a modified Fc-fusion protein may facilitate pan-HPV vaccine development.

  10. Monoclonal antibodies in oncology. Review article

    Energy Technology Data Exchange (ETDEWEB)

    Chan, S Y.T.; Sikora, K

    1986-05-01

    Monoclonal antibodies (MCAs) can be used to differentiate between normal and neoplastic cells and thus exploited for diagnostic and, ultimately, therapeutic gain. The evidence for the existence of human tumour antigens is reviewed. Several areas of diagnosis are already benefiting from the application of the monoclonal technology. Immunohistology can help the pathologist with difficult diagnostic problems. New classifications of lymphoma and leukaemia can be based on specific surface molecules. Similarly, the detection of shed tumour antigens is already established as part of the routine assessment of many patients with common solid tumours. Isotopically labeled monoclonal antibodies have been used to localise primary and metastatic tumours. The use of antibodies in this way is not only a promising diagnostic tool but also the first step in studying the possibility of arming antibodies to provide therapeutic agents. Such trials are currently in progress. 69 refs.; 7 figs.; 3 tabs.

  11. Labeling by ( sup 3 H)1,3-di(2-tolyl)guanidine of two high affinity binding sites in guinea pig brain: Evidence for allosteric regulation by calcium channel antagonists and pseudoallosteric modulation by sigma ligands

    Energy Technology Data Exchange (ETDEWEB)

    Rothman, R.B.; Reid, A.; Mahboubi, A.; Kim, C.H.; De Costa, B.R.; Jacobson, A.E.; Rice, K.C. (National Institute of Mental Health, Bethesda, MD (USA))

    1991-02-01

    Equilibrium binding studies with the sigma receptor ligand ({sup 3}H)1,3-di(2-tolyl)guanidine (({sup 3}H)DTG) demonstrated two high affinity binding sites in membranes prepared from guinea pig brain. The apparent Kd values of DTG for sites 1 and 2 were 11.9 and 37.6 nM, respectively. The corresponding Bmax values were 1045 and 1423 fmol/mg of protein. Site 1 had high affinity for (+)-pentazocine, haloperidol, (R)-(+)-PPP, carbepentane, and other sigma ligands, suggesting a similarity with the dextromethorphan/sigma 1 binding site described by Musacchio et al. (Life Sci. 45:1721-1732 (1989)). Site 2 had high affinity for DTG and haloperidol (Ki = 36.1 nM) and low affinity for most other sigma ligands. Kinetic experiments demonstrated that ({sup 3}H)DTG dissociated in a biphasic manner from both site 1 and site 2. DTG and haloperidol increased the dissociation rate of ({sup 3}H)DTG from site 1 and site 2, demonstrating the presence of pseudoallosteric interactions. Inorganic calcium channel blockers such as Cd2+ selectively increased the dissociation rate of ({sup 3}H)DTG from site 2, suggesting an association of this binding site with calcium channels.

  12. Labeling by [3H]1,3-di(2-tolyl)guanidine of two high affinity binding sites in guinea pig brain: Evidence for allosteric regulation by calcium channel antagonists and pseudoallosteric modulation by sigma ligands

    International Nuclear Information System (INIS)

    Rothman, R.B.; Reid, A.; Mahboubi, A.; Kim, C.H.; De Costa, B.R.; Jacobson, A.E.; Rice, K.C.

    1991-01-01

    Equilibrium binding studies with the sigma receptor ligand [ 3 H]1,3-di(2-tolyl)guanidine ([ 3 H]DTG) demonstrated two high affinity binding sites in membranes prepared from guinea pig brain. The apparent Kd values of DTG for sites 1 and 2 were 11.9 and 37.6 nM, respectively. The corresponding Bmax values were 1045 and 1423 fmol/mg of protein. Site 1 had high affinity for (+)-pentazocine, haloperidol, (R)-(+)-PPP, carbepentane, and other sigma ligands, suggesting a similarity with the dextromethorphan/sigma 1 binding site described by Musacchio et al. [Life Sci. 45:1721-1732 (1989)]. Site 2 had high affinity for DTG and haloperidol (Ki = 36.1 nM) and low affinity for most other sigma ligands. Kinetic experiments demonstrated that [ 3 H]DTG dissociated in a biphasic manner from both site 1 and site 2. DTG and haloperidol increased the dissociation rate of [ 3 H]DTG from site 1 and site 2, demonstrating the presence of pseudoallosteric interactions. Inorganic calcium channel blockers such as Cd2+ selectively increased the dissociation rate of [ 3 H]DTG from site 2, suggesting an association of this binding site with calcium channels

  13. Interactions of dopaminergic agonists and antagonists with dopaminergic D3 binding sites in rat striatum. Evidence that [3H]dopamine can label a high affinity agonist-binding state of the D1 dopamine receptor

    International Nuclear Information System (INIS)

    Leff, S.E.; Creese, I.

    1985-01-01

    The interactions of dopaminergic agonists and antagonists with 3 H-agonist labeled D3 dopaminergic binding sites of rat striatum have been characterized by radioligand-binding techniques. When the binding of [ 3 H]dopamine and [ 3 H]apomorphine to D2 dopamine receptors is blocked by the inclusion of D2 selective concentrations of unlabeled spiroperidol or domperidone, these ligands appear to label selectively the previously termed D3 binding site. Antagonist/[ 3 H]dopamine competition curves are of uniformly steep slope (nH . 1.0), suggesting the presence of a single D3 binding site. The relative potencies of antagonists to inhibit D3 specific [ 3 H]dopamine binding are significantly correlated with their potencies to block D1 dopamine receptors as measured by the inhibition of both dopamine-stimulated adenylate cyclase and [ 3 H]flupentixol-binding activities. The affinities of agonists to inhibit D3 specific [ 3 H]dopamine binding are also correlated with estimates of these agonists affinities for the high affinity binding component of agonist/[ 3 H]flupentixol competition curves. Both D3 specific [ 3 H] dopamine binding and the high affinity agonist-binding component of dopamine/[ 3 H]flupentixol competition curves show a similar sensitivity to guanine nucleotides. Taken together, these data strongly suggest that the D3 binding site is related to a high affinity agonist-binding state of the D1 dopamine receptor

  14. Tumor detection using radiolabeled monoclonal antibodies

    International Nuclear Information System (INIS)

    Moldofsky, P.J.; Powe, J.; Hammond, N.D.

    1987-01-01

    Radioisotope conjugated to monoclonal antibody products has been used for imaging tumors targeted by the antibody. As imaging progresses, new sets of procedural and technical questions arise. In this chapter, we discuss several current problems in imaging tumor with radiolabeled monoclonal antibody. These include (1) methods for selection of specific antibody and, once the particular antibody is selected, which fragment form is to be used; (2) imaging procedures: what are the optimum imaging parameters, such as optimum time for imaging after administration of tracer and considerations regarding background subtraction; and (3) noninvasive quantitative techniques: quantitation of localization of antibody indirectly from quantitative information in the images.100 references

  15. Thermodynamic and kinetic approaches for evaluation of monoclonal antibody - Lipoprotein interactions.

    Science.gov (United States)

    Multia, Evgen; Sirén, Heli; Andersson, Karl; Samuelsson, Jörgen; Forssén, Patrik; Fornstedt, Torgny; Öörni, Katariina; Jauhiainen, Matti; Riekkola, Marja-Liisa

    2017-02-01

    Two complementary instrumental techniques were used, and the data generated was processed with advanced numerical tools to investigate the interactions between anti-human apoB-100 monoclonal antibody (anti-apoB-100 Mab) and apoB-100 containing lipoproteins. Partial Filling Affinity Capillary Electrophoresis (PF-ACE) combined with Adsorption Energy Distribution (AED) calculations provided information on the heterogeneity of the interactions without any a priori model assumptions. The AED calculations evidenced a homogenous binding site distribution for the interactions. Quartz Crystal Microbalance (QCM) studies were used to evaluate thermodynamics and kinetics of the Low-Density Lipoprotein (LDL) and anti-apoB-100 Mab interactions. High affinity and selectivity were observed, and the emerging data sets were analysed with so called Interaction Maps. In thermodynamic studies, the interaction between LDL and anti-apoB-100 Mab was found to be predominantly enthalpy driven. Both techniques were also used to study antibody interactions with Intermediate-Density (IDL) and Very Low-Density (VLDL) Lipoproteins. By screening affinity constants for IDL-VLDL sample in a single injection we were able to distinguish affinity constants for both subpopulations using the numerical Interaction Map tool. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. AL amyloid imaging and therapy with a monoclonal antibody to a cryptic epitope on amyloid fibrils.

    Directory of Open Access Journals (Sweden)

    Jonathan S Wall

    Full Text Available The monoclonal antibody 2A4 binds an epitope derived from a cleavage site of serum amyloid protein A (sAA containing a -Glu-Asp- amino acid pairing. In addition to its reactivity with sAA amyloid deposits, the antibody was also found to bind amyloid fibrils composed of immunoglobulin light chains. The antibody binds to synthetic fibrils and human light chain (AL amyloid extracts with high affinity even in the presence of soluble light chain proteins. Immunohistochemistry with biotinylated 2A4 demonstrated positive reaction with ALκ and ALλ human amyloid deposits in various organs. Surface plasmon resonance analyses using synthetic AL fibrils as a substrate revealed that 2A4 bound with a K(D of ∼10 nM. Binding was inhibited in the presence of the -Glu-Asp- containing immunogen peptide. Radiolabeled 2A4 specifically localized with human AL amyloid extracts implanted in mice (amyloidomas as evidenced by single photon emission (SPECT imaging. Furthermore, co-localization of the radiolabeled mAb with amyloid was shown in biodistribution and micro-autoradiography studies. Treatment with 2A4 expedited regression of ALκ amyloidomas in mice, likely mediated by the action of macrophages and neutrophils, relative to animals that received a control antibody. These data indicate that the 2A4 mAb might be of interest for potential imaging and immunotherapy in patients with AL amyloidosis.

  17. DETECTION AND PURIFICATION OF TYROSINE-SULFATED PROTEINS USING A NOVEL ANTI-SULFOTYROSINE MONOCLONAL ANTIBODY*

    Science.gov (United States)

    Hoffhines, Adam J.; Damoc, Eugen; Bridges, Kristie G.; Leary, Julie A.; Moore, Kevin L.

    2006-01-01

    Protein-tyrosine O-sulfation is a post-translational modification mediated by one of two Golgi tyrosylprotein sulfotransferases (TPST-1 & TPST-2) that catalyze the transfer of sulfate to tyrosine residues in secreted and transmembrane proteins. Tyrosine sulfation plays a role in protein-protein interactions in several well-defined systems. Although dozens of tyrosine-sulfated proteins are known, many more are likely to exist and await description. Advancing our understanding of the importance of tyrosine sulfation in biological systems requires the development of new tools for the detection and study of tyrosine-sulfated proteins. We have developed a novel anti-sulfotyrosine monoclonal antibody, called PSG2, that binds with high affinity and exquisite specificity to sulfotyrosine residues in peptides and proteins independent of sequence context. We show that it can detect tyrosinesulfated proteins in complex biological samples and can be used as a probe to assess the role of tyrosine sulfation in protein function. We also demonstrate the utility of PSG2 in the purification of tyrosine-sulfated proteins from crude tissue samples. Finally, Western blot analysis using PSG2 indicates that certain sperm/epididymal proteins are undersulfated in Tpst2−/− mice. This indicates that TPST-1 and TPST-2 have distinct macromolecular substrate specificities and provides clues as to the molecular mechanism of the infertility of Tpst2−/− males. PSG2 should be widely applicable for identification of tyrosine-sulfated proteins in other systems and organisms. PMID:17046811

  18. Inhibition of fibroblast growth factor receptor 3-dependent lung adenocarcinoma with a human monoclonal antibody

    Directory of Open Access Journals (Sweden)

    Yongjun Yin

    2016-05-01

    Full Text Available Activating mutations in fibroblast growth factor receptor 3 (FGFR3 have been identified in multiple types of human cancer and in congenital birth defects. In human lung cancer, fibroblast growth factor 9 (FGF9, a high-affinity ligand for FGFR3, is overexpressed in 10% of primary resected non-small cell lung cancer (NSCLC specimens. Furthermore, in a mouse model where FGF9 can be induced in lung epithelial cells, epithelial proliferation and ensuing tumorigenesis is dependent on FGFR3. To develop new customized therapies for cancers that are dependent on FGFR3 activation, we have used this mouse model to evaluate a human monoclonal antibody (D11 with specificity for the extracellular ligand-binding domain of FGFR3, that recognizes both human and mouse forms of the receptor. Here, we show that D11 effectively inhibits signaling through FGFR3 in vitro, inhibits the growth of FGFR3-dependent FGF9-induced lung adenocarcinoma in mice, and reduces tumor-associated morbidity. Given the potency of FGF9 in this mouse model and the absolute requirement for signaling through FGFR3, this study validates the D11 antibody as a potentially useful and effective reagent for treating human cancers or other pathologies that are dependent on activation of FGFR3.

  19. Stability of monoclonal antibodies at high-concentration

    DEFF Research Database (Denmark)

    Neergaard, Martin S; Nielsen, Anders D; Parshad, Henrik

    2014-01-01

    Few studies have so far directly compared the impact of antibody subclass on protein stability. This case study investigates two mAbs (one IgG1 and one IgG4 ) with identical variable region. Investigations of mAbs that recognize similar epitopes are necessary to identify possible differences betw...

  20. Generation of a Monoclonal Antibody against Mycoplasma spp. following Accidental Contamination during Production of a Monoclonal Antibody against Lawsonia intracellularis

    OpenAIRE

    Hwang, Jeong-Min; Lee, Ji-Hye; Yeh, Jung-Yong

    2012-01-01

    This report describes Mycoplasma contamination of Lawsonia intracellularis cultures that led to the unintended acquisition of a monoclonal antibody against Mycoplasma spp. during the attempted generation of a monoclonal antibody against L. intracellularis.

  1. Strain differentiation of polioviruses with monoclonal antibodies.

    NARCIS (Netherlands)

    A.D.M.E. Osterhaus (Albert); A.L. van Wezel; A.J.H. Stegmann; J.A.A.M. van Asten (Jack)

    1984-01-01

    textabstractPanels of monoclonal antibodies raised against different poliovirus type 1, 2 and 3 strains, were tested in a micro-neutralization test and in a micro-enzyme linked immunosorbent assay against a large number of poliovirus strains. The results were compared with those obtained with the

  2. Monoclonal antibodies reactive with hairy cell leukemia

    NARCIS (Netherlands)

    Visser, L; Shaw, A; Slupsky, J; Vos, H; Poppema, S

    Monoclonal antibodies reactive with hairy cell leukemia were developed to aid in the diagnosis of this subtype of B cell chronic lymphocytic leukemia and to gain better insight into the origin of hairy cells. Three antibodies were found to be of value in the diagnosis of hairy cell leukemia.

  3. Monoclonal antibody technologies and rapid detection assays

    Science.gov (United States)

    Novel methodologies and screening strategies will be outlined on the use of hybridoma technology for the selection of antigen specific monoclonal antibodies. The development of immunoassays used for diagnostic detection of prions and bacterial toxins will be discussed and examples provided demonstr...

  4. Monoclonal antibody therapy of inflammatory bowel disease

    NARCIS (Netherlands)

    van Deventer, S. J.; Camoglio, L.

    1996-01-01

    Several anti-inflammatory drugs have therapeutic efficacy in inflammatory bowel disease, but their targets remain incompletely characterized. The development of monoclonal antibodies that either recognize epitopes on immune-competent cells, or neutralize pro-inflammatory cytokines, has helped to

  5. Monoclonal Antibody Therapy for Advanced Neuroblastoma

    Science.gov (United States)

    NCI is sponsoring two clinical trials of a monoclonal antibody called ch14.18, in combination with other drugs, to see if the antibody may be helpful for children or young adults (up to age 21) with relapsed or refractory neuroblastoma.

  6. Dynamics evaluation of total IgG, IgG1 and IgG2a in the serum of mice immunized with radioattenuated paracoccidioides brasiliensis yeast cells

    International Nuclear Information System (INIS)

    Martins, Estefania M.N.; Andrade, Antero S.R.; Reis, Bernardo S.; Goes, Alfredo M.

    2007-01-01

    Paracoccidioides brasiliensis is the fungus agent of paracoccidioidomycosis, a deep-seated systemic infection of humans. Up to the moment no vaccine has still been reported. The potential of gamma radiation for pathogens attenuation and vaccine development was explored in this work. In our laboratory we developed radioattenuated yeast cells of P. brasiliensis and the aim of the present work was to evaluate the antibody production dynamics in mice immunized with this cells. Were analyzed the IgG antibodies titers as well as the type of response by analyzing the IgG1 and IgG2a antibody pattern in the course of infection. The mice were divided in two groups that were immunized one time and two times respectively. The mice infected with the virulent P. brasiliensis showed a high level of antibody production while the infection with the radioattenuated yeast did not significantly change the antibody level. The level of IgG raised in both immunized groups after the challenge. In the group immunized one time was not observed a significant difference between the levels of both subclasses when compared with the control. After the challenge of the group immunized two times the IgG2a levels increased significantly when analyzed 90 days post challenge. We concluded that a pattern related to the disease control was apparent in the group submitted to two immunizations. The mice had not developed a totally polarized pattern of TH1/TH2 response but a trend to a TH1 response was evident. (author)

  7. Dynamics evaluation of total IgG, IgG1 and IgG2a in the serum of mice immunized with radioattenuated paracoccidioides brasiliensis yeast cells

    Energy Technology Data Exchange (ETDEWEB)

    Martins, Estefania M.N.; Andrade, Antero S.R. [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil)]. E-mail: estefaniabio@yahoo.com.br; antero@cdtn.br; Reis, Bernardo S.; Goes, Alfredo M. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Bioquimica e Imunologia]. E-mail: brsgarbi@mono.icb.ufmg.br; goes@mono.icb.ufmg.br

    2007-07-01

    Paracoccidioides brasiliensis is the fungus agent of paracoccidioidomycosis, a deep-seated systemic infection of humans. Up to the moment no vaccine has still been reported. The potential of gamma radiation for pathogens attenuation and vaccine development was explored in this work. In our laboratory we developed radioattenuated yeast cells of P. brasiliensis and the aim of the present work was to evaluate the antibody production dynamics in mice immunized with this cells. Were analyzed the IgG antibodies titers as well as the type of response by analyzing the IgG1 and IgG2a antibody pattern in the course of infection. The mice were divided in two groups that were immunized one time and two times respectively. The mice infected with the virulent P. brasiliensis showed a high level of antibody production while the infection with the radioattenuated yeast did not significantly change the antibody level. The level of IgG raised in both immunized groups after the challenge. In the group immunized one time was not observed a significant difference between the levels of both subclasses when compared with the control. After the challenge of the group immunized two times the IgG2a levels increased significantly when analyzed 90 days post challenge. We concluded that a pattern related to the disease control was apparent in the group submitted to two immunizations. The mice had not developed a totally polarized pattern of TH1/TH2 response but a trend to a TH1 response was evident. (author)

  8. Discovery and characterization of antibody variants using mass spectrometry-based comparative analysis for biosimilar candidates of monoclonal antibody drugs.

    Science.gov (United States)

    Li, Wenhua; Yang, Bin; Zhou, Dongmei; Xu, Jun; Ke, Zhi; Suen, Wen-Chen

    2016-07-01

    Liquid chromatography mass spectrometry (LC-MS) is the most commonly used technique for the characterization of antibody variants. MAb-X and mAb-Y are two approved IgG1 subtype monoclonal antibody drugs recombinantly produced in Chinese hamster ovary (CHO) cells. We report here that two unexpected and rare antibody variants have been discovered during cell culture process development of biosimilars for these two approved drugs through intact mass analysis. We then used comprehensive mass spectrometry-based comparative analysis including reduced light, heavy chains, and domain-specific mass as well as peptide mapping analysis to fully characterize the observed antibody variants. The "middle-up" mass comparative analysis demonstrated that the antibody variant from mAb-X biosimilar candidate was caused by mass variation of antibody crystalline fragment (Fc), whereas a different variant with mass variation in antibody antigen-binding fragment (Fab) from mAb-Y biosimilar candidate was identified. Endoproteinase Lys-C digested peptide mapping and tandem mass spectrometry analysis further revealed that a leucine to glutamine change in N-terminal 402 site of heavy chain was responsible for the generation of mAb-X antibody variant. Lys-C and trypsin coupled non-reduced and reduced peptide mapping comparative analysis showed that the formation of the light-heavy interchain trisulfide bond resulted in the mAb-Y antibody variant. These two cases confirmed that mass spectrometry-based comparative analysis plays a critical role for the characterization of monoclonal antibody variants, and biosimilar developers should start with a comprehensive structural assessment and comparative analysis to decrease the risk of the process development for biosimilars. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Facilitation of syngeneic stem cell engraftment by anti-class I monoclonal antibody pretreatment of unirradiated recipients

    International Nuclear Information System (INIS)

    Voralia, M.; Semeluk, A.; Wegmann, T.G.

    1987-01-01

    We have established a murine model of syngeneic bone marrow transplantation based on the use of monoclonal antibody as the sole conditioning regimen in unirradiated recipients. Administration of a single injection of monoclonal antibody directed against major histocompatibility complex-encoded class I determinants facilitated permanent hemopoietic stem cell engraftment without any apparent side-effects. Whereas untreated hosts exhibited a maximal chimerism of 15% at donor cell doses of up to 12 X 10(7) bone marrow cells, pretreatment by 2 mg of anti-class I antibody one week prior to transplantation of 3 X 10(7) syngeneic bone marrow cells resulted in a mean donor representation of about 80%. The antibody can be given up to four weeks prior to transplantation, and the degree of donor engraftment observed is a function of the dose of antibody administered. The fact that specific antibody enhanced engraftment in two strain combinations indicates that antibody is the active agent in facilitating engraftment and that facilitation is not strain-restricted. Anti-class I antibodies of the IgG2a, but not IgG1, isotype are effective in promoting engraftment. Although the isotype requirement suggests a role for antibody-mediated cytotoxicity in promoting stem cell engraftment, the extensive time-frame of facilitation suggests that other effects of the antibody may also be involved. The model of syngeneic bone marrow transplantation we describe here will be useful in studying the mechanisms regulating stem cell engraftment and may have potential clinical application as an approach to autologous marrow transplantation

  10. High-yield production of a human monoclonal IgG by rhizosecretion in hydroponic tobacco cultures.

    Science.gov (United States)

    Madeira, Luisa M; Szeto, Tim H; Henquet, Maurice; Raven, Nicole; Runions, John; Huddleston, Jon; Garrard, Ian; Drake, Pascal M W; Ma, Julian K-C

    2016-02-01

    Rhizosecretion of recombinant pharmaceuticals from in vitro hydroponic transgenic plant cultures is a simple, low cost, reproducible and controllable production method. Here, we demonstrate the application and adaptation of this manufacturing platform to a human antivitronectin IgG1 monoclonal antibody (mAb) called M12. The rationale for specific growth medium additives was established by phenotypic analysis of root structure and by LC-ESI-MS/MS profiling of the total protein content profile of the hydroponic medium. Through a combination of optimization approaches, mAb yields in hydroponic medium reached 46 μg/mL in 1 week, the highest figure reported for a recombinant mAb in a plant secretion-based system to date. The rhizosecretome was determined to contain 104 proteins, with the mAb heavy and light chains the most abundant. This enabled evaluation of a simple, scalable extraction and purification protocol and demonstration that only minimal processing was necessary prior to protein A affinity chromatography. MALDI-TOF MS revealed that purified mAb contained predominantly complex-type plant N-glycans, in three major glycoforms. The binding of M12 purified from hydroponic medium to vitronectin was comparable to its Chinese hamster ovary (CHO)-derived counterpart. This study demonstrates that in vitro hydroponic cultivation coupled with recombinant protein rhizosecretion can be a practical, low-cost production platform for monoclonal antibodies. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  11. Effect of Surfactants on Mechanical, Thermal, and Photostability of a Monoclonal Antibody.

    Science.gov (United States)

    Agarkhed, Meera; O'Dell, Courtney; Hsieh, Ming-Ching; Zhang, Jingming; Goldstein, Joel; Srivastava, Arvind

    2018-01-01

    The purpose of this work was to evaluate the effect of commonly used surfactants (at 0.01% w/v concentration) on mechanical, thermal, and photostability of a monoclonal antibody (MAb1) of IgG1 sub-class and to evaluate the minimum concentration of surfactant (Polysorbate 80) required in protecting MAb1 from mechanical stress. Surfactants evaluated were non-ionic surfactants, Polysorbate 80, Polysorbate 20, Pluronic F-68 (polyoxyethylene-polyoxypropylene block polymer), Brij 35 (polyoxyethylene lauryl ether), Triton X-100, and an anionic surfactant, Caprylic acid (1-Heptanecarboxylic acid). After evaluating effect of surfactants and determining stabilizing effect of Polysorbate 80 against mechanical stress without compromising thermal and photostability of MAb1, the minimum concentration of Polysorbate 80 required for mechanical stability was further examined. Polysorbate 80 concentration was varied from 0 to 0.02%. Mechanical stability was evaluated by agitation of MAb1 at 300 rotations per minute at room temperature for 72 h. Samples were analyzed for purity by SEC-HPLC, turbidity by absorbance at 350 nm, visible particles by visual inspection, and sub-visible particles by light obscuration technique on a particle analyzer. All non-ionic surfactants tested showed a similar effect in protecting against mechanical stress and did not exhibit any significant negative effect on thermal and photostability. However, Caprylic acid had a slightly negative effect on mechanical and photostability when compared to the non-ionic surfactants or sample without surfactant. This work demonstrated that polysorbate 80 is better than other surfactants tested and that a concentration of at least 0.005% (w/v) Polysorbate 80 is needed to protect MAb1 against mechanical stress.

  12. Production and characterization of monoclonal antibodies specific to pangasius catfish, basa, and tra.

    Science.gov (United States)

    Gajewski, K G; Chen, Y-T; Hsieh, Y-H P

    2009-04-01

    Four IgG (subclass IgG1) class monoclonal antibodies (MAbs) strongly reactive to Asian farm-raised Pangasius catfish, tra (Pangasius hypophthalmus) and basa (Pangasius bocourti), have been developed. These MAbs were raised by immunizing an animal with thermal-stable crude sarcoplasmic protein extract of cooked tra. The MAbs were selected by screening hybridoma clones against more than 70 common fish and meat protein extracts. Two MAbs, T7E10 and T1G11, were found to be specific to the Asian Pangasius catfish, tra, and basa, with no cross-reactions with any of the common fish and meat species or with the food additive proteins (bovine serum albumin, soy proteins, milk proteins, egg proteins, and gelatin) tested. MAb T7E10 recognized 2 antigenic proteins (molecular weight approximately 36 and 75 kDa) in raw and cooked tra and basa extracts, while T1G11 bound to several proteins (molecular weight between 13 and 18 kDa) in tra and basa extracts. Two other MAbs, F7B8 and F1G11, recognized a common protein (36 KDa) and cross-reacted with all the fish extracts tested and with several mammalian species. These MAbs can be employed individually or in combination in various formats of immunoassays for rapid identification of Pangasius catfish, either raw or cooked. They can also be used to study the biological, biochemical, and physiological aspects of thermal-stable antigenic proteins. This is the first study identifying these thermal-stable antigenic proteins present in Pangasius catfish as species-specific biomarkers.

  13. Unbiased in-depth characterization of CEX fractions from a stressed monoclonal antibody by mass spectrometry.

    Science.gov (United States)

    Griaud, François; Denefeld, Blandine; Lang, Manuel; Hensinger, Héloïse; Haberl, Peter; Berg, Matthias

    2017-07-01

    Characterization of charge-based variants by mass spectrometry (MS) is required for the analytical development of a new biologic entity and its marketing approval by health authorities. However, standard peak-based data analysis approaches are time-consuming and biased toward the detection, identification, and quantification of main variants only. The aim of this study was to characterize in-depth acidic and basic species of a stressed IgG1 monoclonal antibody using comprehensive and unbiased MS data evaluation tools. Fractions collected from cation ion exchange (CEX) chromatography were analyzed as intact, after reduction of disulfide bridges, and after proteolytic cleavage using Lys-C. Data of both intact and reduced samples were evaluated consistently using a time-resolved deconvolution algorithm. Peptide mapping data were processed simultaneously, quantified and compared in a systematic manner for all MS signals and fractions. Differences observed between the fractions were then further characterized and assigned. Time-resolved deconvolution enhanced pattern visualization and data interpretation of main and minor modifications in 3-dimensional maps across CEX fractions. Relative quantification of all MS signals across CEX fractions before peptide assignment enabled the detection of fraction-specific chemical modifications at abundances below 1%. Acidic fractions were shown to be heterogeneous, containing antibody fragments, glycated as well as deamidated forms of the heavy and light chains. In contrast, the basic fractions contained mainly modifications of the C-terminus and pyroglutamate formation at the N-terminus of the heavy chain. Systematic data evaluation was performed to investigate multiple data sets and comprehensively extract main and minor differences between each CEX fraction in an unbiased manner.

  14. Development of monoclonal antibodies to rohu [Labeo rohita] immunoglobulins for use in immunoassays.

    Science.gov (United States)

    Rathore, Gaurav; Kumar, Gokhlesh; Sood, Neeraj; Kapoor, D; Lakra, W S

    2008-12-01

    Serum immunoglobulins [Ig] of rohu [Labeo rohita] were purified by affinity chromatography using bovine serum albumin as capture ligand. The purified rohu Ig [r-Ig] had a molecular weight [MW] of 880 kDa as determined with gel filtration chromatography. The heavy chain of r-Ig had an MW of 77.8 kDa and that of light chain was 26.4 kDa in SDS-PAGE. Purified r-Ig was used for the production of two anti-rohu Ig monoclonal antibodies [D7 and H4] that belonged to subclass IgG2b and IgG1, respectively. Both the MAbs were specific to heavy chain of r-Ig as seen in Western blotting. Anti-rohu Ig MAb was used as a diagnostic reagent in ELISA and immunocytochemical assays to demonstrate its application for sero-surveillance and for immunological studies in rohu. A competitive ELISA was used to demonstrate the antigenic relatedness of r-Ig with whole serum Ig of other fish species. Cross reactivity of anti-rohu Ig MAb was observed with serum Ig of Catla catla and Cirrihinus mrigala. No reactivity to serum Ig of Ophiocephalus striatus and Clarias gariepinus was seen. Anti-rohu Ig MAb was found to be suitable for the detection of pathogen specific [Edwardsiella tarda] antibodies in serum of immunized rohu by an indirect ELISA. In flow cytometry using D7 MAb, the mean percentage [+/-SE] of Ig positive cells in spleen and blood of rohu were found to be 64.85% [+/-2.34] and 51.84% [+/-2.55] of gated lymphocytes, respectively. Similarly, D7 MAb also stained 52.84% [+/-1.30] and 10.5% of gated lymphocytes in kidney and thymus, respectively. The anti-rohu Ig MAbs also showed specific staining of Ig bearing cells in spleen sections by the indirect immunoperoxidase test.

  15. Anti-leukemic activity and tolerability of anti-human CD47 monoclonal antibodies

    Science.gov (United States)

    Pietsch, E C; Dong, J; Cardoso, R; Zhang, X; Chin, D; Hawkins, R; Dinh, T; Zhou, M; Strake, B; Feng, P-H; Rocca, M; Santos, C Dos; Shan, X; Danet-Desnoyers, G; Shi, F; Kaiser, E; Millar, H J; Fenton, S; Swanson, R; Nemeth, J A; Attar, R M

    2017-01-01

    CD47, a broadly expressed cell surface protein, inhibits cell phagocytosis via interaction with phagocyte-expressed SIRPα. A variety of hematological malignancies demonstrate elevated CD47 expression, suggesting that CD47 may mediate immune escape. We discovered three unique CD47-SIRPα blocking anti-CD47 monoclonal antibodies (mAbs) with low nano-molar affinity to human and cynomolgus monkey CD47, and no hemagglutination and platelet aggregation activity. To characterize the anti-cancer activity elicited by blocking CD47, the mAbs were cloned into effector function silent and competent Fc backbones. Effector function competent mAbs demonstrated potent activity in vitro and in vivo, while effector function silent mAbs demonstrated minimal activity, indicating that blocking CD47 only leads to a therapeutic effect in the presence of Fc effector function. A non-human primate study revealed that the effector function competent mAb IgG1 C47B222-(CHO) decreased red blood cells (RBC), hematocrit and hemoglobin by >40% at 1 mg/kg, whereas the effector function silent mAb IgG2σ C47B222-(CHO) had minimal impact on RBC indices at 1 and 10 mg/kg. Taken together, our findings suggest that targeting CD47 is an attractive therapeutic anti-cancer approach. However, the anti-cancer activity observed with anti-CD47 mAbs is Fc effector dependent as are the side effects observed on RBC indices. PMID:28234345

  16. Investigation of the Influence of Protein-Losing Enteropathy on Monoclonal Antibody Pharmacokinetics in Mice.

    Science.gov (United States)

    Yang, Yujie; Li, Tommy R; Balthasar, Joseph P

    2017-11-01

    Protein losing enteropathy (PLE), which is characterized by substantial loss of plasma proteins into the gastrointestinal (GI) tract, is a complication of a variety of GI diseases, including inflammatory bowel disease. Clinical studies have found that the clearance of monoclonal antibodies (mAb) is often increased in subjects with diseases known to cause PLE; however, direct relationships between PLE and mAb pharmacokinetics have not been demonstrated. This study employed a murine model of colitis to examine the influence of PLE on mAb pharmacokinetics. Mice were given dextran sodium sulfate (DSS, 2% w/v) supplemented tap water as drinking source for 6 days to induce colitis and PLE. Mice were then intravenously injected with 8C2, a murine IgG1 mAb. 8C2 plasma concentrations were measured up to 14 days post injection. Fecal alpha-1-antitrypsin (A1AT) clearance was measured as biomarker for PLE. DSS-treated mice developed PLE of clinically relevant severity. They also showed a transient increase in 8C2 plasma clearance and a decrease in 8C2 plasma exposure. The area under the 8C2 plasma concentration-time curve for the length of the study (AUC 0-14d ) reduced from 1368 ± 255 to 594 ± 224 day μg/ml following DSS treatment (p = 0.001). A quantitative relationship between A1AT clearance and 8C2 clearance was obtained via population pharmacokinetic modeling. DSS treatment substantially increased 8C2 clearance and reduced 8C2 exposure. Increased mAb plasma clearance was highly correlated with A1AT fecal clearance, suggesting the possible utility of A1AT fecal clearance as a mechanistic biomarker to predict the pharmacokinetics of therapeutic antibodies.

  17. Monoclonal antibodies and coupling reagents to cell membrane proteins for leukocyte labeling

    International Nuclear Information System (INIS)

    McAfee, J.G.; Gagne, G.; Subramanian, G.; Schneider, R.F.

    1984-01-01

    Current gamma-emitting agents for tagging leukocytes, In-111 oxine or tropolone, label all cell types indiscriminantly, and nuclear localization in lymphocytes results in radiation damage. Coupling reagents and murine monoclonal antibodies (Mab) specific for cell surface antigens of human leukocytes were tried as cell labeling agents to avoid nuclear localization. 10/sup 8/ mixed human leukocytes in Hepes buffer were added to tubes coated with 5 mg of dry cyclic dianhydride of DTPA for 15 minutes at room temperature. After washing, 0.1 ml of In-111 Cl in ACD (pH 6.8) was added. After 30 minutes, a cell labeling yield of 23% was obtained. Washing the cells in an elutriation centrifuge showed that this label was irreversible. Mab for cell surface antigens of human granulocytes were labeled with 300 μCi of I-125 using the Iodobead technic and unbound activity was removed by gel column chromatography. 1-10 μg were added to 10/sup 8/ mixed leukocytes in 0.5 ml plasma or saline for 1 hr. With Mab anti-leu M4 (clone G7 E11), an IgM, the cell labeling yield was 21%, irreversible, and specific for granulocytes. With anti-human leukocyte Mab NEI-042 (clone 9.4), and IgG2a, and anti-granulocyte Mab MAS-065 (clone FMCl1) an IgG1, the cell labeling was relatively unstable. Labeling of leukocyte subpopulations with Mab is feasible, and the binding of multivalent IgM is stronger than that of other immunoglobulins. DTPA cyclic anhydride is firmly bound to cell membranes, but the labeling is non-specific

  18. Monoclonal antibodies to hyphal exoantigens derived from the opportunistic pathogen Aspergillus terreus.

    Science.gov (United States)

    Nayak, Ajay P; Green, Brett J; Janotka, Erika; Hettick, Justin M; Friend, Sherri; Vesper, Steve J; Schmechel, Detlef; Beezhold, Donald H

    2011-09-01

    Aspergillus terreus has been difficult to identify in cases of aspergillosis, and clinical identification has been restricted to the broad identification of aspergillosis lesions in affected organs or the detection of fungal carbohydrates. As a result, there is a clinical need to identify species-specific biomarkers that can be used to detect invasive A. terreus disease. Monoclonal antibodies (MAbs) were developed to a partially purified preparation of cytolytic hyphal exoantigens (HEA) derived from A. terreus culture supernatant (CSN). Twenty-three IgG1 isotype murine MAbs were developed and tested for cross-reactivity against hyphal extracts of 54 fungal species. Sixteen MAbs were shown to be specific for A. terreus. HEA were detected in conidia, hyphae, and in CSN of A. terreus. HEA were expressed in high levels in the hyphae during early stages of A. terreus growth at 37°C, whereas at room temperature the expression of HEA peaked by days 4 to 5. Expression kinetics of HEA in CSN showed a lag, with peak levels at later time points at room temperature and 37°C than in hyphal extracts. Serum spiking experiments demonstrated that human serum components do not inhibit detection of the HEA epitopes by MAb enzyme-linked immunosorbent assay (ELISA). Immunoprecipitation and proteomic analysis demonstrated that MAbs 13E11 and 12C4 immunoprecipitated a putative uncharacterized leucine aminopeptidase (Q0CAZ7), while MAb 19B2 recognized a putative dipeptidyl-peptidase V (DPP5). Studies using confocal laser scanning microscopy showed that the uncharacterized leucine aminopeptidase mostly localized to extracellular matrix structures while dipeptidyl-peptidase V was mostly confined to the cytoplasm.

  19. An Immunoglobulin G1 Monoclonal Antibody Highly Specific to the Wall of Cryptosporidium Oocysts

    Science.gov (United States)

    Weir, C.; Vesey, G.; Slade, M.; Ferrari, B.; Veal, D. A.; Williams, K.

    2000-01-01

    The detection of Cryptosporidium oocysts in drinking water is critically dependent on the quality of immunofluorescent reagents. Experiments were performed to develop a method for producing highly specific antibodies to Cryptosporidium oocysts that can be used for water testing. BALB/c mice were immunized with six different antigen preparations and monitored for immunoglobulin G (IgG) and IgM responses to the surface of Cryptosporidium oocysts. One group of mice received purified oocyst walls, a second group received a soluble protein preparation extracted from the outside of the oocyst wall, and the third group received whole inactivated oocysts. Three additional groups were immunized with sequentially prepared oocyst extracts to provide for a comparison of the immune response. Mice injected with the soluble protein extract demonstrated an IgG response to oocysts surface that was not seen in the whole-oocyst group. Mice injected with whole oocysts showed an IgM response only, while mice injected with purified oocyst walls showed little increase in IgM or IgG levels. Of the additional reported preparations only one, BME (2-mercaptoethanol treated), produced a weak IgM response to the oocyst wall. A mouse from the soluble oocyst extract group yielding a high IgG response was utilized to produce a highly specific IgG1 monoclonal antibody (Cry104) specific to the oocyst surface. Comparative flow cytometric analysis indicated that Cry104 has a higher avidity and specificity to oocysts in water concentrates than other commercially available antibodies. PMID:10973448

  20. Epitope mapping of functional domains of human factor V with human and mouse monoclonal antibodies

    International Nuclear Information System (INIS)

    Annamalai, A.E.; Rao, A.K.; Chiu, H.C.; Wang, D.; Dutta-Roy, A.K.; Colman, R.W.

    1986-01-01

    The authors previously described two human monoclonal antibodies (MAbs) which inactivated factor V. The authors have now purified the predominant antibody (H2) on protein A Sepharose using a pH gradient and typed it as IgG 1 ,. Immunoprecipitation of 125 I-human factor Va with H2 demonstrated specificity for the heavy chain (D), Mr = 105,000. The authors compared using ELISA the competitive binding to factor Va, of H2, H1 and two mouse MAbs, B38 (directed to E) and B10 (to activation peptide, Cl). All four antibodies recognized distinct epitopes in factor V with steric overlap in some cases. Factor Xa showed a concentration dependent competition for binding of H1, H2 and B38 but not B10 to factor V/Va in ELISA. All MAbs bound to factor V/Va in the absence of Ca ++ . However, Ca ++ at 8 mM increased the binding of H1 and H2 to 165% and 360% and did not have any effect on the binding of either mouse MAbs. Prothrombin at a concentration of up to 400 μg/ml did not inhibit binding of any of these antibodies. Thus, both the light (E) and heavy (D) chains of factor Va but not the activation peptide (Cl) interact with factor Xa as defined by the MAbs. In addition, sites on both chains for Ca ++ are recognized by particular MAbs (H1 and H2). These studies increase their knowledge of the interactions of factor V domains in the formation of prothrombinase complex

  1. Establishment of H2Mab-119, an Anti-Human Epidermal Growth Factor Receptor 2 Monoclonal Antibody, Against Pancreatic Cancer.

    Science.gov (United States)

    Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Chang, Yao-Wen; Harada, Hiroyuki; Suzuki, Hiroyoshi; Kaneko, Mika K; Kato, Yukinari

    2017-12-01

    Human epidermal growth factor receptor 2 (HER2) is overexpressed in breast cancer and is associated with poor clinical outcomes. In addition, HER2 expression has been reported in other cancers, such as gastric, colorectal, lung, and pancreatic cancers. An anti-HER2 humanized antibody, trastuzumab, leads to significant survival benefits in patients with HER2-overexpressing breast cancers and gastric cancers. Herein, we established a novel anti-HER2 monoclonal antibody (mAb), H 2 Mab-119 (IgG 1 , kappa), and characterized its efficacy against pancreatic cancers using flow cytometry, Western blot, and immunohistochemical analyses. H 2 Mab-119 reacted with pancreatic cancer cell lines, such as KLM-1, Capan-2, and MIA PaCa-2, but did not react with PANC-1 in flow cytometry analysis. Western blot analysis also revealed a moderate signal for KLM-1 and a weak signal for MIA PaCa-2, although H 2 Mab-119 reacted strongly with LN229/HER2 cells. Finally, immunohistochemical analyses with H 2 Mab-119 revealed sensitive and specific reactions against breast and colon cancers but did not react with pancreatic cancers, indicating that H 2 Mab-119 is useful for detecting HER2 overexpression in pancreatic cancers using flow cytometry and Western blot analyses.

  2. H2Mab-77 is a Sensitive and Specific Anti-HER2 Monoclonal Antibody Against Breast Cancer.

    Science.gov (United States)

    Itai, Shunsuke; Fujii, Yuki; Kaneko, Mika K; Yamada, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Chang, Yao-Wen; Handa, Saori; Takahashi, Maki; Suzuki, Hiroyoshi; Harada, Hiroyuki; Kato, Yukinari

    2017-08-01

    Human epidermal growth factor receptor 2 (HER2) plays a critical role in the progression of breast cancers, and HER2 overexpression is associated with poor clinical outcomes. Trastuzumab is an anti-HER2 humanized antibody that leads to significant survival benefits in patients with HER2-positive metastatic breast cancers. In this study, we developed novel anti-HER2 monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. Initially, we expressed the full length or ectodomain of HER2 in LN229 glioblastoma cells and then immunized mice with ectodomain of HER2 or LN229/HER2, and performed the first screening by enzyme-linked immunosorbent assays using ectodomain of HER2. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical analyses (fourth screening). Among 100 mAb clones, only three mAbs reacted with HER2 in Western blot, and clone H 2 Mab-77 (IgG 1 , kappa) was selected. Finally, immunohistochemical analyses with H 2 Mab-77 showed sensitive and specific reactions against breast cancer cells, warranting the use of H 2 Mab-77 to detect HER2 in pathological analyses of breast cancers.

  3. Enavatuzumab, a Humanized Anti-TWEAK Receptor Monoclonal Antibody, Exerts Antitumor Activity through Attracting and Activating Innate Immune Effector Cells

    Directory of Open Access Journals (Sweden)

    Shiming Ye

    2017-01-01

    Full Text Available Enavatuzumab is a humanized IgG1 anti-TWEAK receptor monoclonal antibody that was evaluated in a phase I clinical study for the treatment of solid malignancies. The current study was to determine whether and how myeloid effector cells were involved in postulated mechanisms for its potent antitumor activity in xenograft models. The initial evidence for a role of effector cells was obtained in a subset of tumor xenograft mouse models whose response to enavatuzumab relied on the binding of Fc of the antibody to Fcγ receptor. The involvement of effector cells was further confirmed by immunohistochemistry, which revealed strong infiltration of CD45+ effector cells into tumor xenografts in responding models, but minimal infiltration in nonresponders. Consistent with the xenograft studies, human effector cells preferentially migrated toward in vivo-responsive tumor cells treated by enavatuzumab in vitro, with the majority of migratory cells being monocytes. Conditioned media from enavatuzumab-treated tumor cells contained elevated levels of chemokines, which might be responsible for enavatuzumab-triggered effector cell migration. These preclinical studies demonstrate that enavatuzumab can exert its potent antitumor activity by actively recruiting and activating myeloid effectors to kill tumor cells. Enavatuzumab-induced chemokines warrant further evaluation in clinical studies as potential biomarkers for such activity.

  4. Increased half-life and enhanced potency of Fc-modified human PCSK9 monoclonal antibodies in primates.

    Directory of Open Access Journals (Sweden)

    Yijun Shen

    Full Text Available Blocking proprotein convertase subtilisin kexin type 9 (PCSK9 binding to low-density lipoprotein receptor (LDLR can profoundly lower plasma LDL levels. Two anti-PCKS9 monoclonal antibodies (mAbs, alirocumab and evolocumab, were approved by the FDA in 2015. The recommended dose is 75 mg to 150 mg every two weeks for alirocumab and 140mg every two weeks or 420 mg once a month for evolocumab. This study attempted to improve the pharmacokinetic properties of F0016A, an IgG1 anti-PCKS9 mAb, to generate biologically superior molecules. We engineered several variants with two or three amino acid substitutions in the Fc fragment based on prior knowledge. The Fc-modified mAbs exhibited increased binding to FcRn, resulting in prolonged serum half-life and enhanced efficacy in vivo. These results demonstrate that Fc-modified anti-PCKS9 antibodies may enable less frequent or lower dosing of antibodies by improved recycling into the blood.

  5. Lessons from the Crystal Structure of the S. aureus Surface Protein Clumping Factor A in Complex With Tefibazumab, an Inhibiting Monoclonal Antibody

    Directory of Open Access Journals (Sweden)

    Vannakambadi K. Ganesh

    2016-11-01

    Full Text Available The Staphylococcus aureus fibrinogen binding MSCRAMM (Microbial Surface Components Recognizing Adhesive Matrix Molecules, ClfA (clumping factor A is an important virulence factor in staphylococcal infections and a component of several vaccines currently under clinical evaluation. The mouse monoclonal antibody aurexis (also called 12-9, and the humanized version tefibazumab are therapeutic monoclonal antibodies targeting ClfA that in combination with conventional antibiotics were effective in animal models but showed less impressive efficacy in a limited Phase II clinical trial. We here report the crystal structure and a biochemical characterization of the ClfA/tefibazumab (Fab complex. The epitope for tefibazumab is located to the “top” of the N3 subdomain of ClfA and partially overlaps with a previously unidentified second binding site for fibrinogen. A high-affinity binding of ClfA to fibrinogen involves both an interaction at the N3 site and the previously identified docking of the C-terminal segment of the fibrinogen γ-chain in the N2N3 trench. Although tefibazumab binds ClfA with high affinity we observe a modest IC50 value for the inhibition of fibrinogen binding to the MSCRAMM. This observation, paired with a common natural occurring variant of ClfA that is not effectively recognized by the mAb, may partly explain the modest effect tefibazumab showed in the initial clinic trail. This information will provide guidance for the design of the next generation of therapeutic anti-staphylococcal mAbs targeting ClfA.

  6. Monoclonal antibodies to murine thrombospondin-1 and thrombospondin-2 reveal differential expression patterns in cancer and low antigen expression in normal tissues

    International Nuclear Information System (INIS)

    Bujak, Emil; Pretto, Francesca; Ritz, Danilo; Gualandi, Laura; Wulhfard, Sarah; Neri, Dario

    2014-01-01

    There is a considerable interest for the discovery and characterization of tumor-associated antigens, which may facilitate antibody-based pharmacodelivery strategies. Thrombospondin-1 and thrombospondin-2 are homologous secreted proteins, which have previously been reported to be overexpressed during remodeling typical for wound healing and tumor progression and to possibly play a functional role in cell proliferation, migration and apoptosis. To our knowledge, a complete immunohistochemical characterization of thrombospondins levels in normal rodent tissues has not been reported so far. Using antibody phage technology, we have generated and characterized monoclonal antibodies specific to murine thrombospondin-1 and thrombospondin-2, two antigens which share 62% aminoacid identity. An immunofluorescence analysis revealed that both antigens are virtually undetectable in normal mouse tissues, except for a weak staining of heart tissue by antibodies specific to thrombospondin-1. The analysis also showed that thrombospondin-1 was strongly expressed in 5/7 human tumors xenografted in nude mice, while it was only barely detectable in 3/8 murine tumors grafted in immunocompetent mice. By contrast, a high-affinity antibody to thrombospondin-2 revealed a much lower level of expression of this antigen in cancer specimens. Our analysis resolves ambiguities related to conflicting reports on thrombosponding expression in health and disease. Based on our findings, thrombospondin-1 (and not thrombospondin-2) may be considered as a target for antibody-based pharmacodelivery strategies, in consideration of its low expression in normal tissues and its upregulation in cancer. - Highlights: • High affinity monoclonal antibodies to murine and human TSP1 and 2 were raised. • Both antigens are virtually undetectable in normal mouse tissues. • Strong positivity of human tumor xenografts for TSP1 was detected. • Study revealed much lower level of TSP2 expression in cancer specimens

  7. Monoclonal antibodies to murine thrombospondin-1 and thrombospondin-2 reveal differential expression patterns in cancer and low antigen expression in normal tissues

    Energy Technology Data Exchange (ETDEWEB)

    Bujak, Emil [Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Vladimir-Prelog-Weg 2, CH-8093 Zurich (Switzerland); Pretto, Francesca; Ritz, Danilo; Gualandi, Laura; Wulhfard, Sarah [Philochem AG, Libernstrasse 3, CH-8112 Otelfingen (Switzerland); Neri, Dario, E-mail: neri@pharma.ethz.ch [Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Vladimir-Prelog-Weg 2, CH-8093 Zurich (Switzerland)

    2014-09-10

    There is a considerable interest for the discovery and characterization of tumor-associated antigens, which may facilitate antibody-based pharmacodelivery strategies. Thrombospondin-1 and thrombospondin-2 are homologous secreted proteins, which have previously been reported to be overexpressed during remodeling typical for wound healing and tumor progression and to possibly play a functional role in cell proliferation, migration and apoptosis. To our knowledge, a complete immunohistochemical characterization of thrombospondins levels in normal rodent tissues has not been reported so far. Using antibody phage technology, we have generated and characterized monoclonal antibodies specific to murine thrombospondin-1 and thrombospondin-2, two antigens which share 62% aminoacid identity. An immunofluorescence analysis revealed that both antigens are virtually undetectable in normal mouse tissues, except for a weak staining of heart tissue by antibodies specific to thrombospondin-1. The analysis also showed that thrombospondin-1 was strongly expressed in 5/7 human tumors xenografted in nude mice, while it was only barely detectable in 3/8 murine tumors grafted in immunocompetent mice. By contrast, a high-affinity antibody to thrombospondin-2 revealed a much lower level of expression of this antigen in cancer specimens. Our analysis resolves ambiguities related to conflicting reports on thrombosponding expression in health and disease. Based on our findings, thrombospondin-1 (and not thrombospondin-2) may be considered as a target for antibody-based pharmacodelivery strategies, in consideration of its low expression in normal tissues and its upregulation in cancer. - Highlights: • High affinity monoclonal antibodies to murine and human TSP1 and 2 were raised. • Both antigens are virtually undetectable in normal mouse tissues. • Strong positivity of human tumor xenografts for TSP1 was detected. • Study revealed much lower level of TSP2 expression in cancer specimens

  8. A variety of human monoclonal antibodies against epidermal growth factor receptor isolated from a phage antibody library.

    Science.gov (United States)

    Kurosawa, Gene; Kondo, Mariko; Kurosawa, Yoshikazu

    2016-11-04

    When the technology for constructing human antibody (Ab) libraries using a phage-display system was developed, many researchers in Ab-related fields anticipated that it would be widely applied to the development of pharmaceutical drugs against various diseases, including cancers. However, successful examples of such applications are very limited. Moreover, researchers who utilize phage-display technology now show divergent ways of thinking about phage Ab libraries. For example, there is debate about what should be the source of V H and V L genes for the construction of libraries to cover the whole repertoire of Abs present in the human body. In the immune system, the introduction of mutations into V genes followed by selection based on binding activity, termed Ab maturation, is required for the production of Abs exhibiting high affinity to the antigen (Ag). Therefore, introduction of mutations and selection are required for isolation of Abs with high affinity after isolation of clones from phage Ab libraries. We constructed a large human Ab library termed AIMS, developed a screening method termed ICOS, and succeeded in isolating many human monoclonal Abs (mAbs) that specifically and strongly bind to various tumor-associated Ags. Eight anti-EGFR mAbs were included, which we characterized. These mAbs showed various different activities against EGFR-expressing cancer cells. In this paper, we describe these data and discuss the possibility and necessity that the mAbs isolated from the AIMS library might be developed as therapeutic drugs against cancers without introduction of mutations. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Molecular cloning of a second subunit of the receptor for human granulocyte - macrophage colony-stimulating factor (GM-CSF): Reconstitution of a high-affinity GM-CSF receptor

    International Nuclear Information System (INIS)

    Hayashida, Kazuhiro; Kitamura, Toshio; Gorman, D.M.; Miyajima, Atsushi; Arai, Kenichi; Yokota, Takashi

    1990-01-01

    Using the mouse interleukin 3 (IL-3) receptor cDNA as a probe, the authors obtained a monologous cDNA (KH97) from a cDNA library of a human hemopoietic cell line, TF-1. The protein encoded by the KH97 cDNA has 56% amino acid sequence identity with the mouse IL-3 receptor and retains features common to the family of cytokine receptors. Fibroblasts transfected with the KH97 cDNA expressed a protein of 120 kDa but did not bind any human cytokines, including IL-3 and granulocyte - macrophage colony-stimulating factor (GM-CSF). Interestingly, cotransfection of cDNAs for KH97 and the low-affinity human GM-CSF receptor in fibroblasts resulted in formation of a high-affinity receptor for GM-CSF. The dissociation rate of GM-CSF from the reconstituted high-affinity receptor was slower than that from the low-affinity site, whereas the association rate was unchanged. Cross-linking of 125 I-labeled GM-CSF to fibroblasts cotransfected with both cDNAs revealed the same cross-linking patterns as in TF-1 cells - i.e., two major proteins of 80 and 120 kDa which correspond to the low-affinity GM-CSF receptor and the KH97 protein, respectively. These results indicate that the high-affinity GM-CSF receptor is composed of at least two components in a manner analogous to the IL-2 receptor. They therefore propose to designate the low-affinity GM-CSF receptor and the KH97 protein as the α and β subunits of the GM-CSF receptor, respectively

  10. Recent developments in monoclonal antibody radiolabeling techniques

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, S.C.; Mease, R.C.

    1989-01-01

    Monoclonal antibodies (MAbs) have shown the potential to serve as selective carriers of radionuclides to specific in vivo antigens. Accordingly, there has been an intense surge of research activity in an effort to develop and evaluate MAb-based radiopharmaceuticals for tumor imaging (radioimmunoscintigraphy) and therapy (radioimmunotherapy), as well as for diagnosing nonmalignant diseases. A number of problems have recently been identified, related to the MAbs themselves and to radiolabeling techniques, that comprise both the selectivity and the specificity of the in vivo distribution of radiolabeled MAbs. This paper will address some of these issues and primarily discuss recent developments in the techniques for radiolabeling monoclonal antibodies that may help resolve problems related to the poor in vivo stability of the radiolabel and may thus produce improved biodistribution. Even though many issues are identical with therapeutic radionuclides, the discussion will focus mainly on radioimmunoscintigraphic labels. 78 refs., 6 tabs.

  11. Quantitative imaging with radiolabeled monoclonal antibodies

    International Nuclear Information System (INIS)

    Moldofsky, P.J.; Hammond, N.D.

    1988-01-01

    The ability to image tumor by using radiolabeled monoclonal antibody products has been widely demonstrated. The questions of safety and efficacy remain open and require further experience, but at least in some clinical situations radioimmunoimaging has provided clinically useful information. Imaging tumor with radiolabeled monoclonal and polyclonal antibodies has been widely reported, and several summaries have recently appeared. For extensive review of recent clinical imaging the reader is referred to these excellent sources. Having demonstrated the possibility of imaging tumor with radiolabeled antibody, the question now apparent is: will the imaging modality provide information new and different from the already available with established techniques in computed tomography, magnetic resonance imaging, and standard nuclear medicine?

  12. [Monoclonal antibodies in diagnosis of acute leukemias].

    Science.gov (United States)

    Krawczyńska, A; Robak, T

    1996-01-01

    Immunophenotyping has become an essential component for the study of acute myeloblastic (AML) and lymphoblastic (ALL) leukaemias. The recent development of highly specific monoclonal antibodies (Mc Ab) to differentiation antigens (CD) of haematopoetic cells have made it readily available to clinical laboratories in most major hospitals. Immunophenotyping complements standard morphology by providing information on lineage, stage of differentiation and clonality. In addition some of the flow cytometry findings have independent prognostic significance. Monoclonal antibodies useful in defining lineage (B-cell versus T-cell) and stages of differentiation of ALL. It can be also used in identifying characteristic feature of AML and aiding in lineage determination in acute leukaemias that are morphologically undifferentiated. Surface immunophenotyping is especially helpful for recognizing mixed lineage acute leukaemia and diagnosing certain rare entities such as erythroleukaemia (M6), acute megakaryocytic leukaemia (M7) and minimally differentiation acute myeloid leukaemia.

  13. Recent developments in monoclonal antibody radiolabeling techniques

    International Nuclear Information System (INIS)

    Srivastava, S.C.; Mease, R.C.

    1989-01-01

    Monoclonal antibodies (MAbs) have shown the potential to serve as selective carriers of radionuclides to specific in vivo antigens. Accordingly, there has been an intense surge of research activity in an effort to develop and evaluate MAb-based radiopharmaceuticals for tumor imaging (radioimmunoscintigraphy) and therapy (radioimmunotherapy), as well as for diagnosing nonmalignant diseases. A number of problems have recently been identified, related to the MAbs themselves and to radiolabeling techniques, that comprise both the selectivity and the specificity of the in vivo distribution of radiolabeled MAbs. This paper will address some of these issues and primarily discuss recent developments in the techniques for radiolabeling monoclonal antibodies that may help resolve problems related to the poor in vivo stability of the radiolabel and may thus produce improved biodistribution. Even though many issues are identical with therapeutic radionuclides, the discussion will focus mainly on radioimmunoscintigraphic labels. 78 refs., 6 tabs

  14. Taking aim at cancer with monoclonal antibodies

    International Nuclear Information System (INIS)

    Klausner, A.

    1986-01-01

    Conjugating radioisotopes to monoclonal antibodies could have certain advantages in cancer therapy. Radioactive compounds have the double-edged ability to kill cells that are up to centimeter or more away. This is a plausible way to overcome tumor heterogeneity, but it also means that normal cells near the tumor could be affected. Hybritech (San Diego, CA) has been supplying antibody linked to the radioisotope yttrium-90 for a number of clinical trials. Work at Johns Hopkins University (Baltimore, MD) has focused on polyclonal antibodies to hepatoma. Monoclonal antibodies will be used there soon, and trials could be expanded eventually to include breast, lung, and prostate cancer as well. Hybritech also expects that the yttrium-antibody conjugates developed with NCI will enter the clinic later this year for treating leukemia and lymphoma systems; treatments for melanomas should follow

  15. Monoclonal TCR-redirected tumor cell killing.

    Science.gov (United States)

    Liddy, Nathaniel; Bossi, Giovanna; Adams, Katherine J; Lissina, Anna; Mahon, Tara M; Hassan, Namir J; Gavarret, Jessie; Bianchi, Frayne C; Pumphrey, Nicholas J; Ladell, Kristin; Gostick, Emma; Sewell, Andrew K; Lissin, Nikolai M; Harwood, Naomi E; Molloy, Peter E; Li, Yi; Cameron, Brian J; Sami, Malkit; Baston, Emma E; Todorov, Penio T; Paston, Samantha J; Dennis, Rebecca E; Harper, Jane V; Dunn, Steve M; Ashfield, Rebecca; Johnson, Andy; McGrath, Yvonne; Plesa, Gabriela; June, Carl H; Kalos, Michael; Price, David A; Vuidepot, Annelise; Williams, Daniel D; Sutton, Deborah H; Jakobsen, Bent K

    2012-06-01

    T cell immunity can potentially eradicate malignant cells and lead to clinical remission in a minority of patients with cancer. In the majority of these individuals, however, there is a failure of the specific T cell receptor (TCR)–mediated immune recognition and activation process. Here we describe the engineering and characterization of new reagents termed immune-mobilizing monoclonal TCRs against cancer (ImmTACs). Four such ImmTACs, each comprising a distinct tumor-associated epitope-specific monoclonal TCR with picomolar affinity fused to a humanized cluster of differentiation 3 (CD3)-specific single-chain antibody fragment (scFv), effectively redirected T cells to kill cancer cells expressing extremely low surface epitope densities. Furthermore, these reagents potently suppressed tumor growth in vivo. Thus, ImmTACs overcome immune tolerance to cancer and represent a new approach to tumor immunotherapy.

  16. New monoclonal antibody to human apolipoprotein J

    Czech Academy of Sciences Publication Activity Database

    Čapková, Jana; Geussová, Gizela; Pěknicová, Jana

    2002-01-01

    Roč. 2002, č. 48 (2002), s. 40-42 ISSN 0015-5500 R&D Projects: GA ČR GV524/96/K162 Grant - others:NFDK-MAOB(XE) 1985-NFDK-MAOB Institutional research plan: CEZ:AV0Z5052915 Keywords : apo J * human spermatoza * monoclonal antibody Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.615, year: 2002

  17. Polyclonal and monoclonal antibodies in clinic.

    Science.gov (United States)

    Wootla, Bharath; Denic, Aleksandar; Rodriguez, Moses

    2014-01-01

    Immunoglobulins (Ig) or antibodies are heavy plasma proteins, with sugar chains added to amino-acid residues by N-linked glycosylation and occasionally by O-linked glycosylation. The versatility of antibodies is demonstrated by the various functions that they mediate such as neutralization, agglutination, fixation with activation of complement and activation of effector cells. Naturally occurring antibodies protect the organism against harmful pathogens, viruses and infections. In addition, almost any organic chemical induces antibody production of antibodies that would bind specifically to the chemical. These antibodies are often produced from multiple B cell clones and referred to as polyclonal antibodies. In recent years, scientists have exploited the highly evolved machinery of the immune system to produce structurally and functionally complex molecules such as antibodies from a single B clone, heralding the era of monoclonal antibodies. Most of the antibodies currently in the clinic, target components of the immune system, are not curative and seek to alleviate symptoms rather than cure disease. Our group used a novel strategy to identify reparative human monoclonal antibodies distinct from conventional antibodies. In this chapter, we discuss the therapeutic relevance of both polyclonal and monoclonal antibodies in clinic.

  18. Establishment of EMab-134, a Sensitive and Specific Anti-Epidermal Growth Factor Receptor Monoclonal Antibody for Detecting Squamous Cell Carcinoma Cells of the Oral Cavity.

    Science.gov (United States)

    Itai, Shunsuke; Yamada, Shinji; Kaneko, Mika K; Chang, Yao-Wen; Harada, Hiroyuki; Kato, Yukinari

    2017-12-01

    Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, activates downstream signaling cascades in many tumors. In this study, we established novel anti-EGFR monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. We immunized mice with a combination of the extracellular domain of EGFR and EGFR-overexpressing LN229 glioblastoma cells (LN229/EGFR) and performed the first screening using enzyme-linked immunosorbent assay. Next, we selected mAbs using flow cytometry. Among 156 established clones, two mAbs, EMab-51 (IgG 1 , kappa) and EMab-134 (IgG 1 , kappa), reacted with EGFR in Western blot analysis; EMab-134 showed a much higher sensitivity compared with EMab-51. We compared the binding affinities of EMab-51 and EMab-134 using flow cytometry; the calculated K D values for EMab-51 and EMab-134 against SAS cells/HSC-2 cells were 9.2 × 10 -9 M/9.9 × 10 -9 M and 2.6 × 10 -9 M/8.3 × 10 -9 M, respectively, indicating that EMab-134 has a higher affinity to EGFR-expressing cells. Immunohistochemical analysis of EMab-51 and EMab-134 showed sensitive and specific reactions against oral cancer cells; EMab-134 demonstrated a much higher sensitivity (36/38 cases; 94.7%) to oral squamous cell carcinomas compared with EMab-51 (6/38 cases; 15.8%). This novel anti-EGFR mAb, EMab-134, could be advantageous for detecting EGFR in the pathological analysis of EGFR-expressing cancers.

  19. Biodistribution of an anti-interleukin 2 receptor monoclonal antibody in rat recipients of a heart allograft, and its use as a rejection marker in gamma scintigraphy

    International Nuclear Information System (INIS)

    Thedrez, P.; Paineau, J.; Jacques, Y.; Chatal, J.F.; Pelegrin, A.; Bouchaud, C.; Soulillou, J.P.

    1989-01-01

    Anti-interleukin-2 receptor monoclonal antibodies have been shown to prevent allograft rejection. This paper reports on the biodistribution of a mouse MoAb directed at the 55 Kd alpha chain of rat interleukin-2 receptor (IL2-R) during allograft rejection. Only a low percentage (approximately 1%) of intact 125I-labeled MoAb was detected in the rejected graft, and irrelevant control IgG1 was found at a similar level. This suggests that most of the injected intact MoAb bound to graft tissue via its monomorphic Fc segment. In contrast, OX39 F(ab')2 fragments showed a preferential localization in the rejected allograft and did not bind to the LEW-to-LEW syngeneic heart graft. Irrelevant F(ab')2 did not concentrate in the allogeneic graft. Accordingly, F(ab')2 fragments from OX39 or irrelevant MoAb were used for gamma-scintigraphy on allograft recipients together with biodistribution studies. Results show that scintigraphy was able to detect allograft accumulation of 131I OX39 F(ab')2, whereas no imaging was obtained when OX39 F(ab')2 was used in the syngeneic combination or when irrelevant 131-IgG1 F(ab')2 was given to allograft recipients. This method, applied to the clinical situation, could be of interest for detection of early graft rejection episodes by immunoscintigraphy using reagents specific for activation determinants on lymphocyte membranes, such as anti-interleukin-2 receptor MoAb

  20. Complement-mediated bactericidal activity of anti-factor H binding protein monoclonal antibodies against the meningococcus relies upon blocking factor H binding.

    Science.gov (United States)

    Giuntini, Serena; Reason, Donald C; Granoff, Dan M

    2011-09-01

    Binding of the complement-downregulating protein factor H (fH) to the surface of the meningococcus is important for survival of the organism in human serum. The meningococcal vaccine candidate factor H binding protein (fHbp) is an important ligand for human fH. While some fHbp-specific monoclonal antibodies (MAbs) block binding of fH to fHbp, the stoichiometry of blocking in the presence of high serum concentrations of fH and its effect on complement-mediated bactericidal activity are unknown. To investigate this question, we constructed chimeric antibodies in which the human IgG1 constant region was paired with three murine fHbp-specific binding domains designated JAR 3, JAR 5, and MAb502. By surface plasmon resonance, the association rates for binding of all three MAbs to immobilized fHbp were >50-fold higher than that for binding of fH to fHbp, and the MAb dissociation rates were >500-fold lower than that for fH. While all three MAbs elicited similar C1q-dependent C4b deposition on live bacteria (classical complement pathway), only those antibodies that inhibited binding of fH to fHbp (JAR 3 and JAR 5) had bactericidal activity with human complement. MAb502, which did not inhibit fH binding, had complement-mediated bactericidal activity only when tested with fH-depleted human complement. When an IgG1 anti-fHbp MAb binds to sparsely exposed fHbp on the bacterial surface, there appears to be insufficient complement activation for bacteriolysis unless fH binding also is inhibited. The ability of fHbp vaccines to elicit protective antibodies, therefore, is likely to be enhanced if the antibody repertoire is of high avidity and includes fH-blocking activity.

  1. The C-terminal SH2 domain of p85 accounts for the high affinity and specificity of the binding of phosphatidylinositol 3-kinase to phosphorylated platelet-derived growth factor beta receptor.

    Science.gov (United States)

    Klippel, A; Escobedo, J A; Fantl, W J; Williams, L T

    1992-01-01

    Upon stimulation by its ligand, the platelet-derived growth factor (PDGF) receptor associates with the 85-kDa subunit of phosphatidylinositol (PI) 3-kinase. The 85-kDa protein (p85) contains two Src homology 2 (SH2) domains and one SH3 domain. To define the part of p85 that interacts with the PDGF receptor, a series of truncated p85 mutants was analyzed for association with immobilized PDGF receptor in vitro. We found that a fragment of p85 that contains a single Src homology domain, the C-terminal SH2 domain (SH2-C), was sufficient for directing the high-affinity interaction with the receptor. Half-maximal binding of SH2-C to the receptor was observed at an SH2-C concentration of 0.06 nM. SH2-C, like full-length p85, was able to distinguish between wild-type PDGF receptor and a mutant receptor lacking the PI 3-kinase binding site. An excess of SH2-C blocked binding of full-length p85 and PI 3-kinase to the receptor but did not interfere with the binding of two other SH2-containing proteins, phospholipase C-gamma and GTPase-activating protein. These results demonstrate that a region of p85 containing a single SH2 domain accounts both for the high affinity and specificity of binding of PI 3-kinase to the PDGF receptor. Images PMID:1312663

  2. Assay for the specificity of monoclonal antibodies in crossed immunoelectrophoresis

    DEFF Research Database (Denmark)

    Skjødt, K; Schou, C; Koch, C

    1984-01-01

    A method is described based on crossed immunoelectrophoresis of a complex antigen mixture in agarose gel followed by incubation of the gel with the monoclonal antibody. The bound monoclonal antibody is detected by the use of a secondary enzyme-labelled antibody. Using this technique we have been ...... I molecules. In other experiments using the same technique we demonstrated the reaction of a monoclonal antibody specific for chicken Ig light chains. Udgivelsesdato: 1984-Aug-3...

  3. Development and Characterization of Canine Distemper Virus Monoclonal Antibodies.

    Science.gov (United States)

    Liu, Yuxiu; Hao, Liying; Li, Xiangdong; Wang, Linxiao; Zhang, Jianpo; Deng, Junhua; Tian, Kegong

    2017-06-01

    Five canine distemper virus monoclonal antibodies were developed by immunizing BALB/c mice with a traditional vaccine strain Snyder Hill. Among these monoclonal antibodies, four antibodies recognized both field and vaccine strains of canine distemper virus without neutralizing ability. One monoclonal antibody, 1A4, against hemagglutinin protein of canine distemper virus was found to react only with vaccine strain virus but not field isolates, and showed neutralizing activity to vaccine strain virus. These monoclonal antibodies could be very useful tools in the study of the pathogenesis of canine distemper virus and the development of diagnostic reagents.

  4. Lack of radioimmunodetection and complications associated with monoclonal anticarcinoembryonic antigen antibody cross-reactivity with an antigen on circulating cells

    International Nuclear Information System (INIS)

    Dillman, R.O.; Beauregard, J.C.; Sobol, R.E.; Royston, I.; Bartholomew, R.M.; Hagan, P.S.; Halpern, S.E.

    1984-01-01

    Characterization of several high-affinity murine monoclonal anticarcinoembryonic antigen (CEA) antibodies suggested good specificity except for cross-reactivity with an antigen on granulocytes and erythrocytes which was different from the previously described normal cross-reacting antigen of granulocytes. In vivo studies in athymic mice using an indium conjugate of an anti-CEA monoclonal antibody (MoAb) revealed excellent specific uptake in colorectal carcinoma xenografts. Studies were conducted in humans to determine the limitations produced by the cross-reactivity with granulocytes and erythrocytes. Patients with metastatic colorectal cancer received 3 to 6 mg of anti-CEA MoAb over 10 min or 2 hr. In five of six trials, the MoAb infusion was associated with a 40 to 90% decrease in circulating granulocytes and systemic toxicity including fever, rigors, and emesis. One patient had no change in cell count and had no toxicity. Radionuclide scans with 111 In-anti-CEA MoAb showed marked uptake in the spleen when cells were eliminated, and in the liver, especially when pretreatment CEA levels were high. Metastatic tumor sites failed to concentrate the isotope. This study emphasizes the potential limitations for radioimmunodetection and/or radioimmunotherapy imposed by reactivity with circulating cells, and suggests that certain toxic reactions associated with MoAb infusions are related to destruction of circulating cells rather than allergic reactions to mouse protein. It also emphasizes how variables such as dose and binding affinity of antibody, radioisotope used, and assessment at different observation points can obscure lack of antibody specificity

  5. Lack of radioimmunodetection and complications associated with monoclonal anticarcinoembryonic antigen antibody cross-reactivity with an antigen on circulating cells

    Energy Technology Data Exchange (ETDEWEB)

    Dillman, R.O.; Beauregard, J.C.; Sobol, R.E.; Royston, I.; Bartholomew, R.M.; Hagan, P.S.; Halpern, S.E.

    1984-05-01

    Characterization of several high-affinity murine monoclonal anticarcinoembryonic antigen (CEA) antibodies suggested good specificity except for cross-reactivity with an antigen on granulocytes and erythrocytes which was different from the previously described normal cross-reacting antigen of granulocytes. In vivo studies in athymic mice using an indium conjugate of an anti-CEA monoclonal antibody (MoAb) revealed excellent specific uptake in colorectal carcinoma xenografts. Studies were conducted in humans to determine the limitations produced by the cross-reactivity with granulocytes and erythrocytes. Patients with metastatic colorectal cancer received 3 to 6 mg of anti-CEA MoAb over 10 min or 2 hr. In five of six trials, the MoAb infusion was associated with a 40 to 90% decrease in circulating granulocytes and systemic toxicity including fever, rigors, and emesis. One patient had no change in cell count and had no toxicity. Radionuclide scans with /sup 111/In-anti-CEA MoAb showed marked uptake in the spleen when cells were eliminated, and in the liver, especially when pretreatment CEA levels were high. Metastatic tumor sites failed to concentrate the isotope. This study emphasizes the potential limitations for radioimmunodetection and/or radioimmunotherapy imposed by reactivity with circulating cells, and suggests that certain toxic reactions associated with MoAb infusions are related to destruction of circulating cells rather than allergic reactions to mouse protein. It also emphasizes how variables such as dose and binding affinity of antibody, radioisotope used, and assessment at different observation points can obscure lack of antibody specificity.

  6. Nuclear oncology with monoclonal antibodies and peptides

    International Nuclear Information System (INIS)

    Hosono, Makoto

    1998-01-01

    Imaging and therapy using radiolabeled monoclonal antibodies have proved useful in many clinical studies. However, immunogenicity of mouse antibodies to human and insufficient tumor-to-normal tissue ratios remained to be solved. Chimerization and humanization by genetic engineering, and multistep targeting techniques have enabled lower immunogenicity and higher tumor-to-normal tissue contrast. Peptides like somatostatin-analogs have been reportedly useful in imaging tumors, which are either somatostatin receptor positive or negative. Elevated normal tissue accumulation of radiolabeled peptides is a drawback in aiming internal radiation therapy. (author). 51 refs

  7. Monoclonal Idiotope Vaccine against Streptococcus pneumoniae Infection

    Science.gov (United States)

    McNamara, Mary K.; Ward, Ronald E.; Kohler, Heinz

    1984-12-01

    A monoclonal anti-idiotope antibody coupled to a carrier protein was used to immunize BALB/c mice against a lethal Streptococcus pneumoniae infection. Vaccinated mice developed a high titer of antibody to phosphorylcholine, which is known to protect against infection with Streptococcus pneumoniae. Measurement of the median lethal dose of the bacteria indicated that anti-idiotope immunization significantly increased the resistance of BALB/c mice to the bacterial challenge. Antibody to an idiotope can thus be used as an antigen substitute for the induction of protective immunity.

  8. Synthesis, in vitro validation and in vivo pharmacokinetics of [{sup 125}I]N-[2-(4-iodophenyl)ethyl]-N-methyl-2-(1-piperidinyl) ethylamine: A high-affinity ligand for imaging sigma receptor positive tumors

    Energy Technology Data Exchange (ETDEWEB)

    John, Christy S; Gulden, Mary E; Vilner, Bertold J; Bowen, Wayne D

    1996-08-01

    N-[2-(4-iodophenyl)ethyl]-N-methyl-2-(1-piperidinyl)ethylamine, IPEMP, and the corresponding bromo derivative, BrPEMP, have been synthesized and characterized. Both BrPEMP and IPEMP were evaluated for sigma-1 and sigma-2 subtype receptor affinities and found to possess very high affinities for both receptor subtypes. The precursor for radioiodination n-tributylstannylphenylethylpiperidinylethylamine was prepared from its bromo derivative by palladium-catalyzed stannylation reaction. Radioiodinated 4-[{sup 125}I]PEMP was readily prepared in high yields and high specific activity by oxidative iododestannylation reaction using chloramine-T as oxidizing agent. Sites labeled by 4-[{sup 125}I]PEMP in guinea pig brain membranes showed high affinity for BD1008, haloperidol, and (+)-pentazocine (Ki = 5.06 {+-} 0.40, 32.6 {+-} 2.75, and 48.1 {+-} 8.60 nM, respectively), which is consistent with sigma receptor pharmacology. Competition binding studies of 4-[{sup 125}I]PEMP in melanoma (A375) and MCF-7 breast cancer cells showed a high affinity, dose-dependent inhibition of binding with known sigma ligand N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl) ethylamine, BD1008 (Ki = 5, 11 nM, respectively), supporting the labeling of sigma sites in these cells. Haloperidol, however showed a weaker (Ki 100-200 nM) affinity for the sites labeled by 4-[{sup 125}I]PEMP in these cells. Biodistribution studies of 4-[{sup 125}I]PEMP in rats showed a fast clearance of this radiopharmaceutical from blood, liver, lung, and other organs. A co-injection of 4-IPEMP with 4-[{sup 125}I]PEMP resulted in 37%, 69%, and 35% decrease in activity in liver, kidney, and brain (organs possessing sigma receptors), respectively at 1-h postinjection. These results suggest that 4-[{sup 125}I]PEMP is a promising radiopharmaceutical for pursuing further studies in animal models with tumors.

  9. Humanization and characterization of an anti-ricin neutralization monoclonal antibody.

    Directory of Open Access Journals (Sweden)

    Wei-Gang Hu

    Full Text Available Ricin is regarded as a high terrorist risk for the public due to its high toxicity and ease of production. Currently, there is no therapeutic or vaccine available against ricin. D9, a murine monoclonal antibody developed previously in our laboratory, can strongly neutralize ricin and is therefore a good candidate for humanization. Humanization of D9 variable regions was achieved by a complementarity-determining region grafting approach. The humanized D9 (hD9 variable regions were further grafted onto human heavy and light chain constant regions to assemble the complete antibody gene. A foot-and-mouth-disease virus-derived 2A self-processing sequence was introduced between heavy and light chain DNA sequences to cleave the recombinant protein into a functional full-length antibody molecule from a single open reading frame driven by a single promoter in an adenoviral vector. After expression in mammalian cells and purification, the hD9 was demonstrated to have equimolar expression of the full-length antibody heavy and light chains. More importantly, the hD9 exhibited high affinity to ricin with K(D of 1.63 nM, comparable to its parental murine D9 (2.55 nM. In a mouse model, intraperitoneal (i.p. administration of hD9, at a low dose of 5 µg per mouse, 4 hours after the i.p. challenge with 5×LD50 ricin was found to rescue 100% of the mice. In addition, administered 6 hours post-challenge, hD9 could still rescue 50% of the mice. The hD9 has the potential to be used for prophylactic or therapeutic purposes against ricin poisoning.

  10. Affinities and densities of high-affinity [3H]muscimol (GABA-A) binding sites and of central benzodiazepine receptors are unchanged in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy

    International Nuclear Information System (INIS)

    Butterworth, R.F.; Lavoie, J.; Giguere, J.F.; Pomier-Layrargues, G.

    1988-01-01

    The integrity of GABA-A receptors and of central benzodiazepine receptors was evaluated in membrane preparations from prefrontal cortex and caudate nuclei obtained at autopsy from nine cirrhotic patients who died in hepatic coma and an equal number of age-matched control subjects. Histopathological studies revealed Alzheimer Type II astrocytosis in all cases in the cirrhotic group; controls were free from neurological, psychiatric or hepatic diseases. Binding to GABA-A receptors was studied using [ 3 H]muscimol as radioligand. The integrity of central benzodiazepine receptors was evaluated using [ 3 H]flunitrazepam and [ 3 H]Ro15-1788. Data from saturation binding assays was analyzed by Scatchard plot. No modifications of either affinities (Kd) or densities (Bmax) of [ 3 H]muscimol of central benzodiazepine binding sites were observed. These findings do not support recent suggestions that alterations of either high-affinity GABA or benzodiazepine receptors play a significant role in the pathogenesis of hepatic encephalopathy

  11. Aggregates in monoclonal antibody manufacturing processes.

    Science.gov (United States)

    Vázquez-Rey, María; Lang, Dietmar A

    2011-07-01

    Monoclonal antibodies have proved to be a highly successful class of therapeutic products. Large-scale manufacturing of pharmaceutical antibodies is a complex activity that requires considerable effort in both process and analytical development. If a therapeutic protein cannot be stabilized adequately, it will lose partially or totally its therapeutic properties or even cause immunogenic reactions thus potentially further endangering the patients' health. The phenomenon of protein aggregation is a common issue that compromises the quality, safety, and efficacy of antibodies and can happen at different steps of the manufacturing process, including fermentation, purification, final formulation, and storage. Aggregate levels in drug substance and final drug product are a key factor when assessing quality attributes of the molecule, since aggregation might impact biological activity of the biopharmaceutical. In this review it is analyzed how aggregates are formed during monoclonal antibody industrial production, why they have to be removed and the manufacturing process steps that are designed to either minimize or remove aggregates in the final product. Copyright © 2011 Wiley Periodicals, Inc.

  12. Kinetics of intralymphatically delivered monoclonal antibodies

    International Nuclear Information System (INIS)

    Wahl, R.L.; Geatti, O.; Liebert, M.; Beers, B.; Jackson, G.; Laino, L.; Kronberg, S.; Wilson, B.S.; Beierwaltes, W.H.

    1985-01-01

    Radiolabeled monoclonal antibody (MoAb) administration subcutaneously (sq), so that preferential uptake is to the lymphatics, holds significant promise for the detection of lymph node metastases. Only limited information is available about clearance rates of intralymphatically administered MoAbs. I-131 labeled intact IgG (225.28S), F(ab's)2 (225.28S) or IgM (FT162) were administered sq to anesthetized Balb/C mice. Eight mice were studied with each MoAb, 4 with a foot-pad injection, 4 with an anterior abdominal injection. Gamma camera images were collected into a computer, over the first 6 hrs after injection with the animals anesthetized and immobile. Animals were then allowed to move about freely. Additional images were then acquired out to 48 hrs. Regions of interest wre selected over the injection site and the kinetics of antibody egress determined. Clearance rates from local sq injection sites are influenced by motion and somewhat by location. The class and fragment status of the MoAb appear relatively less important in determining clearance rates from sq injections than they are in determining whole-body clearance after iv injections. Additional studies using Fab fragments and additional monoclonals will be useful in extending these observations

  13. Bone marrow dosimetry for monoclonal antibody therapy

    International Nuclear Information System (INIS)

    Bigler, R.E.; Zanzonico, P.B.; Leonard, R.

    1986-01-01

    Immunoglobulins must permeate through the basement membrane of capillaries in order to enter the extracellular space (ECS) of tissue. Since the process is quite slow, the blood plasma activity in various organs contributes considerably to the radiation dose of the dose-limiting tissues. In bone marrow the basement membrane is absent and the blood circulation is functionally open. Therefore, blood plasma and marrow ECS maintain equal concentrations of labeled immunoglobulins. A combination of factors including intravenous administration, slow absorption into most tissues, slow breakdown and elimination of labeled immunoglobulin, and rapid entry into bone marrow ECS as well as known radiosensitivity of marrow led the authors to expect this tissue would prove to be the primary tissue at risk for systemic monoclonal antibody therapy. They have developed and applied in a Phase I clinical study of 131 I labeled CEA antibody a procedure for estimation of radiation dose to red bone marrow. Serieal measurements of blood plasma and total body retention are carried out. Binding of labeled antibody to the cellular components of blood is verified to be very low. They have observed bone marrow depression at doses greater than 400 rad. If no special procedures are used to reconstitute marrow after radiation treatment, this level represents a much greater than generally recognized limitation to radiolabeled monoclonal antibody therapy. 25 references, 4 tables

  14. Emerging monoclonal antibodies against Clostridium difficile infection.

    Science.gov (United States)

    Péchiné, Séverine; Janoir, Claire; Collignon, Anne

    2017-04-01

    Clostridium difficile infections are characterized by a high recurrence rate despite antibiotic treatments and there is an urgent need to develop new treatments such as fecal transplantation and immonotherapy. Besides active immunotherapy with vaccines, passive immunotherapy has shown promise, especially with monoclonal antibodies. Areas covered: Herein, the authors review the different assays performed with monoclonal antibodies against C. difficile toxins and surface proteins to treat or prevent primary or recurrent episodes of C. difficile infection in animal models and in clinical trials as well. Notably, the authors lay emphasis on the phase III clinical trial (MODIFY II), which allowed bezlotoxumab to be approved by the Food and Drug Administration and the European Medicines Agency. They also review new strategies for producing single domain antibodies and nanobodies against C. difficile and new approaches to deliver them in the digestive tract. Expert opinion: Only two human Mabs against TcdA and TcdB have been tested alone or in combination in clinical trials. However, many animal model studies have provided rationale for the use of Mabs and nanobodies in C. difficile infection and pave the way for further clinical investigation.

  15. Slc5a8, a Na+-coupled high-affinity transporter for short-chain fatty acids, is a conditional tumour suppressor in colon that protects against colitis and colon cancer under low-fibre dietary conditions.

    Science.gov (United States)

    Gurav, Ashish; Sivaprakasam, Sathish; Bhutia, Yangzom D; Boettger, Thomas; Singh, Nagendra; Ganapathy, Vadivel

    2015-07-15

    Mammalian colon harbours trillions of bacteria under physiological conditions; this symbiosis is made possible because of a tolerized response from the mucosal immune system. The mechanisms underlying this tolerogenic phenomenon remain poorly understood. In the present study we show that Slc5a8 (solute carrier gene family 5a, member 8), a Na(+)-coupled high-affinity transporter in colon for the bacterial fermentation product butyrate, plays a critical role in this process. Among various immune cells in colon, dendritic cells (DCs) are unique not only in their accessibility to luminal contents but also in their ability to induce tolerogenic phenotype in T-cells. We found that DCs exposed to butyrate express the immunosuppressive enzymes indoleamine 2,3-dioxygenase 1 (IDO1) and aldehyde dehydrogenase 1A2 (Aldh1A2), promote conversion of naive T-cells into immunosuppressive forkhead box P3(+) (FoxP3(+)) Tregs (regulatory T-cells) and suppress conversion of naive T-cells into pro-inflammatory interferon (IFN)-γ-producing cells. Slc5a8-null DCs do not induce IDO1 and Aldh1A2 and do not generate Tregs or suppress IFN-γ-producing T-cells in response to butyrate. We also provide in vivo evidence for an obligatory role for Slc5a8 in suppression of IFN-γ-producing T-cells. Furthermore, Slc5a8 protects against colitis and colon cancer under conditions of low-fibre intake but not when dietary fibre intake is optimal. This agrees with the high-affinity nature of the transporter to mediate butyrate entry into cells. We conclude that Slc5a8 is an obligatory link between dietary fibre and mucosal immune system via the bacterial metabolite butyrate and that this transporter is a conditional tumour suppressor in colon linked to dietary fibre content. © 2015 Authors; published by Portland Press Limited.

  16. Identification of the ligand-binding subunit of the human 5-hydroxytryptamine1A receptor with N-(p-azido-m-[125I] iodophenethyl)spiperone, a high affinity radioiodinated photoaffinity probe

    International Nuclear Information System (INIS)

    Raymond, J.R.; Fargin, A.; Lohse, M.J.; Regan, J.W.; Senogles, S.E.; Lefkowitz, R.J.; Caron, M.G.

    1989-01-01

    The ligand-binding subunit of the human 5-hydroxytryptamine1A (5-HT1A) receptor transiently expressed in COS-7 cells and of the native human 5-HT1A receptor derived from hippocampus and frontal cortex were identified by photoaffinity labeling with N-(p-azido-m-[125I]iodophenethyl)spiperone [( 125I]N3-NAPS), previously characterized as a high affinity radioiodinated D2-dopamine receptor probe. The identity of the ligand-binding subunit was confirmed by immunoprecipitation with an antipeptide rabbit antiserum, JWR21, raised against a synthetic peptide derived from the predicted amino acid sequence of the putative third intracellular loop of the human 5-HT1A receptor. In transiently transfected COS-7 cells expressing 14 +/- 3 pmol/mg of protein human 5-HT1A receptors, a single broad 75-kDa band was photoaffinity labeled by [125I]N3-NAPS. This band displayed the expected pharmacology of the 5-HT1A receptor, as evidenced by the ability of a series of competing ligands to block [125I]N3-NAPS photoincorporation. Moreover, antiserum JWR21 specifically and quantitatively immunoprecipitated the 75-kDa photoaffinity-labeled band from a soluble extract of the transfected COS-7 cell membranes, further confirming its identity. Finally, utilizing a combination of photoaffinity labeling and immunoprecipitation, the native ligand-binding subunit of 62-64 kDa was identified in human hippocampus and frontal cortex. The availability of the high specific activity, high affinity, photoaffinity ligand [125I]N3-NAPS and of a potent immunoprecipitating antiserum (JWR21) should greatly facilitate the biochemical characterization of the human 5-HT1A receptor

  17. Feasibility study of the Fab fragment of a monoclonal antibody against tissue factor as a diagnostic tool.

    Science.gov (United States)

    Tsumura, Ryo; Sato, Ryuta; Furuya, Fumiaki; Koga, Yoshikatsu; Yamamoto, Yoshiyuki; Fujiwara, Yuki; Yasunaga, Masahiro; Matsumura, Yasuhiro

    2015-12-01

    Tissue factor (TF) is expressed strongly in various types of cancer, especially cancers that are often refractory to treatment, such as pancreatic cancer. In this study, we compared the differences in the biophysical and pharmacological properties of whole IgG and the Fab fragment of anti-human TF monoclonal antibody (1849 antibodies), in order to determine their suitability for application in the diagnosis and treatment of cancers. In the biophysical examination, we investigated the characteristics of 1849-whole IgG and 1849-Fab by SPR sensing and confocal fluorescence microscopy analysis using recombinant human TF antigen and TF-overexpressing human pancreatic cancer cell line, BxPC3, respectively. After conjugation with Alexa-Flour-647, in vivo imaging was conducted in mice bearing BxPC3 xenograft tumors. Furthermore, the distribution of the conjugates in tumors and major organs was evaluated by ex vivo study. The in vitro experiments showed that 1849 antibodies had high affinity against TF antigen. In addition, 1849-Fab showed a faster dissociation rate from the antigen than 1849-whole IgG. In mice, 1849-Fab-Alexa-Flour-647 showed rapid renal clearance and faster tumor accumulation, achieving a high contrast signal over nearby normal tissues in the early phase and enhanced tumor penetration after administration. On the other hand, 1849-whole IgG-Alexa-Flour-647 showed slow clearance from the blood and sustained high tumor accumulation. These results suggest that 1849-Fab may be a useful tool for pancreatic cancer diagnosis.

  18. Label Free QCM Immunobiosensor for AFB1 Detection Using Monoclonal IgA Antibody as Recognition Element

    Directory of Open Access Journals (Sweden)

    Özlem Ertekin

    2016-08-01

    Full Text Available This study introduces the use of an IgA isotype aflatoxin (AF specific monoclonal antibody for the development of a highly sensitive Quartz Crystal Microbalance (QCM immunobiosensor for the detection of AF in inhibitory immunoassay format. The higher molecular weight of IgA antibodies proved an advantage over commonly used IgG antibodies in label free immunobiosensor measurements. IgA and IgG antibodies with similar affinity for AF were used in the comparative studies. Sensor surface was prepared by covalent immobilization of AFB1, using self assembled monolayer (SAM formed on gold coated Quartz Crystal, with 1-Ethyl-3-(3-dimethylaminopropyl carbodiimide/N-hydroxy succinimide (EDC/NHS method using a diamine linker. Nonspecific binding to the surface was decreased by minimizing the duration of EDC/NHS activation. Sensor surface was chemically blocked after AF immobilization without any need for protein blocking. This protein free sensor chip endured harsh solutions with strong ionic detergent at high pH, which is required for the regeneration of the high affinity antibody-antigen interaction. According to the obtained results, the detection range with IgA antibodies was higher than IgG antibodies in QCM immunosensor developed for AFB1.

  19. Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates demonstrate tumor specificity and anti-tumor activity.

    Science.gov (United States)

    Prendergast, Jillian M; Galvao da Silva, Ana Paula; Eavarone, David A; Ghaderi, Darius; Zhang, Mai; Brady, Dane; Wicks, Joan; DeSander, Julie; Behrens, Jeff; Rueda, Bo R

    Targeted therapeutics that can differentiate between normal and malignant tumor cells represent the ideal standard for the development of a successful anti-cancer strategy. The Sialyl-Thomsen-nouveau antigen (STn or Sialyl-Tn, also known as CD175s) is rarely seen in normal adult tissues, but it is abundantly expressed in many types of human epithelial cancers. We have identified novel antibodies that specifically target with high affinity the STn glycan independent of its carrier protein, affording the potential to recognize a wider array of cancer-specific sialylated proteins. A panel of murine monoclonal anti-STn therapeutic antibodies were generated and their binding specificity and efficacy were characterized in vitro and in in vivo murine cancer models. A subset of these antibodies were conjugated to monomethyl auristatin E (MMAE) to generate antibody-drug conjugates (ADCs). These ADCs demonstrated in vitro efficacy in STn-expressing cell lines and significant tumor growth inhibition in STn-expressing tumor xenograft cancer models with no evidence of overt toxicity.

  20. Development of a second generation monoclonal immunoradiometric assay. Increased sensitivity leads to enhanced detection of hepatitis B viral infection

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, H; Wands, J R; Kameda, H

    1988-09-13

    The authors have developed and employed a second generation monoclonal immunoradiometric assay (M2-IRMA) using antibodies of high affinity for epitopes that reside on hepatitis B surface antigen (HBsAg). This assay is capable of detecting as little as 15 pg/ml of HBsAg in serum. Improvements in sensitivity over a first generation immunoradiometric assay (MI-IRMA) was achieved by increasing the sample volume and time of incubation, and subjecting the reaction to a mechanical rotary device. 164 subjects with chronic hepatitis, 105 with cirrhosis, 67 with hepatocellular carcinoma, six with acute hepatitis A, seven with acute hepatitis B, 167 chronic carriers of hepatitis B virus (HBV) and 235 healthy individuals from Japan were studied and the results of the M2-IRMA were compared to a conventional polyclonal radioimmunoassay (P-RIA). By using a more sensitive assay design (M2-IRMA), a significant number of additional cases of HBV infection heretofore unsuspected in the etiology of chronic liver disease were identified. It is concluded that improvement in assay sensitivity for HBsAg is important in the serologic diagnosis of HBV in patients with chronic hepatitis, cirrhosis and hepatocellular carcinoma. 14 refs.; 6 figs.; 6 tabs.

  1. Crossreactivity of boar sperm monoclonal antibodies with human ...

    African Journals Online (AJOL)

    Monoclonal antibodies against the head (H mabs) and tail (Tmabs) of boar spermatozoa were produced. Spermatozoa from boar, stallion, bull, human, ram, goat and rabbit were independently incubated with the monoclonal antibodies and later stained by immunofluorescence method. There were positive reactions of the ...

  2. A monoclonal antibody that specifically recognizes m6A nucleoside

    OpenAIRE

    Espuny, Ruth; Castro, Ana; Codony, Carles; Eritja Casadellà, Ramón; Bach-Elias, Montse

    1998-01-01

    A hybridoma against the nucleoside m6A has been obtained from mouse spleen. This hybridoma was named H65 and it secretes monoclonal antibodies anti-m6A. The competition assays showed that the monoclonal antibody was highly specific for m6A nucleoside.

  3. Monoclonal antibody PAL-E specific for endothelium

    NARCIS (Netherlands)

    Schlingemann, R. O.; Dingjan, G. M.; Emeis, J. J.; Blok, J.; Warnaar, S. O.; Ruiter, D. J.

    1985-01-01

    A monoclonal antibody, PAL-E, is described that is specific for endothelial cells. The monoclonal antibody, an IgG2a, markedly stains endothelium of capillaries, medium-sized and small veins, and venules in frozen sections of human and some animal tissues tested. It reacts not at all or only weakly

  4. Treatment with anti-interferon-δ monoclonal antibodies modifies experimental autoimmune encephalomyelitis in interferon-δ receptor knockout mice

    DEFF Research Database (Denmark)

    Espejo, C.; Penkowa, Milena; Saez-Torres, I.

    2001-01-01

    Neuroinflammation, neuronal degeneration, regeneration, monoclonal antibodies, multiple schlerosis......Neuroinflammation, neuronal degeneration, regeneration, monoclonal antibodies, multiple schlerosis...

  5. Induction and characterization of monoclonal anti-idiotypic antibodies reactive with idiotopes of canine parvovirus neutralizing monoclonal antibodies.

    NARCIS (Netherlands)

    G.F. Rimmelzwaan (Guus); J. van Es (Johan); G.A. Drost; F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert)

    1991-01-01

    textabstractMonoclonal anti-idiotypic (anti-Id) antibodies (Ab2) were generated against idiotypes (Id) of canine parvovirus (CPV) specific monoclonal antibodies (MoAbs). The binding of most of these anti-Id antibodies to their corresponding Id could be inhibited by antigen, thus classifying these

  6. SPECT assay of radiolabeled monoclonal antibodies

    International Nuclear Information System (INIS)

    Jaszczak, R.J.

    1992-02-01

    The long-term goal of this research project is to develop methods to improve the utility of single photon emission computed tomography (SPECI) to quantify the biodistribution of monoclonal antibodies (MoAbs) labeled with clinically relevant radionuclides ( 123 I, 131 I, and 111 In) and with another radionuclide, 211 At, recently used in therapy. We describe here our progress in developing quantitative SPECT methodology for 111 In and 123 I. We have focused our recent research thrusts on the following aspects of SPECT: (1) The development of improved SPECT hardware, such as improved acquisition geometries. (2) The development of better reconstruction methods that provide accurate compensation for the physical factors that affect SPECT quantification. (3) The application of carefully designed simulations and experiments to validate our hardware and software approaches

  7. Production and characterization of a monoclonal antibody against recombinant cathepsin L1 of Fasciola gigantica.

    Science.gov (United States)

    Anuracpreeda, Panat; Srirakam, Thippawan; Pandonlan, Sudarat; Changklungmoa, Narin; Chotwiwatthanakun, Charoonroj; Tinikul, Yotsawan; Poljaroen, Jaruwan; Meemon, Krai; Sobhon, Prasert

    2014-08-01

    Monoclonal antibodies (MoAbs) against a recombinant cathepsin L1 of Fasciola gigantica (rFgCatL1) were produced in vitro by fusion of BALB/c mice spleen cells immunized with rFgCatL1 and mouse myeloma cells. Reactivity and specificity of these MoAbs were evaluated by indirect ELISA and immunoblotting techniques. Seven MoAb clones were selected from the stable hybridoma clones, namely 1E10, 1F5, 3D11, 4B10, 4D3, 4E3 and 5E7. Clones 1E10, 1F5 and 3D11 were IgM, whereas clones 4B10, 4D3, 4E3 and 5E7 were IgG1. All MoAbs had kappa light chain isotypes. All MoAbs reacted with rCatL1 at molecular weight (MW) 30kDa and with the native CatL1 at MW 27kDa in whole body (WB) extracts of metacercariae (Met), newly excysted juveniles (NEJ), 1, 3, 5-week-old juveniles (Ju), adult WB and adult excretory-secretory (ES) fractions, but not with adult tegumental antigens (TA). All of these MoAbs showed no cross-reactions with antigens of other parasites commonly found in ruminants and human, including Paramphistomum cervi, Eurytrema pancreaticum, Gigantocotyle explanatum, Schistosoma spindale, Schistosoma mansoni, Moniezia benedeni, Avitellina centripunctata, Trichuris sp., Haemonchus placei and Setaria labiato-papillosa. Localization of CatL1 in each developmental stages of F. gigantica by immunoperoxidase technique, using these MoAbs as probes, indicated that CatL1 was present at high concentration in the caecal epithelium and caecal lumen of metacercariae, NEJ, 1, 3, 5-week-old juveniles and adult fluke. This finding indicated that CatL1 is a copiously expressed parasite protein that is released into the ES, thus CatL1 and its MoAb could be a good candidate for immunodiagnosis of fasciolosis in ruminant and human. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Human breast tumor imaging using 111In labeled monoclonal antibody: Anthymic mouse model

    International Nuclear Information System (INIS)

    Ban An Khaw; Massachusetts General Hospital, Boston; Bailes, J.S.; Schneider, S.L.; Lancaster, J.; Lasher, J.C.; McGuire, W.L.; Powers, J.; Strauss, H.W.

    1988-01-01

    The monoclonal antibody (MoAb) 323/A3, an IgG1, was raised against the human breast tumor cell line MCF-7 and recognized a 43 Kd membrane associated glycoprotein. Histochemical studies with the antibody detected 75% of metastatic lymph nodes, 59% of primary breast tumors, and showed some staining in 20% of benign breast lesions. For radionuclide imaging, the MoAb 323/A3 was labeled with both 125 I and 111 In, via covalently coupled diethylenetriaminepentaacetic acid (DTPA) by the mixed anhydride method. The antibody activity of the DTPA modified 323/A3 was assessed by an immunoassay using viable and fixed MCF-7 target cells. Male athymic nude mice bearing BT-20 human mammary tumors were injected with dual 125 I/ 111 In labeled DTPA 323/A3 via the tail veins. The animals were imaged with a gamma camera equipped with a pinhole collimator at 1-3 h, 1, 2, 3, 4 and 5 days after the tracer administration. On day 5 or 6, the animals were killed, and the biodistribution of the radiotracers was determined for the blood, thyroid, heart, lungs, liver, spleen, kidneys, gastro-intestinal tract and tumor. Target to blood ratio at 6 days for the 111 In tracer was 24:1 in the group with a mean tumor weight of 0.492 g, and 13:1 in another group with a mean tumor weight of 0.1906 g (day 5). However, the 125 I activity showed only 3.6:1 and 5.4:1 target to blood ratios in the corresponding groups. The larger tumors localized less 111 I tracer (27.13%±7.57% injected dose/g, Mean±SD) than the smaller tumors (52.75%±22.25% ID/g). Analysis of the gamma images showed that the maximum tracer concentration occurred in the tumors at about 2 to 3 days after intravenous tracer administration. The excellent tumor resolution observed with BT-20 tumors may be due to increased 43 Kd glycoprotein antigen density in this tumor cell line. (orig.)

  9. Kinetic data of in-vivo labeled granulocytes in humans with a murine Tc-99m-labelled monoclonal antibody

    International Nuclear Information System (INIS)

    Becker, W.; Boerner, W.; Borst, U.; Schaefer, R.; Fischbach, W.; Pasurka, B.

    1989-01-01

    Twenty-five patients were examined in vivo with 99m Tc labelled monoclonal antibodies; 15 with suspected infections with an antigranulocyte antibody (BW 250/183), 10 with suspected recurrence of a colorectal carcinoma with an anti CEA antibody (BW 431/26). Both antibodies were IgG1 isotypes. In the patients with suspected infections no change of the peripheral leukocyte count could be observed after the antibody injection (1 mg, n=9; 0.05 mg, n=1; 0.25 mg, n=6). In 2 patients examined with the anti CEA antibody (2 mg), a significant decrease of the peripheral leukocyte count could be observed. The recovery rate of the 99m Tc antibody labelled granulocytes was calculated to be about 10%. The increase of the antibody-antigen binding was calculated to be 0.2%/min. In vivo the organ distribution curves demonstrated an increase of 99m Tc activity over spleen and bone marrow of 1.1%/min, which was interpreted as antigen-antibody reactivity. The organ distribution curves of the anti granulocyte antibody over spleen and bone marrow showed typical binding characteristics to the local granulocyte epitopes. The curves over other organs showed a simple perfusion pattern. The curves of the anti CEA antibody showed a perfusion pattern over all the examined organs. A sham dialysis model in one patient with renal insufficiency undergoing regular dialysis treatment demonstrated the viability of 99m Tc antibody labelled granulocytes in vivo. The kinetic patterns of the 99m Tc antibody in patients with Crohn's disease were interpreted as CEA binding of the antibody in the bowel wall. (orig.)

  10. Generation and characterization of monoclonal antibodies specific for 18 kDa antigen from Taenia solium cysticerci.

    Science.gov (United States)

    Zhang, Shaohua; Luo, Xuenong; Guo, Aijiang; Zhu, Xueliang; Cai, Xuepeng

    2016-07-01

    The gene encoding a mature 18 kDa glycoprotein of Taenia solium cysticerci (Ts18) was cloned and bacterially expressed with a His-tagged fusion protein. Monoclonal antibodies (MAbs) against the recombinant Ts18 antigen were generated in vitro by routine murine hybridoma technique of fusing splenocytes, from BALB/c mice immunized with the vesicular fluid of T. solium cysticerci (TsVF), with mouse myeloma cells (SP2/0). The reactivity and specificity of these MAbs were evaluated by indirect ELISA and immunoblotting techniques. Three stable hybridoma clones, namely 3B11, 6C5, and 6G4, were screened using His-Ts18-based ELISA, and these showed two IgG1 isotypes and one IgM isotype. All MAbs reacted with His-Ts18 at molecular weight (MW) 12.8 kDa and the native antigen at MW 18 kDa in TsVF and whole larval extracts (WLE). In a dot blotting test, MAbs 6C5 and 6G4 showed no obvious cross-reactivity with heterologous vesicular fluids from other taeniid species, including Taenia saginata (TsaVF), Taenia pisiformis (TpVF), Taenia hydatigena (ThVF), Taenia multiceps (TmVF), and Echinococcus granulosus (EgVF). Immunofluorescent assays showed that MAb 6C5 specifically reacted with the Ts18 expressed from pEGFP-N1-Ts18-transfected HeLa cells. Immunolocalization analysis, using MAb 6C5 as a probe, indicated that Ts18 was present at high concentrations in the region of the larval sucker and spiral canal. The results indicate that the Ts18 protein is an abundantly secreted parasite protein and MAbs against it might provide a step forward for improving the diagnosis of porcine cysticercosis. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Monoclonal antibody against recombinant Fasciola gigantica cathepsin L1H could detect juvenile and adult cathepsin Ls of Fasciola gigantica.

    Science.gov (United States)

    Wongwairot, Sirima; Kueakhai, Pornanan; Changklungmoa, Narin; Jaikua, Wipaphorn; Sansri, Veerawat; Meemon, Krai; Songkoomkrong, Sineenart; Riengrojpitak, Suda; Sobhon, Prasert

    2015-01-01

    Cathepsin Ls (CatLs), the major cysteine protease secreted by Fasciola spp., are important for parasite digestion and tissue invasion. Fasciola gigantica cathepsin L1H (FgCatL1H) is the isotype expressed in the early stages for migration and invasion. In the present study, a monoclonal antibody (MoAb) against recombinant F. gigantica cathepsin L1H (rFgCatL1H) was produced by hybridoma technique using spleen cells from BALB/c mice immunized with recombinant proFgCatL1H (rproFgCatL1H). This MoAb is an immunoglobulin (Ig)G1 with κ light chain isotype. The MoAb reacted specifically with rproFgCatL1H, the native FgCatL1H at a molecular weight (MW) 38 to 48 kDa in the extract of whole body (WB) of metacercariae and newly excysted juvenile (NEJ) and cross-reacted with rFgCatL1 and native FgCatLs at MW 25 to 28 kDa in WB of 2- and 4-week-old juveniles, adult, and adult excretory-secretory (ES) fractions by immunoblotting and indirect ELISA. It did not cross-react with antigens in WB fractions from other parasites, including Gigantocotyle explanatum, Paramphistomum cervi, Gastrothylax crumenifer, Eurytrema pancreaticum, Setaria labiato-papillosa, and Fischoederius cobboldi. By immunolocalization, MoAb against rFgCatL1H reacted with the native protein in the gut of metacercariae and NEJ and also cross-reacted with CatL1 in 2- and 4-week-old juveniles and adult F. gigantica. Therefore, FgCatL1H and its MoAb may be used for immunodiagnosis of both early and late fasciolosis in ruminants and humans.

  12. Detection of experimental infections with 99mTc-labeled monoclonal antibodies against TNF-α and interleukin-8

    International Nuclear Information System (INIS)

    Welling, Mick; Feitsma, Hans I.J.; Calame, Wim; Pauwels, Ernest K.J.

    1997-01-01

    This study was designed to assess monoclonal antibodies (MAbs) directed against tumor necrosis factor-α (TNF-α) (anti-TNF) or interleukin-8 (anti-IL-8) as radioactive agents for the detection of Staphylococcus aureus- or Klebsiella pneumoniae-infected thighs in mice. At 5 min (acute infection) or 20 h (established) post-infection, 20 μg of the 99m Tc-labeled MAbs were injected. At various time intervals, the accumulation of the radiotracer in the infected thighs was assessed and expressed as a target-to-nontarget (T/NT) ratio. The binding of 99m Tc-labeled MAbs to circulating mononuclear cells and granulocytes was quantitated 20 h after injection. The pharmacokinetics of the MAbs, in relation to the control agents 99m Tc-labeled polyclonal human immunoglobulin (IgG) and a 99m Tc-labeled nonspecific IgG1 MAb, were also studied. In acute infections, 99m Tc-anti-TNF accumulated to a higher extent (p 99m Tc-IgG and was higher at 0.25 h in K. pneumoniae-infected mice (p 99m Tc-IgG. In established S. aureus and K. pneumoniae infections, 99m Tc-anti-IL-8 detected the infection more intensely than 99m Tc-IgG until 1 h after injection. In both S. aureus and K. pneumoniae infections, localization of sites of infection correlates (p 99m Tc-labeled MAbs to granulocytes and mononuclear cells in both acute and established infections. It was concluded that 99m Tc-labeled MAbs, directed against TNF-α and IL-8, accumulate in bacterial infections in mice to a higher extent than does 99m Tc-IgG after infection and is related to the binding of the antibodies to blood leukocytes. With these 99m Tc-labeled MAbs, information might be gained about the development of an infection

  13. Indium-111-labeled B72. 3 monoclonal antibody in the detection and staging of breast cancer: A phase 1 study

    Energy Technology Data Exchange (ETDEWEB)

    Lamki, L.M.; Buzdar, A.U.; Singletary, S.E.; Rosenblum, M.G.; Bhadkamkar, V.; Esparza, L.; Podoloff, D.A.; Zukiwski, A.; Hortobagyi, G.N.; Murray, J.L. (Department of Nuclear Medicine, University of Texas M.D. Anderson Cancer Center, Houston (USA))

    1991-07-01

    Sixteen patients with primary breast cancer were studied with a pancarcinoma monoclonal antibody B72.3, an IgG1 molecule directed against tumor-associated glycoprotein (TAG-72) present in several tumors. Five millicuries of 111In was used to label 0.2 mg (six patients), or 2 mg (six patients), or 20 mg using the site-directed bifunctional DTPA method (at carbohydrate moiety). Digital, planar, and SPECT images were obtained at 2, 48, 72 and 96 hr when possible. HAMA levels were obtained before the Mab infusion and at 1, 3, and 6 wk postinfusion. Fourteen of 14 known primary breast lesions were detected by imaging (100% sensitivity). Two fibrocystic lesions were negative. Seven of 14 patients had lymph node metastases by histologic methods, but all were missed by radioimmunoscintigraphy. Tumor uptake of Mab ranged 0.00054%-0.0038% of the ID/g. The tumor-to-normal breast tissue ratio was 4.3 {plus minus} 0.91 (mean {plus minus} s.e.m.). Lymph nodes localization of 111In-B72.3 by tissue analysis was similar for tumor-bearing and normal nodes (0.0039 {plus minus} 0.0023 versus 0.0025 {plus minus} 0.0019). Pharmacokinetics revealed mean plasma half-life of 33.3-41.2 hr for the different doses. There was no statistical difference between any of the pharmacokinetic parameters of different doses. HAMA was positive only in 17% of the patients. The study suggests that this antibody has 100% sensitivity for primary breast cancers, but very poor detection rate of metastatic lesions in axillary lymph nodes; thus making it of questionable value in the initial staging process of this disease.

  14. A direct qPCR method for residual DNA quantification in monoclonal antibody drugs produced in CHO cells.

    Science.gov (United States)

    Hussain, Musaddeq

    2015-11-10

    Chinese hamster ovary (CHO) cells are the host cell of choice for manufacturing of monoclonal antibody (mAb) drugs in the biopharmaceutical industry. Host cell DNA is an impurity of such manufacturing process and must be controlled and monitored in order to ensure drug purity and safety. A conventional method for quantification of host residual DNA in drug requires extraction of DNA from the mAb drug substance with subsequent quantification of the extracted DNA using real-time PCR (qPCR). Here we report a method where the DNA extraction step is eliminated prior to qPCR. In this method, which we have named 'direct resDNA qPCR', the mAb drug substance is digested with a protease called KAPA in a 96-well PCR plate, the protease in the digest is then denatured at high temperature, qPCR reagents are added to the resultant reaction wells in the plate along with standards and controls in other wells of the same plate, and the plate subjected to qPCR for analysis of residual host DNA in the samples. This direct resDNA qPCR method for CHO is sensitive to 5.0fg of DNA with high precision and accuracy and has a wide linear range of determination. The method has been successfully tested with four mAbs drug, two IgG1 and two IgG4. Both the purified drug substance as well as a number of process intermediate samples, e.g., bioreactor harvest, Protein A column eluate and ion-exchange column eluates were tested. This method simplifies the residual DNA quantification protocol, reduces time of analysis and leads to increased assay sensitivity and development of automated high-throughput methods. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Monoclonal antibody to a subset of human monocytes found only in the peripheral blood and inflammatory tissues

    Energy Technology Data Exchange (ETDEWEB)

    Zwadlo, G.; Schlegel, R.; Sorg, C.

    1986-07-15

    A monoclonal antibody is described that was generated by immunizing mice with cultured human blood monocytes. The antibody (27E10) belongs to the IgG1 subclass and detects a surface antigen at M/sub r/ 17,000 that is found on 20% of peripheral blood monocytes. The antigen is increasingly expressed upon culture of monocytes, reaching a maximum between days 2 and 3. Stimulation of monocytes with interferon-..gamma.. (IFN-..gamma..), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), and lipopolysaccharide (LPS) Ylalanine (fMLP) increased the 27E10 antigen density. The amount of 27E10-positive cells is not or is only weakly affected. The antigen is absent from platelets, lymphotyces, and all tested human cell lines, yet it cross-reacts with 15% of freshly isolated granulocytes. By using the indirect immunoperoxidase technique, the antibody is found to be negative on cryostat sections of normal human tissue (skin, lung, and colon) and positive on only a few monocyte-like cells in liver and on part of the cells of the splenic red pulp. In inflammatory tissue, however, the antibody is positive on monocytes/macrophages and sometimes on endothelial cells and epidermal cells, depending on the stage and type of inflammation, e.g., BCG ranulomas are negative, whereas psoriasis vulgaris, atopic dermatitis, erythrodermia, pressure urticaria, and periodontitis contain positively staining cells. In contact eczemas at different times after elicitation (6 hr, 24 hr, and 72 hr), the 27E10 antigen is seen first after 24 hr on a few infiltrating monocytes/macrophages, which increase in numbers after 72 hr.

  16. Monoclonal antibodies and Fc fragments for treating solid tumors

    Directory of Open Access Journals (Sweden)

    Eisenbeis AM

    2012-01-01

    Full Text Available Andrea M Eisenbeis, Stefan J GrauDepartment of Neurosurgery, University Hospital of Cologne, Cologne, GermanyAbstract: Advances in biotechnology, better understanding of pathophysiological processes, as well as the identification of an increasing number of molecular markers have facilitated the use of monoclonal antibodies and Fc fragments in various fields in medicine. In this context, a rapidly growing number of these substances have also emerged in the field of oncology. This review will summarize the currently approved monoclonal antibodies used for the treatment of solid tumors with a focus on their clinical application, biological background, and currently ongoing trials.Keywords: targeted therapy, monoclonal antibodies, cancer, biological therapy

  17. The detection of ovarian cancer using 123I monoclonal antibody

    International Nuclear Information System (INIS)

    Granowska, M.; Britton, K.E.; Shepherd, J.

    1984-01-01

    The technique of the production of monoclonal antibodies is described. Antibodies show reactivity with epithelial surfaces of cancer of breast, colon and ovary. The iodogen reaction is used for labelling monoclonal antibodies with 123 I. Description of labelling technique and quality control. After intravenous injection of 74 MBq 123 I-labelled monoclonal antibody (0.5 mg) static camera images of the abdomen were recorded at 10 min, 4 and 22 hours in anterior and posterior position. 20 out of 22 patients with ovarian cancer with and without metastases were correctly diagnosed and confirmed at surgery. (author)

  18. Monoclonal gammopathy: a diagnosis for to keep in mind

    International Nuclear Information System (INIS)

    Howland Alvarez, Ivon; Figueredo Peguero, Yrving; Luna Conde, Clara

    2011-01-01

    How to identify monoclonal gammopathies at risk for progression has been studied for the last year. 40 patients were studied in which a monoclonal band had been detected, in some of the cases de novo. The electrophoresis was performed in the Hydrasys system. Of the total of electrophoresis carried out, the 14% was monoclonal gammopathy. In 36% a diagnostic assumption was not stated. Most frequent diagnosis in the group of patients with a diagnosis was multiple myeloma. Average age of patients was 61.5 years and there were differences among percentages for sex

  19. Development of EMab-51, a Sensitive and Specific Anti-Epidermal Growth Factor Receptor Monoclonal Antibody in Flow Cytometry, Western Blot, and Immunohistochemistry.

    Science.gov (United States)

    Itai, Shunsuke; Kaneko, Mika K; Fujii, Yuki; Yamada, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Handa, Saori; Chang, Yao-Wen; Suzuki, Hiroyoshi; Harada, Hiroyuki; Kato, Yukinari

    2017-10-01

    The epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases and is involved in cell growth and differentiation. EGFR homodimers or heterodimers with other HER members, such as HER2 and HER3, activate downstream signaling cascades in many cancers. In this study, we developed novel anti-EGFR monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. First, we expressed the full-length or ectodomain of EGFR in LN229 glioblastoma cells and then immunized mice with LN229/EGFR or ectodomain of EGFR, and performed the first screening using enzyme-linked immunosorbent assays. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical (fourth screening) analyses. Among 100 mAbs, only one clone EMab-51 (IgG 1 , kappa) reacted with EGFR in Western blot analysis. Finally, immunohistochemical analyses with EMab-51 showed sensitive and specific reactions against oral cancer cells, warranting the use of EMab-51 to detect EGFR in pathological analyses of EGFR-expressing cancers.

  20. Broad-spectrum inhibition of HIV-1 by a monoclonal antibody directed against a gp120-induced epitope of CD4.

    Science.gov (United States)

    Burastero, Samuele E; Frigerio, Barbara; Lopalco, Lucia; Sironi, Francesca; Breda, Daniela; Longhi, Renato; Scarlatti, Gabriella; Canevari, Silvana; Figini, Mariangela; Lusso, Paolo

    2011-01-01

    To penetrate susceptible cells, HIV-1 sequentially interacts with two highly conserved cellular receptors, CD4 and a chemokine receptor like CCR5 or CXCR4. Monoclonal antibodies (MAbs) directed against such receptors are currently under clinical investigation as potential preventive or therapeutic agents. We immunized Balb/c mice with molecular complexes of the native, trimeric HIV-1 envelope (Env) bound to a soluble form of the human CD4 receptor. Sera from immunized mice were found to contain gp120-CD4 complex-enhanced antibodies and showed broad-spectrum HIV-1-inhibitory activity. A proportion of MAbs derived from these mice preferentially recognized complex-enhanced epitopes. In particular, a CD4-specific MAb designated DB81 (IgG1Κ) was found to preferentially bind to a complex-enhanced epitope on the D2 domain of human CD4. MAb DB81 also recognized chimpanzee CD4, but not baboon or macaque CD4, which exhibit sequence divergence in the D2 domain. Functionally, MAb DB81 displayed broad HIV-1-inhibitory activity, but it did not exert suppressive effects on T-cell activation in vitro. The variable regions of the heavy and light chains of MAb DB81 were sequenced. Due to its broad-spectrum anti-HIV-1 activity and lack of immunosuppressive effects, a humanized derivative of MAb DB81 could provide a useful complement to current preventive or therapeutic strategies against HIV-1.

  1. Broad-spectrum inhibition of HIV-1 by a monoclonal antibody directed against a gp120-induced epitope of CD4.

    Directory of Open Access Journals (Sweden)

    Samuele E Burastero

    Full Text Available To penetrate susceptible cells, HIV-1 sequentially interacts with two highly conserved cellular receptors, CD4 and a chemokine receptor like CCR5 or CXCR4. Monoclonal antibodies (MAbs directed against such receptors are currently under clinical investigation as potential preventive or therapeutic agents. We immunized Balb/c mice with molecular complexes of the native, trimeric HIV-1 envelope (Env bound to a soluble form of the human CD4 receptor. Sera from immunized mice were found to contain gp120-CD4 complex-enhanced antibodies and showed broad-spectrum HIV-1-inhibitory activity. A proportion of MAbs derived from these mice preferentially recognized complex-enhanced epitopes. In particular, a CD4-specific MAb designated DB81 (IgG1Κ was found to preferentially bind to a complex-enhanced epitope on the D2 domain of human CD4. MAb DB81 also recognized chimpanzee CD4, but not baboon or macaque CD4, which exhibit sequence divergence in the D2 domain. Functionally, MAb DB81 displayed broad HIV-1-inhibitory activity, but it did not exert suppressive effects on T-cell activation in vitro. The variable regions of the heavy and light chains of MAb DB81 were sequenced. Due to its broad-spectrum anti-HIV-1 activity and lack of immunosuppressive effects, a humanized derivative of MAb DB81 could provide a useful complement to current preventive or therapeutic strategies against HIV-1.

  2. Generation and characterization of tabalumab, a human monoclonal antibody that neutralizes both soluble and membrane-bound B-cell activating factor

    Directory of Open Access Journals (Sweden)

    Manetta J

    2014-08-01

    Full Text Available Joseph Manetta, Holly Bina, Paul Ryan, Niles Fox, Derrick R Witcher, Kristine Kikly Biotechnology Discovery Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, USA Abstract: B-cell activating factor (BAFF is a B-cell survival factor with a key role in B-cell homeostasis and tolerance. Dysregulated BAFF expression may contribute to autoimmune diseases or B-cell malignancies via effects on abnormal B-lymphocyte activation, proliferation, survival, and immunoglobulin secretion. Monoclonal antibodies were generated against human BAFF, characterized for species specificity and affinity, and screened for the ability to neutralize both membrane-bound and soluble BAFF. In addition, studies were undertaken to determine the relative potency of membrane-bound and soluble BAFF. Tabalumab has a high affinity for human, cynomolgus monkey, and rabbit BAFF. No binding to mouse BAFF was detected. Tabalumab was able to neutralize soluble human, cynomolgus monkey, or rabbit BAFF with equal potency. Our data demonstrate that membrane-bound BAFF can be a more potent stimulus for B-cells than soluble BAFF, and tabalumab also neutralized membrane-bound BAFF. Tabalumab prevented BAFF from binding to BAFF receptors and demonstrated pharmacodynamic effects in human BAFF transgenic mice. Tabalumab is a high-affinity human antibody with neutralizing activity against membrane-bound and soluble BAFF. Given our findings that membrane-bound BAFF can have greater in vitro potency than soluble BAFF, neutralization of both forms of BAFF is likely to be important for optimal therapeutic effect. Keywords: autoimmunity, B-cell malignancies, B-cell survival factor, BAFF

  3. Monoclonal gammopathy: a diagnosis for to keep in mind; Gammapatia monoclonal: un diagnostico a tener en cuenta

    Energy Technology Data Exchange (ETDEWEB)

    Howland Alvarez, Ivon; Figueredo Peguero, Yrving; Luna Conde, Clara, E-mail: ihowlanda@infomed.sld.cu [Centro de Investigaciones Medico Quirurgicas, La Habana (Cuba); others, and

    2011-07-01

    How to identify monoclonal gammopathies at risk for progression has been studied for the last year. 40 patients were studied in which a monoclonal band had been detected, in some of the cases de novo. The electrophoresis was performed in the Hydrasys system. Of the total of electrophoresis carried out, the 14% was monoclonal gammopathy. In 36% a diagnostic assumption was not stated. Most frequent diagnosis in the group of patients with a diagnosis was multiple myeloma. Average age of patients was 61.5 years and there were differences among percentages for sex.

  4. [International classification of various types of monoclonal antibodies].

    Science.gov (United States)

    Scheen, A J

    2009-01-01

    Significant advances in the development of monoclonal antibodies ("mabs") have been acknowledged during the last two decades. Successive developments led to the marketing of murine antibodies ("o-mab" first, followed by chimeric antibodies ("xi-mab"), humanised antibodies ("zu-mab") and, finally, human monoclonal antibodies ("u-mab"). In order to facilitate the distinction between the various monoclonal antibodies used in clinical practice, an international nomenclature has been proposed with the use of a specific suffix corresponding to the origine/source of "mabs" preceded by an infix referring to the medicine's target. The efforts in developing new types of monoclonal antibodies aimed at improving their pharmacokinetics (longer half-life), pharmacodynamics (better efficacy because of stronger affinity to human receptor), and safety profile (less antigenic and immunogenic reactions). These progresses could be obtained thanks to the remarkable development of molecular biotechnology.

  5. Monoclonal antibody 6E4 against human GAPDHS protein

    Czech Academy of Sciences Publication Activity Database

    Dorosh, Andriy

    2011-01-01

    Roč. 30, č. 3 (2011), s. 321-321 ISSN 1554-0014 Institutional research plan: CEZ:AV0Z50520701 Keywords : Monoclonal antibody * GAPDHS Subject RIV: EI - Biotechnology ; Bionics Impact factor: 0.417, year: 2011

  6. DEVELOPMENT OF MONOCLONAL ANTIBODIES AGAINST FATHEAD MINNOW (PIMEPHALES PROMELAS) VITELLOGENIN

    Science.gov (United States)

    We have obtained a panel of monoclonal antibodies directed against fathead minnow vitellogenin (Vtg) for use in sensitive ELISAs to quantify the response of exposure in vivo to estrogen or estrogen mimics.

  7. Monoclonal antibodies in clinical diagnosis: A brief review application

    African Journals Online (AJOL)

    SERVER

    2008-04-17

    Apr 17, 2008 ... More than 100 different monoclonal antibody diagnostic products are ... are produced by in vitro and in vivo method but have advantages and some disadvantages. .... replication and differentiation, advancing our knowledge.

  8. Biodistribution mechanisms of therapeutic monoclonal antibodies in health and disease.

    Science.gov (United States)

    Tabrizi, Mohammad; Bornstein, Gadi Gazit; Suria, Hamza

    2010-03-01

    The monoclonal antibody market continues to witness an impressive rate of growth and has become the leading source of expansion in the biologic segment within the pharmaceutical industry. Currently marketed monoclonal antibodies target a diverse array of antigens. These antigens are distributed in a variety of tissues such as tumors, lungs, synovial fluid, psoriatic plaques, and lymph nodes. As the concentration of drug at the proximity of the biological receptor determines the magnitude of the observed pharmacological responses, a significant consideration in effective therapeutic application of monoclonal antibodies is a thorough understanding of the processes that regulate antibody biodistribution. Monoclonal antibody distribution is affected by factors such as molecular weight, blood flow, tissue and tumor heterogeneity, structure and porosity, target antigen density, turnover rate, and the target antigen expression profile.

  9. Monastrol, a 3,4-dihydropyrimidin-2(1H)-thione, as structural scaffold for the development of modulators for GHB high-affinity binding sites and α1β2δ GABAA receptors

    DEFF Research Database (Denmark)

    Damgaard, Maria; Al-Khawaja, Anas; Nittegaard-Nielsen, Mia

    2017-01-01

    -affinity binding and is furthermore reported as an allosteric modulator selective for the α1β2δ GABAARs. Therefore, structural determinants for selectivity at the two targets were investigated. 39 structural diverse monastrol analogues were synthesized by employing the Biginelli cyclocondensation and examined......-affinity binding. However, three analogues of monastrol (11, 12 and 24) enhanced the maximal binding of [(3)H]NCS-382 to a higher maximal level than seen for monastrol itself. Selected compounds were further characterized as modulators at α1β2δ, α1β2γ2s and α1β2 GABAARs. Most of these modulators were shown to have...... δ-specific GABA-potentiating effects. The dual effect shown for monastrol to modulate the GHB high-affinity binding and α1β2δ GABAAR activity was also shown for the compounds 11, 18 and 24. Compound 29 displayed minimal modulatory effect on GABAARs and therefore appears to be a GHB high...

  10. HAMS: High-Affinity Mass Spectrometry Screening. A High-Throughput Screening Method for Identifying the Tightest-Binding Lead Compounds for Target Proteins with No False Positive Identifications.

    Science.gov (United States)

    Imaduwage, Kasun P; Go, Eden P; Zhu, Zhikai; Desaire, Heather

    2016-11-01

    A major challenge in drug discovery is the identification of high affinity lead compounds that bind a particular target protein; these leads are typically identified by high throughput screens. Mass spectrometry has become a detection method of choice in drug screening assays because the target and the ligand need not be modified. Label-free assays are advantageous because they can be developed more rapidly than assays requiring labels, and they eliminate the risk of the label interfering with the binding event. However, in commonly used MS-based screening methods, detection of false positives is a major challenge. Here, we describe a detection strategy designed to eliminate false positives. In this approach, the protein and the ligands are incubated together, and the non-binders are separated for detection. Hits (protein binders) are not detectable by MS after incubation with the protein, but readily identifiable by MS when the target protein is not present in the incubation media. The assay was demonstrated using three different proteins and hundreds of non-inhibitors; no false positive hits were identified in any experiment. The assay can be tuned to select for ligands of a particular binding affinity by varying the quantity of protein used and the immobilization method. As examples, the method selectively detected inhibitors that have K i values of 0.2 μM, 50 pM, and 700 pM. These findings demonstrate that the approach described here compares favorably with traditional MS-based screening methods. Graphical Abstract ᅟ.

  11. Identification and typing of herpes simplex viruses with monoclonal antibodies.

    OpenAIRE

    Balachandran, N; Frame, B; Chernesky, M; Kraiselburd, E; Kouri, Y; Garcia, D; Lavery, C; Rawls, W E

    1982-01-01

    Monoclonal antibodies which reacted with type-specific antigens of herpes simplex virus type 2 or with antigens shared by herpes simplex virus types 1 and 2 were used in an indirect immunofluorescence assay to type virus isolates and to detect viral antigens in cells obtained from herpetic lesions. Complete concordance was obtained for 42 isolates typed by endonuclease restriction analysis of viral DNA and by indirect immunofluorescence with monoclonal antibodies. Examination of a limited num...

  12. Rapid screening of monoclonal antibodies: new 'microstick' radioimmunoassay

    International Nuclear Information System (INIS)

    Scheinberg, D.A.; Strand, M.; Wilsnack, R.

    1983-01-01

    A new system for assaying monoclonal antibodies consisting of an 8 x 12 array of sticks which fits into a 96-well microtiter plate is described. Tests using virus specific monoclonal antibodies and virus proteins demonstrated sensitivity equivalent to the conventional microtiter plate assay. Antibody production, antigen specific antibody, and immunoglobulin isotypes could be measured under sterile conditions directly in the original fusion mixture wells and much greater rapidity than with the microtiter plate assay. (Auth.)

  13. Rat Monoclonal Antibodies Specific for LST1 Proteins

    OpenAIRE

    Schiller, Christian; Nitschké, Maximilian J. E.; Seidl, Alexander; Kremmer, Elisabeth; Weiss, Elisabeth H.

    2009-01-01

    The LST1 gene is located in the human MHC class III region and encodes transmembrane and soluble isoforms that have been suggested to play a role in the regulation of the immune response and are associated with inflammatory diseases such as rheumatoid arthritis. Here we describe the generation and characterization of the first monoclonal antibodies against LST1. Two hybridoma lines secreting monoclonal antibodies designated 7E2 and 8D12 were established. The 7E2 antibody detects recombinant a...

  14. Preparation and characterization of chimeric CD19 monoclonal antibody

    International Nuclear Information System (INIS)

    Zola, H.; Macardle, P.J.; Bradford, T.; Weedon, H.; Yasui, H.; Kurosawa, Y.

    1991-01-01

    CD19 antibodies have been suggested as candidates for immunological attack on leukemic and lymphoma cells of the B lineage because the antigen is restricted to the B lineage. With the potential use of FMC63 in immunotherapy in mind a mouse-human chimera was produced in which the genes coding for the VDJ region of the heavy chain and the VJ region of the light chain derive from the FMC63 mouse hybridoma, while the C region genes code for human IgG1. The genes have been transfected back into a mouse myeloma line, which secretes low levels of immunoglobulin. (Ig). This Ig was purified and biotinylated in order to determine the specificity of the antibody. The chimeric antibody has a reaction profile concordant with the original FMC63 antibody, but has the properties of a human IgG1, including the ability to fix human complement. However, the antibody is not cytotoxic in vitro in the presence of complement or cells capable of mediating antibody-dependent cellular cytotoxicity. Possible reasons for this and ways of using the antibody are discussed. 47 refs., 7 figs

  15. Breast cancer imaging with mouse monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Major, P.; Wang Taqui; Unger, M.; Rosenthall, L.

    1989-10-01

    The localization of /sup 111/In-labelled MA5 monoclonal antibody, reactive with a breast tumor associated antigen, was studied in 17 patients. MA5 was selected because (1) it reacts with >95% of primary and metastatic lesions, (2) the recognized antigen is present on the cell surface in vivo and (3) MA5 gives excellent localization in human breast tumor xenografts. Each patient received 2 mg antibody labeled with 5 mCi /sup 111/In and in some cases, 3 mg or 18 mg unlabeled carrier antibody. No serious allergic reactions were noted. There was a large uptake in the liver, less significant uptake in the spleen and bone and minimal accumulation in the bowel. Bone lesions, primary tumors, soft tissue recurrences and lung metastases larger than 3 cm diameter were imaged, while only 1 lesion smaller than 3 cm was detected. Non specific accumulation of tracer was noted at the site of a port-a-cath, in a hematoma, in fibrocystic lesions, and at sites of previous radiation treatment. Extensive fibrosis and poor vascularization characteristic of breast tumors may explain in part the limited sensitivity of the imaging. (orig.).

  16. Monoclonal antibodies based on hybridoma technology.

    Science.gov (United States)

    Yagami, Hisanori; Kato, Hiroshi; Tsumoto, Kanta; Tomita, Masahiro

    2013-03-01

    Based on the size and scope of the present global market for medicine, monoclonal antibodies (mAbs) have a very promising future, with applications for cancers through autoimmune ailments to infectious disease. Since mAbs recognize only their target antigens and not other unrelated proteins, pinpoint medical treatment is possible. Global demand is dramatically expanding. Hybridoma technology, which allows production of mAbs directed against antigens of interest is therefore privileged. However, there are some pivotal points for further development to generate therapeutic antibodies. One is selective generation of human mAbs. Employment of transgenic mice producing human antibodies would overcome this problem. Another focus is recognition sites and conformational epitopes in antigens may be just as important as linear epitopes, especially when membrane proteins such as receptors are targeted. Recognition of intact structures is of critical importance for medical purposes. In this review, we describe patent related information for therapeutic mAbs based on hybridoma technology and also discuss new advances in hybridoma technology that facilitate selective production of stereospecific mAbs.

  17. Monoclonal antibody to DNA containing thymine glycol

    Energy Technology Data Exchange (ETDEWEB)

    Leadon, S A; Hanawalt, P C [Stanford Univ., CA (USA). Dept. of Biological Sciences

    1983-08-01

    Exposure of DNA to ionizing or near ultraviolet radiation modifies thymine to form ring-saturated products. One of the major products formed is 5,6-dihydroxy-5.6-dihydrothymine (thymine glycol). Thymine glycol can also be selectively formed by oxidizing DNA with OsO/sub 4/. We have isolated hybrids that produce monoclonal antibodies against thymine glycol by fusing mouse myeloma cells (P3X63-Ag8-6.5.3) with spleen cells from BALB/c mice immunized with OsO/sub 4/-oxidized poly(dT) complexed with methylated bovine serum albumin. This report describes the characterization of the antibody from one hybridoma using a competitive enzyme-linked immunosorbent assay (ELISA). The antibody reacted with both single- and double-stranded DNA treated with OsO/sub 4/, and with OsO/sub 4/-treated poly(dA-dT) and poly(dT); it did not crossreact with unmodified or apurinic DNA. It also reacted with DNA treated with H/sub 2/O/sub 2/ or with ..gamma..-rays at doses as low as 250 rad. We were able to detect 2 fmoles of thymine glycol in OsO/sub 4/-treated DNA and could quantitate 1 thymine glycol per 220000 thymines. Using the antibody and the ELISA, the formation and removal of thymine glycol was examined in cultures of African green monkey cells irradiated with 25 krad of ..gamma..-rays. The antibody reactive sites produced by irradiation (8.5 per 10/sup 6/ thymines) were efficiently removed from the cellular DNA.

  18. Novel monoclonal treatments in severe asthma.

    Science.gov (United States)

    Meteran, Howraman; Meteran, Hanieh; Porsbjerg, Celeste; Backer, Vibeke

    2017-12-01

    To provide a general overview of the current biological treatments and discuss their potential anti-asthmatic effects. We reviewed articles in PubMed found using the search words "Asthma/therapy AND antibodies, monoclonal/therapeutic use AND cytokines." Only articles published in English since 2000 were considered. The search identified 29 studies; 8 additional studies were found by hand search, generating 37 studies. Of the 37 studies investigating biological treatments of asthma, 5 were on the effects of anti-IgE (omalizumab); 12 on anti-IL-5; 8 on anti-IL-13; 5 on anti-IL-4R-α; 3 on anti-IL-9; one on TNF-α; one on anti-IL-2R-α; one on TSLP (Thymic Stromal Lymphopoietin); and one on OX40L. Sample sizes ranged from 3 to 943 participants. Studies of therapies targeting IgE, IL-2, IL4R-α, IL-5, and IL-13 showed some efficacy, whereas those targeting TSLP, IL-9, and TNF-α lacked convincing effectiveness. Research on the biological treatment of asthma shows promising results. While anti-IgE (omalizumab) has been used in the treatment of asthma for some years, anti-IL-5 has recently been approved for use. The efficacy of results of other large studies with a longer duration is needed to draw a firm conclusion. Such studies should not only focus on clinical outcomes, but also consider asthma-related quality of life. Knowledge on the asthma phenotypes and identification of biomarkers associated with these will be useful for physicians considering the right treatment for the asthma patient.

  19. Generation and characterization of ixekizumab, a humanized monoclonal antibody that neutralizes interleukin-17A

    Directory of Open Access Journals (Sweden)

    Liu L

    2016-04-01

    Full Text Available Ling Liu,1 Jirong Lu,1 Barrett W Allan,2 Ying Tang,2 Jonathan Tetreault,1 Chi-kin Chow,1 Barbra Barmettler,2 James Nelson,2 Holly Bina,1 Lihua Huang,3 Victor J Wroblewski,4 Kristine Kikly1 1Biotechnology Discovery Research, Indianapolis, IN, 2Applied Molecular Evolution, Lilly Biotechnology Center, San Diego, CA, 3Bioproduct Research and Development, 4Drug Disposition, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, USA Abstract: Interleukin (IL-17A exists as a homodimer (A/A or as a heterodimer (A/F with IL-17F. IL-17A is expressed by a subset of T-cells, called Th17 cells, at inflammatory sites. Most cell types can respond to the local production of IL-17A because of the near ubiquitous expression of IL-17A receptors, IL-17RA and IL-17RC. IL-17A stimulates the release of cytokines and chemokines designed to recruit and activate both neutrophils and memory T-cells to the site of injury or inflammation and maintain a proinflammatory state. IL-17A-producing pathogenic T-cells contribute to the pathogenesis of autoimmune diseases, including psoriasis, psoriatic arthritis, rheumatoid arthritis, and ankylosing spondylitis. This study describes the generation and characterization of ixekizumab, a humanized IgG4 variant IL-17A-neutralizing antibody. Ixekizumab binds human and cynomolgus monkey IL-17A with high affinity and binds rabbit IL-17A weakly but does not bind to rodent IL-17A or other IL-17 family members. Ixekizumab effectively inhibits the interaction between IL-17A and its receptor in binding assays and potently blocks IL-17A-induced GRO or KC secretion in cell-based assays. In an in vivo mouse pharmcodynamic model, ixekizumab blocks human IL-17A-induced mouse KC secretion. These data provide a comprehensive preclinical characterization of ixekizumab, for which the efficacy and safety have been demonstrated in human clinical trials in psoriasis and psoriatic arthritis.Keywords: ixekizumab, IL-17A monoclonal antibody

  20. Time resolved native ion-mobility mass spectrometry to monitor dynamics of IgG4 Fab arm exchange and "bispecific" monoclonal antibody formation.

    Science.gov (United States)

    Debaene, François; Wagner-Rousset, Elsa; Colas, Olivier; Ayoub, Daniel; Corvaïa, Nathalie; Van Dorsselaer, Alain; Beck, Alain; Cianférani, Sarah

    2013-10-15

    Monoclonal antibodies (mAbs) and derivatives such as antibody-drug conjugates (ADC) and bispecific antibodies (bsAb), are the fastest growing class of human therapeutics. Most of the therapeutic antibodies currently on the market and in clinical trials are chimeric, humanized, and human immunoglobulin G1 (IgG1). An increasing number of IgG2s and IgG4s that have distinct structural and functional properties are also investigated to develop products that lack or have diminished antibody effector functions compared to IgG1. Importantly, wild type IgG4 has been shown to form half molecules (one heavy chain and one light chain) that lack interheavy chain disulfide bonds and form intrachain disulfide bonds. Moreover, IgG4 undergoes a process of Fab-arm exchange (FAE) in which the heavy chains of antibodies of different specificities can dissociate and recombine in bispecific antibodies both in vitro and in vivo. Here, native mass spectrometry (MS) and time-resolved traveling wave ion mobility MS (TWIM-MS) were used for the first time for online monitoring of FAE and bsAb formation using Hz6F4-2v3 and natalizumab, two humanized IgG4s which bind to human Junctional Adhesion Molecule-A (JAM-A) and alpha4 integrin, respectively. In addition, native MS analysis of bsAb/JAM-A immune complexes revealed that bsAb can bind up to two antigen molecules, confirming that the Hz6F4 family preferentially binds dimeric JAM-A. Our results illustrate how IM-MS can rapidly assess bsAb structural heterogeneity and be easily implemented into MS workflows for bsAb production follow up and bsAb/antigen complex characterization. Altogether, these results provide new MS-based methodologies for in-depth FAE and bsAb formation monitoring. Native MS and IM-MS will play an increasing role in next generation biopharmaceutical product characterization like bsAbs, antibody mixtures, and antibody-drug conjugates (ADC) as well as for biosimilar and biobetter antibodies.

  1. High-Affinity Low-Capacity and Low-Affinity High-Capacity N-Acetyl-2-Aminofluorene (AAF) Macromolecular Binding Sites Are Revealed During the Growth Cycle of Adult Rat Hepatocytes in Primary Culture.

    Science.gov (United States)

    Koch, Katherine S; Moran, Tom; Shier, W Thomas; Leffert, Hyam L

    2018-05-01

    Long-term cultures of primary adult rat hepatocytes were used to study the effects of N-acetyl-2-aminofluorene (AAF) on hepatocyte proliferation during the growth cycle; on the initiation of hepatocyte DNA synthesis in quiescent cultures; and, on hepatocyte DNA replication following the initiation of DNA synthesis. Scatchard analyses were used to identify the pharmacologic properties of radiolabeled AAF metabolite binding to hepatocyte macromolecules. Two classes of growth cycle-dependent AAF metabolite binding sites-a high-affinity low-capacity site (designated Site I) and a low-affinity high-capacity site (designated Site II)-associated with two spatially distinct classes of macromolecular targets, were revealed. Based upon radiolabeled AAF metabolite binding to purified hepatocyte genomic DNA or to DNA, RNA, proteins, and lipids from isolated nuclei, Site IDAY 4 targets (KD[APPARENT] ≈ 2-4×10-6 M and BMAX[APPARENT] ≈ 6 pmol/106 cells/24 h) were consistent with genomic DNA; and with AAF metabolized by a nuclear cytochrome P450. Based upon radiolabeled AAF binding to total cellular lysates, Site IIDAY 4 targets (KD[APPARENT] ≈ 1.5×10-3 M and BMAX[APPARENT] ≈ 350 pmol/106 cells/24 h) were consistent with cytoplasmic proteins; and with AAF metabolized by cytoplasmic cytochrome P450s. DNA synthesis was not inhibited by concentrations of AAF that saturated DNA binding in the neighborhood of the Site I KD. Instead, hepatocyte DNA synthesis inhibition required higher concentrations of AAF approaching the Site II KD. These observations raise the possibility that carcinogenic DNA adducts derived from AAF metabolites form below concentrations of AAF that inhibit replicative and repair DNA synthesis.

  2. In Vitro and In Vivo Evaluation of Alexa Fluor 680-Bombesin[7–14]NH2 Peptide Conjugate, a High-Affinity Fluorescent Probe with High Selectivity for the Gastrin-Releasing Peptide Receptor

    Directory of Open Access Journals (Sweden)

    Lixin Ma

    2007-05-01

    Full Text Available Gastrin-releasing peptide (GRP receptors are overexpressed on several types of human cancer cells, including breast, prostate, small cell lung, and pancreatic cancers. Bombesin (BBN, a 14–amino acid peptide that is an analogue of human GRP, binds to GRP receptors with very high affinity and specificity. The aim of this study was to develop a new fluorescent probe based on BBN having high tumor uptake and optimal pharmacokinetics for specific targeting and optical imaging of human breast cancer tissue. In this study, solid-phase peptide synthesis was used to produce H2N-glycylglycylglycine-BBN[7–14]NH2 peptide with the following general sequence: H2N-G-G-G-Q-W-A-V-G-H-L-M-(NH2. This conjugate was purified by reversed-phase high-performance liquid chromatography and characterized by electrospray-ionization mass spectra. The fluorescent probe Alexa Fluor 680-G-G-G-BBN[7–14]NH2 conjugate was prepared by reaction of Alexa Fluor 680 succinimidyl ester to H2N-G-G-G-BBN[7–14]NH2 in dimethylformamide (DMF. In vitro competitive binding assays, using 125I-Tyr4-BBN as the radiolabeling gold standard, demonstrated an inhibitory concentration 50% value of 7.7 ± 1.4 nM in human T-47D breast cancer cells. Confocal fluorescence microscopy images of Alexa Fluor 680-G-G-G-BBN[7–14]NH2 in human T-47D breast cancer cells indicated specific uptake, internalization, and receptor blocking of the fluorescent bioprobe in vitro. In vivo investigations in SCID mice bearing xenografted T-47D breast cancer lesions demonstrated the ability of this new conjugate to specifically target tumor tissue with high selectivity and affinity.

  3. Monoclonal antibodies to drosophila cytochrome P-450's

    International Nuclear Information System (INIS)

    Sundseth, S.S.; Kennel, S.J.; Waters, L.C.

    1987-01-01

    Hybridomas producing monoclonal antibodies were prepared by the fusion of SP2/0 myeloma cells and spleen cells from a female BALB/c mouse immunized by cytochrome P-450-A and P-450-B purified from Drosophila Hikone-R (BG) microsomes. P-450-A and P-450-B are electrophoretically distinct subsets of Drosophila P-450. P-450-A is ubiquitous among strains tested, while P-450-B is present in only a few strains displaying unique enzyme activities and increased insecticide resistance. The Oregon-R strain contains only cytochromes P-450-A and is susceptible to insecticides. The authors Hikone-R (BG) strain expresses both cytochromes P-450-A and P-450-B and is insecticide resistant. Antibody producing hybridomas were detected in a solid-phase radioimmunoassay (RIA) by binding to Hikone-R (BG) or Oregon-R microsomes. Four independent hybridomas were identified as producing monoclonal antibodies that recognized proteins in the P-450 complex by immunoblot experiments. Three monoclonal antibodies recognized P-450-A proteins, while one monoclonal antibody bound predominantly P-450-B. This monoclonal antibody also recognized southern armyworm (Spodoptera eridania, Cramer) microsomal proteins

  4. C-kit-targeted imaging of gastrointestinal stromal tumor using radiolabeled anti-c-kit monoclonal antibody in a mouse tumor model

    International Nuclear Information System (INIS)

    Sogawa, Chizuru; Tsuji, Atsushi B.; Sudo, Hitomi; Sugyo, Aya; Yoshida, Chisato; Odaka, Kenichi; Uehara, Tomoya; Arano, Yasushi; Koizumi, Mitsuru; Saga, Tsuneo

    2010-01-01

    Introduction: Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor arising from the gastrointestinal tract and highly expresses mutated c-kit. We aimed to develop a specific and sensitive method for detecting GISTs using radiolabeled anti-c-kit monoclonal antibody. Methods: A mutated c-kit-expressing cell clone was established by transfecting an expressing vector of mutated c-kit gene into HEK293 human embryonic kidney cells. The tumors were developed by inoculating c-kit-expressing cells into nude mice. 125 I- and 111 In-labeled anti-c-kit antibodies (12A8 and 41A11) were evaluated in vitro by cell binding, competitive inhibition and cellular internalization assays, and in vivo by biodistribution and imaging studies in tumor-bearing mice. Results: Both 125 I- and 111 In-labeled antibodies showed specific binding with c-kit-expressing cells with high affinity (dissociation constants = 2.2-7.1x10 9 M -1 ). Internalization assay showed that 125 I-labeled antibodies were rapidly internalized and dehalogenated, with the release of 125 I from the cells, resulting in reduction of cell-associated radioactivity with time. In contrast, 111 In-labeled antibody was internalized but did not result in the reduced radioactivity associated with tumor cells. Reflecting this phenomenon, the in vivo tumor uptake of 125 I-labeled antibody was low on Day 1, further decreasing with time, while tumor uptake of 111 In-labeled antibody was high on Day 1, further increasing with time. The xenografted tumor was clearly visualized by scintigraphy after injection of 111 In-labeled antibody. Conclusion: The anti-c-kit monoclonal antibody labeled with a metal radionuclide would be promising for c-kit-targeted imaging of GISTs.

  5. Detection of experimental thrombi in rabbits with an 131I-labelled fibrin-specific monoclonal antibody

    International Nuclear Information System (INIS)

    Walker, K.Z.; Milner, L.J.; Boniface, G.R.

    1990-01-01

    The detection of thrombi in rabbits has been investigated with 131 I-labelled DD-3B6/22, a monoclonal antibody (Mab) reactive at high affinity (Kd=2.68x10 -10 M) with human D Dimer (DD). DD-3B6/22 bound well to both 'fresh' and 'aged' human clots in an in vitro assay but showed poor binding to rabbit clots. However, reactivity was restored to rabbit blood if it was seeded, before clotting, with human DD covalently coupled to Sepharose beads. Thus, a rabbit model was developed in which blood was allowed to clot around DD-Sepharose beads introduced into the jugular vein. Gamma camera imaging showed that intact 131 I-labelled DD-3B6/22 localised to these clots within 24 h. Uptake at this time was 0.202%±0.012% injected dose per gram (%ID/g) compared with 0.086±0.018%ID/g after injection of control antibody. 131 I-labelled F(ab') 2 fragments of DD-3B6/22 allowed earlier scintigraphic detection of the clot which was evident 4 h after injection. Uptake in the clot at 24 h was 0.154±0.038% ID/g compared with 0.109±0.027% ID/g for a control F(ab') 2 . As antigen levels in the clot are estimated to be less than 300 μg DD, thus representing a very small human clot, the DD-3B6/22 Mab would appear to have a good potential for the sensitive detection of thrombi in a clinical setting. (orig.)

  6. An intelligent identification algorithm for the monoclonal picking instrument

    Science.gov (United States)

    Yan, Hua; Zhang, Rongfu; Yuan, Xujun; Wang, Qun

    2017-11-01

    The traditional colony selection is mainly operated by manual mode, which takes on low efficiency and strong subjectivity. Therefore, it is important to develop an automatic monoclonal-picking instrument. The critical stage of the automatic monoclonal-picking and intelligent optimal selection is intelligent identification algorithm. An auto-screening algorithm based on Support Vector Machine (SVM) is proposed in this paper, which uses the supervised learning method, which combined with the colony morphological characteristics to classify the colony accurately. Furthermore, through the basic morphological features of the colony, system can figure out a series of morphological parameters step by step. Through the establishment of maximal margin classifier, and based on the analysis of the growth trend of the colony, the selection of the monoclonal colony was carried out. The experimental results showed that the auto-screening algorithm could screen out the regular colony from the other, which meets the requirement of various parameters.

  7. Exploration of novel strategies to enhance monoclonal antibodies targeting

    International Nuclear Information System (INIS)

    Khawli, L.A.; Epstein, A.L.

    1997-01-01

    This paper highlights the major obstacles and prospects of antibody targeting for the radio imaging and therapy of human malignant lymphomas and more challenging solid tumors. To improve the therapeutic potential of monoclonal antibodies, the authors have focused their attention on the development of new and successful methods to augment antibody uptake in the tumor. These approaches include the use of radiolabeled streptavidin to target biotinylated monoclonal antibodies already bound to tumor, pretreatment with vasoactive immunoconjugates, and the use of chemically modified antibodies. Because of the promising preclinical data obtained with these three newer approaches, plans are underway to test them in the clinic. More generally, these approaches are applicable to the use of other monoclonal antibody/tumor systems for the diagnosis and therapy of human cancers and related diseases

  8. Radioimmunoimaging of experimental gliomas using radiolabelled monoclonal antibodies

    International Nuclear Information System (INIS)

    Glaessner, H.

    1986-01-01

    The biodistribution and tumour uptake of radiolabelled (131 I) glioma-seeking monoclonal antibodies (14 AC1) and their F(ab') 2 fragments were investigated in nude mice having received glioma transplants. Radioimmunoimaging by external scintigraphy at 48 and 96 hours pointed to a superior tumour localisation by the fragments that was clearly related to the dose. Wholebody determinations of the biokinetic behaviour led to the following results: Faster clearance anc more ready elimination from the blood pool for the fragments, preferential uptake in the tumour; intact antibodies; binding in the liver, spleen and lungs. The study confirmed the value of fragments of monoclonal antibodies in the diagnosis of tumours and pointed to the possibility of using intact monoclonal antibodies as carriers of radioisotopes and cytotoxic drugs within the scope of therapeutic programmes. (TRV) [de

  9. Efficacy of Wnt-1 monoclonal antibody in sarcoma cells

    International Nuclear Information System (INIS)

    Mikami, Iwao; Koizumi, Kiyoshi; Jablons, David M; You, Liang; He, Biao; Xu, Zhidong; Batra, Sonny; Lee, Amie Y; Mazieres, Julien; Reguart, Noemi; Uematsu, Kazutsugu

    2005-01-01

    Sarcomas are one of the most refractory diseases among malignant tumors. More effective therapies based on an increased understanding of the molecular biology of sarcomas are needed as current forms of therapy remain inadequate. Recently, it has been reported that Wnt-1/β-catenin signaling inhibits apoptosis in several cancers. In this study, we investigated the efficacy of a monoclonal anti-Wnt-1 antibody in sarcoma cells. We treated cell lines A-204, SJSA-1, and fresh primary cultures of lung metastasis of sarcoma with a monoclonal anti-Wnt-1 antibody. Wnt-1 siRNA treatment was carried out in A-204. We assessed cell death using Crystal Violet staining. Apoptosis induction was estimated by flow cytometry analysis (Annexin V and PI staining). Cell signaling changes were determined by western blotting analysis. We detected Wnt-1 expression in all tissue samples and cell lines. Significant apoptosis induction was found in monoclonal anti-Wnt-1 antibody treated cells compared to control monoclonal antibody treated cells (p < 0.02). Similarly, we observed increased apoptosis in Wnt-1 siRNA treated cells. Blockade of Wnt-1 signaling in both experiments was confirmed by analyzing intracellular levels of Dishevelled-3 and of cytosolic β-catenin. Furthermore, the monoclonal anti-Wnt-1 antibody also induced cell death in fresh primary cultures of metastatic sarcoma in which Wnt-1 signaling was active. Our results indicate that Wnt-1 blockade by either monoclonal antibody or siRNA induces cell death in sarcoma cells. These data suggest that Wnt-1 may be a novel therapeutic target for the treatment of a subset of sarcoma cells in which Wnt-1/β-catenin signaling is active

  10. Making Recombinant Monoclonal Antibody And Radiolabelling For Medical Purpose

    International Nuclear Information System (INIS)

    Nguyen Thi Thu; Duong Van Dong; Vo Thi Cam Hoa; Bui Van Cuong; Chu Van Khoa; Vu Bich Huong; Le Quang Huan

    2008-01-01

    Recombinant monoclonal antibody labeling with 131 I specific to tumor cell has been studied and prepared for treatment of Hodgkin lymphoma. In this study, a recombinant monoclonal antibody with two specific properties is a hybrid molecule created by coupling an antibody variable fragments with peptide melittin. The gene coding the antibody fragment has been obtained from human synthetic Fv libraries using for panning and screening on populations of lymphocytes fragmented from human blood cells with Hodgkin diseases. The gene encoding peptit melittin has been cloned from honeybee Apis cerana DNA. The gene coding recombinant monoclonal antibody has been expressed in E.coli BL21 (DE3) at 37 o C and was induced with 0.6 mM IPTG. The recombinant compound has been purified by affinity chromatography with HiTrap affinity column. The obtained recombinant monoclonal antibody has showed cytolytic activities when added to cell culture medium for LU cancer cell line with the amount of 100 - 200 mg/ml. This monoclonal antibody is labeled with 131 I using chloramine T procedure. ChT mass for the oxidation of 50 μg monoclonal antibody in 76 MBq was 10 μg. Sodium metabisulfite was used as a reducing agent. Reaction time was above 3 mins. The radiochemical purity was determined using electrophoresis and TLC methods. Radiochemical yield was > 97%. Radiochemical purity after purification was > 99%. Nuclear purity was > 99%. Stability of the label antibody was 12 days. This is the product promise potential used in the diagnostic and therapeutic of Hodgkin lymphoma. (author)

  11. Monoclonal anti-melanoma antibodies and their possible clinical use

    International Nuclear Information System (INIS)

    Hellstroem, K.E.; Hellstroem, Ingegerd; Washington Univ., Seattle; Washington Univ., Seattle

    1985-01-01

    Cell surface antigens of human melanoma, as defined by monoclonal antibodies, are discussed and in particular the three antigens p97, a GD3 ganglioside and a proteoglycan. The potential diagnostic uses of antibodies to melanoma antigens are reviewed including in vitro diagnosis by immuno-histology, in vitro diagnosis by serum assays and in vivo diagnosis by tumour imaging using radioactively labelled antibodies. The potential therapeutic uses of monoclonal antibodies to melanoma antigens are also reviewed including targets for antibody therapy, the use of antibodies alone, radiolabelled antibodies, antibody-toxin conjugates, antibody-drug conjugates, anti-idiotypic antibodies and vaccines. (UK)

  12. ERBB oncogene proteins as targets for monoclonal antibodies.

    Science.gov (United States)

    Polanovski, O L; Lebedenko, E N; Deyev, S M

    2012-03-01

    General properties of the family of tyrosine kinase ERBB receptors are considered in connection with their role in the generation of cascades of signal transduction in normal and tumor cells. Causes of acquisition of oncogene features by genes encoding these receptors and their role in tumorigenesis are analyzed. Anti-ERBB monoclonal antibodies approved for therapy are described in detail, and mechanisms of their antitumor activity and development of resistance to them are reviewed. The existing and the most promising strategies for creating and using monoclonal antibodies and their derivatives for therapy of cancer are discussed.

  13. A dual-mode surface display system for the maturation and production of monoclonal antibodies in glyco-engineered Pichia pastoris.