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Sample records for human antibody binding

  1. Antigen-binding radioimmunoassays for human IgG antibodies to bovine ν-lactoglobulin

    International Nuclear Information System (INIS)

    Turner, M.W.; Paganelli, R.; Levinsky, R.J.; Williams, A.

    1983-01-01

    A double antibody antigen-binding assay for the detection of human IgG antibodies to the bovine milk allergen ν-lactoglobulin is described. The levels of such antibodies in patients with established cows' milk protein intolerance were significantly higher than the levels observed in a healthy control group (P<0.01). The assay showed excellent correlation with a solid phase antigen binding assay (rsub(s) = 0.8, P<0.001). (Auth.)

  2. Human vaccination against Plasmodium vivax Duffy-binding protein induces strain-transcending antibodies

    OpenAIRE

    Payne, Ruth O.; Silk, Sarah E.; Elias, Sean C.; Milne, Kathryn H.; Rawlinson, Thomas A.; Llewellyn, David; Shakri, A. Rushdi; Jin, Jing; Labb?, Genevi?ve M.; Edwards, Nick J.; Poulton, Ian D.; Roberts, Rachel; Farid, Ryan; J?rgensen, Thomas; Alanine, Daniel G.W.

    2017-01-01

    BACKGROUND: Plasmodium vivax is the most widespread human malaria geographically; however, no effective vaccine exists. Red blood cell invasion by the P. vivax merozoite depends on an interaction between the Duffy antigen receptor for chemokines (DARC) and region II of the parasite's Duffy-binding protein (PvDBP_RII). Naturally acquired binding-inhibitory antibodies against this interaction associate with clinical immunity, but it is unknown whether these responses can be induced by human vac...

  3. Agonistic Human Antibodies Binding to Lecithin-Cholesterol Acyltransferase Modulate High Density Lipoprotein Metabolism*

    Science.gov (United States)

    Gunawardane, Ruwanthi N.; Fordstrom, Preston; Piper, Derek E.; Masterman, Stephanie; Siu, Sophia; Liu, Dongming; Brown, Mike; Lu, Mei; Tang, Jie; Zhang, Richard; Cheng, Janet; Gates, Andrew; Meininger, David; Chan, Joyce; Carlson, Tim; Walker, Nigel; Schwarz, Margrit; Delaney, John; Zhou, Mingyue

    2016-01-01

    Drug discovery opportunities where loss-of-function alleles of a target gene link to a disease-relevant phenotype often require an agonism approach to up-regulate or re-establish the activity of the target gene. Antibody therapy is increasingly recognized as a favored drug modality due to multiple desirable pharmacological properties. However, agonistic antibodies that enhance the activities of the target enzymes are rarely developed because the discovery of agonistic antibodies remains elusive. Here we report an innovative scheme of discovery and characterization of human antibodies capable of binding to and agonizing a circulating enzyme lecithin cholesterol acyltransferase (LCAT). Utilizing a modified human LCAT protein with enhanced enzymatic activity as an immunogen, we generated fully human monoclonal antibodies using the XenoMouseTM platform. One of the resultant agonistic antibodies, 27C3, binds to and substantially enhances the activity of LCAT from humans and cynomolgus macaques. X-ray crystallographic analysis of the 2.45 Å LCAT-27C3 complex shows that 27C3 binding does not induce notable structural changes in LCAT. A single administration of 27C3 to cynomolgus monkeys led to a rapid increase of plasma LCAT enzymatic activity and a 35% increase of the high density lipoprotein cholesterol that was observed up to 32 days after 27C3 administration. Thus, this novel scheme of immunization in conjunction with high throughput screening may represent an effective strategy for discovering agonistic antibodies against other enzyme targets. 27C3 and other agonistic human anti-human LCAT monoclonal antibodies described herein hold potential for therapeutic development for the treatment of dyslipidemia and cardiovascular disease. PMID:26644477

  4. The antibody response against human and chimeric anti-TNF therapeutic antibodies primarily targets the TNF binding region

    NARCIS (Netherlands)

    van Schie, K. A.; Hart, M. H.; de Groot, E. R.; Kruithof, S.; Aarden, L. A.; Wolbink, G. J.; Rispens, T.

    2015-01-01

    In a subset of patients, anti tumour necrosis factor (TNF) therapeutic antibodies are immunogenic, resulting in the formation of antidrug antibodies (ADAs). Neutralising ADAs compete with TNF for its binding site and reduces the effective serum concentration, causing clinical non-response. It is

  5. Differences in human skin between the epidermal growth factor receptor distribution detected by EGF binding and monoclonal antibody recognition

    DEFF Research Database (Denmark)

    Green, M R; Couchman, J R

    1985-01-01

    , the eccrine sweat glands, capillary system, and the hair follicle outer root sheath, generally similar in pattern to that previously reported for full-thickness rat skin and human epidermis. The same areas also bound EGF-R1 but in addition the monoclonal antibody recognized a cone of melanin containing......Two methods have been used to examine epidermal growth factor (EGF) receptor distribution in human scalp and foreskin. The first employed [125I]EGF viable explants and autoradiography to determine the EGF binding pattern while the second used a monoclonal antibody to the human EGF receptor to map...... whether EGF-R1 could recognize molecules unrelated to the EGF receptor, the EGF binding and EGF-R1 recognition profiles were compared on cultures of SVK14 cells, a SV40 transformed human keratinocyte cell line. EGF binding and EGF-R1 monoclonal antibody distribution on these cells was found to be similar...

  6. Determining the binding affinity of therapeutic monoclonal antibodies towards their native unpurified antigens in human serum.

    Directory of Open Access Journals (Sweden)

    Christine Bee

    Full Text Available Monoclonal antibodies (mAbs are a growing segment of therapeutics, yet their in vitro characterization remains challenging. While it is essential that a therapeutic mAb recognizes the native, physiologically occurring epitope, the generation and selection of mAbs often rely on the use of purified recombinant versions of the antigen that may display non-native epitopes. Here, we present a method to measure both, the binding affinity of a therapeutic mAb towards its native unpurified antigen in human serum, and the antigen's endogenous concentration, by combining the kinetic exclusion assay and Biacore's calibration free concentration analysis. To illustrate the broad utility of our method, we studied a panel of mAbs raised against three disparate soluble antigens that are abundant in the serum of healthy donors: proprotein convertase subtilisin/kexin type 9 (PCSK9, progranulin (PGRN, and fatty acid binding protein (FABP4. We also determined the affinity of each mAb towards its purified recombinant antigen and assessed whether the interactions were pH-dependent. Of the six mAbs studied, three did not appear to discriminate between the serum and recombinant forms of the antigen; one mAb bound serum antigen with a higher affinity than recombinant antigen; and two mAbs displayed a different affinity for serum antigen that could be explained by a pH-dependent interaction. Our results highlight the importance of taking pH into account when measuring the affinities of mAbs towards their serum antigens, since the pH of serum samples becomes increasingly alkaline upon aerobic handling.

  7. Broad and potent HIV-1 neutralization by a human antibody that binds the gp41-gp120 interface

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Jinghe; Kang, Byong H.; Pancera, Marie; Lee, Jeong Hyun; Tong, Tommy; Feng, Yu; Imamichi, Hiromi; Georgiev, Ivelin S.; Chuang, Gwo-Yu; Druz, Aliaksandr; Doria-Rose, Nicole A.; Laub, Leo; Sliepen, Kwinten; van Gils, Marit J.; de la Peña, Alba Torrents; Derking, Ronald; Klasse, Per-Johan; Migueles, Stephen A.; Bailer, Robert T.; Alam, Munir; Pugach, Pavel; Haynes, Barton F.; Wyatt, Richard T.; Sanders, Rogier W.; Binley, James M.; Ward, Andrew B.; Mascola, John R.; Kwong, Peter D.; Connors, Mark [NIH

    2015-10-15

    The isolation of human monoclonal antibodies is providing important insights into the specificities that underlie broad neutralization of HIV-1 (reviewed in ref. 1). Here we report a broad and extremely potent HIV-specific monoclonal antibody, termed 35O22, which binds a novel HIV-1 envelope glycoprotein (Env) epitope. 35O22 neutralized 62% of 181 pseudoviruses with a half-maximum inhibitory concentration (IC50) <50 μg ml-1. The median IC50 of neutralized viruses was 0.033 μg ml-1, among the most potent thus far described. 35O22 did not bind monomeric forms of Env tested, but did bind the trimeric BG505 SOSIP.664. Mutagenesis and a reconstruction by negative-stain electron microscopy of the Fab in complex with trimer revealed that it bound to a conserved epitope, which stretched across gp120 and gp41. The specificity of 35O22 represents a novel site of vulnerability on HIV Env, which serum analysis indicates to be commonly elicited by natural infection. Binding to this new site of vulnerability may thus be an important complement to current monoclonal-antibody-based approaches to immunotherapies, prophylaxis and vaccine design.

  8. Monoclonal antibodies to the reactive centre loop (RCL) of human corticosteroid-binding globulin (CBG) can protect against proteolytic cleavage.

    Science.gov (United States)

    Lewis, John G; Elder, Peter A

    2017-07-01

    Corticosteroid-binding globulin (CBG) binds most of the cortisol in circulation and is a non-functional member of the family of serine protease inhibitors (serpins) with an exposed elastase sensitive reactive centre loop (RCL). The RCL can be cleaved by human neutrophil elastase, released from activated neutrophils, and can also be cleaved at nearby site(s) by elastase released by Pseudomonas aeruginosa, and at two further sites, also within the RCL, by bovine chymotrypsin. Cleavage of the RCL results in a conformational change accompanied by a marked decrease in affinity for cortisol and hence its release at the site of proteolysis. These cleavages are irreversible and the similar half-lives of cleaved and intact CBG could mean that there may be some advantage in slowing the rate of CBG cleavage in acute inflammation thereby increasing the proportion of intact CBG in circulation. Here we show, for the first time, that pre-incubation of tethered human CBG with two monoclonal antibodies to the RCL of CBG protects against cleavage by all three enzymes. Furthermore, in plasma, pre-incubation with both RCL monoclonal antibodies delays neutrophil elastase cleavage of the RCL and one of these RCL monoclonal antibodies also delays bovine chymotrypsin cleavage of the RCL. These findings may provide a basis and rationale for the concept of the use of RCL antibodies as therapeutic agents to effectively increase the proportion of intact CBG in circulation which may be of benefit in acute inflammation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Genotoxic effect and antigen binding characteristics of SLE auto-antibodies to peroxynitrite-modified human DNA.

    Science.gov (United States)

    Khan, Md Asad; Alam, Khursheed; Mehdi, Syed Hassan; Rizvi, M Moshahid A

    2017-12-01

    Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease characterized by auto-antibodies against native deoxyribonucleic acid after modification and is one of the reasons for the development of SLE. Here, we have evaluated the structural perturbations in human placental DNA by peroxynitrite using spectroscopy, thermal denaturation and high-performance liquid chromatography (HPLC). Peroxynitrite is a powerful potent bi-functional oxidative/nitrative agent that is produced both endogenously and exogenously. In experimental animals, the peroxynitrite-modified DNA was found to be highly immunogenic. The induced antibodies showed cross-reactions with different types of DNA and nitrogen bases that were modified with peroxynitrite by inhibition ELISA. The antibody activity was inhibited by approximately 89% with its immunogen as the inhibitor. The antigen-antibodies interaction between induced antibodies with peroxynitrite-modified DNA showed retarded mobility as compared to the native form. Furthermore, significantly increased binding was also observed in SLE autoantibodies with peroxynitrite-modified DNA than native form. Moreover, DNA isolated from lymphocyte of SLE patients revealed significant recognition of anti-peroxynitrite-modified DNA immunoglobulin G (IgG). Our data indicates that DNA modified with peroxynitrite presents unique antigenic determinants that may induce autoantibody response in SLE. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Tabhu: tools for antibody humanization

    DEFF Research Database (Denmark)

    Olimpieri, Pier Paolo; Marcatili, Paolo; Tramontano, Anna

    2015-01-01

    Antibodies are rapidly becoming essential tools in the clinical practice, given their ability to recognize their cognate antigens with high specificity and affinity, and a high yield at reasonable costs in model animals. Unfortunately, when administered to human patients, xenogeneic antibodies can...... elicit unwanted and dangerous immunogenic responses. Antibody humanization methods are designed to produce molecules with a better safety profile still maintaining their ability to bind the antigen. This can be accomplished by grafting the non-human regions determining the antigen specificity...... and time-consuming experiments. Here we present tools for antibody humanization (Tabhu) a web server for antibody humanization. Tabhu includes tools for human template selection, grafting, back-mutation evaluation, antibody modelling and structural analysis, helping the user in all the critical steps...

  11. Conserved epitope on several human vitamin K-dependent proteins: location of the antigenic site and influence of metal ions on antibody binding

    International Nuclear Information System (INIS)

    Church, W.R.; Messier, T.; Howard, P.R.; Amiral, J.; Meyer, D.; Mann, K.G.

    1988-01-01

    A murine monoclonal antibody (designated H-11) produced by injecting mice with purified human protein C was found to bind several human vitamin K-dependent proteins. Using a solid-phase competitive radioimmunoassay with antibody immobilized onto microtiter plates, binding of 125 I-labeled protein C to the antibody was inhibited by increasing amounts of protein C, prothrombin, and Factors X and VII over a concentration range of 1 x 10 -8 to 1 x 10 -6 M. Chemical treatment of prothrombin with a variety of agents did not destroy the antigenic site recognized by the antibody as measured by immunoblotting of prothrombin or prothrombin derivative immobilized onto nitrocellulose. Immunoblotting of purified vitamin K-dependent polypeptides with the monoclonal antibody following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose indicated that the antigenic site was found on the light chains of protein C and Factor X. The exact location of the antigenic determinant for antibody H-11 was established using synthetic peptides. Comparison of protein sequences of bovine and human vitamin K-dependent proteins suggests that the sequence Phe-Leu-Glu-Glu-Xaa-Arg/Lys is required for antibody binding. Increasing concentrations of Ca 2+ , Mg 2+ , or Mn 2+ partially inhibited binding of 125 I-protein C to the antibody in a solid-phase assay system with half-maximal binding observed at divalent metal ion concentrations of 2, 4, and 0.6 mM, respectively. The antigenic site thus recognized by monoclonal antibody H-11 is located at the amino-terminal region in the highly conserved γ-carboxyglutamic acid-containing domains of several, but not all, vitamin K-dependent proteins

  12. Interleukin 1-induced down-regulation of antibody binding to CD4 molecules on human lymphocytes

    DEFF Research Database (Denmark)

    Tvede, N; Christensen, L D; Ødum, Niels

    1988-01-01

    Interleukin 1 (IL-1) is involved in the early activation of T lymphocytes. The CD4 antigen, described as a phenotypic marker of helper T cells, is also important in early T-cell activation by its ability to bind to MHC class II molecules on antigen-presenting cells, and to transmit positive (and...

  13. Human monoclonal antibodies: the residual challenge of antibody immunogenicity.

    Science.gov (United States)

    Waldmann, Herman

    2014-01-01

    One of the major reasons for seeking human monoclonal antibodies has been to eliminate immunogenicity seen with rodent antibodies. Thus far, there has yet been no approach which absolutely abolishes that risk for cell-binding antibodies. In this short article, I draw attention to classical work which shows that monomeric immunoglobulins are intrinsically tolerogenic if they can be prevented from creating aggregates or immune complexes. Based on these classical studies two approaches for active tolerization to therapeutic antibodies are described.

  14. Characteristics Studies of 125I- and total PSA antibody's Binding with prostate specific antigen (PSA) in Human Uterus Tumors

    International Nuclear Information System (INIS)

    Al-Mudaffar, S.; Al-Salihi, J.

    2005-01-01

    Two groups of uterus tumors (benign and malignant) postmenopausal patients were used to investigate the presence of prostate specific antigen (PSA). Preliminary experiments were performed to follow the binding of '1 25 I-anti total PSA antibody with PSA in uterus tissues homogenates of the two groups with their corresponding antigen and found to be (8.8,7.1%) for benign and malignant tumors, respectively. An Immuno Radio Metric Assay (IRMA) procedure was developed for measuring PSA in benign and malignant uterus tumors homogenates. The optimum conditions of the binding of 125 I-anti total PSA antibody with PSA were as follows: PSA concentration (150,200 μg protein),tracer antibody concentration (125,250 μg protein), p H (7.6,7.2), temp (15,25?C) and time (1.5 hrs) for postmenopausal benign and malignant uterus tumors tissue homogenates, respectively. The use of different concentrations of Na + and Mg 2+ ions were shown to cause an increase in the binding at concentration of (125,75 mΜ) of Na 1+ ions (75,225 mΜ) of Mg 2+ ions for benign and malignant uterus tumors homogenates, respectively, while the use of different concentrations of urea and polyethylene glycol (PEG) Caused a decrease in the binding with the increase in the concentration of each of urea and PEG in the both cases

  15. Tabhu: tools for antibody humanization.

    KAUST Repository

    Olimpieri, Pier Paolo

    2014-10-09

    SUMMARY: Antibodies are rapidly becoming essential tools in the clinical practice, given their ability to recognize their cognate antigens with high specificity and affinity, and a high yield at reasonable costs in model animals. Unfortunately, when administered to human patients, xenogeneic antibodies can elicit unwanted and dangerous immunogenic responses. Antibody humanization methods are designed to produce molecules with a better safety profile still maintaining their ability to bind the antigen. This can be accomplished by grafting the non-human regions determining the antigen specificity into a suitable human template. Unfortunately, this procedure may results in a partial or complete loss of affinity of the grafted molecule that can be restored by back-mutating some of the residues of human origin to the corresponding murine ones. This trial-and-error procedure is hard and involves expensive and time-consuming experiments. Here we present tools for antibody humanization (Tabhu) a web server for antibody humanization. Tabhu includes tools for human template selection, grafting, back-mutation evaluation, antibody modelling and structural analysis, helping the user in all the critical steps of the humanization experiment protocol. AVAILABILITY: http://www.biocomputing.it/tabhu CONTACT: anna.tramontano@uniroma1.it, pierpaolo.olimpieri@uniroma1.it SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

  16. Penetration and binding of monoclonal antibody in human osteosarcoma multicell spheroids. Comparison of confocal laser scanning microscopy and autoadiography

    International Nuclear Information System (INIS)

    Hjelstuen, M.H.; Rasch-Halvorsen, K.; Brekken, C.; Bruland, Oe.; Davies, C. de L.

    1996-01-01

    Penetration and binding of monoclonal antibody (MAb) in multicell osteosarcoma spheroids have been studied by autoradiography and confocal laser scanning microscopy (CLSM). Optical sectioning of the 3-dimensional spheroids was performed by CLSM. Owing to attenuation of fluorescence intensity, FITC-labelled MAb could not be detected at depths greater than 60 μm within the spheroids. The antibody uptake seen in autoradiographs and CLSM images 60 μm within the spheroids were essentially identical. MAb had reached all parts of the spheroids within 6 h. Quantitative measurements of the fluorescence intensity of FITC-labelled MAb seen in confocal images and measurements of MAb bound per cell using flow cytometry, showed that maximum uptake was reached after 6 h. The possibility to perform both quantatitive and qualitative measurements makes CLSM a promising method for studying antibody uptake in thick tissue samples. (orig.)

  17. Combined roles of human IgG subclass, alternative complement pathway activation, and epitope density in the bactericidal activity of antibodies to meningococcal factor h binding protein.

    Science.gov (United States)

    Giuntini, Serena; Reason, Donald C; Granoff, Dan M

    2012-01-01

    Meningococcal vaccines containing factor H binding protein (fHbp) are in clinical development. fHbp binds human fH, which enables the meningococcus to resist complement-mediated bacteriolysis. Previously, we found that chimeric human IgG1 mouse anti-fHbp monoclonal antibodies (MAbs) had human complement-mediated bactericidal activity only if the MAb inhibited fH binding. Since IgG subclasses differ in their ability to activate complement, we investigated the role of human IgG subclasses on antibody functional activity. We constructed chimeric MAbs in which three different murine fHbp-specific binding domains were each paired with human IgG1, IgG2, or IgG3. Against a wild-type group B isolate, all three IgG3 MAbs, irrespective of their ability to inhibit fH binding, had bactericidal activity that was >5-fold higher than the respective IgG1 MAbs, while the IgG2 MAbs had the least activity. Against a mutant with increased fHbp expression, the anti-fHbp MAbs elicited greater C4b deposition (classical pathway) and greater bactericidal activity than against the wild-type strain, and the IgG1 MAbs had similar or greater activity than the respective IgG3 MAbs. The bactericidal activity against both wild-type and mutant strains also was dependent, in part, on activation of the alternative complement pathway. Thus, at lower epitope density in the wild-type strain, the IgG3 anti-fHbp MAbs had the greatest bactericidal activity. At a higher epitope density in the mutant, the IgG1 MAbs had similar or greater bactericidal activity than the IgG3 MAbs, and the activity was less dependent on the inhibition of fH binding than at a lower epitope density.

  18. Reshaping Human Antibodies: Grafting an Antilysozyme Activity

    Science.gov (United States)

    Verhoeyen, Martine; Milstein, Cesar; Winter, Greg

    1988-03-01

    The production of therapeutic human monoclonal antibodies by hybridoma technology has proved difficult, and this has prompted the ``humanizing'' of mouse monoclonal antibodies by recombinant DNA techniques. It was shown previously that the binding site for a small hapten could be grafted from the heavy-chain variable domain of a mouse antibody to that of a human myeloma protein by transplanting the hypervariable loops. It is now shown that a large binding site for a protein antigen (lysozyme) can also be transplanted from mouse to human heavy chain. The success of such constructions may be facilitated by an induced-fit mechanism.

  19. Modified cytokeratins expressed on the surface of carcinoma cells undergo endocytosis upon binding of human monoclonal antibody and its recombinant Fab fragment

    DEFF Research Database (Denmark)

    Ditzel, H J; Garrigues, U; Andersen, C B

    1997-01-01

    display selection and the human Fab fragment was expressed in bacteria. Analysis by confocal laser scanning microscopy demonstrated that COU-1 bound in a uniform punctate pattern to the surface of viable carcinoma cells stained at 4 degrees C, and binding increased significantly when cells were cultured...... was significantly reduced. Similar results were obtained using intact IgM COU-1 and the recombinant Fab fragment. Immunohistological studies indicated that COU-1, in contrast to murine monoclonal antibodies against normal cytokeratin 8 and 18, could differentiate between malignant and normal colon epithelia...

  20. Improved decision making for prioritizing tumor targeting antibodies in human xenografts: Utility of fluorescence imaging to verify tumor target expression, antibody binding and optimization of dosage and application schedule.

    Science.gov (United States)

    Dobosz, Michael; Haupt, Ute; Scheuer, Werner

    2017-01-01

    Preclinical efficacy studies of antibodies targeting a tumor-associated antigen are only justified when the expression of the relevant antigen has been demonstrated. Conventionally, antigen expression level is examined by immunohistochemistry of formalin-fixed paraffin-embedded tumor tissue section. This method represents the diagnostic "gold standard" for tumor target evaluation, but is affected by a number of factors, such as epitope masking and insufficient antigen retrieval. As a consequence, variances and discrepancies in histological staining results can occur, which may influence decision-making and therapeutic outcome. To overcome these problems, we have used different fluorescence-labeled therapeutic antibodies targeting human epidermal growth factor receptor (HER) family members and insulin-like growth factor-1 receptor (IGF1R) in combination with fluorescence imaging modalities to determine tumor antigen expression, drug-target interaction, and biodistribution and tumor saturation kinetics in non-small cell lung cancer xenografts. For this, whole-body fluorescence intensities of labeled antibodies, applied as a single compound or antibody mixture, were measured in Calu-1 and Calu-3 tumor-bearing mice, then ex vivo multispectral tumor tissue analysis at microscopic resolution was performed. With the aid of this simple and fast imaging method, we were able to analyze the tumor cell receptor status of HER1-3 and IGF1R, monitor the antibody-target interaction and evaluate the receptor binding sites of anti-HER2-targeting antibodies. Based on this, the most suitable tumor model, best therapeutic antibody, and optimal treatment dosage and application schedule was selected. Predictions drawn from obtained imaging data were in excellent concordance with outcome of conducted preclinical efficacy studies. Our results clearly demonstrate the great potential of combined in vivo and ex vivo fluorescence imaging for the preclinical development and characterization of

  1. Tabhu: tools for antibody humanization.

    KAUST Repository

    Olimpieri, Pier Paolo; Marcatili, Paolo; Tramontano, Anna

    2014-01-01

    for antibody humanization. Tabhu includes tools for human template selection, grafting, back-mutation evaluation, antibody modelling and structural analysis, helping the user in all the critical steps of the humanization experiment protocol. AVAILABILITY: http

  2. Anti-citrullinated protein antibodies promote apoptosis of mature human Saos-2 osteoblasts via cell-surface binding to citrullinated heat shock protein 60.

    Science.gov (United States)

    Lu, Ming-Chi; Yu, Chia-Li; Yu, Hui-Chun; Huang, Hsien-Bin; Koo, Malcolm; Lai, Ning-Sheng

    2016-01-01

    We hypothesized that anti-citrullinated protein antibodies (ACPAs) react with osteoblast surface citrullinated proteins and affect cell function, leading to joint damage in patients with rheumatoid arthritis (RA). First, we purified ACPAs by cyclic citrullinated peptide (CCP)-conjugated affinity column chromatography. The cognate antigens of ACPAs on Saos-2 cells, a sarcoma osteogenic cell line generated from human osteoblasts, were probed by ACPAs, and the reactive bands were analyzed using proteomic analyses. We found that ACPAs bind to Saos-2 cell membrane, and several protein candidates, including HSP60, were identified. We then cloned and purified recombinant heat shock protein 60 (HSP60) and citrullinated HSP60 (citHSP60) and investigated the effect of ACPAs on Saos-2 cell. We confirmed that HSP60 obtained from Saos-2 cell membrane were citrullinated and reacted with ACPAs, which induces Saos-2 cells apoptosis via binding to surface-expressed citHSP60 through Toll-like receptor 4 signaling. ACPAs promoted interleukin (IL)-6 and IL-8 expression in Saos-2 cells. Finally, sera from patients with RA and healthy controls were examined for their titers of anti-HSP60 and anti-citHSP60 antibodies using an enzyme-linked immunosorbent assay. The radiographic change in patients with RA was evaluated using the Genant-modified Sharp scoring system. Patients with RA showed higher sera titers of anti-citHSP60, but not anti-HSP60, antibodies when compared with controls. In addition, the anti-citHSP60 level was positively associated with increased joint damage in patients with RA. In conclusion, Saos-2 cell apoptosis was mediated by ACPAs via binding to cell surface-expressed citHSP60 and the titer of anti-citHSP60 in patients with RA positively associated with joint damage. Copyright © 2015 Elsevier GmbH. All rights reserved.

  3. Recombinant Protein Truncation Strategy for Inducing Bactericidal Antibodies to the Macrophage Infectivity Potentiator Protein of Neisseria meningitidis and Circumventing Potential Cross-Reactivity with Human FK506-Binding Proteins

    OpenAIRE

    Bielecka, Magdalena K.; Devos, Nathalie; Gilbert, Mélanie; Hung, Miao-Chiu; Weynants, Vincent; Heckels, John E.; Christodoulides, Myron

    2014-01-01

    A recombinant macrophage infectivity potentiator (rMIP) protein of Neisseria meningitidis induces significant serum bactericidal antibody production in mice and is a candidate meningococcal vaccine antigen. However, bioinformatics analysis of MIP showed some amino acid sequence similarity to human FK506-binding proteins (FKBPs) in residues 166 to 252 located in the globular domain of the protein. To circumvent the potential concern over generating antibodies that could recognize human protein...

  4. Crystal structure and size-dependent neutralization properties of HK20, a human monoclonal antibody binding to the highly conserved heptad repeat 1 of gp41.

    Science.gov (United States)

    Sabin, Charles; Corti, Davide; Buzon, Victor; Seaman, Mike S; Lutje Hulsik, David; Hinz, Andreas; Vanzetta, Fabrizia; Agatic, Gloria; Silacci, Chiara; Mainetti, Lara; Scarlatti, Gabriella; Sallusto, Federica; Weiss, Robin; Lanzavecchia, Antonio; Weissenhorn, Winfried

    2010-11-18

    The human monoclonal antibody (mAb) HK20 neutralizes a broad spectrum of primary HIV-1 isolates by targeting the highly conserved heptad repeat 1 (HR1) of gp41, which is transiently exposed during HIV-1 entry. Here we present the crystal structure of the HK20 Fab in complex with a gp41 mimetic 5-Helix at 2.3 Å resolution. HK20 employs its heavy chain CDR H2 and H3 loops to bind into a conserved hydrophobic HR1 pocket that is occupied by HR2 residues in the gp41 post fusion conformation. Compared to the previously described HR1-specific mAb D5, HK20 approaches its epitope with a different angle which might favor epitope access and thus contribute to its higher neutralization breadth and potency. Comparison of the neutralization activities of HK20 IgG, Fab and scFv employing both single cycle and multiple cycle neutralization assays revealed much higher potencies for the smaller Fab and scFv over IgG, implying that the target site is difficult to access for complete antibodies. Nevertheless, two thirds of sera from HIV-1 infected individuals contain significant titers of HK20-inhibiting antibodies. The breadth of neutralization of primary isolates across all clades, the higher potencies for C-clade viruses and the targeting of a distinct site as compared to the fusion inhibitor T-20 demonstrate the potential of HK20 scFv as a therapeutic tool.

  5. [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

  6. Circulating chromatin-anti-chromatin antibody complexes bind with high affinity to dermo-epidermal structures in murine and human lupus nephritis

    DEFF Research Database (Denmark)

    Fismen, S; Hedberg, A; Fenton, K A

    2009-01-01

    Murine and human lupus nephritis are characterized by glomerular deposits of electron-dense structures (EDS). Dominant components of EDS are chromatin fragments and IgG antibodies. Whether glomerular EDS predispose for similar deposits in skin is unknown. We analysed (i) whether dermo-epidermal i......Murine and human lupus nephritis are characterized by glomerular deposits of electron-dense structures (EDS). Dominant components of EDS are chromatin fragments and IgG antibodies. Whether glomerular EDS predispose for similar deposits in skin is unknown. We analysed (i) whether dermo......-epidermal immune complex deposits have similar molecular composition as glomerular deposits, (ii) whether chromatin fragments bind dermo-epidermal structures, and (iii) whether deposits in nephritic glomeruli predispose for accumulation of similar deposits in skin. Paired skin and kidney biopsies from nephritic...... (NZBxNZW)F1 and MRL-lpr/lpr mice and from five patients with lupus nephritis were analysed by immunofluorescence, immune electron microscopy (IEM) and co-localization TUNEL IEM. Affinity of chromatin fragments for membrane structures was determined by surface plasmon resonance. Results demonstrated (i...

  7. Crystal structure of the antigen-binding fragment of a monoclonal antibody specific for the multidrug-resistance-linked ABC transporter human P-glycoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Esser, Lothar; Shukla, Suneet; Zhou, Fei; Ambudkar, Suresh V.; Xia, Di

    2016-07-27

    P-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancers that plays important roles in the pharmacokinetics of a large number of drugs. The drug-resistance phenotype of P-gp can be modulated by the monoclonal antibody UIC2, which specifically recognizes human P-gp in a conformation-dependent manner. Here, the purification, sequence determination and high-resolution structure of the Fab fragment of UIC2 (UIC2/Fab) are reported. Purified UIC2/Fab binds human P-gp with a 1:1 stoichiometry. Crystals of UIC2/Fab are triclinic (space groupP1), with unit-cell parametersa= 40.67,b= 44.91,c= 58.09 Å, α = 97.62, β = 99.10, γ = 94.09°, and diffracted X-rays to 1.6 Å resolution. The structure was determined by molecular replacement and refined to 1.65 Å resolution. The asymmetric unit contains one molecule of UIC2/Fab, which exhibits a positively charged antigen-binding surface, suggesting that it might recognize an oppositely charged extracellular epitope of P-gp.

  8. Crystal structure and size-dependent neutralization properties of HK20, a human monoclonal antibody binding to the highly conserved heptad repeat 1 of gp41.

    Directory of Open Access Journals (Sweden)

    Charles Sabin

    Full Text Available The human monoclonal antibody (mAb HK20 neutralizes a broad spectrum of primary HIV-1 isolates by targeting the highly conserved heptad repeat 1 (HR1 of gp41, which is transiently exposed during HIV-1 entry. Here we present the crystal structure of the HK20 Fab in complex with a gp41 mimetic 5-Helix at 2.3 Å resolution. HK20 employs its heavy chain CDR H2 and H3 loops to bind into a conserved hydrophobic HR1 pocket that is occupied by HR2 residues in the gp41 post fusion conformation. Compared to the previously described HR1-specific mAb D5, HK20 approaches its epitope with a different angle which might favor epitope access and thus contribute to its higher neutralization breadth and potency. Comparison of the neutralization activities of HK20 IgG, Fab and scFv employing both single cycle and multiple cycle neutralization assays revealed much higher potencies for the smaller Fab and scFv over IgG, implying that the target site is difficult to access for complete antibodies. Nevertheless, two thirds of sera from HIV-1 infected individuals contain significant titers of HK20-inhibiting antibodies. The breadth of neutralization of primary isolates across all clades, the higher potencies for C-clade viruses and the targeting of a distinct site as compared to the fusion inhibitor T-20 demonstrate the potential of HK20 scFv as a therapeutic tool.

  9. Binding Affinity, Specificity and Comparative Biodistribution of the Parental Murine Monoclonal Antibody MX35 (Anti-NaPi2b) and Its Humanized Version Rebmab200

    DEFF Research Database (Denmark)

    Lindegren, Sture; Andrade, Luciana N S; Bäck, Tom

    2015-01-01

    The aim of this preclinical study was to evaluate the characteristics of the monoclonal antibody Rebmab200, which is a humanized version of the ovarian-specific murine antibody MX35. This investigation contributes to the foundation for future clinical α-radioimmunotherapy of minimal residual...

  10. A Cross-Reactive Human Single-Chain Antibody for Detection of Major Fish Allergens, Parvalbumins, and Identification of a Major IgE-Binding Epitope.

    Directory of Open Access Journals (Sweden)

    Merima Bublin

    Full Text Available Fish allergy is associated with moderate to severe IgE-mediated reactions to the calcium binding parvalbumins present in fish muscle. Allergy to multiple fish species is caused by parvalbumin-specific cross-reactive IgE recognizing conserved epitopes. In this study, we aimed to produce cross-reactive single chain variable fragment (scFv antibodies for the detection of parvalbumins in fish extracts and the identification of IgE epitopes. Parvalbumin-specific phage clones were isolated from the human ETH-2 phage display library by three rounds of biopanning either against cod parvalbumin or by sequential biopanning against cod (Gad m 1, carp (Cyp c 1 and rainbow trout (Onc m 1 parvalbumins. While biopanning against Gad m 1 resulted in the selection of clones specific exclusively for Gad m 1, the second approach resulted in the selection of clones cross-reacting with all three parvalbumins. Two clones, scFv-gco9 recognizing all three parvalbumins, and scFv-goo8 recognizing only Gad m 1 were expressed in the E. coli non-suppressor strain HB2151 and purified from the periplasm. scFv-gco9 showed highly selective binding to parvalbumins in processed fish products such as breaded cod sticks, fried carp and smoked trout in Western blots. In addition, the scFv-gco9-AP produced as alkaline phosphatase fusion protein, allowed a single-step detection of the parvalbumins. In competitive ELISA, scFv-gco9 was able to inhibit binding of IgE from fish allergic patients' sera to all three β-parvalbumins by up to 80%, whereas inhibition by scFv-goo8 was up to 20%. 1H/15N HSQC NMR analysis of the rGad m 1:scFv-gco9 complex showed participation of amino acid residues conserved among these three parvalbumins explaining their cross-reactivity on a molecular level. In this study, we have demonstrated an approach for the selection of cross-reactive parvalbumin-specific antibodies that can be used for allergen detection and for mapping of conserved epitopes.

  11. A Cross-Reactive Human Single-Chain Antibody for Detection of Major Fish Allergens, Parvalbumins, and Identification of a Major IgE-Binding Epitope.

    Science.gov (United States)

    Bublin, Merima; Kostadinova, Maria; Fuchs, Julian E; Ackerbauer, Daniela; Moraes, Adolfo H; Almeida, Fabio C L; Lengger, Nina; Hafner, Christine; Ebner, Christof; Radauer, Christian; Liedl, Klaus R; Valente, Ana Paula; Breiteneder, Heimo

    2015-01-01

    Fish allergy is associated with moderate to severe IgE-mediated reactions to the calcium binding parvalbumins present in fish muscle. Allergy to multiple fish species is caused by parvalbumin-specific cross-reactive IgE recognizing conserved epitopes. In this study, we aimed to produce cross-reactive single chain variable fragment (scFv) antibodies for the detection of parvalbumins in fish extracts and the identification of IgE epitopes. Parvalbumin-specific phage clones were isolated from the human ETH-2 phage display library by three rounds of biopanning either against cod parvalbumin or by sequential biopanning against cod (Gad m 1), carp (Cyp c 1) and rainbow trout (Onc m 1) parvalbumins. While biopanning against Gad m 1 resulted in the selection of clones specific exclusively for Gad m 1, the second approach resulted in the selection of clones cross-reacting with all three parvalbumins. Two clones, scFv-gco9 recognizing all three parvalbumins, and scFv-goo8 recognizing only Gad m 1 were expressed in the E. coli non-suppressor strain HB2151 and purified from the periplasm. scFv-gco9 showed highly selective binding to parvalbumins in processed fish products such as breaded cod sticks, fried carp and smoked trout in Western blots. In addition, the scFv-gco9-AP produced as alkaline phosphatase fusion protein, allowed a single-step detection of the parvalbumins. In competitive ELISA, scFv-gco9 was able to inhibit binding of IgE from fish allergic patients' sera to all three β-parvalbumins by up to 80%, whereas inhibition by scFv-goo8 was up to 20%. 1H/15N HSQC NMR analysis of the rGad m 1:scFv-gco9 complex showed participation of amino acid residues conserved among these three parvalbumins explaining their cross-reactivity on a molecular level. In this study, we have demonstrated an approach for the selection of cross-reactive parvalbumin-specific antibodies that can be used for allergen detection and for mapping of conserved epitopes.

  12. The 1.7 Å X-ray crystal structure of the porcine factor VIII C2 domain and binding analysis to anti-human C2 domain antibodies and phospholipid surfaces.

    Directory of Open Access Journals (Sweden)

    Caileen M Brison

    Full Text Available The factor VIII C2 domain is essential for binding to activated platelet surfaces as well as the cofactor activity of factor VIII in blood coagulation. Inhibitory antibodies against the C2 domain commonly develop following factor VIII replacement therapy for hemophilia A patients, or they may spontaneously arise in cases of acquired hemophilia. Porcine factor VIII is an effective therapeutic for hemophilia patients with inhibitor due to its low cross-reactivity; however, the molecular basis for this behavior is poorly understood. In this study, the X-ray crystal structure of the porcine factor VIII C2 domain was determined, and superposition of the human and porcine C2 domains demonstrates that most surface-exposed differences cluster on the face harboring the "non-classical" antibody epitopes. Furthermore, antibody-binding results illustrate that the "classical" 3E6 antibody can bind both the human and porcine C2 domains, although the inhibitory titer to human factor VIII is 41 Bethesda Units (BU/mg IgG versus 0.8 BU/mg IgG to porcine factor VIII, while the non-classical G99 antibody does not bind to the porcine C2 domain nor inhibit porcine factor VIII activity. Further structural analysis of differences between the electrostatic surface potentials suggest that the C2 domain binds to the negatively charged phospholipid surfaces of activated platelets primarily through the 3E6 epitope region. In contrast, the G99 face, which contains residue 2227, should be distal to the membrane surface. Phospholipid binding assays indicate that both porcine and human factor VIII C2 domains bind with comparable affinities, and the human K2227A and K2227E mutants bind to phospholipid surfaces with similar affinities as well. Lastly, the G99 IgG bound to PS-immobilized factor VIII C2 domain with an apparent dissociation constant of 15.5 nM, whereas 3E6 antibody binding to PS-bound C2 domain was not observed.

  13. Detection of human antibodies binding with smooth and rough LPSs from Proteus mirabilis O3 strains S1959, R110, R45.

    Science.gov (United States)

    Gleńska-Olender, J; Durlik, K; Konieczna, I; Kowalska, P; Gawęda, J; Kaca, W

    2017-11-01

    Bacteria of the genus Proteus of the family Enterobacteriaceae are facultative human pathogens responsible mainly for urinary tract and wound infections, bacteremia and the development of rheumatoid arthritis (RA). We have analyzed and compared by ELISA the titer of antibodies in plasmas of healthy individuals and in sera of rheumatoid arthritis patients recognizing a potential host cross-reactive epitope (lysine-galacturonic acid epitopes) present in Proteus lipopolysaccharide (LPS). In our experiments LPSs isolated from two mutants of smooth Proteus mirabilis 1959 (O3), i.e. strains R110 and R45, were used. R110 (Ra type mutant) is lacking the O-specific polysaccharide, but possesses a complete core oligosaccharide, while R45 (Re type) has a reduced core oligosaccharide and contains two 3-deoxy-D-manno-oct-2-ulosonic acid residues and one of 4-amino-4-deoxy-L-arabinopyranose residues. Titer of P. mirabilis S1959 LPS-specific-antibodies increased with the age of blood donors. RA and blood donors' sera contained antibodies against S and Ra and Re type of P. mirabilis O3 LPSs. Antibodies recognizing lysine-galacturonic acid epitopes of O3 LPS were detected by ELISA in some plasmas of healthy individuals and sera of rheumatoid arthritis patients. RA patients antibodies reacting with P. mirabilis S1959 S and R LPSs may indicate a potential role of anti-LPS antibodies in molecular mimicry in RA diseases.

  14. Binding of antibodies to the extractable nuclear antigens SS-A/Ro and SS-B/La is induced on the surface of human keratinocytes by ultraviolet light (UVL): Implications for the pathogenesis of photosensitive cutaneous lupus

    International Nuclear Information System (INIS)

    Furukawa, F.; Kashihara-Sawami, M.; Lyons, M.B.; Norris, D.A.

    1990-01-01

    Autoantibodies to the non-histone nucleoprotein antigens SS-A/Ro, SS-B/La, and RNP are highly associated with photosensitive cutaneous lupus erythematosus (LE). In order to better understand the potential mechanisms of ultraviolet (UV) light on photosensitivity in patients with cutaneous LE, we designed immunopathologic in vitro and in vivo experiments to evaluate the effects of UV on the binding of such autoantibodies to the surface of human keratinocytes, one major target of immunologic damage in photosensitive LE. Short-term 2% paraformaldehyde fixation of suspensions of cultured human keratinocytes previously incubated with monospecific antiserum probes enabled the detection of ENA expression on the cell surface by flow-cytometry analysis. UVB light (280-320 nm) induced the binding of monospecific antibody probes for SS-A/Ro and SS-B/La on keratinocytes in a dose-dependent pattern with maximal induction observed at the dose of 200 mJ/cm2 UVB. Binding of SS-A/Ro, SS-B/La, and RNP antibody was augmented strongly, but binding of anti-Sm was very weak. In contrast, UVA (320-400 nm) light had no effect on the induction of binding of these antibody probes. Identical results were seen by standard immunofluorescence techniques. Hydroxyurea-treated keratinocytes showed similar induction of those antigens by UVB irradiation, which suggested that ENA expression on cultured keratinocytes by UVB were cell-cycle independent. Tunicamycin, an inhibitor of glycosylation of proteins, reduced UVB light effect on the SS-A/Ro and SS-B/La antigen's expression. These in vitro FACS analyses revealed that ENA augmentation on the keratinocyte cell surface was dose dependent, UVB dependent, glycosylation dependent, and cell-cycle independent. In vivo ENA augmentation on the keratinocyte surface was examined in suction blister epidermal roofs

  15. New perspectives on recombinant human antibodies

    NARCIS (Netherlands)

    J. de Kruif (John); A.-R. van der Vuurst de Vries (Anne); L. Cilenti (L.); E. Boel (E.); W. van Ewijk (Willem); T. Logtenberg (Ton)

    1996-01-01

    textabstractThe limited potential of murine monoclonal antibodies for human immunotherapy has driven recent progress in recombinant antibody technology. Here, de Kruif and colleagues report on advances in the development and use of phage-antibody-display libraries.

  16. Radioimmunological proof of thyroglobulin antibodies in humans by the use of a double antibody method

    International Nuclear Information System (INIS)

    Waller, V.

    1982-01-01

    Thyroid antibodies, especially thyroglobulin antibodies, allow themselves to be proven with the double antibody method, in competitive radio binding assays and with the solid phase technique. These methods offer advantages relative to sensitivity and quantifiability. In this work a sensitive radioimmunoassay as a double antibody method was worked out whereby a 125 I-thyroglobulin/thyroglobulin antibody immune complex was precipitated out using anti-human immunoglobulin. The measured results from the radioimmunoassay show a good correlation with the results of the immune histological findings. A high to very high Tg antibody level occurs with autoimmune thyroiditis (80%), primary hypothyroidism (74%) and hyperthyroidism (70%). The control values with healthy people came to less than 5% specific binding. In correlation with the results of other authors this method is advantageous relative to test start and evaluation procedures. (orig.) [de

  17. Mapping of monoclonal antibody- and receptor-binding domains on human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) using a surface plasmon resonance-based biosensor.

    Science.gov (United States)

    Laricchia-Robbio, L; Liedberg, B; Platou-Vikinge, T; Rovero, P; Beffy, P; Revoltella, R P

    1996-10-01

    An automated surface plasmon resonance (SPR)-based biosensor system has been used for mapping antibody and receptor-binding regions on the recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) molecule. A rabbit antimouse IgG1-Fc antibody (RAM.Fc) was coupled to an extended carboxymethylated-hydrogel matrix attached to a gold surface in order to capture an anti-rhGM-CSF monoclonal antibody (MAb) injected over the sensing layer. rhGM-CSF was subsequently injected and allowed to bind to this antibody. Multisite binding assays were then performed, by flowing sequentially other antibodies and peptides over the surface, and the capacity of the latter to interact with the entrapped rhGM-CSF in a multimolecular complex was monitored in real time with SPR. Eleven MAb (all IgG1K), were analyzed: respectively, four antipeptide MAb raised against three distinct epitopes of the cytokine (two clones against residues 14-24, that includes part of the first alpha-helix toward the N-terminal region; one clone against peptide 30-41, an intrahelical loop; and one clone against residues 79-91, including part of the third alpha-helix) and seven antiprotein MAbs raised against the entire rhGM-CSF, whose target native epitopes are still undetermined. In addition, the binding capacity to rhGM-CSF of a synthetic peptide, corresponding to residues 238-254 of the extracellular human GM-CSF receptor alpha-chain, endowed with rhGM-CSF binding activity, was tested. The results from experiments performed with the biosensor were compared with those obtained by a sandwich enzyme-linked immunosorbent assay (ELISA), using the same reagents. The features of the biosensor technology (fully automated, measure in real time, sharpened yes/no response, less background disturbances, no need for washing step or labeling of the reagent) offered several advantages in these studies of MAb immunoreactivity and epitope mapping, giving a much better resolution and enabling more distinct

  18. A Human Antibody That Binds to the Sixth Ig-Like Domain of VCAM-1 Blocks Lung Cancer Cell Migration In Vitro

    Directory of Open Access Journals (Sweden)

    Mi Ra Kim

    2017-03-01

    Full Text Available Vascular cell adhesion molecule-1 (VCAM-1 is closely associated with tumor progression and metastasis. However, the relevance and role of VCAM-1 in lung cancer have not been clearly elucidated. In this study, we found that VCAM-1 was highly overexpressed in lung cancer tissue compared with that of normal lung tissue, and high VCAM-1 expression correlated with poor survival in lung cancer patients. VCAM-1 knockdown reduced migration of A549 human lung cancer cells into Matrigel, and competitive blocking experiments targeting the Ig-like domain 6 of VCAM-1 (VCAM-1-D6 demonstrated that the VCAM-1-D6 domain was critical for VCAM-1 mediated A549 cell migration into Matrigel. Next, we developed a human monoclonal antibody specific to human and mouse VCAM-1-D6 (VCAM-1-D6 huMab, which was isolated from a human synthetic antibody library using phage display technology. Finally, we showed that VCAM-1-D6 huMab had a nanomolar affinity for VCAM-1-D6 and that it potently suppressed the migration of A549 and NCI-H1299 lung cancer cell lines into Matrigel. Taken together, these results suggest that VCAM-1-D6 is a key domain for regulating VCAM-1-mediated lung cancer invasion and that our newly developed VCAM-1-D6 huMab will be a useful tool for inhibiting VCAM-1-expressing lung cancer cell invasion.

  19. Monoclonal Antibodies, Derived from Humans Vaccinated with the RV144 HIV Vaccine Containing the HVEM Binding Domain of Herpes Simplex Virus (HSV) Glycoprotein D, Neutralize HSV Infection, Mediate Antibody-Dependent Cellular Cytotoxicity, and Protect Mice from Ocular Challenge with HSV-1.

    Science.gov (United States)

    Wang, Kening; Tomaras, Georgia D; Jegaskanda, Sinthujan; Moody, M Anthony; Liao, Hua-Xin; Goodman, Kyle N; Berman, Phillip W; Rerks-Ngarm, Supachai; Pitisuttithum, Punnee; Nitayapan, Sorachai; Kaewkungwal, Jaranit; Haynes, Barton F; Cohen, Jeffrey I

    2017-10-01

    The RV144 HIV vaccine trial included a recombinant HIV glycoprotein 120 (gp120) construct fused to a small portion of herpes simplex virus 1 (HSV-1) glycoprotein D (gD) so that the first 40 amino acids of gp120 were replaced by the signal sequence and the first 27 amino acids of the mature form of gD. This region of gD contains most of the binding site for HVEM, an HSV receptor important for virus infection of epithelial cells and lymphocytes. RV144 induced antibodies to HIV that were partially protective against infection, as well as antibodies to HSV. We derived monoclonal antibodies (MAbs) from peripheral blood B cells of recipients of the RV144 HIV vaccine and showed that these antibodies neutralized HSV-1 infection in cells expressing HVEM, but not the other major virus receptor, nectin-1. The MAbs mediated antibody-dependent cellular cytotoxicity (ADCC), and mice that received the MAbs and were then challenged by corneal inoculation with HSV-1 had reduced eye disease, shedding, and latent infection. To our knowledge, this is the first description of MAbs derived from human recipients of a vaccine that specifically target the HVEM binding site of gD. In summary, we found that monoclonal antibodies derived from humans vaccinated with the HVEM binding domain of HSV-1 gD (i) neutralized HSV-1 infection in a cell receptor-specific manner, (ii) mediated ADCC, and (iii) reduced ocular disease in virus-infected mice. IMPORTANCE Herpes simplex virus 1 (HSV-1) causes cold sores and neonatal herpes and is a leading cause of blindness. Despite many trials, no HSV vaccine has been approved. Nectin-1 and HVEM are the two major cellular receptors for HSV. These receptors are expressed at different levels in various tissues, and the role of each receptor in HSV pathogenesis is not well understood. We derived human monoclonal antibodies from persons who received the HIV RV144 vaccine that contained the HVEM binding domain of HSV-1 gD fused to HIV gp120. These antibodies were

  20. 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.

  1. Beyond Antibodies as Binding Partners: The Role of Antibody Mimetics in Bioanalysis.

    Science.gov (United States)

    Yu, Xiaowen; Yang, Yu-Ping; Dikici, Emre; Deo, Sapna K; Daunert, Sylvia

    2017-06-12

    The emergence of novel binding proteins or antibody mimetics capable of binding to ligand analytes in a manner analogous to that of the antigen-antibody interaction has spurred increased interest in the biotechnology and bioanalytical communities. The goal is to produce antibody mimetics designed to outperform antibodies with regard to binding affinities, cellular and tumor penetration, large-scale production, and temperature and pH stability. The generation of antibody mimetics with tailored characteristics involves the identification of a naturally occurring protein scaffold as a template that binds to a desired ligand. This scaffold is then engineered to create a superior binder by first creating a library that is then subjected to a series of selection steps. Antibody mimetics have been successfully used in the development of binding assays for the detection of analytes in biological samples, as well as in separation methods, cancer therapy, targeted drug delivery, and in vivo imaging. This review describes recent advances in the field of antibody mimetics and their applications in bioanalytical chemistry, specifically in diagnostics and other analytical methods.

  2. Distance between two binding sites of the same antibody molecule

    International Nuclear Information System (INIS)

    Cser, L.; Gladkikh, I.A.; Ostanevich, Y.M.; Franek, F.; Novotny, J.; Nezlin, R.S.

    1978-01-01

    Neutron small-angle scattering experiments are reported, aimed at determining the distance between the two binding sites of the same antibody molecule employing complexes of anti-Dnp antibody with an antigenically univalent, high molecular weight ligand. Although the distance values could be determined only with a large statistical error, the data allowed the conclusion that the geometrical parameters of the complexes formed with the early (i.e., precipitating) antibody are significantly different from those of the complexes formed with the late (i.e, non-precipitating) antibody. The data suggest that the precipitating antibody complexed with a high molecular weight antigen assumes an extended shape with an antigen to antigen distance of 35.8 +- 1.3 nm. (Auth.)

  3. A glycoconjugate antigen based on the recognition motif of a broadly neutralizing human immunodeficiency virus antibody, 2G12, is immunogenic but elicits antibodies unable to bind to the self glycans of gp120

    DEFF Research Database (Denmark)

    Astronomo, Rena D; Lee, Hing-Ken; Scanlan, Christopher N

    2008-01-01

    The glycan shield of human immunodeficiency virus type 1 (HIV-1) gp120 contributes to viral evasion from humoral immune responses. However, the shield is recognized by the HIV-1 broadly neutralizing antibody (Ab), 2G12, at a relatively conserved cluster of oligomannose glycans. The discovery of 2G......12 raises the possibility that a carbohydrate immunogen may be developed that could elicit 2G12-like neutralizing Abs and contribute to an AIDS vaccine. We have previously dissected the fine specificity of 2G12 and reported that the synthetic tetramannoside (Man(4)) that corresponds to the D1 arm...

  4. Human antibody technology and the development of antibodies against cytomegalovirus.

    Science.gov (United States)

    Ohlin, Mats; Söderberg-Nauclér, Cecilia

    2015-10-01

    Cytomegalovirus (CMV) is a virus that causes chronic infections in a large set of the population. It may cause severe disease in immunocompromised individuals, is linked to immunosenescence and implied to play an important role in the pathogenesis of cardiovascular diseases and cancer. Modulation of the immune system's abilities to manage the virus represent a highly viable therapeutic option and passive immunotherapy with polyclonal antibody preparations is already in clinical use. Defined monoclonal antibodies offer many advantages over polyclonal antibodies purified from serum. Human CMV-specific monoclonal antibodies have consequently been thoroughly investigated with respect to their potential in the treatment of diseases caused by CMV. Recent advances in human antibody technology have substantially expanded the breadth of antibodies for such applications. This review summarizes the fundamental basis for treating CMV disease by use of antibodies, the basic technologies to be used to develop such antibodies, and relevant human antibody specificities available to target this virus. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Mechanism of Binding to Ebola Virus Glycoprotein by the ZMapp, ZMAb, and MB-003 Cocktail Antibodies

    OpenAIRE

    Davidson, Edgar; Bryan, Christopher; Fong, Rachel H.; Barnes, Trevor; Pfaff, Jennifer M.; Mabila, Manu; Rucker, Joseph B.; Doranz, Benjamin J.

    2015-01-01

    Cocktails of monoclonal antibodies (MAbs) that target the surface glycoprotein (GP) of Ebola virus (EBOV) are effective in nonhuman primate models and have been used under emergency compassionate-treatment protocols in human patients. However, the amino acids that form the detailed binding epitopes for the MAbs in the ZMapp, ZMAb, and the related MB-003 cocktails have yet to be identified. Other binding properties that define how each MAb functionally interacts with GP—such as affinity, epito...

  6. Recombinant protein truncation strategy for inducing bactericidal antibodies to the macrophage infectivity potentiator protein of Neisseria meningitidis and circumventing potential cross-reactivity with human FK506-binding proteins.

    Science.gov (United States)

    Bielecka, Magdalena K; Devos, Nathalie; Gilbert, Mélanie; Hung, Miao-Chiu; Weynants, Vincent; Heckels, John E; Christodoulides, Myron

    2015-02-01

    A recombinant macrophage infectivity potentiator (rMIP) protein of Neisseria meningitidis induces significant serum bactericidal antibody production in mice and is a candidate meningococcal vaccine antigen. However, bioinformatics analysis of MIP showed some amino acid sequence similarity to human FK506-binding proteins (FKBPs) in residues 166 to 252 located in the globular domain of the protein. To circumvent the potential concern over generating antibodies that could recognize human proteins, we immunized mice with recombinant truncated type I rMIP proteins that lacked the globular domain and the signal leader peptide (LP) signal sequence (amino acids 1 to 22) and contained the His purification tag at either the N or C terminus (C-term). The immunogenicity of truncated rMIP proteins was compared to that of full (i.e., full-length) rMIP proteins (containing the globular domain) with either an N- or C-terminal His tag and with or without the LP sequence. By comparing the functional murine antibody responses to these various constructs, we determined that C-term His truncated rMIP (-LP) delivered in liposomes induced high levels of antibodies that bound to the surface of wild-type but not Δmip mutant meningococci and showed bactericidal activity against homologous type I MIP (median titers of 128 to 256) and heterologous type II and III (median titers of 256 to 512) strains, thereby providing at least 82% serogroup B strain coverage. In contrast, in constructs lacking the LP, placement of the His tag at the N terminus appeared to abrogate bactericidal activity. The strategy used in this study would obviate any potential concerns regarding the use of MIP antigens for inclusion in bacterial vaccines. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  7. Recombinant Protein Truncation Strategy for Inducing Bactericidal Antibodies to the Macrophage Infectivity Potentiator Protein of Neisseria meningitidis and Circumventing Potential Cross-Reactivity with Human FK506-Binding Proteins

    Science.gov (United States)

    Bielecka, Magdalena K.; Devos, Nathalie; Gilbert, Mélanie; Hung, Miao-Chiu; Weynants, Vincent; Heckels, John E.

    2014-01-01

    A recombinant macrophage infectivity potentiator (rMIP) protein of Neisseria meningitidis induces significant serum bactericidal antibody production in mice and is a candidate meningococcal vaccine antigen. However, bioinformatics analysis of MIP showed some amino acid sequence similarity to human FK506-binding proteins (FKBPs) in residues 166 to 252 located in the globular domain of the protein. To circumvent the potential concern over generating antibodies that could recognize human proteins, we immunized mice with recombinant truncated type I rMIP proteins that lacked the globular domain and the signal leader peptide (LP) signal sequence (amino acids 1 to 22) and contained the His purification tag at either the N or C terminus (C-term). The immunogenicity of truncated rMIP proteins was compared to that of full (i.e., full-length) rMIP proteins (containing the globular domain) with either an N- or C-terminal His tag and with or without the LP sequence. By comparing the functional murine antibody responses to these various constructs, we determined that C-term His truncated rMIP (−LP) delivered in liposomes induced high levels of antibodies that bound to the surface of wild-type but not Δmip mutant meningococci and showed bactericidal activity against homologous type I MIP (median titers of 128 to 256) and heterologous type II and III (median titers of 256 to 512) strains, thereby providing at least 82% serogroup B strain coverage. In contrast, in constructs lacking the LP, placement of the His tag at the N terminus appeared to abrogate bactericidal activity. The strategy used in this study would obviate any potential concerns regarding the use of MIP antigens for inclusion in bacterial vaccines. PMID:25452551

  8. Neutralisation and binding of VHS virus by monovalent antibody fragments

    DEFF Research Database (Denmark)

    Cupit, P.M.; Lorenzen, Niels; Strachan, G.

    2001-01-01

    We have previously reported the cloning and characterisation of the heavy and light chain variable domain genes encoding three monoclonal antibodies (Mabs) that bind viral haemorrhagic septicaemia virus (VHSV). Two of these antibodies, 3F1H10 and 3F1A2 both neutralised the virus though 3F1A2...... appeared to recognise a broader range of virus isolates. The variable domains of these two antibodies differ by only four residues (Lorenzen et al., 2000a. Fish Shellfish Immunol. 10, 129-142). To further study the mechanism of neutralisation, Fab fragments as well as a series of recombinant bacterial...... single chain antibody (scAb) fragments were generated from the three anti-VHSV Mabs and their variable domain genes, respectively. Fabs and scAbs derived from the neutralising Mabs were both able to neutralise the VHSV type 1 isolate DK-F1. In addition, a series of scAb fragments were produced using...

  9. Binding of monoclonal antibody to protein antigen in fluid phase or bound to solid supports

    Energy Technology Data Exchange (ETDEWEB)

    Kennel, S J

    1982-01-01

    Rat monoclonal antibody (MoAb) to fragment D (FgD) of human fibrinogen was used to characterize the direct binding of antibody to protein in solution or bound to solid supports. Purified IgG, F(ab')/sub 2/ and Fab' were prepared from ascites fluid of hybridoma 104-14B which is a fusion product of spleen cells from a rat immunized with FgD and the mouse myeloma cell line, P3-X63-Ag8. Two-dimensional electrophoresis of radioiodinated antibody preparations demonstrated the presence of hybrid immunoglobulin molecules, but only structures having rat heavy and rat light chains had active antibody combinig sites. The affinity constant for IgG as well as F(ab')/sub 2/ and Fab', 6x10/sup 9/ M/sup -1/, was identical when tested using fluid phase antigen (/sup 125/I-labeled FgD). Affinity constants determined for direct binding of iodinated IgG using FgD immobilized on solid supports showed a slight dependence on the antigen concentration used in the measurement. These values ranged from 0.5x10/sup 9/ M/sup -1/ at high antigen concentrations (1.3x10/sup -7/ M) to 9x10/sup 9/ M/sup -1/ at low antigen concentration (1.3x10/sup -10/ M). Binding constants for F(ab')/sub 2/ and Fab' gave similar results indicating that binding was homogeneous and univalent. The capacity of solid state antigen to bind antibody varied with the method used to bind FgD to the solid support. FgD bound directly to polystyrene plates was least efficient at binding labeled antibody; FgD bound to plates through intermediate carriers poly(L-lysine) was only slightly more efficient, while antigen bound to Sepharose beads by cyanogen bromide activation was the most active.

  10. Specific binding of beta-endorphin to normal human erythrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Chenet, B.; Hollis, V. Jr.; Kang, Y.; Simpkins, C.

    1986-03-05

    Beta-endorphin (BE) exhibits peripheral functions which may not be mediated by interactions with receptors in the brain. Recent studies have demonstrated binding of BE to both opioid and non-opioid receptors on lymphocytes and monocytes. Abood has reported specific binding of /sup 3/H-dihydromorphine in erythrocytes. Using 5 x 10/sup -11/M /sup 125/I-beta-endorphin and 10/sup -5/M unlabeled BE, they have detected 50% specific binding to human erythrocytes. This finding is supported by results from immunoelectron microscopy using rabbit anti-BE antibody and biotinylated secondary antibody with avidin-biotin complexes horseradish peroxidase. Binding is clearly observed and is confined to only one side of the cells. Conclusions: (1) BE binding to human erythrocytes was demonstrated by radioreceptor assay and immunoelectron microscopy, and (2) BE binding sites exist on only one side of the cells.

  11. Probing cocaine-antibody interactions in buffer and human serum.

    Directory of Open Access Journals (Sweden)

    Muthu Ramakrishnan

    Full Text Available Despite progress in cocaine immunotherapy, the kinetic and thermodynamic properties of antibodies which bind to cocaine and its metabolites are not well understood. It is also not clear how the interactions between them differ in a complex matrix such as the serum present in the human body. In the present study, we have used microscale thermophoresis (MST, isothermal titration calorimetry (ITC, and surface plasmon resonance (SPR we have evaluated the affinity properties of a representative mouse monoclonal (mAb08 as well as those of polyclonal antibodies purified from vaccinated mouse and human patient serum.MST analysis of fluorescently tagged mAb08 binding to cocaine reveals an approximately 15 fold decrease in its equilibrium dissociation constant in 20-50% human serum compared with that in saline buffer. A similar trend was also found using enriched polyclonal antibodies purified from vaccinated mice and patient serum, for which we have used fluorescently tagged bovine serum albumin conjugated to succinyl norcocaine (BSA-SNC. This conjugate closely mimics both cocaine and the hapten used to raise these antibodies. The ITC data also revealed that cocaine has a moderate affinity of about 2 µM to 20% human serum and very little interaction with human serum albumin or nonspecific human IgG at that concentration range. In a SPR inhibition experiment, the binding of mAb08 to immobilized BSA-SNC was inhibited by cocaine and benzoylecgonine in a highly competitive manner, whereas the purified polyclonal antibodies from vaccinated humans and mice, revealed preferential selectivity to pharmacologically active cocaine but not to the inactive metabolite benzoylecgonine. We have also developed a simple binding model to simulate the challenges associated with cocaine immunotherapy using the variable quantitative and kinetic properties of the antibodies.High sensitivity calorimetric determination of antibody binding to cocaine and its metabolites provide

  12. Antibodies to lactalbumin interfere with its radioimmunoassay in human plasma

    Energy Technology Data Exchange (ETDEWEB)

    Stevens, U; Laurence, D J.R. [Royal Marsden Hospital, London (UK); Ormerod, M G [Institute of Cancer Research, Sutton (UK). Surrey Branch

    1978-01-01

    Two radioimmunoassays for human lactalbumin have been established using a rabbit antiserum. One assay uses a second antibody to separate bound from free label; the other uses polyethylene glycol to precipitate gamma globulin non-specifically. It is confirmed that about half the normal human population have a substance in their blood which inhibits the binding of lactalbumin to the rabbit antibody. Comparison of the two assays has demonstrated that this material is not lactalbumin but a naturally occurring antibody. It is shown that it is in the IgG fraction of human plasma and is probably a cross-reacting antibody to bovine lactalbumin. None out of fifteen males and fourteen out of fifty eight non-pregnant, non-lacatating females had low levels of lactalbumin in their blood (0.6-2.0 ng/ml). The assay could not detect a statistically significant difference between normal women and those with either benign breast disease or metastatic mammary carcinoma.

  13. 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.

  14. Immunoscintigraphy of metastases with radiolabelled human antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Al-Azzawi, F.; Smith, J.; Stimson, W.H.

    1987-02-28

    It was concluded that Epstein-Barr virus transformation of committed lymphocytes offers great potential in the production of antitumour antibodies of human origin. An outline case report is presented where the human I/sup 131/ labelled antibody was used as a targeting agent to delineate the extent of secondary growth in the liver. (U.K.).

  15. Conference scene: progress with promising human antibodies.

    Science.gov (United States)

    Larrick, James W

    2012-03-01

    Antibodies and antibody-based therapeutics have become big business, with annual sales over US$50 billion, accounting for >6% of worldwide pharmaceutical revenues. Ten molecules have blockbuster status (>US$1 billion), with six generating more than US$6 billion in sales. In excess of 300 products based on this rapidly maturing technology are in clinical trials. The generation and manufacture of human antibodies is now routine, although the cost of goods remains an issue. Optimizing combinations of antibodies with other therapeutics (e.g., chemotherapy) is a major short-term goal, while target validation and product differentiation remain significant hurdles if growth is to continue. Some of the notable highlights of the recent 16th International Conference on Human Antibodies and Hybridomas meeting in Cannes, France are described below. The conference was sponsored by the international journal Human Antibodies, in association with the Integrative Medical Sciences Association (IMSA). The Program Chairman was Professor Mark Glassy, IMSA, San Diego, CA, USA.

  16. Dynamics behind affinity maturation of an anti-HCMV antibody family influencing antigen binding

    KAUST Repository

    Di Palma, Francesco; Tramontano, Anna

    2017-01-01

    The investigation of antibody affinity maturation and its effects on antigen binding is important with respect to understanding the regulation of the immune response. To shed light on this crucial process, we analyzed two Igs neutralizing the human cytomegalovirus: the primary germline antibody M2J1 and its related mature antibody 8F9. Both antibodies target the AD-2S1 epitope of the gB envelope protein and are considered to establish similar interactions with the cognate antigen. We used molecular dynamics simulations to understand the effect of mutations on the antibody–antigen interactions. The results provide a qualitative explanation for the increased 8F9 peptide affinity compared with that of M2J1. The emerging atomistic-detailed description of these complexes reveals the molecular effects of the somatic hypermutations occurring during affinity maturation.

  17. Dynamics behind affinity maturation of an anti-HCMV antibody family influencing antigen binding

    KAUST Repository

    Di Palma, Francesco

    2017-08-03

    The investigation of antibody affinity maturation and its effects on antigen binding is important with respect to understanding the regulation of the immune response. To shed light on this crucial process, we analyzed two Igs neutralizing the human cytomegalovirus: the primary germline antibody M2J1 and its related mature antibody 8F9. Both antibodies target the AD-2S1 epitope of the gB envelope protein and are considered to establish similar interactions with the cognate antigen. We used molecular dynamics simulations to understand the effect of mutations on the antibody–antigen interactions. The results provide a qualitative explanation for the increased 8F9 peptide affinity compared with that of M2J1. The emerging atomistic-detailed description of these complexes reveals the molecular effects of the somatic hypermutations occurring during affinity maturation.

  18. Passive vaccination with a human monoclonal antibody: generation of antibodies and studies for efficacy in Bacillus anthracis infections.

    Science.gov (United States)

    vor dem Esche, Ulrich; Huber, Maria; Zgaga-Griesz, Andrea; Grunow, Roland; Beyer, Wolfgang; Hahn, Ulrike; Bessler, Wolfgang G

    2011-07-01

    A major difficulty in creating human monoclonal antibodies is the lack of a suitable myeloma cell line to be used for fusion experiments. In order to create fully human monoclonal antibodies for passive immunization, the human mouse heteromyeloma cell line CB-F7 was evaluated. Using this cell line, we generated human monoclonal antibodies against Bacillus anthracis toxin components. Antibodies against protective antigen (PA) and against lethal factor (LF) were obtained using peripheral blood lymphocytes (PBLs) from persons vaccinated with the UK anthrax vaccine. PBL were fused with the cell line CB-F7. We obtained several clones producing PA specific Ig and one clone (hLF1-SAN) producing a monoclonal antibody (hLF1) directed against LF. The LF binding antibody was able to neutralize Anthrax toxin activity in an in vitro neutralization assay, and preliminary in vivo studies in mice also indicated a trend towards protection. We mapped the epitope of the antibody binding to LF by dot blot analysis and ELIFA using 80 synthetic LF peptides of 20 amino acid lengths with an overlapping range of 10 amino acids. Our results suggest the binding of the monoclonal antibody to the peptide regions 121-150 or 451-470 of LF. The Fab-fragment of the antibody hLF1 was cloned in Escherichia coli and could be useful as part of a fully human monoclonal antibody for the treatment of Anthrax infections. In general, our studies show the applicability of the CB-F7 line to create fully human monoclonal antibodies for vaccination. Copyright © 2010 Elsevier GmbH. All rights reserved.

  19. A single dose of a neuron-binding human monoclonal antibody improves brainstem NAA concentrations, a biomarker for density of spinal cord axons, in a model of progressive multiple sclerosis.

    Science.gov (United States)

    Wootla, Bharath; Denic, Aleksandar; Watzlawik, Jens O; Warrington, Arthur E; Rodriguez, Moses

    2015-04-29

    Intracerebral infection of susceptible mouse strains with Theiler's murine encephalomyelitis virus (TMEV) results in chronic demyelinating disease with progressive axonal loss and neurologic dysfunction similar to progressive forms of multiple sclerosis (MS). We previously showed that as the disease progresses, a marked decrease in brainstem N-acetyl aspartate (NAA; metabolite associated with neuronal integrity) concentrations, reflecting axon health, is measured. We also demonstrated stimulation of neurite outgrowth by a neuron-binding natural human antibody, IgM12. Treatment with either the serum-derived or recombinant human immunoglobulin M 12 (HIgM12) preserved functional motor activity in the TMEV model. In this study, we examined IgM-mediated changes in brainstem NAA concentrations and central nervous system (CNS) pathology. (1)H-magnetic resonance spectroscopy (MRS) showed that treatment with HIgM12 significantly increased brainstem NAA concentrations compared to controls in TMEV-infected mice. Pathologic analysis demonstrated a significant preservation of axons in the spinal cord of animals treated with HIgM12. This study links drug efficacy of slowing deficits with axon preservation and NAA concentrations in the brainstem in a model of progressive MS. HIgM12-mediated changes of NAA concentrations in the brainstem are a surrogate marker of axon injury/preservation throughout the spinal cord. This study provides proof-of-concept that a neuron-reactive human IgM can be therapeutic and provides a biomarker for clinical trials.

  20. HYDROXYCHLOROQUINE REDUCES BINDING OF ANTIPHOSPHOLIPID ANTIBODIES TO SYNCYTIOTROPHOBLASTS AND RESTORES ANNEXIN A5 EXPRESSION

    Science.gov (United States)

    Wu, Xiao-Xuan; Guller, Seth; Rand, Jacob H.

    2011-01-01

    Objectives Antibody-mediated disruption of the annexin A5 (AnxA5) anticoagulant shield has been posited to be a thrombogenic mechanism in the antiphospholipid syndrome. We recently showed that the antimalarial drug, hydroxychloroquine, dissociates antiphospholipid immune complexes and restores AnxA5 binding to planar phospholipid bilayer. Using quantitative immunoassays, we demonstrated similar effects on BeWo trophoblasts. We therefore investigated the effects of the drug on localization of AnxA5 in primary cultures of human placental syncytiotrophoblasts (SCTs). Study Laser confocal microscopy with computer-based morphometric analysis was used to localize AnxA5 and antiphospholipid antibodies on SCTs exposed to polyclonal and monoclonal antiphospholipid and control IgGs. Results Hydroxychloroquine reversed the effects of the antiphospholipid antibodies on the SCTs by markedly reducing IgG binding and restoring AnxA5 expression. Conclusions These results provide the first morphologic evidence for this effect of hydroxychloroquine on human placental SCTs and support the possibility of novel treatments that target antiphospholipid antibody binding. PMID:21871597

  1. Heparin-associated thrombocytopenia: antibody binding specificity to platelet antigens.

    Science.gov (United States)

    Lynch, D M; Howe, S E

    1985-11-01

    Sera from four patients with heparin-associated thrombocytopenia (HAT) were evaluated by a quantitative enzyme-linked immunosorbent assay (ELISA) to detect heparin-dependent serum platelet-bindable immunoglobulin (S-PBIg) and by Western blotting and immunoprecipitation to investigate the specificity of the antibody binding. All HAT sera showed mildly increased S-PBIg (mean, 7.8 fg per platelet; normal, less than 6.0 fg per platelet) to intact target platelets in the ELISA, which was markedly increased in the presence of heparin (mean, 20.9 fg per platelet). This increase was 20-fold greater than normal control sera, which showed a mean differential increase of only 0.5 fg per platelet. Immunoglobulin binding specificity to platelet antigens was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of platelet lysate with transfer of the platelet fractions onto nitrocellulose strips (Western blotting) and subsequent immunoassay using HAT and normal sera. In the presence of heparin, the four HAT patients demonstrated increased binding of immunoglobulin to platelet antigens of apparent molecular weights of 180, 124, and 82 kd. Radiolabeled heparin when incubated with HAT sera, normal sera, or albumin blanks bound to platelet proteins of the same apparent molecular weights. These observations are consistent with current hypotheses suggesting that HAT antibody is directed to heparin-platelet complexes or, alternatively, that heparin induces conformational change of antigenic sites on the platelet membrane.

  2. Thermodynamic model of binding of flexible bivalent haptens to antibody

    Energy Technology Data Exchange (ETDEWEB)

    Dembo, M; Goldstein, B

    1978-01-01

    Studies by Wilder et al. of the binding of Fab' fragments to small haptens have shown that the cross-linking constant (the equilibrium constant for binding an additional Fab' fragment to a hapten-Fab' complex) is strongly dependent on the length of the hapten. We present a simple model for predicting the relationship between the intermolecular cross-linking constant and the monovalent hapten-antibody binding constant. In particular we used the model to obtain the dependence of the cross-linking constant on the length of th hapten, the depth to which the hapten fills th Fab' binding site, and the size of the Fab' fragment. To test the model, we devised expressions which allowed us to analyze the data of Wilder et al. From their data we determined the values of two parameters which we took to be unknown in the theory, the size of the Fab' fragment and the depth to which the hapten fills the Fab' binding site. The values arrived at in this way agreed well with published measurements of these parameters.

  3. A novel polyclonal antibody against human cytomegalovirus ...

    African Journals Online (AJOL)

    Future research should be directed to epitope screening of synthetic HMCV peptides, which could help to understand HCMV infection and virus-neutralising antibodies more fully and to prepare HCMV vaccines and antiviral drugs. Key words: Human cytomegalovirus, AD169 strain, Towne strains, polyclonal antibody.

  4. Monoclonal antibodies to meningococcal factor H binding protein with overlapping epitopes and discordant functional activity.

    Science.gov (United States)

    Giuntini, Serena; Beernink, Peter T; Reason, Donald C; Granoff, Dan M

    2012-01-01

    Meningococcal factor H binding protein (fHbp) is a promising vaccine candidate. Anti-fHbp antibodies can bind to meningococci and elicit complement-mediated bactericidal activity directly. The antibodies also can block binding of the human complement down-regulator, factor H (fH). Without bound fH, the organism would be expected to have increased susceptibility to bacteriolysis. Here we describe bactericidal activity of two anti-fHbp mAbs with overlapping epitopes in relation to their different effects on fH binding and bactericidal activity. Both mAbs recognized prevalent fHbp sequence variants in variant group 1. Using yeast display and site-specific mutagenesis, binding of one of the mAbs (JAR 1, IgG3) to fHbp was eliminated by a single amino acid substitution, R204A, and was decreased by K143A but not by R204H or D142A. The JAR 1 epitope overlapped that of previously described mAb (mAb502, IgG2a) whose binding to fHbp was eliminated by R204A or R204H substitutions, and was decreased by D142A but not by K143A. Although JAR 1 and mAb502 appeared to have overlapping epitopes, only JAR 1 inhibited binding of fH to fHbp and had human complement-mediated bactericidal activity. mAb502 enhanced fH binding and lacked human complement-mediated bactericidal activity. To control for confounding effects of different mouse IgG subclasses on complement activation, we created chimeric mAbs in which the mouse mAb502 or JAR 1 paratopes were paired with human IgG1 constant regions. While both chimeric mAbs showed similar binding to fHbp, only JAR 1, which inhibited fH binding, had human complement-mediated bactericidal activity. The lack of human complement-mediated bactericidal activity by anti-fHbp mAb502 appeared to result from an inability to inhibit binding of fH. These results underscore the importance of inhibition of fH binding for anti-fHbp mAb bactericidal activity.

  5. Effect of iodination site on binding of radiolabeled ligand by insulin antibodies and insulin autoantibodies

    International Nuclear Information System (INIS)

    Diaz, J.L.; Wilkin, T.J.

    1988-01-01

    Four human insulins and four porcine insulins, each monoiodinated to the same specific activity at one of the four tyrosine residues (A14, A19, B16, B26) and purified by reversed-phase liquid chromatography, were tested in a radiobinding assay against a panel of insulin-antibody (IA)-positive sera from 10 insulin-treated diabetics and insulin-autoantibody-positive (IAA) sera from 10 nondiabetics. Of the 10 IAA-positive sera, five were fully cross reactive with both insulin species, and five were specific for human insulin. The rank order of binding of sera with the four ligands from each species was random for IA (mean rank values of 1.9 for A14, 2.0 for A19, 2.5 for B16, and 3.6 for B26 from a possible ranking range of 1 to 4), but more consistent for non-human-insulin-specific IAA (mean rank values 1.3 for A14, 3.8 for A19, 1.7 for B16, and 3.2 for B26 for labeled human insulins; 1.2 for A14, 4.0 for A19, 1.8 for B16, and 3.0 for B26 for labeled porcine insulins). The rank order of binding was virtually uniform for human-insulin-specific IAA (mean values 1.2 for A14, 3.0 for A19, 1.8 for B16, and 4.0 for B26). The influence of iodination site on the binding of labeled insulin appears to be dependent on the proximity of the labeled tyrosine to the antibody binding site and the clonal diversity, or restriction, of insulin-binding antibodies in the test serum. When IA and IAA are measured, the implications of this study regarding the choice of assay ligand may be important

  6. Targeting of human glioma xenografts in vivo utilizing radiolabeled antibodies

    International Nuclear Information System (INIS)

    Williams, J.A.; Wessels, B.W.; Wharam, M.D.; Order, S.E.; Wanek, P.M.; Poggenburg, J.K.; Klein, J.L.

    1990-01-01

    Radiolabeled antibodies provide a potential basis for selective radiotherapy of human gliomas. We have measured tumor targeting by radiolabeled monoclonal and polyclonal antibodies directed against neuroectodermal and tumor-associated antigens in nude mice bearing human glioma xenografts. Monoclonal P96.5, a mouse IgG2a immunoglobulin, defines an epitope of a human melanoma cell surface protein, and specifically binds the U-251 human glioma as measured by immunoperoxidase histochemistry. 111In-radiolabeled P96.5 specifically targets the U-251 human glioma xenograft and yields 87.0 microCuries (microCi) of tumor activity per gram per 100 microCi injected activity compared to 4.5 microCi following administration of radiolabeled irrelevant monoclonal antibody. Calculations of targeting ratios demonstrate deposited dose to be 11.6 times greater with radiolabeled P96.5 administration compared to irrelevant monoclonal antibody. The proportion of tumor dose found in normal organs is less than 10%, further supporting specific targeting of the human glioma xenograft by this antibody. Monoclonal antibody ZME018, which defines a second melanoma-associated antigen, and polyclonal rabbit antiferritin, which defines a tumor-associated antigen, demonstrate positive immunoperoxidase staining of the tumor, but comparatively decreased targeting. When compared to the 111In-radiolabeled antibody, 90Y-radiolabeled P96.5 demonstrates comparable tumor targeting and percentages of tumor dose found in normal organs. To test the therapeutic potential of 90Y-radiolabeled P96.5, tumors and normal sites were implanted with miniature thermoluminescent dosimeters (TLD). Seven days following administration of 100 microCi 90Y-radiolabeled P96.5, average absorbed doses of 3770, 980, 353, and 274 cGy were observed in tumor, liver, contralateral control site, and total body, respectively

  7. Anti-Hemagglutinin Antibody Derived Lead Peptides for Inhibitors of Influenza Virus Binding.

    Directory of Open Access Journals (Sweden)

    Henry Memczak

    Full Text Available Antibodies against spike proteins of influenza are used as a tool for characterization of viruses and therapeutic approaches. However, development, production and quality control of antibodies is expensive and time consuming. To circumvent these difficulties, three peptides were derived from complementarity determining regions of an antibody heavy chain against influenza A spike glycoprotein. Their binding properties were studied experimentally, and by molecular dynamics simulations. Two peptide candidates showed binding to influenza A/Aichi/2/68 H3N2. One of them, termed PeB, with the highest affinity prevented binding to and infection of target cells in the micromolar region without any cytotoxic effect. PeB matches best the conserved receptor binding site of hemagglutinin. PeB bound also to other medical relevant influenza strains, such as human-pathogenic A/California/7/2009 H1N1, and avian-pathogenic A/Mute Swan/Rostock/R901/2006 H7N1. Strategies to improve the affinity and to adapt specificity are discussed and exemplified by a double amino acid substituted peptide, obtained by substitutional analysis. The peptides and their derivatives are of great potential for drug development as well as biosensing.

  8. Molecular immune recognition of botulinum neurotoxin B. The light chain regions that bind human blocking antibodies from toxin-treated cervical dystonia patients. Antigenic structure of the entire BoNT/B molecule.

    Science.gov (United States)

    Atassi, M Zouhair; Jankovic, Joseph; Steward, Lance E; Aoki, K Roger; Dolimbek, Behzod Z

    2012-01-01

    We recently mapped the regions on the heavy (H) chain of botulinum neurotoxin, type B (BoNT/B) recognized by blocking antibodies (Abs) from cervical dystonia (CD) patients who develop immunoresistance during toxin treatment. Since blocking could also be effected by Abs directed against regions on the light (L) chain, we have mapped here the L chain, using the same 30 CD antisera. We synthesized, purified and characterized 32 19-residue L chain peptides that overlapped successively by 5 residues (peptide L32 overlapped with peptide N1 of the H chain by 12 residues). In a given patient, Abs against the L chain seemed less intense than those against H chain. Most sera recognized a limited set of L chain peptides. The levels of Abs against a given region varied with the patient, consistent with immune responses to each epitope being under separate MHC control. The peptides most frequently recognized were: L13, by 30 of 30 antisera (100%); L22, by 23 of 30 (76.67%); L19, by 15 of 30 (50.00%); L26, by 11 of 30 (36.70%); and L14, by 12 of 30 (40.00%). The activity of L14 probably derives from its overlap with L13. The levels of Ab binding decreased in the following order: L13 (residues 169-187), L22 (295-313), L19 (253-271), and L26 (351-369). Peptides L12 (155-173), L18 (239-257), L15 (197-215), L1 (1-19) and L23 (309-327) exhibited very low Ab binding. The remaining peptides had little or no Ab-binding activity. The antigenic regions are analyzed in terms of their three-dimensional locations and the enzyme active site. With the previous localization of the antigenic regions on the BoNT/B H chain, the human Ab recognition of the entire BoNT/B molecule is presented and compared to the recognition of BoNT/A by human blocking Abs. Copyright © 2011. Published by Elsevier GmbH.

  9. In vitro and in vivo properties of human/mouse chimeric monoclonal antibody specific for common acute lymphocytic leukemia antigen

    International Nuclear Information System (INIS)

    Saga, T.; Endo, K.; Koizumi, M.; Kawamura, Y.; Watanabe, Y.; Konishi, J.; Ueda, R.; Nishimura, Y.; Yokoyama, M.; Watanabe, T.

    1990-01-01

    A human/mouse chimeric monoclonal antibody specific for a common acute lymphocytic leukemia antigen was efficiently obtained by ligating human heavy-chain enhancer element to the chimeric heavy- and light-chain genes. Cell binding and competitive inhibition assays of both radioiodine and indium-111- (111In) labeled chimeric antibodies demonstrated in vitro immunoreactivity identical with that of the parental murine monoclonal antibodies. The biodistribution of the radiolabeled chimeric antibody in tumor-bearing nude mice was similar to that of the parental murine antibody. Tumor accumulation of radioiodinated parental and chimeric antibodies was lower than that of 111 In-labeled antibodies, probably because of dehalogenation of the radioiodinated antibodies. Indium-111-labeled chimeric antibody clearly visualized xenografted tumor. These results suggest that a human/mouse chimeric antibody can be labeled with 111 In and radioiodine without the loss of its immunoreactivity, and that chimeric antibody localizes in vivo in the same way as the parental murine antibody

  10. Generation of human antibody fragments against Streptococcus mutans using a phage display chain shuffling approach

    Directory of Open Access Journals (Sweden)

    Barth Stefan

    2005-01-01

    Full Text Available Abstract Background Common oral diseases and dental caries can be prevented effectively by passive immunization. In humans, passive immunotherapy may require the use of humanized or human antibodies to prevent adverse immune responses against murine epitopes. Therefore we generated human single chain and diabody antibody derivatives based on the binding characteristics of the murine monoclonal antibody Guy's 13. The murine form of this antibody has been used successfully to prevent Streptococcus mutans colonization and the development of dental caries in non-human primates, and to prevent bacterial colonization in human clinical trials. Results The antibody derivatives were generated using a chain-shuffling approach based on human antibody variable gene phage-display libraries. Like the parent antibody, these derivatives bound specifically to SAI/II, the surface adhesin of the oral pathogen S. mutans. Conclusions Humanization of murine antibodies can be easily achieved using phage display libraries. The human antibody fragments bind the antigen as well as the causative agent of dental caries. In addition the human diabody derivative is capable of aggregating S. mutans in vitro, making it a useful candidate passive immunotherapeutic agent for oral diseases.

  11. Site-directed antibody immobilization using a protein A-gold binding domain fusion protein for enhanced SPR immunosensing.

    Science.gov (United States)

    de Juan-Franco, Elena; Caruz, Antonio; Pedrajas, J R; Lechuga, Laura M

    2013-04-07

    We have implemented a novel strategy for the oriented immobilization of antibodies onto a gold surface based on the use of a fusion protein, the protein A-gold binding domain (PAG). PAG consists of a gold binding peptide (GBP) coupled to the immunoglobulin-binding domains of staphylococcal protein A. This fusion protein provides an easy and fast oriented immobilization of antibodies preserving its native structure, while leaving the antigen binding sites (Fab) freely exposed. Using this immobilization strategy, we have demonstrated the performance of the immunosensing of the human Growth Hormone by SPR. A limit of detection of 90 ng mL(-1) was obtained with an inter-chip variability lower than 7%. The comparison of this method with other strategies for the direct immobilization of antibodies over gold surfaces has showed the enhanced sensitivity provided by the PAG approach.

  12. Anti-idiotypes against a monoclonal anti-haloperidol antibody bind to dopamine receptor

    International Nuclear Information System (INIS)

    Elazar, Z.; Kanety, H.; Schreiber, M.; Fuchs, S.

    1988-01-01

    Anti-idiotypic antibodies were raised in rabbits by immunization with a monoclonal anti-haloperidol antibody. Some of these anti-idiotypic antibodies bind in a concentration dependent manner to bovine striatal membranes. Following affinity purification, these antibodies inhibit haloperidol binding to striatal membranes and deplete [ 3 H]-spiperone binding sites from a solubilized preparation of striatal membranes. It is thus concluded that these anti-idiotypic antibodies are an internal image of haloperidol and as such can interact with D 2 -dopamine receptors

  13. SINGLE CHAIN VARIABLE FRAGMENTS OF ANTIBODIES AGAINST DIPHTHERIA TOXIN B-SUBUNIT ISOLATED FROM PHAGE DISPLAY HUMAN ANTIBODY LIBRARY

    Directory of Open Access Journals (Sweden)

    Oliinyk O. S.

    2014-02-01

    Full Text Available Diphtheria toxin is an exoantigen of Corynebacterium diphtheriae that inhibits protein synthesis and kills sensitive cells. The aim of this study was to obtain human recombinant single-chain variable fragment (scFv antibodies against receptor-binding B subunit of diphtheria toxin. 12 specific clones were selected after three rounds of a phage display naїve (unimmunized human antibody library against recombinant B-subunit. scFv DNA inserts from these 12 clones were digested with MvaI, and 6 unique restriction patterns were found. Single-chain antibodies were expressed in Escherichia coli XL1-blue. The recombinant proteins were characterized by immunoblotting of bacterial extracts and detection with an anti-E-tag antibody. The toxin B-subunit-binding function of the single-chain antibody was shown by ELISA. The affinity constants for different clones were found to be from 106 to 108 М–1. Due to the fact, that these antibody fragments recognized epitopes in the receptor-binding Bsubunit of diphtheria toxin, further studies are interesting to evaluate their toxin neutralization properties and potential for therapeutic applications. Obtained scFv-antibodies can also be used for detection and investigation of biological properties of diphtheria toxin.

  14. Collaborative enhancement of antibody binding to distinct PECAM-1 epitopes modulates endothelial targeting.

    Directory of Open Access Journals (Sweden)

    Ann-Marie Chacko

    Full Text Available Antibodies to platelet endothelial cell adhesion molecule-1 (PECAM-1 facilitate targeted drug delivery to endothelial cells by "vascular immunotargeting." To define the targeting quantitatively, we investigated the endothelial binding of monoclonal antibodies (mAbs to extracellular epitopes of PECAM-1. Surprisingly, we have found in human and mouse cell culture models that the endothelial binding of PECAM-directed mAbs and scFv therapeutic fusion protein is increased by co-administration of a paired mAb directed to an adjacent, yet distinct PECAM-1 epitope. This results in significant enhancement of functional activity of a PECAM-1-targeted scFv-thrombomodulin fusion protein generating therapeutic activated Protein C. The "collaborative enhancement" of mAb binding is affirmed in vivo, as manifested by enhanced pulmonary accumulation of intravenously administered radiolabeled PECAM-1 mAb when co-injected with an unlabeled paired mAb in mice. This is the first demonstration of a positive modulatory effect of endothelial binding and vascular immunotargeting provided by the simultaneous binding a paired mAb to adjacent distinct epitopes. The "collaborative enhancement" phenomenon provides a novel paradigm for optimizing the endothelial-targeted delivery of therapeutic agents.

  15. Human plasminogen binding protein tetranectin

    DEFF Research Database (Denmark)

    Kastrup, J S; Rasmussen, H; Nielsen, B B

    1997-01-01

    The recombinant human plasminogen binding protein tetranectin (TN) and the C-type lectin CRD of this protein (TN3) have been crystallized. TN3 crystallizes in the tetragonal space group P4(2)2(1)2 with cell dimensions a = b = 64.0, c = 75.7 A and with one molecule per asymmetric unit. The crystals...... to at least 2.5 A. A full data set has been collected to 3.0 A. The asymmetric unit contains one monomer of TN. Molecular replacement solutions for TN3 and TN have been obtained using the structure of the C-type lectin CRD of rat mannose-binding protein as search model. The rhombohedral space group indicates...

  16. [Construction of human phage antibody library and screening for human monoclonal antibodies of amylin].

    Science.gov (United States)

    Gong, Qian; Li, Chang-ying; Chang, Ji-wu; Zhu, Tie-hong

    2012-06-01

    To screen monoclonal antibodies to amylin from a constructed human phage antibody library and identify their antigenic specificity and combining activities. The heavy chain Fd fragment and light chain of human immunoglobulin genes were amplified from peripheral blood lymphocytes of healthy donors using RT-PCR, and then inserted into phagemid pComb3XSS to generate a human phage antibody library. The insertion of light chain or heavy chain Fd genes were identified by PCR after the digestion of Sac I, Xba I, Xho Iand Spe I. One of positive clones was analyzed by DNA sequencing. The specific anti-amylin clones were screened from antibody library against human amylin antigens and then the positive clones were determined by Phage-ELISA analysis. A Fab phage antibody library with 0.8×10(8); members was constructed with the efficacy of about 70%. DNA sequence analysis indicated V(H); gene belonged to V(H);3 gene family and V(λ); gene belonged to the V(λ); gene family. Using human amylin as panning antigen, specific anti-amylin Fab antibodies were enriched by screening the library for three times. Phage-ELISA assay showed the positive clones had very good specificity to amylin antigen. The successful construction of a phage antibody library and the identification of anti-amylin Fab antibodies provide a basis for further study and preparation of human anti-amylin antibodies.

  17. Radiolabeled Humanized Anti-CD3 Monoclonal Antibody Visilizumab for Imaging Human T-Lymphocytes

    NARCIS (Netherlands)

    Malviya, Gaurav; D'Alessandria, Calogero; Bonanno, Elena; Vexler, Vladimir; Massari, Roberto; Trotta, Carlo; Scopinaro, Francesco; Dierckx, Rudi; Signore, Alberto

    2009-01-01

    Visilizumab is an IgG(2) humanized monoclonal antibody (mAb) characterized by non-Fc gamma R binding and specific to the CD3 antigen, expressed on more than 95% of circulating resting T-lymphocytes and on activated T-lymphocytes homing in inflamed tissues. We hypothesized that the use of a

  18. Radioimmunodetection of human melanoma tumor xenografts with human monoclonal antibodies

    International Nuclear Information System (INIS)

    Gomibuchi, Makoto; Saxton, R.E.; Lake, R.R.; Katano, Mitsuo; Irie, R.F.

    1986-01-01

    A human IgM monoclonal antibody has been established that defines a tumor-associated membrane antigen expressed on human melanoma cells. The antigen has been identified as the ganglioside GD2. In this paper, the authors describe the potential usefulness of the human monoclonal antibody for radioimaging. Nude mice bearing tumors derived from a human melanoma cell line were used as a model. Antibody activity was degradated significantly after labeling with 131 I by the use of a modified chloramine-T method. After testing various concentrations, labeled antibody of a specific activity of 2.8μCi/μg produced the best results. Balb/c nude mice bearing a GD2-positive M14 melanoma cell line were injected with 10-30μg of labeled antibody, and its radiolocalization in different organs and in the whole body were evaluated. The best tumor image was obtained on Day 6. The labeled antibody uptake ratio between tumor and muscle was 9.2:1; the ratio between tumor and liver was 1.4:1. These studies represent the first report of experimental tumor imaging with human monoclonal antibody. Human monoclonals will probably prove to be superior reagents for tumor imaging in melanoma patients if the problem of anti-body radiolysis is resolved. (author)

  19. Rifampicin-dependent antibodies bind a similar or identical epitope to glycoprotein IX-specific quinine-dependent antibodies

    NARCIS (Netherlands)

    Burgess, Janette K.; Lopez, Jose A.; Gaudry, Leonie E.; Chong, Beng H.

    2000-01-01

    The drug-dependent antibody of a patient with rifampicin-induced thrombocytopenia was characterized using the antigen-capture enzyme-linked immunosorbent assay (MAIPA assay), flow cytometry, and immunoprecipitation. The antibody was found to bind glycoprotein (GP) Ib-IX but not GPIIb-IIIa because

  20. Fusion proteins of HIV-1 envelope glycoprotein gp120 with CD4-induced antibodies showed enhanced binding to CD4 and CD4 binding site antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Weizao, E-mail: chenw3@mail.nih.gov [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); Feng, Yang [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); Wang, Yanping [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); The Basic Research Program, Science Applications International Corporation-Frederick, Inc., National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); Zhu, Zhongyu; Dimitrov, Dimiter S. [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States)

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Some recombinant HIV-1 gp120s do not preserve their conformations on gp140s. Black-Right-Pointing-Pointer We hypothesize that CD4i antibodies could induce conformational changes in gp120. Black-Right-Pointing-Pointer CD4i antibodies enhance binding of CD4 and CD4bs antibodies to gp120. Black-Right-Pointing-Pointer CD4i antibody-gp120 fusion proteins could have potential as vaccine immunogens. -- Abstract: Development of successful AIDS vaccine immunogens continues to be a major challenge. One of the mechanisms by which HIV-1 evades antibody-mediated neutralizing responses is the remarkable conformational flexibility of its envelope glycoprotein (Env) gp120. Some recombinant gp120s do not preserve their conformations on gp140s and functional viral spikes, and exhibit decreased recognition by CD4 and neutralizing antibodies. CD4 binding induces conformational changes in gp120 leading to exposure of the coreceptor-binding site (CoRbs). In this study, we test our hypothesis that CD4-induced (CD4i) antibodies, which target the CoRbs, could also induce conformational changes in gp120 leading to better exposed conserved neutralizing antibody epitopes including the CD4-binding site (CD4bs). We found that a mixture of CD4i antibodies with gp120 only weakly enhanced CD4 binding. However, such interactions in single-chain fusion proteins resulted in gp120 conformations which bound to CD4 and CD4bs antibodies better than the original or mutagenically stabilized gp120s. Moreover, the two molecules in the fusion proteins synergized with each other in neutralizing HIV-1. Therefore, fusion proteins of gp120 with CD4i antibodies could have potential as components of HIV-1 vaccines and inhibitors of HIV-1 entry, and could be used as reagents to explore the conformational flexibility of gp120 and mechanisms of entry and immune evasion.

  1. Preparation and validation of radio iodinated recombinant human IL-10 for the measurement of natural human antibodies against IL-10

    DEFF Research Database (Denmark)

    de Lemos Rieper, Carina; Galle, Pia; Svenson, Morten

    2009-01-01

    activity of 75 cpm/pg. Validation of the tracer confirmed preserved antibody epitopes and receptor binding ability. A robust Radio Immuno Assay (RIA) was developed and validated to detect natural human anti-IL-10 antibodies based on the formation of (125)I-labeled IL-10-IgG complexes in solution...

  2. Lentivirus display: stable expression of human antibodies on the surface of human cells and virus particles.

    Directory of Open Access Journals (Sweden)

    Ran Taube

    Full Text Available BACKGROUND: Isolation of human antibodies using current display technologies can be limited by constraints on protein expression, folding and post-translational modifications. Here we describe a discovery platform that utilizes self-inactivating (SIN lentiviral vectors for the surface display of high-affinity single-chain variable region (scFv antibody fragments on human cells and lentivirus particles. METHODOLOGY/PRINCIPAL FINDINGS: Bivalent scFvFc human antibodies were fused in frame with different transmembrane (TM anchoring moieties to allow efficient high-level expression on human cells and the optimal TM was identified. The addition of an eight amino acid HIV-1 gp41 envelope incorporation motif further increased scFvFc expression on human cells and incorporation into lentiviral particles. Both antibody-displaying human cells and virus particles bound antigen specifically. Sulfation of CDR tyrosine residues, a property recently shown to broaden antibody binding affinity and antigen recognition was also demonstrated. High level scFvFc expression and stable integration was achieved in human cells following transduction with IRES containing bicistronic SIN lentivectors encoding ZsGreen when scFvFc fusion proteins were expressed from the first cassette. Up to 10(6-fold enrichment of antibody expressing cells was achieved with one round of antigen coupled magnetic bead pre-selection followed by FACS sorting. Finally, the scFvFc displaying human cells could be used directly in functional biological screens with remarkable sensitivity. CONCLUSIONS/SIGNIFICANCE: This antibody display platform will complement existing technologies by virtue of providing properties unique to lentiviruses and antibody expression in human cells, which, in turn, may aid the discovery of novel therapeutic human mAbs.

  3. A camelid single-domain antibody neutralizes botulinum neurotoxin A by blocking host receptor binding

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Guorui; Lam, Kwok-ho; Weisemann, Jasmin; Peng, Lisheng; Krez, Nadja; Perry, Kay; Shoemaker, Charles B.; Dong, Min; Rummel, Andreas; Jin, Rongsheng (BCH); (Cornell); (Tufts CTSI); (UCI); (MHH)

    2017-08-07

    Antibody treatment is currently the only available countermeasure for botulism, a fatal illness caused by flaccid paralysis of muscles due to botulinum neurotoxin (BoNT) intoxication. Among the seven major serotypes of BoNT/A-G, BoNT/A poses the most serious threat to humans because of its high potency and long duration of action. Prior to entering neurons and blocking neurotransmitter release, BoNT/A recognizes motoneurons via a dual-receptor binding process in which it engages both the neuron surface polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Previously, we identified a potent neutralizing antitoxin against BoNT/A1 termed ciA-C2, derived from a camelid heavy-chain-only antibody (VHH). In this study, we demonstrate that ciA-C2 prevents BoNT/A1 intoxication by inhibiting its binding to neuronal receptor SV2. Furthermore, we determined the crystal structure of ciA-C2 in complex with the receptor-binding domain of BoNT/A1 (HCA1) at 1.68 Å resolution. The structure revealed that ciA-C2 partially occupies the SV2-binding site on HCA1, causing direct interference of HCA1 interaction with both the N-glycan and peptide-moiety of SV2. Interestingly, this neutralization mechanism is similar to that of a monoclonal antibody in clinical trials, despite that ciA-C2 is more than 10-times smaller. Taken together, these results enlighten our understanding of BoNT/A1 interactions with its neuronal receptor, and further demonstrate that inhibiting toxin binding to the host receptor is an efficient countermeasure strategy.

  4. Site-specific photoconjugation of antibodies using chemically synthesized IgG-binding domains.

    Science.gov (United States)

    Perols, Anna; Karlström, Amelie Eriksson

    2014-03-19

    Site-specific labeling of antibodies can be performed using the immunoglobulin-binding Z domain, derived from staphylococcal protein A (SpA), which has a well-characterized binding site in the Fc region of antibodies. By introducing a photoactivable probe in the Z domain, a covalent bond can be formed between the Z domain and the antibody by irradiation with UV light. The aim of this study was to improve the conjugation yield for labeling of different subclasses of IgG having different sequence composition, using a photoactivated Z domain variant. Four different variants of the Z domain (Z5BPA, Z5BBA, Z32BPA, and Z32BBA) were synthesized to investigate the influence of the position of the photoactivable probe and the presence of a flexible linker between the probe and the protein. For two of the variants, the photoreactive benzophenone group was introduced as part of an amino acid side chain by incorporation of the unnatural amino acid benzoylphenylalanine (BPA) during peptide synthesis. For the other two variants, the photoreactive benzophenone group was attached via a flexible linker by coupling of benzoylbenzoic acid (BBA) to the ε-amino group of a selectively deprotected lysine residue. Photoconjugation experiments using human IgG1, mouse IgG1, and mouse IgG2A demonstrated efficient conjugation for all antibodies. It was shown that differences in linker length had a large impact on the conjugation efficiency for labeling of mouse IgG1, whereas the positioning of the photoactivable probe in the sequence of the protein had a larger effect for mouse IgG2A. Conjugation to human IgG1 was only to a minor extent affected by position or linker length. For each subclass of antibody, the best variant tested using a standard conjugation protocol resulted in conjugation efficiencies of 41-66%, which corresponds to on average approximately one Z domain attached to each antibody. As a combination of the two best performing variants, Z5BBA and Z32BPA, a Z domain variant with

  5. Selection and characterization of a human neutralizing antibody to human fibroblast growth factor-2

    International Nuclear Information System (INIS)

    Tao, Jun; Xiang, Jun-Jian; Li, Dan; Deng, Ning; Wang, Hong; Gong, Yi-Ping

    2010-01-01

    Compelling evidences suggest that fibroblast growth factor-2 (FGF-2) plays important roles in tumor growth, angiogenesis and metastasis. Molecules blocking the FGF-2 signaling have been proposed as anticancer agents. Through screening of a human scFv phage display library, we have isolated several human single-chain Fv fragments (scFvs) that bind to human FGF-2. After expression and purification in bacteria, one scFv, named 1A2, binds to FGF-2 with a high affinity and specificity, and completes with FGF-2 binding to its receptor. This 1A2 scFv was then cloned into the pIgG1 vector and expressed in 293T cells. The purified hIgG1-1A2 antibody showed a high binding affinity of 8 x 10 -9 M to rhFGF-2. In a set of vitro assays, it inhibited various biological activities of FGF-2 such as the proliferation, migration and tube formation of human umbilical vein endothelial cells. More importantly, hIgG1-1A2 antibody also efficiently blocked the growth while inducing apoptosis of glioma cells. For the first time, we generated a human anti-FGF-2 antibody with proven in vitro anti-tumor activity. It may therefore present a new therapeutic candidate for the treatment of cancers that are dependent on FGF-2 signaling for growth and survival.

  6. Characterisation of peptide microarrays for studying antibody-antigen binding using surface plasmon resonance imagery.

    Directory of Open Access Journals (Sweden)

    Claude Nogues

    Full Text Available BACKGROUND: Non-specific binding to biosensor surfaces is a major obstacle to quantitative analysis of selective retention of analytes at immobilized target molecules. Although a range of chemical antifouling monolayers has been developed to address this problem, many macromolecular interactions still remain refractory to analysis due to the prevalent high degree of non-specific binding. We describe how we use the dynamic process of the formation of self assembling monolayers and optimise physical and chemical properties thus reducing considerably non-specific binding and allowing analysis of specific binding of analytes to immobilized target molecules. METHODOLOGY/PRINCIPAL FINDINGS: We illustrate this approach by the production of specific protein arrays for the analysis of interactions between the 65kDa isoform of human glutamate decarboxylase (GAD65 and a human monoclonal antibody. Our data illustrate that we have effectively eliminated non-specific interactions with the surface containing the immobilised GAD65 molecules. The findings have several implications. First, this approach obviates the dubious process of background subtraction and gives access to more accurate kinetic and equilibrium values that are no longer contaminated by multiphase non-specific binding. Second, an enhanced signal to noise ratio increases not only the sensitivity but also confidence in the use of SPR to generate kinetic constants that may then be inserted into van't Hoff type analyses to provide comparative DeltaG, DeltaS and DeltaH values, making this an efficient, rapid and competitive alternative to ITC measurements used in drug and macromolecular-interaction mechanistic studies. Third, the accuracy of the measurements allows the application of more intricate interaction models than simple Langmuir monophasic binding. CONCLUSIONS: The detection and measurement of antibody binding by the type 1 diabetes autoantigen GAD65 represents an example of an antibody

  7. Binding of antibodies to dsDNA by UVB-irradiated spermatozoa in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Wollina, U; Schaarschmidt, H; Thiel, W; Knopf, B

    1986-08-01

    Human spermatozoa were exposed to monochromatic UVB light (300 nm) at a dosage range from 1-10/sup 3/ J/m/sup 2/ (xenon-mercury short-arc lamp) and 10/sup -2/-10/sup -1/ J/m/sup 2/ (xenon lamp). UVB irradiation >= 10 J/m/sup 2/ resulted in a subsequent binding of antibodies to double-stranded (ds) DNA. A strong homogeneous head fluorescence was visible. There was no immunoglobulin class restriction. The results were discussed in regards to lupus antigen expression by UV light.

  8. Binding-site analysis of opioid receptors using monoclonal anti-idiotypic antibodies

    International Nuclear Information System (INIS)

    Conroy, W.G.

    1988-01-01

    Structural relatedness between the variable region of anti-ligand antibodies and opioid binding sites allowed the generation of anti-idiotypic antibodies which recognized opioid receptors. The IgG 3 k antibodies which bound to opioid receptors were obtained when an anti-morphine antiserum was the idiotype. Both antibodies bound to opioid receptors, but only one of these blocked the binding of [ 3 H]naloxone. The antibody which did not inhibit the binding of [ 3 H]naloxone was itself displaced from the receptor by opioid ligands. The unique binding properties displayed by this antibody indicated that anti-idiotypic antibodies are not always a perfect image of the original ligand, and therefore may be more useful than typical ligands as probes for the receptor. An auto-anti-idiotypic technique was successfully used to obtain anti-opioid receptor antibodies. Another IgG 3 k antibody that blocked the binding of [ 3 H]naloxone to rat brain opioid receptors was obtained when a mouse was immunized with naloxone conjugated to bovine serum albumin. These data confirmed that an idiotype-anti-idiotype network which can generate an anti-receptor antibody normally functions when an opioid ligand is introduced into an animal in an immunogenic form

  9. Human antibody recognition of antigenic site IV on Pneumovirus fusion proteins.

    Science.gov (United States)

    Mousa, Jarrod J; Binshtein, Elad; Human, Stacey; Fong, Rachel H; Alvarado, Gabriela; Doranz, Benjamin J; Moore, Martin L; Ohi, Melanie D; Crowe, James E

    2018-02-01

    Respiratory syncytial virus (RSV) is a major human pathogen that infects the majority of children by two years of age. The RSV fusion (F) protein is a primary target of human antibodies, and it has several antigenic regions capable of inducing neutralizing antibodies. Antigenic site IV is preserved in both the pre-fusion and post-fusion conformations of RSV F. Antibodies to antigenic site IV have been described that bind and neutralize both RSV and human metapneumovirus (hMPV). To explore the diversity of binding modes at antigenic site IV, we generated a panel of four new human monoclonal antibodies (mAbs) and competition-binding suggested the mAbs bind at antigenic site IV. Mutagenesis experiments revealed that binding and neutralization of two mAbs (3M3 and 6F18) depended on arginine (R) residue R429. We discovered two R429-independent mAbs (17E10 and 2N6) at this site that neutralized an RSV R429A mutant strain, and one of these mAbs (17E10) neutralized both RSV and hMPV. To determine the mechanism of cross-reactivity, we performed competition-binding, recombinant protein mutagenesis, peptide binding, and electron microscopy experiments. It was determined that the human cross-reactive mAb 17E10 binds to RSV F with a binding pose similar to 101F, which may be indicative of cross-reactivity with hMPV F. The data presented provide new concepts in RSV immune recognition and vaccine design, as we describe the novel idea that binding pose may influence mAb cross-reactivity between RSV and hMPV. Characterization of the site IV epitope bound by human antibodies may inform the design of a pan-Pneumovirus vaccine.

  10. A collagen-binding EGFR antibody fragment targeting tumors with a collagen-rich extracellular matrix

    OpenAIRE

    Hui Liang; Xiaoran Li; Bin Wang; Bing Chen; Yannan Zhao; Jie Sun; Yan Zhuang; Jiajia Shi; He Shen; Zhijun Zhang; Jianwu Dai

    2016-01-01

    Many tumors over-express collagen, which constitutes the physical scaffold of tumor microenvironment. Collagen has been considered to be a target for cancer therapy. The collagen-binding domain (CBD) is a short peptide, which could bind to collagen and achieve the sustained release of CBD-fused proteins in collagen scaffold. Here, a collagen-binding EGFR antibody fragment was designed and expressed for targeting the collagen-rich extracellular matrix in tumors. The antibody fragment (Fab) of ...

  11. Specific binding of antigen-antibody in physiological environments: Measurement, force characteristics and analysis

    Science.gov (United States)

    Gu, Xin; Zhou, Jun; Zhou, Lu; Xie, Shusen; Petti, Lucia; Wang, Shaomin; Wang, Fuyan

    2018-05-01

    The specific recognition of the antigen by the antibody is the crucial step in immunoassays. Measurement and analysis of the specific recognition, including the ways in which it is influenced by external factors are of paramount significance for the quality of the immunoassays. Using prostate-specific antigen (PSA)/anti-PSA antibody and α-fetoprotein (AFP) /anti-AFP antibody as examples, we have proposed a novel solution for measuring the binding forces between the antigens and their corresponding antibodies in different physiological environments by combining laminar flow control technology and optical tweezers technology. On the basis of the experimental results, the different binding forces of PSA/anti-PSA antibody and AFP/anti-AFP antibody in the same phosphate-buffered saline (PBS) environments are analysed by comparing the affinity constant of the two antibodies and the number of antigenic determinants of the two antigens. In different electrolyte environments, the changes of the binding force of antigens-antibodies are explained by the polyelectrolyte effect and hydrophobic interaction. Furthermore, in different pH environments, the changes of binding forces of antigens-antibodies are attributed to the role of the denaturation of protein. The study aims to recognise the antigen-antibody immune mechanism, thus ensuring further understanding of the biological functions of tumour markers, and it promises to be very useful for the clinical diagnosis of early-stage cancer.

  12. Optimization of the Small Glycan Presentation for Binding a Tumor-Associated Antibody

    DEFF Research Database (Denmark)

    Kveton, Filip; Blšáková, Anna; Hushegyi, Andras

    2017-01-01

    on the immobilization of the Tn antigen on a mixed self-assembled monolayer (SAM) (2D biosensor) and the third one utilizing a layer of a human serum albumin (HSA) for the immobilization of a glycan forming a 3D interface. Results showed that the 3D interface with the immobilized Tn antigen is the most effective...... bioreceptive surface for binding its analyte. The 3D impedimetric glycan biosensor exhibited a limit of detection of 1.4 aM, a wide linear range (6 orders of magnitude), and high assay reproducibility with an average relative standard deviation of 4%. The buildup of an interface was optimized using various...... techniques with the visualization of the glycans on the biosensor surface by atomic force microscopy. The study showed that the 3D biosensor is not only the most sensitive compared to other two biosensor platforms but that the Tn antigen on the 3D biosensor surface is more accessible for antibody binding...

  13. Different mechanisms are involved in the antibody mediated inhibition of ligand binding to the urokinase receptor

    DEFF Research Database (Denmark)

    List, K; Høyer-Hansen, G; Rønne, E

    1999-01-01

    Certain monoclonal antibodies are capable of inhibiting the biological binding reactions of their target proteins. At the molecular level, this type of effect may be brought about by completely different mechanisms, such as competition for common binding determinants, steric hindrance or interfer......Certain monoclonal antibodies are capable of inhibiting the biological binding reactions of their target proteins. At the molecular level, this type of effect may be brought about by completely different mechanisms, such as competition for common binding determinants, steric hindrance......) can be employed as a highly useful tool to characterize the inhibitory mechanism of specific antagonist antibodies. Two inhibitory antibodies against uPAR, mAb R3 and mAb R5, were shown to exhibit competitive and non-competitive inhibition, respectively, of ligand binding to the receptor. The former...

  14. Effects of altered FcγR binding on antibody pharmacokinetics in cynomolgus monkeys

    Science.gov (United States)

    Leabman, Maya K; Meng, Y Gloria; Kelley, Robert F; DeForge, Laura E; Cowan, Kyra J; Iyer, Suhasini

    2013-01-01

    Antibody interactions with Fcγ receptors (FcγRs), like FcγRIIIA, play a critical role in mediating antibody effector functions and thereby contribute significantly to the biologic and therapeutic activity of antibodies. Over the past decade, considerable work has been directed towards production of antibodies with altered binding affinity to FcγRs and evaluation of how the alterations modulate their therapeutic activity. This has been achieved by altering glycosylation status at N297 or by engineering modifications in the crystallizable fragment (Fc) region. While the effects of these modifications on biologic activity and efficacy have been examined, few studies have been conducted to understand their effect on antibody pharmacokinetics (PK). We present here a retrospective analysis in which we characterize the PK of three antibody variants with decreased FcγR binding affinity caused by amino acid substitutions in the Fc region (N297A, N297G, and L234A/L235A) and three antibody variants with increased FcγRIIIA binding affinity caused by afucosylation at N297, and compare their PK to corresponding wild type antibody PK in cynomolgus monkeys. For all antibodies, PK was examined at a dose that was known to be in the linear range. Since production of the N297A and N297G variants in Chinese hamster ovary cells results in aglycosylated antibodies that do not bind to FcγRs, we also examined the effect of expression of an aglycosylated antibody, without sequence change(s), in E. coli. All the variants demonstrated similar PK compared with that of the wild type antibodies, suggesting that, for the six antibodies presented here, altered FcγR binding affinity does not affect PK. PMID:24492343

  15. 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

  16. Clinical application of antibody monoclonal humanized anti-EGFrnimotuzumab labeled

    International Nuclear Information System (INIS)

    Perera Pintado, Alejandro; Peña Quián, Yamilé; Batista Cuéllar, Juan F.; Prats Capote, Anaís; Torres Aroche, Leonel A.; Casacó Santana, Caridad; Sánchez Mendosa, Elvia L.; Sánchez González, Yolaine; Romero Collado, Susana; Quesada Pozo, Rodobaldo; Valladares Oviedo, Lourdes; Masquida García, Elsa M.; Leyva Montaña, René; Casacó, Angel; Ramos Suzarte, Mayra; Crombet, Tania

    2016-01-01

    Most malignant tumors are of epithelial origin. These are characterized by overexpression of the receptor of epidermal growth factor (EGFR), which the neoplastic cells escape the regulatory mechanisms are allowed, so its high concentration of membrane is generally associated with a poor prognosis . By binding an antibody specifically to this receptor, preventing binding of EGF latter and activation mechanism tyrosine kinase inhibiting cell mitosis and apoptosis causing tumor cell. For this reason, the EGFr has been considered as an attractive target for anti-tumor therapy. The humanized monoclonal antibody anti-EGFr nimotuzumab was developed by the Center of Molecular Immunology (Havana, Cuba). Numerous clinical trials have been developed in the Department of Clinical Research Center Isotopes (Cuba), in which it has been applied this antibody, both labeled with 99mTc for immuno gammagraphic detection of tumors, as labeled with 188 Re for radioimmunotherapy of gliomas high degree of malignancy. The aim of this paper is to show the experience of the Department of Clinical Research of CENTIS in various clinical trials with marking for both immuno gammagraphics detection of tumors, such as for radioimmunotherapy nimotuzumab. (author)

  17. Generation of “LYmph Node Derived Antibody Libraries” (LYNDAL) for selecting fully human antibody fragments with therapeutic potential.

    Science.gov (United States)

    Diebolder, Philipp; Keller, Armin; Haase, Stephanie; Schlegelmilch, Anne; Kiefer, Jonathan D; Karimi, Tamana; Weber, Tobias; Moldenhauer, Gerhard; Kehm, Roland; Eis-Hübinger, Anna M; Jäger, Dirk; Federspil, Philippe A; Herold-Mende, Christel; Dyckhoff, Gerhard; Kontermann, Roland E; Arndt, Michaela A E; Krauss, Jürgen

    2014-01-01

    The development of efficient strategies for generating fully human monoclonal antibodies with unique functional properties that are exploitable for tailored therapeutic interventions remains a major challenge in the antibody technology field. Here, we present a methodology for recovering such antibodies from antigen-encountered human B cell repertoires. As the source for variable antibody genes, we cloned immunoglobulin G (IgG)-derived B cell repertoires from lymph nodes of 20 individuals undergoing surgery for head and neck cancer. Sequence analysis of unselected “LYmph Node Derived Antibody Libraries” (LYNDAL) revealed a naturally occurring distribution pattern of rearranged antibody sequences, representing all known variable gene families and most functional germline sequences. To demonstrate the feasibility for selecting antibodies with therapeutic potential from these repertoires, seven LYNDAL from donors with high serum titers against herpes simplex virus (HSV) were panned on recombinant glycoprotein B of HSV-1. Screening for specific binders delivered 34 single-chain variable fragments (scFvs) with unique sequences. Sequence analysis revealed extensive somatic hypermutation of enriched clones as a result of affinity maturation. Binding of scFvs to common glycoprotein B variants from HSV-1 and HSV-2 strains was highly specific, and the majority of analyzed antibody fragments bound to the target antigen with nanomolar affinity. From eight scFvs with HSV-neutralizing capacity in vitro,the most potent antibody neutralized 50% HSV-2 at 4.5 nM as a dimeric (scFv)2. We anticipate our approach to be useful for recovering fully human antibodies with therapeutic potential.

  18. Direct binding of radioiodinated monoclonal antibody to tumor cells: significance of antibody purity and affinity for drug targeting or tumor imaging

    International Nuclear Information System (INIS)

    Kennel, S.J.; Foote, L.J.; Lankford, P.K.; Johnson, M.; Mitchell, T.; Braslawsky, G.R.

    1983-01-01

    For MoAb to be used efficiently for drug targeting and tumor imaging, the fraction of antibody binding to tumor cells must be maximized. The authors have studied the binding of 125 I MoAb in three different tumor systems. The fraction of antibody that could be bound to the cell surface was directly proportional to the antibody purity. The affinity constant also limits the fraction of antibody that can bind to cells at a given antigen concentration. Rearrangement of the standard expression for univalent equilibrium binding between two reactants shows that in antigen excess, the maximum fraction of antibody that can bind =Ka[Ag total]/1 + Ka[Ag total]. Binding data using four different MoAb with three cell systems confirm this relationship. Estimates for reasonable concentrations of tumor antigens in vivo indicate that antibodies with binding constants less than 10 8 M -1 are not likely to be useful for drug targeting or tumor imaging

  19. 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

  20. Neutralization of botulinum neurotoxin by a human monoclonal antibody specific for the catalytic light chain.

    Directory of Open Access Journals (Sweden)

    Sharad P Adekar

    2008-08-01

    Full Text Available Botulinum neurotoxins (BoNT are a family of category A select bioterror agents and the most potent biological toxins known. Cloned antibody therapeutics hold considerable promise as BoNT therapeutics, but the therapeutic utility of antibodies that bind the BoNT light chain domain (LC, a metalloprotease that functions in the cytosol of cholinergic neurons, has not been thoroughly explored.We used an optimized hybridoma method to clone a fully human antibody specific for the LC of serotype A BoNT (BoNT/A. The 4LCA antibody demonstrated potent in vivo neutralization when administered alone and collaborated with an antibody specific for the HC. In Neuro-2a neuroblastoma cells, the 4LCA antibody prevented the cleavage of the BoNT/A proteolytic target, SNAP-25. Unlike an antibody specific for the HC, the 4LCA antibody did not block entry of BoNT/A into cultured cells. Instead, it was taken up into synaptic vesicles along with BoNT/A. The 4LCA antibody also directly inhibited BoNT/A catalytic activity in vitro.An antibody specific for the BoNT/A LC can potently inhibit BoNT/A in vivo and in vitro, using mechanisms not previously associated with BoNT-neutralizing antibodies. Antibodies specific for BoNT LC may be valuable components of an antibody antidote for BoNT exposure.

  1. A Rational Engineering Strategy for Designing Protein A-Binding Camelid Single-Domain Antibodies

    Science.gov (United States)

    Henry, Kevin A.; Sulea, Traian; van Faassen, Henk; Hussack, Greg; Purisima, Enrico O.; MacKenzie, C. Roger; Arbabi-Ghahroudi, Mehdi

    2016-01-01

    Staphylococcal protein A (SpA) and streptococcal protein G (SpG) affinity chromatography are the gold standards for purifying monoclonal antibodies (mAbs) in therapeutic applications. However, camelid VHH single-domain Abs (sdAbs or VHHs) are not bound by SpG and only sporadically bound by SpA. Currently, VHHs require affinity tag-based purification, which limits their therapeutic potential and adds considerable complexity and cost to their production. Here we describe a simple and rapid mutagenesis-based approach designed to confer SpA binding upon a priori non-SpA-binding VHHs. We show that SpA binding of VHHs is determined primarily by the same set of residues as in human mAbs, albeit with an unexpected degree of tolerance to substitutions at certain core and non-core positions and some limited dependence on at least one residue outside the SpA interface, and that SpA binding could be successfully introduced into five VHHs against three different targets with no adverse effects on expression yield or antigen binding. Next-generation sequencing of llama, alpaca and dromedary VHH repertoires suggested that species differences in SpA binding may result from frequency variation in specific deleterious polymorphisms, especially Ile57. Thus, the SpA binding phenotype of camelid VHHs can be easily modulated to take advantage of tag-less purification techniques, although the frequency with which this is required may depend on the source species. PMID:27631624

  2. Characterization of human monoclonal antibodies that neutralize multiple poliovirus serotypes.

    Science.gov (United States)

    Puligedda, Rama Devudu; Kouiavskaia, Diana; Al-Saleem, Fetweh H; Kattala, Chandana Devi; Nabi, Usman; Yaqoob, Hamid; Bhagavathula, V Sandeep; Sharma, Rashmi; Chumakov, Konstantin; Dessain, Scott K

    2017-10-04

    Following the eradication of wild poliovirus (PV), achieving and maintaining a polio-free status will require eliminating potentially pathogenic PV strains derived from the oral attenuated vaccine. For this purpose, a combination of non-cross-resistant drugs, such as small molecules and neutralizing monoclonal antibodies (mAbs), may be ideal. We previously isolated chimpanzee and human mAbs capable of neutralizing multiple PV types (cross-neutralization). Here, we describe three additional human mAbs that neutralize types 1 and 2 PV and one mAb that neutralizes all three types. Most bind conformational epitopes and have unusually long heavy chain complementarity determining 3 domains (HC CDR3). We assessed the ability of the mAbs to neutralize A12 escape mutant PV strains, and found that the neutralizing activities of the mAbs were disrupted by different amino acid substitutions. Competitive binding studies further suggested that the specific mAb:PV interactions that enable cross-neutralization differ among mAbs and serotypes. All of the cloned mAbs bind PV in the vicinity of the "canyon", a circular depression around the 5-fold axis of symmetry through which PV recognizes its cellular receptor. We were unable to generate escape mutants to two of the mAbs, suggesting that their epitopes are important for the PV life cycle. These data indicate that PV cross-neutralization involves binding to highly conserved structures within the canyon that binds to the cellular receptor. These may be facilitated by the long HC CDR3 domains, which may adopt alternative binding configurations. We propose that the human and chimpanzee mAbs described here could have potential as anti-PV therapeutics. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Structure of a Human Astrovirus Capsid-Antibody Complex and Mechanistic Insights into Virus Neutralization

    Energy Technology Data Exchange (ETDEWEB)

    Bogdanoff, Walter A.; Campos, Jocelyn; Perez, Edmundo I.; Yin, Lu; Alexander, David L.; DuBois, Rebecca M. (UCSC)

    2016-11-02

    ABSTRACT

    Human astroviruses (HAstVs) are a leading cause of viral diarrhea in young children, the immunocompromised, and the elderly. There are no vaccines or antiviral therapies against HAstV disease. Several lines of evidence point to the presence of protective antibodies in healthy adults as a mechanism governing protection against reinfection by HAstV. However, development of anti-HAstV therapies is hampered by the gap in knowledge of protective antibody epitopes on the HAstV capsid surface. Here, we report the structure of the HAstV capsid spike domain bound to the neutralizing monoclonal antibody PL-2. The antibody uses all six complementarity-determining regions to bind to a quaternary epitope on each side of the dimeric capsid spike. We provide evidence that the HAstV capsid spike is a receptor-binding domain and that the antibody neutralizes HAstV by blocking virus attachment to cells. We identify patches of conserved amino acids that overlap the antibody epitope and may comprise a receptor-binding site. Our studies provide a foundation for the development of therapies to prevent and treat HAstV diarrheal disease.

    IMPORTANCEHuman astroviruses (HAstVs) infect nearly every person in the world during childhood and cause diarrhea, vomiting, and fever. Despite the prevalence of this virus, little is known about how antibodies in healthy adults protect them against reinfection. Here, we determined the crystal structure of a complex of the HAstV capsid protein and a virus-neutralizing antibody. We show that the antibody binds to the outermost spike domain of the capsid, and we provide evidence that the antibody blocks virus attachment to human cells. Importantly, our findings suggest that a subunit-based vaccine focusing the immune system on the HAstV capsid spike domain could be effective in protecting children against HAstV disease.

  4. Antibody binding constants from Farr test and other radioimmunoassays. A theoretical and experimental analysis

    International Nuclear Information System (INIS)

    Engel, J.; Schalch, W.

    1980-01-01

    For the reaction of monovalently reacting antibody (116-700pIEF) with its antigen (streptococcal group A-variant polysaccharide), an apparent binding constant Ksub(a) was derived by the ammonium sulfate precipitation technique (Farr assay) which was 40 times larger than the true binding constant K = 10 6 M -1 determined by fluorescence titration and equilibrium dialysis. For monovalently reacting antibodies the time needed for re-equilibration of the binding reaction is short as compared to the time of ammonium sulfate incubation. A thermodynamic analysis was therefore performed for the case of complete equilibration of all components in solution and in the ammonium sulfate precipitate. It was found that in this limiting case Ksub(a)/K is equal to the ratio of the solubilities of the antibody and the antibody complex corrected by the activity coefficients of the components in the precipitate. For other antibody-antigen reactions in which the antibody reacts with both binding sites to the same antigen molecule, re-equilibration of the binding reaction in solution is much slower. For such systems a disturbance of the binding reaction by the precipitation is less likely and correct binding constants may be obtained by the Farr technique or other radioimmunoassays involving precipitation. (author)

  5. New Insights into the Functional Behavior of Antibodies as Revealed by Binding Studies on an Anti-Uranium Monoclonal Antibody

    International Nuclear Information System (INIS)

    Blake, Diane A.; Xia Li; Haini Yu; Blake, Robert C.

    2004-01-01

    As part of an ongoing effort to develop immunoassays for chelated uranium(VI) on a hand-held flow fluorimeter, an anti-uranium monoclonal antibody designated as 8A11 was fluorescently labeled using two different strategies. When 8A11 was coupled via reactive lysines to either ALEXATM 488 or Cy5TM, the resulting fluorescent antibody conjugate exhibited positive cooperativity in the presence of its antigen, U(VI) chelated with 2,9-dicarboxy-1,10-phenanthroline (U(VI)-DCP). That is, when one of the two binding sites on the covalently modified 8A11 was occupied with bound antigen, the affinity of the remaining site on the antibody for U(VI)-DCP appeared to increase. Unmodified 8A11 bound U(VI)-DCP with the expected hyperbolic dependence on the concentration of antigen, consistent with independent and equal binding of ligand at both sites. Proteolytic cleavage of the fluorescently conjugated 8A11 to produce the fluorescent monovalent Fab fragment yielded an active preparation that now bound U(VI)-DCP with no evidence of positive cooperativity. Although, in principle, any divalent antibody has the potential to exhibit positive cooperativity in its binding interactions with its antigen, very little literature precedent for this type of behavior exists. Native 8A11 was also noncovalently labeled with highly fluorescent ZENONTM reagents. These reagents are fluorescently-labeled Fab fragments of goat anti-mouse antibodies that bind to the Fc portion of 8A11. These high-affinity, monovalent fluorescent reagents permitted the intact 8A11 mouse antibody to be labeled in situ with no covalent modifications. Incubation of the 8A11 with ZENON 647 produced a fluorescent protein complex that showed an 8-fold higher affinity for U(VI)-DCP than did the free 8A11 alone. Again, very few literature precedents exist for this phenomenon, where agents that bind to the Fc portion of an intact antibody change the affinity of the antibody for the antigen at the structurally distant Fab portion

  6. Development and characterization of polyclonal antibodies against the linker region of the telomere-binding protein TRF2

    Directory of Open Access Journals (Sweden)

    Nadya V. Ilicheva

    2018-03-01

    Full Text Available Background: TRF2 (telomeric repeat binding factor 2 is an essential component of the telomere-binding protein complex shelterin. TRF2 induces the formation of a special structure of telomeric DNA and counteracts activation of DNA damage-response pathways telomeres. TRF2 has a poorly characterized linker region (udTRF2 between its homodimerization and DNA-binding domains. Some lines of evidence have shown that this region could be involved in TRF2 interaction with nuclear lamina. Results: In this study, the fragment of the TERF2 gene encoding udTRF2 domain of telomere-binding protein TRF2 was produced by PCR and cloned into the pET32a vector. The resulting plasmid pET32a-udTRF2 was used for the expression of the recombinant udTRF2 in E. coli RosettaBlue (DE3. The protein was isolated and purified using ammonium sulfate precipitation followed by ion-exchange chromatography. The purified recombinant protein udTRF2 was injected into guinea pigs to generate polyclonal antibodies. The ability of anti-udTRF2 antibodies to bind endogenous TRF2 in human skin fibroblasts was tested by western blotting and immunofluorescent staining. Conclusions: In this study, the recombinant protein udTRF2 and antibodies to it were generated. Both protein and antibodies will provide a useful tool for investigation of the functions of the udTRF2 domain and its role in the interaction between TRF2 and nuclear lamina. Keywords: Chromosomes, Molecular cloning, Nuclear lamina, Nucleoprotein complexes, Polyclonal antibodies, Recombinant polypeptide, Shelterin, Telomere-binding protein TRF2, Telomeres, Telomeric DNA, TTAGGG repeats

  7. Heparin binding domain of antithrombin III: Characterization using a synthetic peptide directed polyclonal antibody

    International Nuclear Information System (INIS)

    Smith, J.W.; Dey, B.; Knauer, D.J.

    1990-01-01

    Antithrombin III (ATIII) is a plasma-borne serine protease inhibitor that apparently forms covalent complexes with thrombin. The interaction between ATIII and thrombin is enhanced several thousandfold by the glycosaminoglycan, heparin. The authors have previously proposed that the heparin binding site of ATIII residues within a region extending from amino acid residues 114-156. Computer-assisted analysis of this region revealed the presence of a 22 amino acid domain (residues 124-145), part of which shows a strong potential for the formation of an amphipathic helix: hydrophobic on one face and highly positively charged on the other. In the presence studies, polyclonal antisera were generated against a synthetic peptide corresponding to residues 124-145 in native human ATIII. Affinity-purified IgG from these antisera, as well as monovalent Fab's derived from them, specifically blocked the binding of heparin to ATIII. Additionally, occupancy of the heparin binding site by these same monovalent and bivalent IgG's at least partially substituted for heparin, accelerating linkage formation between ATIII and thrombin. These results provide the first immunological evidence that region 124-145 is directly involved in the binding of heparin to ATIII and that an antibody-induced conformational change within this region can mediate ATIII activation

  8. Radiometric immunosorbent assay for the detection of anti-hormone-binding protein antibodies

    International Nuclear Information System (INIS)

    Pierce, E.A.; Dame, M.C.; DeLuca, H.F.

    1986-01-01

    A radiometric immunosorbent assay (RISA) for the detection of monoclonal antibodies to hormone-binding proteins has been developed. The assay involves incubating hybridoma supernatants in microtiter wells that have been coated with goat anti-mouse IgG antibodies. Any mouse IgG in the test supernatant is thus specifically retained in the wells. Radioactive ligand-binding protein complexes are then incubated in the wells. The presence of anti-binding protein antibodies in the supernatant is indicated by specific retention of radioactive ligand-binding protein complexes in the wells. Crude antigen preparations, such as tissue homogenates, can be used to detect antibodies. The assay is capable of detecting antibody at concentrations 20 ng/ml (approx. 100 pM IgG). The RISA has been used successfully to screen for monoclonal antibodies to the intracellular receptor for 1,25-dihydroxyvitamin D 3 and should be useful for the detection of antibodies to ligand-binding proteins in general

  9. Basic studies on the tumor imaging using antibodies to human alpha-fetoprotein

    International Nuclear Information System (INIS)

    Sakahara, Harumi; Endo, Keigo; Nakashima, Tetsuo; Ohta, Hitoya; Torizuka, Kanji

    1984-01-01

    Using polyclonal antibodies to human α-fetoprotein (AFP), the effect of iodination on the antibody activity and tumor accumulation of radioiodinated antibodies in tumor bearing nude mice were examined. Antibodies, obtained from horse antiserum and purified by affinity chromatography, were iodinated by the chloramine-T method and their antibody activity was evaluated using RIA and Scatchard plot analysis. When high concentrations of chloramine-T were used or more than 2.6 iodine atoms were incorporated per antibody molecule, the antigen binding capacity rather than the affinity constant was affected by the iodination. The antibody activity was completely destroyed at an iodine to antibody molar ratio of 15.4. Antibodies, however, which were iodinated under low concentrations of chloramine-T and contained less than 0.8 iodines per antibody molecule, showed almost full retention of their antibody activity. Nude mice transplanted with AFP producing human testicular tumor or AFP non-producing human urinary bladder tumor were administered intravenously with 131 I-labeled antibodies to human AFP. Scintigrams were taken at 1, 2, 4 and 7 days after the injection of labeled antibodies. At day 7, nude mice were sacrificed and organs and tumor were removed, weighed and counted. In nude mice bearing testicular tumor, tumor image became gradually clear with decreasing background activity and tumor to blood ratio, obtained, was 0.82 for testicular tumor compared to 0.42 for bladder tumor. These results indicated a specific in vivo localization of 131 I-labeled antihuman AFP antibodies in AFP producing tumor. (author)

  10. 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

  11. Investigations on antibody binding to a micro-cantilever coated with a BAM pesticide residue

    DEFF Research Database (Denmark)

    Bache, Michael; Taboryski, Rafael Jozef; Schmid, Silvan

    2011-01-01

    -BAM antibody is measured using the CantiLab4© system from Cantion A/S with four gold-coated cantilevers and piezo resistive readout. The detection mechanism is in principle label-free, but fluorescent-marked antibodies have been used to subsequently verify the binding on the cantilever surface. The bending...

  12. Topographic antigenic determinants recognized by monoclonal antibodies on human choriogonadotropin beta-subunit

    International Nuclear Information System (INIS)

    Bidart, J.M.; Troalen, F.; Salesse, R.; Bousfield, G.R.; Bohuon, C.J.; Bellet, D.H.

    1987-01-01

    We describe a first attempt to study the antibody-combining sites recognized by monoclonal antibodies raised against the beta-subunit of human choriogonadotropin (hCG). Two groups of antibodies were first defined by their ability to recognize only the free beta-subunit or the free and combined subunit. Antibodies FBT-11 and FBT-11-L bind only to hCG beta-subunit but not to hCG, whereas antibodies FBT-10 and D1E8 bind to both the beta-subunit and the hormone. In both cases, the antigenic determinants were localized to the core of the protein (residues 1-112), indicating the weak immunogenicity of the specific carboxyl-terminal extension of hCG-beta. Nine synthetic peptides spanning different regions of hCG-beta and lutropin-beta were assessed for their capacity to inhibit antibody binding. A synthetic peptide inclusive of the NH2-terminal region (residues 1-7) of the hCG beta-subunit was found to inhibit binding to the radiolabeled subunit of a monoclonal antibody specific for free hCG-beta (FBT-11). Further delineation of the antigenic site recognized by this antibody provided evidence for the involvement of fragment 82-92. Moreover, monoclonal antibody FBT-11 inhibited the recombination of hCG-beta to hCG-alpha, indicating that its antigenic determinant might be located nearby or in the hCG-beta portion interacting with the alpha-subunit. Binding of monoclonal antibody FBT-10, corresponding to the second antigenic determinant, was weakly inhibited by fragment 82-105 and did not impair the recombination of the hCG beta-subunit to the hCG alpha-subunit. Its combining site appeared to be located in a region of the intact native choriogonadotropin present at the surface of the hormone-receptor complex

  13. 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

  14. 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.

  15. Binding of corroded ions to human saliva.

    Science.gov (United States)

    Mueller, H J

    1985-05-01

    Employing equilibrium dialysis, the binding abilities of Cu, Al, Co and Cr ions from corroded Cu-Al and Co-Cr dental casting alloys towards human saliva and two of its gel chromatographic fractions were determined. Results indicate that both Cu and Co bind to human saliva i.e. 0.045 and 0.027 mg/mg protein, respectively. Besides possessing the largest binding ability, Cu also possessed the largest binding capacity. The saturation of Cu binding was not reached up to the limit of 0.35 mg protein/ml employed in the tests, while Co reached full saturation at about 0.2 mg protein/ml. Chromium showed absolutely no binding to human saliva while Al ions did not pass through the dialysis membranes. Compared to the binding with solutions that were synthetically made up to contain added salivary-type proteins, it is shown that the binding to human saliva is about 1 order of magnitude larger, at least for Cu ions.

  16. Binding induced conformational changes of proteins correlate with their intrinsic fluctuations: a case study of antibodies

    Directory of Open Access Journals (Sweden)

    Keskin Ozlem

    2007-05-01

    Full Text Available Abstract Background How antibodies recognize and bind to antigens can not be totally explained by rigid shape and electrostatic complimentarity models. Alternatively, pre-existing equilibrium hypothesis states that the native state of an antibody is not defined by a single rigid conformation but instead with an ensemble of similar conformations that co-exist at equilibrium. Antigens bind to one of the preferred conformations making this conformation more abundant shifting the equilibrium. Results Here, two antibodies, a germline antibody of 36–65 Fab and a monoclonal antibody, SPE7 are studied in detail to elucidate the mechanism of antibody-antigen recognition and to understand how a single antibody recognizes different antigens. An elastic network model, Anisotropic Network Model (ANM is used in the calculations. Pre-existing equilibrium is not restricted to apply to antibodies. Intrinsic fluctuations of eight proteins, from different classes of proteins, such as enzymes, binding and transport proteins are investigated to test the suitability of the method. The intrinsic fluctuations are compared with the experimentally observed ligand induced conformational changes of these proteins. The results show that the intrinsic fluctuations obtained by theoretical methods correlate with structural changes observed when a ligand is bound to the protein. The decomposition of the total fluctuations serves to identify the different individual modes of motion, ranging from the most cooperative ones involving the overall structure, to the most localized ones. Conclusion Results suggest that the pre-equilibrium concept holds for antibodies and the promiscuity of antibodies can also be explained this hypothesis: a limited number of conformational states driven by intrinsic motions of an antibody might be adequate to bind to different antigens.

  17. Human Neutralizing Monoclonal Antibody Inhibition of Middle East Respiratory Syndrome Coronavirus Replication in the Common Marmoset.

    Science.gov (United States)

    Chen, Zhe; Bao, Linlin; Chen, Cong; Zou, Tingting; Xue, Ying; Li, Fengdi; Lv, Qi; Gu, Songzhi; Gao, Xiaopan; Cui, Sheng; Wang, Jianmin; Qin, Chuan; Jin, Qi

    2017-06-15

    Middle East respiratory syndrome coronavirus (MERS-CoV) infection in humans is highly lethal, with a fatality rate of 35%. New prophylactic and therapeutic strategies to combat human infections are urgently needed. We isolated a fully human neutralizing antibody, MCA1, from a human survivor. The antibody recognizes the receptor-binding domain of MERS-CoV S glycoprotein and interferes with the interaction between viral S and the human cellular receptor human dipeptidyl peptidase 4 (DPP4). To our knowledge, this study is the first to report a human neutralizing monoclonal antibody that completely inhibits MERS-CoV replication in common marmosets. Monotherapy with MCA1 represents a potential alternative treatment for human infections with MERS-CoV worthy of evaluation in clinical settings. © Crown copyright 2017.

  18. A radioisotope dilution assay for unlabelled vitamin B12-intrinsic factor complex employing the binding intrinsic factor antibody: probable evidence for two types of binding antibody

    International Nuclear Information System (INIS)

    Jacob, E.; O'Brien, H.A.W.; Mollin, D.L.

    1977-01-01

    A new radioisotope dilution assay for vitamin B 12 -intrinsic factor complex is described. The method is based on the use of the binding type intrinsic antibody (the binding reagent), which when combined with the intrinsic factor-vitamin B 12 complex (labelled ligand), is quantitatively adsorbed onto zirconium phosphate gel pH 6.25. The new assay has been shown to provide a measure of intrinsic factor comparable with other intrinsic factor assays, but it has the important advantage of being able to measure the unlabelled vitamin B 12 -intrinsic factor complex (unlabelled ligand), and will, therefore, be valuable in the study of physiological events in the gastrointestinal tract. During the study, it was found that there is some evidence for at least two types of binding intrinsic factor antibody: One which combines preferentially with the intrinsic factor-vitamin B 12 complex and one which combines equally well with this complex or with free intrinsic factor. (author)

  19. Heparin-independent, PF4-dependent binding of HIT antibodies to platelets: implications for HIT pathogenesis.

    Science.gov (United States)

    Padmanabhan, Anand; Jones, Curtis G; Bougie, Daniel W; Curtis, Brian R; McFarland, Janice G; Wang, Demin; Aster, Richard H

    2015-01-01

    Antibodies specific for platelet factor 4 (PF4)/heparin complexes are the hallmark of heparin-induced thrombocytopenia and thrombosis (HIT), but many antibody-positive patients have normal platelet counts. The basis for this is not fully understood, but it is believed that antibodies testing positive in the serotonin release assay (SRA) are the most likely to cause disease. We addressed this issue by characterizing PF4-dependent binding of HIT antibodies to intact platelets and found that most antibodies testing positive in the SRA, but none of those testing negative, bind to and activate platelets when PF4 is present without any requirement for heparin (P HIT antibodies recognize PF4 in a complex with heparin, only a subset of these antibodies recognize more subtle epitopes induced in PF4 when it binds to CS, the major platelet glycosaminoglycan. Antibodies having this property could explain "delayed HIT" seen in some individuals after discontinuation of heparin and the high risk for thrombosis that persists for weeks in patients recovered from HIT. © 2015 by The American Society of Hematology.

  20. Molecular characterization of two sub-family specific monoclonal antibodies to meningococcal Factor H binding protein

    Directory of Open Access Journals (Sweden)

    C. Lo Passo

    2018-04-01

    Full Text Available Factor H binding protein (FHbp is a component of two licensed vaccines for prevention of sepsis and meningitis caused by serogroup B meningococci. FHbp binds human Factor H (FH, which contributes to evasion of host immunity and FHbp sequence variants can be classified into two sub-families. Antibodies against FHbp elicit complement-mediated killing and can inhibit recruitment of FH to the bacterial surface. We report epitope mapping studies of two murine IgG mAbs, designated JAR 31 and JAR 36, isolated from a mouse immunized with FHbp in sub-family A, which is present in ∼30–40% of invasive isolates. In the present study, we tested the reactivity of mAbs JAR 31 and JAR 36 with seven natural FHbp sequence variants from different phylogenic groups. We screened bacteriophage-displayed peptide libraries to identify amino acid residues contributing to the JAR 36 epitope. Based on the reactivities of mAbs JAR 31 and JAR 36 with the seven FHbp variants, and the frequent occurrences of aspartate (D and lysine (K residues in the JAR 36-bound phage peptides, we selected six residues in the carboxyl-terminal region of FHbp for replacement with alanine (A. The D201A and K203A substitutions respectively eliminated and decreased binding of mAbs JAR 31 and JAR 36 to FHbp. These substitutions did not affect binding of the control mAb JAR 33 or of human FH. JAR 31 or JAR 36 mediated cooperative complement-mediated bactericidal activity with other anti-FHbp mAbs. The identification of two amino acid residues involved in the epitopes recognized by these anti-FHbp mAbs may contribute to a more complete understanding of the spatial requirements for cooperative anti-FHbp mAb bactericidal activity. Keywords: Biochemistry, Immunology, Microbiology, Molecular biology

  1. HIV-1 binding and neutralizing antibodies of injecting drug users

    Directory of Open Access Journals (Sweden)

    E.P. Ouverney

    2005-09-01

    Full Text Available Previous studies have demonstrated a stronger seroreactivity against some synthetic peptides responsible for inducing neutralizing antibodies in injecting drug users (IDU compared to that of individuals sexually infected with HIV-1 (S, but the effectiveness in terms of the neutralizing ability of these antibodies has not been evaluated. Our objective was to study the humoral immune response of IDU by determining the specificity of their antibodies and the presence of neutralizing antibodies. The neutralization capacity against the HIV-1 isolate MN (genotype B, the primary HIV-1 isolate 95BRRJ021 (genotype F, and the seroreactivity with peptides known to induce neutralizing antibodies, from the V2 and V3 loops of different HIV-1 subtypes, were analyzed. Seroreactivity indicates that IDU plasma are more likely to recognize a broader range of peptides than S plasma, with significantly higher titers, especially of V3 peptides. Similar neutralization frequencies of the MN isolate were observed in plasma of the IDU (16/47 and S (20/60 groups in the 1:10 dilution. The neutralization of the 95BRRJ021 isolate was more frequently observed for plasma from the S group (15/23 than from the IDU group (15/47, P = 0.0108. No correlation between neutralization and seroreactivity with the peptides tested was observed. These results suggest that an important factor responsible for the extensive and broad humoral immune response observed in IDU is their infection route. There was very little difference in neutralizing antibody response between the IDU and S groups despite their differences in seroreactivity and health status.

  2. Identification of actin binding protein, ABP-280, as a binding partner of human Lnk adaptor protein.

    Science.gov (United States)

    He, X; Li, Y; Schembri-King, J; Jakes, S; Hayashi, J

    2000-08-01

    Human Lnk (hLnk) is an adaptor protein with multiple functional domains that regulates T cell activation signaling. In order to identify cellular Lnk binding partners, a yeast two-hybrid screening of human spleen cDNA library was carried out using human hLnk as bait. A polypeptide sequence identical to the C-terminal segment of the actin binding protein (ABP-280) was identified as a hLnk binding protein. The expressed hLnk and the FLAG tagged C-terminal 673 amino acid residues of ABP-280 or the endogenous ABP-280 in COS-7 cells could be co-immunoprecipitated using antibodies either to hLnk, FLAG or ABP-280, respectively. Furthermore, immunofluorescence confocal microscope showed that hLnk and ABP-280 co-localized at the plasma membrane and at juxtanuclear region of COS-7 cells. In Jurkat cells, the endogenous hLnk also associates with the endogenous ABP-280 indicating that the association of these two proteins is physiological. The interacting domains of both proteins were mapped using yeast two-hybrid assays. Our results indicate that hLnk binds to the residues 2006-2454 (repeats 19-23C) of ABP-280. The domain in hLnk that associates with ABP-280 was mapped to an interdomain region of 56 amino acids between pleckstrin homology and Src homology 2 domains. These results suggest that hLnk may exert its regulatory role through its association with ABP-280.

  3. Human Cell Line-Derived Monoclonal IgA Antibodies for Cancer Immunotherapy

    Directory of Open Access Journals (Sweden)

    Felix Hart

    2017-05-01

    Full Text Available IgA antibodies have great potential to improve the functional diversity of current IgG antibody-based cancer immunotherapy options. However, IgA production and purification is not well established, which can at least in part be attributed to the more complex glycosylation as compared to IgG antibodies. IgA antibodies possess up to five N-glycosylation sites within their constant region of the heavy chain as compared to one site for IgG antibodies. The human GlycoExpress expression system was developed to produce biotherapeutics with optimized glycosylation and used here to generate a panel of IgA isotype antibodies directed against targets for solid (TA-mucin 1, Her2, EGFR, Thomsen–Friedenreich and hematological (CD20 cancer indications. The feasibility of good manufacturing practice was shown by the production of 11 g IgA within 35 days in a one liter perfusion bioreactor, and IgA antibodies in high purity were obtained after purification. The monoclonal IgA antibodies possessed a high sialylation degree, and no non-human glycan structures were detected. Kinetic analysis revealed increased avidity antigen binding for IgA dimers as compared to monomeric antibodies. The IgA antibodies exhibited potent Fab- and Fc-mediated functionalities against cancer cell lines, whereby especially granulocytes are recruited. Therefore, for patients who do not sufficiently benefit from therapeutic IgG antibodies, IgA antibodies may complement current regiment options and represent a promising strategy for cancer immunotherapy. In conclusion, a panel of novel biofunctional IgA antibodies with human glycosylation was successfully generated.

  4. Generation of human Fab antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences.

    Science.gov (United States)

    Andris-Widhopf, Jennifer; Steinberger, Peter; Fuller, Roberta; Rader, Christoph; Barbas, Carlos F

    2011-09-01

    The development of therapeutic antibodies for use in the treatment of human diseases has long been a goal for many researchers in the antibody field. One way to obtain these antibodies is through phage-display libraries constructed from human lymphocytes. This protocol describes the construction of human Fab (fragment antigen binding) antibody libraries. In this method, the individual rearranged heavy- and light-chain variable regions are amplified separately and are linked through a series of overlap polymerase chain reaction (PCR) steps to give the final Fab products that are used for cloning.

  5. 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.)

  6. Characterization of Binding Epitopes of CA125 Monoclonal Antibodies

    DEFF Research Database (Denmark)

    Marcos-Silva, Lara; Narimatsu, Yoshiki; Halim, Adnan

    2014-01-01

    The most used cancer serum biomarker is the CA125 immunoassay for ovarian cancer that detects the mucin glycoprotein MUC16. Several monoclonal antibodies (mAbs) including OC125 and M11 are used in CA125 assays. However, despite considerable efforts, our knowledge of the molecular characteristics...

  7. Conformational Occlusion of Blockade Antibody Epitopes, a Novel Mechanism of GII.4 Human Norovirus Immune Evasion.

    Science.gov (United States)

    Lindesmith, Lisa C; Mallory, Michael L; Debbink, Kari; Donaldson, Eric F; Brewer-Jensen, Paul D; Swann, Excel W; Sheahan, Timothy P; Graham, Rachel L; Beltramello, Martina; Corti, Davide; Lanzavecchia, Antonio; Baric, Ralph S

    2018-01-01

    Extensive antigenic diversity within the GII.4 genotype of human norovirus is a major driver of pandemic emergence and a significant obstacle to development of cross-protective immunity after natural infection and vaccination. However, human and mouse monoclonal antibody studies indicate that, although rare, antibodies to conserved GII.4 blockade epitopes are generated. The mechanisms by which these epitopes evade immune surveillance are uncertain. Here, we developed a new approach for identifying conserved GII.4 norovirus epitopes. Utilizing a unique set of virus-like particles (VLPs) representing the in vivo -evolved sequence diversity within an immunocompromised person, we identify key residues within epitope F, a conserved GII.4 blockade antibody epitope. The residues critical for antibody binding are proximal to evolving blockade epitope E. Like epitope F, antibody blockade of epitope E was temperature sensitive, indicating that particle conformation regulates antibody access not only to the conserved GII.4 blockade epitope F but also to the evolving epitope E. These data highlight novel GII.4 mechanisms to protect blockade antibody epitopes, map essential residues of a GII.4 conserved epitope, and expand our understanding of how viral particle dynamics may drive antigenicity and antibody-mediated protection by effectively shielding blockade epitopes. Our data support the notion that GII.4 particle breathing may well represent a major mechanism of humoral immune evasion supporting cyclic pandemic virus persistence and spread in human populations. IMPORTANCE In this study, we use norovirus virus-like particles to identify key residues of a conserved GII.4 blockade antibody epitope. Further, we identify an additional GII.4 blockade antibody epitope to be occluded, with antibody access governed by temperature and particle dynamics. These findings provide additional support for particle conformation-based presentation of binding residues mediated by a particle

  8. Many Routes to an Antibody Heavy-Chain CDR3: Necessary, Yet Insufficient, for Specific Binding

    Science.gov (United States)

    D’Angelo, Sara; Ferrara, Fortunato; Naranjo, Leslie; Erasmus, M. Frank; Hraber, Peter; Bradbury, Andrew R. M.

    2018-01-01

    Because of its great potential for diversity, the immunoglobulin heavy-chain complementarity-determining region 3 (HCDR3) is taken as an antibody molecule’s most important component in conferring binding activity and specificity. For this reason, HCDR3s have been used as unique identifiers to investigate adaptive immune responses in vivo and to characterize in vitro selection outputs where display systems were employed. Here, we show that many different HCDR3s can be identified within a target-specific antibody population after in vitro selection. For each identified HCDR3, a number of different antibodies bearing differences elsewhere can be found. In such selected populations, all antibodies with the same HCDR3 recognize the target, albeit at different affinities. In contrast, within unselected populations, the majority of antibodies with the same HCDR3 sequence do not bind the target. In one HCDR3 examined in depth, all target-specific antibodies were derived from the same VDJ rearrangement, while non-binding antibodies with the same HCDR3 were derived from many different V and D gene rearrangements. Careful examination of previously published in vivo datasets reveals that HCDR3s shared between, and within, different individuals can also originate from rearrangements of different V and D genes, with up to 26 different rearrangements yielding the same identical HCDR3 sequence. On the basis of these observations, we conclude that the same HCDR3 can be generated by many different rearrangements, but that specific target binding is an outcome of unique rearrangements and VL pairing: the HCDR3 is necessary, albeit insufficient, for specific antibody binding. PMID:29568296

  9. Bispecific antibodies targeting human CD73

    DEFF Research Database (Denmark)

    2017-01-01

    The present invention relates to a bispecific antibody targeting CD73. In particular, the present invention relates to a bispecific antibody targeting different epitopes on CD73 or a bispecific antibody targeting an epitope on CD73 and an epitope on a different antigen.......The present invention relates to a bispecific antibody targeting CD73. In particular, the present invention relates to a bispecific antibody targeting different epitopes on CD73 or a bispecific antibody targeting an epitope on CD73 and an epitope on a different antigen....

  10. Agonistic Human Monoclonal Antibodies against Death Receptor 4 (DR4) | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    The National Cancer Institute is seeking parties interested in licensing human monoclonal antibodies (mAbs) that bind to death receptor 4 ("DR4"). The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its functional receptors, DR4 and DR5, have been recognized as promising targets for cancer treatment.

  11. Monoclonal antibody to the rat glucocorticoid receptor. Relationship between the immunoreactive and DNA-binding domain

    International Nuclear Information System (INIS)

    Eisen, L.P.; Reichman, M.E.; Thompson, E.B.; Gametchu, B.; Harrison, R.W.; Eisen, H.J.

    1985-01-01

    The region of the glucocorticoid receptor that reacted with a monoclonal antibody (BUGR-1) was identified. In order to identify the immunoreactive region, the rat liver glucocorticoid receptor was subjected to limited proteolysis; immunoreactive fragments were identified by Western blotting. The monoclonal antibody reacted with both the undigested Mr approximately 97,000 receptor subunit and a Mr approximately 45,000 fragment containing the steroid-binding and DNA-binding domains. Digestion by trypsin also produced two steroid-binding fragments of Mr approximately 27,000 and 31,000 which did not react with the antibody and an immunoreactive Mr approximately 16,000 fragment. This Mr approximately 16,000 fragment was shown to bind to DNA-cellulose, indicating that it contained a DNA-binding domain of the receptor. The undigested receptor must have steroid associated with it to undergo activation to a DNA-binding form. However, the Mr approximately 16,000 immunoreactive fragment binds to DNA-cellulose even if it is obtained by digestion of the steroid-free holoreceptor which does not itself bind to DNA

  12. (TH) diazepam binding to human granulocytes

    Energy Technology Data Exchange (ETDEWEB)

    Bond, P.A.; Cundall, R.L.; Rolfe, B.

    1985-07-08

    (TH)-diazepam binds to sites on human granulocyte membranes, with little or no binding to platelets or lymphocytes. These (TH)-diazepam binding sites are of the peripheral type, being strongly inhibited by R05-4864 (Ki=6.23nM) but only weakly by clonazepam (Ki=14 M). Binding of (TH) diazepam at 0 is saturable, specific and stereoselective. Scatchard analysis indicates a single class of sites with Bmax of 109 +/- 17f moles per mg of protein and K/sub D/ of 3.07 +/- 0.53nM. Hill plots of saturation experiments gave straight lines with a mean Hill coefficient of 1.03 +/- 0.014. Binding is time dependent and reversible and it varies linearly with granulocyte protein concentration over the range 0.025-0.300 mg of protein. 11 references, 3 figures, 1 table.

  13. DNA-mediated strand displacement facilitates sensitive electronic detection of antibodies in human serums.

    Science.gov (United States)

    Dou, Baoting; Yang, Jianmei; Shi, Kai; Yuan, Ruo; Xiang, Yun

    2016-09-15

    We describe here the development of a sensitive and convenient electronic sensor for the detection of antibodies in human serums. The sensor is constructed by self-assembly formation of a mixed monolayer containing the small molecule epitope conjugated double stranded DNA probes on gold electrode. The target antibody binds the epitope on the dsDNA probe and lowers the melting temperature of the duplex, which facilitates the displacement of the antibody-linked strand of the duplex probe by an invading methylene blue-tagged single stranded DNA (MB-ssDNA) through the strand displacement reaction and leads to the capture of many MB-ssDNA on the sensor surface. Subsequent electrochemical oxidation of the methylene blue labels results in amplified current response for sensitive monitoring of the antibodies. The antibody assay conditions are optimized and the sensor exhibits a linear range between 1.0 and 25.0nM with a detection limit of 0.67nM for the target antibody. The sensor is also selective and can be employed to detect the target antibodies in human serum samples. With the advantages of using small molecule epitope as the antibody recognition element over traditional antigen, the versatile manipulability of the DNA probes and the unique properties of the electrochemical transduction technique, the developed sensor thus hold great potential for simple and sensitive detection of different antibodies and other proteins in real samples. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Endocytosis of Integrin-Binding Human Picornaviruses

    Directory of Open Access Journals (Sweden)

    Pirjo Merilahti

    2012-01-01

    Full Text Available Picornaviruses that infect humans form one of the largest virus groups with almost three hundred virus types. They include significant enteroviral pathogens such as rhino-, polio-, echo-, and coxsackieviruses and human parechoviruses that cause wide range of disease symptoms. Despite the economic importance of picornaviruses, there are no antivirals. More than ten cellular receptors are known to participate in picornavirus infection, but experimental evidence of their role in cellular infection has been shown for only about twenty picornavirus types. Three enterovirus types and one parechovirus have experimentally been shown to bind and use integrin receptors in cellular infection. These include coxsackievirus A9 (CV-A9, echovirus 9, and human parechovirus 1 that are among the most common and epidemic human picornaviruses and bind to αV-integrins via RGD motif that resides on virus capsid. In contrast, echovirus 1 (E-1 has no RGD and uses integrin α2β1 as cellular receptor. Endocytosis of CV-A9 has recently been shown to occur via a novel Arf6- and dynamin-dependent pathways, while, contrary to collagen binding, E-1 binds inactive β1 integrin and enters via macropinocytosis. In this paper, we review what is known about receptors and endocytosis of integrin-binding human picornaviruses.

  15. Computational identification of antigen-binding antibody fragments.

    Science.gov (United States)

    Burkovitz, Anat; Leiderman, Olga; Sela-Culang, Inbal; Byk, Gerardo; Ofran, Yanay

    2013-03-01

    Determining which parts of the Ab are essential for Ag recognition and binding is crucial for understanding B cell-mediated immunity. Identification of fragments of Abs that maintain specificity to the Ag will also allow for the development of improved Ab-based therapy and diagnostics. In this article, we show that structural analysis of Ab-Ag complexes reveals which fragments of the Ab may bind the Ag on their own. In particular, it is possible to predict whether a given CDR is likely to bind the Ag as a peptide by analyzing the energetic contribution of each CDR to Ag binding and by assessing to what extent the interaction between that CDR and the Ag depends on other CDRs. To demonstrate this, we analyzed five Ab-Ag complexes and predicted for each of them which of the CDRs may bind the Ag on its own as a peptide. We then show that these predictions are in agreement with our experimental analysis and with previously published experimental results. These findings promote our understanding of the modular nature of Ab-Ag interactions and lay the foundation for the rational design of active CDR-derived peptides.

  16. Construction of human antibody gene libraries and selection of antibodies by phage display.

    Science.gov (United States)

    Frenzel, André; Kügler, Jonas; Wilke, Sonja; Schirrmann, Thomas; Hust, Michael

    2014-01-01

    Antibody phage display is the most commonly used in vitro selection technology and has yielded thousands of useful antibodies for research, diagnostics, and therapy.The prerequisite for successful generation and development of human recombinant antibodies using phage display is the construction of a high-quality antibody gene library. Here, we describe the methods for the construction of human immune and naive scFv gene libraries.The success also depends on the panning strategy for the selection of binders from these libraries. In this article, we describe a panning strategy that is high-throughput compatible and allows parallel selection in microtiter plates.

  17. At least two Fc Neu5Gc residues of monoclonal antibodies are required for binding to anti-Neu5Gc antibody

    OpenAIRE

    Yu, Chuanfei; Gao, Kai; Zhu, Lei; Wang, Wenbo; Wang, Lan; Zhang, Feng; Liu, Chunyu; Li, Meng; Wormald, Mark R.; Rudd, Pauline M.; Wang, Junzhi

    2016-01-01

    Two non-human glycan epitopes, galactose-Į-1,3-galactose (Į-gal) and Neu5Gc-Į-2-6-galactose (Neu5Gc) have been shown to be antigenic when attached to Fab oligosaccharides of monoclonal antibodies (mAbs) , while Į-gal attached to Fc glycans were not. However, the antigenicity of Neu5Gc on the Fc glycans remains unclear in the context that most mAbs carry only Fc glycans. After studying two clinical mAbs carrying significant amounts of Fc Neu5Gc, we show that their binding activity with anti-Ne...

  18. 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.

  19. 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.

  20. Generation and analyses of human synthetic antibody libraries and their application for protein microarrays

    DEFF Research Database (Denmark)

    Säll, Anna; Walle, Maria; Wingren, Christer

    2016-01-01

    in a high-throughput manner. To address this we designed and constructed two human synthetic antibody fragment (scFv) libraries denoted HelL-11 and HelL-13. By the use of phage display technology, in total 466 unique scFv antibodies specific for 114 different antigens were generated. The specificities......Antibody-based proteomics offers distinct advantages in the analysis of complex samples for discovery and validation of biomarkers associated with disease. However, its large-scale implementation requires tools and technologies that allow development of suitable antibody or antibody fragments...... for diagnostics and therapeutics. In addition, this work provides a great example on how a synthetic approach can be used to optimize library designs. By having precise control of the diversity introduced into the antigen-binding sites, synthetic libraries offer increased understanding of how different diversity...

  1. Mouse monoclonal antibodies against human c-Mpl and characterization for flow cytometry applications.

    Science.gov (United States)

    Abbott, Christina; Huang, Guo; Ellison, Aaron R; Chen, Ching; Arora, Taruna; Szilvassy, Stephen J; Wei, Ping

    2010-04-01

    Mouse monoclonal antibodies (MAbs) against human c-Mpl, the cognate receptor for thrombopoietin (TPO), were generated using hybridoma technology and characterized by various assays to demonstrate their specificity and affinity. Two such MAbs, 1.6 and 1.75, were determined to be superior for flow cytometry studies and exhibited double-digit picomolar (pM) affinities to soluble human c-Mpl protein. Both MAbs specifically bound to cells engineered to overexpress human c-Mpl protein, immortalized human hematopoietic cell lines that express endogenous c-Mpl, primary human bone marrow and peripheral blood-derived CD34(+) cells, and purified human platelets. No binding was detected on cell lines that did not express c-Mpl. Receptor competition and siRNA knock-down studies further confirmed the specificity of antibodies 1.6 and 1.75 for human c-Mpl. In contrast to these newly generated MAbs, none of eight commercially available anti-c-Mpl antibodies tested were found to bind specifically to human c-Mpl and were thus shown to be unsuitable for flow cytometry studies. Monoclonal antibodies 1.6 and 1.75 will therefore be useful flow cytometry reagents to detect cell surface c-Mpl expression.

  2. Isolation of a monoclonal antibody from a phage display library binding the rhesus macaque MHC class I allomorph Mamu-A1*001.

    Directory of Open Access Journals (Sweden)

    Nathan Holman

    Full Text Available Monoclonal antibodies that bind to human leukocyte antigen (HLA are useful tools for HLA-typing, tracking donor-recipient chimerisms after bone marrow transplants, and characterizing specific major histocompatibility complexes (MHC on cell surfaces. Unfortunately, equivalent reagents are not available for rhesus macaques, which are commonly used animal as models in organ transplant and infectious disease research. To address this deficiency, we isolated an antibody that recognizes the common Indian rhesus macaque MHC class I molecule, Mamu-A1*001. We induced Mamu-A1*001-binding antibodies by alloimmunizing a female Mamu-A1*001-negative rhesus macaque with peripheral blood mononuclear cells (PBMC from a male Mamu-A1*001-positive donor. A Fab phage display library was constructed with PBMC from the alloimmunized macaque and panned to isolate an antibody that binds to Mamu-A1*001 but not to other common rhesus macaque MHC class I molecules. The isolated antibody distinguishes PBMC from Mamu-A1*001-positive and -negative macaques. Additionally, the Mamu-A1*001-specific antibody binds the cynomolgus macaque MHC class I ortholog Mafa-A1*001:01 but not variants Mafa-A1*001:02/03, indicating a high degree of binding specificity. The Mamu-A1*001-specific antibody will be useful for identifying Mamu-A1*001-positive rhesus macaques, for detecting Mamu-A1*001-positive cells in populations of Mamu-A1*001-negative cells, and for examining disease processes that alter expression of Mamu-A1*001 on cell surfaces. Moreover, the alloimmunization process we describe will be useful for isolating additional MHC allomorph-specific monoclonal antibodies or antibodies against other polymorphic host proteins which are difficult to isolate with traditional technologies.

  3. Epitope mapping and characterization of a novel CD4-induced human monoclonal antibody capable of neutralizing primary HIV-1 strains

    International Nuclear Information System (INIS)

    Xiang Shihua; Wang Liping; Abreu, Mariam; Huang, C.-C.; Kwong, Peter D.; Rosenberg, Eric; Robinson, James E.; Sodroski, Joseph

    2003-01-01

    Human immunodeficiency virus (HIV-1) enters target cells by binding its gp120 exterior envelope glycoprotein to CD4 and one of the chemokine receptors, CCR5 or CXCR4. CD4-induced (CD4i) antibodies bind gp120 more efficiently after CD4 binding and block the interaction with the chemokine receptor. Examples of CD4i antibodies are limited, and the prototypes of the CD4i antibodies exhibit only weak neutralizing activity against primary, clinical HIV-1 isolates. Here we report the identification of a novel antibody, E51, that exhibits CD4-induced binding to gp120 and neutralizes primary HIV-1 more efficiently than the prototypic CD4i antibodies. The E51 antibody blocks the interaction of gp120-CD4 complexes with CCR5 and binds to a highly conserved, basic gp120 element composed of the β19-strand and surrounding structures. Thus, on primary HIV-1 isolates, this gp120 region, which has been previously implicated in chemokine receptor binding, is accessible to a subset of CD4i antibodies

  4. 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

  5. Novel peptide ligand with high binding capacity for antibody purification

    DEFF Research Database (Denmark)

    Lund, L. N.; Gustavsson, P. E.; Michael, R.

    2012-01-01

    Small synthetic ligands for protein purification have become increasingly interesting with the growing need for cheap chromatographic materials for protein purification and especially for the purification of monoclonal antibodies (mAbs). Today, Protein A-based chromatographic resins are the most...... commonly used capture step in mAb down stream processing; however, the use of Protein A chromatography is less attractive due to toxic ligand leakage as well as high cost. Whether used as an alternative to the Protein A chromatographic media or as a subsequent polishing step, small synthetic peptide...... ligands have an advantage over biological ligands; they are cheaper to produce, ligand leakage by enzymatic degradation is either eliminated or significantly reduced, and they can in general better withstand cleaning in place (CIP) conditions such as 0.1 M NaOH. Here, we present a novel synthetic peptide...

  6. Molluskan Hemocyanins Activate the Classical Pathway of the Human Complement System through Natural Antibodies.

    Science.gov (United States)

    Pizarro-Bauerle, Javier; Maldonado, Ismael; Sosoniuk-Roche, Eduardo; Vallejos, Gerardo; López, Mercedes N; Salazar-Onfray, Flavio; Aguilar-Guzmán, Lorena; Valck, Carolina; Ferreira, Arturo; Becker, María Inés

    2017-01-01

    Molluskan hemocyanins are enormous oxygen-carrier glycoproteins that show remarkable immunostimulatory properties when inoculated in mammals, such as the generation of high levels of antibodies, a strong cellular reaction, and generation of non-specific antitumor immune responses in some types of cancer, particularly for superficial bladder cancer. These proteins have the ability to bias the immune response toward a T h 1 phenotype. However, despite all their current uses with beneficial clinical outcomes, a clear mechanism explaining these properties is not available. Taking into account reports of natural antibodies against the hemocyanin of the gastropod Megathura crenulata [keyhole limpet hemocyanin (KLH)] in humans as well as other vertebrate species, we report here for the first time, the presence, in sera from unimmunized healthy donors, of antibodies recognizing, in addition to KLH, two other hemocyanins from gastropods with documented immunomodulatory capacities: Fisurella latimarginata hemocyanin (FLH) and Concholepas concholepas hemocyanin (CCH). Through an ELISA screening, we found IgM and IgG antibodies reactive with these hemocyanins. When the capacity of these antibodies to bind deglycosylated hemocyanins was studied, no decreased interaction was detected. Moreover, in the case of FLH, deglycosylation increased antibody binding. We evaluated through an in vitro complement deposition assay whether these antibodies activated the classical pathway of the human complement system. The results showed that all three hemocyanins and their deglycosylated counterparts elicited this activation, mediated by C1 binding to immunoglobulins. Thus, this work contributes to the understanding on how the complement system could participate in the immunostimulatory properties of hemocyanins, through natural, complement-activating antibodies reacting with these proteins. Although a role for carbohydrates cannot be completely ruled out, in our experimental setting

  7. FcγRII-binding Centyrins mediate agonism and antibody-dependent cellular phagocytosis when fused to an anti-OX40 antibody.

    Science.gov (United States)

    Zhang, Di; Whitaker, Brian; Derebe, Mehabaw G; Chiu, Mark L

    2018-04-01

    Immunostimulatory antibodies against the tumor necrosis factor receptors (TNFR) are emerging as promising cancer immunotherapies. The agonism activity of such antibodies depends on crosslinking to Fc gamma RIIB receptor (FcγRIIB) to enable the antibody multimerization that drives TNFR activation. Previously, Fc engineering was used to enhance the binding of such antibodies to Fcγ receptors. Here, we report the identification of Centyrins as alternative scaffold proteins with binding affinities to homologous FcγRIIB and FcγRIIA, but not to other types of Fcγ receptors. One Centyrin, S29, was engineered at distinct positions of an anti-OX40 SF2 antibody to generate bispecific and tetravalent molecules named as mAbtyrins. Regardless of the position of S29 on the SF2 antibody, SF2-S29 mAbtyrins could bind FcγRIIB and FcγRIIA specifically while maintaining binding to OX40 receptors. In a NFκB reporter assay, attachment of S29 Centyrin molecules at the C-termini, but not the N-termini, resulted in SF2 antibodies with increased agonism owing to FcγRIIB crosslinking. The mAbtyrins also showed agonism in T-cell activation assays with immobilized FcγRIIB and FcγRIIA, but this activity was confined to mAbtyrins with S29 specifically at the C-termini of antibody heavy chains. Furthermore, regardless of the position of the molecule, S29 Centyrin could equip an otherwise Fc-silent antibody with antibody-dependent cellular phagocytosis activity without affecting the antibody's intrinsic antibody-dependent cell-meditated cytotoxicity and complement-dependent cytotoxicity. In summary, the appropriate adoption FcγRII-binding Centyrins as functional modules represents a novel strategy to engineer therapeutic antibodies with improved functionalities.

  8. Characterisation of monoclonal antibodies for human luteinising hormone, and mapping of antigenic determinants on the hormone

    International Nuclear Information System (INIS)

    Soos, M.; Siddle, K.

    1983-01-01

    Twelve mouse monoclonal antibodies for human luteinising hormone were produced. The affinities varied from 4 X 10 7 to 1 X 10 10 l/mol. The specificity of each antibody was assessed by determining the relative reactivities with luteinising hormone, thyroid stimulating hormone, follicle stimulating hormone and chorionic gonadotrophin. Six antibodies bound to the α-subunit as shown by similar reactivity with all hormones, and the remainder to the β-subunit as shown by specificity for luteinising hormone. This latter group of antibodies cross-reacted only weakly with thyroid stimulating hormone (approximately 10%) and follicle stimulating hormone (approximately 3%). Three of these antibodies also showed low reactivity towards chorionic gonadotrophin (<10%), though the others did not (80-300%). The ability of different antibodies to bind simultaneously to luteinising hormone was examined and it was shown that several distinct antigenic determinants existed on both subunits. The characterisation of monoclonal binding sites is discussed in relation to the use of antibodies in two-site immunoradiometric assays. (Auth.)

  9. Generation and analyses of human synthetic antibody libraries and their application for protein microarrays.

    Science.gov (United States)

    Säll, Anna; Walle, Maria; Wingren, Christer; Müller, Susanne; Nyman, Tomas; Vala, Andrea; Ohlin, Mats; Borrebaeck, Carl A K; Persson, Helena

    2016-10-01

    Antibody-based proteomics offers distinct advantages in the analysis of complex samples for discovery and validation of biomarkers associated with disease. However, its large-scale implementation requires tools and technologies that allow development of suitable antibody or antibody fragments in a high-throughput manner. To address this we designed and constructed two human synthetic antibody fragment (scFv) libraries denoted HelL-11 and HelL-13. By the use of phage display technology, in total 466 unique scFv antibodies specific for 114 different antigens were generated. The specificities of these antibodies were analyzed in a variety of immunochemical assays and a subset was further evaluated for functionality in protein microarray applications. This high-throughput approach demonstrates the ability to rapidly generate a wealth of reagents not only for proteome research, but potentially also for diagnostics and therapeutics. In addition, this work provides a great example on how a synthetic approach can be used to optimize library designs. By having precise control of the diversity introduced into the antigen-binding sites, synthetic libraries offer increased understanding of how different diversity contributes to antibody binding reactivity and stability, thereby providing the key to future library optimization. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  10. Rheumatoid factor interference in immunogenicity assays for human monoclonal antibody therapeutics.

    Science.gov (United States)

    Tatarewicz, Suzanna; Miller, Jill M; Swanson, Steven J; Moxness, Michael S

    2010-05-31

    Rheumatoid factors (RFs) are endogenous human antibodies that bind to human gamma globulins. RFs demonstrate preferential binding to aggregated gamma globulins and are involved in the clearing mechanism of immune complexes. Immunoassays designed to measure human anti-human antibodies (HAHA) after administration of monoclonal antibody therapeutics are thus vulnerable to interference from RFs. When using a sensitive electrochemiluminescent (ECL) bridging immunoassay, samples from subjects with rheumatoid arthritis demonstrated much higher baseline reactivity than healthy subjects. Interference was found to be dependent on the aggregation state of the therapeutic antibody that had been conjugated with the detection reagent (ruthenium). Size exclusion high performance liquid chromatography (SE-HPLC) demonstrated that of the total integrated peaks, as little as 0.55% high molecular weight aggregates (>600kDa) were sufficient to cause increased reactivity. Stability studies of the ruthenium and biotin conjugated therapeutic antibody indicated that storage time, temperature and buffer formulation were critical in maintaining the integrity of the reagents. Through careful SE-HPLC monitoring we were able to choose appropriate storage and buffer conditions which led to a reduction in the false reactivity rate in therapeutic-naïve serum from a rheumatoid arthritis population.

  11. 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

  12. Solid phase measurements of antibody and lectin binding to xenogenic carbohydrate antigens

    DEFF Research Database (Denmark)

    Kirkeby, Svend; André, Sabine; Gabius, Hans-Joachim

    2004-01-01

    OBJECTIVES: In future pig-to-man xenotransplantation it is important to master tools that identify potentially xenogenic alphagalactose (Galalpha) antigens in the doner tissue. DESIGN AND METHODS: We have measured the binding potentials of Galalpha detecting lectins and antibodies, including...

  13. Investigations on antibody binding to a micro-cantilever coated with a BAM pesticide residue

    Directory of Open Access Journals (Sweden)

    Aamand Jens

    2011-01-01

    Full Text Available Abstract The attachment of an antibody to an antigen-coated cantilever has been investigated by repeated experiments, using a cantilever-based detection system by Cantion A/S. The stress induced by the binding of a pesticide residue BAM (2,6 dichlorobenzamide immobilized on a cantilever surface to anti-BAM antibody is measured using the CantiLab4© system from Cantion A/S with four gold-coated cantilevers and piezo resistive readout. The detection mechanism is in principle label-free, but fluorescent-marked antibodies have been used to subsequently verify the binding on the cantilever surface. The bending and increase in mass of each cantilever has also been investigated using a light interferometer and a Doppler Vibrometer. The system has been analyzed during repeated measurements to investigate whether the CantiLab4© system is a suited platform for a pesticide assay system.

  14. Chimeric bispecific OC/TR monoclonal antibody mediates lysis of tumor cells expressing the folate-binding protein (MOv18) and displays decreased immunogenicity in patients

    NARCIS (Netherlands)

    Luiten, R. M.; Warnaar, S. O.; Sanborn, D.; Lamers, C. H.; Bolhuis, R. L.; Litvinov, S. V.; Zurawski, V. R.; Coney, L. R.

    1997-01-01

    The bispecific OC/TR monoclonal antibody (mAb) cross-links the CD3 molecule on T cells with the human folate-binding protein (FBP), which is highly expressed on nonmucinous ovarian carcinomas. Clinical trials of patients with ovarian carcinoma with the OC/TR mAb have shown some complete and partial

  15. Fully human monoclonal antibodies from antibody secreting cells after vaccination with Pneumovax®23 are serotype specific and facilitate opsonophagocytosis.

    Science.gov (United States)

    Smith, Kenneth; Muther, Jennifer J; Duke, Angie L; McKee, Emily; Zheng, Nai-Ying; Wilson, Patrick C; James, Judith A

    2013-05-01

    B lymphocyte memory generates antibody-secreting cells (ASCs) that represent a source of protective antibodies that may be exploited for therapeutics. Here we vaccinated four donors with Pneumovax®23 and produced human monoclonal antibodies (hmAbs) from ASCs. We have cloned 137 hmAbs and the specificities of these antibodies encompass 19 of the 23 serotypes in the vaccine, as well as cell wall polysaccharide (CWPS). Although the majority of the antibodies are serotype specific, 12% cross-react with two serotypes. The Pneumovax®23 ASC antibody sequences are highly mutated and clonal, indicating an anamnestic response, even though this was a primary vaccination. Hmabs from 64% of the clonal families facilitate opsonophagocytosis. Although 9% of the total antibodies bind to CWPS impurity in the vaccine, none of these clonal families showed opsonophagocytic activity. Overall, these studies have allowed us to address unanswered questions in the field of human immune responses to polysaccharide vaccines, including the cross-reactivity of individual antibodies between serotypes and the percentage of antibodies that are protective after vaccination with Pneumovax®23. Copyright © 2012 Elsevier GmbH. All rights reserved.

  16. A collagen-binding EGFR antibody fragment targeting tumors with a collagen-rich extracellular matrix.

    Science.gov (United States)

    Liang, Hui; Li, Xiaoran; Wang, Bin; Chen, Bing; Zhao, Yannan; Sun, Jie; Zhuang, Yan; Shi, Jiajia; Shen, He; Zhang, Zhijun; Dai, Jianwu

    2016-02-17

    Many tumors over-express collagen, which constitutes the physical scaffold of tumor microenvironment. Collagen has been considered to be a target for cancer therapy. The collagen-binding domain (CBD) is a short peptide, which could bind to collagen and achieve the sustained release of CBD-fused proteins in collagen scaffold. Here, a collagen-binding EGFR antibody fragment was designed and expressed for targeting the collagen-rich extracellular matrix in tumors. The antibody fragment (Fab) of cetuximab was fused with CBD (CBD-Fab) and expressed in Pichia pastoris. CBD-Fab maintained antigen binding and anti-tumor activity of cetuximab and obtained a collagen-binding ability in vitro. The results also showed CBD-Fab was mainly enriched in tumors and had longer retention time in tumors in A431 s.c. xenografts. Furthermore, CBD-Fab showed a similar therapeutic efficacy as cetuximab in A431 xenografts. Although CBD-Fab hasn't showed better therapeutic effects than cetuximab, its smaller molecular and special target may be applicable as antibody-drug conjugates (ADC) or immunotoxins.

  17. The peanut lectin-binding glycoproteins of human epidermal keratinocytes

    International Nuclear Information System (INIS)

    Morrison, A.I.; Keeble, S.; Watt, F.M.

    1988-01-01

    The peanut lectin (PNA) is known to bind more strongly to keratinocytes that are undergoing terminal differentiation than to proliferating keratinocytes. In order to investigate the significance of this change in cell-surface carbohydrate authors have identified the PNA-binding glycoproteins of cultured human keratinocytes and antibodies against them. Two heavily glycosylated bands of 110 and 250 kDa were resolved by PAGE of [ 14 C]galactose- or [ 14 C]mannose- and [ 14 C]glucosamine-labeled cell extracts eluted with galactose from PNA affinity columns. The higher molecular weight band was also detected on PNA blots of unlabeled cell extracts transferred to nitrocellulose. Both bands were sensitive to pronase digestion, but only the 250-kDa band was digested with trypsin. A rabbit antiserum that we prepared (anti-PNA-gp) immunoprecipitated both bands from cell extracts. In contrast to PNA, anti-PNA-gp bound equally to proliferating and terminally differentiating cells, indicating that some epitope(s) of the PNA-binding glycoproteins is present on the cell surface prior to terminal differentiation. When keratinocytes grown as a monolayer in low-calcium medium were switched to medium containing 2 mM calcium ions in order to induce desmosome formation and stratification, there was a dramatic redistribution of the PNA-binding glycoproteins, which became concentrated at the boundaries between cells. This may suggest a role for the glycoproteins in cell-cell interactions during stratification

  18. Sensitive radioimmunoassay for detection of antibodies to recombinant human interferon-alpha A

    International Nuclear Information System (INIS)

    Palleroni, A.V.; Trown, P.W.

    1986-01-01

    A radioimmunoassay (RIA) for the detection of antibodies to recombinant human leukocyte interferon A (rHuIFN-alpha A) in human serum has been developed and validated against the standard antiviral neutralization bioassay (ANB). The assay measures the binding of 125 I-labeled rHuIFN-alpha A to immunoglobulins in serum. Aliquots of patients' sera are incubated with 125 I-rHuIFN-alpha A and the complexes formed between antibodies in the sera and the 125 I-rHuIFN-alpha A are precipitated with goat anti-human IgG serum. The radioactivity in the immune precipitate is a measure of the quantity of antibody (if present) in the serum. The sensitivity of this RIA is 5 ng of IgG/ml of serum

  19. Human chorionic ganodotropin binding sites in the human endometrium

    International Nuclear Information System (INIS)

    Bhattacharya, S.; Banerjee, J.; Sen, S.; Manna, P.R.

    1993-01-01

    The existence of high-affinity and low-capacity specific binding sites for luteinizing hormone/human chorionic gonadotropin (hCG) has been reported in porcine, rabbit and rat uteri. The authors have identified the hCG binding sites in the human endometrium collected from 35-42-year-old ovulatory and anovulatory women. The binding characteristics of hCG to endometrial tissue preparations from ovulatory and anovulatory women showed saturability with high affinity and low capacity. Scatchard plot analysis showed the dissociation constant of specific binding sites in the ovulatory women to be 3.5x10 -10 mol/l and in anovulatory women to be 3.1x10 -10 mol/l. The maximum binding capacity varied considerably between ovulatory and anovulatory endometrium. Among the divalent metal ions tested Zn 2+ effected a remarkable increase in [ 125 I]hCG binding to the endometrium, whereas Mn 2+ showed a marginal increase and other metal ions did not have any effect. Data obtained with human endometrium indicate an influence of the functional state of the ovary on [ 125 I]hCG binding to endometrium. 14 refs., 3 figs

  20. Meningococcal factor H-binding protein vaccines with decreased binding to human complement factor H have enhanced immunogenicity in human factor H transgenic mice.

    Science.gov (United States)

    Rossi, Raffaella; Granoff, Dan M; Beernink, Peter T

    2013-11-04

    Factor H-binding protein (fHbp) is a component of a meningococcal vaccine recently licensed in Europe for prevention of serogroup B disease, and a second vaccine in clinical development. The protein specifically binds human factor H (fH), which down-regulates complement activation and enhances resistance to bactericidal activity. There are conflicting data from studies in human fH transgenic mice on whether binding of human fH to fHbp vaccines decreases immunogenicity, and whether mutant fHbp vaccines with decreased fH binding have enhanced immunogenicity. fHbp can be classified into two sub-families based on sequence divergence and immunologic cross-reactivity. Previous studies of mutant fHbp vaccines with low fH binding were from sub-family B, which account for approximately 60% of serogroup B case isolates. In the present study, we evaluated the immunogenicity of two mutant sub-family A fHbp vaccines containing single substitutions, T221A or D211A, which resulted in 15- or 30-fold lower affinity for human fH, respectively, than the corresponding control wild-type fHbp vaccine. In transgenic mice with high serum concentrations of human fH, both mutant vaccines elicited significantly higher IgG titers and higher serum bactericidal antibody responses than the control fHbp vaccine that bound human fH. Thus, mutations introduced into a sub-family A fHbp antigen to decrease fH binding can increase protective antibody responses in human fH transgenic mice. Collectively the data suggest that mutant fHbp antigens with decreased fH binding will result in superior vaccines in humans. Copyright © 2013 Elsevier Ltd. All rights reserved.

  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. Human serum albumin binding of certain antimalarials

    Science.gov (United States)

    Marković, Olivera S.; Cvijetić, Ilija N.; Zlatović, Mario V.; Opsenica, Igor M.; Konstantinović, Jelena M.; Terzić Jovanović, Nataša V.; Šolaja, Bogdan A.; Verbić, Tatjana Ž.

    2018-03-01

    Interactions between eight in-house synthesized aminoquinolines, along with well-known chloroquine, and human serum albumin (HSA) have been studied by fluorescence spectroscopy. The synthesized aminoquinolines, despite being structurally diverse, were found to be very potent antimalarials. Fluorescence measurements indicate that three compounds having additional thiophene or benzothiophene substructure bind more strongly to HSA than other studied compounds. Competitive binding experiments indicate that these three compounds bind significantly stronger to warfarin compared to diazepam binding site. Fluorescence quenching at three temperatures (20, 25, and 37 °C) was analyzed using classical Stern-Volmer equation, and a static quenching mechanism was proposed. The enthalpy and entropy changes upon sulphur-containing compound-HSA interactions were calculated using Van't Hoff equation. Positive values of enthalpy and entropy changes indicate that non-specific, hydrophobic interactions are the main contributors to HSA-compound interaction. Molecular docking and calculated lipophilicity descriptors indicate the same, pointing out that the increased lipophilicity of sulphur-containing compounds might be a reason for their better binding to HSA. Obtained results might contribute to design of novel derivatives with improved pharmacokinetic properties and drug efficacy.

  3. Human antibody fragments specific for the epidermal growth factor receptor selected from large non-immunised phage display libraries.

    Science.gov (United States)

    Souriau, Christelle; Rothacker, Julie; Hoogenboom, Hennie R; Nice, Edouard

    2004-09-01

    Antibodies to EGFR have been shown to display anti-tumour effects mediated in part by inhibition of cellular proliferation and angiogenesis, and by enhancement of apoptosis. Humanised antibodies are preferred for clinical use to reduce complications with HAMA and HAHA responses frequently seen with murine and chimaeric antibodies. We have used depletion and subtractive selection strategies on cells expressing the EGFR to sample two large antibody fragment phage display libraries for the presence of human antibodies which are specific for the EGFR. Four Fab fragments and six scFv fragments were identified, with affinities of up to 2.2nM as determined by BIAcore analysis using global fitting of the binding curves to obtain the individual rate constants (ka and kd). This overall approach offers a generic screening method for the identification of growth factor specific antibodies and antibody fragments from large expression libraries and has potential for the rapid development of new therapeutic and diagnostic reagents.

  4. A novel polyclonal antibody against human cytomegalovirus ...

    African Journals Online (AJOL)

    User

    2011-05-09

    May 9, 2011 ... The identification of the synthetic peptide antibody was confirmed by ... cell virus transmission and fusion of infected cells, as well ..... Cytomegalovirus and Epstein-. Barr virus subtypes-The search for clinical significance.

  5. Binding of HIV-1 gp41-directed neutralizing and non-neutralizing fragment antibody binding domain (Fab and single chain variable fragment (ScFv antibodies to the ectodomain of gp41 in the pre-hairpin and six-helix bundle conformations.

    Directory of Open Access Journals (Sweden)

    John M Louis

    Full Text Available We previously reported a series of antibodies, in fragment antigen binding domain (Fab formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066 and non-neutralizing (8062 antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥ 150-fold in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.

  6. Humanized versus murine anti-human epidermal growth factor receptor monoclonal antibodies for immunoscintigraphic studies

    Energy Technology Data Exchange (ETDEWEB)

    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

    2000-02-01

    The anti-human epidermal growth factor receptor (EGF-R) humanized antibody h-R3 (IgG{sub 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 {mu}g/100 {mu}Ci of {sup 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 {sup 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.

  7. 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

  8. Native Mass Spectrometry, Ion mobility, and Collision-Induced Unfolding Categorize Malaria Antigen/Antibody Binding

    Science.gov (United States)

    Huang, Yining; Salinas, Nichole D.; Chen, Edwin; Tolia, Niraj H.; Gross, Michael L.

    2017-09-01

    Plasmodium vivax Duffy Binding Protein (PvDBP) is a promising vaccine candidate for P. vivax malaria. Recently, we reported the epitopes on PvDBP region II (PvDBP-II) for three inhibitory monoclonal antibodies (2D10, 2H2, and 2C6). In this communication, we describe the combination of native mass spectrometry and ion mobility (IM) with collision induced unfolding (CIU) to study the conformation and stabilities of three malarial antigen-antibody complexes. These complexes, when collisionally activated, undergo conformational changes that depend on the location of the epitope. CIU patterns for PvDBP-II in complex with antibody 2D10 and 2H2 are highly similar, indicating comparable binding topology and stability. A different CIU fingerprint is observed for PvDBP-II/2C6, indicating that 2C6 binds to PvDBP-II on an epitope different from 2D10 and 2H2. This work supports the use of CIU as a means of classifying antigen-antibody complexes by their epitope maps in a high throughput screening workflow. [Figure not available: see fulltext.

  9. Antibody-Unfolding and Metastable-State Binding in Force Spectroscopy and Recognition Imaging

    Science.gov (United States)

    Kaur, Parminder; Qiang-Fu; Fuhrmann, Alexander; Ros, Robert; Kutner, Linda Obenauer; Schneeweis, Lumelle A.; Navoa, Ryman; Steger, Kirby; Xie, Lei; Yonan, Christopher; Abraham, Ralph; Grace, Michael J.; Lindsay, Stuart

    2011-01-01

    Force spectroscopy and recognition imaging are important techniques for characterizing and mapping molecular interactions. In both cases, an antibody is pulled away from its target in times that are much less than the normal residence time of the antibody on its target. The distribution of pulling lengths in force spectroscopy shows the development of additional peaks at high loading rates, indicating that part of the antibody frequently unfolds. This propensity to unfold is reversible, indicating that exposure to high loading rates induces a structural transition to a metastable state. Weakened interactions of the antibody in this metastable state could account for reduced specificity in recognition imaging where the loading rates are always high. The much weaker interaction between the partially unfolded antibody and target, while still specific (as shown by control experiments), results in unbinding on millisecond timescales, giving rise to rapid switching noise in the recognition images. At the lower loading rates used in force spectroscopy, we still find discrepancies between the binding kinetics determined by force spectroscopy and those determined by surface plasmon resonance—possibly a consequence of the short tethers used in recognition imaging. Recognition imaging is nonetheless a powerful tool for interpreting complex atomic force microscopy images, so long as specificity is calibrated in situ, and not inferred from equilibrium binding kinetics. PMID:21190677

  10. Origin, diversity and maturation of human antiviral antibodies analyzed by high-throughput sequencing

    Directory of Open Access Journals (Sweden)

    Ponraj ePrabakaran

    2012-08-01

    Full Text Available Our understanding of how antibodies are generated and function could help develop effective vaccines and antibody-based therapeutics against viruses such as HIV-1, SARS Coronavirus (CoV, and Hendra and Nipah viruses (henipaviruses. Although broadly neutralizing antibodies (bnAbs against the HIV-1 were observed in patients, elicitation of such bnAbs remains a major challenge when compared to other viral targets. We previously hypothesized that HIV-1 could have evolved a strategy to evade the immune system due to absent or very weak binding of germline antibodies to the conserved epitopes that may not be sufficient to initiate and/or maintain an effective immune response. To further explore our hypothesis, we used the 454 sequence analysis of a large naïve library of human IgM antibodies which had been used for selecting antibodies against SARS Coronavirus (CoV receptor-binding domain (RBD, and soluble G proteins (sG of Hendra and Nipah viruses (henipaviruses. We found that the human IgM repertoires from the 454 sequencing have diverse germline usages, recombination patterns, junction diversity and a lower extent of somatic mutation. In this study, we identified germline intermediates of antibodies specific to HIV-1 and other viruses as observed in normal individuals, and compared their genetic diversity and somatic mutation level along with available structural and functional data. Further computational analysis will provide framework for understanding the underlying genetic and molecular determinants related to maturation pathways of antiviral bnAbs that could be useful for applying novel approaches to the design of effective vaccine immunogens and antibody-based therapeutics.

  11. Development and characterization of human monoclonal antibodies that neutralize multiple TGFβ isoforms.

    Science.gov (United States)

    Bedinger, Daniel; Lao, Llewelyn; Khan, Shireen; Lee, Steve; Takeuchi, Toshihiko; Mirza, Amer M

    2016-01-01

    Transforming growth factor (TGF)β levels are elevated in, and drive the progression of, numerous disease states such as advanced metastatic cancer and systemic and ocular fibrosis. There are 3 main isoforms, TGFβ1, 2, and 3. As multiple TGFβ isoforms are involved in disease processes, maximal therapeutic efficacy may require neutralization of 2 or more of the TGFβ isoforms. Fully human antibody phage display libraries were used to discover a number of antibodies that bind and neutralize various combinations of TGFβ1, 2 or 3. The primary panning did not yield any uniformly potent pan-isoform neutralizing antibodies; therefore, an antibody that displayed potent TGFβ 1, 2 inhibition, but more modest affinity versus TGFβ3, was affinity matured by shuffling with a light chain sub-library and further screening. This process yielded a high affinity pan-isoform neutralizing clone. Antibodies were analyzed and compared by binding affinity, as well as receptor and epitope competition by surface plasmon resonance methods. The antibodies were also shown to neutralize TGFβ effects in vitro in 3 assays: 1) interleukin (IL)-4 induced HT-2 cell proliferation; 2) TGFβ-mediated IL-11 release by A549 cells; and 3) decreasing SMAD2 phosphorylation in Detroit 562 cells. The antibodies' potency in these in vitro assays correlated well with their isoform-specific affinities. Furthermore, the ability of the affinity-matured clone to decrease tumor burden in a Detroit 562 xenograft study was superior to that of the parent clone. This affinity-matured antibody acts as a very potent inhibitor of all 3 main isoforms of TGFβ and may have utility for therapeutic intervention in human disease.

  12. Fluorescence correlation spectroscopy to study antibody binding and stoichiometry of complexes

    Science.gov (United States)

    Swift, Kerry M.; Matayoshi, Edmund D.

    2008-02-01

    FCS (fluorescence correlation spectroscopy) was used to study the association at the single molecule level of tumor necrosis factor alpha (TNF-α) and two of its protein antagonists Humira (TM) (adalimumab), a fully humanized monoclonal antibody, and Enbrel (TM) (etanercept), a soluble form of the TNF receptor. Single molecule approaches potentially have the advantage not only of enhanced sensitivity, but also of observing at equilibrium the details that would otherwise be lost in classical ensemble experiments where heterogeneity is averaged. We prepared fluorescent conjugates of the protein drugs and their biological target, the trimeric soluble form of TNF-α. The bivalency of adalimumab and the trimeric nature of TNF-α potentially allow several forms of associative complexes that may differ in stoichiometry. Detailed knowledge of this reaction may be relevant to understanding adalimumab's pharmacological properties. Our FCS data showed that a single trimeric TNF-α can bind up to three adalimumab molecules. Under some conditions even larger complexes are formed, apparently the result of cross-linking of TNF-α trimers by adalimumab. In addition, distinct differences between Humira and Enbrel were observed in their association with TNF-α.

  13. Field flow fractionation for assessing neonatal Fc receptor and Fcγ receptor binding to monoclonal antibodies in solution.

    Science.gov (United States)

    Pollastrini, Joey; Dillon, Thomas M; Bondarenko, Pavel; Chou, Robert Y-T

    2011-07-01

    Analysis of the strength and stoichiometry of immunoglobulin G (IgG) binding to neonatal Fc receptor (FcRn) and Fcγ receptor (FcγR) is important for evaluating the pharmacokinetics and effector functions of therapeutic monoclonal antibody (mAb) products, respectively. The current standard for assessing FcγR and FcRn binding is composed of cell-based and surface plasmon resonance (SPR) assays. In this work, asymmetrical flow field flow fractionation (AF4) was evaluated to establish the true stoichiometry of IgG binding in solution. AF4 and liquid chromatography-mass spectrometry (LC-MS) were applied to directly observe IgG/FcγR and IgG/FcRn complexes, which were not observed using nonequilibrium size exclusion chromatography (SEC) analysis. Human serum albumin (HSA), an abundant component of human blood and capable of binding FcRn, was studied in combination with FcRn and IgG. AF4 demonstrated that the majority of large complexes of IgG/FcRn/HSA were at an approximate 1:2:1 molar ratio. In addition, affinity measurements of the complex were performed in the sub-micromolar affinity range. A significant decrease in binding was detected for IgG molecules with increased oxidation in the Fc region. AF4 was useful in detecting weak binding between full-length IgG/Fc fragments and Fc receptors and the effect of chemical modifications on binding. AF4 is a useful technique in the assessment of mAb product quality attributes. Copyright © 2011 Elsevier Inc. All rights reserved.

  14. Fab-dsFv: A bispecific antibody format with extended serum half-life through albumin binding.

    Science.gov (United States)

    Davé, Emma; Adams, Ralph; Zaccheo, Oliver; Carrington, Bruce; Compson, Joanne E; Dugdale, Sarah; Airey, Michael; Malcolm, Sarah; Hailu, Hanna; Wild, Gavin; Turner, Alison; Heads, James; Sarkar, Kaushik; Ventom, Andrew; Marshall, Diane; Jairaj, Mark; Kopotsha, Tim; Christodoulou, Louis; Zamacona, Miren; Lawson, Alastair D; Heywood, Sam; Humphreys, David P

    2016-10-01

    An antibody format, termed Fab-dsFv, has been designed for clinical indications that require monovalent target binding in the absence of direct Fc receptor (FcR) binding while retaining substantial serum presence. The variable fragment (Fv) domain of a humanized albumin-binding antibody was fused to the C-termini of Fab constant domains, such that the VL and VH domains were individually connected to the Cκ and CH1 domains by peptide linkers, respectively. The anti-albumin Fv was selected for properties thought to be desirable to ensure a durable serum half-life mediated via FcRn. The Fv domain was further stabilized by an inter-domain disulfide bond. The bispecific format was shown to be thermodynamically and biophysically stable, and retained good affinity and efficacy to both antigens simultaneously. In in vivo studies, the serum half-life of Fab-dsFv, 2.6 d in mice and 7.9 d in cynomolgus monkeys, was equivalent to Fab'-PEG.

  15. Two-site sandwich radioimmunoassay of human gamma interferon with monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Tanaka, E; Imai, M; Usuda, S; Tachibana, K; Okamoto, H; Ohike, Y; Nakamura, T; Miyakawa, Y; Mayumi, M [Jichi Medical School, Minamikawachi, Tochigi (Japan)

    1985-03-18

    Two monoclonal antibodies were raised against human gamma interferon (IFN-..gamma..) derived from E. coli harboring the recombinant cDNA for IFN-..gamma.., and one against a synthetic peptide representing its C-terminus amino acid sequence of 20 residues. The monoclonal antibody against the synthetic peptide reacted either with IFN-..gamma.. or the synthetic peptide. One monoclonal anti-IFN-..gamma.. did not react with the synthetic peptide, while the other showed a weak binding with the peptide. A 2-site '1-step' radioimmunoassay was developed. The assay was rapid with a sensitivity capable of detecting a few ng/ml of IFN-..gamma...

  16. Monoclonal antibodies to human factor VII: a one step immunoradiometric assay for VII:Ag.

    OpenAIRE

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

    1988-01-01

    Three mouse monoclonal antibodies (RFF-VII/1, RFF-VII/2, and RFF-VII/3) which bind specifically to different epitopes on human factor VII antigen were raised. Two of the antibodies, RFF-VII/1 and RFF-VII/2, bound strongly to factor VII antigen (VII:Ag), but only RFF-VII/1 and RFF-VII/3 were potent inhibitors of factor VII coagulation activity (VII:C). RFF-VII/1 and RFF-VII/2 were used in a one step, double monoclonal immunoradiometric assay for VII:Ag. This was highly reproducible and detecte...

  17. Antibodies against human cytochrome P-450db1 in autoimmune hepatitis type II.

    OpenAIRE

    Zanger, U M; Hauri, H P; Loeper, J; Homberg, J C; Meyer, U A

    1988-01-01

    In a subgroup of children with chronic active hepatitis, circulating autoantibodies occur that bind to liver and kidney endoplasmic reticulum (anti-liver/kidney microsome antibody type I or anti-LKM1). Anti-LKM1 titers follow the severity of the disease and the presence of these antibodies serves as a diagnostic marker for this autoimmune hepatitis type II. We demonstrate that anti-LKM1 IgGs specifically inhibit the hydroxylation of bufuralol in human liver microsomes. Using two assay systems...

  18. Three-site sandwich radioimmunoassay with monoclonal antibodies for a sensitive determination of human alpha-fetoprotein

    International Nuclear Information System (INIS)

    Nomura, M.; Imai, M.; Takahashi, K.; Kumakura, T.; Tachibana, K.; Aoyagi, S.; Usuda, S.; Nakamura, T.; Miyakawa, Y.; Mayumi, M.

    1983-01-01

    Utilizing monoclonal antibodies against human alpha-fetoprotein, 3 distinct antigenic determinants were identified. These antigenic determinants, provisionally designated a, b and c, were arranged in such a manner that the binding of one determinant with the corresponding antibody did not inhibit, or only barely inhibited the binding of antibodies directed to the other 2 determinants. Monoclonal antibodies with 3 different specificities were, therefore, applied to develop a sandwich-type solid-phase radioimmunoassay of the antigen in which wells were coated with anti-a, and radiolabeled anti-b together with radiolabeled anti-c was employed to detect the bound antigen. The 3-site sandwich radioimmunoassay involving 3 different determinants gave a higher sensitivity than 2-site assays in which only anti-b or anti-c was employed as a radiolabeled reagent, because the radioactivity of the 2 labeled antibodies was added on the antigen bound to immobilized anti-a. (Auth.)

  19. Human IgG lacking effector functions demonstrate lower FcRn-binding and reduced transplacental transport

    NARCIS (Netherlands)

    Stapleton, Nigel M.; Armstrong-Fisher, Sylvia S.; Andersen, Jan Terje; van der Schoot, C. Ellen; Porter, Charlene; Page, Kenneth R.; Falconer, Donald; de Haas, Masja; Williamson, Lorna M.; Clark, Michael R.; Vidarsson, Gestur; Armour, Kathryn L.

    2018-01-01

    We have previously generated human IgG1 antibodies that were engineered for reduced binding to the classical Fcγ receptors (FcγRI-III) and C1q, thereby eliminating their destructive effector functions (constant region G1Δnab). In their potential use as blocking agents, favorable binding to the

  20. Human Immunodeficiency Virus Proteins Mimic Human T Cell Receptors Inducing Cross-Reactive Antibodies

    Directory of Open Access Journals (Sweden)

    Robert Root-Bernstein

    2017-10-01

    Full Text Available Human immunodeficiency virus (HIV hides from the immune system in part by mimicking host antigens, including human leukocyte antigens. It is demonstrated here that HIV also mimics the V-β-D-J-β of approximately seventy percent of about 600 randomly selected human T cell receptors (TCR. This degree of mimicry is greater than any other human pathogen, commensal or symbiotic organism studied. These data suggest that HIV may be evolving into a commensal organism just as simian immunodeficiency virus has done in some types of monkeys. The gp120 envelope protein, Nef protein and Pol protein are particularly similar to host TCR, camouflaging HIV from the immune system and creating serious barriers to the development of safe HIV vaccines. One consequence of HIV mimicry of host TCR is that antibodies against HIV proteins have a significant probability of recognizing the corresponding TCR as antigenic targets, explaining the widespread observation of lymphocytotoxic autoantibodies in acquired immunodeficiency syndrome (AIDS. Quantitative enzyme-linked immunoadsorption assays (ELISA demonstrated that every HIV antibody tested recognized at least one of twelve TCR, and as many as seven, with a binding constant in the 10−8 to 10−9 m range. HIV immunity also affects microbiome tolerance in ways that correlate with susceptibility to specific opportunistic infections.

  1. Binding of alpha-fetoprotein by immobilized monoclonal antibodies during episodes of zero-gravity obtained by parabolic flight

    Science.gov (United States)

    Spooner, Brian S.; Guikema, James A.; Barnes, Grady

    1990-01-01

    Alpha-fetoprotein (AFP), a single-chain polypeptide which is synthesized by the liver and yolk sac of the human fetus, provided a model ligand for assessing the effects of microgravity on ligand binding to surface-immobilized model receptor molecules. Monoclonal antibodies, used as receptors for AFP, were immobilized by covalent attachment to latex microparticles. Zero gravity environment was obtained by parabolic flight aboard NASA 930, a modified KC-135 aircraft. Buring the onset of an episode of zero gravity, ligand and receptor were mixed. Timed incubation (20 s) was terminated by centrifugation, the supernatant removed, and microparticies were assessed for bound AFP by immunochemical methods. The extent of binding was not influenced by microgravity, when compared with 1-G controls, which suggests that aberrant cellular activities observed in microgravity are not the simple expression of altered macromolecular interactions.

  2. Effect of trastuzumab interchain disulfide bond cleavage on Fcγ receptor binding and antibody-dependent tumour cell phagocytosis.

    Science.gov (United States)

    Suzuki, Mami; Yamanoi, Ayaka; Machino, Yusuke; Ootsubo, Michiko; Izawa, Ken-ichi; Kohroki, Junya; Masuho, Yasuhiko

    2016-01-01

    The Fc domain of human IgG1 binds to Fcγ receptors (FcγRs) to induce effector functions such as phagocytosis. There are four interchain disulfide bonds between the H and L chains. In this study, the disulfide bonds within the IgG1 trastuzumab (TRA), which is specific for HER2, were cleaved by mild S-sulfonation or by mild reduction followed by S-alkylation with three different reagents. The cleavage did not change the binding activities of TRA to HER2-bearing SK-BR-3 cells. The binding activities of TRA to FcγRIIA and FcγRIIB were greatly enhanced by modification with mild reduction and S-alkylation with ICH2CONH2 or N-(4-aminophenyl) maleimide, while the binding activities of TRA to FcγRI and FcγRIIIA were decreased by any of the four modifications. However, the interchain disulfide bond cleavage by the different modifications did not change the antibody-dependent cell-mediated phagocytosis (ADCP) of SK-BR-3 cells by activated THP-1 cells. The order of FcγR expression levels on the THP-1 cells was FcγRII > FcγRI > FcγRIII and ADCP was inhibited by blocking antibodies against FcγRI and FcγRII. These results imply that the effect of the interchain disulfide bond cleavage on FcγRs binding and ADCP is dependent on modifications of the cysteine residues and the FcγR isotypes. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  3. Specific binding of large aggregates of amphiphilic molecules to the respective antibodies.

    Science.gov (United States)

    Nabok, Alexei; Tsargorodskaya, Anna; Holloway, Alan; Starodub, Nikolay F; Demchenko, Anna

    2007-07-31

    The Binding of nonylphenol to respective antibodies immobilized on solid substrates was studied with the methods of total internal reflection ellipsometry (TIRE) and QCM (quartz crystal microbalance) impedance spectroscopy. The binding reaction was proved to be highly specific having an association constant of KA=1.6x10(6) mol(-1) L and resulted in an increase in both the adsorbed layer thickness of 23 nm and the added mass of 18.3 microg/cm2 at saturation. The obtained responses of both TIRE and QCM methods are substantially higher than anticipated for the immune binding of single molecules of nonylphenol. The mechanism of binding of large aggregates of nonylphenol was suggested instead. Modeling of the micelle of amphiphilic nonylphenol molecules in aqueous solutions yielded a micelle size of about 38 nm. The mechanism of binding of large molecular aggregates to respective antibodies can be extended to other hydrophobic low-molecular-weight toxins such as T-2 mycotoxin. The formation of large molecular aggregates of nonylphenol and T-2 mycotoxin molecules on the surface was proved by the AFM study.

  4. Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment.

    Science.gov (United States)

    Kirley, Terence L; Norman, Andrew B

    2015-01-01

    Variations of post-translational modifications are important for stability and in vivo behavior of therapeutic antibodies. A recombinant humanized anti-cocaine monoclonal antibody (h2E2) was characterized for heterogeneity of N-linked glycosylation and disulfide bonds. In addition, charge heterogeneity, which is partially due to the presence or absence of C-terminal lysine on the heavy chains, was examined. For cocaine overdose therapy, Fab fragments may be therapeutic, and thus, a simplified method of generation, purification, and characterization of the Fab fragment generated by Endoproteinase Lys-C digestion was devised. Both the intact h2E2 antibody and purified Fab fragments were analyzed for their affinities for cocaine and 2 of its metabolites, benzoylecgonine and cocaethylene, by fluorescence quenching of intrinsic antibody tyrosine and tryptophan fluorescence resulting from binding of these drugs. Binding constants obtained from fluorescence quenching measurements are in agreement with recently published radioligand and ELISA binding assays. The dissociation constants determined for the h2E2 monoclonal and its Fab fragment are approximately 1, 5, and 20 nM for cocaethylene, cocaine, and benzoylecgonine, respectively. Tryptophan fluorescence quenching (emission at 330 nm) was measured after either excitation of tyrosine and tryptophan (280 nm) or selective excitation of tryptophan alone (295 nm). More accurate binding constants are obtained using tryptophan selective excitation at 295 nm, likely due to interfering absorption of cocaine and metabolites at 280 nm. These quenching results are consistent with multiple tryptophan and tyrosine residues in or near the predicted binding location of cocaine in a previously published 3-D model of this antibody's variable region.

  5. Chloramine-T induced binding of monoclonal antibody B72. 3 to concanavalin-A

    Energy Technology Data Exchange (ETDEWEB)

    Cole, W.C.; Jhingran, S.G. (Methodist Hospital, Houston, TX (United States) Baylor Coll. of Medicine, Houston, TX (United States))

    1993-07-01

    The effects of chloramine-T (CT) on monoclonal antibody B72.3 were studied with particular reference to Con-A lectin binding. After exposure to chloramine-T concentrations from 0.8 to 4.0 mg/mL (115-574 mol CT/mol B72.3), B72.3 showed progressive binding to agarose-linked Con-A. This behavior was paralleled by decreasing immunoreactivity and increasing fragmentation and aggregation of B72.3 demonstrated by SDS-PAGE and size exclusion HPLC. (Author).

  6. Isolation of human anti-serum albumin Fab antibodies with an extended serum-half life.

    Science.gov (United States)

    Kang, Hyeon-Ju; Kim, Hye-Jin; Cha, Sang-Hoon

    2016-01-01

    The serum albumin (SA) has been exploited to generate long-acting biotherapeutics by taking advantage of the FcRn-mediated recycling mechanism in a direct or an indirect way. Since Fab fragments have been proven to be clinically safe for human usage, we assumed that human anti-SA Fab antibodies could have a great potential as a carrier molecule to extend the serum half-life of therapeutic proteins. We, herein, had attempted to isolate anti-SA Fab antibodies from HuDVFab-8L antibody library via a phage display technology, and identified eight discrete human Fab antibodies. One of the Fab antibodies, SL335, showed the strongest binding reactivity to human SA with nM range of affinity at both pH 6 and pH 7.4, and cross-reacted to SAs from various species including rat, mouse, canine and monkey. The in vivo pharmacokinetic assay using a rat model indicated that SL335 has approximately 10 fold longer serum half-life and 26 to 44-fold increase in AUC0 → ∞ compared to the negative control Fab molecule in both intravenous and subcutaneous administrations. Knowing that Fabs have proven to be safe in clinics for a long time, SL335 seems to have a great potential in generating long-acting protein drugs by tagging effector molecules with either chemical conjugation or genetic fusion. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Development of broad-spectrum human monoclonal antibodies for rabies post-exposure prophylaxis

    International Nuclear Information System (INIS)

    Benedictis, P. de; Minola, A.; Rota, E.; Aiello, R.; Zecchin, B.; Salomoni, A.; Foglierini, M.; Agatic, G.; Vanzetta, F.; Lavenir, R.; Lepelletier, A.; Bentley, E.; Weiss, R.; Cattoli, G.

    2016-01-01

    Full text: Currently available rabies post-exposure prophylaxis (PEP) for use in humans includes equine or human rabies immunoglobulins (RIG). The replacement of RIG with an equally or more potent and safer product is strongly encouraged due to the high costs and limited availability of existing RIG. In this study, we identified two broadly neutralizing human monoclonal antibodies that represent a valid and affordable alternative to RIG in rabies PEP. Memory B cells from four selected vaccinated donors were immortalized and monoclonal antibodies were tested for neutralizing activity and epitope specificity. Two antibodies, identified as RVC20 and RVC58 (binding to antigenic site I and III, respectively), were selected for their potency and broad-spectrum reactivity. In vitro, RVC20 and RVC58 were able to neutralize all 35 rabies virus (RABV) and 25 non-RABV lyssaviruses. They showed higher potency and breath compared to antibodies under clinical development (namely CR57, CR4098, and RAB1) and commercially available human RIG. In vivo, the RVC20–RVC58 cocktail protected Syrian hamsters from a lethal RABV challenge and did not affect the endogenous hamster post-vaccination antibody response. (author)

  8. Human immunodeficiency virus (HIV) specific antibodies among ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-03-20

    Mar 20, 2009 ... Key words: HIV-1/2 antibody prevalence, pregnant women, commercial sex workers, risk factors, Nigeria. INTRODUCTION. There are two .... Africa. However, among Japanese and Chilean female. SWs, Miyazaki et al. .... STIs (P = 0.0001, OR = 6.0), level of education (P = 0.0001, OR = 40.7) and age (P ...

  9. Clearance of 131I-labeled murine monoclonal antibody from patients' blood by intravenous human anti-murine immunoglobulin antibody

    International Nuclear Information System (INIS)

    Stewart, J.S.; Sivolapenko, G.B.; Hird, V.; Davies, K.A.; Walport, M.; Ritter, M.A.; Epenetos, A.A.

    1990-01-01

    Five patients treated with intraperitoneal 131I-labeled mouse monoclonal antibody for ovarian cancer also received i.v. exogenous polyclonal human anti-murine immunoglobulin antibody. The pharmacokinetics of 131I-labeled monoclonal antibody in these patients were compared with those of 28 other patients receiving i.p.-radiolabeled monoclonal antibody for the first time without exogenous human anti-murine immunoglobulin, and who had no preexisting endogenous human anti-murine immunoglobulin antibody. Patients receiving i.v. human anti-murine immunoglobulin antibody demonstrated a rapid clearance of 131I-labeled monoclonal antibody from their circulation. The (mean) maximum 131I blood content was 11.4% of the injected activity in patients receiving human anti-murine immunoglobulin antibody compared to 23.3% in patients not given human anti-murine immunoglobulin antibody. Intravenous human anti-murine immunoglobulin antibody decreased the radiation dose to bone marrow (from 131I-labeled monoclonal antibody in the vascular compartment) 4-fold. Following the injection of human anti-murine immunoglobulin antibody, 131I-monoclonal/human anti-murine immunoglobulin antibody immune complexes were rapidly transported to the liver. Antibody dehalogenation in the liver was rapid, with 87% of the injected 131I excreted in 5 days. Despite the efficient hepatic uptake of immune complexes, dehalogenation of monoclonal antibody was so rapid that the radiation dose to liver parenchyma from circulating 131I was decreased 4-fold rather than increased. All patients developed endogenous human anti-murine immunoglobulin antibody 2 to 3 weeks after treatment

  10. Simplifying complex sequence information: a PCP-consensus protein binds antibodies against all four Dengue serotypes.

    Science.gov (United States)

    Bowen, David M; Lewis, Jessica A; Lu, Wenzhe; Schein, Catherine H

    2012-09-14

    Designing proteins that reflect the natural variability of a pathogen is essential for developing novel vaccines and drugs. Flaviviruses, including Dengue (DENV) and West Nile (WNV), evolve rapidly and can "escape" neutralizing monoclonal antibodies by mutation. Designing antigens that represent many distinct strains is important for DENV, where infection with a strain from one of the four serotypes may lead to severe hemorrhagic disease on subsequent infection with a strain from another serotype. Here, a DENV physicochemical property (PCP)-consensus sequence was derived from 671 unique sequences from the Flavitrack database. PCP-consensus proteins for domain 3 of the envelope protein (EdomIII) were expressed from synthetic genes in Escherichia coli. The ability of the purified consensus proteins to bind polyclonal antibodies generated in response to infection with strains from each of the four DENV serotypes was determined. The initial consensus protein bound antibodies from DENV-1-3 in ELISA and Western blot assays. This sequence was altered in 3 steps to incorporate regions of maximum variability, identified as significant changes in the PCPs, characteristic of DENV-4 strains. The final protein was recognized by antibodies against all four serotypes. Two amino acids essential for efficient binding to all DENV antibodies are part of a discontinuous epitope previously defined for a neutralizing monoclonal antibody. The PCP-consensus method can significantly reduce the number of experiments required to define a multivalent antigen, which is particularly important when dealing with pathogens that must be tested at higher biosafety levels. Copyright © 2012 Elsevier Ltd. All rights reserved.

  11. Naturally Acquired Antibodies to Plasmodium vivax Duffy Binding Protein (DBP) in Rural Brazilian Amazon

    Science.gov (United States)

    Souza-Silva, Flávia A.; da Silva-Nunes, Mônica; Sanchez, Bruno A. M.; Ceravolo, Isabela P.; Malafronte, Rosely S.; Brito, Cristiana F. A.; Ferreira, Marcelo U.; Carvalho, Luzia H.

    2010-01-01

    Duffy binding protein (DBP), a leading malaria vaccine candidate, plays a critical role in Plasmodium vivax erythrocyte invasion. Sixty-eight of 366 (18.6%) subjects had IgG anti-DBP antibodies by enzyme-linked immunosorbent assay (ELISA) in a community-based cross-sectional survey in the Brazilian Amazon Basin. Despite continuous exposure to low-level malaria transmission, the overall seroprevalence decreased to 9.0% when the population was reexamined 12 months later. Antibodies from 16 of 50 (36.0%) subjects who were ELISA-positive at the baseline were able to inhibit erythrocyte binding to at least one of two DBP variants tested. Most (13 of 16) of these subjects still had inhibitory antibodies when reevaluated 12 months later. Cumulative exposure to malaria was the strongest predictor of DBP seropositivity identified by multiple logistic regression models in this population. The poor antibody recognition of DBP elicited by natural exposure to P. vivax in Amazonian populations represents a challenge to be addressed by vaccine development strategies. PMID:20133990

  12. Clinical experience in humans with radiolabeled antibody for tumor detection

    International Nuclear Information System (INIS)

    Morrison, R.T.; Lyster, D.M.; Szasz, I.; Alcorn, L.N.; Huckell, V.F.; Rhodes, B.; Breslow, K.; Burchiel, S.

    1982-01-01

    I-131 and Tc-99m labeled polyclonal or monoclonal antibody and fragments of antibody, specific to human chorionic gonadotropin (hCG) or to a melanoma cell surface antigen (MCSA) were injected into proven cancer patients. Using standard homeostasis parameters, and scanning techniques, the safety and efficacy of each antibody was evaluated. Antibody fragments were expected to clear faster from the circulation allowing for earlier imaging and a better target-to-non-target ratio. The technetium label may perturb the antiboby's kinetics so that clearance is more rapid for both whole antibody and fragments. After a statistical evaluation of all parameters measured pre and post injection it was concluded that no acute toxicity reactions were present in any patient studied. Scan results were not acceptable for a tumor detecting procedure used in routine practice. Tumor upake was seen in less than 10% of scans

  13. Technical note: Protozoa-specific antibodies raised in sheep plasma bind to their target protozoa in the rumen.

    Science.gov (United States)

    Williams, Y J; Rea, S M; Popovski, S; Skillman, L C; Wright, A-D G

    2014-12-01

    Binding of IgG antibodies to Entodinium spp. in the rumen of sheep (Ovis aries) was investigated by adding IgG, purified from plasma, directly into the rumen. Plasma IgG was sourced from sheep that had or had not been immunized with a vaccine containing whole fixed Entodinium spp. cells. Ruminal fluid was sampled approximately 2 h after each antibody dosing. Binding of protozoa by a specific antibody was detected using an indirect fluorescent antibody test. An antibody titer in the ruminal fluid was determined by ELISA, and the concentration of ruminal fluid ammonia-N and ruminal pH were also determined. Entodinium spp. and total protozoa from IgG-infused sheep were enumerated by microscopic counts. Two-hourly additions of IgG maintained a low antibody titer in the rumen for 12 h and the binding of the antibody to the rumen protozoa was demonstrated. Increased ammonia-N concentrations and altered ruminal fluid pH patterns indicated that additional fermentation of protein was occurring in the rumen after addition of IgG. No reduction in numbers of Entodinium spp. was observed (P>0.05). Although binding of antibodies to protozoa has been demonstrated in the rumen, it is unclear how much cell death occurred. On the balance of probability, it would appear that the antibody was degraded or partially degraded, and the impact of this on protozoal populations and the measurement of a specific titer is also unclear.

  14. Human peripheral blood monocytes display surface antigens recognized by monoclonal antinuclear antibodies

    International Nuclear Information System (INIS)

    Holers, V.M.; Kotzin, B.L.

    1985-01-01

    The authors used monoclonal anti-nuclear autoantibodies and indirect immunofluorescence to examine normal human peripheral blood mononuclear leukocytes for the presence of cell surface nuclear antigens. Only one monoclonal anti-histone antibody (MH-2) was found to bind to freshly isolated PBL, staining approximately 10% of large cells. However, after cells were placed into culture for 16-24 h, a high percentage (up to 60%) of large-sized cells were recognized by an anti-DNA (BWD-1) and several different antihistone monoclonal antibodies (BWH-1, MH-1, and MH-2). These antibodies recognize separate antigenic determinants on chromatin and histones extracted from chromatin. The histone antigen-positive cells were viable, and the monoclonal antibodies could be shown to be binding to the cell surface and not to the nucleus. Using monoclonal antibodies specific for monocytes and T cells, and complement-mediated cytotoxicity, the cells bearing histone antigens were shown to be primarily monocytes. The appearance of histone and DNA antigen-positive cells was nearly completely inhibited by the addition of low concentrations of cycloheximide at initiation of the cultures. In contrast, little effect on the percentage of positive cells was detected if cells were exposed to high doses of gamma irradiation before culture. These data further support the existence of cell surface nuclear antigens on selected cell subsets, which may provide insight into the immunopathogenesis of systemic lupus erythematosus and related autoimmune diseases

  15. Human immunodeficiency virus (HIV) specific antibodies among ...

    African Journals Online (AJOL)

    obtained from each sample was tested using parallel testing algorithm with DETERMINE® HIV-1/2 and HIV-1/2 STAT-PAK® test was used for statistical analysis of the data. The overall prevalence of HIV-1/2 antibodies was 29.1% (n = 199). Seroprevalence of 39.4 and 19.0% were observed for the CSWs and the PW, ...

  16. Comparison of in vitro cell binding characteristics of four monoclonal antibodies and their individual tumor localization properties in mice

    International Nuclear Information System (INIS)

    Andrew, S.M.; Johnstone, R.W.; Russell, S.M.; McKenzie, I.F.; Pietersz, G.A.

    1990-01-01

    Although many antibodies are being used for imaging studies, it is not clear which in vitro properties of antibodies will best reflect their in vivo characteristics. The ability to correlate in vitro binding characteristics of monoclonal antibodies to tumor antigens with their in vivo localization characteristics, particularly with respect to tumor localization properties, is desirable for rapid selection of monoclonal antibodies with potential for clinical use. The in vitro binding characteristics of three monoclonal antibodies to the murine Ly-2.1 antigen and one to the Ly-3.1 antigen have been studied on cultured tumor cells bearing these antigens. The association and dissociation rate constants, apparent affinity, and immunoreactivity of each antibody in vitro were compared with their ability to localize the s.c. tumors from the same cell line growing in Ly-2.1-/Ly-3.1-mice. The antibody with the highest affinity and fastest association rate localized to tumor at the earliest time (16-20 h after injection) and had the highest percentage of the injected dose/g in the tumor (greater than 25%). The antibody with the lowest affinity showed significantly less localization to tumor cells, compared with the other three antibodies. The ranking of the antibodies by affinity agreed with the ranking in terms of their ability to localize to tumors, but the in vitro immunoreactivity of the antibodies, as measured by a cell binding assay, did not correlate with their tumor localization properties. Immunoscintigraphic studies did not precisely correlate with biodistribution data or in vitro binding characteristics, because tumors could be satisfactorily imaged with each antibody, although it was noted that the antibody with the highest affinity gave the best image

  17. Identification of a human monoclonal antibody to replace equine diphtheria antitoxin for treatment of diphtheria intoxication.

    Science.gov (United States)

    Sevigny, Leila M; Booth, Brian J; Rowley, Kirk J; Leav, Brett A; Cheslock, Peter S; Garrity, Kerry A; Sloan, Susan E; Thomas, William; Babcock, Gregory J; Wang, Yang

    2013-11-01

    Diphtheria antitoxin (DAT) has been the cornerstone of the treatment of Corynebacterium diphtheriae infection for more than 100 years. Although the global incidence of diphtheria has declined steadily over the last quarter of the 20th century, the disease remains endemic in many parts of the world, and significant outbreaks still occur. DAT is an equine polyclonal antibody that is not commercially available in the United States and is in short supply globally. A safer, more readily available alternative to DAT would be desirable. In the current study, we obtained human monoclonal antibodies (hMAbs) directly from antibody-secreting cells in the circulation of immunized human volunteers. We isolated a panel of diverse hMAbs that recognized diphtheria toxoid, as well as a variety of recombinant protein fragments of diphtheria toxin. Forty-five unique hMAbs were tested for neutralization of diphtheria toxin in in vitro cytotoxicity assays with a 50% effective concentration of 0.65 ng/ml for the lead candidate hMAb, 315C4. In addition, 25 μg of 315C4 completely protected guinea pigs from intoxication in an in vivo lethality model, yielding an estimated relative potency of 64 IU/mg. In comparison, 1.6 IU of DAT was necessary for full protection from morbidity and mortality in this model. We further established that our lead candidate hMAb binds to the receptor-binding domain of diphtheria toxin and physically blocks the toxin from binding to the putative receptor, heparin-binding epidermal growth factor-like growth factor. The discovery of a specific and potent human neutralizing antibody against diphtheria toxin holds promise as a potential therapeutic.

  18. Combining Phage and Yeast Cell Surface Antibody Display to Identify Novel Cell Type-Selective Internalizing Human Monoclonal Antibodies.

    Science.gov (United States)

    Bidlingmaier, Scott; Su, Yang; Liu, Bin

    2015-01-01

    Using phage antibody display, large libraries can be generated and screened to identify monoclonal antibodies with affinity for target antigens. However, while library size and diversity is an advantage of the phage display method, there is limited ability to quantitatively enrich for specific binding properties such as affinity. One way of overcoming this limitation is to combine the scale of phage display selections with the flexibility and quantitativeness of FACS-based yeast surface display selections. In this chapter we describe protocols for generating yeast surface antibody display libraries using phage antibody display selection outputs as starting material and FACS-based enrichment of target antigen-binding clones from these libraries. These methods should be widely applicable for the identification of monoclonal antibodies with specific binding properties.

  19. Discovery Of Human Antibodies Against Spitting Cobra Toxins

    DEFF Research Database (Denmark)

    Bojsen-Møller, Laura; Lohse, Brian; Harrison, Robert

    Current snakebite envenoming treatment options consist of animal-derived antisera and are associated with severe adverse reactions due to the heterologous nature of the animal-derived antibodies present in these antisera, and the presence of therapeutically irrelevant antibodies. The African...... spitting cobras are among the most medically important snakes in sub-Saharan regions due to the severity of the clinical outcomes caused by their cytotoxic venom, which is derived from cytotoxins of the 3FTx toxin family and PLA2. Here we report the results of our progress in identifying human antibodies...... targeting relevant toxins from the venom of the black necked spitting cobra (Naja nigricolis)....

  20. Entamoeba histolytica and E. dispar trophozoites in the liver of hamsters: in vivo binding of antibodies and complement

    Directory of Open Access Journals (Sweden)

    Gomes Maria A

    2010-03-01

    Full Text Available Abstract Background Human amoebiasis is caused by the parasitic protozoan Entamoeba histolytica that lives in the large intestine of hosts, where can produce asymptomatic colonization until severe invasive infections with blood diarrhea and spreading to other organs. The amoebic abscesses in liver are the most frequent form of amoebiasis outside intestine and still there are doubts about the pathogenic mechanisms involved in their formation. In this study we evaluated the in situ binding of antibodies, C3 and C9 complement components on trophozoites, in livers of hamsters infected with E. histolytica or E. dispar. These parameters were correlated with the extension of the hepatic lesions observed in these animals and with trophozoites survivor. Methods Hamsters were inoculated intra-hepatically with 100,000 trophozoites of E. histolytica or E. dispar strain and necropsied 12, 24, 48, 72, 144 and 192 h after inoculation. Antibodies, C3 and C9 binding to trophozoites were detected by immunohistochemistry. The estimation of the necrosis area and the number of labeled trophozoites was performed using digital morphometry analysis. Results In the liver sections of animals inoculated with the amoebas, the binding of antibodies to E. histolytica trophozoites was significantly lower than to E. dispar trophozoites. Trophozoites of E. dispar were also more frequently vacuolated and high labeled cellular debris observed in the lesions. Positive diffuse reaction to C3 complement component was more intense in livers of animals inoculated with E. histolytica after 24 and 72 h of infection. C3(+ and C9(+ trophozoites were detected in the vascular lumen, granulomas and inside and in the border of necrotic areas of both infected group animals. C3(+ and C9(+ trophozoite debris immunostaining was higher in livers of E. dispar than in livers of E. histolytica. A positive correlation between necrotic areas and number of C9(+ trophozoites was observed in animals

  1. Antioxidant flavonoids bind human serum albumin

    Science.gov (United States)

    Kanakis, C. D.; Tarantilis, P. A.; Polissiou, M. G.; Diamantoglou, S.; Tajmir-Riahi, H. A.

    2006-10-01

    Human serum albumin (HSA) is a principal extracellular protein with a high concentration in blood plasma and carrier for many drugs to different molecular targets. Flavonoids are powerful antioxidants and prevent DNA damage. The antioxidative protections are related to their binding modes to DNA duplex and complexation with free radicals in vivo. However, flavonoids are known to inhibit the activities of several enzymes such as calcium phospholipid-dependent protein kinase, tyrosine protein kinase from rat lung, phosphorylase kinase, phosphatidylinositol 3-kinase and DNA topoisomerases that exhibit the importance of flavonoid-protein interaction. This study was designed to examine the interaction of human serum albumin (HSA) with quercetin (que), kaempferol (kae) and delphinidin (del) in aqueous solution at physiological conditions, using constant protein concentration of 0.25 mM (final) and various drug contents of 1 μM-1 mM. FTIR and UV-vis spectroscopic methods were used to determine the polyphenolic binding mode, the binding constant and the effects of flavonoid complexation on protein secondary structure. The spectroscopic results showed that flavonoids are located along the polypeptide chains through H-bonding interactions with overall affinity constant of Kque = 1.4 × 10 4 M -1, Kkae = 2.6 × 10 5 M -1 and Kdel = 4.71 × 10 5 M -1. The protein secondary structure showed no alterations at low pigment concentration (1 μM), whereas at high flavonoid content (1 mM), major reduction of α-helix from 55% (free HSA) to 42-46% and increase of β-sheet from 15% (free HSA) to 17-19% and β-anti from 7% (free HSA) to 10-20% occurred in the flavonoid-HSA adducts. The major reduction of HSA α-helix is indicative of a partial protein unfolding upon flavonoid interaction.

  2. Sensitive chain specific radioimmunoassay for human immunoglobulins using monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Sikora, K; Alderson, T St.J.; Ellis, J [Ludwig Institute for Cancer Research, Cambridge (UK)

    1983-02-25

    A sensitive radioimmunoassay is described for human immunoglobulins. This solid-phase assay uses commercially available monoclonal antibodies and is specific for different Ig chain types. Levels of less than 20 ng/ml Ig are detectable. The assay is suitable for the analysis of human hybridoma supernatants.

  3. Human antibody and antigen response to IncA antibody of Chlamydia trachomatis.

    Science.gov (United States)

    Tsai, P Y; Hsu, M C; Huang, C T; Li, S Y

    2007-01-01

    The high prevalence of C. trachomatis worldwide has underscored the importance of identifying specific immunogenic antigens in facilitating diagnosis as well as vaccine development. The aim of this study is to evaluate IncA antibody and antigen production in natural human infections. Our temporal expression study showed that IncA transcription and protein expression could be detected as early as 4 hours after the start of infection. Antibody responses could be detected in urine and genital swab samples from C. trachomatis-positive patients. It is especially interesting to note that the IncA antigen could be detected in urine. In conclusion, we have identified IncA as an important antigen in human. The potential applicability of the IncA antibody or antigen in the diagnosis as well as to vaccine development for C. trachomatis is also discussed.

  4. Structural Basis for Escape of Human Astrovirus from Antibody Neutralization: Broad Implications for Rational Vaccine Design

    Energy Technology Data Exchange (ETDEWEB)

    Bogdanoff, Walter A.; Perez, Edmundo I.; López, Tomás; Arias, Carlos F.; DuBois, Rebecca M. (UNAM-Mexico); (UCSC)

    2017-10-25

    ABSTRACT

    Human astroviruses are recognized as a leading cause of viral diarrhea worldwide in children, immunocompromised patients, and the elderly. There are currently no vaccines available to prevent astrovirus infection; however, antibodies developed by healthy individuals during previous infection correlate with protection from reinfection, suggesting that an effective vaccine could be developed. In this study, we investigated the molecular mechanism by which several strains of human astrovirus serotype 2 (HAstV-2) are resistant to the potent HAstV-2-neutralizing monoclonal antibody PL-2 (MAb PL-2). Sequencing of the HAstV-2 capsid genes reveals mutations in the PL-2 epitope within the capsid's spike domain. To understand the molecular basis for resistance from MAb PL-2 neutralization, we determined the 1.35-Å-resolution crystal structure of the capsid spike from one of these HAstV-2 strains. Our structure reveals a dramatic conformational change in a loop within the PL-2 epitope due to a serine-to-proline mutation, locking the loop in a conformation that sterically blocks binding and neutralization by MAb PL-2. We show that mutation to serine permits loop flexibility and recovers MAb PL-2 binding. Importantly, we find that HAstV-2 capsid spike containing a serine in this loop is immunogenic and elicits antibodies that neutralize all HAstV-2 strains. Taken together, our results have broad implications for rational selection of vaccine strains that do not contain prolines in antigenic loops, so as to elicit antibodies against diverse loop conformations.

    IMPORTANCEHuman astroviruses (HAstVs) infect nearly every person in the world during childhood and cause diarrhea, vomiting, and fever. In this study, we investigated how several strains of HAstV are resistant to a virus-neutralizing monoclonal antibody. We determined the crystal structure of the capsid protein spike domain from one of these HAstV strains and found that

  5. Blocking immunosuppression by human Tregs in vivo with antibodies targeting integrin αVβ8.

    Science.gov (United States)

    Stockis, Julie; Liénart, Stéphanie; Colau, Didier; Collignon, Amandine; Nishimura, Stephen L; Sheppard, Dean; Coulie, Pierre G; Lucas, Sophie

    2017-11-21

    Human regulatory T cells (Tregs) suppress other T cells by converting the latent, inactive form of TGF-β1 into active TGF-β1. In Tregs, TGF-β1 activation requires GARP, a transmembrane protein that binds and presents latent TGF-β1 on the surface of Tregs stimulated through their T cell receptor. However, GARP is not sufficient because transduction of GARP in non-Treg T cells does not induce active TGF-β1 production. RGD-binding integrins were shown to activate TGF-β1 in several non-T cell types. Here we show that αVβ8 dimers are present on stimulated human Tregs but not in other T cells, and that antibodies against αV or β8 subunits block TGF-β1 activation in vitro. We also show that αV and β8 interact with GARP/latent TGF-β1 complexes in human Tregs. Finally, a blocking antibody against β8 inhibited immunosuppression by human Tregs in a model of xenogeneic graft-vs.-host disease induced by the transfer of human T cells in immunodeficient mice. These results show that TGF-β1 activation on the surface of human Tregs implies an interaction between the integrin αVβ8 and GARP/latent TGF-β1 complexes. Immunosuppression by human Tregs can be inhibited by antibodies against GARP or against the integrin β8 subunit. Such antibodies may prove beneficial against cancer or chronic infections.

  6. Experiments on the radioimmunological determination of the triiodothyronine binding capacity of human blood serum

    International Nuclear Information System (INIS)

    Traub, H.W.J.

    1980-01-01

    Bovine serum antibodies have been obtained from rabbits. Constant amounts of antibodies have been incubated with increasing amounts of antigens. The maximum value found for T 3 binding to the antigen-antibody complex was 66 μg RA/mg antigen. Human serum samples containing 0,05 ng 125 J-T 3 /μl were incubated with 600 μg RA, and the antigen was precipitated with 8,4 mg anti-RA. The mean value obtained for 30 enthyreotic test persons was a binding to RA of 7.9 +- 0,52% 125 J-T 3 . Comparative measurements of patient and standard serum samples shared only slight differences. This means that the measured data do not reflect the thyroid function. (orig./MG) [de

  7. Human anti-plague monoclonal antibodies protect mice from Yersinia pestis in a bubonic plague model.

    Directory of Open Access Journals (Sweden)

    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.

  8. Ovarian carcinoma glyco-antigen targeted by human IgM antibody.

    Directory of Open Access Journals (Sweden)

    Yi Chen

    Full Text Available Epithelial Ovarian Cancer (EOC cells expression of a novel carbohydrate antigen was defined using a human VH4-34 encoded IgM monoclonal antibody (mAb216. MAb216 binds to a poly N-acetyllactosamine epitope expressed on B cells and kills normal and malignant B cells in vitro and in vivo. EOC patient ascites and EOC cell lines were used to study the anti tumor effect of mAb216. Various assays were used to characterize the epitope and demonstrate antibody-mediated binding and cytotoxicity in EOC. Drug and antibody combination effects were determined by calculating the combination index values using the Chou and Talalay method. MAb216 displays direct antibody mediated cytotoxicity on a population of human EOC tumor and ascites samples and EOC cell lines, which express high amounts of poly N-acetyllactosamine epitope, carried by CD147/CD98. Eighty four percent of patient samples, including platin resistant, had a tumor population that bound the monoclonal antibody. The binding pattern of mAb216 and mechanism of cytotoxicity was similar to that seen on normal and malignant B cells with unique general membrane disruption and "pore" formation. In vitro incubation with mAb216 and cisplatin enhanced killing of OVCAR3 cell line. In EOC cell lines percent cytotoxicity correlated with percent expression of epitope. Although in vitro data shows specific EOC cytotoxicity, for possible treatment of EOC MAb216 would need to be evaluated in a clinical trial with or without chemotherapy.

  9. At least two Fc Neu5Gc residues of monoclonal antibodies are required for binding to anti-Neu5Gc antibody.

    Science.gov (United States)

    Yu, Chuanfei; Gao, Kai; Zhu, Lei; Wang, Wenbo; Wang, Lan; Zhang, Feng; Liu, Chunyu; Li, Meng; Wormald, Mark R; Rudd, Pauline M; Wang, Junzhi

    2016-01-29

    Two non-human glycan epitopes, galactose-α-1,3-galactose (α-gal) and Neu5Gc-α-2-6-galactose (Neu5Gc) have been shown to be antigenic when attached to Fab oligosaccharides of monoclonal antibodies (mAbs) , while α-gal attached to Fc glycans was not. However, the antigenicity of Neu5Gc on the Fc glycans remains unclear in the context that most mAbs carry only Fc glycans. After studying two clinical mAbs carrying significant amounts of Fc Neu5Gc, we show that their binding activity with anti-Neu5Gc antibody resided in a small subset of mAbs carrying two or more Fc Neu5Gc, while mAbs harboring only one Neu5Gc showed no reactivity. Since most Neu5Gc epitopes were distributed singly on the Fc of mAbs, our results suggest that the potential antigenicity of Fc Neu5Gc is low. Our study could be referenced in the process design and optimization of mAb production in murine myeloma cells and in the quality control of mAbs for industries and regulatory authorities.

  10. Beyond CDR-grafting: Structure-guided humanization of framework and CDR regions of an anti-myostatin antibody.

    Science.gov (United States)

    Apgar, James R; Mader, Michelle; Agostinelli, Rita; Benard, Susan; Bialek, Peter; Johnson, Mark; Gao, Yijie; Krebs, Mark; Owens, Jane; Parris, Kevin; St Andre, Michael; Svenson, Kris; Morris, Carl; Tchistiakova, Lioudmila

    2016-10-01

    Antibodies are an important class of biotherapeutics that offer specificity to their antigen, long half-life, effector function interaction and good manufacturability. The immunogenicity of non-human-derived antibodies, which can be a major limitation to development, has been partially overcome by humanization through complementarity-determining region (CDR) grafting onto human acceptor frameworks. The retention of foreign content in the CDR regions, however, is still a potential immunogenic liability. Here, we describe the humanization of an anti-myostatin antibody utilizing a 2-step process of traditional CDR-grafting onto a human acceptor framework, followed by a structure-guided approach to further reduce the murine content of CDR-grafted antibodies. To accomplish this, we solved the co-crystal structures of myostatin with the chimeric (Protein Databank (PDB) id 5F3B) and CDR-grafted anti-myostatin antibody (PDB id 5F3H), allowing us to computationally predict the structurally important CDR residues as well as those making significant contacts with the antigen. Structure-based rational design enabled further germlining of the CDR-grafted antibody, reducing the murine content of the antibody without affecting antigen binding. The overall "humanness" was increased for both the light and heavy chain variable regions.

  11. An efficiently cleaved HIV-1 clade C Env selectively binds to neutralizing antibodies.

    Directory of Open Access Journals (Sweden)

    Saikat Boliar

    Full Text Available An ideal HIV-1 Env immunogen is expected to mimic the native trimeric conformation for inducing broadly neutralizing antibody responses. The native conformation is dependent on efficient cleavage of HIV-1 Env. The clade B isolate, JRFL Env is efficiently cleaved when expressed on the cell surface. Here, for the first time, we report the identification of a native clade C Env, 4-2.J41 that is naturally and efficiently cleaved on the cell surface as confirmed by its biochemical and antigenic characteristics. In addition to binding to several conformation-dependent neutralizing antibodies, 4-2.J41 Env binds efficiently to the cleavage-dependent antibody PGT151; thus validating its native cleaved conformation. In contrast, 4-2.J41 Env occludes non-neutralizing epitopes. The cytoplasmic-tail of 4-2.J41 Env plays an important role in maintaining its conformation. Furthermore, codon optimization of 4-2.J41 Env sequence significantly increases its expression while retaining its native conformation. Since clade C of HIV-1 is the prevalent subtype, identification and characterization of this efficiently cleaved Env would provide a platform for rational immunogen design.

  12. Current status of cancer immunodetection with radiolabeled human monoclonal antibodies.

    Science.gov (United States)

    De Jager, R; Abdel-Nabi, H; Serafini, A; Pecking, A; Klein, J L; Hanna, M G

    1993-04-01

    The use of radiolabeled murine monoclonal antibodies (MoAbs) for cancer immunodetection has been limited by the development of human antimouse antibodies (HAMA). Human monoclonal antibodies do not elicit a significant human antihuman (HAHA) response. The generation and production of human monoclonal antibodies met with technical difficulties that resulted in delaying their clinical testing. Human monoclonal antibodies of all isotypes have been obtained. Most were immunoglobulin (Ig) M directed against intracellular antigens. Two antibodies, 16.88 (IgM) and 88BV59 (IgG3k), recognize different epitopes on a tumor-associated antigen, CTA 16.88, homologous to cytokeratins 8, 18, and 19. CTA 16.88 is expressed by most epithelial-derived tumors including carcinomas of the colon, pancreas, breast, ovary, and lung. The in vivo targeting by these antibodies is related to their localization in nonnecrotic areas of tumors. Repeated administration of 16.88 over 5 weeks to a cumulative dose of 1,000 mg did not elicit a HAHA response. Two of 53 patients developed a low titer of HAHA 1 to 3 months after a single administration of 88BV59. Planar imaging of colorectal cancer with Iodine-131 (131I)-16.88 was positive in two studies in 9 of 12 and 16 of 20 patients preselected by immunohistochemistry. Tumors less than 2 cm in diameter are usually not detected. The lack of immunogenicity and long tumor residence time (average = 17 days) makes 16.88 a good candidate for therapy. Radioimmunlymphoscintigraphy with indium-111 (111In)-LiLo-16.88 administered by an intramammary route was used in the presurgical staging of primary breast cancer. The negative predictive value of lymph node metastases for tumors less than 3 cm was 90.5%. Planar and single photon emission computed tomography imaging of colorectal carcinoma with technetium-99m (99mTc) 88BV59 was compared with computed tomography (CT) scan in 36 surgical patients. The antibody scan was more sensitive than the CT scan in detecting

  13. Molecular evolution of broadly neutralizing Llama antibodies to the CD4-binding site of HIV-1.

    Science.gov (United States)

    McCoy, Laura E; Rutten, Lucy; Frampton, Dan; Anderson, Ian; Granger, Luke; Bashford-Rogers, Rachael; Dekkers, Gillian; Strokappe, Nika M; Seaman, Michael S; Koh, Willie; Grippo, Vanina; Kliche, Alexander; Verrips, Theo; Kellam, Paul; Fassati, Ariberto; Weiss, Robin A

    2014-12-01

    To date, no immunization of humans or animals has elicited broadly neutralizing sera able to prevent HIV-1 transmission; however, elicitation of broad and potent heavy chain only antibodies (HCAb) has previously been reported in llamas. In this study, the anti-HIV immune responses in immunized llamas were studied via deep sequencing analysis using broadly neutralizing monoclonal HCAbs as a guides. Distinct neutralizing antibody lineages were identified in each animal, including two defined by novel antibodies (as variable regions called VHH) identified by robotic screening of over 6000 clones. The combined application of five VHH against viruses from clades A, B, C and CRF_AG resulted in neutralization as potent as any of the VHH individually and a predicted 100% coverage with a median IC50 of 0.17 µg/ml for the panel of 60 viruses tested. Molecular analysis of the VHH repertoires of two sets of immunized animals showed that each neutralizing lineage was only observed following immunization, demonstrating that they were elicited de novo. Our results show that immunization can induce potent and broadly neutralizing antibodies in llamas with features similar to human antibodies and provide a framework to analyze the effectiveness of immunization protocols.

  14. Calcium-binding proteins from human platelets

    International Nuclear Information System (INIS)

    Gogstad, G.O.; Krutnes, M.B.; Solum, N.O.

    1983-01-01

    Calcium-binding platelet proteins were examined by crossed immunoelectrophoresis of solubilized platelets against antibodies to whole platelets followed by incubation of the immunoplates with 45 Ca 2 + and autoradiography. When the immunoplates had been pretreated with EDTA at pH 9.0 in order to remove divalent cations, three immunoprecipitates were markedly labelled with 45 Ca 2 + . These corresponded to the glycoprotein IIb-IIIa complex, glycoprotein Ia and a presently unidentified antigen termed G18. These antigens were membrane-bound and surface-oriented. When an excess of EDTA was introduced in the incubation media the results revealed that the glycoprotein IIb-IIIa complex and antigen G18, but not glycoprotein Ia, contained sites with a stronger affinity for calcium than has EDTA at pH 7.4 Immunoprecipitates of the separate glycoproteins IIb and IIIa both bound calcium in the same manner as the glycoprotein IIb-IIIa complex. As another approach, platelet-rich plasma was incubated with 45 Ca 2 + prior to crossed immunoelectrophoresis of the solubilized platelets. A single immunoprecipitate was wekly labelled. This did not correspond to any of the immunoprecipitates which were visible after staining with Coomassie blue. The labelling of this antigen was markedly increased when the platelt-rich plasma had been preincubated with EDTA and in this case a weak labelling of the glycoprotein IIB-IIIa precipitate also became apparent. No increased incorporation of calcium occured in any of these immunoprecipitates when the platelets were aggregated with ADP in the presence of 45 Ca 2 + . (orig.)

  15. 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)

  16. A human PrM antibody that recognizes a novel cryptic epitope on dengue E glycoprotein.

    Directory of Open Access Journals (Sweden)

    Annie Hoi Yi Chan

    Full Text Available Dengue virus (DENV is a major mosquito-borne pathogen infecting up to 100 million people each year; so far no effective treatment or vaccines are available. Recently, highly cross-reactive and infection-enhancing pre-membrane (prM-specific antibodies were found to dominate the anti-DENV immune response in humans, raising concern over vaccine candidates that contain native dengue prM sequences. In this study, we have isolated a broadly cross-reactive prM-specific antibody, D29, during a screen with a non-immunized human Fab-phage library against the four serotypes of DENV. The antibody is capable of restoring the infectivity of virtually non-infectious immature DENV (imDENV in FcγR-bearing K562 cells. Remarkably, D29 also cross-reacted with a cryptic epitope on the envelope (E protein located to the DI/DII junction as evidenced by site-directed mutagenesis. This cryptic epitope, while inaccessible to antibody binding in a native virus particle, may become exposed if E is not properly folded. These findings suggest that generation of anti-prM antibodies that enhance DENV infection may not be completely avoided even with immunization strategies employing E protein alone or subunits of E proteins.

  17. Mycobacterium leprae antigens involved in human immune responses. I. Identification of four antigens by monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Britton, W.J.; Hellqvist, L.; Basten, A.; Raison, R.L.

    1985-12-01

    Four distinct antigens were identified in soluble sonicates of Mycobacterium leprae by using a panel of 11 monoclonal antibodies. Cross-reactivity studies with other mycobacterial species were conducted by using ELISA and immunoblot assays, and demonstrated that determinants on two of the antigens were present in many mycobacteria, whereas the other two were limited in distribution. Competitive inhibition experiments with radiolabeled monoclonal antibodies showed cross-inhibition between antibodies identifying two of the four antigenicbands. These two bands, of M/sub tau/ 4.5 to 6 KD and 30 to 40 KD, were resistant to protease treatment after immunoblotting. In contrast the two other bands of 16 and 70 KD were protease-sensitive. Although all four bands reacted with some human lepromatous leprosy sera in immunoblots, the 4.5 to 6 KD and 30 to 40 KD bands were most prominent. Lepromatous leprosy sera also inhibited the binding of radiolabeled monoclonal antibodies to each of the four antigens, with the mean titer causing 50% inhibition being higher for antibodies reacting with the 4.5 to 6 KD and 30 to 40 KD bands. These findings indicated that all four antigens were involved in the human B cell response to M. leprae.

  18. Characterization of Human Colorectal Cancer MDR1/P-gp Fab Antibody

    Directory of Open Access Journals (Sweden)

    Xuemei Zhang

    2013-01-01

    Full Text Available In this study, the peptide sized 21 kDa covering P-gp transmembrane region was first prepared for generating a novel mouse monoclonal antibody Fab fragment with biological activity against multiple drug resistance protein P-gp21 by phage display technology. Phage-displayed antibody library prepared from mice spleen tissues was selected against the recombinant protein P-gp21 with five rounds of panning. A number of clones expressing Fab bound to P-gp21, showing neutralized activity in vitro, were isolated and screened by enzyme-linked immunosorbent assay based on its recognition properties to P-gp21 and human colorectal cancer tissue homogenate, resulting in identification of an optimal recombinant Fab clone (Number 29. Further characterization by recloning number 29 into an expression vector showed significant induction of the Fab antibody in the clone number 29 by Isopropyl β-D-1-thiogalactopyranoside (IPTG. After purified by HiTrap Protein L, the specificity of the Fab antibody to P-gp21 was also confirmed. Not only was the targeted region of this monoclonal Fab antibody identified as a 16-peptide epitope (ALKDKKELEGSGKIAT comprising residues 883–898 within the transmembrane (TM domain of human P-gp, but also the binding ability with it was verified. The clinical implication of our results for development of personalized therapy of colorectal cancer will be further studied.

  19. Mycobacterium leprae antigens involved in human immune responses. I. Identification of four antigens by monoclonal antibodies

    International Nuclear Information System (INIS)

    Britton, W.J.; Hellqvist, L.; Basten, A.; Raison, R.L.

    1985-01-01

    Four distinct antigens were identified in soluble sonicates of Mycobacterium leprae by using a panel of 11 monoclonal antibodies. Cross-reactivity studies with other mycobacterial species were conducted by using ELISA and immunoblot assays, and demonstrated that determinants on two of the antigens were present in many mycobacteria, whereas the other two were limited in distribution. Competitive inhibition experiments with radiolabeled monoclonal antibodies showed cross-inhibition between antibodies identifying two of the four antigenicbands. These two bands, of M/sub tau/ 4.5 to 6 KD and 30 to 40 KD, were resistant to protease treatment after immunoblotting. In contrast the two other bands of 16 and 70 KD were protease-sensitive. Although all four bands reacted with some human lepromatous leprosy sera in immunoblots, the 4.5 to 6 KD and 30 to 40 KD bands were most prominent. Lepromatous leprosy sera also inhibited the binding of radiolabeled monoclonal antibodies to each of the four antigens, with the mean titer causing 50% inhibition being higher for antibodies reacting with the 4.5 to 6 KD and 30 to 40 KD bands. These findings indicated that all four antigens were involved in the human B cell response to M. leprae

  20. Human antibody response to a strain-specific HIV-1 gp120 epitope associated with cell fusion inhibition

    NARCIS (Netherlands)

    Goudsmit, J.; Boucher, C. A.; Meloen, R. H.; Epstein, L. G.; Smit, L.; van der Hoek, L.; Bakker, M.

    1988-01-01

    PEPSCAN analysis, performed using 536 overlapping nonapeptides derived from the HTLV-III B nucleotide sequence of the region encoding the external envelope protein of 120 kDa (gp120), identified in the V3 region of gp120 a major binding site for antibodies of HIV-1-infected humans. The minimal amino

  1. Identification of antigen-specific human monoclonal antibodies using high-throughput sequencing of the antibody repertoire.

    Science.gov (United States)

    Liu, Ju; Li, Ruihua; Liu, Kun; Li, Liangliang; Zai, Xiaodong; Chi, Xiangyang; Fu, Ling; Xu, Junjie; Chen, Wei

    2016-04-22

    High-throughput sequencing of the antibody repertoire provides a large number of antibody variable region sequences that can be used to generate human monoclonal antibodies. However, current screening methods for identifying antigen-specific antibodies are inefficient. In the present study, we developed an antibody clone screening strategy based on clone dynamics and relative frequency, and used it to identify antigen-specific human monoclonal antibodies. Enzyme-linked immunosorbent assay showed that at least 52% of putative positive immunoglobulin heavy chains composed antigen-specific antibodies. Combining information on dynamics and relative frequency improved identification of positive clones and elimination of negative clones. and increase the credibility of putative positive clones. Therefore the screening strategy could simplify the subsequent experimental screening and may facilitate the generation of antigen-specific antibodies. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Identification of epitopes within integrin β4 for binding of auto-antibodies in ocular cicatricial and mucous membrane pemphigoid: preliminary report.

    Science.gov (United States)

    Rashid, Khwaja Aftab; Foster, C Stephen; Ahmed, A Razzaque

    2013-11-19

    To identify the epitopes on human β4 integrin to which the sera of patients with ocular cicatricial pemphigoid (OCP) and mucous membrane pemphigoid (MMP) without ocular involvement bind. Fragments of the intracellular domain of the β4 molecule were cloned, expressed, purified and peptides were synthesized. Antibodies to various fragments and peptides were produced in rabbits. Binding specificity was determined via Western blot and blocking experiments. Test sera and controls were injected into neonatal BALB/c mice for in vivo passive transfer. Sera from patients with OCP, MMP, and both OCP and MMP were bound to cloned fragments of IC3.0. Its subcloned fragments IC3.4 (1489 aa-1572 aa) and IC3.4.1 (1489 aa-1510 aa) were bound with the sera from patients with OCP only. Subcloned fragments IC3.6 (1573 aa-1822 aa) and IC3.6.1 (1689 aa-1702 aa) were bound with MMP sera only. No cross-reactivity in binding was observed. Immuno-affinity-purified sera from patients with OCP, MMP, and rabbit antibodies to IC3.0, IC3.4, IC3.4.1, IC3.6, and IC3.6.1, when injected in neonatal BALB/c mice, produced subepidermal blisters in their skin. These preliminary observations identified IC3.4.1 as the possible epitope for the binding of OCP auto-antibody and IC3.6.1 as the possible epitope for the binding of MMP auto-antibody without ocular disease. Antibodies specific to these peptides produced blisters when injected in mice. Still-unidentified epitopes may exist. These observations may enhance our understanding of the role of β4 integrin in the pathobiology of OCP and MMP. Early diagnosis may be possible if serologic tests with specificity and sensitivity can be developed.

  3. Induction of human immunodeficiency virus neutralizing antibodies using fusion complexes.

    Science.gov (United States)

    Zipeto, Donato; Matucci, Andrea; Ripamonti, Chiara; Scarlatti, Gabriella; Rossolillo, Paola; Turci, Marco; Sartoris, Silvia; Tridente, Giuseppe; Bertazzoni, Umberto

    2006-05-01

    Human immunodeficiency virus-1 (HIV-1) infects cells by membrane fusion that is mediated by the envelope proteins gp120/gp41 and the cellular receptors CD4 and CCR5. During this process, some conserved viral epitopes are temporarily exposed and may induce a neutralizing antibody response when fixed in the fusogenic conformation. These transient structures are conserved and may be effective antigens for use in an anti-HIV-1 vaccine. In this study we tested different conditions of preparation of fusion complexes inducing neutralizing antibodies against both R5 and X4 tropic HIV-1 strains. Cell lines expressing HIV-1 gp120/gp41 and CD4-CCR5 were prepared and conditions for producing fusion complexes were tested. Complexes produced at different temperature and fixative combinations were used to immunize mice. Results indicated that (a) fusion complexes prepared at either 21 degrees C, 30 degrees C or 37 degrees C were immunogenic and induced neutralizing antibodies against both R5 and X4 HIV-1 heterologous isolates; (b) after extensive purification of antibodies there was no cytotoxic effect; (c) complexes prepared at 37 degrees C were more immunogenic and induced higher titers of neutralizing antibodies than complexes prepared at either 21 degrees C or 30 degrees C; (d) the fixative used did not affect the titer of neutralizing antibodies except for glutaraldehyde which was ineffective; (e) the neutralizing activity was retained after CD4-CCR5 antibody removal. The production of higher titers of neutralizing antibody with fusion complexes prepared at 37 degrees C, as compared to lower temperatures, may be related to the induction of antibodies against many different conformation intermediates that subsequently act synergistically at different steps in the fusion process.

  4. Cetuximab in combination with anti-human IgG antibodies efficiently down-regulates the EGF receptor by macropinocytosis

    International Nuclear Information System (INIS)

    Berger, Christian; Madshus, Inger Helene; Stang, Espen

    2012-01-01

    The monoclonal antibody C225 (Cetuximab) blocks binding of ligand to the epidermal growth factor receptor (EGFR). In addition, it is known that incubation with C225 induces endocytosis of the EGFR. This endocytosis has previously been shown to be increased when C225 is combined with an additional monoclonal anti-EGFR antibody. However, the effects of antibody combinations on EGFR activation, endocytosis, trafficking and degradation have been unclear. By binding a secondary antibody to the C225-EGFR complex, we here demonstrate that a combination of antibodies can efficiently internalize and degrade the EGFR. Although the combination of antibodies activated the EGFR kinase and induced ubiquitination of the EGFR, the kinase activity was not required for internalization of the EGFR. In contrast to EGF-induced EGFR down-regulation, the antibody combination efficiently degraded the EGFR without initiating downstream proliferative signaling. The antibody-induced internalization of EGFR was found not to depend on clathrin and/or dynamin, but depended on actin polymerization, suggesting induction of macropinocytosis. Macropinocytosis may cause internalization of large membrane areas, and this could explain the highly efficient internalization of the EGFR induced by combination of antibodies. -- Highlight: ► Cetuximab induced endocytosis of EGFR increases upon combination with anti-human IgG. ► Antibody combination causes internalization of EGFR by macropinocytosis. ► Antibody-induced internalization of EGFR is independent of EGFR kinase activity. ► Antibody combination may have a zipper effect and cross-link EGFRs on neighboring cells.

  5. Cetuximab in combination with anti-human IgG antibodies efficiently down-regulates the EGF receptor by macropinocytosis

    Energy Technology Data Exchange (ETDEWEB)

    Berger, Christian [Department of Pathology, Oslo University Hospital, Rikshospitalet, Post box 4950 Nydalen, 0424 Oslo (Norway); Madshus, Inger Helene [Institute of Pathology, University of Oslo, Rikshospitalet, 0027 Oslo (Norway); Department of Pathology, Oslo University Hospital, Rikshospitalet, Post box 4950 Nydalen, 0424 Oslo (Norway); Stang, Espen, E-mail: espsta@rr-research.no [Department of Pathology, Oslo University Hospital, Rikshospitalet, Post box 4950 Nydalen, 0424 Oslo (Norway)

    2012-12-10

    The monoclonal antibody C225 (Cetuximab) blocks binding of ligand to the epidermal growth factor receptor (EGFR). In addition, it is known that incubation with C225 induces endocytosis of the EGFR. This endocytosis has previously been shown to be increased when C225 is combined with an additional monoclonal anti-EGFR antibody. However, the effects of antibody combinations on EGFR activation, endocytosis, trafficking and degradation have been unclear. By binding a secondary antibody to the C225-EGFR complex, we here demonstrate that a combination of antibodies can efficiently internalize and degrade the EGFR. Although the combination of antibodies activated the EGFR kinase and induced ubiquitination of the EGFR, the kinase activity was not required for internalization of the EGFR. In contrast to EGF-induced EGFR down-regulation, the antibody combination efficiently degraded the EGFR without initiating downstream proliferative signaling. The antibody-induced internalization of EGFR was found not to depend on clathrin and/or dynamin, but depended on actin polymerization, suggesting induction of macropinocytosis. Macropinocytosis may cause internalization of large membrane areas, and this could explain the highly efficient internalization of the EGFR induced by combination of antibodies. -- Highlight: Black-Right-Pointing-Pointer Cetuximab induced endocytosis of EGFR increases upon combination with anti-human IgG. Black-Right-Pointing-Pointer Antibody combination causes internalization of EGFR by macropinocytosis. Black-Right-Pointing-Pointer Antibody-induced internalization of EGFR is independent of EGFR kinase activity. Black-Right-Pointing-Pointer Antibody combination may have a zipper effect and cross-link EGFRs on neighboring cells.

  6. A plasmid containing the human metallothionein II gene can function as an antibody-assisted electrophoretic biosensor for heavy metals.

    Science.gov (United States)

    Wooten, Dennis C; Starr, Clarise R; Lyon, Wanda J

    2016-01-01

    Different forms of heavy metals affect biochemical systems in characteristic ways that cannot be detected with typical metal analysis methods like atomic absorption spectrometry. Further, using living systems to analyze interaction of heavy metals with biochemical systems can be laborious and unreliable. To generate a reliable easy-to-use biologically-based biosensor system, the entire human metallothionein-II (MT-II) gene was incorporated into a plasmid (pUC57-MT) easily replicated in Escherichia coli. In this system, a commercial polyclonal antibody raised against human metal-responsive transcription factor-1 protein (MTF-1 protein) could modify the electrophoretic migration patterns (i.e. cause specific decreases in agarose gel electrophoretic mobility) of the plasmid in the presence or absence of heavy metals other than zinc (Zn). In the study here, heavy metals, MTF-1 protein, and polyclonal anti-MTF-1 antibody were used to assess pUC57-MT plasmid antibody-assisted electrophoretic mobility. Anti-MTF-1 antibody bound both MTF-1 protein and pUC57-MT plasmid in a non-competitive fashion such that it could be used to differentiate specific heavy metal binding. The results showed that antibody-inhibited plasmid migration was heavy metal level-dependent. Zinc caused a unique mobility shift pattern opposite to that of other metals tested, i.e. Zn blocked the antibody ability to inhibit plasmid migration, despite a greatly increased affinity for DNA by the antibody when Zn was present. The Zn effect was reversed/modified by adding MTF-1 protein. Additionally, antibody inhibition of plasmid mobility was resistant to heat pre-treatment and trypsinization, indicating absence of residual DNA extraction-resistant bacterial DNA binding proteins. DNA binding by anti-DNA antibodies may be commonly enhanced by xenobiotic heavy metals and elevated levels of Zn, thus making them potentially effective tools for assessment of heavy metal bioavailability in aqueous solutions and

  7. Development of new versions of anti-human CD34 monoclonal antibodies with potentially reduced immunogenicity

    International Nuclear Information System (INIS)

    Qian Weizhu; Wang Ling; Li Bohua; Wang Hao; Hou Sheng; Hong Xueyu; Zhang Dapeng; Guo Yajun

    2008-01-01

    Despite the widespread clinical use of CD34 antibodies for the purification of human hematopoietic stem/progenitor cells, all the current anti-human CD34 monoclonal antibodies (mAbs) are murine, which have the potential to elicit human antimouse antibody (HAMA) immune response. In the present study, we developed three new mouse anti-human CD34 mAbs which, respectively, belonged to class I, class II and class III CD34 epitope antibodies. In an attempt to reduce the immunogenicity of these three murine mAbs, their chimeric antibodies, which consisted of mouse antibody variable regions fused genetically to human antibody constant regions, were constructed and characterized. The anti-CD34 chimeric antibodies were shown to possess affinity and specificity similar to that of their respective parental murine antibodies. Due to the potentially better safety profiles, these chimeric antibodies might become alternatives to mouse anti-CD34 antibodies routinely used for clinical application

  8. Development of a sensitive and specific epitope-blocking ELISA for universal detection of antibodies to human enterovirus 71 strains.

    Directory of Open Access Journals (Sweden)

    Fang He

    Full Text Available BACKGROUND: Human Enterovirus 71 (EV71 is a common cause of hand, foot and mouth disease (HFMD in young children. It is often associated with severe neurological diseases and mortalities in recent outbreaks across the Asia Pacific region. Currently, there is no efficient universal antibody test available to detect EV71 infections. METHODOLOGY/PRINCIPAL FINDING: In the present study, an epitope-blocking ELISA was developed to detect specific antibodies to human EV71 viruses in human or animal sera. The assay relies on a novel monoclonal antibody (Mab 1C6 that specifically binds to capsid proteins in whole EV71 viruses without any cross reaction to any EV71 capsid protein expressed alone. The sensitivity and specificity of the epitope-blocking ELISA for EV71 was evaluated and compared to microneutralization using immunized animal sera to multiple virus genotypes of EV71 and coxsackieviruses. Further, 200 serum sample from human individuals who were potentially infected with EV71 viruses were tested in both the blocking ELISA and microneutralization. Results indicated that antibodies to EV71 were readily detected in immunized animals or human sera by the epitope blocking ELISA whereas specimens with antibodies to other enteroviruses yielded negative results. This assay is not only simpler to perform but also shows higher sensitivity and specificity as compared to microneutralization. CONCLUSION: The epitope-blocking ELISA based on a unique Mab 1C6 provided highly sensitive and 100% specific detection of antibodies to human EV71 viruses in human sera.

  9. A simple physiologically based pharmacokinetic model evaluating the effect of anti-nicotine antibodies on nicotine disposition in the brains of rats and humans

    Energy Technology Data Exchange (ETDEWEB)

    Saylor, Kyle, E-mail: saylor@vt.edu; Zhang, Chenming, E-mail: chzhang2@vt.edu

    2016-09-15

    Physiologically based pharmacokinetic (PBPK) modeling was applied to investigate the effects of anti-nicotine antibodies on nicotine disposition in the brains of rats and humans. Successful construction of both rat and human models was achieved by fitting model outputs to published nicotine concentration time course data in the blood and in the brain. Key parameters presumed to have the most effect on the ability of these antibodies to prevent nicotine from entering the brain were selected for investigation using the human model. These parameters, which included antibody affinity for nicotine, antibody cross-reactivity with cotinine, and antibody concentration, were broken down into different, clinically-derived in silico treatment levels and fed into the human PBPK model. Model predictions suggested that all three parameters, in addition to smoking status, have a sizable impact on anti-nicotine antibodies' ability to prevent nicotine from entering the brain and that the antibodies elicited by current human vaccines do not have sufficient binding characteristics to reduce brain nicotine concentrations. If the antibody binding characteristics achieved in animal studies can similarly be achieved in human studies, however, nicotine vaccine efficacy in terms of brain nicotine concentration reduction is predicted to meet threshold values for alleviating nicotine dependence. - Highlights: • Modelling of nicotine disposition in the presence of anti-nicotine antibodies • Key vaccine efficacy factors are evaluated in silico in rats and in humans. • Model predicts insufficient antibody binding in past human nicotine vaccines. • Improving immunogenicity and antibody specificity may lead to vaccine success.

  10. A simple physiologically based pharmacokinetic model evaluating the effect of anti-nicotine antibodies on nicotine disposition in the brains of rats and humans

    International Nuclear Information System (INIS)

    Saylor, Kyle; Zhang, Chenming

    2016-01-01

    Physiologically based pharmacokinetic (PBPK) modeling was applied to investigate the effects of anti-nicotine antibodies on nicotine disposition in the brains of rats and humans. Successful construction of both rat and human models was achieved by fitting model outputs to published nicotine concentration time course data in the blood and in the brain. Key parameters presumed to have the most effect on the ability of these antibodies to prevent nicotine from entering the brain were selected for investigation using the human model. These parameters, which included antibody affinity for nicotine, antibody cross-reactivity with cotinine, and antibody concentration, were broken down into different, clinically-derived in silico treatment levels and fed into the human PBPK model. Model predictions suggested that all three parameters, in addition to smoking status, have a sizable impact on anti-nicotine antibodies' ability to prevent nicotine from entering the brain and that the antibodies elicited by current human vaccines do not have sufficient binding characteristics to reduce brain nicotine concentrations. If the antibody binding characteristics achieved in animal studies can similarly be achieved in human studies, however, nicotine vaccine efficacy in terms of brain nicotine concentration reduction is predicted to meet threshold values for alleviating nicotine dependence. - Highlights: • Modelling of nicotine disposition in the presence of anti-nicotine antibodies • Key vaccine efficacy factors are evaluated in silico in rats and in humans. • Model predicts insufficient antibody binding in past human nicotine vaccines. • Improving immunogenicity and antibody specificity may lead to vaccine success.

  11. Single-Domain Antibodies As Therapeutics against Human Viral Diseases

    Directory of Open Access Journals (Sweden)

    Yanling Wu

    2017-12-01

    Full Text Available In full-size formats, monoclonal antibodies have been highly successful as therapeutics against cancer and immune diseases. However, their large size leads to inaccessibility of some epitopes and relatively high production costs. As an alternative, single-domain antibodies (sdAbs offer special advantages compared to full-size antibodies, including smaller size, larger number of accessible epitopes, relatively low production costs and improved robustness. Currently, sdAbs are being developed against a number of viruses, including human immunodeficiency virus-1 (HIV-1, influenza viruses, hepatitis C virus (HCV, respiratory syncytial virus (RSV, and enteric viruses. Although sdAbs are very potent inhibitors of viral infections, no sdAbs have been approved for clinical use against virial infection or any other diseases. In this review, we discuss the current state of research on sdAbs against viruses and their potential as therapeutics against human viral diseases.

  12. A murine monoclonal anti-idiotypic antibody detects a common idiotope on human, mouse and rabbit antibodies to allergen Lol p IV.

    Science.gov (United States)

    Zhou, E M; Dzuba-Fischer, J M; Rector, E S; Sehon, A H; Kisil, F T

    1991-09-01

    A syngeneic mouse monoclonal anti-idiotypic antibody (anti-Id), designated as B1/1, was generated against a monoclonal antibody (MoAb 91) specific for Ryegrass pollen allergen Lol p IV. This anti-Id recognized an idiotope (Id) that was also present on other monoclonal antibodies with the same specificity as MoAb 91. Observations that (i) the anti-Id inhibited the binding of MoAb 91 to Lol p IV and (ii) the Id-anti-Id interaction could be inhibited by Lol p IV indicated that the Id was located within or near the antigen combining site. These properties served to characterize B1/1 as an internal image anti-Id. Evidence that an immune response in different species to Lol p IV elicits the formation of antibodies which express a common Id was provided by the observations that (i) the Id-anti-Id interactions could be inhibited by mouse, human and rabbit antisera to Lol p IV and (ii) the binding of these antisera to Lol p IV could be inhibited by the anti-Id. Interestingly, the internal image anti-Id B1/1 also recognized an Id on a monoclonal antibody which was directed to an epitope of Lol p IV, different from that recognized by MoAb 91.

  13. Performance evaluation of phage-displayed synthetic human single-domain antibody libraries: A retrospective analysis.

    Science.gov (United States)

    Henry, Kevin A; Tanha, Jamshid

    2018-05-01

    Fully human synthetic single-domain antibodies (sdAbs) are desirable therapeutic molecules but their development is a considerable challenge. Here, using a retrospective analysis of in-house historical data, we examined the parameters that impact the outcome of screening phage-displayed synthetic human sdAb libraries to discover antigen-specific binders. We found no evidence for a differential effect of domain type (V H or V L ), library randomization strategy, incorporation of a stabilizing disulfide linkage or sdAb display format (monovalent vs. multivalent) on the probability of obtaining any antigen-binding human sdAbs, instead finding that the success of library screens was primarily related to properties of target antigens, especially molecular mass. The solubility and binding affinity of sdAbs isolated from successful screens depended both on properties of the sdAb libraries (primarily domain type) and the target antigens. Taking attrition of sdAbs with major manufacturability concerns (aggregation; low expression) and sdAbs that do not recognize native cell-surface antigens as independent probabilities, we calculate the overall likelihood of obtaining ≥1 antigen-binding human sdAb from a single library-target screen as ~24%. Successful library-target screens should be expected to yield ~1.3 human sdAbs on average, each with average binding affinity of ~2 μM. Copyright © 2018 Elsevier B.V. All rights reserved.

  14. Generation of a haptoglobin-hemoglobin complex-specific Fab antibody blocking the binding of the complex to CD163

    DEFF Research Database (Denmark)

    Horn, Ivo R; Nielsen, Marianne Jensby; Madsen, Mette

    2003-01-01

    During intravascular hemolysis hemoglobin (Hb) binds to haptoglobin (Hp) leading to endocytosis of the complex by the macrophage receptor, CD163. In the present study, we used a phage-display Fab antibody strategy to explore if the complex formation between Hp and Hb leads to exposure of antigenic...... epitopes specific for the complex. By Hp-Hb-affinity screening of a phage-Fab library, we isolated a phage clone against the ligand complex. Surface plasmon resonance analyses of the Fab part expressed as a recombinant protein revealed a high affinity binding (KD = 3.9 nm) to Hp-Hb, whereas no binding...... was measured for non-complexed Hp or Hb. The Fab antibody completely inhibited the binding of 125I-labeled Hp-Hb complexes to CD163 and blocked their uptake in CD163-transfected cells. In conclusion, we have raised a receptor-blocking antibody specifically recognizing the Hp-Hb complex. In addition to provide...

  15. IgY antibodies in human nutrition for disease prevention.

    Science.gov (United States)

    Müller, Sandra; Schubert, Andreas; Zajac, Julia; Dyck, Terry; Oelkrug, Christopher

    2015-10-20

    Oral administration of preformed specific antibodies is an attractive approach against infections of the digestive system in humans and animals in times of increasing antibiotic resistances. Previous studies showed a positive effect of egg yolk IgY antibodies on bacterial intoxications in animals and humans. Immunization of chickens with specific antigens offers the possibility to create various forms of antibodies. Research shows that orally applied IgY's isolated from egg yolks can passively cure or prevent diseases of the digestive system. The use of these alternative therapeutic drugs provides further advantages: (1) The production of IgY's is a non-invasive alternative to current methods; (2) The keeping of chickens is inexpensive; (3) The animals are easy to handle; (4) It avoids repetitive bleeding of laboratory animals; (5) It is also very cost effective regarding the high IgY concentration within the egg yolk. Novel targets of these antigen specific antibodies are Helicobacter pylori and also molecules involved in signaling pathways in gastric cancer. Furthermore, also dental caries causing bacteria like Streptococcus mutans or opportunistic Pseudomonas aeruginosa in cystic fibrosis patients are possible targets. Therefore, IgY's included in food for human consumption may be able to prevent or cure human diseases.

  16. Discovery of human antibodies against black cobra toxins

    DEFF Research Database (Denmark)

    Øhlenschlæger, Mia; Andersen, Mikael Rørdam; Lohse, Brian

    Snakebite envenoming represents a major health threat intropical parts of the developing world1. Animal-derivedantisera currently constitute the only effective treatment option,but are associated with severe side effects due toincompatibility with the human immune system. We aim atdiscovering hum...... antibodies that target the medically mostimportant toxins from N. melanoleuca venom using phagedisplay technology....

  17. A radioimmunoassay for human antibody specific for microbial antigens

    International Nuclear Information System (INIS)

    Tew, J.G.; Burmeister, J.; Greene, E.J.; Pflaumer, S.K.; Goldstein, J.

    1977-01-01

    A simple and sensitive method for detecting and quantitating antibody specific or microbial antigens is described. Bacterial, fungal, parasitic or viral antigens attached to bromoacetyl cellulose or the intact cells themselves were added to a series of two-fold dilutions of human serum. After a short incubation period, which allowed human antibody to attach to the antigens, the complex was thoroughly washed and carbon-14 labeled anti-human light chain antibody was added to each dilution. The resulting complex was washed, collected on a filter pad, placed in a scintillation vial and radioassayed. The relationship between radioactivity bound and -log 2 of the serum dilution was linear. The endpoint for each assay and a confidence interval was calculated by doing inverse prediction from simple linear regression. Results obtained using this assay indicated the presence of antibody in a pool of normal human sera specific for herpes virus and for both cell surface and intracellular antigens of Streptococcus mutans, Naegleria fowleri and Cryptococcus neoformans. In general the dominant response was against the intracellular antigens rather than cell surface antigens

  18. Senile amyloidosis and neuron binding antibody in the aging Syrian hamster

    International Nuclear Information System (INIS)

    Blumenthal, H.T.; Musacchia, X.J.

    1985-01-01

    The effects of age, sex, and irradiation on the genesis of amyloidosis, neuron-binding antibody (NBA), and the concomitant appearance of these two phenomena were studied in a colony of Syrian hamsters. In nonirradiated controls amyloidosis increased in prevalence with age after 12 months, and prevalence was higher in females than in males. Irradiation had the effect of advancing the appearance of amyloidosis to the 7-12 months group but did not intensify the amyloidotic process. IgG binding to the nucleus or cytoplasm of neurons was rare, and, despite the fact that IgM and IgA binding to these structures was present in about one-third of the animals, there was neither an aging nor an irradiation effect. The only statistically significant findings with respect to the concomitant occurrence of amyloid and NBA were negative correlations between nuclear IgM and IgA binding and amyloidosis. Of the various species thus far studied, the hamster is the first in which there has been no aging effect in respect to NBA

  19. Receptor-binding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine

    International Nuclear Information System (INIS)

    He Yuxian; Zhou Yusen; Liu Shuwen; Kou Zhihua; Li Wenhui; Farzan, Michael; Jiang Shibo

    2004-01-01

    The spike (S) protein of severe acute respiratory syndrome (SARS) coronavirus (CoV), a type I transmembrane envelope glycoprotein, consists of S1 and S2 domains responsible for virus binding and fusion, respectively. The S1 contains a receptor-binding domain (RBD) that can specifically bind to angiotensin-converting enzyme 2 (ACE2), the receptor on target cells. Here we show that a recombinant fusion protein (designated RBD-Fc) containing 193-amino acid RBD (residues 318-510) and a human IgG1 Fc fragment can induce highly potent antibody responses in the immunized rabbits. The antibodies recognized RBD on S1 domain and completely inhibited SARS-CoV infection at a serum dilution of 1:10,240. Rabbit antisera effectively blocked binding of S1, which contains RBD, to ACE2. This suggests that RBD can induce highly potent neutralizing antibody responses and has potential to be developed as an effective and safe subunit vaccine for prevention of SARS

  20. 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.

  1. Monoclonal Antibody Production against Human Spermatozoal Surface Antigens

    Directory of Open Access Journals (Sweden)

    M Jedi-Tehrani

    2005-10-01

    Full Text Available Introduction: As monoclonal antibodies are potential tools for characterization of soluble or cellular surface antigens, use of these proteins has always been considered in infertility and reproduction research. Therefore, in this study, monoclonal antibodies against human sperm surface antigens were produced. Material and Methods: To produce specific clones against human sperm surface antigens, proteins were extracted using solubilization methods. Balb/c mice were immunized intraperitoneally with the proteins using complete Freund’s adjuvant in the first injection and incomplete Adjuvant in the following booster injections. Hybridoma cells producing ASA were cloned by limiting dilution. Results: Five stable ASA producing hybridoma clones were achieved and their antibody isotypes were determined by ELISA. All the isotypes were of IgG class. Their cross reactivity with rat and mice spermatozoa was examined but they did not have any cross reactivity. Conclusion: The produced antibodies can be used in further studies to characterize and evaluate each of the antigens present on human sperm surface and determining their role in fertilization.

  2. Production and characterization of anti-human IgG F(ab')2 antibody fragment.

    Science.gov (United States)

    Valedkarimi, Zahra; Nasiri, Hadi; Aghebati-Maleki, Leili; Abdolalizadeh, Jalal; Esparvarinha, Mojghan; Majidi, Jafar

    2018-04-10

    In present study an optimized protocol for the separation of antibodies into antigen-binding fragments F(ab')2 using pepsin digestion was investigated. The production of these fragments is a consequential step in the development of medical research, treatment and diagnosis. For production of polyclonal antibody rabbit received antigen in four steps. The rabbit serum at 1/128000 dilution showed high absorbance in reaction with human IgG at the designed ELISA method. Rabbit IgG was purified by Ion-Exchange Chromatography (IEC) method. Purity was assessed by SDS-PAGE method. In non-reduced condition only one band was seen in about 150 kDa MW position and in reduced form, two bands were seen in 50 and 25 kDa MW positions. Rabbit IgG was digested by pepsin enzyme. The antibody fragments solution was applied to Gel filtration column to isolate the F(ab')2. Non-reduced SDS-PAGE for determining the purity of F(ab')2 fragment resulted in one band in 100 kDa corresponds to F(ab')2 fragment and a band in 150 kDa MW position corresponds to undigested IgG antibodies. The activities of FITC conjugated F(ab')2 fragment and commercial ones were compared using flowcytometry method. The activity results implied that the FITC conjugated- anti human F(ab')2 fragment worked as efficiently as the commercial one.

  3. 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.)

  4. Secretion of autoimmune antibodies in the human subcutaneous adipose tissue.

    Science.gov (United States)

    Frasca, Daniela; Diaz, Alain; Romero, Maria; Thaller, Seth; Blomberg, Bonnie B

    2018-01-01

    The adipose tissue (AT) contributes to systemic and B cell intrinsic inflammation, reduced B cell responses and secretion of autoimmune antibodies. In this study we show that adipocytes in the human obese subcutaneous AT (SAT) secrete several pro-inflammatory cytokines and chemokines, which contribute to the establishment and maintenance of local and systemic inflammation, and consequent suboptimal immune responses in obese individuals, as we have previously shown. We also show that pro-inflammatory chemokines recruit immune cells expressing the corresponding receptors to the SAT, where they also contribute to local and systemic inflammation, secreting additional pro-inflammatory mediators. Moreover, we show that the SAT generates autoimmune antibodies. During the development of obesity, reduced oxygen and consequent hypoxia and cell death lead to further release of pro-inflammatory cytokines, "self" protein antigens, cell-free DNA and lipids. All these stimulate class switch and the production of autoimmune IgG antibodies which have been described to be pathogenic. In addition to hypoxia, we have measured cell cytotoxicity and DNA damage mechanisms, which may also contribute to the release of "self" antigens in the SAT. All these processes are significantly elevated in the SAT as compared to the blood. We definitively found that fat-specific IgG antibodies are secreted by B cells in the SAT and that B cells express mRNA for the transcription factor T-bet and the membrane marker CD11c, both involved in the production of autoimmune IgG antibodies. Finally, the SAT also expresses RNA for cytokines known to promote Germinal Center formation, isotype class switch, and plasma cell differentiation. Our results show novel mechanisms for the generation of autoimmune antibody responses in the human SAT and allow the identification of new pathways to possibly manipulate in order to reduce systemic inflammation and autoantibody production in obese individuals.

  5. Low prevalence of antibodies and other plasma factors binding to CC chemokines and IL-2 in HIV-positive patients

    DEFF Research Database (Denmark)

    Meyer, C N; Svenson, M; Schade Larsen, C

    2000-01-01

    of HIV-infected patients were therefore assessed by radioimmunoassay and radioreceptor assay. IgG from 4/505 HIV patients and 9/2000 healthy controls (p>0.05) bound rMIP-1alpha and rMIP-1beta, but not rRANTES. No other plasma factors bound the chemokines. The antibodies inhibited receptor binding of both...... chemokines. There was no association between presence of antibodies and disease stage or HIV progression rate. Three of 11 patients treated with rIL-2 developed IgG antibodies suppressing cellular binding and growth promotion of rIL-2. Hence, circulating factors, including antibodies MIP-1alpha/MIP-1beta...

  6. Localization of gonadotropin binding sites in human ovarian neoplasms

    International Nuclear Information System (INIS)

    Nakano, R.; Kitayama, S.; Yamoto, M.; Shima, K.; Ooshima, A.

    1989-01-01

    The binding of human luteinizing hormone and human follicle-stimulating hormone to ovarian tumor biopsy specimens from 29 patients was analyzed. The binding sites for human luteinizing hormone were demonstrated in one tumor of epithelial origin (mucinous cystadenoma) and in one of sex cord-stromal origin (theca cell tumor). The binding sites for human follicle-stimulating hormone were found in three tumors of epithelial origin (serous cystadenoma and mucinous cystadenoma) and in two of sex cord-stromal origin (theca cell tumor and theca-granulosa cell tumor). The surface-binding autoradiographic study revealed that the binding sites for gonadotropins were localized in the stromal tissue. The results suggest that gonadotropic hormones may play a role in the growth and differentiation of a certain type of human ovarian neoplasms

  7. 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.

  8. Phase I study of anticolon cancer humanized antibody A33.

    Science.gov (United States)

    Welt, Sydney; Ritter, Gerd; Williams, Clarence; Cohen, Leonard S; John, Mary; Jungbluth, Achim; Richards, Elizabeth A; Old, Lloyd J; Kemeny, Nancy E

    2003-04-01

    Humanized A33 (huA33; IgG1) monoclonal antibody detects a determinant expressed by 95% of colorectal cancers and can activate immune cytolytic mechanisms. The present study was designed to (a) define the toxicities and maximum tolerated dose of huA33 and (b) determine huA33 immunogenicity. Patients (n = 11) with advanced chemotherapy-resistant colorectal cancer received 4-week cycles of huA33 at 10, 25, or 50 mg/m(2)/week. Serum samples were analyzed using biosensor technology for evidence of human antihuman antibody (HAHA) response. Eight of 11 patients developed a HAHA response. Significant toxicity was limited to four patients who developed high HAHA titers. In two of these cases, infusion-related reactions such as fevers, rigors, facial flushing, and changes in blood pressure were observed, whereas in the other two cases, toxicity consisted of skin rash, fever, or myalgia. Of three patients who remained HAHA negative, one achieved a radiographic partial response, with reduction of serum carcinoembryonic antigen from 80 to 3 ng/ml. Four patients had radiographic evidence of stable disease (2, 4, 6, and 12 months), with significant reductions (>25%) in serum carcinoembryonic antigen levels in two cases. The complementarity-determining region-grafted huA33 antibody is immunogenic in the majority of colon cancer patients (73%). HAHA activity can be measured reproducibly and quantitatively by BIACORE analysis. Whereas the huA33 construct tested here may be too immunogenic for further clinical development, the antitumor effects observed in the absence of antibody-mediated toxicity and in this heavily pretreated patient population warrant clinical testing of other IgG1 humanized versions of A33 antibody.

  9. The Plasmodium falciparum erythrocyte invasion ligand Pfrh4 as a target of functional and protective human antibodies against malaria.

    Directory of Open Access Journals (Sweden)

    Linda Reiling

    Full Text Available BACKGROUND: Acquired antibodies are important in human immunity to malaria, but key targets remain largely unknown. Plasmodium falciparum reticulocyte-binding-homologue-4 (PfRh4 is important for invasion of human erythrocytes and may therefore be a target of protective immunity. METHODS: IgG and IgG subclass-specific responses against different regions of PfRh4 were determined in a longitudinal cohort of 206 children in Papua New Guinea (PNG. Human PfRh4 antibodies were tested for functional invasion-inhibitory activity, and expression of PfRh4 by P. falciparum isolates and sequence polymorphisms were determined. RESULTS: Antibodies to PfRh4 were acquired by children exposed to P. falciparum malaria, were predominantly comprised of IgG1 and IgG3 subclasses, and were associated with increasing age and active parasitemia. High levels of antibodies, particularly IgG3, were strongly predictive of protection against clinical malaria and high-density parasitemia. Human affinity-purified antibodies to the binding region of PfRh4 effectively inhibited erythrocyte invasion by P. falciparum merozoites and antibody levels in protected children were at functionally-active concentrations. Although expression of PfRh4 can vary, PfRh4 protein was expressed by most isolates derived from the cohort and showed limited sequence polymorphism. CONCLUSIONS: Evidence suggests that PfRh4 is a target of antibodies that contribute to protective immunity to malaria by inhibiting erythrocyte invasion and preventing high density parasitemia. These findings advance our understanding of the targets and mechanisms of human immunity and evaluating the potential of PfRh4 as a component of candidate malaria vaccines.

  10. Laminin binding protein, 34/67 laminin receptor, carries stage-specific embryonic antigen-4 epitope defined by monoclonal antibody Raft.2

    International Nuclear Information System (INIS)

    Katagiri, Yohko U.; Kiyokawa, Nobutaka; Nakamura, Kyoko; Takenouchi, Hisami; Taguchi, Tomoko; Okita, Hajime; Umezawa, Akihiro; Fujimoto, Junichiro

    2005-01-01

    We previously produced monoclonal antibodies against the detergent-insoluble microdomain, i.e., the raft microdomain, of the human renal cancer cell line ACHN. Raft.2, one of these monoclonal antibodies, recognizes sialosyl globopentaosylceramide, which has the stage-specific embryonic antigen (SSEA)-4 epitope. Although the mouse embryonal carcinoma (EC) cell line F9 does not express SSEA-4, some F9 cells stained with Raft.2. Western analysis and matrix-assisted laser desorption ionization-time of flight mass spectrometry identified the Raft.2 binding molecule as laminin binding protein (LBP), i.e., 34/67 laminin receptor. Weak acid treatment or digestion with Clostridium perfringens sialidase reduced Raft.2 binding to LBP on nitrocellulose sheets and [ 14 C]galactose was incorporated into LBP, indicating LBP to have a sialylated carbohydrate moiety. Subcellular localization analysis by sucrose density-gradient centrifugation and examination by confocal microscopy revealed LBP to be localized on the outer surface of the plasma membrane. An SSEA-4-positive human EC cell line, NCR-G3 cells, also expressed Raft.2-binding LBP

  11. Structural Characterisation Reveals Mechanism of IL-13-Neutralising Monoclonal Antibody Tralokinumab as Inhibition of Binding to IL-13Rα1 and IL-13Rα2.

    Science.gov (United States)

    Popovic, B; Breed, J; Rees, D G; Gardener, M J; Vinall, L M K; Kemp, B; Spooner, J; Keen, J; Minter, R; Uddin, F; Colice, G; Wilkinson, T; Vaughan, T; May, R D

    2017-01-20

    Interleukin (IL)-13 is a pleiotropic T helper type 2 cytokine frequently associated with asthma and atopic dermatitis. IL-13-mediated signalling is initiated by binding to IL-13Rα1, which then recruits IL-4Rα to form a heterodimeric receptor complex. IL-13 also binds to IL-13Rα2, considered as either a decoy or a key mediator of fibrosis. IL-13-neutralising antibodies act by preventing IL-13 binding to IL-13Rα1, IL-4Rα and/or IL-13Rα2. Tralokinumab (CAT-354) is an IL-13-neutralising human IgG4 monoclonal antibody that has shown clinical benefit in patients with asthma. To decipher how tralokinumab inhibits the effects of IL-13, we determined the structure of tralokinumab Fab in complex with human IL-13 to 2 Å resolution. The structure analysis reveals that tralokinumab prevents IL-13 from binding to both IL-13Rα1 and IL-13Rα2. This is supported by biochemical ligand-receptor interaction assay data. The tralokinumab epitope is mainly composed of residues in helices D and A of IL-13. It is mostly light chain complementarity-determining regions that are driving paratope interactions; the variable light complementarity-determining region 2 plays a key role by providing residue contacts for a network of hydrogen bonds and a salt bridge in the core of binding. The key residues within the paratope contributing to binding were identified as Asp50, Asp51, Ser30 and Lys31. This study demonstrates that tralokinumab prevents the IL-13 pharmacodynamic effect by binding to IL-13 helices A and D, thus preventing IL-13 from interacting with IL-13Rα1 and IL-13Rα2. Copyright © 2016 AstraZeneca. Published by Elsevier Ltd.. All rights reserved.

  12. Multiple binding modes of ibuprofen in human serum albumin identified by absolute binding free energy calculations

    KAUST Repository

    Evoli, Stefania

    2016-11-10

    Human serum albumin possesses multiple binding sites and transports a wide range of ligands that include the anti-inflammatory drug ibuprofen. A complete map of the binding sites of ibuprofen in albumin is difficult to obtain in traditional experiments, because of the structural adaptability of this protein in accommodating small ligands. In this work, we provide a set of predictions covering the geometry, affinity of binding and protonation state for the pharmaceutically most active form (S-isomer) of ibuprofen to albumin, by using absolute binding free energy calculations in combination with classical molecular dynamics (MD) simulations and molecular docking. The most favorable binding modes correctly reproduce several experimentally identified binding locations, which include the two Sudlow\\'s drug sites (DS2 and DS1) and the fatty acid binding sites 6 and 2 (FA6 and FA2). Previously unknown details of the binding conformations were revealed for some of them, and formerly undetected binding modes were found in other protein sites. The calculated binding affinities exhibit trends which seem to agree with the available experimental data, and drastically degrade when the ligand is modeled in a protonated (neutral) state, indicating that ibuprofen associates with albumin preferentially in its charged form. These findings provide a detailed description of the binding of ibuprofen, help to explain a wide range of results reported in the literature in the last decades, and demonstrate the possibility of using simulation methods to predict ligand binding to albumin.

  13. Human liver aldehyde dehydrogenase: coenzyme binding

    International Nuclear Information System (INIS)

    Kosley, L.L.; Pietruszko, R.

    1987-01-01

    The binding of [U- 14 C] NAD to mitochondrial (E2) and cytoplasmin(E1) aldehyde dehydrogenase was measured by gel filtration and sedimentation techniques. The binding data for NAD and (E1) yielded linear Scatchard plots giving a dissociation constant of 25 (+/- 8) uM and the stoichiometry of 2 mol of NAD bound per mol of E1. The binding data for NAD and (E2) gave nonlinear Scatchard plots. The binding of NADH to E2 was measured via fluorescence enhancement; this could not be done with E1 because there was no signal. The dissociation constant for E2 by this technique was 0.7 (+/- 0.4) uM and stoichiometry of 1.0 was obtained. The binding of [U- 14 C] NADH to (E1) and (E2) was also measured by the sedimentation technique. The binding data for (E1) and NADH gave linear Scatchard plots giving a dissociation constant of 13 (+/- 6) uM and the stoichiometry of 2.0. The binding data for NADH to (E2) gave nonlinear Scatchard plots. With (E1), the dissociation constants for both NAD and NADH are similar to those determined kinetically, but the stoichiometry is only half of that found by stopped flow technique. With (E2) the dissociation constant by fluorometric procedure was 2 orders of magnitude less than that from catalytic reaction

  14. Immunotherapy of Alzheimer's disease (AD): from murine models to anti-amyloid beta (Abeta) human monoclonal antibodies.

    Science.gov (United States)

    Geylis, Valeria; Steinitz, Michael

    2006-01-01

    The deposition of amyloid beta (Abeta) protein is a key pathological feature in Alzheimer's disease (AD). In murine models of AD, both active and passive immunization against Abeta induce a marked reduction in amyloid brain burden and an improvement in cognitive functions. Preliminary results of a prematurely terminated clinical trial where AD patients were actively vaccinated with aggregated Abeta bear resemblance to those documented in murine models. Passive immunization of AD patients with anti-Abeta antibodies, in particular human antibodies, is a strategy that provides a more cautious management and control of any undesired side effects. Sera of all healthy adults contain anti-Abeta IgG autoimmune antibodies. Hence antigen-committed human B-cells are easily immortalized by Epstein-Barr virus (EBV) into anti-Abeta secreting cell lines. Two anti-Abeta human monoclonal antibodies which we recently prepared bind to the N-terminus of Abeta peptide and were shown to stain amyloid plaques in non-fixed brain sections from an AD patient. It is anticipated that specifically selected anti-Abeta human monoclonal antibodies could reduce and inhibit deposits of amyloid in brain while avoiding the cognitive decline that characterizes AD. In the future, this type of antibody may prove to be a promising immune therapy for the disease.

  15. The Duffy binding protein (PkDBPαII) of Plasmodium knowlesi from Peninsular Malaysia and Malaysian Borneo show different binding activity level to human erythrocytes.

    Science.gov (United States)

    Lim, Khai Lone; Amir, Amirah; Lau, Yee Ling; Fong, Mun Yik

    2017-08-11

    The zoonotic Plasmodium knowlesi is a major cause of human malaria in Malaysia. This parasite uses the Duffy binding protein (PkDBPαII) to interact with the Duffy antigen receptor for chemokines (DARC) receptor on human and macaque erythrocytes to initiate invasion. Previous studies on P. knowlesi have reported distinct Peninsular Malaysia and Malaysian Borneo PkDBPαII haplotypes. In the present study, the differential binding activity of these haplotypes with human and macaque (Macaca fascicularis) erythrocytes was investigated. The PkDBPαII of Peninsular Malaysia and Malaysian Borneo were expressed on the surface of COS-7 cells and tested with human and monkey erythrocytes, with and without anti-Fy6 (anti-Duffy) monoclonal antibody treatment. Binding activity level was determined by counting the number of rosettes formed between the transfected COS-7 cells and the erythrocytes. Anti-Fy6 treatment was shown to completely block the binding of human erythrocytes with the transfected COS-7 cells, thus verifying the specific binding of human DARC with PkDBPαII. Interestingly, the PkDBPαII of Peninsular Malaysia displayed a higher binding activity with human erythrocytes when compared with the Malaysian Borneo PkDBPαII haplotype (mean number of rosettes formed = 156.89 ± 6.62 and 46.00 ± 3.57, respectively; P < 0.0001). However, no difference in binding activity level was seen in the binding assay using M. fascicularis erythrocytes. This study is the first report of phenotypic difference between PkDBPαII haplotypes. The biological implication of this finding is yet to be determined. Therefore, further studies need to be carried out to determine whether this differential binding level can be associated with severity of knowlesi malaria in human.

  16. Detection of antibodies to bacterial cell wall peptidoglycan in human sera

    International Nuclear Information System (INIS)

    Heymer, B.; Schleifer, K.H.; Read, S.; Zabriskie, J.B.; Krause, R.M.

    1976-01-01

    A radioimmunoassay has been developed for the measurement of antibodies to peptidoglycan in human sera including patients with rheumatic feaver and juvenile rheumatoid arthritis. The assay is based on the percentage of binding of the hapten 125 I-L-Ala-γ-D-Glu-L-Lys-D-Ala-D-Ala, the major peptide determinant of peptidoglycan. Because of differences in the avidity of the antibodies in different sera, the amount of antibody was expressed as pentapeptide hapten-binding capacity (pentapeptide-HBC in ng/ml of serum). Fourteen out of 105 normal blood donors had a pentapeptide-HBC value greater than or equal to 75 ng/ml serum. Values in healthy children 5 to 18 years of age were less than or equal to 50 ng/ml. Sixty-eight percent of the individuals with rheumatic fever had values greater than or equal to 75 ng/ml, an indication that streptococcal infections can stimulate an immune response to peptidoglycan. Thirty-five percent of the patients with juvenile rheumatoid arthritis had values greater than or equal to 75 ng/ml. Such a finding points to a possible association between bacterial infections and juvenile rheumatoid arthritis

  17. An insight into the thermodynamic characteristics of human thrombopoietin complexation with TN1 antibody

    Science.gov (United States)

    Shibazaki, Chie; Adachi, Motoyasu; Honjo, Eijiro; Tamada, Taro; Maeda, Yoshitake; Tahara, Tomoyuki; Kato, Takashi; Miyazaki, Hiroshi; Blaber, Michael; Kuroki, Ryota

    2016-01-01

    Abstract Human thrombopoietin (hTPO) primarily stimulates megakaryocytopoiesis and platelet production and is neutralized by the mouse TN1 antibody. The thermodynamic characteristics of TN1 antibody–hTPO complexation were analyzed by isothermal titration calorimetry (ITC) using an antigen‐binding fragment (Fab) derived from the TN1 antibody (TN1‐Fab). To clarify the mechanism by which hTPO is recognized by TN1‐Fab the conformation of free TN1‐Fab was determined to a resolution of 2.0 Å using X‐ray crystallography and compared with the hTPO‐bound form of TN1‐Fab determined by a previous study. This structural comparison revealed that the conformation of TN1‐Fab does not substantially change after hTPO binding and a set of 15 water molecules is released from the antigen‐binding site (paratope) of TN1‐Fab upon hTPO complexation. Interestingly, the heat capacity change (ΔCp) measured by ITC (−1.52 ± 0.05 kJ mol−1 K−1) differed significantly from calculations based upon the X‐ray structure data of the hTPO‐bound and unbound forms of TN1‐Fab (−1.02 ∼ 0.25 kJ mol−1 K−1) suggesting that hTPO undergoes an induced‐fit conformational change combined with significant desolvation upon TN1‐Fab binding. The results shed light on the structural biology associated with neutralizing antibody recognition. PMID:27419667

  18. Carbon-13 NMR study of switch variant anti-dansyl antibodies: Antigen binding and domain-domain interactions

    Energy Technology Data Exchange (ETDEWEB)

    Kato, Koichi; Matsunaga, Chigusa; Odaka, Asano; Yamato, Sumie; Takaha, Wakana; Shimada, Ichio; Arata, Yoji (Univ. of Tokyo (Japan))

    1991-07-02

    A {sup 13}C NMR study is reported of switch variant anti-dansyl antibodies, which possess the identical V{sub H}, V{sub L}, and C{sub L} domains in conjunction with highly homologous but not identical heavy-chain constant regions. Each of the antibodies has been selectively labeled with {sup 13}C at the carbonyl carbon of Trp, Tyr, His, or Cys residue by growing hybridoma cells in serum-free medium. Spectral assignments have been made by folowing the procedure described previously for the switch variant antibodies labeled with (1-{sup 13}C)Met. On the basis of the spectral data collected for the antibodies and their proteolytic fragments, the authors discuss how {sup 13}C NMR spectroscopy can be used for the structural analyses of antigen binding and also of domain-domain interactions in the antibody molecule.

  19. Carbon-13 NMR study of switch variant anti-dansyl antibodies: Antigen binding and domain-domain interactions

    International Nuclear Information System (INIS)

    Kato, Koichi; Matsunaga, Chigusa; Odaka, Asano; Yamato, Sumie; Takaha, Wakana; Shimada, Ichio; Arata, Yoji

    1991-01-01

    A 13 C NMR study is reported of switch variant anti-dansyl antibodies, which possess the identical V H , V L , and C L domains in conjunction with highly homologous but not identical heavy-chain constant regions. Each of the antibodies has been selectively labeled with 13 C at the carbonyl carbon of Trp, Tyr, His, or Cys residue by growing hybridoma cells in serum-free medium. Spectral assignments have been made by folowing the procedure described previously for the switch variant antibodies labeled with [1- 13 C]Met. On the basis of the spectral data collected for the antibodies and their proteolytic fragments, the authors discuss how 13 C NMR spectroscopy can be used for the structural analyses of antigen binding and also of domain-domain interactions in the antibody molecule

  20. Mu opioid receptor binding sites in human brain

    International Nuclear Information System (INIS)

    Pilapil, C.; Welner, S.; Magnan, J.; Zamir, N.; Quirion, R.

    1986-01-01

    Our experiments focused on the examination of the distribution of mu opioid receptor binding sites in normal human brain using the highly selective ligand [ 3 H]DAGO, in both membrane binding assay and in vitro receptor autoradiography. Mu opioid binding sites are very discretely distributed in human brain with high densities of sites found in the posterior amygdala, caudate, putamen, hypothalamus and certain cortical areas. Moreover the autoradiographic distribution of [ 3 H]DAGO binding sites clearly reveals the discrete lamination (layers I and III-IV) of mu sites in cortical areas

  1. Maturation of Shark Single-Domain (IgNAR) Antibodies: Evidence for Induced-Fit Binding

    Energy Technology Data Exchange (ETDEWEB)

    Stanfield, R.L.; Dooley, H.; Verdino, P.; Flajnik, M.F.; Wilson, I.A.; /Scripps Res. Inst. /Maryland U.

    2007-07-13

    Sharks express an unusual heavy-chain isotype called IgNAR, whose variable regions bind antigen as independent soluble domains. To further probe affinity maturation of the IgNAR response, we structurally characterized the germline and somatically matured versions of a type II variable (V) region, both in the presence and absence of its antigen, hen egg-white lysozyme. Despite a disulfide bond linking complementarity determining regions (CDRs) 1 and 3, both germline and somatically matured V regions displayed significant structural changes in these CDRs upon complex formation with antigen. Somatic mutations in the IgNAR V region serve to increase the number of contacts with antigen, as reflected by a tenfold increase in affinity, and one of these mutations appears to stabilize the CDR3 region. In addition, a residue in the HV4 loop plays an important role in antibody-antigen interaction, consistent with the high rate of somatic mutations in this non-CDR loop.

  2. Vaccine induced antibodies to the first variable loop of human immunodeficiency virus type 1 gp120, mediate antibody-dependent virus inhibition in macaques.

    Science.gov (United States)

    Bialuk, Izabela; Whitney, Stephen; Andresen, Vibeke; Florese, Ruth H; Nacsa, Janos; Cecchinato, Valentina; Valeri, Valerio W; Heraud, Jean-Michel; Gordon, Shari; Parks, Robyn Washington; Montefiori, David C; Venzon, David; Demberg, Thorsten; Guroff, Marjorie Robert-; Landucci, Gary; Forthal, Donald N; Franchini, Genoveffa

    2011-12-09

    The role of antibodies directed against the hyper variable envelope region V1 of human immunodeficiency virus type 1 (HIV-1), has not been thoroughly studied. We show that a vaccine able to elicit strain-specific non-neutralizing antibodies to this region of gp120 is associated with control of highly pathogenic chimeric SHIV(89.6P) replication in rhesus macaques. The vaccinated animal that had the highest titers of antibodies to the amino terminus portion of V1, prior to challenge, had secondary antibody responses that mediated cell killing by antibody-dependent cellular cytotoxicity (ADCC), as early as 2 weeks after infection and inhibited viral replication by antibody-dependent cell-mediated virus inhibition (ADCVI), by 4 weeks after infection. There was a significant inverse correlation between virus level and binding antibody titers to the envelope protein, (R=-0.83, p=0.015), and ADCVI (R=-0.84 p=0.044). Genotyping of plasma virus demonstrated in vivo selection of three SHIV(89.6P) variants with changes in potential N-linked glycosylation sites in V1. We found a significant inverse correlation between virus levels and titers of antibodies that mediated ADCVI against all the identified V1 virus variants. A significant inverse correlation was also found between neutralizing antibody titers to SHIV(89.6) and virus levels (R=-0.72 p=0.0050). However, passive inoculation of purified immunoglobulin from animal M316, the macaque that best controlled virus, to a naïve macaque, resulted in a low serum neutralizing antibodies and low ADCVI activity that failed to protect from SHIV(89.6P) challenge. Collectively, while our data suggest that anti-envelope antibodies with neutralizing and non-neutralizing Fc(R-dependent activities may be important in the control of SHIV replication, they also demonstrate that low levels of these antibodies alone are not sufficient to protect from infection. Published by Elsevier Ltd.

  3. Broad neutralizing human monoclonal antibodies against influenza virus from vaccinated healthy donors

    Energy Technology Data Exchange (ETDEWEB)

    Kubota-Koketsu, Ritsuko; Mizuta, Hiroyuki [Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871 (Japan); Oshita, Masatoshi; Ideno, Shoji [Osaka Research Laboratory, Benesis Corporation, Yodogawa-ku, Osaka 532-6505 (Japan); Yunoki, Mikihiro [Osaka Research Laboratory, Benesis Corporation, Yodogawa-ku, Osaka 532-6505 (Japan); Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871 (Japan); Kuhara, Motoki [Ina Laboratory, Medical and Biological Laboratories Corporation, Ltd., Ina, Nagano 396-0002 (Japan); Yamamoto, Naomasa [Department of Biochemistry, School of Pharmaceutical Sciences, Ohu University, Koriyama, Fukushima 963-8611 (Japan); Okuno, Yoshinobu [Kanonji Institute, The Research Foundation for Microbial Diseases of Osaka University, Kanonji, Kagawa 768-0061 (Japan); Ikuta, Kazuyoshi, E-mail: ikuta@biken.osaka-u.ac.jp [Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871 (Japan)

    2009-09-11

    Human monoclonal antibodies (HuMAbs) prepared from patients with viral infections could provide information on human epitopes important for the development of vaccines as well as potential therapeutic applications. Through the fusion of peripheral blood mononuclear cells from a total of five influenza-vaccinated volunteers, with newly developed murine-human chimera fusion partner cells, named SPYMEG, we obtained 10 hybridoma clones stably producing anti-influenza virus antibodies: one for influenza A H1N1, four for influenza A H3N2 and five for influenza B. Surprisingly, most of the HuMAbs showed broad reactivity within subtype and four (two for H3N2 and two for B) showed broad neutralizing ability. Importantly, epitope mapping revealed that the two broad neutralizing antibodies to H3N2 derived from different donors recognized the same epitope located underneath the receptor-binding site of the hemagglutinin globular region that is highly conserved among H3N2 strains.

  4. Broad neutralizing human monoclonal antibodies against influenza virus from vaccinated healthy donors

    International Nuclear Information System (INIS)

    Kubota-Koketsu, Ritsuko; Mizuta, Hiroyuki; Oshita, Masatoshi; Ideno, Shoji; Yunoki, Mikihiro; Kuhara, Motoki; Yamamoto, Naomasa; Okuno, Yoshinobu; Ikuta, Kazuyoshi

    2009-01-01

    Human monoclonal antibodies (HuMAbs) prepared from patients with viral infections could provide information on human epitopes important for the development of vaccines as well as potential therapeutic applications. Through the fusion of peripheral blood mononuclear cells from a total of five influenza-vaccinated volunteers, with newly developed murine-human chimera fusion partner cells, named SPYMEG, we obtained 10 hybridoma clones stably producing anti-influenza virus antibodies: one for influenza A H1N1, four for influenza A H3N2 and five for influenza B. Surprisingly, most of the HuMAbs showed broad reactivity within subtype and four (two for H3N2 and two for B) showed broad neutralizing ability. Importantly, epitope mapping revealed that the two broad neutralizing antibodies to H3N2 derived from different donors recognized the same epitope located underneath the receptor-binding site of the hemagglutinin globular region that is highly conserved among H3N2 strains.

  5. Antibodies against human cytochrome P-450db1 in autoimmune hepatitis type II.

    Science.gov (United States)

    Zanger, U M; Hauri, H P; Loeper, J; Homberg, J C; Meyer, U A

    1988-11-01

    In a subgroup of children with chronic active hepatitis, circulating autoantibodies occur that bind to liver and kidney endoplasmic reticulum (anti-liver/kidney microsome antibody type I or anti-LKM1). Anti-LKM1 titers follow the severity of the disease and the presence of these antibodies serves as a diagnostic marker for this autoimmune hepatitis type II. We demonstrate that anti-LKM1 IgGs specifically inhibit the hydroxylation of bufuralol in human liver microsomes. Using two assay systems with different selectivity for the two cytochrome P-450 isozymes catalyzing bufuralol metabolism in human liver, we show that anti-LKM1 exclusively recognizes cytochrome P-450db1. Immunopurification of the LKM1 antigen from solubilized human liver microsomes resulted in an electrophoretically homogenous protein that had the same molecular mass (50 kDa) as purified P-450db1 and an identical N-terminal amino acid sequence. Recognition of both purified P-450db1 and the immunoisolated protein on western blots by several monoclonal antibodies confirmed the identity of the LKM1 antigen with cytochrome P-450db1. Cytochrome P-450db1 has been identified as the target of a common genetic polymorphism of drug oxidation. However, the relationship between the polymorphic cytochrome P-450db1 and the appearance of anti-LKM1 autoantibodies as well as their role in the pathogenesis of chronic active hepatitis remains speculative.

  6. Dengue-Immune Humans Have Higher Levels of Complement-Independent Enhancing Antibody than Complement-Dependent Neutralizing Antibody.

    Science.gov (United States)

    Yamanaka, Atsushi; Konishi, Eiji

    2017-09-25

    Dengue is the most important arboviral disease worldwide. We previously reported that most inhabitants of dengue-endemic countries who are naturally immune to the disease have infection-enhancing antibodies whose in vitro activity does not decrease in the presence of complement (complement-independent enhancing antibodies, or CiEAb). Here, we compared levels of CiEAb and complement-dependent neutralizing antibodies (CdNAb) in dengue-immune humans. A typical antibody dose-response pattern obtained in our assay system to measure the balance between neutralizing and enhancing antibodies showed both neutralizing and enhancing activities depending on serum dilution factor. The addition of complement to the assay system increased the activity of neutralizing antibodies at lower dilutions, indicating the presence of CdNAb. In contrast, similar dose-response curves were obtained with and without complement at higher dilutions, indicating higher levels of CiEAb than CdNAb. For experimental support for the higher CiEAb levels, a cocktail of mouse monoclonal antibodies against dengue virus type 1 was prepared. The antibody dose-response curves obtained in this assay, with or without complement, were similar to those obtained with human serum samples when a high proportion of D1-V-3H12 (an antibody exhibiting only enhancing activity and thus a model for CiEAb) was used in the cocktail. This study revealed higher-level induction of CiEAb than CdNAb in humans naturally infected with dengue viruses.

  7. A potent human neutralizing antibody Fc-dependently reduces established HBV infections.

    Science.gov (United States)

    Li, Dan; He, Wenhui; Liu, Ximing; Zheng, Sanduo; Qi, Yonghe; Li, Huiyu; Mao, Fengfeng; Liu, Juan; Sun, Yinyan; Pan, Lijing; Du, Kaixin; Ye, Keqiong; Li, Wenhui; Sui, Jianhua

    2017-09-26

    Hepatitis B virus (HBV) infection is a major global health problem. Currently-available therapies are ineffective in curing chronic HBV infection. HBV and its satellite hepatitis D virus (HDV) infect hepatocytes via binding of the preS1 domain of its large envelope protein to sodium taurocholate cotransporting polypeptide (NTCP). Here, we developed novel human monoclonal antibodies that block the engagement of preS1 with NTCP and neutralize HBV and HDV with high potency. One antibody, 2H5-A14, functions at picomolar level and exhibited neutralization-activity-mediated prophylactic effects. It also acts therapeutically by eliciting antibody-Fc-dependent immunological effector functions that impose durable suppression of viral infection in HBV-infected mice, resulting in reductions in the levels of the small envelope antigen and viral DNA, with no emergence of escape mutants. Our results illustrate a novel antibody-Fc-dependent approach for HBV treatment and suggest 2H5-A14 as a novel clinical candidate for HBV prevention and treatment of chronic HBV infection.

  8. Radioimmunoassay of bovine, ovine and porcine luteinizing hormone with a monoclonal antibody and a human tracer

    Energy Technology Data Exchange (ETDEWEB)

    Fosberg, M; Tagle, R; Madej, A; Molina, J R; Carlsson, M -A

    1993-01-01

    A radioimmunoassay for bovine (bLH), ovine (oLH) and porcine (pLH) luteinizing hormone was developed using a human [sup 125]ILH tracer from a commercial kit and a monoclonal antibody (518B7) specific for LH but with low species specificity. Standard curves demonstrated similar binding kinetics when bLH, oLH and pLH were incubated with tracer and antibody for 2 h at room temperature. A 30-min delay in the addition of the tracer gave sufficient sensitivity when analysing pLH. Separation of antibody-bound LH from free hormone was achieved by using second antibody-coated micro Sepharose beads. The assay was validated and the performance compared with that of an RIA currently in use for determination of bLH (coefficient of correlation: 0.99 and 0.98). Regardless of the standards used, intra-assay coefficients of variation were <10% for LH concentrations exceeding 1 [mu]g/L. The inter-assay coefficients of variation were <15%. The assay was used for clinical evaluation demonstrating the pre-ovulatory LH surge in two cyclic cows, LH pulsatility in an oophorectomized ewe and LH response to GnRH injection in a boar. (au) (7 refs.).

  9. Swine Influenza Virus Antibodies in Humans, Western Europe, 2009

    Science.gov (United States)

    Gerloff, Nancy A.; Kremer, Jacques R.; Charpentier, Emilie; Sausy, Aurélie; Olinger, Christophe M.; Weicherding, Pierre; Schuh, John; Van Reeth, Kristien

    2011-01-01

    Serologic studies for swine influenza viruses (SIVs) in humans with occupational exposure to swine have been reported from the Americas but not from Europe. We compared levels of neutralizing antibodies against 3 influenza viruses—pandemic (H1N1) 2009, an avian-like enzootic subtype H1N1 SIV, and a 2007–08 seasonal subtype H1N1—in 211 persons with swine contact and 224 matched controls in Luxembourg. Persons whose profession involved contact with swine had more neutralizing antibodies against SIV and pandemic (H1N1) 2009 virus than did the controls. Controls also had antibodies against these viruses although exposure to them was unlikely. Antibodies against SIV and pandemic (H1N1) 2009 virus correlated with each other but not with seasonal subtype H1N1 virus. Sequential exposure to variants of seasonal influenza (H1N1) viruses may have increased chances for serologic cross-reactivity with antigenically distinct viruses. Further studies are needed to determine the extent to which serologic responses correlate with infection. PMID:21392430

  10. Generation and Characterization of Novel Human IRAS Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Bo Wang

    2009-01-01

    Full Text Available Imidazoline receptors were first proposed by Bousquet et al., when they studied antihypertensive effect of clonidine. A strong candidate for I1R, known as imidazoline receptor antisera-selected protein (IRAS, has been cloned from human hippocampus. We reported that IRAS mediated agmatine-induced inhibition of opioid dependence in morphine-dependent cells. To elucidate the functional and structure properties of I1R, we developed the newly monoclonal antibody against the N-terminal hIRAS region including the PX domain (10–120aa through immunization of BALB/c mice with the NusA-IRAS fusion protein containing an IRAS N-terminal (10–120aa. Stable hybridoma cell lines were established and monoclonal antibodies specifically recognized full-length IRAS proteins in their native state by immunoblotting and immunoprecipitation. Monoclonal antibodies stained in a predominantly punctate cytoplasmic pattern when applied to IRAS-transfected HEK293 cells by indirect immunofluorescence assays and demonstrated excellent reactivity in flow immunocytometry. These monoclonal antibodies will provide powerful reagents for the further investigation of hIRAS protein functions.

  11. 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.

  12. Light-chain residue 95 is critical for antigen binding and multispecificity of monoclonal antibody G2.

    Science.gov (United States)

    Usui, Daiki; Inaba, Satomi; Kamatari, Yuji O; Ishiguro, Naotaka; Oda, Masayuki

    2017-09-02

    The monoclonal antibody, G2, specifically binds to the immunogen peptide derived from the chicken prion protein, Pep18mer, and two chicken proteins derived peptides, Pep8 and Pep395; G2 binds with equal affinity to Pep18mer. The amino acid sequences of the three peptides are completely different, and so the recognition mechanism of G2 is unique and interesting. We generated a single-chain Fv (scFv) antibody of G2, and demonstrated its correct folding with an antigen binding function similar to intact G2 antibody. We also generated a Pro containing mutant of G2 scFv at residue 95 of the light chain, and analyzed its antigen binding using a surface plasmon biosensor. The mutant lost its binding ability to Pep18mer, but remained those to Pep8 and Pep395. The results clearly indicate residue 95 as being critical for multispecific antigen binding of G2 at the site generated from the junctional diversity introduced at the joints between the V and J gene segments. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Intramolecular trimerization, a novel strategy for making multispecific antibodies with controlled orientation of the antigen binding domains

    DEFF Research Database (Denmark)

    Álvarez-Cienfuegos, Ana; Alanes, Natalia Nuñez del Prado; Compte, Marta

    2016-01-01

    Here, we describe a new strategy that allows the rapid and efficient engineering of mono and multispecific trivalent antibodies. By fusing single-domain antibodies from camelid heavy-chain-only immunoglobulins (VHHs) to the N-terminus of a human collagen XVIII trimerization domain (TIEXVIII) we p...

  14. Epitopes in α8β1 and other RGD-binding integrins delineate classes of integrin-blocking antibodies and major binding loops in α subunits.

    Science.gov (United States)

    Nishimichi, Norihisa; Kawashima, Nagako; Yokosaki, Yasuyuki

    2015-09-09

    Identification of epitopes for integrin-blocking monoclonal antibodies (mAbs) has aided our understanding of structure-function relationship of integrins. We mapped epitopes of chicken anti-integrin-α8-subunit-blocking mAbs by mutational analyses, examining regions that harboured all mapped epitopes recognized by mAbs against other α-subunits in the RGD-binding-integrin subfamily. Six mAbs exhibited blocking function, and these mAbs recognized residues on the same W2:41-loop on the top-face of the β-propeller. Loop-tips sufficiently close to W2:41 (face was identified as an additional component of the epitope of one antibody, clone YZ5. Binding sequences on the two loops were conserved in virtually all mammals, and that on W3:34 was also conserved in chickens. These indicate 1) YZ5 binds both top and bottom loops, and the binding to W3:34 is by interactions to conserved residues between immunogen and host species, 2) five other blocking mAbs solely bind to W2:41 and 3) the α8 mAbs would cross-react with most mammals. Comparing with the mAbs against the other α-subunits of RGD-integrins, two classes were delineated; those binding to "W3:34 and an top-loop", and "solely W2:41", accounting for 82% of published RGD-integrin-mAbs.

  15. Mycoplasma infection of cell lines can simulate the expression of Fc receptors by binding of the carbohydrate moiety of antibodies.

    Science.gov (United States)

    Lemke, H; Krausse, R; Lorenzen, J; Havsteen, B

    1985-05-01

    During the production of Fc receptor (FcR)-bearing hybridomas it was observed with a particular monoclonal anti-sheep red blood cell antibody (anti-SRBC 1/5, IgG1) that the contamination with Mycoplasma arginini of in vitro cultured cell lines leads to an apparent FcR activity. This property did not correspond with the serological typing since other antibodies of the same isotype could not support FcR rosette formation. Another mycoplasma strain M. orale lacked this property. Analysis of the binding reaction revealed that M. arginini contains a lectin which binds the carbohydrate moiety of the anti-SRBC 1/5 antibody, i.e. anti-SRBC 1/5 synthesized under the influence of tunicamycin or deglycosylated by NaIO4 oxidation did not support rosette formation. These data suggest that binding of antibodies to certain mycoplasma strains may be a pathogenic factor during mycoplasma infections by masking the microorganisms with the host's own defense molecules. The experiments with M. arginini-infected cell lines gain immunological importance since we obtained identical results with staphylococcal protein A, as another bacteriological FcR, and cell lines expressing intrinsic membrane FcR. Although it is an open question whether the glycoconjugates are directly bound by the FcR or else by influencing the three-dimensional structure of the antibodies, it seems possible that FcR in general may be lectins.

  16. Neutralisation of HIV-1 cell-cell spread by human and llama antibodies.

    Science.gov (United States)

    McCoy, Laura E; Groppelli, Elisabetta; Blanchetot, Christophe; de Haard, Hans; Verrips, Theo; Rutten, Lucy; Weiss, Robin A; Jolly, Clare

    2014-10-02

    Direct cell-cell spread of HIV-1 is a very efficient mode of viral dissemination, with increasing evidence suggesting that it may pose a considerable challenge to controlling viral replication in vivo. Much current vaccine research involves the study of broadly neutralising antibodies (bNabs) that arise during natural infection with the aims of eliciting such antibodies by vaccination or incorporating them into novel therapeutics. However, whether cell-cell spread of HIV-1 can be effectively targeted by bNabs remains unclear, and there is much interest in identifying antibodies capable of efficiently neutralising virus transmitted by cell-cell contact. In this study we have tested a panel of bNAbs for inhibition of cell-cell spread, including some not previously evaluated for inhibition of this mode of HIV-1 transmission. We found that three CD4 binding site antibodies, one from an immunised llama (J3) and two isolated from HIV-1-positive patients (VRC01 and HJ16) neutralised cell-cell spread between T cells, while antibodies specific for glycan moieties (2G12, PG9, PG16) and the MPER (2F5) displayed variable efficacy. Notably, while J3 displayed a high level of potency during cell-cell spread we found that the small size of the llama heavy chain-only variable region (VHH) J3 is not required for efficient neutralisation since recombinant J3 containing a full-length human heavy chain Fc domain was significantly more potent. J3 and J3-Fc also neutralised cell-cell spread of HIV-1 from primary macrophages to CD4+ T cells. In conclusion, while bNabs display variable efficacy at preventing cell-cell spread of HIV-1, we find that some CD4 binding site antibodies can inhibit this mode of HIV-1 dissemination and identify the recently described llama antibody J3 as a particularly potent inhibitor. Effective neutralisation of cell-cell spread between physiologically relevant cell types by J3 and J3-Fc supports the development of VHH J3 nanobodies for therapeutic or

  17. The biodistribution of mouse monoclonal antibody ONS-M21 and the application for imaging diagnosis with its humanized antibody

    International Nuclear Information System (INIS)

    Ohkawa, Motohisa

    1997-01-01

    The mouse monoclonal antibody ONS-M21 combines with medulloblastomas and several gliomas specifically. And also we had already produced it humanized antibody. This study investigated the in vivo biodistribution of ONS-M21 and the application for imaging diagnosis using its humanized antibody. The nude mice (BALB/c nu/nu) bearing human medulloblastoma ONS-76 cells subcutaneously were injected 125 I-labeled ONS-M21 antibody via their tail vein. The radioactivities of their normal organs and the s.c. tumor were counted with γ-counter. And their autoradiograph (ARG) 6 hours after this administration was compared with gadolinium enhanced T1-weighted magnetic resonance image (Gd-T1-MRI). The brain tumor models transplanted ONS-76 cells stereotaxically was made by the nude rats (F344/N Jcl-rnu). And compared with MRI and ARG after the administration of 125 I-labeled humanized antibody into these models. The ARG indicated the accumulation of 125I -labeled ONS-M21 in the tumors which was detected by Gd-T1-MRI study. In this study, 125 I-labeled ONS-M21 remained in the tumor longer than the other normal organs. The mouse monoclonal antibody ONS-M21 have specific affinity for ONS-76 tumor in vivo. Then this humanized antibody is considerable to apply the imaging diagnosis of the malignant brain tumors. (author)

  18. Prolonged in vivo residence times of llama single-domain antibody fragments in pigs by binding to porcine immunoglobulins

    NARCIS (Netherlands)

    Harmsen, M.M.; Solt, van C.B.; Fijten, H.P.D.; Setten, van M.C.

    2005-01-01

    The therapeutic parenteral application of llama single-domain antibody fragments (VHHs) is hampered by their small size, resulting in a fast elimination from the body. Here we describe a method to increase the serum half-life of VHHs in pigs by fusion to another VHH binding to porcine immunoglobulin

  19. Stimulation of chymosin secretion by simultaneous expression with chymosin-binding llama single-domain antibody fragments in yeast

    NARCIS (Netherlands)

    Harmsen, M.M.; Smits, C.B.; Geus, de B.

    2002-01-01

    We studied the effect of coexpression of chymosin and chymosin-binding llama single-domain antibody fragments (VHHs) on the secretion of chymosin by Saccharomyces cerevisiae cells. A VHH expression library containing chymosin-specific VHHs was obtained by immunization of a llama and coexpressed with

  20. Low prevalence of antibodies and other plasma factors binding to CC chemokines and IL-2 in HIV-positive patients

    DEFF Research Database (Denmark)

    Meyer, C N; Svenson, M; Larsen, Carsten Schade

    2000-01-01

    Neutralizing cytokine antibodies are found in healthy and diseased individuals, including patients treated with recombinant cytokines. Identification of CCR-5 as co-receptor for HIV has focused interest on CC chemokines and their potential therapeutic use. Chemokine-binding components in plasma...

  1. The original Pathologische Anatomie Leiden-Endothelium monoclonal antibody recognizes a vascular endothelial growth factor binding site within neuropilin-1

    NARCIS (Netherlands)

    Jaalouk, Diana E.; Ozawa, Nfichael G.; Sun, Jessica; Lahdenranta, Johanna; Schlingemann, Reinier O.; Pasqualini, Renata; Arap, Wadih

    2007-01-01

    For two decades, the antigen recognized by the Pathologische Anatomie Leiden-Endothelium (PAL-E) monoclonal antibody, a standard vascular endothelial cell marker, has remained elusive. Here, we used a combinatorial phage display-based approach ("epitope mapping") to select peptides binding to the

  2. 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.

  3. Radioimmunodetection of human tumor xenografts by monoclonal antibody F(ab')/sub 2/ fragments

    Energy Technology Data Exchange (ETDEWEB)

    Herlyn, D.; Munz, D.L.; Herlyn, M.; Koprowski, H.; Powe, J.; Alavi, A.; Meinken, G.E.; Srivastava, S.C.

    1986-01-01

    Procedures are described for the radiolocalization of human tumors by murine monoclonal antibodies (MAb) in animal model systems. Visualization of tumor xenografts was clearer in nude mice compared to experimentally immunosuppressed mice due to the higher tumor viability. MAb localization in tumor tissue was greatly enhanced when F(ab')/sub 2/ fragments rather than intact antibody molecules were used. Although tumors could be visualized with /sup 131/I-, /sup 123/I-or /sup 111/In-labeled MAb fragments without background subtraction, tumor-to-background ratios of radioactivity were highest for /sup 131/I-labeled fragments. /sup 131/I-labeled F(ab')/sub 2/ fragments of eight MAb against human colorectal carcinoma, melanoma or lung carcinoma localized specifically only in those tumors that bound the MAb in vitro and not in unrelated tumors. Radiolabeled fragments of MAb with other specificities (anti-hepatitis virus MAb) did not localize in tumors. All MAb that inhibited tumor growth in nude mice effectively localized these tumors by ..gamma..-scintigraphy. Some MAb were effective in localizing tumors but ineffective in inhibiting their growth. The ability of the specific radiolabeled F(ab')/sub 2/ fragments to localize in tumor grafts correlated significantly with MAb binding affinity and density of antigenic sites on tumor cells together, but not with either in vitro binding parameter alone.

  4. Development and Validation of an Enzyme-Linked Immunosorbent Assay for the Detection of Binding Anti-Drug Antibodies against Interferon Beta

    DEFF Research Database (Denmark)

    Ingenhoven, Kathleen; Kramer, Daniel; Jensen, Poul Erik Hyldgaard

    2017-01-01

    to be 26 ng/mL using commercially available polyclonal rabbit antihuman IFN-β in human sera as the positive control. CONCLUSION: An ultrasensitive ELISA for IFN-β-binding ADA testing has been validated. This will form the basis to assess anti-biopharmaceutical immunization toward IFN-β with regards to its......OBJECTIVE: To develop and validate a method for the detection of binding anti-drug antibodies (ADAs) against interferon beta (IFN-β) in human serum as part of a European initiative (ABIRISK) aimed at the prediction and analysis of clinical relevance of anti-biopharmaceutical immunization...... to minimize the risk. METHOD: A two-tiered bridging enzyme-linked immunosorbent assay (ELISA) format was selected and validated according to current recommendations. Screening assay: ADA in serum samples form complexes with immobilized IFN-β and biotinylated IFN-β, which are then detected using HRP labeled...

  5. Antibody Binding Alters the Characteristics and Contents of Extracellular Vesicles Released by Histoplasma capsulatum

    Energy Technology Data Exchange (ETDEWEB)

    Baltazar, Ludmila M.; Nakayasu, Ernesto S.; Sobreira, Tiago; Choi, Hyungwon; Casadevall, Arturo; Nimrichter, Leonardo; Nosanchuk, Joshua D.

    2016-03-30

    ABSTRACT

    Histoplasma capsulatumproduces extracellular vesicles containing virulence-associated molecules capable of modulating host machinery, benefiting the pathogen. Treatment ofH. capsulatumcells with monoclonal antibodies (MAbs) can change the outcome of infection in mice. We evaluated the sizes, enzymatic contents, and proteomic profiles of the vesicles released by fungal cells treated with either protective MAb 6B7 (IgG1) or nonprotective MAb 7B6 (IgG2b), both of which bindH. capsulatumheat shock protein 60 (Hsp60). Our results showed that treatment with either MAb was associated with changes in size and vesicle loading. MAb treatments reduced vesicle phosphatase and catalase activities compared to those of vesicles from untreated controls. We identified 1,125 proteins in vesicles, and 250 of these manifested differences in abundance relative to that of proteins in vesicles isolated from yeast cells exposed to Hsp60-binding MAbs, indicating that surface binding of fungal cells by MAbs modified protein loading in the vesicles. The abundance of upregulated proteins in vesicles upon MAb 7B6 treatment was 44.8% of the protein quantities in vesicles from fungal cells treated with MAb 6B7. Analysis of orthologous proteins previously identified in vesicles from other fungi showed that different ascomycete fungi have similar proteins in their extracellular milieu, many of which are associated with virulence. Our results demonstrate that antibody binding can modulate fungal cell responses, resulting in differential loading of vesicles, which could alter fungal cell susceptibility to host defenses. This finding provides additional evidence that antibody binding modulates microbial physiology and suggests a new function for specific immunoglobulins through alterations of fungal secretion.

    IMPORTANCEDiverse fungal species release extracellular vesicles, indicating that this is a

  6. 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

  7. 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.

  8. Critical epitopes in the nucleocapsid protein of SFTS virus recognized by a panel of SFTS patients derived human monoclonal antibodies.

    Directory of Open Access Journals (Sweden)

    Li Yu

    Full Text Available BACKGROUND: SFTS virus (SFTSV is a newly discovered pathogen to cause severe fever with thrombocytopenia syndrome (SFTS in human. Successful control of SFTSV epidemic requires better understanding of the antigen target in humoral immune responses to the new bunyavirus infection. METHODOLOGY/PRINCIPAL FINDINGS: We have generated a combinatorial Fab antibody phage library from two SFTS patients recovered from SFTSV infection. To date, 94 unique human antibodies have been generated and characterized from over 1200 Fab antibody clones obtained by screening the library with SFTS purified virions. All those monoclonal antibodies (MAbs recognized the nucleocapsid (N protein of SFTSV while none of them were reactive to the viral glycoproteins Gn or Gc. Furthermore, over screening 1000 mouse monoclonal antibody clones derived from SFTSV virions immunization, 462 clones reacted with N protein, while only 16 clones were reactive to glycoprotein. Furthermore, epitope mapping of SFTSV N protein was performed through molecular simulation, site mutation and competitive ELISA, and we found that at least 4 distinct antigenic epitopes within N protein were recognized by those human and mouse MAbs, in particular mutation of Glu10 to Ala10 abolished or significantly reduced the binding activity of nearly most SFTS patients derived MAbs. CONCLUSIONS/SIGNIFICANCE: The large number of human recombinant MAbs derived from SFTS patients recognized the viral N protein indicated the important role of the N protein in humoral responses to SFTSV infection, and the critical epitopes we defined in this study provided molecular basis for detection and diagnosis of SFTSV infection.

  9. Development and characterization of a pre-treatment procedure to eliminate human monoclonal antibody therapeutic drug and matrix interference in cell-based functional neutralizing antibody assays.

    Science.gov (United States)

    Xu, Weifeng; Jiang, Hao; Titsch, Craig; Haulenbeek, Jonathan R; Pillutla, Renuka C; Aubry, Anne-Françoise; DeSilva, Binodh S; Arnold, Mark E; Zeng, Jianing; Dodge, Robert W

    2015-01-01

    Biological therapeutics can induce an undesirable immune response resulting in the formation of anti-drug antibodies (ADA), including neutralizing antibodies (NAbs). Functional (usually cell-based) NAb assays are preferred to determine NAb presence in patient serum, but are often subject to interferences from numerous serum factors, such as growth factors and disease-related cytokines. Many functional cell-based NAb assays are essentially drug concentration assays that imply the presence of NAbs by the detection of small changes in functional drug concentration. Any drug contained in the test sample will increase the total amount of drug in the assay, thus reducing the sensitivity of NAb detection. Biotin-drug Extraction with Acid Dissociation (BEAD) has been successfully applied to extract ADA, thereby removing drug and other interfering factors from human serum samples. However, to date there has been no report to estimate the residual drug level after BEAD treatment when the drug itself is a human monoclonal antibody; mainly due to the limitation of traditional ligand-binding assays. Here we describe a universal BEAD optimization procedure for human monoclonal antibody (mAb) drugs by using a LC-MS/MS method to simultaneously measure drug (a mutant human IgG4), NAb positive control (a mouse IgG), and endogenous human IgGs as an indicator of nonspecific carry-over in the BEAD eluate. This is the first report demonstrating that residual human mAb drug level in clinical sample can be measured after BEAD pre-treatment, which is critical for further BEAD procedure optimization and downstream immunogenicity testing. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Expression cloning and production of human heavy-chain-only antibodies from murine transgenic plasma cells

    NARCIS (Netherlands)

    D.D. Drabek (Dubravka); R. Janssens (Rick); Boer, E. (Ernie de); Rademaker, R. (Rik); Kloess, J. (Johannes); J.J. Skehel (John ); Grosveld, F. (Frank)

    2016-01-01

    textabstractSeveral technologies have been developed to isolate human antibodies against different target antigens as a source of potential therapeutics, including hybridoma technology, phage and yeast display systems. For conventional antibodies, this involves either random pairing of VH and

  11. Surfactant protein D binds to human immunodeficiency virus (HIV) envelope protein gp120 and inhibits HIV replication

    DEFF Research Database (Denmark)

    Meschi, Joseph; Crouch, Erika C; Skolnik, Paul

    2005-01-01

    The envelope protein (gp120) of human immunodeficiency virus (HIV) contains highly conserved mannosylated oligosaccharides. These glycoconjugates contribute to resistance to antibody neutralization, and binding to cell surface lectins on macrophages and dendritic cells. Mannose-binding lectin (MBL......) binds to gp120 and plays a role in defence against the virus. In this study it is demonstrated that surfactant protein D (SP-D) binds to gp120 and inhibits HIV infectivity at significantly lower concentrations than MBL. The binding of SP-D was mediated by its calcium-dependent carbohydrate......-binding activity and was dependent on glycosylation of gp120. Native dodecameric SP-D bound to HIV gp120 more strongly than native trimeric SP-D. Since one common polymorphic form of SP-D is predominantly expressed as trimers and associated with lower blood levels, these individuals may have less effective innate...

  12. Design and Characterization of a Human Monoclonal Antibody that Modulates Mutant Connexin 26 Hemichannels Implicated in Deafness and Skin Disorders

    Directory of Open Access Journals (Sweden)

    Liang Xu

    2017-09-01

    Full Text Available Background: Mutations leading to changes in properties, regulation, or expression of connexin-made channels have been implicated in 28 distinct human hereditary diseases. Eight of these result from variants of connexin 26 (Cx26, a protein critically involved in cell-cell signaling in the inner ear and skin. Lack of non-toxic drugs with defined mechanisms of action poses a serious obstacle to therapeutic interventions for diseases caused by mutant connexins. In particular, molecules that specifically modulate connexin hemichannel function without affecting gap junction channels are considered of primary importance for the study of connexin hemichannel role in physiological as well as pathological conditions. Monoclonal antibodies developed in the last three decades have become the most important class of therapeutic biologicals. Recombinant methods permit rapid selection and improvement of monoclonal antibodies from libraries with large diversity.Methods: By screening a combinatorial library of human single-chain fragment variable (scFv antibodies expressed in phage, we identified a candidate that binds an extracellular epitope of Cx26. We characterized antibody action using a variety of biochemical and biophysical assays in HeLa cells, organotypic cultures of mouse cochlea and human keratinocyte-derived cells.Results: We determined that the antibody is a remarkably efficient, non-toxic, and completely reversible inhibitor of hemichannels formed by connexin 26 and does not affect direct cell-cell communication via gap junction channels. Importantly, we also demonstrate that the antibody efficiently inhibits hyperative mutant Cx26 hemichannels implicated in autosomal dominant non-syndromic hearing impairment accompanied by keratitis and hystrix-like ichthyosis-deafness (KID/HID syndrome. We solved the crystal structure of the antibody, identified residues that are critical for binding and used molecular dynamics to uncover its mechanism of action

  13. Detection of vitamin D binding protein on the surface of cytotrophoblasts isolated from human placentae

    International Nuclear Information System (INIS)

    Nestler, J.E.; McLeod, J.F.; Kowalski, M.A.; Strauss, J.F. III; Haddad, J.G. Jr.

    1987-01-01

    Vitamin D binding protein (DBP), a Mr 56,000-58,000 alpha 2-glycoprotein, is the major serum protein involved in the transport of vitamin D sterols. Recently it has been suggested that DBP may also be involved in immunoglobulin G binding to cells. Because the trophoblast is involved in the transport of molecules such as vitamin D and immunoglobulin G to the fetus, we asked whether DBP could be detected on the surface of human placental trophoblast cells. Cytotrophoblasts purified from human term placentae were fixed and made permeant with Triton X-100 and examined by indirect immunofluorescence after incubation with a monoclonal antibody to DBP. Greater than 90% of these cells stained positively, whereas no staining was observed with nonimmune antiserum. The presence of DBP on/in the surface of cytotrophoblasts could also be demonstrated by fluorescent cytometry. When cell surface-associated proteins of cytotrophoblasts were radioiodinated, a Mr 57,000 radiolabeled protein could be immunoisolated from the cell lysate with a purified monospecific polyclonal antibody to DBP. Immunoisolation of this radiolabeled protein was prevented by the addition of excess unlabeled human DBP to the cell lysate before incubation with antibody. This Mr 57,000 radiolabeled protein could also be isolated by affinity chromatography selecting for proteins that bind to globular actin. When cytotrophoblasts were incubated with [ 35 S]methionine for 3 or 18 h, active synthesis of DBP could not be demonstrated by immunoisolation techniques. These studies demonstrate the presence of DBP on the surface of well washed, human cytotrophoblasts. This DBP may be maternally derived, since active synthesis of DBP could not be demonstrated

  14. Production and characterisation of monoclonal antibodies against RAI3 and its expression in human breast cancer

    International Nuclear Information System (INIS)

    Jörißen, Hannah; Klockenbring, Torsten; Bektas, Nuran; Dahl, Edgar; Hartmann, Arndt; Haaf, Anette ten; Di Fiore, Stefano; Kiefer, Hans; Thess, Andreas; Barth, Stefan

    2009-01-01

    RAI3 is an orphan G-protein coupled receptor (GPCR) that has been associated with malignancy and may play a role in the proliferation of breast cancer cells. Although its exact function in normal and malignant cells remains unclear and evidence supporting its role in oncogenesis is controversial, its abundant expression on the surface of cancer cells would make it an interesting target for the development of antibody-based therapeutics. To investigate the link with cancer and provide more evidence for its role, we carried out a systematic analysis of RAI3 expression in a large set of human breast cancer specimens. We expressed recombinant human RAI3 in bacteria and reconstituted the purified protein in liposomes to raise monoclonal antibodies using classical hybridoma techniques. The specific binding activity of the antibodies was confirmed by enzyme-linked immunosorbent assay (ELISA), western blot and immunocytochemistry. We carried out a systematic immunohistochemical analysis of RAI3 expression in human invasive breast carcinomas (n = 147) and normal breast tissues (n = 44) using a tissue microarray. In addition, a cDNA dot blot hybridisation assay was used to investigate a set of matched normal and cancerous breast tissue specimens (n = 50) as well as lymph node metastases (n = 3) for RAI3 mRNA expression. The anti-RAI3 monoclonal antibodies bound to recombinant human RAI3 protein with high specificity and affinity, as shown by ELISA, western blot and ICC. The cDNA dot blot and immunohistochemical experiments showed that both RAI3 mRNA and RAI3 protein were abundantly expressed in human breast carcinoma. However, there was no association between RAI3 protein expression and prognosis based on overall and recurrence-free survival. We have generated a novel, highly-specific monoclonal antibody that detects RAI3 in formaldehyde-fixed paraffin-embedded tissue. This is the first study to report a systematic analysis of RAI3 expression in normal and cancerous human

  15. Humanization of JAA-F11, a Highly Specific Anti-Thomsen-Friedenreich Pancarcinoma Antibody and In Vitro Efficacy Analysis

    Directory of Open Access Journals (Sweden)

    Swetha Tati

    2017-09-01

    Full Text Available JAA-F11 is a highly specific mouse monoclonal to the Thomsen-Friedenreich Antigen (TF-Ag which is an alpha-O-linked disaccharide antigen on the surface of ~80% of human carcinomas, including breast, lung, colon, bladder, ovarian, and prostate cancers, and is cryptic on normal cells. JAA-F11 has potential, when humanized, for cancer immunotherapy for multiple cancer types. Humanization of JAA-F11, was performed utilizing complementarity determining regions grafting on a homology framework. The objective herein is to test the specificity, affinity and biology efficacy of the humanized JAA-F11 (hJAA-F11. Using a 609 target glycan array, 2 hJAA-F11 constructs were shown to have excellent chemical specificity, binding only to TF-Ag alpha-linked structures and not to TF-Ag beta-linked structures. The relative affinity of these hJAA-F11 constructs for TF-Ag was improved over the mouse antibody, while T20 scoring predicted low clinical immunogenicity. The hJAA-F11 constructs produced antibody-dependent cellular cytotoxicity in breast and lung tumor lines shown to express TF-Ag by flow cytometry. Internalization of hJAA-F11 into cancer cells was also shown using a surface binding ELISA and confirmed by immunofluorescence microscopy. Both the naked hJAA-F11 and a maytansine-conjugated antibody (hJAA-F11-DM1 suppressed in vivo tumor progression in a human breast cancer xenograft model in SCID mice. Together, our results support the conclusion that the humanized antibody to the TF-Ag has potential as an adjunct therapy, either directly or as part of an antibody drug conjugate, to treat breast cancer, including triple negative breast cancer which currently has no targeted therapy, as well as lung cancer.

  16. Antibody Prophylaxis Against Dengue Virus 2 Infection in Non-Human Primates.

    Science.gov (United States)

    Simmons, Monika; Putnak, Robert; Sun, Peifang; Burgess, Timothy; Marasco, Wayne A

    2016-11-02

    Passive immunization with anti-dengue virus (DENV) immune serum globulin (ISG) or monoclonal antibodies (Mabs) may serve to supplement or replace vaccination for short-term dengue immune prophylaxis. In the present study, we sought to establish proof-of-concept by evaluating several DENV-neutralizing antibodies for their ability to protect rhesus macaques against viremia following live virus challenge, including human anti-dengue ISG, and a human Mab (Mab11/wt) and its genetically engineered variant (Mab11/mutFc) that is unable to bind to cells with Fc gamma receptors (FcγR) and potentiate antibody-dependent enhancement (ADE). In the first experiment, groups of animals received ISG or Mab11/wt at low doses (3-10 mg/kg) or a saline control followed by challenge with DENV-2 at day 10 or 30. After passive immunization, only low-titered circulating virus-neutralizing antibody titers were measured in both groups, which were undetectable by day 30. After challenge at day 10, a reduction in viremia duration compared with the control was seen only in the ISG group (75%). However, after a day 30 challenge, no reduction in viremia was observed in both immunized groups. In a second experiment to test the effect of higher antibody doses on short-term protection, groups received either ISG, Mab11/wt, Mab11/mutFc (each at 25 mg/kg) or saline followed by challenge with DENV-2 on day 10. Increased virus-neutralizing antibody titers were detected in all groups at day 5 postinjection, with geometric mean titers (GMTs) of 464 (ISG), 313 (Mab11/wt), and 309 (Mab11/mutFc). After challenge, there was complete protection against viremia in the group that received ISG, and a reduction in viremia duration of 89% and 83% in groups that received Mab11/wt and Mab11/mutFc, respectively. An in vitro ADE assay in Fcγ receptor-bearing K562 cells with sera collected immediately before challenge showed increased DENV-2 infection levels in the presence of both ISG and Mab11/wt, which peaked at a

  17. Projecting human pharmacokinetics of therapeutic antibodies from nonclinical data: What have we learned?

    OpenAIRE

    Deng, Rong; Iyer, Suhasini; Theil, Frank-Peter; Mortensen, Deborah L; Fielder, Paul J; Prabhu, Saileta

    2011-01-01

    The pharmacokinetics (PK) of therapeutic antibodies is determined by target and non-target mediated mechanisms. These antibody-specific factors need to be considered during prediction of human PK based upon preclinical information. Principles of allometric scaling established for small molecules using data from multiple animal species cannot be directly applied to antibodies. Here, different methods for projecting human clearance (CL) from animal PK data for 13 therapeutic monoclonal antibodi...

  18. Immune recognition of botulinum neurotoxin B: antibody-binding regions on the heavy chain of the toxin.

    Science.gov (United States)

    Dolimbek, Behzod Z; Steward, Lance E; Aoki, K Roger; Atassi, M Zouhair

    2008-02-01

    The purpose of this work was to map the continuous regions recognized by human, horse and mouse anti-botulinum neurotoxin B (BoNT/B) antibodies (Abs). We synthesized a panel of sixty 19-residue peptides (peptide C31 was 24 residues) that overlapped consecutively by 5 residues and together encompassed the entire heavy chain of BoNT/B (H/B, residues 442-1291). Abs from the three host species recognized similar, but not identical, peptides. There were also peptides recognized by two or only by one host species. Where a peptide was recognized by Abs of more than one host species, these Abs were at different levels among the species. Human, horse and mouse Abs bound, although in different amounts, to regions within peptides 736-754, 778-796, 848-866, 932-950, 974-992, 1058-1076 and 1128-1146. Human and horse Abs bound to peptides 890-908 and 1170-1188. Human and mouse Abs recognized peptides 470-488/484-502 overlap, 638-656, 722-740, 862-880, 1030-1048, 1072-1090, 1240-1258 and 1268-1291. We concluded that the antigenic regions localized with the three antisera are quite similar, exhibiting in some cases a small shift to the left or to the right. This is consistent with what is known about protein immune recognition. In the three-dimensional structure, the regions recognized on H/B by anti-BoNT/B Abs occupied surface locations and analysis revealed no correlation between these surface locations and surface electrostatic potential, hydrophilicity, hydrophobicity, or temperature factor. A region that bound mouse Abs overlapped with a recently defined site on BoNT/B that binds to mouse and rat synaptotagmin II, thus providing a molecular explanation for the blocking (protecting) activity of these Abs. The regions thus localized afford candidates for incorporation into a synthetic vaccine design.

  19. Host-Primed Ebola Virus GP Exposes a Hydrophobic NPC1 Receptor-Binding Pocket, Revealing a Target for Broadly Neutralizing Antibodies.

    Science.gov (United States)

    Bornholdt, Zachary A; Ndungo, Esther; Fusco, Marnie L; Bale, Shridhar; Flyak, Andrew I; Crowe, James E; Chandran, Kartik; Saphire, Erica Ollmann

    2016-02-23

    The filovirus surface glycoprotein (GP) mediates viral entry into host cells. Following viral internalization into endosomes, GP is cleaved by host cysteine proteases to expose a receptor-binding site (RBS) that is otherwise hidden from immune surveillance. Here, we present the crystal structure of proteolytically cleaved Ebola virus GP to a resolution of 3.3 Å. We use this structure in conjunction with functional analysis of a large panel of pseudotyped viruses bearing mutant GP proteins to map the Ebola virus GP endosomal RBS at molecular resolution. Our studies indicate that binding of GP to its endosomal receptor Niemann-Pick C1 occurs in two distinct stages: the initial electrostatic interactions are followed by specific interactions with a hydrophobic trough that is exposed on the endosomally cleaved GP1 subunit. Finally, we demonstrate that monoclonal antibodies targeting the filovirus RBS neutralize all known filovirus GPs, making this conserved pocket a promising target for the development of panfilovirus therapeutics. Ebola virus uses its glycoprotein (GP) to enter new host cells. During entry, GP must be cleaved by human enzymes in order for receptor binding to occur. Here, we provide the crystal structure of the cleaved form of Ebola virus GP. We demonstrate that cleavage exposes a site at the top of GP and that this site binds the critical domain C of the receptor, termed Niemann-Pick C1 (NPC1). We perform mutagenesis to find parts of the site essential for binding NPC1 and map distinct roles for an upper, charged crest and lower, hydrophobic trough in cleaved GP. We find that this 3-dimensional site is conserved across the filovirus family and that antibody directed against this site is able to bind cleaved GP from every filovirus tested and neutralize viruses bearing those GPs. Copyright © 2016 Bornholdt et al.

  20. Regional and systemic distribution of anti-tumor x anti-CD3 heteroaggregate antibodies and cultured human peripheral blood lymphocytes in a human colon cancer xenograft

    International Nuclear Information System (INIS)

    Nelson, H.; Ramsey, P.S.; Kerr, L.A.; McKean, D.J.; Donohue, J.H.

    1990-01-01

    Anti-tumor antibody (317G5) covalently coupled to an anti-CD3 antibody (OKT3) produces a heteroaggregate (HA) antibody that can target PBL to lyse tumor cells expressing the appropriate tumor Ag. The i.v. and i.p. distribution of radiolabeled HA antibody 317G5 x OKT3 and of radiolabeled cultured human PBL were studied in athymic nude mice bearing solid intraperitoneal tumor established from the human colon tumor line, LS174T. Mice were injected with 125I-labeled HA antibody, 125I-labeled anti-tumor mAb, or 111In-labeled PBL, and at designated timepoints tissues were harvested and measured for radioactivity. 125I-317G5 x OKT3 localized specifically to tumor sites. Tumor radioactivity levels (percent injected dose/gram) were lower with 125I-317G5 x OKT3 HA antibody than with 125I-317G5 anti-tumor mAb, but were similar to levels reported for other anti-tumor mAb. The major difference in radioactivity levels observed between i.v. and i.p. administration of 125I-317G5 x OKT3 was an increase in hepatic radioactivity after i.v. HA antibody administration. HA antibodies produced from F(ab')2 fragments, which exhibit decreased m. w. and decreased Fc receptor-mediated binding, demonstrated improved tumor:tissue ratios as compared to intact antibody HA. 125I-317G5 F(ab')2 x OKT3 F(ab')2 antibody levels were equivalent to intact HA antibody levels in tumor, but were lower than intact HA antibody levels in the blood, bowel, and liver. Tumor:bowel ratios (20:1 at 48 h) were highest when 317G5 F(ab')2 x OKT3 F(ab')2 was injected i.p. Autoradiography confirmed that anti-tumor x anti-CD3 HA antibodies localized specifically to intraperitoneal tumor; that i.p. administered HA antibodies penetrated tumor directly; and that i.v. administered HA antibodies distributed along tumor vasculature

  1. Radioimmunoimaging of experimental thrombi in dogs using technetium-99m-labeled monoclonal antibody fragments reactive with human platelets

    International Nuclear Information System (INIS)

    Som, P.; Oster, Z.H.; Zamora, P.O.; Yamamoto, K.; Sacker, D.F.; Brill, A.B.; Newell, K.D.; Rhodes, B.A.

    1986-01-01

    Monoclonal antibody 50H.19, which reacts with human platelets, was converted to fragments, pretinned, and made into kits for subsequent radiolabeling with /sup 99m/Tc. The antibody, which cross-reacts with dog platelets, was used to evaluate in vitro binding to blood clots and in vivo in experimental thrombi in dogs. After radiolabeling, 97.4 +/- 6.4% of the /sup 99m/Tc was antibody-associated. The preparations retained immunoreactivity, as determined by: binding studies using whole blood and determining the ratio of cell-to-plasma radioactivity (ratios of 57.6-61.2) and binding of the antibody to clots (clot/serum ratios were 57.2-74.6%). Approximately 50% of the radioactivity was cleared from the blood in 3-6 min and 18-24% was excreted in urine within 3 hr. Experimental thrombi in dogs could be visualized consistently within 2-3 hr postinjection in peripheral veins and arteries, pulmonary arteries, and the right ventricle. In addition, damage to blood vessel intima without visible thrombi could also be detected. This method has the following advantages: short and simple pre-imaging preparation, and rapid visualization of thrombi with no need for blood-pool subtraction or delayed imaging

  2. Binding of (/sup 3/H)imipramine to human platelet membranes with compensation for saturable binding to filters and its implication for binding studies with brain membranes

    Energy Technology Data Exchange (ETDEWEB)

    Phillips, O.M.; Wood, K.M.; Williams, D.C.

    1984-08-01

    Apparent specific binding of (/sup 3/H)imipramine to human platelet membranes at high concentrations of imipramine showed deviation from that expected of a single binding site, a result consistent with a low-affinity binding site. The deviation was due to displaceable, saturable binding to the glass fibre filters used in the assays. Imipramine, chloripramine, desipramine, and fluoxetine inhibited binding to filters whereas 5-hydroxytryptamine and ethanol were ineffective. Experimental conditions were developed that eliminated filter binding, allowing assay of high- and low-affinity binding to membranes. Failure to correct for filter binding may lead to overestimation of binding parameters, Bmax and KD for high-affinity binding to membranes, and may also be misinterpreted as indicating a low-affinity binding component in both platelet and brain membranes. Low-affinity binding (KD less than 2 microM) of imipramine to human platelet membranes was demonstrated and its significance discussed.

  3. Characterization of Palytoxin Binding to HaCaT Cells Using a Monoclonal Anti-Palytoxin Antibody

    Directory of Open Access Journals (Sweden)

    Chiara Florio

    2013-02-01

    Full Text Available Palytoxin (PLTX is the reference compound for a group of potent marine biotoxins, for which the molecular target is Na+/K+-ATPase. Indeed, ouabain (OUA, a potent blocker of the pump, is used to inhibit some PLTX effects in vitro. However, in an effort to explain incomplete inhibition of PLTX cytotoxicity, some studies suggest the possibility of two different binding sites on Na+/K+-ATPase. Hence, this study was performed to characterize PLTX binding to intact HaCaT keratinocytes and to investigate the ability of OUA to compete for this binding. PLTX binding to HaCaT cells was demonstrated by immunocytochemical analysis after 10 min exposure. An anti-PLTX monoclonal antibody-based ELISA showed that the binding was saturable and reversible, with a Kd of 3 × 10−10 M. However, kinetic experiments revealed that PLTX binding dissociation was incomplete, suggesting an additional, OUA-insensitive, PLTX binding site. Competitive experiments suggested that OUA acts as a negative allosteric modulator against high PLTX concentrations (0.3–1.0 × 10−7 M and possibly as a non-competitive antagonist against low PLTX concentrations (0.1–3.0 × 10−9 M. Antagonism was supported by PLTX cytotoxicity inhibition at OUA concentrations that displaced PLTX binding (1 × 10−5 M. However, this inhibition was incomplete, supporting the existence of both OUA-sensitive and -insensitive PLTX binding sites.

  4. Naturally occurring anti-glycan antibodies binding to Globo H-expressing cells identify ovarian cancer patients.

    Science.gov (United States)

    Pochechueva, Tatiana; Alam, Shahidul; Schötzau, Andreas; Chinarev, Alexander; Bovin, Nicolai V; Hacker, Neville F; Jacob, Francis; Heinzelmann-Schwarz, Viola

    2017-02-10

    Glycosphingolipids are important compounds of the plasma membrane of mammalian cells and a number of them have been associated with malignant transformation and progression, reinforcing tumour aggressiveness and metastasis. Here we investigated the levels of naturally occurring anti-glycan antibodies to Globo H in blood plasma obtained from high-grade serous ovarian cancer patients (SOC) and women without gynaecological malignancies (control) using suspension glycan array technology employing chemically synthesized glycans as antibody targets. We found that anti-human Globo H IgG antibodies were able to significantly discriminate SOC from controls (P anti-Globo H antibodies highly correlated (r = 0.992). The incubation of plasma-derived anti-glycan antibodies with chemically synthesized (presented on fluorescence microspheres) and native Globo H (expressed on Globo H-positive cell lines) revealed strong reactivity of naturally occurring human anti-Globo H antibodies towards its antigen expressed on ovarian cancer cells. Our data demonstrate that human plasma-derived antibodies to Globo H as well as the presence of the antigen might be considered as therapeutic option in ovarian cancer.

  5. Antitumor activity of chLpMab-2, a human-mouse chimeric cancer-specific antihuman podoplanin antibody, via antibody-dependent cellular cytotoxicity.

    Science.gov (United States)

    Kaneko, Mika K; Yamada, Shinji; Nakamura, Takuro; Abe, Shinji; Nishioka, Yasuhiko; Kunita, Akiko; Fukayama, Masashi; Fujii, Yuki; Ogasawara, Satoshi; Kato, Yukinari

    2017-04-01

    Human podoplanin (hPDPN), a platelet aggregation-inducing transmembrane glycoprotein, is expressed in different types of tumors, and it binds to C-type lectin-like receptor 2 (CLEC-2). The overexpression of hPDPN is involved in invasion and metastasis. Anti-hPDPN monoclonal antibodies (mAbs) such as NZ-1 have shown antitumor and antimetastatic activities by binding to the platelet aggregation-stimulating (PLAG) domain of hPDPN. Recently, we developed a novel mouse anti-hPDPN mAb, LpMab-2, using the cancer-specific mAb (CasMab) technology. In this study we developed chLpMab-2, a human-mouse chimeric anti-hPDPN antibody, derived from LpMab-2. chLpMab-2 was produced using fucosyltransferase 8-knockout (KO) Chinese hamster ovary (CHO)-S cell lines. By flow cytometry, chLpMab-2 reacted with hPDPN-expressing cancer cell lines including glioblastomas, mesotheliomas, and lung cancers. However, it showed low reaction with normal cell lines such as lymphatic endothelial and renal epithelial cells. Moreover, chLpMab-2 exhibited high antibody-dependent cellular cytotoxicity (ADCC) against PDPN-expressing cells, despite its low complement-dependent cytotoxicity. Furthermore, treatment with chLpMab-2 abolished tumor growth in xenograft models of CHO/hPDPN, indicating that chLpMab-2 suppressed tumor development via ADCC. In conclusion, chLpMab-2 could be useful as a novel antibody-based therapy against hPDPN-expressing tumors. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  6. Structural Basis of Human Parechovirus Neutralization by Human Monoclonal Antibodies

    NARCIS (Netherlands)

    Shakeel, Shabih; Westerhuis, Brenda M.; Ora, Ari; Koen, Gerrit; Bakker, Arjen Q.; Claassen, Yvonne; Wagner, Koen; Beaumont, Tim; Wolthers, Katja C.; Butcher, Sarah J.

    2015-01-01

    Since it was first recognized in 2004 that human parechoviruses (HPeV) are a significant cause of central nervous system and neonatal sepsis, their clinical importance, primarily in children, has started to emerge. Intravenous immunoglobulin treatment is the only treatment available in such

  7. Immunoscintigraphy of human tumors transplanted in nude mice with radiolabeled anti-ras p21 monoclonal antibodies

    International Nuclear Information System (INIS)

    Katoh, Y.; Nakata, K.; Kohno, K.; Shima, M.; Satoh, A.; Kusumoto, Y.; Ishii, N.; Kohji, T.; Shiku, H.; Nagataki, S.

    1990-01-01

    Anti-ras p21 monoclonal antibody (RASK-3) was used for immunoscintigraphy of human cancer cell lines in nude mice. Iodine-125-labeled RASK-3 was injected into nude mice with either human colon cancers (FCC-1 or BM-314) or lung cancer (KNS-62). Clear images were obtained in all three cancers 7 days after the injection of antibody. No localization of 125 I-labeled control monoclonal antibody was observed. The ratio of tissue/blood radioactivity and % ID/g in the tumor were significantly higher than other organs by Day 8. The specific localization index examined by 131 I-RASK-3 and 125 I-control monoclonal antibody was also higher in the tumor than in other tissues. In the in vitro study, binding of RASK-3 to tumor cells increased significantly by treatment of cells with either lysolecithin or periodate-lysine-paraformaldehyde, which confirmed the intracellular localization of ras p21. The mechanism by which anti-ras p21 antibodies accumulate in tumor sites could be the necrotic changes in tumor cells or changes in membrane permeability of non-necrotic cells. These results provide a strong rationale for the utilization of ras p21 as a target antigen in the imaging of a variety of human cancers

  8. Kinetics of Antibody Binding to Membranes of Living Bacteria Measured by a Photonic Crystal-Based Biosensor

    Directory of Open Access Journals (Sweden)

    Ekaterina Rostova

    2016-10-01

    Full Text Available Optical biosensors based on photonic crystal surface waves (PC SWs offer a possibility to study binding interactions with living cells, overcoming the limitation of rather small evanescent field penetration depth into a sample medium that is characteristic for typical optical biosensors. Besides this, simultaneous excitation of s- and p-polarized surface waves with different penetration depths is realized here, permitting unambiguous separation of surface and volume contributions to the measured signal. PC-based biosensors do not require a bulk signal correction, compared to widely used surface plasmon resonance-based devices. We developed a chitosan-based protocol of PC chip functionalization for bacterial attachment and performed experiments on antibody binding to living bacteria measured in real time by the PCSW-based biosensor. Data analysis reveals specific binding and gives the value of the dissociation constant for monoclonal antibodies (IgG2b against bacterial lipopolysaccharides equal to KD = 6.2 ± 3.4 nM. To our knowledge, this is a first demonstration of antibody-binding kinetics to living bacteria by a label-free optical biosensor.

  9. Covalent Binding of Antibodies to Cellulose Paper Discs and Their Applications in Naked-eye Colorimetric Immunoassays.

    Science.gov (United States)

    Peng, Yanfen; Gelder, Victor Van; Amaladoss, Anburaj; Patel, Kadamb Haribhai

    2016-10-21

    This report presents two methods for the covalent immobilization of capture antibodies on cellulose filter paper grade No. 1 (medium-flow filter paper) discs and grade No. 113 (fast-flow filter paper) discs. These cellulose paper discs were grafted with amine functional groups through a silane coupling technique before the antibodies were immobilized on them. Periodate oxidation and glutaraldehyde cross-linking methods were used to graft capture antibodies on the cellulose paper discs. In order to ensure the maximum binding capacity of the capture antibodies to their targets after immobilization, the effects of various concentrations of sodium periodate, glutaraldehyde, and capture antibodies on the surface of the paper discs were investigated. The antibodies that were coated on the amine-functionalized cellulose paper discs through a glutaraldehyde cross-linking agent showed enhanced binding activity to the target when compared to the periodate oxidation method. IgG (in mouse reference serum) was used as a reference target in this study to test the application of covalently immobilized antibodies through glutaraldehyde. A new paper-based, enzyme-linked immunosorbent assay (ELISA) was successfully developed and validated for the detection of IgG. This method does not require equipment, and it can detect 100 ng/ml of IgG. The fast-flow filter paper was more sensitive than the medium-flow filter paper. The incubation period of this assay was short and required small sample volumes. This naked-eye, colorimetric immunoassay can be extended to detect other targets that are identified with conventional ELISA.

  10. Production of human anti-HLA monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Walker, M.C.; Mercier, F.; Roger, J.; Varin, M.

    1986-03-01

    Only 40% of the several hundred anti-HLA murine monoclonal antibodies (MAbs) that have been made detect HLA-A,B,C or DR specificities previously defined by human alloantisera, the range of recognized specificities is very narrow, and few of the MAbs have proven useful as tissue typing reagents. In hopes of obtaining HLA typing reagents, the authors are developing a protocol for the production of human anti-HLA MAbs from HLA-antigen (Ag) immunized peripheral blood B cells of volunteering renal patients, immunized to one or more HLA Ags through therapeutic blood transfusions. A simple enrichment of the donor B cells has not been sufficient for anti-HLA MAb production, the authors are currently delineating the conditions necessary for increasing the number of HLA-specific donor B cells by in vitro stimulation with cells expressing the HLA Ag to which the B cell donor is immunized. For the production of MAbs, the stimulated B cells are transformed with Epstein-Barr virus and subsequently fused with KR-4 lymphoblastoid cells. Hybridomas are selected by HAT and Ouabain. Supernatants are screened for anti-HLA activity against lymphocyte targets expressing the original immunizing HLA Ag by complement mediated /sup 51/Cr release assay. Antibody specificity is determined by the complement-dependent microcytotoxicity test used for HLA typing.

  11. Anti-idiotypic antibodies to poliovirus antibodies in commercial immunoglubulin preparations, human serum and milk.

    NARCIS (Netherlands)

    M. Hahn-Zoric; B. Carlsson; S. Jeansson; H.P. Ekre; A.D.M.E. Osterhaus (Albert); D. Roberton; L.A. Hanson

    1993-01-01

    textabstractOur previous studies have suggested that fetal antibody production can be induced by maternal antiidiotypic antibodies transferred to the fetus via the placenta. We tested commercial Ig, sera, and milk for the presence of anti-idiotypic antibodies to poliovirus type 1, using affinity

  12. Therapeutic efficacy of antibodies lacking Fcγ receptor binding against lethal dengue virus infection is due to neutralizing potency and blocking of enhancing antibodies [corrected].

    Directory of Open Access Journals (Sweden)

    Katherine L Williams

    2013-02-01

    Full Text Available Dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS are life-threatening complications following infection with one of the four serotypes of dengue virus (DENV. At present, no vaccine or antiviral therapies are available against dengue. Here, we characterized a panel of eight human or mouse-human chimeric monoclonal antibodies (MAbs and their modified variants lacking effector function and dissected the mechanism by which some protect against antibody-enhanced lethal DENV infection. We found that neutralizing modified MAbs that recognize the fusion loop or the A strand epitopes on domains II and III of the envelope protein, respectively, act therapeutically by competing with and/or displacing enhancing antibodies. By analyzing these relationships, we developed a novel in vitro suppression-of-enhancement assay that predicts the ability of modified MAbs to act therapeutically against antibody-enhanced disease in vivo. These studies provide new insight into the biology of DENV pathogenesis and the requirements for antibodies to treat lethal DENV disease.

  13. Pretargeting vs. direct targeting of human betalox5 islet cells subcutaneously implanted in mice using an anti-human islet cell antibody

    International Nuclear Information System (INIS)

    Liu Guozheng; Dou Shuping; Akalin, Ali; Rusckowski, Mary; Streeter, Philip R.; Shultz, Leonard D.; Greiner, Dale L.

    2012-01-01

    Introduction: We previously demonstrated MORF/cMORF pretargeting of human islets and betalox 5 cells (a human beta cell line) transplanted subcutaneously in mice with the anti-human islet antibody, HPi1. We now compare pretargeting with direct targeting in the beta cell transplant model to evaluate the degree to which target/non-target (T/NT) ratios may be improved by pretargeting. Methods: Specific binding of an anti-human islet antibody HPi1 to the beta cells transplanted subcutaneously in mice was examined against a negative control antibody. We then compared pretargeting by MORF-HPi1 plus 111 In-labeled cMORF to direct targeting by 111 In-labeled HPi1. Results: HPi1 binding to betalox5 human cells in the transplant was shown by immunofluorescence. Normal organ 111 In backgrounds by pretargeting were always lower, although target accumulations were similar. More importantly, the transplant to pancreas and liver ratios was, respectively, 26 and 10 by pretargeting as compared to 9 and 0.6 by direct targeting. Conclusions: Pretargeting greatly improves the T/NT ratios, and based on the estimated endocrine to exocrine ratio within a pancreas, pretargeting may be approaching the sensitivity required for successful imaging of human islets within this organ.

  14. Enhancement of antibody-dependent cell-mediated cytotoxicity by endowing IgG with FcαRI (CD89) binding.

    Science.gov (United States)

    Borrok, M Jack; Luheshi, Nadia M; Beyaz, Nurten; Davies, Gareth C; Legg, James W; Wu, Herren; Dall'Acqua, William F; Tsui, Ping

    2015-01-01

    Fc effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) are crucial to the efficacy of many antibody therapeutics. In addition to IgG, antibodies of the IgA isotype can also promote cell killing through engagement of myeloid lineage cells via interactions between the IgA-Fc and FcαRI (CD89). Herein, we describe a unique, tandem IgG1/IgA2 antibody format in the context of a trastuzumab variable domain that exhibits enhanced ADCC and ADCP capabilities. The IgG1/IgA2 tandem Fc format retains IgG1 FcγR binding as well as FcRn-mediated serum persistence, yet is augmented with myeloid cell-mediated effector functions via FcαRI/IgA Fc interactions. In this work, we demonstrate anti-human epidermal growth factor receptor-2 antibodies with the unique tandem IgG1/IgA2 Fc can better recruit and engage cytotoxic polymorphonuclear (PMN) cells than either the parental IgG1 or IgA2. Pharmacokinetics of IgG1/IgA2 in BALB/c mice are similar to the parental IgG, and far surpass the poor serum persistence of IgA2. The IgG1/IgA2 format is expressed at similar levels and with similar thermal stability to IgG1, and can be purified via standard protein A chromatography. The tandem IgG1/IgA2 format could potentially augment IgG-based immunotherapeutics with enhanced PMN-mediated cytotoxicity while avoiding many of the problems associated with developing IgAs.

  15. Antitumor and cytotoxic properties of a humanized antibody specific for the GM3(Neu5Gc) ganglioside.

    Science.gov (United States)

    Dorvignit, Denise; García-Martínez, Liliana; Rossin, Aurélie; Sosa, Katya; Viera, Justo; Hernández, Tays; Mateo, Cristina; Hueber, Anne-Odile; Mesa, Circe; López-Requena, Alejandro

    2015-12-01

    Gangliosides are sialic acid-bearing glycosphingolipids expressed on all mammalian cell membranes, and participate in several cellular processes. During malignant transformation their expression changes, both at the quantitative and qualitative levels. Of particular interest is the overexpression by tumor cells of Neu5Gc-gangliosides, which are absent, or detected in trace amounts, in human normal cells. The GM3(Neu5Gc) ganglioside in particular has been detected in many human tumors, and it is considered one of the few tumor specific antigen. We previously demonstrated that a humanized antibody specific for this molecule, named 14F7hT, retained the binding and cytotoxic properties of the mouse antibody. In this work, we confirm that 14F7hT exerts a non-apoptotic cell death mechanism in vitro and shows its potent in vivo antitumor activity on a solid mouse myeloma model. Also, we demonstrate, in contrast to the murine counterpart, the capacity of this antibody to induce antibody-dependent cell-mediated cytotoxicity using human effector cells, which increases its potential for the treatment of GM3(Neu5Gc)-expressing human tumors. Copyright © 2015 Elsevier GmbH. All rights reserved.

  16. Repeated Vaccination of Cows with HIV Env gp140 during Subsequent Pregnancies Elicits and Sustains an Enduring Strong Env-Binding and Neutralising Antibody Response.

    Directory of Open Access Journals (Sweden)

    Behnaz Heydarchi

    Full Text Available An important feature of a potential vaccine against HIV is the production of broadly neutralising antibodies (BrNAbs capable of potentially blocking infectivity of a diverse array of HIV strains. BrNAbs naturally arise in some HIV infected individuals after several years of infection and their serum IgG can neutralise various HIV strains across different subtypes. We previously showed that vaccination of cows with HIV gp140 AD8 trimers resulted in a high titre of serum IgG against HIV envelope (Env that had strong BrNAb activity. These polyclonal BrNAbs concentrated into the colostrum during the late stage of pregnancy and can be harvested in vast quantities immediately after calving. In this study, we investigated the effect of prolonged HIV gp140 vaccination on bovine colostrum IgG HIV Env-binding and BrNAb activity over subsequent pregnancies. Repeated immunisation led to a maintained high titre of HIV Env specific IgG in the colostrum batches, but this did not increase through repeated cycles. Colostrum IgG from all batches also strongly competed with sCD4 binding to gp140 Env trimer and with human-derived monoclonal VRC01 and b12 BrNAbs that bind the CD4 binding site (CD4bs. Furthermore, competition neutralisation assays using RSC3 Env gp120 protein core and a derivative CD4bs mutant, RSC3 Δ371I/P363N, showed that CD4bs neutralising antibodies contribute to the neutralising activity of all batches of purified bovine colostrum IgG. This result indicates that the high IgG titre/avidity of anti-CD4bs antibodies with BrNAb activity was achieved during the first year of vaccination and was sustained throughout the years of repeated vaccinations in the cow tested. Although IgG of subsequent colostrum batches may have a higher avidity towards the CD4bs, the overall breadth in neutralisation was not enhanced. This implies that the boosting vaccinations over 4 years elicited a polyclonal antibody response that maintained the proportion of both

  17. Biotin IgM Antibodies in Human Blood: A Previously Unknown Factor Eliciting False Results in Biotinylation-Based Immunoassays

    Science.gov (United States)

    Chen, Tingting; Hedman, Lea; Mattila, Petri S.; Jartti, Laura; Jartti, Tuomas; Ruuskanen, Olli; Söderlund-Venermo, Maria; Hedman, Klaus

    2012-01-01

    Biotin is an essential vitamin that binds streptavidin or avidin with high affinity and specificity. As biotin is a small molecule that can be linked to proteins without affecting their biological activity, biotinylation is applied widely in biochemical assays. In our laboratory, IgM enzyme immuno assays (EIAs) of µ-capture format have been set up against many viruses, using as antigen biotinylated virus like particles (VLPs) detected by horseradish peroxidase-conjugated streptavidin. We recently encountered one serum sample reacting with the biotinylated VLP but not with the unbiotinylated one, suggesting in human sera the occurrence of biotin-reactive antibodies. In the present study, we search the general population (612 serum samples from adults and 678 from children) for IgM antibodies reactive with biotin and develop an indirect EIA for quantification of their levels and assessment of their seroprevalence. These IgM antibodies were present in 3% adults regardless of age, but were rarely found in children. The adverse effects of the biotin IgM on biotinylation-based immunoassays were assessed, including four inhouse and one commercial virus IgM EIAs, showing that biotin IgM do cause false positivities. The biotin can not bind IgM and streptavidin or avidin simultaneously, suggesting that these biotin-interactive compounds compete for the common binding site. In competitive inhibition assays, the affinities of biotin IgM antibodies ranged from 2.1×10−3 to 1.7×10−4 mol/L. This is the first report on biotin antibodies found in humans, providing new information on biotinylation-based immunoassays as well as new insights into the biomedical effects of vitamins. PMID:22879954

  18. 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

  19. The biological characteristics of anti-CD71 mouse/human chimeric antibody

    International Nuclear Information System (INIS)

    Wang Shuo; Jiang Lin; Lei Ping; Zhu Huifen; Shen Guanxin; Cui Wuren; Wang Yanggong

    2002-01-01

    Objective: To study the biological characteristics of an anti-CD71 mouse/human chimeric antibody (D2C). Methods: Analysis of the chimeric Ab production in culture supernatant was made by the standard concentration curve method with ELISA. The antibody was purified by DEAE-Sephredax-A50 ion-exchange chromatography and was confirmed by SDS-PAGE. The competition inhibition studies for binding to the same epitope on CD71 were performed between the chimeric Ab(D2C) in the culture supernatant was about 0.5-5 μg/ml in 5-day cultures when seeded at 1 x 10 5 cells/5ml compared with 12.5-25 μg/ml in the supernatant from their parental monoclonal Ab(7579). The purified chimeric Ab(D2C) from mouse ascetics was 1-2 mg/ml. The SDS-PAGE analysis of purified chimeric Ab(D2C) with discontinuous system confirmed two protein bands of 55 kDa and 25 kDa. It was clear that both chimeric Ab(D2C) and murine monoclonal Ab (7579) compete effectively to join the same epitope of CD71 each other. The chimeric antibody's affinity constant (Ka), quantitated by Scatchard analysis, is about 9.34-9.62 x 10 9 L/mol. Conclusion: The chimeric Ab(D2C) produced from the transfectomas is stable. The binding capacity of the chimeric Ab(D2C) to the antigen (CD71) was retained

  20. Reactivity of eleven anti-human leucocyte monoclonal antibodies with lymphocytes from several domestic animals

    DEFF Research Database (Denmark)

    Aasted, Bent; Blixenkrone-Møller, Merete; Larsen, Else Bang

    1988-01-01

    Nine commercially available monoclonal antibodies and two monoclonal antibodies from The American Type Culture Collection, raised against various human leucocyte surface antigens, were tested on lymphocytes from cow, sheep, goat, swine, horse, cat, dog, mink, and rabbit as well as man. Four...... antibodies bound to lymphocytes from some of the animals. These were the antibodies against CD8 and CD4 antigen, the antibody to C3b-receptor, and the antibody to the HLA-DR antigen. The CD8 antigen-reactive antibody reacted with lymphocytes from mink, cat, dog, and sheep, while the CD4 antigen......-reactive antibody reacted with lymphocytes from mink. The anti-C3b-R antibody reacted with lymphocytes from horse, swine, dog, and cat, and the anti-HLA-DR reacted with lymphocytes from cow, goat, sheep, horse, dog, cat, and mink....

  1. UV-induced DNA-binding proteins in human cells

    International Nuclear Information System (INIS)

    Glazer, P.M.; Greggio, N.A.; Metherall, J.E.; Summers, W.C.

    1989-01-01

    To investigate the response of human cells to DNA-damaging agents such as UV irradiation, the authors examined nuclear protein extracts of UV-irradiated HeLa cells for the presence of DNA-binding proteins. Electrophoretically separated proteins were transferred to a nitrocellulose filter that was subsequently immersed in a binding solution containing radioactively labeled DNA probes. Several DNA-binding proteins were induced in HeLa cells after UV irradiation. These included proteins that bind predominantly double-stranded DNA and proteins that bind both double-stranded and single-stranded DNA. The binding proteins were induced in a dose-dependent manner by UV light. Following a dose of 12 J/m 2 , the binding proteins in the nuclear extracts increased over time to a peak in the range of 18 hr after irradiation. Experiments with metabolic inhibitors (cycloheximide and actinomycin D) revealed that de novo synthesis of these proteins is not required for induction of the binding activities, suggesting that the induction is mediated by protein modification

  2. Effects of exercise on insulin binding to human muscle

    International Nuclear Information System (INIS)

    Bonen, A.; Tan, M.H.; Clune, P.; Kirby, R.L.

    1985-01-01

    A procedure was developed to measure insulin binding to human skeletal muscle obtained via the percutaneous muscle biopsy technique. With this method the effects of exercise on insulin binding were investigated. Subjects (n = 9) exercised for 60 min on a bicycle ergometer at intensities ranging from 20-86% maximum O 2 consumption (VO 2 max). Blood samples were obtained before, during, and after exercise and analyzed for glucose and insulin. Muscle samples (250 mg) for the vastus lateralis were obtained 30 min before exercise, at the end of exercise, and 60 min after exercise. Two subjects rested during the experimental period. There was no linear relationship between exercise intensities and the changes in insulin binding to human muscle. At rest (n = 2) and at exercise intensities below 60% VO 2 max (n = 5) no change in insulin binding occurred (P greater than 0.05). However, when exercise occurred at greater than or equal to 69% VO 2 max (n = 4), a pronounced decrement in insulin binding (30-50%) was observed (P less than 0.05). This persisted for 60 min after exercise. These results indicate that insulin binding in human muscle is not altered by 60 min of exercise at less than or equal to 60% VO 2 max but that a marked decrement occurs when exercise is greater than or equal to 69% VO 2 max

  3. Identification of a CD4-Binding-Site Antibody to HIV that Evolved Near-Pan Neutralization Breadth

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Jinghe; Kang, Byong H.; Ishida, Elise; Zhou, Tongqing; Griesman, Trevor; Sheng, Zizhang; Wu, Fan; Doria-Rose, Nicole A.; Zhang, Baoshan; McKee, Krisha; O’Dell, Sijy; Chuang, Gwo-Yu; Druz, Aliaksandr; Georgiev, Ivelin S.; Schramm, Chaim A.; Zheng, Anqi; Joyce, M.  Gordon; Asokan, Mangaiarkarasi; Ransier, Amy; Darko, Sam; Migueles, Stephen A.; Bailer, Robert T.; Louder, Mark K.; Alam, S.  Munir; Parks, Robert; Kelsoe, Garnett; Von Holle, Tarra; Haynes, Barton F.; Douek, Daniel C.; Hirsch, Vanessa; Seaman, Michael S.; Shapiro, Lawrence; Mascola, John R.; Kwong, Peter D.; Connors, Mark

    2016-11-01

    Detailed studies of the broadly neutralizing antibodies (bNAbs) that underlie the best available examples of the humoral immune response to HIV are providing important information for the development of therapies and prophylaxis for HIV-1 infection. Here, we report a CD4-binding site (CD4bs) antibody, named N6, that potently neutralized 98% of HIV-1 isolates, including 16 of 20 that were resistant to other members of its class. N6 evolved a mode of recognition such that its binding was not impacted by the loss of individual contacts across the immunoglobulin heavy chain. In addition, structural analysis revealed that the orientation of N6 permitted it to avoid steric clashes with glycans, which is a common mechanism of resistance. Thus, an HIV-1-specific bNAb can achieve potent, near-pan neutralization of HIV-1, making it an attractive candidate for use in therapy and prophylaxis.

  4. Informing the Human Plasma Protein Binding of ...

    Science.gov (United States)

    The free fraction of a xenobiotic in plasma (Fub) is an important determinant of chemical adsorption, distribution, metabolism, elimination, and toxicity, yet experimental plasma protein binding data is scarce for environmentally relevant chemicals. The presented work explores the merit of utilizing available pharmaceutical data to predict Fub for environmentally relevant chemicals via machine learning techniques. Quantitative structure-activity relationship (QSAR) models were constructed with k nearest neighbors (kNN), support vector machines (SVM), and random forest (RF) machine learning algorithms from a training set of 1045 pharmaceuticals. The models were then evaluated with independent test sets of pharmaceuticals (200 compounds) and environmentally relevant ToxCast chemicals (406 total, in two groups of 238 and 168 compounds). The selection of a minimal feature set of 10-15 2D molecular descriptors allowed for both informative feature interpretation and practical applicability domain assessment via a bounded box of descriptor ranges and principal component analysis. The diverse pharmaceutical and environmental chemical sets exhibit similarities in terms of chemical space (99-82% overlap), as well as comparable bias and variance in constructed learning curves. All the models exhibit significant predictability with mean absolute errors (MAE) in the range of 0.10-0.18 Fub. The models performed best for highly bound chemicals (MAE 0.07-0.12), neutrals (MAE 0

  5. Method and cell lines for the production of monoclonal antibodies to human glycophorin A

    Science.gov (United States)

    Bigbee, W.L.; Fong, S.S.N.; Jensen, R.H.; Vanderlaan, M.

    Cloned mouse hybridoma cell lines have been established which continuously produce antibodies that differentiate between the M and N forms of human glycophorin A. These antibodies have potential application as human blood group reagents, as markers for terminally differentiated erythroid cells and as immunofluorescent labels of somatically variant human erythrocytes.

  6. Development of human antibody fragments using antibody phage display for the detection and diagnosis of Venezuelan equine encephalitis virus (VEEV

    Directory of Open Access Journals (Sweden)

    Hust Michael

    2008-09-01

    Full Text Available Abstract Background Venezuelan equine encephalitis virus (VEEV belongs to the Alphavirus group. Several species of this family are also pathogenic to humans and are recognized as potential agents of biological warfare and terrorism. The objective of this work was the generation of recombinant antibodies for the detection of VEEV after a potential bioterrorism assault or an natural outbreak of VEEV. Results In this work, human anti-VEEV single chain Fragments variable (scFv were isolated for the first time from a human naïve antibody gene library using optimized selection processes. In total eleven different scFvs were identified and their immunological specificity was assessed. The specific detection of the VEEV strains TC83, H12/93 and 230 by the selected antibody fragments was proved. Active as well as formalin inactivated virus particles were recognized by the selected antibody fragments which could be also used for Western blot analysis of VEEV proteins and immunohistochemistry of VEEV infected cells. The anti-VEEV scFv phage clones did not show any cross-reactivity with Alphavirus species of the Western equine encephalitis virus (WEEV and Eastern equine encephalitis virus (EEEV antigenic complex, nor did they react with Chikungunya virus (CHIKV, if they were used as detection reagent. Conclusion For the first time, this study describes the selection of antibodies against a human pathogenic virus from a human naïve scFv antibody gene library using complete, active virus particles as antigen. The broad and sensitive applicability of scFv-presenting phage for the immunological detection and diagnosis of Alphavirus species was demonstrated. The selected antibody fragments will improve the fast identification of VEEV in case of a biological warfare or terroristic attack or a natural outbreak.

  7. Interaction of complement-solubilized immune complexes with CR1 receptors on human erythrocytes. The binding reaction

    DEFF Research Database (Denmark)

    Jepsen, H H; Svehag, S E; Jarlbaek, L

    1986-01-01

    showed no binding. IC solubilized in 50% human serum in the presence of autologous RBC bound rapidly to RBC-CR1, with maximal binding within less than 1 min at 37 degrees C. Release of CR1-bound IC under these conditions occurred slowly, requiring more than 30 min. Only binding of 'partially' solubilized...... of an intact classical pathway in preparing the IC for binding to RBC-CR1. C-solubilized IC could be absorbed to solid-phase conglutinin or antibody to C3c and C4c, and these ligands were able to inhibit the binding of solubilized IC to RBC. Heparin also exerted a marked, dose-dependent inhibitory effect...

  8. Characterization of a Novel Humanized Anti-CD20 Antibody with Potent Anti-Tumor Activity against Non-Hodgkin's Lymphoma

    Directory of Open Access Journals (Sweden)

    Haifeng Zhang

    2013-09-01

    Full Text Available Background: Rituximab, a mouse Fab and human Fc chimeric antibody, has been widely used to treat Non-Hodgkin's lymphoma (NHL. However, only 48% of patients respond to the treatment and complete response rate is below 10%. Also, immunogenicity was reported in 17-20% patients receiving the treatment, making it unsuitable for long term diseases such as autoimmune disorders. It has been a hot research field to “humanize” rituximab toward improved efficacy and reduced immunogenicity. Methods: In this study, an advanced antibody humanization technology was applied to the sequence of the anti-CD20 antibody 2B8, its sequence of which was based on the original murine monoclonal antibody of rituximab in Roche. The complementarity-determining regions (CDRs of the humanized antibodies were further optimized through computer-aided molecular dock. Results: Five novel humanized anti-CD20 antibodies 1-5(1635, 1534, 3637, 1634 and 1536 were generated and their immunogenicity was significantly decreased when compared to rituximab. The novel humanized anti-CD20 antibodies 1-5 retained the binding activity of their murine counterpart, as demonstrated by the fluorescence-activated cell-sorting analysis (FACS. When compared to rituximab, the humanized antibodies still have the similar properties on both complement-dependent cytotoxicity (CDC and antibody-dependent cell-mediated cytotoxicity (ADCC. Furthermore, its anti-tumor efficacy in xenograft model is comparable to that of rituximab. Conclusion: The humanized anti-CD20 antibodies 1-5 have lower immunogenicity than rituximab. And at the same time, they still retain the anti-tumor effect both in vitro and vivo.

  9. Production and purification of polyclonal antibody against F(ab')2 fragment of human immunoglobulin G

    OpenAIRE

    Nasiri, Hadi; Valedkarimi, Zahra; Aghebati-Maleki, Leili; Abdolalizadeh, Jalal; Kazemi, Tohid; Esparvarinha, Mojghan; Majidi, Jafar

    2017-01-01

    Antibodies are essential tools of biomedical and biochemical researches. Polyclonal antibodies are produced against different epitopes of antigens. Purified F(ab')2 can be used for animal’s immunization to produce polyclonal antibodies. Human immunoglobulin G (IgG) was purified by ion exchange chromatography method. In all stages verification method of the purified antibodies was sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purified IgG was digested by pepsin enzyme a...

  10. Detection of antibodies against Turkey astrovirus in humans.

    Science.gov (United States)

    Meliopoulos, Victoria A; Kayali, Ghazi; Burnham, Andrew; Oshansky, Christine M; Thomas, Paul G; Gray, Gregory C; Beck, Melinda A; Schultz-Cherry, Stacey

    2014-01-01

    Astroviruses are a leading cause of gastroenteritis in mammals and birds worldwide. Although historically thought to be species-specific, increasing evidence suggests that astroviruses may cross species barriers. In this report, we used enzyme-linked immunosorbent assays to screen sera from three distinct human cohorts involved in influenza studies in Memphis, TN or Chapel Hill, NC, and Midwestern poultry abattoir workers for antibodies to turkey astrovirus type 2 (TAstV-2). Surprisingly, 26% of one cohort's population was TAstV-2 positive as compared to 0 and 8.9% in the other cohorts. This cohort was composed of people with exposure to turkeys in the Midwestern United States including abattoir workers, turkey growers, and non-occupationally exposed participants. The odds of testing positive for antibodies against turkey astrovirus among abattoir workers were approximately 3 times higher than the other groups. These studies suggest that people with contact to turkeys can develop serological responses to turkey astrovirus. Further work is needed to determine if these exposures result in virus replication and/or clinical disease.

  11. Detection of antibodies against Turkey astrovirus in humans.

    Directory of Open Access Journals (Sweden)

    Victoria A Meliopoulos

    Full Text Available Astroviruses are a leading cause of gastroenteritis in mammals and birds worldwide. Although historically thought to be species-specific, increasing evidence suggests that astroviruses may cross species barriers. In this report, we used enzyme-linked immunosorbent assays to screen sera from three distinct human cohorts involved in influenza studies in Memphis, TN or Chapel Hill, NC, and Midwestern poultry abattoir workers for antibodies to turkey astrovirus type 2 (TAstV-2. Surprisingly, 26% of one cohort's population was TAstV-2 positive as compared to 0 and 8.9% in the other cohorts. This cohort was composed of people with exposure to turkeys in the Midwestern United States including abattoir workers, turkey growers, and non-occupationally exposed participants. The odds of testing positive for antibodies against turkey astrovirus among abattoir workers were approximately 3 times higher than the other groups. These studies suggest that people with contact to turkeys can develop serological responses to turkey astrovirus. Further work is needed to determine if these exposures result in virus replication and/or clinical disease.

  12. Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of the CGRP Binding Monoclonal Antibody LY2951742 (Galcanezumab in Healthy Volunteers

    Directory of Open Access Journals (Sweden)

    David Monteith

    2017-10-01

    Full Text Available Background: Calcitonin gene-related peptide (CGRP is pivotal in the pathophysiology of migraine headaches and represents a promising target for migraine treatment. The humanized monoclonal antibody galcanezumab (LY2951742 binds to CGRP and may be effective in migraine prophylaxis.Objectives: The primary objective was to evaluate the safety and tolerability of single and multiple doses of galcanezumab in humans. Secondary objectives included assessing the pharmacokinetics and evaluating target engagement.Methods: A double-blind, randomized, placebo-controlled study (NCT 01337596 with single escalating and multiple subcutaneous (SC doses of galcanezumab was performed in healthy male volunteers. Single doses of 1, 5, 25, 75, 200, and 600 mg of galcanezumab (n = 7/dose or placebo (n = 2/dose were injected SC in six consecutive cohorts of nine subjects each. One cohort of nine subjects received multiple (4 150 mg doses of galcanezumab or placebo every other week. Target engagement was evaluated by measuring inhibition of capsaicin-induced increase in dermal blood flow (DBF.Findings: Sixty-three subjects were randomized and included in the safety analyses. Galcanezumab was well tolerated in single doses (1–600 mg SC and consecutive doses (150 mg SC. There was no dose-dependent difference in type or frequency of treatment-emergent adverse events, and no clinically meaningful difference when compared with placebo. Pharmacokinetics were linear. Galcanezumab induced a robust, dose-dependent, and durable inhibition of capsaicin-induced increase in DBF, supporting the continued clinical development of galcanezumab for prophylaxis in migraine patients.

  13. The PD-1/PD-L1 complex resembles the antigen-binding Fv domains of antibodies and T cell receptors

    Energy Technology Data Exchange (ETDEWEB)

    Lin, David Yin-wei; Tanaka, Yoshimasa; Iwasaki, Masashi; Gittis, Apostolos G.; Su, Hua-Poo; Mikami, Bunzo; Okazaki, Taku; Honjo, Tasuku; Minato, Nagahiro; Garboczi, David N. (NIH); (Kyoto)

    2008-07-29

    Signaling through the programmed death 1 (PD-1) inhibitory receptor upon binding its ligand, PD-L1, suppresses immune responses against autoantigens and tumors and plays an important role in the maintenance of peripheral immune tolerance. Release from PD-1 inhibitory signaling revives 'exhausted' virus-specific T cells in chronic viral infections. Here we present the crystal structure of murine PD-1 in complex with human PD-L1. PD-1 and PD-L1 interact through the conserved front and side of their Ig variable (IgV) domains, as do the IgV domains of antibodies and T cell receptors. This places the loops at the ends of the IgV domains on the same side of the PD-1/PD-L1 complex, forming a surface that is similar to the antigen-binding surface of antibodies and T cell receptors. Mapping conserved residues allowed the identification of residues that are important in forming the PD-1/PD-L1 interface. Based on the structure, we show that some reported loss-of-binding mutations involve the PD-1/PD-L1 interaction but that others compromise protein folding. The PD-1/PD-L1 interaction described here may be blocked by antibodies or by designed small-molecule drugs to lower inhibitory signaling that results in a stronger immune response. The immune receptor-like loops offer a new surface for further study and potentially the design of molecules that would affect PD-1/PD-L1 complex formation and thereby modulate the immune response.

  14. Binding behavior of CRP and anti-CRP antibody analyzed with SPR and AFM measurement

    International Nuclear Information System (INIS)

    Lee, Soo-Keun; Kim, Hyun-Chul; Cho, Sang-Joon; Jeong, Sang Won; Jeon, Won Bae

    2008-01-01

    Atomic force microscope (AFM) was exploited to take picture of the molecular topology of C-reactive protein (CRP) in phosphate-buffered saline (PBS) solution. An explicit molecular image of CRP demonstrated a pentagonal structure composed of five subunits. Dimensions of the doughnut-shaped CRP molecule measured by AFM were about 25 nm in outside diameter and 10 nm in central pore diameter, and the height of CRP molecule was about 4 nm which was comparable to the value determined by X-ray crystallography. Bis(N-succinimido)-11,11'-dithiobis (undecyl succinate) (DSNHS) was synthesized for use as a linker for immobilizing anti-CRP antibody (anti-CRP) onto the gold surface of a surface plasmon resonance (SPR) sensor chip. DSNHS formed self-assembled monolayer (SAM) on the gold surface. By use of an AFM tip, a pattern of ditch was engraved within the SAM of DSNHS, and anti-CRP was immobilized on the engraved SAM through replacement of N-hydroxysuccinimide group on the outside surface of DSNHS by the amine group of anti-CRP. Formation of CRP/anti-CRP complex on the gold surface of SPR sensor chip was clearly demonstrated by measuring SPR angle shift. A consecutive series of SAM, SAM/anti-CRP, and SAM/anti-CRP/CRP complexes was generated on a SPR sensor chip, and the changes in depth of the ditch were monitored by taking AFM images of the complexes. Comparative analysis of the depth differences indicates that binding of CRP to anti-CRP occurs in a planar mode

  15. Palmitate and stearate binding to human serum albumin. Determination of relative binding constants

    DEFF Research Database (Denmark)

    Vorum, H; Fisker, K; Honoré, B

    1997-01-01

    Multiple binding equilibria of two apparently insoluble ligands, palmitate and stearate, to defatted human serum albumin were studied in a 66 mM sodium phosphate buffer (pH 7.4) at 37 degrees C, by determination of dialytic exchange rates of ligands among identical equilibrium solutions. The expe...

  16. Thrombin binding to human brain and spinal cord

    International Nuclear Information System (INIS)

    McKinney, M.; Snider, R.M.; Richelson, E.

    1983-01-01

    Thrombin, a serine protease that regulates hemostasis, has been shown to stimulate the formation of cGMP in murine neuroblastoma cells. The nervous system in vivo thus may be postulated to respond to this blood-borne factor after it breaches the blood-brain barrier, as in trauma. Human alpha-thrombin was radiolabeled with 125I and shown to bind rapidly, reversibly, and with high affinity to human brain and spinal cord. These findings indicate the presence of specific thrombin-binding sites in nervous tissue and may have important clinical implications

  17. Polyclonal and monoclonal antibodies specific for the six-helix bundle of the human respiratory syncytial virus fusion glycoprotein as probes of the protein post-fusion conformation

    International Nuclear Information System (INIS)

    Palomo, Concepción; Mas, Vicente; Vázquez, Mónica; Cano, Olga; Luque, Daniel; Terrón, María C.; Calder, Lesley J.; Melero, José A.

    2014-01-01

    Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRSV F occurs via refolding from a pre-fusion form to a highly stable post-fusion state involving large conformational changes of the F trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of hRSV F , we have prepared polyclonal (α-6HB) and monoclonal (R145) rabbit antibodies specific for the 6HB. Among other applications, these antibodies were used to explore the requirements of 6HB formation by isolated protein segments or peptides and by truncated mutants of the F protein. Site-directed mutagenesis and electron microscopy located the R145 epitope in the post-fusion hRSV F at a site distantly located from previously mapped epitopes, extending the repertoire of antibodies that can decorate the F molecule. - Highlights: • Antibodies specific for post-fusion respiratory syncytial virus fusion protein are described. • Polyclonal antibodies were obtained in rabbit inoculated with chimeric heptad repeats. • Antibody binding required assembly of a six-helix bundle in the post-fusion protein. • A monoclonal antibody with similar structural requirements is also described. • Binding of this antibody to the post-fusion protein was visualized by electron microscopy

  18. Polyclonal and monoclonal antibodies specific for the six-helix bundle of the human respiratory syncytial virus fusion glycoprotein as probes of the protein post-fusion conformation

    Energy Technology Data Exchange (ETDEWEB)

    Palomo, Concepción; Mas, Vicente; Vázquez, Mónica; Cano, Olga [Unidad de Biología Viral, Centro Nacional de Microbiología, Madrid (Spain); CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain); Luque, Daniel; Terrón, María C. [Unidad de Microscopía Electrónica y Confocal, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain); Calder, Lesley J. [National Institute for Medical Research, MRC, Mill Hill, London NW7 1AA (United Kingdom); Melero, José A., E-mail: jmelero@isciii.es [Unidad de Biología Viral, Centro Nacional de Microbiología, Madrid (Spain); CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain)

    2014-07-15

    Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRSV{sub F} occurs via refolding from a pre-fusion form to a highly stable post-fusion state involving large conformational changes of the F trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of hRSV{sub F}, we have prepared polyclonal (α-6HB) and monoclonal (R145) rabbit antibodies specific for the 6HB. Among other applications, these antibodies were used to explore the requirements of 6HB formation by isolated protein segments or peptides and by truncated mutants of the F protein. Site-directed mutagenesis and electron microscopy located the R145 epitope in the post-fusion hRSV{sub F} at a site distantly located from previously mapped epitopes, extending the repertoire of antibodies that can decorate the F molecule. - Highlights: • Antibodies specific for post-fusion respiratory syncytial virus fusion protein are described. • Polyclonal antibodies were obtained in rabbit inoculated with chimeric heptad repeats. • Antibody binding required assembly of a six-helix bundle in the post-fusion protein. • A monoclonal antibody with similar structural requirements is also described. • Binding of this antibody to the post-fusion protein was visualized by electron microscopy.

  19. Characterization of a cocaine binding protein in human placenta

    International Nuclear Information System (INIS)

    Ahmed, M.S.; Zhou, D.H.; Maulik, D.; Eldefrawi, M.E.

    1990-01-01

    [ 3 H]-Cocaine binding sites are identified in human placental villus tissue plasma membranes. These binding sites are associated with a protein and show saturable and specific binding of [ 3 H]-cocaine with a high affinity site of 170 fmole/mg protein. The binding is lost with pretreatment with trypsin or heat. The membrane bound protein is solubilized with the detergent 3-(3-cholamidopropyl)dimethyl-ammonio-1-propane sulphonate (CHAPS) with retention of its saturable and specific binding of [ 3 H]-cocaine. The detergent-protein complex migrates on a sepharose CL-6B gel chromatography column as a protein with an apparent molecular weight of 75,900. The protein has an S 20,w value of 5.1. The binding of this protein to norcocaine, pseudococaine, nomifensine, imipramine, desipramine, amphetamine and dopamine indicates that it shares some, but not all, the properties of the brain cocaine receptor. The physiologic significance of this protein in human placenta is currently unclear

  20. Beta 3 and PDI proteins isolated from human platelets bind with ECwt rotavirus in vitro

    International Nuclear Information System (INIS)

    Mayorga, Diana; Rubio, Linda; Guerrero-Fonseca, Carlos A; Acosta-Losada, Orlando

    2010-01-01

    Commercial integrin Beta 3 is currently not available and commercial PDI is too expensive, which is making access difficult to these proteins needed for conducting experiments aimed at the establishment of possible interactions between integrin Beta 3 and PDI and wild type rotavirus strains. Objective. To explore a methodology allowing isolation of proteins Beta 3 and PDI from human platelets to be used as antigens in the generation of rabbit polyclonal antibodies useful in the assessment of interactions between these proteins and rotavirus ECwt. Materials and methods. Proteins Beta 3 and PDI from human platelet lysates were separated using preparative electrophoresis under reducing conditions and then eluted. Interactions of these proteins with rotavirus ECwt were analyzed using co-immunoprecipitation, Western blotting and capture ELISA. Results. Proteins from human platelet lysates were separated by preparative electrophoresis under reducing conditions. The identification of proteins Beta 3 and PDI present in a gel slice was performed through their reaction with commercial antibodies in a Western blotting analysis. Protein purity was established after electro elution from a gel slice. Polyclonal antibodies against protein Beta 3 were generated in rabbit. Incubation of eluted proteins Beta 3 and PDI with rotavirus ECwt showed in co-immunoprecipitation and ELISA assays that these proteins bound virus in vitro. The same binding was showed to occur when rotavirus was incubated with isolated small intestinal villi from suckling mice. Conclusions. Relatively high amounts of proteins Beta 3 and PDI were partially purified from human platelets by preparative electrophoresis. The isolation of these proteins allowed the generation of polyclonal antibodies against Beta 3 in addition to the establishment of the in vitro interaction of proteins Beta 3 and PDI with rotavirus ECwt. This interaction was also demonstrated in vivo after incubating the virus with isolated small

  1. 125I-Clq-binding and specific antibodies as indicators of pulmonary disease activity in cystic fibrosis

    International Nuclear Information System (INIS)

    Moss, R.B.; Hsu, Y.P.; Lewiston, N.J.

    1981-01-01

    We studied the incidence and levels of circulating immune complexes by the 125 I-Clq-binding assay in patients with cystic fibrosis in relation to clinical respiratory status and specific IgG and IgE antibodies to Pseudomonas aeruginosa. Staphylococcus aureus, Aspergillus fumigatus, and Candida albicans. Overall prevalence of CIC was 43%, but 86% of serially studied patients had evidence of CIC at some time. Patients with acute respiratory exacerbations and deteriorating pulmonary function had a higher incidence of CIC (76%) as compared to stable patients (36%, P less than 0.01), as well as significantly higher levels of CIC. Acute exacerbations were also associated with significant increases in IgG antibody to Pseudomonas (P less than 0.005) but not in other antibodies. CIC did not correlate with Pseudomonas-specific IgG nor with any other specific antibody studied. A variety of age-related differences in specific antibody levels were seen. The episodic appearance of CIC is common in CF and is usually associated with exacerbation of lung disease

  2. Monoclonal Antibodies Against Fusicoccin with Binding Characteristics Similar to the Putative Fusicoccin Receptor of Higher Plants 1

    Science.gov (United States)

    Feyerabend, Martin; Weiler, Elmar W.

    1987-01-01

    Monoclonal antibodies were raised against fusicoccin. The toxin, linked to bovine serum albumin through its t-pentenyl moiety, served as immunogen. Hybridomas secreting anti-fusicoccin antibodies were screened by radioimmunoassay employing a novel radioactive derivative, [3H]-nor-fusicoccin-alcohol of high specific activity (1.5 × 1014Bq/mole). The two monoclonal antibodies reported here are of high apparent affinity for fusicoccin (0.71 × 10−9 molar and 1.85 × 10−9 molar). This is comparable to the apparent affinity of rabbit antiserum raised against the same type of conjugate (9.3 × 10−9 molar). A method for the single step purification of the monoclonal antibodies from ascites fluid is reported. A solid-phase immunoassay, using alkaline phosphatase as enzyme, exhibits a measuring range from 0.1 to 1.5 picomoles (about 70 picograms to 1 nanogram) of fusicoccin. The displacement of [3H]-nor-fusicoccin-alcohol from the antibodies by compounds structurally related to fusicoccin exhibits similar selectivity as a microsomal binding assay with the same tracer as radiolabeled probe. Images Fig. 2 PMID:16665786

  3. Intrinsic thermodynamics of inhibitor binding to human carbonic anhydrase IX.

    Science.gov (United States)

    Linkuvienė, Vaida; Matulienė, Jurgita; Juozapaitienė, Vaida; Michailovienė, Vilma; Jachno, Jelena; Matulis, Daumantas

    2016-04-01

    Human carbonic anhydrase 9th isoform (CA IX) is an important marker of numerous cancers and is increasingly interesting as a potential anticancer drug target. Various synthetic aromatic sulfonamide-bearing compounds are being designed as potent inhibitors of CA IX. However, sulfonamide compound binding to CA IX is linked to several reactions, the deprotonation of the sulfonamide amino group and the protonation of the CA active site Zn(II)-bound hydroxide. These linked reactions significantly affect the affinities and other thermodynamic parameters such as enthalpies and entropies of binding. The observed and intrinsic affinities of compound binding to CA IX were determined by the fluorescent thermal shift assay. The enthalpies and entropies of binding were determined by the isothermal titration calorimetry. The pKa of CA IX was determined to be 6.8 and the enthalpy of CA IX-Zn(II)-bound hydroxide protonation was -24 kJ/mol. These values enabled the analysis of intrinsic thermodynamics of a library of compounds binding to CA IX. The most strongly binding compounds exhibited the intrinsic affinity of 0.01 nM and the observed affinity of 2 nM. The intrinsic thermodynamic parameters of compound binding to CA IX helped to draw the compound structure to thermodynamics relationship. It is important to distinguish the intrinsic from observed parameters of any disease target protein interaction with its inhibitors as drug candidates when drawing detailed compound structure to thermodynamics correlations. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Spectroscopic characterization of furosemide binding to human carbonic anhydrase II.

    Science.gov (United States)

    Ranjbar, Samira; Ghobadi, Sirous; Khodarahmi, Reza; Nemati, Houshang

    2012-05-01

    This study reports the interaction between furosemide and human carbonic anhydrase II (hCA II) using fluorescence, UV-vis and circular dichroism (CD) spectroscopy. Fluorescence data indicated that furosemide quenches the intrinsic fluorescence of the enzyme via a static mechanism and hydrogen bonding and van der Walls interactions play the major role in the drug binding. The binding average distance between furosemide and hCA II was estimated on the basis of the theory of Förster energy transfer. Decrease of protein surface hydrophobicity was also documented upon furosemide binding. Chemical modification of hCA II using N-bromosuccinimide indicated decrease of the number of accessible tryptophans in the presence of furosemide. CD results suggested the occurance of some alterations in α-helical content as well as tertiary structure of hCA II upon drug binding. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. Comparison of printed glycan array, suspension array and ELISA in the detection of human anti-glycan antibodies.

    Science.gov (United States)

    Pochechueva, Tatiana; Jacob, Francis; Goldstein, Darlene R; Huflejt, Margaret E; Chinarev, Alexander; Caduff, Rosemarie; Fink, Daniel; Hacker, Neville; Bovin, Nicolai V; Heinzelmann-Schwarz, Viola

    2011-12-01

    Anti-glycan antibodies represent a vast and yet insufficiently investigated subpopulation of naturally occurring and adaptive antibodies in humans. Recently, a variety of glycan-based microarrays emerged, allowing high-throughput profiling of a large repertoire of antibodies. As there are no direct approaches for comparison and evaluation of multi-glycan assays we compared three glycan-based immunoassays, namely printed glycan array (PGA), fluorescent microsphere-based suspension array (SA) and ELISA for their efficacy and selectivity in profiling anti-glycan antibodies in a cohort of 48 patients with and without ovarian cancer. The ABO blood group glycan antigens were selected as well recognized ligands for sensitivity and specificity assessments. As another ligand we selected P(1), a member of the P blood group system recently identified by PGA as a potential ovarian cancer biomarker. All three glyco-immunoassays reflected the known ABO blood groups with high performance. In contrast, anti-P(1) antibody binding profiles displayed much lower concordance. Whilst anti-P(1) antibody levels between benign controls and ovarian cancer patients were significantly discriminated using PGA (p=0.004), we got only similar results using SA (p=0.03) but not for ELISA. Our findings demonstrate that whilst assays were largely positively correlated, each presents unique characteristic features and should be validated by an independent patient cohort rather than another array technique. The variety between methods presumably reflects the differences in glycan presentation and the antigen/antibody ratio, assay conditions and detection technique. This indicates that the glycan-antibody interaction of interest has to guide the assay selection. © The Author(s) 2011. This article is published with open access at Springerlink.com

  6. Unique biological properties of catalytic domain directed human anti-CAIX antibodies discovered through phage-display technology.

    Directory of Open Access Journals (Sweden)

    Chen Xu

    2010-03-01

    Full Text Available Carbonic anhydrase IX (CAIX, gene G250/MN-encoded transmembrane protein is highly expressed in various human epithelial tumors such as renal clear cell carcinoma (RCC, but absent from the corresponding normal tissues. Besides the CA signal transduction activity, CAIX may serve as a biomarker in early stages of oncogenesis and also as a reliable marker of hypoxia, which is associated with tumor resistance to chemotherapy and radiotherapy. Although results from preclinical and clinical studies have shown CAIX as a promising target for detection and therapy for RCC, only a limited number of murine monoclonal antibodies (mAbs and one humanized mAb are available for clinical testing and development. In this study, paramagnetic proteoliposomes of CAIX (CAIX-PMPLs were constructed and used for anti-CAIX antibody selection from our 27 billion human single-chain antibody (scFv phage display libraries. A panel of thirteen human scFvs that specifically recognize CAIX expressed on cell surface was identified, epitope mapped primarily to the CA domain, and affinity-binding constants (KD determined. These human anti-CAIX mAbs are diverse in their functions including induction of surface CAIX internalization into endosomes and inhibition of the carbonic anhydrase activity, the latter being a unique feature that has not been previously reported for anti-CAIX antibodies. These human anti-CAIX antibodies are important reagents for development of new immunotherapies and diagnostic tools for RCC treatment as well as extending our knowledge on the basic structure-function relationships of the CAIX molecule.

  7. Strong antitumor activities of IgG3 antibodies to a human melanoma-associated ganglioside

    International Nuclear Information System (INIS)

    Hellstroem, I.; Brankovan, V.; Hellstroem, K.E.

    1985-01-01

    Three mouse monoclonal IgG3 antibodies, 2B2, IF4, and MG-21, recognize a G/sub D3/ ganglioside antigen that is expressed at the cell surface of most human melanomas. All three antibodies mediate antibody-dependent cellular cytotoxicity (ADCC) in vitro when tested with human lymphocytes or effector cells in a 2-hr or 4-hr 51 Cr-release test, and one antibody, MG-21, also gives strong complement-dependent cytotoxicity with human serum. Antibody 2B2, which gives ADDC also in the presence of mouse lymphocytes, inhibited the outgrowth of a human melanoma in nude mice, but antibody IF4, which showed no ADCC with mouse lymphocyte effectors, did not

  8. Trichinella spiralis Calreticulin Binds Human Complement C1q As an Immune Evasion Strategy.

    Science.gov (United States)

    Zhao, Limei; Shao, Shuai; Chen, Yi; Sun, Ximeng; Sun, Ran; Huang, Jingjing; Zhan, Bin; Zhu, Xinping

    2017-01-01

    As a multicellular parasitic nematode, Trichinella spiralis regulates host immune responses by producing a variety of immunomodulatory molecules to escape from host immune attack, but the mechanisms underlying the immune evasion are not well understood. Here, we identified that T. spiralis calreticulin ( Ts -CRT), a Ca 2+ -binding protein, facilitated T. spiralis immune evasion by interacting with the first component of human classical complement pathway, C1q. In the present study, Ts -CRT was found to be expressed on the surface of different developmental stages of T. spiralis as well as in the secreted products of adult and muscle larval worms. Functional analysis identified that Ts -CRT was able to bind to human C1q, resulting in the inhibition of C1q-initiated complement classical activation pathway reflected by reduced C4/C3 generation and C1q-dependent lysis of antibody-sensitized sheep erythrocytes. Moreover, recombinant Ts -CRT (r Ts -CRT) binding to C1q suppressed C1q-induced THP-1-derived macrophages chemotaxis and reduced monocyte-macrophages release of reactive oxygen intermediates (ROIs). Blocking Ts -CRT on the surface of newborn larvae (NBL) of T. spiralis with anti- Ts -CRT antibody increased the C1q-mediated adherence of monocyte-macrophages to larvae and impaired larval infectivity. All of these results suggest that T. spiralis -expressed Ts -CRT plays crucial roles in T. spiralis immune evasion and survival in host mostly by directly binding to host complement C1q, which not only reduces C1q-mediated activation of classical complement pathway but also inhibits the C1q-induced non-complement activation of macrophages.

  9. A fully synthetic human Fab antibody library based on fixed VH/VL framework pairings with favorable biophysical properties

    Science.gov (United States)

    Tiller, Thomas; Schuster, Ingrid; Deppe, Dorothée; Siegers, Katja; Strohner, Ralf; Herrmann, Tanja; Berenguer, Marion; Poujol, Dominique; Stehle, Jennifer; Stark, Yvonne; Heßling, Martin; Daubert, Daniela; Felderer, Karin; Kaden, Stefan; Kölln, Johanna; Enzelberger, Markus; Urlinger, Stefanie

    2013-01-01

    This report describes the design, generation and testing of Ylanthia, a fully synthetic human Fab antibody library with 1.3E+11 clones. Ylanthia comprises 36 fixed immunoglobulin (Ig) variable heavy (VH)/variable light (VL) chain pairs, which cover a broad range of canonical complementarity-determining region (CDR) structures. The variable Ig heavy and Ig light (VH/VL) chain pairs were selected for biophysical characteristics favorable to manufacturing and development. The selection process included multiple parameters, e.g., assessment of protein expression yield, thermal stability and aggregation propensity in fragment antigen binding (Fab) and IgG1 formats, and relative Fab display rate on phage. The framework regions are fixed and the diversified CDRs were designed based on a systematic analysis of a large set of rearranged human antibody sequences. Care was taken to minimize the occurrence of potential posttranslational modification sites within the CDRs. Phage selection was performed against various antigens and unique antibodies with excellent biophysical properties were isolated. Our results confirm that quality can be built into an antibody library by prudent selection of unmodified, fully human VH/VL pairs as scaffolds. PMID:23571156

  10. Lectin binding profiles of SSEA-4 enriched, pluripotent human embryonic stem cell surfaces

    Science.gov (United States)

    Venable, Alison; Mitalipova, Maisam; Lyons, Ian; Jones, Karen; Shin, Soojung; Pierce, Michael; Stice, Steven

    2005-01-01

    Background Pluripotent human embryonic stem cells (hESCs) have the potential to form every cell type in the body. These cells must be appropriately characterized prior to differentiation studies or when defining characteristics of the pluripotent state. Some developmentally regulated cell surface antigens identified by monoclonal antibodies in a variety of species and stem cell types have proven to be side chains of membrane glycolipids and glycoproteins. Therefore, to examine hESC surfaces for other potential pluripotent markers, we used a panel of 14 lectins, which were chosen based on their specificity for a variety of carbohydrates and carbohydrate linkages, along with stage specific embryonic antigen-4 (SSEA-4), to determine binding quantitation by flow cytometry and binding localization in adherent colonies by immunocytochemistry. Results Enriching cells for SSEA-4 expression increased the percentage of SSEA-4 positive cells to 98–99%. Using enriched high SSEA-4-expressing hESCs, we then analyzed the binding percentages of selected lectins and found a large variation in binding percentages ranging from 4% to 99% binding. Lycopersicon (tomato)esculetum lectin (TL), Ricinus communis agglutinin (RCA), and Concanavalin A (Con A) bound to SSEA-4 positive regions of hESCs and with similar binding percentages as SSEA-4. In contrast, we found Dolichos biflorus agglutinin (DBA) and Lotus tetragonolobus lectin (LTL) did not bind to hESCs while Phaseolus vulgaris leuco-agglutinin (PHA-L), Vicia villosa agglutinin (VVA), Ulex europaeus agglutinin (UEA), Phaseolus vulgaris erythro-agglutinin (PHA-E), and Maackia amurensis agglutinin (MAA) bound partially to hESCs. These binding percentages correlated well with immunocytochemistry results. Conclusion Our results provide information about types of carbohydrates and carbohydrate linkages found on pluripotent hESC surfaces. We propose that TL, RCA and Con A may be used as markers that are associated with the pluripotent

  11. Lectin binding profiles of SSEA-4 enriched, pluripotent human embryonic stem cell surfaces

    Directory of Open Access Journals (Sweden)

    Shin Soojung

    2005-07-01

    Full Text Available Abstract Background Pluripotent human embryonic stem cells (hESCs have the potential to form every cell type in the body. These cells must be appropriately characterized prior to differentiation studies or when defining characteristics of the pluripotent state. Some developmentally regulated cell surface antigens identified by monoclonal antibodies in a variety of species and stem cell types have proven to be side chains of membrane glycolipids and glycoproteins. Therefore, to examine hESC surfaces for other potential pluripotent markers, we used a panel of 14 lectins, which were chosen based on their specificity for a variety of carbohydrates and carbohydrate linkages, along with stage specific embryonic antigen-4 (SSEA-4, to determine binding quantitation by flow cytometry and binding localization in adherent colonies by immunocytochemistry. Results Enriching cells for SSEA-4 expression increased the percentage of SSEA-4 positive cells to 98–99%. Using enriched high SSEA-4-expressing hESCs, we then analyzed the binding percentages of selected lectins and found a large variation in binding percentages ranging from 4% to 99% binding. Lycopersicon (tomatoesculetum lectin (TL, Ricinus communis agglutinin (RCA, and Concanavalin A (Con A bound to SSEA-4 positive regions of hESCs and with similar binding percentages as SSEA-4. In contrast, we found Dolichos biflorus agglutinin (DBA and Lotus tetragonolobus lectin (LTL did not bind to hESCs while Phaseolus vulgaris leuco-agglutinin (PHA-L, Vicia villosa agglutinin (VVA, Ulex europaeus agglutinin (UEA, Phaseolus vulgaris erythro-agglutinin (PHA-E, and Maackia amurensis agglutinin (MAA bound partially to hESCs. These binding percentages correlated well with immunocytochemistry results. Conclusion Our results provide information about types of carbohydrates and carbohydrate linkages found on pluripotent hESC surfaces. We propose that TL, RCA and Con A may be used as markers that are associated with the

  12. Epitopes in α8β1 and other RGD-binding integrins delineate classes of integrin-blocking antibodies and major binding loops in α subunits

    OpenAIRE

    Norihisa Nishimichi; Nagako Kawashima; Yasuyuki Yokosaki

    2015-01-01

    Identification of epitopes for integrin-blocking monoclonal antibodies (mAbs) has aided our understanding of structure-function relationship of integrins. We mapped epitopes of chicken anti-integrin-α8-subunit-blocking mAbs by mutational analyses, examining regions that harboured all mapped epitopes recognized by mAbs against other α-subunits in the RGD-binding-integrin subfamily. Six mAbs exhibited blocking function, and these mAbs recognized residues on the same W2:41-loop on the top-face o...

  13. Structural and Histone Binding Ability Characterizations of Human PWWP Domains

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Hong; Zeng, Hong; Lam, Robert; Tempel, Wolfram; Amaya, Maria F.; Xu, Chao; Dombrovski, Ludmila; Qiu, Wei; Wang, Yanming; Min, Jinrong (Toronto); (Penn)

    2013-09-25

    The PWWP domain was first identified as a structural motif of 100-130 amino acids in the WHSC1 protein and predicted to be a protein-protein interaction domain. It belongs to the Tudor domain 'Royal Family', which consists of Tudor, chromodomain, MBT and PWWP domains. While Tudor, chromodomain and MBT domains have long been known to bind methylated histones, PWWP was shown to exhibit histone binding ability only until recently. The PWWP domain has been shown to be a DNA binding domain, but sequence analysis and previous structural studies show that the PWWP domain exhibits significant similarity to other 'Royal Family' members, implying that the PWWP domain has the potential to bind histones. In order to further explore the function of the PWWP domain, we used the protein family approach to determine the crystal structures of the PWWP domains from seven different human proteins. Our fluorescence polarization binding studies show that PWWP domains have weak histone binding ability, which is also confirmed by our NMR titration experiments. Furthermore, we determined the crystal structures of the BRPF1 PWWP domain in complex with H3K36me3, and HDGF2 PWWP domain in complex with H3K79me3 and H4K20me3. PWWP proteins constitute a new family of methyl lysine histone binders. The PWWP domain consists of three motifs: a canonical {beta}-barrel core, an insertion motif between the second and third {beta}-strands and a C-terminal {alpha}-helix bundle. Both the canonical {beta}-barrel core and the insertion motif are directly involved in histone binding. The PWWP domain has been previously shown to be a DNA binding domain. Therefore, the PWWP domain exhibits dual functions: binding both DNA and methyllysine histones.

  14. Increased levels of IgG antibodies against human HSP60 in patients with spondyloarthritis

    DEFF Research Database (Denmark)

    Hjelholt, Astrid; Carlsen, Thomas Gelsing; Deleuran, Bent Winding

    2013-01-01

    Introduction: Spondyloarthritis (SpA) comprises a heterogeneous group of inflammatory diseases, with strong association to human leukocyte antigen (HLA)-B27. SpA is suggested triggered by bacterial infection, and bacterial heat shock protein (HSP) seems to be a strong T cell antigen. Since...... against human HSP60, but not antibodies against bacterial HSP60, were elevated in the SpA group compared with the control group. Association between IgG3 antibodies against human HSP60 and BASMI was shown in HLA-B27+ patients. Only weak correlation between antibodies against bacterial and human HSP60...... was seen, and there was no indication of cross-reaction. Conclusion: These results suggest that antibodies against human HSP60 is associated with SpA, however, the theory that antibodies against human HSP60 is a specific part of the aetiology, through cross-reaction to bacterial HSP60, cannot be supported...

  15. Extra-epitopic hepatitis C virus polymorphisms confer resistance to broadly neutralizing antibodies by modulating binding to scavenger receptor B1.

    Science.gov (United States)

    El-Diwany, Ramy; Cohen, Valerie J; Mankowski, Madeleine C; Wasilewski, Lisa N; Brady, Jillian K; Snider, Anna E; Osburn, William O; Murrell, Ben; Ray, Stuart C; Bailey, Justin R

    2017-02-01

    Broadly-neutralizing monoclonal antibodies (bNAbs) may guide vaccine development for highly variable viruses including hepatitis C virus (HCV), since they target conserved viral epitopes that could serve as vaccine antigens. However, HCV resistance to bNAbs could reduce the efficacy of a vaccine. HC33.4 and AR4A are two of the most potent anti-HCV human bNAbs characterized to date, binding to highly conserved epitopes near the amino- and carboxy-terminus of HCV envelope (E2) protein, respectively. Given their distinct epitopes, it was surprising that these bNAbs showed similar neutralization profiles across a panel of natural HCV isolates, suggesting that some viral polymorphisms may confer resistance to both bNAbs. To investigate this resistance, we developed a large, diverse panel of natural HCV envelope variants and a novel computational method to identify bNAb resistance polymorphisms in envelope proteins (E1 and E2). By measuring neutralization of a panel of HCV pseudoparticles by 10 μg/mL of each bNAb, we identified E1E2 variants with resistance to one or both bNAbs, despite 100% conservation of the AR4A binding epitope across the panel. We discovered polymorphisms outside of either binding epitope that modulate resistance to both bNAbs by altering E2 binding to the HCV co-receptor, scavenger receptor B1 (SR-B1). This study is focused on a mode of neutralization escape not addressed by conventional analysis of epitope conservation, highlighting the contribution of extra-epitopic polymorphisms to bNAb resistance and presenting a novel mechanism by which HCV might persist even in the face of an antibody response targeting multiple conserved epitopes.

  16. 92R Monoclonal Antibody Inhibits Human CCR9+ Leukemia Cells Growth in NSG Mice Xenografts.

    Science.gov (United States)

    Somovilla-Crespo, Beatriz; Martín Monzón, Maria Teresa; Vela, Maria; Corraliza-Gorjón, Isabel; Santamaria, Silvia; Garcia-Sanz, Jose A; Kremer, Leonor

    2018-01-01

    CCR9 is as an interesting target for the treatment of human CCR9 + -T cell acute lymphoblastic leukemia, since its expression is limited to immature cells in the thymus, infiltrating leukocytes in the small intestine and a small fraction of mature circulating T lymphocytes. 92R, a new mouse mAb (IgG2a isotype), was raised using the A-isoform of hCCR9 as immunogen. Its initial characterization demonstrates that binds with high affinity to the CCR9 N-terminal domain, competing with the previously described 91R mAb for receptor binding. 92R inhibits human CCR9 + tumor growth in T and B-cell deficient Rag2 -/- mice. In vitro assays suggested complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity as possible in vivo mechanisms of action. Unexpectedly, 92R strongly inhibited tumor growth also in a model with compromised NK and complement activities, suggesting that other mechanisms, including phagocytosis or apoptosis, might also be playing a role on 92R-mediated tumor elimination. Taken together, these data contribute to strengthen the hypothesis of the immune system's opportunistic nature.

  17. Structural basis for the binding of the neutralizing antibody, 7D11, to the poxvirus L1 protein

    International Nuclear Information System (INIS)

    Su, Hua-Poo; Golden, Joseph W.; Gittis, Apostolos G.; Hooper, Jay W.; Garboczi, David N.

    2007-01-01

    Medical countermeasures to prevent or treat smallpox are needed due to the potential use of poxviruses as biological weapons. Safety concerns with the currently available smallpox vaccine indicate a need for research on alternative poxvirus vaccine strategies. Molecular vaccines involving the use of proteins and/or genes and recombinant antibodies are among the strategies under current investigation. The poxvirus L1 protein, encoded by the L1R open reading frame, is the target of neutralizing antibodies and has been successfully used as a component of both protein subunit and DNA vaccines. L1-specific monoclonal antibodies (e.g., mouse monoclonal antibody mAb-7D11, mAb-10F5) with potent neutralizing activity bind L1 in a conformation-specific manner. This suggests that proper folding of the L1 protein used in molecular vaccines will affect the production of neutralizing antibodies and protection. Here, we co-crystallized the Fab fragment of mAb-7D11 with the L1 protein. The crystal structure of the complex between Fab-7D11 and L1 reveals the basis for the conformation-specific binding as recognition of a discontinuous epitope containing two loops that are held together by a disulfide bond. The structure of this important conformational epitope of L1 will contribute to the development of molecular poxvirus vaccines and also provides a novel target for anti-poxvirus drugs. In addition, the sequence and structure of Fab-7D11 will contribute to the development of L1-targeted immunotherapeutics

  18. Small GTP-binding proteins in human endothelial cells

    NARCIS (Netherlands)

    de Leeuw, H. P.; Koster, P. M.; Calafat, J.; Janssen, H.; van Zonneveld, A. J.; van Mourik, J. A.; Voorberg, J.

    1998-01-01

    Small GTP-binding proteins of the Ras superfamily control an extensive number of intracellular events by alternating between GDP- and GTP-bound conformation. The presence of members of this protein family was examined in human umbilical vein endothelial cells employing RT-PCR. Sequence analysis of

  19. Proteomic analysis of heparin-binding proteins from human seminal ...

    Indian Academy of Sciences (India)

    Prakash

    (MALDI TOF/MS) for protein analysis of human HBPs. We resolved 70 ... Thus, the combined effects of seminal plasma components support the survival of ...... The BBXB motif of RANTES is the principal site for heparin binding and controls ...

  20. Production and characterization of human anti-V3 monoclonal antibodies from the cells of HIV-1 infected Indian donors

    Directory of Open Access Journals (Sweden)

    Andrabi Raiees

    2012-09-01

    Full Text Available Abstract Background Analysis of human monoclonal antibodies (mAbs developed from HIV-1 infected donors have enormously contributed to the identification of neutralization sensitive epitopes on the HIV-1 envelope glycoprotein. The third variable region (V3 is a crucial target on gp120, primarily due to its involvement in co-receptor (CXCR4 or CCR5 binding and presence of epitopes recognized by broadly neutralizing antibodies. Methods Thirty-three HIV-1 seropositive drug naive patients (18 males and 15 females within the age range of 20–57 years (median = 33 years were recruited in this study for mAb production. The mAbs were selected from EBV transformed cultures with conformationally constrained Cholera-toxin-B containing V3C (V3C-CTB fusion protein. We tested the mAbs for their binding with HIV-1 derived proteins and peptides by ELISA and for neutralization against HIV-1 viruses by TZM-bl assays. Results We isolated three anti-V3 mAbs, 277, 903 and 904 from the cells of different individuals. The ELISA binding revealed a subtype-C and subtype-A specific binding of antibody 277 and 903 while mAb 904 exhibited cross reactivity also with subtype-B V3. Epitope mapping of mAbs with overlapping V3 peptides showed exclusive binding to V3 crown. The antibodies displayed high and low neutralizing activity against 2/5 tier 1 and 1/6 tier 2 viruses respectively. Overall, we observed a resistance of the tier 2 viruses to neutralization by the anti-V3 mAbs, despite the exposure of the epitopes recognized by these antibodies on two representative native viruses (Du156.12 and JRFL, suggesting that the affinity of mAb might equally be crucial for neutralization, as the epitope recognition. Conclusions Our study suggests that the anti-V3 antibodies derived from subtype-C infected Indian patients display neutralization potential against tier 1 viruses while such activity may be limited against more resistant tier 2 viruses. Defining the fine epitope

  1. Addressing the Immunogenicity of the Cargo and of the Targeting Antibodies with a Focus on Deimmunized Bacterial Toxins and on Antibody-Targeted Human Effector Proteins

    Science.gov (United States)

    Grinberg, Yehudit; Benhar, Itai

    2017-01-01

    Third-generation immunotoxins are composed of a human, or humanized, targeting moiety, usually a monoclonal antibody or an antibody fragment, and a non-human effector molecule. Due to the non-human origin of the cytotoxic domain, these molecules stimulate potent anti-drug immune responses, which limit treatment options. Efforts are made to deimmunize such immunotoxins or to combine treatment with immunosuppression. An alternative approach is using the so-called “human cytotoxic fusion proteins”, in which antibodies are used to target human effector proteins. Here, we present three relevant approaches for reducing the immunogenicity of antibody-targeted protein therapeutics: (1) reducing the immunogenicity of the bacterial toxin, (2) fusing human cytokines to antibodies to generate immunocytokines and (3) addressing the immunogenicity of the targeting antibodies. PMID:28574434

  2. Tumor necrosis factor-alpha binding capacity and anti-infliximab antibodies measured by fluid-phase radioimmunoassays as predictors of clinical efficacy of infliximab in Crohn's disease

    DEFF Research Database (Denmark)

    Ainsworth, Mark A; Bendtzen, Klaus; Brynskov, Jørn

    2007-01-01

    To investigate if the combined assessment of anti-infliximab antibodies (Ab) and the degree of TNF-alpha binding capacity (TNF-alpha-BC) afforded by infliximab may predict the response to infliximab treatment in patients with Crohn's disease (CD).......To investigate if the combined assessment of anti-infliximab antibodies (Ab) and the degree of TNF-alpha binding capacity (TNF-alpha-BC) afforded by infliximab may predict the response to infliximab treatment in patients with Crohn's disease (CD)....

  3. Expression of human protein S100A7 (psoriasin, preparation of antibody and application to human larynx squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    Barbieri Manuela R

    2011-11-01

    Full Text Available Abstract Background Up-regulation of S100A7 (Psoriasin, a small calcium-binding protein, is associated with the development of several types of carcinomas, but its function and possibility to serve as a diagnostic or prognostic marker have not been fully defined. In order to prepare antibodies to the protein for immunohistochemical studies we produced the recombinant S100A7 protein in E. coli. mRNA extracted from human tracheal tumor tissue which was amplified by RT-PCR to provide the region coding for the S100A7 gene. The amplified fragment was cloned in the vector pCR2.1-TOPO and sub-cloned in the expression vector pAE. The protein rS100A7 (His-tag was expressed in E. coli BL21::DE3, purified by affinity chromatography on an Ni-NTA column, recovered in the 2.0 to 3.5 mg/mL range in culture medium, and used to produce a rabbit polyclonal antibody anti-rS100A7 protein. The profile of this polyclonal antibody was evaluated in a tissue microarray. Results The rS100A7 (His-tag protein was homogeneous by SDS-PAGE and mass spectrometry and was used to produce an anti-recombinant S100A7 (His-tag rabbit serum (polyclonal antibody anti-rS100A7. The molecular weight of rS100A7 (His-tag protein determined by linear MALDI-TOF-MS was 12,655.91 Da. The theoretical mass calculated for the nonapeptide attached to the amino terminus is 12,653.26 Da (delta 2.65 Da. Immunostaining with the polyclonal anti-rS100A7 protein generated showed reactivity with little or no background staining in head and neck squamous cell carcinoma cells, detecting S100A7 both in nucleus and cytoplasm. Lower levels of S100A7 were detected in non-neoplastic tissue. Conclusions The polyclonal anti-rS100A7 antibody generated here yielded a good signal-to-noise contrast and should be useful for immunohistochemical detection of S100A7 protein. Its potential use for other epithelial lesions besides human larynx squamous cell carcinoma and non-neoplastic larynx should be explored in future.

  4. Chimeric Anti-Human Podoplanin Antibody NZ-12 of Lambda Light Chain Exerts Higher Antibody-Dependent Cellular Cytotoxicity and Complement-Dependent Cytotoxicity Compared with NZ-8 of Kappa Light Chain.

    Science.gov (United States)

    Kaneko, Mika K; Abe, Shinji; Ogasawara, Satoshi; Fujii, Yuki; Yamada, Shinji; Murata, Takeshi; Uchida, Hiroaki; Tahara, Hideaki; Nishioka, Yasuhiko; Kato, Yukinari

    2017-02-01

    Podoplanin (PDPN), a type I transmembrane 36-kDa glycoprotein, is expressed not only in normal cells, such as renal epithelial cells (podocytes), lymphatic endothelial cells, and pulmonary type I alveolar cells, but also in cancer cells, including brain tumors and lung squamous cell carcinomas. Podoplanin activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelets, and the podoplanin/CLEC-2 interaction facilitates blood/lymphatic vessel separation. We previously produced neutralizing anti-human podoplanin monoclonal antibody (mAb), clone NZ-1 (rat IgG 2a , lambda), which neutralizes the podoplanin/CLEC-2 interaction and inhibits platelet aggregation and cancer metastasis. Human-rat chimeric antibody, NZ-8, was previously developed using variable regions of NZ-1 and human constant regions of heavy chain (IgG 1 ) and light chain (kappa chain). Although NZ-8 showed high antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against human podoplanin-expressing cancer cells, the binding affinity of NZ-8 was lower than that of NZ-1. Herein, we produced a novel human-rat chimeric antibody, NZ-12, the constant regions of which consist of IgG 1 heavy chain and lambda light chain. Using flow cytometry, we demonstrated that the binding affinity of NZ-12 was much higher than that of NZ-8. Furthermore, ADCC and CDC activities of NZ-12 were significantly increased against glioblastoma cell lines (LN319 and D397) and lung cancer cell line (PC-10). These results suggested that NZ-12 could become a promising therapeutic antibody against podoplanin-expressing brain tumors and lung cancers.

  5. Monoclonal antibody OKM5 inhibits the in vitro binding of Plasmodium falciparum-infected erythrocytes to monocytes, endothelial, and C32 melanoma cells

    International Nuclear Information System (INIS)

    Barnwell, J.W.; Ockenhouse, C.F.; Knowles, D.M. II

    1985-01-01

    Plasmodium falciparum-infected erythrocytes bind in vitro to human endothelial cells, monocytes, and a certain melanoma cell line. Evidence suggests that this interaction is mediated by similar mechanisms which lead to the sequestration of parasitized erythrocytes in vivo through their attachment to endothelial cells of small blood vessels. They show here the monoclonal antibody OKM5, previously shown to react with the membranes of endothelial cells, monocyte,s and platelets, also reacts with the C32 melanoma cell line which also binds P. falciparum-infected erythrocytes. At relatively low concentrations, OKM5 inhibits and reverses the in vitro adherence of infected erythrocytes to target cells. As with monocytes, OKM5 antibody recognizes an 125 I-labeled protein of approximately 88 Kd on the surface of C32 melanoma cells. It seems likely, therefore, that the 88 Kd polypeptide plays a role in cytoadherence, possibly as the receptor or part of a receptor for a ligand on the surface of infected erythrocytes

  6. Monoclonal antibodies directed to human insulin-like growth factor I (IGF I)

    International Nuclear Information System (INIS)

    Laubli, U.K.; Baier, W.; Celio, M.R.; Binz, H.; Humbel, R.E.

    1982-01-01

    Mouse hybridomas secreting antibodies to human insulin-like growth factor I (IGF I) were produced by fusion of spleen cells of hyperimmunised mice with FO mouse-myeloma cells. Eight clones producing antibodies against human IGF I have been isolated, two of which have been characterised. One was used in a radioimmunoassay, the other for immunopurification of IGF. (Auth.)

  7. Binding of recombinant HIV coat protein gp120 to human monocytes

    International Nuclear Information System (INIS)

    Finbloom, D.S.; Hoover, D.L.; Meltzer, M.S.

    1991-01-01

    Inasmuch as the exact level of CD4 Ag expression on macrophages is controversial and because HIV may interact with macrophages in a manner different from that on T cells, we analyzed the binding of gp120 to freshly isolated and cultured monocytes. rgp120 was iodinated using the lactoperoxidase method to a sp. act. of 600 Ci/mmol. Highly purified monocytes (greater than 90%) were isolated from the leukapheresed blood of normal volunteers by Ficoll-Hypaque sedimentation followed by countercurrent centrifugal elutriation and cultured 7 days in DMEM supplemented with 1000 U/ml macrophage CSF in 10% human serum. Whereas MOLT/4 cells consistently bound freshly prepared 125I-rgp120 at 80% specificity with 5100 +/- 700 mol/cell, MCSF cultured monocytes bound rgp120 at only 0 to 20% specificity and 420 +/- 200 mol/cell. Most of the radioactivity bound by these cells could not be blocked by the addition of unlabeled rgp120. In contrast, the U937 myeloid cell line bound rgp120 with 50% specificity and about 2500 mol/cell. Whereas the antibody OKT4a (anti-CD4) blocked 80% of the binding on MOLT/4 cells and 50% on U937 cells, binding was only inhibited on the average of 6% on cultured monocytes. When soluble rCD4 was used as an inhibitor, binding to MOLT/4 cells was blocked by 80%. In contrast, binding to cultured monocytes was inhibited by 28%. HIV infectivity was blocked by similar concentrations of OKT4a. These observations suggest that although most binding of gp120 to cultured monocytes is not to the CD4 determinant, several hundred molecules do bind to a CD4-like molecule which promotes virus entry and replication

  8. Human Monoclonal Islet Cell Antibodies From a Patient with Insulin- Dependent Diabetes Mellitus Reveal Glutamate Decarboxylase as the Target Antigen

    Science.gov (United States)

    Richter, Wiltrud; Endl, Josef; Eiermann, Thomas H.; Brandt, Michael; Kientsch-Engel, Rosemarie; Thivolet, Charles; Jungfer, Herbert; Scherbaum, Werner A.

    1992-09-01

    The autoimmune phenomena associated with destruction of the β cell in pancreatic islets and development of type 1 (insulin-dependent) diabetes mellitus (IDDM) include circulating islet cell antibodies. We have immortalized peripheral blood lymphocytes from prediabetic individuals and patients with newly diagnosed IDDM by Epstein-Barr virus transformation. IgG-positive cells were selected by anti-human IgG-coupled magnetic beads and expanded in cell culture. Supernatants were screened for cytoplasmic islet cell antibodies using the conventional indirect immunofluorescence test on cryostat sections of human pancreas. Six islet cell-specific B-cell lines, originating from a patient with newly diagnosed IDDM, could be stabilized on a monoclonal level. All six monoclonal islet cell antibodies (MICA 1-6) were of the IgG class. None of the MICA reacted with human thyroid, adrenal gland, anterior pituitary, liver, lung, stomach, and intestine tissues but all six reacted with pancreatic islets of different mammalian species and, in addition, with neurons of rat cerebellar cortex. MICA 1-6 were shown to recognize four distinct antigenic epitopes in islets. Islet cell antibody-positive diabetic sera but not normal human sera blocked the binding of the monoclonal antibodies to their target epitopes. Immunoprecipitation of 35S-labeled human islet cell extracts revealed that a protein of identical size to the enzyme glutamate decarboxylase (EC 4.1.1.15) was a target of all MICA. Furthermore, antigen immunotrapped by the MICA from brain homogenates showed glutamate decarboxylase enzyme activity. MICA 1-6 therefore reveal glutamate decarboxylase as the predominant target antigen of cytoplasmic islet cell autoantibodies in a patient with newly diagnosed IDDM.

  9. Rapid isolation of antibody from a synthetic human antibody library by repeated fluorescence-activated cell sorting (FACS.

    Directory of Open Access Journals (Sweden)

    Sung Sun Yim

    Full Text Available Antibodies and their derivatives are the most important agents in therapeutics and diagnostics. Even after the significant progress in the technology for antibody screening from huge libraries, it takes a long time to isolate an antibody, which prevents a prompt action against the spread of a disease. Here, we report a new strategy for isolating desired antibodies from a combinatorial library in one day by repeated fluorescence-activated cell sorting (FACS. First, we constructed a library of synthetic human antibody in which single-chain variable fragment (scFv was expressed in the periplasm of Escherichia coli. After labeling the cells with fluorescent antigen probes, the highly fluorescent cells were sorted by using a high-speed cell sorter, and these cells were reused without regeneration in the next round of sorting. After repeating this sorting, the positive clones were completely enriched in several hours. Thus, we screened the library against three viral antigens, including the H1N1 influenza virus, Hepatitis B virus, and Foot-and-mouth disease virus. Finally, the potential antibody candidates, which show K(D values between 10 and 100 nM against the target antigens, could be successfully isolated even though the library was relatively small (∼ 10(6. These results show that repeated FACS screening without regeneration of the sorted cells can be a powerful method when a rapid response to a spreading disease is required.

  10. Monoclonal antibodies to human chorionic gonadotropin and their application to two-site sandwich radioimmunoassay

    International Nuclear Information System (INIS)

    Mizuchi, A.; Iio, M.; Miyachi, Y.

    1984-01-01

    Monoclonal antibodies were prepared against human chorionic gonadotropin (HCG). One monoclonal antibody recognized a conformational determinant expressed only on native HCG molecule and another monoclonal antibody had the specificity for the epitopes located on the β-subunit of HCG. Monoclonal antibodies reacting with different antigenic determinants on the HCG molecule were used to develop a simplified 2-site sandwich radioimmunoassay in which one monoclonal antibody was immobilized and another labeled with 125 iodine. This assay was highly specific for HCG and there was no cross-reactivity with α,β-subunit of HCG, luteinizing hormone and follicle stimulating hormone. (Auth.)

  11. 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.

  12. Use of thermodynamic coupling between antibody-antigen binding and phospholipid acyl chain phase transition energetics to predict immunoliposome targeting affinity.

    Science.gov (United States)

    Klegerman, Melvin E; Zou, Yuejiao; Golunski, Eva; Peng, Tao; Huang, Shao-Ling; McPherson, David D

    2014-09-01

    Thermodynamic analysis of ligand-target binding has been a useful tool for dissecting the nature of the binding mechanism and, therefore, potentially can provide valuable information regarding the utility of targeted formulations. Based on a consistent coupling of antibody-antigen binding and gel-liquid crystal transition energetics observed for antibody-phosphatidylethanolamine (Ab-PE) conjugates, we hypothesized that the thermodynamic parameters and the affinity for antigen of the Ab-PE conjugates could be effectively predicted once the corresponding information for the unconjugated antibody is determined. This hypothesis has now been tested in nine different antibody-targeted echogenic liposome (ELIP) preparations, where antibody is conjugated to dipalmitoylphosphatidylethanolamine (DPPE) head groups through a thioether linkage. Predictions were satisfactory (affinity not significantly different from the population of values found) in five cases (55.6%), but the affinity of the unconjugated antibody was not significantly different from the population of values found in six cases (66.7%), indicating that the affinities of the conjugated antibody tended not to deviate appreciably from those of the free antibody. While knowledge of the affinities of free antibodies may be sufficient to judge their suitability as targeting agents, thermodynamic analysis may still provide valuable information regarding their usefulness for specific applications.

  13. A natural human monoclonal antibody targeting Staphylococcus Protein A protects against Staphylococcus aureus bacteremia

    Science.gov (United States)

    Varshney, Avanish K.; Sunley, Kevin M.; Bowling, Rodney A.; Kwan, Tzu-Yu; Mays, Heather R.; Rambhadran, Anu; Zhang, Yanfeng; Martin, Rebecca L.; Cavalier, Michael C.; Simard, John

    2018-01-01

    Staphylococcus aureus can cause devastating and life-threatening infections. With the increase in multidrug resistant strains, novel therapies are needed. Limited success with active and passive immunization strategies have been attributed to S. aureus immune evasion. Here, we report on a monoclonal antibody, 514G3, that circumvents a key S. aureus evasion mechanism by targeting the cell wall moiety Protein A (SpA). SpA tightly binds most subclasses of immunoglobulins via their Fc region, neutralizing effector function. The organism can thus shield itself with a protective coat of serum antibodies and render humoral immunity ineffective. The present antibody reactivity was derived from an individual with natural anti-SpA antibody titers. The monoclonal antibody is of an IgG3 subclass, which differs critically from other immunoglobulin subclasses since its Fc is not bound by SpA. Moreover, it targets a unique epitope on SpA that allows it to bind in the presence of serum antibodies. Consequently, the antibody opsonizes S. aureus and maintains effector function to enable natural immune mediated clearance. The data presented here provide evidence that 514G3 antibody is able to successfully rescue mice from S. aureus mediated bacteremia. PMID:29364906

  14. Designing two-in-one antibodies.

    Science.gov (United States)

    Valladares, Ignacio Garcia; Espinoza, Luis R

    2009-09-01

    Evaluation of: Bostrom J, Shang-Fan Y, Kan D et al.: Variants of the antibody Herceptin that interact with HER2 and VEGF at the antigen binding site. Science 323, 1610-1614 (2009). The longstanding held notion that one antibody equals one antigen and, hence, one function has been challenged in recent years. Improved technology in antibody production, especially the accumulation of sequence data of immunoglobulin genes and the advent of PCR have made it possible to clone antibody gene repertoires. The current paper provides further challenge to the notion of one antibody = one antigen by developing 'two-in-one' antibodies with an antigen-binding site that binds two distinct proteins with high affinity. A therapeutic variant antibody of Herceptin (Genentech, CA, USA) was isolated that binds the human EGF receptor (HER)2 and also to VEGF. This development may represent a breakthrough discovery and may have significant implications in the therapy of malignant, infectious, allergic and autoimmune disorders.

  15. 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.

  16. Human broadly neutralizing antibodies to the envelope glycoprotein complex of hepatitis C virus

    DEFF Research Database (Denmark)

    Giang, Erick; Dorner, Marcus; Prentoe, Jannick C

    2012-01-01

    , and an effective vaccine should target conserved T- and B-cell epitopes of the virus. Conserved B-cell epitopes overlapping the CD81 receptor-binding site (CD81bs) on the E2 viral envelope glycoprotein have been reported previously and provide promising vaccine targets. In this study, we isolated 73 human m......Abs recognizing five distinct antigenic regions on the virus envelope glycoprotein complex E1E2 from an HCV-immune phage-display antibody library by using an exhaustive-panning strategy. Many of these mAbs were broadly neutralizing. In particular, the mAb AR4A, recognizing a discontinuous epitope outside the CD81......bs on the E1E2 complex, has an exceptionally broad neutralizing activity toward diverse HCV genotypes and protects against heterologous HCV challenge in a small animal model. The mAb panel will be useful for the design and development of vaccine candidates to elicit broadly neutralizing antibodies...

  17. Thermodynamic analysis of allosamidin binding to the human chitotriosidase

    Energy Technology Data Exchange (ETDEWEB)

    Eide, Kristine Bistrup; Lundmark, Silje Thoresen [Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P.O. Box 5003, N-1432 Ås (Norway); Sakuda, Shohei [Department of Applied Biological Chemistry, University of Tokyo, Bunkyo-Ku, Tokyo 113 (Japan); Sørlie, Morten, E-mail: morten.sorlie@umb.no [Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P.O. Box 5003, N-1432 Ås (Norway)

    2013-08-10

    Highlights: • Large differences in thermodynamic signatures for family 18 chitinase inhibition. • Allosamidin binds tight to HCHT. • Binding driven by enthalpy change and desolvation. - Abstract: Human chitotriosidase (HCHT) is one of two active family 18 chitinases produced by humans, the other being acidic mammalian chitinase (AMCase). The enzyme is thought to be part of the innate human defense mechanism against fungal parasites. Recently, it has been shown that levels of HCHT bioactivity and protein are significantly increased in the circulation and lungs of systemic sclerosis patients and for this reason is a suggested therapeutic target. For this reason, we have undertaken a detailed thermodynamic investigation using isothermal titration calorimetry of the binding interaction of HCHT with the well-known family 18 chitinase inhibitor allosamidin. The binding is shown to be strong (K{sub d} = 0.20 ± 0.03 μM and ΔG{sub r}° = −38.9 ± 0.4 kJ/mol) and driven by favorable changes in enthalpy (ΔH{sub r}° = −50.2 ± 1.2 kJ/mol) and solvation entropy (−TΔS{sub solv}° = −41.8 ± 4.4 kJ/mol). It is accompanied with a large penalty in conformational entropy change (−TΔS{sub conf}° = 43.1 ± 4.2 kJ/mol)

  18. Protein adsorption/desorption and antibody binding stoichiometry on silicon interferometric biosensors examined with TOF-SIMS

    KAUST Repository

    Gajos, Katarzyna

    2018-03-05

    Time-of-flight secondary ion mass spectrometry has been employed to examine, with biomolecular discrimination, sensing arm areas (20 μm x 600 μm) of integrated onto silicon chips Mach-Zehnder interferometers aiming to optimize their biofunctionalization with regard to indirect immunochemical (competitive) detection of ochratoxin A. Sensing areas are examined after: modification with (3-aminopropyl)triethoxysilane, spotting of OTA-ovalbumin conjugate (probe) from solutions with different concentration, blocking with bovine serum albumin, reaction with OTA-specific mouse monoclonal antibody followed by goat anti-mouse IgG secondary antibody. Component mass loadings of all proteins involved in immunodetection are determined from TOF-SIMS micro-analysis combined with ellipsometry of planar surfaces. These data show that partial desorption of surface-bound probe and blocking protein takes place upon primary immunoreaction to a degree that depends on probe concentration in spotting solution. Taking into account this desorption, apparent binding stoichiometry of both antibodies in immune complexes formed onto chip surface is determined more accurately than the respective evaluation based on real-time sensor response. In addition, mass loadings for probe and secondary antibody is observed to saturate for optimum probe concentrations. Also, principal component analysis of TOF-SIMS data could resolve both immunoreactions and biofunctionalization and discriminate surfaces prepared with optimum probe concentrations from those prepared using suboptimum ones.

  19. Protein adsorption/desorption and antibody binding stoichiometry on silicon interferometric biosensors examined with TOF-SIMS

    KAUST Repository

    Gajos, Katarzyna; Budkowski, Andrzej; Petrou, Panagiota; Pagkali, Varvara; Awsiuk, Kamil; Rysz, Jakub; Bernasik, Andrzej; Misiakos, Konstantinos; Raptis, Ioannis; Kakabakos, Sotirios

    2018-01-01

    Time-of-flight secondary ion mass spectrometry has been employed to examine, with biomolecular discrimination, sensing arm areas (20 μm x 600 μm) of integrated onto silicon chips Mach-Zehnder interferometers aiming to optimize their biofunctionalization with regard to indirect immunochemical (competitive) detection of ochratoxin A. Sensing areas are examined after: modification with (3-aminopropyl)triethoxysilane, spotting of OTA-ovalbumin conjugate (probe) from solutions with different concentration, blocking with bovine serum albumin, reaction with OTA-specific mouse monoclonal antibody followed by goat anti-mouse IgG secondary antibody. Component mass loadings of all proteins involved in immunodetection are determined from TOF-SIMS micro-analysis combined with ellipsometry of planar surfaces. These data show that partial desorption of surface-bound probe and blocking protein takes place upon primary immunoreaction to a degree that depends on probe concentration in spotting solution. Taking into account this desorption, apparent binding stoichiometry of both antibodies in immune complexes formed onto chip surface is determined more accurately than the respective evaluation based on real-time sensor response. In addition, mass loadings for probe and secondary antibody is observed to saturate for optimum probe concentrations. Also, principal component analysis of TOF-SIMS data could resolve both immunoreactions and biofunctionalization and discriminate surfaces prepared with optimum probe concentrations from those prepared using suboptimum ones.

  20. Protein adsorption/desorption and antibody binding stoichiometry on silicon interferometric biosensors examined with TOF-SIMS

    Science.gov (United States)

    Gajos, Katarzyna; Budkowski, Andrzej; Petrou, Panagiota; Pagkali, Varvara; Awsiuk, Kamil; Rysz, Jakub; Bernasik, Andrzej; Misiakos, Konstantinos; Raptis, Ioannis; Kakabakos, Sotirios

    2018-06-01

    Time-of-flight secondary ion mass spectrometry has been employed to examine, with biomolecular discrimination, sensing arm areas (20 μm × 600 μm) of integrated onto silicon chips Mach-Zehnder interferometers aiming to optimize their biofunctionalization with regard to indirect immunochemical (competitive) detection of ochratoxin A. Sensing areas are examined after: modification with (3-aminopropyl)triethoxysilane, spotting of OTA-ovalbumin conjugate (probe) from solutions with different concentration, blocking with bovine serum albumin, reaction with OTA-specific mouse monoclonal antibody followed by goat anti-mouse IgG secondary antibody. Component mass loadings of all proteins involved in immunodetection are determined from TOF-SIMS micro-analysis combined with ellipsometry of planar surfaces. These data show that partial desorption of surface-bound probe and blocking protein takes place upon primary immunoreaction to a degree that depends on probe concentration in spotting solution. Taking into account this desorption, apparent binding stoichiometry of both antibodies in immune complexes formed onto chip surface is determined more accurately than the respective evaluation based on real-time sensor response. In addition, mass loadings for probe and secondary antibody is observed to saturate for optimum probe concentrations. Also, principal component analysis of TOF-SIMS data could resolve both immunoreactions and biofunctionalization and discriminate surfaces prepared with optimum probe concentrations from those prepared using suboptimum ones.

  1. Vaccine-Elicited Tier 2 HIV-1 Neutralizing Antibodies Bind to Quaternary Epitopes Involving Glycan-Deficient Patches Proximal to the CD4 Binding Site.

    Directory of Open Access Journals (Sweden)

    Ema T Crooks

    2015-05-01

    Full Text Available Eliciting broad tier 2 neutralizing antibodies (nAbs is a major goal of HIV-1 vaccine research. Here we investigated the ability of native, membrane-expressed JR-FL Env trimers to elicit nAbs. Unusually potent nAb titers developed in 2 of 8 rabbits immunized with virus-like particles (VLPs expressing trimers (trimer VLP sera and in 1 of 20 rabbits immunized with DNA expressing native Env trimer, followed by a protein boost (DNA trimer sera. All 3 sera neutralized via quaternary epitopes and exploited natural gaps in the glycan defenses of the second conserved region of JR-FL gp120. Specifically, trimer VLP sera took advantage of the unusual absence of a glycan at residue 197 (present in 98.7% of Envs. Intriguingly, removing the N197 glycan (with no loss of tier 2 phenotype rendered 50% or 16.7% (n = 18 of clade B tier 2 isolates sensitive to the two trimer VLP sera, showing broad neutralization via the surface masked by the N197 glycan. Neutralizing sera targeted epitopes that overlap with the CD4 binding site, consistent with the role of the N197 glycan in a putative "glycan fence" that limits access to this region. A bioinformatics analysis suggested shared features of one of the trimer VLP sera and monoclonal antibody PG9, consistent with its trimer-dependency. The neutralizing DNA trimer serum took advantage of the absence of a glycan at residue 230, also proximal to the CD4 binding site and suggesting an epitope similar to that of monoclonal antibody 8ANC195, albeit lacking tier 2 breadth. Taken together, our data show for the first time that strain-specific holes in the glycan fence can allow the development of tier 2 neutralizing antibodies to native spikes. Moreover, cross-neutralization can occur in the absence of protecting glycan. Overall, our observations provide new insights that may inform the future development of a neutralizing antibody vaccine.

  2. A single point in protein trafficking by Plasmodium falciparum determines the expression of major antigens on the surface of infected erythrocytes targeted by human antibodies.

    Science.gov (United States)

    Chan, Jo-Anne; Howell, Katherine B; Langer, Christine; Maier, Alexander G; Hasang, Wina; Rogerson, Stephen J; Petter, Michaela; Chesson, Joanne; Stanisic, Danielle I; Duffy, Michael F; Cooke, Brian M; Siba, Peter M; Mueller, Ivo; Bull, Peter C; Marsh, Kevin; Fowkes, Freya J I; Beeson, James G

    2016-11-01

    Antibodies to blood-stage antigens of Plasmodium falciparum play a pivotal role in human immunity to malaria. During parasite development, multiple proteins are trafficked from the intracellular parasite to the surface of P. falciparum-infected erythrocytes (IEs). However, the relative importance of different proteins as targets of acquired antibodies, and key pathways involved in trafficking major antigens remain to be clearly defined. We quantified antibodies to surface antigens among children, adults, and pregnant women from different malaria-exposed regions. We quantified the importance of antigens as antibody targets using genetically engineered P. falciparum with modified surface antigen expression. Genetic deletion of the trafficking protein skeleton-binding protein-1 (SBP1), which is involved in trafficking the surface antigen PfEMP1, led to a dramatic reduction in antibody recognition of IEs and the ability of human antibodies to promote opsonic phagocytosis of IEs, a key mechanism of parasite clearance. The great majority of antibody epitopes on the IE surface were SBP1-dependent. This was demonstrated using parasite isolates with different genetic or phenotypic backgrounds, and among antibodies from children, adults, and pregnant women in different populations. Comparisons of antibody reactivity to parasite isolates with SBP1 deletion or inhibited PfEMP1 expression suggest that PfEMP1 is the dominant target of acquired human antibodies, and that other P. falciparum IE surface proteins are minor targets. These results establish SBP1 as part of a critical pathway for the trafficking of major surface antigens targeted by human immunity, and have key implications for vaccine development, and quantifying immunity in populations.

  3. Neisseria meningitidis and Escherichia coli are protected from leukocyte phagocytosis by binding to erythrocyte complement receptor 1 in human blood

    DEFF Research Database (Denmark)

    Brekke, O. L.; Hellerud, B. C.; Christiansen, D.

    2011-01-01

    The initial interaction of Gram-negative bacteria with erythrocytes and its implications on leukocyte phagocytosis and oxidative burst in human whole blood were examined. Alexa-labeled Escherichia coli, wild-type H44/76 N. meningitidis and the H44/76lpxA lipopolysaccharide (LPS)-deficient mutant...... antagonist (C5aRa) and a complement receptor 1 (CR1)-blocking antibody (3D9) were examined. Most bacteria (80%) immediately bound to erythrocytes. The binding gradually declined over time, with a parallel increase in phagocytosis. Complement inhibition with compstatin reduced erythrocyte binding...... and bacterial C3 opsonization. In contrast, the C5aRa efficiently reduced phagocytosis, but did not affect the binding of bacteria to erythrocytes. The anti-CR1 blocking mAb dose-dependently reduced bacterial binding to erythrocytes to nil, with subsequent increased phagocytosis and oxidative burst. LPS had...

  4. Novel Prostate Specific Antigen plastic antibody designed with charged binding sites for an improved protein binding and its application in a biosensor of potentiometric transduction

    International Nuclear Information System (INIS)

    Rebelo, Tânia S.C.R.; Santos, C.; Costa-Rodrigues, J.; Fernandes, M.H.; Noronha, João P.; Sales, M. Goreti F.

    2014-01-01

    Graphical abstract: EF13-201, Novel Prostate Specific Antigen plastic antibody designed with charged binding sites for an improved protein binding and its application in a biosensor of potentiometric transduction. - Abstract: This work shows that the synthesis of protein plastic antibodies tailored with selected charged monomers around the binding site enhances protein binding. These charged receptor sites are placed over a neutral polymeric matrix, thus inducing a suitable orientation the protein reception to its site. This is confirmed by preparing control materials with neutral monomers and also with non-imprinted template. This concept has been applied here to Prostate Specific Antigen (PSA), the protein of choice for screening prostate cancer throughout the population, with serum levels >10 ng/mL pointing out a high probability of associated cancer. Protein Imprinted Materials with charged binding sites (C/PIM) have been produced by surface imprinting over graphene layers to which the protein was first covalently attached. Vinylbenzyl(trimethylammonium chloride) and vinyl benzoate were introduced as charged monomers labelling the binding site and were allowed to self-organize around the protein. The subsequent polymerization was made by radical polymerization of vinylbenzene. Neutral PIM (N/PIM) prepared without oriented charges and non imprinted materials (NIM) obtained without template were used as controls. These materials were used to develop simple and inexpensive potentiometric sensor for PSA. They were included as ionophores in plasticized PVC membranes, and tested over electrodes of solid or liquid conductive contacts, made of conductive carbon over a syringe or of inner reference solution over micropipette tips. The electrodes with charged monomers showed a more stable and sensitive response, with an average slope of -44.2 mV/decade and a detection limit of 5.8 × 10 −11 mol/L (2 ng/mL). The corresponding non-imprinted sensors showed lower

  5. Isolation of Panels of Llama Single-Domain Antibody Fragments Binding All Nine Neuraminidase Subtypes of Influenza A Virus

    Directory of Open Access Journals (Sweden)

    Guus Koch

    2013-04-01

    Full Text Available Avian influenza A virus comprises sixteen hemagglutinin (HA and nine neuraminidase (NA subtypes (N1–N9. To isolate llama single-domain antibody fragments (VHHs against all N subtypes, four llamas were immunized with mixtures of influenza viruses. Selections using influenza virus yielded predominantly VHHs binding to the highly immunogenic HA and nucleoprotein. However, selection using enzymatically active recombinant NA (rNA protein enabled us to isolate NA binding VHHs. Some isolated VHHs cross-reacted to other N subtypes. These were subsequently used for the capture of N subtypes that could not be produced as recombinant protein (rN6 or were enzymatically inactive (rN1, rN5 in phage display selection, yielding novel VHHs. In total we isolated 188 NA binding VHHs, 64 of which were expressed in yeast. Most VHHs specifically recognize a single N subtype, but some VHHs cross-react with other N-subtypes. At least one VHH bound to all N subtypes, except N4, identifying a conserved antigenic site. Thus, this work (1 describes methods for isolating NA binding VHHs, (2 illustrates the suitability of llama immunization with multiple antigens for retrieving many binders against different antigens and (3 describes 64 novel NA binding VHHs, including a broadly reactive VHH, which can be used in various assays for influenza virus subtyping, detection or serology.

  6. A three-layer immunoradiometric assay for determination of IgG subclass antibodies in Human Sera (''IgG subclass RAST'')

    International Nuclear Information System (INIS)

    Djurup, R.; Soendergaard, I.; Weeke, B.; University of Copenhagen, Denmark); Magnusson, C.G.M.

    1984-01-01

    We report the development of a three-layer immunoradiometric assay (TIRA) for measurement of IgG antibodies of all four subclasses in human sera. The first layer consists of diluted human serum, the second layer is monoclonal mouse antibodies to human IgG subclasses, and the third layer is 125 I-labelled rabbit anti-mouse IgG. Monoclonal anti-IgGI, anti-IgG3 and anti-IgG4 reacted only with their complementary IgG subclass, whereas the anti-IgG2 showed slight cross-reactivity to immunoglobins of other subclasses and classes and to light chain proteins. The observed cross-reactivity was found to be without importance, when the TIRA was applied to measurement of IgG subclass antibodies. Equipotency was established by use of appropriate dilutions of the monoclonal antibodies, and the assay was calibrated by use of human reference serum. The TIRA therefore permits reliable inter-individual and intra-individual comparisons of the IgG antibody response in all four subclasses. Non-specific binding obtained with pooled normal human serum was below 0.33%. Inter-assay coefficient of variation was between 18 and 27%. The TIRA was applied to measurement of IgG subclass antibodies to timothy grass pollen in sera from grass pollen allergies undergoing immunotherapy. (author)

  7. Serum protein inhibition of thyrotropin binding to human thyroid tissue

    International Nuclear Information System (INIS)

    Beall, G.N.; Chopra, I.J.; Solomon, D.H.; Kruger, S.R.

    1978-01-01

    We used a modificaton of the TSH radioreceptor assay to detect TSH-binding inhibition (TBI) activity in serum and serum fractions from normal subjects and patients with Graves' disease. TBI activity is present in normal IgG prepared by DEAE-Sephadex chromatography and in normal globulins prepared by precipitation at 1.6 M ammonium sulfate. Other normal serum proteins also had TBI activity when large concentrations were tested. Gel filtration chromatography and powder block electrophoresis were used to prepare fractions of normal and Graves' disease sera. In these fractions from normal serum, TBI activity was found in both γ-globulin and α-globulin-albumin fractions electrophoretically and in both 7S and 4S peaks from gel filtration. TBI activity from Graves' disease patients' sera was similarly distributed, but relatively more TBI accompanied the electrophoretic γ-globulins. Sepharose Protein-A and anti-IgG were used as immunoabsorbents to isolate and purify IgG from normal and Graves' disease sera. TBI activity in IgG was proportional to the IgG concentration, indicating that the TBI which migrates as a γ-globulin electrophoretically is an IgG and thus may possibly be an antibody. Inhibitory activity found in normal serum globulins and in the non-IgG fractions of both normal and abnormal sera seriously interferes with attempts to use the TSH radioreceptor assay to study the hypothesized anti-TSH receptor antibody in the serum of patients with Graves' disease

  8. Human pentraxin 3 binds to the complement regulator c4b-binding protein.

    Directory of Open Access Journals (Sweden)

    Anne Braunschweig

    Full Text Available The long pentraxin 3 (PTX3 is a soluble recognition molecule with multiple functions including innate immune defense against certain microbes and the clearance of apoptotic cells. PTX3 interacts with recognition molecules of the classical and lectin complement pathways and thus initiates complement activation. In addition, binding of PTX3 to the alternative complement pathway regulator factor H was shown. Here, we show that PTX3 binds to the classical and lectin pathway regulator C4b-binding protein (C4BP. A PTX3-binding site was identified within short consensus repeats 1-3 of the C4BP α-chain. PTX3 did not interfere with the cofactor activity of C4BP in the fluid phase and C4BP maintained its complement regulatory activity when bound to PTX3 on surfaces. While C4BP and factor H did not compete for PTX3 binding, the interaction of C4BP with PTX3 was inhibited by C1q and by L-ficolin. PTX3 bound to human fibroblast- and endothelial cell-derived extracellular matrices and recruited functionally active C4BP to these surfaces. Whereas PTX3 enhanced the activation of the classical/lectin pathway and caused enhanced C3 deposition on extracellular matrix, deposition of terminal pathway components and the generation of the inflammatory mediator C5a were not increased. Furthermore, PTX3 enhanced the binding of C4BP to late apoptotic cells, which resulted in an increased rate of inactivation of cell surface bound C4b and a reduction in the deposition of C5b-9. Thus, in addition to complement activators, PTX3 interacts with complement inhibitors including C4BP. This balanced interaction on extracellular matrix and on apoptotic cells may prevent excessive local complement activation that would otherwise lead to inflammation and host tissue damage.

  9. A review of human anti-globulin antibody (HAGA, HAMA, HACA, HAHA) responses to monoclonal antibodies. Not four letter words.

    Science.gov (United States)

    Mirick, G R; Bradt, B M; Denardo, S J; Denardo, G L

    2004-12-01

    The United States Food and Drug Administration (FDA) has approved unconjugated monoclonal antibodies (MAbs) for immunotherapy (IT) of B-cell lymphoma, breast cancer and acute myeloid leukemia. More recently, approval has been given for conjugated ZevalinTM ((90)yttrium ibritumomab tiuxetan, IDEC-Y2B8, Biogen Idec, Cambridge, MA) and BexxarTM ((131)I-tositumomab, Corixa, Corp., Seattle, WA and GlaxoSmithKline, Philadelphia, PA) anti-CD20 MAbs for use in radioimmunotherapy (RIT) of non-Hodgkin's lymphoma (NHL), thus redefining the standard care of cancer patients. Because of, and despite a lack of basis for concern about allergic reactions due to human antibody responses to these foreign proteins, assays were developed to determine HAGA (human anti-globulin antibody) levels that developed in patient sera following treatment with MAbs. Strategies were also devised to ''humanize'' MAbs and to temporarily block patient immune function with drugs in order to decrease the seroconversion rates, with considerable success. On the other hand, a survival advantage has been observed in some patients who developed a HAGA following treatment. This correlates with development of an anti-idiotype antibody cascade directed toward the MAbs used to treat these patients. What follows is a selective review of HAGA and its effect on cancer treatment over the past 2 decades.

  10. A review of human anti-globulin antibody (HAGA, HAMA, HACA, HAHA) responses to monoclonal antibodies. Not four letter words

    International Nuclear Information System (INIS)

    Mirick, G. R.; Bradt, B. M.; Denardo, S. J.; Denardo, G. L.

    2004-01-01

    The United States Food and Drugs Administration (FDA) has approved unconjugated monoclonal antibodies (MAbs) for immunotherapy (IT) of B-cell lymphoma, breast cancer and acute myeloid leukemia. More recently, approval has been given for conjugated ZevalinTM ( 9 0yttrium ibritumomab tiuxetan, IDEC-Y2B8, Biogen Idec, Cambridge, MA) and BexxarTM ( 1 31I-tositumomab, Corixa, Corp., Seattle, WA and GlaxoSmithKline, Philadelphia, PA) antiCD20 MAns for use in radioimmunotherapy (RIT) of non-Hodgikin's lymphoma (NHL), thus redefining the standard care of cancer patients. Because of, and despite a lack of basis for concern about allergic reactions due to human antibody responses to these foreign proteins, essays were developed to determine HAGE (human anti-globulin antibody) levels that developed in patient sera following treatment with MAbs. Strategies were also devised to humanize MAbs and to temporarily block patient immune function with drugs in order to decrease the seroconversion rates, with considerable success. On the other hand, a survival advantage has been observed in some patients who developed a HAGA following treatment. This correlates with development of an anti-idiotype antibody cascade directed toward the MAbs used to treat these patients. What follows is a selective review of HAGA and its effect on cancer treatment over the past 2 decades

  11. A review of human anti-globulin antibody (HAGA, HAMA, HACA, HAHA) responses to monoclonal antibodies. Not four letter words

    Energy Technology Data Exchange (ETDEWEB)

    Mirick, G. R.; Bradt, B. M.; Denardo, S. J.; Denardo, G. L. [Calfornia Univ., Sacramento (United States). Davis Medical Center

    2004-12-01

    The United States Food and Drugs Administration (FDA) has approved unconjugated monoclonal antibodies (MAbs) for immunotherapy (IT) of B-cell lymphoma, breast cancer and acute myeloid leukemia. More recently, approval has been given for conjugated ZevalinTM ({sup 9}0yttrium ibritumomab tiuxetan, IDEC-Y2B8, Biogen Idec, Cambridge, MA) and BexxarTM ({sup 1}31I-tositumomab, Corixa, Corp., Seattle, WA and GlaxoSmithKline, Philadelphia, PA) antiCD20 MAns for use in radioimmunotherapy (RIT) of non-Hodgikin's lymphoma (NHL), thus redefining the standard care of cancer patients. Because of, and despite a lack of basis for concern about allergic reactions due to human antibody responses to these foreign proteins, essays were developed to determine HAGE (human anti-globulin antibody) levels that developed in patient sera following treatment with MAbs. Strategies were also devised to humanize MAbs and to temporarily block patient immune function with drugs in order to decrease the seroconversion rates, with considerable success. On the other hand, a survival advantage has been observed in some patients who developed a HAGA following treatment. This correlates with development of an anti-idiotype antibody cascade directed toward the MAbs used to treat these patients. What follows is a selective review of HAGA and its effect on cancer treatment over the past 2 decades.

  12. [Construction of the lentiviral expression vector for anti-p185(erbB2) mouse/human chimeric antibody].

    Science.gov (United States)

    Liu, Fang; Li, Li; Zhang, Wei; Wang, Qi

    2013-04-01

    This research was to construct the lentiviral expression vector for anti- p185(erbB2) mouse/human chimeric antibody and to determine the expression of the chimeric antibody gene in 293T cells transfected with this vector. The genes (vL and vH) coding light and heavy chain of variable regions of anti-p185(erbB2) mAb and the constant regions of human IgG1 (kappa and gamma1) were cloned with PCR method. The target genes were assembled by three-primers PCR method to obtain the chimeric light chain (L) and the chimeric heavy chain (H). Both chains inserted into the down stream and upper stream of IRES gene of the plasmid pVAX1/IRES respectively. We digested the plasmid pVAX1/ H-IRES-L with endoenzyme and subcloned H-IRES-L into the lentiviral vector pWPI. The enzyme digestion and sequence analysis showed that the lentiviral expression vector pWPI/H-IRES-L was constructed correctly. Then, it was transfected into 293T cells and after 48h, GFP protein expression in 293T cells were detected by fluorescent microscope and the chimeric antibody expression was detected by RT-PCR and direct ELISA. The results showed that after 293T cells were transfected with recombination plasmid, both light and heavy chains of the chimeric antibody genes could express together. The chimeric antibody expressed could bind to p185(erbB2) specifically. This research may lay a sound foundation for further study of anti-p185(erbB2) engineered antibody.

  13. Imparting albumin-binding affinity to a human protein by mimicking the contact surface of a bacterial binding protein.

    Science.gov (United States)

    Oshiro, Satoshi; Honda, Shinya

    2014-04-18

    Attachment of a bacterial albumin-binding protein module is an attractive strategy for extending the plasma residence time of protein therapeutics. However, a protein fused with such a bacterial module could induce unfavorable immune reactions. To address this, we designed an alternative binding protein by imparting albumin-binding affinity to a human protein using molecular surface grafting. The result was a series of human-derived 6 helix-bundle proteins, one of which specifically binds to human serum albumin (HSA) with adequate affinity (KD = 100 nM). The proteins were designed by transferring key binding residues of a bacterial albumin-binding module, Finegoldia magna protein G-related albumin-binding domain (GA) module, onto the human protein scaffold. Despite 13-15 mutations, the designed proteins maintain the original secondary structure by virtue of careful grafting based on structural informatics. Competitive binding assays and thermodynamic analyses of the best binders show that the binding mode resembles that of the GA module, suggesting that the contacting surface of the GA module is mimicked well on the designed protein. These results indicate that the designed protein may act as an alternative low-risk binding module to HSA. Furthermore, molecular surface grafting in combination with structural informatics is an effective approach for avoiding deleterious mutations on a target protein and for imparting the binding function of one protein onto another.

  14. Postbooster Antibodies from Humans as Source of Diphtheria Antitoxin.

    Science.gov (United States)

    Bermejo-Martin, Jesús F; Avila-Alonso, Ana; González-Rivera, Milagros; Tamayo, Eduardo; Eiros, Jose María; Almansa, Raquel

    2016-07-01

    Diphtheria antitoxin for therapeutic use is in limited supply. A potential source might be affinity-purified antibodies originally derived from plasma of adults who received a booster dose of a vaccine containing diphtheria toxoid. These antibodies might be useful for treating even severe cases of diphtheria.

  15. Anti-influenza Hyperimmune Immunoglobulin Enhances Fc-functional Antibody Immunity during Human Influenza Infection.

    Science.gov (United States)

    Vanderven, Hillary A; Wragg, Kathleen; Ana-Sosa-Batiz, Fernanda; Kristensen, Anne B; Jegaskanda, Sinthujan; Wheatley, Adam K; Wentworth, Deborah; Wines, Bruce D; Hogarth, P Mark; Rockman, Steve; Kent, Stephen J

    2018-05-31

    New treatments for severe influenza are needed. Passive transfer of influenza-specific hyperimmune pooled immunoglobulin (Flu-IVIG) boosts neutralising antibody responses to past strains in influenza-infected subjects. The effect of Flu-IVIG on antibodies with Fc-mediated functions, which may target diverse influenza strains, is unclear. We studied the capacity of Flu-IVIG, relative to standard IVIG, to bind to Fc receptors and mediate antibody-dependent cellular cytotoxicity in vitro. The effect of Flu-IVIG infusion, compared to placebo infusion, was examined in serial plasma samples from 24 subjects with confirmed influenza infection in the INSIGHT FLU005 pilot study. Flu-IVIG contains higher concentrations of Fc-functional antibodies than IVIG against a diverse range of influenza hemagglutinins. Following infusion of Flu-IVIG into influenza-infected subjects, a transient increase in Fc-functional antibodies was present for 1-3 days against infecting and non-infecting strains of influenza. Flu-IVIG contains antibodies with Fc-mediated functions against influenza virus and passive transfer of Flu-IVIG increases anti-influenza Fc-functional antibodies in the plasma of influenza-infected subjects. Enhancement of Fc-functional antibodies to a diverse range of influenza strains suggests that Flu-IVIG infusion could prove useful in the context of novel influenza virus infections, when there may be minimal or no neutralising antibodies in the Flu-IVIG preparation.

  16. Immunochemical identification of human trophoblast membrane antigens using monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Brown, P J; Molloy, C M; Johnson, P M [Liverpool Univ. (UK). Dept. of Immunology

    1983-11-01

    Human trophoblast membrane antigens recognised by monoclonal antibodies (H310, H315, H316 and H317) have been identified using combinations of radioimmunoprecipitation, SDS-PAGE, electroblotting, chromatographic and ELISA-type techniques. H317 is known to identify heat-stable placental-type alkaline phosphatase and accordingly was shown to react with a protein of subunit Msub(r) of 68000. H310 and H316 both recognise an antigen with a subunit Msub(r) of 34000 under reducing conditions. In non-reducing conditions, the H310/316 antigen gave oligomers of a component of Msub(r) 62000. It is unknown whether this 62000 dalton component is a dimer of the 34000 dalton protein with either itself or a second protein chain of presumed Msub(r) around 28000. H315 recognises an antigen with subunit Msub(r) of 36000; in non-reducing conditions this component readily associates to oligomeric structures. The epitope recognised by H315 may be sensitive to SDS. The two proteins recognised by H310/316 and H315 have been termed the p34 and p36 trophoblast membrane proteins, respectively.

  17. Human antiiodothyronine antibodies in patients with thyroid disorders and their effect on RIA of Iodothyronines

    International Nuclear Information System (INIS)

    Merlin, P.; Balsamo, A.; Mongardi, L.; Rapetti, C.; de Filippis, V.

    1983-01-01

    Human antiiodothyronine antibodies have been reported to occur with several thyroid conditions, associated or not with anti-thyroglobulin and/or anti-microsomes antibodies. These antibodies interfere in RIA of iodothyronines (T 3 ), giving an underestimation or an overestimation of total hormone levels when using a non-specific precipitation method (e.g. charcoal, PEG) or a specific method (e.g. double antibody), respectively. The presence of anti-iodothyronine antibodies was investigated in seven thyroid patients. The effect of the human anti-T 3 in RIA of total T 3 was ckecked by using different precipitation methods; the results showed that in the presence of circulating antibodies the only reliable method for the evaluation of total hormone is the RIA of serum ethanol extract

  18. A recombinant dromedary antibody fragment (VHH or nanobody) directed against human Duffy antigen receptor for chemokines.

    Science.gov (United States)

    Smolarek, Dorota; Hattab, Claude; Hassanzadeh-Ghassabeh, Gholamreza; Cochet, Sylvie; Gutiérrez, Carlos; de Brevern, Alexandre G; Udomsangpetch, Rachanee; Picot, Julien; Grodecka, Magdalena; Wasniowska, Kazimiera; Muyldermans, Serge; Colin, Yves; Le Van Kim, Caroline; Czerwinski, Marcin; Bertrand, Olivier

    2010-10-01

    Fy blood group antigens are carried by the Duffy antigen receptor for chemokines (DARC), a red cells receptor for Plasmodium vivax broadly implicated in human health and diseases. Recombinant VHHs, or nanobodies, the smallest intact antigen binding fragment derivative from the heavy chain-only antibodies present in camelids, were prepared from a dromedary immunized against DARC N-terminal extracellular domain and selected for DARC binding. A described VHH, CA52, does recognize native DARC on cells. It inhibits P. vivax invasion of erythrocytes and displaces interleukin-8 bound to DARC. The targeted epitope overlaps the well-defined DARC Fy6 epitope. K (D) of CA52-DARC equilibrium is sub-nanomolar, hence ideal to develop diagnostic or therapeutic compounds. Immunocapture by immobilized CA52 yielded highly purified DARC from engineered K562 cells. This first report on a VHH with specificity for a red blood cell protein exemplifies VHHs' potentialities to target, to purify, and to modulate the function of cellular markers.

  19. Directed Selection of Recombinant Human Monoclonal Antibodies to Herpes Simplex Virus Glycoproteins from Phage Display Libraries

    Science.gov (United States)

    Sanna, Pietro Paolo; Williamson, R. Anthony; de Logu, Alessandro; Bloom, Floyd E.; Burton, Dennis R.

    1995-07-01

    Human monoclonal antibodies have considerable potential in the prophylaxis and treatment of viral disease. However, only a few such antibodies suitable for clinical use have been produced to date. We have previously shown that large panels of human recombinant monoclonal antibodies against a plethora of infectious agents, including herpes simplex virus types 1 and 2, can be established from phage display libraries. Here we demonstrate that facile cloning of recombinant Fab fragments against specific viral proteins in their native conformation can be accomplished by panning phage display libraries against viral glycoproteins "captured" from infected cell extracts by specific monoclonal antibodies immobilized on ELISA plates. We have tested this strategy by isolating six neutralizing recombinant antibodies specific for herpes simplex glycoprotein gD or gB, some of which are against conformationally sensitive epitopes. By using defined monoclonal antibodies for the antigen-capture step, this method can be used for the isolation of antibodies to specific regions and epitopes within the target viral protein. For instance, monoclonal antibodies to a nonneutralizing epitope can be used in the capture step to clone antibodies to neutralizing epitopes, or antibodies to a neutralizing epitope can be used to clone antibodies to a different neutralizing epitope. Furthermore, by using capturing antibodies to more immunodominant epitopes, one can direct the cloning to less immunogenic ones. This method should be of value in generating antibodies to be used both in the prophylaxis and treatment of viral infections and in the characterization of the mechanisms of antibody protective actions at the molecular level.

  20. 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.

  1. Visualization of specific binding sites of benzodiazepine in human brain

    International Nuclear Information System (INIS)

    Shinotoh, H.; Yamasaki, T.; Inoue, O.; Itoh, T.; Suzuki, K.; Hashimoto, K.; Tateno, Y.; Ikehira, H.

    1986-01-01

    Using 11C-labeled Ro15-1788 and positron emission tomography, studies of benzodiazepine binding sites in the human brain were performed on four normal volunteers. Rapid and high accumulation of 11C activity was observed in the brain after i.v. injection of [11C]Ro15-1788, the maximum of which was within 12 min. Initial distribution of 11C activity in the brain was similar to the distribution of the normal cerebral blood flow. Ten minutes after injection, however, a high uptake of 11C activity was observed in the cerebral cortex and moderate uptake was seen in the cerebellar cortex, the basal ganglia, and the thalamus. The accumulation of 11C activity was low in the brain stem. This distribution of 11C activity was approximately parallel to the known distribution of benzodiazepine receptors. Saturation experiments were performed on four volunteers with oral administration of 0.3-1.8 mg/kg of cold Ro15-1788 prior to injection. Initial distribution of 11C activity following injection peaked within 2 min and then the accumulation of 11C activity decreased rapidly and remarkably throughout the brain. The results indicated that [11C] Ro15-1788 associates and dissociates to specific and nonspecific binding sites rapidly and has a high ratio of specific receptor binding to nonspecific binding in vivo. Carbon-11 Ro15-1788 is a suitable radioligand for the study of benzodiazepine receptors in vivo in humans

  2. High-throughput screening of monoclonal antibodies against plant cell wall glycans by hierarchical clustering of their carbohydrate microarray binding profiles

    DEFF Research Database (Denmark)

    Moller, Isabel Eva; Marcus, Susan E.; Haeger, Ash

    2008-01-01

    Antibody-producing hybridoma cell lines were created following immunisation with a crude extract of cell wall polymers from the plant Arabidopsis thaliana. In order to rapidly screen the specificities of individual monoclonal antibodies (mAbs), their binding to microarrays containing 50 cell wall...... investigated using subsequent immunochemical and biochemical analyses and two novel mAbs are described in detail. mAb LM13 binds to an arabinanase-sensitive pectic epitope and mAb LM14, binds to an epitope occurring on arabinogalactan-proteins. Both mAbs display novel patterns of recognition of cell walls...

  3. Antibody responses in humans infected with newly emerging strains of West Nile Virus in Europe.

    Directory of Open Access Journals (Sweden)

    Stefan Chabierski

    Full Text Available Infection with West Nile Virus (WNV affects an increasing number of countries worldwide. Although most human infections result in no or mild flu-like symptoms, the elderly and those with a weakened immune system are at higher risk for developing severe neurological disease. Since its introduction into North America in 1999, WNV has spread across the continental United States and caused annual outbreaks with a total of 36,000 documented clinical cases and ∼1,500 deaths. In recent years, outbreaks of neuroinvasive disease also have been reported in Europe. The WNV strains isolated during these outbreaks differ from those in North America, as sequencing has revealed that distinct phylogenetic lineages of WNV concurrently circulate in Europe, which has potential implications for the development of vaccines, therapeutics, and diagnostic tests. Here, we studied the human antibody response to European WNV strains responsible for outbreaks in Italy and Greece in 2010, caused by lineage 1 and 2 strains, respectively. The WNV structural proteins were expressed as a series of overlapping fragments fused to a carrier-protein, and binding of IgG in sera from infected persons was analyzed. The results demonstrate that, although the humoral immune response to WNV in humans is heterogeneous, several dominant peptides are recognized.

  4. Production of a Human Antibody Library in the Phage-Display Vector pSEX81.

    Science.gov (United States)

    Welschof, M; Little, M; Dörsam, H

    1998-01-01

    Human monoclonal antibodies (MAbs) are more suitable than MAbs of animal origin for clinical applications because of lower hypersensitivity reactions, less formation of circulating immune complexes and lower anti-immunoglobulin responses The classical production of human MAbs via the hybridoma technique or Epstein-Barr virus (EBV) transformation is limited by the instability of cell lines, low antibody production, and the problems of imununizing humans with certain antigens (1,2). A promising alternative 1s the production of human recombinant antibodies (3). Recombinant DNA technology has made it possible to clone human antibody genes in vectors and to generate antibody expression libraries (4-7). One approach has been to amplify and recombine the IgG repertoire of an "immunized" donor. This has been used to isolate several antibodies related to diseases (8,9). In order to obtain more universal antibody libraries the naive IgM repertoire of several "unimmunized" donors were pooled (10,12). The complexity of the combinatorial libraries has been further increased by creating the so-called "semisynthetic" antibody libraries (22-14).

  5. Safety testing of monoclonal antibodies in non-human primates: Case studies highlighting their impact on human risk assessment.

    Science.gov (United States)

    Brennan, Frank R; Cavagnaro, Joy; McKeever, Kathleen; Ryan, Patricia C; Schutten, Melissa M; Vahle, John; Weinbauer, Gerhard F; Marrer-Berger, Estelle; Black, Lauren E

    2018-01-01

    Monoclonal antibodies (mAbs) are improving the quality of life for patients suffering from serious diseases due to their high specificity for their target and low potential for off-target toxicity. The toxicity of mAbs is primarily driven by their pharmacological activity, and therefore safety testing of these drugs prior to clinical testing is performed in species in which the mAb binds and engages the target to a similar extent to that anticipated in humans. For highly human-specific mAbs, this testing often requires the use of non-human primates (NHPs) as relevant species. It has been argued that the value of these NHP studies is limited because most of the adverse events can be predicted from the knowledge of the target, data from transgenic rodents or target-deficient humans, and other sources. However, many of the mAbs currently in development target novel pathways and may comprise novel scaffolds with multi-functional domains; hence, the pharmacological effects and potential safety risks are less predictable. Here, we present a total of 18 case studies, including some of these novel mAbs, with the aim of interrogating the value of NHP safety studies in human risk assessment. These studies have identified mAb candidate molecules and pharmacological pathways with severe safety risks, leading to candidate or target program termination, as well as highlighting that some pathways with theoretical safety concerns are amenable to safe modulation by mAbs. NHP studies have also informed the rational design of safer drug candidates suitable for human testing and informed human clinical trial design (route, dose and regimen, patient inclusion and exclusion criteria and safety monitoring), further protecting the safety of clinical trial participants.

  6. Boronated monoclonal antibody conjugates for neutron capture therapy

    International Nuclear Information System (INIS)

    Borg, D.C.; Elmore, J.J. Jr.; Ferrone, S.

    1986-01-01

    This paper describes the effectiveness of 10 B-labeled monoclonal antibodies against Colo-38 human melanoma in vitro. The authors obtained high boron to antibody ratios while maintaining antibody activity by using dextran intermediate carriers to link 10 B to the antibody. They developed a double cell quasi-competitive binding bioassay to minimize the effects of nonspecific binding of boronated complexes to cells. 1 fig., 2 tabs

  7. Disassembly and reassembly of human papillomavirus virus-like particles produces more virion-like antibody reactivity

    Directory of Open Access Journals (Sweden)

    Zhao Qinjian

    2012-02-01

    Full Text Available Abstract Background Human papillomavirus (HPV vaccines based on major capsid protein L1 are licensed in over 100 countries to prevent HPV infections. The yeast-derived recombinant quadrivalent HPV L1 vaccine, GARDASIL(R, has played an important role in reducing cancer and genital warts since its introduction in 2006. The L1 proteins self-assemble into virus-like particles (VLPs. Results VLPs were subjected to post-purification disassembly and reassembly (D/R treatment during bioprocessing to improve VLP immunoreactivity and stability. The post-D/R HPV16 VLPs and their complex with H16.V5 neutralizing antibody Fab fragments were visualized by cryo electron microscopy, showing VLPs densely decorated with antibody. Along with structural improvements, post-D/R VLPs showed markedly higher antigenicity to conformational and neutralizing monoclonal antibodies (mAbs H16.V5, H16.E70 and H263.A2, whereas binding to mAbs recognizing linear epitopes (H16.J4, H16.O7, and H16.H5 was greatly reduced. Strikingly, post-D/R VLPs showed no detectable binding to H16.H5, indicating that the H16.H5 epitope is not accessible in fully assembled VLPs. An atomic homology model of the entire HPV16 VLP was generated based on previously determined high-resolution structures of bovine papillomavirus and HPV16 L1 pentameric capsomeres. Conclusions D/R treatment of HPV16 L1 VLPs produces more homogeneous VLPs with more virion-like antibody reactivity. These effects can be attributed to a combination of more complete and regular assembly of the VLPs, better folding of L1, reduced non-specific disulfide-mediated aggregation and increased stability of the VLPs. Markedly different antigenicity of HPV16 VLPs was observed upon D/R treatment with a panel of monoclonal antibodies targeting neutralization sensitive epitopes. Multiple epitope-specific assays with a panel of mAbs with different properties and epitopes are required to gain a better understanding of the immunochemical

  8. Serological analysis of human anti-human antibody responses in colon cancer patients treated with repeated doses of humanized monoclonal antibody A33.

    Science.gov (United States)

    Ritter, G; Cohen, L S; Williams, C; Richards, E C; Old, L J; Welt, S

    2001-09-15

    Mouse monoclonal antibody A33 (mAb A33) recognizes a M(r) 43,000 cell surface glycoprotein (designated A33) expressed in human colonic epithelium and colon cancer but absent from most other normal tissues. In patients, mAb A33 localizes with high specificity to colon cancer and is retained for up to 6 weeks in the cancer but cleared rapidly from normal colon (5-6 days). As a carrier of (125)I or (131)I, mAb A33 has shown antitumor activity. Induction of strong human anti-mouse antibody (immunoglobulin; HAMA) responses in patients, however, limits the use of the murine mAb A33 to very few injections. A humanized version of this antibody (huAb A33) has been prepared for Phase I and II clinical studies in patients with colon cancer. In those studies, immunogenicity of huAb A33 has been monitored using a novel, highly sensitive BIACORE method, which allows measurement of human anti-human antibodies (HAHAs) without the use of secondary reagents. We found that 63% (26 of 41) of the patients treated with repeated doses of huAb A33 developed HAHAs against a conformational antigenic determinant located in the V(L) and V(H) regions of huAb A33. Detailed serological analysis showed two distinct types of HAHAs. HAHA of type I (49% of patients) was characterized by an early onset with peak HAHA levels after 2 weeks of treatment, which declined with ongoing huAb A33 treatment. HAHA of type II (17% of patients) was characterized by a typically later onset of HAHA than in type I and by progressively increasing HAHA levels with each subsequent huAb A33 administration. Colon cancer patients with type I HAHAs did not develop infusion-related adverse events. In contrast, HAHA of type II was indicative of infusion-related adverse events. By using this new method, we were able to distinguish these two types of HAHAs in patients while on antibody treatment, allowing patients to be removed from study prior to the onset of severe infusion-related adverse events.

  9. Structural Basis for Recognition of Human Enterovirus 71 by a Bivalent Broadly Neutralizing Monoclonal Antibody.

    Directory of Open Access Journals (Sweden)

    Xiaohua Ye

    2016-03-01

    Full Text Available Enterovirus 71 (EV71 is the main pathogen responsible for hand, foot and mouth disease with severe neurological complications and even death in young children. We have recently identified a highly potent anti-EV71 neutralizing monoclonal antibody, termed D5. Here we investigated the structural basis for recognition of EV71 by the antibody D5. Four three-dimensional structures of EV71 particles in complex with IgG or Fab of D5 were reconstructed by cryo-electron microscopy (cryo-EM single particle analysis all at subnanometer resolutions. The most critical EV71 mature virion-Fab structure was resolved to a resolution of 4.8 Å, which is rare in cryo-EM studies of virus-antibody complex so far. The structures reveal a bivalent binding pattern of D5 antibody across the icosahedral 2-fold axis on mature virion, suggesting that D5 binding may rigidify virions to prevent their conformational changes required for subsequent RNA release. Moreover, we also identified that the complementary determining region 3 (CDR3 of D5 heavy chain directly interacts with the extremely conserved VP1 GH-loop of EV71, which was validated by biochemical and virological assays. We further showed that D5 is indeed able to neutralize a variety of EV71 genotypes and strains. Moreover, D5 could potently confer protection in a mouse model of EV71 infection. Since the conserved VP1 GH-loop is involved in EV71 binding with its uncoating receptor, the scavenger receptor class B, member 2 (SCARB2, the broadly neutralizing ability of D5 might attribute to its inhibition of EV71 from binding SCARB2. Altogether, our results elucidate the structural basis for the binding and neutralization of EV71 by the broadly neutralizing antibody D5, thereby enhancing our understanding of antibody-based protection against EV71 infection.

  10. Maximizing in vivo target clearance by design of pH-dependent target binding antibodies with altered affinity to FcRn.

    Science.gov (United States)

    Yang, Danlin; Giragossian, Craig; Castellano, Steven; Lasaro, Marcio; Xiao, Haiguang; Saraf, Himanshu; Hess Kenny, Cynthia; Rybina, Irina; Huang, Zhong-Fu; Ahlberg, Jennifer; Bigwarfe, Tammy; Myzithras, Maria; Waltz, Erica; Roberts, Simon; Kroe-Barrett, Rachel; Singh, Sanjaya

    2017-10-01

    Antibodies with pH-dependent binding to both target antigens and neonatal Fc receptor (FcRn) provide an alternative tool to conventional neutralizing antibodies, particularly for therapies where reduction in antigen level is challenging due to high target burden. However, the requirements for optimal binding kinetic framework and extent of pH dependence for these antibodies to maximize target clearance from circulation are not well understood. We have identified a series of naturally-occurring high affinity antibodies with pH-dependent target binding properties. By in vivo studies in cynomolgus monkeys, we show that pH-dependent binding to the target alone is not sufficient for effective target removal from circulation, but requires Fc mutations that increase antibody binding to FcRn. Affinity-enhanced pH-dependent FcRn binding that is double-digit nM at pH 7.4 and single-digit nM at pH 6 achieved maximal target reduction when combined with similar target binding affinities in reverse pH directions. Sustained target clearance below the baseline level was achieved 3 weeks after single-dose administration at 1.5 mg/kg. Using the experimentally derived mechanistic model, we demonstrate the essential kinetic interplay between target turnover and antibody pH-dependent binding during the FcRn recycling, and identify the key components for achieving maximal target clearance. These results bridge the demand for improved patient dosing convenience with the "know-how" of therapeutic modality by design.

  11. Addressing the Immunogenicity of the Cargo and of the Targeting Antibodies with a Focus on Deimmunized Bacterial Toxins and on Antibody-Targeted Human Effector Proteins

    OpenAIRE

    Grinberg, Yehudit; Benhar, Itai

    2017-01-01

    Third-generation immunotoxins are composed of a human, or humanized, targeting moiety, usually a monoclonal antibody or an antibody fragment, and a non-human effector molecule. Due to the non-human origin of the cytotoxic domain, these molecules stimulate potent anti-drug immune responses, which limit treatment options. Efforts are made to deimmunize such immunotoxins or to combine treatment with immunosuppression. An alternative approach is using the so-called ?human cytotoxic fusion protein...

  12. The Isolation of Novel Phage Display-Derived Human Recombinant Antibodies Against CCR5, the Major Co-Receptor of HIV

    Science.gov (United States)

    Shimoni, Moria; Herschhorn, Alon; Britan-Rosich, Yelena; Kotler, Moshe; Benhar, Itai

    2013-01-01

    Abstract Selecting for antibodies against specific cell-surface proteins is a difficult task due to many unrelated proteins that are expressed on the cell surface. Here, we describe a method to screen antibody-presenting phage libraries against native cell-surface proteins. We applied this method to isolate antibodies that selectively recognize CCR5, which is the major co-receptor for HIV entry (consequently, playing a pivotal role in HIV transmission and pathogenesis). We employed a phage screening strategy by using cells that co-express GFP and CCR5, along with an excess of control cells that do not express these proteins (and are otherwise identical to the CCR5-expressing cells). These control cells are intended to remove most of the phages that bind the cells nonspecifically; thus leading to an enrichment of the phages presenting anti-CCR5-specific antibodies. Subsequently, the CCR5-presenting cells were quantitatively sorted by flow cytometry, and the bound phages were eluted, amplified, and used for further successive selection rounds. Several different clones of human single-chain Fv antibodies that interact with CCR5-expressing cells were identified. The most specific monoclonal antibody was converted to a full-length IgG and bound the second extracellular loop of CCR5. The experimental approach presented herein for screening for CCR5-specific antibodies can be applicable to screen antibody-presenting phage libraries against any cell-surface expressed protein of interest. PMID:23941674

  13. Human IgG lacking effector functions demonstrate lower FcRn-binding and reduced transplacental transport.

    Science.gov (United States)

    Stapleton, Nigel M; Armstrong-Fisher, Sylvia S; Andersen, Jan Terje; van der Schoot, C Ellen; Porter, Charlene; Page, Kenneth R; Falconer, Donald; de Haas, Masja; Williamson, Lorna M; Clark, Michael R; Vidarsson, Gestur; Armour, Kathryn L

    2018-03-01

    We have previously generated human IgG1 antibodies that were engineered for reduced binding to the classical Fcγ receptors (FcγRI-III) and C1q, thereby eliminating their destructive effector functions (constant region G1Δnab). In their potential use as blocking agents, favorable binding to the neonatal Fc receptor (FcRn) is important to preserve the long half-life typical of IgG. An ability to cross the placenta, which is also mediated, at least in part, by FcRn is desirable in some indications, such as feto-maternal alloimmune disorders. Here, we show that G1Δnab mutants retain pH-dependent binding to human FcRn but that the amino acid alterations reduce the affinity of the IgG1:FcRn interaction by 2.0-fold and 1.6-fold for the two antibodies investigated. The transport of the modified G1Δnab mutants across monolayers of human cell lines expressing FcRn was approximately 75% of the wild-type, except that no difference was observed with human umbilical vein endothelial cells. G1Δnab mutation also reduced transport in an ex vivo placenta model. In conclusion, we demonstrate that, although the G1Δnab mutations are away from the FcRn-binding site, they have long-distance effects, modulating FcRn binding and transcellular transport. Our findings have implications for the design of therapeutic human IgG with tailored effector functions. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

  14. Functional human antibody CDR fusions as long-acting therapeutic endocrine agonists.

    Science.gov (United States)

    Liu, Tao; Zhang, Yong; Liu, Yan; Wang, Ying; Jia, Haiqun; Kang, Mingchao; Luo, Xiaozhou; Caballero, Dawna; Gonzalez, Jose; Sherwood, Lance; Nunez, Vanessa; Wang, Danling; Woods, Ashley; Schultz, Peter G; Wang, Feng

    2015-02-03

    On the basis of the 3D structure of a bovine antibody with a well-folded, ultralong complementarity-determining region (CDR), we have developed a versatile approach for generating human or humanized antibody agonists with excellent pharmacological properties. Using human growth hormone (hGH) and human leptin (hLeptin) as model proteins, we have demonstrated that functional human antibody CDR fusions can be efficiently engineered by grafting the native hormones into different CDRs of the humanized antibody Herceptin. The resulting Herceptin CDR fusion proteins were expressed in good yields in mammalian cells and retain comparable in vitro biological activity to the native hormones. Pharmacological studies in rodents indicated a 20- to 100-fold increase in plasma circulating half-life for these antibody agonists and significantly extended in vivo activities in the GH-deficient rat model and leptin-deficient obese mouse model for the hGH and hLeptin antibody fusions, respectively. These results illustrate the utility of antibody CDR fusions as a general and versatile strategy for generating long-acting protein therapeutics.

  15. Antibodies to the human T-cell lymphoma/leukemia virus type I in Dutch haemophiliacs

    NARCIS (Netherlands)

    Goudsmit, J.; Miedema, F.; Breederveld, C.; Terpstra, F.; Roos, M.; Schellekens, P.; Melief, C.

    1986-01-01

    95 Dutch haemophiliacs were tested for antibodies to membrane antigens on cells infected with human T-cell leukemia virus type I (HTLV-I-MA) by indirect immunofluorescence and to purified HTLV-I by enzyme-linked immunosorbent assay. Antibodies to HTLV-I-MA were present in 8 of 95 (8%) haemophiliacs,

  16. Human Monoclonal Antibodies Targeting Glypican-2 in Neuroblastoma | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    Researchers at the National Cancer Institute’s Laboratory of Molecular Biology (NCI LMB) have developed and isolated several single domain monoclonal human antibodies against GPC2. NCI seeks parties interested in licensing or co-developing GPC2 antibodies and/or conjugates.

  17. Production and characterisation of monoclonal antibodies against native and disassembled human catalase

    NARCIS (Netherlands)

    Wiemer, E. A.; Ofman, R.; Middelkoop, E.; de Boer, M.; Wanders, R. J.; Tager, J. M.

    1992-01-01

    Catalase isolated from human erythrocytes was used to immunise mice, in order to generate hybridomas producing specific monoclonal antibodies to the enzyme. Hybridomas secreting anti-(catalase) antibodies were identified by a modified enzyme-linked immunosorbent assay (ELISA) using either

  18. 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

  19. Separation of hemagglutination-inhibiting immunoglobulin M antibody to rubella virus in human serum by high-performance liquid chromatography.

    OpenAIRE

    Kobayashi, N; Suzuki, M; Nakagawa, T; Matumoto, M

    1986-01-01

    High-performance liquid chromatography was successfully used to separate hemagglutination-inhibiting immunoglobulin M (IgM) rubella virus antibody from IgG rubella virus antibody in human serum. The fractionation by high-performance liquid chromatography was as effective as sucrose density gradient centrifugation in separating IgM antibody from IgG antibody.

  20. Deoxyribonucleic-binding homeobox proteins are augmented in human cancer

    DEFF Research Database (Denmark)

    Wewer, U M; Mercurio, A M; Chung, S Y

    1990-01-01

    Homeobox genes encode sequence-specific DNA-binding proteins that are involved in the regulation of gene expression during embryonic development. In this study, we examined the expression of homeobox proteins in human cancer. Antiserum was obtained against a synthetic peptide derived from...... was then isolated and used to elicit a rabbit antiserum. In immunostaining, both antisera reacted with the nuclei of cultured tumor cells. In tissue sections of human carcinoma, nuclear immunoreactivity was observed in the tumor cells in 40 of 42 cases examined. Adjacent normal epithelial tissue obtained from......, the presence of the homeobox transcript in human carcinoma was documented by in situ hybridization and RNase protection mapping. These results demonstrate that human cancer is associated with the expression of homeobox proteins. Such homeobox proteins, as well as other regulatory proteins, could be involved...

  1. In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis.

    Science.gov (United States)

    Salinas-Jazmín, Nohemi; González-González, Edith; Vásquez-Bochm, Luz X; Pérez-Tapia, Sonia M; Velasco-Velázquez, Marco A

    2017-05-04

    Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

  2. The Tetherin Antagonism of the Ebola Virus Glycoprotein Requires an Intact Receptor-Binding Domain and Can Be Blocked by GP1-Specific Antibodies.

    Science.gov (United States)

    Brinkmann, Constantin; Nehlmeier, Inga; Walendy-Gnirß, Kerstin; Nehls, Julia; González Hernández, Mariana; Hoffmann, Markus; Qiu, Xiangguo; Takada, Ayato; Schindler, Michael; Pöhlmann, Stefan

    2016-12-15

    The glycoprotein of Ebola virus (EBOV GP), a member of the family Filoviridae, facilitates viral entry into target cells. In addition, EBOV GP antagonizes the antiviral activity of the host cell protein tetherin, which may otherwise restrict EBOV release from infected cells. However, it is unclear how EBOV GP antagonizes tetherin, and it is unknown whether the GP of Lloviu virus (LLOV), a filovirus found in dead bats in Northern Spain, also counteracts tetherin. Here, we show that LLOV GP antagonizes tetherin, indicating that tetherin may not impede LLOV spread in human cells. Moreover, we demonstrate that appropriate processing of N-glycans in tetherin/GP-coexpressing cells is required for tetherin counteraction by EBOV GP. Furthermore, we show that an intact receptor-binding domain (RBD) in the GP1 subunit of EBOV GP is a prerequisite for tetherin counteraction. In contrast, blockade of Niemann-Pick disease type C1 (NPC1), a cellular binding partner of the RBD, did not interfere with tetherin antagonism. Finally, we provide evidence that an antibody directed against GP1, which protects mice from a lethal EBOV challenge, may block GP-dependent tetherin antagonism. Our data, in conjunction with previous reports, indicate that tetherin antagonism is conserved among the GPs of all known filoviruses and demonstrate that the GP1 subunit of EBOV GP plays a central role in tetherin antagonism. Filoviruses are reemerging pathogens that constitute a public health threat. Understanding how Ebola virus (EBOV), a highly pathogenic filovirus responsible for the 2013-2016 Ebola virus disease epidemic in western Africa, counteracts antiviral effectors of the innate immune system might help to define novel targets for antiviral intervention. Similarly, determining whether Lloviu virus (LLOV), a filovirus detected in bats in northern Spain, is inhibited by innate antiviral effectors in human cells might help to determine whether the virus constitutes a threat to humans. The

  3. Radioimmunoassay for Epstein-Barr Virus (EBV)-associated Nuclear Antigen (EBNA). Binding of iodinated antibodies to antigen immobilized in polyacrylamide gel

    International Nuclear Information System (INIS)

    Dolken, G.; Klein, G.

    1977-01-01

    A solid-phase radioimmunoassay was developed for the EBV-associated nuclear antigen (EBNA). Total homogenates of EBV-DNA and EBNA positive or negative cells were polymerized in polyacrylamide gel and compared for their ability to bind 125 I-IgG prepared from anti-EBNA positive and anti-EBNA negative sera. EBNA specific binding was demonstrated and confirmed by serological and cellular specificity controls. The assay allows the quantitation of antigen or antibody even in the presence of detergents and is suitable for biochemical characterization of the antigen. Reciprocal blocking studies with extracts from different cell lines showed quantitative and qualitative differences. One part of the EBNA specificiti(es) present in the human Burkitt lymphoma derived lines RAJI, DAUDI and AW-RAMOS was lacking in B96-8, a marmoset line carrying EBV derived from a human infectious mononucleosis line. This result may reflect differences in the viral genomes derived from Burkitt lymphoma and infectious mononucleosis lines or differences in the host cells. (author)

  4. Development and Validation of an Enzyme-Linked Immunosorbent Assay for the Detection of Binding Anti-Drug Antibodies against Interferon Beta

    Directory of Open Access Journals (Sweden)

    Kathleen Ingenhoven

    2017-07-01

    Full Text Available ObjectiveTo develop and validate a method for the detection of binding anti-drug antibodies (ADAs against interferon beta (IFN-β in human serum as part of a European initiative (ABIRISK aimed at the prediction and analysis of clinical relevance of anti-biopharmaceutical immunization to minimize the risk.MethodA two-tiered bridging enzyme-linked immunosorbent assay (ELISA format was selected and validated according to current recommendations. Screening assay: ADA in serum samples form complexes with immobilized IFN-β and biotinylated IFN-β, which are then detected using HRP labeled Streptavidin and TMB substrate. Confirmation assay: Screen “putative positive” samples are tested in the presence of excess drug (preincubation of sera with 0.3 µg/mL of soluble IFN-β and percentage of inhibition is calculated.ResultsThe assay is precise, and the sensitivity of the assay was confirmed to be 26 ng/mL using commercially available polyclonal rabbit antihuman IFN-β in human sera as the positive control.ConclusionAn ultrasensitive ELISA for IFN-β-binding ADA testing has been validated. This will form the basis to assess anti-biopharmaceutical immunization toward IFN-β with regards to its clinical relevance and may allow for the development of predictive tools, key aims within the ABIRISK consortium.

  5. Anti-fibrin antibody binding in valvular vegetations and kidney lesions during experimental endocarditis.

    Science.gov (United States)

    Yokota, M; Basi, D L; Herzberg, M C; Meyer, M W

    2001-01-01

    In Streptococcus sanguinis (sanguis) induced experimental endocarditis, we sought evidence that the development of aortic valvular vegetation depends on the availability of fibrin. Endocarditis was induced in New Zealand white rabbits by catheter placement into the left ventricle and inoculation of the bacteria. Fibrin was localized in the developing vegetation with 99mTechnetium (Tc)-labeled anti-fibrin antibody one or three days later. When rabbit anti-fibrin antibody was given intravenously on day 1, the mass of aortic valvular vegetation was significantly reduced at day 3; infusion of non-specific rabbit IgG showed no effect. The 99mTc-labeled anti-fibrin antibody also labeled kidneys that showed macroscopic subcapsular hemorrhage. To learn if the deposition of fibrin in the kidneys was a consequence of endocarditis required a comparison of farm-bred and specific pathogen-free rabbits before and after the induction of endocarditis. Before induction, the kidneys of farm-bred rabbits were labeled, but specific pathogen-free rabbits were free of labeling and signs of macroscopic hemorrhage. After 3 days of endocarditis, kidneys of 10 of 14 specific pathogen-free rabbits labeled with 99mTc-labeled anti-fibrin antibody and showed hemorrhage. Kidney lesions were suggested to be a frequent sequellae of S. sanguinis infective endocarditis. For the first time, fibrin was shown to be required for the continued development of aortic valvular vegetations.

  6. Next Generation Antibody Therapeutics Using Bispecific Antibody Technology.

    Science.gov (United States)

    Igawa, Tomoyuki

    2017-01-01

    Nearly fifty monoclonal antibodies have been approved to date, and the market for monoclonal antibodies is expected to continue to grow. Since global competition in the field of antibody therapeutics is intense, we need to establish novel antibody engineering technologies to provide true benefit for patients, with differentiated product values. Bispecific antibodies are among the next generation of antibody therapeutics that can bind to two different target antigens by the two arms of immunoglobulin G (IgG) molecule, and are thus believed to be applicable to various therapeutic needs. Until recently, large scale manufacturing of human IgG bispecific antibody was impossible. We have established a technology, named asymmetric re-engineering technology (ART)-Ig, to enable large scale manufacturing of bispecific antibodies. Three examples of next generation antibody therapeutics using ART-Ig technology are described. Recent updates on bispecific antibodies against factor IXa and factor X for the treatment of hemophilia A, bispecific antibodies against a tumor specific antigen and T cell surface marker CD3 for cancer immunotherapy, and bispecific antibodies against two different epitopes of soluble antigen with pH-dependent binding property for the elimination of soluble antigen from plasma are also described.

  7. A simple method for assessment of human anti-Neu5Gc antibodies applied to Kawasaki disease.

    Directory of Open Access Journals (Sweden)

    Vered Padler-Karavani

    Full Text Available N-glycolylneuraminic acid (Neu5Gc is an immunogenic sugar of dietary origin that metabolically incorporates into diverse native glycoconjugates in humans. Anti-Neu5Gc antibodies are detected in all human sera, though with variable levels and epitope-recognition profiles. These antibodies likely play a role in several inflammation-mediated pathologies including cardiovascular diseases and cancer. In cancer, they have dualistic and opposing roles, either stimulating or repressing disease, as a function of their dose, and some of these antibodies serve as carcinoma biomarkers. Thus, anti-Neu5Gc antibodies may signify risk of inflammation-mediated diseases, and changes in their levels could potentially be used to monitor disease progression and/or response to therapy. Currently, it is difficult to determine levels of anti-Neu5Gc antibodies in individual human samples because these antibodies recognize multiple Neu5Gc-epitopes. Here we describe a simple and specific method for detection and overall estimation of human anti-Neu5Gc antibodies. We exploit the difference between two mouse models that differ only by Neu5Gc-presence (wild-type or Neu5Gc-absence (Cmah(-/- knockout. We characterize mouse serum from both strains by HPLC, lectin and mass-spectrometry analysis and show the target Neu5Gc-epitopes. We then use Cmah(-/- knockout sera to inhibit all non-Neu5Gc-reactivity followed by binding to wild-type sera to detect overall anti-Neu5Gc response in a single assay. We applied this methodology to characterize and quantify anti-Neu5Gc IgG and IgA in sera of patients with Kawasaki disease (KD at various stages compared to controls. KD is an acute childhood febrile disease characterized by inflammation of coronary arteries that untreated may lead to coronary artery aneurysms with risk of thrombosis and myocardial infarction. This estimated response is comparable to the average of detailed anti-Neu5Gc IgG profile analyzed by a sialoglycan microarray

  8. Novel antibody binding determinants on the capsid surface of serotype O foot-and-mouth disease virus

    Science.gov (United States)

    Asfor, Amin S.; Upadhyaya, Sasmita; Knowles, Nick J.; King, Donald P.; Paton, David J.

    2014-01-01

    Five neutralizing antigenic sites have been described for serotype O foot-and-mouth disease viruses (FMDV) based on monoclonal antibody (mAb) escape mutant studies. However, a mutant virus selected to escape neutralization of mAb binding at all five sites was previously shown to confer complete cross-protection with the parental virus in guinea pig challenge studies, suggesting that amino acid residues outside the mAb binding sites contribute to antibody-mediated in vivo neutralization of FMDV. Comparison of the ability of bovine antisera to neutralize a panel of serotype O FMDV identified three novel putative sites at VP2-74, VP2-191 and VP3-85, where amino acid substitutions correlated with changes in sero-reactivity. The impact of these positions was tested using site-directed mutagenesis to effect substitutions at critical amino acid residues within an infectious copy of FMDV O1 Kaufbeuren (O1K). Recovered viruses containing additional mutations at VP2-74 and VP2-191 exhibited greater resistance to neutralization with both O1K guinea pig and O BFS bovine antisera than a virus that was engineered to include only mutations at the five known antigenic sites. The changes at VP2-74 and VP3-85 are adjacent to critical amino acids that define antigenic sites 2 and 4, respectively. However VP2-191 (17 Å away from VP2-72), located at the threefold axis and more distant from previously identified antigenic sites, exhibited the most profound effect. These findings extend our knowledge of the surface features of the FMDV capsid known to elicit neutralizing antibodies, and will improve our strategies for vaccine strain selection and rational vaccine design. PMID:24584474

  9. Computational identification of epitopes in the glycoproteins of novel bunyavirus (SFTS virus) recognized by a human monoclonal antibody (MAb 4-5)

    Science.gov (United States)

    Zhang, Wenshuai; Zeng, Xiaoyan; Zhang, Li; Peng, Haiyan; Jiao, Yongjun; Zeng, Jun; Treutlein, Herbert R.

    2013-06-01

    In this work, we have developed a new approach to predict the epitopes of antigens that are recognized by a specific antibody. Our method is based on the "multiple copy simultaneous search" (MCSS) approach which identifies optimal locations of small chemical functional groups on the surfaces of the antibody, and identifying sequence patterns of peptides that can bind to the surface of the antibody. The identified sequence patterns are then used to search the amino-acid sequence of the antigen protein. The approach was validated by reproducing the binding epitope of HIV gp120 envelop glycoprotein for the human neutralizing antibody as revealed in the available crystal structure. Our method was then applied to predict the epitopes of two glycoproteins of a newly discovered bunyavirus recognized by an antibody named MAb 4-5. These predicted epitopes can be verified by experimental methods. We also discuss the involvement of different amino acids in the antigen-antibody recognition based on the distributions of MCSS minima of different functional groups.

  10. Transient human anti-mouse antibodies (HAMA) interference in CA 125 measurements during monitoring of ovarian cancer patients treated with murine monoclonal antibody.

    NARCIS (Netherlands)

    Oei, A.L.M.; Sweep, F.C.; Massuger, L.F.A.G.; Olthaar, A.J.; Thomas, C.M.G.

    2008-01-01

    OBJECTIVE: To investigate the influence of human anti-mouse antibodies (HAMA) on serial CA 125 measurements in serum of patients with epithelial ovarian cancer following single intraperitoneal (IP) therapy with Yttrium-90-labeled human milk fat globule 1 murine monoclonal antibody ((90)Y-muHMFG1) as

  11. Chromosomal localization of the human diazepam binding inhibitor gene

    International Nuclear Information System (INIS)

    DeBernardi, M.A.; Crowe, R.R.; Mocchetti, I.; Shows, T.B.; Eddy, R.L.; Costa, E.

    1988-01-01

    The authors have used in situ chromosome hybridization and human-mouse somatic cell hybrids to map the gene(s) for human diazepam binding inhibitor (DBI), an endogenous putative modulator of the γ-aminobutyric acid receptor acting at the allosteric regulatory center of this receptor that includes the benzodiazepine recognition site. In 784 chromosome spreads hybridized with human DBI cDNA, the distribution of 1,476 labeled sites revealed a significant clustering of autoradiographic grains (11.3% of total label) on the long arm of chromosome 2 (2q). Furthermore, 63.5% of the grains found on 2q were located on 2q12-21, suggesting regional mapping of DBI gene(s) to this segment. Secondary hybridization signals were frequently observed on other chromosomes and they were statistically significant mainly for chromosomes 5, 6, 11, and 14. In addition, DNA from 32 human-mouse cell hybrids was digested with BamHI and probed with human DBI cDNA. A 3.5-kilobase band, which probably represents the human DBI gene, was assigned to chromosome 2. Four higher molecular weight bands, also detected in BamHI digests, could not be unequivocally assigned. A chromosome 2 location was excluded for the 27-, 13-, and 10-kilobase bands. These results assign a human DBI gene to chromosome 2 (2q12-21) and indicate that three of the four homologous sequences detected by the human DBI probe are located on three other chromosomes

  12. Antibody Treatment of Ebola and Sudan Virus Infection via a Uniquely Exposed Epitope within the Glycoprotein Receptor Binding Site

    Science.gov (United States)

    2016-06-14

    defective particles. For example, studies performed with anti-EBOV equine immunoglobulin clearly showed rapid reduction in EBOV specific equine IgG...titer during the peak of viremia in NHPs, while total equine IgG titer remained constant (Jahrling et al., 1996), suggesting high antibody consumption... therapy of Ebola hemorrhagic fever. J Infect Dis 179 Suppl 1, S248-258. Coughlin, M.M., and Prabhakar, B.S. (2012). Neutralizing human monoclonal

  13. A human monoclonal antibody drug and target discovery platform for B-cell chronic lymphocytic leukemia based on allogeneic hematopoietic stem cell transplantation and phage display.

    Science.gov (United States)

    Baskar, Sivasubramanian; Suschak, Jessica M; Samija, Ivan; Srinivasan, Ramaprasad; Childs, Richard W; Pavletic, Steven Z; Bishop, Michael R; Rader, Christoph

    2009-11-12

    Allogeneic hematopoietic stem cell transplantation (alloHSCT) is the only potentially curative treatment available for patients with B-cell chronic lymphocytic leukemia (B-CLL). Here, we show that post-alloHSCT antibody repertoires can be mined for the discovery of fully human monoclonal antibodies to B-CLL cell-surface antigens. Sera collected from B-CLL patients at defined times after alloHSCT showed selective binding to primary B-CLL cells. Pre-alloHSCT sera, donor sera, and control sera were negative. To identify post-alloHSCT serum antibodies and subsequently B-CLL cell-surface antigens they recognize, we generated a human antibody-binding fragment (Fab) library from post-alloHSCT peripheral blood mononuclear cells and selected it on primary B-CLL cells by phage display. A panel of Fab with B-CLL cell-surface reactivity was strongly enriched. Selection was dominated by highly homologous Fab predicted to bind the same antigen. One Fab was converted to immunoglobulin G1 and analyzed for reactivity with peripheral blood mononuclear cells from B-CLL patients and healthy volunteers. Cell-surface antigen expression was restricted to primary B cells and up-regulated in primary B-CLL cells. Mining post-alloHSCT antibody repertoires offers a novel route to discover fully human monoclonal antibodies and identify antigens of potential therapeutic relevance to B-CLL and possibly other cancers. Trials described herein were registered at www.clinicaltrials.gov as nos. NCT00055744 and NCT00003838.

  14. Direct binding to antigen-coated beads refines the specificity and cross-reactivity of four monoclonal antibodies that recognize polymorphic epitopes of HLA class I molecules.

    Science.gov (United States)

    Hilton, H G; Parham, P

    2013-04-01

    Monoclonal antibodies with specificity for human leukocyte antigen (HLA) class I determinants of HLA were originally characterized using serological assays in which the targets were cells expressing three to six HLA class I variants. Because of this complexity, the specificities of the antibodies were defined indirectly by correlation. Here we use a direct binding assay, in which the targets are synthetic beads coated with 1 of 111 HLA class I variants, representing the full range of HLA-A, -B and -C variation. We studied one monoclonal antibody with monomorphic specificity (W6/32) and four with polymorphic specificity (MA2.1, PA2.1, BB7.2 and BB7.1) and compared the results with those obtained previously. W6/32 reacted with all HLA class I variants. MA2.1 not only exhibits high specificity for HLA-A*02, -B*57 and -B*58, but also exhibited cross-reactivity with HLA-A*11 and -B*15:16. At low concentration (1 µg/ml), PA2.1 and BB7.2 were both specific for HLA-A*02 and -A*69, and at high concentration (50 µg/ml) exhibited significant cross-reactions with HLA-A*68, -A*23 and -A*24. BB7.1 exhibits specificity for HLA-B*07 and -B*42, as previously described, but reacts equally well with HLA-B*81, a rare allotype defined some 16 years after the description of BB7.1. The results obtained with cell-based and bead-based assays are consistent and, in combination with amino acid sequence comparison, increase understanding of the polymorphic epitopes recognized by the MA2.1, PA2.1, BB7.2 and BB7.1 antibodies. Comparison of two overlapping but distinctive bead sets from two sources gave similar results, but the overall levels of binding were significantly different. Several weaker reactions were observed with only one of the bead sets. © 2013 John Wiley & Sons A/S.

  15. Development of an affinity-matured humanized anti-epidermal growth factor receptor antibody for cancer immunotherapy.

    Science.gov (United States)

    Nakanishi, Takeshi; Maru, Takamitsu; Tahara, Kazuhiro; Sanada, Hideaki; Umetsu, Mitsuo; Asano, Ryutaro; Kumagai, Izumi

    2013-02-01

    We showed previously that humanization of 528, a murine anti-epidermal growth factor receptor (EGFR) antibody, causes reduced affinity for its target. Here, to improve the affinity of the humanized antibody for use in cancer immunotherapy, we constructed phage display libraries focused on the complementarity-determining regions (CDRs) of the antibody and carried out affinity selection. Two-step selections using libraries constructed in a stepwise manner enabled a 32-fold affinity enhancement of humanized 528 (h528). Thermodynamic analysis of the interactions between the variable domain fragment of h528 (h528Fv) mutants and the soluble extracellular domain of EGFR indicated that the h528Fv mutants obtained from the first selection showed a large increase in negative enthalpy change due to binding, resulting in affinity enhancement. Furthermore, mutants from the second selection showed a decrease in entropy loss, which led to further affinity maturation. These results suggest that a single mutation in the heavy chain variable domain (i.e. Tyr(52) to Trp) enthalpically contributed for overcoming the energetic barrier to the antigen-antibody interaction, which was a major hurdle for the in vitro affinity maturation of h528. We reported previously that the humanized bispecific diabody hEx3 Db, which targets EGFR and CD3, shows strong anti-tumor activity. hEx3 Db mutants, in which the variable domains of h528 were replaced with those of the affinity-enhanced mutants, were prepared and characterized. In a growth inhibition assay of tumor cells, the hEx3 Db mutants showed stronger anti-tumor activity than that of hEx3 Db, suggesting that affinity enhancement of h528Fv enhances the anti-tumor activity of the bispecific diabody.

  16. Detection of cyclobutane thymine dimers in DNA of human cells with monoclonal antibodies raised against a thymine dimer-containing tetranucleotide

    Energy Technology Data Exchange (ETDEWEB)

    Roza, L; Wulp, K.J.M. van der; MacFarlane, S J; Lohman, P H.M.; Baan, R A

    1988-11-01

    A hybrid cell line (hybridoma) has been isolated after fusion between mouse-plasmacytoma cells and spleen cells from mice immunized with a thymine dimer-containing tetranucleotide coupled to a carrier protein. Monoclonal antibodies produced by this hybridoma were characterized by testing the effect of various inhibitors in a competitive enzyme-linked immunosorbent assay (ELISA). The antibodies have a high specificity for thymine dimers in single-stranded DNA or poly(dT), but do not bind UV-irradiated d(TpC)/sub 5/. Less binding is observed with short thymine dimer-containing sequences. In vitro treatment of UV-irradiated DNA with photoreactivating enzyme in the presence of light, or with Micrococcus luteus UV-endonuclease results in disappearance of antigenicity. Antibody-binding to DNA isolated from UV-irradiated human fibroblasts (at 254 nm) is linear with dose. Removal of thymine dimers in these cells during a post-irradiation incubation, as detected with the antibodies, is fast initially but the rate rapidly decreases (about 50% residual dimers at 20 h after 10 J/m/sup 2/). The induction of thymine dimers in human skin irradiated with low doses of UV-B, too, was demonstrated immunochemically, by ELISA as well as by quantitative immunofluorescence microscopy.

  17. Porphyromonas endodontalis binds, reduces and grows on human hemoglobin.

    Science.gov (United States)

    Zerr, M; Drake, D; Johnson, W; Cox, C D

    2001-08-01

    Porphyromonas endodontalis is a black-pigmented, obligate anaerobic rod-shaped bacterium implicated as playing a major role in endodontic infections. We have previously shown that P. endodontalis requires the porphyrin nucleus, preferably supplied as hemoglobin, as a growth supplement. The bacteria also actively transport free iron, although this activity does not support growth in the absence of a porphyrin source. The purpose of this study was to further investigate the binding and subsequent utilization of human hemoglobin by P. endodontalis. P. endodontalis binds hemoglobin and reduces the Fe(III) porphyrin, resulting in a steady accumulation of ferrous hemoglobin. Reduction of methemoglobin was similar to the extracellular reduction of nitrobluetetrazolium in the presence of oxidizable substrate. Turbidimetric and viable cell determinations showed that P. endodontalis grew when supplied only hemoglobin. Therefore, we conclude that hemoglobin appears to serve as a sole carbon and nitrogen source, and that these bacteria reduce extracellular compounds at the expense of oxidized substrates.

  18. A commercial ELISA detects high levels of human H5 antibody but cross-reacts with influenza A antibodies.

    Science.gov (United States)

    Stelzer-Braid, Sacha; Wong, Bruce; Robertson, Peter; Lynch, Garry W; Laurie, Karen; Shaw, Robert; Barr, Ian; Selleck, Paul W; Baleriola, Cristina; Escott, Ros; Katsoulotos, Gregory; Rawlinson, William D

    2008-10-01

    Commercial serological assays to determine influenza A H5N1 infection are available, although the accuracy and reproducibility of these are not reported in detail. This study aimed to assess the validity of a commercial ELISA H5 hemagglutinin (HA) antibody kit. A commercial ELISA for detection of antibodies towards influenza A H5 HA was evaluated using human sera from vaccinated individuals. The ELISA was used to screen 304 sera with elevated influenza A complement fixation titres collected between the period 1995-2007. The ELISA was found to be accurate for sera with high levels of anti-H5 antibodies, and would be useful in clinical settings where a rapid result is required. Thirteen of the stored sera were positive using the ELISA, but were confirmed as negative for H5N1 exposure using further serological tests. Absorption studies suggested that antibodies towards seasonal H3N2 and H1N1 influenza may cross-react with H5 antigen, giving false positive results with the ELISA.

  19. Comparison of (/sup 125/I)beta-endorphin binding to rat brain and NG108-15 cells using a monoclonal antibody directed against the opioid receptor

    Energy Technology Data Exchange (ETDEWEB)

    Bidlack, J.M.; O' Malley, W.E.; Schulz, R.

    1988-02-01

    The properties of (/sup 125/I)beta h-endorphin-binding sites from rat brain membranes and membranes from the NG108-15 cell line were compared using a monoclonal antibody directed against the opioid receptor and opioid peptides as probes. The binding of (/sup 125/I)beta h-endorphin to both rat brain and NG108-15 membranes yielded linear Scatchard plots with Kd values of 1.2 nM and 1.5 nM, respectively, and Bmax values of 865 fmol/mg rat brain membrane protein and 1077 fmol/mg NG108-15 membrane protein. A monoclonal antibody, OR-689.2.4, capable of inhibiting mu and delta binding but not kappa binding to rat brain membranes, noncompetitively inhibited the binding of 1 nM (/sup 125/I)beta h-endorphin to rat brain and NG108-15 membranes with an IC50 value of 405 nM for rat brain membranes and 543 nM for NG108-15 membranes. The monoclonal antibody also inhibited the binding of 3 nM (/sup 3/H) (D-penicillamine2, D-penicillamine5) enkephalin to NG108-15 membranes with an IC50 value of 370 nM. In addition to blocking the binding of (/sup 125/I)beta h-endorphin to brain membranes, the antibody also displaced (/sup 125/I)beta h-endorphin from membranes. Site-specific opioid peptides had large variations in their IC50 values depending on whether they were inhibiting (/sup 125/I)beta h-endorphin binding to rat brain or the NG108-15 membranes. When the peptides were tested with the monoclonal antibody for their combined ability to inhibit (/sup 125/I)beta h-endorphin binding to both membrane preparations, the peptides and antibody blocked binding as though they were acting at allosterically coupled sites, not two totally independent sites. These studies suggest that mu-, delta-, and beta-endorphin-binding sites share some sequence homology with the 35,000-dalton protein that the antibody is directed against.

  20. Differential neutralizing activities of a single domain camelid antibody (VHH specific for ricin toxin's binding subunit (RTB.

    Directory of Open Access Journals (Sweden)

    Cristina Herrera

    Full Text Available Ricin, a member of the A-B family of ribosome-inactivating proteins, is classified as a Select Toxin by the Centers for Disease Control and Prevention because of its potential use as a biothreat agent. In an effort to engineer therapeutics for ricin, we recently produced a collection of alpaca-derived, heavy-chain only antibody VH domains (VHH or "nanobody" specific for ricin's enzymatic (RTA and binding (RTB subunits. We reported that one particular RTB-specific VHH, RTB-B7, when covalently linked via a peptide spacer to different RTA-specific VHHs, resulted in heterodimers like VHH D10/B7 that were capable of passively protecting mice against a lethal dose challenge with ricin. However, RTB-B7 itself, when mixed with ricin at a 1 ∶ 10 toxin:antibody ratio did not afford any protection in vivo, even though it had demonstrable toxin-neutralizing activity in vitro. To better define the specific attributes of antibodies associated with ricin neutralization in vitro and in vivo, we undertook a more thorough characterization of RTB-B7. We report that RTB-B7, even at 100-fold molar excess (toxin:antibody was unable to alter the toxicity of ricin in a mouse model. On the other hand, in two well-established cytotoxicity assays, RTB-B7 neutralized ricin with a 50% inhibitory concentration (IC50 that was equivalent to that of 24B11, a well-characterized and potent RTB-specific murine monoclonal antibody. In fact, RTB-B7 and 24B11 were virtually identical when compared across a series of in vitro assays, including adherence to and neutralization of ricin after the toxin was pre-bound to cell surface receptors. RTB-B7 differed from both 24B11 and VHH D10/B7 in that it was relatively less effective at blocking ricin attachment to receptors on host cells and was not able to form high molecular weight toxin:antibody complexes in solution. Whether either of these activities is important in ricin toxin neutralizing activity in vivo remains to be determined.

  1. Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics

    Directory of Open Access Journals (Sweden)

    Peter Bannas

    2017-11-01

    Full Text Available Monoclonal antibodies have revolutionized cancer therapy. However, delivery to tumor cells in vivo is hampered by the large size (150 kDa of conventional antibodies. The minimal target recognition module of a conventional antibody is composed of two non-covalently associated variable domains (VH and VL. The proper orientation of these domains is mediated by their hydrophobic interface and is stabilized by their linkage to disulfide-linked constant domains (CH1 and CL. VH and VL domains can be fused via a genetic linker into a single-chain variable fragment (scFv. scFv modules in turn can be fused to one another, e.g., to generate a bispecific T-cell engager, or they can be fused in various orientations to antibody hinge and Fc domains to generate bi- and multispecific antibodies. However, the inherent hydrophobic interaction of VH and VL domains limits the stability and solubility of engineered antibodies, often causing aggregation and/or mispairing of V-domains. Nanobodies (15 kDa and nanobody-based human heavy chain antibodies (75 kDa can overcome these limitations. Camelids naturally produce antibodies composed only of heavy chains in which the target recognition module is composed of a single variable domain (VHH or Nb. Advantageous features of nanobodies include their small size, high solubility, high stability, and excellent tissue penetration in vivo. Nanobodies can readily be linked genetically to Fc-domains, other nanobodies, peptide tags, or toxins and can be conjugated chemically at a specific site to drugs, radionuclides, photosensitizers, and nanoparticles. These properties make them particularly suited for specific and efficient targeting of tumors in vivo. Chimeric nanobody-heavy chain antibodies combine advantageous features of nanobodies and human Fc domains in about half the size of a conventional antibody. In this review, we discuss recent developments and perspectives for applications of nanobodies and nanobody

  2. Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics.

    Science.gov (United States)

    Bannas, Peter; Hambach, Julia; Koch-Nolte, Friedrich

    2017-01-01

    Monoclonal antibodies have revolutionized cancer therapy. However, delivery to tumor cells in vivo is hampered by the large size (150 kDa) of conventional antibodies. The minimal target recognition module of a conventional antibody is composed of two non-covalently associated variable domains (VH and VL). The proper orientation of these domains is mediated by their hydrophobic interface and is stabilized by their linkage to disulfide-linked constant domains (CH1 and CL). VH and VL domains can be fused via a genetic linker into a single-chain variable fragment (scFv). scFv modules in turn can be fused to one another, e.g., to generate a bispecific T-cell engager, or they can be fused in various orientations to antibody hinge and Fc domains to generate bi- and multispecific antibodies. However, the inherent hydrophobic interaction of VH and VL domains limits the stability and solubility of engineered antibodies, often causing aggregation and/or mispairing of V-domains. Nanobodies (15 kDa) and nanobody-based human heavy chain antibodies (75 kDa) can overcome these limitations. Camelids naturally produce antibodies composed only of heavy chains in which the target recognition module is composed of a single variable domain (VHH or Nb). Advantageous features of nanobodies include their small size, high solubility, high stability, and excellent tissue penetration in vivo . Nanobodies can readily be linked genetically to Fc-domains, other nanobodies, peptide tags, or toxins and can be conjugated chemically at a specific site to drugs, radionuclides, photosensitizers, and nanoparticles. These properties make them particularly suited for specific and efficient targeting of tumors in vivo . Chimeric nanobody-heavy chain antibodies combine advantageous features of nanobodies and human Fc domains in about half the size of a conventional antibody. In this review, we discuss recent developments and perspectives for applications of nanobodies and nanobody-based human heavy

  3. Antibodies to Anthrax Toxin in Humans and Guinea Pigs and Their Relevance to Protective Immunity

    National Research Council Canada - National Science Library

    Turnbull, P

    1988-01-01

    ... stimulation of more than just production of antibody to PA. Titers to the three components in sera of individuals with histories of clinically diagnosed anthrax as well as from human vaccinees are included in the report.

  4. Generation of human scFvs antibodies recognizing a prion protein epitope expressed on the surface of human lymphoblastoid cells

    Directory of Open Access Journals (Sweden)

    Imperiale Valentina

    2007-07-01

    Full Text Available Abstract Background A hallmark of prion disease is the transformation of normal cellular prion protein (PrPc into an infectious disease-associated isoform, (PrPsc. Anti-prion protein monoclonal antibodies are invaluable for structure-function studies of PrP molecules. Furthermore recent in vitro and in vivo studies indicate that anti-PrP monoclonal antibodies can prevent the incorporation of PrPc into propagating prions. In the present article, we show two new human phage antibodies, isolated on recombinant hamster prion protein (rHaPrP. Results We adopted an antibody phage display strategy to isolate specific human antibodies directed towards rHaPrP which has been used as a bait for panning the synthetic ETH-2 antibody phage library. Two phage antibodies clones named MA3.B4 and MA3.G3 were isolated and characterized under genetic biochemical and immunocytochemical aspects. The clones were found to recognize the prion protein in ELISA studies. In flow-cytometry studies, these human single chain Fragment variable (scFv phage-antibodies show a well defined pattern of reactivity on human lymphoblastoid and myeloid cells. Conclusion Sequence analysis of the gene encoding for the antibody fragments and antigen recognition patterns determined by flow-cytometry analysis indicate that the isolated scFvs recognize novel epitopes in the PrPc molecule. These new anti PrPc human antibodies are unique reagents for prion protein detection and may represent a biologic platform to develop new reagents to treat PrPsc associated disease.

  5. Comprehensive human transcription factor binding site map for combinatory binding motifs discovery.

    Directory of Open Access Journals (Sweden)

    Arnoldo J Müller-Molina

    Full Text Available To know the map between transcription factors (TFs and their binding sites is essential to reverse engineer the regulation process. Only about 10%-20% of the transcription factor binding motifs (TFBMs have been reported. This lack of data hinders understanding gene regulation. To address this drawback, we propose a computational method that exploits never used TF properties to discover the missing TFBMs and their sites in all human gene promoters. The method starts by predicting a dictionary of regulatory "DNA words." From this dictionary, it distills 4098 novel predictions. To disclose the crosstalk between motifs, an additional algorithm extracts TF combinatorial binding patterns creating a collection of TF regulatory syntactic rules. Using these rules, we narrowed down a list of 504 novel motifs that appear frequently in syntax patterns. We tested the predictions against 509 known motifs confirming that our system can reliably predict ab initio motifs with an accuracy of 81%-far higher than previous approaches. We found that on average, 90% of the discovered combinatorial binding patterns target at least 10 genes, suggesting that to control in an independent manner smaller gene sets, supplementary regulatory mechanisms are required. Additionally, we discovered that the new TFBMs and their combinatorial patterns convey biological meaning, targeting TFs and genes related to developmental functions. Thus, among all the possible available targets in the genome, the TFs tend to regulate other TFs and genes involved in developmental functions. We provide a comprehensive resource for regulation analysis that includes a dictionary of "DNA words," newly predicted motifs and their corresponding combinatorial patterns. Combinatorial patterns are a useful filter to discover TFBMs that play a major role in orchestrating other factors and thus, are likely to lock/unlock cellular functional clusters.

  6. Human anti-luteinizing hormone-releasing hormone antibodies in patients treated with synthetic luteinizing hormone-releasing hormone

    International Nuclear Information System (INIS)

    Meakin, J.L.; Keogh, E.J.; Martin, C.E.

    1985-01-01

    One hundred sixty-three patients who were given synthetic LH-RH therapeutically underwent monitoring of serum IgG anti-LH-RH antibodies. Five of the patients showed specific binding to antibodies. Development of anti-LH-RH antibodies was not limited to those patients with a congenital deficiency of LH-RH. Urticarial responses occurred in four patients, only one of whom had IgG antibodies. Patients who had IgG antibodies or an urticarial response underwent monitoring of their serum IgE anti-LH-RH antibodies, but none had a positive binding response. The refractory state which has been reported in patients in whom similar antibodies to LH-RH develop was not invariably observed among these patients

  7. Human antibodies to immunoglobulin A (IgA)

    International Nuclear Information System (INIS)

    Munster, P.J.J. van; Nadorp, J.H.S.M.; Shuurman, H.J.

    1978-01-01

    In the sera of 12 out of 27 individuals with IgA deficiency (serum level below 0.02 mg IgA/m) class-specific anti-IgA antibodies were demonstrated by haemagglutination. These sera showed false-positive results in a solid-phase inhibition radioimmunoassay (RIST) (apparent IgA concentration between 0.6 and 13.7 μg IgA/ml) indicating that the RIST is not an appropriate test for the analysis of serum of IgA seficient individuals. A modification of the RIST is proposed (titration RIA) that permits differentiation between low levels of IgA and class-specific anti-IgA antibodies. With this test IgA deficient individuals could be classified as those with low but detectable levels of IgA and those with class-specific anti-IgA antibodies. A computer procedure was developed to calculate both the amount and the avidity (K) of the anti-IgA antibodies and to simulate the assay system. The K value calculated from experimental points proved to be an overestimation of the K value which fitted most adequately in the simulation. The comparison of the results with clinical findings indicated a possible correlation between the amount and the avidity of the anti-IgA antibodies and the appearence of anaphylactic reactions after transfusion of IgA. (Auth.)

  8. A human monoclonal antibody drug and target discovery platform for B-cell chronic lymphocytic leukemia based on allogeneic hematopoietic stem cell transplantation and phage display

    OpenAIRE

    Baskar, Sivasubramanian; Suschak, Jessica M.; Samija, Ivan; Srinivasan, Ramaprasad; Childs, Richard W.; Pavletic, Steven Z.; Bishop, Michael R.; Rader, Christoph

    2009-01-01

    Allogeneic hematopoietic stem cell transplantation (alloHSCT) is the only potentially curative treatment available for patients with B-cell chronic lymphocytic leukemia (B-CLL). Here, we show that post-alloHSCT antibody repertoires can be mined for the discovery of fully human monoclonal antibodies to B-CLL cell-surface antigens. Sera collected from B-CLL patients at defined times after alloHSCT showed selective binding to primary B-CLL cells. Pre-alloHSCT sera, donor sera, and control sera w...

  9. Detection of anti neutrophil antibodies by radio-iodinated protein A binding test

    International Nuclear Information System (INIS)

    Cartron, J.; Muller, J.Y.; Tchernia, G.; Paule, B.; Varet, B.

    1983-01-01

    The granulocyte associated IgG in normal and neutropenic subjects has been determined by a direct quantitative assay using radiolabeled staphylococcal protein A. This assay allows to postulate an immunological mechanism to explain the neutropenia in 19 cases of neutropenias associated with malfunctions of the immune system and in 4 cases of idiopathic neutropenias. Discussed in this report is the possible interaction of immune complexes bound in vivo to the granulocytes. By an immunofluorescence test, it has been possible to detect IgG or IgM antibodies in only 52% of patients with a positive direct assay. The determination of granulocyte-associated IgG is therefore a better indicator for defining an auto-immune neutropenia than the detection of free antibodies in the sera [fr

  10. Tumor-specific binding of radiolabeled G-22 monoclonal antibody in glioma patients

    Energy Technology Data Exchange (ETDEWEB)

    Yoshida, Jun; Wakabayashi, Toshihiko; Mizuno, Masaaki; Sugita, Kenichiro; Oshima, Motoo; Tadokoro, Masanori; Sakuma, Sadayuki [Nagoya Univ. (Japan). Faculty of Medicine; Seo, Hisao

    1992-03-01

    Iodine-131-labeled G-22 monoclonal antibody F(ab'){sub 2} fragment reacting specifically with a glioma-associated surface glycoprotein was administered to 12 glioma patients to investigate its use in radioimaging of intracranial gliomas. No immediate or delayed side effects were attributable to antibody injection. Nine patients received the radiolabeled complex intravenously. The images of low-grade gliomas were generally poor and disappeared within 4 days. High-contrast images were obtained beyond the 7th day in high-grade gliomas except one case in the pineal region. Three patients received intraventricular or intratumoral administration. Clear images of all tumors were demonstrated from the 2nd until later than the 7th day. One patient with cerebrospinal fluid (CSF) dissemination of brainstem glioma demonstrated negative CSF cytology after intraventricular administration. (author).

  11. Flow cytometry-based assay to evaluate human serum MUC1-Tn antibodies

    DEFF Research Database (Denmark)

    Van Elssen, Catharina H M J; Clausen, Henrik; Germeraad, Wilfred T V

    2011-01-01

    Mucin-1 (MUC1) is a heavily O-glycosylated, transmembrane protein that is expressed on the apical surface of most secretory epithelia. In malignantly transformed epithelia, MUC1 has lost its apical distribution, is underglycosylated and is secreted into the circulation. Due to the underglycosylat......Mucin-1 (MUC1) is a heavily O-glycosylated, transmembrane protein that is expressed on the apical surface of most secretory epithelia. In malignantly transformed epithelia, MUC1 has lost its apical distribution, is underglycosylated and is secreted into the circulation. Due...... to detect antibodies binding to the underglycosylated MUC1 protein. This cellular system is complementary to the previously published methods to detect MUC1 serum antibodies, since the antibodies to the native protein are evaluated and therefore it can be effectively used for MUC1 antibody monitoring...... in vaccination studies as well as for functional assays....

  12. Radioimmunoscintigraphy of human pancreatic carcinoma xenografts in nude mice with 131I-labeled monoclonal antibody

    International Nuclear Information System (INIS)

    Tsuda, Takatoshi; Koshiba, H.; Usui, T.; Kubota, M.; Kikuchi, Kokichi; Morita, Kazuo

    1990-01-01

    Encouraged by reports of radioimmunoimaging of colorectal carcinomas and by examining an immunohistochemical report on resected pancreas cancer tissues, we studied the diagnostic potential of radioimmunoimaging with the radioiodinelabeled monoclonal antibody (MoAb; HC-1) to a human pancreas cancer cell line (HGC25) was labeled with radioiodine and injected into athymic nude mice implanted with human pancreas cancer cells. Antibody HC-1 was cleared from the circulation and accumulated significantly in the implanted tumor sites. (author)

  13. Conformational occlusion of blockade antibody