WorldWideScience

Sample records for antibody fragments specific

  1. Characterization of Tumor-Avid Antibody Fragments Genetically Engineered for Mono-Specific Radionuclide Chelation

    International Nuclear Information System (INIS)

    Quinn, T.P.

    2003-01-01

    The successful clinical application of targeted-radiopharmaceuticals depends on the development of molecules that optimize tumor specific radionuclide deposition and minimize non-specific organ irradiation. To this end, this proposal outlines a research effort to identify and evaluate novel antibodies and antibody fragments that bind breast tumors. The tumor-avid antibodies will be investigated for as imaging and therapeutic agents and to gain a better understanding of the pharmacokinetics and metabolism of radiolabeled tumor-avid antibody fragments through the use of site-specifically labeled molecules. Antibodies or antibody fragments, that bind breast carcinoma carbohydrate antigens, will be obtained from hybridoma or bacteriophage library screening. More specifically, antibody fragments that bind the carcinoma-associated Thomsen-Friedenreich (T) antigen will be radiolabeled with 99m Tc and 188 Re at a natural amino acid chelation site and will be investigated in vivo for their abilities to target human breast tumors. In addition, site-specific radiolabeled antibody fragments will be biosynthesized using misacylated suppressor tRNAs. Homogeneously radiolabeled populations of antibody fragments will be used to investigate the effects of radionuclide location and chelation chemistries on their biodistribution and metabolism. It is hypothesized that site-specifically radiolabeled antibody fragments will possess enhanced tumor imaging and therapeutic properties due to optimal label location and conjugation chemistries. New insights into the factors that govern antibody metabolism in vivo are also expected from this work. Results from these studies should enhance our ability to design and synthesize radiolabeled antibody fragments that have improved pharmacokinetic properties. The studies in this proposal involve basic research into the development of antibody-based radiopharmaceuticals, with the ultimate goal of application in humans. This type of basic nuclear

  2. 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.

  3. Human antibody fragments specific for Bothrops jararacussu venom reduce the toxicity of other Bothrops sp. venoms.

    Science.gov (United States)

    Roncolato, Eduardo Crosara; Pucca, Manuela Berto; Funayama, Jaqueline Carlos; Bertolini, Thaís Barboza; Campos, Lucas Benício; Barbosa, José Elpidio

    2013-01-01

    Approximately 20,000 snakebites are registered each year in Brazil. The classical treatment for venomous snakebite involves the administration of sera obtained from immunized horses. Moreover, the production and care of horses is costly, and the use of heterologous sera can cause hypersensitivity reactions. The production of human antibody fragments by phage display technology is seen as a means of overcoming some of these disadvantages. The studies here attempted to test human monoclonal antibodies specific to Bothrops jararacussu against other Bothrops sp. venoms, using the Griffin.1 library of human single-chain fragment-variable (scFv) phage antibodies. Using the Griffin.1 phage antibody library, this laboratory previously produced scFvs capable of inhibiting the phospholipase and myotoxic activities of Bothrops jararacussu venom. The structural and functional similarities of the various forms of phospholipase A2 (PLA₂) in Bothrops venom served as the basis for the present study wherein the effectiveness of those same scFvs were evaluated against B. jararaca, B. neuwiedi, and B. moojeni venoms. Each clone was found to recognize all three Bothrops venoms, and purified scFvs partially inhibited their in vitro phospholipase activity. In vivo assays demonstrated that the scFv clone P2B7 reduced myotoxicity and increased the survival of animals that received the test venoms. The results here indicate that the scFv P2B7 is a candidate for inclusion in a mixture of specific antibodies to produce a human anti-bothropic sera. This data demonstrates that the human scFv P2B7 represents an alternative therapeutic approach to heterologous anti-bothropic sera available today.

  4. Fab antibody fragment-functionalized liposomes for specific targeting of antigen-positive cells

    Czech Academy of Sciences Publication Activity Database

    Ohradanova-Repic, A.; Nogueira, E.; Hartl, I.; Gomes, A.C.; Preto, A.; Steinhuber, E.; Muehlgrabner, V.; Repic, M.; Kuttke, M.; Zwirzitz, A.; Prouza, M.; Suchánek, M.; Wozniak-Knopp, G.; Hořejší, Václav; Schabbauer, G.; Cavaco-Paulo, A.; Stockinger, H.

    2018-01-01

    Roč. 14, č. 1 (2018), s. 123-130 ISSN 1549-9634 Institutional support: RVO:68378050 Keywords : Active targeting * Liposome functionalization * Immunoliposome * Antibody engineering * Recombinant Fab antibody fragment Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Cell biology Impact factor: 5.720, year: 2016

  5. Isolation and characterisation of Ebolavirus-specific recombinant antibody fragments from murine and shark immune libraries.

    Science.gov (United States)

    Goodchild, Sarah A; Dooley, Helen; Schoepp, Randal J; Flajnik, Martin; Lonsdale, Stephen G

    2011-09-01

    Members of the genus Ebolavirus cause fulminating outbreaks of disease in human and non-human primate populations with a mortality rate up to 90%. To facilitate rapid detection of these pathogens in clinical and environmental samples, robust reagents capable of providing sensitive and specific detection are required. In this work recombinant antibody libraries were generated from murine (single chain variable domain fragment; scFv) and nurse shark, Ginglymostoma cirratum (IgNAR V) hosts immunised with Zaire ebolavirus. This provides the first recorded IgNAR V response against a particulate antigen in the nurse shark. Both murine scFv and shark IgNAR V libraries were panned by phage display technology to identify useful antibodies for the generation of immunological detection reagents. Two murine scFv were shown to have specificity to the Zaire ebolavirus viral matrix protein VP40. Two isolated IgNAR V were shown to bind to the viral nucleoprotein (NP) and to capture viable Zaire ebolavirus with a high degree of sensitivity. Assays developed with IgNAR V cross-reacted to Reston ebolavirus, Sudan ebolavirus and Bundibugyo ebolavirus. Despite this broad reactivity, neither of IgNAR V showed reactivity to Côte d'Ivoire ebolavirus. IgNAR V was substantially more resistant to irreversible thermal denaturation than murine scFv and monoclonal IgG in a comparative test. The demonstrable robustness of the IgNAR V domains may offer enhanced utility as immunological detection reagents in fieldable biosensor applications for use in tropical or subtropical countries where outbreaks of Ebolavirus haemorrhagic fever occur. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

  6. Site-specific chemical modification of antibody fragments using traceless cleavable linkers.

    Science.gov (United States)

    Bernardes, Gonçalo J L; Steiner, Martina; Hartmann, Isabelle; Neri, Dario; Casi, Giulio

    2013-11-01

    Antibody-drug conjugates (ADCs) are promising agents for the selective delivery of cytotoxic drugs to specific cells (for example, tumors). In this protocol, we describe two strategies for the precise modification at engineered C- or N-terminal cysteines of antibodies in IgG, diabody and small immunoprotein (SIP) formats that yield homogenous ADCs. In this protocol, cemadotin derivatives are used as model drugs, as these agents have a potent cytotoxic activity and are easy to synthesize. However, other drugs with similar functional groups could be considered. In the first approach, a cemadotin derivative containing a sulfhydryl group results in a mixed disulfide linkage. In the second approach, a cemadotin derivative containing an aldehyde group is joined via a thiazolidine linkage. The procedures outlined are robust, enabling the preparation of ADCs with a defined number of drugs per antibody in a time frame between 7 and 24 h.

  7. Baculovirus display of functional antibody Fab fragments.

    Science.gov (United States)

    Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

    2015-08-01

    The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

  8. The Antiviral Mechanism of an Influenza A Virus Nucleoprotein-Specific Single-Domain Antibody Fragment

    Energy Technology Data Exchange (ETDEWEB)

    Hanke, Leo; Knockenhauer, Kevin E.; Brewer, R. Camille; van Diest, Eline; Schmidt, Florian I.; Schwartz, Thomas U.; Ploegh, Hidde L. (Whitehead); (MIT)

    2016-12-13

    Alpaca-derived single-domain antibody fragments (VHHs) that target the influenza A virus nucleoprotein (NP) can protect cells from infection when expressed in the cytosol. We found that one such VHH, αNP-VHH1, exhibits antiviral activity similar to that of Mx proteins by blocking nuclear import of incoming viral ribonucleoproteins (vRNPs) and viral transcription and replication in the nucleus. We determined a 3.2-Å crystal structure of αNP-VHH1 in complex with influenza A virus NP. The VHH binds to a nonconserved region on the body domain of NP, which has been associated with binding to host factors and serves as a determinant of host range. Several of the NP/VHH interface residues determine sensitivity of NP to antiviral Mx GTPases. The structure of the NP/αNP-VHH1 complex affords a plausible explanation for the inhibitory properties of the VHH and suggests a rationale for the antiviral properties of Mx proteins. Such knowledge can be leveraged for much-needed novel antiviral strategies.

    IMPORTANCEInfluenza virus strains can rapidly escape from protection afforded by seasonal vaccines or acquire resistance to available drugs. Additional ways to interfere with the virus life cycle are therefore urgently needed. The influenza virus nucleoprotein is one promising target for antiviral interventions. We have previously isolated alpaca-derived single-domain antibody fragments (VHHs) that protect cells from influenza virus infection if expressed intracellularly. We show here that one such VHH exhibits antiviral activities similar to those of proteins of the cellular antiviral defense (Mx proteins). We determined the three-dimensional structure of this VHH in complex with the influenza virus nucleoprotein and identified the interaction site, which overlaps regions that determine sensitivity of the virus to Mx proteins. Our data define a new vulnerability of influenza virus, help us to better understand the cellular antiviral mechanisms, and

  9. Selection and Characterization of Single Chain Antibody Fragments Specific for Hsp90 as a Potential Cancer Targeting Molecule

    Directory of Open Access Journals (Sweden)

    Edyta Petters

    2015-08-01

    Full Text Available Heat shock proteins play an essential role in facilitating malignant transformation and they have been recognized as important factors in human cancers. One of the key elements of the molecular chaperones machinery is Hsp90 and it has recently become a target for anticancer therapeutic approaches. The potential and importance of Hsp90-directed agents becomes apparent when one realizes that disruption of Hsp90 function may influence over 200 oncogenic client proteins. Here, we described the selection and characterization of Hsp90-specific antibody fragments from commercially available Tomlinson I and J phage display libraries. The affinities of Hsp90-binding scFv variants were measured using SPR method. Then, based on the best clone selected, we performed the affinity maturation procedure and obtained valuable Hsp90-specific clones. The selected binders were expressed and applied for immunostaining, ELISA and SPR analysis using model cancer cell lines. All performed experiments confirmed the ability of selected antibodies to interact with the Hsp90. Therefore, the presented Hsp90-specific scFv, might be a starting point for the development of a novel antibody-based strategy targeting cancer.

  10. Construction and expression of a functional monoclonal antibody SZ-51 specific for GMP-140 chimeric fab fragment in Escherichia coli

    International Nuclear Information System (INIS)

    Gu Jianming; Zhang Xiaomin; Xia Lijun; Wan Haiying; Liu Yue; Li Peixia; Ruan Changgeng

    1996-04-01

    The variable region cDNAs of a monoclonal antibody SZ-51 specific for α-granule membrane protein (GMP-140) on the surface of activated human platelets were spliced with the constant region cDNA of the heavy chain CH1 and light chain k of human Ig G by means of the gene recombination techniques. The above recombinant gene was amplified by the polymerase chain reaction (PCR). The expression vector of phage plasmid pHEN1 SZ-51 Fab/Hu was constructed. The pHEN1-51 Fab/Hu was introduced into non-suppressor E. coli HB2151. The amount of expression of SZ-51 chimeric Fab/Hu measured by quantitative ELISA was about 500 μg/L. Western blot demonstrated that the SZ-51 chimeric Fab fragment could specifically bind to GMP-140. (2 figs.)

  11. Refolding Technologies for Antibody Fragments

    Directory of Open Access Journals (Sweden)

    Tsutomu Arakawa

    2014-05-01

    Full Text Available Refolding is one of the production technologies for pharmaceutical grade antibody fragments. Detergents and denaturants are primarily used to solubilize the insoluble proteins. The solubilized and denatured proteins are refolded by reducing the concentration of the denaturants or detergents. Several refolding technologies have been used for antibody fragments, comprising dilution, dialysis, solid phase solvent exchange and size exclusion chromatography, as reviewed here. Aggregation suppressor or folding-assisting agents, including arginine hydrochloride, ionic liquids and detergents or denaturants at low concentrations, are included in the refolding solvent to enhance refolding yield.

  12. Isolation of Mal d 1 and Api g 1 - specific recombinant antibodies from mouse IgG Fab fragment libraries - Mal d 1-specific antibody exhibits cross-reactivity against Bet v 1.

    Science.gov (United States)

    Haka, Jaana; Niemi, Merja H; Iljin, Kristiina; Reddy, Vanga Siva; Takkinen, Kristiina; Laukkanen, Marja-Leena

    2015-05-27

    Around 3-5% of the population suffer from IgE-mediated food allergies in Western countries and the number of food-allergenic people is increasing. Individuals with certain pollen allergies may also suffer from a sensitisation to proteins in the food products. As an example a person sensitised to the major birch pollen allergen, Bet v 1, is often sensitised to its homologues, such as the major allergens of apple, Mal d 1, and celery, Api g 1, as well. Development of tools for the reliable, sensitive and quick detection of allergens present in various food products is essential for allergic persons to prevent the consumption of substances causing mild and even life-threatening immune responses. The use of monoclonal antibodies would ensure the specific detection of the harmful food content for a sensitised person. Mouse IgG antibody libraries were constructed from immunised mice and specific recombinant antibodies for Mal d 1 and Api g 1 were isolated from the libraries by phage display. More detailed characterisation of the resulting antibodies was carried out using ELISA, SPR experiments and immunoprecipitation assays. The allergen-specific Fab fragments exhibited high affinity towards the target recombinant allergens. Furthermore, the Fab fragments also recognised native allergens from natural sources. Interestingly, isolated Mal d 1-specific antibody bound also to Bet v 1, the main allergen eliciting the cross-reactivity syndrome between the birch pollen and apple. Despite the similarities in Api g 1 and Bet v 1 tertiary structures, the isolated Api g 1-specific antibodies showed no cross-reactivity to Bet v 1. Here, high-affinity allergen-specific recombinant antibodies were isolated with interesting binding properties. With further development, these antibodies can be utilised as tools for the specific and reliable detection of allergens from different consumable products. This study gives new preliminary insights to elucidate the mechanism behind the pollen

  13. In vitro neutralisation of rotavirus infection by two broadly specific recombinant monovalent llama-derived antibody fragments

    NARCIS (Netherlands)

    F. Aladin (Farah); A.W.C. Einerhand (Sandra); J. Bouma (Janneke); S. Bezemer (Sandra); P. Hermans (Pim); D. Wolvers (Danielle); K. Bellamy (Kate); L.G.J. Frenken (Leon); J. Gray (Jim); M. Iturriza-Gómara (Miren)

    2012-01-01

    textabstractRotavirus is the main cause of viral gastroenteritis in young children. Therefore, the development of inexpensive antiviral products for the prevention and/or treatment of rotavirus disease remains a priority. Previously we have shown that a recombinant monovalent antibody fragment

  14. Specific Conjugation of the Hinge Region for Homogeneous Preparation of Antibody Fragment-Drug Conjugate: A Case Study for Doxorubicin-PEG-anti-CD20 Fab' Synthesis.

    Science.gov (United States)

    Zhou, Zhan; Zhang, Jing; Zhang, Yan; Ma, Guanghui; Su, Zhiguo

    2016-01-20

    Conventional preparation strategies for antibody-drug conjugates (ADCs) result in heterogeneous products with various molecular sizes and species. In this study, we developed a homogeneous preparation strategy by site-specific conjugation of the anticancer drug with an antibody fragment. The model drug doxorubicin (DOX) was coupled to the Fab' fragment of anti-CD20 IgG at its permissive sites through a heterotelechelic PEG linker, generating an antibody fragment-drug conjugate (AFDC). Anti-CD20 IgG was digested and reduced specifically with β-mercaptoethylamine to generate the Fab' fragment with two free mercapto groups in its hinge region. Meanwhile, DOX was conjugated with α-succinimidylsuccinate ω-maleimide polyethylene glycol (NHS-PEG-MAL) to form MAL-PEG-DOX, which was subsequently linked to the free mercapto containing Fab' fragment to form a Fab'-PEG-DOX conjugate. The dual site-specific bioconjugation was achieved through the combination of highly selective reduction of IgG and introduction of heterotelechelic PEG linker. The resulting AFDC provides an utterly homogeneous product, with a definite ratio of one fragment to two drugs. Laser confocal microscopy and cell ELISA revealed that the AFDC could accumulate in the antigen-positive Daudi tumor cell. In addition, the Fab'-PEG-DOX retained appreciable targeting ability and improved antitumor activity, demonstrating an excellent therapeutic effect on the lymphoma mice model for better cure rate and significantly reduced side effects.

  15. Selective targeting of tumour neovasculature by a radiohalogenated human antibody fragment specific for the ED-B domain of fibronectin

    International Nuclear Information System (INIS)

    Demartis, S.; Tarli, L.; Neri, D.; Borsi, L.; Zardi, L.

    2001-01-01

    Angiogenesis is a characteristic feature of many aggressive tumours and other disorders. Antibodies capable of binding to new blood vessels, but not to mature vessels, could be used as selective targeting agents for immunoscintigraphic and radioimmunotherapeutic applications. Here we show that scFv(L19), a recombinant human antibody fragment with sub-nanomolar affinity for the ED-B domain of fibronectin, a marker of angiogenesis, can be stably labelled with iodine-125 and astatine-211 with full retention of immunoreactivity, using a trimethyl-stannyl benzoate bifunctional derivative. Biodistribution studies in mice bearing two different types of tumour grafted subcutaneously, followed by ex vivo micro-autoradiographic analysis, revealed that scFv(L19) rapidly localises around tumour blood vessels, but not around normal vessels. Four hours after intravenous injection of the stably radioiodinated scFv(L19), tumour to blood ratios were 6:1 in mice bearing the F9 murine teratocarcinoma and 9:1 in mice bearing an FE8 rat sarcoma. As expected, all other organs (including kidney) contained significantly less radioactivity than the tumour. Since the ED-B domain of fibronectin has an identical sequence in mouse and man, scFv(L19) is a pan-species antibody and the results presented here suggest clinical utility of radiolabelled scFv(L19) for the scintigraphic detection of angiogenesis in vivo. Furthermore, it should now be possible to investigate scFv(L19) for the selective delivery of 211 At to the tumour neovasculature, causing the selective death of tumour endothelial cells and tumour collapse. (orig.)

  16. An efficient method for isolating antibody fragments against small peptides by antibody phage display

    DEFF Research Database (Denmark)

    Duan, Zhi; Siegumfeldt, Henrik

    2010-01-01

    We generated monoclonal scFv (single chain variable fragment) antibodies from an antibody phage display library towards three small synthetic peptides derived from the sequence of s1-casein. Key difficulties for selection of scFv-phages against small peptides were addressed. Small peptides do....... The scFvs were sequenced and characterized, and specificity was characterized by ELISA. The methods developed in this study are universally applicable for antibody phage display to efficiently produce antibody fragments against small peptides....

  17. Escherichia coli F4 fimbriae specific lama single-domain antibody fragments effectively inhibit bacterial adhesion in vitro but poorly protect against diarrhea

    NARCIS (Netherlands)

    Harmsen, M.M.; Solt, van C.B.; Hoogendoorn, A.; Zijderveld, van F.G.; Niewold, T.A.; Meulen, van der J.

    2005-01-01

    Oral administration of polyclonal antibodies directed against enterotoxigenic Escherichia coli (ETEC) F4 fimbriae is used to protect against piglet post-weaning diarrhoea. For cost reasons, we aim to replace these polyclonal antibodies by recombinant llama single-domain antibody fragments (VHHs)

  18. Microbials for the production of monoclonal antibodies and antibody fragments.

    Science.gov (United States)

    Spadiut, Oliver; Capone, Simona; Krainer, Florian; Glieder, Anton; Herwig, Christoph

    2014-01-01

    Monoclonal antibodies (mAbs) and antibody fragments represent the most important biopharmaceutical products today. Because full length antibodies are glycosylated, mammalian cells, which allow human-like N-glycosylation, are currently used for their production. However, mammalian cells have several drawbacks when it comes to bioprocessing and scale-up, resulting in long processing times and elevated costs. By contrast, antibody fragments, that are not glycosylated but still exhibit antigen binding properties, can be produced in microbial organisms, which are easy to manipulate and cultivate. In this review, we summarize recent advances in the expression systems, strain engineering, and production processes for the three main microbials used in antibody and antibody fragment production, namely Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Crystallization and preliminary X-ray diffraction analysis of the Fab fragment of WO2, an antibody specific for the Aβ peptides associated with Alzheimer’s disease

    Energy Technology Data Exchange (ETDEWEB)

    Wun, Kwok S. [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Miles, Luke A. [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Department of Chemical and Biomolecular Engineering, The University of Melbourne, Victoria 3010 (Australia); Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 30 Flemington Road, Parkville, Victoria 3010 (Australia); Crespi, Gabriela A. N. [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Wycherley, Kaye [WEHI Biotechnology Centre, La Trobe R& D Park, Bundoora, Victoria 3086 (Australia); Ascher, David B. [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Barnham, Kevin J.; Cappai, Roberto [Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 30 Flemington Road, Parkville, Victoria 3010 (Australia); Department of Pathology, The University of Melbourne, Victoria 3010 (Australia); The Mental Health Research Institute of Victoria, Parkville, Victoria 3052 (Australia); Beyreuther, Konrad [ZMBH, University of Heidelberg, Heidelberg (Germany); Masters, Colin L. [Department of Pathology, The University of Melbourne, Victoria 3010 (Australia); The Mental Health Research Institute of Victoria, Parkville, Victoria 3052 (Australia); Parker, Michael W. [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 30 Flemington Road, Parkville, Victoria 3010 (Australia); McKinstry, William J., E-mail: wjmckinstry@hotmail.com [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Department of Medicine (St Vincent’s Hospital), The University of Melbourne, 41 Victoria Parade, Fitzroy 3065 (Australia)

    2008-05-01

    Crystallization and X-ray diffraction data collection of the Fab fragment of the monoclonal antibody WO2 in the absence or presence of amyloid β peptides associated with Alzheimer’s disease are reported. The murine monoclonal antibody WO2 specifically binds the N-terminal region of the amyloid β peptide (Aβ) associated with Alzheimer’s disease. This region of Aβ has been shown to be the immunodominant B-cell epitope of the peptide and hence is considered to be a basis for the development of immunotherapeutic strategies against this prevalent cause of dementia. Structural studies have been undertaken in order to characterize the molecular basis for antibody recognition of this important epitope. Here, details of the crystallization and X-ray analysis of the Fab fragment of the unliganded WO2 antibody in two crystal forms and of the complexes that it forms with the truncated Aβ peptides Aβ{sub 1–16} and Aβ{sub 1–28} are presented. These crystals were all obtained using the hanging-drop vapour-diffusion method at 295 K. Crystals of WO2 Fab were grown in polyethylene glycol solutions containing ZnSO{sub 4}; they belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1} and diffracted to 1.6 Å resolution. The complexes of WO2 Fab with either Aβ{sub 1–@}@{sub 16} or Aβ{sub 1–28} were cocrystallized from polyethylene glycol solutions. These two complex crystals grew in the same space group, P2{sub 1}2{sub 1}2{sub 1}, and diffracted to 1.6 Å resolution. A second crystal form of WO2 Fab was grown in the presence of the sparingly soluble Aβ{sub 1–42} in PEG 550 MME. This second form belonged to space group P2{sub 1} and diffracted to 1.9 Å resolution.

  20. 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

  1. Effects of Fab' fragments of specific egg yolk antibody (IgY-Fab') against Shewanella putrefaciens on the preservation of refrigerated turbot.

    Science.gov (United States)

    Zhang, Qian; Lin, Hong; Sui, Jianxin; Wang, Jingxue; Cao, Limin

    2015-01-01

    In our previous studies the specific egg yolk antibody (IgY) against Shewanella putrefaciens (one of the specific spoilage organisms for marine products during aerobic chilling storage) demonstrated significant activity to prolong the shelf life of refrigerated fish. The exploitation of the antigen-binding fragment plus the hinge region (IgY-Fab') is now considered a promising method for improving the efficiency of such natural antimicrobial agents. The antimicrobial activity of IgY-Fab' against S. putrefaciens was investigated using refrigerated turbot as samples. By microbial, chemical and sensory tests, it was shown to be able to effectively inhibit bacterial growth and prolong the shelf life of samples, with an efficiency evaluated significantly higher than that of whole IgY with the same molarity. The interaction between IgY agents and S. putrefaciens cells was also investigated, and the IgY-Fab' showed a much greater ability to damage cell membranes than the whole IgY. Compared to whole IgY with the same molarity, IgY-Fab' demonstrated higher and more durable antimicrobial efficiency. Such a result was assumed to be closely related to its structural properties (such as the much lower molecular weight), which may enhance its ability to influence physiological activities of antigen bacteria, especially the property or/and structure of cell membranes. © 2014 Society of Chemical Industry.

  2. Structural Basis for the Specific Neutralization of Stx2a with a Camelid Single Domain Antibody Fragment

    Directory of Open Access Journals (Sweden)

    Robert Alvin Bernedo-Navarro

    2018-03-01

    Full Text Available Background: Shiga toxin-producing Escherichia coli (STEC are a subset of pathogens leading to illnesses such as diarrhea, hemolytic uremic syndrome and even death. The Shiga toxins are the main virulence factors and divided in two groups: Stx1 and Stx2, of which the latter is more frequently associated with severe pathologies in humans. Results: An immune library of nanobodies (Nbs was constructed after immunizing an alpaca with recombinant Shiga toxin-2a B subunit (rStx2aB, to retrieve multiple rStx2aB-specific Nbs. The specificity of five Nbs towards rStx2aB was confirmed in ELISA and Western blot. Nb113 had the highest affinity (9.6 nM and its bivalent construct exhibited a 100-fold higher functional affinity. The structure of the Nb113 in complex with rStx2aB was determined via X-ray crystallography. The crystal structure of the Nb113–rStx2aB complex revealed that five copies of Nb113 bind to the rStx2aB pentamer and that the Nb113 epitope overlaps with the Gb3 binding site, thereby providing a structural basis for the neutralization of Stx2a by Nb113 that was observed on Vero cells. Finally, the tandem-repeated, bivalent Nb1132 exhibits a higher toxin neutralization capacity compared to monovalent Nb113. Conclusions: The Nb of highest affinity for rStx2aB is also the best Stx2a and Stx2c toxin neutralizing Nb, especially in a bivalent format. This lead Nb neutralizes Stx2a by competing for the Gb3 receptor. The fusion of the bivalent Nb1132 with a serum albumin specific Nb is expected to combine high toxin neutralization potential with prolonged blood circulation.

  3. 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.

  4. How protein recognizes ladder-like polycyclic ethers. Interactions between ciguatoxin (CTX3C) fragments and its specific antibody 10C9.

    Science.gov (United States)

    Ui, Mihoko; Tanaka, Yoshikazu; Tsumuraya, Takeshi; Fujii, Ikuo; Inoue, Masayuki; Hirama, Masahiro; Tsumoto, Kouhei

    2008-07-11

    Ciguatoxins are a family of marine toxins composed of transfused polycyclic ethers. It has not yet been clarified at the atomic level on the pathogenic mechanism of these toxins or the interaction between a polycyclic ether compounds and a protein. Using the crystal structures of anti-ciguatoxin antibody 10C9 Fab in ligand-free form and in complexes with ABCD-ring (CTX3C-ABCD) and ABCDE-ring (CTX3C-ABCDE) fragments of the antigen CTX3C at resolutions of 2.6, 2.4, and 2.3 angstroms, respectively, we elucidated the mechanism of the interaction between the polycyclic ethers and the antibody. 10C9 Fab has an extraordinarily large and deep binding pocket at the center of the variable region, where CTX3C-ABCD or CTX3C-ABCDE binds longitudinally in the pocket via hydrogen bonds and van der Waals interactions. Upon antigen-antibody complexation, 10C9 Fab adjusts to the antigen fragments by means of rotational motion in the variable region. In addition, the antigen fragment lacking the E-ring induces a large motion in the constant region. Consequently, the thermostability of 10C9 Fab is enhanced by 10 degrees C upon complexation with CTX3C-ABCDE but not with CTX3C-ABCD. The crystal structures presented in this study also show that 10C9 Fab recoginition of CTX3C antigens requires molecular rearrangements over the entire antibody structure. These results further expand the fundamental understanding of the mechanism by which ladder-like polycyclic ethers are recognized and may be useful for the design of novel therapeutic agents by antibodies, marine toxins, or new diagnostic reagents for the detection and targeting of members of the polycyclic ether family.

  5. Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single-Domain Antibody Fragments.

    Directory of Open Access Journals (Sweden)

    Nidiane D R Prado

    Full Text Available Antivenoms, produced using animal hyperimmune plasma, remains the standard therapy for snakebites. Although effective against systemic damages, conventional antivenoms have limited efficacy against local tissue damage. Additionally, the hypersensitivity reactions, often elicited by antivenoms, the high costs for animal maintenance, the difficulty of producing homogeneous lots, and the instability of biological products instigate the search for innovative products for antivenom therapy. In this study, camelid antibody fragments (VHH with specificity to Bothropstoxin I and II (BthTX-I and BthTX-II, two myotoxic phospholipases from Bothrops jararacussu venom, were selected from an immune VHH phage display library. After biopanning, 28 and 6 clones recognized BthTX-I and BthTX-II by ELISA, respectively. Complementarity determining regions (CDRs and immunoglobulin frameworks (FRs of 13 VHH-deduced amino acid sequences were identified, as well as the camelid hallmark amino acid substitutions in FR2. Three VHH clones (KF498607, KF498608, and KC329718 were capable of recognizing BthTX-I by Western blot and showed affinity constants in the nanomolar range against both toxins. VHHs inhibited the BthTX-II phospholipase A2 activity, and when tested for cross-reactivity, presented specificity to the Bothrops genus in ELISA. Furthermore, two clones (KC329718 and KF498607 neutralized the myotoxic effects induced by B. jararacussu venom, BthTX-I, BthTX-II, and by a myotoxin from Bothrops brazili venom (MTX-I in mice. Molecular docking revealed that VHH CDRs are expected to bind the C-terminal of both toxins, essential for myotoxic activity, and to epitopes in the BthTX-II enzymatic cleft. Identified VHHs could be a biotechnological tool to improve the treatment for snake envenomation, an important and neglected world public health problem.

  6. Noninvasive detection of cardiac repair after acute myocardial infarction in rats by 111In fab fragment of monoclonal antibody specific for tenascin-C

    International Nuclear Information System (INIS)

    Okada, Kenichi; Uehara, Tomoya; Arano, Yasushi

    2008-01-01

    Left ventricular (LV) remodeling after acute myocardial infarction (MI) causes heart failure, and thus it is important to evaluate cardiac repair as the early stage of LV remodeling. Tenascin-C (TNC), an extracellular matrix glycoprotein, is transiently and abundantly expressed in the heart during the early stage of tissue remodeling after MI. However, it is not expressed in healthy adult heart. This study was undertaken to develop a new noninvasive diagnostic technique to detect cardiac repair after acute MI using 111 In Fab fragment of a monoclonal antibody specific for TNC. 111 In-anti-TNC-Fab was injected intravenously in 13 rats at 1 (D1, n=3), 3 (D3, n=5), and 5 (D5, n=5) days after producing MI and in 5 sham-operated rats (S). We performed autoradiography and dual-isotope single-photon emission computed tomography imaging (SPECT) of 111 In-anti-TNC-Fab and 99m Tc methoxyisobutyl isonitrile (MIBI). The radioactivity in the heart was significantly higher in D (D1, 0.45±0.06% injected-dose/g; D3, 0.64±0.12; D5, 0.38±0.07) than S (0.27±0.06, P 111 In-anti-TNC-Fab, which was complementary to the perfusion image. The results of the present study indicated that we can localize the infarcted region in the heart by ex vivo and in vivo imaging methods using 111 In-anti-TNC-Fab, and suggested the potential usefulness of noninvasive detection of cardiac repair. (author)

  7. Enhancement of antitumor activity by using a fully human gene encoding a single-chain fragmented antibody specific for carcinoembryonic antigen

    Directory of Open Access Journals (Sweden)

    Shibaguchi H

    2017-08-01

    Full Text Available Hirotomo Shibaguchi,1,* Naixiang Luo,1,* Naoto Shirasu,1,* Motomu Kuroki,2 Masahide Kuroki1 1Department of Biochemistry, Faculty of Medicine, Fukuoka University, Fukuoka, Japan; 2School of Nursing, Faculty of Medicine, Fukuoka University, Fukuoka, Japan *These authors equally contributed to this work Abstract: Human leukocyte antigen and/or costimulatory molecules are frequently lacking in metastatic tumor cells, and thus tumor cells are able to escape from the immune system. Although lymphocytes with a chimeric antigen receptor (CAR is a promising approach for overcoming this challenge in cancer immunotherapy, administration of modified T cells alone often demonstrates little efficacy in patients. Therefore, in order to enhance the antitumor activity of immune cells in the cancer microenvironment, we used lymphocytes expressing CAR in combination with a fusion protein of IL-2 that contained the single-chain fragmented antibody (scFv specific for the carcinoembryonic antigen. Among a series of CAR constructs, with or without a spacer and the intracellular domain of CD28, the CAR construct containing CD8α, CD28, and CD3ζ most effectively activated and expressed INF-γ in CAR-bearing T cells. Furthermore, in comparison with free IL-2, the combination of peripheral blood mononuclear cells expressing CAR and the fusion protein containing IL-2 significantly enhanced the antitumor activity against MKN-45 cells, a human gastric cancer cell line. In conclusion, this novel combination therapy of CAR and a fusion protein consisting of a functional cytokine and a fully human scFv may be a promising approach for adoptive cancer immunotherapy. Keywords: chimeric antigen receptor, fusion protein, human scFv, CEA, combination therapy

  8. Microbial platform technology for recombinant antibody fragment production: A review.

    Science.gov (United States)

    Gupta, Sanjeev Kumar; Shukla, Pratyoosh

    2017-02-01

    Recombinant antibody fragments are being used for the last few years as an important therapeutic protein to cure various critical and life threatening human diseases. Several expression platforms now days employed for the production of these recombinant fragments, out of which bacterial system has emerged a promising host for higher expression. Since, a small antibody fragment unlike full antibody does not require human-like post-translational modification therefore it is potentially expressed in prokaryotic production system. Recently, small antibody fragments such as scFvs (single-chain variable fragments) and Fabs (antibody fragments) which does not require glycosylation are successfully produced in bacteria and have commercially launched for therapeutic use as these fragments shows better tissue penetration and less immunogenic to human body compared to full-size antibody. Recently developed Wacker's ESETEC secretion technology is an efficient technology for the expression and secretion of the antibody fragment (Fab) exceeded up to 4.0 g/L while scFv up to 3.5 g/L into the fermentation broth. The Pfenex system and pOP prokaryotic expression vector are another platform used for the considerably good amount of antibody fragment production successfully. In this review, we summarize the recent progress on various expression platforms and cloning approaches for the production of different forms of antibody fragments in E. coli.

  9. Generation of a rabbit single-chain fragment variable (scFv) antibody for specific detection of Bradyrhizobium sp. DOA9 in both free-living and bacteroid forms.

    Science.gov (United States)

    Vu, Nguyen Xuan; Pruksametanan, Natcha; Srila, Witsanu; Yuttavanichakul, Watcharin; Teamtisong, Kamonluck; Teaumroong, Neung; Boonkerd, Nantakorn; Tittabutr, Panlada; Yamabhai, Montarop

    2017-01-01

    A simple and reliable method for the detection of specific nitrogen-fixing bacteria in both free-living and bacteroid forms is essential for the development and application of biofertilizer. Traditionally, a polyclonal antibody generated from an immunized rabbit was used for detection. However, the disadvantages of using a polyclonal antibody include limited supply and cross-reactivity to related bacterial strains. This is the first report on the application of phage display technology for the generation of a rabbit recombinant monoclonal antibody for specific detection and monitoring of nitrogen-fixing bacteria in both free-living form and in plant nodules. Bradyrhizobium sp. DOA9, a broad host range soil bacteria, originally isolated from the root nodules of Aeschynomene americana in Thailand was used as a model in this study. A recombinant single-chain fragment variable (scFv) antibody library was constructed from the spleen of a rabbit immunized with DOA9. After three rounds of biopanning, one specific phage-displayed scFv antibody, designated bDOA9rb8, was identified. Specific binding of this antibody was confirmed by phage enzyme-linked immunosorbent assay (phage ELISA). The phage antibody could bind specifically to DOA9 in both free-living cells (pure culture) and bacteroids inside plant nodules. In addition to phage ELISA, specific and robust immunofluorescence staining of both free-living and bacteroid forms could also be observed by confocal-immunofluorescence imaging, without cross-reactivity with other tested bradyrhizobial strains. Moreover, specific binding of free scFv to DOA9 was also demonstrated by ELISA. This recombinant antibody can also be used for the study of the molecular mechanism of plant-microbe interactions in the future.

  10. Cloning, bacterial expression and crystallization of Fv antibody fragments

    Science.gov (United States)

    E´, Jean-Luc; Boulot, Ginette; Chitarra, V´ronique; Riottot, Marie-Madeleine; Souchon, H´le`ne; Houdusse, Anne; Bentley, Graham A.; Narayana Bhat, T.; Spinelli, Silvia; Poljak, Roberto J.

    1992-08-01

    The variable Fv fragments of antibodies, cloned in recombinant plasmids, can be expressed in bacteria as functional proteins having immunochemical properties which are very similar or identical with those of the corresponding parts of the parent eukaryotic antibodies. They offer new possibilities for the study of antibody-antigen interactions since the crystals of Fv fragments and of their complexes with antigen reported here diffract X-rays to a higher resolution that those obtained with the cognate Fab fragments. The Fv approach should facilitate the structural study of the combining site of antibodies and the further characterization of antigen-antibody interactions by site-directed mutagenesis experiments.

  11. A simple and robust approach to immobilization of antibody fragments.

    Science.gov (United States)

    Ikonomova, Svetlana P; He, Ziming; Karlsson, Amy J

    2016-08-01

    Antibody fragments, such as the single-chain variable fragment (scFv), have much potential in research and diagnostics because of their antigen-binding ability similar to a full-sized antibody and their ease of production in microorganisms. Some applications of antibody fragments require immobilization on a surface, and we have established a simple immobilization method that is based on the biotin-streptavidin interaction and does not require a separate purification step. We genetically fused two biotinylation tags-the biotin carboxyl carrier protein (BCCP) or the AviTag minimal sequence-to six different scFvs (scFv13R4, scFvD10, scFv26-10, scFv3, scFv5, and scFv12) for site-specific biotinylation in vivo by endogenous biotin ligases produced by Escherichia coli. The biotinylated scFvs were immobilized onto streptavidin-coated plates directly from cell lysates, and immobilization was detected through enzyme-linked immunosorbent assays. All scFvs fusions were successfully immobilized, and scFvs biotinylated via the BCCP tag tended to immobilize better than those biotinylated via the AviTag, even when biotinylation efficiency was improved with the biotin ligase BirA. The ability of immobilized scFvs to bind antigens was confirmed using scFv13R4 and scFvD10 with their respective targets β-galactosidase and bacteriophage lambda head protein D (gpD). The immobilized scFv13R4 bound to β-galactosidase at the same level for both biotinylation tags when the surface was saturated with the scFv, and immobilized scFvs retained their functionality for at least 100days after immobilization. The simplicity and robustness of our method make it a promising approach for future applications that require antibody fragment immobilization. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Cell-free synthesis of functional antibody fragments to provide a structural basis for antibody-antigen interaction.

    Directory of Open Access Journals (Sweden)

    Takayoshi Matsuda

    Full Text Available Growing numbers of therapeutic antibodies offer excellent treatment strategies for many diseases. Elucidation of the interaction between a potential therapeutic antibody and its target protein by structural analysis reveals the mechanism of action and offers useful information for developing rational antibody designs for improved affinity. Here, we developed a rapid, high-yield cell-free system using dialysis mode to synthesize antibody fragments for the structural analysis of antibody-antigen complexes. Optimal synthesis conditions of fragments (Fv and Fab of the anti-EGFR antibody 059-152 were rapidly determined in a day by using a 30-μl-scale unit. The concentration of supplemented disulfide isomerase, DsbC, was critical to obtaining soluble antibody fragments. The optimal conditions were directly applicable to a 9-ml-scale reaction, with linear scalable yields of more than 1 mg/ml. Analyses of purified 059-152-Fv and Fab showed that the cell-free synthesized antibody fragments were disulfide-bridged, with antigen binding activity comparable to that of clinical antibodies. Examination of the crystal structure of cell-free synthesized 059-152-Fv in complex with the extracellular domain of human EGFR revealed that the epitope of 059-152-Fv broadly covers the EGF binding surface on domain III, including residues that formed critical hydrogen bonds with EGF (Asp355EGFR, Gln384EGFR, H409EGFR, and Lys465EGFR, so that the antibody inhibited EGFR activation. We further demonstrated the application of the cell-free system to site-specific integration of non-natural amino acids for antibody engineering, which would expand the availability of therapeutic antibodies based on structural information and rational design. This cell-free system could be an ideal antibody-fragment production platform for functional and structural analysis of potential therapeutic antibodies and for engineered antibody development.

  13. Designer genes. Recombinant antibody fragments for biological imaging

    Energy Technology Data Exchange (ETDEWEB)

    Wu, A.M.; Yazaki, P.J. [Beckman Research Institute of the City of Hope, Duarte, CA (United States). Dept. of Molecular Biology

    2000-09-01

    Monoclonal antibodies (MAbs), with high specificity and high affinity for their target antigens, can be utilized for delivery of agents such as radionuclides, enzymes, drugs or toxins in vivo. However, the implementation of radiolabeled antibodies as magic bullets for detection and treatment of diseases such as cancer has required addressing several shortcomings of murine MAbs. These include their immunogenicity, sub-optimal targeting and pharmacokinetic properties, and practical issues of production and radiolabeling. Genetic engineering provides a powerful approach for redesigning antibodies for use in oncologic applications in vivo. Recombinant fragments have been produced that retain high affinity for target antigens, and display a combination of rapid, high-level tumor targeting with concomitant clearance from normal tissues and the circulation in animal models. An important first step was cloning and engineering of antibody heavy and light chain variable domains into single-chain Fvs (molecular weight, 25-17 kDa), in which the variable regions are joined via a synthetic linker peptide sequence. Although scFvs themselves showed limited tumor uptake in preclinical and clinical studies, they provide a useful building block for intermediate sized recombinant fragments. Covalently linked dimers or non-covalent dimers of scFvs (also known as diabodies) show improved targeting and clearance properties due to their higher molecular weight (55kDa) and increased avidity. Further gains can be made by generation of larger recombinant fragments, such as the minibody, an scFv-C{sub H}3 fusion protein that self-assembles into a bivalent dimer of 80 kDa. A systematic evaluation of scFv, diabody, minibody, and intact antibody (based on comparison of tumor uptakes, tumor: blood activity ratios, and calculation of an Imaging Figure of Merit) can form the basis for selection of combinations of recombinant fragments and radionuclides for imaging applications. Ease of engineering

  14. The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi

    NARCIS (Netherlands)

    Joosten, V.; Lokman, C.; Hondel, C.A.M.J.J. van den; Punt, P.J.

    2003-01-01

    In this review we will focus on the current status and views concerning the production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi. We will focus on single-chain antibody fragment production (scFv and VHH) by these lower eukaryotes and the possible applications

  15. Designer genes. Recombinant antibody fragments for biological imaging

    International Nuclear Information System (INIS)

    Wu, A.M.; Yazaki, P.J.

    2000-01-01

    Monoclonal antibodies (MAbs), with high specificy and high affinity for their target antigens, can be utilized for delivery of agents such as radionuclides, enzymes, drugs or toxins in vivo. However, the implementation of radiolabeled antibodies as magic bullets for detection and treatment of diseases such as cancer has required addressing several shortcomings of murine MAbs. These include their immunogenicity, sub-optimal targeting and pharmacokinetic properties, and practical issues of production and radiolabeling. Genetic engineering provides a powerful approach for redesigning antibodies for use in oncologic applications in vivo. Recombinant fragments have been produced that retain high affinity for target antigens, and display a combination of rapid, high-level tumor targeting with concomitant clearance from normal tissues and the circulation in animal models. An important first step was cloning and engineering of antibody heavy and light chain variable domains into single-chain Fvs (molecular weight, 25-17 kDa), in which the variable regions are joined via a synthetic linker peptide sequence. Although scFvs themselves showed limited tumor uptake in preclinical and clinical studies, they provide a useful building block for intermediate sized recombinant fragments. Covalently linked dimers or non-covalent dimers of scFvs (also known as diabodies) show improved targeting and clearance properties due to their higher molecular weight (55kDa) and increased avidity. Further gains can be made by generation of larger recombinant fragments, such as the minibody, an scFv-C H 3 fusion protein that self-assembles into a bivalent dimer of 80 kDa. A systematic evaluation of scFv, diabody, minibody, and intact antibody (based on comparison of tumor uptakes, tumor: blood activity ratios, and calculation of an Imaging Figure of Merit) can form the basis for selection of combinations of recombinant fragments and radionuclides for imaging applications. Ease of engineering and

  16. Development of an immunoassay for determination of 2,4-dichlorophenoxyacetic acid (2,4-D) based upon the recombinant Fab fragment of 2,4-D specific antibody

    Science.gov (United States)

    Nguyen, Van C.; Nguyen, Thi D. T.; Dau, Hung A.; Tham, Thu N.; Quyen, Dinh T.; Bachmman, Till; Schmid, Rolf D.

    2001-09-01

    To develop an immunoassay and further an immunosensor for 2,4-D based upon recombinant antibody, the Fab fragments of 2,4-D specific antibody were expressed in E. coli. Western blotting analysis of the periplasmic cell fractions shown that under the non-reducing condition only a single protein band at a molecular mass of 45-kDa, corresponding to the whole Fab fragment was detected. Antigen binding activity for 2,4-D was found only in the extract of cells bearing the 2,4-D plasmid. An immunoassay based on the competitive reaction of 2,4-D and enzyme tracer with 2,4-D Fab fragments immobilized on micro titer plates via rabbit anti-mouse IgC was developed. Using this assay, 2,4-D could be detected at concentration range of 0.5 (mu) g/1 to 10(mu) g/1. The center point of the 2,4-D test was found at a concentration of 5 (mu) g/l. The assay was applied for detection of 2,4-D in spiked orange samples, resulting in recovery rate of 90 percent. The immunoassay could be applied to monitor human exposure to 2,4-D from contamination in fruit samples.

  17. Construction of bifunctional molecules specific to antigen and antibody’s Fc-fragment by fusion of scFv-antibodies with staphylococcal protein A

    Directory of Open Access Journals (Sweden)

    Kolibo D. V.

    2009-06-01

    Full Text Available Aim. To develop approach for detection of scFv and their complexes with antigens. Methods. The fusion proteins, which include sequences of scFv and staphylococcal protein A, were constructed and the obtained bifunctional molecules were immunochemically analysed. Results. It was shown, that scFv fused with protein A and their complexes with antigens are effectively recognized by labelled immunoglobulins with unrestricted antigenic specificity. Conclusions. The fusion of scFv with protein A fragment is a perspective approach to increase the efficiency of application in ELISA. The obtained scFv, fused with protein A, could be used for development of test-systems for the detection of diphtheria toxin.

  18. Thrombus imaging with indium-111 and iodine-131-labeled fibrin-specific monoclonal antibody and its F(ab')2 and Fab fragments

    International Nuclear Information System (INIS)

    Rosebrough, S.F.; Grossman, Z.D.; McAfee, J.G.

    1988-01-01

    We have previously reported successful imaging of fresh (2-4 hr old) and aged (1-5 days old) canine thrombi with 131 I-labeled intact monoclonal antibody (MAb) specific for fibrin. We now report thrombus imaging with 131 I-labeled F(ab')2 and Fab and 111 In-labeled intact MAb, F(ab')2, and Fab. Indium-111-labeled F(ab')2 proved to be the best imaging agent due to less nonspecific binding in the liver than whole IgG. Image quality was improved by the higher administered dose permissible with 111 In and its better physical characteristics for imaging, compared to 131 I. Immunofluorescence of fresh human histologic sections showed intact MAb and F(ab')2 binding to thrombi, pulmonary emboli, and atherosclerotic plaques, strengthening the feasibility of clinical thrombus imaging

  19. Improved production and function of llama heavy chain antibody fragments by molecular evolution

    NARCIS (Netherlands)

    Linden, van der R.H.; Geus, de B.; Frenken, G.J.; Peters, H.; Verrips, C.T.

    2000-01-01

    The aim of this study was to improve production level of llama heavy chain antibody fragments (V (HH)) in Saccharomyces cerevisiae while retaining functional characteristics. For this purpose, the DNA shuffling technique was used on llama V (HH) fragments specific for the azo-dye reactive red-6. In

  20. 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...

  1. Antibody Fragments as Potential Biopharmaceuticals for Cancer Therapy: Success and Limitations.

    Science.gov (United States)

    Kholodenko, Roman V; Kalinovsky, Daniel V; Doronin, Igor I; Ponomarev, Eugene D; Kholodenko, Irina V

    2017-08-17

    Monoclonal antibodies (mAbs) are an important class of therapeutic agents approved for the therapy of many types of malignancies. However, in certain cases applications of conventional mAbs have several limitations in anticancer immunotherapy. These limitations include insufficient efficacy and adverse effects. The antigen-binding fragments of antibodies have a considerable potential to overcome the disadvantages of conventional mAbs, such as poor penetration into solid tumors and Fc-mediated bystander activation of the immune system. Fragments of antibodies retain antigen specificity and part of functional properties of conventional mAbs and at the same time have much better penetration into the tumors and a greatly reduced level of adverse effects. Recent advantages in antibody engineering allowed to produce different types of antibody fragments with improved structure and properties for efficient elimination of tumor cells. These molecules opened up new perspectives for anticancer therapy. Here we will overview the structural features of the various types of antibody fragments and their applications for anticancer therapy as separate molecules and as part of complex conjugates or structures. Mechanisms of antitumor action of antibody fragments as well as their advantages and disadvantages for clinical application will be discussed in this review. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  2. Use of a fragment of glycoprotein G-2 produced in the baculovirus expression system for detecting herpes simplex virus type 2-specific antibodies

    NARCIS (Netherlands)

    Ikoma, M; Liljeqvist, JA; Glazenburg, KL; The, TH; Welling-Wester, S; Groen, J.

    Fragments of glycoprotein G (gG-2(281-594His)), comprising residues 281 to 594 of herpes simplex virus type 2 (HSV-2), glycoprotein G of HSV-1 (gG-1(t26-189His)), and glycoprotein D of HSV-1 (gD-1(1-313)), were expressed in the baculovirus expression system to develop an assay for the detection of

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

    Science.gov (United States)

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

    2009-11-01

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

  4. Monoclonal antibodies and Fc fragments for treating solid tumors

    Directory of Open Access Journals (Sweden)

    Eisenbeis AM

    2012-01-01

    Full Text Available Andrea M Eisenbeis, Stefan J GrauDepartment of Neurosurgery, University Hospital of Cologne, Cologne, GermanyAbstract: Advances in biotechnology, better understanding of pathophysiological processes, as well as the identification of an increasing number of molecular markers have facilitated the use of monoclonal antibodies and Fc fragments in various fields in medicine. In this context, a rapidly growing number of these substances have also emerged in the field of oncology. This review will summarize the currently approved monoclonal antibodies used for the treatment of solid tumors with a focus on their clinical application, biological background, and currently ongoing trials.Keywords: targeted therapy, monoclonal antibodies, cancer, biological therapy

  5. Generation of single domain antibody fragments derived from camelids and generation of manifold constructs.

    Science.gov (United States)

    Vincke, Cécile; Gutiérrez, Carlos; Wernery, Ulrich; Devoogdt, Nick; Hassanzadeh-Ghassabeh, Gholamreza; Muyldermans, Serge

    2012-01-01

    Immunizing a camelid (camels and llamas) with soluble, properly folded proteins raises an affinity-matured immune response in the unique camelid heavy-chain only antibodies (HCAbs). The peripheral blood lymphocytes of the immunized animal are used to clone the antigen-binding antibody fragment from the HCAbs in a phage display vector. A representative aliquot of the library of these antigen-binding fragments is used to retrieve single domain antigen-specific binders by successive rounds of panning. These single domain antibody fragments are cloned in tandem to generate manifold constructs (bivalent, biparatopic or bispecific constructs) to increase their functional affinity, to increase specificity, or to connect two independent antigen molecules.

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

    Science.gov (United States)

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

    2017-05-26

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

  7. 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.

  8. Chimeric recombinant antibody fragments in cardiac troponin I immunoassay.

    Science.gov (United States)

    Hyytiä, Heidi; Heikkilä, Taina; Brockmann, Eeva-Christine; Kekki, Henna; Hedberg, Pirjo; Puolakanaho, Tarja; Lövgren, Timo; Pettersson, Kim

    2015-03-01

    To introduce a novel nanoparticle-based immunoassay for cardiac troponin I (cTnI) utilizing chimeric antibody fragments and to demonstrate that removal of antibody Fc-part and antibody chimerization decrease matrix related interferences. A sandwich-type immunoassay for cTnI based on recombinant chimeric (mouse variable/human constant) antigen binding (cFab) antibodies and intrinsically fluorescent nanoparticles was developed. To test whether using chimeric antibody fragments helps to avoid matrix related interferences, samples (n=39) with known amounts of triglycerides, bilirubin, rheumatoid factor (RF) or human anti-mouse antibodies (HAMAs) were measured with the novel assay, along with a previously published nanoparticle-based research assay with the same antibody epitopes. The limit of detection (LoD) was 3.30ng/L. Within-laboratory precision for 29ng/L and 2819ng/L cTnI were 13.7% and 15.9%, respectively. Regression analysis with Siemens ADVIA Centaur® yielded a slope (95% confidence intervals) of 0.18 (0.17-1.19) and a y-intercept of 1.94 (-1.28-3.91) ng/L. When compared to a previously published nanoparticle-based assay, the novel assay showed substantially reduced interference in the tested interference prone samples, 15.4 vs. 51.3%. A rheumatoid factor containing sample was decreased from 241ng/L to fragments enabled the development of a sensitive (LoD=3.3ng/L) immunoassay for the detection of cTnI and decreased matrix related interferences, thus resulting in a lower number of falsely elevated cTnI-values. Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  9. Crystallization and diffraction properties of the Fab fragment of 3B5H10, an antibody specific for disease-causing polyglutamine stretches

    International Nuclear Information System (INIS)

    Peters-Libeu, Clare; Newhouse, Yvonne; Krishnan, Preethi; Cheung, Kenneth; Brooks, Elizabeth; Weisgraber, Karl; Finkbeiner, Steven

    2005-01-01

    Optimization of crystallization conditions and cryoprotectants decreased the anisotropy of the diffraction obtained from 3B5H10 Fab crystals. Dehydration improved the resolution of cryoprotected 3B5H10 crystals from 2.6 to 1.9 Å, but changed the space group of the crystals from P2 1 2 1 2 to P2 1 . Because it binds soluble forms of proteins with disease-associated polyglutamine expansions, the antibody 3B5H10 is a powerful tool for studying polyglutamine-related diseases. Crystals of the 3B5H10 Fab (47 kDa) were obtained by vapor diffusion at room temperature from PEG 3350. However, the initial crystals gave highly anisotropic diffraction patterns. After optimization of the crystallization conditions and cryoprotectants, a nearly isotropic diffraction pattern at 2.6 Å resolution was achieved for crystals with unit-cell parameters a = 133.26, b = 79.52, c = 41.49 Å and space group P2 1 2 1 2. Dehydrated crystals diffracted isotropically to 1.9 Å with unit-cell parameters a = 123.65, b = 78.25, c = 42.26 Å, β = 90.3° and space group P2 1

  10. Crystallization and diffraction properties of the Fab fragment of 3B5H10, an antibody specific for disease-causing polyglutamine stretches

    Energy Technology Data Exchange (ETDEWEB)

    Peters-Libeu, Clare; Newhouse, Yvonne; Krishnan, Preethi; Cheung, Kenneth; Brooks, Elizabeth [Gladstone Institute of Neurological Disease, 1650 Owens Street, San Francisco, CA 94158 (United States); Weisgraber, Karl [Gladstone Institute of Neurological Disease, 1650 Owens Street, San Francisco, CA 94158 (United States); Department of Pathology, University of California, San Francisco, CA 94143 (United States); Finkbeiner, Steven, E-mail: sfinkbeiner@gladstone.ucsf.edu [Gladstone Institute of Neurological Disease, 1650 Owens Street, San Francisco, CA 94158 (United States); Departments of Neurology, Physiology and Neuroscience and Biomedical Sciences Program, University of California, San Francisco, CA 94143 (United States)

    2005-12-01

    Optimization of crystallization conditions and cryoprotectants decreased the anisotropy of the diffraction obtained from 3B5H10 Fab crystals. Dehydration improved the resolution of cryoprotected 3B5H10 crystals from 2.6 to 1.9 Å, but changed the space group of the crystals from P2{sub 1}2{sub 1}2 to P2{sub 1}. Because it binds soluble forms of proteins with disease-associated polyglutamine expansions, the antibody 3B5H10 is a powerful tool for studying polyglutamine-related diseases. Crystals of the 3B5H10 Fab (47 kDa) were obtained by vapor diffusion at room temperature from PEG 3350. However, the initial crystals gave highly anisotropic diffraction patterns. After optimization of the crystallization conditions and cryoprotectants, a nearly isotropic diffraction pattern at 2.6 Å resolution was achieved for crystals with unit-cell parameters a = 133.26, b = 79.52, c = 41.49 Å and space group P2{sub 1}2{sub 1}2. Dehydrated crystals diffracted isotropically to 1.9 Å with unit-cell parameters a = 123.65, b = 78.25, c = 42.26 Å, β = 90.3° and space group P2{sub 1}.

  11. Engineering Venom’s Toxin-Neutralizing Antibody Fragments and Its Therapeutic Potential

    Directory of Open Access Journals (Sweden)

    Larissa M. Alvarenga

    2014-08-01

    Full Text Available Serum therapy remains the only specific treatment against envenoming, but anti-venoms are still prepared by fragmentation of polyclonal antibodies isolated from hyper-immunized horse serum. Most of these anti-venoms are considered to be efficient, but their production is tedious, and their use may be associated with adverse effects. Recombinant antibodies and smaller functional units are now emerging as credible alternatives and constitute a source of still unexploited biomolecules capable of neutralizing venoms. This review will be a walk through the technologies that have recently been applied leading to novel antibody formats with better properties in terms of homogeneity, specific activity and possible safety.

  12. 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.

  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. Isolation and expression of recombinant antibody fragments to the biological warfare pathogen Brucella melitensis.

    Science.gov (United States)

    Hayhurst, Andrew; Happe, Scott; Mabry, Robert; Koch, Zephyr; Iverson, Brent L; Georgiou, George

    2003-05-01

    Brucella melitensis is a highly infectious animal pathogen able to cause a recurring debilitating disease in humans and is therefore high on the list of biological warfare agents. Immunoglobulin genes from mice immunized with gamma-irradiated B. melitensis strain 16M were used to construct a library that was screened by phage display against similarly prepared bacteria. The selected phage particles afforded a strong enzyme-linked immunosorbent assay (ELISA) signal against gamma-irradiated B. melitensis cells. However, extensive efforts to express the respective single chain antibody variable region fragment (scFv) in soluble form failed due to: (i) poor solubility and (ii) in vivo degradation of the c-myc tag used for the detection of the recombinant antibodies. Both problems could be addressed by: (i) fusing a human kappa light chain constant domain (Ck) chain to the scFv to generate single chain antibody fragment (scAb) antibody fragments and (ii) by co-expression of the periplasmic chaperone Skp. While soluble, functional antibodies could be produced in this manner, phage-displaying scFvs or scAbs were still found to be superior ELISA reagents for immunoassays, due to the large signal amplification afforded by anti-phage antibodies. The isolated phage antibodies were shown to be highly specific to B. melitensis and did not recognize Yersinia pseudotuberculosis in contrast to the existing diagnostic monoclonal YST 9.2.1.

  15. Antibody or Antibody Fragments: Implications for Molecular Imaging and Targeted Therapy of Solid Tumors

    Directory of Open Access Journals (Sweden)

    Katerina T. Xenaki

    2017-10-01

    Full Text Available The use of antibody-based therapeutics has proven very promising for clinical applications in cancer patients, with multiple examples of antibodies and antibody–drug conjugates successfully applied for the treatment of solid tumors and lymphomas. Given reported recurrence rates, improvements are clearly still necessary. A major factor limiting the efficacy of antibody-targeted cancer therapies may be the incomplete penetration of the antibody or antibody–drug conjugate into the tumor. Incomplete tumor penetration also affects the outcome of molecular imaging, when using such targeting agents. From the injection site until they arrive inside the tumor, targeting molecules are faced with several barriers that impact intratumoral distribution. The primary means of antibody transport inside tumors is based on diffusion. The diffusive penetration inside the tumor is influenced by both antibody properties, such as size and binding affinity, as well as tumor properties, such as microenvironment, vascularization, and targeted antigen availability. Engineering smaller antibody fragments has shown to improve the rate of tumor uptake and intratumoral distribution. However, it is often accompanied by more rapid clearance from the body and in several cases also by inherent destabilization and reduction of the binding affinity of the antibody. In this perspective, we discuss different cancer targeting approaches based on antibodies or their fragments. We carefully consider how their size and binding properties influence their intratumoral uptake and distribution, and how this may affect cancer imaging and therapy of solid tumors.

  16. Recombinant fragment of an antibody tailored for direct radioiodination

    Czech Academy of Sciences Publication Activity Database

    Sedláček, Juraj; Fábry, Milan; Sieglová, Irena; Král, Vlastimil; Uhnáková, Bronislava; Mudra, M.; Kronrád, L.; Sawicka, A.; Mikolajczak, R.; Řezáčová, Pavlína

    2012-01-01

    Roč. 55, č. 1 (2012), s. 52-56 ISSN 0362-4803 R&D Projects: GA MPO 2A-2TP1/076; GA MŠk 1M0505 Institutional research plan: CEZ:AV0Z50520514 Keywords : I125 labelling * single-chain antibody variable fragment * tyrosine-rich polypeptide segment * fusion protein Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.240, year: 2012

  17. Fragmentation, labeling and biodistribution studies of KS1/4, a monoclonal antibody

    International Nuclear Information System (INIS)

    Mohd, S.B.

    1987-01-01

    In this study, an IgG2a (KS1/4), a monoclonal antibody (MoAb) specific against a human lung adenocarcinoma (UCLA P-3) was successfully fragmented enzymatically to yield F(ab') 2 and Fab by using pepsin and papain, respectively. The kinetic of fragmentation of the MoAb was compared to that of human immunoglobulin G (IgG). A similar pattern of fragmentation was observed with both antibodies with a higher percentage yield of the F(ab') 2 and Fab obtained upon the fragmentation of the IgG by the enzymes. The KS1/4 and the two fragments were labeled with three different radionuclides, namely iodine-131, indium-111 and selenium-75. The radioiodination of the MoAb and the fragments was carried out by using a modified chloramine-T method. Radiometal labeling of the MoAb and the fragments with indium-111 was performed by using DTPA as a bifunctional chelating agent, while intrinsic labeling of the MoAb was done by culturing the hybridoma in the presence of 75 Se-methionine. The biodistribution of the radiolabeled MoAb, F(ab') 2 and Fab fragments were performed by injecting the preparations intravenously into nude mice bearing human lung adenocarcinoma

  18. Composition and method for detecting cancer with technetium labeled antibody fragments

    International Nuclear Information System (INIS)

    Burchiel, S. W.; Crockford, D. R.; Rhodes, B. A.

    1984-01-01

    F(ab') 2 or Fab fragments of antibodies to: (a) human chorionic gonadotropin (hCG), hCG alpha subunit, hCG beta subunit, or an hCG-like material; or (b) other tumor specific or tumor associated molecules, to include carcinoembryonic antigen (CEA), alpha fetoprotein (AFP), human melanoma associated antigens, human sarcoma associated antigens or other antigens, are radiolabeled with technetium-99m (Tc-99m). When the F(ab') 2 or Fab fragments of antibody to such tumor associated antigens are injected intravenously into a patient, the radiolabeled composition accumulates at tumor sites. The accumulation of the cancer seeking radiopharmaceutical at tumor sites permits detection by external gamma scintigraphy. Thus, the composition is useful in the monitoring, localization and detection of cancer in the body. In an alternative composition, a double antibody approach to tumor localization using radiolabeled F(ab') 2 or Fab fragments is utilized. In this approach, a tumor specific antibody in the form of IgG, F(ab') 2 or Fab is first administered to a patient intravenously. Following a sufficient period of time, a second antibody in the form of F(ab') 2 or Fab is administered. The second antibody is radiolabeled with Tc-99m and has the property that it is reactive with the first antibody. This double antibody method has the advantage over a single antibody approach in that smaller tumors can be localized and detected and that the total amount of radioactive trace localized at the cancer site is increased

  19. Comparative imaging and biodistribution studies with an anti-CEA monoclonal antibody and its F(ab)2 and Fab fragments in mice with colon carcinoma xenografts

    International Nuclear Information System (INIS)

    Andrew, S.M.; Pimm, M.V.; Baldwin, R.W.; Perkins, A.C.

    1986-01-01

    An IgG1 mouse monoclonal antibody directed against CEA has been digested with papain to yield F(ab) 2 and Fab fragments. Following radioiodination, intact antibody and fragments showed specific binding to cells of a CEA-producing tumour, although the immune reactivities of the fragments were lower than that of intact antibody. Gamma scintigraphy of nude mice bearing CEA producing human tumour xenografts and injected with 131 I-labelled fragments showed earlier and superior imaging of tumours than did 131 I-intact antibody, and this was most marked with the Fab fragment. Sequential dissection analyses showed that this was due to earlier and higher tumour-to-blood ratios with fragments than with intact antibody, but in absolute terms the degree of localization of both fragment types was significantly lower than that of intact antibody. (orig.)

  20. 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.

  1. Scanning force microscopy and fluorescence microscopy of microcontact printed antibodies and antibody fragments.

    Science.gov (United States)

    LaGraff, John R; Chu-LaGraff, Quynh

    2006-05-09

    Unlabeled primary immunoglobulin G (IgG) antibodies and its F(ab')2 and Fc fragments were attached to oxygen-plasma-cleaned glass substrates using either microcontact printing (MCP) or physical adsorption during bath application from dilute solutions. Fluorescently labeled secondary IgGs were then bound to surface-immobilized IgG, and the relative surface coverage was determined by measuring the fluorescence intensity. Results indicated that the surface coverage of IgG increased with increasing protein solution concentration for both MCP and bath-applied IgG and that a greater concentration of IgG was transferred to a glass substrate using MCP than during physisorption during bath applications. Scanning force microscopy (SFM) showed that patterned MCP IgG monolayers were 5 nm in height, indicating that IgG molecules lie flat on the substrate. After incubation with a secondary IgG, the overall line thickness increased to around 15 nm, indicating that the secondary IgG was in a more vertical orientation with respect to the substrate. The surface roughness of these MCP patterned IgG bilayers as measured by SFM was observed to increase with increasing surface coverage. Physisorption of IgG to both unmodified patterned polydimethylsiloxane (PDMS) stamps and plasma-cleaned glass substrates was modeled by Langmuir adsorption kinetics yielding IgG binding constants of K(MCP) = 1.7(2) x 10(7) M(-1) and K(bath) = 7.8(7) x 10(5) M(-1), respectively. MCP experiments involving primary F(ab')2 and Fc fragments incubated in fluorescently labeled fragment-specific secondary IgGs were carried out to test for the function and orientation of IgG. Finally, possible origins of MCP stamping defects such as pits, pull outs, droplets, and reverse protein transfer are discussed.

  2. 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.

  3. Production of Monoclonal Antibodies specific for Progesterone

    OpenAIRE

    YÜCEL, Fatıma

    2014-01-01

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

  4. Single Chain Antibody Fragment against Venom from the Snake Daboia russelii formosensis

    Directory of Open Access Journals (Sweden)

    Chi-Hsin Lee

    2017-10-01

    Full Text Available Russell’s vipers containing hemotoxic and neurotoxic venom commonly cause snake envenomation. Horse-derived antivenom is a specific antidote, but its production is expensive and has side effects. Developing a cost-effective and more tolerable therapeutic strategy is favorable. In this study, using glutaraldehyde-attenuated Daboia russelii formosensis (DRF venom proteins to immunize chickens, polyclonal yolk-immunoglobulin (IgY antibodies were generated and showed a specific binding affinity. Phage display technology was used to generate two antibody libraries of single-chain variable fragments (scFvs containing 3.4 × 107 and 5.5 × 107 transformants, respectively. Phage-based ELISA indicated that specific clones were enriched after bio-panning. The nucleotide sequences of scFv-expressing clones were analyzed and classified into six groups in the short linker and four groups in the long linker. These scFv antibodies specifically bound to DRF proteins, but not other venom proteins. Mass spectrometric data suggested that these scFv antibodies may recognize phospholipase A2 RV-4 or RV-7. In vivo studies showed that anti-DRF IgY exhibited complete protective effects and mixed scFv antibodies increased the survival rate and time of mice challenged with a lethal dose of DRF proteins. These antibodies can be potentially applied in a rapid diagnostic method or for treatment in the future.

  5. Single Chain Antibody Fragment against Venom from the Snake Daboia russelii formosensis.

    Science.gov (United States)

    Lee, Chi-Hsin; Lee, Yu-Ching; Lee, Yueh-Lun; Leu, Sy-Jye; Lin, Liang-Tzung; Chen, Chi-Ching; Chiang, Jen-Ron; Mwale, Pharaoh Fellow; Tsai, Bor-Yu; Hung, Ching-Sheng; Yang, Yi-Yuan

    2017-10-27

    Russell's vipers containing hemotoxic and neurotoxic venom commonly cause snake envenomation. Horse-derived antivenom is a specific antidote, but its production is expensive and has side effects. Developing a cost-effective and more tolerable therapeutic strategy is favorable. In this study, using glutaraldehyde-attenuated Daboia russelii formosensis (DRF) venom proteins to immunize chickens, polyclonal yolk-immunoglobulin (IgY) antibodies were generated and showed a specific binding affinity. Phage display technology was used to generate two antibody libraries of single-chain variable fragments (scFvs) containing 3.4 × 10⁷ and 5.5 × 10⁷ transformants, respectively. Phage-based ELISA indicated that specific clones were enriched after bio-panning. The nucleotide sequences of scFv-expressing clones were analyzed and classified into six groups in the short linker and four groups in the long linker. These scFv antibodies specifically bound to DRF proteins, but not other venom proteins. Mass spectrometric data suggested that these scFv antibodies may recognize phospholipase A2 RV-4 or RV-7. In vivo studies showed that anti-DRF IgY exhibited complete protective effects and mixed scFv antibodies increased the survival rate and time of mice challenged with a lethal dose of DRF proteins. These antibodies can be potentially applied in a rapid diagnostic method or for treatment in the future.

  6. Altered specificity of single-chain antibody fragments bound to pandemic H1N1-2009 influenza virus after conversion of the phage-bound to the soluble form

    Directory of Open Access Journals (Sweden)

    Kaku Yoshihiro

    2012-09-01

    Full Text Available Abstract Background In 2009, a novel influenza A/H1N1 virus (H1N1pdm quickly spread worldwide and co-circulated with then-existing seasonal H1N1 virus (sH1N1. Distinguishing between these 2 viruses was necessary to better characterize the epidemiological properties of the emergent virus, including transmission patterns, pathogenesis, and anti-influenza drug resistance. This situation prompted us to develop a point-of-care virus differentiation system before entering the 2009–2010 influenza season. Aiming to establish H1N1pdm-specific detection tools rapidly, we employed phage display libraries to select H1N1pdm-specific single-chain variable fragments (scFvs. Findings Human single-fold scFv libraries (Tomlinson I + J underwent selection for the ability to bind H1N1pdm virus particles. Three rounds of panning brought 1152 phage-bound scFvs, of which 58 clones reacted with H1N1pdm specifically or preferentially over sH1N1 in an enzyme-linked immunosorbent assay (ELISA. After conversion of the scFvs to soluble form, 7 clones demonstrating high/stable expression were finally obtained. However, all the soluble scFvs except No. 29 were found to have lost their specificity/preference for H1N1pdm in ELISA. The specificity/preference of No. 29 was also confirmed by immunofluorescence assay and immunoprecipitation, and the viral nucleoprotein was identified by ELISA as its target protein. The change in specificity associated with scFv conversion from phage-bound to soluble form could be due to loss of phage scaffold pIII protein, which likely provides structural support for the scFv antigen-binding site. It is also possible that the similar antigenic properties of H1N1pdm and sH1N1 led to the observed alterations in scFv specificity. Discussion Using a phage display library, we obtained 7 soluble scFv clones reactive against H1N1pdm; however, only 1 showed specificity/preference toward H1N1pdm. Our results confirmed that using phage display

  7. PET imaging of osteosarcoma in dogs using a fluorine-18-labeled monoclonal antibody fab fragment

    International Nuclear Information System (INIS)

    Page, R.L.; Garg, P.K.; Gard, S.

    1994-01-01

    Four dogs with histologically confirmed osteogenic sarcoma were studied with PET following intravenous injection of the 18 F-labeled Fab fragment of TP-3, a monoclonal antibody specific for human and canine osteosarcomas. The antibody fragment was labeled using the N-succinimidyl (8-(4'-( 18 F)fluorobenzyl)amino)suberate acylation agent. Blood clearance of activity was biphasic in all dogs but half-times were variable (T 1/2β = 2-13 hr). Catabolism of labeled Fab was reflected by the decrease in protein-associated activity in serum from more than 90% at 1 min to 60%-80% at 4 hr. PET images demonstrated increased accumulation of 18 F at the primary tumor site relative to normal contralateral bone in one dog as early as 15 min after injection. Biopsies obtained after euthanasia indicated higher uptake at the edges of the tumor as observed on the PET scans. Tumor uptake was 1-3 x 10 -3 % injected dose/g, a level similar to that reported for other Fab fragments in human tumors. In the three dogs with metastatic disease, early PET images reflected activity in the blood pool but later uptake was observed in suspected metastatic sites. These results, although preliminary, suggest that PET imaging of 18 F-labeled antibody fragments is feasible and that dogs with spontaneous tumors could be a valuable model for preclinical research with radioimmunoconjugates. 34 refs., 6 figs., 2 tabs

  8. PET imaging of osteosarcoma in dogs using a fluorine-18-labeled monoclonal antibody fab fragment

    Energy Technology Data Exchange (ETDEWEB)

    Page, R.L.; Garg, P.K.; Gard, S. [North Carolina State Univ., Raleigh, NC (United States)]|[Duke Univ. Medical Center, Durham, NC (United States)]|[North Carolina and Norke Radium Hospital, Oslo (Norway)] [and others

    1994-09-01

    Four dogs with histologically confirmed osteogenic sarcoma were studied with PET following intravenous injection of the {sup 18}F-labeled Fab fragment of TP-3, a monoclonal antibody specific for human and canine osteosarcomas. The antibody fragment was labeled using the N-succinimidyl (8-(4{prime}-({sup 18}F)fluorobenzyl)amino)suberate acylation agent. Blood clearance of activity was biphasic in all dogs but half-times were variable (T{sub 1/2{beta}} = 2-13 hr). Catabolism of labeled Fab was reflected by the decrease in protein-associated activity in serum from more than 90% at 1 min to 60%-80% at 4 hr. PET images demonstrated increased accumulation of {sup 18}F at the primary tumor site relative to normal contralateral bone in one dog as early as 15 min after injection. Biopsies obtained after euthanasia indicated higher uptake at the edges of the tumor as observed on the PET scans. Tumor uptake was 1-3 x 10{sup -3}% injected dose/g, a level similar to that reported for other Fab fragments in human tumors. In the three dogs with metastatic disease, early PET images reflected activity in the blood pool but later uptake was observed in suspected metastatic sites. These results, although preliminary, suggest that PET imaging of {sup 18}F-labeled antibody fragments is feasible and that dogs with spontaneous tumors could be a valuable model for preclinical research with radioimmunoconjugates. 34 refs., 6 figs., 2 tabs.

  9. Raising an Antibody Specific to Breast Cancer Subpopulations Using Phage Display on Tissue Sections

    DEFF Research Database (Denmark)

    Larsen, Simon Asbjørn; Meldgaard, Theresa; Fridriksdottir, Agla Jael Rubner

    2016-01-01

    BACKGROUND/AIM: Primary tumors display a great level of intra-tumor heterogeneity in breast cancer. The current lack of prognostic and predictive biomarkers limits accurate stratification and the ability to predict response to therapy. The aim of the present study was to select recombinant antibody...... fragments specific against breast cancer subpopulations, aiding the discovery of novel biomarkers. MATERIALS AND METHODS: Recombinant antibody fragments were selected by phage display. A novel shadowstick technology enabled the direct selection using tissue sections of antibody fragments specific against...

  10. Use of an anti-platelet monoclonal antibody F (ab')2 fragment for imaging thrombus

    International Nuclear Information System (INIS)

    Loutfi, I.; Stuttle, A.W.J.; Peters, A.M.; George, P.; Lavender, J.P.; Lumley, P.

    1990-01-01

    Ten patients with suspected thrombus have been studied using 111 In-labelled F (ab')2 fragments of P256, a monoclonal antibody which recognizes an epitope on the platelet membrane glycoprotein IIb/IIIa complex. The F (ab')2 fragment was radiolabelled with 111 In via diethylenetri-aminepentamacetic acid to give a specific activity of up to 190 MBq (5mCi) mg - 1 without impairment of immunoreactivity. In vitro platelet aggregation studies showed that the F (ab')2 fragment caused less platelet aggregation than the whole antibody on a molar ratio and was without significant effect upon the sensitivity of platelets to a range of aggregating agents. Platalets were labelled in ten patients by intravenous injection of approximately 100 μg P256 F (ab')2. Of the ten patients studies, six showed localization of activity consistent with platelet accumulation. Localization was clearly seen when associated with thrombus of the lower limbs (three patients: deep vein thrombosis; one patient: aortofemoral graft), and was apparent although less marked in two other cases, one of aortic aneurysm and one of carotid stenosis. Use of radiolabelled P256 F (ab')2 offers a means of non-invasive detection of thrombus which, from in vitro studies, would appear to have less direct effect of platelet behaviour than the whole antibody. (author). 9 refs. 8 figs. 1 tab

  11. Monoclonal antibodies from rats immunized with fragment D of human fibrinogen

    International Nuclear Information System (INIS)

    Kennel, S.J.; Chen, J.P.; Lankford, P.K.; Foote, L.J.

    1981-01-01

    Fischer rats were immunized with fragment D (Fg-D) of human fibrinogen (Fg) to obtain antibody specific for neoantigens unique to this molecule. Absorption of serum with whole Fg indicated that some of the antibody produced reacted preferentially with Fg-D. Hybridoma cultures were prepared by fusion of immune rat spleen cells with mouse myeloma P3-X63-Ag8. Monoclonal antibodies obtained from these cultures fell into two classes: (a) Those reacting equally well with Fg and Fg-D. (b) Those reacting preferentially but not absolutely wth Fg-D. Antibody from hybridoma 104-14, a member of the first group had an affinity for Fg-D of 1.5 x 10 9 M -1 while antibodies from 106-59 and 106-71 (group 2) demonstrated much lower affinities of 1.0 x 10 7 and 4.7 x 10 6 M -1 , respectively. The cross reactivity of antibodies in the second group indicated that they react with protein conformations that are altered during production of Fg-D from Fg

  12. Isolation of llama antibody fragments for prevention of dandruff by phage display in shampoo

    NARCIS (Netherlands)

    Dolk, E.; Vaart, M. van der; Lutje Hulsik, D.; Vriend, G.; Haard, H. de; Spinelli, S.; Cambillau, C.; Frenken, L.; Verrips, T.

    As part of research exploring the feasibility of using antibody fragments to inhibit the growth of organisms implicated in dandruff, we isolated antibody fragments that bind to a cell surface protein of Malassezia furfur in the presence of shampoo. We found that phage display of llama

  13. Immunotherapy with GD2 specific monoclonal antibodies

    International Nuclear Information System (INIS)

    Cheung, N.K.V.; Medof, E.M.; Munn, D.

    1988-01-01

    Targeted immunotherapy focuses anti-tumor activity of antibodies and effector cells, which are actively developed by the host or adoptively transferred, onto tumor cells and into tumor sites. Such tumor selective therapy can be more specific and efficient. The value of such an approach is evident in the classical interaction of antibodies. This paper reports that the ganglioside G D2 is an ideal antigen for specific tumor targeting because of its relative lack of heterogeneity among human neuroblastoma, its high density on tumor cells, its lack of antigen modulation upon binding to antibody, and its restricted distribution in normal tissues

  14. Localization of tumors in vivo by scintigraphic identification of Clostridium butyricum using 131I-labelled antibodies and F(ab')2-antibody fragments

    International Nuclear Information System (INIS)

    Vogt, R.; Mehnert, W.H.; Schmidt, H.E.; Altenbrunn, H.J.; Akademie der Wissenschaften der DDR, Berlin-Buch. Zentralinstitut fuer Isotopen- und Strahlenforschung)

    1979-01-01

    Tumor-bearing mice injected with clostridial spores show enrichment and germination of the spores within the tumor. 131 I-labelled anti-Clostridium-antibodies and anti-Clostridium-F(ab') 2 -fragments were used for a possible localization of tumors in vivo by scintiscanning. The application of the antibody revealed increased radioactivity in the tumors of mice pretreated with spores as well as in animals without pretreatment. In using F(ab') 2 -fragments instead of total antibody neither the apparently unspecific increase of radioactivity in not pretreated mice nor the specific fixation of labelled F(ab') 2 -fragments to clostridial rods in the tumors of pretreated animals could be demonstrated. The results are discussed with respect to further investigation

  15. [Molecular dynamics of immune complex of photoadduct-containing DNA with Fab-Anti-DNA antibody fragment].

    Science.gov (United States)

    Akberova, N I; Zhmurov, A A; Nevzorova, T A; Litvinov, R I

    2016-01-01

    Antibodies to DNA play an important role in the pathogenesis of autoimmune diseases. The elucidation of structural mechanisms of both the antigen recognition and the interaction of anti-DNA antibodies with DNA will help to understand the role of DNA-containing immune complexes in various pathologies and can provide a basis for new treatment modalities. Moreover, the DNA-antibody complex is an analog of specific intracellular DNA-protein interactions. In this work, we used in silico molecular dynamic simulations of bimolecular complexes of the dsDNA segment containing the Fab fragment of an anti-DNA antibody to obtain the detailed thermodynamic and structural characteristics of dynamic intermolecular interactions. Using computationally modified crystal structure of the Fab-DNA complex (PDB ID: 3VW3), we studied the equilibrium molecular dynamics of the 64M-5 antibody Fab fragment associated with the dsDNA fragment containing the thymine dimer, the product of DNA photodamage. Amino acid residues that constitute paratopes and the complementary nucleotide epitopes for the Fab-DNA construct were identified. Stacking and electrostatic interactions were found to play the main role in mediating the most specific antibody-dsDNA contacts, while hydrogen bonds were less significant. These findings may shed light on the formation and properties of pathogenic anti-DNA antibodies in autoimmune diseases, such as systemic lupus erythematosus associated with skin photosensitivity and DNA photodamage.

  16. ECT with /sup 123/I-labeled fragments of anti-CEA monoclonal antibodies in colo-rectal cancer

    International Nuclear Information System (INIS)

    Bischof-Delaloye, A.; Delaloye, B.

    1986-01-01

    The recent progress of tumor localization with labelled antibodies can be attributed to three techniques: 1) use of I-123 as a label; 2) fragmentation of antibodies; 3) tomographic recording and evaluation of patient radiation data. Under these conditions the method yields good sensitivity and specifity indexes (15/16 for primary tumors and local recurrences, 7/10 for metastasis). A strictly prospective study, however, remains mandatory in order to assess the clinical value of this method

  17. Cartilage oligomeric matrix protein specific antibodies are pathogenic

    DEFF Research Database (Denmark)

    Geng, Hui; Nandakumar, Kutty Selva; Pramhed, Anna

    2012-01-01

    -specific monoclonal antibodies (mAbs). METHODS: B cell immunodominant regions on the COMP molecule were measured with a novel enzyme-linked immunosorbent assay using mammalian expressed full-length mouse COMP as well as a panel of recombinant mouse COMP fragments. 18 mAbs specific to COMP were generated......ABSTRACT: INTRODUCTION: Cartilage oligomeric matrix protein (COMP) is a major non-collagenous component of cartilage. Earlier, we developed a new mouse model for rheumatoid arthritis using COMP. This study was undertaken to investigate the epitope specificity and immunopathogenicity of COMP...

  18. Expression and production of llama variable heavy-chain antibody fragments (VHHs) by Aspergillus awamori

    NARCIS (Netherlands)

    Joosten, V.; Gouka, R.J.; Hondel, C.A.M.J.J. van den; Verrips, C.T.; Lokman, B.C.

    2005-01-01

    We report the expression and production of llama variable heavy-chain antibody fragments (VHHs) by Aspergillus awamori. Fragments encoding VHHs were cloned in a suitable Aspergillus expression vector and transformants secreting VHH fragments were analysed for integrated gene copy-numbers, mRNA

  19. Induced refolding of a temperature denatured llama heavy-chain antibody fragment by its antigen

    NARCIS (Netherlands)

    Dolk, E.; Vliet, C. van; Perez, J.M.J.; Vriend, G.; Darbon, H.; Ferrat, G.; Cambillau, C.; Frenken, L.G.J.; Verrips, T.

    2005-01-01

    In a previous study we have shown that llama VHH antibody fragments are able to bind their antigen after a heat shock of 90°C, in contrast to the murine monoclonal antibodies. However, the molecular mechanism by which antibody:antigen interaction occurs under these extreme conditions remains

  20. Evaluation of tumor targeting with radiolabeled F(ab2 fragment of a humanized monoclonal antibody

    Directory of Open Access Journals (Sweden)

    "Babaei MH

    2002-08-01

    Full Text Available Humanized monoclonal antibody U36 and its F(ab'2 fragment, radio labeled with 125I, were tested for tumor localization in nude mice bearing a squamous cell carcinoma xenograft line derived from a head and neck carcinoma. Monoclonal antibody IgG or F(ab'2 fragment were injected in parallel and at days 1, 2 and 3, mice were dissected for determination of isotope biodistribution. IgG as well as F(ab'2 showed highly specific localization in tumor tissue. The mean tumor uptake (n=3 is expressed as the percentage of the injected dose per gram of tumor tissue (%ID/g. %ID/g of IgG was 11.7% at day 1 and decreased to 10.9% at day 3 whereas %ID/g of F(ab'2 was 2.9% at day 1 and decreased on following days. Tumor to blood ratios (T/B at day 1 were 0.86 for IgG and 1.32 for F(ab'2 and reached a maximum at day 3 with values of 4.41 and 1.84 respectively. These findings suggest that the superior tumor to non-tumor ratios in the day of 1 render the F(ab'2 fragment more qualified for specific targeting radioisotopes to tumor xenografts in this exprimental setting.

  1. The future of antibody therapeutics: ADCs bi-specifics and RIT

    International Nuclear Information System (INIS)

    Reichert, J.

    2015-01-01

    Full text of publication follows. Antibodies are widely accepted as remarkably versatile therapeutic agents. As evidence of this, the ∼ 30 antibody products marketed worldwide had total global sales of more than 50 billion dollars in 2012, and the commercial clinical pipeline currently comprises over 350 antibody-based product candidates. In a testament to scientific ingenuity, the investigational molecules (clinical and preclinical) are notably diverse in their composition of matter and include antibodies conjugated to a variety of agents (drugs, radioisotopes), bi-specific antibodies, and fragments or domains of antibodies. The concepts that form the basis of these agents were established decades ago, but advances in technology are now allowing new opportunities for their development. In this presentation, future directions in antibody therapeutics development will be discussed, with a focus on antibody-drug conjugates, bi-specific antibodies and radioimmunotherapy. (author)

  2. T cell receptor-like recognition of tumor in vivo by synthetic antibody fragment.

    Directory of Open Access Journals (Sweden)

    Keith R Miller

    Full Text Available A major difficulty in treating cancer is the inability to differentiate between normal and tumor cells. The immune system differentiates tumor from normal cells by T cell receptor (TCR binding of tumor-associated peptides bound to Major Histocompatibility Complex (pMHC molecules. The peptides, derived from the tumor-specific proteins, are presented by MHC proteins, which then serve as cancer markers. The TCR is a difficult protein to use as a recombinant protein because of production issues and has poor affinity for pMHC; therefore, it is not a good choice for use as a tumor identifier outside of the immune system. We constructed a synthetic antibody-fragment (Fab library in the phage-display format and isolated antibody-fragments that bind pMHC with high affinity and specificity. One Fab, fE75, recognizes our model cancer marker, the Human Epidermal growth factor Receptor 2 (HER2/neu peptide, E75, bound to the MHC called Human Leukocyte Antigen-A2 (HLA-A2, with nanomolar affinity. The fE75 bound selectively to E75/HLA-A2 positive cancer cell lines in vitro. The fE75 Fab conjugated with (64Cu selectively accumulated in E75/HLA-A2 positive tumors and not in E75/HLA-A2 negative tumors in an HLA-A2 transgenic mouse as probed using positron emission tomography/computed tomography (PET/CT imaging. Considering that hundreds to thousands of different peptides bound to HLA-A2 are present on the surface of each cell, the fact that fE75 arrives at the tumor at all shows extraordinary specificity. These antibody fragments have great potential for diagnosis and targeted drug delivery in cancer.

  3. 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

  4. Immunoscintigraphy of human pancreatic carcinoma in nude mice with I-131-F(ab')/sub 2/-fragments of monoclonal antibodies

    International Nuclear Information System (INIS)

    Senekowitsch, R.; Maul, F.D.; Wenisch, H.J.C.; Kriegel, H.; Hor, G.

    1985-01-01

    In the present study radioiodinated F(ab')/sub 2/-fragments of CA19-9 and antibody that reacts specifically with human gastrointestinal cancer were examined for their ability to detect human pancreatic carcinoma hosted in nude mice. Tumor-bearing mice received 80μCi of I-131-F(ab')/sub 2/ with a specific activity of 1.8μCi/μg. All mice were imaged after the injection and every 24hr up to 6 days. The retained radioactivity was also registered with a whole-body counter immediately after imaging. As a control F(ab's)/sub 2/ of a nonspecific antibody were administered in parallel to another group of animals bearing the same tumor. Three animals of each group were killed at 1,2,4 and 8 days for determination of the distribution of both labeled antibody-fragments. On scintigraphic images obtained with the CA19-9-F(ab')/sub 2/ the tumors could be visualized 24hr after injection, the best dilineation however was achieved 96hr p.i.. The biodistribution data exhibited a more rapid blood clearance for the specific fragments compared to that for the unspecific ones. Tumors showed an increase in uptake up to 48hr reaching 1.7% of the injected dose per gram, declining to values of 0.08%/g at day 6 p.i.. The highest tumor-to-blood ratios were found after 96h. They were 7 for the CA19-9-fragments compared to 1.5 for the unspecific fragments. The whole body counting revealed a more rapid excretion for the fragments of the specific monoclonal antibodies than for the unspecific ones. In summary the authors were able to show that CA19-9-F(ab')/sub 2/-fragments can be used for immunodetection of human pancreatic carcinoma hosted in nude mice

  5. 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.

  6. Stability of llama heavy chain antibody fragments under extreme conditions

    NARCIS (Netherlands)

    Dolk, E.

    2004-01-01

    Camelids have next to their normal antibodies, a unique subset of antibodies lacking light chains. The resulting single binding domain, VHH, of these heavy chain antibodies consequently have unique properties. A high stability is one of these properties, which was investigated in this thesis. The

  7. Labeling and stability of radiolabeled antibody fragments by a direct 99mTc-labeling method

    International Nuclear Information System (INIS)

    Pak, K.Y.; Nedelman, M.A.; Tam, S.H.; Wilson, E.; Daddona, P.E.

    1992-01-01

    The in vitro labeling and stability of 99m Tc-labeled antibody Fab' fragments prepared by a direct labeling technique were evaluated. Eight antibody fragments derived from murine IgG1 (N = 5), IgG2a (N = 2) and IgG3 (N = 1) isotypes were labeled with a preformed 99m Tc-D-glucarate complex. No loss of radioactivity incorporation was observed for all the 99m Tc-labeled antibody fragments after 24 h incubation at 37 o C. 99m Tc-labeled antibody fragments (IgG1, N = 2; IgG2a, n = 2; IgG3, N = 1) were stable upon challenge with DTPA, EDTA or acidic pH. Using the affinity chromatography technique, two of the 99m Tc-labeled antibody fragments displayed no loss of immunoreactivity after prolonged incubation in phosphate buffer up to 24 h at 37 o C. Bonding between 99m Tc and antibody fragments was elucidated by challenging with a diamide ditholate (N 2 S 2 ) compound. The Fab' with IgG2a isotype displayed tighter binding to 99m Tc in comparison to Fab' from IgG1 and IgG3 isotype in N 2 S 2 challenge and incubation with human plasma. The in vivo biodistribution of five 99m Tc-labeled fragments were evaluated in normal mice. (Author)

  8. In vivo tumor targeting and imaging with engineered trivalent antibody fragments containing collagen-derived sequences.

    Directory of Open Access Journals (Sweden)

    Angel M Cuesta

    Full Text Available There is an urgent need to develop new and effective agents for cancer targeting. In this work, a multivalent antibody is characterized in vivo in living animals. The antibody, termed "trimerbody", comprises a single-chain antibody (scFv fragment connected to the N-terminal trimerization subdomain of collagen XVIII NC1 by a flexible linker. As indicated by computer graphic modeling, the trimerbody has a tripod-shaped structure with three highly flexible scFv heads radially outward oriented. Trimerbodies are trimeric in solution and exhibited multivalent binding, which provides them with at least a 100-fold increase in functional affinity than the monovalent scFv. Our results also demonstrate the feasibility of producing functional bispecific trimerbodies, which concurrently bind two different ligands. A trimerbody specific for the carcinoembryonic antigen (CEA, a classic tumor-associated antigen, showed efficient tumor targeting after systemic administration in mice bearing CEA-positive tumors. Importantly, a trimerbody that recognizes an angiogenesis-associated laminin epitope, showed excellent tumor localization in several cancer types, including fibrosarcomas and carcinomas. These results illustrate the potential of this new antibody format for imaging and therapeutic applications, and suggest that some laminin epitopes might be universal targets for cancer targeting.

  9. 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 ...

  10. Site-specific fab fragment biotinylation at the conserved nucleotide binding site for enhanced Ebola detection.

    Science.gov (United States)

    Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

    2015-07-01

    The nucleotide binding site (NBS) is a highly conserved region between the variable light and heavy chains at the Fab domains of all antibodies, and a small molecule that we identified, indole-3-butyric acid (IBA), binds specifically to this site. Fab fragment, with its small size and simple production methods compared to intact antibody, is good candidate for use in miniaturized diagnostic devices and targeted therapeutic applications. However, commonly used modification techniques are not well suited for Fab fragments as they are often more delicate than intact antibodies. Fab fragments are of particular interest for sensor surface functionalization but immobilization results in damage to the antigen binding site and greatly reduced activity due to their truncated size that allows only a small area that can bind to surfaces without impeding antigen binding. In this study, we describe an NBS-UV photocrosslinking functionalization method (UV-NBS(Biotin) in which a Fab fragment is site-specifically biotinylated with an IBA-EG11-Biotin linker via UV energy exposure (1 J/cm(2)) without affecting its antigen binding activity. This study demonstrates successful immobilization of biotinylated Ebola detecting Fab fragment (KZ52 Fab fragment) via the UV-NBS(Biotin) method yielding 1031-fold and 2-fold better antigen detection sensitivity compared to commonly used immobilization methods: direct physical adsorption and NHS-Biotin functionalization, respectively. Utilization of the UV-NBS(Biotin) method for site-specific conjugation to Fab fragment represents a proof of concept use of Fab fragment for various diagnostic and therapeutic applications with numerous fluorescent probes, affinity molecules and peptides. © 2015 Wiley Periodicals, Inc.

  11. Antigen-targeting strategies using single-domain antibody fragments

    NARCIS (Netherlands)

    Duarte, Joao Nuno Silva

    2017-01-01

    Antibodies display high selectivity and affinity and have been the preferred platform for antigen targeting. Despite the development of antigen-delivery systems that enable T cell activation, targeting approaches that enhance antibody responses need improvement. This need specially applies to poorly

  12.  Variable fragments of heavy chain antibodies (VHHs: a new magic bullet molecule of medicine?

    Directory of Open Access Journals (Sweden)

    Dorota Smolarek

    2012-06-01

    Full Text Available  Serum of animals belonging to the Camelidae family (camels and llamas contains fully active antibodies that are naturally devoid of light chains. Variable domains derived from heavy chain antibodies (hcAb called VHHs or nanobodies™ can bind antigens as effectively as full-length antibodies and are easy to clone and express. Because of their potential, VHHs are being intensively studied as potential therapeutic, diagnostic and imaging tools. The paper reviews the molecular background of heavy chain antibodies and describes methods of obtaining recombinant fragments of heavy chain antibodies as well as their therapeutic, diagnostic and other applications.

  13. Properties, production and applications of camelid single-domain antibody fragments

    NARCIS (Netherlands)

    Harmsen, M.M.; Haard, de H.J.

    2007-01-01

    Camelids produce functional antibodies devoid of light chains of which the single N-terminal domain is fully capable of antigen binding. These single-domain antibody fragments (VHHs or Nanobodies®) have several advantages for biotechnological applications. They are well expressed in microorganisms

  14. Inhibition of ligand exchange kinetics via active-site trapping with an antibody fragment.

    Science.gov (United States)

    Oyen, David; Steyaert, Jan; Barlow, John N

    2014-04-01

    We describe the first example of an inhibitory antibody fragment (nanobody ca1697) that binds simultaneously to an enzyme (the enzyme dihydrofolate reductase from Escherichia coli) and its bound substrate (folate). Binding of the antibody to the substrate causes a 20-fold reduction in the rate of folate exchange kinetics. This work opens up the prospect of designing new types of antibody-based inhibitors of enzymes and receptors through suitable design of immunogens.

  15. C4d-negative antibody-mediated rejection with high anti-angiotensin II type I receptor antibodies in absence of donor-specific antibodies.

    Science.gov (United States)

    Fuss, Alexander; Hope, Christopher M; Deayton, Susan; Bennett, Greg Donald; Holdsworth, Rhonda; Carroll, Robert P; Coates, P Toby H

    2015-07-01

    Acute antibody-mediated rejection can occur in absence of circulating donor-specific antibodies. Agonistic antibodies targeting the anti-angiotensin II type 1 receptor (anti-AT1 R) are emerging as important non-human leucocyte antigen (HLA) antibodies. Elevated levels of anti-angiotensin II receptor antibodies were first observed in kidney transplant recipients with malignant hypertension and allograft rejection. They have now been studied in three separate kidney transplant populations and associate to frequency of rejection, severity of rejection and graft failure. We report 11 cases of biopsy-proven, Complement 4 fragment d (C4d)-negative, acute rejection occurring without circulating donor-specific anti-HLA antibodies. In eight cases, anti-angiotensin receptor antibodies were retrospectively examined. The remaining three subjects were identified from our centre's newly instituted routine anti-angiotensin receptor antibody screening. All subjects fulfilled Banff 2013 criteria for antibody-mediated rejection and all responded to anti-rejection therapy, which included plasma exchange and angiotensin receptor blocker therapy. These cases support the routine assessment of anti-AT1 R antibodies in kidney transplant recipients to identify subjects at risk. Further studies will need to determine optimal assessment protocol and the effectiveness of pre-emptive treatment with angiotensin receptor blockers. © 2015 Asian Pacific Society of Nephrology.

  16. Immobilization and functional reconstitution of antibody Fab fragment by solid-phase refolding.

    Science.gov (United States)

    Kumada, Yoichi; Hamasaki, Kyoto; Nakagawa, Aya; Sasaki, Eiju; Shirai, Tatsunori; Okumura, Masahiro; Inoue, Manami; Kishimoto, Michimasa

    2013-12-31

    In this study, we demonstrated the successful preparation of a Fab antibody-immobilized hydrophilic polystyrene (phi-PS) plate via one- and two-step solid-phase refolding methods. Both polystyrene-binding peptide (PS-tag)-fused Fd fragment of heavy chain (Fab H-PS) and full-length of light-chain (Fab L-PS) were individually produced in insoluble fractions of Escherichia coli cells, and they were highly purified in the presence of 8M of urea. Antigen-binding activities of Fab antibody immobilized were correctly recovered by the one-step solid-phase refolding method that a mixture of Fab H-PS and Fab L-PS was immobilized in the presence of 0.5-2M urea, followed by surface washing of the phi-PS plate with PBST. These results indicate that by genetic fusion of a PS-tag, a complex between Fab H and Fab L was efficiently immobilized on the surface of a phi-PS plate even in the presence of a low concentration of urea, and was then correctly refolded to retain its high antigen-binding activity via removal of the urea. A two-step solid-phase refolding method whereby Fab H-PS and Fab L-PS were successively refolded on the surface of a phi-PS plate also resulted in Fab antibody formation on the plate. Furthermore, both the binding affinity and the specificity of the Fab antibody produced by the two-step method were highly maintained, according to the results of sandwich ELISA and competitive ELISA using Fab antibody-immobilized plate via two-step solid-phase refolding. Thus, the solid-phase refolding method demonstrated in this study should be quite useful for the preparation of a Fab antibody-immobilized PS surface with high efficiency from individually produced Fab H-PS and Fab L-PS. This method will be applicable to the preparation of a large Fab antibody library on the surface of a PS plate for use in antibody screening. © 2013. Published by Elsevier B.V. All rights reserved.

  17. 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.

  18. Shark Variable New Antigen Receptor (VNAR Single Domain Antibody Fragments: Stability and Diagnostic Applications

    Directory of Open Access Journals (Sweden)

    Stewart Nuttall

    2013-01-01

    Full Text Available The single variable new antigen receptor domain antibody fragments (VNARs derived from shark immunoglobulin new antigen receptor antibodies (IgNARs represent some of the smallest known immunoglobulin-based protein scaffolds. As single domains, they demonstrate favorable size and cryptic epitope recognition properties, making them attractive in diagnosis and therapy of numerous disease states. Here, we examine the stability of VNAR domains with a focus on a family of VNARs specific for apical membrane antigen 1 (AMA-1 from Plasmodium falciparum. The VNARs are compared to traditional monoclonal antibodies (mAbs in liquid, lyophilized and immobilized nitrocellulose formats. When maintained in various formats at 45 °C, VNARs have improved stability compared to mAbs for periods of up to four weeks. Using circular dichroism spectroscopy we demonstrate that VNAR domains are able to refold following heating to 80 °C. We also demonstrate that VNAR domains are stable during incubation under potential in vivo conditions such as stomach acid, but not to the protease rich environment of murine stomach scrapings. Taken together, our results demonstrate the suitability of shark VNAR domains for various diagnostic platforms and related applications.

  19. Fv-clasp: An Artificially Designed Small Antibody Fragment with Improved Production Compatibility, Stability, and Crystallizability.

    Science.gov (United States)

    Arimori, Takao; Kitago, Yu; Umitsu, Masataka; Fujii, Yuki; Asaki, Ryoko; Tamura-Kawakami, Keiko; Takagi, Junichi

    2017-10-03

    Antibody fragments are frequently used as a "crystallization chaperone" to aid structural analysis of complex macromolecules that are otherwise crystallization resistant, but conventional fragment formats have not been designed for this particular application. By fusing an anti-parallel coiled-coil structure derived from the SARAH domain of human Mst1 kinase to the variable region of an antibody, we succeeded in creating a novel chimeric antibody fragment of ∼37 kDa, termed "Fv-clasp," which exhibits excellent crystallization compatibility while maintaining the binding ability of the original IgG molecule. The "clasp" and the engineered disulfide bond at the bottom of the Fv suppressed the internal mobility of the fragment and shielded hydrophobic residues, likely contributing to the high heat stability and the crystallizability of the Fv-clasp. Finally, Fv-clasp antibodies showed superior "chaperoning" activity over conventional Fab fragments, and facilitated the structure determination of an ectodomain fragment of integrin α6β1. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. A novel method for in Situ detection of hydrolyzable casein fragments in a cheese matrix by antibody phage display technique and CLSM

    DEFF Research Database (Denmark)

    Duan, Zhi; Brüggemann, Dagmar Adeline; Siegumfeldt, Henrik

    2009-01-01

    three small synthetic peptides of the alpha(s1)-casein sequence. These peptides traverse enzymatic cleavage sites of casein during cheese ripening. The specificity of the generated anti-peptide antibodies was determined by ELISA and Western blot. Finally, an immunofluorescent labeling protocol......A novel method to monitor in situ hydrolyzable casein fragments during cheese ripening by using immunofluorescent labeling and confocal laser scanning microscopy (CLSM) was developed. Monoclonal single chain variable fragments of antibody (scFvs) were generated by antibody phage display toward...

  1. An unusual cysteine VL87 affects the antibody fragment conformations without interfering with the disulfide bond formation.

    Science.gov (United States)

    Attallah, Carolina; Aguilar, María Fernanda; Garay, A Sergio; Herrera, Fernando E; Etcheverrigaray, Marina; Oggero, Marcos; Rodrigues, Daniel E

    2017-10-01

    The Cys residues are almost perfectly conserved in all antibodies. They contribute significantly to the antibody fragment stability. The relevance of two natural contiguous Cys residues of an anti-recombinant human-follicle stimulation hormone (rhFSH) in a format of single-chain variable fragment (scFv) was studied. This scFv contains 5 Cys residues: V H 22 and V H 92 in the variable heavy chain (V H ) and V L 23, V L 87 and V L 88 in the variable light chain (V L ). The influence of two unusual contiguous Cys at positions V L 87 and V L 88 was studied by considering the wild type fragment and mutant variants: V L -C88S, V L -C87S, V L -C87Y. The analysis was carried out using antigen-binding ability measurement by indirect specific ELISA and a detailed molecular modeling that comprises homology methods, long molecular dynamics simulations and docking. We found that V L -C87 affected the antibody fragment stability without interfering with the disulfide bond formation. The effect of mutating the V L -C87 by a usual residue at this position like Tyr caused distant structural changes at the V H region that confers a higher mobility to the V H -CDR2 and V H -CDR3 loops improving the scFv binding to the antigen. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Estimation of antibodies specific for dextran

    International Nuclear Information System (INIS)

    Matsuuchi, L.; Morrison, S.L.

    1978-01-01

    Methods are described for the isolation and characterization of picogram quantities of anti-dextran antibodies. 14 C-dextrans produced by using the dextransucrases of Leuconostoc mesenteroides strains B1355 and B512 were used in a radioimmunoassay. The specificity of this assay was verified by using cell cytoplasmic lysates from mouse plasmacytomas, J558 (anti-α 1 → 3 dextran) and W3129 (anti-α 1 → 6 dextran). Dextran produced by strain B1355 and insolubilized with epichlorohydrin was used as an immunoabsorbent

  3. Radioimmunological demonstration of DNA specific antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Falck, P [Akademie der Wissenschaften der DDR, Berlin-Buch. Zentralinstitut fuer Isotopen- und Strahlenforschung

    1976-01-01

    Using /sup 125/I chemically labelled denatured (d) and native (n) DNA, specifically binding antibodies were demonstrated in the sera of Lupus erythemathodes patients by means of the Farr technique. (NH/sub 4/)/sub 2/SO/sub 4/ was used to separate the immunologically bound /sup 125/I-d-DNA. For /sup 125/I-n-DNA the use of a secondary antiserum for the precipitation of the primary immune complex is advantageous. The influence of antigen concentration upon the binding rate was studied. Titre determinations can be made with the proposed method.

  4. Antibody or Antibody Fragments : Implications for Molecular Imaging and Targeted Therapy of Solid Tumors

    NARCIS (Netherlands)

    Xenaki, Katerina T; Oliveira, Sabrina; van Bergen En Henegouwen, Paul M P

    2017-01-01

    The use of antibody-based therapeutics has proven very promising for clinical applications in cancer patients, with multiple examples of antibodies and antibody-drug conjugates successfully applied for the treatment of solid tumors and lymphomas. Given reported recurrence rates, improvements are

  5. Direct labelling of monomeric antibody fragments Fab' with 99mTc

    International Nuclear Information System (INIS)

    Li Jun; Wang Shizhen; Yang Ziyi

    1994-01-01

    Direct labelling method and conditions of monomeric antibody Fab' with 99m Tc were investigated. Polyclonal antibody IgG was digested with ficin to produce dimeric fragments F(ab') 2 , which was subsequently reduced to monomeric fragments Fab' with 2-mercaptoethylamine. Finally, Fab' was incubated with sodium gluconate (Sn(II)) kit solution and 99m TcO 4 - eluted at room temperature to form 99m Tc-Fab'. The labelling efficiency was 85%-95%. The stability of labelled products was satisfactory and the elimination rate was faster than 99m Tc-IgG

  6. Characterization of crystals of an antibody-recognition fragment of the cancer differentiation antigen mesothelin in complex with the therapeutic antibody MORAb-009

    International Nuclear Information System (INIS)

    Ma, Jichun; Tang, Wai Kwan; Esser, Lothar; Pastan, Ira; Xia, Di

    2012-01-01

    The therapeutic antibody MORAb-009 disrupts the interaction of mesothelin and the ovarian cancer antigen CA-125. Crystals have been grown of the Fab fragment derived from MORAb-009 and of its complex with an N-terminal fragment of mesothelin. The mesothelin-specific monoclonal antibody MORAb-009 is capable of blocking the binding of mesothelin to CA-125 and displays promising anticancer potential. It is currently undergoing clinical trials. In order to understand the basis of the interaction between MORAb-009 and mesothelin at atomic resolution, both the Fab fragment of MORAb-009 and the complex between the Fab and an N-terminal fragment of mesothelin (residues 7–64) were crystallized. The crystals of the Fab diffracted X-rays to 1.75 Å resolution and had the symmetry of space group P4 1 2 1 2, with unit-cell parameters a = b = 140.6, c = 282.0 Å. The crystals of the mesothelin–Fab complex diffracted to 2.6 Å resolution and belonged to the hexagonal space group P6 4 , with unit-cell parameters a = b = 146.2, c = 80.9 Å. Structural analyses of these molecules are in progress

  7. Selective disulfide reduction for labeling and enhancement of Fab antibody fragments

    International Nuclear Information System (INIS)

    Kirley, Terence L.; Greis, Kenneth D.; Norman, Andrew B.

    2016-01-01

    Many methods have been developed for chemical labeling and enhancement of the properties of antibodies and their common fragments, including the Fab and F(ab’) 2 fragments. Somewhat selective reduction of some antibody disulfide bonds has been previously achieved, yielding antibodies and antibody fragments that can be labeled at defined sites, enhancing their utility and properties. Selective reduction of the two hinge disulfide bonds present in F(ab’) 2 fragments using mild reduction has been useful. However, such reduction is often not quantitative and results in the reduction of multiple disulfide bonds, and therefore subsequent multiple labeling or conjugation sites are neither homogenous nor stoichiometric. Here, a simple and efficient selective reduction of the single disulfide bond linking the partial heavy chain and the intact light chain which compose the Fab fragment is accomplished utilizing tris(2-carboxyethyl)phosphine (TCEP) immobilized on agarose beads. The resultant reduced cysteine residues were labeled with several cysteine-selective fluorescent reagents, as well as by cysteine-directed PEGylation. These two cysteine residues can also be re-ligated by means of a bifunctional cysteine cross-linking agent, dibromobimane, thereby both restoring a covalent linkage between the heavy and light chains at this site, far removed from the antigen binding site, and also introducing a fluorescent probe. There are many other research and clinical uses for these selectively partially reduced Fab fragments, including biotinylation, toxin and drug conjugation, and incorporation of radioisotopes, and this technique enables simple generation of very useful Fab fragment derivatives with many potential applications. - Highlights: • TCEP agarose is effective for selective reduction of a single Fab disulfide bond. • This disulfide is solvent accessible and distant from the antigen binding site. • A variety of buffers of varying pHs can be used, simplifying

  8. Selective disulfide reduction for labeling and enhancement of Fab antibody fragments.

    Science.gov (United States)

    Kirley, Terence L; Greis, Kenneth D; Norman, Andrew B

    2016-11-25

    Many methods have been developed for chemical labeling and enhancement of the properties of antibodies and their common fragments, including the Fab and F(ab') 2 fragments. Somewhat selective reduction of some antibody disulfide bonds has been previously achieved, yielding antibodies and antibody fragments that can be labeled at defined sites, enhancing their utility and properties. Selective reduction of the two hinge disulfide bonds present in F(ab') 2 fragments using mild reduction has been useful. However, such reduction is often not quantitative and results in the reduction of multiple disulfide bonds, and therefore subsequent multiple labeling or conjugation sites are neither homogenous nor stoichiometric. Here, a simple and efficient selective reduction of the single disulfide bond linking the partial heavy chain and the intact light chain which compose the Fab fragment is accomplished utilizing tris(2-carboxyethyl)phosphine (TCEP) immobilized on agarose beads. The resultant reduced cysteine residues were labeled with several cysteine-selective fluorescent reagents, as well as by cysteine-directed PEGylation. These two cysteine residues can also be re-ligated by means of a bifunctional cysteine cross-linking agent, dibromobimane, thereby both restoring a covalent linkage between the heavy and light chains at this site, far removed from the antigen binding site, and also introducing a fluorescent probe. There are many other research and clinical uses for these selectively partially reduced Fab fragments, including biotinylation, toxin and drug conjugation, and incorporation of radioisotopes, and this technique enables simple generation of very useful Fab fragment derivatives with many potential applications. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Screening for single-chain variable fragment antibodies against multiple Cry1 toxins from an immunized mouse phage display antibody library.

    Science.gov (United States)

    Dong, Sa; Bo, Zongyi; Zhang, Cunzheng; Feng, Jianguo; Liu, Xianjin

    2018-04-01

    Single-chain variable fragment (scFv) is a kind of antibody that possess only one chain of the complete antibody while maintaining the antigen-specific binding abilities and can be expressed in prokaryotic system. In this study, scFvs against Cry1 toxins were screened out from an immunized mouse phage displayed antibody library, which was successfully constructed with capacity of 6.25 × 10 7  CFU/mL. Using the mixed and alternative antigen coating strategy and after four rounds of affinity screening, seven positive phage-scFvs against Cry1 toxins were selected and characterized. Among them, clone scFv-3H9 (MG214869) showing relative stable and high binding abilities to six Cry1 toxins was selected for expression and purification. SDS-PAGE indicated that the scFv-3H9 fragments approximately 27 kDa were successfully expressed in Escherichia coli HB2151 strain. The purified scFv-3H9 was used to establish the double antibody sandwich enzyme-linked immunosorbent assay method (DAS-ELISA) for detecting six Cry1 toxins, of which the lowest detectable limits (LOD) and the lowest quantitative limits (LOQ) were 3.14-11.07 and 8.22-39.44 ng mL -1 , respectively, with the correlation coefficient higher than 0.997. The average recoveries of Cry1 toxins from spiked rice leaf samples were ranged from 84 to 95%, with coefficient of variation (CV) less than 8.2%, showing good accuracy for the multi-residue determination of six Cry1 toxins in agricultural samples. This research suggested that the constructed phage display antibody library based on the animal which was immunized with the mixture of several antigens under the same category can be used for the quick and effective screening of generic antibodies.

  10. Rituximab selectively suppresses specific islet antibodies.

    Science.gov (United States)

    Yu, Liping; Herold, Kevan; Krause-Steinrauf, Heidi; McGee, Paula L; Bundy, Brian; Pugliese, Alberto; Krischer, Jeff; Eisenbarth, George S

    2011-10-01

    The TrialNet Study Group evaluated rituximab, a B-cell-depleting monoclonal antibody, for its effect in new-onset patients with type 1A diabetes. Rituximab decreased the loss of C-peptide over the first year of follow-up and markedly depleted B lymphocytes for 6 months after administration. This article analyzes the specific effect of rituximab on multiple islet autoantibodies. A total of 87 patients between the ages of 8 and 40 years received either rituximab or a placebo infusion weekly for four doses close to the onset of diabetes. Autoantibodies to insulin (IAAs), GAD65 (GADAs), insulinoma-associated protein 2 (IA2As), and ZnT8 (ZnT8As) were measured with radioimmunoassays. The primary outcome for this autoantibody analysis was the mean level of autoantibodies during follow-up. Rituximab markedly suppressed IAAs compared with the placebo injection but had a much smaller effect on GADAs, IA2As, and ZnT8As. A total of 40% (19 of 48) of rituximab-treated patients who were IAA positive became IAA negative versus 0 of 29 placebo-treated patients (P 1 year in insulin-treated patients. For the patients receiving insulin for >2 weeks prior to rituximab administration, we cannot assess whether rituximab not only blocks the acquisition of insulin antibodies induced by insulin administration and/or also suppresses preformed insulin autoantibodies. Studies in prediabetic non-insulin-treated patients will likely be needed to evaluate the specific effects of rituximab on levels of IAAs.

  11. Phage-display libraries of murine and human antibody Fab fragments

    DEFF Research Database (Denmark)

    Engberg, J; Andersen, P S; Nielsen, L K

    1996-01-01

    We provide efficient and detailed procedures for construction, expression, and screening of comprehensive libraries of murine or human antibody Fab fragments displayed on the surface of filamentous phage. In addition, protocols for producing and using ultra-electrocompetent cells, for producing Fab...

  12. Improved functional immobilization of llama single-domain antibody fragments to polystyrene surfaces using small peptides

    NARCIS (Netherlands)

    Harmsen, M.M.; Fijten, H.P.D.

    2012-01-01

    We studied the effect of different fusion domains on the functional immobilization of three llama single-domain antibody fragments (VHHs) after passive adsorption to polystyrene in enzyme-linked immunosorbent assays (ELISA). Three VHHs produced without any fusion domain were efficiently adsorbed to

  13. Optimization of the crystallizability of a single-chain antibody fragment

    Czech Academy of Sciences Publication Activity Database

    Škerlová, Jana; Král, Vlastimil; Fábry, Milan; Sedláček, Juraj; Veverka, Václav; Řezáčová, Pavlína

    2014-01-01

    Roč. 70, č. 12 (2014), s. 1701-1706 ISSN 1744-3091 R&D Projects: GA MŠk(CZ) LK11205 Institutional support: RVO:61388963 ; RVO:68378050 Keywords : single-chain antibody fragment * Thermofluor assay * differential scanning fluorimetry * crystallizability optimization * oligomerization * crystallization Subject RIV: CE - Biochemistry Impact factor: 0.527, year: 2014

  14. Antibody engineering using phage display with a coiled-coil heterodimeric Fv antibody fragment.

    Directory of Open Access Journals (Sweden)

    Xinwei Wang

    Full Text Available A Fab-like antibody binding unit, ccFv, in which a pair of heterodimeric coiled-coil domains was fused to V(H and V(L for Fv stabilization, was constructed for an anti-VEGF antibody. The anti-VEGF ccFv showed the same binding affinity as scFv but significantly improved stability and phage display level. Furthermore, phage display libraries in the ccFv format were constructed for humanization and affinity maturation of the anti-VEGF antibody. A panel of V(H frameworks and V(H-CDR3 variants, with a significant improvement in affinity and expressibility in both E. coli and yeast systems, was isolated from the ccFv phage libraries. These results demonstrate the potential application of the ccFv antibody format in antibody engineering.

  15. A novel variable antibody fragment dimerized by leucine zippers with enhanced neutralizing potency against rabies virus G protein compared to its corresponding single-chain variable antibody fragment.

    Science.gov (United States)

    Li, Zhuang; Cheng, Yue; Xi, Hualong; Gu, Tiejun; Yuan, Ruosen; Chen, Xiaoxu; Jiang, Chunlai; Kong, Wei; Wu, Yongge

    2015-12-01

    Fatal rabies can be prevented effectively by post-exposure prophylactic (PEP) with rabies immunoglobulin (RIG). Single-chain variable fragments (scFv), which are composed of a variable heavy chain (VH) and a variable light chain (VL) connected by a peptide linker, can potentially be used to replace RIG. However, in our previous study, a scFv (scFV57S) specific for the rabies virus (RV) G protein showed a lower neutralizing potency than that of its parent IgG due to lower stability and altered peptide assembly pattern. In monoclonal antibodies, the VH and VL interact non-covalently, while in scFvs the VH is connected covalently with the VL by the artificial linker. In this study, we constructed and expressed two peptides 57VL-JUN-HIS and 57VH-FOS-HA in Escherichia coli. The well-known Fos and Jun leucine zippers were utilized to dimerize VH and VL similarly to the IgG counterpart. The two peptides assembled to form zipFv57S in vitro. Due to the greater similarity in structure with IgG, the zipFv57S protein showed a higher binding ability and affinity resulting in notable improvement of in vitro neutralizing activity over its corresponding scFv. The zipFv57S protein was also found to be more stable and showed similar protective rate as RIG in mice challenged with a lethal dose of RV. Our results not only indicated zipFv57S as an ideal alternative for RIG in PEP but also offered a novel and efficient hetero-dimerization pattern of VH and VL leading to enhanced neutralizing potency. Copyright © 2015. Published by Elsevier Ltd.

  16. 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.

  17. Detection of experimental myocarditis by monoclonal antimyosin antibody, Fab fragment

    Energy Technology Data Exchange (ETDEWEB)

    Rezkalla, S.; Kloner, R.A.; Khaw, B.A.; Haber, E.; Fallon, J.T.; Smith, F.E.; Khatib, R.

    1989-02-01

    The purpose of this study was to determine whether monoclonal antimyosin Fab (antigen binding fragment) was capable of labeling hearts with experimental coxsackievirus myocarditis, and to determine whether Fab could be used for detecting myocardial damage in either early or chronic phases of the disease. Sixty-five, 3-week-old cesarean-derived 1 (CD 1) mice were divided into two groups: group I (noninfected animals) and group II (infected with coxsackievirus B3). Mice from each group were killed on days 7, 17, 30, or 90 of infection. Forty-eight hours before killing, mice were injected with monoclonal I-125 antimyosin, Fab (25 microCi/injection) and radioactivity was counted in the heart. Selected heart sections were also examined by autoradiography. Heart radioactivity, count/m/mg (m +/- SEM) on days 7, 17, 30, and 90 of infection was 10.8 +/- 1.7, 21.3 +/- 1.1, 11.2 +/- 3.4, and 12.4 +/- 1.5 for group I, versus 36.7 +/- 8.0 (p less than 0.01), 50.0 +/- 4.5 (p less than 0.001), 33.4 +/- 16.1 (p = NS), and 40.6 +/- 8.5 (p less than 0.01) for group II, respectively. Autoradiography revealed focal uptake within areas of necrotic myocardium. We conclude that I125 Fab may be useful in detecting myocardial damage in the experimental model of murine myocarditis up to day 90 of infection.

  18. Detection of experimental myocarditis by monoclonal antimyosin antibody, Fab fragment

    International Nuclear Information System (INIS)

    Rezkalla, S.; Kloner, R.A.; Khaw, B.A.; Haber, E.; Fallon, J.T.; Smith, F.E.; Khatib, R.

    1989-01-01

    The purpose of this study was to determine whether monoclonal antimyosin Fab (antigen binding fragment) was capable of labeling hearts with experimental coxsackievirus myocarditis, and to determine whether Fab could be used for detecting myocardial damage in either early or chronic phases of the disease. Sixty-five, 3-week-old cesarean-derived 1 (CD 1) mice were divided into two groups: group I (noninfected animals) and group II (infected with coxsackievirus B3). Mice from each group were killed on days 7, 17, 30, or 90 of infection. Forty-eight hours before killing, mice were injected with monoclonal I-125 antimyosin, Fab (25 microCi/injection) and radioactivity was counted in the heart. Selected heart sections were also examined by autoradiography. Heart radioactivity, count/m/mg (m +/- SEM) on days 7, 17, 30, and 90 of infection was 10.8 +/- 1.7, 21.3 +/- 1.1, 11.2 +/- 3.4, and 12.4 +/- 1.5 for group I, versus 36.7 +/- 8.0 (p less than 0.01), 50.0 +/- 4.5 (p less than 0.001), 33.4 +/- 16.1 (p = NS), and 40.6 +/- 8.5 (p less than 0.01) for group II, respectively. Autoradiography revealed focal uptake within areas of necrotic myocardium. We conclude that I125 Fab may be useful in detecting myocardial damage in the experimental model of murine myocarditis up to day 90 of infection

  19. Site-Specific Antibody Functionalization Using Tetrazine-Styrene Cycloaddition.

    Science.gov (United States)

    Umlauf, Benjamin J; Mix, Kalie A; Grosskopf, Vanessa A; Raines, Ronald T; Shusta, Eric V

    2018-05-03

    Biologics, such as antibody-drug conjugates, are becoming mainstream therapeutics. Consequently, methods to functionalize biologics without disrupting their native properties are essential for identifying, characterizing, and translating candidate biologics from the bench to clinical practice. Here, we present a method for site-specific, carboxy-terminal modification of single-chain antibody fragments (scFvs). ScFvs displayed on the surface of yeast were isolated and functionalized by combining intein-mediated expressed protein ligation (EPL) with inverse electron-demand Diels-Alder (IEDDA) cycloaddition using a styrene-tetrazine pair. The high thiol concentration required to trigger EPL can hinder the subsequent chemoselective ligation reactions; therefore, the EPL reaction was used to append styrene to the scFv, limiting tetrazine exposure to damaging thiols. Subsequently, the styrene-functionalized scFv was reacted with tetrazine-conjugated compounds in an IEDDA cycloaddition to generate functionalized scFvs that retain their native binding activity. Rapid functionalization of yeast surface-derived scFv in a site-directed manner could find utility in many downstream laboratory and preclinical applications.

  20. Assay for the specificity of monoclonal antibodies in crossed immunoelectrophoresis

    DEFF Research Database (Denmark)

    Skjødt, K; Schou, C; Koch, C

    1984-01-01

    A method is described based on crossed immunoelectrophoresis of a complex antigen mixture in agarose gel followed by incubation of the gel with the monoclonal antibody. The bound monoclonal antibody is detected by the use of a secondary enzyme-labelled antibody. Using this technique we have been ...... I molecules. In other experiments using the same technique we demonstrated the reaction of a monoclonal antibody specific for chicken Ig light chains. Udgivelsesdato: 1984-Aug-3...

  1. Development of a Recombinant Antibody with Specificity for Chelated Uranyl Ions

    International Nuclear Information System (INIS)

    X. Li; A.M. Kriegel; T.C. Bishop; R.C. Blake; E. Figueiredo; H. Yu; D.A. Blake

    2005-01-01

    The goal of our project is to continue the development of new techniques for rapid, automated identification of radionuclides, metals, and chelators that may contaminant sur face and groundwater at DOE sites. One of the four specific aims of the present project is to develop new technologies in antibody engineering that will enhance our immunosensor program. Recombinant antibodies have potential advantages over monoclonal antibodies produced by standard hybridoma technology. The cloned genes represent a stable, recoverable source for antibody production. In addition, the recombinant format offers opportunities for protein engineering that enhances antibody performance and for studies that relate antibody sequence to binding activity. In this study, a hybridoma that synthesized an antibody (12F6) that recognized a 1:1 complex between 2,9-dicarboxyl-1,10- phenanthroline (DCP) and UO 2 2+ was used as a source of RNA for the development of a recombinant (Fab) 2 fragment. RNA was isolated from the 12F6 hybridoma and the cDNA encoding the entire κ light chain and the linked VH and C1 portions of the heavy chain were amplified from total RNA. cDNA sequences were verified by comparison with the N-terminal amino acid sequences of the light and heavy chains of the native 12F6 monoclonal antibody. A leader sequence and appropriate restriction sites were added to each chain, and the fragments were ligated into a commercial dicistronic vector (pBudCE4.1, Invitrogen, Inc.). COS-1 cells were transfected with this vector and the culture supernatant was assayed for activity and the (Fab) 2 protein. Cells transfected with vector containing 12F6 cDNA synthesized and secreted recombinant (Fab) 2 fragments that bound to the UO 2 2+ -DCP complex with an affinity indistinguishable from that of a (Fab) 2 fragment prepared from the native antibody. Molecular models of the heavy and light chain variable domains were constructed according to the canonical structures method detailed by Morea

  2. 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.

  3. Construction and sequencing analysis of scFv antibody fragment derived from monoclonal antibody against norfloxacin (Nor155

    Directory of Open Access Journals (Sweden)

    J. Mala

    2017-06-01

    Full Text Available Norfloxacin belongs to the group of fluoroquinolone antibiotics which has been approved for treatment in animals. However, its residues in animal products can pose adverse side effects to consumer. Therefore, detection of the residue in different food matrices must be concerned. In this study, a single chain variable fragment (scFv that recognizes norfloxacin antibiotic was constructed. The cDNA was synthesized from total RNA of hybridoma cells against norfloxacin. Genes encoding VH and VL regions of monoclonal antibody against norfloxacin (Nor155 were amplified and size of VH and VL fragments was 402 bp and 363 bp, respectively. The scFv of Nor155 was constructed by an addition of (Gly4Ser3 as a linker between VH and VL regions and subcloned into pPICZαA, an expression vector of Pichia pastoris. The sequence of scFv Nor155 (GenBank No. AJG06891.1 was confirmed by sequencing analysis. The complementarity determining regions (CDR I, II, and III of VH and VL were specified by Kabat method. The obtained recombinant plasmid will be useful for production of scFv antibody against norfloxacin in P. pastoris and further engineer scFv antibody against fluoroquinolone antibiotics.

  4. Technetium-99 labelling of DD-3B6/22 antifibrin monoclonal antibody fragmented Fab' for thrombus imaging

    International Nuclear Information System (INIS)

    Lee, F-T.; Boniface, G.R.; Lambrecht, R.M.; Rylatt, D.B.; Bundesen, P.G.

    1993-01-01

    The antifibrin DD-3B6/22 monoclonal antibody Fab' fragment, a murine immunoglobulin, IgG3, has been labelled with technetium-99m ( 99mTc ) via a transchelation reaction, to specific activity in excess of 30 mCi/mg protein. The radiolabelling of Fab' was dependent on time, temperature, pH, antibody concentrations and nature intermediary transchelation complex used. The resultant radioconjugate was stable in vitro and in vivo. Blood clearance of 99m Tc-Fab' in rat followed two compartment kinetics with the half time of the fast phase being 0.5 h. The main route of excretion was via the kidneys with little uptake indicated by other tissues. The results suggest that the inherent specificity of the antibody, small molecular size, rapid plasma clearance, high specific radioactivity, together with the physical properties of the 99m Tc label, combine to make this labelled monoclonal antibody (MoAb), potentially suitable as a radiopharmaceutical for the scintigraphic detection of thrombi in humans. 17 refs., 3 tabs., 5 figs

  5. Tumour targeting with monovalent fragments of anti-neuroblastoma antibody chCE7

    International Nuclear Information System (INIS)

    Carrel, F.; Novak-Hofer, I.; Ruch, C.; Zimmermann, K.; Amstutz, H.

    1997-01-01

    The in vitro and in vivo behaviour of the monovalent single chain (scFv) and Fab-fragments derived from anti-neuroblastoma antibody chCE7 is reported. When comparing tumour uptake and -retention of radioactivity of 67 Cu-labelled monovalent chCE7 with divalent chCE7 F(ab') 2 the advantage of the radiocopper label over the radioiodine label was more pronounced with the divalent (internalising) F(ab') 2 fragments. (author) 1 fig., 1 ref

  6. Application of 99mTc-labeled chimeric Fab fragments of monoclonal antibody A7 for radioimmunoscintigraphy of pancreatic cancer

    International Nuclear Information System (INIS)

    Matsumura, Hiroomi

    1999-01-01

    Pancreatic cancer is one of the most lethal diseases and its prognosis is still poor. To improve the survival rate, it is essential to develop new technologies for early and definitive diagnosis. In this study, chimeric Fab fragments of monoclonal antibody A7 were successfully radio-labeled with 99m Tc, preventing depression of the antigen-binding activity. 99m Tc-labeled monoclonal antibody A7, 99m Tc-labeled chimeric Fab fragments of monoclonal antibody A7, 99m Tc-labeled normal mouse IgG and 99m Tc-labeled Fab fragments of normal mouse IgG were injected intravenously into nude mice bearing human pancreatic cancer xenografts and the radioactivity was subsequently measured. The tumor accumulation was significantly higher with labeled monoclonal antibody A7 than with normal mouse IgG, and higher with chimeric Fab fragments of monoclonal antibody A7 than with Fab fragments of normal mouse IgG. The tumor/blood ratio of radioactivity increased rapidly over time with chimeric Fab fragments of monoclonal antibody A7. These results suggest that chimeric Fab fragments of monoclonal antibody A7 may be useful for diagnosing pancreatic cancer by means of radioimmunoscintigraphy. (author)

  7. Isolation and characterization of anti c-met single chain fragment variable (scFv) antibodies.

    Science.gov (United States)

    Qamsari, Elmira Safaie; Sharifzadeh, Zahra; Bagheri, Salman; Riazi-Rad, Farhad; Younesi, Vahid; Abolhassani, Mohsen; Ghaderi, Sepideh Safaei; Baradaran, Behzad; Somi, Mohammad Hossein; Yousefi, Mehdi

    2017-12-01

    The receptor tyrosine kinase (RTK) Met is the cell surface receptor for hepatocyte growth factor (HGF) involved in invasive growth programs during embryogenesis and tumorgenesis. There is compelling evidence suggesting important roles for c-Met in colorectal cancer proliferation, migration, invasion, angiogenesis, and survival. Hence, a molecular inhibitor of an extracellular domain of c-Met receptor that blocks c-Met-cell surface interactions could be of great thera-peutic importance. In an attempt to develop molecular inhibitors of c-Met, single chain variable fragment (scFv) phage display libraries Tomlinson I + J against a specific synthetic oligopeptide from the extracellular domain of c-Met receptor were screened; selected scFv were then characterized using various immune techniques. Three c-Met specific scFv (ES1, ES2, and ES3) were selected following five rounds of panning procedures. The scFv showed specific binding to c-Met receptor, and significantly inhibited proliferation responses of a human colorectal carcinoma cell line (HCT-116). Moreover, anti- apoptotic effects of selected scFv antibodies on the HCT-116 cell line were also evaluated using Annexin V/PI assays. The results demonstrated rates of apoptotic cell death of 46.0, 25.5, and 37.8% among these cells were induced by use of ES1, ES2, and ES3, respectively. The results demonstrated ability to successfully isolate/char-acterize specific c-Met scFv that could ultimately have a great therapeutic potential in immuno-therapies against (colorectal) cancers.

  8. The preparation and use of radiolabelled specific helminth antibodies

    International Nuclear Information System (INIS)

    Movsesijan, M.; Jovanovic, B.; Borojevic, D.; Petrovic, M.

    1983-01-01

    Specific antibodies from the serum of sheep infected with Haemonchus contortus were isolated by combination with a ''solid phase antigen'' (soluble antigen coupled to an activated crystalline cellulose). The antibodies were labelled with 125 I while bound to the solid phase then eluted and their potential demonstrated: (1) to determine amounts of specific antibody in unknown sera; (2) to determine amounts of soluble antigen in unknown preparations. (author)

  9. 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.

  10. F(ab'2 antibody fragments against Trypanosoma cruzi calreticulin inhibit its interaction with the first component of human complement

    Directory of Open Access Journals (Sweden)

    LORENA AGUILAR

    2005-01-01

    Full Text Available Trypanosoma cruzi calreticulin (TcCRT, described in our laboratory, retains several important functional features from its vertebrate homologues. We have shown that recombinant TcCRT inhibits the human complement system when it binds to the collagenous portion of C1q. The generation of classical pathway convertases and membrane attack complexes is thus strongly inhibited. In most T. cruzi-infected individuals, TcCRT is immunogenic and mediates the generation of specific antibodies. By reverting the C1q / TcCRT interaction, a parasite immune evasion strategy, these antibodies contribute to the host / parasite equilibrium. In an in vitro correlate of this situation, we show that the C1q / TcCRT interaction is inhibited by F(ab'2 polyclonal anti-TcCRT IgG fragments. It is therefore feasible that in infected humans anti-TcCRT antibodies participate in reverting an important parasite strategy aimed at inhibiting the classical complement pathway. Thus, membrane-bound TcCRT interacts with the collagenous portion C1q, and this C1q is recognized by the CD91-bound host cell CRT, thus facilitating parasite internalization. Based on our in vitro results, it could be proposed that the in vivo interaction between TcCRT and vertebrate C1q could be inhibited by F(ab'2 fragments anti-rTcCRT or against its S functional domain, thus interfering with the internalization process

  11. Multifunctional PSCA antibody fragments for PET and optical prostate cancer imaging

    Science.gov (United States)

    2017-10-01

    that recognize PSCA (prostate stem cell antigen), a cell surface protein highly expressed in prostate cancer. These engineered antibody fragments...operatively. Prostate stem cell antigen (PSCA) is a cell - surface marker overexpressed in primary and metastatic cancers1. In vivo administration of...REPORT: Annual PREPARED FOR: U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012 DISTRIBUTION STATEMENT

  12. Nebulized Anti-IL-13 Monoclonal Antibody Fab' Fragment Reduces Allergen-Induced Asthma

    OpenAIRE

    Hacha, Jonathan; Tomlinson, K; Maertens, Ludovic; Paulissen, Geneviève; Rocks, Natacha; Foidart, Jean-Michel; Noël, Agnès; Palframan, R; Guéders, Maud; Cataldo, Didier

    2012-01-01

    Rationale: Interleukin-13 (IL-13) is a prototypic Th2 cytokine and a central mediator of the complex cascade of events leading to asthmatic phenotype. Indeed, IL-13 plays key roles in IgE synthesis, bronchial hyperresponsiveness, mucus hypersecretion, subepithelial fibrosis and eosinophil infiltration. Objectives: We assessed the potential efficacy of inhaled anti-IL-13 monoclonal antibody Fab' fragment on allergen-induced airway inflammation, hyperresponsiveness and remodeling in an experime...

  13. Radioimmunoimaging using F(ab')2 fragment of monoclonal antibodies against human alpha-fetoprotein

    International Nuclear Information System (INIS)

    Sakahara, Harumi; Endo, Keigo; Nakashima, Tetsuo; Koizumi, Mitsuru; Ohta, Hitoya; Torizuka, Kanji; Okada, Kenichiro; Yoshida, Osamu; Nishi, Shinzo.

    1985-01-01

    Using monoclonal antibodies against human α-fetoprotein (AFP), radioiodinated F(ab') 2 fragments were compared with whole IgG as a radiotracer for radioimmunoimaging of cancer. F(ab') 2 fragments were obtained by pepsin digestion of whole IgG (IgGl). IgG and F(ab') 2 were labeled with 125 I or 131 I by the chloramine-T method with almost full retention of antibody activity. F(ab') 2 fragments were cleared more rapidly from the circulation in normal mice with a half life of 6.3 hours than whole IgG with a half life of 5.5 days. Radioactivity of F(ab') 2 in various organs also decreased faster than IgG. In nude mice transplanted with AFP-producing human testicular tumor, F(ab') 2 fragments demonstrated superior scintigrams to whole IgG at 2 days after the injection, because of the fast disappearance of background radioactivity. Although absolute accumulation of 131 I labeled F(ab') 2 in the tumor was less than that of 131 I labeled IgG, tumor to other organ ratios were much higher with F(ab') 2 than those of IgG. The tumor to blood ratio of 131 I labeled F(ab') 2 was 1.04 at day 2, whereas tumor to blood ratio of 131 I labeled IgG was 0.55 at day 2 and 0.92 at day 4, respectively. These results indicated that for the radiolabeling of monoclonal antibodies, F(ab') 2 fragments would be superior to whole IgG in the radioimmunoimaging of cancer. (author)

  14. Efficient production of Trastuzumab Fab antibody fragments in Brevibacillus choshinensis expression system.

    Science.gov (United States)

    Mizukami, Makoto; Onishi, Hiromasa; Hanagata, Hiroshi; Miyauchi, Akira; Ito, Yuji; Tokunaga, Hiroko; Ishibashi, Matsujiro; Arakawa, Tsutomu; Tokunaga, Masao

    2018-10-01

    The Brevibacillus expression system has been successfully employed for the efficient productions of a variety of recombinant proteins, including enzymes, cytokines, antigens and antibody fragments. Here, we succeeded in secretory expression of Trastuzumab Fab antibody fragments using B. choshinensis/BIC (Brevibacillus in vivocloning) expression system. In the fed-batch high-density cell culture, recombinant Trastuzumab Fab with amino-terminal His-tag (His-BcFab) was secreted at high level, 1.25 g/liter, and Fab without His-tag (BcFab) at ∼145 mg/L of culture supernatant. His-BcFab and BcFab were purified to homogeneity using combination of conventional column chromatographies with a yield of 10-13%. This BcFab preparation exhibited native structure and functions evaluated by enzyme-linked immunosorbent assay, surface plasmon resonance, circular dichroism measurements and size exclusion chromatography. To our knowledge, this is the highest production of Fab antibody fragments in gram-positive bacterial expression/secretion systems. Copyright © 2018 Elsevier Inc. All rights reserved.

  15. Codon-Precise, Synthetic, Antibody Fragment Libraries Built Using Automated Hexamer Codon Additions and Validated through Next Generation Sequencing

    Directory of Open Access Journals (Sweden)

    Laura Frigotto

    2015-05-01

    Full Text Available We have previously described ProxiMAX, a technology that enables the fabrication of precise, combinatorial gene libraries via codon-by-codon saturation mutagenesis. ProxiMAX was originally performed using manual, enzymatic transfer of codons via blunt-end ligation. Here we present Colibra™: an automated, proprietary version of ProxiMAX used specifically for antibody library generation, in which double-codon hexamers are transferred during the saturation cycling process. The reduction in process complexity, resulting library quality and an unprecedented saturation of up to 24 contiguous codons are described. Utility of the method is demonstrated via fabrication of complementarity determining regions (CDR in antibody fragment libraries and next generation sequencing (NGS analysis of their quality and diversity.

  16. 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 ...

  17. Antibody specific epitope prediction-emergence of a new paradigm.

    Science.gov (United States)

    Sela-Culang, Inbal; Ofran, Yanay; Peters, Bjoern

    2015-04-01

    The development of accurate tools for predicting B-cell epitopes is important but difficult. Traditional methods have examined which regions in an antigen are likely binding sites of an antibody. However, it is becoming increasingly clear that most antigen surface residues will be able to bind one or more of the myriad of possible antibodies. In recent years, new approaches have emerged for predicting an epitope for a specific antibody, utilizing information encoded in antibody sequence or structure. Applying such antibody-specific predictions to groups of antibodies in combination with easily obtainable experimental data improves the performance of epitope predictions. We expect that further advances of such tools will be possible with the integration of immunoglobulin repertoire sequencing data. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Cross-Reactivity of Polyclonal Antibodies against Canavalia ensiformis (Jack Bean) Urease and Helicobacter pylori Urease Subunit A Fragments.

    Science.gov (United States)

    Kaminski, Zbigniew Jerzy; Relich, Inga; Konieczna, Iwona; Kaca, Wieslaw; Kolesinska, Beata

    2018-01-01

    Overlapping decapeptide fragments of H. pylori urease subunit A (UreA) were synthesized and tested with polyclonal antibodies against Canavalia ensiformis (Jack bean) urease. The linear epitopes of UreA identified using the dot blot method were then examined using epitope mapping. For this purpose, series of overlapping fragments of UreA, frameshifted ± four amino acid residues were synthesized. Most of the UreA epitopes which reacted with the Jack bean urease polyclonal antibodies had been recognized in previous studies by monoclonal antibodies against H. pylori urease. Fragments 11 - 24, 21 - 33, and 31 - 42 were able to interact with the Jack bean urease antibodies, giving stable immunological complexes. However, the lack of recognition by these antibodies of all the components in the peptide map strongly suggests that a non-continuous (nonlinear) epitope is located on the N-terminal domain of UreA. © 2018 Wiley-VHCA AG, Zurich, Switzerland.

  19. 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, ...

  20. Radioimmunolocalization and selective delivery of radiation in a rat model system: comparison of intact and fragmented antibody

    International Nuclear Information System (INIS)

    Walker, K.Z.; Seymour-Munn, K.; Axiak, S.M.; Raison, R.L.; Basten, A.; Towson, J.E.; Bautovitch, G.J.; Morris, J.

    1988-01-01

    Monoclonal antibody (MoAb) fragments are known to have advantages over intact immunoglobulins for radioimmunoscintigraphy. It is less clear whether they are as effective in the delivery of radioimmunotherapy. The imaging and dosimetric properties of an intact MoAb, K-1-21, reactive against human kappa light chains (LC) were compared with that of its F(ab') 2 and Fab fragments using a normal rat model system. Two days after injection of 131 I-K-1-21 into rats bearing antigen-sepharose implants, gamma camera images showed specific localization of the MoAb to the target (kappa LC) but not to the control (lambda LC) implant. Better images were obtained with K-1-21 F(ab') 2 than with Fab or intact antibody. Mean kappa implant: blood ratios were 8.6 ± 3.9 for Fab, 7.9 ± 1.8 for F(ab') 2 and 2.0 ± 0.3 for intact K-1-21. The improvement associated with the use of 131 I-K-1-21 fragments was, however, achieved at the expense of lower absolute values of activity at the target site. Thus the absorbed dose delivered to the implant by the intact K-1-21 was double that delivered with F(ab') 2 and six times that delivered with Fab. As intact K-1-21 also delivered a greater radiation dose to normal tissues, F(ab') 2 fragments may have the greatest overall advantages for therapy with radionuclide MoAb conjugates. (author)

  1. A monoclonal antibody that specifically recognizes m6A nucleoside

    OpenAIRE

    Espuny, Ruth; Castro, Ana; Codony, Carles; Eritja Casadellà, Ramón; Bach-Elias, Montse

    1998-01-01

    A hybridoma against the nucleoside m6A has been obtained from mouse spleen. This hybridoma was named H65 and it secretes monoclonal antibodies anti-m6A. The competition assays showed that the monoclonal antibody was highly specific for m6A nucleoside.

  2. Monoclonal antibody PAL-E specific for endothelium

    NARCIS (Netherlands)

    Schlingemann, R. O.; Dingjan, G. M.; Emeis, J. J.; Blok, J.; Warnaar, S. O.; Ruiter, D. J.

    1985-01-01

    A monoclonal antibody, PAL-E, is described that is specific for endothelial cells. The monoclonal antibody, an IgG2a, markedly stains endothelium of capillaries, medium-sized and small veins, and venules in frozen sections of human and some animal tissues tested. It reacts not at all or only weakly

  3. 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.

  4. A natively paired antibody library yields drug leads with higher sensitivity and specificity than a randomly paired antibody library.

    Science.gov (United States)

    Adler, Adam S; Bedinger, Daniel; Adams, Matthew S; Asensio, Michael A; Edgar, Robert C; Leong, Renee; Leong, Jackson; Mizrahi, Rena A; Spindler, Matthew J; Bandi, Srinivasa Rao; Huang, Haichun; Tawde, Pallavi; Brams, Peter; Johnson, David S

    2018-04-01

    Deep sequencing and single-chain variable fragment (scFv) yeast display methods are becoming more popular for discovery of therapeutic antibody candidates in mouse B cell repertoires. In this study, we compare a deep sequencing and scFv display method that retains native heavy and light chain pairing with a related method that randomly pairs heavy and light chain. We performed the studies in a humanized mouse, using interleukin 21 receptor (IL-21R) as a test immunogen. We identified 44 high-affinity binder scFv with the native pairing method and 100 high-affinity binder scFv with the random pairing method. 30% of the natively paired scFv binders were also discovered with the randomly paired method, and 13% of the randomly paired binders were also discovered with the natively paired method. Additionally, 33% of the scFv binders discovered only in the randomly paired library were initially present in the natively paired pre-sort library. Thus, a significant proportion of "randomly paired" scFv were actually natively paired. We synthesized and produced 46 of the candidates as full-length antibodies and subjected them to a panel of binding assays to characterize their therapeutic potential. 87% of the antibodies were verified as binding IL-21R by at least one assay. We found that antibodies with native light chains were more likely to bind IL-21R than antibodies with non-native light chains, suggesting a higher false positive rate for antibodies from the randomly paired library. Additionally, the randomly paired method failed to identify nearly half of the true natively paired binders, suggesting a higher false negative rate. We conclude that natively paired libraries have critical advantages in sensitivity and specificity for antibody discovery programs.

  5. Monoclonal Antibody Fragments for Targeting Therapeutics to Growth Plate Cartilage | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    Researchers at The Eunice Kennedy Shriver National Institute on Child Health and Human Development (NICHD) have discovered monoclonal antibodies that bind to matrilin-3, a protein specifically expressed in cartilage tissue, that could be used for treating or inhibiting growth plate disorders, such as a skeletal dysplasia or short stature. The monoclonal antibodies can also be used to target therapeutic agents, such as anti-arthritis agents, to cartilage tissue. NICHD seeks statements of capability or interest from parties interested in collaborative research to co-develop, evaluate, or commercialize treatment of skeletal disorders using targeting antibodies.

  6. Dissection of antibody specificities induced by yellow fever vaccination.

    Directory of Open Access Journals (Sweden)

    Oksana Vratskikh

    Full Text Available The live attenuated yellow fever (YF vaccine has an excellent record of efficacy and one dose provides long-lasting immunity, which in many cases may last a lifetime. Vaccination stimulates strong innate and adaptive immune responses, and neutralizing antibodies are considered to be the major effectors that correlate with protection from disease. Similar to other flaviviruses, such antibodies are primarily induced by the viral envelope protein E, which consists of three distinct domains (DI, II, and III and is presented at the surface of mature flavivirions in an icosahedral arrangement. In general, the dominance and individual variation of antibodies to different domains of viral surface proteins and their impact on neutralizing activity are aspects of humoral immunity that are not well understood. To gain insight into these phenomena, we established a platform of immunoassays using recombinant proteins and protein domains that allowed us to dissect and quantify fine specificities of the polyclonal antibody response after YF vaccination in a panel of 51 vaccinees as well as determine their contribution to virus neutralization by serum depletion analyses. Our data revealed a high degree of individual variation in antibody specificities present in post-vaccination sera and differences in the contribution of different antibody subsets to virus neutralization. Irrespective of individual variation, a substantial proportion of neutralizing activity appeared to be due to antibodies directed to complex quaternary epitopes displayed on the virion surface only but not on monomeric E. On the other hand, DIII-specific antibodies (presumed to have the highest neutralizing activity as well as broadly flavivirus cross-reactive antibodies were absent or present at very low titers. These data provide new information on the fine specificity as well as variability of antibody responses after YF vaccination that are consistent with a strong influence of individual-specific

  7. Study of the viability of technetium-99m labeling of whole antimyosin antibody and its fragment: development of radiopharmaceutical for cardiac survey

    International Nuclear Information System (INIS)

    Carvalho, Guilherme Luiz de Castro

    2007-01-01

    In the acute myocardium infarction, the myocytes cell membrane loses its integrity, allowing the influx of extracellular macromolecules such as circulating antibody into the damaged cell. The use of the specific antibodies against cardiac myosin labeled with 99m Tc allows to determine the localization and extension of myocardial infarction. The purpose of this work was to study the viability of labeling of the antimyosin monoclonal antibody and its fragment F(ab')2 with 99m Tc. Because of the high cost of antimyosin antibody, others antibodies were used to optimize the methodology and the best condition was used for antimyosin antibody. The intact antibody was cleaved by pepsin to produce F(ab') 2 fragment. The F(ab') 2 and the intact antibody were reduced by treatment with Dithiothreitol (DTT) and 2-Mercaptoethanol (2-ME) and labeled with 99m Tc by direct method. Different concentrations of reductant, mixing conditions and incubation times were studied. In the standard condition, incubation at molar ratio 1:1000 (antibody:reducing agent) at room temperature for 30 minutes with continuous rotation (850 rpm), 13.28 - SH groups were formed per molecule. It was studied the influence of p H, of the concentration of stannous chloride (Sn 2+ ) and incubation time in the labeling condition. The better radiochemical yield (90.06 +- 1.53%) was obtained using 2.5 μg of Sn 2+ in p H 4.5 for 60 minutes. The labeling of the fragment F(ab') 2 did not present satisfactory results because of the low yield of the digestion. After purification by PD-10, the biodistribution study was performed and showed that the intact antimyosin antibody labeled with 99m Tc presented fast kinetic compatible with the biodistribution of an intact antibody labeled with 99m Tc. Scintigraphy image of the animal with myocardial infarction was obtained and compared with the image of a normal animal. The studies allow to conclude that the use of fragment F(ab') 2 are not viable, but the use of the labeled

  8. Efficient production of antibody Fab fragment by transient gene expression in insect cells.

    Science.gov (United States)

    Mori, Keita; Hamada, Hirotsugu; Ogawa, Takafumi; Ohmuro-Matsuyama, Yuki; Katsuda, Tomohisa; Yamaji, Hideki

    2017-08-01

    Transient gene expression allows a rapid production of diverse recombinant proteins in early-stage preclinical and clinical developments of biologics. Insect cells have proven to be an excellent platform for the production of functional recombinant proteins. In the present study, the production of an antibody Fab fragment by transient gene expression in lepidopteran insect cells was investigated. The DNA fragments encoding heavy-chain (Hc; Fd fragment) and light-chain (Lc) genes of an Fab fragment were individually cloned into the plasmid vector pIHAneo, which contained the Bombyx mori actin promoter downstream of the B. mori nucleopolyhedrovirus (BmNPV) IE-1 transactivator and the BmNPV HR3 enhancer for high-level expression. Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were co-transfected with the resultant plasmid vectors using linear polyethyleneimine. When the transfection efficiency was evaluated, a plasmid vector encoding an enhanced green fluorescent protein (EGFP) gene was also co-transfected. Transfection and culture conditions were optimized based on both the flow cytometry of the EGFP expression in transfected cells and the yield of the secreted Fab fragments determined by enzyme-linked immunosorbent assay (ELISA). Under optimal conditions, a yield of approximately 120 mg/L of Fab fragments was achieved in 5 days in a shake-flask culture. Transient gene expression in insect cells may offer a promising approach to the high-throughput production of recombinant proteins. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  9. Locking the Elbow: Improved Antibody Fab Fragments as Chaperones for Structure Determination.

    Science.gov (United States)

    Bailey, Lucas J; Sheehy, Kimberly M; Dominik, Pawel K; Liang, Wenguang G; Rui, Huan; Clark, Michael; Jaskolowski, Mateusz; Kim, Yejoon; Deneka, Dawid; Tang, Wei-Jen; Kossiakoff, Anthony A

    2018-02-02

    Antibody Fab fragments have been exploited with significant success to facilitate the structure determination of challenging macromolecules as crystallization chaperones and as molecular fiducial marks for single particle cryo-electron microscopy approaches. However, the inherent flexibility of the "elbow" regions, which link the constant and variable domains of the Fab, can introduce disorder and thus diminish their effectiveness. We have developed a phage display engineering strategy to generate synthetic Fab variants that significantly reduces elbow flexibility, while maintaining their high affinity and stability. This strategy was validated using previously recalcitrant Fab-antigen complexes where introduction of an engineered elbow region enhanced crystallization and diffraction resolution. Furthermore, incorporation of the mutations appears to be generally portable to other synthetic antibodies and may serve as a universal strategy to enhance the success rates of Fabs as structure determination chaperones. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Subcellular localization of estradiol receptor in MCF7 cells studied with nanogold-labelled antibody fragments.

    Science.gov (United States)

    Kessels, M M; Qualmann, B; Thole, H H; Sierralta, W D

    1998-01-01

    Ultrastructural localization studies of estradiol receptor in hormone-deprived and hormone-stimulated MCF7 cells were done using F(ab') fragments of three different antibodies (#402, 13H2, HT277) covalently linked to nanogold. These ultra-small, non-charged immunoreagents, combined with a size-enlargement by silver enhancement, localized estradiol receptor in both nuclear and cytoplasmic areas of non-stimulated target cells; stimulation with the steroid induced a predominantly nuclear labelling. In the cytoplasm of resting cells, tagging was often observed at or in the proximity of stress fibers. In the nucleus a large proportion of receptor was found inside the nucleolus, specially with the reagent derived from antibody 13H2. We postulate that different accessibilities of receptor epitopes account for the different labelling densities observed at cytoskeletal elements and the nucleoli.

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

    Science.gov (United States)

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

    2017-03-01

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

  12. Antigenic specificity of serum antibodies in mice fed soy protein

    DEFF Research Database (Denmark)

    Christensen, Hanne Risager; Bruun, S.W.; Frøkiær, Hanne

    2003-01-01

    Background: Soybean protein is used in a number of food products but unfortunately is also a common cause of food allergy. Upon ingestion of soy protein, healthy mice like other animals and humans generate a soy-specific antibody response in the absence of signs of illness. Not much is known about...... the relationship between the immunogenic proteins involved in this nondeleterious antibody response and the pathological response associated with food allergy. The objective of the present study was to characterize the antigenic specificity of the soy protein-specific antibody response generated in healthy mice...... ingesting soy protein. Methods: Blood from mice fed a soy-containing diet was analyzed using ELISA and immunoblot for antibody reactivity towards various soy protein fractions and pure soy proteins/subunits. Mice bred on a soy-free diet were used as controls. Results: The detectable antigenic specificity...

  13. Oriented Immobilization of Fab Fragments by Site-Specific Biotinylation at the Conserved Nucleotide Binding Site for Enhanced Antigen Detection.

    Science.gov (United States)

    Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

    2015-09-08

    Oriented immobilization of antibodies and antibody fragments has become increasingly important as a result of the efforts to reduce the size of diagnostic and sensor devices to miniaturized dimensions for improved accessibility to the end-user. Reduced dimensions of sensor devices necessitate the immobilized antibodies to conserve their antigen binding activity for proper operation. Fab fragments are becoming more commonly used in small-scaled diagnostic devices due to their small size and ease of manufacture. In this study, we used the previously described UV-NBS(Biotin) method to functionalize Fab fragments with IBA-EG11-Biotin linker utilizing UV energy to initiate a photo-cross-linking reaction between the nucleotide binding site (NBS) on the Fab fragment and IBA-Biotin molecule. Our results demonstrate that immobilization of biotinylated Fab fragments via UV-NBS(Biotin) method generated the highest level of immobilized Fab on surfaces when compared to other typical immobilization methods while preserving antigen binding activity. UV-NBS(Biotin) method provided 432-fold, 114-fold, and 29-fold improved antigen detection sensitivity than physical adsorption, NHS-Biotin, and ε-NH3(+), methods, respectively. Additionally, the limit of detection (LOD) for PSA utilizing Fab fragments immobilized via UV-NBS(Biotin) method was significantly lower than that of the other immobilization methods, with an LOD of 0.4 pM PSA. In summary, site-specific biotinylation of Fab fragments without structural damage or loss in antigen binding activity provides a wide range of application potential for UV-NBS immobilization technique across numerous diagnostic devices and nanotechnologies.

  14. Antibody guided targeting of non-small cell lung cancer using 111In-labeled HMFG1 F(ab')2 fragments

    International Nuclear Information System (INIS)

    Kalofonos, H.P.; Sivolapenko, G.B.; Courtenay-Luck, N.S.

    1988-01-01

    Immunoscintigraphy using F(ab')2 fragments of tumor-associated monoclonal antibody HMFG1 was performed in 14 patients with primary and metastatic non-small cell carcinoma of lung cancer. The antibody was conjugated with diethylenetriamine pentaacetic acid and labeled with 111 In. Quality control studies showed efficient incorporation of 111 In onto antibody (5 mCi/mg), no significant loss of immunoreactivity, and in vitro and in vivo stability. The optimal time for imaging was between 48 and 72 h. Following i.v. administration, serum activity fell rapidly (t1/2a = 2.5 +/- 1.3 (SD) h; t1/2b = 42 +/- 4.5 h). The majority of the radioactivity was associated with the plasma and not with the blood cells. All patients had a significant concentration of 111 In in the liver (approximately 20% of the injected dose, 48 h postadministration). No toxicity was encountered. No human antimurine-IgG antibody was detected in any of the patients within 4 months of follow-up, even in patients receiving two administrations of F(ab')2 fragments. Localization of all primary lesions and the majority (80%) of metastatic lesions was achieved. Seven of 14 patients were also studied using a 111 In-labeled nonspecific antibody (Fab')2 fragment (4C4). In three patients the specificity index was higher than the other four (P less than 0.05). We conclude that although successful targeting of 111 In-labeled (Fab')2 fragments of HMFG1 can be achieved in patients with non-small cell carcinoma of lung, observable tumor localization can also be achieved using a nonspecific antibody

  15. 21 CFR 866.5520 - Immunoglobulin G (Fab fragment specific) immunological test system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Immunoglobulin G (Fab fragment specific... Test Systems § 866.5520 Immunoglobulin G (Fab fragment specific) immunological test system. (a) Identification. An immunoglobulin G (Fab fragment specific) immunological test system is a device that consists...

  16. 21 CFR 866.5540 - Immunoglobulin G (Fd fragment specific) immunological test system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Immunoglobulin G (Fd fragment specific... Test Systems § 866.5540 Immunoglobulin G (Fd fragment specific) immunological test system. (a) Identification. An immunoglobulin G (Fd fragment specific) immunological test system is a device that consists of...

  17. 21 CFR 866.5530 - Immunoglobulin G (Fc fragment specific) immunological test system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Immunoglobulin G (Fc fragment specific... Test Systems § 866.5530 Immunoglobulin G (Fc fragment specific) immunological test system. (a) Identification. An immunoglobulin G (Fc fragment specific) immunological test system is a device that consists of...

  18. Evaluation of selectivity in homologous multimodal chromatographic systems using in silico designed antibody fragment libraries.

    Science.gov (United States)

    Karkov, Hanne Sophie; Woo, James; Krogh, Berit Olsen; Ahmadian, Haleh; Cramer, Steven M

    2015-12-24

    This study describes the in silico design, surface property analyses, production and chromatographic evaluations of a diverse set of antibody Fab fragment variants. Based on previous findings, we hypothesized that the complementarity-determining regions (CDRs) constitute important binding sites for multimodal chromatographic ligands. Given that antibodies are highly diversified molecules and in particular the CDRs, we set out to examine the generality of this result. For this purpose, four different Fab fragments with different CDRs and/or framework regions of the variable domains were identified and related variants were designed in silico. The four Fab variant libraries were subsequently generated by site-directed mutagenesis and produced by recombinant expression and affinity purification to enable examination of their chromatographic retention behavior. The effects of geometric re-arrangement of the functional moieties on the multimodal resin ligands were also investigated with respect to Fab variant retention profiles by comparing two commercially available multimodal cation-exchange ligands, Capto MMC and Nuvia cPrime, and two novel multimodal ligand prototypes. Interestingly, the chromatographic data demonstrated distinct selectivity trends between the four Fab variant libraries. For three of the Fab libraries, the CDR regions appeared as major binding sites for all multimodal ligands. In contrast, the fourth Fab library displayed a distinctly different chromatographic behavior, where Nuvia cPrime and related multimodal ligand prototypes provided markedly improved selectivity over Capto MMC. Clearly, the results illustrate that the discriminating power of multimodal ligands differs between different Fab fragments. The results are promising indications that multimodal chromatography using the appropriate multimodal ligands can be employed in downstream bioprocessing for challenging selective separation of product related variants. Copyright © 2015 Elsevier B

  19. Association of Circulating Transfer RNA fragments with antibody response to Mycoplasma bovis in beef cattle.

    Science.gov (United States)

    Casas, Eduardo; Cai, Guohong; Kuehn, Larry A; Register, Karen B; McDaneld, Tara G; Neill, John D

    2018-03-13

    High throughput sequencing allows identification of small non-coding RNAs. Transfer RNA Fragments are a class of small non-coding RNAs, and have been identified as being involved in inhibition of gene expression. Given their role, it is possible they may be involved in mediating the infection-induced defense response in the host. Therefore, the objective of this study was to identify 5' transfer RNA fragments (tRF5s) associated with a serum antibody response to M. bovis in beef cattle. The tRF5s encoding alanine, glutamic acid, glycine, lysine, proline, selenocysteine, threonine, and valine were associated (P < 0.05) with antibody response against M. bovis. tRF5s encoding alanine, glutamine, glutamic acid, glycine, histidine, lysine, proline, selenocysteine, threonine, and valine were associated (P < 0.05) with season, which could be attributed to calf growth. There were interactions (P < 0.05) between antibody response to M. bovis and season for tRF5 encoding selenocysteine (anticodon UGA), proline (anticodon CGG), and glutamine (anticodon TTG). Selenocysteine is a rarely used amino acid that is incorporated into proteins by the opal stop codon (UGA), and its function is not well understood. Differential expression of tRF5s was identified between ELISA-positive and negative animals. Production of tRF5s may be associated with a host defense mechanism triggered by bacterial infection, or it may provide some advantage to a pathogen during infection of a host. Further studies are needed to establish if tRF5s could be used as a diagnostic marker of chronic exposure.

  20. Immunoradiometric assay for cytomegalovirus-specific IgG antibodies

    International Nuclear Information System (INIS)

    Klapper, P.E.; Cleator, G.M.; Prinja-Wolks, D.; Morris, D.J.

    1990-01-01

    An immunoradiometric assay (radio-immunosorbent test; RIST) for the detection of IgG antibodies to human herpesvirus 4 [human cytomegalovirus (CMV)] has been developed. The technique utilizes CMV antigen passively adsorbed to a polyvinyl microtitration plate and a radiolabelled murine monoclonal anti-human IgG antibody to detect binding of human antibody to the 'solid phase' reagent. The assay was optimized, and its specifity confirmed by testing paired acute and convalescent sera from patients with acute CMV or other human herpesvirus infections. To determine the assay's sensitivity 1433 blood donor sera were examined. The RIST was more sensitive than a standard complement fixation (CFT). Use of a monoclonal anti-human IgG antibody in the RIST reduced non-specific binding to the control uninfected cell antigen such that blood donor sera could be tested in the assay using only a CMV antigen without generating an unacceptable false positive rate. (author). 23 refs.; 1 tab

  1. Anti-coagulation effect of Fc fragment against anti-β2-GP1 antibodies in mouse models with APS.

    Science.gov (United States)

    Xie, Weidong; Zhang, Yaou; Bu, Cunya; Sun, Shijing; Hu, Shaoliang; Cai, Guoping

    2011-01-01

    Anti-beta (2)-glycoprotein I (anti-β2-GP1) is one of the important pathogenesis factors responsible for thrombosis formation in patients with antiphospholipid syndrome (APS). Administration of intravenous immunoglobulin (IVIg) is a common method used to inhibit the abnormal antibody levels and decrease the mortality of APS in emergency situations. We hypothesize that the Fc fragment of IgG is the molecular structure responsible for these effects. The present study investigates the beneficial effects of both recombinant and natural human Fc fragments of heterogeneous IgG against human anti-β2-GP1 antibodies in mouse models with APS. Results showed that both recombinant and natural human Fc fragments moderately but significantly decreased the levels of serum anti-β2-GP1 antibodies and had anti-coagulation effects in human β2-GP1-immunized mice. Furthermore, both recombinant and natural human Fc fragments inhibited thrombosis formation and decreased mortality in mouse models infused intravenously with human anti-β2GP1 antibodies from patients with APS. Findings suggest that the Fc fragment might be one of the active structural units of heterogeneous IgG. Thus, recombinant human Fc fragment administration may be a useful treatment for individuals with APS. Copyright © 2010 Elsevier B.V. All rights reserved.

  2. Identification and evaluation of nextgeneration PTM-specific antibodies

    DEFF Research Database (Denmark)

    Persson, Nina Emilia

    -chain fragment variable (scFv)clones. Two different analyses are performed on the same microarray. There is no need for anypurification or enrichment before screening. In the first analysis, the ability of the individualscFv clone to bind to the soluble form of the antigens is evaluated. Favouring selection....... Including antibodies target several different categories of antigens suchas proteins, glycoproteins and glycolipids. Glycoproteins have become highlighted in cancerresearch since they are frequently involved in the initiation and spreading of cancer. One form ofglycosylation of proteins is the O...... for the patients. Severalmonoclonal antibodies have been generated against the Tn- and STn-antigens, but none has yetreached approval for therapeutic or diagnostic use. Indicating the need for a new generation ofantibodies against this type aberrant glycosylation.The two major techniques used for the production...

  3. Radioimmunoassay of class-specific antibodies (RIACA): chicken antibodies to DNP

    International Nuclear Information System (INIS)

    Viljanen, M.K.; Granfors, K.; Toivanen, P.

    1977-01-01

    A radioimmunological method for the quantitation of class-specific antibodies has been developed. The method allows the quantitation of nanogram per ml concentrations of IgG and IgM-anti-DNP antibodies without any physical or chemical pretreatment of the sample. DNP was coupled covalently to a cyanogen bromide activated paper disk with the augmentation of lysine molecule. Anti-DNP antibodies were allowed to react with the coupled DNP and then quantitated by their capacity to bind 125 I-labelled anti-chicken-μ or anti-chicken-γ. The inter-assay variation coefficients ranged from 8.1 to 14.7% and the mean standard deviations of duplicate determinations were about 11%. The combination of this method with the exact immunoradiometric quantitation of the total serum IgM and IgG, and with an immunoabsorption technique, makes it possible to quantitate class-specific antibodies on weight units

  4. A Strategy for Generating a Broad-Spectrum Monoclonal Antibody and Soluble Single-Chain Variable Fragments against Plant Potyviruses

    Science.gov (United States)

    Liu, Han-Lin; Lin, Wei-Fang; Hu, Wen-Chi; Lee, Yung-An

    2015-01-01

    Potyviruses are major pathogens that often cause mixed infection in calla lilies. To reduce the time and cost of virus indexing, a detection method for the simultaneous targeting of multiple potyviruses was developed by generating a broad-spectrum monoclonal antibody (MAb) for detecting the greatest possible number of potyviruses. The conserved 121-amino-acid core regions of the capsid proteins of Dasheen mosaic potyvirus (DsMV), Konjak mosaic potyvirus (KoMV), and Zantedeschia mild mosaic potyvirus (ZaMMV) were sequentially concatenated and expressed as a recombinant protein for immunization. After hybridoma cell fusion and selection, one stable cell line that secreted a group-specific antibody, named C4 MAb, was selected. In the reaction spectrum test, the C4 MAb detected at least 14 potyviruses by indirect enzyme-linked immunosorbent assay (I-ELISA) and Western blot analysis. Furthermore, the variable regions of the heavy (VH) and light (VL) chains of the C4 MAb were separately cloned and constructed as single-chain variable fragments (scFvs) for expression in Escherichia coli. Moreover, the pectate lyase E (PelE) signal peptide of Erwinia chrysanthemi S3-1 was added to promote the secretion of C4 scFvs into the medium. According to Western blot analysis and I-ELISA, the soluble C4 scFv (VL-VH) fragment showed a binding specificity similar to that of the C4 MAb. Our results demonstrate that a recombinant protein derived from fusion of the conserved regions of viral proteins has the potential to produce a broad-spectrum MAb against a large group of viruses and that the PelE signal peptide can improve the secretion of scFvs in E. coli. PMID:26209665

  5. Site-specific proteolytic degradation of IgG monoclonal antibodies expressed in tobacco plants.

    Science.gov (United States)

    Hehle, Verena K; Lombardi, Raffaele; van Dolleweerd, Craig J; Paul, Mathew J; Di Micco, Patrizio; Morea, Veronica; Benvenuto, Eugenio; Donini, Marcello; Ma, Julian K-C

    2015-02-01

    Plants are promising hosts for the production of monoclonal antibodies (mAbs). However, proteolytic degradation of antibodies produced both in stable transgenic plants and using transient expression systems is still a major issue for efficient high-yield recombinant protein accumulation. In this work, we have performed a detailed study of the degradation profiles of two human IgG1 mAbs produced in plants: an anti-HIV mAb 2G12 and a tumour-targeting mAb H10. Even though they use different light chains (κ and λ, respectively), the fragmentation pattern of both antibodies was similar. The majority of Ig fragments result from proteolytic degradation, but there are only a limited number of plant proteolytic cleavage events in the immunoglobulin light and heavy chains. All of the cleavage sites identified were in the proximity of interdomain regions and occurred at each interdomain site, with the exception of the VL /CL interface in mAb H10 λ light chain. Cleavage site sequences were analysed, and residue patterns characteristic of proteolytic enzymes substrates were identified. The results of this work help to define common degradation events in plant-produced mAbs and raise the possibility of predicting antibody degradation patterns 'a priori' and designing novel stabilization strategies by site-specific mutagenesis. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  6. Efficient heterologous expression and secretion in Aspergillus oryzae of a llama variable heavy-chain antibody fragment V(HH) against EGFR.

    Science.gov (United States)

    Okazaki, Fumiyoshi; Aoki, Jun-ichi; Tabuchi, Soichiro; Tanaka, Tsutomu; Ogino, Chiaki; Kondo, Akihiko

    2012-10-01

    We have constructed a filamentous fungus Aspergillus oryzae that secretes a llama variable heavy-chain antibody fragment (V(HH)) that binds specifically to epidermal growth factor receptor (EGFR) in a culture medium. A major improvement in yield was achieved by fusing the V(HH) with a Taka-amylase A signal sequence (sTAA) and a segment of 28 amino acids from the N-terminal region of Rhizopus oryzae lipase (N28). The yields of secreted, immunologically active anti-EGFR V(HH) reached 73.8 mg/1 in a Sakaguchi flask. The V(HH) fragments were released from the sTAA or N28 proteins by an indigenous A. oryzae protease during cultivation. The purified recombinant V(HH) fragment was specifically recognized and could bind to the EGFR with a high affinity.

  7. Prion-Specific Antibodies Produced in Wild-Type Mice

    DEFF Research Database (Denmark)

    Heegaard, Peter M. H.; Bergström, Ann-Louise; Andersen, Heidi Gertz

    2015-01-01

    Peptide-specific antibodies produced against synthetic peptides are of high value in probing protein structure and function, especially when working with challenging proteins, including not readily available, non-immunogenic, toxic, and/or pathogenic proteins. Here, we present a straightforward...... method for production of mouse monoclonal antibodies (MAbs) against peptides representing two sites of interest in the bovine prion protein (boPrP), the causative agent of bovine spongiform encephalopathy ("mad cow disease") and new variant Creutzfeldt-Jakob's disease (CJD) in humans, as well......-peptide antibodies, even against peptides very homologous to murine protein sequences. In general, using the strategies described here for selecting, synthesizing, and conjugating peptides and immunizing 4-5 mice with 2-3 different peptides, high-titered antibodies reacting with the target protein are routinely...

  8. A novel affinity purification method to isolate peptide specific antibodies

    DEFF Research Database (Denmark)

    Karlsen, Alan E; Lernmark, A; Kofod, Hans

    1990-01-01

    Site-specific, high affinity polyclonal antisera are effectively and successfully produced by immunizing rabbits with synthetic peptides. The use of these antisera in subsequent immune analysis is often limited because of non-specific binding. We describe a new and simple method to effectively...... affinity-purify anti-peptide antibodies. To test our system, rabbits were immunized with model peptides representing sequences of the putative rabbit growth hormone receptor and several HLA-DQ beta-chain molecules. Polystyrene plastic beads were coated with peptides. Immune serum was incubated...... with the beads and after a wash step the bound antibodies were eluted in 1 M acetic acid. The eluted material was composed predominantly of intact immunoglobulin as evidenced by the presence of heavy and light chain bands in SDS-PAGE. The eluted antibodies were peptide specific in ELISA and bound only to intact...

  9. Development of an EGFRvIII specific recombinant antibody

    Directory of Open Access Journals (Sweden)

    Li Gordon

    2010-10-01

    Full Text Available Abstract Background EGF receptor variant III (EGFRvIII is the most common variant of the EGF receptor observed in human tumors. It results from the in frame deletion of exons 2-7 and the generation of a novel glycine residue at the junction of exons 1 and 8. This novel juxtaposition of amino acids within the extra-cellular domain of the EGF receptor creates a tumor specific and immunogenic epitope. EGFRvIII expression has been seen in many tumor types including glioblastoma multiforme (GBM, breast adenocarcinoma, non-small cell lung carcinoma, ovarian adenocarcinoma and prostate cancer, but has been rarely observed in normal tissue. Because this variant is tumor specific and highly immunogenic, it can be used for both a diagnostic marker as well as a target for immunotherapy. Unfortunately many of the monoclonal and polyclonal antibodies directed against EGFRvIII have cross reactivity to wild type EGFR or other non-specific proteins. Furthermore, a monoclonal antibody to EGFRvIII is not readily available to the scientific community. Results In this study, we have developed a recombinant antibody that is specific for EGFRvIII, has little cross reactivity for the wild type receptor, and which can be easily produced. We initially designed a recombinant antibody with two anti-EGFRvIII single chain Fv's linked together and a human IgG1 Fc component. To enhance the specificity of this antibody for EGFRvIII, we mutated tyrosine H59 of the CDRH2 domain and tyrosine H105 of the CDRH3 domain to phenylalanine for both the anti-EGFRvIII sequence inserts. This mutated recombinant antibody, called RAbDMvIII, specifically detects EGFRvIII expression in EGFRvIII expressing cell lines as well as in EGFRvIII expressing GBM primary tissue by western blot, immunohistochemistry (IHC and immunofluorescence (IF and FACS analysis. It does not recognize wild type EGFR in any of these assays. The affinity of this antibody for EGFRvIII peptide is 1.7 × 107 M-1 as

  10. Data on atherosclerosis specific antibody conjugation to nanoemulsions

    Directory of Open Access Journals (Sweden)

    Geoffrey Prévot

    2017-12-01

    Full Text Available This article present data related to the publication entitled “Iron oxide core oil-in-water nanoemulsion as tracer for atherosclerosis MPI and MRI imaging” (Prévot et al., 2017 [1]. Herein we describe the engineering in the baculovirus-insect cell system and purification processes of the human scFv-Fc TEG4-2C antibody, specific of platelets within the atheroma plaque. For molecular targeting purpose, atheroma specific antibody was conjugated to nanoemulsions (NEs using a heterobifunctional linker (DSPE-PEG-maleimide. Atheroma labelling was assayed by immunochemistry on arterial sections from rabbits.

  11. A double antibody radioimmunoassay specific for placental alkaline phosphatase

    International Nuclear Information System (INIS)

    Dass, S.; Bagshawe, K.D.

    1984-01-01

    Placental alkaline phosphatase (PLAP) is normally found in enzymically measurable amounts in second and third trimester pregnancy serum. Its occurrence in sera and tumours from patients with malignant disease has led to the development of methods to specifically identify and quantitate the enzyme. Recently immunological techniques have been used, employing antibodies raised to purified PLAP; these include solid phase radioimmunoassays and enzyme-immunoassay. The development of a sensitive, specific, automated double-antibody radioimmunoassay for the measurement of PLAP in serum is reported. (Auth.)

  12. Evaluation of radioiodinated and radiocopper labeled monovalent fragments of monoclonal antibody chCE7 for targeting of neuroblastoma

    International Nuclear Information System (INIS)

    Carrel, Francois; Amstutz, Hanspeter; Novak-Hofer, Ilse; Schubiger, P. August

    1997-01-01

    Monovalent fragments of antineuroblastoma antibody mAb chCE7 were evaluated for their in vitro and in vivo tumor cell binding properties. Single chain fragments were constructed from the variable region genes cloned from hybridoma cells, expressed in E.coli and purified by metal chelate affinity chromatography. Radioiodinated CE7-scFv fragments were found to bind with high affinity (K d ∼10 -9 M) to target cells in vitro but formed aggregates at 37 deg. C, and bound to serum proteins in vitro and in vivo. Circular Dichroism spectra revealed the protein to be in a conformationally altered form and no permanent 'refolding' could be achieved. In contrast, chCE7-Fab fragments were found to bind to target tumor cells with similar affinity than the parent mAb chCE7 (K d ∼10 -10 M), showed no tendency to aggregate and were stable in serum both in vitro and in vivo. Kinetics of association and dissociation of radioiodinated scFv and Fab fragments were found to be rapid. Radioiodination with the Iodogen method led to impaired immunoreactivity which was found to further increase the off- rates of radioiodinated fragments from tumor cells. Radioiodination with the Bolton-Hunter reagent as well as labeling of chCE7-Fab fragments with 67 Cu via the macrocyclic CPTA ligand led to fully immunoreactive Fab fragments. Radioiodinated and radiocopper labeled monovalent CE7 fragments did not internalize into target tumor cells as the parent mAb and its F(ab') 2 fragment. A comparison of the biodistribution in tumor bearing nude mice of the radiocopper labeled monovalent, non internalizing Fab fragments with the internalizing divalent F(ab') 2 fragments showed in both cases high levels of radioactivity in the kidneys. Concerning tumor uptake, radioactivity from both internalizing and non internalizing fragments remained associated with tumor tissue for longer times than in case of the corresponding radioiodinated fragments. When compared with the radioiodinated forms, tumor uptake

  13. Determination of allergen specificity by heavy chains in grass pollen allergen-specific IgE antibodies.

    Science.gov (United States)

    Gadermaier, Elisabeth; Flicker, Sabine; Lupinek, Christian; Steinberger, Peter; Valenta, Rudolf

    2013-04-01

    Affinity and clonality of allergen-specific IgE antibodies are important determinants for the magnitude of IgE-mediated allergic inflammation. We sought to analyze the contribution of heavy and light chains of human allergen-specific IgE antibodies for allergen specificity and to test whether promiscuous pairing of heavy and light chains with different allergen specificity allows binding and might affect affinity. Ten IgE Fabs specific for 3 non-cross-reactive major timothy grass pollen allergens (Phl p 1, Phl p 2, and Phl p 5) obtained by means of combinatorial cloning from patients with grass pollen allergy were used to construct stable recombinant single chain variable fragments (ScFvs) representing the original Fabs and shuffled ScFvs in which heavy chains were recombined with light chains from IgE Fabs with specificity for other allergens by using the pCANTAB 5 E expression system. Possible ancestor genes for the heavy chain and light chain variable region-encoding genes were determined by using sequence comparison with the ImMunoGeneTics database, and their chromosomal locations were determined. Recombinant ScFvs were tested for allergen specificity and epitope recognition by means of direct and sandwich ELISA, and affinity by using surface plasmon resonance experiments. The shuffling experiments demonstrate that promiscuous pairing of heavy and light chains is possible and maintains allergen specificity, which is mainly determined by the heavy chains. ScFvs consisting of different heavy and light chains exhibited different affinities and even epitope specificity for the corresponding allergen. Our results indicate that allergen specificity of allergen-specific IgE is mainly determined by the heavy chains. Different heavy and light chain pairings in allergen-specific IgE antibodies affect affinity and epitope specificity and thus might influence clinical reactivity to allergens. Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by

  14. Packing motifs as predictors of the propensity of antibody fragments to crystallize

    Science.gov (United States)

    Edmundson, Allen B.; DeWitt, Christina R.; Goldsteen, Benjamin Z.; Ramsland, Paul A.

    1999-01-01

    A recurring theme in the crystallization of antibody fragments in our laboratory has been a packing pattern involving formation of intermolecular, antiparallel β-pleated sheets across two-fold axes. The most common motif is the antiparallel stacking of constant (C) domains of light (L) chain dimers or Fab molecules. Here, cross-molecule six-stranded sheets are produced by hydrogen-bonding interactions of three-residue polypeptide segments (triads), in the i, i+2 and i+4 positions of the final strands (designated 3-3) of the three-chain layers from two adjacent molecules. In the variable (V) domains the triads are supplied by the first strands (4-1) of the four-chain layers and the resulting cross-molecule sheets contain eight strands. The latter type of packing is more likely to be seen in crystals of Fv fragments (V domains only) than in those of L chain dimers or Fabs. Amongst the triads from either the V or C domains, there are on average four sets of backbone carbonyl and amide groups within hydrogen bonding distance (chain dimers, Fab and Fvs are likely to crystallize in these packing patterns.

  15. Crystallization and preliminary crystallographic studies of the single-chain variable fragment of antibody chA21 in complex with an N-terminal fragment of ErbB2

    International Nuclear Information System (INIS)

    Liu, Yang; Zhou, Huihao; Zhu, Juanjuan; Gao, Yongxiang; Niu, Liwen; Liu, Jing; Teng, Maikun

    2009-01-01

    An antibody–antigen complex consisting of a single-chain variable fragment of the potential therapeutic antibody chA21 and an N-terminal fragment (residues 1–192) of the human ErbB2 extracellular domain was expressed, purified and crystallized. X-ray diffraction data were collected to 2.45 Å resolution. ErbB2 is a transmembrane tyrosine kinase, the overexpression of which causes abnormality and disorder in cell signalling and leads to cell transformation. Previously, an anti-ErbB2 single-chain chimeric antibody chA21 that specifically inhibits the growth of ErbB2-overexpressing cancer cells in vitro and in vivo was developed. Here, an antibody–antigen complex consisting of the single-chain variable fragment (scFv) of chA21 and an N-terminal fragment (residues 1–192, named EP I) of the ErbB2 extracellular domain was crystallized using the sitting-drop vapour-diffusion method. An X-ray diffraction data set was collected to 2.45 Å resolution from a single flash-cooled crystal; the crystal belonged to space group P2 1 2 1 2 1

  16. 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.

  17. In vitro and in vivo tumor models for studies of distribution of radiolabelled monoclonal antibodies and fragments

    International Nuclear Information System (INIS)

    Buchegger, F.; Halpern, S.E.; Sutherland, R.M.; Schreyer, M.; Mach, J.P.; Rochester Univ., NY

    1986-01-01

    Colon carcinoma multicellular spheroids were incubated in vitro with radiolabelled MAbs. The more rapid penetration of fragments as compared to intact MAbs was clearly demonstrated. For the study of antibody localization in tumors in vivo, the model of nude mice with ligated kidneys was used. Although very artificial, this model allowed to demonstrate that, without urinary excretion, Fab fragments accumulated more rapidly into the tumor than intact MAbs and disappeared faster from the blood. This difference was less striking for F(ab') 2 fragments. In the liver a decreased accumulation of both types of fragments as compared to intact MAbs was observed. Concerning radio-immunotherapy we think that Fab fragments are not useful because of their too short half-life the circulation and in tumor and because they will probably be too toxic for the kidneys. Intact MAbs and F(ab') 2 fragments have each their advantages. Intact MAbs show highest tumor accumulation in mice without ligated kidney, however, they remain mostly on the periphery of tumor nodules, as shown by autoradiography. F(ab') 2 fragments have been found to penetrate deeper into the tumor and to accumulate less in the liver. It might be therefore an advantage to combine intact MAbs with F(ab') 2 fragments, so that in the tumor two different regions could be attacked whereas in normal tissues toxicity could be distributed to different organs such as to the liver with intact MAbs and to the kidney with F(ab') 2 fragments. (orig.) [de

  18. I-125 input into antibodies molecules specific to australian antigen

    International Nuclear Information System (INIS)

    Abdukayumov, A. M.; Chistyakov, P.G.; Garajshina, G. R.

    1999-01-01

    There are experimental data on I-125 input into antibodies molecules specific to superficial antigen of hepatitis B virus (australian antigen). Three ways of input are submitted: with the help of T chloramine usage, Bolton-Hunter Reagent and with the help of iodogen. There are also comparative characteristics of iodized products obtained: molar radioactivity, radiochemical frequency, immuno - reactivity. The report also discusses advantages and disadvantages of the used methods for inputting I-125 into antibodies to australian antigen in order to study the possibility of creating radio immunological test system for detecting superficial antigen of B hepatitis

  19. Cultivation of Pichia pastoris carrying the scFv anti LDL (- antibody fragment. Effect of preculture carbon source

    Directory of Open Access Journals (Sweden)

    Cesar Andres Diaz Arias

    Full Text Available Abstract Antibodies and antibody fragments are nowadays among the most important biotechnological products, and Pichia pastoris is one of the most important vectors to produce them as well as other recombinant proteins. The conditions to effectively cultivate a P. pastoris strain previously genetically modified to produce the single-chain variable fragment anti low density lipoprotein (- under the control of the alcohol oxidase promoter have been investigated in this study. In particular, it was evaluated if, and eventually how, the carbon source (glucose or glycerol used in the preculture preceding cryopreservation in 20% glycerol influences both cell and antibody fragment productions either in flasks or in bioreactor. Although in flasks the volumetric productivity of the antibody fragment secreted by cells precultured, cryopreserved and reactivated in glycerol was 42.9% higher compared with cells precultured in glucose, the use of glycerol in bioreactor led to a remarkable shortening of the lag phase, thereby increasing it by no less than thrice compared to flasks. These results are quite promising in comparison with those reported in the literature for possible future industrial applications of this cultivation, taking into account that the overall process time was reduced by around 8 h.

  20. 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

  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. Expression and purification of a novel therapeutic single-chain variable fragment antibody against BNP from inclusion bodies of Escherichia coli.

    Science.gov (United States)

    Bu, Dawei; Zhou, Yuwei; Tang, Jian; Jing, Fang; Zhang, Wei

    2013-12-01

    Abnormal brain natriuretic peptide (BNP) secretion is regarded as the dominating mechanism of cerebral salt wasting syndrome (CSW), which results from a renal loss of sodium and water during intracranial disease leading to hyponatremia. Scale preparation of therapeutic single-chain variable fragment (scFv) that can neutralize elevated circulating BNP may have potential value for clinical use. In this report, we used a recently isolated humanized anti-BNP scFv fragment (3C1) as model antibody (Ab) to evaluate the potential of scale production of this therapeutic protein. The truncated gene encoding for scFv fragment cloned in pET22b (+) was mainly overexpressed as inclusion bodies in Escherichia coli (E. coli) Rosetta (DE3) pLysS cells. The insoluble fragment was solubilized and purified by Ni-NTA agarose resin under denaturation conditions, and recovered via an effective refolding buffer containing 50 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1 mM EDTA, 0.5 M arginine, 2 mM GSH, 1 mM GSSG, and 5% glycerol. The refolded scFv fragment was concentrated by PEG20000, and dialyzed in PBS (containing 5% glycerol, pH 7.4). The final yield was approximately 10.2 mg active scFv fragment per liter of culture (3.4 g wet weight cells). The scFv fragment was more than 95% pure assessed by SDS-PAGE assay. Recombinant scFv fragment with His tag displayed its immunoreactivity with anti-His tag Ab by western blotting. ELISA showed the scFv fragment specifically bound to BNP, and it displayed similar activity as the traditional anti-BNP monoclonal Ab (mAb). Thus, the current strategy allows convenient small-scale production of this therapeutic protein. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. Preparation and radiolabeling of humanized anti-HER1 monoclonal antibody nimotuzumab Fab' fragment with 68Ga and 90Y

    International Nuclear Information System (INIS)

    Alonso Martinez, L. M.; Xiques Castillo, A.; Leyva Montanna, R.; Perez-Malo Cruz, M.; Zamora Barrabi, M.; Manresa Sanchez, Y.

    2013-01-01

    Antibody-based targeted delivery of radioisotopes to malignant tissues is a promising approach in cancer diagnostics and therapy. However, intact antibody molecules are large glycoproteins (∼150 kDa) that have limited application in molecular imaging and therapy due to their relatively slow clearance from the circulation leading to a high background signal rather both cases the sensitivity can be increased with the use of enzymatically produced Fab' fragments. In this work, the ability to get labeled with 62 Ga and 90 Y of a monoclonal antibody (mAb) Fab' fragment against the transmembrane receptor tyrosine kinase HER-1 was studied for future applications in PET imaging and radioimmunotherapy of tumors. In order to obtain the Fab' fragment the mAb was cleaved with pepsin in molar excess. After separating the reaction mixture in two steps using affinity and ion-exchange chromatography, the Fab' fragment was finally obtained by reduction of the F(ab') 2 with a molar excess of 2-mercaptoethanol followed by a size exclusion purification step. The Fab' fragment was derivatized with 1,4,7,10-tetraaza cyclododecane-1,4,7,10-tetraacetic acid mono N-hydroxysuccinimide commercial ester (DOTA-NHS-ester) applying a simple procedure and the number of DOTA groups linked to Fab' were determinate. The labeling of the conjugate with 68 Ga and 90 Y from 'in-house generators yielded radiochemically pure probes that can become a suitable radioimmunoconjugated in a near future. (Author)

  4. Aptamers, antibody scFv, and antibody Fab' fragments: An overview and comparison of three of the most versatile biosensor biorecognition elements.

    Science.gov (United States)

    Crivianu-Gaita, Victor; Thompson, Michael

    2016-11-15

    The choice of biosensing elements is crucial for the development of the optimal biosensor. Three of the most versatile biosensing elements are antibody single-chain Fv fragments (scFv), antibody fragment-antigen binding (Fab') units, and aptamers. This article provides an overview of these three biorecognition elements with respects to their synthesis/engineering, various immobilization techniques, and examples of their use in biosensors. Furthermore, the final section of the review compares and contrasts their characteristics (time/cost of development, ease and variability of immobilization, affinity, stability) illustrating their advantages and disadvantages. Overall, scFv fragments are found to display the highest customizability (i.e. addition of functional groups, immobilizing peptides, etc.) due to recombinant synthesis techniques. If time and cost are an issue in the development of the biosensor, Fab' fragments should be chosen as they are relatively cheap and can be developed quickly from whole antibodies (several days). However, if there are sufficient funds and time is not a factor, aptamers should be utilized as they display the greatest affinity towards their target analytes and are extremely stable (excellent biosensor regenerability). Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Determination of specific IgG antibody by crossed radioimmunoelectrophoresis

    International Nuclear Information System (INIS)

    Nordvall, S.L.; Uhlin, T.; Einarsson, R.

    1983-01-01

    A crossed radioimmunoelectrophoretic method was developed for detection of honey bee venom specific IgG antibodies in patient sera. At the serum concentration 1/200 the contrast between specific binding and backgroud was the most favourable. The detection limit was fairly low, approximately 30 kU/l(IgG RAST units). A reference system based on the reference kits in Phadebas IgG-RAST was elaborated. (author)

  6. Determination of specific IgG antibody by crossed radioimmunoelectrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Nordvall, S.L. (Dept. of Paediatrics, University Hospital, Uppsala, Sweden); Uhlin, T.; Einarsson, R. (Allergy Research, Pharmacia Diagnostics AB, Uppsala, Sweden)

    1983-01-01

    A crossed radioimmunoelectrophoretic method was developed for detection of honey bee venom specific IgG antibodies in patient sera. At the serum concentration 1/200 the contrast between specific binding and backgroud was the most favourable. The detection limit was fairly low, approximately 30 kU/l(IgG RAST units). A reference system based on the reference kits in Phadebas IgG-RAST was elaborated.

  7. 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

  8. 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).

  9. Fab fragments of ovine antibody to colchicine enhance its clearance in the rat.

    Science.gov (United States)

    Peake, Philip W; Pianta, Timothy J; Succar, Lena; Fernando, Mangalee; Buckley, Nicholas A; Endre, Zoltan H

    2015-06-01

    Colchicine is an anti-inflammatory alkaloid used for the treatment of acute gout, but has a narrow therapeutic index. Colchicine overdoses are relatively rare, but have high mortality requiring rapid treatment. To evaluate the ability of a newly available ovine fragment antigen-binding (Fab) antibody to colchicine (ColchiFab(™)) to protect rats against renal and other injury 24 h after colchicine ingestion. Rats were gavaged with colchicine (5 mg/kg), then 2 h later injected intraperitoneally with 5 ml of sterile saline, or Fab anti-colchicine, a newly available ovine antibody to colchicine. Samples of blood were taken at 1, 2, 5 and 24 h after gavage, and urine was collected from 5 to 24 h after gavage. Concentrations of colchicine in tissue, blood and urine were measured by liquid chromatography/mass spectrometry, concentrations of Fab anti-colchicine, urinary neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 or KIM-1 by enzyme-linked immunosorbent assay or ELISA, while concentrations of creatine kinase and creatinine (Cr) were measured enzymatically. Colchicine equilibrated rapidly throughout the body and increased serum creatine kinase. Fab anti-colchicine also rapidly redistributed to the blood and remained at high concentrations over 24 h. Fab anti-colchicine caused a rapid 7.1-fold increase in serum colchicine level, followed by excretion of both colchicine and Fab anti-colchicine through the urine. This was associated with the accumulation of colchicine in the kidney, a reversal of colchicine-induced diarrhoea, and increasing urinary NGAL level; from 168 ± 48 to 477 ± 255 ng/mmol Cr [mean ± standard deviation or SD]. Fab anti-colchicine greatly increased the clearance of colchicine, although increasing NGAL level suggested the presence of mild kidney damage. These data suggest clinical utility for Fab anti-colchicine in the treatment of colchicine overdose.

  10. Secretory signal peptide modification for optimized antibody-fragment expression-secretion in Leishmania tarentolae

    Directory of Open Access Journals (Sweden)

    Klatt Stephan

    2012-07-01

    Full Text Available Abstract Background Secretory signal peptides (SPs are well-known sequence motifs targeting proteins for translocation across the endoplasmic reticulum membrane. After passing through the secretory pathway, most proteins are secreted to the environment. Here, we describe the modification of an expression vector containing the SP from secreted acid phosphatase 1 (SAP1 of Leishmania mexicana for optimized protein expression-secretion in the eukaryotic parasite Leishmania tarentolae with regard to recombinant antibody fragments. For experimental design the online tool SignalP was used, which predicts the presence and location of SPs and their cleavage sites in polypeptides. To evaluate the signal peptide cleavage site as well as changes of expression, SPs were N-terminally linked to single-chain Fragment variables (scFv’s. The ability of L. tarentolae to express complex eukaryotic proteins with highly diverse post-translational modifications and its easy bacteria-like handling, makes the parasite a promising expression system for secretory proteins. Results We generated four vectors with different SP-sequence modifications based on in-silico analyses with SignalP in respect to cleavage probability and location, named pLTEX-2 to pLTEX-5. To evaluate their functionality, we cloned four individual scFv-fragments into the vectors and transfected all 16 constructs into L. tarentolae. Independently from the expressed scFv, pLTEX-5 derived constructs showed the highest expression rate, followed by pLTEX-4 and pLTEX-2, whereas only low amounts of protein could be obtained from pLTEX-3 clones, indicating dysfunction of the SP. Next, we analysed the SP cleavage sites by Edman degradation. For pLTEX-2, -4, and -5 derived scFv’s, the results corresponded to in-silico predictions, whereas pLTEX-3 derived scFv’s contained one additional amino-acid (AA. Conclusions The obtained results demonstrate the importance of SP-sequence optimization for efficient

  11. Ultra scaledown to predict filtering centrifugation of secreted antibody fragments from fungal broth.

    Science.gov (United States)

    Boulding, N; Yim, S S S; Keshavarz-Moore, E; Ayazi Shamlou, P; Berry, M

    2002-08-20

    Extracellularly expressed anti-hen egg lysozyme single-chain antibody fragments (scFv) produced by Aspergillus awamori were recovered using filtering centrifugation. Two filtering centrifuges with 0.5- and 30-L capacities were used to represent laboratory- and pilot-scale equipment, respectively. Critical regime analysis using the computational fluid dynamics (CFD) technique provided information about the local energy dissipation rates in both units. Experimental data indicated loss of scFv activity for energy dissipation rates above about 2.0 x 10(4) W kg(-1). This loss of activity increased in the presence of gas-liquid interfaces during filtering centrifugation. An ultra scaledown filtering centrifuge with a maximum working volume of 35 mL was designed to mimic the operating conditions identified by the critical regime analysis for the laboratory- and pilot-plant-scale units. The recovered scFv activity levels and the separation performance of the three units were comparable when operated at equal maximum energy dissipation rates. Copyright 2002 Wiley Periodicals, Inc.

  12. Llama-Derived Single Domain Antibodies Specific for Abrus Agglutinin

    Science.gov (United States)

    Goldman, Ellen R.; Anderson, George P.; Zabetakis, Dan; Walper, Scott; Liu, Jinny L.; Bernstein, Rachael; Calm, Alena; Carney, James P.; O’Brien, Thomas W.; Walker, Jennifer L.; Garber, Eric A. E.

    2011-01-01

    Llama derived single domain antibodies (sdAb), the recombinantly expressed variable heavy domains from the unique heavy-chain only antibodies of camelids, were isolated from a library derived from llamas immunized with a commercial abrin toxoid preparation. Abrin is a potent toxin similar to ricin in structure, sequence and mechanism of action. The selected sdAb were evaluated for their ability to bind to commercial abrin as well as abrax (a recombinant abrin A-chain), purified abrin fractions, Abrus agglutinin (a protein related to abrin but with lower toxicity), ricin, and unrelated proteins. Isolated sdAb were also evaluated for their ability to refold after heat denaturation and ability to be used in sandwich assays as both capture and reporter elements. The best binders were specific for the Abrus agglutinin, showing minimal binding to purified abrin fractions or unrelated proteins. These binders had sub nM affinities and regained most of their secondary structure after heating to 95 °C. They functioned well in sandwich assays. Through gel analysis and the behavior of anti-abrin monoclonal antibodies, we determined that the commercial toxoid preparation used for the original immunizations contained a high percentage of Abrus agglutinin, explaining the selection of Abrus agglutinin binders. Used in conjunction with anti-abrin monoclonal and polyclonal antibodies, these reagents can fill a role to discriminate between the highly toxic abrin and the related, but much less toxic, Abrus agglutinin and distinguish between different crude preparations. PMID:22174977

  13. Targeted Multiplex Imaging Mass Spectrometry with Single Chain Fragment Variable (scfv) Recombinant Antibodies

    Science.gov (United States)

    Thiery, Gwendoline; Mernaugh, Ray L.; Yan, Heping; Spraggins, Jeffrey M.; Yang, Junhai; Parl, Fritz F.; Caprioli, Richard M.

    2012-10-01

    Recombinant scfv antibodies specific for CYP1A1 and CYP1B1 P450 enzymes were combined with targeted imaging mass spectrometry to simultaneously detect the P450 enzymes present in archived, paraffin-embedded, human breast cancer tissue sections. By using CYP1A1 and CYP1B1 specific scfv, each coupled to a unique reporter molecule (i.e., a mass tag) it was possible to simultaneously detect multiple antigens within a single tissue sample with high sensitivity and specificity using mass spectrometry. The capability of imaging multiple antigens at the same time is a significant advance that overcomes technical barriers encountered when using present day approaches to develop assays that can simultaneously detect more than a single antigen in the same tissue sample.

  14. Imaging of myocardial infarction in dogs and humans using monoclonal antibodies specific for human myosin heavy chains

    International Nuclear Information System (INIS)

    Leger, J.; Chevalier, J.; Larue, C.; Gautier, P.; Planchenault, J.; Aumaitre, E.; Messner, P.; Puech, P.; Saccavini, J.C.; Pau, B.

    1991-01-01

    The use of three different monoclonal antibodies specific for human ventricular myosin heavy chains in the visualization of the location and extent of necrosis in dogs with experimental acute myocardial infarction and in humans is described. Using a classic immunohistochemical method or ex vivo analysis of heart slices in dogs with acute myocardial infarction subjected to intravenous injection of unlabeled antimyosin antibodies or antimyosin antibodies labeled with indium-111, it was observed that all antibody fragments specifically reached the targeted necrotic zone less than 2 h after antibody injection and remained bound for up to 24 h. In a limited but significant number of cases (5 of the 12 humans and 11 of 43 dogs), it was possible to image the necrotic zone in vivo as early as 2 to 4 h after antibody injection. In other cases, individual blood clearance variations retarded or even prevented in vivo necrosis detection. Higher antimyosin fixation values were obtained in the necrotic zones in dogs with a rapid blood clearance relative to that of the other dogs. It is concluded that antimyosin antibodies always reached necrotic areas within 2 h. If blood clearance was rapid, in vivo imaging of the necrotic area was possible 2 to 6 h after necrosis, even in humans. In some cases, however, uncontrolled individual variations in the timing required for sufficient blood clearance hampered this rapid in vivo detection of myocardial necrosis

  15. Effect of polyethylene glycol conjugation on conformational and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab').

    Science.gov (United States)

    Roque, Cristopher; Sheung, Anthony; Rahman, Nausheen; Ausar, S Fernando

    2015-02-02

    We have investigated the effects of site specific "hinge" polyethylene glycol conjugation (PEGylation) on thermal, pH, and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab') using a variety of biophysical techniques. The results obtained by circular dichroism (CD), ultraviolet (UV) absorbance, and fluorescence spectroscopy suggested that the physical stability of the Fab' is maximized at pH 6-7 with no apparent differences due to PEGylation. Temperature-induced aggregation experiments revealed that PEGylation was able to increase the transition temperature, as well as prevent the formation of visible and subvisible aggregates. Statistical comparison of the three-index empirical phase diagram (EPD) revealed significant differences in thermal and pH stability signatures between Fab' and PEG-Fab'. Upon mechanical stress, micro-flow imaging (MFI) and measurement of the optical density at 360 nm showed that the PEG-Fab' had significantly higher resistance to surface-induced aggregation compared to the Fab'. Analysis of the interaction parameter, kD, indicated repulsive intermolecular forces for PEG-Fab' and attractive forces for Fab'. In conclusion, PEGylation appears to protect Fab' against thermal and mechanical stress-induced aggregation, likely due to a steric hindrance mechanism.

  16. 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.

  17. Measuring Response to Therapy by Near-Infrared Imaging of Tumors Using a Phosphatidylserine-Targeting Antibody Fragment

    Directory of Open Access Journals (Sweden)

    Jian Gong

    2013-06-01

    Full Text Available Imaging tumors and their response to treatment could be a valuable biomarker toward early assessment of therapy in patients with cancer. Phosphatidylserine (PS is confined to the inner leaflet of the plasma membrane in normal cells but is externalized on tumor vascular endothelial cells (ECs and tumor cells, and PS exposure is further enhanced in response to radiation and chemotherapy. In the present study, we evaluated the potential of a PS-targeting human F(ab'2 antibody fragment, PGN650, to detect exposure of PS in tumor-bearing mice. Tumor uptake of PGN650 was measured by near-infrared optical imaging in human tumor xenografts in immunodeficient mice. PGN650 specifically targeted tumors and was shown to target CD31-positive ECs and tumor cells. Tumor uptake of PGN650 was significantly higher in animals pretreated with docetaxel. The peak tumor to normal tissue (T/N ratio of probe was observed at 24 hours postinjection of probe, and tumor binding was detected for at least 120 hours. In repeat dose studies, PGN650 uptake in tumors was significantly higher following pretreatment with docetaxel compared to baseline uptake prior to treatment. PGN650 may be a useful probe to detect PS exposed in tumors and to monitor enhanced PS exposure to optimize therapeutic agents to treat tumors.

  18. Phage display selection of fully human antibody fragments to inhibit growth-promoting effects of glycine-extended gastrin 17 on human colorectal cancer cells.

    Science.gov (United States)

    Khajeh, Shirin; Tohidkia, Mohammad Reza; Aghanejad, Ayuob; Mehdipour, Tayebeh; Fathi, Farzaneh; Omidi, Yadollah

    2018-06-09

    Glycine-extended gastrin 17 (G17-Gly), a dominant processing intermediate of gastrin gene, has been implicated in the development or maintenance of colorectal cancers (CRCs). Hence, neutralizing G17-Gly activity by antibody entities can provide a potential therapeutic strategy in the patients with CRCs. To this end, we isolated fully human antibody fragments from a phage antibody library through biopanning against different epitopes of G17-Gly in order to obtain the highest possible antibody diversity. ELISA screening and sequence analysis identified 2 scFvs and 4 V L antibody fragments. Kinetic analysis of the antibody fragments by SPR revealed K D values to be in the nanomolar range (87.9-334 nM). The selected anti-G17-Gly antibody fragments were analyzed for growth inhibition and apoptotic assays in a CRC cell line, HCT-116, which is well-characterized for expressing gastrin intermediate species but not amidated gastrin. The antibody fragments exhibited significant inhibition of HCT-116 cells proliferation ranging from 36.5 to 73% of controls. Further, Annexin V/PI staining indicated that apoptosis rates of scFv H8 and V L G8 treated cells were 45.8 and 63%, respectively. Based on these results, we for the first time, demonstrated the isolation of anti-G17-Gly human scFv and V L antibodies with potential therapeutic applications in G17-Gly-responsive tumors.

  19. Immunocytochemistry by electron spectroscopic imaging using well defined boronated monovalent antibody fragments.

    Science.gov (United States)

    Kessels, M M; Qualmann, B; Sierralta, W D

    1996-01-01

    Contributing to the rapidly developing field of immunoelectron microscopy a new kind of markers has been created. The element boron, incorporated as very stable carborane clusters into different kinds of peptides, served as a marker detectable by electron spectroscopic imaging (ESI)--an electron microscopic technique with high-resolution potential. Covalently linked immunoreagents conspicuous by the small size of both antigen recognizing part and marker moiety are accessible by using peptide concepts for label construction and their conjugation with Fab' fragments. Due to a specific labeling of the free thiol groups of the Fab' fragments, the antigen binding capacity was not affected by the attachment of the markers and the resulting immunoprobes exhibited an elongated shape with the antigen combining site and the label located at opposite ends. The labeling densities observed with these reagents were found to be significantly higher than those obtained by using conventional colloidal gold methods. Combined with digital image processing and analysis systems, boron-based ESI proved to be a powerful approach in ultrastructural immunocytochemistry employing pre- and post-embedding methods.

  20. Selection of diethylstilbestrol-specific single-chain antibodies from a non-immunized mouse ribosome display library.

    Directory of Open Access Journals (Sweden)

    Yanan Sun

    Full Text Available Single chain variable fragments (scFvs against diethylstilbestrol (DES were selected from the splenocytes of non-immunized mice by ribosome display technology. A naive library was constructed and engineered to allow in vitro transcription and translation using an E. coli lysate system. Alternating selection in solution and immobilization in microtiter wells was used to pan mRNA-ribosome-antibody (ARM complexes. After seven rounds of ribosome display, the expression vector pTIG-TRX containing the selected specific scFv DNAs were transformed into Escherichia coli BL21 (DE3 for expression. Twenty-six positive clones were screened and five clones had high antibody affinity and specificity to DES as evidenced by indirect competitive ELISA. Sequence analysis showed that these five DES-specific scFvs had different amino acid sequences, but the CDRs were highly similar. Surface plasmon resonance (SPR analysis was used to determine binding kinetics of one clone (30-1. The measured K(D was 3.79 µM. These results indicate that ribosome display technology can be used to efficiently isolate hapten-specific antibody (Ab fragments from a naive library; this study provides a methodological framework for the development of novel immunoassays for multiple environmental pollutants with low molecular weight detection using recombinant antibodies.

  1. Isolation of monoclonal antibodies with predetermined conformational epitope specificity.

    Directory of Open Access Journals (Sweden)

    Anton M Sholukh

    Full Text Available Existing technologies allow isolating antigen-specific monoclonal antibodies (mAbs from B cells. We devised a direct approach to isolate mAbs with predetermined conformational epitope specificity, using epitope mimetics (mimotopes that reflect the three-dimensional structure of given antigen subdomains. We performed differential biopanning using bacteriophages encoding random peptide libraries and polyclonal antibodies (Abs that had been affinity-purified with either native or denatured antigen. This strategy yielded conformational mimotopes. We then generated mimotope-fluorescent protein fusions, which were used as baits to isolate single memory B cells from rhesus monkeys (RMs. To amplify RM immunoglobulin variable regions, we developed RM-specific PCR primers and generated chimeric simian-human mAbs with predicted epitope specificity. We established proof-of-concept of our strategy by isolating mAbs targeting the conformational V3 loop crown of HIV Env; the new mAbs cross-neutralized viruses of different clades. The novel technology allows isolating mAbs from RMs or other hosts given experimental immunogens or infectious agents.

  2. Use and limitations of radiolabelled anti-CEA antibodies and their fragments for photoscanning detection of human colorectal carcinomas

    Energy Technology Data Exchange (ETDEWEB)

    Mach, J P; Forni, M; Ritschard, J; Buchegger, F; Carrel, S; Widgren, S; Donath, A; Alberto, P [Lausanne Univ. (Switzerland)

    1980-08-01

    Fifty-three patients with histologically proven carcinoma were injected with highly purified (/sup 131/I)-labelled goat antibodies or fragments of antibodies against carcinoembryonic antigen (CEA). Each patient was tested by external photoscanning 4, 24, 36, 48h after injection. In 22 patients (16 of 38 injected with intact antibodies, 5 of 13 with F(ab')/sub 2/ fragments and 1 of 2 with Fab' fragments), an increased concentration of /sup 131/I radioactivity corresponding to the previously known tumor location was detected by photoscanning 36-48 h after injection. Blood pool and secreted radioactivity was determined in all patients by injecting 15 min after scanning, (sup(99m)Tc)-labeled normal serum albumin and free sup(99m)TcO/sub 4//sup -/. The computerized subtraction of sup(99m)Tc from /sup 131/I radioactivity enhanced the definition of tumor localization in the 22 positive patients. However, in spite of the computerized subtraction, interpretation of the scans remained doubtful for 12 patients and was entirely negative for 19 additional patients.

  3. 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...

  4. The clinical syndrome of specific antibody deficiency in children.

    Science.gov (United States)

    Boyle, R J; Le, C; Balloch, A; Tang, M L-K

    2006-12-01

    Specific antibody deficiency (SAD) is an immune deficiency which has been reported in adults and children with recurrent respiratory tract infections; however, the clinical features of SAD are not well described. This study evaluated formally the clinical syndrome of SAD, by comparing the clinical features of children with SAD and those of children with recurrent infection but normal immune function tests. SAD was defined as an adequate IgG antibody response to less than 50% of 12 pneumococcal serotypes tested following 23-valent unconjugated pneumococcal immunization. An adequate IgG antibody response was defined as a post-immunization titre of >or= 1.3 microg/ml or >or= four times the preimmunization value. Seventy-four children with recurrent infection were evaluated where immune deficiencies other than SAD had been excluded. Eleven (14.9%) of these children had SAD. Clinical features differed between the group with SAD and the group with normal antibody responses. A history of otitis media, particularly in association with chronic otorrhoea was associated with SAD [relative risk (RR) of SAD in those with chronic otorrhoea 4.64 (P = 0.02)]. SAD was associated with allergic disease, particularly allergic rhinitis [RR of SAD in those with allergic rhinitis 3.77 (P = 0.04)]. These two clinical associations of SAD were independent in this study [RR of chronic otorrhoea in those with allergic rhinitis 0.85 (P = 0.28)]. SAD was not an age-related phenomenon in this population. SAD has a distinct clinical phenotype, presenting as recurrent infection associated with chronic otorrhoea and/or allergic disease, and the condition should be sought in children with these features.

  5. Serum and urine analysis of the aminoterminal procollagen peptide type III by radioimmunoassay with antibody Fab fragments.

    Science.gov (United States)

    Rohde, H; Langer, I; Krieg, T; Timpl, R

    1983-09-01

    A radioimmunoassay based on antibody Fab fragments was developed for the aminoterminal peptide Col 1-3 of bovine type III procollagen. This assay does not distinguish the intact aminopropeptide Col 1-3 from its globular fragment Col 1. Parallel inhibition profiles were observed with human serum and urine allowing the simultaneous quantitative determination of intact and fragmented antigens in these samples. Most of the material has a size similar to that of fragment Col 1 indicating that the aminopropeptide is degraded under physiologic conditions. The concentration of aminopeptide in normal sera was in the range 15-63 ng/ml. Daily excretion was found to be in the range 30-110 micrograms. More than 50% of patients with alcoholic hepatitis and liver cirrhosis showed elevated serum levels of aminopropeptide by the Fab assay. Elevated concentrations were detected more frequently with an antibody radioimmunoassay which measures mainly the intact form of the aminopropeptide. It is suggested that analysis of patients material by both assays could improve their diagnostic application.

  6. Functional Characterization of ATM Kinase Using Acetylation-Specific Antibodies.

    Science.gov (United States)

    Sun, Yingli; Du, Fengxia

    2017-01-01

    The activation of ATM is critical in the DNA double strand breaks repair pathway. Acetylation of ATM by Tip60 histone acetyltransferase (HAT) plays a key role in the activation of ATM kinase activity in response to DNA damage. ATM forms a stable complex with Tip60 through the FATC domain of ATM. Tip60 acetylates lysine3016 of ATM, and this acetylation induces the activation of ATM. Several techniques are included in the study of ATM acetylation by Tip60, such as in vitro kinase assay, systematic mutagenesis, western blots. Here, we describe how to study the acetylation of ATM using acetylation-specific antibodies.

  7. PET Imaging of Abdominal Aortic Aneurysm with 64Cu-Labeled Anti-CD105 Antibody Fab Fragment.

    Science.gov (United States)

    Shi, Sixiang; Orbay, Hakan; Yang, Yunan; Graves, Stephen A; Nayak, Tapas R; Hong, Hao; Hernandez, Reinier; Luo, Haiming; Goel, Shreya; Theuer, Charles P; Nickles, Robert J; Cai, Weibo

    2015-06-01

    The critical challenge in abdominal aortic aneurysm (AAA) research is the accurate diagnosis and assessment of AAA progression. Angiogenesis is a pathologic hallmark of AAA, and CD105 is highly expressed on newly formed vessels. Our goal was to use (64)Cu-labeled anti-CD105 antibody Fab fragment for noninvasive assessment of angiogenesis in the aortic wall in a murine model of AAA. Fab fragment of TRC105, a mAb that specifically binds to CD105, was generated by enzymatic papain digestion and conjugated to NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) for (64)Cu labeling. The binding affinity/specificity of NOTA-TRC105-Fab was evaluated by flow cytometry and various ex vivo studies. BALB/c mice were anesthetized and treated with calcium phosphate to induce AAA and underwent weekly PET scans using (64)Cu-NOTA-TRC105-Fab. Biodistribution and autoradiography studies were also performed to confirm the accuracy of PET results. NOTA-TRC105-Fab exhibited high purity and specifically bound to CD105 in vitro. Uptake of (64)Cu-NOTA-TRC105-Fab increased from a control level of 3.4 ± 0.1 to 9.5 ± 0.4 percentage injected dose per gram (%ID/g) at 6 h after injection on day 5 and decreased to 7.2 ± 1.4 %ID/g on day 12, which correlated well with biodistribution and autoradiography studies (i.e., much higher tracer uptake in AAA than normal aorta). Of note, enhanced AAA contrast was achieved, due to the minimal background in the abdominal area of mice. Degradation of elastic fibers and highly expressed CD105 were observed in ex vivo studies. (64)Cu-NOTA-TRC105-Fab cleared rapidly through the kidneys, which enabled noninvasive PET imaging of the aorta with enhanced contrast and showed increased angiogenesis (CD105 expression) during AAA. (64)Cu-NOTA-TRC105-Fab PET may potentially be used for future diagnosis and prognosis of AAA. © 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

  8. Monoclonal antibodies specific to heat-treated porcine blood.

    Science.gov (United States)

    Raja Nhari, Raja Mohd Hafidz; Hamid, Muhajir; Rasli, Nurmunirah Mohamad; Omar, Abdul Rahman; El Sheikha, Aly Farag; Mustafa, Shuhaimi

    2016-05-01

    Porcine blood is potentially being utilized in food as a binder, gelling agent, emulsifier or colorant. However, for certain communities, the usage of animal blood in food is strictly prohibited owing to religious concerns and health reasons. This study reports the development of monoclonal antibodies (MAbs) against heat-treated soluble proteins (HSPs) of autoclaved porcine blood; characterization of MAbs against blood, non-blood and plasma from different animal species using qualitative indirect non-competitive enzyme-linked immunosorbent assay (ELISA); and immunoblotting of antigenic components in HSPs of porcine blood. Fifteen MAbs are specific to heat-treated and raw porcine blood and not cross-reacted with other animal blood and non-blood proteins (meat and non-meat). Twelve MAbs are specific to porcine plasma, while three MAbs specific to porcine plasma are cross-reacted with chicken plasma. Immunoblotting revealed antigenic protein bands (∼60, ∼85-100 and ∼250 kDa) in porcine blood and plasma recognized by the MAbs. Selection of MAbs that recognized 60 kDa HSPs of porcine blood and plasma as novel monoclonal antibodies would be useful for detection of porcine plasma in processed food using the immunoassay method. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

  9. Production, purification, crystallization and preliminary X-ray diffraction analysis of the HIV-2-neutralizing V3 loop-specific Fab fragment 7C8

    International Nuclear Information System (INIS)

    Uchtenhagen, Hannes; Sourial, Samer; Friemann, Rosmarie; Ehnlund, Mariethe; Spetz, Anna-Lena; Harris, Robert A.; Madhurantakam, Chaithanya; Achour, Adnane

    2009-01-01

    Neutralizing Fab fragments of the HIV-2-binding murine antibody 7C8 were generated after purification from hybridoma cell-culture supernatant. Crystallization conditions were determined and diffraction data were collected to 2.7 Å resolution. 7C8 is a mouse monoclonal antibody that is specific for the third hypervariable loop (V3 loop) of the human immunodeficiency virus type 2 (HIV-2) associated protein gp125. Fab fragments of 7C8 effectively neutralize HIV-2. 7C8 was expressed and purified from a hybridoma cell line in order to establish the molecular basis underlying the specificity of the 7C8 antibody for the V3 loop as well as the specific role of the elongated third complementarity-determining region of the heavy chain (CDRH3). The antibody was digested with papain and Fab fragments were purified using size-exclusion chromatography. Hanging-drop vapour-diffusion crystallization techniques were employed and the protein was crystallized in 50 mM ammonium sulfate, 100 mM Tris–HCl pH 8.5, 25%(w/v) PEG 8000 and 2.5%(w/v) PEG 400 at 275 K. The analysed crystals belonged to the rhombohedral space group P3 2 21, with unit-cell parameters a = b = 100.1, c = 196.8 Å, and diffracted to 2.7 Å resolution

  10. A novel antihuman C3d monoclonal antibody with specificity to the C3d complement split product

    DEFF Research Database (Denmark)

    Rasmussen, Karina Juhl; Skjødt, Mikkel-Ole; Vitved, Lars

    2017-01-01

    The complement component C3 and the cleavage products of C3b/iC3b, C3c and C3d are used as biomarkers in clinical diagnostics. Currently, no specific antibodies are able to differentiate C3d from other fragments, although such a distinction could be very valuable considering that they may reflect...... different pathophysiological mechanisms. We have developed a rat antihuman C3d monoclonal antibody with specificity to the end sequence of the N-terminal region of C3d. The antibody can therefore only bind to C3d when it manifests itself as the final end product of cleaved C3. We believe...

  11. Detection and specifity of class specific antibodies to whole bacteria cells using a solid phase radioimmunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Czerkinsky, C.; Rees, A.S.; Bergimeier, L.A.; Challacombe, S.J. (Guy' s Hospital Medical and Dental Schools, London (UK))

    1983-07-01

    A solid phase radioimmunoassay has been developed which can be used for the detection of isotype specific antibodies to whole bacteria and other particulate antigens, and is applicable to a variety of species. Bacteria are bound to the solid phase by the use either of antibodies, or of methyl glyoxal. Both methods result in a sensitive and reproducible assay, and bacteria do not appear to desorb from the solid phase. The specificity of antibodies to whole bacteria was examined by absorption of antisera with various species of bacteria and retesting, or by determining the binding of antisera to various bacteria bound to the solid phase. Both methods revealed specificity for the bacteria examined. Inhibition studies showed that antibodies to Streptococcus mutans whole cells could be inhibited by purified cell surface antigens glucosyltransferase and antigen I/II, but only minimally by lipoteichoic acid, c polysaccharide or dextran. In murine antisera antibodies of the IgG, IgM, and IgA classes could be detected at amounts of less than 1 ng/ml.

  12. A novel fusion protein of IP10-scFv retains antibody specificity and chemokine function

    Energy Technology Data Exchange (ETDEWEB)

    Junqing, Guo; Liu, Chen; Hongwu, Ai; Jiannian, Jing; Jiyong, Zhou; Chuyu, Zhang; Shangyou, You

    2004-07-23

    We combined the specificity of tumor-specific antibody with the chemokine function of interferon-{gamma} inducible protein 10 (IP-10) to recruit immune effector cells in the vicinity of tumor cells. A novel fusion protein of IP10-scFv was constructed by fusing mouse IP-10 to V{sub H} region of single-chain Fv fragment (scFv) against acidic isoferritin (AIF), and expressed in NS0 murine myeloma cells. The IP10-scFv fusion protein was shown to maintain the specificity of the antiAIF scFv with similar affinity constant, and bind to the human hepatocarcinoma SMMC 7721 cells secreting AIF as well as the activated mouse T lymphocytes expressing CXCR3 receptor. Furthermore, the IP10-scFv protein either in solution or bound on the surface of SMMC 7721 cells induced significant chemotaxis of mouse T cells in vitro. The results indicate that the IP10-scFv fusion protein possesses both bioactivities of the tumor-specific antibody and IP-10 chemokine, suggesting its possibility to induce an enhanced immune response against the residual tumor cells in vivo.

  13. A novel fusion protein of IP10-scFv retains antibody specificity and chemokine function

    International Nuclear Information System (INIS)

    Guo Junqing; Chen Liu; Ai Hongwu; Jing Jiannian; Zhou Jiyong; Zhang Chuyu; You Shangyou

    2004-01-01

    We combined the specificity of tumor-specific antibody with the chemokine function of interferon-γ inducible protein 10 (IP-10) to recruit immune effector cells in the vicinity of tumor cells. A novel fusion protein of IP10-scFv was constructed by fusing mouse IP-10 to V H region of single-chain Fv fragment (scFv) against acidic isoferritin (AIF), and expressed in NS0 murine myeloma cells. The IP10-scFv fusion protein was shown to maintain the specificity of the antiAIF scFv with similar affinity constant, and bind to the human hepatocarcinoma SMMC 7721 cells secreting AIF as well as the activated mouse T lymphocytes expressing CXCR3 receptor. Furthermore, the IP10-scFv protein either in solution or bound on the surface of SMMC 7721 cells induced significant chemotaxis of mouse T cells in vitro. The results indicate that the IP10-scFv fusion protein possesses both bioactivities of the tumor-specific antibody and IP-10 chemokine, suggesting its possibility to induce an enhanced immune response against the residual tumor cells in vivo

  14. Micro solid-phase radioimmunoassay for detection of herpesvirus type-specific antibody: specificity and sensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Adler-Storthz, K.; Matson, D.O.; Adam, E.; Dreesman, G.R. (Baylor Univ., Houston, TX (USA). Coll. of Medicine)

    1983-02-01

    The specificity and sensitivity of a micro solid-phase radioimmunoassay (micro-SPRIA) that detects type-specific IgG antibody to herpes simplex virus types 1 and 2 (HSV1 and HSV2) were evaluated. Glycoproteins VP123 (molecular weight, 123,000) of HSV1 and VP119 (molecular weight, 119,000) of HSV2 were found to display the greatest degree of antigenic type-specificity of several HSV antigens tested with the micro-SPRIA technique. When testing a group of sera, negative for anti-HSV antibodies by microneutralization, in the micro-SPRIA, a range of negative reactivities was noted, suggesting that cut-points should be determined for each antigen preparation. The micro-SPRIA detected appropriate antibody activity in patients with recurrent infection and a marked agreement was noted in comparison to detection of anti-HSV antibodies measured with the microneutralization test. The type-specificity of the micro-SPRIA was substantiated by the independence of test results using VP119 and VP123 antigens for a random group of positive sera. The assay is rapid, specific, and sensitive and allows the testing of multiple serum samples with a standardized set of reagents.

  15. Generation of human antibody fragments recognizing distinct epitopes of the nucleocapsid (N SARS-CoV protein using a phage display approach

    Directory of Open Access Journals (Sweden)

    Grasso Felicia

    2005-09-01

    Full Text Available Abstract Background Severe acute respiratory syndrome (SARS-CoV is a newly emerging virus that causes SARS with high mortality rate in infected people. Successful control of the global SARS epidemic will require rapid and sensitive diagnostic tests to monitor its spread, as well as, the development of vaccines and new antiviral compounds including neutralizing antibodies that effectively prevent or treat this disease. Methods The human synthetic single-chain fragment variable (scFv ETH-2 phage antibody library was used for the isolation of scFvs against the nucleocapsid (N protein of SARS-CoV using a bio panning-based strategy. The selected scFvs were characterized under genetics-molecular aspects and for SARS-CoV N protein detection in ELISA, western blotting and immunocytochemistry. Results Human scFv antibodies to N protein of SARS-CoV can be easily isolated by selecting the ETH-2 phage library on immunotubes coated with antigen. These in vitro selected human scFvs specifically recognize in ELISA and western blotting studies distinct epitopes in N protein domains and detect in immunohistochemistry investigations SARS-CoV particles in infected Vero cells. Conclusion The human scFv antibodies isolated and described in this study represent useful reagents for rapid detection of N SARS-CoV protein and SARS virus particles in infected target cells.

  16. 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.

  17. Critical contribution of aromatic rings to specific recognition of polyether rings. The case of ciguatoxin CTX3C-ABC and its specific antibody 1C49.

    Science.gov (United States)

    Tsumoto, Kouhei; Yokota, Akiko; Tanaka, Yoshikazu; Ui, Mihoko; Tsumuraya, Takeshi; Fujii, Ikuo; Kumagai, Izumi; Nagumo, Yoko; Oguri, Hiroki; Inoue, Masayuki; Hirama, Masahiro

    2008-05-02

    To address how proteins recognize polyether toxin compounds, we focused on the interaction between the ABC ring compound of ciguatoxin 3C and its specific antibody, 1C49. Surface plasmon resonance analyses indicated that Escherichia coli-expressed variable domain fragments (Fv) of 1C49 had the high affinity constants and slow dissociation constants typical of antigen-antibody interactions. Linear van't Hoff analyses suggested that the interaction is enthalpy-driven. We resolved the crystal structure of 1C49 Fv bound to ABC ring compound of ciguatoxin 3C at a resolution of 1.7A. The binding pocket of the antibody had many aromatic rings and bound the antigen by shape complementarity typical of hapten-antibody interactions. Three hydrogen bonds and many van der Waals interactions were present. We mutated several residues of the antibody to Ala, and we used surface plasmon resonance to analyze the interactions between the mutated antibodies and the antigen. This analysis identified Tyr-91 and Trp-96 in the light chain as hot spots for the interaction, and other residues made incremental contributions by conferring enthalpic advantages and reducing the dissociation rate constant. Systematic mutation of Tyr-91 indicated that CH-pi and pi-pi interactions between the aromatic ring at this site and the antigen made substantial contributions to the association, and van der Waals interactions inhibited dissociation, suggesting that aromaticity and bulkiness are critical for the specific recognition of polyether compounds by proteins.

  18. Comparison of Six Automated Treponema-Specific Antibody Assays.

    Science.gov (United States)

    Park, Borae G; Yoon, Jihoon G; Rim, John Hoon; Lee, Anna; Kim, Hyon-Suk

    2016-01-01

    Six different Treponema (TP)-specific immunoassays were compared to the fluorescent treponemal antibody absorption (FTA-ABS) test. A total of 615 samples were tested. The overall percent agreement, analytical sensitivity, and analytical specificity of each assay compared to the FTA-ABS test were as follows: Architect Syphilis TP, 99.2%, 96.8%, and 100%; Cobas Syphilis, 99.8%, 99.4%, and 100%; ADVIA Centaur Syphilis, 99.8%, 99.4%, and 100%; HISCL Anti-TP assay kit, 99.7%, 98.7%, and 100%; Immunoticles Auto3 TP, 99.0%, 97.5%, and 99.6%; Mediace TPLA, 98.0%, 98.1%, and 98.0%. All results that were discrepant between the TP-specific assays were associated with samples from noninfectious cases (11 immunoassay false positives and 7 from previous syphilis cases). Our study demonstrated that TP-specific immunoassays generally showed high sensitivities, specificities, and percentages of agreement compared to FTA-ABS, with rare cases of false-positive or false-negative results. Therefore, most TP-specific immunoassays are acceptable for use in screening for syphilis. However, it is important to perform a thorough review of a patient's clinical and treatment history for interpreting the results of syphilis serology. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  19. Extracorporeal life support and digoxin-specific Fab fragments for successful management of Taxus baccata intoxication with low output and ventricular arrhythmia.

    Science.gov (United States)

    Farag, Mina; Badowski, Dominika; Koschny, Ronald; Skopp, Gisela; Brcic, Andreas; Szabo, Gabor B

    2017-12-01

    Yew plants are evergreen shrubs which are widely spread throughout the northern hemisphere. Taxane alkaloid derivatives, mainly taxine B, represent the main toxins of Taxus baccata and are highly cardiotoxic. Due to the lack of randomized clinical trials, case reports on accidental or suicidal yew intoxications build the only source of knowledge of clinical treatment options. We report the case of a suicidal yew ingestion admitted to our hospital under prolonged cardiopulmonary resuscitation due to pulseless electrical activity. Extra-corporeal life support (ECLS) was established to maintain adequate organ perfusion. Repeated administration of digoxin-specific Fab antibody fragments, which cross-react with taxine, was associated with an immediate conversion from asystole to broad-complex bradycardia and a gradual normalization of the electrocardiogram (ECG). This was paralleled by a recovery of the cardiac function and weaning from the ECLS. The taxine metabolite 3,5-dimethoxyphenol could be detected by mass spectrometry before but not after the first Fab-fragment treatment. In contrast, the total amount of taxine (including the neutralized, Fab fragment-bound fraction) was increased after each Fab fragment administration, suggesting an accumulation of neutralized, since antibody-bound taxine in the blood by anti-digoxin Fab fragments. In conclusion, the successful clinical course of this case suggests a benefit of an early anti-digoxin Fab-fragment administration for the treatment of yew intoxication. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Antibody index and specific antibody quotient in horses after intragastric administration of Sarcocystis neurona sporocysts.

    Science.gov (United States)

    Heskett, Katherine A; Mackay, Robert J

    2008-03-01

    To investigate the use of a specific antibody index (AI) that relates Sarcocystis neurona-specific IgG quotient (Q(SN)) to total IgG quotient (Q(IgG)) for the detection of the anti-S neurona antibody fraction of CNS origin in CSF samples obtained from horses after intragastric administration of S neurona sporocysts. 18 adult horses. 14 horses underwent intragastric inoculation (day 0) with S neurona sporocysts, and 4 horses remained unchallenged; blood and CSF samples were collected on days - 1 and 84. For purposes of another study, some challenged horses received intermittent administration of ponazuril (20 mg/kg, PO). Sarcocystis neurona-specific IgG concentrations in CSF (SN(CSF)) and plasma (SN(plasma)) were measured via a direct ELISA involving merozoite lysate antigen and reported as ELISA units (EUs; arbitrary units based on a nominal titer for undiluted immune plasma of 100,000 EUs/mL). Total IgG concentrations in CSF (IgG(CSF)) and plasma (IgG(plasma)) were quantified via a sandwich ELISA and a radial immunodiffusion assay, respectively; Q(SN), Q(IgG), and AI were calculated. Following sporocyst challenge, mean +/- SEM SN(CSF) and SN(plasma) increased significantly (from 8.8 +/- 1.0 EUs/mL to 270.0 +/- 112.7 EUs/mL and from 1,737 +/- 245 EUs/mL to 43,169 +/- 13,770 EUs/mL, respectively). Challenge did not affect total IgG concentration, Q(SN), Q(IgG), or AI. S neurona-specific IgG detected in CSF samples from sporocyst-challenged horses appeared to be extraneural in origin; thus, this experimental challenge may not reliably result in CNS infection. Calculation of a specific AI may have application to the diagnosis of S neurona-associated myeloencephalitis in horses.

  1. Radiolabeled monoclonal antibody 15 and its fragments for localization and imaging of xenografts of human lung cancer

    International Nuclear Information System (INIS)

    Endo, K.; Kamma, H.; Ogata, T.

    1988-01-01

    Monoclonal antibody (MAb) 15 and its F(ab')2 and Fab fragments were radioiodinated, and their biodistribution and imaging were compared in BALB/c nude mice bearing a xenograft of a human lung cancer (TKB-2). Association constants for 125I-labeled MAb 15 IgG, F(ab')2, and Fab were 1.9 X 10(9), 1.8 X 10(9), and 3.7 X 10(8) M-1, respectively. Immunoreactive fractions ranged from 0.59 to 0.50. Cultured TKB-2 cells expressed 1.1 X 10(4) binding sites/cell for MAb 15 IgG in vitro. The binding of a control antibody and the binding of its fragments to TKB-2 cells were less than 3% of the input doses. The mice with the TKB-2 tumors were given simultaneous injections of 10 microCi of 131I-labeled MAb 15 or its fragments and 10 microCi of 125I-labeled control IgG or its fragments. With MAb 15 IgG, the percentage of the injected dose bound per gram of tissue (ID/g) of the tumor was 3.68% at day 7, when the localization index (LI) was 4.38. At day 2 after MAb 15 F(ab')2 injection, 1.12% of the ID/g was localized in the tumor and the LI was 3.04. After MAb 15 Fab injection, the percentage of the ID/g of the tumor was 0.31% and the LI was 2.58 at day 1. MAb 15 IgG, F(ab')2, and Fab cleared from the blood early, with a half-life of 33, 16, and 9 hours, respectively. The distributions of MAb 15 and its fragments in the normal organs did not differ from those of the control. Radioimaging with 100 microCi of 131I-labeled MAb 15 and its fragments showed that 42%, 44%, and 32% of the total-body count were localized in the tumor with IgG at day 7, F(ab')2 at day 2, or Fab at day 1, respectively. Because the radioactivity remaining in the tumor with Fab was low, the image was insufficient. Throughout the period, less than 10% of the control IgG and its fragments remained in the tumor. Microautoradiography confirmed the binding of MAb 15 and its fragments to the tumor cells

  2. Specificity of anti-phospholipid antibodies in infectious mononucleosis: a role for anti-cofactor protein antibodies

    Science.gov (United States)

    Sorice, M; Pittoni, V; Griggi, T; Losardo, A; Leri, O; Magno, M S; Misasi, R; Valesini, G

    2000-01-01

    The antigen specificity of anti-phospholipid antibodies in infectious mononucleosis (IM) was studied using ELISA for the detection of anti-β2-glycoprotein I (β2-GPI), anti-annexin V, anti-protein S and anti-prothrombin antibodies and TLC immunostaining for the detection of anti-phospholipid antibodies. This technique enabled us to look at antibodies reacting to ‘pure’ phospholipid antigens in the absence of protein contamination. Sera from 46 patients with IM, 18 with systemic lupus erythematosus (SLE), 21 with primary anti-phospholipid antibody syndrome (PAPS), 50 with Helicobacter pylori infection and 30 healthy blood donors were tested. This study highlights anti-phospholipid antibodies in patients with IM as specific ‘pure’ anti-cardiolipin antibodies, while in PAPS and SLE patients anti-phosphatidylserine and anti-phosphatidylethanolamine antibodies were also found. This investigation also shows that the anti-cardiolipin antibodies found in IM can be present with anti-cofactor protein antibodies. The higher prevalence of anti-cofactor antibodies found in IM sera than in Helicobacter pylori sera may be due to the immunostimulatory effect and/or the polyclonal activation often observed in course of Epstein–Barr virus infection. However, anti-β2-GPI and, to a lesser extent, anti-prothrombin antibodies occur with a significantly lower prevalence in IM than in PAPS patients. This finding suggests that these antibodies should be regarded as the expression of the broad autoimmune syndrome involving the phospholipid-binding plasma proteins. PMID:10792380

  3. Assessment of Digoxin-Specific Fab Fragment Dosages in Digoxin Poisoning.

    Science.gov (United States)

    Nordt, Sean Patrick; Clark, Richard F; Machado, Carol; Cantrell, F Lee

    2016-01-01

    Digoxin poisoning still remains a common cause of morbidity and mortality. Fortunately, digoxin-specific Fab fragments are commercially available as an antidote. However, these Fab fragments are several thousand dollars per vial. There is a standardized formula to calculate appropriate Fab fragment dosage based on the serum digoxin concentration. This can greatly reduce the amount of Fab fragment administered. There is also an empiric dosing guideline recommending 6-10 vials be given; however, this may result in higher amounts of Fab fragments being administered than required. We performed this study to assess the amounts of digoxin-specific Fab fragments administered in the treatment of digoxin poisonings recorded in a poison control system database from January 1, 2000, to December 31, 2009, in which digoxin serum concentrations were available. This was a retrospective study of 278 patients, 107 with acute poisonings (group A) and 171 following chronic poisoning (group B). In group A, the calculated Fab dose was higher than the calculated dose based on available concentrations in 39 (36%) of group A and 15 (9%) of group B patients. The average wholesale price cost of the excessive dosages ranged from $4818 to as high as $50,589 per patient. Our data suggests that clinician education on digoxin poisoning and the use of the standardized formula to calculate the Fab dose may decrease over utilization and decrease costs associated with the administration of digoxin-specific Fab fragments in the treatment of digoxin poisonings.

  4. Antigen-Specific Antibody Glycosylation Is Regulated via Vaccination

    NARCIS (Netherlands)

    Mahan, Alison E.; Jennewein, Madeleine F.; Suscovich, Todd; Dionne, Kendall; Tedesco, Jacquelynne; Chung, Amy W.; Streeck, Hendrik; Pau, Maria; Schuitemaker, Hanneke; Francis, Don; Fast, Patricia; Laufer, Dagna; Walker, Bruce D.; Baden, Lindsey; Barouch, Dan H.; Alter, Galit

    2016-01-01

    Antibody effector functions, such as antibody-dependent cellular cytotoxicity, complement deposition, and antibody-dependent phagocytosis, play a critical role in immunity against multiple pathogens, particularly in the absence of neutralizing activity. Two modifications to the IgG constant domain

  5. Selecting highly structure-specific antibodies using structured synthetic mimics of the cystine knot protein sclerostin

    NARCIS (Netherlands)

    Back, J.W.; Frisch, C.; Van Pee, K.; Boschert, V.; van Vught, R.; Puijk, W.; Mueller, T. D.; Knappik, A.; Timmerman, P.

    2012-01-01

    Antibodies directed against specific regions of a protein have traditionally been raised against full proteins, protein domains or simple unstructured peptides, containing contiguous stretches of primary sequence. We have used a new approach of selecting antibodies against restrained peptides

  6. 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.

  7. Bivalent fragment of the ior-CEA1 antibody. A challenge to the positive CEA tumors radioimmunotherapy

    International Nuclear Information System (INIS)

    Ravelo, Rolando; Sanchez, Iradia; Pimentel, Gilmara; Oliva, Juan; Perez, Lincidio; Ayala, Marta; Bell, Hansell; Gavilondo, Jorge

    2006-01-01

    The directed radiotherapy of the solid tumors with fragments recombinants of radiolabelled antibodies is a topic of current investigation, so much at preclinical level as clinical. This work describes the preclinical characterization of a new fragment type diabody of the AcMo ior CEA1 that has been labelled with 131 I for their use in the diagnosis and the therapy of CEA positive tumors. The radiolabelling methodology used allows the incorporation of more than 90% of the radio iodine to the molecule without committing the capacity of recognition of its antigen significantly. The combination of the favourable properties pharmacy kinetic and high selective accumulation in the tumor, they make of the diabody anti CEA an appropriate candidate for the radioimmunodiagnosis and the radioimmunotherapy of tumors that expresses CEA (Author)

  8. 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

  9. Isolation and characterisation of a human-like antibody fragment (scFv that inactivates VEEV in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Torsten Rülker

    Full Text Available Venezuelan equine encephalitis virus (VEEV belongs to the Alphavirus genus and several species of this family are pathogenic to humans. The viruses are classified as potential agents of biological warfare and terrorism and sensitive detection as well as effective prophylaxis and antiviral therapies are required.In this work, we describe the isolation of the anti-VEEV single chain Fragment variable (scFv, ToR67-3B4, from a non-human primate (NHP antibody gene library. We report its recloning into the bivalent scFv-Fc format and further immunological and biochemical characterisation.The scFv-Fc ToR67-3B4 recognised viable as well as formalin and ß-propionolactone (ß-Pl inactivated virus particles and could be applied for immunoblot analysis of VEEV proteins and immuno-histochemistry of VEEV infected cells. It detected specifically the viral E1 envelope protein of VEEV but did not react with reduced viral glycoprotein preparations suggesting that recognition depends upon conformational epitopes. The recombinant antibody was able to detect multiple VEEV subtypes and displayed only marginal cross-reactivity to other Alphavirus species except for EEEV. In addition, the scFv-Fc fusion described here might be of therapeutic use since it successfully inactivated VEEV in a murine disease model. When the recombinant antibody was administered 6 hours post challenge, 80% to 100% of mice survived lethal VEEV IA/B or IE infection. Forty to sixty percent of mice survived when scFv-Fc ToR67-3B4 was applied 6 hours post challenge with VEEV subtypes II and former IIIA. In combination with E2-neutralising antibodies the NHP antibody isolated here could significantly improve passive protection as well as generic therapy of VEE.

  10. Development of an Indirect Competitive Enzyme-Linked Immunosorbent Assay for Glycocholic Acid Based on Chicken Single-Chain Variable Fragment Antibodies.

    Science.gov (United States)

    Cui, Xiping; Vasylieva, Natalia; Wu, Panpan; Barnych, Bogdan; Yang, Jun; Shen, Ding; He, Qiyi; Gee, Shirley J; Zhao, Suqing; Hammock, Bruce D

    2017-10-17

    Glycocholic acid (GCA) is an important metabolite of bile acids, whose urine levels are expected to be a specific diagnostic biomarker for hepatocellular carcinoma (HCC). A high-throughput immunoassay for determination of GCA would be of significant advantage and useful for primary diagnosis, surveillance, and early detection of HCC. Single-chain variable fragment (scFv) antibodies have several desirable characteristics and are an attractive alternative to traditional antibodies for the immunoassay. Because chicken antibodies possess single heavy and light variable functional domains, they are an ideal framework for simplified generation of recombinant antibodies for GCA detection. However, chicken scFvs have rarely been used to detect GCA. In this study, a scFv library was generated from chickens immunized with a GCA hapten coupled to bovine serum albumin (BSA), and anti-GCA scFvs were isolated by a phage-displayed method. Compared to the homologous coating antigen, use of a heterologous coating antigen resulted in about an 85-fold improvement in sensitivity of the immunoassay. This assay, under optimized conditions, had a linear range of 0.02-0.18 μg/mL, with an IC 50 of 0.06 μg/mL. The assay showed negligible cross-reactivity with various related bile acids, except for taurocholic acid. The detection of GCA from spiked human urine samples ranged from 86.7% to 123.3%. These results, combined with the advantages of scFv antibodies, indicated that a chicken scFv-based enzyme-linked immunosorbent assay is a suitable method for high-throughput screening of GCA in human urine.

  11. Production of a phage-displayed single chain variable fragment ...

    African Journals Online (AJOL)

    Abstract. Purpose: To develop specific single chain variable fragments (scFv) against ... libraries. The binding ability of the selected scFv antibody fragments against the IBDV particles was ..... Hermelink H, Koscielniak E. A human recombinant.

  12. Timothy-specific IgG antibody levels vary with the pollen seasons.

    Science.gov (United States)

    Nordvall, S L; Larsson, P H; Johansson, S G

    1986-11-01

    Serum samples were collected from eight grass pollen hypersensitive children during a 4-year period. The sera were assayed for contents of timothy-specific IgE antibodies by RAST. Timothy-specific IgG and IgA antibodies were quantified by a refined ELISA in which covalent binding of the antigen to the polystyrene solid phase had been performed. IgG antibodies were also assayed by a Sepharose-protein-A technique with radiolabelled timothy allergens as the antigen. It was possible to register clearcut seasonal variations with postseasonally boosted antibody levels not only of timothy-specific IgE but also of IgG antibody. Both IgG1 and IgG4 antibodies specific for timothy showed seasonal variations of a similar degree. It was not possible to register seasonal variations of the same magnitude of timothy-specific IgA antibodies.

  13. Human oxidation-specific antibodies reduce foam cell formation and atherosclerosis progression

    DEFF Research Database (Denmark)

    Tsimikas, Sotirios; Miyanohara, Atsushi; Hartvigsen, Karsten

    2011-01-01

    We sought to assess the in vivo importance of scavenger receptor (SR)-mediated uptake of oxidized low-density lipoprotein (OxLDL) in atherogenesis and to test the efficacy of human antibody IK17-Fab or IK17 single-chain Fv fragment (IK17-scFv), which lacks immunologic properties of intact antibod...... antibodies other than the ability to inhibit uptake of OxLDL by macrophages, to inhibit atherosclerosis....

  14. An integrated top-down and bottom-up proteomic approach to characterize the antigen-binding fragment of antibodies.

    Science.gov (United States)

    Dekker, Lennard; Wu, Si; Vanduijn, Martijn; Tolić, Nikolai; Stingl, Christoph; Zhao, Rui; Luider, Theo; Paša-Tolić, Ljiljana

    2014-05-01

    We have previously shown that different individuals exposed to the same antigen produce antibodies with identical mutations in their complementarity determining regions (CDR), suggesting that CDR tryptic peptides can serve as biomarkers for disease diagnosis and prognosis. Complete Fabs derived from disease specific antibodies have even higher potential; they could potentially be used for disease treatment and are required to identify the antigens toward which the antibodies are directed. However, complete Fab sequence characterization via LC-MS analysis of tryptic peptides (i.e. bottom-up) has proven to be impractical for mixtures of antibodies. To tackle this challenge, we have developed an integrated bottom-up and top-down MS approach, employing 2D chromatography coupled with Fourier transform mass spectrometry (FTMS), and applied this approach for full characterization of the variable parts of two pharmaceutical monoclonal antibodies with sensitivity comparable to the bottom-up standard. These efforts represent an essential step toward the identification of disease specific antibodies in patient samples with potentially significant clinical impact. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Rat Monoclonal Antibodies Specific for LST1 Proteins

    OpenAIRE

    Schiller, Christian; Nitschké, Maximilian J. E.; Seidl, Alexander; Kremmer, Elisabeth; Weiss, Elisabeth H.

    2009-01-01

    The LST1 gene is located in the human MHC class III region and encodes transmembrane and soluble isoforms that have been suggested to play a role in the regulation of the immune response and are associated with inflammatory diseases such as rheumatoid arthritis. Here we describe the generation and characterization of the first monoclonal antibodies against LST1. Two hybridoma lines secreting monoclonal antibodies designated 7E2 and 8D12 were established. The 7E2 antibody detects recombinant a...

  16. Characterization of Endotrypanum Parasites Using Specific Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Ramos Franco Antonia Maria

    1997-01-01

    Full Text Available A large number of Endotrypanum stocks (representing an heterogeneous population of strains have been screened against a panel of monoclonal antibodies (MAbs derived for selected species of Endotrypanum or Leishmania, to see whether this approach could be used to group/differentiate further among these parasites. Using different immunological assay systems, MAbs considered specific for the genus Endotrypanum (E-24, CXXX-3G5-F12 or strain M6159 of E. schaudinni (E-2, CXIV-3C7-F5 reacted variably according to the test used but in the ELISA or immunofluorescence assay both reacted with all the strains tested. Analyses using these MAbs showed antigenic diversity occurring among the Endotrypanum strains, but no qualitative or quantitative reactivity pattern could be consistently related to parasite origin (i.e., host species involved or geographic area of isolation. Western blot analyses of the parasites showed that these MAbs recognized multiple components. Differences existed either in the epitope density or molecular forms associated with the antigenic determinants and therefore allowed the assignment of the strains to specific antigenic groups. Using immunofluorescence or ELISA assay, clone E-24 produced reaction with L. equatorensis (which is a parasite of sloth and rodent, but not with other trypanosomatids examined. Interestingly, the latter parasite and the Endotrypanum strains cross-reacted with a number of MAbs that were produced against members of the L. major-L. tropica complex

  17. Generation of high-affinity, internalizing anti-FGFR2 single-chain variable antibody fragment fused with Fc for targeting gastrointestinal cancers.

    Science.gov (United States)

    Borek, Aleksandra; Sokolowska-Wedzina, Aleksandra; Chodaczek, Grzegorz; Otlewski, Jacek

    2018-01-01

    Fibroblast growth factor receptors (FGFRs) are promising targets for antibody-based cancer therapies, as their substantial overexpression has been found in various tumor cells. Aberrant activation of FGF receptor 2 (FGFR2) signaling through overexpression of FGFR2 and/or its ligands, mutations, or receptor amplification has been reported in multiple cancer types, including gastric, colorectal, endometrial, ovarian, breast and lung cancer. In this paper, we describe application of the phage display technology to produce a panel of high affinity single chain variable antibody fragments (scFvs) against the extracellular ligand-binding domain of FGFR2 (ECD_FGFR2). The binders were selected from the human single chain variable fragment scFv phage display libraries Tomlinson I + J and showed high specificity and binding affinity towards human FGFR2 with nanomolar KD values. To improve the affinity of the best binder selected, scFvF7, we reformatted it to a bivalent diabody format, or fused it with the Fc region (scFvF7-Fc). The scFvF7-Fc antibody construct presented the highest affinity for FGFR2, with a KD of 0.76 nM, and was selectively internalized into cancer cells overexpressing FGFR2, Snu-16 and NCI-H716. Finally, we prepared a conjugate of scFvF7-Fc with the cytotoxic drug monomethyl-auristatin E (MMAE) and evaluated its cytotoxicity. The conjugate delivered MMAE selectively to FGFR2-positive tumor cells. These results indicate that scFvF7-Fc-vcMMAE is a highly potent molecule for the treatment of cancers with FGFR2 overexpression.

  18. Radioiodinated iodobenzoyl conjugates of a monoclonal antibody Fab fragment. In vivo comparisons with chloramine-T-labeled Fab

    International Nuclear Information System (INIS)

    Wilbur, D.S.; Hadley, S.W.; Grant, L.M.; Hylarides, M.D.

    1991-01-01

    A comparative investigation of the biodistributions of radioiodinated p- and m-iodobenzoyl conjugates of a monoclonal antibody Fab fragment, NR-LU-10 Fab, and the same antibody Fab fragment radioiodinated by the chloramine-T (ChT) method has been carried out in mice. Coinjected, dual-isotope studies in athymic mice with tumor xenografts have demonstrated that there are only minor differences in the in vivo distributions of the iodobenzoyl-labeled Fabs, except in the excretory organs, kidneys, and intestines, where major differences were observed. Similarly, coinjection of either the p-iodobenzoyl or m-iodobenzoyl conjugate of NR-LU-10 Fab with the Fab radioiodinated with ChT/radioiodide into BALB/c mice provided additional data that indicated that the two iodobenzoyl conjugates distributed similar in a number of selected tissues. The tissue-distribution differences of the regioisomeric iodobenzoyl conjugates in relation to the ChT-radioiodinated Fab were large for the stomach and neck, consistent with previous studies. The most notable difference between the two iodobenzoyl conjugates was the kidney activity, where the m-iodobenzoyl conjugate was similar to the directly labeled Fab, but the p-iodobenzoyl-conjugated Fab was higher by nearly a factor of 2

  19. Potent neutralization of VEGF biological activities with a fully human antibody Fab fragment directed against VEGF receptor 2

    International Nuclear Information System (INIS)

    Miao, H.-Q.; Hu, Kun; Jimenez, Xenia; Navarro, Elizabeth; Zhang, Haifan; Lu Dan; Ludwig, Dale L.; Balderes, Paul; Zhu Zhenping

    2006-01-01

    Compelling evidence suggest that vascular endothelial growth factor (VEGF) and its receptors, especially receptor 2 (VEGFR2, or kinase insert domain-containing receptor, KDR), play a critical role in angiogenesis under both physiological and pathological conditions, including cancer and angiogenic retinopathies such as age-related macular degeneration (AMD). To this end, inhibition of angiogenesis with antagonists to either VEGF or KDR has yielded significant therapeutic efficacy both in preclinical studies in animal models and in clinical trials in patients with cancer and AMD. We previously reported the identification of a high affinity, fully human anti-KDR antibody fragment, 1121B Fab, through a highly stringent affinity maturation process with a Fab originally isolated from a naive human antibody phage display library. In this study, we demonstrate that 1121B Fab is able to strongly block KDR/VEGF interaction, resulting in potent inhibition of an array of biological activities of VEGF, including activation of the receptor and its signaling pathway, intracellular calcium mobilization, and migration and proliferation of endothelial cells. Taken together, our data lend strong support to the further development of 1121B Fab fragment as an anti-angiogenesis agent in both cancer and angiogenic retinopathies

  20. Potential for novel MUC1 glycopeptide-specific antibody in passive cancer immunotherapy

    DEFF Research Database (Denmark)

    Madsen, Caroline B; Wandall, Hans H; Pedersen, Anders Elm

    2013-01-01

    MUC1 is an important target for antibodies in passive cancer immunotherapy. Antibodies against mucin glycans or mucin peptide backbone alone may give rise to cross reactivity with normal tissues. Therefore, attempts to identify antibodies against cancer-specific MUC1 glycopeptide epitopes havebeen...

  1. Selection of monoclonal anti-CEA antibody fragments for tumor detection by immunoscintigraphy

    International Nuclear Information System (INIS)

    Mach, J.P.; Buchegger, F.

    1986-01-01

    It is described how individual MAb directed against carcinoembryotic antigen (CEA) is selected which does not crossreact with granulocytes and gives the best tumor localization in the model of nude mice grafted with human colon carcinoma. Using this model, the superiority of F(ab')/sub 2/ and particularly Fab fragments from high affinity MAb for the localization of relatively small tumor nodules is demonstrated. These MAb fragments are also successfully used in an ongoing clinical trial for the detection of primary and metastatic colorectal carcinomas

  2. Structure and pathogenicity of antibodies specific for citrullinated collagen type II in experimental arthritis

    DEFF Research Database (Denmark)

    Uysal, Hüseyin; Bockermann, Robert; Nandakumar, Kutty S

    2009-01-01

    Antibodies to citrulline-modified proteins have a high diagnostic value in rheumatoid arthritis (RA). However, their biological role in disease development is still unclear. To obtain insight into this question, a panel of mouse monoclonal antibodies was generated against a major triple helical...... is indeed citrullinated in vivo. The structure determination of a Fab fragment of one of these antibodies in complex with a citrullinated peptide showed a surprising beta-turn conformation of the peptide and provided information on citrulline recognition. Based on these findings, we propose...

  3. Mouse-specific antibody responses to a monoclonal antibody during repeated immunoscintigraphy investigations: Comparison of antibody titres and imaging studies in a rat model

    International Nuclear Information System (INIS)

    Pimm, M.V.; Gribben, S.J.; Markham, A.J.; Perkins, A.C.

    1990-01-01

    As a model for human mouse-specific antibody responses in patients undergoing immunoscintigraphy, we have investigated in rats the production of mouse-specific antibodies (MA) to the mouse monoclonal antibody 791T/36. At intervals of between 5 and 16 weeks the rats were given repeated cycles of intravenous (IV) injections of antibody with or without a simultaneous intradermal (ID) injection. The IV dose was 60 μg/kg, a dose similar to that used in many clinical immunoscintigraphy studies. The ID injection was 2 μg, which mimicks the skin test dose often given in clinical imaging protocols. The study was carried out with both 131 I-labelled antibody and with antibody labelled with 111 In by DTPA chelation. MA was measured with a passive haemagglutination assay using sheep red blood cells coated with the monoclonal antibody. Of rats given ID injections of unlabelled antibody at the same time as the IV imaging doses, 9/20 produced MA during 4 cycles of injections. In contrast, only 2/16 rats given only the IV dose produced MA. Both 131 I- and 111 In-labelled antibody appeared equally immunogenic with 5/18 and 6/18 overall responders, respectively. The production of MA was associated with a significant perturbation in the biodistribution of the IV dose of labelled antibody as seen by gamma-camera imaging of the rats given 111 In-labelled antibody. There was clearance of immune complexes to the liver, this organ accumulating up to 90% of the whole body count rate of radiolabel. MA titres of between 1/100 and 1/78000 caused equal perturbation of biodistribution, although below 1/100 the effect was more variable. (orig.)

  4. Specificity and polyreactivity of the antibody response during natural HIV-1 infection

    OpenAIRE

    Wang, Xin

    2006-01-01

    The specificity and polyreactivity of the antibody response in natural HIV-1 infection were studied. First, to investigate the overall antibody response, overlapping linear peptides were used to screen sera taken from HIV-1-infected individuals. The polyclonal antibody response was relatively stable during long-term infection, compared with acute infection, and mostly directed against immunodominant regions. Low level, transient antibody responses were detected against membrane proximal exter...

  5. Restriction fragment polymorphism (RFLP) of a "new" HLA-DP specificity, CDP-HEI

    DEFF Research Database (Denmark)

    Hyldig-Nielsen, J J; Ødum, Niels; Morling, Niels

    1988-01-01

    Southern blotting with a DP beta cDNA probe of MspI digested DNA from 83 healthy unrelated individuals revealed a 1.8 kb fragment present in all four individuals (and no others) possessing the newly determined DP specificity, CDP-HEI.......Southern blotting with a DP beta cDNA probe of MspI digested DNA from 83 healthy unrelated individuals revealed a 1.8 kb fragment present in all four individuals (and no others) possessing the newly determined DP specificity, CDP-HEI....

  6. Site-specific fragmentation of polystyrene molecule using size-selected Ar gas cluster ion beam

    International Nuclear Information System (INIS)

    Moritani, Kousuke; Mukai, Gen; Hashinokuchi, Michihiro; Mochiji, Kozo

    2009-01-01

    The secondary ion mass spectrum (SIMS) of a polystyrene thin film was investigated using a size-selected Ar gas cluster ion beam (GCIB). The fragmentation in the SIM spectrum varied by kinetic energy per atom (E atom ); the E atom dependence of the secondary ion intensity of the fragment species of polystyrene can be essentially classified into three types based on the relationship between E atom and the dissociation energy of a specific bonding site in the molecule. These results indicate that adjusting E atom of size-selected GCIB may realize site-specific bond breaking within a molecule. (author)

  7. Localization by whole-body autoradiography of intact and fragmented radiolabeled antibodies in a metastatic human colonic cancer model

    International Nuclear Information System (INIS)

    Fand, Irwin; Sharkey, R.M.; Grundy, J.P.; Goldenberg, D.M.

    1992-01-01

    In this report, we have employed macroautoradiography to compare the tumor targeting of 125 I-labeled anti-carcinoembryonic antigen (CEA) MAb (NP-4) to 125 I-labeled anti-colon-specific antigen-p (CSAp) MAb (Mu-9) and their labeled F(ab') 2 and Fab' fragments, in nude mice each bearing large dorsal human colonic tumor xenografts, and small nodular tumors in the liver and lungs. Using intact MAbs (NP-4 and Mu-9), clearance of background radioactivity was delayed to 3-7 days post-treatment. Treatment with F(ab') 2 and Fab' fragments of both NP-4 and Mu-9 MAbs, however, promoted clearance of background 125 I-radioactivity which was well advanced by 6-24 h and complete by 24-48 h after injection. Localization of 125 I-radioactivity in large and micrometastatic tumor perimeters was the most characteristic uptake pattern observed for both intact and fragmented MAbs. Qualitative analysis of macroautoradiographic images and quantitative densitometry indicated that the higher tumor-to-blood ratios achieved with labeled F(ab') 2 and Fab' fragments at early time points, compared to labeled whole immunoglobulin, appeared to be more a function of rapid plasma clearance, tumor mass, location of xenografts and specific tumor growth patterns than increased tumor penetrance by lower molecular weight univalent and bivalent immune fragments. (Author)

  8. Use of epitope libraries to identify exon-specific monoclonal antibodies for characterization of altered dystrophins in muscular dystrophy

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen thi Man; Morris, G.E. (North East Wales Inst., Clwyd (United Kingdom))

    1993-06-01

    The majority of mutations in Xp21-linked muscular dystrophy (MD) can be identified by PCR or Southern blotting, as deletions or duplications of groups of exons in the dystrophin gene, but it is not always possible to predict how much altered dystrophin, if any, will be produced. Use of exon-specific monoclonal antibodies (mAbs) on muscle biopsies from MD patients can, in principle, provide information on both the amount of altered dystrophin produced and, when dystrophin is present, the nature of the genetic deletion or point mutation. For this purpose, mAbs which recognize regions of dystrophin encoded by known exons and whose binding is unaffected by the absence of adjacent exons are required. To map mAbs to specific exons, random [open quotes]libraries[close quotes] of expressed dystrophin fragments were created by cloning DNAseI digestion fragments of a 4.3-kb dystrophin cDNA into a pTEX expression vector. The libraries were then used to locate the epitopes recognized by 48 mAbs to fragments of 25--60 amino acids within the 1,434-amino-acid dystrophin fragment used to produce the antibodies. This is sufficiently detailed to allow further refinement by using synthetic peptides and, in many cases, to identify the exon in the DMD (Duchenne MD) gene which encodes the epitope. To illustrate their use in dystrophin analysis, a Duchenne patient with a frameshift deletion of exons 42 and 43 makes a truncated dystrophin encoded by exons 1--41, and the authors now show that this can be detected in the sarcolemma by mAbs up to and including those specific for exon 41 epitopes but not by mAbs specific for exon 43 or later epitopes. 38 refs., 2 figs., 4 tabs.

  9. Feasibility study of the Fab fragment of a monoclonal antibody against tissue factor as a diagnostic tool.

    Science.gov (United States)

    Tsumura, Ryo; Sato, Ryuta; Furuya, Fumiaki; Koga, Yoshikatsu; Yamamoto, Yoshiyuki; Fujiwara, Yuki; Yasunaga, Masahiro; Matsumura, Yasuhiro

    2015-12-01

    Tissue factor (TF) is expressed strongly in various types of cancer, especially cancers that are often refractory to treatment, such as pancreatic cancer. In this study, we compared the differences in the biophysical and pharmacological properties of whole IgG and the Fab fragment of anti-human TF monoclonal antibody (1849 antibodies), in order to determine their suitability for application in the diagnosis and treatment of cancers. In the biophysical examination, we investigated the characteristics of 1849-whole IgG and 1849-Fab by SPR sensing and confocal fluorescence microscopy analysis using recombinant human TF antigen and TF-overexpressing human pancreatic cancer cell line, BxPC3, respectively. After conjugation with Alexa-Flour-647, in vivo imaging was conducted in mice bearing BxPC3 xenograft tumors. Furthermore, the distribution of the conjugates in tumors and major organs was evaluated by ex vivo study. The in vitro experiments showed that 1849 antibodies had high affinity against TF antigen. In addition, 1849-Fab showed a faster dissociation rate from the antigen than 1849-whole IgG. In mice, 1849-Fab-Alexa-Flour-647 showed rapid renal clearance and faster tumor accumulation, achieving a high contrast signal over nearby normal tissues in the early phase and enhanced tumor penetration after administration. On the other hand, 1849-whole IgG-Alexa-Flour-647 showed slow clearance from the blood and sustained high tumor accumulation. These results suggest that 1849-Fab may be a useful tool for pancreatic cancer diagnosis.

  10. Single chain variable fragment antibodies block aggregation and toxicity induced by familial ALS-linked mutant forms of SOD1.

    Science.gov (United States)

    Ghadge, Ghanashyam D; Pavlovic, John D; Koduvayur, Sujatha P; Kay, Brian K; Roos, Raymond P

    2013-08-01

    Approximately 10% of amyotrophic lateral sclerosis (ALS) cases are familial (known as FALS) with an autosomal dominant inheritance pattern, and ~25% of FALS cases are caused by mutations in Cu/Zn superoxide dismutase (SOD1). There is convincing evidence that mutant SOD1 (mtSOD1) kills motor neurons (MNs) because of a gain-of-function toxicity, most likely related to aggregation of mtSOD1. A number of recent reports have suggested that antibodies can be used to treat mtSOD1-induced FALS. To follow up on the use of antibodies as potential therapeutics, we generated single chain fragments of variable region antibodies (scFvs) against SOD1, and then expressed them as 'intrabodies' within a motor neuron cell line. In the present study, we describe isolation of human scFvs that interfere with mtSOD1 in vitro aggregation and toxicity. These scFvs may have therapeutic potential in sporadic ALS, as well as FALS, given that sporadic ALS may also involve abnormalities in the SOD1 protein or activity. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. Species- and sex-specific connectivity effects of habitat fragmentation in a suite of woodland birds.

    Science.gov (United States)

    Amos, Nevil; Harrisson, Katherine A; Radford, James Q; White, Matt; Newell, Graeme; Mac Nally, Ralph; Sunnucks, Paul; Pavlova, Alexandra

    2014-06-01

    Loss of functional connectivity following habitat loss and fragmentation could drive species declines. A comprehensive understanding of fragmentation effects on functional connectivity of an ecological assemblage requires investigation of multiple species with different mobilities, at different spatial scales, for each sex, and in different landscapes. Based on published data on mobility and ecological responses to fragmentation of 10 woodland-dependent birds, and using simulation studies, we predicted that (1) fragmentation would impede dispersal and gene flow of eight "decliners" (species that disappear from suitable patches when landscape-level tree cover falls below species-specific thresholds), but not of two "tolerant" species (whose occurrence in suitable habitat patches is independent of landscape tree cover); and that fragmentation effects would be stronger (2) in the least mobile species, (3) in the more philopatric sex, and (4) in the more fragmented region. We tested these predictions by evaluating spatially explicit isolation-by-landscape-resistance models of gene flow in fragmented landscapes across a 50 x 170 km study area in central Victoria, Australia, using individual and population genetic distances. To account for sex-biased dispersal and potential scale- and configuration-specific effects, we fitted models specific to sex and geographic zones. As predicted, four of the least mobile decliners showed evidence of reduced genetic connectivity. The responses were strongly sex specific, but in opposite directions in the two most sedentary species. Both tolerant species and (unexpectedly) four of the more mobile decliners showed no reduction in gene flow. This is unlikely to be due to time lags because more mobile species develop genetic signatures of fragmentation faster than do less mobile ones. Weaker genetic effects were observed in the geographic zone with more aggregated vegetation, consistent with gene flow being unimpeded by landscape

  12. Development of single chain variable fragment (scFv) antibodies against Xylella fastidiosa subsp. pauca by phage display.

    Science.gov (United States)

    Yuan, Qing; Jordan, Ramon; Brlansky, Ronald H; Istomina, Olga; Hartung, John

    2015-10-01

    Xylella fastidiosa is a member of the gamma proteobacteria. It is fastidious, insect-vectored and xylem-limited and causes a variety of diseases, some severe, on a wide range of economically important perennial crops, including grape and citrus. Antibody based detection assays are commercially available for X. fastidiosa, and are effective at the species, but not at the subspecies level. We have made a library of scFv antibody fragments directed against X. fastidiosa subsp. pauca strain 9a5c (citrus) by using phage display technology. Antibody gene repertoires were PCR-amplified using 23 primers for the heavy chain variable region (V(H)) and 21 primers for the light chain variable region (V(L)). The V(H) and V(L) were joined by overlap extension PCR, and then the genes of the scFv library were ligated into the phage vector pKM19. The library contained 1.2×10(7) independent clones with full-length scFv inserts. In each of 3cycles of affinity-selection with 9a5c, about 1.0×10(12) phage were used for panning with 4.1×10(6), 7.1×10(6), 2.1×10(7) phage recovered after the first, second and third cycles, respectively. Sixty-six percent of clones from the final library bound X. fastidiosa 9a5c in an ELISA. Some of these scFv antibodies recognized strain 9a5c and did not recognize X. fastidiosa strains that cause Pierce's disease of grapevine. Published by Elsevier B.V.

  13. Restriction fragment polymorphism (RFLP) of a "new" HLA-DP specificity, CDP-HEI

    DEFF Research Database (Denmark)

    Hyldig-Nielsen, J J; Ødum, Niels; Morling, Niels

    1988-01-01

    Southern blotting with a DP beta cDNA probe of MspI digested DNA from 83 healthy unrelated individuals revealed a 1.8 kb fragment present in all four individuals (and no others) possessing the newly determined DP specificity, CDP-HEI....

  14. A Recombinant Antibody with the Antigen-Specific, Major Histocompatibility Complex-Restricted Specificity of T Cells

    Science.gov (United States)

    Andersen, Peter S.; Stryhn, Anette; Hansen, Bjarke E.; Fugger, Lars; Engberg, Jan; Buus, Soren

    1996-03-01

    Specific recognition of peptide/major histocompatibility complex (MHC) molecule complexes by the T-cell receptor is a key reaction in the specific immune response. Antibodies against peptide/MHC complexes would therefore be valuable tools in studying MHC function and T-cell recognition and might lead to novel approaches in immunotherapy. However, it has proven difficult to generate antibodies with the specificity of T cells by conventional hybridoma techniques. Here we report that the phage display technology is a feasible alternative to generate antibodies recognizing specific, predetermined peptide/MHC complexes.

  15. Monoclonal antibodies against pregnancy-specific β1-glycoprotein (SP1) in immunohistochemistry and radioimmunoassay

    International Nuclear Information System (INIS)

    Wahlstroem, T.; Heikinheimo, M.

    1983-01-01

    Monoclonal mouse antibodies against pregnancy-specific beta-1-glycoprotein (SP 1 ) have been studied for their suitability in immunoperoxidase staining and radioimmunoassay methodologies. These antibodies were useful in staining normal placentas, hydatidiform moles, invasive moles and choriocarcinomas. They showed good specificity, with minimal background staining, and will thus be superior to conventional polyclonal antisera in immunohistochemistry. However, the presently tested monoclonal anti-SP 1 antibodies were found not to be suitable for radioimmunoassay. (Auth.)

  16. Radioimmunoassay for detection of VP1 specific neutralizing antibodies of foot and mouse disease virus

    International Nuclear Information System (INIS)

    Patzer, E.J.; Jackson, M.L.; Moore, D.M.

    1985-01-01

    A solid-phase radioimmunoassay was developed for the detection of antibodies against a specific region of the VP1 protein of the A24 and O1 serotypes of foot and mouth disease virus. The antibody titers from the radioimmunoassay showed a positive correlation with neutralizing antibody titers determined by a mouse protection assay. The specificity of the assay resides in the peptide used as antigen. The assay is rapid, reproducible and does not require the use of whole virions. (orig.)

  17. Synthetic oligonucleotide antigens modified with locked nucleic acids detect disease specific antibodies

    DEFF Research Database (Denmark)

    Samuelsen, Simone V; Solov'yov, Ilia A.; Balboni, Imelda M.

    2016-01-01

    New techniques to detect and quantify antibodies to nucleic acids would provide a significant advance over current methods, which often lack specificity. We investigate the potential of novel antigens containing locked nucleic acids (LNAs) as targets for antibodies. Particularly, employing...... molecular dynamics we predict optimal nucleotide composition for targeting DNA-binding antibodies. As a proof of concept, we address a problem of detecting anti-DNA antibodies that are characteristic of systemic lupus erythematosus, a chronic autoimmune disease with multiple manifestations. We test the best...... that the novel method is a promising tool to create antigens for research and point-of-care monitoring of anti-DNA antibodies....

  18. Targeting lysine specific demethylase 4A (KDM4A) tandem TUDOR domain - A fragment based approach.

    Science.gov (United States)

    Upadhyay, Anup K; Judge, Russell A; Li, Leiming; Pithawalla, Ron; Simanis, Justin; Bodelle, Pierre M; Marin, Violeta L; Henry, Rodger F; Petros, Andrew M; Sun, Chaohong

    2018-06-01

    The tandem TUDOR domains present in the non-catalytic C-terminal half of the KDM4A, 4B and 4C enzymes play important roles in regulating their chromatin localizations and substrate specificities. They achieve this regulatory role by binding to different tri-methylated lysine residues on histone H3 (H3-K4me3, H3-K23me3) and histone H4 (H4-K20me3) depending upon the specific chromatin environment. In this work, we have used a 2D-NMR based fragment screening approach to identify a novel fragment (1a), which binds to the KDM4A-TUDOR domain and shows modest competition with H3-K4me3 binding in biochemical as well as in vitro cell based assays. A co-crystal structure of KDM4A TUDOR domain in complex with 1a shows that the fragment binds stereo-specifically to the methyl lysine binding pocket forming a network of strong hydrogen bonds and hydrophobic interactions. We anticipate that the fragment 1a can be further developed into a novel allosteric inhibitor of the KDM4 family of enzymes through targeting their C-terminal tandem TUDOR domain. Copyright © 2018 Elsevier Ltd. All rights reserved.

  19. High specificity but low sensitivity of mutation-specific antibodies against EGFR mutations in non-small-cell lung cancer

    DEFF Research Database (Denmark)

    Bondgaard, Anna-Louise; Høgdall, Estrid; Mellemgaard, Anders

    2014-01-01

    of more sensitive methods including real-time PCR (RT-PCR). Immunohistochemistry with mutation-specific antibodies might be a promising detection method. We evaluated 210 samples with NSCLC from an unselected Caucasian population. Extracted DNA was analyzed for EGFR mutations by RT-PCR (Therascreen EGFR......, and staining score (multipum of intensity (graded 0-3) and percentages (0-100%) of stained tumor cells) was calculated. Positivity was defined as staining score >0. Specificity of exon19 antibody was 98.8% (95% confidence interval=95.9-99.9%) and of exon21 antibody 97.8% (95% confidence interval=94...... was demonstrated. However, sensitivity was low, especially for exon19 deletions, and thus these antibodies cannot yet be used as screening method for EGFR mutations in NSCLC. Refinement of sensitivity for the mutation-specific antibodies is warranted to improve molecular diagnosis using EGFR immunohistochemistry....

  20. Secretion of an immunoreactive single-chain variable fragment antibody against mouse interleukin 6 by Lactococcus lactis.

    Science.gov (United States)

    Shigemori, Suguru; Ihara, Masaki; Sato, Takashi; Yamamoto, Yoshinari; Nigar, Shireen; Ogita, Tasuku; Shimosato, Takeshi

    2017-01-01

    Interleukin 6 (IL-6) is an important pathogenic factor in development of various inflammatory and autoimmune diseases and cancer. Blocking antibodies against molecules associated with IL-6/IL-6 receptor signaling are an attractive candidate for the prevention or therapy of these diseases. In this study, we developed a genetically modified strain of Lactococcus lactis secreting a single-chain variable fragment antibody against mouse IL-6 (IL6scFv). An IL6scFv-secretion vector was constructed by cloning an IL6scFv gene fragment into a lactococcal secretion plasmid and was electroporated into L. lactis NZ9000 (NZ-IL6scFv). Secretion of recombinant IL6scFv (rIL6scFv) by nisin-induced NZ-IL6scFv was confirmed by western blotting and was optimized by tuning culture conditions. We found that rIL6scFv could bind to commercial recombinant mouse IL-6. This result clearly demonstrated the immunoreactivity of rIL6scFv. This is the first study to engineer a genetically modified strain of lactic acid bacteria (gmLAB) that produces a functional anti-cytokine scFv. Numerous previous studies suggested that mucosal delivery of biomedical proteins using gmLAB is an effective and low-cost way to treat various disorders. Therefore, NZ-IL6scFv may be an attractive tool for the research and development of new IL-6 targeting agents for various inflammatory and autoimmune diseases as well as for cancer.

  1. Therapeutic Activity of an Engineered Synthetic Killer Antiidiotypic Antibody Fragment against Experimental Mucosal and Systemic Candidiasis

    OpenAIRE

    Polonelli, Luciano; Magliani, Walter; Conti, Stefania; Bracci, Luisa; Lozzi, Luisa; Neri, Paolo; Adriani, Daniela; De Bernardis, Flavia; Cassone, Antonio

    2003-01-01

    Peptides derived from the sequence of a single-chain, recombinant, antiidiotypic antibody (IdAb; KT-scFv) acting as a functional internal image of a microbicidal, wide-spectrum yeast killer toxin (KT) were synthesized and studied for their antimicrobial activity by using the KT-susceptible Candida albicans as model organism. A decapeptide containing the first three amino acids (SAS) of the light chain CDR1 was selected and optimized by alanine replacement of a single residue. This peptide exe...

  2. 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.

  3. 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.

  4. Suppression of Aggrus/podoplanin-induced platelet aggregation and pulmonary metastasis by a single-chain antibody variable region fragment

    International Nuclear Information System (INIS)

    Miyata, Kenichi; Takagi, Satoshi; Sato, Shigeo; Morioka, Hiroshi; Shiba, Kiyotaka; Minamisawa, Tamiko; Takami, Miho; Fujita, Naoya

    2014-01-01

    Almost all highly metastatic tumor cells possess high platelet aggregating abilities, thereby form large tumor cell-platelet aggregates in the microvasculature. Embolization of tumor cells in the microvasculature is considered to be the first step in metastasis to distant organs. We previously identified the platelet aggregation-inducing factor expressed on the surfaces of highly metastatic tumor cells and named as Aggrus. Aggrus was observed to be identical to the marker protein podoplanin (alternative names, T1α, OTS-8, and others). Aggrus is frequently overexpressed in several types of tumors and enhances platelet aggregation by interacting with the platelet receptor C-type lectin-like receptor 2 (CLEC-2). Here, we generated a novel single-chain antibody variable region fragment (scFv) by linking the variable regions of heavy and light chains of the neutralizing anti-human Aggrus monoclonal antibody MS-1 with a flexible peptide linker. Unfortunately, the generated KM10 scFv failed to suppress Aggrus-induced platelet aggregation in vitro. Therefore, we performed phage display screening and finally obtained a high-affinity scFv, K-11. K-11 scFv was able to suppress Aggrus-induced platelet aggregation in vitro. Moreover, K-11 scFv prevented the formation of pulmonary metastasis in vivo. These results suggest that K-11 scFv may be useful as metastasis inhibitory scFv and is expected to aid in the development of preclinical and clinical examinations of Aggrus-targeted cancer therapies

  5. A Recombinant Secondary Antibody Mimic as a Target-specific Signal Amplifier and an Antibody Immobilizer in Immunoassays.

    Science.gov (United States)

    Min, Junseon; Song, Eun Kyung; Kim, Hansol; Kim, Kyoung Taek; Park, Tae Joo; Kang, Sebyung

    2016-04-11

    We construct a novel recombinant secondary antibody mimic, GST-ABD, which can bind to the Fc regions of target-bound primary antibodies and acquire multiple HRPs simultaneously. We produce it in tenth of mg quantities with a bacterial overexpression system and simple purification procedures, significantly reducing the manufacturing cost and time without the use of animals. GST-ABD is effectively conjugated with 3 HRPs per molecule on an average and selectively bind to the Fc region of primary antibodies derived from three different species (mouse, rabbit, and rat). HRP-conjugated GST-ABD (HRP-GST-ABD) is successfully used as an alternative to secondary antibodies to amplify target-specific signals in both ELISA and immunohistochemistry regardless of the target molecules and origin of primary antibodies used. GST-ABD also successfully serves as an anchoring adaptor on the surface of GSH-coated plates for immobilizing antigen-capturing antibodies in an orientation-controlled manner for sandwich-type indirect ELISA through simple molecular recognition without any complicated chemical modification.

  6. The use of a cocktail of single chain Fv antibody fragments to improve the in vitro and in vivo targeting of melanoma

    International Nuclear Information System (INIS)

    Pacifico, M.D.; Pearl, R.A.; Kupsch, J.M.

    2006-01-01

    Radio scintigraphy using single chain antibody fragments (scFvs) offers a potenti al means of early detection of melanoma metastases. However, previous studies have shown suboptimal levels of tumour localization and nonspecific background accumulation which may be due to antigen heterogeneity. We aimed to improve tumour localization by using a cocktail of different scFvs targeting different epitopes on melanoma cells. We have previously developed three scFvs against distinct and highly tumour-specific melanoma cell-surface antigens by chain shuffling and antibody phage selection on melanoma cells. Three scFvs, RAFT3, B3 and B4 were labeled with 1 25I odine and tested both individually and as a cocktail in a nude mouse xenograft model far human melanoma. Results demonstrated improved tumour localization in vivo when compared to the individual scFvs. Tumour uptake of the cocktail at l hour was 24.220% ID/g (injected dose/gram) compared with 2.854%, 2.263% and 1.355% far B4, RAFT3 and B3, respectively, when injected individually. In addition, the cocktail exhibited significantly superior tumour to normal tissue ratios far muscle and spleen (p<0.05). A combination or cocktail of scFv clones may have an advantage aver individual scFvs far melanoma targeting in patients because of heterogeneity in the expression of different epitopes of antigens on melanoma cells

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

    NARCIS (Netherlands)

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

    1995-01-01

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

  8. Characterization of antibodies specific for UV-damaged DNA by ELISA

    Energy Technology Data Exchange (ETDEWEB)

    Eggset, G; Volden, G; Krokan, H

    1987-04-01

    The specificity of affinity purified antibodies raised against UV-irradiated DNA was examined using an enzyme-linked immunosorbent assay. DNA irradiated with UV doses higher than needed for saturation with pyrimidine dimers bound increasing amounts of antibody. Photosensitized DNA, containing high amounts of pyrimidine dimers, showed very poor binding of antibody. When UV-irradiated DNA was given a second dose of 340-nm UV light, the binding of antibodies was inhibited. Taken together, this indicates a major specificity for (6-4)-photoproducts, which are photochemically reversed by UV light in the 340-nm region. The antibodies also showed little but detectable binding to pyrimidine glycols produced in DNA by oxidation with OsO/sub 4/. Previously, we have used these antibodies for the detection of UV-induced DNA damage and its repair in human skin in vivo. These findings indicate that (6-4)-photoproducts, considered highly mutagenic, are repaired in human skin.

  9. Characterization of antibodies specific for UV-damaged DNA by ELISA

    International Nuclear Information System (INIS)

    Eggset, G.; Volden, G.; Krokan, H.; Norsk Hydro Research Centre, Porsgrunn

    1987-01-01

    The specificity of affinity purified antibodies raised against UV-irradiated DNA was examined using an enzyme-linked immunosorbent assay. DNA irradiated with UV doses higher than needed for saturation with pyrimidine dimers bound increasing amounts of antibody. Photosensitized DNA, containing high amounts of pyrimidine dimers, showed very poor binding of antibody. When UV-irradiated DNA was given a second dose of 340-nm UV light, the binding of antibodies was inhibited. Taken together, this indicates a major specificity for (6-4)-photoproducts, which are photochemically reversed by UV light in the 340-nm region. The antibodies also showed little but detectable binding to pyrimidine glycols produced in DNA by oxidation with OsO 4 . Previously, we have used these antibodies for the detection of UV-induced DNA damage and its repair in human skin in vivo. These findings indicate that (6-4)-photoproducts, considered highly mutagenic, are repaired in human skin. (author)

  10. NMR Detection of Semi-Specific Antibody Interactions in Serum Environments

    Directory of Open Access Journals (Sweden)

    Saeko Yanaka

    2017-09-01

    Full Text Available Although antibody functions are executed in heterogeneous blood streams characterized by molecular crowding and promiscuous intermolecular interaction, detailed structural characterizations of antibody interactions have thus far been performed under homogeneous in vitro conditions. NMR spectroscopy potentially has the ability to study protein structures in heterogeneous environments, assuming that the target protein can be labeled with NMR-active isotopes. Based on our successful development of isotope labeling of antibody glycoproteins, here we apply NMR spectroscopy to characterize antibody interactions in heterogeneous extracellular environments using mouse IgG-Fc as a test molecule. In human serum, many of the HSQC peaks originating from the Fc backbone exhibited attenuation in intensity of various magnitudes. Similar spectral changes were induced by the Fab fragment of polyclonal IgG isolated from the serum, but not by serum albumin, indicating that a subset of antibodies reactive with mouse IgG-Fc exists in human serum without preimmunization. The metaepitopes recognized by serum polyclonal IgG cover the entire molecular surface of Fc, including the binding sites to Fc receptors and C1q. In-serum NMR observation will offer useful tools for the detailed characterization of biopharamaceuticals, including therapeutic antibodies in physiologically relevant heterogeneous environments, also giving deeper insight into molecular recognition by polyclonal antibodies in the immune system.

  11. Detection and quantification of microcystins (cyanobacterial hepatotoxins) with recombinant antibody fragments isolated from a naïve human phage display library.

    Science.gov (United States)

    McElhiney, J; Lawton, L A; Porter, A J

    2000-12-01

    Single-chain antibody fragments against the cyanobacterial hepatotoxin microcystin-LR were isolated from a naive human phage display library and expressed in Escherichia coli. In competition enzyme-linked immunosorbent assay (ELISA), the most sensitive antibody clone selected from the library detected free microcystin-LR with an IC(50) value of 4 microM. It was found to cross react with three other microcystin variants - microcystin-RR, microcystin-LW and microcystin-LF - and detected microcystins in extracts of the cyanobacterium Microcystis aeruginosa, found to contain the toxins by high-performance liquid chromatography (HPLC). The quantification of microcystins in these extracts by ELISA and HPLC showed good correlation. Although the antibody isolated in this study was considerably less sensitive than the polyclonal and monoclonal antibodies already available for microcystin detection, phage display technology represents a cheaper, more rapid alternative for the production of anti-microcystin antibodies than the methods currently in use.

  12. Staphylococcal enterotoxin-specific IgE antibodies in atopic dermatitis.

    Science.gov (United States)

    Ide, Fumihito; Matsubara, Tomoyo; Kaneko, Miho; Ichiyama, Takashi; Mukouyama, Tokuko; Furukawa, Susumu

    2004-06-01

    The authors clarified the clinical significance of the measurement of serum concentrations of specific IgE antibodies to staphylococcal enterotoxin (SE) A- and SEB in atopic dermatitis (AD). The serum concentrations of SEA- and SEB-specific IgE antibodies in 140 pediatric patients with AD were measured with an immuno CAP -radioallergosorbent test system (RAST). To check the cross-reaction of specific IgE antibodies to SEA/SEB and other allergens, the CAP RAST fluorescent enzyme immunoassay inhibition test was performed. Forty-seven patients (33.6%) tested positive for either SEA- or SEB-specific IgE antibodies. School children showed higher positive rates of SEA/SEB-specific IgE antibodies than infants or young children. The patients with severe AD and those with exacerbation of symptoms in summer, had higher positive rates of SEA/SEB-specific IgE antibodies than patients with mild AD or those with exacerbation in winter. In addition, the positive rates of specific IgE antibodies to both dog-dander and cat-dander were higher in patients with positive SEA/SEB-specific IgE antibodies than in patients with negative ones. No cross-reactions occurred among specific IgE antibodies to SEA/SEB and dog/cat dander with one patient's serum, which had positive IgE-specific antibodies against cat/dog dander and SEA/SEB. The positive rate of SEA/SEB-specific IgE antibodies in the patients with dogs and/or cats as pets was 48.4%, which was higher than in those with no pets. Atopic dermatitis patients who exhibit high positive rates of SEA/SEB-specific IgE antibodies were found to be school children, severe cases, cases with high serum concentrations of total IgE, cases with exacerbation in summer, and cases with dogs and/or cats as pets. The measurement of serum concentrations of specific IgE antibodies to SEA and SEB, thus has some value for evaluating AD patients.

  13. Generation and characterization of recombinant human antibodies specific for native laminin epitopes. Potential application in cancer therapy. Cancer Immunol. Immunother

    DEFF Research Database (Denmark)

    Sanz, Laura; Kristensen, Peter; Russell, Stephen J.

    2001-01-01

    of human-derived antibody fragments able to modulate laminin-regulated biological functions would allow the development of new strategies to improve treatment of cancer patients. In this report, we explore the use of phage display technology to isolate human anti-laminin antibody fragments. A library...... to mouse, rat and human laminin. and show strong immunohistochemical reactivity with basement membranes in human and murine tissue sections. Their properties make them ideal candidates for in vivo applications....

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

    NARCIS (Netherlands)

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

    1993-01-01

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

  15. Pharmacokinetics and tumor targeting of 131I-labeled F(ab')2 fragments of the chimeric monoclonal antibody G250: preclinical and clinical pilot studies.

    NARCIS (Netherlands)

    Brouwers, A.H.; Mulders, P.F.A.; Oosterwijk, E.; Buijs, W.C.A.M.; Corstens, F.H.M.; Boerman, O.C.; Oyen, W.J.G.

    2004-01-01

    INTRODUCTION: Clinical and animal studies of chimeric monoclonal antibody G250 (moAb cG250) for the targeting of clear-cell renal cell carcinoma (RCC), to date, have been with the intact IgG form. To determine whether F(ab')2 fragments are more suited for radioimmunotherapy (RIT) than intact IgG,

  16. Production of bifunctional proteins by Aspergillus awamori: Llama variable heavy chain antibody fragment (V-HH) R9 coupled to Arthromyces ramosus peroxidase (ARP)

    NARCIS (Netherlands)

    Joosten, V.; Roelofs, M.S.; Dries, van den N.; Goosen, T.; Verrips, C.T.; Hondel, van den C.A.M.J.J.; Lokman, B.C.

    2005-01-01

    The Arthromyces ramosus peroxidase gene (arp) was genetically fused to either the 5'- or 3'-terminal ends of the gene encoding llama variable heavy chain antibody fragment V-HH R9, resulting in the fusion expression cassettes ARP-R9 or R9-ARP. Aspergillus awamori transformants were obtained which

  17. Production of bifunctional proteins by Aspergillus awamori: Llama variable heavy chain antibody fragment (VHH) R9 coupled to Arthromyces ramosus peroxidase (ARP)

    NARCIS (Netherlands)

    Joosten, V.; Roelofs, M.S.; Dries, N. van den; Goosen, T.; Verrips, C.T.; Hondel, C.A.M.J.J. van den; Lokman, B.C.

    2005-01-01

    The Arthromyces ramosus peroxidase gene (arp) was genetically fused to either the 5′- or 3′-terminal ends of the gene encoding llama variable heavy chain antibody fragment VHH R9, resulting in the fusion expression cassettes ARP-R9 or R9-ARP. Aspergillus awamori transformants were obtained which

  18. Polyfunctional HIV-Specific Antibody Responses Are Associated with Spontaneous HIV Control.

    Directory of Open Access Journals (Sweden)

    Margaret E Ackerman

    2016-01-01

    Full Text Available Elite controllers (ECs represent a unique model of a functional cure for HIV-1 infection as these individuals develop HIV-specific immunity able to persistently suppress viremia. Because accumulating evidence suggests that HIV controllers generate antibodies with enhanced capacity to drive antibody-dependent cellular cytotoxicity (ADCC that may contribute to viral containment, we profiled an array of extra-neutralizing antibody effector functions across HIV-infected populations with varying degrees of viral control to define the characteristics of antibodies associated with spontaneous control. While neither the overall magnitude of antibody titer nor individual effector functions were increased in ECs, a more functionally coordinated innate immune-recruiting response was observed. Specifically, ECs demonstrated polyfunctional humoral immune responses able to coordinately recruit ADCC, other NK functions, monocyte and neutrophil phagocytosis, and complement. This functionally coordinated response was associated with qualitatively superior IgG3/IgG1 responses, whereas HIV-specific IgG2/IgG4 responses, prevalent among viremic subjects, were associated with poorer overall antibody activity. Rather than linking viral control to any single activity, this study highlights the critical nature of functionally coordinated antibodies in HIV control and associates this polyfunctionality with preferential induction of potent antibody subclasses, supporting coordinated antibody activity as a goal in strategies directed at an HIV-1 functional cure.

  19. Isolation of Single-Domain Antibody Fragments That Preferentially Detect Intact (146S Particles of Foot-and-Mouth Disease Virus for Use in Vaccine Quality Control

    Directory of Open Access Journals (Sweden)

    Michiel M. Harmsen

    2017-08-01

    Full Text Available Intact (146S foot-and-mouth disease virus (FMDVs can dissociate into specific (12S viral capsid degradation products. FMD vaccines normally consist of inactivated virions. Vaccine quality is dependent on 146S virus particles rather than 12S particles. We earlier isolated two llama single-domain antibody fragments (VHHs that specifically recognize 146S particles of FMDV strain O1 Manisa and shown their potential use in quality control of FMD vaccines during manufacturing. These 146S-specific VHHs were specific for particular O serotype strains and did not bind strains from other FMDV serotypes. Here, we describe the isolation of 146S-specific VHHs against FMDV SAT2 and Asia 1 strains by phage display selection from llama immune libraries. VHHs that bind both 12S and 146S particles were readily isolated but VHHs that bind specifically to 146S particles could only be isolated by phage display selection using prior depletion for 12S particles. We obtained one 146S-specific VHH—M332F—that binds to strain Asia 1 Shamir and several VHHs that preferentially bind 146S particles of SAT2 strain SAU/2/00, from which we selected VHH M379F for further characterization. Both M332F and M379F did not bind FMDV strains from other serotypes. In a sandwich enzyme-linked immunosorbent assay (ELISA employing unlabeled and biotinylated versions of the same VHH M332F showed high specificity for 146S particles but M379F showed lower 146S-specificity with some cross-reaction with 12S particles. These ELISAs could detect 146S particle concentrations as low as 2.3–4.6 µg/l. They can be used for FMD vaccine quality control and research and development, for example, to identify virion stabilizing excipients.

  20. Isoschizomers and amplified fragment length polymorphism for the detection of specific cytosine methylation changes.

    Science.gov (United States)

    Ruiz-García, Leonor; Cabezas, Jose Antonio; de María, Nuria; Cervera, María-Teresa

    2010-01-01

    Different molecular techniques have been developed to study either the global level of methylated cytosines or methylation at specific gene sequences. One of them is a modification of the Amplified Fragment Length Polymorphism (AFLP) technique that has been used to study methylation of anonymous CCGG sequences in different fungi, plant and animal species. The main variation of this technique is based on the use of isoschizomers with different methylation sensitivity (such as HpaII and MspI) as a frequent cutter restriction enzyme. For each sample, AFLP analysis is performed using both EcoRI/HpaII and EcoRI/MspI digested samples. Comparative analysis between EcoRI/HpaII and EcoRI/MspI fragment patterns allows the identification of two types of polymorphisms: (1) "Methylation-insensitive polymorphisms" that show common EcoRI/HpaII and EcoRI/MspI patterns but are detected as polymorphic amplified fragments among samples; and (2) "Methylation-sensitive polymorphisms" that are associated with amplified fragments differing in their presence or absence or in their intensity between EcoRI/HpaII and EcoRI/MspI patterns. This chapter describes a detailed protocol of this technique and discusses modifications that can be applied to adjust the technology to different species of interest.

  1. Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates demonstrate tumor specificity and anti-tumor activity.

    Science.gov (United States)

    Prendergast, Jillian M; Galvao da Silva, Ana Paula; Eavarone, David A; Ghaderi, Darius; Zhang, Mai; Brady, Dane; Wicks, Joan; DeSander, Julie; Behrens, Jeff; Rueda, Bo R

    Targeted therapeutics that can differentiate between normal and malignant tumor cells represent the ideal standard for the development of a successful anti-cancer strategy. The Sialyl-Thomsen-nouveau antigen (STn or Sialyl-Tn, also known as CD175s) is rarely seen in normal adult tissues, but it is abundantly expressed in many types of human epithelial cancers. We have identified novel antibodies that specifically target with high affinity the STn glycan independent of its carrier protein, affording the potential to recognize a wider array of cancer-specific sialylated proteins. A panel of murine monoclonal anti-STn therapeutic antibodies were generated and their binding specificity and efficacy were characterized in vitro and in in vivo murine cancer models. A subset of these antibodies were conjugated to monomethyl auristatin E (MMAE) to generate antibody-drug conjugates (ADCs). These ADCs demonstrated in vitro efficacy in STn-expressing cell lines and significant tumor growth inhibition in STn-expressing tumor xenograft cancer models with no evidence of overt toxicity.

  2. Radioimmunoimaging of human colon carcinoma grafted into nudemice using 131I-labeled monoclonal anticea antibody and its F(ab')2 fragments

    International Nuclear Information System (INIS)

    Liu Guangda

    1988-01-01

    131 I-labeled monoclonal anti-CEA antibody and its F(ab') 2 fragments were injected into nude mice bearing human colon carcinoma xenografts for tumor localization and radioimmunoimaging studies. Transplanted tumors were visualized in 12 hours after injection of the labeled anti-CEA or its F(ab') 2 by gamma camera. Biodistribution data indicated that F(ab') 2 fragments were cleared more rapidly from blood (T 1/2 = 13.3 h for F(ab') 2 , T 1/2 = 21.1 h for intact antibody) over 6-24 h and had higher tumor blood ratios. The intact antibody was concentrated in the tumor better than F(ab') 2 . In double-label experiments, a nonspecific localization of the control ( 125 I-labeled anti-HCG) in the tumor was also observed

  3. Epidemiology of myasthenia gravis with anti-muscle specific kinase antibodies in the Netherlands

    NARCIS (Netherlands)

    Niks, Erik H.; Kuks, Jan B. M.; Verschuuren, Jan J. G. M.

    The epidemiology of myasthenia gravis subtypes and the frequency of antibodies to muscle-specific kinase (MuSK) was studied in patients with generalised myasthenia gravis without anti-acetylcholine receptor antibodies who had an onset of symptoms between 1990 and 2004 in a well-defined region in the

  4. Rapid profiling of the antigen regions recognized by serum antibodies using massively parallel sequencing of antigen-specific libraries.

    KAUST Repository

    Domina, Maria; Lanza Cariccio, Veronica; Benfatto, Salvatore; D'Aliberti, Deborah; Venza, Mario; Borgogni, Erica; Castellino, Flora; Biondo, Carmelo; D'Andrea, Daniel; Grassi, Luigi; Tramontano, Anna; Teti, Giuseppe; Felici, Franco; Beninati, Concetta

    2014-01-01

    There is a need for techniques capable of identifying the antigenic epitopes targeted by polyclonal antibody responses during deliberate or natural immunization. Although successful, traditional phage library screening is laborious and can map only some of the epitopes. To accelerate and improve epitope identification, we have employed massive sequencing of phage-displayed antigen-specific libraries using the Illumina MiSeq platform. This enabled us to precisely identify the regions of a model antigen, the meningococcal NadA virulence factor, targeted by serum antibodies in vaccinated individuals and to rank hundreds of antigenic fragments according to their immunoreactivity. We found that next generation sequencing can significantly empower the analysis of antigen-specific libraries by allowing simultaneous processing of dozens of library/serum combinations in less than two days, including the time required for antibody-mediated library selection. Moreover, compared with traditional plaque picking, the new technology (named Phage-based Representation OF Immuno-Ligand Epitope Repertoire or PROFILER) provides superior resolution in epitope identification. PROFILER seems ideally suited to streamline and guide rational antigen design, adjuvant selection, and quality control of newly produced vaccines. Furthermore, this method is also susceptible to find important applications in other fields covered by traditional quantitative serology.

  5. Rapid profiling of the antigen regions recognized by serum antibodies using massively parallel sequencing of antigen-specific libraries.

    Directory of Open Access Journals (Sweden)

    Maria Domina

    Full Text Available There is a need for techniques capable of identifying the antigenic epitopes targeted by polyclonal antibody responses during deliberate or natural immunization. Although successful, traditional phage library screening is laborious and can map only some of the epitopes. To accelerate and improve epitope identification, we have employed massive sequencing of phage-displayed antigen-specific libraries using the Illumina MiSeq platform. This enabled us to precisely identify the regions of a model antigen, the meningococcal NadA virulence factor, targeted by serum antibodies in vaccinated individuals and to rank hundreds of antigenic fragments according to their immunoreactivity. We found that next generation sequencing can significantly empower the analysis of antigen-specific libraries by allowing simultaneous processing of dozens of library/serum combinations in less than two days, including the time required for antibody-mediated library selection. Moreover, compared with traditional plaque picking, the new technology (named Phage-based Representation OF Immuno-Ligand Epitope Repertoire or PROFILER provides superior resolution in epitope identification. PROFILER seems ideally suited to streamline and guide rational antigen design, adjuvant selection, and quality control of newly produced vaccines. Furthermore, this method is also susceptible to find important applications in other fields covered by traditional quantitative serology.

  6. Rapid profiling of the antigen regions recognized by serum antibodies using massively parallel sequencing of antigen-specific libraries.

    KAUST Repository

    Domina, Maria

    2014-12-04

    There is a need for techniques capable of identifying the antigenic epitopes targeted by polyclonal antibody responses during deliberate or natural immunization. Although successful, traditional phage library screening is laborious and can map only some of the epitopes. To accelerate and improve epitope identification, we have employed massive sequencing of phage-displayed antigen-specific libraries using the Illumina MiSeq platform. This enabled us to precisely identify the regions of a model antigen, the meningococcal NadA virulence factor, targeted by serum antibodies in vaccinated individuals and to rank hundreds of antigenic fragments according to their immunoreactivity. We found that next generation sequencing can significantly empower the analysis of antigen-specific libraries by allowing simultaneous processing of dozens of library/serum combinations in less than two days, including the time required for antibody-mediated library selection. Moreover, compared with traditional plaque picking, the new technology (named Phage-based Representation OF Immuno-Ligand Epitope Repertoire or PROFILER) provides superior resolution in epitope identification. PROFILER seems ideally suited to streamline and guide rational antigen design, adjuvant selection, and quality control of newly produced vaccines. Furthermore, this method is also susceptible to find important applications in other fields covered by traditional quantitative serology.

  7. Intramuscular Immunisation with Chlamydial Proteins Induces Chlamydia trachomatis Specific Ocular Antibodies.

    Directory of Open Access Journals (Sweden)

    Alexander Badamchi-Zadeh

    Full Text Available Ocular infection with Chlamydia trachomatis can cause trachoma, which is the leading cause of blindness due to infection worldwide. Despite the large-scale implementation of trachoma control programmes in the majority of countries where trachoma is endemic, there remains a need for a vaccine. Since C. trachomatis infects the conjunctival epithelium and stimulates an immune response in the associated lymphoid tissue, vaccine regimens that enhance local antibody responses could be advantageous. In experimental infections of non-human primates (NHPs, antibody specificity to C. trachomatis antigens was found to change over the course of ocular infection. The appearance of major outer membrane protein (MOMP specific antibodies correlated with a reduction in ocular chlamydial burden, while subsequent generation of antibodies specific for PmpD and Pgp3 correlated with C. trachomatis eradication.We used a range of heterologous prime-boost vaccinations with DNA, Adenovirus, modified vaccinia Ankara (MVA and protein vaccines based on the major outer membrane protein (MOMP as an antigen, and investigated the effect of vaccine route, antigen and regimen on the induction of anti-chlamydial antibodies detectable in the ocular lavage fluid of mice.Three intramuscular vaccinations with recombinant protein adjuvanted with MF59 induced significantly greater levels of anti-MOMP ocular antibodies than the other regimens tested. Intranasal delivery of vaccines induced less IgG antibody in the eye than intramuscular delivery. The inclusion of the antigens PmpD and Pgp3, singly or in combination, induced ocular antigen-specific IgG antibodies, although the anti-PmpD antibody response was consistently lower and attenuated by combination with other antigens.If translatable to NHPs and/or humans, this investigation of the murine C. trachomatis specific ocular antibody response following vaccination provides a potential mouse model for the rapid and high throughput

  8. Epitope and functional specificity of monoclonal antibodies to mouse gamma interferon: the synthetic peptide approach

    International Nuclear Information System (INIS)

    Russell, J.K.; Hayes, M.P.; Carter, J.M.; Torres, B.A.; Dunn, B.M.; Johnson, H.M.

    1986-01-01

    Four anti-recombinant mouse gamma interferon (α-IFNγ) monoclonal antibodies were generated using hamster spleen cells. Binding of 125 I-IFNγ by these protein A-bound antibodies was specifically blocked by cold IFNγ. Binding by three of these antibodies was also blocked by a synthetic peptide corresponding to the N-terminal 1-39 amino acids of IFNγ, while a corresponding C-terminal (95-133) peptide had no effect on binding. One of the N-terminal specific monoclonal antibodies inhibited both the antiviral and macrophage priming (for tumor cell killing) activities of IFNγ, while the other two had no effect on either biological function. Blocking experiments with cold IFNγ and N-terminal peptide suggest that the epitope specificities of the monoclonal antibodies could be determined by the conformational or topographic structure of IFNγ. Polyclonal antibodies to either the N-terminal or C-terminal peptides also inhibited both the antiviral and macrophage priming activities of IFNγ. All of the antibodies that inhibited IFNγ function also blocked binding of IFNγ to membrane receptor on cells, while antibodies that did not inhibit function also did not block binding. The data suggest that both the N-terminal and C-terminal domains of IFNγ play an important role in its antiviral and macrophage priming functions, possibly in a cooperative manner

  9. Characterization of a monoclonal antibody with specificity for holo-transcobalamin

    Directory of Open Access Journals (Sweden)

    Fedosov Sergey N

    2006-01-01

    Full Text Available Abstract Background Holotranscobalamin, cobalamin-saturated transcobalamin, is the minor fraction of circulating cobalamin (vitamin B12, which is available for cellular uptake and hence is physiologically relevant. Currently, no method allows simple, direct quantification of holotranscobalamin. We now report on the identification and characterization of a monoclonal antibody with a unique specificity for holotranscobalamin. Methods The specificity and affinity of the monoclonal antibodies were determined using surface plasmon resonance and recombinant transcobalamin as well as by immobilizing the antibodies on magnetic microspheres and using native transcobalamin in serum. The epitope of the holotranscobalamin specific antibody was identified using phage display and comparison to a de novo generated three-dimensional model of transcobalamin using the program Rosetta. A direct assay for holotrnscobalamin in the ELISA format was developed using the specific antibody and compared to the commercial assay HoloTC RIA. Results An antibody exhibiting >100-fold specificity for holotranscobalamin over apotranscobalamin was identified. The affinity but not the specificity varied inversely with ionic strength and pH, indicating importance of electrostatic interactions. The epitope was discontinuous and epitope mapping of the antibody by phage display identified two similar motifs with no direct sequence similarity to transcobalamin. A comparison of the motifs with a de novo generated three-dimensional model of transcobalamin identified two structures in the N-terminal part of transcobalamin that resembled the motif. Using this antibody an ELISA based prototype assay was developed and compared to the only available commercial assay for measuring holotranscobalamin, HoloTC RIA. Conclusion The identified antibody possesses a unique specificity for holotranscobalamin and can be used to develop a direct assay for the quantification of holotranscobalamin.

  10. Targeted in vivo inhibition of specific protein-protein interactions using recombinant antibodies.

    Directory of Open Access Journals (Sweden)

    Matej Zábrady

    Full Text Available With the growing availability of genomic sequence information, there is an increasing need for gene function analysis. Antibody-mediated "silencing" represents an intriguing alternative for the precise inhibition of a particular function of biomolecules. Here, we describe a method for selecting recombinant antibodies with a specific purpose in mind, which is to inhibit intrinsic protein-protein interactions in the cytosol of plant cells. Experimental procedures were designed for conveniently evaluating desired properties of recombinant antibodies in consecutive steps. Our selection method was successfully used to develop a recombinant antibody inhibiting the interaction of ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 3 with such of its upstream interaction partners as the receiver domain of CYTOKININ INDEPENDENT HISTIDINE KINASE 1. The specific down-regulation of the cytokinin signaling pathway in vivo demonstrates the validity of our approach. This selection method can serve as a prototype for developing unique recombinant antibodies able to interfere with virtually any biomolecule in the living cell.

  11. 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

  12. Presence of specific IgG antibody to grain dust does not go with respiratory symptoms.

    Science.gov (United States)

    Park, H S; Suh, C H; Nahm, D H; Kim, H Y

    1999-02-01

    A high prevalence of work-related symptoms in relation to grain dust exposure has been reported in grain dust workers, but the role of the specific IgG antibody is unknown. To study the possible role of specific IgG (sIgG) and specific IgG4 (sIgG4) in the development of work-related symptoms, sIgG and sIgG4 subclass antibodies against grain dust antigens were determined by ELISA in sera from 43 workers and 27 non-exposed controls. They were compared with results of specific IgE antibodies, exposure intensity and the presence of respiratory symptoms. SIgG and sIgG4 antibodies were detectable in almost all sera of exposed workers, and the prevalence were significantly higher than those of controls (pgrain dust exposure and may unlikely play a role in the etiology of respiratory symptoms.

  13. Functional improvement of antibody fragments using a novel phage coat protein III fusion system

    DEFF Research Database (Denmark)

    Jensen, Kim Bak; Larsen, Martin; Pedersen, Jesper Søndergaard

    2002-01-01

    Functional expressions of proteins often depend on the presence of host specific factors. Frequently recombinant expression strategies of proteins in foreign hosts, such as bacteria, have been associated with poor yields or significant loss of functionality. Improvements in the performance of het......(s) of the filamentous phage coat protein III. Furthermore, it will be shown that the observed effect is neither due to improved stability nor increased avidity....

  14. Red blood cell antibodies in pregnancy and their clinical consequences: synergistic effects of multiple specificities.

    Science.gov (United States)

    Nordvall, Maria; Dziegiel, Morten; Hegaard, Hanne Kristine; Bidstrup, Mogens; Jonsbo, Finn; Christensen, Birgit; Hedegaard, Morten

    2009-10-01

    The objective was to determine clinical consequences of various specificities for the infant/fetus. The population was patients referred between 1998 and 2005 to the tertiary center because of detected red blood cell (RBC) alloimmunization. Altogether 455 infants were delivered by 390 alloimmunized women. This was a retrospective cohort study. Data were obtained from the blood bank register and the obstetric and neonatal database. As indicators of hemolytic activity of the antibodies, the frequency of the therapeutic interventions intrauterine transfusion, exchange transfusion, and simple transfusion was used. Anti-D was the most common antibody (46.6%), followed by anti-K (15.4%). A combination of antibodies was detected in 27%. All three types of therapeutic intervention were significantly more frequent in women with anti-D plus an additional antibody than in women with anti-D as the sole antibody. The anti-D titer closely paralleled the clinical importance of the antibody. One case of anti-s with a titer of 512 required all three types of transfusion. Anti-D was the single most frequent and harmful specificity closely followed by anti-K. Combinations of antibody specificities were more harmful than single specificities, and a potentially synergistic effect should be considered.

  15. High production of llama variable heavy-chain antibody fragment (VHH) fused to various reader proteins by Aspergillus oryzae.

    Science.gov (United States)

    Hisada, Hiromoto; Tsutsumi, Hiroko; Ishida, Hiroki; Hata, Yoji

    2013-01-01

    Llama variable heavy-chain antibody fragment (VHH) fused to four different reader proteins was produced and secreted in culture medium by Aspergillus oryzae. These fusion proteins consisted of N-terminal reader proteins, VHH, and a C-terminal his-tag sequence which facilitated purification using one-step his-tag affinity chromatography. SDS-PAGE analysis of the deglycosylated purified fusion proteins confirmed that the molecular weight of each corresponded to the expected sum of VHH and the respective reader proteins. The apparent high molecular weight reader protein glucoamylase (GlaB) was found to be suitable for efficient VHH production. The GlaB-VHH-His protein bound its antigen, human chorionic gonadotropin, and was detectable by a new ELISA-based method using a coupled assay with glucoamylase, glucose oxidase, peroxidase, maltose, and 3,3',5,5'-tetramethylbenzidine as substrates. Addition of potassium phosphate to the culture medium induced secretion of 0.61 mg GlaB-VHH-His protein/ml culture medium in 5 days.

  16. Localization of mammary tumors in vivo with 131I-labeled Fab fragments of antibodies against mouse mammary epithelial (MME) antigens

    International Nuclear Information System (INIS)

    Wilbanks, T.; Peterson, J.A.; Miller, S.; Kaufman, L.; Ortendahl, D.; Ceriani, R.L.

    1981-01-01

    The Fab fragments of antibodies against cell-type-specific surface antigens of mouse mammary epithelial cells (MME-antigens) were used to localize mammary tumors successfully. The radioiodine-labeled anti-MME (Fab) was injected into mice carrying simulated mammary metastases, and after 24 hours the amount of label per gram of excised tissue was several times greater in the tumor than in liver, brain, lung, or muscle. Kidney showed considerable accumulation of label but this appeared to be nonspecific. Kinetic studies revealed a rapid elimination of labeled Fab in the urine with only 1% of the injected dose remaining in the entire blood pool after 24 hours. Wit a high-purity germanium camera, mammary tumors were clearly located ty the 131 I-labeled anti-MME (Fab), and normalization to /sup 99m/Tc-pertechnetate distribution in the animal increased the specificity. The density of 131 I-label was fourfold greater over the mammary tumor than over comparable areas of the mouse. No accumulation of 131 I-anti-MME (Fab) was observed in nonmammary tumors nor in mammary tumors when labeled nonspecific Fab was used. An analogous system using an antihuman mammary epithelial antiserum is being developed for localization of breast metastases in humans

  17. Inner-shell excitation and site specific fragmentation of poly(methylmethacrylate) thin film

    Science.gov (United States)

    Tinone, Marcia C. K.; Tanaka, Kenichiro; Maruyama, Junya; Ueno, Nobuo; Imamura, Motoyasu; Matsubayashi, Nobuyuki

    1994-04-01

    Soft x-ray excitations in the 250-600 eV photon energy range on poly(methylmethacrylate) (PMMA) result in ionic fragmentation of the original polymer with the most intense ions corresponding to CH+3, H+, CH+2, CH+, CHO+, and COOCH+3. The photon energy dependence of ion desorption from thin films of PMMA was measured to investigate the primary steps in radiation induced decomposition following carbon and oxygen 1s electron excitations using monochromatic pulsed-synchrotron radiation. It was clearly found that the decomposition depends on the nature of the electronic states created in the excited species. The fragmentation pattern changes depending on the transitions of the 1s electron to a Rydberg orbital, an unoccupied molecular orbital or the ionization continuum. Moreover, the fragmentation occurs specifically around the site of the atom where the optical excitation takes place. Excitations from carbon and oxygen 1s to σ* states seem to be specially efficient for ion production as observed in the case of CH+3, CH+2, and CH+ at 288.7 and 535.6 eV, and in the case of CHO+ at 539.3 eV.

  18. Potential Role of Specific Antibodies as Important Vaccine Induced Protective Mechanism against Aeromonas salmonicida in Rainbow Trout

    DEFF Research Database (Denmark)

    Rømer Villumsen, Kasper; Dalsgaard, Inger; Holten-Andersen, Lars

    2012-01-01

    of specific antibodies in plasma was monitored using ELISA. A significant increase in specific antibody levels was seen in fish vaccinated with both vaccines during the 18 weeks between vaccination and challenge. Within 3 days post challenge, a significant decrease in specific antibodies occurred...

  19. Anti-Idiotypic Antibodies Specific to prM Monoantibody Prevent Antibody Dependent Enhancement of Dengue Virus Infection

    Directory of Open Access Journals (Sweden)

    Miao Wang

    2017-05-01

    Full Text Available Dengue virus (DENV co-circulates as four serotypes (DENV1-4. Primary infection only leads to self-limited dengue fever. But secondary infection with another serotype carries a higher risk of increased disease severity, causing life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS. Serotype cross-reactive antibodies facilitate DENV infection in Fc-receptor-bearing cells by promoting virus entry via Fcγ receptors (FcγR, a process known as antibody dependent enhancement (ADE. Most studies suggested that enhancing antibodies were mainly specific to the structural premembrane protein (prM of DENV. However, there is still no effective drugs or vaccines to prevent ADE. In this study, we firstly confirmed that both DENV-2 infected human sera (anti-DENV-2 and DENV-2 prM monoclonal antibody (prM mAb could significantly enhance DENV-1 infection in K562 cells. Then we developed anti-idiotypic antibodies (prM-AIDs specific to prM mAb by immunizing of Balb/c mice. Results showed that these polyclonal antibodies can dramatically reduce ADE phenomenon of DENV-1 infection in K562 cells. To further confirm the anti-ADE effect of prM-AIDs in vivo, interferon-α and γ receptor-deficient mice (AG6 were used as the mouse model for DENV infection. We found that administration of DENV-2 prM mAb indeed caused a higher DENV-1 titer as well as interleukin-10 (IL-10 and alaninea minotransferase (ALT in mice infected with DENV-1, similar to clinical ADE symptoms. But when we supplemented prM-AIDs to DENV-1 challenged AG6 mice, the viral titer, IL-10 and ALT were obviously decreased to the negative control level. Of note, the number of platelets in peripheral blood of prM-AIDs group were significantly increased at day 3 post infection with DENV-1 compared that of prM-mAb group. These results confirmed that our prM-AIDs could prevent ADE not only in vitro but also in vivo, suggested that anti-idiotypic antibodies might be a new choice to be considered to

  20. Anti-Idiotypic Antibodies Specific to prM Monoantibody Prevent Antibody Dependent Enhancement of Dengue Virus Infection.

    Science.gov (United States)

    Wang, Miao; Yang, Fan; Huang, Dana; Huang, Yalan; Zhang, Xiaomin; Wang, Chao; Zhang, Shaohua; Zhang, Renli

    2017-01-01

    Dengue virus (DENV) co-circulates as four serotypes (DENV1-4). Primary infection only leads to self-limited dengue fever. But secondary infection with another serotype carries a higher risk of increased disease severity, causing life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Serotype cross-reactive antibodies facilitate DENV infection in Fc-receptor-bearing cells by promoting virus entry via Fcγ receptors (FcγR), a process known as antibody dependent enhancement (ADE). Most studies suggested that enhancing antibodies were mainly specific to the structural premembrane protein (prM) of DENV. However, there is still no effective drugs or vaccines to prevent ADE. In this study, we firstly confirmed that both DENV-2 infected human sera (anti-DENV-2) and DENV-2 prM monoclonal antibody (prM mAb) could significantly enhance DENV-1 infection in K562 cells. Then we developed anti-idiotypic antibodies (prM-AIDs) specific to prM mAb by immunizing of Balb/c mice. Results showed that these polyclonal antibodies can dramatically reduce ADE phenomenon of DENV-1 infection in K562 cells. To further confirm the anti-ADE effect of prM-AIDs in vivo , interferon-α and γ receptor-deficient mice (AG6) were used as the mouse model for DENV infection. We found that administration of DENV-2 prM mAb indeed caused a higher DENV-1 titer as well as interleukin-10 (IL-10) and alaninea minotransferase (ALT) in mice infected with DENV-1, similar to clinical ADE symptoms. But when we supplemented prM-AIDs to DENV-1 challenged AG6 mice, the viral titer, IL-10 and ALT were obviously decreased to the negative control level. Of note, the number of platelets in peripheral blood of prM-AIDs group were significantly increased at day 3 post infection with DENV-1 compared that of prM-mAb group. These results confirmed that our prM-AIDs could prevent ADE not only in vitro but also in vivo , suggested that anti-idiotypic antibodies might be a new choice to be considered to treat

  1. Immunization Elicits Antigen-Specific Antibody Sequestration in Dorsal Root Ganglia Sensory Neurons

    Science.gov (United States)

    Gunasekaran, Manojkumar; Chatterjee, Prodyot K.; Shih, Andrew; Imperato, Gavin H.; Addorisio, Meghan; Kumar, Gopal; Lee, Annette; Graf, John F.; Meyer, Dan; Marino, Michael; Puleo, Christopher; Ashe, Jeffrey; Cox, Maureen A.; Mak, Tak W.; Bouton, Chad; Sherry, Barbara; Diamond, Betty; Andersson, Ulf; Coleman, Thomas R.; Metz, Christine N.; Tracey, Kevin J.; Chavan, Sangeeta S.

    2018-01-01

    The immune and nervous systems are two major organ systems responsible for host defense and memory. Both systems achieve memory and learning that can be retained, retrieved, and utilized for decades. Here, we report the surprising discovery that peripheral sensory neurons of the dorsal root ganglia (DRGs) of immunized mice contain antigen-specific antibodies. Using a combination of rigorous molecular genetic analyses, transgenic mice, and adoptive transfer experiments, we demonstrate that DRGs do not synthesize these antigen-specific antibodies, but rather sequester primarily IgG1 subtype antibodies. As revealed by RNA-seq and targeted quantitative PCR (qPCR), dorsal root ganglion (DRG) sensory neurons harvested from either naïve or immunized mice lack enzymes (i.e., RAG1, RAG2, AID, or UNG) required for generating antibody diversity and, therefore, cannot make antibodies. Additionally, transgenic mice that express a reporter fluorescent protein under the control of Igγ1 constant region fail to express Ighg1 transcripts in DRG sensory neurons. Furthermore, neural sequestration of antibodies occurs in mice rendered deficient in neuronal Rag2, but antibody sequestration is not observed in DRG sensory neurons isolated from mice that lack mature B cells [e.g., Rag1 knock out (KO) or μMT mice]. Finally, adoptive transfer of Rag1-deficient bone marrow (BM) into wild-type (WT) mice or WT BM into Rag1 KO mice revealed that antibody sequestration was observed in DRG sensory neurons of chimeric mice with WT BM but not with Rag1-deficient BM. Together, these results indicate that DRG sensory neurons sequester and retain antigen-specific antibodies released by antibody-secreting plasma cells. Coupling this work with previous studies implicating DRG sensory neurons in regulating antigen trafficking during immunization raises the interesting possibility that the nervous system collaborates with the immune system to regulate antigen-mediated responses. PMID:29755449

  2. Immunization Elicits Antigen-Specific Antibody Sequestration in Dorsal Root Ganglia Sensory Neurons

    Directory of Open Access Journals (Sweden)

    Manojkumar Gunasekaran

    2018-04-01

    Full Text Available The immune and nervous systems are two major organ systems responsible for host defense and memory. Both systems achieve memory and learning that can be retained, retrieved, and utilized for decades. Here, we report the surprising discovery that peripheral sensory neurons of the dorsal root ganglia (DRGs of immunized mice contain antigen-specific antibodies. Using a combination of rigorous molecular genetic analyses, transgenic mice, and adoptive transfer experiments, we demonstrate that DRGs do not synthesize these antigen-specific antibodies, but rather sequester primarily IgG1 subtype antibodies. As revealed by RNA-seq and targeted quantitative PCR (qPCR, dorsal root ganglion (DRG sensory neurons harvested from either naïve or immunized mice lack enzymes (i.e., RAG1, RAG2, AID, or UNG required for generating antibody diversity and, therefore, cannot make antibodies. Additionally, transgenic mice that express a reporter fluorescent protein under the control of Igγ1 constant region fail to express Ighg1 transcripts in DRG sensory neurons. Furthermore, neural sequestration of antibodies occurs in mice rendered deficient in neuronal Rag2, but antibody sequestration is not observed in DRG sensory neurons isolated from mice that lack mature B cells [e.g., Rag1 knock out (KO or μMT mice]. Finally, adoptive transfer of Rag1-deficient bone marrow (BM into wild-type (WT mice or WT BM into Rag1 KO mice revealed that antibody sequestration was observed in DRG sensory neurons of chimeric mice with WT BM but not with Rag1-deficient BM. Together, these results indicate that DRG sensory neurons sequester and retain antigen-specific antibodies released by antibody-secreting plasma cells. Coupling this work with previous studies implicating DRG sensory neurons in regulating antigen trafficking during immunization raises the interesting possibility that the nervous system collaborates with the immune system to regulate antigen-mediated responses.

  3. Novel Monoclonal Antibodies Recognizing Human Prostate-Specific Membrane Antigen (PSMA) as Research and Theranostic Tools.

    Science.gov (United States)

    Nováková, Zora; Foss, Catherine A; Copeland, Benjamin T; Morath, Volker; Baranová, Petra; Havlínová, Barbora; Skerra, Arne; Pomper, Martin G; Barinka, Cyril

    2017-05-01

    Prostate-specific membrane antigen (PSMA) is a validated target for the imaging and therapy of prostate cancer. Here, we report the detailed characterization of four novel murine monoclonal antibodies (mAbs) recognizing human PSMA as well as PSMA orthologs from different species. Performance of purified mAbs was assayed using a comprehensive panel of in vitro experimental setups including Western blotting, immunofluorescence, immunohistochemistry, ELISA, flow cytometry, and surface-plasmon resonance. Furthermore, a mouse xenograft model of prostate cancer was used to compare the suitability of the mAbs for in vivo applications. All mAbs demonstrate high specificity for PSMA as documented by the lack of cross-reactivity to unrelated human proteins. The 3F11 and 1A11 mAbs bind linear epitopes spanning residues 226-243 and 271-288 of human PSMA, respectively. 3F11 is also suitable for the detection of PSMA orthologs from mouse, pig, dog, and rat in experimental setups where the denatured form of PSMA is used. 5D3 and 5B1 mAbs recognize distinct surface-exposed conformational epitopes and are useful for targeting PSMA in its native conformation. Most importantly, using a mouse xenograft model of prostate cancer we show that both the intact 5D3 and its Fab fragment are suitable for in vivo imaging. With apparent affinities of 0.14 and 1.2 nM as determined by ELISA and flow cytometry, respectively, 5D3 has approximately 10-fold higher affinity for PSMA than the clinically validated mAb J591 and, therefore, is a prime candidate for the development of next-generation theranostics to target PSMA. Prostate 77:749-764, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  4. Characterization of a monoclonal antibody that specifically inhibits triosephosphate isomerase activity of Taenia solium.

    Science.gov (United States)

    Víctor, Sanabria-Ayala; Yolanda, Medina-Flores; Araceli, Zavala-Carballo; Lucía, Jiménez; Abraham, Landa

    2013-08-01

    In the present study, we obtained and characterized partially a monoclonal antibody (4H11D10B11 mAb) against triosephosphate isomerase from Taenia solium (TTPI). This antibody recognized the enzyme by both ELISA and western blot and was able to inhibit its enzymatic activity in 74%. Moreover, the antigen-binding fragments (Fabs), products of digestion of the monoclonal antibody with papain, retained almost the same inhibitory effect. We determined the binding site by ELISA; synthetic peptides containing sequences from different non-conserved regions of the TTPI were confronted to the 4H11D10B11 mAb. The epitope recognized by the monoclonal antibody was located on peptide TTPI-56 (ATPAQAQEVHKVVRDWIRKHVDAGIADKARI), and an analysis of mimotopes, obtained with the 4H11D10B11 mAb, suggests that the epitope spans the sequence WIRKHVDAGIAD, residues 193-204 of the enzyme. This epitope is located within helix 6, next to loop 6, an essential active loop during catalysis. The antibody did not recognize triosephosphate isomerase from man and pig, definitive and intermediary hosts of T. solium, respectively. Furthermore, it did not bind to the catalytic site, since kinetic analysis demonstrated that inhibition had a non-competitive profile. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. A recombinant antibody with the antigen-specific, major histocompatibility complex-restricted specificity of T cells

    DEFF Research Database (Denmark)

    Andersen, P S; Stryhn, A; Hansen, B E

    1996-01-01

    Specific recognition of peptide/major histocompatibility complex (MHC) molecule complexes by the T-cell receptor is a key reaction in the specific immune response. Antibodies against peptide/MHC complexes would therefore be valuable tools in studying MHC function and T-cell recognition and might ...

  6. Application of Food-specific IgG Antibody Detection in Allergy Dermatosis

    Directory of Open Access Journals (Sweden)

    Yine Hu

    2015-01-01

    Full Text Available The application of food-specific IgG antibody detection in allergy dermatoses was explored. 181 patients with allergy dermatoses were diagnosed from January to September 2014 and 20 healthy subjects were selected. Fourteen kinds of food-specific IgG antibodies were detected by ELISA method among all the subjects. The positive rates of IgG antibody of the patient group and the healthy group were respectively 65.2% and 5.0%. The positive rates of IgG antibody of egg, milk, shrimp and crab took a large proportion in three groups of patients with three kinds of allergy dermatoses of urticaria, eczema and allergic dermatitis, the proportion of which was respectively 70.2%, 77.8% and 71.7%. Among urticaria and allergic dermatitis patients with positive antibody, the positive rate of children was significantly higher than that of adults (p0.05. Allergy dermatoses are closely related to food-specific IgG antibodies, and the allergy dermatoses patients have a high incidence rate of food intolerance; detecting IgG antibody in the serum of patients is of great significance for the diagnosis and treatment of allergy dermatoses.

  7. 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...

  8. Titres of Specific Antibodies against Toxoplasma gondii in Goats and their Kids

    Directory of Open Access Journals (Sweden)

    Ľubica Mišurová

    2009-01-01

    Full Text Available The aim of our study was to perform repeated determination of specific antibody levels in mothers and their kids in order to assess indirectly the possibility of vertical transmission of toxoplasmosis in goats. Twenty-eight goats with their kids were included in the study. The following variables were assessed: number of born kids in relation to antibody titres of goats; levels of specific antibodies in the blood of goats and kids; and concentrations of immunoglobulins (Ig, total protein (TP and total globulins (G in order to define the end of colostral immunity and the start of active production of antibodies in kids under 69 days of age. Specific antibodies against Toxoplasma gondii in goats were detected by IFAT in titres ranging from 0 to 1 280. Out of a total of 28 animals, 5 goats were negative (17.9% and 23 goats were seropositive (82.1%. The goats delivered 42 kids. A total ratio of number of kids to number of mothers was 1.5. Partial evaluation of results in goats without positive titre against T. gondii before parturition and goats with positive titre showed that negative goats tended to have more kids (p p < 0.01 of monitored non-specific immunity indicators. During this period, we observed increased titres of specific antibodies against toxoplasmosis in 20 kids (5 kids 41 days old, 5 kids 55 days old, and 10 kids 69 days old and thus we could assume the possibility of vertical transmission of toxoplasmosis.

  9. Specificity of antibodies directed against the cytolethal distending toxin of Haemophilus ducreyi in patients with chancroid.

    Science.gov (United States)

    Mbwana, Judica; Ahmed, Hinda J; Ahlman, Karin; Sundaeus, Vivian; Dahlén, Gunnar; Lyamuya, Eligius; Lagergård, Teresa

    2003-09-01

    Antibodies specific for the cytolethal-distending toxin of Haemophilus ducreyi (HdCDT) complex and for the CdtA, CdtB, and CdtC components were measured by ELISA in the sera of 50 patients with culture and/or PCR proven chancroid, 42 patients with periodontitis, 50 blood donors from Tanzania, 50 blood donors from Sweden. In addition, the biological activity e.g. neutralization capacity of the sera were tested. Our results demonstrate that majority of chancroid patients and healthy individuals had detectable levels of serum antibodies to HdCDT complex and to separate toxin components. However, high levels (> or =100 units) of antibodies to HdCDT complex were significantly more prevalent in the sera of patients with both chancroid and periodontitis than in the sera of the corresponding controls (P=0.001 and P=0.04, respectively). In the sera of the 50 patients with chancroid, antibodies to CdtA, CdtB, and CdtC were detected in 50, 35, and 34 individuals, respectively. Antibodies to CdtC, being less frequently detected than the antibodies to other components, show a good correlation with the neutralizing capacity of sera. High levels of neutralizing antibodies (> or =160) were detected in only 22 and 2% of the patients with chancroid and periodontitis, respectively. The data suggest that the low levels of anti-HdCDT antibodies, which include neutralizing antibodies, may contribute to limited protection in chancroid and since anti-HdCDT antibodies, may be detected in healthy individuals and in patients with certain disease conditions (e.g. periodontitis), they may not be specific markers for chancroid infection.

  10. Isolation and characterization of antigen-specific alpaca (Lama pacos) VHH antibodies by biopanning followed by high-throughput sequencing.

    Science.gov (United States)

    Miyazaki, Nobuo; Kiyose, Norihiko; Akazawa, Yoko; Takashima, Mizuki; Hagihara, Yosihisa; Inoue, Naokazu; Matsuda, Tomonari; Ogawa, Ryu; Inoue, Seiya; Ito, Yuji

    2015-09-01

    The antigen-binding domain of camelid dimeric heavy chain antibodies, known as VHH or Nanobody, has much potential in pharmaceutical and industrial applications. To establish the isolation process of antigen-specific VHH, a VHH phage library was constructed with a diversity of 8.4 × 10(7) from cDNA of peripheral blood mononuclear cells of an alpaca (Lama pacos) immunized with a fragment of IZUMO1 (IZUMO1PFF) as a model antigen. By conventional biopanning, 13 antigen-specific VHHs were isolated. The amino acid sequences of these VHHs, designated as N-group VHHs, were very similar to each other (>93% identity). To find more diverse antibodies, we performed high-throughput sequencing (HTS) of VHH genes. By comparing the frequencies of each sequence between before and after biopanning, we found the sequences whose frequencies were increased by biopanning. The top 100 sequences of them were supplied for phylogenic tree analysis. In total 75% of them belonged to N-group VHHs, but the other were phylogenically apart from N-group VHHs (Non N-group). Two of three VHHs selected from non N-group VHHs showed sufficient antigen binding ability. These results suggested that biopanning followed by HTS provided a useful method for finding minor and diverse antigen-specific clones that could not be identified by conventional biopanning. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  11. Use of AN Eosinophil Specific Monoclonal Antibody in Assessing Eosinophil Function.

    Science.gov (United States)

    Minkoff, Marjorie Sue

    A monoclonal antibody to an eosinophil specific determinant is very important in assessing eosinophil function during helminthic infection. Eosinophils induced by Schistosoma mansoni infection in BALB/c mice were used to induce C57B1/6 immunocytes for production of hybridomas secreting eosinophil monoclonal antibodies. These antibodies were shown to react with an eosinophil surface epitope but not with neutrophils or macrophages as determined by ELISA, immunodiffusion, immunofluorescence, and immunoblot assay. Affinity chromatography with eosinophil chemotactic factor-sepharose consistently selected out a { rm M_ R} 67,000 protein from solubilized eosinophil membrane antigens but not from neutrophil and macrophage antigens. In vitro studies showed that the eosinophil-specific monoclonal antibodies abrogated antibody-dependent eosinophil -mediated killing of S. mansoni schistosomula using mouse, rat or human eosinophils. Neutrophil and macrophage killing activities were unaffected. The monoclonal antibodies effected complement-dependent lysis of mouse and rat eosinophils but not of human eosinophils. ECF-treated eosinophils showed enhanced killing of schistosomula which was blocked by the monoclonal antibody. Murine and human eosinophils preincubated with monoclonal antibody exhibited decreased chemotaxis to ECF at optimal chemotactic concentrations. The monoclonal antibody also blocked eosinophil binding to ECF- sepharose beads. In vivo induction of peripheral blood eosinophilia by injection of S. mansoni eggs was suppressed by injections of monoclonal antibodies 2CD13 and 2QD45 in mouse and rat experimental models. Eosinophilia induced by keyhole limpet hemocyanin- cyclophosphamide treatment was also suppressed by monoclonal antibody in both murine and rat systems. Pulmonary granulomas in mice given egg injection and monoclonal antibody were smaller and contained fewer eosinophils than those granulomas from mice given eggs only. In immuno-biochemical studies, the

  12. 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

  13. Bone marrow dosimetry in rats using direct tissue counting after injection of radio-iodinated intact monoclonal antibodies or F(ab')2 fragments

    International Nuclear Information System (INIS)

    Buchegger, F.; Chalandon, Y.; Pelegrin, A.; Hardman, N.; Mach, J.P.

    1991-01-01

    Normal rats were injected intravenously with 131I- and 125I-labeled intact murine and chimeric mouse-human monoclonal antibodies directed against carcinoembryonic antigen or with the corresponding F(ab')2 fragments. At different times after injection, individual animals were killed and radioactivity of blood and major organs, including bones and bone marrow, was determined. Ratios comparing radioactivity concentration in different tissues with that of bone marrow were calculated and found to remain stable during several effective half-lives of the antibodies. Mean bone marrow radioactivity was 35% (range, 29%-40%) of that of blood and 126% (range, 108%-147%) of that of liver after injection of intact Mabs or F(ab')2 fragments. In nude rats bearing human colon carcinoma xenografts producing carcinoembryonic antigen, relative bone marrow radioactivity was slightly lower than that in normal rats

  14. High contrast tumor imaging with radio-labeled antibody Fab fragments tailored for optimized pharmacokinetics via PASylation.

    Science.gov (United States)

    Mendler, Claudia T; Friedrich, Lars; Laitinen, Iina; Schlapschy, Martin; Schwaiger, Markus; Wester, Hans-Jürgen; Skerra, Arne

    2015-01-01

    Although antigen-binding fragments (Fabs) of antibodies constitute established tracers for in vivo radiodiagnostics, their functionality is hampered by a very short circulation half-life. PASylation, the genetic fusion with a long, conformationally disordered amino acid chain comprising Pro, Ala and Ser, provides a convenient way to expand protein size and, consequently, retard renal filtration. Humanized αHER2 and αCD20 Fabs were systematically fused with 100 to 600 PAS residues and produced in E. coli. Cytofluorimetric titration analysis on tumor cell lines confirmed that antigen-binding activities of the parental antibodies were retained. The radio-iodinated PASylated Fabs were studied by positron emission tomography (PET) imaging and biodistribution analysis in mouse tumor xenograft models. While the unmodified αHER2 and αCD20 Fabs showed weak tumor uptake (0.8% and 0.2% ID/g, respectively; 24 h p.i.) tumor-associated radioactivity was boosted with increasing PAS length (up to 9 and 26-fold, respectively), approaching an optimum for Fab-PAS400. Remarkably, 6- and 5-fold higher tumor-to-blood ratios compared with the unmodified Fabs were measured in the biodistribution analysis (48 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200, respectively. These findings were confirmed by PET studies, showing high imaging contrast in line with tumor-to-blood ratios of 12.2 and 5.7 (24 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200. Even stronger tumor signals were obtained with the corresponding αCD20 Fabs, both in PET imaging and biodistribution analysis, with an uptake of 2.8% ID/g for Fab-PAS100 vs. 0.24% ID/g for the unmodified Fab. Hence, by engineering Fabs via PASylation, plasma half-life can be tailored to significantly improve tracer uptake and tumor contrast, thus optimally matching reagent/target interactions.

  15. Targeting osteomyelitis with complete [99mTc]besilesomab and fragmented [99mTc]sulesomab antibodies: kinetic evaluations

    International Nuclear Information System (INIS)

    GRATZ, Stefan; KEMKE, Bendix; KEIZE, Patrik; KAMPEN, Wim U.; LUSTER, Markus; HÖFFKEN, Helmut

    2016-01-01

    The aim of this retrospective study was to compare the targeting of “pure” osteomyelitis (i.e., without surrounding soft tissue infection) by directly 99mTc-labelled complete immunoglobulin G (IgG) monoclonal antibody (MAb) ([99mTc]besilesomab) and by directly 99mTc-labelled fragment antigen-binding (FAb) MAb ([99mTc]sulesomab) in relation to their kinetic fate. A total of 73 patients with “pure” osteomyelitis were examined with [99mTc]besilesomab, (Scintimun®, IBA/CIS bio international, Saclay, France; N.=38) and [99mTc]sulesomab (LeukoScan®, Immunomedics Inc., Morris Plains, NJ, USA; N.=35). Kinetic data were deduced from whole-body and single-photon emission computed tomographic scans, performed 10 minutes to 24 hour p.i. (region-of-interest technique [ROI]). In targeting “pure” osteomyelitis, sensitivities at 1-4 hours were found to be higher for [99mTc]sulesomab (44% and 80% for [99mTc]besilesomab and [99mTc]sulesomab, respectively) but at significantly lower target/background (T/B) ratios than with [99mTc]besilesomab (1.8±0.3 versus 1.4±0.5 for [99mTc]besilesomab and [99mTc]sulesomab respectively; P<0.01). With [99mTc]besilesomab, there was a continuous osteomyelitis uptake over 24 hours, whereas with [99mTc]sulesomab, the maximal uptake occurred mostly within 1-4 hours, with subsequent clearance being slower for antigen-bound activity than for nonspecific background. Hence, diagnosis was possible mostly after 4h with [99mTc]sulesomab but often not before 24 hours with [99mTc]besilesomab, the later increasing significantly (P<0.01) in sensitivity (87% and 84% for [99mTc]besilesomab and [99mTc]sulesomab, respectively). These results show that the higher sensitivity of [99mTc]sulesomab in osteomyelitis targeting at earlier p.i. times does not rely on an increased antibody uptake but on a more rapid clearance of nonspecific background activity due to faster metabolism and excretion. Intact [99mTc]besilesomab show a slow, continuous uptake

  16. Rapid production of antigen-specific monoclonal antibodies from a variety of animals

    Directory of Open Access Journals (Sweden)

    Kurosawa Nobuyuki

    2012-09-01

    Full Text Available Abstract Background Although a variety of animals have been used to produce polyclonal antibodies against antigens, the production of antigen-specific monoclonal antibodies from animals remains challenging. Results We propose a simple and rapid strategy to produce monoclonal antibodies from a variety of animals. By staining lymph node cells with an antibody against immunoglobulin and a fluorescent dye specific for the endoplasmic reticulum, plasma/plasmablast cells were identified without using a series of antibodies against lineage markers. By using a fluorescently labeled antigen as a tag for a complementary cell surface immunoglobulin, antigen-specific plasma/plasmablast cells were sorted from the rest of the cell population by fluorescence-activated cell sorting. Amplification of cognate pairs of immunoglobulin heavy and light chain genes followed by DNA transfection into 293FT cells resulted in the highly efficient production of antigen-specific monoclonal antibodies from a variety of immunized animals. Conclusions Our technology eliminates the need for both cell propagation and screening processes, offering a significant advantage over hybridoma and display strategies.

  17. Activity, specificity, and titer of naturally occurring canine anti-DEA 7 antibodies.

    Science.gov (United States)

    Spada, Eva; Proverbio, Daniela; Baggiani, Luciana; Canzi, Ilaria; Perego, Roberta

    2016-11-01

    The reported prevalence of naturally occurring anti-dog erythrocyte antigen (DEA) 7 antibodies in DEA 7-negative dogs is as high as 50%. Characterization of these antibodies may better define their importance in canine transfusion medicine. We determined in vitro activity, specificity, and titer of anti-DEA 7 antibodies in DEA 7-negative dogs. Plasma samples from 317 DEA 7-negative dogs were cross-matched with DEA 7-positive red blood cells (RBCs) using gel column technology. Agglutination occurred with DEA 7-positive RBCs but not with DEA 7-negative RBCs in 73 samples (23%), which were hence classified as containing anti-DEA 7 antibodies. These samples were evaluated for hemolytic and agglutinating activity, strength of agglutination, and antibody specificity and titers. All samples showed agglutination but none showed hemolysis. Gel agglutination was graded as 1+ for 20 samples (27%), 2+ for 49 samples (67%), 3+ for 4 samples (6%); no samples were graded 4+. The agglutination titer was DEA 7 antibodies were found in 23% of DEA 7-negative dogs. The presence of naturally occurring anti-DEA 7 antibodies suggests that cross-matching of canine blood recipients is advisable, even at first transfusion, to minimize delayed transfusion reactions. © 2016 The Author(s).

  18. Identification and characterization of a thermally cleaved fragment of monoclonal antibody-A detected by sodium dodecyl sulfate-capillary gel electrophoresis.

    Science.gov (United States)

    Kubota, Kei; Kobayashi, Naoki; Yabuta, Masayuki; Ohara, Motomu; Naito, Toyohiro; Kubo, Takuya; Otsuka, Koji

    2017-06-05

    This report describes a novel, comprehensive approach to identifying a fragment peak of monoclonal antibody-A (mAb-A), detected by sodium dodecyl sulfate-capillary gel electrophoresis (SDS-cGE). The fragment migrated close to the internal standard (10kDa marker) of SDS-cGE and increased about 0.5% under a 25°C condition for 6 months. Generally, identification of fragments observed in SDS-cGE is challenging to carry out due to the difficulty of collecting analytical amounts of fractionations from the capillary. In this study, in-gel digestion peptide mapping and reversed phase liquid chromatography-mass spectrometry (RPLC-MS) were employed to elucidate the structure of the fragment. In addition, a Gelfree 8100 fractionation system was newly introduced to collect the fragment and the fraction was applied to the structural analysis of a mAb for the first time. These three analytical methods showed comparable results, proving that the fragment was a fraction of heavy chain HC1-104. The fragment contained complementarity determining regions (CDRs), which are significant to antigen binding, and thus would affect the efficacy of mAb-A. In addition, SDS-cGE without the 10kDa marker was demonstrated to clarify the increased amount of the fragment, and the experiment revealed that the fragment increases 0.2% per year in storage at 5°C. The combination of the three analytical methodologies successfully identified the impurity peak detected by SDS-cGE, providing information critical to assuring the quality and stability of the biotherapeutics. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Application of 125I radioimmunoassay to measure inhibition of precipitin reactions using carbohydrate-specific antibodies

    International Nuclear Information System (INIS)

    Boullanger, P.H.; Nagpurkar, A.; Noujaim, A.A.; Lemieux, R.U.

    1978-01-01

    Antibodies raised to an artificial antigen with β-D-galactopyranosyl groups as antigenic determinants were purified using an immunoadsorbent prepared from the hapten involved in the synthesis of the antigen. In order to study the specificity of these antibodies, 125 I radiolabelling of either the artificial antigen or the antibody was used in the study of inhibitions of the precipitin reaction. The method, involving labelling of the artificial antigen and counting radioactivity in the supernatant, was found to be more accurate and faster than the usual methods based on measuring the amount of protein precipitated by chemical or spectroscopic methods. (author)

  20. ELISA with double antigen sandwich for screening specific serum anti-TP antibody in blood donors

    International Nuclear Information System (INIS)

    Wang Yiqing; Shi Zhixu

    2002-01-01

    Objective: To select a sensitive and specific laboratory examination suitable for screening serum anti-TP antibody in blood donors. Methods: The serum anti-TP antibody in 11271 blood donors were detected using ELISA with double antigen sandwich and the outcomes were compared with those using RPR assay. The conflicting specimen were confirmed by repeating the test with TPHA assay. Results: The positive rates of serum anti-TP antibody by ELISA with double antigen sandwich and RPR was 0.36% (41/11271) and 0.26% (29/11271), respectively. The coincidence of the detecting outcomes by ELISA with double antigen sandwich and RPR with TPHA was 97.5% (40/41) and 63.41%(26/41) respectively. Conclusion: Compared with RPR assay, ELISA with double antigen sandwich has higher sensibility and specificity for screening serum anti-TP antibody in blood donors

  1. The detection and specifity of class specific antibodies to whole bacteria cells using a solid phase radioimmunoassay

    International Nuclear Information System (INIS)

    Czerkinsky, C.; Rees, A.S.; Bergimeier, L.A.; Challacombe, S.J.

    1983-01-01

    A solid phase radioimmunoassay has been developed which can be used for the detection of isotype specific antibodies to whole bacteria and other particulate antigens, and is applicable to a variety of species. Bacteria are bound to the solid phase by the use either of antibodies, or of methyl glyoxal. Both methods result in a sensitive and reproducible assay, and bacteria do not appear to desorb from the solid phase. The specificity of antibodies to whole bacteria was examined by absorption of antisera with various species of bacteria and retesting, or by determining the binding of antisera to various bacteria bound to the solid phase. Both methods revealed specificity for the bacteria examined. Inhibition studies showed that antibodies to Streptococcus mutans whole cells could be inhibited by purified cell surface antigens glucosyltransferase and antigen I/II, but only minimally by lipoteichoic acid, c polysaccharide or dextran. In murine antisera antibodies of the IgG, IgM, and IgA classes could be detected at amounts of less than 1 ng/ml. (author)

  2. Radioimmunoimaging of human breast carcinoma xenografts in nude mouse model with 111In-labeled new monoclonal antibody EBA-1 and F(ab')2 fragments

    International Nuclear Information System (INIS)

    Yemul, Shrishailam; Leon, J.A.; Pozniakoff, Ted; Esser, P.D.; Estabrook, Alison; Met-Path Inc., Teterboro, NJ

    1993-01-01

    Radioimmunoimaging characteristics of a new monoclonal antibody EBA-1 and its F(ab') 2 fragments utilizing nu/nu mice bearing human breast carcinoma xenografts are described. 111 In-DPTA conjugates of EBA-1 localized with tumor/blood ratios of 0.99 ± 0.10 (P 2 radioconjugates at 48 h. These results suggest that EBA-1 and its F(ab') 2 might be useful reagents in radioimmunoimaging and radioimmunotherapy. (author)

  3. Specificity of anti-tau antibodies when analyzing mice models of Alzheimer's disease: problems and solutions.

    Directory of Open Access Journals (Sweden)

    Franck R Petry

    Full Text Available Aggregates of hyperphosphorylated tau protein are found in a group of diseases called tauopathies, which includes Alzheimer's disease. The causes and consequences of tau hyperphosphorylation are routinely investigated in laboratory animals. Mice are the models of choice as they are easily amenable to transgenic technology; consequently, their tau phosphorylation levels are frequently monitored by Western blotting using a panel of monoclonal/polyclonal anti-tau antibodies. Given that mouse secondary antibodies can recognize endogenous mouse immunoglobulins (Igs and the possible lack of specificity with some polyclonal antibodies, non-specific signals are commonly observed. Here, we characterized the profiles of commonly used anti-tau antibodies in four different mouse models: non-transgenic mice, tau knock-out (TKO mice, 3xTg-AD mice, and hypothermic mice, the latter a positive control for tau hyperphosphorylation. We identified 3 tau monoclonal antibody categories: type 1, characterized by high non-specificity (AT8, AT180, MC1, MC6, TG-3, type 2, demonstrating low non-specificity (AT270, CP13, CP27, Tau12, TG5, and type 3, with no non-specific signal (DA9, PHF-1, Tau1, Tau46. For polyclonal anti-tau antibodies, some displayed non-specificity (pS262, pS409 while others did not (pS199, pT205, pS396, pS404, pS422, A0024. With monoclonal antibodies, most of the interfering signal was due to endogenous Igs and could be eliminated by different techniques: i using secondary antibodies designed to bind only non-denatured Igs, ii preparation of a heat-stable fraction, iii clearing Igs from the homogenates, and iv using secondary antibodies that only bind the light chain of Igs. All of these techniques removed the non-specific signal; however, the first and the last methods were easier and more reliable. Overall, our study demonstrates a high risk of artefactual signal when performing Western blotting with routinely used anti-tau antibodies, and proposes

  4. Activated human nasal epithelial cells modulate specific antibody response against bacterial or viral antigens.

    Directory of Open Access Journals (Sweden)

    Chiou-Yueh Yeh

    Full Text Available Nasal mucosa is an immune responsive organ evidenced by eliciting both specific local secretory IgA and systemic IgG antibody responses with intra-nasal administration of antigens. Nevertheless, the role of nasal epithelial cells in modulating such responses is unclear. Human nasal epithelial cells (hNECs obtained from sinus mucosa of patients with chronic rhinosinusitis were cultured in vitro and firstly were stimulated by Lactococcus lactis bacterium-like particles (BLPs in order to examine their role on antibody production. Secondly, both antigens of immunodominant protein IDG60 from oral Streptococcus mutans and hemagglutinin (HA from influenza virus were tested to evaluate the specific antibody response. Stimulated hNECs by BLPs exhibited a significant increase in the production of interleukin-6 (IL-6, and thymic stromal lymphopoietin (TSLP. Conditioned medium of stimulated hNECs has effects on enhancing the proliferation of CD4+ T cells together with interferon-γ and IL-5 production, increasing the costimulatory molecules on dendritic cells and augmenting the production of IDG60 specific IgA, HA specific IgG, IgA by human peripheral blood lymphocytes. Such production of antigen specific IgG and IgA is significantly counteracted in the presence of IL-6 and TSLP neutralizing antibodies. In conclusion, properly stimulated hNECs may impart immuno-modulatory effects on the antigen-specific antibody response at least through the production of IL-6 and TSLP.

  5. Adjuvant-Mediated Epitope Specificity and Enhanced Neutralizing Activity of Antibodies Targeting Dengue Virus Envelope Protein

    Directory of Open Access Journals (Sweden)

    Denicar Lina Nascimento Fabris Maeda

    2017-09-01

    Full Text Available The heat-labile toxins (LT produced by enterotoxigenic Escherichia coli display adjuvant effects to coadministered antigens, leading to enhanced production of serum antibodies. Despite extensive knowledge of the adjuvant properties of LT derivatives, including in vitro-generated non-toxic mutant forms, little is known about the capacity of these adjuvants to modulate the epitope specificity of antibodies directed against antigens. This study characterizes the role of LT and its non-toxic B subunit (LTB in the modulation of antibody responses to a coadministered antigen, the dengue virus (DENV envelope glycoprotein domain III (EDIII, which binds to surface receptors and mediates virus entry into host cells. In contrast to non-adjuvanted or alum-adjuvanted formulations, antibodies induced in mice immunized with LT or LTB showed enhanced virus-neutralization effects that were not ascribed to a subclass shift or antigen affinity. Nonetheless, immunosignature analyses revealed that purified LT-adjuvanted EDIII-specific antibodies display distinct epitope-binding patterns with regard to antibodies raised in mice immunized with EDIII or the alum-adjuvanted vaccine. Notably, the analyses led to the identification of a specific EDIII epitope located in the EF to FG loop, which is involved in the entry of DENV into eukaryotic cells. The present results demonstrate that LT and LTB modulate the epitope specificity of antibodies generated after immunization with coadministered antigens that, in the case of EDIII, was associated with the induction of neutralizing antibody responses. These results open perspectives for the more rational development of vaccines with enhanced protective effects against DENV infections.

  6. Functional characterization of two scFv-Fc antibodies from an HIV controller selected on soluble HIV-1 Env complexes: a neutralizing V3- and a trimer-specific gp41 antibody.

    Directory of Open Access Journals (Sweden)

    Maria Trott

    Full Text Available HIV neutralizing antibodies (nAbs represent an important tool in view of prophylactic and therapeutic applications for HIV-1 infection. Patients chronically infected by HIV-1 represent a valuable source for nAbs. HIV controllers, including long-term non-progressors (LTNP and elite controllers (EC, represent an interesting subgroup in this regard, as here nAbs can develop over time in a rather healthy immune system and in the absence of any therapeutic selection pressure. In this study, we characterized two particular antibodies that were selected as scFv antibody fragments from a phage immune library generated from an LTNP with HIV neutralizing antibodies in his plasma. The phage library was screened on recombinant soluble gp140 envelope (Env proteins. Sequencing the selected peptide inserts revealed two major classes of antibody sequences. Binding analysis of the corresponding scFv-Fc derivatives to various trimeric and monomeric Env constructs as well as to peptide arrays showed that one class, represented by monoclonal antibody (mAb A2, specifically recognizes an epitope localized in the pocket binding domain of the C heptad repeat (CHR in the ectodomain of gp41, but only in the trimeric context. Thus, this antibody represents an interesting tool for trimer identification. MAb A7, representing the second class, binds to structural elements of the third variable loop V3 and neutralizes tier 1 and tier 2 HIV-1 isolates of different subtypes with matching critical amino acids in the linear epitope sequence. In conclusion, HIV controllers are a valuable source for the selection of functionally interesting antibodies that can be selected on soluble gp140 proteins with properties from the native envelope spike.

  7. Tumour localization and pharmacokinetics of iodine-125 human monoclonal IgM antibody (COU-1) and its monomeric and half-monomeric fragments analysed in nude mice grafted with human tumour

    International Nuclear Information System (INIS)

    Ditzel, H.; Erb, K.; Rasmussen, J.W.; Jensenius, J.C.

    1992-01-01

    Human monoclonal IgM antibodies reactive with cancer-associated antigens may not have the optimal imaging capability due to their large size. Fragmentation of human IgM is less than straight-forward due to the loss of immunoreactivity. From the human monoclonal IgM antibody COU-1 we have prepared monomeric and half-monomeric fragments, which retain the ability to bind to colon cancer cells in vitro. The pharmacokinetics and tumour localization were evaluated in nude mice bearing human colon adenocarcinoma and human melanoma grafts. Faster clearance from the circulation was seen for the smaller half-monomeric fragment with a half-life (rapid phase/slow phase) of 2 h/16 h compared with the intact antibody, 4 h/25 h, and the monomeric fragment, 3 h/27 h. Intact COU-1 as well as the fragments accumulated in the colon tumour graft. Higher amounts of radioactivity were found in the colon tumour as compared to normal organs for intact COU-1 at days 4 and 6, for the monomeric fragment at day 4, and for the half-monomeric fragment at day 2 after injection. This investigation demonstrates the favourable biodistribution of the half monomeric COU-1 fragment. The fast clearance of this fragment resulted in a tumour-to-muscle ratio as high as 22 on day 2 after injection. Also, only this fragment gave a positive tumour-to-blood ratio. Normal IgM and its fragments were used as controls. Radioimmunoscintigraphy demonstrated the colon tumour discriminatory properties of each of the three iodine-labelled antibody preparations. The results compare favourably with previously reported investigations of the localization of human monoclonal antibodies and suggest that fragments of human monoclonal IgM antibodies may be useful tools for the immunodetection of cancer in patients. (orig.)

  8. Specific Monoclonal Antibody Overcomes the Salmonella enterica Serovar Typhimurium's Adaptive Mechanisms of Intramacrophage Survival and Replication.

    Directory of Open Access Journals (Sweden)

    Swarmistha Devi Aribam

    Full Text Available Salmonella-specific antibodies play an important role in host immunity; however, the mechanisms of Salmonella clearance by pathogen-specific antibodies remain to be completely elucidated since previous studies on antibody-mediated protection have yielded inconsistent results. These inconsistencies are at least partially attributable to the use of polyclonal antibodies against Salmonella antigens. Here, we developed a new monoclonal antibody (mAb-449 and identified its related immunogen that protected BALB/c mice from infection with Salmonella enterica serovar Typhimurium. In addition, these data indicate that the mAb-449 immunogen is likely a major protective antigen. Using in vitro infection studies, we also analyzed the mechanism by which mAb-449 conferred host protection. Notably, macrophages infected with mAb-449-treated S. Typhimurium showed enhanced pathogen uptake compared to counterparts infected with control IgG-treated bacteria. Moreover, these macrophages produced elevated levels of pro-inflammatory cytokine TNFα and nitric oxide, indicating that mAb-449 enhanced macrophage activation. Finally, the number of intracellular bacteria in mAb-449-activated macrophages decreased considerably, while the opposite was found in IgG-treated controls. Based on these findings, we suggest that, although S. Typhimurium has the potential to survive and replicate within macrophages, host production of a specific antibody can effectively mediate macrophage activation for clearance of intracellular bacteria.

  9. A Novel, Rapid Assay for Detection and Differentiation of Serotype-Specific Antibodies to Venezuelan Equine Encephalitis Complex Alphaviruses

    National Research Council Canada - National Science Library

    Wang, Eryu; Paessler, Slobodan; Smith, Darci R; Coffey, Lark L; Kang, Wenli; Estrada-Franco, Jose; Weaver, Scott C; Aguilar, Patricia V; Pfeffer, Martin; Olson, James

    2005-01-01

    ... of Venezuelan equine encephalitis (VEE) virus. Two monoclonal antibodies that differentially recognize epizootic versus enzootic VEE virus epitopes were used to measure the serotype-specific blocking abilities of antibodies in sera of naturally...

  10. Selection of cholera toxin specific IgNAR single-domain antibodies from a naïve shark library.

    Science.gov (United States)

    Liu, Jinny L; Anderson, George P; Delehanty, James B; Baumann, Richard; Hayhurst, Andrew; Goldman, Ellen R

    2007-03-01

    Shark immunoglobulin new antigen receptor (IgNAR, also referred to as NAR) variable domains (Vs) are single-domain antibody (sdAb) fragments containing only two hypervariable loop structures forming 3D topologies for a wide range of antigen recognition and binding. Their small size ( approximately 12kDa) and high solubility, thermostability and binding specificity make IgNARs an exceptional alternative source of engineered antibodies for sensor applications. Here, two new shark NAR V display libraries containing >10(7) unique clones from non-immunized (naïve) adult spiny dogfish (Squalus acanthias) and smooth dogfish (Mustelus canis) sharks were constructed. The most conserved consensus sequences derived from random clone sequence were compared with published nurse shark (Ginglymostoma cirratum) sequences. Cholera toxin (CT) was chosen for panning one of the naïve display libraries due to its severe pathogenicity and commercial availability. Three very similar CT binders were selected and purified soluble monomeric anti-CT sdAbs were characterized using Luminex(100) and traditional ELISA assays. These novel anti-CT sdAbs selected from our newly constructed shark NAR V sdAb library specifically bound to soluble antigen, without cross reacting with other irrelevant antigens. They also showed superior heat stability, exhibiting slow loss of activity over the course of one hour at high temperature (95 degrees C), while conventional antibodies lost all activity in the first 5-10min. The successful isolation of target specific sdAbs from one of our non-biased NAR libraries, demonstrate their ability to provide binders against an unacquainted antigen of interest.

  11. Radiolabeled antibody imaging

    International Nuclear Information System (INIS)

    Wahl, R.L.

    1987-01-01

    Radiolabeled antibodies, in particular monoclonal antibodies, offer the potential for the specific nuclear imaging of malignant and benign diseases in man. If this imaging potential is realized, they may also have a large role in cancer treatment. This paper reviews: (1) what monoclonal antibodies are and how they differ from polyclonal antibodies, (2) how they are produced and radiolabeled, (3) the results of preclinical and clinical trials in cancer imaging, including the utility of SPECT and antibody fragments, (4) the role of antibodies in the diagnosis of benign diseases, (5) alternate routes of antibody delivery, (6) the role of these agents in therapy, and (7) whether this technology ''revolutionizes'' the practice of nuclear radiology, or has a more limited complementary role in the imaging department

  12. HIV-specific Fc effector function early in infection predicts the development of broadly neutralizing antibodies.

    Directory of Open Access Journals (Sweden)

    Simone I Richardson

    2018-04-01

    Full Text Available While the induction of broadly neutralizing antibodies (bNAbs is a major goal of HIV vaccination strategies, there is mounting evidence to suggest that antibodies with Fc effector function also contribute to protection against HIV infection. Here we investigated Fc effector functionality of HIV-specific IgG plasma antibodies over 3 years of infection in 23 individuals, 13 of whom developed bNAbs. Antibody-dependent cellular phagocytosis (ADCP, complement deposition (ADCD, cellular cytotoxicity (ADCC and cellular trogocytosis (ADCT were detected in almost all individuals with levels of activity increasing over time. At 6 months post-infection, individuals with bNAbs had significantly higher levels of ADCD and ADCT that correlated with antibody binding to C1q and FcγRIIa respectively. In addition, antibodies from individuals with bNAbs showed more IgG subclass diversity to multiple HIV antigens which also correlated with Fc polyfunctionality. Germinal center activity represented by CXCL13 levels and expression of activation-induced cytidine deaminase (AID was found to be associated with neutralization breadth, Fc polyfunctionality and IgG subclass diversity. Overall, multivariate analysis by random forest classification was able to group bNAb individuals with 85% sensitivity and 80% specificity based on the properties of their antibody Fc early in HIV infection. Thus, the Fc effector function profile predicted the development of neutralization breadth in this cohort, suggesting that intrinsic immune factors within the germinal center provide a mechanistic link between the Fc and Fab of HIV-specific antibodies.

  13. HIV-specific Fc effector function early in infection predicts the development of broadly neutralizing antibodies.

    Science.gov (United States)

    Richardson, Simone I; Chung, Amy W; Natarajan, Harini; Mabvakure, Batsirai; Mkhize, Nonhlanhla N; Garrett, Nigel; Abdool Karim, Salim; Moore, Penny L; Ackerman, Margaret E; Alter, Galit; Morris, Lynn

    2018-04-01

    While the induction of broadly neutralizing antibodies (bNAbs) is a major goal of HIV vaccination strategies, there is mounting evidence to suggest that antibodies with Fc effector function also contribute to protection against HIV infection. Here we investigated Fc effector functionality of HIV-specific IgG plasma antibodies over 3 years of infection in 23 individuals, 13 of whom developed bNAbs. Antibody-dependent cellular phagocytosis (ADCP), complement deposition (ADCD), cellular cytotoxicity (ADCC) and cellular trogocytosis (ADCT) were detected in almost all individuals with levels of activity increasing over time. At 6 months post-infection, individuals with bNAbs had significantly higher levels of ADCD and ADCT that correlated with antibody binding to C1q and FcγRIIa respectively. In addition, antibodies from individuals with bNAbs showed more IgG subclass diversity to multiple HIV antigens which also correlated with Fc polyfunctionality. Germinal center activity represented by CXCL13 levels and expression of activation-induced cytidine deaminase (AID) was found to be associated with neutralization breadth, Fc polyfunctionality and IgG subclass diversity. Overall, multivariate analysis by random forest classification was able to group bNAb individuals with 85% sensitivity and 80% specificity based on the properties of their antibody Fc early in HIV infection. Thus, the Fc effector function profile predicted the development of neutralization breadth in this cohort, suggesting that intrinsic immune factors within the germinal center provide a mechanistic link between the Fc and Fab of HIV-specific antibodies.

  14. Novel camelid antibody fragments targeting recombinant nucleoprotein of Araucaria hantavirus: a prototype for an early diagnosis of Hantavirus Pulmonary Syndrome.

    Directory of Open Access Journals (Sweden)

    Soraya S Pereira

    Full Text Available In addition to conventional antibodies, camelids produce immunoglobulins G composed exclusively of heavy chains in which the antigen binding site is formed only by single domains called VHH. Their particular characteristics make VHHs interesting tools for drug-delivery, passive immunotherapy and high-throughput diagnosis. Hantaviruses are rodent-borne viruses of the Bunyaviridae family. Two clinical forms of the infection are known. Hemorrhagic Fever with Renal Syndrome (HFRS is present in the Old World, while Hantavirus Pulmonary Syndrome (HPS is found on the American continent. There is no specific treatment for HPS and its diagnosis is carried out by molecular or serological techniques, using mainly monoclonal antibodies or hantavirus nucleoprotein (N to detect IgM and IgG in patient serum. This study proposes the use of camelid VHHs to develop alternative methods for diagnosing and confirming HPS. Phage display technology was employed to obtain VHHs. After immunizing one Lama glama against the recombinant N protein (prNΔ₈₅ of a Brazilian hantavirus strain, VHH regions were isolated to construct an immune library. VHHs were displayed fused to the M13KO7 phage coat protein III and the selection steps were performed on immobilized prNΔ₈₅. After selection, eighty clones recognized specifically the N protein. These were sequenced, grouped based mainly on the CDRs, and five clones were analyzed by western blot (WB, surface plasmon resonance (SPR device, and ELISA. Besides the ability to recognize prNΔ85 by WB, all selected clones showed affinity constants in the nanomolar range. Additionaly, the clone KC329705 is able to detect prNΔ₈₅ in solution, as well as the native viral antigen. Findings support the hypothesis that selected VHHs could be a powerful tool in the development of rapid and accurate HPS diagnostic assays, which are essential to provide supportive care to patients and reduce the high mortality rate associated with

  15. [Fingerprints identification of Gynostemma pentaphyllum by RAPD and cloning and analysis of its specific DNA fragment].

    Science.gov (United States)

    Jiang, Jun-fu; Li, Xiong-ying; Wu, Yao-sheng; Luo, Yu; Zhao, Rui-qiang; Lan, Xiu-wan

    2009-02-01

    To identify the resources of Gynostemma pentaphyllum and its spurious breed plant Cayratia japonica at level of DNA. Two random primers ( WGS001, WGS004) screened were applied to do random amplification with genomic DNA extracted from Gynostemma pentaphyllum and Cayratia japonica which were collected from different habitats. After amplificated with WGS004, one characteristic fragment about 500 bp which was common to all Gynostemma pentaphyllum samples studied but not to Cayratia japonica was cloned and sequenced. Then these sequences obtained were analyzed for identity and compared by Blastn program in GenBank. There were obvious different bands amplified by above two primers in their fingerprints of genomic DNA. On the basis of these different bands of DNA fingerprints, they could distinguish Gynostemma pentaphyllum and Cayratia japonica obviously. Sequence alignment of seven cloned bands showed that their identities ranged from 45.7% - 94.5%. There was no similar genome sequences searched in GenBank. This indicated that these seven DNA fragments had not been reported before and they should be new sequences. RAPD technique can be used for the accurate identification of Gynostemma pentaphyllum and its counterfeit goods Cayratia japonica. Besides, these specific DNA sequences for Gynostemmna pentaphyllum in this study are useful for the further research on identification of species and assisted selection breeding in Gynostemma pentaphyllum.

  16. Rhinovirus-induced VP1-specific Antibodies are Group-specific and Associated With Severity of Respiratory Symptoms

    Directory of Open Access Journals (Sweden)

    Katarzyna Niespodziana

    2015-01-01

    Interpretation: Our results demonstrate that increases of antibodies towards the VP1 N-terminus are group-specific and associated with severity of respiratory symptoms and suggest that it may be possible to develop serological tests for identifying causative RV groups.

  17. Indirect micro-immunofluorescence test for detecting type-specific antibodies to herpes simplex virus.

    Science.gov (United States)

    Forsey, T; Darougar, S

    1980-02-01

    A rapid indirect micro-immunofluorescence test capable of detecting and differentiating type-specific antibodies to herpes simplex virus is described. The test proved highly sensitive and, in 80 patients with active herpes ocular infection, antibody was detected in 94%. No anti-herpes antibody was detected in a control group of 20 patients with adenovirus infections. Testing of animal sera prepared against herpes simplex virus types 1 and 2 and of human sera from cases of ocular and genital herpes infections showed that the test can differentiate antibodies to the infecting serotypes. Specimens of whole blood, taken by fingerprick, and eye secretions, both collected on cellulose sponges, could be tested by indirect micro-immunofluorescence. Anti-herpes IgG, IgM, and IgA can also be detected.

  18. Specific antibodies to porcine zona pellucida detected by quantitative radioimmunoassay in both fertile and infertile women

    International Nuclear Information System (INIS)

    Kurachi, H.; Wakimoto, H.; Sakumoto, T.; Aono, T.; Kurachi, K.

    1984-01-01

    The specific radioimmunoassay system was developed for the titration of the antibodies to porcine zona pellucida (ZP) in human sera by using 125 I-labeled purified porcine ZP as antigen, which is known to have cross-reactivity with human ZP. The antibodies in human sera were detected in 3 of 11 (27%) women with unexplained infertility, in 16 of 48 (33%) amenorrheic patients, in 4 of 12 (33%) fertile women, and in 3 of 10 (30%) men. Moreover, antibody titers in infertile women were no higher than those in fertile women and in men. These results seem to suggest that the antibodies in human sera that cross-react with porcine ZP may not be an important factor in causing infertility in women

  19. Generation and Characterization of Inhibitory Antibodies Specific to Guinea Pig CXCR1 and CXCR2.

    Science.gov (United States)

    Tanaka, Kento; Yoshimura, Chigusa; Shiina, Tetsuo; Terauchi, Tomoko; Yoshitomi, Tomomi; Hirahara, Kazuki

    2017-04-01

    CXCR1 and CXCR2 are chemokine receptors that have different selectivity of chemokine ligands, but the distinct role of each receptor is not clearly understood. This is due to the absence of specific inhibitors in guinea pigs, which are the appropriate species for investigation of CXCR1 and CXCR2 because of their functional similarity to humans. In this study, we generated and evaluated monoclonal antibodies that specifically bound to guinea pig CXCR1 (gpCXCR1) and guinea pig CXCR2 (gpCXCR2) for acquisition of specific inhibitors. To assess the activity of antibodies, we established CHO-K1 cells stably expressing either gpCXCR1 or gpCXCR2 (CHO/gpCXCR1 or CHO/gpCXCR2). CHO/gpCXCR1 showed migration in response to guinea pig interleukin (IL)-8, and CHO/gpCXCR2 showed migration in response to both guinea pig IL-8 and guinea pig growth-regulated oncogene α. The receptor selectivities of the chemokines of guinea pigs were the same as the human orthologs. The inhibitory activities of the anti-gpCXCR1 and anti-gpCXCR2 monoclonal antibodies on cell migration were observed in a concentration-dependent manner. In conclusion, we successfully obtained inhibitory antibodies specific to gpCXCR1 and gpCXCR2. These inhibitory antibodies will be useful to clarify the physiological roles of CXCR1 and CXCR2 in guinea pigs.

  20. Targeting the autolysis loop of urokinase-type plasminogen activator with conformation-specific monoclonal antibodies.

    Science.gov (United States)

    Botkjaer, Kenneth A; Fogh, Sarah; Bekes, Erin C; Chen, Zhuo; Blouse, Grant E; Jensen, Janni M; Mortensen, Kim K; Huang, Mingdong; Deryugina, Elena; Quigley, James P; Declerck, Paul J; Andreasen, Peter A

    2011-08-15

    Tight regulation of serine proteases is essential for their physiological function, and unbalanced states of protease activity have been implicated in a variety of human diseases. One key example is the presence of uPA (urokinase-type plasminogen activator) in different human cancer types, with high levels correlating with a poor prognosis. This observation has stimulated efforts into finding new principles for intervening with uPA's activity. In the present study we characterize the so-called autolysis loop in the catalytic domain of uPA as a potential inhibitory target. This loop was found to harbour the epitopes for three conformation-specific monoclonal antibodies, two with a preference for the zymogen form pro-uPA, and one with a preference for active uPA. All three antibodies were shown to have overlapping epitopes, with three common residues being crucial for all three antibodies, demonstrating a direct link between conformational changes of the autolysis loop and the creation of a catalytically mature active site. All three antibodies are potent inhibitors of uPA activity, the two pro-uPA-specific ones by inhibiting conversion of pro-uPA to active uPA and the active uPA-specific antibody by shielding the access of plasminogen to the active site. Furthermore, using immunofluorescence, the conformation-specific antibodies mAb-112 and mAb-12E6B10 enabled us to selectively stain pro-uPA or active uPA on the surface of cultured cells. Moreover, in various independent model systems, the antibodies inhibited tumour cell invasion and dissemination, providing evidence for the feasibility of pharmaceutical intervention with serine protease activity by targeting surface loops that undergo conformational changes during zymogen activation. © The Authors Journal compilation © 2011 Biochemical Society

  1. Targeting the autolysis loop of urokinase-type plasminogen activator with conformation-specific monoclonal antibodies

    DEFF Research Database (Denmark)

    Bøtkjær, Kenneth Alrø; Fogh, Sarah; Bekes, Erin C

    2011-01-01

    Tight regulation of serine proteases is essential for their physiological function, and unbalanced states of protease activity have been implicated in a variety of human diseases. One key example is the presence of uPA (urokinase-type plasminogen activator) in different human cancer types......, demonstrating a direct link between conformational changes of the autolysis loop and the creation of a catalytically mature active site. All three antibodies are potent inhibitors of uPA activity, the two pro-uPA-specific ones by inhibiting conversion of pro-uPA to active uPA and the active u......PA-specific antibody by shielding the access of plasminogen to the active site. Furthermore, using immunofluorescence, the conformation-specific antibodies mAb-112 and mAb-12E6B10 enabled us to selectively stain pro-uPA or active uPA on the surface of cultured cells. Moreover, in various independent model systems...

  2. Antibodies to dopamine: radioimmunological study of specificity in relation to immunocytochemistry

    Energy Technology Data Exchange (ETDEWEB)

    Geffard, M.; Kah, O.; Onteniente, B.; Seguela, P.; Le Moal, M.; Delaage, M.

    1984-06-01

    Two classes of anti-3,4- dihydroxyphenylethylamine (dopamine) antibodies were raised in rabbits using dopamine conjugated to albumin either via formaldehyde or via glutaraldehyde. Each was usable for immunohistochemical detection of dopamine neurons provided that the tissue was fixed by the homologous cross-linking agent. However, anti-dopamine-glutaraldehyde antibodies turned out to be of more general use because of the better fixative properties of glutaraldehyde which fixed dopamine in rat and in teleost, whereas formaldehyde only worked in lower vertebrates (such as goldfish) and not in rat brain. The specificity of anti-dopamine-glutaraldehyde antibodies was firmly established by competition experiments in equilibrium dialysis, using an immunoreactive tritiated derivative synthesized by coupling dopamine to N-alpha-acetyl-L-lysine N-methylamide via glutaraldehyde. Specificity studies in vitro and immunohistological results demonstrating the specific staining of dopaminergic neurons were found to correlate well.

  3. Tau passive immunotherapy in mutant P301L mice: antibody affinity versus specificity.

    Directory of Open Access Journals (Sweden)

    Cristina d'Abramo

    Full Text Available The use of antibodies to treat neurodegenerative diseases has undergone rapid development in the past decade. To date, immunotherapeutic approaches to Alzheimer's disease have mostly targeted amyloid beta as it is a secreted protein that can be found in plasma and CSF and is consequently accessible to circulating antibodies. Few recent publications have suggested the utility of treatment of tau pathology with monoclonal antibodies to tau. Our laboratory has begun a systematic study of different classes of tau monoclonal antibodies using mutant P301L mice. Three or seven months old mutant tau mice were inoculated weekly with tau monoclonal antibodies at a dose of 10 mg/Kg, until seven or ten months of age were reached respectively. Our data strongly support the notion that in P301L animals treated with MC1, a conformational monoclonal antibody specific for PHF-tau, the rate of development of tau pathology is effectively reduced, while injecting DA31, a high affinity tau sequence antibody, does not exert such benefit. MC1 appears superior to DA31 in overall effects, suggesting that specificity is more important than affinity in therapeutic applications. Unfortunately the survival rate of the P301L treated mice was not improved when immunizing either with MC1 or PHF1, a high affinity phospho-tau antibody previously reported to be efficacious in reducing pathological tau. These data demonstrate that passive immunotherapy in mutant tau models may be efficacious in reducing the development of tau pathology, but a great deal of work remains to be done to carefully select the tau epitopes to target.

  4. Protection by meningococcal outer membrane protein PorA-specific antibodies and a serogroup B capsular polysaccharide-specific antibody in complement-sufficient and C6-deficient infant rats

    NARCIS (Netherlands)

    Toropainen, Maija; Saarinen, Leena; Vidarsson, Gestur; Käyhty, Helena

    2006-01-01

    The relative contributions of antibody-induced complement-mediated bacterial lysis and antibody/complement-mediated phagocytosis to host immunity against meningococcal infections are currently unclear. Further, the in vivo effector functions of antibodies may vary depending on their specificity and

  5. Antibody class capture assay (ACCA) for rubella-specific IgM antibody.

    Science.gov (United States)

    Isaac, M; Payne, R A

    1982-01-01

    Enzyme-linked immunosorbent assays for IgM antirubella were carried out on 1,546 sera, using an IgM capture method with a F (ab')2 conjugate (ACCA). Under the conditions described, sera containing IgM antirubella bound up to 15 times as much enzyme activity as negative specimens. Paired serum specimens from 27 patients, serial serum specimens from 6 patients, and single serum specimens from 15 patients who had had recent rubella were examined by the haemagglutination inhibition test (HAI) in the presence and absence of 2-mercaptoethanol following sucrose density gradient centrifugation (SDGC). ACCA confirmed all the results found with HAI following SDGC. Specimens were examined from ten patients with congenital rubella; ACCA confirmed the results found with both immunofluorescence following SDGC and radioimmunoassay. Pre- and post-vaccination specimens from 123 patients who had been vaccinated against rubella were examined. An IgM response could only be demonstrated in the 57 cases when IgG was absent in the first specimen. The specificity of the assay was confirmed by testing 31 serum specimens from rubella immune patients that also contained rheumatoid factor, 163 serum specimens from patients with acute infections other than rubella, and 12 serum specimens from infants with miscellaneous neonatal abnormalities other than congenital rubella. The ACCA proved a simple, sensitive, and specific test for IgM antirubella and the results compared favourably with those obtained by the SDGC technique.

  6. Generation of Recombinant Monoclonal Antibodies from Immunised Mice and Rabbits via Flow Cytometry and Sorting of Antigen-Specific IgG+ Memory B Cells.

    Directory of Open Access Journals (Sweden)

    Dale O Starkie

    Full Text Available Single B cell screening strategies, which avoid both hybridoma fusion and combinatorial display, have emerged as important technologies for efficiently sampling the natural antibody repertoire of immunized animals and humans. Having access to a range of methods to interrogate different B cell subsets provides an attractive option to ensure large and diverse panels of high quality antibody are produced. The generation of multiple antibodies and having the ability to find rare B cell clones producing IgG with unique and desirable characteristics facilitates the identification of fit-for-purpose molecules that can be developed into therapeutic agents or research reagents. Here, we describe a multi-parameter flow cytometry single-cell sorting technique for the generation of antigen-specific recombinant monoclonal antibodies from single IgG+ memory B cells. Both mouse splenocytes and rabbit PBMC from immunised animals were used as a source of B cells. Reagents staining both B cells and other unwanted cell types enabled efficient identification of class-switched IgG+ memory B cells. Concurrent staining with antigen labelled separately with two spectrally-distinct fluorophores enabled antigen-specific B cells to be identified, i.e. those which bind to both antigen conjugates (double-positive. These cells were then typically sorted at one cell per well using FACS directly into a 96-well plate containing reverse transcriptase reaction mix. Following production of cDNA, PCR was performed to amplify cognate heavy and light chain variable region genes and generate transcriptionally-active PCR (TAP fragments. These linear expression cassettes were then used directly in a mammalian cell transfection to generate recombinant antibody for further testing. We were able to successfully generate antigen-specific recombinant antibodies from both the rabbit and mouse IgG+ memory B cell subset within one week. This included the generation of an anti-TNFR2 blocking

  7. Incomplete separation of radioiodinated thyroid hormones in serum using specific antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Perrild, H; Skovsted, L; Korsgaard Christensen, L [Department of Internal Medicine and Endocrinology, Herlev Hospital, DK-2730 Herlev, Denmark

    1980-01-01

    Alkaline Sephadex G-25 columns were used to separate labelled 3,5,3',5'-thyroxine, 3,5,3'-triiodothyronine, 3,3',5'-triiodothyronine and 3,3'-diiodothyronine from the serum binding proteins followed by a quantitative elution of each hormone by coupling to its respective antibody. It is shown that although these antibodies (diluted 1:1500-1:100 000) in our radioimmunoassays are highly specific they show a high degree of non-specific binding when they are used in the concentrations necessary to get a maximal recovery of the hormones in column separating experiments.

  8. ImmunoPET of tissue factor expression in triple-negative breast cancer with a radiolabeled antibody Fab fragment

    International Nuclear Information System (INIS)

    Shi, Sixiang; Hong, Hao; Orbay, Hakan; Yang, Yunan; Ohman, Jakob D.; Graves, Stephen A.; Nickles, Robert J.; Liu, Bai; Wong, Hing C.; Cai, Weibo

    2015-01-01

    To date, there is no effective therapy for triple-negative breast cancer (TNBC), which has a dismal clinical outcome. Upregulation of tissue factor (TF) expression leads to increased patient morbidity and mortality in many solid tumor types, including TNBC. Our goal was to employ the Fab fragment of ALT-836, a chimeric anti-human TF mAb, for PET imaging of TNBC, which can be used to guide future TNBC therapy. ALT-836-Fab was generated by enzymatic papain digestion. SDS-PAGE and FACS studies were performed to evaluate the integrity and TF binding affinity of ALT-836-Fab before NOTA conjugation and 64 Cu-labeling. Serial PET imaging and biodistribution studies were carried out to evaluate the tumor targeting efficacy and pharmacokinetics in the MDA-MB-231 TNBC model, which expresses high levels of TF on the tumor cells. Blocking studies, histological assessment, as well as RT-PCR were performed to confirm TF specificity of 64 Cu-NOTA-ALT-836-Fab. ALT-836-Fab was produced with high purity, which exhibited superb TF binding affinity and specificity. Serial PET imaging revealed rapid and persistent tumor uptake of 64 Cu-NOTA-ALT-836-Fab (5.1 ± 0.5 %ID/g at 24 h post-injection; n = 4) and high tumor/muscle ratio (7.0 ± 1.2 at 24 h post-injection; n = 4), several-fold higher than that of the blocking group and tumor models that do not express significant level of TF, which was confirmed by biodistribution studies. TF specificity of the tracer was also validated by histology and RT-PCR. 64 Cu-NOTA-ALT-836-Fab exhibited prominent tissue factor targeting efficiency in MDA-MB-231 TNBC model. The use of a Fab fragment led to fast tumor uptake and good tissue/muscle ratio, which may be translated into same-day immunoPET imaging in the clinical setting to improve TNBC patient management. (orig.)

  9. ImmunoPET of tissue factor expression in triple-negative breast cancer with a radiolabeled antibody Fab fragment.

    Science.gov (United States)

    Shi, Sixiang; Hong, Hao; Orbay, Hakan; Graves, Stephen A; Yang, Yunan; Ohman, Jakob D; Liu, Bai; Nickles, Robert J; Wong, Hing C; Cai, Weibo

    2015-07-01

    To date, there is no effective therapy for triple-negative breast cancer (TNBC), which has a dismal clinical outcome. Upregulation of tissue factor (TF) expression leads to increased patient morbidity and mortality in many solid tumor types, including TNBC. Our goal was to employ the Fab fragment of ALT-836, a chimeric anti-human TF mAb, for PET imaging of TNBC, which can be used to guide future TNBC therapy. ALT-836-Fab was generated by enzymatic papain digestion. SDS-PAGE and FACS studies were performed to evaluate the integrity and TF binding affinity of ALT-836-Fab before NOTA conjugation and (64)Cu-labeling. Serial PET imaging and biodistribution studies were carried out to evaluate the tumor targeting efficacy and pharmacokinetics in the MDA-MB-231 TNBC model, which expresses high levels of TF on the tumor cells. Blocking studies, histological assessment, as well as RT-PCR were performed to confirm TF specificity of (64)Cu-NOTA-ALT-836-Fab. ALT-836-Fab was produced with high purity, which exhibited superb TF binding affinity and specificity. Serial PET imaging revealed rapid and persistent tumor uptake of (64)Cu-NOTA-ALT-836-Fab (5.1 ± 0.5 %ID/g at 24 h post-injection; n = 4) and high tumor/muscle ratio (7.0 ± 1.2 at 24 h post-injection; n = 4), several-fold higher than that of the blocking group and tumor models that do not express significant level of TF, which was confirmed by biodistribution studies. TF specificity of the tracer was also validated by histology and RT-PCR. (64)Cu-NOTA-ALT-836-Fab exhibited prominent tissue factor targeting efficiency in MDA-MB-231 TNBC model. The use of a Fab fragment led to fast tumor uptake and good tissue/muscle ratio, which may be translated into same-day immunoPET imaging in the clinical setting to improve TNBC patient management.

  10. ImmunoPET of tissue factor expression in triple-negative breast cancer with a radiolabeled antibody Fab fragment

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Sixiang [University of Wisconsin, Materials Science Program, Madison, WI (United States); Hong, Hao; Orbay, Hakan; Yang, Yunan; Ohman, Jakob D. [University of Wisconsin, Department of Radiology, Madison, WI (United States); Graves, Stephen A.; Nickles, Robert J. [University of Wisconsin, Department of Medical Physics, Madison, WI (United States); Liu, Bai; Wong, Hing C. [Altor BioScience, Miramar, FL (United States); Cai, Weibo [University of Wisconsin, Materials Science Program, Madison, WI (United States); University of Wisconsin, Department of Radiology, Madison, WI (United States); University of Wisconsin, Department of Medical Physics, Madison, WI (United States); University of Wisconsin Carbone Cancer Center, Madison, WI (United States); University of Wisconsin, Departments of Radiology and Medical Physics, Madison, WI (United States)

    2015-07-15

    To date, there is no effective therapy for triple-negative breast cancer (TNBC), which has a dismal clinical outcome. Upregulation of tissue factor (TF) expression leads to increased patient morbidity and mortality in many solid tumor types, including TNBC. Our goal was to employ the Fab fragment of ALT-836, a chimeric anti-human TF mAb, for PET imaging of TNBC, which can be used to guide future TNBC therapy. ALT-836-Fab was generated by enzymatic papain digestion. SDS-PAGE and FACS studies were performed to evaluate the integrity and TF binding affinity of ALT-836-Fab before NOTA conjugation and {sup 64}Cu-labeling. Serial PET imaging and biodistribution studies were carried out to evaluate the tumor targeting efficacy and pharmacokinetics in the MDA-MB-231 TNBC model, which expresses high levels of TF on the tumor cells. Blocking studies, histological assessment, as well as RT-PCR were performed to confirm TF specificity of {sup 64}Cu-NOTA-ALT-836-Fab. ALT-836-Fab was produced with high purity, which exhibited superb TF binding affinity and specificity. Serial PET imaging revealed rapid and persistent tumor uptake of {sup 64}Cu-NOTA-ALT-836-Fab (5.1 ± 0.5 %ID/g at 24 h post-injection; n = 4) and high tumor/muscle ratio (7.0 ± 1.2 at 24 h post-injection; n = 4), several-fold higher than that of the blocking group and tumor models that do not express significant level of TF, which was confirmed by biodistribution studies. TF specificity of the tracer was also validated by histology and RT-PCR. {sup 64}Cu-NOTA-ALT-836-Fab exhibited prominent tissue factor targeting efficiency in MDA-MB-231 TNBC model. The use of a Fab fragment led to fast tumor uptake and good tissue/muscle ratio, which may be translated into same-day immunoPET imaging in the clinical setting to improve TNBC patient management. (orig.)

  11. Agonistic effects of a monoclonal antibody specific for the interleukin-2 receptor

    International Nuclear Information System (INIS)

    Eardley, D.D.; Makrides, V.

    1986-01-01

    Interleukin-2 (IL-2) mediated immune responses can be blocked by monoclonal antibodies to the IL-2 receptor. The monoclonal antibody, M720, is defined as specific for the IL-2 receptor because it blocks 35 S-IL-2 binding to Con A blasts, reacts with lymphoblasts but not resting splenocytes, and inhibits IL-2 induced proliferation to mitogen, antigen, or allogeneic stimuli. Under appropriate culture conditions, the IL-2 receptor-specific antibody can act like IL-2 in that it will induce proliferation in T cells in the absence of additional antigen or mitogen. This agonistic effect is dependent on time, dose of antibody, and requires fetal calf serum (FCS) in the media. Because the FCS is not mitogenic by itself, the authors propose that the FCS components act as incomplete mitogen to induce appearance of IL-2 receptors but lack a factor which would push the majority of the cells into the S phase of the cell cycle. This factor is usually IL-2, but in the authors experiments, the IL-2 receptor-specific antibody can provide the same stimulus. These data indicate that factors like FCS can induce IL-2 receptors, but without additional IL-2 or receptor triggering, the cells will not proceed through the synthetic and proliferative phases of cell growth

  12. Influenza A virus H5-specific antibodies in mute swans (Cygnus olor) in the USA.

    Science.gov (United States)

    Kistler, Whitney M; Stallknecht, David E; Lebarbenchon, Camille; Pedersen, Kerri; Marks, David R; Mickley, Randy; DeLiberto, Thomas J; Yabsley, Michael J

    2015-04-01

    The use of serologic assays for influenza A virus (IAV) surveillance in wild birds has increased because of the availability of commercial enzyme-linked immunosorbent assays (ELISAs). Recently, an H5-specific blocking ELISA (bELISA) was shown to reliably detect H5-specific antibodies to low- and high-pathogenic H5 viruses in experimentally infected waterfowl. Mute Swans (Cygnus olor) were frequently associated with highly pathogenic H5N1 outbreaks in Europe and may have a similar role if highly pathogenic H5N1 is introduced into North America. We measured the prevalence of antibodies to the nucleoprotein and H5 protein in Mute Swans using three serologic assays. We collected 340 serum samples from Mute Swans in Michigan, New Jersey, New York, and Rhode Island, US. We detected antibodies to the IAV nucleoprotein in 66.2% (225/340) of the samples. We detected H5-specific antibodies in 62.9% (214/340) and 18.8% (64/340) using a modified H5 bELISA protocol and hemagglutination inhibition (HI) assay, respectively. The modified H5 bELISA protocol detected significantly more positive samples than did the manufacturer's protocol. We also tested 46 samples using virus neutralization. Neutralization results had high agreement with the modified H5 bELISA protocol and detected a higher prevalence than did the HI assay. These results indicate that North American Mute Swans have high nucleoprotein and H5 antibody prevalences.

  13. Radioimmunoassay of serum antibodies with B-streptococcus specificity in pregnant women and infants

    International Nuclear Information System (INIS)

    Frey, C.W.

    1980-01-01

    In a specific competitive radioimmunoassay of purified rabbit antibodies, labeled with iodine 125 against group- and type-antigens of streptococcus agalactiae (streptococci type B), we investigated the amount of serum anti-bodies providing specificity of streptococci type B in not preselected pregnant women, newborn and babies with colonies of streptococci type B or with diseases due to streptococci type B and in some of their mothers. These antibodies could be detected in 26 of 45 pregnant women and in 3 of 7 children with colonies of streptococci type B. 5 of 18 newborn with the ''early-onset'' type of infection and 6 of 7 of their mothers provided antibodies with specificity of streptococci type B as did one of two newborn with the ''late onset'' type of infection. Contrary to the supposition of Baker and Kasper and in accordance with the findings of Wilkinson, the ''risk group'' cannot be determined only by detecting the antibodies against streptococci type B. The risk group comprises those persons in whom the colonisation of streptococci agalactiae leads to the frequently life-threatening infecton of neonatals with streptococci type B. (orig.) [de

  14. Selection and characterization of naturally occurring single-domain (IgNAR) antibody fragments from immunized sharks by phage display.

    Science.gov (United States)

    Dooley, Helen; Flajnik, Martin F; Porter, Andrew J

    2003-09-01

    The novel immunoglobulin isotype novel antigen receptor (IgNAR) is found in cartilaginous fish and is composed of a heavy-chain homodimer that does not associate with light chains. The variable regions of IgNAR function as independent domains similar to those found in the heavy-chain immunoglobulins of Camelids. Here, we describe the successful cloning and generation of a phage-displayed, single-domain library based upon the variable domain of IgNAR. Selection of such a library generated from nurse sharks (Ginglymostoma cirratum) immunized with the model antigen hen egg-white lysozyme (HEL) enabled the successful isolation of intact antigen-specific binders matured in vivo. The selected variable domains were shown to be functionally expressed in Escherichia coli, extremely stable, and bind to antigen specifically with an affinity in the nanomolar range. This approach can therefore be considered as an alternative route for the isolation of minimal antigen-binding fragments with favorable characteristics.

  15. Single-dilution enzyme-linked immunosorbent assay for quantification of antigen-specific salmonid antibody

    Science.gov (United States)

    Alcorn, S.W.; Pascho, R.J.

    2000-01-01

    An enzyme-linked immunosorbent assay (ELISA) was developed on the basis of testing a single dilution of serum to quantify the level of antibody to the p57 protein of Renibaclerium salmoninarum in sockeye salmon (Oncorhynchus nerka). The levels of antibody were interpolated from a standard curve constructed by relating the optical densities (OD) produced by several dilutions of a high-titer rainbow trout (O. mykiss) antiserum to the p57 protein. The ELISA OD values produced by as many as 36 test sera on each microplate were compared with the standard curve to calculate the antigen-specific antibody activity. Repeated measurements of 36 samples on 3 microplates on each of 6 assay dates indicated that the mean intraassay coefficient of variation (CV) was 6.68% (range, 0-23%) and the mean interassay CV was 8.29% (range, 4-16%). The antibody levels determined for the serum sample from 24 sockeye salmon vaccinated with a recombinant p57 protein generally were correlated with the levels determined by endpoint titration (r2 = 0.936) and with results from another ELISA that was based on extrapolation of antibody levels from a standard curve (r2 = 0.956). The single-dilution antibody ELISA described here increases the number of samples that can be tested on each microplate compared with immunoassays based on analysis of several dilutions of each test serum. It includes controls for interassay standardization and can be used to test fish weighing <3 g.

  16. Subtype-Specific Influenza A Virus Antibodies in Canada Geese (Branta canadensis)

    Science.gov (United States)

    Kistler, Whitney M.; Stallknecht, David E.; DeLiberto, Thomas J.; Van Why, Kyle; Yabsley, Michael J.

    2015-01-01

    Historically, surveillance for influenza A viruses (IAVs) in wild birds has relied on viral detection assays. This was largely due to poor performance of serological assays in wild birds; however, recently developed commercial serological assays have improved the ability to detect IAV antibodies in wild birds. Serological surveillance for IAV antibodies in Canada geese (Branta canadensis) has shown that, despite a low prevalence of virus isolations, Canada geese are frequently exposed to IAVs and that exposure increases with latitude, which follows virus isolation prevalence patterns observed in dabbling ducks. The objectives of this study were to further evaluate IAV antibodies in Canada geese using a subtype-specific serological assay to determine if Canada geese are exposed to subtypes that commonly circulate in dabbling ducks. We collected serum samples from Canada geese in Minnesota, New Jersey, Pennsylvania, and Wisconsin and tested for antibodies to IAVs using a blocking ELISA. Positive samples were further tested by hemagglutination inhibition for 10 hemagglutinin IAV subtypes (H1–H10). Overall, we detected antibodies to NP in 24% (714/2,919) of geese. Antibodies to H3, H4, H5, and H6 subtypes predominated, with H5 being detected most frequently. A decrease in H5 HI antibody prevalence and titers was observed from 2009 to 2012. We also detected similar exposure pattern in Canada geese from New Jersey, Minnesota, Washington and Wisconsin. Based on the published literature, H3, H4, and H6 viruses are the most commonly reported IAVs from dabbling ducks. These results indicate that Canada geese also are frequently exposed to viruses of the same HA subtypes; however, the high prevalence of antibodies to H5 viruses was not expected as H5 IAVs are generally not well represented in reported isolates from ducks. PMID:25845755

  17. [Immunochemical Detection of Azospirilla in Soil with Genus-Specific Antibodies].

    Science.gov (United States)

    Shirokov, A A; Krasov, A I; Selivanov, N Yu; Burygin, G L; Shchegolev, S Yu; Matora, L Yu

    2015-01-01

    Immunoelectrophoresis and immunodiffusion analysis with antibodies to whole intact cells of the type strain of nitrogen-fixing soil bacteria Azospirillum brasilense Sp7 revealed at least three conservative surface immunogenic proteins of azospirilla. Cross-reactions with these proteins made it possible to use the above antibodies for detection of azospirilla as a genus-specific probe conjugated with horseradish peroxidase as an enzymatic label. Direct immune-enzyme analysis of soil suspensions (typical chernozem, Saratov oblast) confirmed applicability of the conjugates based on genus-specific antibodies to the surface proteins of azospirilla for direct detection of this bacterial genus in environmental samples. These results provide a basis for broad application of this method for analysis of Azospirillum occurrence in soil.

  18. Echinococcus granulosus: the potential use of specific radiolabelled antibodies in diagnosis by immunoscintigraphy

    Energy Technology Data Exchange (ETDEWEB)

    Rogan, M.T.; Morris, D.L.; Pritchard, D.I.; Perkins, A.C. (Nottingham Univ. (UK))

    1990-05-01

    Diagnosis of hydatid disease in man is frequently dependent on the imaging of cysts in situ by techniques such as ultrasonography and CAT scans. Such methods are useful but are not specific and can lead to errors in diagnosis. The present work reports preliminary experiments on the development of a specific imaging technique for hydatid cysts using radiolabelled antibodies. A purified preparation of antigen B of hydatid fluid was used to raise polyclonal antisera in rabbits and the resulting affinity-purified IgG labelled with {sup 131}I. Gerbils with an established Echinococcus granulosus infection were injected intraperitoneally with the labelled antibody and imaged 48 h later with a gamma camera. Hydatid cysts could be identified within the peritoneal cavity and post-mortem assessment of activity showed the cysts to contain approximately four times as much activity as the surrounding organs thereby indicating successful targeting of the antibody to the cysts. (author).

  19. Echinococcus granulosus: the potential use of specific radiolabelled antibodies in diagnosis by immunoscintigraphy

    International Nuclear Information System (INIS)

    Rogan, M.T.; Morris, D.L.; Pritchard, D.I.; Perkins, A.C.

    1990-01-01

    Diagnosis of hydatid disease in man is frequently dependent on the imaging of cysts in situ by techniques such as ultrasonography and CAT scans. Such methods are useful but are not specific and can lead to errors in diagnosis. The present work reports preliminary experiments on the development of a specific imaging technique for hydatid cysts using radiolabelled antibodies. A purified preparation of antigen B of hydatid fluid was used to raise polyclonal antisera in rabbits and the resulting affinity-purified IgG labelled with 131 I. Gerbils with an established Echinococcus granulosus infection were injected intraperitoneally with the labelled antibody and imaged 48 h later with a gamma camera. Hydatid cysts could be identified within the peritoneal cavity and post-mortem assessment of activity showed the cysts to contain approximately four times as much activity as the surrounding organs thereby indicating successful targeting of the antibody to the cysts. (author)

  20. Muscle-Specific Tyrosine Kinase and Myasthenia Gravis Owing to Other Antibodies.

    Science.gov (United States)

    Rivner, Michael H; Pasnoor, Mamatha; Dimachkie, Mazen M; Barohn, Richard J; Mei, Lin

    2018-05-01

    Around 20% of patients with myasthenia gravis are acetylcholine receptor antibody negative; muscle-specific tyrosine kinase antibodies (MuSK) were identified as the cause of myasthenia gravis in 30% to 40% of these cases. Anti MuSK myasthenia gravis is associated with specific clinical phenotypes. One is a bulbar form with fewer ocular symptoms. Others show an isolated head drop or symptoms indistinguishable from acetylcholine receptor-positive myasthenia gravis. These patients usually respond well to immunosuppressive therapy, but not as well to cholinesterase inhibitors. Other antibodies associated with myasthenia gravis, including low-density lipoprotein receptor-related protein 4, are discussed. Copyright © 2018 Elsevier Inc. All rights reserved.

  1. Method of stably radiolabeling antibodies with technetium and rhenium

    International Nuclear Information System (INIS)

    Paik, C.H.; Reba, R.C.; Eckelman, W.C.

    1987-01-01

    A method is described for labeling antibodies or antibody fragments with radionuclides of technetium or rhenium to obtain stable labeling, comprising: reacting a reduced radioisotope of technetium or rhenium with an antibody or antibody fragment, or a diethylenetriaminepentaacetic acid conjugated antibody or antibody fragment, in the presence of free or carrier-bound diethylenetriaminepentaacetic acid (DTPA). The amount of DTPA is sufficient to substantially completely inhibit binding of the reduced technetium or rhenium to nonstable binding sites of the antibody or antibody fragment, or the DTPA-conjugated antibody or antibody fragment. The resultant stably labeled antibody or antibody fragment, or DTPA[conjugated antibody or antibody fragment is recovered

  2. 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.

  3. Development and immunochemical evaluation of a novel chicken IgY antibody specific for KLK6

    Directory of Open Access Journals (Sweden)

    Sotiropoulou Georgia

    2012-12-01

    Full Text Available Abstract Background Human kallikrein-related peptidase 6 (KLK6 has been implicated in various types of cancer and in neurodegenerative and demyelinating diseases including multiple sclerosis. Further, anti-KLK6 antibodies attenuated disease manifestations in the mouse model of multiple sclerosis. Availability of specific antibodies against KLK6 is fundamental to the development of improved diagnostic and/or immunotherapeutic applications. Here, we exploited the enhanced immunogenicity of mammalian proteins in avian species to generate a polyclonal antibody against KLK6. Results Chicken were immunized with recombinant KLK6 and antibodies Y (IgYs were purified from egg yolk with a simple procedure and evaluated for KLK6 detection by ELISA and Western blot using recombinant proteins and human cell lysates and supernatants. The anti-KLK6 Y polyclonal exhibited high affinity for KLK6 with a detection limit of 30 fmol. On the other hand, the widely used rabbit polyclonal antibody that was raised against the same recombinant KLK6 had a detection limit of 300 fmol. Moreover, the IgYs did not display any crossreactivity with recombinant KLKs or endogenous KLKs and other cellular proteins. Conclusions Based on its high specificity and sensitivity the developed anti-KLK6 IgY is expected to aid the development of improved diagnostic tools for the detection of KLK6 in biological and clinical samples.

  4. Monoclonal Antibody Analysis and Insecticidal Spectrum of Three Types of Lepidopteran-Specific Insecticidal Crystal Proteins of Bacillus thuringiensis

    Science.gov (United States)

    Höfte, Herman; Van Rie, Jeroen; Jansens, Stefan; Van Houtven, Annemie; Vanderbruggen, Hilde; Vaeck, Mark

    1988-01-01

    We have investigated the protein composition and the insecticidal spectrum of crystals of 29 Bacillus thuringiensis strains active against lepidopteran larvae. All crystals contained proteins of 130 to 140 kilodaltons (kDa) which could be grouped into three types by the molecular weight of the protoxin and the trypsin-activated core fragment. Proteins of the three types showed a characteristic insecticidal spectrum when tested against five lepidopteran species. Type A crystal proteins were protoxins of 130 or 133 kDa, which were processed into 60-kDa toxins by trypsin. Several genes encoding crystal proteins of this type have been cloned and sequenced earlier. They are highly conserved in the N-terminal half of the toxic fragment and were previously classified in three subtypes (the 4.5-, 5.3-, and 6.6-kilobase subtypes) based on the restriction map of their genes. The present study shows that different proteins of these three subtypes were equally toxic against Manduca sexta and Pieris brassicae and had no detectable activity against Spodoptera littoralis. However, the 4.5-, 5.3-, and 6.6-kilobase subtypes differed in their toxicity against Heliothis virescens and Mamestra brassicae. Type B crystal proteins consisted of 140-kDa protoxins with a 55-kDa tryptic core fragment. These were only active against one of the five insect species tested (P. brassicae). The protoxin and the trypsin-activated toxin of type C were 135- and 63-kDa proteins, respectively. Proteins of this type were associated with high toxicity against S. littoralis and M. brassicae. A panel of 35 monoclonal antibodies was used to compare the structural characteristics of crystal proteins of the three different types and subtypes. Each type of protein could be associated with a typical epitope structure, indicating an unambiguous correlation between antigenic structure and insect specificity. Images PMID:16347711

  5. Re-188 labelling of DD-3B6/22 Fab' monoclonal antibody fragment for radio immuno therapy

    International Nuclear Information System (INIS)

    Schmidt, P.F.; Smith, S.V.; Bundesen, P.

    1996-01-01

    The chemical similarity of technetium and rhenium has created much interest in the nuclear medicine field to make a 'matched pair' of radiopharmaceuticals for radioimmuno- diagnosis and therapy. Clinical trials with the 99 mTc-DD-3B6/22 Fab' has shown promise in the diagnosis of ovarian cancer. The design of the analogous therapeutic agent with rhenium-188 (155 keV γ 15 % abundant, β E max 2.1 MeV, T 1/2 17 h) is under investigation. The present study describes the approach taken for direct radiolabelling of the DD-3B6/22 Fab' with carrier-free 188 Re and its biological evaluation in balb/c and nude mice. The effect of temperature, pH and antibody concentration on the amount and rate of transchelation was also evaluated. The final product had a specific activity of 35 mCi/mg with an immunoreactive fraction of 77%. Stability of the product was assessed under various conditions: temperature, presence and absence of an inert atmosphere and presence of ascorbic acid (stabilised). Pharmacokinetics of the final product was evaluated in balb/c and nude mice transplanted with both D-dimer (+Ve) and Glycine (-Ve) beads. Results show that 188 Re DD-3B6/22 Fab' clears rapidly from the blood (α = 2.4 hr, β = 3.5 hr) and is excreted through the renal system. Localisation to subcutaneous antigen beads shows specific uptake to the D-dimer (antigen) beads was achieved within 6 h (0.23% ID) and was maintained for 24 hour post injection. Specificity to antigen implants was 5:1 (P 99m Tc DD-3B6/22 Fab' in mice. The radiolabelling procedures are congenial for therapeutic levels and hence the authors believe that the 188 Re DD-3B6/22 Fab' has some potential for use in treatment of ovarian cancer

  6. Localized conformational interrogation of antibody and antibody-drug conjugates by site-specific carboxyl group footprinting.

    Science.gov (United States)

    Pan, Lucy Yan; Salas-Solano, Oscar; Valliere-Douglass, John F

    Establishing and maintaining conformational integrity of monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) during development and manufacturing is critical for ensuring their clinical efficacy. As presented here, we applied site-specific carboxyl group footprinting (CGF) for localized conformational interrogation of mAbs. The approach relies on covalent labeling that introduces glycine ethyl ester tags onto solvent-accessible side chains of protein carboxylates. Peptide mapping is used to monitor the labeling kinetics of carboxyl residues and the labeling kinetics reflects the conformation or solvent-accessibility of side chains. Our results for two case studies are shown here. The first study was aimed at defining the conformational changes of mAbs induced by deglycosylation. We found that two residues in C H 2 domain (D268 and E297) show significantly enhanced side chain accessibility upon deglycosylation. This site-specific result highlighted the advantage of monitoring the labeling kinetics at the amino acid level as opposed to the peptide level, which would result in averaging out of highly localized conformational differences. The second study was designed to assess conformational effects brought on by conjugation of mAbs with drug-linkers. All 59 monitored carboxyl residues displayed similar solvent-accessibility between the ADC and mAb under native conditions, which suggests the ADC and mAb share similar side chain conformation. The findings are well correlated and complementary with results from other assays. This work illustrated that site-specific CGF is capable of pinpointing local conformational changes in mAbs or ADCs that might arise during development and manufacturing. The methodology can be readily implemented within the industry to provide comprehensive conformational assessment of these molecules.

  7. The metabolic enhancer piracetam attenuates mitochondrion-specific endonuclease G translocation and oxidative DNA fragmentation.

    Science.gov (United States)

    Gupta, Sonam; Verma, Dinesh Kumar; Biswas, Joyshree; Rama Raju, K Siva; Joshi, Neeraj; Wahajuddin; Singh, Sarika

    2014-08-01

    This study was performed to investigate the involvement of mitochondrion-specific endonuclease G in piracetam (P)-induced protective mechanisms. Studies have shown the antiapoptotic effects of piracetam but the mechanism of action of piracetam is still an enigma. To assess the involvement of endonuclease G in piracetam-induced protective effects, astrocyte glial cells were treated with lipopolysaccharide (LPS) and piracetam. LPS treatment caused significantly decreased viability, mitochondrial activity, oxidative stress, chromatin condensation, and DNA fragmentation, which were attenuated by piracetam cotreatment. Cotreatment of astrocytes with piracetam showed its significantly time-dependent absorption as observed with high-performance liquid chromatography. Astrocytes treated with piracetam alone showed enhanced mitochondrial membrane potential (MMP) in comparison to control astrocytes. However, in LPS-treated cells no significant alteration in MMP was observed in comparison to control cells. Protein and mRNA levels of the terminal executor of the caspase-mediated pathway, caspase-3, were not altered significantly in LPS or LPS + piracetam-treated astrocytes, whereas endonuclease G was significantly translocated to the nucleus in LPS-treated astrocytes. Piracetam cotreatment attenuated the LPS-induced endonuclease G translocation. In conclusion this study indicates that LPS treatment of astrocytes caused decreased viability, oxidative stress, mitochondrial dysfunction, chromatin condensation, DNA damage, and translocation of endonuclease G to the nucleus, which was inhibited by piracetam cotreatment, confirming that the mitochondrion-specific endonuclease G is one of the factors involved in piracetam-induced protective mechanisms. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Characterization of ductal and lobular breast carcinomas using novel prolactin receptor isoform specific antibodies

    Directory of Open Access Journals (Sweden)

    Heger Christopher D

    2010-12-01

    Full Text Available Abstract Background Prolactin is a polypeptide hormone responsible for proliferation and differentiation of the mammary gland. More recently, prolactin's role in mammary carcinogenesis has been studied with greater interest. Studies from our laboratory and from others have demonstrated that three specific isoforms of the prolactin receptor (PRLR are expressed in both normal and cancerous breast cells and tissues. Until now, reliable isoform specific antibodies have been lacking. We have prepared and characterized polyclonal antibodies against each of the human PRLR isoforms that can effectively be used to characterize human breast cancers. Methods Rabbits were immunized with synthetic peptides of isoform unique regions and immune sera affinity purified prior to validation by Western blot and immunohistochemical analyses. Sections of ductal and lobular carcinomas were stained with each affinity purified isoform specific antibody to determine expression patterns in breast cancer subclasses. Results We show that the rabbit antibodies have high titer and could specifically recognize each isoform of PRLR. Differences in PRLR isoform expression levels were observed and quantified using histosections from xenografts of established human breast cancer cells lines, and ductal and lobular carcinoma human biopsy specimens. In addition, these results were verified by real-time PCR with isoform specific primers. While nearly all tumors contained LF and SF1b, the majority (76% of ductal carcinoma biopsies expressed SF1a while the majority of lobular carcinomas lacked SF1a staining (72% and 27% had only low levels of expression. Conclusions Differences in the receptor isoform expression profiles may be critical to understanding the role of PRL in mammary tumorigenesis. Since these antibodies are specifically directed against each PRLR isoform, they are valuable tools for the evaluation of breast cancer PRLR content and have potential clinical importance in

  9. Screening for hen's egg and chicken meat specific IgE antibodies in ...

    African Journals Online (AJOL)

    Background: Allergy to hen's egg and meat contributes significantly to the manifestations of food allergy all over the world. Objectives: This study was performed to assess the presence of hen's egg and meat specific IgE antibodies among patients investigated for various allergic disorders. Methods. This is a retrospective ...

  10. Generating Isoform-Specific Antibodies : Lessons from Nucleocytoplasmic Glycoprotein Skp1

    NARCIS (Netherlands)

    West, Christopher M.; Van Der Wel, Hanke; Chinoy, Zoiesha; Boons, Geert Jan; Gauthier, Ted J.; Taylor, Carol M.; Xu, Yuechi

    2015-01-01

    Antibodies that discriminate protein isoforms differing by modifications at specific amino acids have revolutionized studies of their functions. Skp1 is a novel nucleocytoplasmic glycoprotein that is hydroxylated at proline-143 and then O-glycosylated by a pentasaccharide attached via a GlcNAcα1,

  11. Docking of B-cell epitope antigen to specific hepatitis B antibody

    Indian Academy of Sciences (India)

    The interaction of pres1 region of hepatitis B virus B-cell epitope antigen with specific hepatitis B neutralizing monoclonal antibody was examined by docking study. We modelled the 3D complex structure of B-cell epitope antigen residues CTTPAQGNSMFPSCCCTKPTDGNCY by homology modelling and docked it with the ...

  12. Development of an Enzyme Linked Immunosorbent Assay to Detect Chicken Parvovirus Specific Antibodies

    Science.gov (United States)

    Here we report the development and application of an enzyme linked immunosorbent assay to detect parvovirus-specific antibodies in chicken sera. We used an approach previously described for other parvoviruses to clone and express viral structural proteins in insect cells from recombinant baculovirus...

  13. The schistosoma-specific antibody response after treatment in non-immune travellers

    DEFF Research Database (Denmark)

    Duus, Liv Marie; Christensen, Anders Vittrup; Navntoft, Dorte

    2009-01-01

    Egg detection is the gold standard in diagnosing and controlling treatment in schistosomiasis, but sensitivity is poor in lightly infected individuals, whereas Schistosoma-specific antibodies are more sensitive. The purpose of the study was to evaluate use of Gut Associated Antigen (GAA...

  14. A sensitive chain specific radioimmunoassay for human immunoglobulins using monoclonal antibodies

    International Nuclear Information System (INIS)

    Sikora, K.; Alderson, T.St.J.; Ellis, J.

    1983-01-01

    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. (Auth.)

  15. Directional Selection for Specific Sheep Cell Antibody Responses Affects Natural Rabbit Agglutinins of Chickens

    NARCIS (Netherlands)

    Cotter, P.F.; Ayoub, J.; Parmentier, H.K.

    2005-01-01

    Agglutination data from generations 8 through 19 indicate that bidirectional selection for specific SRBC antibody responses was successful in a line cross of ISA × Warren medium heavy layers. After 11 generations titers of the high SRBC selected line (H line) were nearly 1:32,000; those of the low

  16. Development of a dipstick assay for detection of Leishmania-specific canine antibodies

    NARCIS (Netherlands)

    Schallig, Henk D. F. H.; Cardoso, Luís; Hommers, Marieke; Kroon, Nel; Belling, Guus; Rodrigues, Manuela; Semião-Santos, Saul J.; Vetter, Hans

    2004-01-01

    A dipstick assay, based on Leishmania infantum antigen, for the rapid detection of Leishmania-specific antibodies in canine serum samples was developed and evaluated. After determination of optimal dipstick test conditions, test performance was compared with two existing serological tests, i.e., the

  17. Determining Vaccination Frequency in Farmed Rainbow Trout Using Vibrio anguillarum O1 Specific Serum Antibody Measurements

    DEFF Research Database (Denmark)

    Holten-Andersen, Lars; Dalsgaard, Inger; Nylén, Jørgen

    2012-01-01

    Background Despite vaccination with a commercial vaccine with a documented protective effect against Vibrio anguillarum O1 disease outbreaks caused by this bacterium have been registered among rainbow trout at Danish fish farms. The present study examined specific serum antibody levels as a valid...

  18. Evaluation of six immunoassays for detection of dengue virus-specific immunoglobulin M and G antibodies

    NARCIS (Netherlands)

    J. Groen (Jan); P. Koraka (Penelope); J. Velzing (Jans); C. Copra (Cederick); A.D.M.E. Osterhaus (Albert)

    2000-01-01

    textabstractThe performance of six commercially available immunoassay systems for the detection of dengue virus-specific immunoglobulin M (IgM) and IgG antibodies in serum was evaluated. These included two IgM and IgG enzyme immunoassays (EIA) from MRL Laboratories and PanBio, a rapid

  19. Screening for hen's egg and chicken meat specific IgE antibodies in ...

    African Journals Online (AJOL)

    Abstract. Background: Allergy to hen's egg and meat contributes significantly to the manifestations of food allergy all over the world. Objectives: This study was performed to assess the presence of hen's egg and meat specific IgE antibodies among patients investigated for various allergic disorders. Methods. This is a ...

  20. The effects of variations in the specificities of the antibody components on a two-site immunoradiometric assay for ferritin

    International Nuclear Information System (INIS)

    Cowan, S.I.; Stagg, B.H.; Niemann, E.

    1977-01-01

    Variations in the sub-unit antigenic structure of ferritins derived from various human tissues are reflected in the differing specificities of antisera raised against these ferritin preparations. In this study it was shown that antibody specificity played an important role in determining the sensitivity and overall binding of labelled antibody in a two-site immunoradiometric assay for ferritin. Homologous assay systems, in which solid phase and radiolabelled antibodies were of similar specificities, were generally less sensitive and showed lower binding than heterologous assay systems, in which solid phase and labelled antibodies were of different specificities. The source of the ferritin which was used as assay standard also played an important part in determining the sensitivity and overall binding in homologous antibody systems, spleen ferritin standards yielding assays superior to those obtained with placenta or liver ferritin standards. However, these differences between standards were not seen in a heterologous system employing solid phase antibodies directed against liver ferritin and labelled antibodies directed against placenta ferritin. The nature of the ferritin used to prepare immunoadsorbant for the purification of antibodies prior to radioiodination also affected the assay characteristics; antibodies prepared on spleen ferritin immunoadsorbant being more reactive than antibodies prepared on placenta ferritin immunoadsorbant, which in turn were more reactive then antibodies prepared on liver ferritin immunoadsorbant. (orig.) [de

  1. Radioimmunoassay of class-specific antibodies to Streptococcus mutans in monkey serum and saliva

    International Nuclear Information System (INIS)

    Walker, J.; Colman, G.; Huges, M.

    1979-01-01

    A radioimmunoassay (RIA) has been developed to measure class-specific antibodies to Streptococcus mutans in the serum and saliva of monkeys (Macaca fascicularis). Antihuman immunoglobulin antibodies purified by affinity chromatography on immobilised monkey immoglobulins and labelled with 125 I were employed. Formalised cells of S. mutans and an extract of culture supernatant adsorbed to polystyrene wells were used as solid-phase antigens. The coefficients of variation of IgG, IgA, and IgM assays were less than or equal to 10% for both antigen systems. It is shown that this RIA is a sensitive, reproducible and quantitative method. (Auth.)

  2. Radioimmunoassay for the detection of virus-specific IgA antibodies in saliva

    International Nuclear Information System (INIS)

    Friedman, M.G.

    1982-01-01

    The use of a sensitive and versatile radioimmunoassay (RIA) for detection of mumps-specific IgA and measles-specific IgA in unconcentrated saliva samples is described. The samples were obtained either by expectoration or by swabbing of the oral cavity, with or without stimulation of secretion, and were inactivated and clarified before testing. Mumps-specific IgA antibodies were detected as early as one day after onset of illness and peaked at 1-2 weeks after onset. Measles-specific salivary IgA antibodies were detected in 15-month old children 2-3 weeks after immunization. These results suggest that the RIA technique may be useful for early diagnosis of viral infections and for confirmation of response to immunization without the need for a blood sample, as well as for the study of the secretory immune response in very young and older subjects. (Auth.)

  3. Fc-specific biotinylation of antibody using an engineered photoactivatable Z-Biotin and its biosensing application.

    Science.gov (United States)

    Yang, Hong-Ming; Bao, Ru-Meng; Yu, Chang-Mei; Lv, Yan-Na; Zhang, Wei-Fen; Tang, Jin-Bao

    2017-01-01

    The development of a site-specific and covalent attachment methodology is crucial for antibody-biotin conjugates to preserve the antigen-binding ability of antibodies and yield homogeneous products. In this study, an engineered photoactivatable Z-domain variant [an UV-active amino acid benzoylphenylalanine (Bpa) was genetically incorporated into the Z-domain] carrying one biotin molecule (Z Bpa -Biotin) was prepared by employing aminoacyl-tRNA synthetase/suppressor tRNA and Avitag/BirA techniques. The site-specific and covalent attachment of IgG-biotin conjugates, viz. photo-biotinylated IgG, was successfully achieved after UV exposure by combining the inherent Fc-binding capability of the Z-domain with the formation of covalent bond by the photo-crosslinker. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis assay showed that more than 90% of IgGs conjugated with Z Bpa -Biotin molecules suffered 3 h UV irradiation. Further pepsin digestion analysis confirmed that the Z Bpa -Biotin was conjugated to the Fc fragment of IgG without interference. We took the tumor biomarker carcinoembryoic antigen (CEA) as model to evaluate the detection efficiency of the site-specific photo-biotinylated IgG in biosensing application using surface plasmon resonance (SPR) technology. The photo-biotinylated IgG coated surface gave a limit of detection (LOD) of 2 ng mL -1 , is 5-fold lower than that of the randomly NHS-biotinylated IgG (10 ng mL -1 ). Given that the (strept)avidin-biotin complex is extensively used in immunoassays, the proposed method for biotinylated IgG provides a powerful approach to further expand related applications. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Variable Domain N-Linked Glycans Acquired During Antigen-Specific Immune Responses Can Contribute to Immunoglobulin G Antibody Stability

    Directory of Open Access Journals (Sweden)

    Fleur S. van de Bovenkamp

    2018-04-01

    Full Text Available Immunoglobulin G (IgG can contain N-linked glycans in the variable domains, the so-called Fab glycans, in addition to the Fc glycans in the CH2 domains. These Fab glycans are acquired following introduction of N-glycosylation sites during somatic hypermutation and contribute to antibody diversification. We investigated whether Fab glycans may—in addition to affecting antigen binding—contribute to antibody stability. By analyzing thermal unfolding profiles of antibodies with or without Fab glycans, we demonstrate that introduction of Fab glycans can improve antibody stability. Strikingly, removal of Fab glycans naturally acquired during antigen-specific immune responses can deteriorate antibody stability, suggesting in vivo selection of stable, glycosylated antibodies. Collectively, our data show that variable domain N-linked glycans acquired during somatic hypermutation can contribute to IgG antibody stability. These findings indicate that introducing Fab glycans may represent a mechanism to improve therapeutic/diagnostic antibody stability.

  5. Immunoscintigraphy of adenocarcinomas by means of 111In-labelled F(ab')2 fragments of anti-CEA monoclonal antibody F023C5

    International Nuclear Information System (INIS)

    Riva, P.; Paganelli, G.; Callegaro, L.

    1988-01-01

    F(ab') 2 fragments of F023C5, an anti-CEA monoclonal antibody, were conjugated to diethylenetriamine pentaacetic acid (DTPA) and converted into a ready to use reagent for instant 111 In-labelling. The resulting 111 In radiopharmaceutical was administered intravenously and tested for its ability to image (at 48-72 h after administration) 31 primary and 85 metastatic carcinoma lesions in 70 adenocarcinoma patients (26 gastrointestinal, 18 breast and 26 lung tumour patients) whose serum CEA was elevated in 43 cases and normal in the other 27. (author)

  6. Experimental study on 211At labelled monoclonal antibody 3H11 and its Fab fragment radioimmunotherapy for human gastric cancer xenografts in nude mice

    International Nuclear Information System (INIS)

    Jin Jiannan; Liu Ning; Zhang Shuyuan; Zhang Shiyuan; Luo Deyuan; Zhou Maolun

    1996-01-01

    Experimental radioimmunotherapy investigation of α-emitting radionuclide 211 At labelled anti-gastric cancer monoclonal antibody 3H11 and its Fab fragment for nude mice carrying human gastric cancer xenografts was conducted. Three i.p. injections of 14.8 or 22.2 kBq/g mouse were given, once every 5 days. The results showed that the growth of tumor xenografts was inhibited efficiently. The most evident therapy effect was observed at 15 days after treatment, and the tumor inhibition rates were 65% and 72%, respectively. No radiation injury of important organs was found

  7. Analyzing Protein Changes in Guinea Pig Tissue Lysates Using Non-guinea Pig Specific Antibodies: Procedures for Western Blotting and Examples Using 16 Individual Antibodies for Common CNS Proteins

    National Research Council Canada - National Science Library

    Johnson, Erik A; Daugherty, Kelly S

    2006-01-01

    ... behavioral and protein changes due to the absence of guinea pig-specific antibodies. We have developed a procedure to determine the specificity of commercially available, non-guinea pig-specific antibodies in guinea pig lysates...

  8. In-depth analysis of subclass-specific conformational preferences of IgG antibodies

    Directory of Open Access Journals (Sweden)

    Xinsheng Tian

    2015-01-01

    Full Text Available IgG subclass-specific differences in biological function and in vitro stability are often referred to variations in the conformational flexibility, while this flexibility has rarely been characterized. Here, small-angle X-ray scattering data from IgG1, IgG2 and IgG4 antibodies, which were designed with identical variable regions, were thoroughly analysed by the ensemble optimization method. The extended analysis of the optimized ensembles through shape clustering reveals distinct subclass-specific conformational preferences, which provide new insights for understanding the variations in physical/chemical stability and biological function of therapeutic antibodies. Importantly, the way that specific differences in the linker region correlate with the solution structure of intact antibodies is revealed, thereby visualizing future potential for the rational design of antibodies with designated physicochemical properties and tailored effector functions. In addition, this advanced computational approach is applicable to other flexible multi-domain systems and extends the potential for investigating flexibility in solutions of macromolecules by small-angle X-ray scattering.

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

    International Nuclear Information System (INIS)

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

    1984-01-01

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

  10. Wildtype p53-specific Antibody and T-Cell Responses in Cancer Patients

    DEFF Research Database (Denmark)

    Pedersen, Anders Elm; Stryhn, Anette; Justesen, Sune

    2011-01-01

    patients. Detection of antibodies against wt p53 protein has been used as a diagnostic and prognostic marker and discovery of new T-cell epitopes has enabled design of cancer vaccination protocols with promising results. Here, we identified wt p53-specific antibodies in various cancer patients......(264-272) in breast cancer patients and against HLA-A*01:01 binding peptide wt p53(226-234) and HLA-B*07:02 binding peptide wt p53(74-82) in renal cell cancer and breast cancer patients, respectively. Finally, we analyzed antibody and T-cell responses against wt p53 15-mer peptides in patients with metastatic renal...

  11. Antibody fragments directed against different portions of the human neural cell adhesion molecule L1 act as inhibitors or activators of L1 function.

    Directory of Open Access Journals (Sweden)

    Yan Wang

    Full Text Available The neural cell adhesion molecule L1 plays important roles in neuronal migration and survival, neuritogenesis and synaptogenesis. L1 has also been found in tumors of different origins, with levels of L1 expression correlating positively with the metastatic potential of tumors. To select antibodies targeting the varied functions of L1, we screened the Tomlinson library of recombinant human antibody fragments to identify antibodies binding to recombinant human L1 protein comprising the entire extracellular domain of human L1. We obtained four L1 binding single-chain variable fragment antibodies (scFvs, named I4, I6, I13, and I27 and showed by enzyme-linked immunosorbent assay (ELISA that scFvs I4 and I6 have high affinity to the immunoglobulin-like (Ig domains 1-4 of L1, while scFvs I13 and I27 bind strongly to the fibronectin type III homologous (Fn domains 1-3 of L1. Application of scFvs I4 and I6 to human SK-N-SH neuroblastoma cells reduced proliferation and transmigration of these cells. Treatment of SK-N-SH cells with scFvs I13 and I27 enhanced cell proliferation and migration, neurite outgrowth, and protected against the toxic effects of H(2O(2 by increasing the ratio of Bcl-2/Bax. In addition, scFvs I4 and I6 inhibited and scFvs I13 and I27 promoted phosphorylation of src and Erk. Our findings indicate that scFvs reacting with the immunoglobulin-like domains 1-4 inhibit L1 functions, whereas scFvs interacting with the fibronectin type III domains 1-3 trigger L1 functions of cultured neuroblastoma cells.

  12. Chemokine Receptor-Specific Antibodies in Cancer Immunotherapy: Achievements and Challenges

    Science.gov (United States)

    Vela, Maria; Aris, Mariana; Llorente, Mercedes; Garcia-Sanz, Jose A.; Kremer, Leonor

    2015-01-01

    The 1990s brought a burst of information regarding the structure, expression pattern, and role in leukocyte migration and adhesion of chemokines and their receptors. At that time, the FDA approved the first therapeutic antibodies for cancer treatment. A few years later, it was reported that the chemokine receptors CXCR4 and CCR7 were involved on directing metastases to liver, lung, bone marrow, or lymph nodes, and the over-expression of CCR4, CCR6, and CCR9 by certain tumors. The possibility of inhibiting the interaction of chemokine receptors present on the surface of tumor cells with their ligands emerged as a new therapeutic approach. Therefore, many research groups and companies began to develop small molecule antagonists and specific antibodies, aiming to neutralize signaling from these receptors. Despite great expectations, so far, only one anti-chemokine receptor antibody has been approved for its clinical use, mogamulizumab, an anti-CCR4 antibody, granted in Japan to treat refractory adult T-cell leukemia and lymphoma. Here, we review the main achievements obtained with anti-chemokine receptor antibodies for cancer immunotherapy, including discovery and clinical studies, proposed mechanisms of action, and therapeutic applications. PMID:25688243

  13. Rise and fall of an anti-MUC1 specific antibody.

    Directory of Open Access Journals (Sweden)

    Holger Thie

    2011-01-01

    Full Text Available So far, human antibodies with good affinity and specificity for MUC1, a transmembrane protein overexpressed on breast cancers and ovarian carcinomas, and thus a promising target for therapy, were very difficult to generate.A human scFv antibody was isolated from an immune library derived from breast cancer patients immunised with MUC1. The anti-MUC1 scFv reacted with tumour cells in more than 80% of 228 tissue sections of mamma carcinoma samples, while showing very low reactivity with a large panel of non-tumour tissues. By mutagenesis and phage display, affinity of scFvs was increased up to 500fold to 5,7×10(-10 M. Half-life in serum was improved from below 1 day to more than 4 weeks and was correlated with the dimerisation tendency of the individual scFvs. The scFv bound to T47D and MCF-7 mammalian cancer cell lines were recloned into the scFv-Fc and IgG format resulting in decrease of affinity of one binder. The IgG variants with the highest affinity were tested in mouse xenograft models using MCF-7 and OVCAR tumour cells. However, the experiments showed no significant decrease in tumour growth or increase in the survival rates. To study the reasons for the failure of the xenograft experiments, ADCC was analysed in vitro using MCF-7 and OVCAR3 target cells, revealing a low ADCC, possibly due to internalisation, as detected for MCF-7 cells.Antibody phage display starting with immune libraries and followed by affinity maturation is a powerful strategy to generate high affinity human antibodies to difficult targets, in this case shown by the creation of a highly specific antibody with subnanomolar affinity to a very small epitope consisting of four amino acids. Despite these "best in class" binding parameters, the therapeutic success of this antibody was prevented by the target biology.

  14. Monoclonal antibody-dendrimer conjugates enable radiolabeling of antibody with markedly high specific activity with minimal loss of immunoreactivity

    Energy Technology Data Exchange (ETDEWEB)

    Kobayashi, H.; Togashi, K. [Kyoto Univ. (Japan). School of Medicine; Sato, N.; Saga, T.; Nakamoto, Y.; Ishimori, T.; Konishi, J. [Dept. of Nuclear Medicine and Medical Imaging, Kyoto Univ. Graduate School of Medicine, Kyoto (Japan); Toyama, S. [Inst. for Virus Research, Kyoto Univ., Kyoto (Japan); Brechbiel, M.W. [Chemistry Section, Radiation Oncology Branch, National Cancer Inst., National Inst. of Health, Bethesda, Md. (United States)

    2000-09-01

    For the purpose of radioimmunotherapy, labelling of monoclonal antibody with high specific activity is often necessary, especially when using a radionuclide with a shorter half-life. Polyamine dendrimers (PAMAM) are novel synthetic polymeric molecules with large numbers of amine residues on their spherical surface. In order to bind large numbers of radiometals to single antibody molecules, the generation-4 PAMAM (G4), which has 64 amines, was conjugated with 43 molecules of 2-(p-isothiocyanatobenzyl)-6-methyl-diethylene triamine penta-acetic acid (1B4M), a derivative of DTPA. This product [G4-(1B4M){sub 43}] was then conjugated with OST7, a murine monoclonal IgG{sub 1}. We evaluated the achievable specific activity for {sup 111}In labeling, immunore-activity, biodistribution, and tumor targeting in mice of the {sup 111}In- or {sup 153}Gd-OST7-G4-(1B4M){sub 43} as compared with radiolabeled OST7-1B4M or 56C-1B4M. The maximum specific activity of {sup 111}In-OST7-G4-(1B4M){sub 43} and {sup 111}In-OST7-1B4M was 470 and 8.7 GBq/mg (12,700 and 263 mCi/mg), respectively. Immunoreactivity of radiolabeled OST7-G4-(1B4M){sub 43} and OST7-1B4M, as determined by the binding to KT005 cells expressing the antigen, was respectively 91% and 84% of that of {sup 125}I-labelled OST7. Biodistribution studies for preparations with maximum specific activity in normal mice 3 h after injection showed that {sup 111}In- or {sup 153}Gd-OST7-G4-(1B4M){sub 43} cleared faster from the blood and accumulated more in the liver than did {sup 111}In- or {sup 153}Gd-OST7-1B4M. The dendrimer 1B4M [G4-(1B4M){sub 64}] itself showed similar saturation effects with metals. The radioactivity in all the other organs reflected the rapid clearance of radioactivity from the blood. {sup 153}Gd-OST7-G4-(1B4M){sub 43} showed specific accumulation in the KT005 tumor. In conclusion, we could successfully bind 49 times as many metal atoms to an antibody molecule as is possible with conventional metal labeling for

  15. 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.

  16. Re-engineering therapeutic antibodies for Alzheimer's disease as blood-brain barrier penetrating bi-specific antibodies.

    Science.gov (United States)

    Pardridge, William M

    2016-12-01

    Therapeutic antibodies are large molecule drugs that do not cross the blood-brain barrier (BBB). Therefore, drug development of therapeutic antibodies for Alzheimer's disease (AD) requires that these molecules be re-engineered to enable BBB delivery. This is possible by joining the therapeutic antibody with a transporter antibody, resulting in the engineering of a BBB-penetrating bispecific antibody (BSA). Areas covered: The manuscript covers transporter antibodies that cross the BBB via receptor-mediated transport systems on the BBB, such as the insulin receptor or transferrin receptor. Furthermore, it highlights therapeutic antibodies for AD that target the Abeta amyloid peptide, beta secretase-1, or the metabotropic glutamate receptor-1. BSAs are comprised of both the transporter antibody and the therapeutic antibody, as well as IgG constant region, which can induce immune tolerance or trigger transport via Fc receptors. Expert opinion: Multiple types of BSA molecular designs have been used to engineer BBB-penetrating BSAs, which differ in valency and spatial orientation of the transporter and therapeutic domains of the BSA. The plasma pharmacokinetics and dosing regimens of BSAs differ from that of conventional therapeutic antibodies. BBB-penetrating BSAs may be engineered in the future as new treatments of AD, as well as other neural disorders.

  17. Drug delivery systems--2. Site-specific drug delivery utilizing monoclonal antibodies.

    Science.gov (United States)

    Ranade, V V

    1989-10-01

    Monoclonal antibodies (MAbs) are purified antibodies produced by a single clone of cells. They are engineered to recognize and bind to a single specific antigen. Accordingly, when administered, MAbs home in on a particular circulating protein or on cells that bear the correct antigenic signature on their surfaces. It is the specificity of MAbs that has made them valuable tools for health professions. Following the discovery of Kohler and Milstein regarding the method of somatic cell hybridization, a number of investigators have successfully adopted this technique to obtain T-lymphocyte hybrid cell lines by fusion of activated T (thymus derived) lymphocytes with a T lymphoma cell line leading to an immortalization of a specific differentiated function. The hybrids thus obtained were subsequently shown to produce homogeneous effector molecules with a wide variety of immune functions such as enhancement or suppression of antibody responses, generation of helper T cells, suppressor T cells and cytotoxic T cells. Study of these regulatory molecules has been further shown to provide a greater insight into the genetic, biochemical and molecular mechanisms responsible for cellular development, and the interaction and triggering of various cell types. The successful application of hybridoma technology has now resulted into several advances in the understanding the mechanism and treatment of diseases, especially cancer and development of vaccines, promotion of organ transplantation and therapy against parasites as well. Since monoclonal antibodies could be made in unlimited supply, they have been used in genetic studies such as mRNA and gene isolation, chromosomal isolation of specific genes, immunoglobulin structure, detection of new or rare immunoglobulin gene products, structural studies of enzymes and other proteins and structural and population studies of protein polymorphisms. In some instances, the monoclonal antibodies have been found to replace conventional antisera

  18. PET Imaging of Macrophage Mannose Receptor-Expressing Macrophages in Tumor Stroma Using 18F-Radiolabeled Camelid Single-Domain Antibody Fragments.

    Science.gov (United States)

    Blykers, Anneleen; Schoonooghe, Steve; Xavier, Catarina; D'hoe, Kevin; Laoui, Damya; D'Huyvetter, Matthias; Vaneycken, Ilse; Cleeren, Frederik; Bormans, Guy; Heemskerk, Johannes; Raes, Geert; De Baetselier, Patrick; Lahoutte, Tony; Devoogdt, Nick; Van Ginderachter, Jo A; Caveliers, Vicky

    2015-08-01

    Tumor-associated macrophages constitute a major component of the stroma of solid tumors, encompassing distinct subpopulations with different characteristics and functions. We aimed to identify M2-oriented tumor-supporting macrophages within the tumor microenvironment as indicators of cancer progression and prognosis, using PET imaging. This can be realized by designing (18)F-labeled camelid single-domain antibody fragments (sdAbs) specifically targeting the macrophage mannose receptor (MMR), which has been identified as an important biomarker on this cell population. Cross-reactive anti-MMR sdAbs were generated after immunization of an alpaca with the extracellular domains of both human and mouse MMR. The lead binder was chosen on the basis of comparisons of binding affinity and in vivo pharmacokinetics. The PET tracer (18)F-fluorobenzoate (FB)-anti-MMR sdAb was developed using the prosthetic group N-succinimidyl-4-(18)F-fluorobenzoate ((18)F-SFB), and its biodistribution, tumor-targeting potential, and specificity in terms of macrophage and MMR targeting were evaluated in mouse tumor models. Four sdAbs were selected after affinity screening, but only 2 were found to be cross-reactive for human and mouse MMR. The lead anti-MMR 3.49 sdAb, bearing an affinity of 12 and 1.8 nM for mouse and human MMR, respectively, was chosen for its favorable in vivo biodistribution profile and tumor-targeting capacity. (18)F-FB-anti-MMR 3.49 sdAb was synthesized with a 5%-10% radiochemical yield using an automated and optimized protocol. In vivo biodistribution analyses showed fast clearance via the kidneys and retention in MMR-expressing organs and tumor. The kidney retention of the fluorinated sdAb was 20-fold lower than a (99m)Tc-labeled counterpart. Compared with MMR- and C-C chemokine receptor 2-deficient mice, significantly higher uptake was observed in tumors grown in wild-type mice, demonstrating the specificity of the (18)F tracer for MMR and macrophages, respectively. Anti

  19. Specific Antibodies Reacting with SV40 Large T Antigen Mimotopes in Serum Samples of Healthy Subjects.

    Directory of Open Access Journals (Sweden)

    Mauro Tognon

    Full Text Available Simian Virus 40, experimentally assayed in vitro in different animal and human cells and in vivo in rodents, was classified as a small DNA tumor virus. In previous studies, many groups identified Simian Virus 40 sequences in healthy individuals and cancer patients using PCR techniques, whereas others failed to detect the viral sequences in human specimens. These conflicting results prompted us to develop a novel indirect ELISA with synthetic peptides, mimicking Simian Virus 40 capsid viral protein antigens, named mimotopes. This immunologic assay allowed us to investigate the presence of serum antibodies against Simian Virus 40 and to verify whether Simian Virus 40 is circulating in humans. In this investigation two mimotopes from Simian Virus 40 large T antigen, the viral replication protein and oncoprotein, were employed to analyze for specific reactions to human sera antibodies. This indirect ELISA with synthetic peptides from Simian Virus 40 large T antigen was used to assay a new collection of serum samples from healthy subjects. This novel assay revealed that serum antibodies against Simian Virus 40 large T antigen mimotopes are detectable, at low titer, in healthy subjects aged from 18-65 years old. The overall prevalence of reactivity with the two Simian Virus 40 large T antigen peptides was 20%. This new ELISA with two mimotopes of the early viral regions is able to detect in a specific manner Simian Virus 40 large T antigen-antibody responses.

  20. Donor-specific anti-HLA antibodies with antibody-mediated rejection and long-term outcomes following heart transplantation.

    Science.gov (United States)

    Clerkin, Kevin J; Farr, Maryjane A; Restaino, Susan W; Zorn, Emmanuel; Latif, Farhana; Vasilescu, Elena R; Marboe, Charles C; Colombo, Paolo C; Mancini, Donna M

    2017-05-01

    Donor-specific anti-HLA antibodies (DSA) are common after heart transplantation and are associated with rejection, cardiac allograft vasculopathy, and mortality. A noninvasive diagnostic test for pathologic antibody-mediated rejection (pAMR) does not exist. From January 1, 2010, through August 31, 2013, 221 consecutive adult patients underwent heart transplantation and were followed through October 1, 2015. The primary objective was to determine whether the presence of DSA could detect AMR at the time of pathologic diagnosis. Secondary analyses included association of DSA (stratified by major histocompatibility complex class and de novo status) during AMR with new graft dysfunction, graft loss (mortality or retransplantation), and development of cardiac allograft vasculopathy. During the study period, 69 patients (31.2%) had DSA (24% had de novo DSA), and there were 74 episodes of pAMR in 38 patients. Sensitivity of DSA at any mean fluorescence intensity to detect concurrent pAMR was only 54.3%. The presence of any DSA during pAMR increased the odds of graft dysfunction (odds ratio = 5.37; 95% confidence interval [CI], 1.34-21.47; p = 0.018), adjusting for age, sex, and timing of AMR. Circulating class II DSA after transplantation increased risk of future pAMR (hazard ratio = 2.97; 95% CI, 1.31-6.73; p = 0.009). Patients who developed de novo class II DSA had 151% increased risk of graft loss (contingent on 30-day survival) compared with patients who did not have DSA (95% CI, 1.11-5.69; p = 0.027). DSA were inadequate to diagnose pAMR. Class II DSA provided prognostic information regarding future pAMR, graft dysfunction with pAMR, and graft loss. Copyright © 2017 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.

  1. Tumor detection using radiolabeled monoclonal antibodies

    International Nuclear Information System (INIS)

    Moldofsky, P.J.; Powe, J.; Hammond, N.D.

    1987-01-01

    Radioisotope conjugated to monoclonal antibody products has been used for imaging tumors targeted by the antibody. As imaging progresses, new sets of procedural and technical questions arise. In this chapter, we discuss several current problems in imaging tumor with radiolabeled monoclonal antibody. These include (1) methods for selection of specific antibody and, once the particular antibody is selected, which fragment form is to be used; (2) imaging procedures: what are the optimum imaging parameters, such as optimum time for imaging after administration of tracer and considerations regarding background subtraction; and (3) noninvasive quantitative techniques: quantitation of localization of antibody indirectly from quantitative information in the images.100 references

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-07-01

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

  3. PrP(Sc-specific antibodies with the ability to immunodetect prion oligomers.

    Directory of Open Access Journals (Sweden)

    Mourad Tayebi

    Full Text Available The development of antibodies with binding capacity towards soluble oligomeric forms of PrPSc recognised in the aggregation process in early stage of the disease would be of paramount importance in diagnosing prion diseases before extensive neuropathology has ensued. As blood transfusion appears to be efficient in the transmission of the infectious prion agent, there is an urgent need to develop reagents that would specifically recognize oligomeric forms of the abnormally folded prion protein, PrPSc.To that end, we show that anti-PrP monoclonal antibodies (called PRIOC mAbs derived from mice immunised with native PrP-coated microbeads are able to immunodetect oligomers/multimers of PrPSc. Oligomer-specific immunoreactivity displayed by these PRIOC mAbs was demonstrated as large aggregates of immunoreactive deposits in prion-permissive neuroblastoma cell lines but not in equivalent non-infected or prn-p(0/0 cell lines. In contrast, an anti-monomer PrP antibody displayed diffuse immunoreactivity restricted to the cell membrane. Furthermore, our PRIOC mAbs did not display any binding with monomeric recombinant and cellular prion proteins but strongly detected PrPSc oligomers as shown by a newly developed sensitive and specific ELISA. Finally, PrioC antibodies were also able to bind soluble oligomers formed of Aβ and α-synuclein. These findings demonstrate the potential use of anti-prion antibodies that bind PrPSc oligomers, recognised in early stage of the disease, for the diagnosis of prion diseases in blood and other body fluids.

  4. Donor-Specific Anti-HLA Antibodies in Huntington's Disease Recipients of Human Fetal Striatal Grafts.

    Science.gov (United States)

    Porfirio, Berardino; Paganini, Marco; Mazzanti, Benedetta; Bagnoli, Silvia; Bucciantini, Sandra; Ghelli, Elena; Nacmias, Benedetta; Putignano, Anna Laura; Rombolà, Giovanni; Saccardi, Riccardo; Lombardini, Letizia; Di Lorenzo, Nicola; Vannelli, Gabriella B; Gallina, Pasquale

    2015-01-01

    Fetal grafting in a human diseased brain was thought to be less immunogenic than other solid organ transplants, hence the minor impact on the efficacy of the transplant. How much prophylactic immune protection is required for neural allotransplantation is also debated. High-sensitive anti-HLA antibody screening in this field has never been reported. Sixteen patients with Huntington's disease underwent human fetal striatal transplantation in the frame of an open-label observational trial, which is being carried out at Florence University. All patients had both brain hemispheres grafted in two separate robotic-stereotactic procedures. The trial started in February 2006 with the first graft to the first patient (R1). R16 was given his second graft on March 2011. All patients received triple immunosuppressive treatment. Pre- and posttransplant sera were analyzed for the presence of anti-HLA antibodies using the multiplexed microsphere-based suspension array Luminex xMAP technology. Median follow-up was 38.5 months (range 13-85). Six patients developed anti-HLA antibodies, which turned out to be donor specific. Alloimmunization occurred in a time window of 0-49 months after the first neurosurgical procedure. The immunogenic determinants were non-self-epitopes from mismatched HLA antigens. These determinants were both public epitopes shared by two or more HLA molecules and private epitopes unique to individual HLA molecules. One patient had non-donor-specific anti-HLA antibodies in her pretransplant serum sample, possibly due to previous sensitization events. Although the clinical significance of donor-specific antibodies is far from being established, particularly in the setting of neuronal transplantation, these findings underline the need of careful pre- and posttransplant immunogenetic evaluation of patients with intracerebral grafts.

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

    International Nuclear Information System (INIS)

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

    2008-01-01

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

  6. Prevalence of parvovirus B19 specific antibody in pregnant women with spontaneous abortion.

    Science.gov (United States)

    Rahbar, Nahid; Vali Zadeh, Saeid; Ghorbani, Raheb; Kheradmand, Pegah

    2015-01-01

    Human parvovirus B19 is a very common viral infection especially in school-aged children. The infection during pregnancy can affect the fetus due to lack of mother's immunity. Although, there is still no evidence of fetal teratogenic effects with parvovirus B19, but non-immune fetal hydrops and abortion may be caused by vertical transmission of the virus during pregnancy. This study was aimed to assess the prevalence of parvovirus B19-specific antibody (IgM) in pregnant women who had a spontaneous abortion. This cross-sectional study was carried out in all pregnant women who referred due to a spontaneous abortion. All demographic information such as age, occupation, and gestational age, last history of abortion, gravity, and presence of children below the age of six was recorded and a blood sample was provided for all the women. Then, the blood samples were tested to assay parvovirus B19-specific antibody (IgM) by EuroImmune ELISA kit. Among 94 pregnant women with the mean age of 28.4 years who had a spontaneous abortion, parvovirus B19 specific antibody (IgM) was detected in 17 participants (18.1%). Meanwhile, 14 women (14.9%) were suspected for presence of the antibody in their blood sample. There was no significant difference between the presence of antibody and age of pregnant women, occupation, gestational age, number of previous abortion, presence of children below the age of six and number of pregnancy. These findings revealed that a high percentage of pregnant women are probably non-immune against parvovirus B19, and also there might be a number of spontaneous abortions in which parvovirus infection caused fetal death.  However, more studies are needed to prove the absolute role of parvovirus B19 in these abortions.

  7. Lemur species-specific metapopulation responses to habitat loss and fragmentation.

    Directory of Open Access Journals (Sweden)

    Travis S Steffens

    Full Text Available Determining what factors affect species occurrence is vital to the study of primate biogeography. We investigated the metapopulation dynamics of a lemur community consisting of eight species (Avahi occidentalis, Propithecus coquereli, Microcebus murinus, Microcebus ravelobensis, Lepilemur edwardsi, Cheirogaleus medius, Eulemur mongoz, and Eulemur fulvus within fragmented tropical dry deciduous forest habitat in Ankarafantsika National Park, Madagascar. We measured fragment size and isolation of 42 fragments of forest ranging in size from 0.23 to 117.7 ha adjacent to continuous forest. Between June and November 2011, we conducted 1218 surveys and observed six of eight lemur species (M. murinus, M. ravelobensis, C. medius, E. fulvus, P. coquereli, and L. edwardsi in the 42 fragments. We applied among patch incidence function models (IFMs with various measures of dispersal and a mainland-island IFM to lemur species occurrence, with the aim of answering the following questions: 1 Do lemur species in dry deciduous forest fragments form metapopulations? 2 What are the separate effects of area (extinction risk and connectivity/isolation (colonization potential within a lemur metapopulation? 3 Within simulated metapopulations over time, how do area and connectivity/isolation affect occurrence? and 4 What are the conservation implications of our findings? We found that M. murinus formed either a mainland-island or an among patch metapopulation, M. ravelobensis formed a mainland-island metapopulation, C. medius and E. fulvus formed among patch metapopulations, and neither P. coquereli or L. edwardsi formed a metapopulation. Metapopulation dynamics and simulations suggest that area was a more consistent positive factor determining lemur species occurrence than fragment isolation and is crucial to the maintenance of lemur populations within this fragmented landscape. Using a metapopulation approach to lemur biogeography is critical for understanding how

  8. Prevention of Herpes Simplex Virus Induced Stromal Keratitis by a Glycoprotein B-Specific Monoclonal Antibody

    Science.gov (United States)

    Krawczyk, Adalbert; Dirks, Miriam; Kasper, Maren; Buch, Anna; Dittmer, Ulf; Giebel, Bernd; Wildschütz, Lena; Busch, Martin; Goergens, Andre; Schneweis, Karl E.; Eis-Hübinger, Anna M.; Sodeik, Beate; Heiligenhaus, Arnd; Roggendorf, Michael; Bauer, Dirk

    2015-01-01

    The increasing incidence of acyclovir (ACV) and multidrug-resistant strains in patients with corneal HSV-1 infections leading to Herpetic Stromal Keratitis (HSK) is a major health problem in industrialized countries and often results in blindness. To overcome this obstacle, we have previously developed an HSV-gB-specific monoclonal antibody (mAb 2c) that proved to be highly protective in immunodeficient NOD/SCID-mice towards genital infections. In the present study, we examined the effectivity of mAb 2c in preventing the immunopathological disease HSK in the HSK BALB/c mouse model. Therefore, mice were inoculated with HSV-1 strain KOS on the scarified cornea to induce HSK and subsequently either systemically or topically treated with mAb 2c. Systemic treatment was performed by intravenous administration of mAb 2c 24 h prior to infection (pre-exposure prophylaxis) or 24, 40, and 56 hours after infection (post-exposure immunotherapy). Topical treatment was performed by periodical inoculations (5 times per day) of antibody-containing eye drops as control, starting at 24 h post infection. Systemic antibody treatment markedly reduced viral loads at the site of infection and completely protected mice from developing HSK. The administration of the antiviral antibody prior or post infection was equally effective. Topical treatment had no improving effect on the severity of HSK. In conclusion, our data demonstrate that mAb 2c proved to be an excellent drug for the treatment of corneal HSV-infections and for prevention of HSK and blindness. Moreover, the humanized counterpart (mAb hu2c) was equally effective in protecting mice from HSV-induced HSK when compared to the parental mouse antibody. These results warrant the future development of this antibody as a novel approach for the treatment of corneal HSV-infections in humans. PMID:25587898

  9. Prevention of herpes simplex virus induced stromal keratitis by a glycoprotein B-specific monoclonal antibody.

    Directory of Open Access Journals (Sweden)

    Adalbert Krawczyk

    Full Text Available The increasing incidence of acyclovir (ACV and multidrug-resistant strains in patients with corneal HSV-1 infections leading to Herpetic Stromal Keratitis (HSK is a major health problem in industrialized countries and often results in blindness. To overcome this obstacle, we have previously developed an HSV-gB-specific monoclonal antibody (mAb 2c that proved to be highly protective in immunodeficient NOD/SCID-mice towards genital infections. In the present study, we examined the effectivity of mAb 2c in preventing the immunopathological disease HSK in the HSK BALB/c mouse model. Therefore, mice were inoculated with HSV-1 strain KOS on the scarified cornea to induce HSK and subsequently either systemically or topically treated with mAb 2c. Systemic treatment was performed by intravenous administration of mAb 2c 24 h prior to infection (pre-exposure prophylaxis or 24, 40, and 56 hours after infection (post-exposure immunotherapy. Topical treatment was performed by periodical inoculations (5 times per day of antibody-containing eye drops as control, starting at 24 h post infection. Systemic antibody treatment markedly reduced viral loads at the site of infection and completely protected mice from developing HSK. The administration of the antiviral antibody prior or post infection was equally effective. Topical treatment had no improving effect on the severity of HSK. In conclusion, our data demonstrate that mAb 2c proved to be an excellent drug for the treatment of corneal HSV-infections and for prevention of HSK and blindness. Moreover, the humanized counterpart (mAb hu2c was equally effective in protecting mice from HSV-induced HSK when compared to the parental mouse antibody. These results warrant the future development of this antibody as a novel approach for the treatment of corneal HSV-infections in humans.

  10. An Immunosensor Based on Antibody Binding Fragments Attached to Gold Nanoparticles for the Detection of Peptides Derived from Avian Influenza Hemagglutinin H5

    Directory of Open Access Journals (Sweden)

    Urszula Jarocka

    2014-08-01

    Full Text Available This paper concerns the development of an immunosensor for detection of peptides derived from avian influenza hemagglutinin H5. Its preparation consists of successive gold electrode modification steps: (i modification with 1,6-hexanedithiol and gold colloidal nanoparticles; (ii immobilization of antibody-binding fragments (Fab’ of anti-hemagglutinin H5 monoclonal antibodies Mab 6-9-1 via S-Au covalent bonds; and (iii covering the remaining free space on the electrode surfaces with bovine serum albumin. The interactions between Fab’ fragments and hemagglutinin (HA variants have been explored with electrochemical impedance spectroscopy (EIS in the presence of [Fe(CN6]3−/4− as an electroactive marker. The immunosensor was able to recognize three different His-tagged variants of recombinant hemagglutinin from H5N1 viruses: H1 subunit (17–340 residues of A/swan/Poland/305-135V08/2006, the long HA (17–530 residues A/Bar-headed Goose/Qinghai/12/2005 and H1 subunit (1–345 residues of A/Vietnam/1194/2004. The strongest response has been observed for the long variant with detection limit of 2.2 pg/mL and dynamic range from 4.0 to 20.0 pg/mL.

  11. Specific antibodies to detect Tamarillo leaf malformation virus (TALMV) in Tamarillo

    International Nuclear Information System (INIS)

    Gallo Garcia, Yuliana; Marin Montoya, Mauricio; Gutierrez, Pablo Andres

    2011-01-01

    In Colombia, yields of Tamarillo are seriously affected by a complex viral disease known as virosis. This pathology was first reported in 1991 in the north of Antioquia and currently affects all Tamarillo growing regions in the country. Recent works have demonstrated the association of two potyviruses (potyviridae) with this disease: potato virus y (PVY) and Tamarillo leaf malformation virus (TALMV, proposed species). Specific diagnostic tools are required for early asymptomatic detection of these viruses and Tamarillo certification programs. In this study, we report the obtention of TALMV specific antibodies using a 15 residues peptide mimicking the n-terminal coat protein. Specificity and sensitivity of the anti-TALMV antibodies was determined by Elisa and dot-blot using recombinant protein and synthetic peptides as controls. The usefulness of these antibodies was validated from a preliminary trial of TALMV detection in plant samples obtained from Tamarillo crops in eastern Antioquia and results were compared with a TALMV specific coat RT-PCR detection protocol.

  12. Affinity Purification and Comparative Biosensor Analysis of Citrulline-Peptide-Specific Antibodies in Rheumatoid Arthritis

    Directory of Open Access Journals (Sweden)

    Eszter Szarka

    2018-01-01

    Full Text Available Background: In rheumatoid arthritis (RA, anti-citrullinated protein/peptide antibodies (ACPAs are responsible for disease onset and progression, however, our knowledge is limited on ligand binding affinities of autoantibodies with different citrulline-peptide specificity. Methods: Citrulline-peptide-specific ACPA IgGs were affinity purified and tested by ELISA. Binding affinities of ACPA IgGs and serum antibodies were compared by surface plasmon resonance (SPR analysis. Bifunctional nanoparticles harboring a multi-epitope citrulline-peptide and a complement-activating peptide were used to induce selective depletion of ACPA-producing B cells. Results: KD values of affinity-purified ACPA IgGs varied between 10−6 and 10−8 M and inversely correlated with disease activity. Based on their cross-reaction with citrulline-peptides, we designed a novel multi-epitope peptide, containing Cit-Gly and Ala-Cit motifs in two–two copies, separated with a short, neutral spacer. This peptide detected antibodies in RA sera with 66% sensitivity and 98% specificity in ELISA and was recognized by 90% of RA sera, while none of the healthy samples in SPR. When coupled to nanoparticles, the multi-epitope peptide specifically targeted and depleted ACPA-producing B cells ex vivo. Conclusions: The unique multi-epitope peptide designed based on ACPA cross-reactivity might be suitable to develop better diagnostics and novel therapies for RA.

  13. Chimeric antigen receptor (CAR-specific monoclonal antibody to detect CD19-specific T cells in clinical trials.

    Directory of Open Access Journals (Sweden)

    Bipulendu Jena

    Full Text Available Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63. We describe a novel anti-idiotype monoclonal antibody (mAb to detect CD19-specific CAR(+ T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1 was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19(+ tumor targets. This clone can be used to detect CD19-specific CAR(+ T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1 will be useful to investigators implementing CD19-specific CAR(+ T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy.

  14. Mode of inhibition of HIV-1 Integrase by a C-terminal domain-specific monoclonal antibody*

    Directory of Open Access Journals (Sweden)

    Merkel George

    2006-06-01

    Full Text Available Abstract Background To further our understanding of the structure and function of HIV-1 integrase (IN we developed and characterized a library of monoclonal antibodies (mAbs directed against this protein. One of these antibodies, mAb33, which is specific for the C-terminal domain, was found to inhibit HIV-1 IN processing activity in vitro; a corresponding Fv fragment was able to inhibit HIV-1 integration in vivo. Our subsequent studies, using heteronuclear nuclear magnetic resonance spectroscopy, identified six solvent accessible residues on the surface of the C-terminal domain that were immobilized upon binding of the antibody, which were proposed to comprise the epitope. Here we test this hypothesis by measuring the affinity of mAb33 to HIV-1 proteins that contain Ala substitutions in each of these positions. To gain additional insight into the mode of inhibition we also measured the DNA binding capacity and enzymatic activities of the Ala substituted proteins. Results We found that Ala substitution of any one of five of the putative epitope residues, F223, R224, Y226, I267, and I268, caused a decrease in the affinity of the mAb33 for HIV-1 IN, confirming the prediction from NMR data. Although IN derivatives with Ala substitutions in or near the mAb33 epitope exhibited decreased enzymatic activity, none of the epitope substitutions compromised DNA binding to full length HIV-1 IN, as measured by surface plasmon resonance spectroscopy. Two of these derivatives, IN (I276A and IN (I267A/I268A, exhibited both increased DNA binding affinity and uncharacteristic dissociation kinetics; these proteins also exhibited non-specific nuclease activity. Results from these investigations are discussed in the context of current models for how the C-terminal domain interacts with substrate DNA. Conclusion It is unlikely that inhibition of HIV-1 IN activity by mAb33 is caused by direct interaction with residues that are essential for substrate binding. Rather

  15. Radiolabeled Antibody Fragment for Preparation of (177Lu-DOTAm-PAMAM G3.0-F(ab’2 trastuzumab as a Radiopharmaceutical for Cancer Therapy

    Directory of Open Access Journals (Sweden)

    R.D. Haryuni

    2017-06-01

    Full Text Available Several radiolabeled monoclonal antibodies (mAbs have been used as radioimmunotherapy (RIT agents for cancer therapy. The use of mAbs as RIT agents is due to their ability to carry effectors, in the form of radionuclides which emit alpha (α particles, beta (β particles, or auger electrons, and bind specifically to cancer expressed receptor. This paper reports the preparation of radiolabelled trastuzumab in form of (177Lu-DOTAm-PAMAM G3-F(ab'2-trastuzumab, which will be expected as a potential RIT agent for therapy of breast cancer overexpressed human epidermal growth factor receptor 2 (HER2. Due to its reduced molecular weight, the use of F(ab'2-trastuzumab on the aforementioned RIT agent candidate is expected to reach its target much faster compared to the intact trastuzumab. Meanwhile, the role of PAMAM G3 is to increase the specific activity of the radiotherapeutic agent of Lu-177 due to the ability of its 32 –NH2 functional groups that are able to bind many DOTAs (£ 31 which in turn can bind a large number of 177Lu. The preparation was initiated by fragmentation of trastuzumab using pepsin enzyme in 0.02 M acetic acid buffer with a pH of 4.5 to produce F(ab'2-trastuzumab with a purity of 95 % after purification with PD-10 column. The F(ab'2-trastuzumab was then reacted with succinimidyl 4-(N-maleimidomethyl cyclohexane-1-carboxylate (SMCC to produce SMCC-F(ab'2-trastuzumab. The next reaction was to conjugate SMCC-F(ab'2-trastuzumab with DOTA-PAMAM G3.0-SH, which was prepared by reaction NHS-DOTA with PAMAM G3.0 and followed by reacting it with 2-iminothiolane to give (DOTAm-PAMAM G3.0-F(ab'2-trastuzumab. Finally, the (DOTAm-PAMAM G3.0-F(ab'2-trastuzumab was radiolabelled with 177Lu to produce (177Lu-DOTAm-PAMAM G3.0-F(ab'2-trastuzumab, resulting in a radiochemical purity of 98 % after purification with PD-10 column.Received: 31 October 2015; Revised: 30 June 2016; Accepted: 25 September 2016

  16. 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

  17. 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

  18. A Comprehensive Overview on Myositis-Specific Antibodies: New and Old Biomarkers in Idiopathic Inflammatory Myopathy

    Science.gov (United States)

    Satoh, Minoru; Tanaka, Shin; Ceribelli, Angela; Calise, S. John; Chan, Edward K. L.

    2018-01-01

    Autoantibodies specific for idiopathic inflammatory myopathy (myositis-specific autoantibodies (MSAs)) are clinically useful biomarkers to help the diagnosis of polymyositis/dermatomyositis (PM/DM). Many of these are also associated with a unique clinical subset of PM/DM, making them useful in predicting and monitoring certain clinical manifestations. Classic MSAs known for over 30 years include antibodies to Jo-1 (histidyl transfer RNA (tRNA) synthetase) and other aminoacyl tRNA synthetases (ARS), anti-Mi-2, and anti-signal recognition particle (SRP). Anti-Jo-1 is the first autoantibodies to ARS detected in 15–25 % of patients. In addition to anti-Jo-1, antibodies to seven other aminoacyl tRNA synthetases (ARS) have been reported with prevalence, usually 1–5 % or lower. Patients with any antiARS antibodies are associated with anti-synthetase syndrome characterized by myositis, interstitial lung disease (ILD), arthritis, Raynaud’s phenomenon, and others. Several recent studies suggested heterogeneity in clinical features among different anti-ARS antibody-positive patients and anti-ARS may also be found in idiopathic ILD without myositis. Anti-Mi-2 is a classic marker for DM and associated with good response to steroid treatment and good prognosis. Anti-SRP is specific for PM and associated with treatment-resistant myopathy histologically characterized as necrotizing myopathy. In addition to classic MSAs, several new autoantibodies with strong clinical significance have been described in DM. Antibodies to transcription intermediary factor 1γ/α (TIF1γ/α, p155/140) are frequently found in DM associated with malignancy while anti-melanoma differentiation-associated gene 5 (MDA5; CADM140) are associated with clinically amyopathic DM (CADM) complicated by rapidly progressive ILD. Also, anti-MJ/nuclear matrix protein 2 (NXP-2) and anti-small ubiquitin-like modifier-1 (SUMO-1) activating enzyme (SAE) are recognized as new DM-specific autoantibodies. Addition of

  19. Production of Monoclonal Antibodies Specific for Progesterone, Estradiole by Simultaneous Injection of Different Steroids

    OpenAIRE

    YÜCEL, Fatima ŞAHİNGÖZ

    2014-01-01

    We report here the development of hybrid cells producing monoclonal antibodies specific for two different steroid hormones with mixed immunization using hybridoma technology. BALB/c mice were immunized with a mixture of three steroid antigens: progesterone, estradiole and testosterone linked to bovine serum albumine. These mice were used for fusion. In the two fusion experiments, ELISA tests showed that among 645 wells only 2 hybrids reacted with progesterone (MAM 3C2, MAM 3E3) and one o...

  20. [Comparative study of immunoglobulins and specific antibodies in the sera of chronic brucellosis patients].

    Science.gov (United States)

    Kichieva, B N; Chernysheva, M I; Zheludkov, M M; Musaeva, N B

    1982-04-01

    The data on the IgA, IgM and IgG levels in the sera of 89 patients with chronic brucellosis lasting for 1-10 years and longer are presented. The chronic form of brucellosis is characterized by the normal or low level of immunoglobulins. No correlation between the levels of IgG, IgM and the titer of specific antibodies has been established.

  1. Antibody-mediated modulation of cytokinins in tobacco: Organ-specific changes in cytokinin homeostasis

    Czech Academy of Sciences Publication Activity Database

    Gelová, Z.; Hoopen, P.; Novák, Ondřej; Motyka, Václav; Pernisová, M.; Dabravolski, S.; Didi, V.; Tillack, F.; Oklešťková, Jana; Strnad, Miroslav; Hause, B.; Haruštiaková, D.; Conrad, U.; Janda, L.; Hejátko, J.

    2018-01-01

    Roč. 69, č. 3 (2018), s. 441-454 ISSN 0022-0957 R&D Projects: GA MŠk(CZ) LQ1601; GA MŠk(CZ) LO1204; GA MŠk(CZ) LM2015062; GA ČR(CZ) GA16-14649S Institutional support: RVO:61389030 Keywords : Antibody-mediated modulation * biosynthesis * ckx * cytokinin * homeostasis * organ specificity * tobacco Subject RIV: EF - Botanics OBOR OECD: Plant sciences, botany Impact factor: 5.830, year: 2016

  2. On-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry using Fab´antibody fragments for the analysis of serum transthyretin.

    Science.gov (United States)

    Pont, Laura; Benavente, Fernando; Barbosa, José; Sanz-Nebot, Victoria

    2017-08-01

    This paper describes an on-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry (IA-SPE-CE-MS) method using an immunoaffinity sorbent with Fab' antibody fragments (Fab'-IA) for the analysis of serum transthyretin (TTR), a homotetrameric protein (M r ~56,000) involved in different types of amyloidosis. The IA sorbent was prepared by covalent attachment of Fab' fragments obtained from a polyclonal IgG antibody against TTR to succinimidyl silica particles. The Fab'-IA-SPE-CE-MS methodology was first established analyzing TTR standard solutions. Under optimized conditions, repeatability and reproducibility were acceptable, the method was linear between 1 and 25µgmL -1 , limits of detection (LODs) were around 0.5µgmL -1 (50-fold lower than by CE-MS, ~25µgmL -1 ) and different TTR conformations were observed (folded and unfolded). The applicability of the developed method to screen for familial amyloidotic polyneuropathy type I (FAP-I), which is the most common hereditary systemic amyloidosis, was evaluated analyzing serum samples from healthy controls and FAP-I patients. For the analysis of sera, the most abundant proteins were precipitated with 5% (v/v) of phenol before Fab'-IA-SPE-CE-MS. The current method enhanced our previous results for the analysis of TTR using intact antibodies immobilized on magnetic beads. It allowed a slight improvement on LODs (2-fold), the detection of proteoforms found at lower concentrations and the preparation of microcartridges with extended durability. Copyright © 2017. Published by Elsevier B.V.

  3. Dengue serotype cross-reactive, anti-E protein antibodies confound specific immune memory for one year after infection

    Directory of Open Access Journals (Sweden)

    Ying Xiu eToh

    2014-08-01

    Full Text Available Dengue virus has four serotypes and is endemic globally in tropical countries. Neither a specific treatment nor an approved vaccine is available, and correlates of protection are not established. The standard neutralization assay cannot differentiate between serotype-specific and serotype cross-reactive antibodies in patients early after infection, leading to an overestimation of the long-term serotype-specific protection of an antibody response. It is known that the cross-reactive response in patients is temporary but few studies have assessed kinetics and potential changes in serum antibody specificity over time. To better define the specificity of polyclonal antibodies during disease and after recovery, longitudinal samples from patients with primary or secondary DENV-2 infection were collected over a period of one year. We found that serotype cross-reactive antibodies peaked three weeks after infection and subsided within one year. Since secondary patients rapidly produced antibodies specific for the virus envelope (E protein, an E-specific ELISA was superior compared to a virus particle-specific ELISA to identify patients with secondary infections. Dengue infection triggered a massive activation and mobilization of both naïve and memory B cells possibly from lymphoid organs into the blood, providing an explanation for the surge of circulating plasmablasts and the increase in cross-reactive E protein-specific antibodies.

  4. Clinical evaluation of a matrix metalloproteinase-12 cleaved fragment of titin as a cardiovascular-specific serological biomarker

    DEFF Research Database (Denmark)

    Vassiliadis, Efstathios; Melholt Rasmussen, Lars; Byrjalsen, Inger

    2012-01-01

    in a clinical setting, we aimed to develop an assay that could reliably measure fragments of degraded titin in serum and potentially be used in the assessment of cardiac muscle damage. METHODS: A competitive ELISA was developed to specifically measure levels of the titin sequence 12670' NVTVEARLIK 12679...... of coronary calcium (CT-plusCa) or without coronary calcium (CT-noCa). RESULTS: Mean geometric levels of the titin fragment in the CT-noCa group were 506.5 ng/ml (+/-43.88). The CT-plusCa group showed 50.6% higher levels of the marker [763 ng/ml (+/-90.14)] (P ... [792 ng/ml (+/-149)] (P fragment is present in both individuals with undiagnosed and diagnosed CVD. The statistically significant increase in level of the marker in the AMI group is indicative that this neoepitope biomarker may be a useful serological marker in AMI....

  5. Passive immunization of guinea-pigs with llama single-domain antibody fragments against foot-and-mouth disease

    NARCIS (Netherlands)

    Harmsen, M.M.; Solt, van C.B.; Fijten, H.P.D.; Keulen, van L.; Rosalia, R.A.; Weerdmeester, K.; Cornelissen, A.H.M.; Bruin, de M.G.M.; Eble, P.L.; Dekker, A.

    2007-01-01

    Foot-and-mouth disease (FMD) is a highly contagious disease that occasionally causes outbreaks in Europe. There is a need for therapies that provide rapid protection against FMD in outbreak situations. We aim to provide such rapid protection by passive immunization with llama single-domain antibody

  6. Quality control of radiolabeled antibodies through simultaneous determination of antibody concentration and specific activity using time-resolved interaction analysis and reverse kinetic fit

    International Nuclear Information System (INIS)

    Andersson, K.; Mihaylova, D.; Wang, E.; Abrahamsen, L.; Buijs, J.; Bjoerkelund, H.

    2015-01-01

    Full text of publication follows. With the advent of efficient methods for producing proteins that bind to a defined target, the number of radiolabeled proteins, and in particular antibodies, used for medical imaging and cancer therapy is increasing rapidly. In line with this increase, focus should be put on methods for the quality control (QC). Proper antibody quality is of fundamental importance to guarantee safety and consistent efficacy for the patient. Adequate QC procedures exist for small radiolabeled synthetic compounds like FDG, but antibody based radiopharmaceuticals are different. Proteins are much more complex and fragile than the synthetic compounds, and hence require new methods for adequate characterization and QC. Yet another complication is the labeling where there is a risk that a subpopulation of the protein is damaged to the level that it no longer binds the target. Therefore, a new toolbox is required to fulfill the quality characterization of radiolabeled antibodies. We have developed a QC assay for the simultaneous determination of antibody function, concentration and specific activity. The assay is based on time-resolved detection of the antibody interaction with antigen-coated magnetic beads in LigandTracer instruments. The resulting binding curve is evaluated using reverse kinetic fits, where the known interaction parameters of the antibody-antigen interaction are set constant while as the concentration and signal level are fitted. The assay takes approximately 2 hours and the majority of the time constitutes automated data collection in the instrument. The QC assay has been tested on multiple antibody-antigen interactions and consistently provides repeatable results for concentration and specific activity, both with coefficient of variation (CV) less than 15%. We believe that this QC assay can improve the quality of radiolabeled therapeutic antibodies. (authors)

  7. Screening and identification of male-specific DNA fragments in common carps Cyprinus carpio using suppression subtractive hybridization.

    Science.gov (United States)

    Chen, J J; Du, Q Y; Yue, Y Y; Dang, B J; Chang, Z J

    2010-08-01

    In this study, a sex subtractive genomic DNA library was constructed using suppression subtractive hybridization (SSH) between male and female Cyprinus carpio. Twenty-two clones with distinguishable hybridization signals were selected and sequenced. The specific primers were designed based on the sequence data. Those primers were then used to amplify the sex-specific fragments from the genomic DNA of male and female carp. The amplified fragments from two clones showed specificity to males but not to females, which were named as Ccmf2 [387 base pairs (bp)] and Ccmf3 (183 bp), respectively. The sex-specific pattern was analysed in a total of 40 individuals from three other different C. carpio. stocks and grass carp Ctenopharyngodon idella using Ccmf2 and Ccmf3 as dot-blotting probes. The results revealed that the molecular diversity exists on the Y chromosome of C. carpio. No hybridization signals, however, were detected from individuals of C. idella, suggesting that the two sequences are specific to C. carpio. No significant homologous sequences of Ccmf2 and Ccmf3 were found in GenBank. Therefore, it was interpreted that the results as that Ccmf2 and Ccmf3 are two novel male-specific sequences; and both fragments could be used as markers to rapidly and accurately identify the genetic sex of part of C. carpio. This may provide a very efficient selective tool for practically breeding monosex female populations in aquacultural production.

  8. Isolation of scFv antibody fragments against HER2 and CEA tumor antigens from combinatorial antibody libraries derived from cancer patients.

    Science.gov (United States)

    Ayat, Hoda; Burrone, Oscar R; Sadghizadeh, Majid; Jahanzad, Eissa; Rastgou, Nasrin; Moghadasi, Sarrira; Arbabi, Mehdi

    2013-11-01

    Tumor cells expressing HER-2/neu and CEA antigens are potentially ideal targets for antibody-targeted therapy. In this study, two large human combinatorial libraries have been generated from the lymph nodes of breast cancer patients that express HER2 and CEA antigens in their tumors. These 'immune' libraries have been constructed in two different formats of scFv, differing in the length of the peptide linker connecting the two variable VH and VL domains. Libraries derived from these patients may contain a larger pool of anti-tumor antigen antibodies and are useful repertoire for isolating scFvs against any tumor markers. The results of this study showed that we were successful in obtaining human scFvs against HER-2/neu and CEA. For HER-2, cell-panning strategy was performed and resulted in two scFv binders that detected the complete HER-2 receptor on the cell membrane and internalized to the cells. Also, preliminary ELISA data showed that several anti-CEA scFv binders were isolated by panning. Copyright © 2013 The International Alliance for Biological Standardization. All rights reserved.

  9. Age-related changes in the proteoglycans of human skin. Specific cleavage of decorin to yield a major catabolic fragment in adult skin.

    Science.gov (United States)

    Carrino, David A; Onnerfjord, Patrik; Sandy, John D; Cs-Szabo, Gabriella; Scott, Paul G; Sorrell, J Michael; Heinegård, Dick; Caplan, Arnold I

    2003-05-09

    Dramatic changes occur in skin as a function of age, including changes in morphology, physiology, and mechanical properties. Changes in extracellular matrix molecules also occur, and these changes likely contribute to the overall age-related changes in the physical properties of skin. The major proteoglycans detected in extracts of human skin are decorin and versican. In addition, adult human skin contains a truncated form of decorin, whereas fetal skin contains virtually undetectable levels of this truncated decorin. Analysis of this molecule, herein referred to as decorunt, indicates that it is a catabolic fragment of decorin rather than a splice variant. With antibody probes to the core protein, decorunt is found to lack the carboxyl-terminal portion of decorin. Further analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry shows that the carboxyl terminus of decorunt is at Phe(170) of decorin. This result indicates that decorunt represents the amino-terminal 43% of the mature decorin molecule. Such a structure is inconsistent with alternative splicing of decorin and suggests that decorunt is a catabolic fragment of decorin. A neoepitope antiserum, anti-VRKVTF, was generated against the carboxyl terminus of decorunt. This antiserum does not recognize intact decorin in any skin proteoglycan sample tested on immunoblots but recognizes every sample of decorunt tested. The results with anti-VRKVTF confirm the identification of the carboxyl terminus of decorunt. Analysis of collagen binding by surface plasmon resonance indicates that the affinity of decorunt for type I collagen is 100-fold less than that of decorin. This observation correlates with the structural analysis of decorunt, in that it lacks regions of decorin previously shown to be important for interaction with type I collagen. The detection of a catabolic fragment of decorin suggests the existence of a specific catabolic pathway for this proteoglycan. Because of the

  10. Destructive arthritis in a patient with chikungunya virus infection with persistent specific IgM antibodies

    Directory of Open Access Journals (Sweden)

    Receveur Marie-Catherine

    2009-12-01

    Full Text Available Abstract Background Chikungunya fever is an emerging arboviral disease characterized by an algo-eruptive syndrome, inflammatory polyarthralgias, or tenosynovitis that can last for months to years. Up to now, the pathophysiology of the chronic stage is poorly understood. Case presentation We report the first case of CHIKV infection with chronic associated rheumatism in a patient who developed progressive erosive arthritis with expression of inflammatory mediators and persistence of specific IgM antibodies over 24 months following infection. Conclusions Understanding the specific features of chikungunya virus as well as how the virus interacts with its host are essential for the prevention, treatment or cure of chikungunya disease.

  11. Complement-dependent pathogenicity of brain-specific antibodies in cerebrospinal fluid

    DEFF Research Database (Denmark)

    Asgari, Nasrin; Khorooshi, Reza; Lillevang, Søren T

    2013-01-01

    The specificity and potential pathogenicity of autoantibodies vary between neurological diseases. It is often unclear whether their detection in cerebrospinal fluid (CSF) is a consequence or a cause of pathology. The goal was to test whether administration of brain-specific antibodies into CSF...... would be sufficient for pathology. Purified immunoglobulin G from a neuromyelitis optica patient was injected intrathecally with complement to naive mice. Histopathological analysis at 7 days revealed damage to the ependyma, disruption of the CSF parenchymal barrier and pathologic lesions, distant from...

  12. Development of Strongylus vulgaris-specific serum antibodies in naturally infected foals.

    Science.gov (United States)

    Nielsen, M K; Vidyashankar, A N; Gravatte, H S; Bellaw, J; Lyons, E T; Andersen, U V

    2014-03-01

    Strongylus vulgaris is regarded as the most pathogenic helminth parasite infecting horses. Migrating larvae cause pronounced endarteritis and thrombosis in the cranial mesenteric artery and adjacent branches, and thromboembolism can lead to ischemia and infarction of large intestinal segments. A recently developed serum ELISA allows detection of S. vulgaris-specific antibodies during the six-month-long prepatent period. A population of horses has been maintained at the University of Kentucky without anthelmintic intervention since 1979, and S. vulgaris has been documented to be highly prevalent. In 2012, 12 foals were born in this population, and were studied during a 12-month period (March-March). Weekly serum samples were collected to monitor S. vulgaris specific antibodies with the ELISA. Nine colts underwent necropsy at different time points between 90 and 300 days of age. At necropsy, Strongylus spp. and Parascaris equorum were identified to species and stage and enumerated. Initial statistical findings indicate a significant interaction between foal age and ELISA results (pvulgaris-directed maternal antibodies transferred in the colostrum, but then remained ELISA negative during their first three months of life. Foals born in February and March became ELISA positive at about 12 weeks of age, while those born in April and May went positive at about 15 and 21 weeks, respectively. Foal date of birth was significantly associated with ELISA results (pvulgaris burdens (pvulgaris, S. edentatus, and P. equorum burdens (pvulgaris larvae leaving the bloodstream and migrating back to the intestine. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Value of serum TORCH-specific antibody detection in assessment of neonatal jaundice

    Directory of Open Access Journals (Sweden)

    Guang-Hua Dai

    2016-06-01

    Full Text Available Objective: To study the value of serum TORCH-specific antibody detection in assessment of neonatal jaundice. Methods: A total of 70 cases of children with neonatal jaundice were selected as jaundice group, 70 cases of healthy newborn were the control group, and serum serum TORCH-specific antibody content as well as heart function, liver function, kidney function and nerve function indicators were detected. Results: Serum TOX-IgM, RV-IgM, CMV-IgM and HSV-IgM positive rate and content of jaundice group were significantly higher than those of control group; serum CK-MB, cTnI, AST, ALT, Cys-C, RBP, MBP, S100β and NSE content of TORCH-positive children were significantly higher than those of TORCHnegative children, and BDNF, NT-3, NT-4 and NGF content were significantly lower than those of TORCH-negative children; T1WI signal of pallidum MRI of TORCH-positive children was significantly higher than that of TORCH-negative children. Conclusions: Serum TORCHspecific antibodies significantly increase in children with neonatal jaundice and can assess the degree of bilirubin metabolism disorder and the degree of target organ damage.

  14. Atrial cardiomyocyte-specific expression of Cre recombinase driven by an Nppa gene fragment

    NARCIS (Netherlands)

    de Lange, Frederik J.; Moorman, Antoon F. M.; Christoffels, Vincent M.

    2003-01-01

    To study the development of the atria, we produced a transgenic mouse line that expresses Cre under the regulatory control of a 7 kbp fragment of the Natriuretic peptide precursor type A gene (Nppa), from -3 kbp to +4 kbp relative to the transcription start site. Crossing this line with the R26R and

  15. Production of a phage-displayed single chain variable fragment ...

    African Journals Online (AJOL)

    Purpose: To develop specific single chain variable fragments (scFv) against infectious bursal disease virus (IBDV) via phage display technology. Methods: Purified viruses were initially applied for iterative panning rounds of scFv phage display libraries. The binding ability of the selected scFv antibody fragments against the ...

  16. Rise and Fall of an Anti-MUC1 Specific Antibody

    Science.gov (United States)

    Li, Jiandong; von Wasielewski, Reinhard; Bastert, Gunther; Schirrmann, Thomas; Esteves, Isabel Tourais; Behrens, Christian K.; Fournes, Bénédict; Fournier, Nathalie; de Romeuf, Christophe; Hust, Michael; Dübel, Stefan

    2011-01-01

    Background So far, human antibodies with good affinity and specificity for MUC1, a transmembrane protein overexpressed on breast cancers and ovarian carcinomas, and thus a promising target for therapy, were very difficult to generate. Results A human scFv antibody was isolated from an immune library derived from breast cancer patients immunised with MUC1. The anti-MUC1 scFv reacted with tumour cells in more than 80% of 228 tissue sections of mamma carcinoma samples, while showing very low reactivity with a large panel of non-tumour tissues. By mutagenesis and phage display, affinity of scFvs was increased up to 500fold to 5,7×10−10 M. Half-life in serum was improved from below 1 day to more than 4 weeks and was correlated with the dimerisation tendency of the individual scFvs. The scFv bound to T47D and MCF-7 mammalian cancer cell lines were recloned into the scFv-Fc and IgG format resulting in decrease of affinity of one binder. The IgG variants with the highest affinity were tested in mouse xenograft models using MCF-7 and OVCAR tumour cells. However, the experiments showed no significant decrease in tumour growth or increase in the survival rates. To study the reasons for the failure of the xenograft experiments, ADCC was analysed in vitro using MCF-7 and OVCAR3 target cells, revealing a low ADCC, possibly due to internalisation, as detected for MCF-7 cells. Conclusions Antibody phage display starting with immune libraries and followed by affinity maturation is a powerful strategy to generate high affinity human antibodies to difficult targets, in this case shown by the creation of a highly specific antibody with subnanomolar affinity to a very small epitope consisting of four amino acids. Despite these “best in class” binding parameters, the therapeutic success of this antibody was prevented by the target biology. PMID:21264246

  17. The association of heavy and light chain variable domains in antibodies: implications for antigen specificity.

    KAUST Repository

    Chailyan, Anna

    2011-06-28

    The antigen-binding site of immunoglobulins is formed by six regions, three from the light and three from the heavy chain variable domains, which, on association of the two chains, form the conventional antigen-binding site of the antibody. The mode of interaction between the heavy and light chain variable domains affects the relative position of the antigen-binding loops and therefore has an effect on the overall conformation of the binding site. In this article, we analyze the structure of the interface between the heavy and light chain variable domains and show that there are essentially two different modes for their interaction that can be identified by the presence of key amino acids in specific positions of the antibody sequences. We also show that the different packing modes are related to the type of recognized antigen.

  18. Bivalent Llama Single-Domain Antibody Fragments against Tumor Necrosis Factor Have Picomolar Potencies due to Intramolecular Interactions

    Directory of Open Access Journals (Sweden)

    Els Beirnaert

    2017-07-01

    Full Text Available The activity of tumor necrosis factor (TNF, a cytokine involved in inflammatory pathologies, can be inhibited by antibodies or trap molecules. Herein, llama-derived variable heavy-chain domains of heavy-chain antibody (VHH, also called Nanobodies™ were generated for the engineering of bivalent constructs, which antagonize the binding of TNF to its receptors with picomolar potencies. Three monomeric VHHs (VHH#1, VHH#2, and VHH#3 were characterized in detail and found to bind TNF with sub-nanomolar affinities. The crystal structures of the TNF–VHH complexes demonstrate that VHH#1 and VHH#2 share the same epitope, at the center of the interaction area of TNF with its TNFRs, while VHH#3 binds to a different, but partially overlapping epitope. These structures rationalize our results obtained with bivalent constructs in which two VHHs were coupled via linkers of different lengths. Contrary to conventional antibodies, these bivalent Nanobody™ constructs can bind to a single trimeric TNF, thus binding with avidity and blocking two of the three receptor binding sites in the cytokine. The different mode of binding to antigen and the engineering into bivalent constructs supports the design of highly potent VHH-based therapeutic entities.

  19. Construction of a Single Chain Variable Fragment Antibody (scFv) against Carbaryl and Its Interaction with Carbaryl.

    Science.gov (United States)

    Xiuyuan, Zhang; Zhihong, Huang; Lixia, Wang; Xiaonan, Liu

    2015-05-01

    Carbaryl is a low molecular weight insecticide that inhibits cholinesterase. Residues of carbaryl in food and the environment have damaged human health. A high-specificity scFv that can identify carbaryl is still lacking. In the present study, an anti-carbaryl scFv gene was prepared by cloning VL and VH genes from hybridoma cells secreting monoclonal antibody, then VH and VL were fused together using splicing by overlap extension (SOE) PCR with a flexible polypeptide linker connector (Gly4Ser)3, and then the scFv-pET-26b recombinant plasmid was constructed and transformed into E. coli BL21 for expression using IPTG as an inducer. The expressed recombinant protein was identified by SDS-PAGE and ELISA. The three-dimensional structure of the anti-carbaryl scFv was constructed by computer modeling, and carbaryl was docked to the scFv model to obtain the structure of the binding complex. The binding site was composed of Ala51, Ser52, Ile51, Gly54, Ser56, Arg98, and Gly100. This helps to understand the mechanism of interaction between anti-carbaryl antibody and antigen. Furthermore, it provides guidance for in vitro affinity maturation of anti-carbaryl antibody.

  20. Detection of Leptospira DNA in urine and presence of specific antibodies in outdoor cats in Germany.

    Science.gov (United States)

    Weis, Sonia; Rettinger, Anna; Bergmann, Michele; Llewellyn, Julia R; Pantchev, Nikola; Straubinger, Reinhard K; Hartmann, Katrin

    2017-04-01

    Objectives Clinical manifestation of infection with Leptospira species in cats is rare. Nevertheless, cats can develop specific antibodies against the spirochetes after infection. In Canada, Taiwan and the USA it was recently demonstrated that naturally infected cats can also shed DNA from pathogenic Leptospira species in their urine, but the zoonotic potential of infected cats is still unclear. The objective of this study was to demonstrate if outdoor cats in Germany shed DNA from pathogenic Leptospira species in their urine. As a second aim, antibody prevalence was determined. Methods Two hundred and fifteen outdoor cats were prospectively recruited. Urine samples were tested by real-time PCR targeting the lipL32 gene of pathogenic Leptospira species. Antibody titres against eight serovars (Australis, Autumnalis, Bratislava, Canicola, Copenhageni, Grippotyphosa, Pomona, Saxkoebing) belonging to seven serogroups (Australis, Autumnalis, Canicola, Grippotyphosa, Icterohaemorrhagiae, Pomona, Sejroe) were determined by microscopic agglutination test. Results Urine samples from 7/215 cats (3.3%; 95% confidence interval [CI] 0.9-5.7) were PCR-positive. Specific antibodies were detected in 35/195 cats (17.9%; 95% CI: 12.5-23.3) with titres ranging from 1:100 to 1:6400. Australis, Bratislava and Grippotyphosa were the most common serovars. Conclusions and relevance Outdoor cats in Germany can shed DNA from pathogenic Leptospira species. Therefore, outdoor cats should be considered as a possible source of infection for dogs or humans. Further studies are needed to determine the role of Leptospira species as a cause of disease in cats.

  1. Monoclonal antibodies to molluskan hemocyanin from Concholepas concholepas demonstrate common and specific epitopes among subunits.

    Science.gov (United States)

    Oliva, Harold; Moltedo, Bruno; De Ioannes, Pablo; Faunes, Fernando; De Ioannes, Alfredo E; Becker, María Inés

    2002-10-01

    We studied the reactivity of mouse monoclonal antibodies (MAbs) against the hemocyanin from the Chilean marine gastropod Concholepas concholepas (CCH). This protein has been successfully used as a carrier to produce antibodies to haptens and peptides. All MAbs (13) belonging to IgG subclass exhibit dissociation constants (K(d)) from 1 x 10(-7) M to 1 x 10(-9) M. MAbs were characterized by enzyme-linked immunosorbant assay (ELISA) using CCH treated with different procedures, including dissociation into CCH-A and CCH-B subunits, Western blot, enzymatic digestion, chemical deglycosylation, and thermal denaturation. MAbs were classified into three categories, according to subunit specificity by ELISA. The epitope distribution shows that CCH subunits display common epitopes (group I, 5 MAbs, 1H5, 2A8, 3A5, 3B3, and 3E3), as well as specific epitopes for CCH-A subunits (group II, 3 MAbs, 1B8, 4D8, and 8E5) and for CCH-B subunits (group III, 5 MAbs, 1A4, 1E4, 2H10, 3B7, and 7B4). The results can be summarized as follows: (1). six antibodies react with thermal denatured CCH, suggesting that they recognize linear epitopes, whereas seven recognize conformational epitopes; (2). oxidation of carbohydrate moieties does not affect the binding of the MAbs; (3). enzymatic digestion of CCH decreases the reactivity of all antibodies irrespective of the protease used (elastase or trypsin); (4). bringing together the above data, in addition to epitopic complementarity analysis, we identified 12 different epitopes on the CCH molecule recognized by these MAbs. The anti-CCH MAbs presented here can be useful tools to understand the subunit organization of the CCH and its complex structure, which can explain its immunogenic and immunostimulating properties in mammals.

  2. Diagnostic significance of measurements of specific IgG antibodies to Pseudomonas aeruginosa by three different serological methods

    DEFF Research Database (Denmark)

    Pressler, T.; Karpati, F.; Granstrom, M.

    2008-01-01

    to characterize patients with different infection status. Elevated levels of specific anti-Pseudomonas antibodies showed to be the risk factor for developing chronic Pa infection. Due to the specificity of the tests, antibiotic treatment based on serology might be considered in selected cases. There is a window...... of opportunity for suppression and eradication of initial P. aeruginosa infection making measurement of specific anti-Pseudomonas antibodies helpful Udgivelsesdato: 2009/1...

  3. Gag- and env-specific serum antibodies in cats after natural and experimental infection with feline immunodeficiency virus.

    NARCIS (Netherlands)

    G.F. Rimmelzwaan (Guus); C.H.J. Siebelink (Kees); H. Broos; G.A. Drost; K. Weijer (Kees); R. van Herwijnen (Rob); A.D.M.E. Osterhaus (Albert)

    1994-01-01

    textabstractIn order to monitor the antibody response to feline immunodeficiency virus (FIV) in cats, following experimental and natural infection, enzyme-linked immunosorbent assays (ELISAs) were developed using recombinant env and gag proteins and p24-specific monoclonal antibodies. It was shown

  4. Effects of long-term low dose radiation. Epstein-Barr virus-specific antibodies in radiological technologists

    Energy Technology Data Exchange (ETDEWEB)

    Kumagai, Etsuko; Higashida, Yoshiharu; Onomichi, Mitsukazu; Nakamura, Ikuo; Tanoue, Shozo; Tanaka, Ryuji; Kumagai, Takashi; Katsuki, Takato; Sawada, Shozo.

    1988-09-01

    To clarify the long-term effects of occupational exposure to low doses of radiation, Epstein-Barr virus (EBV)-specific antibody titers in sera from 104 radiological technologists (R.T.) and 118 controls in Kumamoto prefecture were measured by the immunofluorescence method. Antibody titers to viral capsid antigen (VCA)-IgG increased with the years of experience as R.T., and the prevalence of abnormal antibody titers to both VCA-IgG and early antigen (EA)-IgG were significantly higher in R.T. with over 15 years of experience or 30 rads of cumulative radiation dose than in the controls. However, there was no correlation between exposure and the frequency of abnormal EBV-associated nuclear antigen (EBNA) antibody titers. The EBV-specific antibody titers of 24 Hiroshima atomic-bomb survivors were also measured. They were similar to those of the R.T. with over 30 years of experience. The EBV-specific antibody titers of R.T. suggest that there may be an impairment of immunologic competence after continuous long-term exposure to low doses of radiation. Also, the correlation of EBV-specific antibody titers and frequency of cells with chromosome aberrations in 53 R.T. was studied. Some correlations were found between the antibody titers to both of the VCA-IgG and EBNA and the frequency of cells with chromosome aberrations.

  5. Detection of specific antibody producing cells in porcine colostrum by in ovo translation of their mRNA

    International Nuclear Information System (INIS)

    Kortbeek-Jacobs, N.; Donk, H. van der

    1978-01-01

    An improved method is described for the determination of antibody producing cells in sows colostrum. The test system comprises in ovo translation of mRNA from swine colostral cells and analysis of the translation products by radioimmunoassay with specific antibodies and antigen. (C.F.)

  6. Higher Plasma Concentration of Food-Specific Antibodies in Persons with Autistic Disorder in Comparison to Their Siblings

    Science.gov (United States)

    Trajkovski, Vladimir; Petlichkovski, Aleksandar; Efinska-Mladenovska, Olivija; Trajkov, Dejan; Arsov, Todor; Strezova, Ana; Ajdinski, Ljubomir; Spiroski, Mirko

    2008-01-01

    Specific IgA, IgG, and IgE antibodies to food antigens in 35 participants with autistic disorder and 21 of their siblings in the Republic of Macedonia were examined. Statistically significant higher plasma concentration of IgA antibodies against alpha-lactalbumin, beta-lactoglobulin, casein, and gliadin were found in the children with autistic…

  7. Effect of maternal dry period length on colostrum immunoglobulin content and natural and specific antibody titers in calves

    NARCIS (Netherlands)

    Mayasari, N.; Vries Reilingh, de G.; Nieuwland, M.G.B.; Remmelink, G.J.; Parmentier, H.K.; Kemp, B.; Knegsel, van A.T.M.

    2015-01-01

    The objective was to study the effect of dry period length in dairy cows on immunoglobulin content and natural antibodies (NAb) titers in colostrum, growth, and plasma natural and specific antibody titers in plasma of calves. Holstein-Friesian dairy cows (n = 167) were randomly assigned to 3 dry

  8. Impact of donor-specific HLA antibodies in transplantation, a review of the literature published in the last three years.

    Science.gov (United States)

    Kaneku, Hugo

    2010-01-01

    This chapter summarizes some of the recent findings published on the role in organ transplantation of HLA antibodies, and--more important--donor-specific HLA antibodies. The negative impact of both, preformed and de novo DSA is now better recognized in recipients of kidney, heart, lung, liver, pancreas, islet cells and bone marrow transplants. An appropriate design of a schedule to monitor HLA antibodies may identify patients at higher risk for immunological events earlier and allow interventions to avoid later graft loss. The value of strategies like preemptive treatment of antibodies and the use of new agents like bortezomib and eculizumab are of interest and need further investigation.

  9. Genus and species-specific IgG and IgM antibodies pulmonary tuberculosis

    International Nuclear Information System (INIS)

    Butt, T.; Abbassi, S.A.; Ahmad, R.N.; Mahmood, A.; Karamat, K.A; Malik, H.S.; Anwar, M.

    2004-01-01

    Objective: To evaluate three different enzyme immunoassays for serological diagnosis of pulmonary tuberculosis and to compare their diagnostic accuracy in different combinations. Subjects and Methods: Sera from patients suffering from pulmonary tuberculosis (n=94) with sputum positive for acid fast bacilli (AFB) and sera from control group of healthy individuals (n=90) with sputum negative for AFB were tested by Pathozyme-Myco G EIA, Pathozyme-TB Complex Plus EIA and Pathozyme Myco M EIA kits for the genus-specific IgG and IgM, and the species-specific IgG antibodies against antigens of Mycobacterium tuberculosis. Results: The detection of IgG against genus-specific antigens by Pathozyme-Myco G had a sensitivity of 46% and a specificity of 93%, of IgG against species-specific antigens by Pathozyme- TB Complex Plus had a sensitivity of 64% and specificity of 97% and of IgM against genus-specific antigens by Pathozyme Myco M had a sensitivity of 67% and specificity of 98%. When the results of these immunoassays were evaluated in combination, their sensitivity improved. Combination of genus-specific IgM and species-specific IgG yielded best results with a sensitivity of 87% and specificity of 93%. Conclusion: The sensitivity of serological diagnosis of tuberculosis is low, but it can be increased by utilizing a combination of several antigens. (author)

  10. Generation of a C3c specific monoclonal antibody and assessment of C3c as a putative inflammatory marker derived from complement factor C3

    DEFF Research Database (Denmark)

    Palarasah, Yaseelan; Skjodt, Karsten; Brandt, Jette

    2010-01-01

    complex (C5b-C9) and quantification of complement split products by precipitation-in-gel techniques (e.g. C3d). We have developed a mouse monoclonal antibody (mAb) that is able to detect fluid phase C3c without interference from other products generated from the complement component C3. The C3c specific m....... The C3c mAb was confirmed to be C3c specific, as it showed no cross-reactivity with native (un-cleaved) C3, with C3b, iC3b, or with C3d. Also, no significant reaction was observed with C3 fragments in factor I deficient sera or plasma. This antibody forms the basis for the generation of a robust ELISA...... that allows for a quick and reliable evaluation of complement activation and consumption as a marker for inflammatory processes. We established the C3c plasma range in 100 healthy Danish blood donors with a mean of 3.47 μg/ml and a range of 2.12-4.92 μg/ml. We believe that such an antibody might...

  11. 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.

  12. Donor Specific Anti-HLA Antibodies with Antibody Mediated Rejection and Long-term Outcomes Following Heart Transplantation

    Science.gov (United States)

    Clerkin, Kevin J.; Farr, Maryjane A.; Restaino, Susan W.; Zorn, Emmanuel; Latif, Farhana; Vasilescu, Elena R.; Marboe, Charles C.; Colombo, Paolo C.; Mancini, Donna M.

    2017-01-01

    Introduction Donor specific anti-HLA antibodies (DSA) are common following heart transplantation and are associated with rejection, cardiac allograft vasculopathy (CAV), and mortality. Currently a non-invasive diagnostic test for pathologic AMR (pAMR) does not exist. Methods 221 consecutive adult patients underwent heart transplantation from January 1st, 2010 through August 31th, 2013 and followed through October 1st, 2015. The primary objective was to determine whether the presence of DSA could detect AMR at the time of pathologic diagnosis. Secondary analyses included the association of DSA (stratified by MHC Class and de-novo status) during AMR with new graft dysfunction, graft loss (mortality or retransplantation), and development of CAV. Results During the study period 69 individual patients (31.2%) had DSA (24% had de-novo DSA) and there were 74 episodes of pAMR in 38 unique patients. The sensitivity of DSA at any MFI to detect concurrent pAMR was only 54.3%. The presence of any DSA during pAMR increased the odds of graft dysfunction (OR 5.37, 95% CI 1.34–21.47, p=0.018), adjusting for age, gender, and timing of AMR. Circulating Class II DSA after transplantation increased the risk of future pAMR (HR 2.97, 95% CI 1.31–6.73, p=0.009). Patients who developed de-novo Class II DSA had a 151% increase in risk of graft loss (contingent on 30-day survival) compared with those who did not have DSA (95% CI 1.11–5.69, p=0.027). Conclusions DSA were inadequate to diagnose pAMR, but Class II DSA provided prognostic information regarding future pAMR, graft dysfunction with pAMR, and graft loss. PMID:27916323

  13. Improvement of western blot test specificity for detecting equine serum antibodies to Sarcocystis neurona.

    Science.gov (United States)

    Rossano, M G; Mansfield, L S; Kaneene, J B; Murphy, A J; Brown, C M; Schott, H C; Fox, J C

    2000-01-01

    Equine protozoal myeloencephalitis (EPM) is a neurological disease of horses and ponies caused by the apicomplexan protozoan parasite Sarcocystis neurona. The purposes of this study were to develop the most stringent criteria possible for a positive test result, to estimate the sensitivity and specificity of the EPM Western blot antibody test, and to assess the ability of bovine antibodies to Sarcocystis cruzi to act as a blocking agent to minimize false-positive results in the western blot test for S. neurona. Sarcocystis neurona merozoites harvested from equine dermal cell culture were heat denatured, and the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a 12-20% linear gradient gel. Separated proteins were electrophoretically transferred to polyvinylidene fluoride membranes and blocked in 1% bovine serum albumin and 0.5% Tween-Tris-buffered saline. Serum samples from 6 horses with S. neurona infections (confirmed by culture from neural tissue) and 57 horses without infections (horses from the Eastern Hemisphere, where S. neurona does not exist) were tested by Western blot. Horses from both groups had reactivity to the 62-, 30-, 16-, 13-, 11-, 10.5-, and 10-kD bands. Testing was repeated with another step. Blots were treated with bovine S. cruzi antibodies prior to loading the equine samples. After this modification of the Western blot test, positive infection status was significantly associated with reactivity to the 30- and 16-kD bands (Pblot had a sample sensitivity of 100% and sample specificity of 98%. It is concluded that the specificity of the Western blot test is improved by blocking proteins not specific to S. neurona and using reactivity to the 30- and 16-kD bands as the criterion for a positive test.

  14. An Immunoglobulin G1 Monoclonal Antibody Highly Specific to the Wall of Cryptosporidium Oocysts

    Science.gov (United States)

    Weir, C.; Vesey, G.; Slade, M.; Ferrari, B.; Veal, D. A.; Williams, K.

    2000-01-01

    The detection of Cryptosporidium oocysts in drinking water is critically dependent on the quality of immunofluorescent reagents. Experiments were performed to develop a method for producing highly specific antibodies to Cryptosporidium oocysts that can be used for water testing. BALB/c mice were immunized with six different antigen preparations and monitored for immunoglobulin G (IgG) and IgM responses to the surface of Cryptosporidium oocysts. One group of mice received purified oocyst walls, a second group received a soluble protein preparation extracted from the outside of the oocyst wall, and the third group received whole inactivated oocysts. Three additional groups were immunized with sequentially prepared oocyst extracts to provide for a comparison of the immune response. Mice injected with the soluble protein extract demonstrated an IgG response to oocysts surface that was not seen in the whole-oocyst group. Mice injected with whole oocysts showed an IgM response only, while mice injected with purified oocyst walls showed little increase in IgM or IgG levels. Of the additional reported preparations only one, BME (2-mercaptoethanol treated), produced a weak IgM response to the oocyst wall. A mouse from the soluble oocyst extract group yielding a high IgG response was utilized to produce a highly specific IgG1 monoclonal antibody (Cry104) specific to the oocyst surface. Comparative flow cytometric analysis indicated that Cry104 has a higher avidity and specificity to oocysts in water concentrates than other commercially available antibodies. PMID:10973448

  15. Structural and biophysical characterization of an epitope-specific engineered Fab fragment and complexation with membrane proteins: implications for co-crystallization.

    Science.gov (United States)

    Johnson, Jennifer L; Entzminger, Kevin C; Hyun, Jeongmin; Kalyoncu, Sibel; Heaner, David P; Morales, Ivan A; Sheppard, Aly; Gumbart, James C; Maynard, Jennifer A; Lieberman, Raquel L

    2015-04-01

    Crystallization chaperones are attracting increasing interest as a route to crystal growth and structure elucidation of difficult targets such as membrane proteins. While strategies to date have typically employed protein-specific chaperones, a peptide-specific chaperone to crystallize multiple cognate peptide epitope-containing client proteins is envisioned. This would eliminate the target-specific chaperone-production step and streamline the co-crystallization process. Previously, protein engineering and directed evolution were used to generate a single-chain variable (scFv) antibody fragment with affinity for the peptide sequence EYMPME (scFv/EE). This report details the conversion of scFv/EE to an anti-EE Fab format (Fab/EE) followed by its biophysical characterization. The addition of constant chains increased the overall stability and had a negligible impact on the antigen affinity. The 2.0 Å resolution crystal structure of Fab/EE reveals contacts with larger surface areas than those of scFv/EE. Surface plasmon resonance, an enzyme-linked immunosorbent assay, and size-exclusion chromatography were used to assess Fab/EE binding to EE-tagged soluble and membrane test proteins: namely, the β-barrel outer membrane protein intimin and α-helical A2a G protein-coupled receptor (A2aR). Molecular-dynamics simulation of the intimin constructs with and without Fab/EE provides insight into the energetic complexities of the co-crystallization approach.

  16. OptMAVEn--a new framework for the de novo design of antibody variable region models targeting specific antigen epitopes.

    Directory of Open Access Journals (Sweden)

    Tong Li

    Full Text Available Antibody-based therapeutics provides novel and efficacious treatments for a number of diseases. Traditional experimental approaches for designing therapeutic antibodies rely on raising antibodies against a target antigen in an immunized animal or directed evolution of antibodies with low affinity for the desired antigen. However, these methods remain time consuming, cannot target a specific epitope and do not lead to broad design principles informing other studies. Computational design methods can overcome some of these limitations by using biophysics models to rationally select antibody parts that maximize affinity for a target antigen epitope. This has been addressed to some extend by OptCDR for the design of complementary determining regions. Here, we extend this earlier contribution by addressing the de novo design of a model of the entire antibody variable region against a given antigen epitope while safeguarding for immunogenicity (Optimal Method for Antibody Variable region Engineering, OptMAVEn. OptMAVEn simulates in silico the in vivo steps of antibody generation and evolution, and is capable of capturing the critical structural features responsible for affinity maturation of antibodies. In addition, a humanization procedure was developed and incorporated into OptMAVEn to minimize the potential immunogenicity of the designed antibody models. As case studies, OptMAVEn was applied to design models of neutralizing antibodies targeting influenza hemagglutinin and HIV gp120. For both HA and gp120, novel computational antibody models with numerous interactions with their target epitopes were generated. The observed rates of mutations and types of amino acid changes during in silico affinity maturation are consistent with what has been observed during in vivo affinity maturation. The results demonstrate that OptMAVEn can efficiently generate diverse computational antibody models with both optimized binding affinity to antigens and reduced

  17. Pinpointing Phosphorylation Sites: Quantitative Filtering and a Novel Site-specific x-Ion Fragment

    DEFF Research Database (Denmark)

    Kelstrup, Christian D; Hekmat, Omid; Francavilla, Chiara

    2011-01-01

    Phosphoproteomics deals with the identification and quantification of thousands of phosphopeptides. Localizing the phosphorylation site is however much more difficult than establishing the identity of a phosphorylated peptide. Further, recent findings have raised doubts of the validity of the site......-phase phosphate rearrangement reactions during collision-induced dissociation (CID) and used these spectra to devise a quantitative filter that by comparing signal intensities of putative phosphorylated fragment ions with their nonphosphorylated counterparts allowed us to accurately pinpoint which fragment ions...... contain a phosphorylated residue and which ones do not. We also evaluated higher-energy collisional dissociation (HCD) and found this to be an accurate method for correct phosphorylation site localization with no gas-phase rearrangements observed above noise level. Analyzing a large set of HCD spectra...

  18. Effects of mutation at the D-JH junction on affinity, specificity, and idiotypy of anti-progesterone antibody DB3.

    Science.gov (United States)

    He, Mingyue; Hamon, Maureen; Liu, Hong; Corper, Adam L; Taussig, Michael J

    2006-09-01

    The crystal structures of the Fab' fragment of the anti-progesterone monoclonal antibody DB3 and its complexes with steroid haptens have shown that the D-JH junctional residue TrpH100 is a key contributor to binding site interactions with ligands. The indole group of TrpH100 also undergoes a significant conformational change between the bound and unliganded states, effectively opening and closing the combining site pocket. In order to explore the effect of substitutions at this position on steroid recognition, we have carried out mutagenesis on a construct encoding a three-domain single-chain fragment (VH/K) of DB3 expressed in Escherichia coli. TrpH100 was replaced by 13 different amino acids or deleted, and the functional and antigenic properties of the mutated fragments were analyzed. Most substitutions, including small, hydrophobic, hydrophilic, neutral, and negatively charged side chains, were reduced or abolished binding to free progesterone, although binding to progesterone-BSA was partially retained. The reduction in antigen binding was paralleled by alteration of the idiotype associated with the DB3 combining site. In contrast, the replacement of TrpH100 by Arg produced a mutant that retained wild-type antibody affinity and idiotype, but with altered specificity. Significant changes in this mutant included increased relative affinities of 10(4)-fold for progesterone-3-carboxymethyloxime and 10-fold for aetiocholanolone. Our results demonstrate an essential role for the junctional residue H100 in determining steroid-binding specificity and combining site idiotype and show that these properties can be changed by a single amino acid substitution at this position.

  19. Donor-specific Anti-HLA antibodies in allogeneic hematopoietic stem cell transplantation

    Directory of Open Access Journals (Sweden)

    Sarah Morin-Zorman

    2016-08-01

    Full Text Available Allogeneic Hematopoietic Stem Cell Transplantation (AHSCT is a curative treatment for a wide variety of hematological diseases. In 30% of the cases, a geno-identical donor is available. Any other situation displays some level of Human Leukocyte Antigen (HLA incompatibility between donor and recipient. Deleterious effects of anti-HLA immunization have long been recognized in solid organ transplant recipients. More recently, anti-HLA immunization was shown to increase the risk of Primary Graft Failure (PGF, a severe complication of AHSCT that occurs in 3 to 4% of matched unrelated donor transplantation and up to 15% in cord blood transplantation and T-cell depleted haplo-identical stem cell transplantation. Rates of PGF in patients with DSA were reported to be between 24 to 83% with the highest rates in haplo-identical and cord blood transplantation recipients. This led to the recommendation of anti-HLA antibody screening to detect Donor Specific Antibodies (DSA in recipients prior to AHSCT. In this review, we highlight the role of anti-HLA antibodies in AHSCT and the mechanisms that may lead to PGF in patients with DSA, and discuss current issues in the field.

  20. Maintenance plasma exchange treatment for muscle specific kinase antibody positive myasthenia gravis patients.

    Science.gov (United States)

    Yamada, Chisa; Teener, James W; Davenport, Robertson D; Cooling, Laura

    2015-10-01

    Anti-muscle specific kinase antibody positive myasthenia gravis (MuSK MG) is often characterized by a relatively severe and progressive course, refractoriness to standard myasthenia gravis (MG) medications, and an increased risk of myasthenic crisis. We report here successful management of three MuSK MG patients using maintenance therapeutic plasma exchange (TPE) treatment for up to 4.5 years. The study was a 5-year retrospective review of all MG patients treated with TPE between 2008 and 2013 at University of Michigan. Inclusion criteria of MuSK MG were positive for anti-MuSK antibodies and a diagnosis of MuSK MG by staff neurologists. Patient data included age, gender, diagnostic testing results, medications, and the dates and response to TPE treatments. A total of 153 MG patients underwent at least one course of TPE between 2008 and 2013. A total of 12 patients (7.8%) were positive for anti-MuSK antibodies. Patients were predominantly female (83.3%) and a median age of onset was 46-years old. Three MuSK MG patients were successfully managed with maintenance TPE. Maintenance TPE may be an effective option for MuSK MG patients. The key of successful maintenance treatment at our institution has been to tailor the TPE frequency for each individual, and to modify the treatment interval in conjunction with medical management. © 2014 Wiley Periodicals, Inc.

  1. Applications of recombinant antibodies in plant pathology.

    Science.gov (United States)

    Ziegler, Angelika; Torrance, Lesley

    2002-09-01

    Summary Advances in molecular biology have made it possible to produce antibody fragments comprising the binding domains of antibody molecules in diverse heterologous systems, such as Escherichia coli, insect cells, or plants. Antibody fragments specific for a wide range of antigens, including plant pathogens, have been obtained by cloning V-genes from lymphoid tissue, or by selection from large naive phage display libraries, thus avoiding the need for immunization. The antibody fragments have been expressed as fusion proteins to create different functional molecules, and fully recombinant assays have been devised to detect plant viruses. The defined binding properties and unlimited cheap supply of antibody fusion proteins make them useful components of standardized immunoassays. The expression of antibody fragments in plants was shown to confer resistance to several plant pathogens. However, the antibodies usually only slowed the progress of infection and durable 'plantibody' resistance has yet to be demonstrated. In future, it is anticipated that antibody fragments from large libraries will be essential tools in high-throughput approaches to post-genomics research, such as the assignment of gene function, characterization of spatio-temporal patterns of protein expression, and elucidation of protein-protein interactions.

  2. An Exopolysaccharide-Deficient Mutant of Lactobacillus rhamnosus GG Efficiently Displays a Protective Llama Antibody Fragment against Rotavirus on Its Surface.

    Science.gov (United States)

    Álvarez, Beatriz; Krogh-Andersen, Kasper; Tellgren-Roth, Christian; Martínez, Noelia; Günaydın, Gökçe; Lin, Yin; Martín, M Cruz; Álvarez, Miguel A; Hammarström, Lennart; Marcotte, Harold

    2015-09-01

    Rotavirus is the leading cause of infantile diarrhea in developing countries, where it causes a high number of deaths among infants. Two vaccines are available, being highly effective in developed countries although markedly less efficient in developing countries. As a complementary treatment to the vaccines, a Lactobacillus strain producing an anti-rotavirus antibody fragment in the gastrointestinal tract could potentially be used. In order to develop such an alternative therapy, the effectiveness of Lactobacillus rhamnosus GG to produce and display a VHH antibody fragment (referred to as anti-rotavirus protein 1 [ARP1]) on the surface was investigated. L. rhamnosus GG is one of the best-characterized probiotic bacteria and has intrinsic antirotavirus activity. Among four L. rhamnosus GG strains [GG (CMC), GG (ATCC 53103), GG (NCC 3003), and GG (UT)] originating from different sources, only GG (UT) was able to display ARP1 on the bacterial surface. The genomic analysis of strain GG (UT) showed that the genes welE and welF of the EPS cluster are inactivated, which causes a defect in exopolysaccharide (EPS) production, allowing efficient display of ARP1 on its surface. Finally, GG (UT) seemed to confer a level of protection against rotavirus-induced diarrhea similar to that of wild-type GG (NCC 3003) in a mouse pup model, indicating that the EPS may not be involved in the intrinsic antirotavirus activity. Most important, GG (EM233), a derivative of GG (UT) producing ARP1, was significantly more protective than the control strain L. casei BL23. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  3. Analysis of the relations between allergen specific LgG antibody and allergic dermatosis of 14 kinds foods

    Directory of Open Access Journals (Sweden)

    Yin’e Hu

    2015-01-01

    Full Text Available To use food-specific IgG antibody detection to explore its application in the allergy dermatoses. 181 patients were included from January 2014 to September 2014. Fourteen food-specific IgG antibodies were detected by ELISA. The positive rates of IgG antibody of the patient group and the healthy group were significantly different. The positive rates of IgG antibody of egg, milk, shrimp and crab took a large proportion in three groups of patients with three kinds of allergy dermatoses of urticaria, eczema and allergic dermatitis, the proportion of which was respectively 70.2%, 77.8% and 71.7%. There was mild and moderate intolerance of food in the allergic dermatitis group while there was no distribution difference of food intolerance in urticaria group and eczema group. Among urticaria and allergic dermatitis patients with positive antibody, the positive rate of children was significantly higher than that of adults while there was no significant difference between children and adults among eczema patients with positive antibody. Allergy dermatoses are closely related to food-specific IgG antibody and the allergy dermatoses patients have a high incidence rate of food intolerance; detecting IgG antibody in patients is of great significance for the diagnosis and treatment of allergy dermatoses.

  4. The Clinical Significance of Specific Antibody Deficiency (SAD) Severity in Chronic Rhinosinusitis (CRS).

    Science.gov (United States)

    Keswani, Anjeni; Dunn, Neha M; Manzur, Angelica; Kashani, Sara; Bossuyt, Xavier; Grammer, Leslie C; Conley, David B; Tan, Bruce K; Kern, Robert C; Schleimer, Robert P; Peters, Anju T

    Despite the increased identification of specific antibody deficiency (SAD) in chronic rhinosinusitis (CRS), little is known about the relationship between SAD severity and the severity and comorbidities of CRS. The prevalence of an impaired antibody response in the general population is also unknown. The objective of this study was to determine if the SAD severity stratification applies to real-life data of patients with CRS. An electronic health record database was used to identify patients with CRS evaluated for humoral immunodeficiency with quantitative immunoglobulins and Streptococcus pneumoniae antibody titers before and after pneumococcal vaccine. SAD severity was defined, according to the guidelines, based on the numbers of titers ≥1.3 μg/dL after vaccination: severe (≤2 serotypes), moderate (3-6 serotypes), and mild (7-10 serotypes). Comorbidities and therapeutic response were assessed. The prevalence of an impaired antibody response in a normal population was assessed. Twenty-four percent of the patients with CRS evaluated for immunodeficiency had SAD, whereas 11% of a normal population had an impaired immune response to polysaccharide vaccination (P SAD. Twenty-four (10%) had severe SAD, 120 (50%) had moderate SAD, and 95 (40%) had mild SAD. Patients with moderate-to-severe SAD had worse asthma, a greater likelihood of pneumonia, and more antibiotic courses in the 2 years after vaccination than patients with mild SAD. This study provides real world data supporting stratification of SAD by severity, demonstrating a significant increase in the comorbid severity of asthma and infections in CRS patients with moderate-to-severe SAD compared with those with mild SAD and those without SAD. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  5. Adhesion of axolemmal fragments to Schwann cells: a signal- and target-specific process closely linked to axolemmal induction of Schwann cell mitosis

    International Nuclear Information System (INIS)

    Sobue, G.; Pleasure, D.

    1985-01-01

    Radioiodinated rat CNS axolemmal fragments adhered to cultured rat Schwann cells by a time-, temperature-, and concentration-dependent process independent of extracellular ionized calcium. Adhesion showed target and signal specificity; axolemmal fragments adhered to endoneurial or dermal fibroblasts to a much lesser extent than to Schwann cells, and plasma membrane fragments from skeletal muscle, erythrocytes, or PNS myelin adhered to Schwann cells to a lesser extent than did axolemmal fragments. Brief trypsinization removed 94 to 97% of bound radioactivity from Schwann cells previously incubated with 125 I-axolemmal fragments for up to 24 hr, indicating that adhesion was largely a surface phenomenon rather than the result of rapid internalization of axolemmal fragments by the Schwann cells. When adhesion was compared to the axolemmal mitogenic response of Schwann cells, the concentration of axolemmal fragments yielding half-maximal adhesion was the same as the concentration producing half-maximal stimulation of Schwann cell mitosis. Trypsin digestion, homogenization, or heating of axolemmal fragments before application to cultured Schwann cells diminished adhesion and axolemmal fragment-induced stimulation of Schwann cell mitosis in a parallel fashion. Whereas adhesion of axolemmal fragments to the surfaces of the cultured Schwann cells reached completion within 4 hr in this assay system, induction of Schwann cell mitosis by the fragments required contact with Schwann cells for a minimum of 6 to 8 hr and reached a maximum when the axolemmal fragments had adhered to the Schwann cells for 24 hr or more

  6. Current diagnostic efficacy of Tc-99m-labeled antitumor antibodies

    International Nuclear Information System (INIS)

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

    1983-01-01

    The authors have recently evaluated technetium 99 labeled antibodies specific to human chorionic gonadotropin (hCG) for the in vivo detection of a variety of human tumors. Both mouse monoclonal and sheep polyclonal antibodies were evaluated in this study. Another antibody specific to hCG studied, is an antigen-agglutinating monoclonal F(ab') 2 fragment. Some preliminary results are reported

  7. In-depth analysis of subclass-specific conformational preferences of IgG antibodies

    DEFF Research Database (Denmark)

    Tian, Xinsheng; Vestergaard, Bente; Thorolfsson, Matthias

    2015-01-01

    IgG subclass-specific differences in biological function and in vitro stability are often referred to variations in the conformational flexibility, while this flexibility has rarely been characterized. Here, small-angle X-ray scattering data from IgG1, IgG2 and IgG4 antibodies, which were designe...... properties and tailored effector functions. In addition, this advanced computational approach is applicable to other flexible multi-domain systems and extends the potential for investigating flexibility in solutions of macromolecules by small-angle X-ray scattering....

  8. Direct evidence that the VEGF-specific antibody bevacizumab has antivascular effects in human rectal cancer

    Science.gov (United States)

    Willett, Christopher G; Boucher, Yves; di Tomaso, Emmanuelle; Duda, Dan G; Munn, Lance L; Tong, Ricky T; Chung, Daniel C; Sahani, Dushyant V; Kalva, Sanjeeva P; Kozin, Sergey V; Mino, Mari; Cohen, Kenneth S; Scadden, David T; Hartford, Alan C; Fischman, Alan J; Clark, Jeffrey W; Ryan, David P; Zhu, Andrew X; Blaszkowsky, Lawrence S; Chen, Helen X; Shellito, Paul C; Lauwers, Gregory Y; Jain, Rakesh K

    2009-01-01

    The effects of vascular endothelial growth factor (VEGF) blockade on the vascular biology of human tumors are not known. Here we show here that a single infusion of the VEGF-specific antibody bevacizumab decreases tumor perfusion, vascular volume, microvascular density, interstitial fluid pressure and the number of viable, circulating endothelial and progenitor cells, and increases the fraction of vessels with pericyte coverage in rectal carcinoma patients. These data indicate that VEGF blockade has a direct and rapid antivascular effect in human tumors. PMID:14745444

  9. ETAC reagents: A new class of sulfhydryl site-specific radiolabelling probes for antibodies

    International Nuclear Information System (INIS)

    del Rosario, R.B.; Brocchini, S.J.; Baron, L.A.; Smith, R.H.; Lawton, R.G.; Wahl, R.L.

    1990-01-01

    A new class of bis-alkylating Michael reagents, equilibrium transfer crosslink reagents, 'ETAC', which combine the techniques of crosslinking with tethering have been synthesized. Following a succession of Michael and retro-Michael additions and elimination of the arylsulfone groups, reduced heavy-heavy and heavy-light disulfide links of an anti-ovarian IgG2a monoclonal antibody, 5G6.4, were site-specifically re-annealed via a 3-carbon bridge having a tether branch containing a designated label

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

    Energy Technology Data Exchange (ETDEWEB)

    Munz, D L; Sandrock, D; Emrich, D [Goettingen Univ. (Germany). Abt. fuer Nuklearmedizin; Morguet, A J; Heim, A; Sold, G; Figulla, H R; Kreuzer, H [Goettingen Univ. (Germany). Abt. fuer Kardiologie und Pulmonologie

    1991-12-01

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

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

    International Nuclear Information System (INIS)

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

    1991-01-01

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

  12. Antibody dynamics in BRSV-infected Danish dairy herds as determined by isotype-specific immunoglobulins

    DEFF Research Database (Denmark)

    Uttenthal, Åse; Larsen, Lars Erik; Philipsen, J.S.

    2000-01-01

    Using specific ELISAs, antibody levels of four different isotypes to bovine respiratory syncytial virus (BRSV) were determined in calves, following experimental BRSV infection. Most calves experienced an increase in the specific IgM and IgG1 titres about 6-10 days after infection with BRSV. The Ig......M titre was transient showing positive titres for only 5-10 days, while specific IgG1 was present for a longer time. IgA was detected concomitantly with IgM but at a lower level. Production of IgG2 anti-BRSV antibodies was detected from 3 weeks after infection. In two closed herds, repeated blood......, another herd with acute BRSV was followed by weekly blood samples in six calves; in both herds IgM and IgG1 was detected shortly after the appearance of clinical signs. Serum samples from 50 Danish dairy herds (453 samples) were tested for immunoglobulins of the isotypes IgG1, IgG2 and IgM. The presence...

  13. Crystallization and preliminary X-ray diffraction analysis of prion protein bound to the Fab fragment of the POM1 antibody

    International Nuclear Information System (INIS)

    Baral, Pravas Kumar; Wieland, Barbara; Swayampakula, Mridula; Polymenidou, Magdalini; Aguzzi, Adriano; Kav, Nat N. V.; James, Michael N. G.

    2011-01-01

    The complex of MoPrP(120–232) and Fab POM1 has been crystallized (space group C2, unit-cell parameters a = 83.68, b = 106.9, c = 76.25 Å, β = 95.6°). Diffraction data to 2.30 Å resolution have been collected using synchrotron radiation. Prion diseases are neurodegenerative diseases that are characterized by the conversion of the cellular prion protein PrP c to the pathogenic isoform PrP sc . Several antibodies are known to interact with the cellular prion protein and to inhibit this transition. An antibody Fab fragment, Fab POM1, was produced that recognizes a structural motif of the C-terminal domain of mouse prion protein. To study the mechanism by which Fab POM1 recognizes and binds the prion molecule, the complex between Fab POM1 and the C-terminal domain of mouse prion (residues 120–232) was prepared and crystallized. Crystals of this binary complex belonged to the monoclinic space group C2, with unit-cell parameters a = 83.68, b = 106.9, c = 76.25 Å, β = 95.6°

  14. Mapping the Binding Interface of VEGF and a Monoclonal Antibody Fab-1 Fragment with Fast Photochemical Oxidation of Proteins (FPOP) and Mass Spectrometry

    Science.gov (United States)

    Zhang, Ying; Wecksler, Aaron T.; Molina, Patricia; Deperalta, Galahad; Gross, Michael L.

    2017-05-01

    We previously analyzed the Fab-1:VEGF (vascular endothelial growth factor) system described in this work, with both native top-down mass spectrometry and bottom-up mass spectrometry (carboxyl-group or GEE footprinting) techniques. This work continues bottom-up mass spectrometry analysis using a fast photochemical oxidation of proteins (FPOP) platform to map the solution binding interface of VEGF and a fragment antigen binding region of an antibody (Fab-1). In this study, we use FPOP to compare the changes in solvent accessibility by quantitating the extent of oxidative modification in the unbound versus bound states. Determining the changes in solvent accessibility enables the inference of the protein binding sites (epitope and paratopes) and a comparison to the previously published Fab-1:VEGF crystal structure, adding to the top-down and bottom-up data. Using this method, we investigated peptide-level and residue-level changes in solvent accessibility between the unbound proteins and bound complex. Mapping these data onto the Fab-1:VEGF crystal structure enabled successful characterization of both the binding region and regions of remote conformation changes. These data, coupled with our previous higher order structure (HOS) studies, demonstrate the value of a comprehensive toolbox of methods for identifying the putative epitopes and paratopes for biotherapeutic antibodies.

  15. Pharmacokinetics of the FO23C5 anti-CEA antibody fragment labelled with 99Tcm and 111In: a comparison in patients

    International Nuclear Information System (INIS)

    Hnatowich, D.J.; Mardirossian, G.; Rusckowski, M.; Roy, S.; Busche, H.; Griffin, T.W.; Brill, A.B.

    1993-01-01

    The FO23C5 anti-carcinoembryonic antigen (CEA) F(ab') 2 antibody was radiolabelled with sup(111)In via diethylenetriaminepentaacetic acid (DTPA) and directly with 99 Tc m by stannous ion and mercaptoethanol antibody reduction to compare the pharmacokinetics of these three agents. Four patients received 15 mCi 99 Tc m -Fab' 1 week before receiving 1 mCi 111 In-F(ab') 2 . Five additional patients received only the 99 Tc m -Fab'. The biodistribution of 99 Tc m was as expected for a labelled Fab' fragment: relative to 111 In, 99 Tc m cleared rapidly from circulation and into kidneys and urine. Liver levels of 111 In and 99 Tc m were surprisingly similar at 1 day although initial 111 In levels were lower and increased while 99 Tc m levels were higher and decreased. Spleen levels were also similar. In 4/9 patients receiving 99 Tc m , hepatobiliary clearance was observed at levels which could confuse interpretation whereas this mode of clearance was observed in only 1/4 patients receiving 111 In. Image quality was superior with 111 In versus 99 Tc m at 1 day postadministration as judged by counting rates and background activity whereas the opposite was true at 2-3 h postadministration. (author)

  16. Multinuclear NMR study of the structure of the Fv fragment of anti-dansyl mouse IgG2a antibody

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, Hideo; Odaka, Asano; Matsunaga, Chigusa; Kato, Koichi; Shimada, Ichio; Arata, Yoji (Univ. of Tokyo (Japan)); Kawaminami, Shunro (Kao Corp., Tochigi (Japan))

    1991-07-02

    A multinuclear NMR study is reported of Fv, which is a minimum antigen-binding unit of immunoglobulin. Fv has been prepared by clostripain digestion of a mouse anti-dansyl IgG2a monoclonal antibody that lacks the entire C{sub H}1 domain. A variety of Fv analogues labeled with {sup 2}H in the aromatic rings and with {sup 13}C and/or {sup 15}N in the peptide bonds have been prepared and used for multinuclear NMR analyses of Fv spectra of Fv sensitively reflect the antigen binding and can be used along with {sup 1}H and {sup 13}C spectral data for the structural analyses of antigen-antibody interactions. Hydrogen-deuterium exchange of the amide protons has been folowed in the absence and presence of DNS-Lys by using the {sup 1}H-{sup 15}N shift correlation spectra. Use of the {beta}-shift observed for the carbonyl carbon resonances has also been helpful in following the hydrogen-deuterium exchange. On the basis of the NMR data obtained, the static and dynamic structure of the Fv fragment in the absence and presence of DNS-Lys has been discussed.

  17. Multinuclear NMR study of the structure of the Fv fragment of anti-dansyl mouse IgG2a antibody

    International Nuclear Information System (INIS)

    Takahashi, Hideo; Odaka, Asano; Matsunaga, Chigusa; Kato, Koichi; Shimada, Ichio; Arata, Yoji; Kawaminami, Shunro

    1991-01-01

    A multinuclear NMR study is reported of Fv, which is a minimum antigen-binding unit of immunoglobulin. Fv has been prepared by clostripain digestion of a mouse anti-dansyl IgG2a monoclonal antibody that lacks the entire C H 1 domain. A variety of Fv analogues labeled with 2 H in the aromatic rings and with 13 C and/or 15 N in the peptide bonds have been prepared and used for multinuclear NMR analyses of Fv spectra of Fv sensitively reflect the antigen binding and can be used along with 1 H and 13 C spectral data for the structural analyses of antigen-antibody interactions. Hydrogen-deuterium exchange of the amide protons has been folowed in the absence and presence of DNS-Lys by using the 1 H- 15 N shift correlation spectra. Use of the β-shift observed for the carbonyl carbon resonances has also been helpful in following the hydrogen-deuterium exchange. On the basis of the NMR data obtained, the static and dynamic structure of the Fv fragment in the absence and presence of DNS-Lys has been discussed

  18. Affinity isolation of antigen-specific circulating B cells for generation of phage display-derived human monoclonal antibodies

    DEFF Research Database (Denmark)

    Ditzel, Henrik

    2009-01-01

    A method is described for affinity isolation of antigen-specific circulating B cells of interest for subsequent generation of immune antibody phage display libraries. This approach should overcome the problem of low yields of monoclonal antibodies of interest in the libraries generated from...... peripheral blood lymphocytes caused by the low abundance of antigen-specific B cells in the circulation. The preselection of B cells is based on the specificity of the surface Ig receptor and is accomplished using the antigen of interest conjugated to magnetic beads. This method should significantly increase...... the frequency of antibody phage particles of interest in the library and allow for efficient isolation monoclonal antibodies with the predefined specificity....

  19. Increased specificity in human cardiac-myosin radioimmunoassay utilizing two monoclonal antibodies in a double sandwich assay

    International Nuclear Information System (INIS)

    Katus, H.A.; Hurrell, J.G.; Matsueda, G.R.; Ehrlich, P.; Zurawski, V.R. Jr.; Khaw, B.-A.; Haber, E.

    1982-01-01

    An immunoradiometric assay that simultaneously measured two different epitopes on the same molecule was devised to differential between cardiac- and skeletal-myosin light chains. Three monoclonal antibodies were examined that were 100% (lC5), 25% (2B9) and 17% (4F10) cross reactive, respectively, between the two antigens. One antibody of the pair to be studied was immobilized to cyanogen bromide-activated Sepharose 4B while the other was iodinated with 125 I using the lactoperoxidase method. The antigen was mixed with the immobilized antibody, the labeled antibody was added and the precipitate then washed and counted in a gamma counter. When both antibodies of the pair to be studied (immobilized and labeled) were the same (2B9), no radioactivity above background was bound to the precipitate, indicating that the second antibody could not bind to an already occupied epitope. When two different antibodies were employed, the specificity of the assay increased over that of a single antibody. The cross reactivity of a pair approximated the product of the cross reactivities of the individual antibodies. Thus, lC5 and 2B9 were 25% cross reactive together, lC5 and 4F10 17% cross reactive, and 2B9 and 4F10 4.3% cross reactive. (author)

  20. A recombinant mimetics of the HIV-1 gp41 prehairpin fusion intermediate fused with human IgG Fc fragment elicits neutralizing antibody response in the vaccinated mice

    International Nuclear Information System (INIS)

    Qi, Zhi; Pan, Chungen; Lu, Hong; Shui, Yuan; Li, Lin; Li, Xiaojuan; Xu, Xueqing; Liu, Shuwen; Jiang, Shibo

    2010-01-01

    Research highlights: → One recombinant mimetics of gp41 prehairpin fusion intermediate (PFI) consisting of gp41 N46 sequence, foldon and IgG Fc, designated N46FdFc, was expressed. → N46FdFc-induced antibodies in mice that neutralized HIV-1 infection, inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. → These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines. -- Abstract: HIV-1 gp41 prehairpin fusion intermediate (PFI) composed of three N-terminal heptad repeats (NHR) plays a crucial role in viral fusion and entry and represents an attractive target for anti-HIV therapeutics (e.g., enfuvirtide) and vaccines. In present study, we constructed and expressed two recombinant gp41 PFI mimetics, designated N46Fd and N46FdFc. N46Fd consists of N46 (residues 536-581) in gp41 NHR and foldon (Fd), a trimerization motif. N46FdFc is composed of N46Fd fused with human IgG Fc fragment as an immunoenhancer. We immunized mice with N46 peptide, N46Fd and N46FdFc, respectively, and found that only N46FdFc elicited neutralizing antibody response in mice against infection by HIV-1 strains IIIB (clade B, X4), 92US657 (clade B, R5), and 94UG103 (clade A, X4R5). Anti-N46FdFc antibodies inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines.