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Sample records for antibody complementarity-determining regions

  1. A complete, multi-level conformational clustering of antibody complementarity-determining regions

    Directory of Open Access Journals (Sweden)

    Dimitris Nikoloudis

    2014-07-01

    Full Text Available Classification of antibody complementarity-determining region (CDR conformations is an important step that drives antibody modelling and engineering, prediction from sequence, directed mutagenesis and induced-fit studies, and allows inferences on sequence-to-structure relations. Most of the previous work performed conformational clustering on a reduced set of structures or after application of various structure pre-filtering criteria. In this study, it was judged that a clustering of every available CDR conformation would produce a complete and redundant repertoire, increase the number of sequence examples and allow better decisions on structure validity in the future. In order to cope with the potential increase in data noise, a first-level statistical clustering was performed using structure superposition Root-Mean-Square Deviation (RMSD as a distance-criterion, coupled with second- and third-level clustering that employed Ramachandran regions for a deeper qualitative classification. The classification of a total of 12,712 CDR conformations is thus presented, along with rich annotation and cluster descriptions, and the results are compared to previous major studies. The present repertoire has procured an improved image of our current CDR Knowledge-Base, with a novel nesting of conformational sensitivity and specificity that can serve as a systematic framework for improved prediction from sequence as well as a number of future studies that would aid in knowledge-based antibody engineering such as humanisation.

  2. Contributions of the complementarity determining regions to the thermal stability of a single-domain antibody.

    Science.gov (United States)

    Zabetakis, Dan; Anderson, George P; Bayya, Nikhil; Goldman, Ellen R

    2013-01-01

    Single domain antibodies (sdAbs) are the recombinantly-expressed variable domain from camelid (or shark) heavy chain only antibodies and provide rugged recognition elements. Many sdAbs possess excellent affinity and specificity; most refold and are able to bind antigen after thermal denaturation. The sdAb A3, specific for the toxin Staphylococcal enterotoxin B (SEB), shows both sub-nanomolar affinity for its cognate antigen (0.14 nM) and an unusually high melting point of 85°C. Understanding the source of sdAb A3's high melting temperature could provide a route for engineering improved melting temperatures into other sdAbs. The goal of this work was to determine how much of sdAb A3's stability is derived from its complementarity determining regions (CDRs) versus its framework. Towards answering this question we constructed a series of CDR swap mutants in which the CDRs from unrelated sdAbs were integrated into A3's framework and where A3's CDRs were integrated into the framework of the other sdAbs. All three CDRs from A3 were moved to the frameworks of sdAb D1 (a ricin binder that melts at 50°C) and the anti-ricin sdAb C8 (melting point of 60°C). Similarly, the CDRs from sdAb D1 and sdAb C8 were moved to the sdAb A3 framework. In addition individual CDRs of sdAb A3 and sdAb D1 were swapped. Melting temperature and binding ability were assessed for each of the CDR-exchange mutants. This work showed that CDR2 plays a critical role in sdAb A3's binding and stability. Overall, results from the CDR swaps indicate CDR interactions play a major role in the protein stability. PMID:24143255

  3. Proline-rich tandem repeats of antibody complementarity-determining regions bind and neutralize human immunodeficiency virus type 1 particles.

    OpenAIRE

    Fontenot, J D; Zacharopoulos, V R; Phillips, D M

    1996-01-01

    The proline-rich tandem repeat domain of human mucin MUC1 forms an extended structure containing large repeating loops that are crested by a turn. We show that the repeating-loop structure of MUC1 can be replaced by an antibody complementarity-determining region loop of a human immunodeficiency virus type 1 (HIV-1)-specific neutralizing antibody to create a chimeric, multivalent, mucin-like, anti-HIV-1 compound. We used 8 residues of an antibody molecule to replace 8 of 20 residues of the MUC...

  4. Antibody complementarity-determining regions (CDRs can display differential antimicrobial, antiviral and antitumor activities.

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    Luciano Polonelli

    Full Text Available BACKGROUND: Complementarity-determining regions (CDRs are immunoglobulin (Ig hypervariable domains that determine specific antibody (Ab binding. We have shown that synthetic CDR-related peptides and many decapeptides spanning the variable region of a recombinant yeast killer toxin-like antiidiotypic Ab are candidacidal in vitro. An alanine-substituted decapeptide from the variable region of this Ab displayed increased cytotoxicity in vitro and/or therapeutic effects in vivo against various bacteria, fungi, protozoa and viruses. The possibility that isolated CDRs, represented by short synthetic peptides, may display antimicrobial, antiviral and antitumor activities irrespective of Ab specificity for a given antigen is addressed here. METHODOLOGY/PRINCIPAL FINDINGS: CDR-based synthetic peptides of murine and human monoclonal Abs directed to: a a protein epitope of Candida albicans cell wall stress mannoprotein; b a synthetic peptide containing well-characterized B-cell and T-cell epitopes; c a carbohydrate blood group A substance, showed differential inhibitory activities in vitro, ex vivo and/or in vivo against C. albicans, HIV-1 and B16F10-Nex2 melanoma cells, conceivably involving different mechanisms of action. Antitumor activities involved peptide-induced caspase-dependent apoptosis. Engineered peptides, obtained by alanine substitution of Ig CDR sequences, and used as surrogates of natural point mutations, showed further differential increased/unaltered/decreased antimicrobial, antiviral and/or antitumor activities. The inhibitory effects observed were largely independent of the specificity of the native Ab and involved chiefly germline encoded CDR1 and CDR2 of light and heavy chains. CONCLUSIONS/SIGNIFICANCE: The high frequency of bioactive peptides based on CDRs suggests that Ig molecules are sources of an unlimited number of sequences potentially active against infectious agents and tumor cells. The easy production and low cost of small

  5. Contributions of the Complementarity Determining Regions to the Thermal Stability of a Single-Domain Antibody

    OpenAIRE

    Zabetakis, Dan; Anderson, George P.; Bayya, Nikhil; Goldman, Ellen R.

    2013-01-01

    Single domain antibodies (sdAbs) are the recombinantly-expressed variable domain from camelid (or shark) heavy chain only antibodies and provide rugged recognition elements. Many sdAbs possess excellent affinity and specificity; most refold and are able to bind antigen after thermal denaturation. The sdAb A3, specific for the toxin Staphylococcal enterotoxin B (SEB), shows both sub-nanomolar affinity for its cognate antigen (0.14 nM) and an unusually high melting point of 85°C. Understanding ...

  6. Characterization of a high-affinity human antibody with a disulfide bridge in the third complementarity-determining region of the heavy chain.

    Science.gov (United States)

    Almagro, Juan Carlos; Raghunathan, Gopalan; Beil, Eric; Janecki, Dariusz J; Chen, Qiang; Dinh, Thai; LaCombe, Ann; Connor, Judy; Ware, Mark; Kim, Paul H; Swanson, Ronald V; Fransson, Johan

    2012-03-01

    Disulfide bridges are common in the antigen-binding site from sharks (new antigen receptor) and camels (single variable heavy-chain domain, VHH), in which they confer both structural diversity and domain stability. In human antibodies, cysteine residues in the third complementarity-determining region of the heavy chain (CDR-H3) are rare but naturally encoded in the IGHD germline genes. Here, by panning a phage display library designed based on human germline genes and synthetic CDR-H3 regions against a human cytokine, we identified an antibody (M3) containing two cysteine residues in the CDR-H3. It binds the cytokine with high affinity (0.4 nM), recognizes a unique epitope on the antigen, and has a distinct neutralization profile as compared with all other antibodies selected from the library. The two cysteine residues form a disulfide bridge as determined by mass spectrometric peptide mapping. Replacing the cysteines with alanines did not change the solubility and stability of the monoclonal antibody, but binding to the antigen was significantly impaired. Three-dimensional modeling and dynamic simulations were employed to explore how the disulfide bridge influences the conformation of CDR-H3 and binding to the antigen. On the basis of these results, we envision that designing human combinatorial antibody libraries to contain intra-CDR or inter-CDR disulfide bridges could lead to identification of human antibodies with unique binding profiles. PMID:22407976

  7. Substitution of Heavy Complementarity Determining Region 3 (CDR-H3) Residues Can Synergistically Enhance Functional Activity of Antibody and Its Binding Affinity to HER2 Antigen.

    Science.gov (United States)

    Moon, Seung Kee; Park, So Ra; Park, Ami; Oh, Hyun Mi; Shin, Hyun Jung; Jeon, Eun Ju; Kim, Seiwhan; Park, Hyun June; Yeon, Young Joo; Yoo, Young Je

    2016-03-31

    To generate a biobetter that has improved therapeutic activity, we constructed scFv libraries via random mutagenesis of several residues of CDR-H3 and -L3 of hu4D5. The scFv clones were isolated from the phage display libraries by stringent panning, and their anti-proliferative activity against HER2-positive cancer cells was evaluated as a primary selection criterion. Consequently, we selected AH06 as a biobetter antibody that had a 7.2-fold increase in anti-proliferative activity (IC50: 0.81 nM) against the gastric cancer cell line NCI-N87 and a 7.4-fold increase in binding affinity (KD: 60 pM) to HER2 compared to hu4D5. The binding energy calculation and molecular modeling suggest that the substitution of residues of CDR-H3 to W98, F100c, A101 and L102 could stabilize binding of the antibody to HER2 and there could be direct hydrophobic interactions between the aromatic ring of W98 and the aliphatic group of I613 within HER2 domain IV as well as the heavy and light chain hydrophobic interactions by residues F100c, A101 and L102 of CDR-H3. Therefore, we speculate that two such interactions were exerted by the residues W98 and F100c. A101 and L102 may have a synergistic effect on the increase in the binding affinity to HER2. AH06 specifically binds to domain IV of HER2, and it decreased the phosphorylation level of HER2 and AKT. Above all, it highly increased the overall level of p27 compared to hu4D5 in the gastric cancer cell line NCI-N82, suggesting that AH06 could potentially be a more efficient therapeutic agent than hu4D5. PMID:26743905

  8. A Combinatorial Approach for the Design of Complementarity-determining Region-derived Peptidomimetics with in Vitro Anti-tumoral Activity*

    OpenAIRE

    Timmerman, Peter; Barderas, Rodrigo; Desmet, Johan; Altschuh, Danièle; Shochat, Susana; Hollestelle, Martine J.; Jo W. M. Höppener; Monasterio, Alberto; Casal, J. Ignacio; Meloen, Rob H.

    2009-01-01

    The great success of therapeutic monoclonal antibodies has fueled research toward mimicry of their binding sites and the development of new strategies for peptide-based mimetics production. Here, we describe a new combinatorial approach for the production of peptidomimetics using the complementarity-determining regions (CDRs) from gastrin17 (pyroEGPWLEEEEEAYGWMDF-NH2) antibodies as starting material for cyclic peptide synthesis in a microarray format. Gastrin17 is a trophic factor in gastroin...

  9. Control of autoantibody affinity by selection against amino acid replacements in the complementarity-determining regions.

    OpenAIRE

    Børretzen, M; Randen, I; Zdárský, E; Førre, O; Natvig, J B; Thompson, K M

    1994-01-01

    Rheumatoid factor (RF) autoantibodies can be produced in healthy individuals after infections or immunizations and thus escape normal tolerization mechanisms. It has not been clear whether such autoantibodies can undergo somatic hypermutation and affinity maturation similar to antibodies to exogenous antigens. We have investigated how these autoantibodies are regulated in normal individuals by analyzing the sequences of monoclonal IgM RFs obtained as hybridomas from donors after immunization....

  10. Heavy chain V region diversity in the duck-billed platypus (Ornithorhynchus anatinus): long and highly variable complementarity-determining region 3 compensates for limited germline diversity.

    Science.gov (United States)

    Johansson, Jeannette; Aveskogh, Maria; Munday, Barry; Hellman, Lars

    2002-05-15

    In this work, to study the emergence of the H chain V region repertoire during mammalian evolution, we present an analysis of 25 independent H chain V regions from a monotreme, the Australian duck-billed platypus, Ornithorhynchus anatinus. All the sequences analyzed were found to form a single branch within the clan III of mammalian V region sequences in a distance tree. However, compared with a classical V gene family this branch was more diversified in sequence. Sequence analysis indicates that the apparent lack of diversity in germline V segments is well compensated for by relatively long and highly diversified D and N nucleotides. In addition, extensive sequence variation was observed in the framework region 3. Furthermore, at least five and possibly seven different J segments seem to be actively used in recombination. Interestingly, internal cysteine bridges in the complementarity-determining region (CDR)3 loop, or between the CDR2 and CDR3 loops, are found in approximately 36% of the platypus V(H) sequences. Such cysteine bridges have also been observed in cow, camel, and shark. Internal cysteine bridges may play a role in stabilizing long and diversified CDR3 and thereby have a role in increasing the affinity of the Ab-Ag interaction. PMID:11994470

  11. Temperature and Stretching Effects on Complementarity Determining Regions (CDRs Conformation and Stability of Nimotuzumab F(ab-Fragment

    Directory of Open Access Journals (Sweden)

    T.S. Humani

    2015-04-01

    Full Text Available Nimotuzumab is a humanized monoclonal antibody (mAb, a potential anticancer against epidermal growth factor receptor (EGFRoverexpressed by glioma, head and neck, lung, ovarium, and colon cancers. The combination of its use with both external and internal beam radiotherapies showed improvement of the therapeutic effect. However, the high molecular weight slows its uptake on tumor cells. In a recent development, nimotuzumab has been fragmented and then labeled using diagnostic and therapeutic radionuclides, such as gallium-68, yttrium-90, lutetium-177, and holmium-166. In that preparation, nimotuzumab is often conditioned in various environments with variations of pH, temperature and the presence of other compounds. In this research, molecular dynamics (MD simulation have been carried out to study the CDRs conformational change of nimotuzumab due to the effect of temperature, and also steered molecular dynamics (SMD simulation to study the stability of nimotuzumab domain as a result of external forces. The simulations were performed using the Not Just Another Molecular Dynamics (NAMD program package and the analysis was performed with the Visual Molecular Dynamics (VMD program package. Based on the stability analysis of each residue on the heavy chain, the active site (CDR3 region that is at residues numbered 98 (Tryptophan and 99 (Phenylalanine has the highest conformational changes. On the light chain, the change occurs at residues numbered 1 (Aspartat, 127 (Serin, and 186 (Tyrosine; and that none of that residues is part of active site or CDRs region of the light chain. The SMD simulation was carried out by fixing the N-terminal end of the heavy chain and applying external forces to the C-terminal end. The pulling was set at a constant velocity of 0.5 Å/ps. The force peak arising at the beginning of the unfolding process is 1226 pN. This force was allegedly caused by the rupture of hydrogen bonds between the heavy chain residue VAL211 (Valine

  12. A combinatorial approach for the design of complementarity-determining region-derived peptidomimetics with in vitro anti-tumoral activity

    NARCIS (Netherlands)

    P. Timmerman; R. Barderas; J. Desmet; D. Altschuh; S. Shochat; M.J. Hollestelle; J.W.M. Höppener; A. Monasterio; J.I. Casal; R.H. Meloen

    2009-01-01

    The great success of therapeutic monoclonal antibodies has fueled research toward mimicry of their binding sites and the development of new strategies for peptide-based mimetics production. Here, we describe a new combinatorial approach for the production of peptidomimetics using the complementarity

  13. Importance of Hypervariable Region 2 for Stability and Affinity of a Shark Single-Domain Antibody Specific for Ebola Virus Nucleoprotein

    Science.gov (United States)

    Anderson, George P.; Teichler, Daniel D.; Zabetakis, Dan; Shriver-Lake, Lisa C.; Liu, Jinny L.; Lonsdale, Stephen G.; Goodchild, Sarah A.; Goldman, Ellen R.

    2016-01-01

    Single-domain antibodies derived from the unique New Antigen Receptor found in sharks have numerous potential applications, ranging from diagnostic reagents to therapeutics. Shark-derived single-domain antibodies possess the same characteristic ability to refold after heat denaturation found in single-domain antibodies derived from camelid heavy-chain-only antibodies. Recently, two shark derived single-domain antibodies specific for the nucleoprotein of Ebola virus were described. Our evaluation confirmed their high affinity for the nucleoprotein, but found their melting temperatures to be low relative to most single-domain antibodies. Our first approach towards improving their stability was grafting antigen-binding regions (complementarity determining regions) of one of these single-domain antibodies onto a high melting temperature shark single-domain antibody. This resulted in two variants: one that displayed excellent affinity with a low melting temperature, while the other had poor affinity but a higher melting temperature. These new proteins, however, differed in only 3 amino acids within the complementarity determining region 2 sequence. In shark single-domain antibodies, the complementarity determining region 2 is often referred to as hypervariable region 2, as this segment of the antibody domain is truncated compared to the sequence in camelid single-domain antibodies and conventional heavy chain variable domains. To elucidate which of the three amino acids or combinations thereof were responsible for the affinity and stability we made the 6 double and single point mutants that covered the intermediates between these two clones. We found a single amino acid change that achieved a 10°C higher melting temperature while maintaining sub nM affinity. This research gives insights into the impact of the shark sdAb hypervariable 2 region on both stability and affinity. PMID:27494523

  14. Importance of Hypervariable Region 2 for Stability and Affinity of a Shark Single-Domain Antibody Specific for Ebola Virus Nucleoprotein.

    Science.gov (United States)

    Anderson, George P; Teichler, Daniel D; Zabetakis, Dan; Shriver-Lake, Lisa C; Liu, Jinny L; Lonsdale, Stephen G; Goodchild, Sarah A; Goldman, Ellen R

    2016-01-01

    Single-domain antibodies derived from the unique New Antigen Receptor found in sharks have numerous potential applications, ranging from diagnostic reagents to therapeutics. Shark-derived single-domain antibodies possess the same characteristic ability to refold after heat denaturation found in single-domain antibodies derived from camelid heavy-chain-only antibodies. Recently, two shark derived single-domain antibodies specific for the nucleoprotein of Ebola virus were described. Our evaluation confirmed their high affinity for the nucleoprotein, but found their melting temperatures to be low relative to most single-domain antibodies. Our first approach towards improving their stability was grafting antigen-binding regions (complementarity determining regions) of one of these single-domain antibodies onto a high melting temperature shark single-domain antibody. This resulted in two variants: one that displayed excellent affinity with a low melting temperature, while the other had poor affinity but a higher melting temperature. These new proteins, however, differed in only 3 amino acids within the complementarity determining region 2 sequence. In shark single-domain antibodies, the complementarity determining region 2 is often referred to as hypervariable region 2, as this segment of the antibody domain is truncated compared to the sequence in camelid single-domain antibodies and conventional heavy chain variable domains. To elucidate which of the three amino acids or combinations thereof were responsible for the affinity and stability we made the 6 double and single point mutants that covered the intermediates between these two clones. We found a single amino acid change that achieved a 10°C higher melting temperature while maintaining sub nM affinity. This research gives insights into the impact of the shark sdAb hypervariable 2 region on both stability and affinity. PMID:27494523

  15. Immunogenicity of an engineered internal image antibody.

    OpenAIRE

    Billetta, R; Hollingdale, M. R.; Zanetti, M

    1991-01-01

    We engineered an antibody expressing in the third complementarity-determining region of its heavy chain variable region a "foreign" epitope, the repetitive tetrapeptide Asn-Ala-Asn-Pro (NANP) of the circumsporozoite protein of Plasmodium falciparum parasite, one of the etiologic agents of malaria in humans. A monoclonal antibody to P. falciparum specific for the (NANP)n amino acid sequence bound to the engineered antibody, and a synthetic (NANP)3 peptide blocked this interaction. Immunization...

  16. A general approach to antibody thermostabilization

    OpenAIRE

    McConnell, Audrey D; Xue ZHANG; Macomber, John L.; Chau, Betty; Sheffer, Joseph C; Rahmanian, Sorena; Hare, Eric; Spasojevic, Vladimir; Horlick, Robert A.; King, David J; Bowers, Peter M.

    2014-01-01

    Antibody engineering to enhance thermostability may enable further application and ease of use of antibodies across a number of different areas. A modified human IgG framework has been developed through a combination of engineering approaches, which can be used to stabilize antibodies of diverse specificity. This is achieved through a combination of complementarity-determining region (CDR)-grafting onto the stable framework, mammalian cell display and in vitro somatic hypermutation (SHM). Thi...

  17. Crystal structure of a shark single-domain antibody V region in complex with lysozyme.

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    Stanfield, Robyn L; Dooley, Helen; Flajnik, Martin F; Wilson, Ian A

    2004-09-17

    Cartilaginous fish are the phylogenetically oldest living organisms known to possess components of the vertebrate adaptive immune system. Key to their immune response are heavy-chain, homodimeric immunoglobulins called new antigen receptors (IgNARs), in which the variable (V) domains recognize antigens with only a single immunoglobulin domain, akin to camelid heavy-chain V domains. The 1.45 angstrom resolution crystal structure of the type I IgNAR V domain in complex with hen egg-white lysozyme (HEL) reveals a minimal antigen-binding domain that contains only two of the three conventional complementarity-determining regions but still binds HEL with nanomolar affinity by means of a binding interface comparable in size to conventional antibodies. PMID:15319492

  18. Cloning and Sequence Analysis of Light Variable Region Gene of Anti-human Retinoblastoma Monoclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    Xiufeng Zhong; Yongping Li; Shuqi Huang; Bo Ning; Chunyan Zhang; Jianliang Zheng; Guanguang Feng

    2002-01-01

    Purpose: To clone the variable region gene of light chain of monoclonal antibody against human retinoblastoma and to analyze the characterization of its nucleotide sequence as well as amino acid sequence.Methods: Total RNA was extracted from 3C6 hybridoma cells secreting specific monoclonal antibody(McAb)against human retinoblastoma(RB), then transcripted reversely into cDNA with olig-dT primers.The variable region of the light chain (VL) gene fragments was amplified using polymeerase chain reaction(PCR) and further cloned into pGEM(R) -T Easy vector. Then, 3C6 VL cDNA was sequenced by Sanger's method.Homologous analysis was done by NCBI BLAST.Results: The complete nucleotide sequence of 3C6 VL cDNA consisted of 321 bp encoding 107 amino acid residues, containing four workframe regions(FRs)and three complementarity-determining regions (CDRs) as well as the typical structure of two cys residues. The sequence is most homological to a member of the Vk9 gene family, and its chain utilizes the Jkl gene segment.Conclusion: The light chain variable region gene of the McAb against human RB was amplified successfully , which belongs to the Vk9 gene family and utilizes Vk-Jk1 gene rearrangement. This study lays a good basis for constructing a recombinant antibody and for making a new targeted therapeutic agents against retinoblastoma.

  19. Antibody remodeling: a general solution to the design of a metal-coordination site in an antibody binding pocket.

    OpenAIRE

    Roberts, V A; Iverson, B L; Iverson, S A; Benkovic, S J; Lerner, R A; Getzoff, E D; Tainer, J A

    1990-01-01

    To develop a general approach to designing cofactor-binding sites for catalytic antibodies, we characterized structural patterns in the binding sites of antibodies and zinc enzymes. Superposition of eight sets of antibody light- and heavy-chain variable domains identified structurally conserved sites within the sequence-variable complementarity determining regions. The pattern for catalytic zinc sites included two ligands close in sequence, a sequence-distant ligand, and a main-chain hydrogen...

  20. Polymerase chain reaction facilitates the cloning, CDR-grafting, and rapid expression of a murine monoclonal antibody directed against the CD18 component of leukocyte integrins.

    OpenAIRE

    Daugherty, B L; DeMartino, J A; Law, M F; Kawka, D W; Singer, I I; Mark, G E

    1991-01-01

    Two novel approaches of recombinant PCR technology were employed to graft the complementarity determining regions from a murine monoclonal antibody (mAb) onto human antibody frameworks. One approach relied on the availability of cloned human variable region templates, whereas the other strategy was dependent only on human variable region protein sequence data. The transient expression of recombinant humanized antibody was driven by the adenovirus major late promoter and was detected 48 hrs po...

  1. Antibody mimetics: promising complementary agents to animal-sourced antibodies.

    Science.gov (United States)

    Baloch, Abdul Rasheed; Baloch, Abdul Wahid; Sutton, Brian J; Zhang, Xiaoying

    2016-01-01

    Despite their wide use as therapeutic, diagnostic and detection agents, the limitations of polyclonal and monoclonal antibodies have inspired scientists to design the next generation biomedical agents, so-called antibody mimetics that offer many advantages over conventional antibodies. Antibody mimetics can be constructed by protein-directed evolution or fusion of complementarity-determining regions through intervening framework regions. Substantial progress in exploiting human, butterfly (Pieris brassicae) and bacterial systems to design and select mimetics using display technologies has been made in the past 10 years, and one of these mimetics [Kalbitor® (Dyax)] has made its way to market. Many challenges lie ahead to develop mimetics for various biomedical applications, especially those for which conventional antibodies are ineffective, and this review describes the current characteristics, construction and applications of antibody mimetics compared to animal-sourced antibodies. The possible limitations of mimetics and future perspectives are also discussed. PMID:25264572

  2. Isolation of anti-toxin single domain antibodies from a semi-synthetic spiny dogfish shark display library

    OpenAIRE

    Goldman Ellen R; Anderson George P; Liu Jinny L

    2007-01-01

    Abstract Background Shark heavy chain antibody, also called new antigen receptor (NAR), consists of one single Variable domain (VH), containing only two complementarity-determining regions (CDRs). The antigen binding affinity and specificity are mainly determined by these two CDRs. The good solubility, excellent thermal stability and complex sequence variation of small single domain antibodies (sdAbs) make them attractive alternatives to conventional antibodies. In this report, we construct a...

  3. 慢性乙型肝炎患者外周血及肝组织中T淋巴细胞受体β链互补决定区3谱系分析%Analysis of the T lymphocyte receptor beta chain complementarity determining region 3 spectratyping in the peripheral blood and hepatic tissue of patients with chronic hepatitis B

    Institute of Scientific and Technical Information of China (English)

    伍绍强; 姚新生; 邱隆敏; 马锐; 毕晓英; 陈宇

    2010-01-01

    目的 分析慢性乙型肝炎(CHB)患者外周血及肝组织T淋巴细胞受体(TCR)β链各家族互补决定区3(CDR3)谱系的变化,了解CHB患者T淋巴细胞克隆增生的情况.方法 采用荧光定量PCR(FQ-PCR)溶解曲线法分析4例健康对照者及11例CHB患者外周血单个核细胞(PBMC)及肝组织T淋巴细胞受体β链可变区(TRBV)基因各家族CDR3谱系漂移情况,比较11例CHB患者TCR β链可变区各家族外周血及肝组织CDR3谱系漂移异常率.率的比较采用卡方检验.结果 11例CHB患者PBMC及肝组织TRBV各家族CDR3溶解曲线谱型图出现数量不等、形态不一的单峰、寡峰、偏峰及极低水平或缺失等现象,肝组织TRBV家族CDR3的谱系漂移异常率明显高于外周血的异常率(x2=23.246,P<0.01),在同一患者的外周血及肝组织中发现部分相同的TRBV亚家族表达,TRBV13.1、BV17和BV22同时被多例CHB患者的外周血及肝组织限制性取用.结论 CHB患者PBMC及肝组织中的T淋巴细胞均存在不同程度的克隆增生,部分TRBV亚家族被多例CHB患者外周血及肝组织同时限制性取用.%Objective To analyse the spectral patterns of complementarity determining region 3 (CDR3) length distribution of T lymphocyte receptor beta chain variable (TRBV) gene families in infiltrating T cells of the liver tissues and the peripheral blood samples of patients with chronic hepatitis B (CHB) in order to evaluate the characteristics of T cell clonal expansion. Methods The spectral patterns drift of TRBV gene families (the monoclonal/oligoclonal TCR β T cells) in the peripheral blood and hepatic tissues from 11 cases of CHB patients were analyzed by the real-time fluorescence quantitative reverse transcription polymerase chain reaction (FQ-PCR) with DNA melting curve analysis, and abnormal rates of TRBV gene families were compared between CHB patients and healthy control. The comparison of rates was done by chi square test. Results The gene melting

  4. Platform for high-throughput antibody selection using synthetically-designed antibody libraries.

    Science.gov (United States)

    Batonick, Melissa; Holland, Erika G; Busygina, Valeria; Alderman, Dawn; Kay, Brian K; Weiner, Michael P; Kiss, Margaret M

    2016-09-25

    Synthetic humanized antibody libraries are frequently generated by random incorporation of changes at multiple positions in the antibody hypervariable regions. Although these libraries have very large theoretical diversities (>10(20)), the practical diversity that can be achieved by transformation of Escherichia coli is limited to about 10(10). To constrain the practical diversity to sequences that more closely mimic the diversity of natural human antibodies, we generated a scFv phage library using entirely pre-defined complementarity determining regions (CDR). We have used this library to select for novel antibodies against four human protein targets and demonstrate that identification of enriched sequences at each of the six CDRs in early selection rounds can be used to reconstruct a consensus antibody with selectivity for the target. PMID:26607994

  5. Distinct antibody species: structural differences creating therapeutic opportunities.

    Science.gov (United States)

    Muyldermans, Serge; Smider, Vaughn V

    2016-06-01

    Antibodies have been a remarkably successful class of molecules for binding a large number of antigens in therapeutic, diagnostic, and research applications. Typical antibodies derived from mouse or human sources use the surface formed by complementarity determining regions (CDRs) on the variable regions of the heavy chain/light chain heterodimer, which typically forms a relatively flat binding surface. Alternative species, particularly camelids and bovines, provide a unique paradigm for antigen recognition through novel domains which form the antigen binding paratope. For camelids, heavy chain antibodies bind antigen with only a single heavy chain variable region, in the absence of light chains. In bovines, ultralong CDR-H3 regions form an independently folding minidomain, which protrudes from the surface of the antibody and is diverse in both its sequence and disulfide patterns. The atypical paratopes of camelids and bovines potentially provide the ability to interact with different epitopes, particularly recessed or concave surfaces, compared to traditional antibodies. PMID:26922135

  6. An antibody with a variable-region coiled-coil "knob" domain.

    Science.gov (United States)

    Zhang, Yong; Goswami, Devrishi; Wang, Danling; Wang, Tsung-Shing Andrew; Sen, Shiladitya; Magliery, Thomas J; Griffin, Patrick R; Wang, Feng; Schultz, Peter G

    2014-01-01

    The X-ray crystal structure of a bovine antibody (BLV1H12) revealed a unique structure in its ultralong heavy chain complementarity determining region 3 (CDR3H) that folds into a solvent-exposed β-strand "stalk" fused to a disulfide crosslinked "knob" domain. We have substituted an antiparallel heterodimeric coiled-coil motif for the β-strand stalk in this antibody. The resulting antibody (Ab-coil) expresses in mammalian cells and has a stability similar to that of the parent bovine antibody. MS analysis of H-D exchange supports the coiled-coil structure of the substituted peptides. Substitution of the knob-domain of Ab-coil with bovine granulocyte colony-stimulating factor (bGCSF) results in a stably expressed chimeric antibody, which proliferates mouse NFS-60 cells with a potency comparable to that of bGCSF. This work demonstrates the utility of this novel coiled-coil CDR3 motif as a means for generating stable, potent antibody fusion proteins with useful pharmacological properties. PMID:24254636

  7. Discovering neutralizing antibodies targeting the stem epitope of H1N1 influenza hemagglutinin with synthetic phage-displayed antibody libraries

    Science.gov (United States)

    Tung, Chao-Ping; Chen, Ing-Chien; Yu, Chung-Ming; Peng, Hung-Pin; Jian, Jhih-Wei; Ma, Shiou-Hwa; Lee, Yu-Ching; Jan, Jia-Tsrong; Yang, An-Suei

    2015-01-01

    Broadly neutralizing antibodies developed from the IGHV1–69 germline gene are known to bind to the stem region of hemagglutinin in diverse influenza viruses but the sequence determinants for the antigen recognition, including neutralization potency and binding affinity, are not clearly understood. Such understanding could inform designs of synthetic antibody libraries targeting the stem epitope on hemagglutinin, leading to artificially designed antibodies that are functionally advantageous over antibodies from natural antibody repertoires. In this work, the sequence space of the complementarity determining regions of a broadly neutralizing antibody (F10) targeting the stem epitope on the hemagglutinin of a strain of H1N1 influenza virus was systematically explored; the elucidated antibody-hemagglutinin recognition principles were used to design a phage-displayed antibody library, which was then used to discover neutralizing antibodies against another strain of H1N1 virus. More than 1000 functional antibody candidates were selected from the antibody library and were shown to neutralize the corresponding strain of influenza virus with up to 7 folds higher potency comparing with the parent F10 antibody. The antibody library could be used to discover functionally effective antibodies against other H1N1 influenza viruses, supporting the notion that target-specific antibody libraries can be designed and constructed with systematic sequence-function information. PMID:26456860

  8. Structural evidence for evolution of shark Ig new antigen receptor variable domain antibodies from a cell-surface receptor

    OpenAIRE

    Streltsov, V. A.; Varghese, J N; Carmichael, J A; Irving, R A; Hudson, P.J.; Nuttall, S D

    2004-01-01

    The Ig new antigen receptors (IgNARs) are single-domain antibodies found in the serum of sharks. Here, we report 2.2- and 2.8-Å structures of the type 2 IgNAR variable domains 12Y-1 and 12Y-2. Structural features include, first, an Ig superfamily topology transitional between cell adhesion molecules, antibodies, and T cell receptors; and, second, a vestigial complementarity-determining region 2 at the “bottom” of the molecule, apparently discontinuous from the antigen-binding paratope and sim...

  9. Synthetic peptides corresponding to third hypervariable region of human monoclonal IgM rheumatoid factor heavy chains define an immunodominant idiotype

    OpenAIRE

    1985-01-01

    Synthetic peptides corresponding to eight individual heavy chain complementarity-determining regions (CDR) of three human monoclonal IgM anti-IgG (rheumatoid factor [RF]) paraproteins elicited rabbit antibodies with markedly different properties. All antisera recognized the immunizing peptide, and several reacted with the isolated IgM heavy chain on immunoblots. However, only the antisera against peptides representing the third CDR bound consistently and specifically to the intact IgM-RF mole...

  10. Large-scale sequence and structural comparisons of human naive and antigen-experienced antibody repertoires.

    Science.gov (United States)

    DeKosky, Brandon J; Lungu, Oana I; Park, Daechan; Johnson, Erik L; Charab, Wissam; Chrysostomou, Constantine; Kuroda, Daisuke; Ellington, Andrew D; Ippolito, Gregory C; Gray, Jeffrey J; Georgiou, George

    2016-05-10

    Elucidating how antigen exposure and selection shape the human antibody repertoire is fundamental to our understanding of B-cell immunity. We sequenced the paired heavy- and light-chain variable regions (VH and VL, respectively) from large populations of single B cells combined with computational modeling of antibody structures to evaluate sequence and structural features of human antibody repertoires at unprecedented depth. Analysis of a dataset comprising 55,000 antibody clusters from CD19(+)CD20(+)CD27(-) IgM-naive B cells, >120,000 antibody clusters from CD19(+)CD20(+)CD27(+) antigen-experienced B cells, and >2,000 RosettaAntibody-predicted structural models across three healthy donors led to a number of key findings: (i) VH and VL gene sequences pair in a combinatorial fashion without detectable pairing restrictions at the population level; (ii) certain VH:VL gene pairs were significantly enriched or depleted in the antigen-experienced repertoire relative to the naive repertoire; (iii) antigen selection increased antibody paratope net charge and solvent-accessible surface area; and (iv) public heavy-chain third complementarity-determining region (CDR-H3) antibodies in the antigen-experienced repertoire showed signs of convergent paired light-chain genetic signatures, including shared light-chain third complementarity-determining region (CDR-L3) amino acid sequences and/or Vκ,λ-Jκ,λ genes. The data reported here address several longstanding questions regarding antibody repertoire selection and development and provide a benchmark for future repertoire-scale analyses of antibody responses to vaccination and disease. PMID:27114511

  11. Cross-neutralization of influenza A viruses mediated by a single antibody loop.

    Science.gov (United States)

    Ekiert, Damian C; Kashyap, Arun K; Steel, John; Rubrum, Adam; Bhabha, Gira; Khayat, Reza; Lee, Jeong Hyun; Dillon, Michael A; O'Neil, Ryann E; Faynboym, Aleksandr M; Horowitz, Michael; Horowitz, Lawrence; Ward, Andrew B; Palese, Peter; Webby, Richard; Lerner, Richard A; Bhatt, Ramesh R; Wilson, Ian A

    2012-09-27

    Immune recognition of protein antigens relies on the combined interaction of multiple antibody loops, which provide a fairly large footprint and constrain the size and shape of protein surfaces that can be targeted. Single protein loops can mediate extremely high-affinity binding, but it is unclear whether such a mechanism is available to antibodies. Here we report the isolation and characterization of an antibody called C05, which neutralizes strains from multiple subtypes of influenza A virus, including H1, H2 and H3. X-ray and electron microscopy structures show that C05 recognizes conserved elements of the receptor-binding site on the haemagglutinin surface glycoprotein. Recognition of the haemagglutinin receptor-binding site is dominated by a single heavy-chain complementarity-determining region 3 loop, with minor contacts from heavy-chain complementarity-determining region 1, and is sufficient to achieve nanomolar binding with a minimal footprint. Thus, binding predominantly with a single loop can allow antibodies to target small, conserved functional sites on otherwise hypervariable antigens. PMID:22982990

  12. Construction of a rationally designed antibody platform for sequencing-assisted selection.

    Science.gov (United States)

    Larman, H Benjamin; Xu, George Jing; Pavlova, Natalya N; Elledge, Stephen J

    2012-11-01

    Antibody discovery platforms have become an important source of both therapeutic biomolecules and research reagents. Massively parallel DNA sequencing can be used to assist antibody selection by comprehensively monitoring libraries during selection, thus greatly expanding the power of these systems. We have therefore constructed a rationally designed, fully defined single-chain variable fragment (scFv) library and analysis platform optimized for analysis with short-read deep sequencing. Sequence-defined oligonucleotide libraries encoding three complementarity-determining regions (L3 from the light chain, H2 and H3 from the heavy chain) were synthesized on a programmable microarray and combinatorially cloned into a single scFv framework for molecular display. Our unique complementarity-determining region sequence design optimizes for protein binding by utilizing a hidden Markov model that was trained on all antibody-antigen cocrystal structures in the Protein Data Bank. The resultant ~10(12)-member library was produced in ribosome-display format, and comprehensively analyzed over four rounds of antigen selections by multiplex paired-end Illumina sequencing. The hidden Markov model scFv library generated multiple binders against an emerging cancer antigen and is the basis for a next-generation antibody production platform. PMID:23064642

  13. Cysteinylation of a monoclonal antibody leads to its inactivation.

    Science.gov (United States)

    McSherry, Troy; McSherry, Jennifer; Ozaeta, Panfilo; Longenecker, Kenton; Ramsay, Carol; Fishpaugh, Jeffrey; Allen, Steven

    2016-01-01

    Post-translational modifications can have a signification effect on antibody stability. A comprehensive approach is often required to best understand the underlying reasons the modification affects the antibody's potency or aggregation state. Monoclonal antibody 001 displayed significant variation in terms of potency, as defined by surface plasmon resonance testing (Biacore), from lot to lot independent of any observable aggregation or degradation, suggesting that a post-translational modification could be driving this variability. Analysis of different antibody lots using analytical hydrophobic interaction chromatography (HIC) uncovered multiple peaks of varying size. Electrospray ionization mass spectrometry (ESI-MS) indicated that the antibody contained a cysteinylation post-translational modification in complementarity-determining region (CDR) 3 of the antibody light chain. Fractionation of the antibody by HIC followed by ESI-MS and Biacore showed that the different peaks were antibody containing zero, one, or two cysteinylation modifications, and that the modification interferes with the ability of the modified antibody arm to bind antigen. Molecular modeling of the modified region shows that this oxidation of an unpaired cysteine in the antibody CDR would block a potential antigen binding pocket, suggesting an inhibition mechanism. PMID:27050640

  14. Recombinant shark natural antibodies to thyroglobulin.

    Science.gov (United States)

    Schluter, Samuel F; Jensen, Ingvill; Ramsland, Paul A; Marchalonis, John J

    2005-01-01

    As cartilaginous fish are the vertebrates most distal from man to produce antibodies, fundamental information regarding conservation and variation of the antigen binding site should be gained by comparing the properties of antibodies directed against the same antigen from the two species. Since monoclonal cell lines cannot be generated using shark B cells, we isolated antigen binding recombinant single chain Fv antibodies (scFv) comprising of the complete variable regions from shark light and heavy chains. Thyroglobulin was used as the selecting antigen as both sharks and humans express natural antibodies to mammalian thyroglobulin in the absence of purposeful immunization. We report that recombinant sandbar shark (Carcharhinus plumbeus) scFvs that bind bovine thyroglobulin consist of heavy chain variable regions (VH) homologous to those of the human VHIII subset and light chain variable regions (VL) homologous to those of the human Vlambda6 subgroup. The homology within the frameworks is sufficient to enable the building of three-dimensional models of the shark VH/VL structure using established human structures as templates. In natural antibodies of both species, the major variability lies in the third complementarity determining region (CDR3) of both VH and VL. PMID:15954089

  15. Novel antimalarial antibodies highlight the importance of the antibody Fc region in mediating protection.

    Science.gov (United States)

    Pleass, Richard J; Ogun, Solabomi A; McGuinness, David H; van de Winkel, Jan G J; Holder, Anthony A; Woof, Jenny M

    2003-12-15

    Parasite drug resistance and difficulties in developing effective vaccines have precipitated the search for alternative therapies for malaria. The success of passive immunization suggests that immunoglobulin (Ig)-based therapies are effective. To further explore the mechanism(s) by which antibody mediates its protective effect, we generated human chimeric IgG1 and IgA1 and a single-chain diabody specific for the C-terminal 19-kDa region of Plasmodium yoelii merozoite surface protein 1 (MSP119), a major target of protective immune responses. These novel human reagents triggered in vitro phagocytosis of merozoites but, unlike their parental mouse IgG2b, failed to protect against parasite challenge in vivo. Therefore, the Fc region appears critical for mediating protection in vivo, at least for this MSP119 epitope. Such antibodies may serve as prototype therapeutic agents, and as useful tools in the development of in vitro neutralization assays with Plasmodium parasites. PMID:12855589

  16. Structural evidence for evolution of shark Ig new antigen receptor variable domain antibodies from a cell-surface receptor.

    Science.gov (United States)

    Streltsov, V A; Varghese, J N; Carmichael, J A; Irving, R A; Hudson, P J; Nuttall, S D

    2004-08-24

    The Ig new antigen receptors (IgNARs) are single-domain antibodies found in the serum of sharks. Here, we report 2.2- and 2.8-A structures of the type 2 IgNAR variable domains 12Y-1 and 12Y-2. Structural features include, first, an Ig superfamily topology transitional between cell adhesion molecules, antibodies, and T cell receptors; and, second, a vestigial complementarity-determining region 2 at the "bottom" of the molecule, apparently discontinuous from the antigen-binding paratope and similar to that observed in cell adhesion molecules. Thus, we suggest that IgNARs originated as cell-surface adhesion molecules coopted to the immune repertoire and represent an evolutionary lineage independent of variable heavy chain/variable light chain type antibodies. Additionally, both 12Y-1 and 12Y-2 form unique crystallographic dimers, predominantly mediated by main-chain framework interactions, which represent a possible model for primordial cell-based interactions. Unusually, the 12Y-2 complementarity-determining region 3 also adopts an extended beta-hairpin structure, suggesting a distinct selective advantage in accessing cryptic antigenic epitopes. PMID:15304650

  17. Intraperitoneal delivery of monoclonal antibodies: enhanced regional delivery advantage using intravenous unlabeled anti-mouse antibody

    International Nuclear Information System (INIS)

    Radiolabeled monoclonal antibodies (MAb) delivered intraperitoneally expose cells in contact with peritoneal fluid to considerably higher levels of MAb than if the MAb dose were given intravenously. This regional delivery advantage for intact MAb is present mainly due to the relatively slow exit of MAb from the peritoneal fluid to the blood. Eventually, following i.p. injection, blood levels of MAb rise resulting in exposure of the animal to high systemic MAb levels and potential toxicity. In this series of experiments, systemic exposure was minimized by the administration of unlabeled goat polyclonal anti-mouse antibody intravenously from 1 1/2 to 6 h following i.p. MAb injection. This maneuver results in the formation of immune complexes with their subsequent clearance and dehalogenation by the reticuloendothelial system, thus minimizing systemic MAb exposure. This approach, of increasing systemic clearance of MAb, did not alter intraperitoneal MAb levels and thus significantly increased the regional delivery advantage to the peritoneal cavity by 70-100%. This approach provides an immunologic rationale for the further enhancement of MAb delivery to i.p. foci of malignant disease and may have diagnostic and therapeutic utility. (author)

  18. Intraperitoneal delivery of monoclonal antibodies: enhanced regional delivery advantage using intravenous unlabeled anti-mouse antibody

    Energy Technology Data Exchange (ETDEWEB)

    Wahl, R.L.; Fisher, S.

    1987-01-01

    Radiolabeled monoclonal antibodies (MAb) delivered intraperitoneally expose cells in contact with peritoneal fluid to considerably higher levels of MAb than if the MAb dose were given intravenously. This regional delivery advantage for intact MAb is present mainly due to the relatively slow exit of MAb from the peritoneal fluid to the blood. Eventually, following i.p. injection, blood levels of MAb rise resulting in exposure of the animal to high systemic MAb levels and potential toxicity. In this series of experiments, systemic exposure was minimized by the administration of unlabeled goat polyclonal anti-mouse antibody intravenously from 1 1/2 to 6 h following i.p. MAb injection. This maneuver results in the formation of immune complexes with their subsequent clearance and dehalogenation by the reticuloendothelial system, thus minimizing systemic MAb exposure. This approach, of increasing systemic clearance of MAb, did not alter intraperitoneal MAb levels and thus significantly increased the regional delivery advantage to the peritoneal cavity by 70-100%. This approach provides an immunologic rationale for the further enhancement of MAb delivery to i.p. foci of malignant disease and may have diagnostic and therapeutic utility.

  19. Rational design of humanized dual-agonist antibodies.

    Science.gov (United States)

    Zhang, Yong; Liu, Yan; Wang, Ying; Schultz, Peter G; Wang, Feng

    2015-01-14

    The ultralong heavy chain complementarity determining region 3 (CDR3H) of bovine antibody BLV1H12 folds into a novel "stalk-knob" structural motif and has been exploited to generate novel agonist antibodies through replacement of the "knob" domain with cytokines and growth factors. By translating this unique "stalk-knob" architecture to the humanized antibody trastuzumab (referred to hereafter by its trade name, Herceptin, Genentech USA), we have developed a versatile approach to the generation of human antibody agonists. Human erythropoietin (hEPO) or granulocyte colony-stimulating factor (hGCSF) was independently fused into CDR3H, CDR2H, or CDR3L of Herceptin using an engineered "stalk" motif. The fusion proteins express in mammalian cells in good yields and have similar in vitro biological activities compared to hEPO and hGCSF. On the basis of these results we then generated a bi-functional Herceptin-CDR fusion protein in which both hEPO and hGCSF were grafted into the heavy- and light-chain CDR3 loops, respectively. This bi-functional antibody fusion exhibited potent EPO and GCSF agonist activities. This work demonstrates the versatility of the CDR-fusion strategy for generating functional human antibody chimeras and provides a novel approach to the development of multi-functional antibody-based therapeutics. PMID:25494484

  20. An Insertion Mutation That Distorts Antibody Binding Site Architecture Enhances Function of a Human Antibody

    Energy Technology Data Exchange (ETDEWEB)

    Krause, Jens C.; Ekiert, Damian C.; Tumpey, Terrence M.; Smith, Patricia B.; Wilson, Ian A.; Crowe, Jr., James E. (Vanderbilt); (Scripps); (CDC)

    2011-09-02

    The structural and functional significance of somatic insertions and deletions in antibody chains is unclear. Here, we demonstrate that a naturally occurring three-amino-acid insertion within the influenza virus-specific human monoclonal antibody 2D1 heavy-chain variable region reconfigures the antibody-combining site and contributes to its high potency against the 1918 and 2009 pandemic H1N1 influenza viruses. The insertion arose through a series of events, including a somatic point mutation in a predicted hot-spot motif, introduction of a new hot-spot motif, a molecular duplication due to polymerase slippage, a deletion due to misalignment, and additional somatic point mutations. Atomic resolution structures of the wild-type antibody and a variant in which the insertion was removed revealed that the three-amino-acid insertion near the base of heavy-chain complementarity-determining region (CDR) H2 resulted in a bulge in that loop. This enlarged CDR H2 loop impinges on adjacent regions, causing distortion of the CDR H1 architecture and its displacement away from the antigen-combining site. Removal of the insertion restores the canonical structure of CDR H1 and CDR H2, but binding, neutralization activity, and in vivo activity were reduced markedly because of steric conflict of CDR H1 with the hemagglutinin antigen.

  1. Using the local immune response from the natural buffalo host to generate an antibody fragment library that binds the early larval stages of Schistosoma japonicum.

    Science.gov (United States)

    Hosking, Christopher G; Driguez, Patrick; McWilliam, Hamish E G; Ilag, Leodevico L; Gladman, Simon; Li, Yuesheng; Piedrafita, David; McManus, Donald P; Meeusen, Els N T; de Veer, Michael J

    2015-09-01

    Antibodies isolated from the local draining inguinal lymph node of field exposed-water buffaloes following challenge with Schistosoma japonicum cercariae showed high reactivity towards S. japonicum antigen preparations and bound specifically to formaldehyde-fixed S. japonicum schistosomules. Using this specific local immune response we produced a series of single-chain antibody Fv domain libraries from the same lymph nodes. Removal of phage that cross reacted with epitopes on adult parasites yielded a single-chain antibody Fv domain-phage library that specifically bound to whole formaldehyde-fixed and live S. japonicum schistosomules. DNA sequencing indicated clear enrichment of the single-chain antibody Fv domain library for buffalo B-cell complementarity determining regions post-selection for schistosomule binding. This study also revealed that long heavy chain complementarity determining regions appear to be an important factor when selecting for antibody binding fragments against schistosomule proteins. The selected single-chain antibody Fv domain-phage were used to probe a schistosome-specific protein microarray, which resulted in the recognition of many proteins expressed across all schistosome life-cycle stages. Following absorption to adult worms, the single-chain antibody Fv domain-phage library showed significantly reduced binding to most proteins, whilst two proteins (NCBI GenBank accession numbers AY915878 and AY815196) showed increased binding. We have thus developed a unique set of host derived single-chain antibody Fv domains comprising buffalo B-cell variable regions that specifically bind to early S. japonicum life-stages. PMID:26116907

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2007-07-13

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

  3. Conformation of the hypervariable region L3 without the key proline residue.

    OpenAIRE

    Guarné, A; Bravo, J.; Calvo, J.; Lozano, F; Vives, J.; I. Fita

    1996-01-01

    The refined structure of the Fab fragment of the monoclonal antibody CRIS-I (IgG2a kappa) against the leukocyte differentiation antigen CD5, determined at 1.9 A resolution with an agreement R-factor of 18.3%, reveals a variant of the canonical conformations proposed for the light chain complementarity determining region L3 (CDR-L3). This is the first Fab structure available with a kappa light chain in which the CDR-L3 lacks the key proline residue in either position 94 or 95. The conformation...

  4. Antibody

    Science.gov (United States)

    An antibody is a protein produced by the body's immune system when it detects harmful substances, called antigens. Examples ... microorganisms (bacteria, fungi, parasites, and viruses) and chemicals. Antibodies may be produced when the immune system mistakenly ...

  5. 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.

  6. Antibody humanization by molecular dynamics simulations-in-silico guided selection of critical backmutations.

    Science.gov (United States)

    Margreitter, Christian; Mayrhofer, Patrick; Kunert, Renate; Oostenbrink, Chris

    2016-06-01

    Monoclonal antibodies represent the fastest growing class of biotherapeutic proteins. However, as they are often initially derived from rodent organisms, there is a severe risk of immunogenic reactions, hampering their applicability. The humanization of these antibodies remains a challenging task in the context of rational drug design. "Superhumanization" describes the direct transfer of the complementarity determining regions to a human germline framework, but this humanization approach often results in loss of binding affinity. In this study, we present a new approach for predicting promising backmutation sites using molecular dynamics simulations of the model antibody Ab2/3H6. The simulation method was developed in close conjunction with novel specificity experiments. Binding properties of mAb variants were evaluated directly from crude supernatants and confirmed using established binding affinity assays for purified antibodies. Our approach provides access to the dynamical features of the actual binding sites of an antibody, based solely on the antibody sequence. Thus we do not need structural data on the antibody-antigen complex and circumvent cumbersome methods to assess binding affinities. © 2016 The Authors Journal of Molecular Recognition Published by John Wiley & Sons Ltd. PMID:26748949

  7. Platelet stimulation by antifibronectin antibodies requires the Fc region of antibody.

    OpenAIRE

    Holderbaum, D; Culp, L. A.; Bensusan, H B; Gershman, H.

    1982-01-01

    Anti-human fibronectin antibodies produced in a goat or in rabbits stimulate the release of serotonin from washed or gelatin/Sepharose-treated human platelets in a dose-dependent manner. This finding led us to propose that fibronectin on the platelet plasma membrane might serve as a collagen receptor on these cells [Bensusan, H. B., Koh, T. L., Henry, K. G., Murray, B. A. & Culp, L. A. (1978) Proc. Natl. Acad. Sci. USA 75, 5864-5868]. To determine whether direct interaction of the antibody wi...

  8. A high-affinity CDR-grafted antibody against influenza A H5N1 viruses recognizes a conserved epitope of H5 hemagglutinin.

    Directory of Open Access Journals (Sweden)

    Feifei Xiong

    Full Text Available Highly pathogenic avian influenza (HPAI H5N1 virus infection is still a potential threat to public health worldwide. While vaccines and antiviral drugs are currently under development, neutralizing antibodies could offer an alternative strategy to prevent and treat H5N1 virus infection. In the present study, we had developed a humanized antibody against H5N1 viruses from mouse-derived hybridoma in order to minimize its immunogenicity for potential clinical application. The humanized antibody hH5M9 was generated by transferring the mouse complementarity determining region (CDR residues together with four key framework region (FR residues onto the FR of the human antibody. This humanized antibody exhibited high affinity and specificity comparable to the parental mouse or chimeric counterpart with broad and strong neutralization activity against all H5N1 clades and subclades except for Egypt clades investigated. Furthermore, through epitope mapping we identified a linear epitope on the top region of hemagglutinin (HA that was H5N1 specific and conserved. Our results for the first time reported a humanized antibody against H5N1 viruses by CDR grafting method. With the expected lower immunogenicity, this humanized antibody was expected to be more efficacious than murine or human-mouse chimeric antibodies for future application in humans.

  9. A Tat-grafted anti-nucleic acid antibody acquires nuclear-localization property and a preference for TAR RNA

    International Nuclear Information System (INIS)

    Highlights: → We generate 'H3Tat-3D8' by grafting Tat48-60 peptide to VH CDR of 3D8 scFv antibody. → H3Tat-3D8 antibody retains nucleic acid binding and hydrolyzing activities. → H3Tat-3D8 acquires a preference for TAR RNA structure. → Properties of Tat48-60 is transferred to an antibody via Tat-grafting into a CDR. -- Abstract: The 3D8 single chain variable fragment (3D8 scFv) is an anti-nucleic acid antibody that can hydrolyze nucleic acids and enter the cytosol of cells without reaching the nucleus. The Tat peptide, derived from the basic region of the HIV-1 Tat protein, translocates to cell nuclei and has TAR RNA binding activity. In this study, we generated a Tat-grafted antibody (H3Tat-3D8) by replacing complementarity-determining region 3 (CDR3) within the VH domain of the 3D8 scFv with a Tat48-60 peptide (GRKKRRQRRRPPQ). H3Tat-3D8 retained the DNA-binding and DNA-hydrolyzing activity of the scFv, and translocated to the nuclei of HeLa cells and preferentially recognized TAR RNA. Thus, the properties associated with the Tat peptide were transferred to the antibody via Tat-grafting without loss of the intrinsic DNA-binding and hydrolyzing activities of the 3D8 scFv antibody.

  10. Epitope Mapping of Anti-Interleukin-13 Neutralizing Antibody CNTO607

    Energy Technology Data Exchange (ETDEWEB)

    Teplyakov, Alexey; Obmolova, Galina; Wu, Sheng-Jiun; Luo, Jinquan; Kang, James; O' Neil, Karyn; Gilliland, Gary L.; (Centocor)

    2009-06-24

    CNTO607 is a neutralizing anti-interleukin-13 (IL-13) human monoclonal antibody obtained from a phage display library. To determine how this antibody inhibits the biological effect of IL-13, we determined the binding epitope by X-ray crystallography. The crystal structure of the complex between CNTO607 Fab and IL-13 reveals the antibody epitope at the surface formed by helices A and D of IL-13. This epitope overlaps with the IL-4Ralpha/IL-13Ralpha1 receptor-binding site, which explains the neutralizing effect of CNTO607. The extensive antibody interface covers an area of 1000 A(2), which is consistent with the high binding affinity. The key features of the interface are the charge and shape complementarity of the molecules that include two hydrophobic pockets on IL-13 that accommodate Phe32 [complementarity-determining region (CDR) L2] and Trp100a (CDR H3) and a number of salt bridges between basic residues of IL-13 and acidic residues of the antibody. Comparison with the structure of the free Fab shows that the CDR residues do not change their conformation upon complex formation, with the exception of two residues in CDR H3, Trp100a and Asp100b, which change rotamer conformations. To evaluate the relative contribution of the epitope residues to CNTO607 binding, we performed alanine-scanning mutagenesis of the A-D region of IL-13. This study confirmed the primary role of electrostatic interactions for antigen recognition.

  11. The diversity of H3 loops determines the antigen-binding tendencies of antibody CDR loops.

    Science.gov (United States)

    Tsuchiya, Yuko; Mizuguchi, Kenji

    2016-04-01

    Of the complementarity-determining regions (CDRs) of antibodies, H3 loops, with varying amino acid sequences and loop lengths, adopt particularly diverse loop conformations. The diversity of H3 conformations produces an array of antigen recognition patterns involving all the CDRs, in which the residue positions actually in contact with the antigen vary considerably. Therefore, for a deeper understanding of antigen recognition, it is necessary to relate the sequence and structural properties of each residue position in each CDR loop to its ability to bind antigens. In this study, we proposed a new method for characterizing the structural features of the CDR loops and obtained the antigen-binding ability of each residue position in each CDR loop. This analysis led to a simple set of rules for identifying probable antigen-binding residues. We also found that the diversity of H3 loop lengths and conformations affects the antigen-binding tendencies of all the CDR loops. PMID:26749247

  12. Co-evolution of affinity and stability of grafted amyloid-motif domain antibodies.

    Science.gov (United States)

    Julian, Mark C; Lee, Christine C; Tiller, Kathryn E; Rabia, Lilia A; Day, Evan K; Schick, Arthur J; Tessier, Peter M

    2015-10-01

    An attractive approach for designing lead antibody candidates is to mimic natural protein interactions by grafting peptide recognition motifs into the complementarity-determining regions (CDRs). We are using this approach to generate single-domain (VH) antibodies specific for amyloid-forming proteins such as the Alzheimer's Aβ peptide. Here, we use random mutagenesis and yeast surface display to improve the binding affinity of a lead VH domain grafted with Aβ residues 33-42 in CDR3. Interestingly, co-selection for improved Aβ binding and VH display on the surface of yeast yields antibody domains with improved affinity and reduced stability. The highest affinity VH domains were strongly destabilized on the surface of yeast as well as unfolded when isolated as autonomous domains. In contrast, stable VH domains with improved affinity were reliably identified using yeast surface display by replacing the display antibody that recognizes a linear epitope tag at the terminus of both folded and unfolded VH domains with a conformational ligand (Protein A) that recognizes a discontinuous epitope on the framework of folded VH domains. Importantly, we find that selection for improved stability using Protein A without simultaneous co-selection for improved Aβ binding leads to strong enrichment for stabilizing mutations that reduce antigen binding. Our findings highlight the importance of simultaneously optimizing affinity and stability to improve the rapid isolation of well-folded and specific antibody fragments. PMID:26386257

  13. Rational design and generation of recombinant control reagents for bispecific antibodies through CDR mutagenesis.

    Science.gov (United States)

    Choi, Bryan D; Gedeon, Patrick C; Kuan, Chien-Tsun; Sanchez-Perez, Luis; Archer, Gary E; Bigner, Darell D; Sampson, John H

    2013-09-30

    Developments in the field of bispecific antibodies have progressed rapidly in recent years, particularly in their potential role for the treatment of malignant disease. However, manufacturing stable molecules has proven to be costly and time-consuming, which in turn has hampered certain aspects of preclinical evaluation including the unavailability of appropriate "negative" controls. Bispecific molecules (e.g., bispecific tandem scFv) exhibit two specificities, often against a tumor antigen as well as an immune-activation ligand such as CD3. While for IgG antibodies, isotype-matched controls are well accepted, when considering smaller antibody fragments it is not possible to adequately control for their biological activity through the use of archetypal isotypes, which differ dramatically in affinity, size, structure, and design. Here, we demonstrate a method for the rapid production of negative control tandem scFvs through complementarity determining region (CDR) mutagenesis, using a recently described bispecific T-cell engager (BiTE) targeting a tumor-specific mutation of the epidermal growth factor receptor (EGFRvIII) as an example. Four independent control constructs were developed by this method through alteration of residues spanning individual CDR domains. Importantly, while target antigen affinity was completely impaired, CD3 binding affinity was conserved in each molecule. These results have a potential to enhance the sophistication by which bispecific antibodies can be evaluated in the preclinical setting and may have broader applications for an array of alternative antibody-derived therapeutic platforms. PMID:23806556

  14. Engineering and characterization of a humanized antibody targeting TNF-α and RANKL.

    Science.gov (United States)

    Wang, Jun; Du, Yuxuan; Qian, Hongyan; Yu, Haitao; Li, Shentao; Zhang, Xulong; Li, Zhanguo; Yuan, Huihui; Zhao, Wenming

    2014-07-18

    To neutralize the pathological activities of tumor necrosis factor-α (TNF-α) and receptor activator of NF-κB ligand (RANKL), we engineered and characterized a humanized 8G12 (h8G12) antibody that targeted TNF-α and RANKL. Standard molecular biological and complementarity determining region (CDR)-grafting techniques were used to engineer the h8G12 antibody, and enzyme-linked immunosorbent assays (ELISAs) and Western blotting were employed to determine its binding activation and specificity. TNF-α-mediated cytotoxicity and RANKL-induced osteoclastogenesis assays were used to evaluate the neutralizing effects of the antibody. The cDNA sequences were established by grafting the murine monoclonal antibody (mAb) 8G12 CDRs into the heavy and light chain (HC and LC) variable regions (VH and VL) of the human mAbs 3DGG_B and 1I9R_L, respectively. The recombinant plasmids were transfected into Chinese hamster ovary (CHO) cells to produce the h8G12 antibody, which could simultaneously recognize TNF-α and RANKL. In addition, the h8G12 antibody reduced the TNF-α-mediated apoptosis of L929 cells by 25.84%. Furthermore, the h8G12 antibody significantly inhibited leukocyte infiltration in a murine allergic contact inflammation model. Concurrent with the inhibition of apoptosis, the h8G12 antibody significantly reduced the number of osteoclast-like cells in a dose-dependent manner. These results demonstrated that the h8G12 antibody neutralized the activities of TNF-α and RANKL and that it might be a potential candidate for the treatment of inflammatory bone diseases, such as rheumatoid arthritis (RA). PMID:24950406

  15. Graves' Disease Mechanisms: The Role of Stimulating, Blocking, and Cleavage Region TSH Receptor Antibodies.

    Science.gov (United States)

    Morshed, S A; Davies, T F

    2015-09-01

    The immunologic processes involved in Graves' disease (GD) have one unique characteristic--the autoantibodies to the TSH receptor (TSHR)--which have both linear and conformational epitopes. Three types of TSHR antibodies (stimulating, blocking, and cleavage) with different functional capabilities have been described in GD patients, which induce different signaling effects varying from thyroid cell proliferation to thyroid cell death. The establishment of animal models of GD by TSHR antibody transfer or by immunization with TSHR antigen has confirmed its pathogenic role and, therefore, GD is the result of a breakdown in TSHR tolerance. Here we review some of the characteristics of TSHR antibodies with a special emphasis on new developments in our understanding of what were previously called "neutral" antibodies and which we now characterize as autoantibodies to the "cleavage" region of the TSHR ectodomain. PMID:26361259

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

    Energy Technology Data Exchange (ETDEWEB)

    Dekker, Leendert J.; Wu, Si; vanDuijn, Martijn M.; Tolic, Nikola; Stingl, Christoph; Zhao, Rui; Luider, Theo N.; Pasa-Tolic, Ljiljana

    2014-05-31

    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 towards 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 towards the identification of disease specific antibodies in patient samples with potentially significant clinical impact.

  17. A broad set of different llama antibodies specific for a 16 kDa heat shock protein of Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Anke K Trilling

    Full Text Available BACKGROUND: Recombinant antibodies are powerful tools in engineering of novel diagnostics. Due to the small size and stable nature of llama antibody domains selected antibodies can serve as a detection reagent in multiplexed and sensitive assays for M. tuberculosis. METHODOLOGY/PRINCIPAL FINDINGS: Antibodies for Mycobacterium tuberculosis (M. tb recognition were raised in Alpaca, and, by phage display, recombinant variable domains of heavy-chain antibodies (VHH binding to M. tuberculosis antigens were isolated. Two phage display selection strategies were followed: one direct selection using semi-purified protein antigen, and a depletion strategy with lysates, aiming to avoid cross-reaction to other mycobacteria. Both panning methods selected a set of binders with widely differing complementarity determining regions. Selected recombinant VHHs were produced in E. coli and shown to bind immobilized lysate in direct Enzymelinked Immunosorbent Assay (ELISA tests and soluble antigen by surface plasmon resonance (SPR analysis. All tested VHHs were specific for tuberculosis-causing mycobacteria (M. tuberculosis, M. bovis and exclusively recognized an immunodominant 16 kDa heat shock protein (hsp. The highest affinity VHH had a dissociation constant (KD of 4 × 10(-10 M. CONCLUSIONS/SIGNIFICANCE: A broad set of different llama antibodies specific for 16 kDa heat shock protein of M. tuberculosis is available. This protein is highly stable and abundant in M. tuberculosis. The VHH that detect this protein are applied in a robust SPR sensor for identification of tuberculosis-causing mycobacteria.

  18. Effects of Darwinian Selection and Mutability on Rate of Broadly Neutralizing Antibody Evolution during HIV-1 Infection.

    Science.gov (United States)

    Sheng, Zizhang; Schramm, Chaim A; Connors, Mark; Morris, Lynn; Mascola, John R; Kwong, Peter D; Shapiro, Lawrence

    2016-05-01

    Accumulation of somatic mutations in antibody variable regions is critical for antibody affinity maturation, with HIV-1 broadly neutralizing antibodies (bnAbs) generally requiring years to develop. We recently found that the rate at which mutations accumulate decreases over time, but the mechanism governing this slowing is unclear. In this study, we investigated whether natural selection and/or mutability of the antibody variable region contributed significantly to observed decrease in rate. We used longitudinally sampled sequences of immunoglobulin transcripts of single lineages from each of 3 donors, as determined by next generation sequencing. We estimated the evolutionary rates of the complementarity determining regions (CDRs), which are most significant for functional selection, and found they evolved about 1.5- to 2- fold faster than the framework regions. We also analyzed the presence of AID hotspots and coldspots at different points in lineage development and observed an average decrease in mutability of less than 10 percent over time. Altogether, the correlation between Darwinian selection strength and evolutionary rate trended toward significance, especially for CDRs, but cannot fully explain the observed changes in evolutionary rate. The mutability modulated by AID hotspots and coldspots changes correlated only weakly with evolutionary rates. The combined effects of Darwinian selection and mutability contribute substantially to, but do not fully explain, evolutionary rate change for HIV-1-targeting bnAb lineages. PMID:27191167

  19. Functional antibody lacking a variable-region disulfide bridge.

    OpenAIRE

    Rudikoff, S.; Pumphrey, J G

    1986-01-01

    In 1981, Auffray et al. [Auffray, C., Sikorav, J. L., Ollo, R. & Rougeon, F. (1981) Ann. Immunol. (Inst. Pasteur) 132D, 77-88] reported a partial cDNA sequence of the heavy chain from the ABPC48 plasmacytoma whose protein product had previously been shown to bind bacterial and grass levan. In the cDNA sequence the second half-cystine of the heretofore invariant disulfide bridge had been replaced by a tyrosine. Since the presence of invariant variable-region disulfide bridges has been consider...

  20. Disjoint combinations profiling (DCP: a new method for the prediction of antibody CDR conformation from sequence

    Directory of Open Access Journals (Sweden)

    Dimitris Nikoloudis

    2014-07-01

    Full Text Available The accurate prediction of the conformation of Complementarity-Determining Regions (CDRs is important in modelling antibodies for protein engineering applications. Specifically, the Canonical paradigm has proved successful in predicting the CDR conformation in antibody variable regions. It relies on canonical templates which detail allowed residues at key positions in the variable region framework or in the CDR itself for 5 of the 6 CDRs. While no templates have as yet been defined for the hypervariable CDR-H3, instead, reliable sequence rules have been devised for predicting the base of the CDR-H3 loop. Here a new method termed Disjoint Combinations Profiling (DCP is presented, which contributes a considerable advance in the prediction of CDR conformations. This novel method is explained and compared with canonical templates and sequence rules in a 3-way blind prediction. DCP achieved 93% accuracy over 951 blind predictions and showed an improvement in cumulative accuracy compared to predictions with canonical templates or sequence rules. In addition to its overall improvement in prediction accuracy, it is suggested that DCP is open to better implementations in the future and that it can improve as more antibody structures are deposited in the databank. In contrast, it is argued that canonical templates and sequence rules may have reached their peak.

  1. Disjoint combinations profiling (DCP): a new method for the prediction of antibody CDR conformation from sequence.

    Science.gov (United States)

    Nikoloudis, Dimitris; Pitts, Jim E; Saldanha, José W

    2014-01-01

    The accurate prediction of the conformation of Complementarity-Determining Regions (CDRs) is important in modelling antibodies for protein engineering applications. Specifically, the Canonical paradigm has proved successful in predicting the CDR conformation in antibody variable regions. It relies on canonical templates which detail allowed residues at key positions in the variable region framework or in the CDR itself for 5 of the 6 CDRs. While no templates have as yet been defined for the hypervariable CDR-H3, instead, reliable sequence rules have been devised for predicting the base of the CDR-H3 loop. Here a new method termed Disjoint Combinations Profiling (DCP) is presented, which contributes a considerable advance in the prediction of CDR conformations. This novel method is explained and compared with canonical templates and sequence rules in a 3-way blind prediction. DCP achieved 93% accuracy over 951 blind predictions and showed an improvement in cumulative accuracy compared to predictions with canonical templates or sequence rules. In addition to its overall improvement in prediction accuracy, it is suggested that DCP is open to better implementations in the future and that it can improve as more antibody structures are deposited in the databank. In contrast, it is argued that canonical templates and sequence rules may have reached their peak. PMID:25071985

  2. Rapid discovery and optimization of therapeutic antibodies against emerging infectious diseases.

    Science.gov (United States)

    Rogers, J; Schoepp, R J; Schröder, O; Clements, T L; Holland, T F; Li, J Q; Li, J; Lewis, L M; Dirmeier, R P; Frey, G J; Tan, X; Wong, K; Woodnutt, G; Keller, M; Reed, D S; Kimmel, B E; Tozer, E C

    2008-08-01

    Using a comprehensive set of discovery and optimization tools, antibodies were produced with the ability to neutralize SARS coronavirus (SARS-CoV) infection in Vero E6 cells and in animal models. These anti-SARS antibodies were discovered using a novel DNA display method, which can identify new antibodies within days. Once neutralizing antibodies were identified, a comprehensive and effective means of converting the mouse sequences to human frameworks was accomplished using HuFR (human framework reassembly) technology. The best variant (61G4) from this screen showed a 3.5-4-fold improvement in neutralization of SARS-CoV infection in vitro. Finally, using a complete site-saturation mutagenesis methodology focused on the CDR (complementarity determining regions), a single point mutation (51E7) was identified that improved the 80% plaque reduction neutralization of the virus by greater than 8-fold. These discovery and evolution strategies can be applied to any emerging pathogen or toxin where a causative agent is known. PMID:18480090

  3. Enhancement and destruction of antibody function by somatic mutation: unequal occurrence is controlled by V gene combinatorial associations.

    Science.gov (United States)

    Chen, C; Roberts, V A; Stevens, S; Brown, M; Stenzel-Poore, M P; Rittenberg, M B

    1995-06-15

    We examined the positive and negative effects of somatic mutation on antibody function using saturation mutagenesis in vitro to mimic the potential of the in vivo process to diversify antibodies. Identical mutations were introduced into the second complementarity determining region of two anti-phosphocholine antibodies, T15 and D16, which share the same germline VH gene sequence. T15 predominates in primary responses and does not undergo affinity maturation. D16 is representative of antibodies that co-dominate in memory responses and do undergo affinity maturation. We previously reported that > 50% of T15 mutants had decreased antigen binding capacity. To test if this high frequency of binding loss was unique to T15 or a consequence of random point mutations applicable to other combining sites, we analyzed the same mutations in D16. We show that D16 suffers a similar loss of function, indicating an equally high potential for B-cell wastage. However, only D16 displayed the capacity for somatic mutation to improve antigen binding, which should enhance its persistence in memory responses. Mutation of residues contacting the haptenic group, as determined by molecular modeling, did not improve binding. Instead, productive mutations occurred in residues that either contacted carrier protein or were distant from the antigen binding site, possibly increasing binding site flexibility through long-range effects. Targeting such residues for mutation should aid in the rational design of improved antibodies. PMID:7796805

  4. Deep Sequencing-guided Design of a High Affinity Dual Specificity Antibody to Target Two Angiogenic Factors in Neovascular Age-related Macular Degeneration.

    Science.gov (United States)

    Koenig, Patrick; Lee, Chingwei V; Sanowar, Sarah; Wu, Ping; Stinson, Jeremy; Harris, Seth F; Fuh, Germaine

    2015-09-01

    The development of dual targeting antibodies promises therapies with improved efficacy over mono-specific antibodies. Here, we engineered a Two-in-One VEGF/angiopoietin 2 antibody with dual action Fab (DAF) as a potential therapeutic for neovascular age-related macular degeneration. Crystal structures of the VEGF/angiopoietin 2 DAF in complex with its two antigens showed highly overlapping binding sites. To achieve sufficient affinity of the DAF to block both angiogenic factors, we turned to deep mutational scanning in the complementarity determining regions (CDRs). By mutating all three CDRs of each antibody chain simultaneously, we were able not only to identify affinity improving single mutations but also mutation pairs from different CDRs that synergistically improve both binding functions. Furthermore, insights into the cooperativity between mutations allowed us to identify fold-stabilizing mutations in the CDRs. The data obtained from deep mutational scanning reveal that the majority of the 52 CDR residues are utilized differently for the two antigen binding function and permit, for the first time, the engineering of several DAF variants with sub-nanomolar affinity against two structurally unrelated antigens. The improved variants show similar blocking activity of receptor binding as the high affinity mono-specific antibodies against these two proteins, demonstrating the feasibility of generating a dual specificity binding surface with comparable properties to individual high affinity mono-specific antibodies. PMID:26088137

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

    Directory of Open Access Journals (Sweden)

    Henry Memczak

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

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

    Science.gov (United States)

    Memczak, Henry; Lauster, Daniel; Kar, Parimal; Di Lella, Santiago; Volkmer, Rudolf; Knecht, Volker; Herrmann, Andreas; Ehrentreich-Förster, Eva; Bier, Frank F; Stöcklein, Walter F M

    2016-01-01

    Antibodies against spike proteins of influenza are used as a tool for characterization of viruses and therapeutic approaches. However, development, production and quality control of antibodies is expensive and time consuming. To circumvent these difficulties, three peptides were derived from complementarity determining regions of an antibody heavy chain against influenza A spike glycoprotein. Their binding properties were studied experimentally, and by molecular dynamics simulations. Two peptide candidates showed binding to influenza A/Aichi/2/68 H3N2. One of them, termed PeB, with the highest affinity prevented binding to and infection of target cells in the micromolar region without any cytotoxic effect. PeB matches best the conserved receptor binding site of hemagglutinin. PeB bound also to other medical relevant influenza strains, such as human-pathogenic A/California/7/2009 H1N1, and avian-pathogenic A/Mute Swan/Rostock/R901/2006 H7N1. Strategies to improve the affinity and to adapt specificity are discussed and exemplified by a double amino acid substituted peptide, obtained by substitutional analysis. The peptides and their derivatives are of great potential for drug development as well as biosensing. PMID:27415624

  7. Codon insertion and deletion functions as a somatic diversification mechanism in human antibody repertoires

    Directory of Open Access Journals (Sweden)

    Zhou Jianhui

    2006-08-01

    Full Text Available Abstract It has been suggested that codon insertion and/or deletion may represent a mechanism that, along with hypermutation, contributes to the affinity maturation of antibodies. We used repertoire cloning to examine human antibodies directed against 3 carbohydrate antigens and 1 protein antigen for the presence of such modifications. We find that both the insertion and deletion of codons occur frequently in antigen-specific responses following vaccination. Codon insertions and deletions were observed most often in the complementarity determining regions, and less frequently in the framework regions, of VH, Vκ, and Vλ gene segments, and involved motifs known to be preferred targets of somatic hypermutation. Clonal lineage analysis shows that these events occur through out the course of the somatic maturation of individual antibody clones. We also determined that these alterations of paratope structure have varying effects on the relative affinity of the binding site for its cognate antigen. Reviewers This article was reviewed by Mark Shlomchik, Deborah Dunn-Walters (nominated by Dr. Andrew Macpherson, and Rachel M. Gerstein. Open peer review Reviewed by Mark Shlomchik, Deborah Dunn-Walters (nominated by Dr. Andrew Macpherson, and Rachel M. Gerstein. For the full reviews, please go to the Reviewers' comments section.

  8. Chimeric antibody with human constant regions and mouse variable regions directed against carcinoma-associated antigen 17-1A

    International Nuclear Information System (INIS)

    The authors have cloned the genomic DNA fragments encoding the heavy and light chain variable regions of monoclonal antibody 17-1A, and they have inserted them into mammalian expression vectors containing genomic DNA segments encoding human γ3 and kappa constant regions. The transfer of these expression vectors containing mouse-human chimeric immunoglobulin genes into Sp2/0 mouse myeloma cells resulted in the production of functional IgG that retained the specific binding to the surface antigen 17-1A expressed on colorectal carcinoma cells

  9. 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

  10. Seroprevalence of Antibodies to Main Porcine Infectious Pathogens in Wild Boars in Some Regions of Russia

    OpenAIRE

    BABORENKO, Elena; KUKUSHKIN, Sergey; DOMSKIY, Igor; MIKHALISHIN, Valery; BAYBIKOV, Taufik

    2009-01-01

    Results of testing 107 serum samples from wild boars (Sus scrofa L., 1758) for thepresence of antibodies to six economically significant porcine infectious disease agents (porcinereproductive and respiratory syndrome (PRRS) virus, porcine parvovirus (PPV), swine influenza virus(SIV) of H1N1 and H3N2 subtypes, Aujeszky’s disease virus (ADV), porcine transmissiblegastroenteritis virus (TGEV) and Mycoplasma hyopneumoniae) are presented in the paper. Wild boarwere sampled in seven regions of Russ...

  11. Antibody Constant Region Peptides Can Display Immunomodulatory Activity through Activation of the Dectin-1 Signalling Pathway

    OpenAIRE

    Elena Gabrielli; Eva Pericolini; Elio Cenci; Claudia Monari; Walter Magliani; Tecla Ciociola; Stefania Conti; Rita Gatti; Francesco Bistoni; Luciano Polonelli; Anna Vecchiarelli

    2012-01-01

    We previously reported that a synthetic peptide with sequence identical to a CDR of a mouse monoclonal antibody specific for difucosyl human blood group A exerted an immunomodulatory activity on murine macrophages. It was therapeutic against systemic candidiasis without possessing direct candidacidal properties. Here we demonstrate that a selected peptide, N10K, putatively deriving from the enzymatic cleavage of the constant region (Fc) of human IgG(1), is able to induce IL-6 secretion and pI...

  12. Molecular features of the complementarity determining region 3 motif of the T cell population and subsets in the blood of patients with chronic severe hepatitis B

    Directory of Open Access Journals (Sweden)

    Yang Jiezuan

    2011-12-01

    Full Text Available Abstract Background T cell receptor (TCR reflects the status and function of T cells. We previously developed a gene melting spectral pattern (GMSP assay, which rapidly detects clonal expansion of the T cell receptor β variable gene (TCRBV in patients with HBV by using quantitative real-time reverse transcription PCR (qRT-PCR with DNA melting curve analysis. However, the molecular profiles of TCRBV in peripheral blood mononuclear cells (PBMCs and CD8+, CD8- cell subsets from chronic severe hepatitis B (CSHB patients have not been well described. Methods Human PBMCs were separated and sorted into CD8+ and CD8- cell subsets using density gradient centrifugation and magnetic activated cell sorting (MACS. The molecular features of the TCRBV CDR3 motif were determined using GMSP analysis; the TCRBV families were cloned and sequenced when the GMSP profile showed a single-peak, indicative of a monoclonal population. Results The number of skewed TCRBV in the CD8+ cell subset was significantly higher than that of the CD8- cell subset as assessed by GMSP analysis. The TCRBV11 and BV7 were expressed more frequently than other members of TCRBV family in PBMCs and CD8+, CD8- subsets. Also the relatively conserved amino acid motifs were detected in the TCRBV22, BV18 and BV11 CDR3 in PBMCs among patients with CSHB. Conclusions The molecular features of the TCRBV CDR3 were markedly different among PBMCs and CD8+, CD8- cell subsets derived from CSHB patients. Analysis of the TCRBV expression in the CD8+ subset was more accurate in assessing the status and function of circulating T cells. The expression of TCRBV11, BV7 and the relatively conserved CDR3 amino acid motifs could also help to predict and treat patients with CSHB.

  13. Monoclonal antibody evidence for structural similarities between the central rod regions of actinin and dystrophin.

    Science.gov (United States)

    Nguyen, T M; Ellis, J M; Ginjaar, I B; van Paassen, M M; van Ommen, G J; Moorman, A F; Cartwright, A J; Morris, G E

    1990-10-15

    A monoclonal antibody, MANDYS141, binds to both dystrophin and actinin on Western blots (SDS-denatured), but only to actinin in frozen sections of human muscle (native conformation). It differs from a polyclonal cross-reacting antiserum in that it binds to several muscle isoforms of actinin (smooth, fast and slow) from man, mouse and chicken and recognises a quite different part of the proposed triple-helical region of dystrophin (amino acids 1750-2248). The results suggest that structural homologies between actinin and dystrophin occur more than once in their central helical regions and provide experimental support for an actinin-like central rod model for dystrophin. PMID:1699800

  14. Fully Human VH Single Domains That Rival the Stability and Cleft Recognition of Camelid Antibodies*

    Science.gov (United States)

    Rouet, Romain; Dudgeon, Kip; Christie, Mary; Langley, David; Christ, Daniel

    2015-01-01

    Human VH single domains represent a promising class of antibody fragments with applications as therapeutic modalities. Unfortunately, isolated human VH domains also generally display poor biophysical properties and a propensity to aggregate. This has encouraged the development of non-human antibody domains as alternative means of antigen recognition and, in particular, camelid (VHH) domains. Naturally devoid of light chain partners, these domains are characterized by favorable biophysical properties and propensity for cleft binding, a highly desirable characteristic, allowing the targeting of cryptic epitopes. In contrast, previously reported structures of human VH single domains had failed to recapitulate this property. Here we report the engineering and characterization of phage display libraries of stable human VH domains and the selection of binders against a diverse set of antigens. Unlike “camelized” human domains, the domains do not rely on potentially immunogenic framework mutations and maintain the structure of the VH/VL interface. Structure determination in complex with hen egg white lysozyme revealed an extended VH binding interface, with complementarity-determining region 3 deeply penetrating into the active site cleft, highly reminiscent of what has been observed for camelid domains. Taken together, our results demonstrate that fully human VH domains can be constructed that are not only stable and well expressed but also rival the cleft binding properties of camelid antibodies. PMID:25737448

  15. Broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza virus hemagglutinin

    Energy Technology Data Exchange (ETDEWEB)

    Whittle, James R.R.; Zhang, Ruijun; Khurana, Surender; King, Lisa R.; Manischewitz, Jody; Golding, Hana; Dormitzer, Philip R.; Haynes, Barton F.; Walter, Emmanuel B.; Moody, M. Anthony; Kepler, Thomas B.; Liao, Hua-Xin; Harrison, Stephen C. (Harvard-Med); (Novartis); (US-FDA); (Duke)

    2011-09-20

    Seasonal antigenic drift of circulating influenza virus leads to a requirement for frequent changes in vaccine composition, because exposure or vaccination elicits human antibodies with limited cross-neutralization of drifted strains. We describe a human monoclonal antibody, CH65, obtained by isolating rearranged heavy- and light-chain genes from sorted single plasma cells, coming from a subject immunized with the 2007 trivalent influenza vaccine. The crystal structure of a complex of the hemagglutinin (HA) from H1N1 strain A/Solomon Islands/3/2006 with the Fab of CH65 shows that the tip of the CH65 heavy-chain complementarity determining region 3 (CDR3) inserts into the receptor binding pocket on HA1, mimicking in many respects the interaction of the physiological receptor, sialic acid. CH65 neutralizes infectivity of 30 out of 36 H1N1 strains tested. The resistant strains have a single-residue insertion near the rim of the sialic-acid pocket. We conclude that broad neutralization of influenza virus can be achieved by antibodies with contacts that mimic those of the receptor.

  16. Length-independent structural similarities enrich the antibody CDR canonical class model

    Science.gov (United States)

    Nowak, Jaroslaw; Baker, Terry; Georges, Guy; Kelm, Sebastian; Klostermann, Stefan; Shi, Jiye; Sridharan, Sudharsan; Deane, Charlotte M.

    2016-01-01

    ABSTRACT Complementarity-determining regions (CDRs) are antibody loops that make up the antigen binding site. Here, we show that all CDR types have structurally similar loops of different lengths. Based on these findings, we created length-independent canonical classes for the non-H3 CDRs. Our length variable structural clusters show strong sequence patterns suggesting either that they evolved from the same original structure or result from some form of convergence. We find that our length-independent method not only clusters a larger number of CDRs, but also predicts canonical class from sequence better than the standard length-dependent approach. To demonstrate the usefulness of our findings, we predicted cluster membership of CDR-L3 sequences from 3 next-generation sequencing datasets of the antibody repertoire (over 1,000,000 sequences). Using the length-independent clusters, we can structurally classify an additional 135,000 sequences, which represents a ∼20% improvement over the standard approach. This suggests that our length-independent canonical classes might be a highly prevalent feature of antibody space, and could substantially improve our ability to accurately predict the structure of novel CDRs identified by next-generation sequencing. PMID:26963563

  17. Length-independent structural similarities enrich the antibody CDR canonical class model.

    Science.gov (United States)

    Nowak, Jaroslaw; Baker, Terry; Georges, Guy; Kelm, Sebastian; Klostermann, Stefan; Shi, Jiye; Sridharan, Sudharsan; Deane, Charlotte M

    2016-01-01

    Complementarity-determining regions (CDRs) are antibody loops that make up the antigen binding site. Here, we show that all CDR types have structurally similar loops of different lengths. Based on these findings, we created length-independent canonical classes for the non-H3 CDRs. Our length variable structural clusters show strong sequence patterns suggesting either that they evolved from the same original structure or result from some form of convergence. We find that our length-independent method not only clusters a larger number of CDRs, but also predicts canonical class from sequence better than the standard length-dependent approach. To demonstrate the usefulness of our findings, we predicted cluster membership of CDR-L3 sequences from 3 next-generation sequencing datasets of the antibody repertoire (over 1,000,000 sequences). Using the length-independent clusters, we can structurally classify an additional 135,000 sequences, which represents a ∼20% improvement over the standard approach. This suggests that our length-independent canonical classes might be a highly prevalent feature of antibody space, and could substantially improve our ability to accurately predict the structure of novel CDRs identified by next-generation sequencing. PMID:26963563

  18. Combining phage display with de novo protein sequencing for reverse engineering of monoclonal antibodies.

    Science.gov (United States)

    Rickert, Keith W; Grinberg, Luba; Woods, Robert M; Wilson, Susan; Bowen, Michael A; Baca, Manuel

    2016-04-01

    The enormous diversity created by gene recombination and somatic hypermutation makes de novo protein sequencing of monoclonal antibodies a uniquely challenging problem. Modern mass spectrometry-based sequencing will rarely, if ever, provide a single unambiguous sequence for the variable domains. A more likely outcome is computation of an ensemble of highly similar sequences that can satisfy the experimental data. This outcome can result in the need for empirical testing of many candidate sequences, sometimes iteratively, to identity one which can replicate the activity of the parental antibody. Here we describe an improved approach to antibody protein sequencing by using phage display technology to generate a combinatorial library of sequences that satisfy the mass spectrometry data, and selecting for functional candidates that bind antigen. This approach was used to reverse engineer 2 commercially-obtained monoclonal antibodies against murine CD137. Proteomic data enabled us to assign the majority of the variable domain sequences, with the exception of 3-5% of the sequence located within or adjacent to complementarity-determining regions. To efficiently resolve the sequence in these regions, small phage-displayed libraries were generated and subjected to antigen binding selection. Following enrichment of antigen-binding clones, 2 clones were selected for each antibody and recombinantly expressed as antigen-binding fragments (Fabs). In both cases, the reverse-engineered Fabs exhibited identical antigen binding affinity, within error, as Fabs produced from the commercial IgGs. This combination of proteomic and protein engineering techniques provides a useful approach to simplifying the technically challenging process of reverse engineering monoclonal antibodies from protein material. PMID:26852694

  19. Precise detection of L. monocytogenes hitting its highly conserved region possessing several specific antibody binding sites.

    Science.gov (United States)

    Jahangiri, Abolfazl; Rasooli, Iraj; Reza Rahbar, Mohammad; Khalili, Saeed; Amani, Jafar; Ahmadi Zanoos, Kobra

    2012-07-21

    Listeria monocytogenes, a facultative intracellular fast-growing Gram-positive food-borne pathogen, can infect immunocompromised individuals leading to meningitis, meningoencephalitis and septicaemias. From the pool of virulence factors of the organism, ActA, a membrane protein, has a critical role in the life cycle of L. monocytogenes. High mortality rate of listeriosis necessitates a sensitive and rapid diagnostic test for precise identification of L. monocytogenes. We used bioinformatic tools to locate a specific conserved region of ActA for designing and developing an antibody-antigen based diagnostic test for the detection of L. monocytogenes. A number of databases were looked for ActA related sequences. Sequences were analyzed with several online software to find an appropriate region for our purpose. ActA protein was found specific to Listeria species with no homologs in other organisms. We finally introduced a highly conserved region within ActA sequence that possess several antibody binding sites specific to L. monocytogenes. This protein sequence can serve as an antigen for designing a relatively cheap, sensitive, and specific diagnostic test for detection of L. monocytogenes. PMID:22575546

  20. Maturation of shark single-domain (IgNAR) antibodies: evidence for induced-fit binding.

    Science.gov (United States)

    Stanfield, Robyn L; Dooley, Helen; Verdino, Petra; Flajnik, Martin F; Wilson, Ian A

    2007-03-23

    Sharks express an unusual heavy-chain isotype called IgNAR, whose variable regions bind antigen as independent soluble domains. To further probe affinity maturation of the IgNAR response, we structurally characterized the germline and somatically matured versions of a type II variable (V) region, both in the presence and absence of its antigen, hen egg-white lysozyme. Despite a disulfide bond linking complementarity determining regions (CDRs) 1 and 3, both germline and somatically matured V regions displayed significant structural changes in these CDRs upon complex formation with antigen. Somatic mutations in the IgNAR V region serve to increase the number of contacts with antigen, as reflected by a tenfold increase in affinity, and one of these mutations appears to stabilize the CDR3 region. In addition, a residue in the HV4 loop plays an important role in antibody-antigen interaction, consistent with the high rate of somatic mutations in this non-CDR loop. PMID:17258766

  1. Analysis of hapten binding and catalytic determinants in a family of catalytic antibodies.

    Science.gov (United States)

    Ulrich, H D; Schultz, P G

    1998-01-01

    We report here the cloning and kinetic analysis of a family of catalytic antibodies raised against a common transition state (TS) analog hapten, which accelerate a unimolecular oxy-Cope rearrangement. Sequence analysis revealed close homologies among the heavy chains of the catalytically active members of this set of antibodies, which derive mainly from a single germline gene, whereas the light chains can be traced back to several different, but related germline genes. The requirements for hapten binding and catalytic activity were determined by the construction of hybrid antibodies. Characterization of the latter antibodies again indicates a strong conservation of binding site structure among the catalytically active clones. The heavy chain was found to be the determining factor for catalytic efficiency, while the light chain exerted a smaller modulating effect that depended on light chain gene usage and somatic mutations. Within the heavy chain, the catalytic activity of a clone, but not hapten binding affinity, depended on the sequence of the third complementarity determining region (CDR). No correlation between high affinity for the hapten and high rate enhancement was found in the oxy-Cope system, a result that stands in contrast to the expectations from transition state theory. A mechanistic explanation for this observation is provided based on the three-dimensional crystal structure of the most active antibody, AZ-28, in complex with the hapten. This study demonstrates the utility of catalytic antibodies in examining the relationship between binding energy and catalysis in the evolution of biological catalysis, as well as expanding our understanding of the molecular basis of an immune response. PMID:9451442

  2. Hypervariable antigenic region 1 of classical swine fever virus E2 protein impacts antibody neutralization.

    Science.gov (United States)

    Liao, Xun; Wang, Zuohuan; Cao, Tong; Tong, Chao; Geng, Shichao; Gu, Yuanxing; Zhou, Yingshan; Li, Xiaoliang; Fang, Weihuan

    2016-07-19

    Envelope glycoprotein E2 of classical swine fever virus (CSFV) is the major antigen that induces neutralizing antibodies and confers protection against CSFV infection. There are three hypervariable antigenic regions (HAR1, HAR2 and HAR3) of E2 that are different between the group 1 vaccine C-strain and group 2 clinical isolates. This study was aimed to characterize the antigenic epitope region recognized by monoclonal antibody 4F4 (mAb-4F4) that is present in the group 2 field isolate HZ1-08, but not in the C-strain, and examine its impact on neutralization titers when antisera from different recombinant viruses were cross-examined. Indirect ELISA with C-strain E2-based chimeric proteins carrying the three HAR regions showed that the mAb-4F4 bound to HAR1 from HZ1-08 E2, but not to HAR2 or HAR3, indicating that the specific epitope is located in the HAR1 region. Of the 6 major residues differences between C-strain and field isolates, Glu713 in the HAR1 region of strain HZ1-08 is critical for mAb-4F4 binding either at the recombinant protein level or using intact recombinant viruses carrying single mutations. C-strain-based recombinant viruses carrying the most antigenic part of E2 or HAR1 from strain HZ1-08 remained non-pathogenic to pigs and induced good antibody responses. By cross-neutralization assay, we observed that the anti-C-strain serum lost most of its neutralization capacity to RecC-HZ-E2 and QZ-14 (subgroup 2.1d field isolate in 2014), and vice versa. More importantly, the RecC-HAR1 virus remained competent in neutralizing ReC-HZ-E2 and QZ-14 strains without compromising the neutralization capability to the recombinant C-strain. Thus, we propose that chimeric C-strain carrying the HAR1 region of field isolates is a good vaccine candidate for classical swine fever. PMID:27317266

  3. Humanization and characterization of an anti-ricin neutralization monoclonal antibody.

    Directory of Open Access Journals (Sweden)

    Wei-Gang Hu

    Full Text Available Ricin is regarded as a high terrorist risk for the public due to its high toxicity and ease of production. Currently, there is no therapeutic or vaccine available against ricin. D9, a murine monoclonal antibody developed previously in our laboratory, can strongly neutralize ricin and is therefore a good candidate for humanization. Humanization of D9 variable regions was achieved by a complementarity-determining region grafting approach. The humanized D9 (hD9 variable regions were further grafted onto human heavy and light chain constant regions to assemble the complete antibody gene. A foot-and-mouth-disease virus-derived 2A self-processing sequence was introduced between heavy and light chain DNA sequences to cleave the recombinant protein into a functional full-length antibody molecule from a single open reading frame driven by a single promoter in an adenoviral vector. After expression in mammalian cells and purification, the hD9 was demonstrated to have equimolar expression of the full-length antibody heavy and light chains. More importantly, the hD9 exhibited high affinity to ricin with K(D of 1.63 nM, comparable to its parental murine D9 (2.55 nM. In a mouse model, intraperitoneal (i.p. administration of hD9, at a low dose of 5 µg per mouse, 4 hours after the i.p. challenge with 5×LD50 ricin was found to rescue 100% of the mice. In addition, administered 6 hours post-challenge, hD9 could still rescue 50% of the mice. The hD9 has the potential to be used for prophylactic or therapeutic purposes against ricin poisoning.

  4. Seroprevalence of Antibodies to Main Porcine Infectious Pathogens in Wild Boars in Some Regions of Russia

    Directory of Open Access Journals (Sweden)

    BABORENKO, Elena

    2009-01-01

    Full Text Available Results of testing 107 serum samples from wild boars (Sus scrofa L., 1758 for thepresence of antibodies to six economically significant porcine infectious disease agents (porcinereproductive and respiratory syndrome (PRRS virus, porcine parvovirus (PPV, swine influenza virus(SIV of H1N1 and H3N2 subtypes, Aujeszky’s disease virus (ADV, porcine transmissiblegastroenteritis virus (TGEV and Mycoplasma hyopneumoniae are presented in the paper. Wild boarwere sampled in seven regions of Russia for diagnostic purposes. The obtained results showed thepresence of antibodies to ADV in 32.5% of samples (83/27, to PPV – in 62% of samples (92/57, toMycoplasma hyopneumoniae – in 52% of samples (98/51. All samples were seronegative to PRRSvirus (107/0, TGEV (91/0 and SIV of H1N1 (89/0 and H3N2 (58/0 subtypes. The researchesdemonstrated the extensive circulation of porcine parvovirus, Aujeszky’s disease virus andMycoplasma hyopneumoniae among Wild boar in some regions of Russia.

  5. Isolation of anti-toxin single domain antibodies from a semi-synthetic spiny dogfish shark display library

    Directory of Open Access Journals (Sweden)

    Goldman Ellen R

    2007-11-01

    Full Text Available Abstract Background Shark heavy chain antibody, also called new antigen receptor (NAR, consists of one single Variable domain (VH, containing only two complementarity-determining regions (CDRs. The antigen binding affinity and specificity are mainly determined by these two CDRs. The good solubility, excellent thermal stability and complex sequence variation of small single domain antibodies (sdAbs make them attractive alternatives to conventional antibodies. In this report, we construct and characterize a diversity enhanced semi-synthetic NAR V display library based on naturally occurring NAR V sequences. Results A semi-synthetic shark sdAb display library with a complexity close to 1e9 was constructed. This was achieved by introducing size and sequence variations in CDR3 using randomized CDR3 primers of three different lengths. Binders against three toxins, staphylococcal enterotoxin B (SEB, ricin, and botulinum toxin A (BoNT/A complex toxoid, were isolated from panning the display library. Soluble sdAbs from selected binders were purified and evaluated using direct binding and thermal stability assays on the Luminex 100. In addition, sandwich assays using sdAb as the reporter element were developed to demonstrate their utility for future sensor applications. Conclusion We demonstrated the utility of a newly created hyper diversified shark NAR displayed library to serve as a source of thermal stable sdAbs against a variety of toxins.

  6. Epiregulin Recognition Mechanisms by Anti-epiregulin Antibody 9E5: STRUCTURAL, FUNCTIONAL, AND MOLECULAR DYNAMICS SIMULATION ANALYSES.

    Science.gov (United States)

    Kado, Yuji; Mizohata, Eiichi; Nagatoishi, Satoru; Iijima, Mariko; Shinoda, Keiko; Miyafusa, Takamitsu; Nakayama, Taisuke; Yoshizumi, Takuma; Sugiyama, Akira; Kawamura, Takeshi; Lee, Young-Hun; Matsumura, Hiroyoshi; Doi, Hirofumi; Fujitani, Hideaki; Kodama, Tatsuhiko; Shibasaki, Yoshikazu; Tsumoto, Kouhei; Inoue, Tsuyoshi

    2016-01-29

    Epiregulin (EPR) is a ligand of the epidermal growth factor (EGF) family that upon binding to its epidermal growth factor receptor (EGFR) stimulates proliferative signaling, especially in colon cancer cells. Here, we describe the three-dimensional structure of the EPR antibody (the 9E5(Fab) fragment) in the presence and absence of EPR. Among the six complementarity-determining regions (CDRs), CDR1-3 in the light chain and CDR2 in the heavy chain predominantly recognize EPR. In particular, CDR3 in the heavy chain dramatically moves with cis-trans isomerization of Pro(103). A molecular dynamics simulation and mutational analyses revealed that Arg(40) in EPR is a key residue for the specific binding of 9E5 IgG. From isothermal titration calorimetry analysis, the dissociation constant was determined to be 6.5 nm. Surface plasmon resonance analysis revealed that the dissociation rate of 9E5 IgG is extremely slow. The superimposed structure of 9E5(Fab)·EPR on the known complex structure of EGF·EGFR showed that the 9E5(Fab) paratope overlaps with Domains I and III on the EGFR, which reveals that the 9E5(Fab)·EPR complex could not bind to the EGFR. The 9E5 antibody will also be useful in medicine as a neutralizing antibody specific for colon cancer. PMID:26627827

  7. Characterization of Therapeutic Monoclonal Antibodies at the Subunit-Level using Middle-Down 193 nm Ultraviolet Photodissociation.

    Science.gov (United States)

    Cotham, Victoria C; Brodbelt, Jennifer S

    2016-04-01

    Monoclonal antibodies (mAbs) are a rapidly advancing class of therapeutic glycoproteins that possess wide clinical utility owing to their biocompatibility, high antigen specificity, and targeted immune stimulation. These therapeutic properties depend greatly on the composition of the immunoglobulin G (IgG) structure, both in terms of primary sequence and post-translational modifications (PTMs); however, large-scale production in cell culture often results in heterogeneous mixtures that can profoundly affect clinical safety and efficacy. This places a high demand on analytical methods that afford comprehensive structural characterization of mAbs to ensure their stringent quality control. Here we report the use of targeted middle-down 193 nm ultraviolet photodissociation (UVPD) to provide detailed primary sequence analysis and PTM site localization of therapeutic monoclonal antibody subunits (∼25 kDa) generated upon digestion with recombinant immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) followed by chemical reduction. Under optimal conditions, targeted UVPD resulted in approximately 60% overall coverage of the IgG sequence, in addition to unambiguous glycosylation site localization and extensive coverage of the antigen-binding complementarity determining regions (CDRs) in a single LC-MS/MS experiment. Combining UVPD and ETD data afforded even deeper sequencing and greater overall characterization of IgG subunits. Overall, this targeted UVPD approach represents a promising new strategy for the comprehensive characterization of antibody-based therapeutics. PMID:26947921

  8. Assessing analytical methods to monitor isoAsp formation in monoclonal antibodies

    OpenAIRE

    CatherineEakin

    2014-01-01

    A ubiquitous post-translational modification observed in proteins is isomerization of aspartic acid to isoaspartic acid (isoAsp). This non-enzymatic post-translational modification occurs spontaneously in proteins and plays a role in aging, autoimmune response, cancer, neurodegeneration, and other diseases. Formation of isoAsp is also a significant issue for recombinant monoclonal antibody based protein therapeutics particularly when isomerization occurs in a complementarity-determining regio...

  9. Somatic diversification in the heavy chain variable region genes expressed by human autoantibodies bearing a lupus-associated nephritogenic anti-DNA idiotype

    Energy Technology Data Exchange (ETDEWEB)

    Demaison, C.; Chastagner, P.; Theze, J.; Zouali, M. (Institut Pasteur, Paris (France))

    1994-01-18

    Monoclonal anti-DNA antibodies bearing a lupus nephritis-associated idiotype were derived from five patients with systemic lupus erythematosus (SLE). Genes encoding their heavy (H)-chain variable (V[sub H]) regions were cloned and sequenced. When compared with their closest V[sub h] germ-line gene relatives, these sequences exhibit a number of silent (S) and replacement (R) substitutions. The ratios of R/S mutations were much higher in the complementarity-determining regions (CDRs) of the antibodies than in the framework regions. Molecular amplification of genomic V[sub H] genes and Southern hybridization with somatic CDR2-specific oligonucleotide probes showed that the configuration of the V[sub H] genes corresponding to V[sub H] sequences in the nephritogenic antibodies is not present in the patient's own germ-line DNA, implying that the B-cell clones underwent somatic mutation in vivo. These findings, together with the characteristics of the diversity and junctional gene elements utilized to form the antibody, indicate that these autoantibodies have been driven through somatic selection processes reminiscent of those that govern antibody responses triggered by exogenous stimuli.

  10. Stress selections on domain antibodies: 'what doesn't kill you makes you stronger'.

    Science.gov (United States)

    Enever, C; Pupecka-Swider, M; Sepp, A

    2015-03-01

    In addition to the desired specificity and affinity for their respective therapeutic targets, antibody-based drugs must also demonstrate an ability to be manufactured and formulated at the concentrations needed for therapeutic application and to remain resistant to aggregation during storage to reduce the risk of induced immunogenicity. Improvements to the thermodynamic stability of the folded state of the protein are considered to be critical for decreasing the aggregation propensity of the protein. In this work, we have improved the biophysical properties of a number of human domain antibodies (dAbs) by identifying mutations which decrease the propensity for dAb self-aggregation without compromising the affinity for their respective target antigen. The mutations were identified by subjecting phage-displayed error-prone PCR-generated libraries to a variety of generic environmental conditions (temperature, pH and protease) followed by antigen capture, facilitating selection for improved thermodynamic stability of the protein. The results indicate that sufficient sequence diversity usually exists within the complementarity determining regions of dAbs to allow for mutations that lead to improvements to biophysical properties with full retention of parent lead biochemical and biological properties. Improved biophysical properties were often accompanied by higher apparent melting temperature values, while alternative selection pressures often identified similar features, suggesting generic nature of these mutations. PMID:25655396

  11. Loop-sequence features and stability determinants in antibody variable domains by high-throughput experiments.

    Science.gov (United States)

    Chang, Hung-Ju; Jian, Jhih-Wei; Hsu, Hung-Ju; Lee, Yu-Ching; Chen, Hong-Sen; You, Jhong-Jhe; Hou, Shin-Chen; Shao, Chih-Yun; Chen, Yen-Ju; Chiu, Kuo-Ping; Peng, Hung-Pin; Lee, Kuo Hao; Yang, An-Suei

    2014-01-01

    Protein loops are frequently considered as critical determinants in protein structure and function. Recent advances in high-throughput methods for DNA sequencing and thermal stability measurement have enabled effective exploration of sequence-structure-function relationships in local protein regions. Using these data-intensive technologies, we investigated the sequence-structure-function relationships of six complementarity-determining regions (CDRs) and ten non-CDR loops in the variable domains of a model vascular endothelial growth factor (VEGF)-binding single-chain antibody variable fragment (scFv) whose sequence had been optimized via a consensus-sequence approach. The results show that only a handful of residues involving long-range tertiary interactions distant from the antigen-binding site are strongly coupled with antigen binding. This implies that the loops are passive regions in protein folding; the essential sequences of these regions are dictated by conserved tertiary interactions and the consensus local loop-sequence features contribute little to protein stability and function. PMID:24268648

  12. Identification of the immunodominant regions of the melanoma antigen tyrosinase by anti-tyrosinase monoclonal antibodies.

    Science.gov (United States)

    Jaanson, Nele; Möll, Kaidi; Kulla, Andres; Ustav, Mart

    2003-10-01

    Tyrosinase, the critical enzyme in melanin synthesis, is also found to be expressed in most malignant melanomas and can serve as a target for the immune response by both CD4+ and CD8+ T-cells. Therefore it could be used as a potential target for therapeutic intervention in tyrosinase-positive melanomas. In order to develop serological reagents for the immunodetection of human tyrosinase and to find the most immunogenic region of the protein, we have raised a panel of monoclonal antibodies (MAbs) against recombinant tyrosinase expressed and purified from bacteria. Epitope mapping revealed the 79 amino acid long stretch between 163 and 241 residues to be the most immunodominant region of the tyrosinase. This region could be further divided into three parts by binding different MAbs. These MAbs were very useful tools for the detection of tyrosinase expression from different constructs in tissue culture cells by immunocytochemistry and in melanocytes by immunohistochemistry. Some of the MAbs that recognized epitopes between 163 and 204 amino acids also recognized an additional distinct protein of about 70 kDa seen on Western blot analysis of transfected and non-transfected COS-7 cells. One of these, the MAb 4B1, was used in immunohistochemistry, and cross reaction with the basement membrane of the human tissue was observed. The analysis of the 4B1 MAb epitope showed that the C-terminal part of that region almost entirely overlaps with the sequence of the recently reported basement membrane protein beta-netrin. PMID:14512789

  13. Leptospira spp. and Toxoplasma gondii antibodies in vampire bats (Desmodus rotundus in Botucatu region, SP, Brazil

    Directory of Open Access Journals (Sweden)

    CB Zetun

    2009-01-01

    Full Text Available The destruction of natural ecosystems has caused several problems to humans and other animals; herein we investigate the close relationship among vampire bats, humans and domestic animals. Toxoplasma gondii and Leptospira spp. infections are two worldwide zoonoses that provoke serious damage to animals. To determine the prevalence of bats seropositive for toxoplasmosis and leptospirosis in the Botucatu region, 204 serum samples of vampire bats (Desmodus rotundus were tested for T. gondii antibodies by modified agglutination test (MAT-t and for Leptospira spp. by microscopic agglutination test (MAT-l. No animal was tested positive for T. gondii while leptospiral positivity was 7.8% for Pyrogenes, Shermani and Javanica serovars, with titers varying from 100 to 1,600. Thus, it was verified that D. rotundus does not play a relevant role in toxoplasmosis epidemiology. However, these bats can be important in the maintenance of Leptospira spp. in the environment.

  14. The Combining Sites of Anti-lipid A Antibodies Reveal a Widely Utilized Motif Specific for Negatively Charged Groups.

    Science.gov (United States)

    Haji-Ghassemi, Omid; Müller-Loennies, Sven; Rodriguez, Teresa; Brade, Lore; Grimmecke, Hans-Dieter; Brade, Helmut; Evans, Stephen V

    2016-05-01

    Lipopolysaccharide dispersed in the blood by Gram-negative bacteria can be a potent inducer of septic shock. One research focus has been based on antibody sequestration of lipid A (the endotoxic principle of LPS); however, none have been successfully developed into a clinical treatment. Comparison of a panel of anti-lipid A antibodies reveals highly specific antibodies produced through distinct germ line precursors. The structures of antigen-binding fragments for two homologous mAbs specific for lipid A, S55-3 and S55-5, have been determined both in complex with lipid A disaccharide backbone and unliganded. These high resolution structures reveal a conserved positively charged pocket formed within the complementarity determining region H2 loops that binds the terminal phosphates of lipid A. Significantly, this motif occurs in unrelated antibodies where it mediates binding to negatively charged moieties through a range of epitopes, including phosphorylated peptides used in diagnostics and therapeutics. S55-3 and S55-5 have combining sites distinct from anti-lipid A antibodies previously described (as a result of their separate germ line origin), which are nevertheless complementary both in shape and charge to the antigen. S55-3 and S55-5 display similar avidity toward lipid A despite possessing a number of different amino acid residues in their combining sites. Binding of lipid A occurs independent of the acyl chains, although the GlcN-O6 attachment point for the core oligosaccharide is buried in the combining site, which explains their inability to recognize LPS. Despite their lack of therapeutic potential, the observed motif may have significant immunological implications as a tool for engineering recombinant antibodies. PMID:26933033

  15. The UW 21/123 monoclonal antibody in the radioimmunological detection of tumour tissue in the head and neck region

    International Nuclear Information System (INIS)

    When incorporated in a radioimmunoassay the UW 21/123 the monoclonal antibody permits the diagnosis of certain squamous cell carcinoma of the cephalocervical region and is particularly reliable in the detection laryngeal carcinomas. It even permits the assessment of tumour cells that are not yet fully developed and thus still escape histological detection. (MBC)

  16. Developmental Pathway of the MPER-Directed HIV-1-Neutralizing Antibody 10E8

    Science.gov (United States)

    Zhang, Baoshan; McKee, Krisha; Longo, Nancy S.; Yang, Yongping; Huang, Jinghe; Parks, Robert; Eudailey, Joshua; Lloyd, Krissey E.; Alam, S. Munir; Haynes, Barton F.; Mullikin, James C.; Connors, Mark; Mascola, John R.; Shapiro, Lawrence; Kwong, Peter D.

    2016-01-01

    Antibody 10E8 targets the membrane-proximal external region (MPER) of HIV-1 gp41, neutralizes >97% of HIV-1 isolates, and lacks the auto-reactivity often associated with MPER-directed antibodies. The developmental pathway of 10E8 might therefore serve as a promising template for vaccine design, but samples from time-of-infection—often used to infer the B cell record—are unavailable. In this study, we used crystallography, next-generation sequencing (NGS), and functional assessments to infer the 10E8 developmental pathway from a single time point. Mutational analysis indicated somatic hypermutation of the 2nd-heavy chain-complementarity determining region (CDR H2) to be critical for neutralization, and structures of 10E8 variants with V-gene regions reverted to genomic origin for heavy-and-light chains or heavy chain-only showed structural differences >2 Å relative to mature 10E8 in the CDR H2 and H3. To understand these developmental changes, we used bioinformatic sieving, maximum likelihood, and parsimony analyses of immunoglobulin transcripts to identify 10E8-lineage members, to infer the 10E8-unmutated common ancestor (UCA), and to calculate 10E8-developmental intermediates. We were assisted in this analysis by the preservation of a critical D-gene segment, which was unmutated in most 10E8-lineage sequences. UCA and early intermediates weakly bound a 26-residue-MPER peptide, whereas HIV-1 neutralization and epitope recognition in liposomes were only observed with late intermediates. Antibody 10E8 thus develops from a UCA with weak MPER affinity and substantial differences in CDR H2 and H3 from the mature 10E8; only after extensive somatic hypermutation do 10E8-lineage members gain recognition in the context of membrane and HIV-1 neutralization. PMID:27299673

  17. TRAIL-R2 Superoligomerization Induced by Human Monoclonal Agonistic Antibody KMTR2.

    Science.gov (United States)

    Tamada, Taro; Shinmi, Daisuke; Ikeda, Masahiro; Yonezawa, Yasushi; Kataoka, Shiro; Kuroki, Ryota; Mori, Eiji; Motoki, Kazuhiro

    2015-01-01

    The fully human monoclonal antibody KMTR2 acts as a strong direct agonist for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 2 (TRAIL-R2), which is capable of inducing apoptotic cell death without cross-linking. To investigate the mechanism of direct agonistic activity induced by KMTR2, the crystal structure of the extracellular region of TRAIL-R2 and a Fab fragment derived from KMTR2 (KMTR2-Fab) was determined to 2.1 Å resolution. Two KMTR2-Fabs assembled with the complementarity-determining region 2 of the light chain via two-fold crystallographic symmetry, suggesting that the KMTR2-Fab assembly tended to enhance TRAIL-R2 oligomerization. A single mutation at Asn53 to Arg located at the two-fold interface in the KMTR2 resulted in a loss of its apoptotic activity, although it retained its antigen-binding activity. These results indicate that the strong agonistic activity, such as apoptotic signaling and tumor regression, induced by KMTR2 is attributed to TRAIL-R2 superoligomerization induced by the interdimerization of KMTR2. PMID:26672965

  18. Identification of a region of beta 2-glycoprotein I critical for lipid binding and anti-cardiolipin antibody cofactor activity.

    OpenAIRE

    Hunt, J E; Simpson, R J; Krilis, S A

    1993-01-01

    beta 2-Glycoprotein I (beta 2-GPI), a phospholipid-binding plasma protein, is an absolute requirement (cofactor) for the binding of autoimmune-type anti-cardiolipin (aCL) antibodies to cardiolipin (CL). The nature of this cofactor activity and the specific regions of the molecule involved have not yet been determined. We have identified a preparation of beta 2-GPI that lacks aCL antibody cofactor activity. Analysis of the structural differences between the active and inactive forms enabled id...

  19. Seroprevalence of antibodies to Rickettsia typhi in the Waikato region of New Zealand.

    Science.gov (United States)

    Lim, M Y; Weinstein, P; Bell, A; Hambling, T; Tompkins, D M; Slaney, D

    2016-08-01

    The first reported New Zealand-acquired case of murine typhus occurred near Auckland in 1989. Since then, 72 locally acquired cases have been recorded from northern New Zealand. By 2008, on the basis of the timing and distribution of cases, it appeared that murine typhus was escalating and spreading southwards. To explore the presence of Rickettsia typhi in the Waikato region, we conducted a seroprevalence study, using indirect immunofluorescence, Western blot, and cross-adsorption assays of blood donor samples. Of 950 human sera from Waikato, 12 (1·3%) had R. typhi antibodies. The seroprevalence for R. typhi was slightly higher in northern Waikato (1·4%) compared to the south (1·2%; no significant difference, χ 2 P = 0·768 at P < 0·05). Our results extend the reported southern range of R. typhi by 140 km and indicate it is endemic in Waikato. Evidence of past Rickettsia felis infections was also detected in six sera. Globally, R. felis is an emerging disease of concern and this pathogen should also be considered when locally acquired rickettsiosis is suspected. If public health interventions are to be implemented to reduce the risk of rickettsioses as a significant public health problem, improvements in rickettsial diagnostics and surveillance will be necessary. PMID:27040715

  20. Antibody constant region peptides can display immunomodulatory activity through activation of the Dectin-1 signalling pathway.

    Directory of Open Access Journals (Sweden)

    Elena Gabrielli

    Full Text Available We previously reported that a synthetic peptide with sequence identical to a CDR of a mouse monoclonal antibody specific for difucosyl human blood group A exerted an immunomodulatory activity on murine macrophages. It was therapeutic against systemic candidiasis without possessing direct candidacidal properties. Here we demonstrate that a selected peptide, N10K, putatively deriving from the enzymatic cleavage of the constant region (Fc of human IgG(1, is able to induce IL-6 secretion and pIkB-α activation. More importantly, it causes an up-regulation of Dectin-1 expression. This leads to an increased activation of β-glucan-induced pSyk, CARD9 and pIkB-α, and an increase in the production of pro-inflammatory cytokines such as IL-6, IL-12, IL-1β and TNF-α. The increased activation of this pathway coincides with an augmented phagocytosis of non opsonized Candida albicans cells by monocytes. The findings suggest that some Fc-peptides, potentially deriving from the proteolysis of immunoglobulins, may cause an unexpected immunoregulation in a way reminiscent of innate immunity molecules.

  1. Site-specific antibodies to human erythropoietin directed toward the NH2-terminal region.

    OpenAIRE

    Sue, J M; Sytkowski, A. J.

    1983-01-01

    Site-specific antibodies to human erythropoietin have been raised in rabbits immunized with a synthetic polypeptide composed of the putative 26 NH2-terminal amino acids of the hormone. The immunogenic peptide was coupled to bovine serum albumin. Antibodies specific for peptide were detected by enzyme-linked immunosorbent assay. They immunoprecipitated both highly purified 125I-labeled erythropoietin and biologically active erythropoietin. The immunoprecipitation of 125I-labeled erythropoietin...

  2. Minimally Mutated HIV-1 Broadly Neutralizing Antibodies to Guide Reductionist Vaccine Design.

    Science.gov (United States)

    Jardine, Joseph G; Sok, Devin; Julien, Jean-Philippe; Briney, Bryan; Sarkar, Anita; Liang, Chi-Hui; Scherer, Erin A; Henry Dunand, Carole J; Adachi, Yumiko; Diwanji, Devan; Hsueh, Jessica; Jones, Meaghan; Kalyuzhniy, Oleksandr; Kubitz, Michael; Spencer, Skye; Pauthner, Matthias; Saye-Francisco, Karen L; Sesterhenn, Fabian; Wilson, Patrick C; Galloway, Denise M; Stanfield, Robyn L; Wilson, Ian A; Burton, Dennis R; Schief, William R

    2016-08-01

    An optimal HIV vaccine should induce broadly neutralizing antibodies (bnAbs) that neutralize diverse viral strains and subtypes. However, potent bnAbs develop in only a small fraction of HIV-infected individuals, all contain rare features such as extensive mutation, insertions, deletions, and/or long complementarity-determining regions, and some are polyreactive, casting doubt on whether bnAbs to HIV can be reliably induced by vaccination. We engineered two potent VRC01-class bnAbs that minimized rare features. According to a quantitative features frequency analysis, the set of features for one of these minimally mutated bnAbs compared favorably with all 68 HIV bnAbs analyzed and was similar to antibodies elicited by common vaccines. This same minimally mutated bnAb lacked polyreactivity in four different assays. We then divided the minimal mutations into spatial clusters and dissected the epitope components interacting with those clusters, by mutational and crystallographic analyses coupled with neutralization assays. Finally, by synthesizing available data, we developed a working-concept boosting strategy to select the mutation clusters in a logical order following a germline-targeting prime. We have thus developed potent HIV bnAbs that may be more tractable vaccine goals compared to existing bnAbs, and we have proposed a strategy to elicit them. This reductionist approach to vaccine design, guided by antibody and antigen structure, could be applied to design candidate vaccines for other HIV bnAbs or protective Abs against other pathogens. PMID:27560183

  3. A novel universal neutralizing monoclonal antibody against enterovirus 71 that targets the highly conserved "knob" region of VP3 protein.

    Directory of Open Access Journals (Sweden)

    Tanja K Kiener

    Full Text Available Hand, foot and mouth disease caused by enterovirus 71(EV71 leads to the majority of neurological complications and death in young children. While putative inactivated vaccines are only now undergoing clinical trials, no specific treatment options exist yet. Ideally, EV71 specific intravenous immunoglobulins could be developed for targeted treatment of severe cases. To date, only a single universally neutralizing monoclonal antibody against a conserved linear epitope of VP1 has been identified. Other enteroviruses have been shown to possess major conformational neutralizing epitopes on both the VP2 and VP3 capsid proteins. Hence, we attempted to isolate such neutralizing antibodies against conformational epitopes for their potential in the treatment of infection as well as differential diagnosis and vaccine optimization. Here we describe a universal neutralizing monoclonal antibody that recognizes a conserved conformational epitope of EV71 which was mapped using escape mutants. Eight escape mutants from different subgenogroups (A, B2, B4, C2, C4 were rescued; they harbored three essential mutations either at amino acid positions 59, 62 or 67 of the VP3 protein which are all situated in the "knob" region. The escape mutant phenotype could be mimicked by incorporating these mutations into reverse genetically engineered viruses showing that P59L, A62D, A62P and E67D abolish both monoclonal antibody binding and neutralization activity. This is the first conformational neutralization epitope mapped on VP3 for EV71.

  4. A region of the insulin receptor important for ligand binding (residues 450-601) is recognized by patients' autoimmune antibodies and inhibitory monoclonal antibodies.

    OpenAIRE

    Zhang, B; Roth, R A

    1991-01-01

    Chimeric receptors containing different portions of the homologous human insulin receptor, insulin-like growth factor I receptor, and insulin receptor-related receptor were utilized to identify the epitopes recognized by various anti-insulin receptor antibodies. The antibodies studied included 12 monoclonal antibodies to the extracellular domain of the human insulin receptor as well as 15 patients' sera with autoimmune anti-insulin receptor antibodies. All of the patients' sera and all 8 mono...

  5. An ultra-specific avian antibody to phosphorylated tau protein reveals a unique mechanism for phosphoepitope recognition.

    Science.gov (United States)

    Shih, Heather H; Tu, Chao; Cao, Wei; Klein, Anne; Ramsey, Renee; Fennell, Brian J; Lambert, Matthew; Ní Shúilleabháin, Deirdre; Autin, Bénédicte; Kouranova, Eugenia; Laxmanan, Sri; Braithwaite, Steven; Wu, Leeying; Ait-Zahra, Mostafa; Milici, Anthony J; Dumin, Jo Ann; LaVallie, Edward R; Arai, Maya; Corcoran, Christopher; Paulsen, Janet E; Gill, Davinder; Cunningham, Orla; Bard, Joel; Mosyak, Lydia; Finlay, William J J

    2012-12-28

    Highly specific antibodies to phosphoepitopes are valuable tools to study phosphorylation in disease states, but their discovery is largely empirical, and the molecular mechanisms mediating phosphospecific binding are poorly understood. Here, we report the generation and characterization of extremely specific recombinant chicken antibodies to three phosphoepitopes on the Alzheimer disease-associated protein tau. Each antibody shows full specificity for a single phosphopeptide. The chimeric IgG pT231/pS235_1 exhibits a K(D) of 0.35 nm in 1:1 binding to its cognate phosphopeptide. This IgG is murine ortholog-cross-reactive, specifically recognizing the pathological form of tau in brain samples from Alzheimer patients and a mouse model of tauopathy. To better understand the underlying binding mechanisms allowing such remarkable specificity, we determined the structure of pT231/pS235_1 Fab in complex with its cognate phosphopeptide at 1.9 Å resolution. The Fab fragment exhibits novel complementarity determining region (CDR) structures with a "bowl-like" conformation in CDR-H2 that tightly and specifically interacts with the phospho-Thr-231 phosphate group, as well as a long, disulfide-constrained CDR-H3 that mediates peptide recognition. This binding mechanism differs distinctly from either peptide- or hapten-specific antibodies described to date. Surface plasmon resonance analyses showed that pT231/pS235_1 binds a truly compound epitope, as neither phosphorylated Ser-235 nor free peptide shows any measurable binding affinity. PMID:23148212

  6. Structural Basis of Neutralization of the Major Toxic Component from the Scorpion Centruroides noxius Hoffmann by a Human-derived Single-chain Antibody Fragment

    Energy Technology Data Exchange (ETDEWEB)

    Canul-Tec, Juan Carlos; Riaño-Umbarila, Lidia; Rudiño-Piñera, Enrique; Becerril, Baltazar; Possani, Lourival D.; Torres-Larios, Alfredo (U. NAM)

    2011-08-09

    It has previously been reported that several single-chain antibody fragments of human origin (scFv) neutralize the effects of two different scorpion venoms through interactions with the primary toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2). Here we present the crystal structure of the complex formed between one scFv (9004G) and the Cn2 toxin, determined in two crystal forms at 2.5 and 1.9 {angstrom} resolution. A 15-residue span of the toxin is recognized by the antibody through a cleft formed by residues from five of the complementarity-determining regions of the scFv. Analysis of the interface of the complex reveals three features. First, the epitope of toxin Cn2 overlaps with essential residues for the binding of {beta}-toxins to its Na+ channel receptor site. Second, the putative recognition of Css2 involves mainly residues that are present in both Cn2 and Css2 toxins. Finally, the effect on the increase of affinity of previously reported key residues during the maturation process of different scFvs can be inferred from the structure. Taken together, these results provide the structural basis that explain the mechanism of the 9004G neutralizing activity and give insight into the process of directed evolution that gave rise to this family of neutralizing scFvs.

  7. Structure and function of broadly reactive antibody PG16 reveal an H3 subdomain that mediates potent neutralization of HIV-1

    Energy Technology Data Exchange (ETDEWEB)

    Pejchal, Robert; Walker, Laura M.; Stanfield, Robyn L.; Phogat, Sanjay K.; Koff, Wayne C.; Poignard, Pascal; Burton, Dennis R.; Wilson, Ian A. (Scripps); (IAVI)

    2010-11-15

    Development of an effective vaccine against HIV-1 will likely require elicitation of broad and potent neutralizing antibodies against the trimeric surface envelope glycoprotein (Env). Monoclonal antibodies (mAbs) PG9 and PG16 neutralize {approx}80% of HIV-1 isolates across all clades with extraordinary potency and target novel epitopes preferentially expressed on Env trimers. As these neutralization properties are ideal for a vaccine-elicited antibody response to HIV-1, their structural basis was investigated. The crystal structure of the antigen-binding fragment (Fab) of PG16 at 2.5 {angstrom} resolution revealed its unusually long, 28-residue, complementarity determining region (CDR) H3 forms a unique, stable subdomain that towers above the antibody surface. A 7-residue 'specificity loop' on the 'hammerhead' subdomain was identified that, when transplanted from PG16 to PG9 and vice versa, accounted for differences in the fine specificity and neutralization of these two mAbs. The PG16 electron density maps also revealed that a CDR H3 tyrosine was sulfated, which was confirmed for both PG9 (doubly) and PG16 (singly) by mass spectral analysis. We further showed that tyrosine sulfation plays a role in binding and neutralization. An N-linked glycan modification is observed in the variable light chain, but not required for antigen recognition. Further, the crystal structure of the PG9 light chain at 3.0 {angstrom} facilitated homology modeling to support the presence of these unusual features in PG9. Thus, PG9 and PG16 use unique structural features to mediate potent neutralization of HIV-1 that may be of utility in antibody engineering and for high-affinity recognition of a variety of therapeutic targets.

  8. Contribution of enhanced engagement of antigen presentation machinery to the clinical immunogenicity of a human interleukin (IL)-21 receptor-blocking therapeutic antibody.

    Science.gov (United States)

    Xue, L; Hickling, T; Song, R; Nowak, J; Rup, B

    2016-01-01

    Reliable risk assessment for biotherapeutics requires accurate evaluation of risk factors associated with immunogenicity. Immunogenicity risk assessment tools were developed and applied to investigate the immunogenicity of a fully human therapeutic monoclonal antibody, ATR-107 [anti-interleukin (IL)-21 receptor] that elicited anti-drug antibodies (ADA) in 76% of healthy subjects in a Phase 1 study. Because the ATR-107 target is expressed on dendritic cells (DCs), the immunogenicity risk related to engagement with DC and antigen presentation pathways was studied. Despite the presence of IL-21R on DCs, ATR-107 did not bind to the DCs more extensively than the control therapeutic antibody (PF-1) that had elicited low clinical ADA incidence. However, ATR-107, but not the control therapeutic antibody, was translocated to the DC late endosomes, co-localized with intracellular antigen-D related (HLA-DR) molecules and presented a dominant T cell epitope overlapping the complementarity determining region 2 (CDR2) of the light chain. ATR-107 induced increased DC activation exemplified by up-regulation of DC surface expression of CD86, CD274 (PD-L1) and CD40, increased expansion of activated DC populations expressing CD86(hi), CD40(hi), CD83(hi), programmed death ligand 1 (PD-L1)(hi), HLA-DR(hi) or CCR7(hi), as well as elevated secretion of tumour necrosis factor (TNF)-α by DCs. DCs exposed to ATR-107 stimulated an autologous T cell proliferative response in human donor cells, in concert with the detection of immunoglobulin (Ig)G-type anti-ATR-107 antibody response in clinical samples. Collectively, the enhanced engagement of antigen presentation machinery by ATR-107 was suggested. The approaches and findings described in this study may be relevant to identifying lower immunogenicity risk targets and therapeutic molecules. PMID:26400440

  9. Development of a mouse monoclonal antibody against the chondroitin sulfate-protein linkage region derived from shark cartilage.

    Science.gov (United States)

    Akatsu, Chizuru; Fongmoon, Duriya; Mizumoto, Shuji; Jacquinet, Jean-Claude; Kongtawelert, Prachya; Yamada, Shuhei; Sugahara, Kazuyuki

    2010-05-01

    Glycosaminoglycans (GAGs) like chondroitin sulfate (CS) and heparan sulfate (HS) are synthesized on the tetrasaccharide linkage region, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser, of proteoglycans. The Xyl can be modified by 2-O-phosphate in both CS and HS, whereas the Gal residues can be sulfated at C-4 and/or C-6 in CS but not in HS. To study the roles of these modifications, monoclonal antibodies were developed against linkage glycopeptides of shark cartilage CS proteoglycans, and one was characterized in detail. This antibody bound hexa- and pentasaccharide-peptides more strongly than unsaturated tetrasaccharide-peptides with the unnatural fourth sugar residue (unsaturated hexuronic acid), suggesting the importance of the fifth and/or fourth saccharide residue GalNAc-5 and/or GlcA-4. Its reactivity was not affected by treatment with chondro-4-sulfatase or alkaline phosphatase, suggesting that 4-O-sulfate on the Gal residues and 2-O-phosphate on the Xyl residue were not recognized. Treatment with weak alkali to cleave the Xyl-Ser linkage completely abolished the binding activity, suggesting the importance of the peptide moiety of the hexasaccharide-peptide for the binding. Based on the amino acid composition and matrix-assisted laser desorption ionization time-of-flight mass spectrometry analyses, it was revealed that the peptide moiety is composed of four amino acids, Ser, Pro, Gly, and Glu. Furthermore, the antibody stained wild-type CHO cells significantly, but much weakly mutant cells deficient in xylosyl- or galactosyltransferase-I required for the biosynthesis of the linkage region. These results suggest that the antibody recognizes the structure GalNAc(+/-6-O-sulfate)-GlcA-Gal-Gal-Xyl-Ser-(Pro, Gly, Glu). The antibody will be a useful tool for investigating the significance of the linkage region in the biosynthesis and/or intracellular transport of different GAG chains especially since such tools to study the linkage region are lacking. PMID:20336366

  10. Protection against syphilis correlates with specificity of antibodies to the variable regions of Treponema pallidum repeat protein K.

    Science.gov (United States)

    Morgan, Cecilia A; Lukehart, Sheila A; Van Voorhis, Wesley C

    2003-10-01

    Syphilis has been recognized as a disease since the late 1400s, yet there is no practical vaccine available. One impediment to the development of a vaccine is the lack of understanding of multiple reinfections in humans despite the development of robust immune responses during the first episode. It has been shown that the Treponema pallidum repeat protein K (TprK) differs in seven discrete variable (V) regions in isolates and that the antibody response during infection is directed to these V regions. Immunization with TprK confers significant protection against infection with the homologous strain. We hypothesize that the antigenic diversity of TprK is involved in immune evasion, which contributes to the lack of heterologous protection. Here, using the rabbit model, we show a correlation between limited heterologous protection and tprK diversity in the challenge inoculum. We demonstrate that antibody responses to the V regions of one TprK molecule show limited cross-reactivity with heterologous TprK V regions. PMID:14500480

  11. Characterization of genetic deletions in Becker muscular dystrophy using monoclonal antibodies against a deletion-prone region of dystrophin

    Energy Technology Data Exchange (ETDEWEB)

    Thanh, L.T.; Man, Nguyen Thi; Morris, G.E. [Wales Institute, Clwyd (United Kingdom)] [and others

    1995-08-28

    We have produced a new panel of 20 monoclonal antibodies (mAbs) against a region of the dystrophin protein corresponding to a deletion-prone region of the Duchenne muscular dystrophy gene (exons 45-50). We show that immunohistochemistry or Western blotting with these {open_quotes}exon-specific{close_quotes} mAbs can provide a valuable addition to Southern blotting or PCR methods for the accurate identification of genetic deletions in Becker muscular dystrophy patients. The antibodies were mapped to the following exons: exon 45 (2 mAbs), exon 46 (6), exon 47 (1), exons 47/48 (4), exons 48-50 (6), and exon 50 (1). PCR amplification of single exons or groups of exons was used both to produce specific dystrophin immunogens and to map the mAbs obtained. PCR-mediated mutagenesis was also used to identify regions of dystrophin important for mAb binding. Because the mAbs can be used to characterize the dystrophin produced by individual muscle fibres, they will also be useful for studying {open_quotes}revertant{close_quotes} fibres in Duchenne muscle and for monitoring the results of myoblast therapy trials in MD patients with deletions in this region of the dystrophin gene. 27 refs., 7 figs., 3 tabs.

  12. Antibody of predetermined specificity to a carboxy-terminal region of H-ras gene products inhibits their guanine nucleotide-binding function.

    OpenAIRE

    Srivastava, S. K.; Lacal, J C; Reynolds, S.H.; Aaronson, S A

    1985-01-01

    The high prevalence of ras oncogenes in human tumors has given increasing impetus to efforts aimed at elucidating the structure and function of their p21 products. To identify functionally important domains of the p21 protein, antibodies were generated against synthetic peptides corresponding to various regions of the protein. Antibodies directed against a synthetic peptide fragment corresponding to amino acid residues 161 to 176 in the carboxy-terminal region of the H-ras-encoded p21 molecul...

  13. 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.

  14. Direct and indirect interactions in the recognition between a cross-neutralizing antibody and the four serotypes of dengue virus.

    Science.gov (United States)

    Lisova, Olesia; Belkadi, Laurent; Bedouelle, Hugues

    2014-04-01

    Dengue fever is the most important vector-borne viral disease. Four serotypes of dengue virus, DENV1 to DENV4, coexist. Secondary infection by a different serotype is a risk factor for severe dengue. Monoclonal antibody mAb4E11 neutralizes the four serotypes of DENV with varying efficacies by recognizing an epitope located within domain-III (ED3) of the viral envelope (E) protein. To better understand the cross-reactivities between mAb4E11 and the four serotypes of DENV, we constructed mutations in both Fab4E11 fragment and ED3, and we searched for indirect interactions in the crystal structures of the four complexes. According to the serotype, 7 to 12 interactions are mediated by one water molecule, 1 to 10 by two water molecules, and several of these interactions are conserved between serotypes. Most interfacial water molecules make hydrogen bonds with both antibody and antigen. Some residues or atomic groups are engaged in both direct and water-mediated interactions. The doubly-indirect interactions are more numerous in the complex of lowest affinity. The third complementarity determining region of the light chain (L-CDR3) of mAb4E11 does not contact ED3. The structures and double-mutant thermodynamic cycles showed that the effects of (hyper)-mutations in L-CDR3 on affinity were caused by conformational changes and indirect interactions with ED3 through other CDRs. Exchanges of residues between ED3 serotypes showed that their effects on affinity were context dependent. Thus, conformational changes, structural context, and indirect interactions should be included when studying cross-reactivity between antibodies and different serotypes of viral antigens for a better design of diagnostics, vaccine, and therapeutic tools against DENV and other Flaviviruses. PMID:24591178

  15. Functional and molecular characterization of human monoclonal antibody reactive with the immunodominant region of HIV type 1 glycoprotein 41.

    Science.gov (United States)

    Cavacini, L A; Emes, C L; Wisnewski, A V; Power, J; Lewis, G; Montefiori, D; Posner, M R

    1998-09-20

    The immunoreactivity, functional activity, and molecular features of a human monoclonal antibody (HMAb), F240, from an HIV-1-infected individual have been studied. Flow cytometric analysis demonstrated that F240 is reactive with cells infected with a broad range of laboratory isolates but not with uninfected cells. Reactivity of F240 is greatly enhanced by preincubation of infected cells with soluble CD4, and to a much lesser extent, with F105, an HMAb reactive with the CD4-binding site of gp120. This enhancement is temperature dependent, with maximum enhancement observed at 37 degrees C, and suggests that the F240 epitope may be more accessible after gp120 has bound to CD4 in vivo. Immunoblot analysis reveals antigen specificity of F240 for gp41 or its precursor gp160. F240 specificity is mapped to the immunodominant region of the gp41 ectodomain by Pepscan analysis. This epitope has been implicated in eliciting nonprotective antibodies that enhance infection in the presence of complement. Consistent with this, F240 failed to neutralize laboratory isolates and enhanced viral infection in a complement-dependent manner. The F240 VH demonstrates extensive somatic mutations compared with the product of its closest homologous germline gene VH3-3.11. Most amino acid substitutions occur in CDR2, characteristic of an antigen-driven response, and in FR3, a phenomenon observed in other anti-HIV-1 envelope HMAbs. Primary structure analysis of the F240 heavy chain revealed strong homology in the CDR domains to an HMAb (3D6) reactive with the same gp41 region, which suggests that these HMAbs could define a potential human antibody clonotype. PMID:9764911

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

    International Nuclear Information System (INIS)

    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

  17. Hepatitis C virus hypervariable region 1 variants presented on hepatitis B virus capsid-like particles induce cross-neutralizing antibodies.

    Directory of Open Access Journals (Sweden)

    Milena Lange

    Full Text Available Hepatitis C virus (HCV infection is still a serious global health burden. Despite improved therapeutic options, a preventative vaccine would be desirable especially in undeveloped countries. Traditionally, highly conserved epitopes are targets for antibody-based prophylactic vaccines. In HCV-infected patients, however, neutralizing antibodies are primarily directed against hypervariable region I (HVRI in the envelope protein E2. HVRI is the most variable region of HCV, and this heterogeneity contributes to viral persistence and has thus far prevented the development of an effective HVRI-based vaccine. The primary goal of an antibody-based HCV vaccine should therefore be the induction of cross-reactive HVRI antibodies. In this study we approached this problem by presenting selected cross-reactive HVRI variants in a highly symmetric repeated array on capsid-like particles (CLPs. SplitCore CLPs, a novel particulate antigen presentation system derived from the HBV core protein, were used to deliberately manipulate the orientation of HVRI and therefore enable the presentation of conserved parts of HVRI. These HVRI-CLPs induced high titers of cross-reactive antibodies, including neutralizing antibodies. The combination of only four HVRI CLPs was sufficient to induce antibodies cross-reactive with 81 of 326 (24.8% naturally occurring HVRI peptides. Most importantly, HVRI CLPs with AS03 as an adjuvant induced antibodies with a 10-fold increase in neutralizing capability. These antibodies were able to neutralize infectious HCVcc isolates and 4 of 19 (21% patient-derived HCVpp isolates. Taken together, these results demonstrate that the induction of at least partially cross-neutralizing antibodies is possible. This approach might be useful for the development of a prophylactic HCV vaccine and should also be adaptable to other highly variable viruses.

  18. A Monoclonal Antibody with Thyrotropin (TSH) Receptor Inverse Agonist and TSH Antagonist Activities Binds to the Receptor Hinge Region as Well as to the Leucine-Rich Domain

    OpenAIRE

    Chen, Chun-Rong; McLachlan, Sandra M.; Rapoport, Basil

    2009-01-01

    Monoclonal antibody CS-17 is a TSH receptor (TSHR) inverse agonist (suppresses constitutive activity) and a TSH antagonist. Elucidation of the CS-17 epitope will provide insight into TSHR structure and function. Present information on its epitope conflicts with recent data regarding another TSHR inverse agonist antibody. To characterize further the CS-17 epitope, we exploited the observation that CS-17 does not recognize a chimeric receptor with TSHR hinge region residues 261–289 replaced wit...

  19. Evolution of an interloop disulfide bond in high-affinity antibody mimics based on fibronectin type III domain and selected by yeast surface display: molecular convergence with single-domain camelid and shark antibodies.

    Science.gov (United States)

    Lipovsek, Dasa; Lippow, Shaun M; Hackel, Benjamin J; Gregson, Melissa W; Cheng, Paul; Kapila, Atul; Wittrup, K Dane

    2007-05-11

    The 10th human fibronectin type III domain ((10)Fn3) is one of several protein scaffolds used to design and select families of proteins that bind with high affinity and specificity to macromolecular targets. To date, the highest affinity (10)Fn3 variants have been selected by mRNA display of libraries generated by randomizing all three complementarity-determining region -like loops of the (10)Fn3 scaffold. The sub-nanomolar affinities of such antibody mimics have been attributed to the extremely large size of the library accessible by mRNA display (10(12) unique sequences). Here we describe the selection and affinity maturation of (10)Fn3-based antibody mimics with dissociation constants as low as 350 pM selected from significantly smaller libraries (10(7)-10(9) different sequences), which were constructed by randomizing only 14 (10)Fn3 residues. The finding that two adjacent loops in human (10)Fn3 provide a large enough variable surface area to select high-affinity antibody mimics is significant because a smaller deviation from wild-type (10)Fn3 sequence is associated with a higher stability of selected antibody mimics. Our results also demonstrate the utility of an affinity-maturation strategy that led to a 340-fold improvement in affinity by maximizing sampling of sequence space close to the original selected antibody mimic. A striking feature of the highest affinity antibody mimics selected against lysozyme is a pair of cysteines on adjacent loops, in positions 28 and 77, which are critical for the affinity of the (10)Fn3 variant for its target and are close enough to form a disulfide bond. The selection of this cysteine pair is structurally analogous to the natural evolution of disulfide bonds found in new antigen receptors of cartilaginous fish and in camelid heavy-chain variable domains. We propose that future library designs incorporating such an interloop disulfide will further facilitate the selection of high-affinity, highly stable antibody mimics from

  20. Long antibody HCDR3s from HIV-naïve donors presented on a PG9 neutralizing antibody background mediate HIV neutralization.

    Science.gov (United States)

    Willis, Jordan R; Finn, Jessica A; Briney, Bryan; Sapparapu, Gopal; Singh, Vidisha; King, Hannah; LaBranche, Celia C; Montefiori, David C; Meiler, Jens; Crowe, James E

    2016-04-19

    Development of broadly neutralizing antibodies (bnAbs) against HIV-1 usually requires prolonged infection and induction of Abs with unusual features, such as long heavy-chain complementarity-determining region 3 (HCDR3) loops. Here we sought to determine whether the repertoires of HIV-1-naïve individuals contain Abs with long HCDR3 loops that could mediate HIV-1 neutralization. We interrogated at massive scale the structural properties of long Ab HCDR3 loops in HIV-1-naïve donors, searching for structured HCDR3s similar to those of the HIV-1 bnAb PG9. We determined the nucleotide sequences encoding 2.3 × 10(7)unique HCDR3 amino acid regions from 70 different HIV-1-naïve donors. Of the 26,917 HCDR3 loops with 30-amino acid length identified, we tested 30 for further study that were predicted to have PG9-like structure when chimerized onto PG9. Three of these 30 PG9 chimeras bound to the HIV-1 gp120 monomer, and two were neutralizing. In addition, we found 14 naturally occurring HCDR3 sequences that acquired the ability to bind to the HIV-1 gp120 monomer when adding 2- to 7-amino acid mutations via computational design. Of those 14 designed Abs, 8 neutralized HIV-1, with IC50values ranging from 0.7 to 98 µg/mL. These data suggest that the repertoire of HIV-1-naïve individuals contains rare B cells that encode HCDR3 loops that bind or neutralize HIV-1 when presented on a PG9 background with relatively few or no additional mutations. Long HCDR3 sequences are present in the HIV-naïve B-cell repertoire, suggesting that this class of bnAbs is a favorable target for rationally designed preventative vaccine efforts. PMID:27044078

  1. Cuban Monoclonal Antibodies for Radioimmunodiagnosis and Radioimmunotherapy of Cancer Diseases

    International Nuclear Information System (INIS)

    The Centre of Molecular Immunology produces monoclonal antibodies for treating cancer diseases. We are mainly focus on two target systems; one is the epidermal growth factor receptor (EGF-R) because there is a tremendous relationship between the EGF/EGF-R system and several human tumours such as lung, head and neck, ovarian breast and brain cancers; the second one is the ganglioside system, the relevance of certain gangliosides in tumour growth and metastatic dissemination has been well documented, GM3(NeuGc) ganglioside is particularly interesting due to its restrictive expression in normal human tissues. Nimotuzumab (h-R3) is a humanized monoclonal antibody (mAb) that was obtained by complementarity-determining regions grafting of a murine mAb (ior egf/r3) to a human framework having remarkable antiproliferative, pro-apoptotic, and antiangiogenic effects. A Phase I clinical trial was performed to evaluate the toxicity and clinical effect of an intracavitary (intracerebral) administration of a single dose of nimotuzumab (h-R3) labelled with increasing doses of 188Re. All patients bearing astrocytomas grade III/IV should be treated previously with conventional therapies and have an EGF-R overexpression in the tumour, demonstrated by immunohistochemical study. Maximal tolerated dose was 3 mg of the h-R3 labelled with 10 mCi of 188Re. The radioimmunoconjugate showed a high retention in the surgical created resection cavity and the brain adjacent tissues with a mean value of 85.5% of the injected dose one hour post-administration. This radioimmunoconjugate may be relatively safe and a promising therapeutic approach for treating high grade gliomas. GM3(NeuGc) ganglioside is particularly interesting due to its restrictive expression in normal human tissues according to immunohistochemical studies, using either polyclonal or monoclonal antibodies. But both immunohistochemical and biochemical methods have strongly suggested its over-expression in human breast and colon

  2. Anti-Staphylococcus aureus single-chain variable region fragments provide protection against mastitis in mice.

    Science.gov (United States)

    Wang, Man; Zhang, Yan; Zhu, Jianguo

    2016-03-01

    Staphylococcus aureus is a leading causative agent of bovine mastitis, which can result in significant economic losses to the dairy industry. However, available vaccines against bovine mastitis do not confer adequate protection, although passive immunization with antibodies may be useful to prevent disease. Hence, we constructed a bovine single-chain variable region fragment (scFv) phage display library using cDNAs from peripheral blood lymphocytes of cows with S. aureus-induced mastitis. After four rounds of selection, eight scFvs that bound S. aureus antigens with high affinity were obtained. The framework regions of the variable domains (VH and VL) of the eight scFvs were highly conserved, and the complementarity-determining regions (CDRs) displayed significant diversity, especially CDR3 of the VH domain. All eight scFvs inhibited S. aureus growth in culture medium. Lactating mice were challenged by injecting S. aureus into the fourth mammary gland. Histopathological analysis showed that treatment with these scFvs prior to bacterial challenge maintained the structure of the mammary acini, decreased infiltration of polymorphonuclear neutrophils, increased levels of interferon-gamma and interleukin-4, and reduced tumor necrosis factor-alpha levels in mammary tissues, as compared with mice treatment with physiological saline (P < 0.05). These novel bovine scFvs may be suitable candidates for therapeutic agents for the prevention of S. aureus-induced bovine mastitis. PMID:26512007

  3. An anti-hapten camelid antibody reveals a cryptic binding site with significant energetic contributions from a nonhypervariable loop

    Energy Technology Data Exchange (ETDEWEB)

    Fanning, Sean W.; Horn, James R. (NIU)

    2014-03-05

    Conventional anti-hapten antibodies typically bind low-molecular weight compounds (haptens) in the crevice between the variable heavy and light chains. Conversely, heavy chain-only camelid antibodies, which lack a light chain, must rely entirely on a single variable domain to recognize haptens. While several anti-hapten VHHs have been generated, little is known regarding the underlying structural and thermodynamic basis for hapten recognition. Here, an anti-methotrexate VHH (anti-MTX VHH) was generated using grafting methods whereby the three complementarity determining regions (CDRs) were inserted onto an existing VHH framework. Thermodynamic analysis of the anti-MTX VHH CDR1-3 Graft revealed a micromolar binding affinity, while the crystal structure of the complex revealed a somewhat surprising noncanonical binding site which involved MTX tunneling under the CDR1 loop. Due to the close proximity of MTX to CDR4, a nonhypervariable loop, the CDR4 loop sequence was subsequently introduced into the CDR1-3 graft, which resulted in a dramatic 1000-fold increase in the binding affinity. Crystal structure analysis of both the free and complex anti-MTX CDR1-4 graft revealed CDR4 plays a significant role in both intermolecular contacts and binding site conformation that appear to contribute toward high affinity binding. Additionally, the anti-MTX VHH possessed relatively high specificity for MTX over closely related compounds aminopterin and folate, demonstrating that VHH domains are capable of binding low-molecular weight ligands with high affinity and specificity, despite their reduced interface.

  4. Antibody mapping and tissue localization of globular and cysteine-rich regions of perlecan domain III

    DEFF Research Database (Denmark)

    Couchman, J R; Ljubimov, A V; Sthanam, M;

    1995-01-01

    Perlecan is the best-characterized basement membrane heparan sulfate proteoglycan. It has a large (approximately 400 KD) core protein consisting of five distinct domains. Domain III, a centrally located domain, contains three globular domains separated by cysteine-rich epidermal growth factor (EGF...... blotting showed that six of the nine MAbs recognized Domain III of perlecan, three of them mapping to globular Subdomain IIIc, and the other three recognized epitopes within the cysteine-rich regions. All six MAbs stained every basement membrane of several mouse organs as well as some connective tissues...

  5. Anti-tumour effects of antibodies targeting the extracellular cysteine-rich region of the receptor tyrosine kinase EphB4.

    Science.gov (United States)

    Stephenson, Sally-Anne; Douglas, Evelyn L; Mertens-Walker, Inga; Lisle, Jessica E; Maharaj, Mohanan S N; Herington, Adrian C

    2015-04-10

    EphB4 is a membrane-bound receptor tyrosine kinase (RTK) commonly over-produced by many epithelial cancers but with low to no expression in most normal adult tissues. EphB4 over-production promotes ligand-independent signaling pathways that increase cancer cell viability and stimulate migration and invasion. Several studies have shown that normal ligand-dependent signaling is tumour suppressive and therefore novel therapeutics which block the tumour promoting ligand-independent signaling and/or stimulate tumour suppressive ligand-dependent signaling will find application in the treatment of cancer. An EphB4-specific polyclonal antibody, targeting a region of 200 amino acids in the extracellular portion of EphB4, showed potent in vitro anti-cancer effects measured by an increase in apoptosis and a decrease in anchorage independent growth. Peptide exclusion was used to identify the epitope targeted by this antibody within the cysteine-rich region of the EphB4 protein, a sequence defined as a potential ligand interacting interface. Addition of antibody to cancer cells resulted in phosphorylation and subsequent degradation of the EphB4 protein, suggesting a mechanism that is ligand mimetic and tumour suppressive. A monoclonal antibody which specifically targets this identified extracellular epitope of EphB4 significantly reduced breast cancer xenograft growth in vivo confirming that EphB4 is a useful target for ligand-mimicking antibody-based anti-cancer therapies. PMID:25831049

  6. Application of two different kinds of sera against the Proteus penneri lipopolysaccharide core region in search of epitopes determining cross-reactions with antibodies

    OpenAIRE

    Palusiak, Agata; Dzieciątkowska, Monika; Sidorczyk, Zygmunt

    2008-01-01

    Introduction: Proteus penneri lipopolysaccharide (LPS) core regions are characterized by a greater structural variability than that observed in other Enterobacteriaceae. This fact and the small amount of published data concerning the serological activity of this part of P. penneri LPS prompted an examination of which fragment might determine cross-reactions with antibodies. To date, such epitopes have been found in the LPS core regions of P. mirabilis and P. vulgaris strains. Materials and Me...

  7. Prevalence of anti-Taenia solium antibodies in sera from outpatients in an Andean region of Ecuador

    Directory of Open Access Journals (Sweden)

    Luis Escalante

    1995-12-01

    Full Text Available Sera from 9,254 individuals that presented at one of three outpatient clinics in Quito, Ecuador were assayed by indirect hemagglutination for the presence of antibodies reactive with antigens from Taenia solium cysts. Immunoblot anlysis of 81 selected sera with IHA titers ranging from 0 to 1,028 showed that a titer of maior ou igual a 32 was suggestive of exposure to the parasite. Nine percent (9 % of the 9,254 patients had titers of 32 or greater. Of 3,503 sera from one clinic, which included sera from food handlers undergoing yearly physicals, 390 (11 % were positive. In addition, a correlation with age was seen in some, but not all, populations. In situations where age-related effects were noted, the highest incidence was seen in the youngest (0-20 years and in the oldest (51-60 years group. Thus, a resurgence of infection after a period of lower prevalence may be developing. Overall, this study shows that cysticercosis is relatively common and potentially a serious health problem in this region.

  8. Mediation of cytotoxic functions by classes and subclasses of sheep antibody reactive with cell surface immunoglobulin idiotypic and constant region determinants

    International Nuclear Information System (INIS)

    Sheep antibodies, reactive with either the idiotypic or constant region antigenic determinants of the immunoglobulin light chain in guinea-pig L2C leukaemic cells, were separated into IgM and into the two subclasses of IgG, IgG1 and IgG2. Antibody of both IgG subclasses inhibited the migration of L2C cells along plastic surfaces; IgM was only weakly inhibitory. Antibody of class IgM and of subclass IgG1 mediated complement cytotoxicity against the L2C cells whereas only that of subclass IgG2 mediated K-cell cytotoxicity; the effector arms were rabbit complement and sheep peripheral leucocytes, respectively. (author)

  9. Nef decreases HIV-1 sensitivity to neutralizing antibodies that target the membrane-proximal external region of TMgp41.

    Directory of Open Access Journals (Sweden)

    Rachel P J Lai

    2011-12-01

    Full Text Available Primate lentivirus nef is required for sustained virus replication in vivo and accelerated progression to AIDS. While exploring the mechanism by which Nef increases the infectivity of cell-free virions, we investigated a functional link between Nef and Env. Since we failed to detect an effect of Nef on the quantity of virion-associated Env, we searched for qualitative changes by examining whether Nef alters HIV-1 sensitivity to agents that target distinct features of Env. Nef conferred as much as 50-fold resistance to 2F5 and 4E10, two potent neutralizing monoclonal antibodies (nAbs that target the membrane proximal external region (MPER of TMgp41. In contrast, Nef had no effect on HIV-1 neutralization by MPER-specific nAb Z13e1, by the peptide inhibitor T20, nor by a panel of nAbs and other reagents targeting gp120. Resistance to neutralization by 2F5 and 4E10 was observed with Nef from a diverse range of HIV-1 and SIV isolates, as well as with HIV-1 virions bearing Env from CCR5- and CXCR4-tropic viruses, clade B and C viruses, or primary isolates. Functional analysis of a panel of Nef mutants revealed that this activity requires Nef myristoylation but that it is genetically separable from other Nef functions such as the ability to enhance virus infectivity and to downregulate CD4. Glycosylated-Gag from MoMLV substituted for Nef in conferring resistance to 2F5 and 4E10, indicating that this activity is conserved in a retrovirus that does not encode Nef. Given the reported membrane-dependence of MPER-recognition by 2F5 and 4E10, in contrast to the membrane-independence of Z13e1, the data here is consistent with a model in which Nef alters MPER recognition in the context of the virion membrane. Indeed, Nef and Glycosylated-Gag decreased the efficiency of virion capture by 2F5 and 4E10, but not by other nAbs. These studies demonstrate that Nef protects lentiviruses from one of the most broadly-acting classes of neutralizing antibodies. This newly

  10. Identification of a new epitope in uPAR as a target for the cancer therapeutic monoclonal antibody ATN-658, a structural homolog of the uPAR binding integrin CD11b (αM.

    Directory of Open Access Journals (Sweden)

    Xiang Xu

    Full Text Available The urokinase plasminogen activator receptor (uPAR plays a role in tumor progression and has been proposed as a target for the treatment of cancer. We recently described the development of a novel humanized monoclonal antibody that targets uPAR and has anti-tumor activity in multiple xenograft animal tumor models. This antibody, ATN-658, does not inhibit ligand binding (i.e. uPA and vitronectin to uPAR and its mechanism of action remains unclear. As a first step in understanding the anti-tumor activity of ATN-658, we set out to identify the epitope on uPAR to which ATN-658 binds. Guided by comparisons between primate and human uPAR, epitope mapping studies were performed using several orthogonal techniques. Systematic site directed and alanine scanning mutagenesis identified the region of aa 268-275 of uPAR as the epitope for ATN-658. No known function has previously been attributed to this epitope Structural insights into epitope recognition were obtained from structural studies of the Fab fragment of ATN-658 bound to uPAR. The structure shows that the ATN-658 binds to the DIII domain of uPAR, close to the C-terminus of the receptor, corroborating the epitope mapping results. Intriguingly, when bound to uPAR, the complementarity determining region (CDR regions of ATN-658 closely mimic the binding regions of the integrin CD11b (αM, a previously identified uPAR ligand thought to be involved in leukocyte rolling, migration and complement fixation with no known role in tumor progression of solid tumors. These studies reveal a new functional epitope on uPAR involved in tumor progression and demonstrate a previously unrecognized strategy for the therapeutic targeting of uPAR.

  11. Use of antibodies specific to defined regions of scorpion α-toxin to study its interaction with its receptor site on the sodium channel

    International Nuclear Information System (INIS)

    Five antibody populations selected by immunoaffinity chromatography for the specificity toward various regions of toxin II of the scorpion Androctonus australis Hector were used to probe the interaction of this protein with its receptor site on the sodium channel. These studies indicate that two antigenic sites, one located around the disulfide bridge 12-63 and one encompassing residues 50-59, are involved in the molecular mechanisms of toxicity neutralization. Fab fragments specific to the region around disulfide bridge 12-63 inhibit binding of the 125I-labeled toxin to its receptor site. Also, these two antigenic regions are inaccessible to the antibodies when the toxin is bound to its receptor site. In contrast, the two other antigenic sites encompassing the only α-helix region (residues 23-32) and a β-turn structure (residues 32-35) are accessible to the respective antibodies when the toxin is bound to its receptor. Together, these data support the recent proposal that a region made of residues that are conserved in the scorpion toxin family is involved in the binding of the toxin to the receptor

  12. Eliciting neutralizing antibodies against the membrane proximal external region of HIV-1 Env by chimeric live attenuated influenza A virus vaccines.

    Science.gov (United States)

    Zang, Yang; Du, Dongchuan; Li, Na; Su, Weiheng; Liu, Xintao; Zhang, Yan; Nie, Jianhui; Wang, Youchun; Kong, Wei; Jiang, Chunlai

    2015-07-31

    Despite significant efforts directed toward research on HIV-1 vaccines, a truly effective immunogen has not been achieved. However, the broadly neutralizing antibodies (BnAbs) 2F5 and 4E10, targeting the highly conserved membrane proximal external region (MPER) of HIV-1, are two promising tools for vaccine development. Here we engrafted the MPER into the linker domain between the trimeric core structure and the transmembrane domain of influenza A virus HA2 to investigate the potential of such chimeric viruses to elicit HIV-1 neutralizing antibodies. In the context of proliferating attenuated influenza A viruses, these HIV-1 neutralizing antibody epitopes could be continuously expressed and mimicked their native conformation to induce humoral immune responses. While MPER-specific antibodies could be detected in serum of guinea pigs vaccinated with the chimeric viruses, they exhibited only weakly neutralizing activities. These antisera from vaccinated animals neutralized viruses of clades B and BC (tier 1), but not of clades AE (tier 1) and C (tier 2). These results suggest that influenza A virus can be used as a vehicle for displaying MPER and inducing BnAbs, but it provides limited protection against HIV-1 infection. In the future development of HIV-1 vaccines by rational design, a more effective live virus vector or multiple antigens should be chosen to facilitate the process of neutralizing antibody maturation. PMID:26126669

  13. Thyroid Antibodies

    Science.gov (United States)

    ... be limited. Home Visit Global Sites Search Help? Thyroid Antibodies Share this page: Was this page helpful? Also known as: Thyroid Autoantibodies; Antithyroid Antibodies; Antimicrosomal Antibody; Thyroid Microsomal Antibody; ...

  14. Development of 2 types of competitive enzyme-linked immunosorbent assay for detecting antibodies to the rinderpest virus using a monoclonal antibody for a specific region of the hemagglutinin protein.

    Science.gov (United States)

    Khamehchian, S; Madani, R; Rasaee, M J; Golchinfar, F; Kargar, R

    2007-06-01

    A competitive enzyme-linked immunosorbent assay (C-ELISA) has been developed and standardized for the detection of antibodies to the rinderpest virus (RPV) in sera from cattle, sheep, and goats. The test is specific for rinderpest because it does not detect antibodies to peste-des-petits-ruminants virus (PPRV). The test depends on the ability of the monoclonal antibody (MAb) directed against the hemagglutinin (H) protein of RPV to compete with the binding of RPV antibodies in the positive serum to the H protein of this virus. This MAb recognized a region from amino acids 575 to 583 on the H protein of RPV that is unique to the RPV H protein and is not present on the hemagglutinin-neuraminidase protein of PPRV. Another C-ELISA (peptide C-ELISA) was set up using this specific region as an antigen. A threshold value of 64.4% inhibition was established for the RPV C-ELISA, with 90 known RPV-negative and 30 RPV-positive serum samples. Using common serum samples, a cutoff value of 43.0% inhibition for the peptide C-ELISA was established. Based on statistical analysis, the overall sensitivity and specificity of the RPV C-ELISA, relative to those of a commercial kit, were found to be 90.00% and 103.33%, respectively. However, the sensitivity and specificity of the peptide C-ELISA were found to be 180.00% and 73.33%, respectively. Although a common MAb in 2 new C-ELISA systems was used, variation in their percent inhibition, due to the use of different antigens, was observed. Taking into consideration the difference in percent inhibition of the 2 described assays and the commercial kit (50%), it was found that the RPV C-ELISA and the peptide C-ELISA are more specific and sensitive tools than the commercial kit for assessing herd immune status and for epidemiologic surveillance. PMID:17668032

  15. THE INVESTIGATION OF BRUCELLA ANTIBODY WITH MILK RING TEST AND AGGLUTINATION TEST IN MILK COLLECTED FROM SAMSUN REGION

    Directory of Open Access Journals (Sweden)

    Goknur TERZI

    2006-06-01

    Full Text Available In this study Brucella antibodies were investigated with agglutination test (Whey-AT and Milk Ring Test (MRT in a total of 100 milk samples as 50 of cow milk and 50 of goat milk collected from center and villages of Samsun. According to MRT Brucella antibodies was positive at 10 samples (20 % of cow milk and 6 samples (12 % of goat milk. In cow milk, 4 (8 % positive, 3 (6 % suspicious and 43 (86 % negative samples; in goat milk 3 (6 % positive, 2 (4 % suspicious and 45 (90 % negative samples were determined according to antibodies titre of serum agglutination test (Whey-AT. [TAF Prev Med Bull 2006; 5(3.000: 196-203

  16. THE INVESTIGATION OF BRUCELLA ANTIBODY WITH MILK RING TEST AND AGGLUTINATION TEST IN MILK COLLECTED FROM SAMSUN REGION

    OpenAIRE

    Goknur TERZI

    2006-01-01

    In this study Brucella antibodies were investigated with agglutination test (Whey-AT) and Milk Ring Test (MRT) in a total of 100 milk samples as 50 of cow milk and 50 of goat milk collected from center and villages of Samsun. According to MRT Brucella antibodies was positive at 10 samples (20 %) of cow milk and 6 samples (12 %) of goat milk. In cow milk, 4 (8 %) positive, 3 (6 %) suspicious and 43 (86 %) negative samples; in goat milk 3 (6 %) positive, 2 (4 %) suspicious and 45 (90 %) negativ...

  17. Study of the Mn-binding sites in photosystem II using antibodies raised against lumenal regions of the D1 and D2 reaction center proteins

    Energy Technology Data Exchange (ETDEWEB)

    Dalmasso, E.A.

    1992-04-01

    The experiments discussed in this thesis focus on identifying the protein segments or specific amino acids which provide ligands to the Mn cluster of photosystem II (PS II). This Mn cluster plays a central role in the oxygen-evolving complex (OEC) of PS II. The Mn cluster is thought to be bound by lumenal regions of the PS II reaction center proteins known as D1 and D2. First, several peptides were synthesized which correspond to specific lumenal segments of the D1 and D2 proteins. Next, polyclonal antibodies were successfully elicited using three of these peptides. The peptides recognized by these antibodies correspond to protein segments of the spinach reaction center proteins: Ile-321 to Ala-344 of D1 (D1-a), Asp-319 to Arg-334 of D1 (D1-b), and Val-300 to Asn-319 of D2 (D2-a). These antibodies were then used in assays which were developed to structurally or functionally probe the potential Mn-binding regions of the D1 and D2 proteins.

  18. Comparison of marmoset and human FSH using synthetic peptides of the β-subunit L2 loop region and anti-peptide antibodies.

    Science.gov (United States)

    Kutteyil, Susha S; Kulkarni, Bhalchandra J; Mojidra, Rahul; Joseph, Shaini; Pathak, Bhakti R; Mahale, Smita D

    2016-06-01

    Follicle stimulating hormone (FSH) is a glycoprotein hormone required for female and male gametogenesis in vertebrates. Common marmoset (Callithrix jacchus) is a New World primate monkey, used as animal model in biomedical research. Observations like, requirement of extremely high dose of human FSH in marmosets for superovulation compared to other primates and generation of antibodies in marmoset against human FSH after repeated superovulation cycles, point towards the possibility that FSH-FSH receptor (FSHR) interaction in marmosets might be different than in the humans. In this study we attempted to understand some of these structural differences using FSH peptides and anti-peptide antibody approach. Based on sequence alignment, in silico modeling and docking studies, L2 loop of FSH β-subunit (L2β) was found to be different between marmoset and human. Hence, peptides corresponding to region 32-50 of marmoset and human L2β loop were synthesized, purified and characterized. The peptides displayed dissimilarity in terms of molecular mass, predicted isoelectric point, predicted charge and in the ability to inhibit hormone-receptor interaction. Polyclonal antibodies generated against both the peptides were found to exhibit specific binding for the corresponding peptide and parent FSH in ELISA and Western blotting respectively and exhibited negligible reactivity to cross-species peptide and FSH in ELISA. The anti-peptide antibody against marmoset FSH was also able to detect native FSH in marmoset plasma samples and pituitary sections. In summary, the L2β loop of marmoset and human FSH has distinct receptor interaction ability and immunoreactivity indicating possibility of subtle conformational and biochemical differences between the two regions which may affect the FSH-FSHR interaction in these two primates. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. PMID:27282136

  19. Production of Monoclonal Antibody Against Recombinant Polypeptide From the Erns Coding Region of the Bovine Viral Diarrhea Virus

    OpenAIRE

    Seyfi Abad Shapouri, Masood Reza; Ekhtelat, Maryam; Ghorbanpoor Najaf Abadi, Masood; Mahmoodi Koohi, Pezhman; Lotfi, Mohsen

    2015-01-01

    Background: Bovine viral diarrhea (BVD) is an economically important cattle disease with a worldwide distribution. Detection and elimination of animals persistently infected (PI) with bovine viral diarrhea virus (BVDV) is essential for the control of BVD and eradication of BVDV. There are usually no pathognomonic clinical signs of BVDV infection. Diagnostic investigations therefore rely on laboratory-based detection of the virus, or virus-induced antigens or antibodies. Objectives: Erns as an...

  20. Anti-Taenia solium metacestode IgG antibodies in serum samples from inhabitants of a central-western region of Brazil

    Directory of Open Access Journals (Sweden)

    Oliveira Heliana B. de

    2006-01-01

    Full Text Available A total of 354 serum samples from inhabitants who frequent the Clinical Laboratory in Catalão, Goiás, in the central-western region of Brazil, were collected from June to August, 2002. The samples were evaluated by indirect immunofluorescence antibody tests and an enzyme linked immunosorbent assay in order to detect anti-Taenia solium metacestode IgG antibodies. Reactive and inconclusive samples were tested by Western blotting (WB. Considering WB as a confirmation, the frequency of antibodies in the serum samples of the above population was 11.3% (CI 5.09 - 17.51. The immunodominant bands most frequently recognized in WB were 64-68 kDa (97.5% and 47-52 kDa (80%. The percentage of seropositivity to cysticercosis was significantly higher for individuals residing in areas without sewage systems (p < 0.0001. In conclusion, the results indicate a probable endemic situation of cysticercosis in this population. These results reinforce the urgent need for control and prevention measures to be taken by the local public health services.

  1. 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. PMID:26700095

  2. Prevalence of antibody to human T cell lymphotropic virus types 1/2 among aboriginal groups inhabiting northern Argentina and the Amazon region of Peru.

    Science.gov (United States)

    Medeot, S; Nates, S; Recalde, A; Gallego, S; Maturano, E; Giordano, M; Serra, H; Reategui, J; Cabezas, C

    1999-04-01

    We carried out a seroepidemiologic survey to define the prevalence of human T cell lymphotropic virus types 1/2 (HTLV-1/2) infections among aboriginal populations from isolated regions of northern Argentina and the Amazon region of Peru. Antibodies against HTLV were measured with agglutination tests and confirmed with by an immunofluorescence assay (IFA) and Western blotting. Five (6.94%) of 72 samples from the Tobas Indians in Argentina were positive by the IFA; two samples were typed as HTLV-1 (2.78%), two as HTLV-2 (2.78%), and one (1.39%) could not be typed because it had similar antibody titers against both viruses. No positive samples were found among 84 Andinos Puneños and 47 Matacos Wichis Indians. Seroprevalences of 2.50% (1 of 40) and 1.43% (1 of 70) for HTLV-1 were observed among Wayku and San Francisco communities in the Amazon region of Peru, and seroprevalences of 4.54% (1 of 22) and 2.38% (1 of 42) for HTLV-2 were observed among Boca Colorada and Galilea communities. No serologic evidence of human immunodeficiency virus (HIV) infection was found among the Indians tested. These results indicated the presence of HTLV-1 and HTLV-2 in the indigenous populations of Argentina and Peru. Moreover, the lack of HIV infection indicates that the virus has probably not yet been introduced into these populations. PMID:10348238

  3. Engineering antibody therapeutics.

    Science.gov (United States)

    Chiu, Mark L; Gilliland, Gary L

    2016-06-01

    The successful introduction of antibody-based protein therapeutics into the arsenal of treatments for patients has within a few decades fostered intense innovation in the production and engineering of antibodies. Reviewed here are the methods currently used to produce antibodies along with how our knowledge of the structural and functional characterization of immunoglobulins has resulted in the engineering of antibodies to produce protein therapeutics with unique properties, both biological and biophysical, that are leading to novel therapeutic approaches. Antibody engineering includes the introduction of the antibody combining site (variable regions) into a host of architectures including bi and multi-specific formats that further impact the therapeutic properties leading to further advantages and successes in patient treatment. PMID:27525816

  4. Development of a monoclonal antibody specific to envelope domain III with broad-spectrum detection of all four dengue virus serotypes.

    Science.gov (United States)

    Kim, Sae-Hae; Kim, Yu Na; Truong, Thang Thua; Thu Thuy, Nguyen Thi; Mai, Le Quynh; Jang, Yong-Suk

    2016-05-13

    Dengue virus (DENV) is a mosquito-borne pathogen that annually infects more than 390 million people in 100 different countries. Symptoms of the viral infection include a relatively weak dengue fever to severe dengue hemorrhagic fever/dengue shock syndrome, which are mortal infectious diseases. As of yet, there is no commercially available vaccine or therapeutic for DENV. Currently, passive immunotherapy using DENV-specific antibody (Ab) is a considered strategy to treat DENV infection. Here, we developed a monoclonal Ab (mAb), EDIIImAb-61, specific to the DENV domain III of the envelope glycoprotein (EDIII) with broad-spectrum detection ability to all four DENV serotypes (DENV-1∼4) to use as a therapeutic Ab. Although EDIII contains non-immunodominant epitopes compared to domains I and II, domain III plays a critical role in host receptor binding. EDIIImAb-61 exhibited cross-reactive binding affinity to all four DENV serotypes that had been isolated from infected humans. To further characterize EDIIImAb-61 and prepare genes for large-scale production using a heterologous expression system, the sequence of the complementarity determining regions was analyzed after cloning the full-length cDNA genes encoding the heavy and light chain of the mAb. Finally, we produced Ab from CHO-K1 cells transfected with the cloned EDIIImAb-61 heavy and light chain genes and confirmed the binding ability of the Ab. Collectively, we conclude that EDIIImAb-61 itself and the recombinant Ab produced using the cloned heavy and light chain gene of EDIIImAb-61 is a candidate for passive immunotherapy against DENV infection. PMID:27059141

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

    Science.gov (United States)

    Prado, Nidiane D. R.; Pereira, Soraya S.; da Silva, Michele P.; Morais, Michelle S. S.; Kayano, Anderson M.; Moreira-Dill, Leandro S.; Luiz, Marcos B.; Zanchi, Fernando B.; Fuly, André L.; E. F. Huacca, Maribel; Fernandes, Cleberson F.; Calderon, Leonardo A.; Zuliani, Juliana P.; Soares, Andreimar M.; Stabeli, Rodrigo G.; F. C. Fernandes, Carla

    2016-01-01

    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. PMID:27028872

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

    Science.gov (United States)

    Prado, Nidiane D R; Pereira, Soraya S; da Silva, Michele P; Morais, Michelle S S; Kayano, Anderson M; Moreira-Dill, Leandro S; Luiz, Marcos B; Zanchi, Fernando B; Fuly, André L; E F Huacca, Maribel; Fernandes, Cleberson F; Calderon, Leonardo A; Zuliani, Juliana P; Pereira da Silva, Luiz H; Soares, Andreimar M; Stabeli, Rodrigo G; F C Fernandes, Carla

    2016-01-01

    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. PMID:27028872

  7. 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.

  8. Antithyroid microsomal antibody

    Science.gov (United States)

    Thyroid antimicrosomal antibody; Antimicrosomal antibody; Microsomal antibody; Thyroid peroxidase antibody; TPOAb ... Granulomatous thyroiditis Hashimoto thyroiditis High levels of these antibodies have also been linked to an increased risk ...

  9. Replacement of lysine residue 1030 in the putative ATP-binding region of the insulin receptor abolishes insulin- and antibody-stimulated glucose uptake and receptor kinase activity

    International Nuclear Information System (INIS)

    To test whether the tyrosine kinase activity of the insulin receptor is crucial for insulin action, the authors have constructed mutations of the human insulin receptor at Lys-1030, which is in the presumed ATP-binding region. By using oligonucleotide-directed mutagenesis, this lysine residue was replaced with either methionine, arginine, or alanine. Chinese hamster ovary cells were transfected by mutant cDNAs and the expressed insulin receptors were characterized. They show here that none of these mutants exhibited insulin-activated autophosphorylation and kinase activity in vitro. They also do not mediate insulin- and antibody-stimulated uptake of 2-deoxyglucose. The tyrosine kinase activity is thus required for a key physiological response of insulin

  10. OptMAVEn – A New Framework for the de novo Design of Antibody Variable Region Models Targeting Specific Antigen Epitopes

    OpenAIRE

    Li, Tong; Pantazes, Robert J; Maranas, Costas D.

    2014-01-01

    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 method...

  11. Sero-Survey of Polio Antibodies during Wild Poliovirus Outbreak in Southern Xinjiang Uygur Autonomous Region, China

    OpenAIRE

    Hai-Bo Wang; Shuang-Li Zhu; Jing-Shan Zheng; Ai-Li Gou; Hui Cui; Yong Zhang; Gui-Jun Ning; Chun-Xiang Fan; Yuan-Sheng Chen; Ke-Li Li; Ping Yuan; Chao Ma; Jing Ma; Hui Zheng; Xin-Chun Fan

    2014-01-01

    Background After being polio free for more than 10 years, an outbreak following importation of wild poliovirus (WPV) was confirmed in Xinjiang Uygur Autonomous Region, China, in 2011. Methods A cross-sectional study was conducted prior to supplementary immunization activities (SIAs), immediately after the confirmation of the WPV outbreak. In selected prefectures, participants aged ≤60 years old who visited hospitals at county-level or above to have their blood drawn for reasons not related to...

  12. The NTS-DBL2X region of VAR2CSA Induces cross-reactive antibodies that inhibit adhesion of several Plasmodium falciparum isolates to chondroitin sulfate A

    DEFF Research Database (Denmark)

    Bigey, Pascal; Gnidehou, Sédami; Doritchamou, Justin;

    2011-01-01

    -adapted parasite lines and field isolates expressing VAR2CSA. Competition enzyme-linked immunosorbent assay (ELISA) was employed to analyze functional resemblance between antibodies induced in animals and those naturally acquired by immune multigravidae. Results. Antibodies targeting the N-terminal sequence (NTS......) up to DBL2X (NTS-DBL2X) efficiently blocked parasite adhesion to chondroitin sulfate A in a manner similar to that of antibodies raised against the entire VAR2CSA extracellular domain. Interestingly, naturally acquired antibodies and those induced by vaccination against NTS-DBL2X target overlapping...

  13. Topographical localization of the C-terminal region of the voltage-dependent sodium channel from Electrophorus electricus using antibodies raised against a synthetic peptide

    Energy Technology Data Exchange (ETDEWEB)

    Gordon, R.D.; Fieles, W.E.; Schotland, D.L.; Hogue-Angeletti, R.; Barchi, R.L.

    1987-01-01

    A peptide corresponding to amino acid residues 1783-1794 near the C terminus of the electric eel sodium channel primary sequence of the eel (Electrophorus electricus) sodium channel has been synthesized and used to raise an antiserum in rabbits. This antiserum specifically recognized the peptide in a solid-phase radioimmunoassay. Specificity of the antiserum for the native channel protein was shown by its specific binding to a 280-kDa protein in immunoblots of eel electroplax membrane proteins. The antiserum also specifically labeled the innervated membrane of the eel electroplax in immunofluorescent studies. The membrane topology of the peptide recognized by this antiserum was proved in binding studies using oriented electroplax membrane vesicles. These vesicles were 98% right-side-out as determined by (/sup 3/H)saxitoxin binding. Binding of the antipeptide antiserum to this fraction was measured before and after permeabilization with 0.01% saponin. Specific binding to intact vesicles was low, but this binding increased 10-fold after permeabilization, implying a cytoplasmic orientation for the peptide. Confirmation for this orientation was then sought by localizing the antibody bound to intact electroplax cells with immunogold electron microscopy. The data imply that the region of the sodium channel primary sequence near the C terminus that is recognized by the anitserum is localized on the cytoplasmic side of the membrane; this localization provides some further constraints on models of sodium channel tertiary structure.

  14. Topographical localization of the C-terminal region of the voltage-dependent sodium channel from Electrophorus electricus using antibodies raised against a synthetic peptide

    International Nuclear Information System (INIS)

    A peptide corresponding to amino acid residues 1783-1794 near the C terminus of the electric eel sodium channel primary sequence of the eel (Electrophorus electricus) sodium channel has been synthesized and used to raise an antiserum in rabbits. This antiserum specifically recognized the peptide in a solid-phase radioimmunoassay. Specificity of the antiserum for the native channel protein was shown by its specific binding to a 280-kDa protein in immunoblots of eel electroplax membrane proteins. The antiserum also specifically labeled the innervated membrane of the eel electroplax in immunofluorescent studies. The membrane topology of the peptide recognized by this antiserum was proved in binding studies using oriented electroplax membrane vesicles. These vesicles were 98% right-side-out as determined by [3H]saxitoxin binding. Binding of the antipeptide antiserum to this fraction was measured before and after permeabilization with 0.01% saponin. Specific binding to intact vesicles was low, but this binding increased 10-fold after permeabilization, implying a cytoplasmic orientation for the peptide. Confirmation for this orientation was then sought by localizing the antibody bound to intact electroplax cells with immunogold electron microscopy. The data imply that the region of the sodium channel primary sequence near the C terminus that is recognized by the anitserum is localized on the cytoplasmic side of the membrane; this localization provides some further constraints on models of sodium channel tertiary structure

  15. Development of a 'mouse and human cross-reactive' affinity-matured exosite inhibitory human antibody specific to TACE (ADAM17) for cancer immunotherapy.

    Science.gov (United States)

    Kwok, Hang Fai; Botkjaer, Kenneth A; Tape, Christopher J; Huang, Yanchao; McCafferty, John; Murphy, Gillian

    2014-06-01

    We previously showed that a human anti-TACE antibody, D1(A12), is a potent inhibitor of TNF-α converting enzyme (TACE) ectodomain proteolysis and has pharmacokinetic properties suitable for studies of the inhibition of TACE-dependent growth factor shedding in relation to possible therapeutic applications. However, the lack of murine TACE immunoreactivity limits pre-clinical in vivo studies to human xenograft models which are poor analogies to in situ pathology and are not considered clinically predictive. Here, to overcome these limitations, we set out to develop a 'mouse and human cross-reactive' specific anti-TACE antibody. We first re-investigated the originally selected anti-TACE ectodomain phage-display clones, and isolated a lead 'mouse-human cross-reactive' anti-TACE scFv, clone A9. We reformatted scFv-A9 into an IgG2 framework for comprehensive biochemical and cellular characterization and further demonstrated that A9 is an exosite TACE inhibitor. However, surface plasmon resonance analysis and quenched-fluorescent (QF) peptide assay indicated that IgG reformatting of A9 caused low binding affinity and an 80-fold reduction in TACE ectodomain inhibition, severely limiting its efficacy. To address this, we constructed second generation phage-display randomization libraries focused on the complementarity-determining region 3, and carried out affinity selections shuffling between human and mouse TACE ectodomain as antigen in addition to an off-rate selection to increase the chance of affinity improvement. The bespoke 'three-step' selections enabled a 100-fold affinity enhancement of A9 IgG, and also improved its IC50 in a QF peptide assay to 0.2 nM. In human and mouse cancer cell assays, matured A9 IgG showed significant cell-surface TACE inhibition as a monotherapy or combination therapy with chemotherapeutic agent. Collectively, these data suggest that we successfully developed an exosite inhibitor of TACE with sub-nanomolar affinity, which possesses both

  16. The hypervariable region of Streptococcus pyogenes M protein escapes antibody attack by antigenic variation and weak immunogenicity

    DEFF Research Database (Denmark)

    Lannergård, Jonas; Gustafsson, Caj Ulrik Mattias; Waldemarsson, Johan;

    2011-01-01

    , we analyzed the clinically important HVR-containing M proteins of the human pathogen Streptococcus pyogenes. Antibodies elicited by M proteins were directed almost exclusively against the C-terminal part and not against the N-terminal HVR. Similar results were obtained for mice and humans with...... invasive S. pyogenes infection. Nevertheless, only anti-HVR antibodies protected efficiently against infection, as shown by passive immunizations. The HVR fused to an unrelated protein elicited no antibodies, implying that it is inherently weakly immunogenic. These data indicate that the M protein HVR...

  17. Uses of monoclonal antibody 8H9

    Science.gov (United States)

    Cheung, Nai-Kong V.

    2013-04-09

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

  18. Bispecific antibodies.

    Science.gov (United States)

    Kontermann, Roland E; Brinkmann, Ulrich

    2015-07-01

    Bispecific antibodies (bsAbs) combine specificities of two antibodies and simultaneously address different antigens or epitopes. BsAbs with 'two-target' functionality can interfere with multiple surface receptors or ligands associated, for example with cancer, proliferation or inflammatory processes. BsAbs can also place targets into close proximity, either to support protein complex formation on one cell, or to trigger contacts between cells. Examples of 'forced-connection' functionalities are bsAbs that support protein complexation in the clotting cascade, or tumor-targeted immune cell recruiters and/or activators. Following years of research and development (R&D), the first bsAb was approved in 2009. Another bsAb entered the market in December 2014 and several more are in clinical trials. Here, we describe the potentials of bsAbs to become the next wave of antibody-based therapies, focusing on molecules in clinical development. PMID:25728220

  19. Combinatorial H3K9acS10ph Histone Modification in IgH Locus S Regions Targets 14-3-3 Adaptors and AID to Specify Antibody Class-Switch DNA Recombination

    Directory of Open Access Journals (Sweden)

    Guideng Li

    2013-11-01

    Full Text Available Class-switch DNA recombination (CSR is central to the antibody response, in that it changes the immunoglobulin heavy chain (IgH constant region, thereby diversifying biological effector functions of antibodies. The activation-induced cytidine deaminase (AID-centered CSR machinery excises and rejoins DNA between an upstream (donor and a downstream (acceptor S region, which precede the respective constant region DNA. AID is stabilized on S regions by 14-3-3 adaptors. These adaptors display a high affinity for 5′-AGCT-3′ repeats, which recur in all S regions. However, how 14-3-3, AID, and the CSR machinery target exclusively the donor and acceptor S regions is poorly understood. Here, we show that histone methyltransferases and acetyltransferases are induced by CD40 or Toll-like receptor signaling and catalyze H3K4me3 and H3K9ac/K14ac histone modifications, which are enriched in S regions but do not specify the S region targets of CSR. By contrast, the combinatorial H3K9acS10ph modification specifically marks the S regions set to recombine and directly recruits 14-3-3 adaptors for AID stabilization there. Inhibition of the enzymatic activity of GCN5 and PCAF histone acetyltransferases reduces H3K9acS10ph in S regions, 14-3-3 and AID stabilization, and CSR. Thus, H3K9acS10ph is a histone code that is “written” specifically in S regions and is “read” by 14-3-3 adaptors to target AID for CSR as an important biological outcome.

  20. A longitudinal study of human antibody responses to Plasmodium falciparum rhoptry-associated protein 1 in a region of seasonal and unstable malaria transmission

    DEFF Research Database (Denmark)

    Fonjungo, P N; Elhassan, I M; Cavanagh, D R; Theander, T G; Hviid, L; Roper, C; Arnot, D E; McBride, J S

    1999-01-01

    Rhoptry-associated protein 1 (RAP1) of Plasmodium falciparum is a nonpolymorphic merozoite antigen that is considered a potential candidate for a malaria vaccine against asexual blood stages. In this longitudinal study, recombinant RAP1 (rRAP1) proteins with antigenicity similar to that of P....... falciparum-derived RAP1 were used to analyze antibody responses to RAP1 over a period of 4 years (1991 to 1995) of 53 individuals naturally exposed to P. falciparum malaria. In any 1 year during the study, between 23 and 39% of individuals who had malaria developed immunoglobulin G (IgG) antibodies...... a few months after the end of malaria transmission, during the dry season, or by the start of the next malaria season. Thus, RAP1 IgG responses were very short-lived. The short duration of RAP1 antibody response may explain the apparent lack of response in a surprisingly high proportion of...

  1. Mechanism of Lethal Toxin Neutralization by a Human Monoclonal Antibody Specific for the PA20 Region of Bacillus anthracis Protective Antigen

    Directory of Open Access Journals (Sweden)

    Jessica Camacho

    2011-08-01

    Full Text Available The primary immunogenic component of the currently approved anthrax vaccine is the protective antigen (PA unit of the binary toxin system. PA-specific antibodies neutralize anthrax toxins and protect against infection. Recent research has determined that in humans, only antibodies specific for particular determinants are capable of effecting toxin neutralization, and that the neutralizing epitopes recognized by these antibodies are distributed throughout the PA monomer. The mechanisms by which the majority of these epitopes effect neutralization remain unknown. In this report we investigate the process by which a human monoclonal antibody specific for the amino-terminal domain of PA neutralizes lethal toxin in an in vitro assay of cytotoxicity, and find that it neutralizes LT by blocking the requisite cleavage of the amino-terminal 20 kD portion of the molecule (PA20 from the remainder of the PA monomer. We also demonstrate that the epitope recognized by this human monoclonal does not encompass the 166RKKR169 furin recognition sequence in domain 1 of PA.

  2. Increased expression of Matrix Metalloproteinase 9 in liver from NZB/W F1 mice received antibody against human parvovirus B19 VP1 unique region protein

    Directory of Open Access Journals (Sweden)

    Hsu Gwo-Jong

    2009-01-01

    Full Text Available Abstract Background Human parvovirus B19 infection has been postulated to the anti-phospholipid syndrome (APS in autoimmunity. However, the influence of anti-B19-VP1u antibody in autoimmune diseases is still obscure. Methods To elucidate the effect of anti-B19-VP1u antibodies in systemic lupus erythematosus (SLE, passive transfer of rabbit anti-B19-VP1u IgG was injected intravenously into NZB/W F1 mice. Results Significant reduction of platelet count and prolonged thrombocytopenia time were detected in anti-B19-VP1u IgG group as compared to other groups, whereas significant increases of anti-B19-VP1u, anti-phospholipid (APhL, and anti-double strand DNA (dsDNA antibody binding activity were detected in anti-B19-VP1u group. Additionally, significant increases of matrix metalloproteinase-9 (MMP9 activity and protein expression were detected in B19-VP1u IgG group. Notably, phosphatidylinositol 3-phosphate kinase (PI3K and phosphorylated extracellular signal-regulated kinase (ERK proteins were involved in the induction of MMP9. Conclusion These experimental results firstly demonstrated the aggravated effects of anti-B19-VP1u antibody in disease activity of SLE.

  3. Cooperativity in virus neutralization by human monoclonal antibodies to two adjacent regions located at the amino terminus of hepatitis C virus E2 glycoprotein

    DEFF Research Database (Denmark)

    Keck, Zhenyong; Wang, Wenyan; Wang, Yong; Lau, Patrick; Carlsen, Thomas H R; Prentoe, Jannick; Xia, Jinming; Patel, Arvind H; Bukh, Jens; Foung, Steven K H

    2013-01-01

    A challenge for hepatitis C virus (HCV) vaccine development is defining conserved epitopes that induce protective antibodies against this highly diverse virus. An envelope glycoprotein (E2) segment located at amino acids (aa) 412 to 423 contains highly conserved neutralizing epitopes. While...

  4. Epitope mapping from real time kinetic studies – Role of cross-linked disulphides and incidental interacting regions in affinity measurements: Study with human chorionic gonadotropin and monoclonal antibodies

    Indian Academy of Sciences (India)

    Nonavinakere Seetharam Srilatha; P Tamil Selvi; Gundlupet Satyanarayana Murthy

    2005-06-01

    Real time kinetic studies were used to map conformational epitopes in human chorionic gonadotropin (hCG) for two monoclonal antibodies (MAbs). The epitopes were identified in the regions (5–14 and 55–62). The association rate constant (+1) was found to be altered by chemical modification of hCG, and the ionic strength of the reaction medium. Based on these changes, we propose the presence of additional interactions away from the epitope-paratope region in the hCG-MAb reaction. We have identified such incidental interacting regions (IIRs) in hCG to be the loop region 35–47 and 60–84. The IIRs contribute significantly towards the of the interaction. Therefore, in a macromolecular interaction of hCG and its MAb, is determined not only by epitopeparatope interaction but also by the interaction of the nonepitopic-nonparatopic IIRs. However, the specificity of the interaction resides exclusively with the epitope-paratope pair.

  5. Prevalence of antibody to hantaviruses in humans and rodents in the Caribbean region of Colombia determined using Araraquara and Maciel virus antigens

    Directory of Open Access Journals (Sweden)

    Camilo Guzmán

    2013-04-01

    Full Text Available We tested sera from 286 agricultural workers and 322 rodents in the department of Córdoba, northeastern Colombia, for antibodies against two hantaviruses. The sera were analysed by indirect ELISA using the lysate of Vero E6 cells infected with Maciel virus (MACV or the N protein of Araraquara virus (ARAV as antigens for the detection of antibodies against hantaviruses. Twenty-four human sera were IgG positive using one or both antigens. We detected anti-MACV IgG antibodies in 10 sera (3.5% and anti-ARAV antibodies in 21 sera (7.34%. Of the 10 samples that were positive for MACV, seven (70% were cross-reactive with ARAV; seven of the 21 ARAV-positive samples were cross-reactive with MACV. Using an ARAV IgM ELISA, two of the 24 human sera (8.4% were positive. We captured 322 rodents, including 210 Cricetidae (181 Zygodontomys brevicauda, 28 Oligoryzomys fulvescens and 1 Oecomys trinitatis, six Heteromys anomalus (Heteromyidae, one Proechimys sp. (Echimyidae and 105 Muridae (34 Rattus rattus and 71 Mus musculus. All rodent sera were negative for both antigens. The 8.4% detection rate of hantavirus antibodies in humans is much higher than previously found in serosurveys in North America, suggesting that rural agricultural workers in northeastern Colombia are frequently exposed to hantaviruses. Our results also indicate that tests conducted with South American hantavirus antigens could have predictive value and could represent a useful alternative for the diagnosis of hantavirus infection in Colombia.

  6. Production of Monoclonal Antibody against Human Nestin.

    Science.gov (United States)

    Hadavi, Reza; Zarnani, Amir Hassan; Ahmadvand, Negah; Mahmoudi, Ahmad Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Sadeghi, Mohammad-Reza; Soltanghoraee, Haleh; Akhondi, Mohammad Mehdi; Tarahomi, Majid; Jeddi-Tehrani, Mahmood; Rabbani, Hodjattallah

    2010-04-01

    We have employed a peptide-based antibody generation protocol for producing antibody against human nestin. Using a 12-mer synthetic peptide from repetitive region of human nestin protein devoid of any N- or O-glyco-sylation sequences, we generated a mouse monoclonal antibody capable of recognizing human, mouse, bovine, and rat nestin. A wide variety of nestin proteins ranging from 140-250 kDa was detected by this antibody. This antibody is highly specific and functional in applications such as ELISA, flow cytometry, immunocytochemistry, and Western blot assays. PMID:23407796

  7. Production of Monoclonal Antibody against Human Nestin

    OpenAIRE

    Hadavi, Reza; Zarnani, Amir Hassan; Ahmadvand, Negah; Mahmoudi, Ahmad Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Sadeghi, Mohammad-Reza; Soltanghoraee, Haleh; Akhondi, Mohammad mehdi; Tarahomi, Majid; Jeddi-Tehrani, Mahmood; Rabbani, Hodjattallah

    2010-01-01

    We have employed a peptide-based antibody generation protocol for producing antibody against human nestin. Using a 12-mer synthetic peptide from repetitive region of human nestin protein devoid of any N- or O-glyco-sylation sequences, we generated a mouse monoclonal antibody capable of recognizing human, mouse, bovine, and rat nestin. A wide variety of nestin proteins ranging from 140–250 kDa was detected by this antibody. This antibody is highly specific and functional in applications such a...

  8. Occurrences of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in sheep from four districts of Tocantins state, Brazilian Legal Amazon Region

    Directory of Open Access Journals (Sweden)

    Andresa Guimarães

    2015-02-01

    Full Text Available Toxoplasmosis and neosporosis have been recognized as economically important diseases with considerable impact on the livestock industry. Little is known concerning the occurrence of Toxoplasma gondii and Neospora caninum in sheep from Tocantins state, Brazil. Here, we investigated antibodies against these parasites and associated factors in 182 sheep from Araguaína, Santa Terezinha do Tocantins, Arguianópolis and Palmeiras do Tocantins districts, Tocantins. Sheep sera were assayed for T. gondii and N. caninum IgG antibodies by indirect fluorescence antibody test (IFAT, using cut-off point at a dilution of 1:40 and 1:25 respectively. The prevalence of seropositive animal for T. gondii was 13.74% and 13.74% for N. caninum. None of the characteristics studied including reproductive problems, presence of cats, presence of dogs and veterinary care (p>0.05 was associated with occurrence of T. gondii or N. caninum infection. Only breed was identified as associated factor for the occurrence of toxoplasmosis in sheep (p<0.05. The present study is the first report on serum occurrence of T. gondii and N. caninum in sheep from the state of Tocantins, Brazil.

  9. A cross-sectional study to estimate the frequency of anti-bovine viral diarrhea virus-1 antibodies in domestic pigs of Mossoró region in the state of Rio Grande do Norte, Brazil

    Directory of Open Access Journals (Sweden)

    Igor Renan Honorato Gatto

    2016-01-01

    Full Text Available ABSTRACT: This study investigated the occurrence of antibodies for BVDV-1 in swine herds located in the region of Mossoró city of the state of Rio Grande do Norte, Brazil. A sample size of 412 animals was estimated assuming unknown prevalence (set at 50%. Virus neutralization assay was used to the detect the presence of antibodies for BVDV-1 and the results found were analysed using multivariable logistic regression model. The obtained prevalence was 4% at animal level and 45% at the animal and herd level. The titers were highly variable between animals and within farms. The multivariable logistic regression analysis showed an association between being housed outside and exposure to BVDV-1 (OR=0.24, 95% CI:0.06, 0.96, P=0.04. Highly correlated data and low prevalence of antibodies at the animal level resulted in insufficient power to detect significant differences with other selected risk factors. In conclusion, the prevalence is within the range reported for other countries.

  10. Monoclonal antibodies

    International Nuclear Information System (INIS)

    Monoclonal antibodies (MAbs) are antibodies having single specificity for a given antigen site (epitope). The development of hybridoma technology and the relative ease by which MAbs can be prepared has revolutionized many aspects of serological applications in diagnosis and differentiation of disease producing agents. The property of monospecificity offers advantages in diagnostic applications over polyclonal sera in that tests can be defined exactly with regard to the antigen detected and the affinity of reaction between the given antigenic site and the monoclonal reagent. In addition, MAbs offer better possibilities for test standardization, because the same reagent can be used in different laboratories. Such an MAb can be supplied by a central laboratory or 'grown' from hybridoma cells, ensuring that the resultant product is identical from laboratory to laboratory and that the part of the test involving the MAb reaction is the same. The methodologies for inoculation regimes, mice, cloning methods, selection of fusion partners, etc., have been validated extensively in developed country laboratories. The decision to establish a MAb production facility must be examined on a strict cost-benefit basis, since it is still expensive to produce a product. There are many MAbs available that should be sought to allow exploitation in developing tests. If a production facility is envisaged, it should produce reagents for national needs, i.e. there should be a clear problem oriented approach whereby exact needs are defined. In the field of veterinary applications, MAbs are the central reagent in many immunoassays based on the enzyme linked immunosorbent assay (ELISA). The development of specific tests for diagnosing diseases is dominated by MAbs and has been fuelled by a strong research base, mainly in developed countries allied to developing countries through the study of related diseases. Thus, there are very many assays dependent on MAbs, some of which form the basis of

  11. Presence of anti-Leishmania (Viannia braziliensis antibodies in blood donors in the West-Central region of the State of Paraná, Brazil

    Directory of Open Access Journals (Sweden)

    Lais de Souza Braga

    2015-10-01

    Full Text Available ABSTRACTINTRODUCTION:Serological screening in blood banks does not include all transmittable diseases. American cutaneous leishmaniasis (ACL has a high detection rate in the municipalities of the State of Paraná.METHODS:This study analyzed the presence of anti- Leishmania braziliensisantibodies in 176 blood donors who live in these endemic areas. The variables were analyzed with the χ2 test and Stata 9.1 software. RESULTS: Twenty (11.4% samples were positive for the presence of anti- L. braziliensisantibodies. CONCLUSIONS: The high percentage of donors with anti- Leishmania spp. antibodies indicates the need to study the risk of ACL transmission through blood donors.

  12. Monoclonal antibodies.

    Science.gov (United States)

    2009-01-01

    The ability to produce and exploit monoclonal antibodies (mAbs) has revolutionized many areas of biological sciences. The unique property of an mAb is that it is a single species of immunoglobulin (IG) molecule. This means that the specificity of the interaction of the paratopes on the IG, with the epitopes on an antigenic target, is the same on every molecule. This property can be used to great benefit in immunoassays to provide tests of defined specificity and sensitivity, which improve the possibilities of standardization. The performance of assays can often be determined relating the actual weight of antibody (hence the number of molecules) to the activity. Often the production of an mAb against a specific epitope is the only way that biological entities can be differentiated. This chapter outlines the areas involving the development of assays based on mAbs. The problems involved address include the physical aspects of mAbs and how they may affect assay design and also the implications of results based on monospecific reagents. Often these are not fully understood, leading to assays that are less than satisfactory, which does not justify the relatively high cost of preparing and screening of mAbs. There are many textbooks and reviews dealing with the preparation of mAbs, the principles involved, and various purification and manipulative methods for the preparation of fragments and conjugation. There has been little general information attempting to summarize the best approaches to assay design using mAbs. Much time can be wasted through bad planning, and this is particularly relevant to mAbs. A proper understanding of some basic principles is essential. It is beyond the scope of this chapter to discuss all aspects, but major areas are highlighted. PMID:19219589

  13. Tabhu: tools for antibody humanization.

    KAUST Repository

    Olimpieri, Pier Paolo

    2014-10-09

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

  14. Plasmodium falciparum: an epitope within a highly conserved region of the 47-kDa amino-terminal domain of the serine repeat antigen is a target of parasite-inhibitory antibodies.

    Science.gov (United States)

    Fox, B A; Xing-Li, P; Suzue, K; Horii, T; Bzik, D J

    1997-02-01

    Previously, the Plasmodium falciparum serine repeat antigen has been shown to be protective in primate models of malaria immunity and also to be a target of in vitro parasite-inhibitory antibodies. To further define parasite-inhibitory epitopes a series of deletions from the amino-terminal 47-kDa domain of the serine repeat antigen (SERA) were constructed as glutathione-S-transferase fusion proteins. Several GST-SERA fusion proteins were used to vaccinate mice with Freund's adjuvant and the resulting immune sera were used to assay for the inhibition of P. falciparum invasion of erythrocytes in vitro. The minimal epitope shown to be the target of invasion-blocking antibodies was SERA amino acids 17-165. Additional GST-SERA deletion constructs of the 47-kDa domain were developed and evaluated for reactivity, by Western immunoblot analysis, with a parasite-inhibitory murine monoclonal antibody (mAb 43E5), a parasite-inhibitory pooled goat polyclonal sera, and a pooled human Nigerian immune serum. The parasite-inhibitory epitope defined by mAb 43E5 was mapped to SERA amino acids 17-110 and, at least, part of the epitope was defined to include amino acids in the region of amino acids 59-72. The parasite-inhibitory epitope recognized by mAb 43E5 appears to be well conserved between diverse geographical isolates of P. falciparum. The results have relevance for malaria vaccine development and suggest that an appropriately designed recombinant SERA antigen produced from a synthetic gene in Escherichia coli may be an effective component of a candidate malaria vaccine. PMID:9030663

  15. Synonymous deoptimization of the foot-and-mouth disease virus P1 coding region causes attenuation in vivo while inducing a strong neutralizing antibody response

    Science.gov (United States)

    Codon bias deoptimization has been previously used to successfully attenuate human pathogens including polio, respiratory syncytial and influenza viruses. We have applied a similar technology to deoptimize the capsid coding region (P1 region) of the cDNA infectious clone of foot-and-mouth disease vi...

  16. 南阳地区40001例患者红细胞血型不规则抗体检测分析%Irregular antibodies of erythrocyte blood group of 40 001 cases in Nanyang region

    Institute of Scientific and Technical Information of China (English)

    徐学新; 韩海心; 杨讯

    2013-01-01

    目的 了解南阳地区患者红细胞血型不规则抗体的发生频率及特异性抗体的分布特点.方法选取就诊的患者40 001(男13 141,女26 860,男∶女=0.489),用卡式微柱凝胶法进行红细胞血型不规则抗体筛查;对检出抗体阳性者鉴定其特异性及Ig类型.结果 40 001例患者中检出红细胞血型不规则抗体者347例(0.87%),其中男68/13 141例(0.52%)、女279/26 860例(1.04%),差异有统计学意义(P<0.05).3960例孕妇检出红细胞血型不规则抗体73例(1.84%).检出较多的特异性抗体依次为抗-M、抗-JK、抗-Lea、抗-P1、抗-Fyb、抗-Leb等.IgM类抗体183例、IgG类抗体74例、IgM+IgG类抗体28例,37℃反应阳性者62例(0.15%,62/40 001).结论 南阳地区患者红细胞血型不规则抗体发生频率和分布特点与其他地区不同,检出率较高,提示对于拟输血者特别是孕妇应例行红细胞血型不规则抗体检测.%Objective To study the incidence and distribution characteristics of irregular antibodies of erythrocyte blood group in patients of Nanyang region, Henan province. Methods 40 001 patients ( male 13 141, female 26 860 ) were detected the erythrocyte irregular antibodies by microcolum gel Coombs test. The positive patients were further identified their antibody specificity, Ig type. Results 347 positives cases( 0. 87% ), included 68 male cases ( 0. 52% ) and 279 female cases ( 1.04% ) were detected. 73 out of 3960 pregnant women ( 1. 84% ); There were 183 cases of IgM type, 74 cases of IgG, 28 cases of IgM + IgG, 62 cases of 37 ℃ positive reaction. The incidence of the specificity type from high to low were anti-M, an-ti-JK,anti-Lea,anti-PI and anti-Fyb and anti-Leb. Conclusion The incidence of erythrocyte irregular antibodies and the specificity type in Nanyang region were different from other regions and countries. This study supported to routine check erythrocyte irregular antibodies for the patients who plan a blood transfusion.

  17. Vaccine-induced Human Antibodies Specific for the Third Variable Region of HIV-1 gp120 Impose Immune Pressure on Infecting Viruses

    Directory of Open Access Journals (Sweden)

    Susan Zolla-Pazner

    2014-11-01

    Full Text Available To evaluate the role of V3-specific IgG antibodies (Abs in the RV144 clinical HIV vaccine trial, which reduced HIV-1 infection by 31.2%, the anti-V3 Ab response was assessed. Vaccinees' V3 Abs were highly cross-reactive with cyclic V3 peptides (cV3s from diverse virus subtypes. Sieve analysis of CRF01_AE breakthrough viruses from 43 vaccine- and 66 placebo-recipients demonstrated an estimated vaccine efficacy of 85% against viruses with amino acids mismatching the vaccine at V3 site 317 (p = 0.004 and 52% against viruses matching the vaccine at V3 site 307 (p = 0.004. This analysis was supported by data showing that vaccinees' plasma Abs were less reactive with I307 when replaced with residues found more often in vaccinees' breakthrough viruses. Simultaneously, viruses with mutations at F317 were less infectious, possibly due to the contribution of F317 to optimal formation of the V3 hydrophobic core. These data suggest that RV144-induced V3-specific Abs imposed immune pressure on infecting viruses and inform efforts to design an HIV vaccine.

  18. Molecular-specific urokinase antibodies

    Science.gov (United States)

    Atassi, M. Zouhair (Inventor); Morrison, Dennis R. (Inventor)

    2009-01-01

    Antibodies have been developed against the different molecular forms of urokinase using synthetic peptides as immunogens. The peptides were synthesized specifically to represent those regions of the urokinase molecules which are exposed in the three-dimensional configuration of the molecule and are uniquely homologous to urokinase. Antibodies are directed against the lysine 158-isoleucine 159 peptide bond which is cleaved during activation from the single-chain (ScuPA) form to the bioactive double chain (54 KDa and 33 KDa) forms of urokinase and against the lysine 135 lysine 136 bond that is cleaved in the process of removing the alpha-chain from the 54 KDa form to produce the 33 KDa form of urokinase. These antibodies enable the direct measurement of the different molecular forms of urokinase from small samples of conditioned medium harvested from cell cultures.

  19. Antibodies and Selection of Monoclonal Antibodies.

    Science.gov (United States)

    Hanack, Katja; Messerschmidt, Katrin; Listek, Martin

    2016-01-01

    Monoclonal antibodies are universal binding molecules with a high specificity for their target and are indispensable tools in research, diagnostics and therapy. The biotechnological generation of monoclonal antibodies was enabled by the hybridoma technology published in 1975 by Köhler and Milstein. Today monoclonal antibodies are used in a variety of applications as flow cytometry, magnetic cell sorting, immunoassays or therapeutic approaches. First step of the generation process is the immunization of the organism with appropriate antigen. After a positive immune response the spleen cells are isolated and fused with myeloma cells in order to generate stable, long-living antibody-producing cell lines - hybridoma cells. In the subsequent identification step the culture supernatants of all hybridoma cells are screened weekly for the production of the antibody of interest. Hybridoma cells producing the antibody of interest are cloned by limited dilution till a monoclonal hybridoma is found. This is a very time-consuming and laborious process and therefore different selection strategies were developed since 1975 in order to facilitate the generation of monoclonal antibodies. Apart from common automation of pipetting processes and ELISA testing there are some promising approaches to select the right monoclonal antibody very early in the process to reduce time and effort of the generation. In this chapter different selection strategies for antibody-producing hybridoma cells are presented and analysed regarding to their benefits compared to conventional limited dilution technology. PMID:27236550

  20. Several regions in the major histocompatibility complex confer risk for anti-CCP-antibody positive rheumatoid arthritis, independent of the DRB1 locus.

    Science.gov (United States)

    Lee, Hye-Soon; Lee, Annette T; Criswell, Lindsey A; Seldin, Michael F; Amos, Christopher I; Carulli, John P; Navarrete, Cristina; Remmers, Elaine F; Kastner, Daniel L; Plenge, Robert M; Li, Wentian; Gregersen, Peter K

    2008-01-01

    Recent evidence suggests that additional risk loci for RA are present in the major histocompatibility complex (MHC), independent of the class II HLA-DRB1 locus. We have now tested a total of 1,769 SNPs across 7.5Mb of the MHC located from 6p22.2 (26.03 Mb) to 6p21.32 (33.59 Mb) derived from the Illumina 550K Beadchip (Illumina, San Diego, CA, USA). For an initial analysis in the whole dataset (869 RA CCP + cases, 1,193 controls), the strongest association signal was observed in markers near the HLA-DRB1 locus, with additional evidence for association extending out into the Class I HLA region. To avoid confounding that may arise due to linkage disequilibrium with DRB1 alleles, we analyzed a subset of the data by matching cases and controls by DRB1 genotype (both alleles matched 1:1), yielding a set of 372 cases with 372 controls. This analysis revealed the presence of at least two regions of association with RA in the Class I region, independent of DRB1 genotype. SNP alleles found on the conserved A1-B8-DR3 (8.1) haplotype show the strongest evidence of positive association (P ~ 0.00005) clustered in the region around the HLA-C locus. In addition, we identified risk alleles that are not present on the 8.1 haplotype, with maximal association signals (P ~ 0.001-0.0027) located near the ZNF311 locus. This latter association is enriched in DRB1*0404 individuals. Finally, several additional association signals were found in the extreme centromeric portion of the MHC, in regions containing the DOB1, TAP2, DPB1, and COL11A2 genes. These data emphasize that further analysis of the MHC is likely to reveal genetic risk factors for rheumatoid arthritis that are independent of the DRB1 shared epitope alleles. PMID:18309376

  1. ANTI DEOXYRIBONUCLEIC ACID AND ANTINUCLEAR ANTIGEN ANTIBODIES IN GRAVES’ DISEASE

    OpenAIRE

    H. Mostafavi

    2005-01-01

    Graves’ disease is an autoimmune disorder characterized by presence of antibodies directed against thyroid stimulating hormone (TSH) receptor or nearby region. Other serological abnormalities like antibodies to double stranded DNA (ds–DNA) and antinuclear antibodies (ANA) have also been observed. We studied antibodies to ds-DNA and ANA in our patients with Graves’ disease and compared them with control group. Sera of 84 patients (29 males, 55 females) with diagnosis of Graves’ disease were pr...

  2. Monoclonal antibodies as diagnostics; an appraisal

    Directory of Open Access Journals (Sweden)

    Siddiqui M

    2010-01-01

    Full Text Available Ever since the development of Hybridoma Technology in 1975 by Kohler and Milstein, our vision for antibodies as tools for research for prevention, detection and treatment of diseases, vaccine production, antigenic characterization of pathogens and in the study of genetic regulation of immune responses and disease susceptibility has been revolutionized. The monoclonal antibodies being directed against single epitopes are homogeneous, highly specific and can be produced in unlimited quantities. In animal disease diagnosis, they are very useful for identification and antigenic characterization of pathogens. Monoclonal antibodies have tremendous applications in the field of diagnostics, therapeutics and targeted drug delivery systems, not only for infectious diseases caused by bacteria, viruses and protozoa but also for cancer, metabolic and hormonal disorders. They are also used in the diagnosis of lymphoid and myeloid malignancies, tissue typing, enzyme linked immunosorbent assay, radio immunoassay, serotyping of microorganisms, immunological intervention with passive antibody, antiidiotype inhibition, or magic bullet therapy with cytotoxic agents coupled with anti mouse specific antibody. Recombinant deoxyribonucleic acid technology through genetic engineering has successfully led to the possibility of reconstruction of monoclonal antibodies viz. chimeric antibodies, humanized antibodies and complementarily determining region grafted antibodies and their enormous therapeutic use.

  3. The roles of APE1, APE2, DNA polymerase β and mismatch repair in creating S region DNA breaks during antibody class switch

    OpenAIRE

    Schrader, Carol E.; Guikema, Jeroen E.J.; Wu, Xiaoming; Stavnezer, Janet

    2008-01-01

    Immunoglobulin class switch recombination (CSR) occurs by an intrachromosomal deletion requiring generation of double-stranded DNA breaks (DSBs) in immunoglobulin switch region DNA. The initial steps of DSB formation have been elucidated: cytosine deamination by activation-induced cytidine deaminase (AID) and the generation of abasic sites by uracil-DNA glycosylase (UNG). We show that abasic sites are converted into single-strand breaks (SSBs) by apurinic/apyrimidinic endonucleases (APE1 and ...

  4. Antiphospholipid Antibody Syndrome

    Science.gov (United States)

    ... page from the NHLBI on Twitter. What Is Antiphospholipid Antibody Syndrome? Antiphospholipid (AN-te-fos-fo-LIP-id) antibody ... weeks or months. This condition is called catastrophic antiphospholipid syndrome (CAPS). People who have APS also are at ...

  5. The antibody mining toolbox

    OpenAIRE

    D'Angelo, Sara; Glanville, Jacob; Ferrara, Fortunato; Naranjo, Leslie; Gleasner, Cheryl D.; Shen, Xiaohong; Bradbury, Andrew RM; Kiss, Csaba

    2013-01-01

    In vitro selection has been an essential tool in the development of recombinant antibodies against various antigen targets. Deep sequencing has recently been gaining ground as an alternative and valuable method to analyze such antibody selections. The analysis provides a novel and extremely detailed view of selected antibody populations, and allows the identification of specific antibodies using only sequencing data, potentially eliminating the need for expensive and laborious low-throughput ...

  6. Heavy chain only antibodies

    DEFF Research Database (Denmark)

    Moghimi, Seyed Moein; Rahbarizadeh, Fatemeh; Ahmadvand, Davoud;

    2013-01-01

    Unlike conventional antibodies, heavy chain only antibodies derived from camel contain a single variable domain (VHH) and two constant domains (CH2 and CH3). Cloned and isolated VHHs possess unique properties that enable them to excel conventional therapeutic antibodies and their smaller antigen...

  7. Hepatitis A virus antibody

    International Nuclear Information System (INIS)

    A description is presented of a radioimmunoassay designed to prove the presence of the antibody against the hepatitis A virus (HA Ab, anti-Ha) using an Abbott HAVAB set. This proof as well as the proof of the antibody against the nucleus of the hepatitis B virus is based on competition between a normal antibody against hepatitis A virus and a 125I-labelled antibody for the binding sites of a specific antigen spread all over the surface of a tiny ball; this is then indirect proof of the antibody under investigation. The method is described of reading the results from the number of impulses per 60 seconds: the higher the titre of the antibody against the hepatitis A virus in the serum examined, the lower the activity of the specimen concerned. The rate is reported of incidence of the antibody against the hepatitis A virus in a total of 68 convalescents after hepatitis A; the antibody was found in 94.1%. The immunoglobulin made from the convalescents' plasma showed the presence of antibodies in dilutions as high as 1:250 000 while the comparable ratio for normal immunoglobulin Norga was only 1:2500. Differences are discussed in the time incidence of the antibodies against the hepatitis A virus, the antibodies against the surface antigen of hepatitis B, and the antibody against the nucleus of the hepatitis V virus. (author)

  8. Monoclonal antibodies and cancer

    International Nuclear Information System (INIS)

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

  9. Risk factor analysis for antibodies to Brucella, Leptospira and C. burnetii among cattle in the Adamawa Region of Cameroon: a cross-sectional study.

    Science.gov (United States)

    Mazeri, Stella; Scolamacchia, Francesca; Handel, Ian G; Morgan, Kenton L; Tanya, Vincent N; Bronsvoort, Barend M deC

    2013-02-01

    Brucellosis, leptospirosis and Q fever are important livestock diseases, commonly responsible for significant production losses, yet their epidemiology in sub-Saharan Africa is largely unknown. Animal reservoirs pose the main risk of transmission to humans, where serious disease can occur. In the developing world setting, the flu-like symptoms of the acute stages of these diseases can be misdiagnosed as malaria, which can result in the administration of the wrong treatment, prolonged disease and increase in antibiotic resistance. Multivariable mixed-effects logistic regression models in this study revealed potential risk factors associated with the aforementioned pathogens in cattle in the Adamawa Region of Cameroon, with wildlife, namely, buffaloes, playing a major role in both Brucella and Coxiella burnetii seropositivity. Cattle mixing with other herds at night and cattle grazing in an area on a route taken by herds on transhumance appear to be positively associated with Leptospira seropositivity, while female cows and whether buffaloes are seen during grazing or transhumance are positively associated with C. burnetii seropositivity. On the other hand, animals that have been on transhumance in the past year and animals belonging to herdsmen of the Fulbe ethnic group appear to be protected against Leptospira and C. burnetii, respectively. Cattle of more than 2 years old appear to have increased odds of being seropositive to either pathogen. Further research is needed to confirm these findings and improve the knowledge of the epidemiology of these three pathogens in Africa, taking particular consideration of the wildlife involvement in the disease transmission. PMID:23117621

  10. Uses of monoclonial antibody 8H9

    Energy Technology Data Exchange (ETDEWEB)

    Cheung, Nai-Kong V.

    2015-06-23

    This invention provides an antibody that binds the same antigen as that of monoclonal antibody 8H9, wherein the heavy chain CDR (Complementary Determining Region)1 comprises NYDIN, heavy chain CDR2 comprises WIFPGDGSTQY, heavy chain CDR3 comprises QTTATWFAY, and the light chain CDR1 comprises RASQSISDYLH, light chain CDR2 comprises YASQSIS, and light chain CDR3 comprises QNGHSFPLT. In another embodiment, there is provided a polypeptide that binds the same antigen as that of monoclonal antibody 8H9, wherein the polypeptide comprises NYDIN, WIFPGDGSTQY, QTTATWFAY, RASQSISDYLH, YASQSIS, and QNGHSFPLT.

  11. IgM Repertoire Biodiversity is Reduced in HIV-1 Infection and Systemic Lupus Erythematosus

    OpenAIRE

    Yin, Li; Hou, Wei; Liu, Li; Cai, Yunpeng; Wallet, Mark Andrew; Gardner, Brent Paul; Chang, Kaifen; Lowe, Amanda Catherine; Rodriguez, Carina Adriana; Sriaroon, Panida; Farmerie, William George; Sleasman, John William; Goodenow, Maureen Michels

    2013-01-01

    Background: HIV-1 infection or systemic lupus erythematosus (SLE) disrupt B cell homeostasis, reduce memory B cells, and impair function of IgG and IgM antibodies. Objective: To determine how disturbances in B cell populations producing polyclonal antibodies relate to the IgM repertoire, the IgM transcriptome in health and disease was explored at the complementarity determining region 3 (CDRH3) sequence level. Methods: 454-deep pyrosequencing in combination with a novel analysis pipelin...

  12. Antissaliva Antibodies of Lutzomyia Longipalpis in area of Visceral Leishmaniasis.

    Science.gov (United States)

    Fraga, Thiago Leite; Fernandes, Magda Freitas; Pontes, Elenir Rose Jardim Cury; Levay, Ana Paula Silva; Almeida da Cunha, Elenice Brandão; França, Adriana de Oliveira; Dorval, Maria Elizabeth Cavalheiros

    2016-07-01

    The aim of the present study was to assess the presence of antissaliva antibodies of Lutzomyia longipalpis in human hosts living in area of visceral leishmaniasis, located in the Center-West region of Brazil. The presence of antissaliva antibodies of L. longipalpis exhibited a strong correlation with the protection and development of antibodies against Leishmania sp. Of the 492 children studied, elevated antissaliva antibodies of L. longipalpis were detected in 38.4% of the participants. There was a higher percentage of positivity (64.7%) among children who exhibited anti-Leishmania sp. antibodies and among those who were positive in the delayed hypersensitivity test (34.8%). PMID:27093167

  13. The second century of the antibody. Molecular perspectives in regulation, pathophysiology, and therapeutic applications.

    OpenAIRE

    Braun, J; Saxon, A; Wall, R; Morrison, S. L.

    1992-01-01

    The modern age of immunology began in 1890 with the discovery of antibodies as a major component of protective immunity. The 2nd century of the antibody begins with a focus on the molecular physiology and pathophysiology of immunoglobulin production. Numerous human variable-region antibody genes have been identified through advances in molecular cloning and anti-variable-region monoclonal antibodies. Some of these variable-region genes are now known to be involved in specific stages of B-lymp...

  14. The Chikungunya Virus Capsid Protein Contains Linear B Cell Epitopes in the N- and C-Terminal Regions that are Dependent on an Intact C-Terminus for Antibody Recognition

    Directory of Open Access Journals (Sweden)

    Lucas Y. H. Goh

    2015-06-01

    Full Text Available Chikungunya virus (CHIKV is an arthropod-borne agent that causes severe arthritic disease in humans and is considered a serious health threat in areas where competent mosquito vectors are prevalent. CHIKV has recently been responsible for several millions of cases of disease, involving over 40 countries. The recent re-emergence of CHIKV and its potential threat to human health has stimulated interest in better understanding of the biology and pathogenesis of the virus, and requirement for improved treatment, prevention and control measures. In this study, we mapped the binding sites of a panel of eleven monoclonal antibodies (mAbs previously generated towards the capsid protein (CP of CHIKV. Using N- and C-terminally truncated recombinant forms of the CHIKV CP, two putative binding regions, between residues 1–35 and 140–210, were identified. Competitive binding also revealed that five of the CP-specific mAbs recognized a series of overlapping epitopes in the latter domain. We also identified a smaller, N-terminally truncated product of native CP that may represent an alternative translation product of the CHIKV 26S RNA and have potential functional significance during CHIKV replication. Our data also provides evidence that the C-terminus of CP is required for authentic antigenic structure of CP. This study shows that these anti-CP mAbs will be valuable research tools for further investigating the structure and function of the CHIKV CP.

  15. Antibodies Against Three Forms of Urokinase

    Science.gov (United States)

    Morrison, Dennis R.; Atassi, M. Zouhair

    2007-01-01

    Antibodies that bind to preselected regions of the urokinase molecule have been developed. These antibodies can be used to measure small quantities of each of three molecular forms of urokinase that could be contained in microsamples or conditioned media harvested from cultures of mammalian cells. Previously available antibodies and assay techniques do not yield both clear distinctions among, and measurements of, all three forms. Urokinase is a zymogen that is synthesized in a single-chain form, called ScuPA, which is composed of 411 amino acid residues (see figure). ScuPA has very little enzyme activity, but it can be activated in two ways: (1) by cleavage of the peptide bond lysine 158/isoleucine 159 and the loss of lysine 158 to obtain the high molecular-weight (HMW) form of the enzyme or (2) by cleavage of the bond lysine 135/lysine 136 to obtain the low-molecular-weight (LMW) form of the enzyme. The antibodies in question were produced in mice and rabbits by use of peptides as immunogens. The peptides were selected to obtain antibodies that bind to regions of ScuPA that include the lysine 158/isoleucine 159 and the lysine 135/lysine 136 bonds. The antibodies include monoclonal and polyclonal ones that yield indications as to whether either of these bonds is intact. The polyclonal antibodies include ones that preferentially bind to the HMW or LMW forms of the urokinase molecule. The monoclonal antibodies include ones that discriminate between the ScuPA and the HMW form. A combination of these molecular-specific antibodies will enable simultaneous assays of the ScuPA, HMW, and LMW forms in the same specimen of culture medium.

  16. ANTI DEOXYRIBONUCLEIC ACID AND ANTINUCLEAR ANTIGEN ANTIBODIES IN GRAVES’ DISEASE

    Directory of Open Access Journals (Sweden)

    H. Mostafavi

    2005-05-01

    Full Text Available Graves’ disease is an autoimmune disorder characterized by presence of antibodies directed against thyroid stimulating hormone (TSH receptor or nearby region. Other serological abnormalities like antibodies to double stranded DNA (ds–DNA and antinuclear antibodies (ANA have also been observed. We studied antibodies to ds-DNA and ANA in our patients with Graves’ disease and compared them with control group. Sera of 84 patients (29 males, 55 females with diagnosis of Graves’ disease were prepared and level of antibodies to ds-DNA and ANA were measured and compared with 41 healthy persons (15 males, 26 females. The level of antibodies to ds-DNA and ANA in patients and control group did not show any significant difference. Our results were different from other studies in other countries. The difference may be explained by difference in our method of antibody measurement or genetic background which needs to be confirmed by HLA studies of our population.

  17. Affinity purification of antibodies

    Science.gov (United States)

    Antibodies are provided in a variety of formats that includes antiserum, hybridoma culture supernatant or ascites. They can all be used successfully in crude form for the detection of target antigens by immunoassay. However, it is advantageous to use purified antibody in defined quantity to facil...

  18. Therapeutic Recombinant Monoclonal Antibodies

    Science.gov (United States)

    Bakhtiar, Ray

    2012-01-01

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

  19. Production Of Human Antibodies

    Science.gov (United States)

    Sammons, David W.; Neil, Garry A.

    1993-01-01

    Process for making human monoclonal antibodies based on combination of techniques. Antibodies made active against specific antigen. Process involves in vivo immunization of human B lymphocyte cells in mice. B cells of interest enriched in vitro before fusion. Method potentially applicable to any antigen. Does not rely on use of Epstein-Barr virus at any step. Human lymphocytes taken from any source.

  20. RBC Antibody Screen

    Science.gov (United States)

    ... the baby is Rh-positive and the mother's antibody status is negative for anti-D, the mother is given additional RhIG. This test also may be used to help diagnose autoimmune-related hemolytic anemia ... when a person produces antibodies against his or her own RBC antigens. This ...

  1. Antibody affinity maturation

    DEFF Research Database (Denmark)

    Skjødt, Mette Louise

    Yeast surface display is an effective tool for antibody affinity maturation because yeast can be used as an all-in-one workhorse to assemble, display and screen diversified antibody libraries. By employing the natural ability of yeast Saccharomyces cerevisiae to efficiently recombine multiple DNA...

  2. Enhancing antibody: a novel component of the immune response.

    OpenAIRE

    Nemazee, D A; Sato, V L

    1982-01-01

    Current descriptions of the immune response identify two classes of antigenic stimuli that result in the production of specific antibody: (i) exogenous antigens and (ii) endogenous variable-region determinants of the immune system. We expand this scheme to include a third class of antigenic stimulus--new determinants created by the binding of antibody to antigen. This paper describes a set of monoclonal antibodies which arose after repeated immunization with antigen alone but which bound anti...

  3. Construction of a human antibody domain (VH) library

    OpenAIRE

    Chen, Weizao; Zhu, Zhongyu; Xiao, Xiaodong; Dimitrov, Dimiter S.

    2009-01-01

    Highly diverse antibody (Fab or scFv) libraries have become vital sources to select antibodies with high affinity and novel properties. Combinatorial strategies provide efficient ways of creating antibody libraries containing a large number of individual clones. These strategies include the reassembly of naturally occurring genes encoding the heavy and light chains from either immune or nonimmune B-cell sources, or introduction of synthetic diversity to either the framework regions (FRs) or t...

  4. Anti-collagen antibodies in sera from rheumatoid arthritis patients.

    OpenAIRE

    Beard, H K; Ryvar, R; Skingle, J; Greenbury, C. L.

    1980-01-01

    Anti-cartilage antibodies, demonstrable by immunofluorescence, were found in 3.3% of rheumatoid arthritis patients. In most of these patients antibodies to type II collagen were detected. In specificity studies on these anti-collagen antibodies, they appeared to be type specific, showing no reaction with collagen types I and III. Denatured type II collagen reacted much less well than native type II, but isolated peptides from different regions of the collagen molecule were differentiated by i...

  5. Selection of Recombinant Human Antibodies.

    Science.gov (United States)

    Tomszak, Florian; Weber, Susanne; Zantow, Jonas; Schirrmann, Thomas; Hust, Michael; Frenzel, André

    2016-01-01

    Since the development of therapeutic antibodies the demand of recombinant human antibodies is steadily increasing. Traditionally, therapeutic antibodies were generated by immunization of rat or mice, the generation of hybridoma clones, cloning of the antibody genes and subsequent humanization and engineering of the lead candidates. In the last few years, techniques were developed that use transgenic animals with a human antibody gene repertoire. Here, modern recombinant DNA technologies can be combined with well established immunization and hybridoma technologies to generate already affinity maturated human antibodies. An alternative are in vitro technologies which enabled the generation of fully human antibodies from antibody gene libraries that even exceed the human antibody repertoire. Specific antibodies can be isolated from these libraries in a very short time and therefore reduce the development time of an antibody drug at a very early stage.In this review, we describe different technologies that are currently used for the in vitro and in vivo generation of human antibodies. PMID:27236551

  6. Antibody discovery: sourcing of monoclonal antibody variable domains.

    Science.gov (United States)

    Strohl, William R

    2014-03-01

    Historically, antibody variable domains for therapeutic antibodies have been sourced primarily from the mouse IgG repertoire, and typically either chimerized or humanized. More recently, human antibodies from transgenic mice producing human IgG, phage display libraries, and directly from human B lymphocytes have been used more broadly as sources of antibody variable domains for therapeutic antibodies. Of the total 36 antibodies approved by major maket regulatory agencies, the variable domain sequences of 26 originate from the mouse. Of these, four are marketed as murine antibodies (of which one is a mouse-rat hybrid IgG antibody), six are mouse-human chimeric antibodies, and 16 are humanized. Ten marketed antibodies have originated from human antibody genes, three isolated from phage libraries of human antibody genes and seven from transgenic mice producing human antibodies. Five antibodies currently in clinical trials have been sourced from camelids, as well as two from non-human primates, one from rat, and one from rabbit. Additional sources of antibody variable domains that may soon find their way into the clinic are potential antibodies from sharks and chickens. Finally, the various methods for retrieval of antibodies from humans, mouse and other sources, including various display technologies and amplification directly from B cells, are described. PMID:24168292

  7. Radiolabelled antibodies in imaging

    International Nuclear Information System (INIS)

    Recent technological advances make it possible to produce pure (monoclonal) antibodies in unlimited quantities without the need for continuous immunization of animals and to label these antibodies with a variety of radionuclides which can be traced by single-photon computed tomography. An outline review of the state of the art is presented, with particular reference to the imaging of myocardial infarcts and to tumour imaging studies using labelled monoclonal antibodies (sup(99m)Tc and 125I). Lengthy bibliography. (U.K.)

  8. The INNs and outs of antibody nonproprietary names.

    Science.gov (United States)

    Jones, Tim D; Carter, Paul J; Plückthun, Andreas; Vásquez, Max; Holgate, Robert G E; Hötzel, Isidro; Popplewell, Andrew G; Parren, Paul W H I; Enzelberger, Markus; Rademaker, Hendrik J; Clark, Michael R; Lowe, David C; Dahiyat, Bassil I; Smith, Victoria; Lambert, John M; Wu, Herren; Reilly, Mary; Haurum, John S; Dübel, Stefan; Huston, James S; Schirrmann, Thomas; Janssen, Richard A J; Steegmaier, Martin; Gross, Jane A; Bradbury, Andrew R M; Burton, Dennis R; Dimitrov, Dimiter S; Chester, Kerry A; Glennie, Martin J; Davies, Julian; Walker, Adam; Martin, Steve; McCafferty, John; Baker, Matthew P

    2016-01-01

    An important step in drug development is the assignment of an International Nonproprietary Name (INN) by the World Health Organization (WHO) that provides healthcare professionals with a unique and universally available designated name to identify each pharmaceutical substance. Monoclonal antibody INNs comprise a -mab suffix preceded by a substem indicating the antibody type, e.g., chimeric (-xi-), humanized (-zu-), or human (-u-). The WHO publishes INN definitions that specify how new monoclonal antibody therapeutics are categorized and adapts the definitions to new technologies. However, rapid progress in antibody technologies has blurred the boundaries between existing antibody categories and created a burgeoning array of new antibody formats. Thus, revising the INN system for antibodies is akin to aiming for a rapidly moving target. The WHO recently revised INN definitions for antibodies now to be based on amino acid sequence identity. These new definitions, however, are critically flawed as they are ambiguous and go against decades of scientific literature. A key concern is the imposition of an arbitrary threshold for identity against human germline antibody variable region sequences. This leads to inconsistent classification of somatically mutated human antibodies, humanized antibodies as well as antibodies derived from semi-synthetic/synthetic libraries and transgenic animals. Such sequence-based classification implies clear functional distinction between categories (e.g., immunogenicity). However, there is no scientific evidence to support this. Dialog between the WHO INN Expert Group and key stakeholders is needed to develop a new INN system for antibodies and to avoid confusion and miscommunication between researchers and clinicians prescribing antibodies. PMID:26716992

  9. Anti-sulfotyrosine antibodies

    Science.gov (United States)

    Bertozzi, Carolyn R.; Kehoe, John; Bradbury, Andrew M.

    2009-09-15

    The invention provides anti-sulfotyrosine specific antibodies capable of detecting and isolating polypeptides that are tyrosine-sulfated. The sulfotyrosine antibodies and antibody fragments of the invention may be used to discriminate between the non-sulfated and sulfated forms of such proteins, using any number of immunological assays, such ELISAs, immunoblots, Western Blots, immunoprecipitations, and the like. Using a phage-display system, single chain antibodies (scFvs) were generated and screened against tyrosine-sulfated synthetic peptide antigens, resulting in the isolation of scFvs that specifically recognize sulfotyrosine-containing peptides and/or demonstrate sulfotyrosine-specific binding in tyrosine sulfated proteins. The VH and VL genes from one such sulfotyrosine-specific scFv were employed to generate a full length, sulfotyrosine-specific immunoglobulin.

  10. HIV Antibody Test

    Science.gov (United States)

    ... be limited. Home Visit Global Sites Search Help? HIV Antibody and HIV Antigen (p24) Share this page: Was this page helpful? Also known as: HIV Screening Tests; AIDS Test; AIDS Screen; HIV Serology; ...

  11. Antinuclear antibody panel

    Science.gov (United States)

    ... blood may be due to: Chronic liver disease Collagen vascular disease Drug-induced lupus erythematosus Myositis (inflammatory muscle disease) ... Saunders; 2011:chap 51. Read More Antibody Arthritis Collagen vascular disease Drug-induced lupus erythematosus Liver disease Scleroderma Systemic ...

  12. PRODUCTION OF MONOCLONAL ANTIBODIES

    Directory of Open Access Journals (Sweden)

    TOLKOVA E.S.

    2015-01-01

    Full Text Available The article considers the use of monoclonal antibodies in immunotherapy and immunodiagnostics of oncological diseases and their production using hybridoma technolody with flow diagram and technological scheme of manufacturing process

  13. PRODUCTION OF MONOCLONAL ANTIBODIES

    OpenAIRE

    TOLKOVA E.S.

    2015-01-01

    The article considers the use of monoclonal antibodies in immunotherapy and immunodiagnostics of oncological diseases and their production using hybridoma technolody with flow diagram and technological scheme of manufacturing process

  14. Expression of Recombinant Antibodies

    OpenAIRE

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transg...

  15. Suppression of contractile force in muscle fibers by antibody to myosin subfragment 2.

    OpenAIRE

    Lovell, S; Karr, T; Harrington, W F

    1988-01-01

    Polyclonal antibody directed against the subfragment-2 region of myosin was purified by affinity chromatography. Skinned muscle fibers that had been preincubated with antibody were able to sustain only 7% of the active isometric force generated by control fibers. The effect of antibody on force production could not be accounted for by inhibition of ATP turnover.

  16. New thrombopoietic growth factors

    OpenAIRE

    Kuter, David J.

    2007-01-01

    Although development of first-generation thrombopoietic growth factors (recombinant human thrombopoietin [TPO] and pegylated recombinant human megakaryocyte growth and development factor [PEG-rHuMGDF]) was stopped due to development of antibodies to PEG-rHuMGDF, nonimmunogenic second-generation thrombopoietic growth factors with unique pharmacologic properties have been developed. TPO peptide mimetics contain TPO receptor-activating peptides inserted into complementarity-determining regions o...

  17. Characterization of antibodies directed against the Ankrd2 human muscle protein

    Directory of Open Access Journals (Sweden)

    Kojić Snežana

    2009-01-01

    Full Text Available In order to study the function of the Ankrd2 protein, for which commercial antibodies are not available, we report the production and analysis of polyclonal antibodies to full-length Ankrd2 and its C-terminal and N-terminal regions, as well as a monoclonal antibody to the C-terminus of the protein. Epitope mapping making use of recombinant deletion mutants showed that an epitope located in region 323-333 aa of Ankrd2 is detected by the monoclonal antibody. The high specificity of all four anti-Ankrd2 antibodies for recombinant and endogenous Ankrd2 protein is also demon­strated.

  18. Epidemiology of myasthenia gravis with anti‐muscle specific kinase antibodies in the Netherlands

    OpenAIRE

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

    2006-01-01

    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 Netherlands. The nationwide prevalence and incidence of myasthenia gravis with anti‐MuSK antibodies were also studied. MuSK antibodies were found in 22% of patients with generalised myasthenia gra...

  19. Prediction of antibody persistency from antibody titres to natalizumab

    DEFF Research Database (Denmark)

    Jensen, Poul Erik H; Koch-Henriksen, Nils; Sellebjerg, Finn; Sørensen, Per S

    2012-01-01

    In a subgroup of patients with multiple sclerosis natalizumab therapy causes generation of anti-natalizumab antibodies that may be transient or persistent. It is recommended to discontinue natalizumab therapy in persistently antibody-positive patients.......In a subgroup of patients with multiple sclerosis natalizumab therapy causes generation of anti-natalizumab antibodies that may be transient or persistent. It is recommended to discontinue natalizumab therapy in persistently antibody-positive patients....

  20. Prediction of Antibody Epitopes

    DEFF Research Database (Denmark)

    Nielsen, Morten; Marcatili, Paolo

    2015-01-01

    self-proteins. Given the sequence or the structure of a protein of interest, several methods exploit such features to predict the residues that are more likely to be recognized by an immunoglobulin.Here, we present two methods (BepiPred and DiscoTope) to predict linear and discontinuous antibody...

  1. Monoclonal antibodies in myeloma

    DEFF Research Database (Denmark)

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

    2015-01-01

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

  2. Clonal relationships between thyroid-stimulating hormone receptor-stimulating antibodies illustrate the effect of hypermutation on antibody function

    DEFF Research Database (Denmark)

    Padoa, Carolyn J; Larsen, Sanne L; Hampe, Christiane S;

    2009-01-01

    Summary Graves' disease is characterized by production of agonist antibodies to the thyroid-stimulating hormone receptor (TSHR), but knowledge of the genetic and somatic events leading to their aberrant production is limited. We describe the genetic analysis of two monoclonal antibodies (mAbs) with......-determining regions (CDRs) and the framework regions. The cloned IGHV and IGLV genes were confirmed to have TSAb properties in experiments in which they were expressed as recombinant Fabs (rFabs). In other experiments, we swapped the IGLV genes with IGHV genes by constructing chimeric rFabs and showed that the...... experimentally immunized mice, multiple pathogenic antibodies to TSHR can arise from a single clone by a series of somatic mutations in the V-region genes and may give an insight into how such antibodies develop spontaneously in autoimmune Graves' disease....

  3. Red Blood Cell Antibody Identification

    Science.gov (United States)

    ... limited. Home Visit Global Sites Search Help? RBC Antibody Identification Share this page: Was this page helpful? Also known as: Alloantibody Identification; Antibody ID, RBC; RBC Ab ID Formal name: Red ...

  4. Anti-insulin antibody test

    Science.gov (United States)

    Insulin antibodies - serum; Insulin Ab test ... Normally, there are no antibodies against insulin in your blood. Normal value ranges may vary slightly among different laboratories. Some labs use different measurements or ...

  5. The Art of Making Antibodies.

    Science.gov (United States)

    Headon, Denis R.

    1986-01-01

    Provides background information for teachers on the nature and production of antibodies. Points out that the production of monoclonal antibodies blends the malignant with the beneficial to create a medical tool of exciting potential. (JN)

  6. Lupus anticoagulants and antiphospholipid antibodies

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/000547.htm Lupus anticoagulants and antiphospholipid antibodies To use the sharing features on this page, please enable JavaScript. Lupus anticoagulants are antibodies against substances in the lining ...

  7. Recombinant antibodies and tumor targeting

    OpenAIRE

    Sheikholvaezin, Ali

    2006-01-01

    Different antibody derived constructs are rapidly advancing as putative tools for treatment of malignant diseases. Antibody engineering has added significant new technologies to modify size, affinities, solubility, stability and biodistribution properties for immunoconjugates. In the present thesis, the aim was to increase our knowledge on how new recombinant antibodies could be tailored to optimize localization to experimental tumors in mice. One hybridoma, producing the monoclonal antibody ...

  8. Antibody Engineering and Therapeutics Conference

    OpenAIRE

    Larrick, James W; Parren, Paul WHI; Huston, James S; Plückthun, Andreas; Bradbury, Andrew; Tomlinson, Ian M; Chester, Kerry A.; Burton, Dennis R.; Adams, Gregory P; Weiner, Louis M.; Scott, Jamie K; Alfenito, Mark R; Veldman, Trudi; Reichert, Janice M

    2013-01-01

    The Antibody Engineering and Therapeutics conference, which serves as the annual meeting of The Antibody Society, will be held in Huntington Beach, CA from Sunday December 8 through Thursday December 12, 2013. The scientific program will cover the full spectrum of challenges in antibody research and development, and provide updates on recent progress in areas from basic science through approval of antibody therapeutics. Keynote presentations will be given by Leroy Hood (Institute of System Bi...

  9. Radiolabeled antibodies as imaging agents

    International Nuclear Information System (INIS)

    The author gives a survey of the progress made on radioimmunodetection. Antibodies may now be more readily used in scintigraphy as a result of the development of labeling methods that apply more suitable radionuclides without significant loss of the antigen-binding activity. Antibodies to tumor-specific or tumor-associated antigens can now be produced in large quantities by monoclonal antibody technology

  10. The investigation of poliomyelitis neutralized antibody level among preschool children in Ningxia hui autonomous region%宁夏2012年学龄前儿童脊髓灰质炎中和抗体水平调查

    Institute of Scientific and Technical Information of China (English)

    陈慧; 刘吉祥; 袁芳; 马江涛; 马学旻; 张颖; 周丽薇; 杨媛媛

    2015-01-01

    Objective To understand the neutralized antibody level of the poliomyelitis among preschool children and evaluate the effectiveness of routine immunization in Ningxia.Methods The poliomyelitis antibody were detected using neutralization test among preschool children of 12 counties in Ningxia.Results The positive rates of neutralized antibody were 98.70 %,98.48%,97.19% and GMT were 1 ∶ 350.28,1 ∶ 241.82,1 ∶ 153.94 for polio Ⅰ,Ⅱ,Ⅲ respectively in 462 serum specimens from 0-6 years children.There were no statistical significance difference of antibody positive rate in those counties.Conclusion The immune level of poliomyelitis among preschool children was hign in Ningxia,and the protective barrier had been built against polio virus.%目的 了解宁夏学龄前儿童脊髓灰质炎(脊灰)抗体水平,评价脊灰糖丸疫苗常规免疫效果.方法 用微量细胞中和试验法对12个县(区)的学龄前儿童进行脊灰中和抗体水平检测.结果 共检测462份0~6岁儿童的血清标本.脊灰Ⅰ、Ⅱ、Ⅲ型中和抗体阳性率分别为98.70%、98.48%和97.19%;抗体几何平均滴度(GMT)分别是1∶350.28、1∶241.82和1∶153.94.城乡、各县(区)间抗体阳性率差异无统计学意义.结论 宁夏学龄前儿童通过常规免疫,对脊灰病毒已形成免疫屏障.

  11. Antibody activity in ankylosing spondylitis sera to two sites on HLA B27.1 at the MHC groove region (within sequence 65-85), and to a Klebsiella pneumoniae nitrogenase reductase peptide (within sequence 181-199)

    OpenAIRE

    1990-01-01

    74 overlapping peptides of varying lengths from Klebsiella pneumoniae nitrogenase reductase (residues 181-199) and from the HLA B27.1 molecule (residues 65-85) were synthesized and tested by ELISA against sera from HLA B27+ ankylosing spondylitis (AS) patients, and sera from HLA B27+ and HLA B27- healthy first-degree relatives. Antibody activity in AS sera to Klebsiella peptides of four to eight amino acids was maximal with the peptide NSRQTDR. Activity to HLA B27 peptides was maximal with th...

  12. CONSTRUCTION AND EXPRESSION OF A HUMAN-MOUSE CHIMERIC ANTIBODY AGAINST HUMAN BLADDER CANCER

    Institute of Scientific and Technical Information of China (English)

    白银; 王琰; 周丽君; 俞莉章

    2001-01-01

    To construct and express a human-mouse chimeric antibody against human bladder cancer. Method: The variable region genes of anti-human bladder cancer monoclonal antibody BDI-1 were cloned by RT-PCR. A human-mouse chimeric antibody expression vector was constructed and transfected into CHO cells. The chimeric antibody against bladder cancer was expressed and characterized. Result: Eukaryotic expression vector of the chimeric antibody against human bladder carcinoma was successfully constructed, and was expressed in eukaryotic cells; the expressed chimeric antibody ch-BDI showed same specificity as its parent McAb against human bladder cancer cells. Conclusion: The constructed chimeric antibody was expressed successfully in eukaryotic cells, and the chimeric antibody had desired affinity against human bladder cancer cells.

  13. Mapping Epitopes on a Protein Antigen by the Proteolysis of Antigen-Antibody Complexes

    Science.gov (United States)

    Jemmerson, Ronald; Paterson, Yvonne

    1986-05-01

    A monoclonal antibody bound to a protein antigen decreases the rate of proteolytic cleavage of the antigen, having the greatest effect on those regions involved in antibody contact. Thus, an epitope can be identified by the ability of the antibody to protect one region of the antigen more than others from proteolysis. By means of this approach, two distinct epitopes, both conformationally well-ordered, were characterized on horse cytochrome c.

  14. Monoclonal antibodies to Pneumocystis carinii

    DEFF Research Database (Denmark)

    Kovacs, J A; Halpern, J L; Lundgren, B; Swan, J C; Parrillo, J E; Masur, H

    1989-01-01

    To increase understanding of the antigenic structure of Pneumocystis carinii, we developed monoclonal antibodies to rat and human P. carinii. The specificity of the antibodies was demonstrated by immunofluorescence and immunoblot studies. Only one of five monoclonal antibodies to rat P. carinii...... reacted with human P. carinii, and none of four monoclonal antibodies to human P. carinii reacted with rat P. carinii. Two antibodies to human P. carinii reacted by immunofluorescence with only one human P. carinii isolate. Immunoblot studies identified major antigens of rat P. carinii with molecular...

  15. [Antibody therapy for Alzheimer's disease].

    Science.gov (United States)

    Tabira, Takeshi; Matsumoto, Shin-Ei; Jin, Haifeng

    2011-11-01

    In order to avoid Abeta-induced autoimmune encephalitis, several monoclonal and polyclonal antibodies are in clinical trials. These are bapineuzumab, solanezumab, ponezumab, gantenerumab, BAN2401, gammaguard and octagam. Since each antibody has a different antigen epitope of Abeta, anti-amyloid activities are different. It is unknown which antibody is effective for Alzheimer disease, and we must wait for the result of clinical trials. Some patients who developed tissue amyloid plaque immuno-reactive (TAPIR) antibody showed slower decline after AN-1792 vaccination. We developed TAPIR-like monoclonal antibody, which was found to react with Abeta oligomers preferentially. PMID:22277519

  16. Tabhu: tools for antibody humanization

    DEFF Research Database (Denmark)

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

    2015-01-01

    and time-consuming experiments. Here we present tools for antibody humanization (Tabhu) a web server for antibody humanization. Tabhu includes tools for human template selection, grafting, back-mutation evaluation, antibody modelling and structural analysis, helping the user in all the critical steps......Antibodies are rapidly becoming essential tools in the clinical practice, given their ability to recognize their cognate antigens with high specificity and affinity, and a high yield at reasonable costs in model animals. Unfortunately, when administered to human patients, xenogeneic antibodies can...

  17. Monoclonal antibody as radiopharmaceutical

    International Nuclear Information System (INIS)

    The purification of anti-CEA monoclonal antibody 4C11 belonging to IgG sub(2a) subclass from mouse ascitis, donated by Ludwig Institute, Brazil was developed. The fragmentation of purified IgG sub(2a) by pepsin digestion and analytical studies by polyacrilamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) were done as preliminary assessment for their specific application in immunoscintigraphy. (author)

  18. Anticardiolipin antibodies in leptospirosis.

    OpenAIRE

    Rugman, F P; Pinn, G.; Palmer, M. F.; Waite, M.; Hay, C. R.

    1991-01-01

    The clinical course and serology of 16 cases of leptospirosis in an area with an unusually high endemic infection rate were studied to gain further insight into the pathology of the secondary immune phase that is typical of the disease. IgG anticardiolipin antibody concentrations were measured by immunoassay and found to be increased in eight serologically confirmed cases with severe complicated disease, compared with eight patients with relatively uncomplicated leptospirosis who had IgG anti...

  19. A monoclonal antibody against leptin.

    Science.gov (United States)

    Mahmoudian, Jafar; Jeddi-Tehrani, Mahmood; Bayat, Ali Ahmad; Mahmoudi, Ahmad Reza; Vojgani, Yasaman; Tavangar, Banafsheh; Hadavi, Reza; Zarei, Saeed

    2012-10-01

    Leptin is an important protein that regulates energy storage and homeostasis in humans and animals. Leptin deficiency results in various abnormalities such as diabetes, obesity, and infertility. Producing a high affinity monoclonal antibody against human leptin provides an important tool to monitor and trace leptin function in different biological fluids. In this study, recombinant human leptin was conjugated to KLH and injected into mice. After immunization, mouse myeloma SP2/0 cells were fused with murine splenocytes followed by selection of antibody-producing hybridoma cells. After screening of different hybridoma colonies by ELISA, a high affinity antibody was selected and purified by affinity chromatography. The affinity constant of the antibody was measured by ELISA. Western blot, immunocytochemistry, and flow cytometry experiments were used to characterize the antibody. The anti-leptin antibody had a high affinity (around 1.13 × 10(-9) M) for its antigen. The saturation of the antibody with leptin (20 moles leptin per 1 mole antibody) in Western blot analysis proved that the antibody had specific binding to its antigen. Immunocytochemistry and flow cytometry on JEG-3 (human placental choriocarcinoma cell) cells revealed that the anti-leptin antibody recognized intracellular leptin. In conclusion, we report here the production and characterization of a murine anti-leptin antibody with high affinity for human leptin. PMID:23098305

  20. Antiphospholipid Antibody and Antiphospholipid Syndrome

    Institute of Scientific and Technical Information of China (English)

    吴竞生

    2008-01-01

    @@ Antiphospholipid antibodies (APA) APA is a big category for all kinds of negative charge phospholipid or lecithin - a protein complex autoantibodies or the same antibody, through its recognition of antigen (target protein) different, and phospholipids or lecithin - protein complex combination of various rely on the interference Phospholipid clotting and anti-coagulation factor, and promote endothelial cells, platelets, complement activation and play a role. APA including lupus anticoagulant(LA) and anticardiolipin antibody (ACA), In addition, there are anti-β2 glycoprotein-I (β2-GPI) antibody, anti-prothrombin (a- PT) antibody, anti-lysophosphatidic acid antibody and anti-phosphatidylserine antibody, and so on. APA as the main target of phospholipid-binding protein, including β2-GPI, prothrombin, annexin, protein C (PC) and protein S (PS), plasminogen, and so on.

  1. A Cayley Tree Immune Network Model with Antibody Dynamics

    CERN Document Server

    Anderson, R W; Perelson, A S; Anderson, Russell W.; Neumann, Avidan U.; Perelson, Alan S.

    1993-01-01

    Abstract: A Cayley tree model of idiotypic networks that includes both B cell and antibody dynamics is formulated and analyzed. As in models with B cells only, localized states exist in the network with limited numbers of activated clones surrounded by virgin or near-virgin clones. The existence and stability of these localized network states are explored as a function of model parameters. As in previous models that have included antibody, the stability of immune and tolerant localized states are shown to depend on the ratio of antibody to B cell lifetimes as well as the rate of antibody complex removal. As model parameters are varied, localized steady-states can break down via two routes: dynamically, into chaotic attractors, or structurally into percolation attractors. For a given set of parameters, percolation and chaotic attractors can coexist with localized attractors, and thus there do not exist clear cut boundaries in parameter space that separate regions of localized attractors from regions of percola...

  2. Antibody therapy for Ebola

    Science.gov (United States)

    Qiu, Xiangguo; Kobinger, Gary P

    2014-01-01

    Ebola viruses can cause severe hemorrhagic fever in humans and nonhuman primates with fatality rates up to 90%, and are identified as biosafety level 4 pathogens and CDC Category A Agents of Bioterrorism. To date, there are no approved therapies and vaccines available to treat these infections. Antibody therapy was estimated to be an effective and powerful treatment strategy against infectious pathogens in the late 19th, early 20th centuries but has fallen short to meet expectations to widely combat infectious diseases. Passive immunization for Ebola virus was successful in 2012, after over 15 years of failed attempts leading to skepticism that the approach would ever be of potential benefit. Currently, monoclonal antibody (mAbs)-based therapies are the most efficient at reversing the progression of a lethal Ebola virus infection in nonhuman primates, which recapitulate the human disease with the highest similarity. Novel combinations of mAbs can even fully cure lethally infected animals after clinical symptoms and circulating virus have been detected, days into the infection. These new developments have reopened the door for using antibody-based therapies for filovirus infections. Furthermore, they are reigniting hope that these strategies will contribute to better control the spread of other infectious agents and provide new tools against infectious diseases. PMID:24503566

  3. Preparation and characterization of polyclonal antibodies against human chaperonin 10

    OpenAIRE

    Somodevilla-Torres, Maria J.; Hillyard, Narelle C.; Morton, Halle; Alewood, Dianne; Halliday, Judy A.; Alewood, Paul F.; Vesey, David A; Walsh, Michael D.; Cavanagh, Alice C.

    2000-01-01

    Abstract Early pregnancy factor (EPF) has been identified as an extracellular homologue of chaperonin 10 (Cpn10), a heat shock protein that functions within the cell as a molecular chaperone. Here, we report the production of polyclonal antibodies directed against several different regions of the human Cpn10 molecule and their application to specific protein quantitation and localization techniques. These antibodies will be valuable tools in further studies to elucidate the mechanisms underly...

  4. Fast track antibody V-gene rescue, recombinant expression in plants and characterization of a PfMSP4-specific antibody

    OpenAIRE

    Kapelski, Stephanie; Boes, Alexander; Spiegel, Holger; de Almeida, Melanie; Klockenbring, Torsten; Reimann, Andreas; Fischer, Rainer; Barth, Stefan; Fendel, Rolf

    2015-01-01

    Background Monoclonal antibodies (mAbs) are essential tools in biological research, diagnosis and therapy, and are conventionally produced in murine hybridoma cell lines. Professional applications of mAbs depend on the steady supply of material. Because hybridoma cultures can stop producing the antibody or even die, preservation of the unique epitope specificity of mAbs by rescue of the sequences encoding the antibody variable domains (V regions) is important. The availability of these sequen...

  5. Second antibody clearance of radiolabeled antibody in cancer radioimmunodetection.

    OpenAIRE

    Sharkey, R M; Primus, F J; Goldenberg, D. M.

    1984-01-01

    The imaging of tumors using radiolabeled antibodies previously has required the implementation of computer-assisted subtraction techniques to reduce background radioactivity. A decrease in radioactivity in the blood of hamsters bearing human colonic tumor xenografts has been achieved by administering a second antibody directed against a radiolabeled primary antibody to carcinoembryonic antigen (CEA). This method was found to reduce the level of blood radioactivity by a factor of 4 within 2 hr...

  6. Analysis of the serum reproductive system related autoantibodies of infertility patients in Tianjin region of China

    OpenAIRE

    Huo, Yan; Xu, Yanying; Wang, Jianmei; Wang, Fang; Liu, Yu; Zhang, Yujuan; Zhang, Bumei

    2015-01-01

    Object: Reproductive system related autoantibodies have been proposed to be associated with natural infertility. However, large scale systematic analysis of these of antibodies has not been conducted. The aim of this study is to analyze the positive rate of antisperm antibody (ASAb), anti-endometrium antibody (EMAb), anti-ovary antibody (AOAb), anti-zona pellucida antibody (AZP) and anticardiolipin antibody (ACA) in infertility patients in Tianjin region of China. Methods: 1305 male and 1711 ...

  7. Antibody Glossary —

    Science.gov (United States)

    The components of the immune system have diverse roles in the initial development of cancers, progression of early-stage malignancies to invasive tumors, establishment of metastatic lesions, tumor dormancy, and response or resistance to therapy. Characterizing the components of the immune system and their functional status in tissues and in tumors requires the use of highly specific reagents. Researchers employ antibodies in a variety of in vitro and in vivo applications to delineate, enrich, or deplete specific immune subsets in order to understand their role(s) in tumorigenesis. This is a glossary of validated reagents and protocols that are useful for functional phenotyping of the immune system in murine cancer models.

  8. The antibody Hijikata Tatsumi

    Directory of Open Access Journals (Sweden)

    Éden Peretta

    2012-11-01

    Full Text Available Considered one of the most influential modern dance representatives in Japan, Tatsumi Hijikata’s work was a milestone in the Japanese post-war experimental artistic scene. Heretic son of his time, he staged a fertile mix of artistic and cultural influences, overlapping subversive elements of European arts and philosophy with radical references from pre-modern Japanese culture. In this way he built the foundations of its unstable antibody, its political-artistic project of dissolution of a organism, both physical and social.

  9. VIRAL ANTIBODIES IN PRESCHOOL CHILDREN

    Directory of Open Access Journals (Sweden)

    S. Saidi

    1974-08-01

    Full Text Available One hundred sera from children 1 - 6 years of age, representative of a large serum collection, were tested for the prevalence of antibodies against different viruses. Hemagglutination-inhibition (HI antibodies were found in 68% for measles; 61 % for rubella; 75'% for influenza A2/Hong Kong/68, 16% for influenza B/Md./59, 0% for group A arboviruses, 10% for group B arboviruses, 3% for phlebotomus fever group and 4% for Congo-Crimean hemorrhagic fever (C-CHF group of arboviruses Poliomyelitis-neutralizing antibodies for type 1, 2 and 3 were 90%; 85% and 84%~ respectively. Antibody to EH virus was detected in 84% of the sera by immuno-fluorescence. None of the sera were positive for hepatitis-B antigen or antibody by immuno-precipitation test. The prevalence of some viral antibodies found in this survey are compared with results obtained from surveys in other parts of the country.

  10. Antibodies to watch in 2015

    OpenAIRE

    Reichert, Janice M

    2014-01-01

    The commercial pipeline of recombinant antibody therapeutics is robust and dynamic. As of early December 2014, a total of 6 such products (vedolizumab, siltuximab, ramucirumab, pembrolizumab, nivolumab, blinatumomab) were granted first marketing approvals in 2014. As discussed in this perspective on antibodies in late-stage development, the outlook for additional approvals, potentially still in 2014 and certainly in 2015, is excellent as marketing applications for 6 antibody therapeutics (sec...

  11. Monoclonal antibodies for treating cancer

    International Nuclear Information System (INIS)

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

  12. Metrics for antibody therapeutics development

    OpenAIRE

    Reichert, Janice M

    2010-01-01

    A wide variety of full-size monoclonal antibodies (mAbs) and therapeutics derived from alternative antibody formats can be produced through genetic and biological engineering techniques. These molecules are now filling the preclinical and clinical pipelines of every major pharmaceutical company and many biotechnology firms. Metrics for the development of antibody therapeutics, including averages for the number of candidates entering clinical study and development phase lengths for mAbs approv...

  13. Empowered Antibody Therapies - IBC conference.

    Science.gov (United States)

    Herold, Jens

    2010-10-01

    The Empowered Antibody Therapies conference, held in Burlingame, CA, USA, included topics covering new therapeutic developments in the field of multispecific antibodies. This conference report highlights selected presentations on DVD-Igs from Abbott Laboratories, ImmTACs from Immunocore, 'Dock-and-Lock' technology from Immunomedics, the bispecific BiTE antibody blinatumomab from Micromet, and Triomabs from TRION Pharma and Fresenius Biotech. PMID:20878591

  14. Characterization of antibodies directed against the Ankrd2 human muscle protein

    OpenAIRE

    Kojić Snežana; Medeot Elisa; Faulkner Georgine

    2009-01-01

    In order to study the function of the Ankrd2 protein, for which commercial antibodies are not available, we report the production and analysis of polyclonal antibodies to full-length Ankrd2 and its C-terminal and N-terminal regions, as well as a monoclonal antibody to the C-terminus of the protein. Epitope mapping making use of recombinant deletion mutants showed that an epitope located in region 323-333 aa of Ankrd2 is detected by the monoclonal antibody. The high specificity of all four ant...

  15. Antibody informatics for drug discovery

    DEFF Research Database (Denmark)

    Shirai, Hiroki; Prades, Catherine; Vita, Randi;

    2014-01-01

    infrastructure for these large data sets has become necessary. In this article, we first identify and discuss the typical obstacles faced during the antibody drug discovery process. We then summarize the current status of three sub-fields of antibody informatics as follows: (i) recent progress in technologies...... (iii) antibody numbering and IMGT. Here, we review “antibody informatics,” which may integrate the above three fields so that bridging the gaps between industrial needs and academic solutions can be accelerated. This article is part of a Special Issue entitled: Recent advances in molecular engineering...

  16. Tumor imaging with monoclonal antibodies

    International Nuclear Information System (INIS)

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

  17. The Proline-Rich Region of Pneumococcal Surface Proteins A and C Contains Surface-Accessible Epitopes Common to All Pneumococci and Elicits Antibody-Mediated Protection against Sepsis▿ ‡

    OpenAIRE

    Daniels, Calvin C; Coan, Patricia; King, Janice; Hale, Joanetha; Benton, Kimberly A.; Briles, David E.; Hollingshead, Susan K.

    2010-01-01

    Pneumococcal surface protein A (PspA) and PspC of Streptococcus pneumoniae are surface virulence proteins that interfere with complement deposition and elicit protective immune responses. The C-terminal halves of PspA and PspC have some structural similarity and contain highly cross-reactive proline-rich (PR) regions. In many PR regions of PspA and PspC, there exists an almost invariant nonproline block (NPB) of about 33 amino acids. Neither the PR regions nor their NPB exhibit the alpha-heli...

  18. Tumor targeting of radiolabeled antibodies using HYNIC chelate

    International Nuclear Information System (INIS)

    There is an increasing interest in the use of labeled antibodies for diagnosis of cancers as well as for therapy. Various radiolabeling methods have been used in order to obtain better tumor specific targeting for detection and therapy. It was generally used to tumor targeted immunotherapy and immunodetection that lym-1, mouse monoclonal antibody, was specific binding to surface antigen of Raji. The 3E8 antibody was produced from humanized anti-TAG-72 monoclonal antibody (AKA) by amino acid change in 95-99 residues of heavy chain complementary determinant regions (HCDRs) 3 using phage displayed library technology. In this study, we are investigating the usefulness of HYNIC chelate as a bifunctional chelating agent in radioimmunodetecton of tumor. Two types of antibodies, Lym-1 and 3E8, were used for the conjugation with HYNIC chelate. Lym-1 and 3E8 are specific antibodies to surface antigen of Non-Hogkin's lymphoma and TAG-72 antigen of colorectal carcinoma, respectively. We prepare HYNIC-antibody conjugates, determine radiolabeling yield with 99mTc and evaluate tumor targeting in tumor bearing nude mice model

  19. Creating Ordered Antibody Arrays with Antibody-Polymer Conjugates

    Science.gov (United States)

    Dong, Xuehui; Obermeyer, Allie; Olsen, Bradley

    Antibodies are a category of functional proteins that play crucial roles in the immune system and have been widely applied in the area of cancer therapeutics, targeting delivery, signal detection, and sensors. Due to the extremely large size and lack of specific functional groups on the surface, it is challenging to functionalize antibodies and manipulate the ordered packing of antibodies in an array with high density and proper orientation, which is critical to achieve outstanding performance in materials. In this work, we demonstrate an efficient and facile approach for preparing antibody-polymer conjugates with two-step sequential ``click'' reaction to form antibody-polymer block copolymers. Highly ordered nanostructures are fabricated based on the principles of block copolymer self-assembly. The nanostructures are studied with both small angle X-ray scattering (SAXS) and transmission electron microscopy (TEM). Lamellae with alternating antibody domain and polymer domain are observed with an overall domain size of ~50 nm. The nanostructure not only increases the packing density and promotes proper orientation of the antibody, but also provides possible channel to facilitate substrate transportation and improves the stability of the antibody.

  20. Characterization for Binding Complex Formation with Site-Directly Immobilized Antibodies Enhancing Detection Capability of Cardiac Troponin I

    OpenAIRE

    Il-Hoon Cho; Sung-Min Seo; Jin-Woo Jeon; Se-Hwan Paek

    2009-01-01

    The enhanced analytical performances of immunoassays that employed site-directly immobilized antibodies as the capture binders have been functionally characterized in terms of antigen-antibody complex formation on solid surfaces. Three antibody species specific to cardiac troponin I, immunoglobulin G (IgG), Fab, and F(ab′)2 were site-directly biotinylated within the hinge region and then immobilized via a streptavidin-biotin linkage. The new binders were more efficient capture antibodies ...

  1. Prevalence Estimates of Antibodies Towards Foot-and-Mouth Disease Virus in Small Ruminants in Uganda

    DEFF Research Database (Denmark)

    Balinda, Sheila Nina; Tjørnehøj, Kirsten; Muwanika, Vincent B.;

    2009-01-01

    summarizes results of serological investigations of sheep and goats for antibodies to FMDV from four districts in 2006 following an FMD outbreak in the region and from an attempted comprehensive random sampling in two districts in 2007. Antibodies were quantified and serotyped using competitive ELISA...... for antibodies towards non-structural proteins (NSP) and structural proteins towards serotype O, and blocking ELISA for antibodies towards the seven serotypes of FMD virus (FMDV). In 2006, sheep and goats in Bushenyi and Isingiro districts were free from antibodies towards FMDV, while herds in Kasese and Mbarara...... districts excluding Kahendero village were all positive for antibodies towards NSP and SP-O. In 2007, mean prevalence estimates of antibodies towards FMDV NSP was 14% in goats and 22% in sheep in Kasese district, while Bushenyi was still free. The difference between these two districts probably reflects...

  2. Characterization of single chain antibody targets through yeast two hybrid

    Directory of Open Access Journals (Sweden)

    Vielemeyer Ole

    2010-08-01

    Full Text Available Abstract Background Due to their unique ability to bind their targets with high fidelity, antibodies are used widely not only in biomedical research, but also in many clinical applications. Recombinant antibodies, including single chain variable fragments (scFv, are gaining momentum because they allow powerful in vitro selection and manipulation without loss of function. Regardless of the ultimate application or type of antibody used, precise understanding of the interaction between the antibody's binding site and its specific target epitope(s is of great importance. However, such data is frequently difficult to obtain. Results We describe an approach that allows detailed characterization of a given antibody's target(s using the yeast two-hybrid system. Several recombinant scFv were used as bait and screened against highly complex cDNA libraries. Systematic sequencing of all retained clones and statistical analysis allowed efficient ranking of the prey fragments. Multiple alignment of the obtained cDNA fragments provided a selected interacting domain (SID, efficiently narrowing the epitope-containing region. Interactions between antibodies and their respective targets were characterized for several scFv. For AA2 and ROF7, two conformation-specific sensors that exclusively bind the activated forms of the small GTPases Rab6 and Rab1 respectively, only fragments expressing the entire target protein's core region were retained. This strongly suggested interaction with a non-linear epitope. For two other scFv, TA10 and SF9, which recognize the large proteins giantin and non-muscle myosin IIA, respectively, precise antibody-binding regions within the target were defined. Finally, for some antibodies, secondary targets within and across species could be revealed. Conclusions Our method, utilizing the yeast two-hybrid technology and scFv as bait, is a simple yet powerful approach for the detailed characterization of antibody targets. It allows precise

  3. Caracterização de amostras do vírus da raiva, isoladas nas regiões Norte e Centro-Oeste do Brasil, com anticorpos monoclonais antilissavírus Antigenic characterization of Brazilian rabies virus isolate North and Central West regions of Brazil with anti-lyssavirus monoclonal antibodies

    Directory of Open Access Journals (Sweden)

    H.B.C.R. Batista

    2008-02-01

    Full Text Available The occurrence of rabies virus antigenic variants in North and Central West regions of Brazil was studied using 61 rabies viruses isolated from different species: 30 from domestic dogs, 20 from cattle, four from horses, two from cats, one from a human and four from unidentified species. The isolates were submitted to antigenic analyses by indirect immunofluorescence with a panel of 12 monoclonal antibodies (Mabs to lyssavirus antigens. Antigenic analyses revealed consistent differences between isolates whose natural hosts were dogs and those of haematophagous bats, often isolated from cattle. Three out of four isolates from horses and one from a domestic dog showed patterns of reactivity found only in viruses of insectivorous bats, indicating that non-haematophagous bats do play a unique role in the transmission of the virus to other species.

  4. Antiphospholipid antibodies and infertility.

    Science.gov (United States)

    Chighizola, C B; de Jesus, G R

    2014-10-01

    Since the late 1980s some publications have proposed that antiphospholipid antibodies (aPL) may have some relationship with infertility, considering reported deleterious effects that aPL exert on trophoblast proliferation and growth. Although not included in current classification criteria for antiphospholipid syndrome, many physicians investigate for aPL in patients with a history of infertility, including antibodies not listed in classification criteria, and most of those patients will receive anticoagulant therapy if any of those antibodies have a result considered positive. A review of literature was conducted searching for studies that investigated the association of aPL and infertility and if aPL positivity alters in vitro fertilization (IVF) outcome. The definition of infertility, routine work-up to exclude other causes of infertility, definition of IVF failure as inclusion criteria and control populations were heterogeneous among studies. Most of them enrolled women over 40 years of age, and exclusion of other confounding factors was also inconsistent. Of 29 studies that assessed aPL positivity rates in infertile women, the majority had small sample sizes, implying a lack of power, and 13 (44.8%) reported higher frequency of aPL in infertile patients compared to controls, but most of them investigated a panel of non-criteria aPL tests, whose clinical significance is highly controversial. Only two studies investigated all three criteria tests, and medium-high titer of anticardiolipin cut-off conforming to international guidelines was used in one study. Considering IVF outcome, there was also disparity in this definition: few studies assessed the live birth rate, others the implantation rate. Of 14 publications that addressed the relationship between aPL and IVF outcome, only two described a detrimental effect of these autoantibodies. In conclusion, available data do not support an association between aPL and infertility, and aPL positivity does not seem to

  5. Detection of antibodies against Babesia bovis and Babesia bigemina in calves from the region of Araguaína, State of Tocantins, Brazil Detecção de anticorpos anti-Babesia bovis e anti-Babesia bigemina em bezerros na região de Araguaína, Estado do Tocantins, Brasil

    Directory of Open Access Journals (Sweden)

    Hébelys Ibiapina da Trindade

    2010-09-01

    Full Text Available The aim of this study was to determine the seroprevalence of antibodies against B. bovis and B. bigemina in calves from the region of Araguaína, State of Tocantins, Brazil. In this research we used sera obtained from 506 calves, from both genders and of 8 to 24 months old, to detect antibodies by indirect Enzyme-Linked Immunosorbent Assay (ELISA-test. Statistical analysis of the data was performed using the Chi-square (χ2 test with Yates correction. The seroprevalence obtained was 90.5 and 91.7% for B. bigemina and B. bovis, respectively, characterizing the region as an area of enzootic stability for the species analyzed. The seroprevalence to B. bovis showed higher positivity among calves 19-24 months old.O objetivo desse estudo foi determinar a soroprevalência de anticorpos anti-B. bovis e anti-B. bigemina em bezerros da região de Araguaína, Estado do Tocantins, Brasil. Nesta pesquisa foram coletadas 506 amostras de soros de bezerros com faixa etária entre 8 e 24 meses, fêmeas e machos, as quais foram processadas pelo ensaio imunoenzimático indireto (ELISA-teste. Como análise estatística utilizou-se o Qui-Quadrado (χ2 com correção de Yates. As prevalências obtidas em bezerros foram de 90,5 e 91,7% para B. bigemina e B. bovis, respectivamente, caracterizando esta região como de estabilidade enzoótica para as espécies analisadas. A soroprevalência para B. bovis apresentou maior positividade entre os bezerros com faixa etária de 19-24 meses.

  6. Targeting of Antibodies using Aptamers

    OpenAIRE

    Missailidis, Sotiris

    2003-01-01

    The chapter presents a methodology for the rapid selection of aptamers against antibody targets. It is a detailed account of the various methodological steps that describe the selection of aptamers, including PCR steps, buffers to be used, target immobilisation, partitioning and amplification of aptamers, clonning and sequencing, to results in high affinity and specificity ligands for the chosen target antibody.

  7. Refolding Technologies for Antibody Fragments

    OpenAIRE

    Tsutomu Arakawa; Daisuke Ejima

    2014-01-01

    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 suppresso...

  8. ANTISPERM ANTIBODIES IN VASOVASOSTOMY

    Directory of Open Access Journals (Sweden)

    Gholamreza Pourmand

    1993-06-01

    Full Text Available Two hundred and forty patients, who had undergone vasectomy from 1977 to 1985 and subsequent vasovasostomy ,were studied for the presence of sperm-specific antibodies by using the Kibrick's gelatin agglutination test. The number of successful pregnancies and the presence of agglutination were also considered in this survey. Sixty-nine pregnancies occurred in total and agglutination was present in 49% out of 51% positive specimens by the Kibrick Test."nThe average sperm motility was slightly higher in the negative Kibrick group than in the positive Kibrick group. The obtained data indicated that there seems to be a relationship between the increased titers and percentage of agglutination in semen samples.

  9. Metrics for antibody therapeutics development.

    Science.gov (United States)

    Reichert, Janice M

    2010-01-01

    A wide variety of full-size monoclonal antibodies (mAbs) and therapeutics derived from alternative antibody formats can be produced through genetic and biological engineering techniques. These molecules are now filling the preclinical and clinical pipelines of every major pharmaceutical company and many biotechnology firms. Metrics for the development of antibody therapeutics, including averages for the number of candidates entering clinical study and development phase lengths for mAbs approved in the United States, were derived from analysis of a dataset of over 600 therapeutic mAbs that entered clinical study sponsored, at least in part, by commercial firms. The results presented provide an overview of the field and context for the evaluation of on-going and prospective mAb development programs. The expansion of therapeutic antibody use through supplemental marketing approvals and the increase in the study of therapeutics derived from alternative antibody formats are discussed. PMID:20930555

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

    International Nuclear Information System (INIS)

    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.)

  11. Epstein-Barr virus antibody test

    Science.gov (United States)

    EBV antibody test; EBV serology ... a lab, where a lab specialist looks for antibodies to the Epstein-Barr virus. In the first stages of an illness, little antibody may be detected. For this reason, the test ...

  12. Measurement of antibodies to tubulin by radioimmunoassay

    International Nuclear Information System (INIS)

    A solid-phase double antibody radioimmunoassay capable of measuring antibody to tubulin, the principal component of microtubules, is described. This assay is simple, combining sensitivity with specificity and also allowing determination of antibody subclasses. (Auth.)

  13. Antibodies - Office of Cancer Clinical Proteomics Research

    Science.gov (United States)

    NCI announces the release of monoclonal antipeptide antibodies from rabbit for distribution on the antibody portal. There are 60 recently added monoclonal antibodies, with 56 generated from mouse and 4 generated from rabbit.

  14. Antibody protection reveals extended epitopes on the human TSH receptor.

    Directory of Open Access Journals (Sweden)

    Rauf Latif

    Full Text Available Stimulating, and some blocking, antibodies to the TSH receptor (TSHR have conformation-dependent epitopes reported to involve primarily the leucine rich repeat region of the ectodomain (LRD. However, successful crystallization of TSHR residues 22-260 has omitted important extracellular non-LRD residues including the hinge region which connects the TSHR ectodomain to the transmembrane domain and which is involved in ligand induced signal transduction. The aim of the present study, therefore, was to determine if TSHR antibodies (TSHR-Abs have non-LRD binding sites outside the LRD. To obtain this information we employed the method of epitope protection in which we first protected TSHR residues 1-412 with intact TSHR antibodies and then enzymatically digested the unprotected residues. Those peptides remaining were subsequently delineated by mass spectrometry. Fourteen out of 23 of the reported stimulating monoclonal TSHR-Ab crystal contact residues were protected by this technique which may reflect the higher binding energies of certain residues detected in this approach. Comparing the protected epitopes of two stimulating TSHR-Abs we found both similarities and differences but both antibodies also contacted the hinge region and the amino terminus of the TSHR following the signal peptide and encompassing cysteine box 1 which has previously been shown to be important for TSH binding and activation. A monoclonal blocking TSHR antibody revealed a similar pattern of binding regions but the residues that it contacted on the LRD were again distinct. These data demonstrated that conformationally dependent TSHR-Abs had epitopes not confined to the LRDs but also incorporated epitopes not revealed in the available crystal structure. Furthermore, the data also indicated that in addition to overlapping contact regions within the LRD, there are unique epitope patterns for each of the antibodies which may contribute to their functional heterogeneity.

  15. Antibody protection reveals extended epitopes on the human TSH receptor.

    Science.gov (United States)

    Latif, Rauf; Teixeira, Avelino; Michalek, Krzysztof; Ali, M Rejwan; Schlesinger, Max; Baliram, Ramkumarie; Morshed, Syed A; Davies, Terry F

    2012-01-01

    Stimulating, and some blocking, antibodies to the TSH receptor (TSHR) have conformation-dependent epitopes reported to involve primarily the leucine rich repeat region of the ectodomain (LRD). However, successful crystallization of TSHR residues 22-260 has omitted important extracellular non-LRD residues including the hinge region which connects the TSHR ectodomain to the transmembrane domain and which is involved in ligand induced signal transduction. The aim of the present study, therefore, was to determine if TSHR antibodies (TSHR-Abs) have non-LRD binding sites outside the LRD. To obtain this information we employed the method of epitope protection in which we first protected TSHR residues 1-412 with intact TSHR antibodies and then enzymatically digested the unprotected residues. Those peptides remaining were subsequently delineated by mass spectrometry. Fourteen out of 23 of the reported stimulating monoclonal TSHR-Ab crystal contact residues were protected by this technique which may reflect the higher binding energies of certain residues detected in this approach. Comparing the protected epitopes of two stimulating TSHR-Abs we found both similarities and differences but both antibodies also contacted the hinge region and the amino terminus of the TSHR following the signal peptide and encompassing cysteine box 1 which has previously been shown to be important for TSH binding and activation. A monoclonal blocking TSHR antibody revealed a similar pattern of binding regions but the residues that it contacted on the LRD were again distinct. These data demonstrated that conformationally dependent TSHR-Abs had epitopes not confined to the LRDs but also incorporated epitopes not revealed in the available crystal structure. Furthermore, the data also indicated that in addition to overlapping contact regions within the LRD, there are unique epitope patterns for each of the antibodies which may contribute to their functional heterogeneity. PMID:22957097

  16. Enhanced antigen detection in immunohistochemical staining using a 'digitized' chimeric antibody.

    Science.gov (United States)

    Eng, Hui-Yan; Wang, Cheng-I; Xue, Yuezhen; Lee, Chia-Yin; Zulkifli, Sarah Binte; Chiam, Poh-Cheang; Ghadessy, Farid J; Lane, David P

    2016-01-01

    The immunohistochemical (IHC) staining of mouse tissue sections using antibodies of mouse origin can result in high nonspecific background due to the staining of endogenous immunoglobulins (Igs) by enzyme-conjugated secondary antibodies. In order to obviate this issue, we developed a chimeric mouse-human anti-p53 monoclonal antibody (MH242) by grafting the variable regions of a known mouse antibody into a human Ig scaffold. This facilitated use of an anti-human secondary antibody, and resulted in near-zero background when compared with its parental mouse monoclonal antibody (PAb242). Furthermore, the chimeric antibody enabled reproducible detection of mutant p53 (homozygous R172H) expression in mouse tissue, an observation hitherto largely equivocal based on the use of existing antibodies. The approach we describe leads to the generation of tractable antibody reagents, whose integrity can be readily verified through DNA sequencing of expressor plasmids. The wide-spread adoption of such 'digitized' antibodies should reduce experimental disparities that can commonly arise through variations in antibody quality. PMID:26508747

  17. Tracking hantavirus nucleocapsid protein using intracellular antibodies

    Directory of Open Access Journals (Sweden)

    Liang Mifang

    2010-11-01

    Full Text Available Abstract Background Hantavirus nucleocapsid (N protein is a multifunctional viral macromolecule involved in multiple stages of the viral replication cycle. The intracellular trafficking of N protein during virus assembly remains unclear. Methods We used N protein-specific intracellular expressed antibodies to track the localization and distribution of Hantaan virus and Seoul virus N protein. The N protein-specific antibody single-chain variable antibody fragments (scFvs, which bind an N-terminal linear epitope (L13F3 and C-terminal conformational domain (H34, were intracellularly expressed in the endoplasmic reticulum (ER by fusion of the SEKDEL retention signal peptide at the carboxyl terminus, and in the cytoplasm (Cyto by deletion of the ER membrane target signal peptide. Stable Vero-E6 cell lines expressing intracellular scFvs were either infected with hantavirus or transfected with an N protein expression plasmid; virus replication and N protein intracellular localization were determined. Result N protein co-localized with scFvs in the ER and cytoplasm with or without viral membrane glycoproteins. Hantavirus replication was inhibited in both the scFvs-ER- and scFvs-Cyto-expressing stable cell lines. Conclusion N protein may be expressed in the ER retention signal peptide of KDEL circulating region (ER/cis-Golgi without the assistance of G protein, and so expression of N protein in both the cytoplasm and within the ER/cis-Golgi plays an important role in virus replication.

  18. New High Affinity Monoclonal Antibodies Recognize Non-Overlapping Epitopes On Mesothelin For Monitoring And Treating Mesothelioma

    OpenAIRE

    Zhang, Yi-Fan; Phung, Yen; Gao, Wei; Kawa, Seiji; Hassan, Raffit; Pastan, Ira; Ho, Mitchell

    2015-01-01

    Mesothelin is an emerging cell surface target in mesothelioma and other solid tumors. Most antibody drug candidates recognize highly immunogenic Region I (296–390) on mesothelin. Here, we report a group of high-affinity non-Region I rabbit monoclonal antibodies. These antibodies do not compete for mesothelin binding with the immunotoxin SS1P that binds Region I of mesothelin. One pair of antibodies (YP218 and YP223) is suitable to detect soluble mesothelin in a sandwich ELISA with high sensit...

  19. Interaction of monoclonal antibodies directed against bromodeoxyuridine with pyrimidine bases, nucleosides, and DNA

    International Nuclear Information System (INIS)

    Although antibodies directed against bromodeoxyuridine (BrdU) are being used in both clinical and basic research laboratories as tools to study and monitor DNA synthesis, little is known about the epitopes with which they react. Four monoclonal antibodies directed against BrdU were produced and were characterized to learn more about the epitopes on BrdU which are important for antibody recognition, to identify compounds other than BrdU which react with the antibodies and which might interfere with immunologic assays for BrdU, and to characterize the reaction of these antibodies with BrdU-containing DNA. By radioimmunoassays, the antibodies generally reacted well with 5-iododeoxyuridine, 5-fluorodeoxyuridine, and 5-nitrouracil. However, none of the antibodies reacted well with uridine - indicating that a substituent on uridine C5 was essential for antibody reactivity - or with 5-bromo or iodo-cytosine, indicating that the region around pyrimidine C4 is important for antibody recognition. Although the antibodies reacted with 5-halogen-substituted uracil bases, the antibodies reacted much better with the corresponding halogenated nucleosides, indicating that the sugar moiety was important for recognition. The presence of a triphosphate group of C'5 of BrdU (i.e., BrdUTP) did not detectably alter antibody recognition. S1 nuclease treatment of purified DNA suggested that all four monoclonal antibodies reacted exclusively with single-stranded regions of BrdU-containing DNA. Comparison of detecting DNA synthesis by [3H]TdR incorporation followed by autoradiography with that by BrdU incorporation followed by indirect immunofluorescence indicated that the latter technique was both an accurate and a sensitive measure of DNA synthesis

  20. Restricted Isotypic Antibody Reactivity to Hepatitis C Virus Synthetic Peptides in Immunocompromised Patients

    Science.gov (United States)

    Devesa, Marisol; de Saez, Arlette; León, Graciela; Sirit, Firelei; Cosson, Clarisa; Bermúdez, Henry; Liprandi, Ferdinando; Noya, Oscar; Pujol, Flor H.

    1999-01-01

    An enzyme immunoassay based on three synthetic peptides from the core, NS4, and NS5 regions of hepatitis C virus allowed the detection of antibodies in 100% of immunocompetent infected patients and in 91% of immunocompromised patients (hemodialysis and hemophiliac patients). Immune impairment seemed to restrict the spectrum of antibody isotypes reacting to the core peptide. PMID:10066669

  1. Antibody Recognition of a Highly Conserved Influenza Virus Epitope

    Energy Technology Data Exchange (ETDEWEB)

    Ekiert, Damian C.; Bhabha, Gira; Elsliger, Marc-André; Friesen, Robert H.E.; Jongeneelen, Mandy; Throsby, Mark; Goudsmit, Jaap; Wilson, Ian A.; Scripps; Crucell

    2009-05-21

    Influenza virus presents an important and persistent threat to public health worldwide, and current vaccines provide immunity to viral isolates similar to the vaccine strain. High-affinity antibodies against a conserved epitope could provide immunity to the diverse influenza subtypes and protection against future pandemic viruses. Cocrystal structures were determined at 2.2 and 2.7 angstrom resolutions for broadly neutralizing human antibody CR6261 Fab in complexes with the major surface antigen (hemagglutinin, HA) from viruses responsible for the 1918 H1N1 influenza pandemic and a recent lethal case of H5N1 avian influenza. In contrast to other structurally characterized influenza antibodies, CR6261 recognizes a highly conserved helical region in the membrane-proximal stem of HA1 and HA2. The antibody neutralizes the virus by blocking conformational rearrangements associated with membrane fusion. The CR6261 epitope identified here should accelerate the design and implementation of improved vaccines that can elicit CR6261-like antibodies, as well as antibody-based therapies for the treatment of influenza.

  2. Directed Selection of Recombinant Human Monoclonal Antibodies to Herpes Simplex Virus Glycoproteins from Phage Display Libraries

    Science.gov (United States)

    Sanna, Pietro Paolo; Williamson, R. Anthony; de Logu, Alessandro; Bloom, Floyd E.; Burton, Dennis R.

    1995-07-01

    Human monoclonal antibodies have considerable potential in the prophylaxis and treatment of viral disease. However, only a few such antibodies suitable for clinical use have been produced to date. We have previously shown that large panels of human recombinant monoclonal antibodies against a plethora of infectious agents, including herpes simplex virus types 1 and 2, can be established from phage display libraries. Here we demonstrate that facile cloning of recombinant Fab fragments against specific viral proteins in their native conformation can be accomplished by panning phage display libraries against viral glycoproteins "captured" from infected cell extracts by specific monoclonal antibodies immobilized on ELISA plates. We have tested this strategy by isolating six neutralizing recombinant antibodies specific for herpes simplex glycoprotein gD or gB, some of which are against conformationally sensitive epitopes. By using defined monoclonal antibodies for the antigen-capture step, this method can be used for the isolation of antibodies to specific regions and epitopes within the target viral protein. For instance, monoclonal antibodies to a nonneutralizing epitope can be used in the capture step to clone antibodies to neutralizing epitopes, or antibodies to a neutralizing epitope can be used to clone antibodies to a different neutralizing epitope. Furthermore, by using capturing antibodies to more immunodominant epitopes, one can direct the cloning to less immunogenic ones. This method should be of value in generating antibodies to be used both in the prophylaxis and treatment of viral infections and in the characterization of the mechanisms of antibody protective actions at the molecular level.

  3. Structure of an isolated unglycosylated antibody CH2 domain

    International Nuclear Information System (INIS)

    The crystal structure of an isolated unglycosylated antibody CH2 domain has been determined at 1.7 Å resolution. The CH2 (CH3 for IgM and IgE) domain of an antibody plays an important role in mediating effector functions and preserving antibody stability. It is the only domain in human immunoglobulins (Igs) which is involved in weak interchain protein–protein interactions with another CH2 domain solely through sugar moieties. The N-linked glycosylation at Asn297 is conserved in mammalian IgGs as well as in homologous regions of other antibody isotypes. To examine the structural details of the CH2 domain in the absence of glycosylation and other antibody domains, the crystal structure of an isolated unglycosylated antibody γ1 CH2 domain was determined at 1.7 Å resolution and compared with corresponding CH2 structures from intact Fc, IgG and Fc receptor complexes. Furthermore, the oligomeric state of the protein in solution was studied using size-exclusion chromatography. The results suggested that the unglycosylated human antibody CH2 domain is a monomer and that its structure is similar to that found in the intact Fc, IgG and Fc receptor complex structures. However, certain structural variations were observed in the Fc receptor-binding sites. Owing to its small size, stability and non-immunogenic Ig template, the CH2-domain structure could be useful for the development by protein design of antibody domains exerting effector functions and/or antigen specificity and as a robust scaffold in protein-engineering applications

  4. Capture ELISA for IgM antibodies against Plasmodium falciparum glutamate rich protein

    DEFF Research Database (Denmark)

    Dziegiel, M; Borre, Mette; Petersen, E; Högh, B; Jepsen, S; Vuust, J; Axelsen, N

    1992-01-01

    -galactosidase fusion protein and used as antigen after purification and biotinylation. Specific IgM antibodies were found in 51% (39/77) of sera from adult Liberians immune to malaria. The binding of IgM antibodies was specific for the malaria portion of the fusion protein and no cross-reactivity was found in sera...... from patients with IgM antibodies due to other diseases. Inhibition studies with a fusion protein containing amino acid residues 816-1134 (GLURP816-1134) representing the carboxy-terminal repeat region suggested a different use of epitopes for IgM antibodies in different individuals....

  5. Protective Antibodies against Placental Malaria and Poor Outcomes during Pregnancy, Benin

    DEFF Research Database (Denmark)

    Ndam, Nicaise Tuikue; Denoeud-Ndam, Lise; Doritchamou, Justin;

    2015-01-01

    . Antibody-mediated immunity to placental malaria is acquired during successive pregnancies, but the target of VAR2CSA-specific protective antibodies is unclear. We assessed VAR2CSA-specific antibodies in pregnant women and analyzed their relationships with protection against placental infection, preterm...... birth, and low birthweight. Antibody responses to the N-terminal region of VAR2CSA during early pregnancy were associated with reduced risks for infections and low birthweight. Among women infected during pregnancy, an increase in CSA binding inhibition was associated with reduced risks for placental...

  6. Stability of monoclonal antibodies at high-concentration

    DEFF Research Database (Denmark)

    Neergaard, Martin S; Nielsen, Anders D; Parshad, Henrik; van de Weert, Marco

    2014-01-01

    Few studies have so far directly compared the impact of antibody subclass on protein stability. This case study investigates two mAbs (one IgG1 and one IgG4 ) with identical variable region. Investigations of mAbs that recognize similar epitopes are necessary to identify possible differences...

  7. Cartilage oligomeric matrix protein specific antibodies are pathogenic

    DEFF Research Database (Denmark)

    Geng, Hui; Nandakumar, Kutty Selva; Pramhed, Anna; Aspberg, Anders; Mattsson, Ragnar; Holmdahl, Rikard

    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 and the...

  8. Antibody fragments: Hope and hype

    OpenAIRE

    Nelson, Aaron L

    2010-01-01

    The antibody molecule is modular and separate domains can be extracted through biochemical or genetic means. It is clear from review of the literature that a wave of novel, antigen-specific molecular forms may soon enter clinical evaluation. This report examines the developmental histories of therapeutics derived from antigen-specific fragments of antibodies produced by recombinant processes. Three general types of fragments were observed, antigen-binding fragments (Fab), single chain variabl...

  9. Functional effects of anticardiolipin antibodies.

    Science.gov (United States)

    Harris, E N; Pierangeli, S S

    1996-10-01

    The 'lupus anticoagulant' phenomenon is the best documented functional effect of antiphospholipid (aPL) antibodies, occurring either by inhibition of the prothrombinase and/or Factor X activation reactions. Understanding the mechanism by which aPL antibodies inhibit phospholipid dependent coagulation reactions may yield important clues about their 'thrombogenic effects' in vivo. We conducted a series of studies to determine the specificity, diversity, and mechanism by which aPL antibodies inhibit phospholipid dependent reactions. Results showed that purified immunoglobulins with lupus anticoagulant and anti-cardiolipin activities were absorbed by negatively charged phospholipids and both activities were recovered from the phospholipid-antibody precipitate. Purified aPL antibodies inhibited the prothrombinase reaction in a plasma free system in which beta 2-glycoprotein 1 (beta 2-GP1) was absent. Affinity purified aPL antibodies had 25-50 times the inhibitory activity of immunoglobulin preparations. The phospholipid binding proteins, beta 2-GPI and placental anticoagulant protein I (PAP I), independently inhibited the prothrombinase reaction, and when these proteins were combined with aPL, inhibition of the prothrombinase reaction was additive. Antibodies of syphilis had no inhibitory effect, partially accounted for by lack of specificity for phosphotidylserine (PS). Although aPL antibodies inhibited the protein C activation reaction, there was no correlation of these activities with inhibition of the prothrombinase reaction. Together, these results show that aPL exert their effects by interaction with negatively charged phospholipids, in particular phosphotidylserine, but lack of correlation between inhibition of the prothrombinase and protein C activation reactions, suggests that the nature of the coagulation protein is also important. PMID:8902763

  10. The antineutrophil antibody in uveitis.

    OpenAIRE

    Young, D W

    1991-01-01

    Ninety eight patients with uveitis of various types were tested for the presence of the antineutrophil antibody or ANCA by an indirect immunofluorescence method. This antibody is found in patients with diseases associated with small vessel vasculitis, including Wegener's granulomatosis and microscopic polyarteritis. Eleven true positive cases were found. A positive test was not associated with the anatomical site of the uveitis but was related to the time course of the disease. In particular ...

  11. Interfacial metal and antibody recognition

    OpenAIRE

    Zhou, Tongqing; Hamer, Dean H.; Hendrickson, Wayne A.; Sattentau, Quentin J.; Kwong, Peter D.

    2005-01-01

    The unique ligation properties of metal ions are widely exploited by proteins, with approximately one-third of all proteins estimated to be metalloproteins. Although antibodies use various mechanisms for recognition, to our knowledge, none has ever been characterized that uses an interfacial metal. We previously described a family of CD4-reactive antibodies, the archetype being Q425. CD4:Q425 engagement does not interfere with CD4:HIV-1 gp120 envelope glycoprotein binding, but it blocks subse...

  12. Pyoderma gangrenosum and anticardiolipin antibody

    Directory of Open Access Journals (Sweden)

    de Godoy Jose Maria

    2006-01-01

    Full Text Available Pyoderma gangrenosum (PG is a rare ulceronecrotic inflammatory cutaneous disorder and is frequently associated with systemic diseases. The authors report a 22-year-old male patient with pyoderma gangrenosum, thrombosis of both popliteal arteries, ischemic stroke and seropositivity for anticardiolipin antibody. Despite intravenous treatment with antibiotics, corticosteroid and heparin, pyoderma gangrenosum caused necrosis of his right lower limb which resulted in amputation. It was concluded that the anticardiolipin antibody may have contributed to the gravity of this case.

  13. Antibodies to watch in 2014.

    Science.gov (United States)

    Reichert, Janice M

    2014-01-01

    Since 2010, mAbs has documented the biopharmaceutical industry's progress in transitioning antibody therapeutics to first Phase 3 clinical studies and regulatory review, and its success at gaining first marketing approvals for antibody-based products. This installment of the "Antibodies to watch" series outlines events anticipated to occur between December 2013 and the end of 2014, including first regulatory actions on marketing applications for vedolizumab, siltuximab, and ramucirumab, as well as the Fc fusion proteins Factor IX-Fc and Factor VIII-Fc; and the submission of first marketing applications for up to five therapeutics (secukinumab, ch14.18, onartuzumab, necitumumab, gevokizumab). Antibody therapeutics in Phase 3 studies are described, with an emphasis on those with study completion dates in 2014, including antibodies targeting interleukin-17a or the interleukin-17a receptor (secukinumab, ixekizumab, brodalumab), proprotein convertase subtilisin/kexin type 9 (alirocumab, evolocumab, bococizumab), and programmed death 1 receptor (lambrolizumab, nivolumab). Five antibodies with US Food and Drug Administration's Breakthrough Therapy designation (obinutuzumab, ofatumumab, lambrolizumab, bimagrumab, daratumumab) are also discussed. PMID:24284914

  14. Avian Diagnostic and Therapeutic Antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Bradley, David Sherman [UND SMHS

    2012-12-31

    A number of infectious agents have the potential of causing significant clinical symptomology and even death, but dispite this, the number of incidence remain below the level that supports producing a vaccine. Therapeutic antibodies provide a viable treatment option for many of these diseases. We proposed that antibodies derived from West Nile Virus (WNV) immunized geese would be able to treat WNV infection in mammals and potential humans. We demonstrated that WNV specific goose antibodies are indeed successful in treating WNV infection both prophylactically and therapeutically in a golden hamster model. We demonstrated that the goose derived antibodies are non-reactogenic, i.e. do not cause an inflammatory response with multiple exposures in mammals. We also developed both a specific pathogen free facility to house the geese during the antibody production phase and a patent-pending purification process to purify the antibodies to greater than 99% purity. Therefore, the success of these study will allow a cost effective rapidly producible therapeutic toward clinical testing with the necessary infrastructure and processes developed and in place.

  15. Radiolabeled monoclonal antibodies: a review

    International Nuclear Information System (INIS)

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

  16. Radioimmunoguided surgery using monoclonal antibody

    International Nuclear Information System (INIS)

    The potential proficiency of radioimmunoguided surgery in the intraoperative detection of tumors was assessed using labeled monoclonal antibody B72.3 in 66 patients with tissue-proved tumor. Monoclonal antibody B72.3 was injected 5 to 42 days preoperatively, and the hand-held gamma-detecting probe was used intraoperatively to detect the presence of tumor. Intraoperative probe counts of less than 20 every 2 seconds, or tumor-to-adjacent normal tissue ratios less than 2:1 were considered negative (system failure). Positive probe counts were detected in 5 of 6 patients with primary colon cancer (83 percent), in 31 of 39 patients with recurrent colon cancer (79 percent), in 4 of 5 patients with gastric cancer (80 percent), in 3 of 8 patients with breast cancer (37.5 percent), and in 4 of 8 patients with ovarian cancer (50 percent) undergoing second-look procedures. Additional patients in each group were scored as borderline positive. Overall, radioimmunoguided surgery using B72.3 identified tumors in 47 patients (71.2 percent), bordered on positive in 6 patients (9.1 percent), and failed to identify tumor in 13 patients (19.7 percent). Improved selection of patients for antigen-positive tumors, the use of higher affinity second-generation antibodies, alternate routes of antibody administration, alternate radionuclides, and more sophisticatedly bioengineered antibodies and antibody combinations should all lead to improvements in radioimmunoguided surgery

  17. Monoclonal antibodies technology. Protocols

    International Nuclear Information System (INIS)

    Full text: Immunization. The first step in preparing useful monoclonal antibodies (MAbs) is to immunize an animal (Balb/c for example) with an appropriate antigen. Methods (only for soluble antigen): Solubilize selected antigen in Phosphate buffer solution (PBS) at pH 7.2-7.4, ideally at a final concentration per animal between 10 to 50 μg/ml. It is recommended that the antigen under consideration be incorporated into the emulsion adjuvants in 1:1 volumetric relation. We commonly use Frend's adjuvant (FA) to prepared immunized solution. The first immunization should be prepared with complete FA, and the another could be prepared with incomplete FA. It is recommended to inject mice with 0.2 ml intraperitoneal (ip) or subcutaneous (sc). Our experience suggests the sc route is the preferred route. A minimum protocol for immunizing mice to generate cells for preparing hybridomas is s follows: immunize sc on day 0, boost sc on day 21, take a trial bleeding on day 26; if antibody titters are satisfactory, boost ip on day 35 with antigen only, and remove the spleen to obtain cells for fusion on day 38. Fusion protocol. The myeloma cell line we are using is X63 Ag8.653. At the moment of fusion myeloma cells need a good viability (at least a 95%). 1. Remove the spleen cells from immunized mice using sterile conditions. An immune spleen should yield between 7 a 10x107 nucleated cells. 2. Place the spleen in 20 ml of serum-free RPMI 1640 in a Petri dish. Using a needle and syringe, inject the spleen with medium to distend and disrupt the spleen stroma and free the nucleated cells. 3. Flush the cell suspension with a Pasteur pipet to disperse clumps of cells. 4. Centrifuge the spleen cell suspension at 250g for 10 min. Resuspend the pellet in serum-free RPMI 1640. Determine cell concentration using Neuhabuer chamber. 5. Mix the myeloma cells and spleen cells in a conical 50-ml tube in serum-free RPMI 1640, 1 x107 spleen cells to 1x106 myeloma cells (ratio 10:1). Centrifuge

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

    International Nuclear Information System (INIS)

    Structural relatedness between the variable region of anti-ligand antibodies and opioid binding sites allowed the generation of anti-idiotypic antibodies which recognized opioid receptors. The IgG3k antibodies which bound to opioid receptors were obtained when an anti-morphine antiserum was the idiotype. Both antibodies bound to opioid receptors, but only one of these blocked the binding of [3H]naloxone. The antibody which did not inhibit the binding of [3H]naloxone was itself displaced from the receptor by opioid ligands. The unique binding properties displayed by this antibody indicated that anti-idiotypic antibodies are not always a perfect image of the original ligand, and therefore may be more useful than typical ligands as probes for the receptor. An auto-anti-idiotypic technique was successfully used to obtain anti-opioid receptor antibodies. Another IgG3k antibody that blocked the binding of [3H]naloxone to rat brain opioid receptors was obtained when a mouse was immunized with naloxone conjugated to bovine serum albumin. These data confirmed that an idiotype-anti-idiotype network which can generate an anti-receptor antibody normally functions when an opioid ligand is introduced into an animal in an immunogenic form

  19. Antibody-protein A conjugated quantum dots for multiplexed imaging of surface receptors in living cells.

    Science.gov (United States)

    Jin, Takashi; Tiwari, Dhermendra K; Tanaka, Shin-Ichi; Inouye, Yasushi; Yoshizawa, Keiko; Watanabe, Tomonobu M

    2010-11-01

    To use quantum dots (QDs) as fluorescent probes for receptor imaging, QD surface should be modified with biomolecules such as antibodies, peptides, carbohydrates, and small-molecule ligands for receptors. Among these QDs, antibody conjugated QDs are the most promising fluorescent probes. There are many kinds of coupling reactions that can be used for preparing antibody conjugated QDs. Most of the antibody coupling reactions, however, are non-selective and time-consuming. In this paper, we report a facile method for preparing antibody conjugated QDs for surface receptor imaging. We used ProteinA as an adaptor protein for binding of antibody to QDs. By using ProteinA conjugated QDs, various types of antibodies are easily attached to the surface of the QDs via non-covalent binding between the F(c) (fragment crystallization) region of antibody and ProteinA. To show the utility of ProteinA conjugated QDs, HER2 (anti-human epidermal growth factor receptor 2) in KPL-4 human breast cancer cells were stained by using anti-HER2 antibody conjugated ProteinA-QDs. In addition, multiplexed imaging of HER2 and CXCR4 (chemokine receptor) in the KPL-4 cells was performed. The result showed that CXCR4 receptors coexist with HER2 receptors in the membrane surface of KPL-4 cells. ProteinA mediated antibody conjugation to QDs is very useful to prepare fluorescent probes for multiplexed imaging of surface receptors in living cells. PMID:20835432

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

    Energy Technology Data Exchange (ETDEWEB)

    Conroy, W.G.

    1988-01-01

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

  1. Antibody Protection Reveals Extended Epitopes on the Human TSH Receptor

    OpenAIRE

    Latif, Rauf; Teixeira, Avelino; Michalek, Krzysztof; Ali, M. Rejwan; Schlesinger, Max; Baliram, Ramkumarie; Morshed, Syed A.; Davies, Terry F.

    2012-01-01

    Stimulating, and some blocking, antibodies to the TSH receptor (TSHR) have conformation-dependent epitopes reported to involve primarily the leucine rich repeat region of the ectodomain (LRD). However, successful crystallization of TSHR residues 22–260 has omitted important extracellular non-LRD residues including the hinge region which connects the TSHR ectodomain to the transmembrane domain and which is involved in ligand induced signal transduction. The aim of the present study, therefore,...

  2. Production of recombinant antibodies using bacteriophages

    OpenAIRE

    Shukra, A. M.; Sridevi, N. V.; Dev Chandran,; Kapil Maithal,

    2014-01-01

    Recombinant antibody fragments such as Fab, scFv, diabodies, triabodies, single domain antibodies and minibodies have recently emerged as potential alternatives to monoclonal antibodies, which can be engineered using phage display technology. These antibodies match the strengths of conventionally produced monoclonal antibodies and offer advantages for the development of immunodiagnostic kits and assays. These fragments not only retain the specificity of the whole monoclonal ...

  3. Human antibody-Fc receptor interactions illuminated by crystal structures.

    Science.gov (United States)

    Woof, Jenny M; Burton, Dennis R

    2004-02-01

    Immunoglobulins couple the recognition of invading pathogens with the triggering of potent effector mechanisms for pathogen elimination. Different immunoglobulin classes trigger different effector mechanisms through interaction of immunoglobulin Fc regions with specific Fc receptors (FcRs) on immune cells. Here, we review the structural information that is emerging on three human immunoglobulin classes and their FcRs. New insights are provided, including an understanding of the antibody conformational adjustments that are required to bring effector cell and target cell membranes sufficiently close for efficient killing and signal transduction to occur. The results might also open up new possibilities for the design of therapeutic antibodies. PMID:15040582

  4. Efficient purification of unique antibodies using peptide affinity-matrix columns

    DEFF Research Database (Denmark)

    Jensen, Liselotte Brix; Riise, Erik; Nielsen, Leif Kofoed;

    2004-01-01

    fragments and had no affinity for other antibodies. Using this peptide matrix MK16 IgG could be purified from cell culture supernatants thereby separating MK16 IgG from bovine IgG normally present in the enriched growth media used for such cells. Investigations of the fine specificity of the ER6.1 peptide......Phage display technology was used to identify peptide ligands with unique specificity for a monoclonal model antibody, MK16, that recognises the human multiple sclerosis associated MHC class II molecule DR2 in complex with a myelin basic protein (MBP)-derived peptide corresponding to residue 85...... demonstrated that it recognised a unique epitope within the heavy chain CDR3 region of the MK16 antibody. Thus, variants of MK16 antibody, which had retained the specificity and affinity of the original antibody but had slightly different amino acid composition in the CDR3 region, were not recognised by the ER...

  5. Docking of B-cell epitope antigen to specific hepatitis B antibody

    Indian Academy of Sciences (India)

    R Rajkannan; E J Padma Malar

    2007-09-01

    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 crystal structure of monoclonal antibody specific for the pres1 region of the hepatitis B virus. At the optimized docked conformation, the interactions between the amino acids of antigen and antibody were examined. It is found that the docked complex is stabilized by 59.3 kcal/mol. The stability of the docked antigen-antibody complex is due to hydrogen bonding and van der Waals interactions. The amino acids of the antigen and antibody responsible for the interaction were identified.

  6. Antibody-Directed Phototherapy (ADP

    Directory of Open Access Journals (Sweden)

    M. Adil Butt

    2013-04-01

    Full Text Available Photodynamic therapy (PDT is a clinically-approved but rather under-exploited treatment modality for cancer and pre-cancerous superficial lesions. It utilises a cold laser or LED to activate a photochemical reaction between a light activated drug (photosensitiser-drug and oxygen to generate cytotoxic oxygen species. These free radical species damage cellular components leading to cell death. Despite its benefits, the complexity, limited potency and side effects of PDT have led to poor general usage. However, the research area is very active with an increasing understanding of PDT-related cell biology, photophysics and significant progress in molecular targeting of disease. Monoclonal antibody therapy is maturing and the next wave of antibody therapies includes antibody-drug conjugates (ADCs, which promise to be more potent and curable. These developments could lift antibody-directed phototherapy (ADP to success. ADP promises to increase specificity and potency and improve drug pharmacokinetics, thus delivering better PDT drugs whilst retaining its other benefits. Whole antibody conjugates with first generation ADP-drugs displayed problems with aggregation, poor pharmacokinetics and loss of immuno-reactivity. However, these early ADP-drugs still showed improved selectivity and potency. Improved PS-drug chemistry and a variety of conjugation strategies have led to improved ADP-drugs with retained antibody and PS-drug function. More recently, recombinant antibody fragments have been used to deliver ADP-drugs with superior drug loading, more favourable pharmacokinetics, enhanced potency and target cell selectivity. These improvements offer a promise of better quality PDT drugs.

  7. Antibodies to watch in 2016.

    Science.gov (United States)

    Reichert, Janice M

    2016-01-01

    The number of novel antibody therapeutics that received first marketing approvals in 2015 met expectations, with 6 (alirocumab (Praluent®), evolocumab (Repatha®), daratumumab (Darzalex®), dinutuximab (Unituxin®), idarucizumab (Praxbind®), mepolizumab (Nucala®)) granted first approvals as of mid-November*. Seven novel antibody therapeutics (begelomab, brodalumab, elotuzumab, ixekizumab, necitumumab, obiltoxaximab, reslizumab) are in regulatory review, and thus a similar number, if not more, are projected to gain first approvals in 2016. Commercial late-stage antibody therapeutics development exceeded expectations by increasing from 39 candidates in Phase 3 studies as of late 2014 to 53 as of late 2015. Of the 53 candidates, transitions to regulatory review by the end of 2016 are projected for 8 (atezolizumab, benralizumab, bimagrumab, durvalumab, inotuzumab ozogamicin, lebrikizumab, ocrelizumab, tremelimumab). Other "antibodies to watch" include 15 candidates (bavituximab, bococizumab, dupilumab, fasinumab, fulranumab, gevokizumab, guselkumab, ibalizumab, LY2951742, onartuzumab, REGN2222, roledumab, romosozumab, sirukumab, Xilonix) undergoing evaluation in Phase 3 studies that have estimated primary completion dates in 2016. As evidenced by the antibody therapeutics discussed in this perspective, the biopharmaceutical industry has a highly active late-stage clinical pipeline that may deliver numerous new products to the global market in the near future. *See Note added in proof for updates through December 31, 2015. PMID:26651519

  8. Autologous antibodies that bind neuroblastoma cells.

    Science.gov (United States)

    Sun, Yujing; Sholler, Giselle S; Shukla, Girja S; Pero, Stephanie C; Carman, Chelsea L; Zhao, Ping; Krag, David N

    2015-11-01

    Antibody therapy of neuroblastoma is promising and our goal is to derive antibodies from patients with neuroblastoma for developing new therapeutic antibodies. The feasibility of using residual bone marrow obtained for clinical indications as a source of tumor cells and a source of antibodies was assessed. From marrow samples, neuroblastoma cells were recovered, grown in cell culture and also implanted into mice to create xenografts. Mononuclear cells from the marrow were used as a source to generate phage display antibody libraries and also hybridomas. Growth of neuroblastoma patient cells was possible both in vitro and as xenografts. Antibodies from the phage libraries and from the monoclonal hybridomas bound autologous neuroblastoma cells with some selectivity. It appears feasible to recover neuroblastoma cells from residual marrow specimens and to generate human antibodies that bind autologous neuroblastoma cells. Expansion of this approach is underway to collect more specimens, optimize methods to generate antibodies, and to evaluate the bioactivity of neuroblastoma-binding antibodies. PMID:26210205

  9. Antisperm antibodies and in vitro fertilization.

    Science.gov (United States)

    Janssen, H J; Bastiaans, B A; Goverde, H J; Hollanders, H M; Wetzels, A A; Schellekens, L A

    1992-08-01

    The purpose of this study was to investigate the influence of antisperm antibodies in the male, the female, or both partners on the outcome of in vitro fertilization treatment. The results in terms of ongoing pregnancies in the male and female antibody-positive group were the same as in the antibody-negative group. In the double antibody-positive group two of the three patients became pregnant. When high levels of antisperm antibodies were present on the spermatozoa, the fertilization rate was significantly reduced. In the female positive group no clear relationship between the antibody titer and the fertilization percentage could be detected. Abnormal semen quality was responsible for a much lower fertilization rate than the presence of antibodies. The conclusion of this study is that in vitro fertilization provides an equal change of conception in couples with antisperm antibodies in comparison with couples with no antibodies if the other semen parameters are normal. PMID:1472812

  10. Remembering antibodies coming of age.

    Science.gov (United States)

    Melchers, Fritz

    2016-01-01

    Fifty years ago, Norbert Hilschmann discovered that antibodies have variable immunoglobulin domains to bind antigens, and constant domains to carry out effector functions in the immune system. Just as this happened, the author of this perspective entered the field of immunology. Ten years later, the genetic basis of antibody variability was discovered by Susumu Tonegawa and his colleagues at the Basel Institute for Immunology, where the author had become a scientific member. At the same time, Georges Köhler, a former graduate student of the author's at the Basel Institute, invented with Cesar Milstein at the Laboratory of Molecular Biology in Cambridge, England, the method to produce monoclonal antibodies. The author describes here his memories connected to these three monumental, paradigm-changing discoveries, which he observed in close proximity. PMID:27144253

  11. Charge-modified single chain antibody constructs of monoclonal antibody CC49: generation, characterization, pharmacokinetics, and biodistribution analysis

    International Nuclear Information System (INIS)

    A novel strategy was developed in which an antibody scFv fragment of the monoclonal antibody (MAb) CC49 was modified by engineering DNA coding sequences to lower its isoelectric point. Negatively charged amino acids were added to the carboxy terminus of the CC49 VH region by adding nucleotide sequences in a polymerase chain reaction (PCR) amplification of the coding sequence of CC49 scFv. Two new DNA constructs coding for CC49 scFv with lower isoelectric points of 5.8 and 5.2 were engineered. These novel strategy-generated, charge-modified antibody constructs were compared for their immunological, pharmacokinetic, and biodistribution properties in athymic mice bearing LS-174T human colon carcinoma xenografts

  12. Cloning murine antibody V-genes with non-degenerate primers and conversion to a recombinant antibody format.

    Science.gov (United States)

    Bialon, Magdalena; Schellenberg, Ludmila; Herzog, Nicolas; Kraus, Stefan; Jörißen, Hannah; Fischer, Rainer; Stein, Christoph; Nähring, Jörg; Barth, Stefan; Püttmann, Christiane

    2014-12-01

    Monoclonal antibodies are produced in cultured hybridoma cell lines, but these cells tend to be unstable; it is therefore necessary to rescue the corresponding genetic information. Here we describe an improved method for the amplification of antibody variable gene (V-gene) information from murine hybridoma cells using a panel of specific, non-degenerate primers. This primer set allows sequences to be rescued from all murine V-genes, except the lambda light chain genes, which rarely contribute to murine immune diversity. We tested the primers against a range of antibodies and recovered specific amplification products in all cases. The heavy and light chain variable regions were subsequently joined by a two-step cloning strategy or by splice overlap extension PCR. PMID:25545205

  13. Antibody Response to Pneumocystis jirovecii

    OpenAIRE

    Daly, Kieran R.; Huang, Laurence; Morris, Alison; Koch, Judy; Crothers, Kristina; Levin, Linda; Eiser, Shary; Satwah, Supriya; Zucchi, Patrizia; Walzer, Peter D.

    2006-01-01

    We conducted a prospective pilot study of the serologic responses to overlapping recombinant fragments of the Pneumocystis jirovecii major surface glycoprotein (Msg) in HIV-infected patients with pneumonia due to P. jirovecii and other causes. Similar baseline geometric mean antibody levels to the fragments measured by an ELISA were found in both groups. Serum antibodies to MsgC in P. jirovecii patients rose to a peak level 3–4 weeks (p50 cells/μL and first episode of pneumocystosis were the ...

  14. Radioimmunotherapy with engineered antibody fragments

    International Nuclear Information System (INIS)

    Authors have developed and begun evaluating radiometal-chelated (213Bi) engineered antibody fragments as radioimmunotherapy agents that target the HER2/neu (c-erbB-2) antigen. The diabody format was found to have 40-fold greater affinity for HER2/neu and to be associated with significantly greater tumor localization than is achieved with scFv molecule. It is shown that short-lived isotopes like 213Bi would be most effective when used in conjunction with antibodies that targeted diffuse malignancies (leukemia or lymphoma) or when used for very rapid pretargeted radioimmunotherapy application in which the radioisotope is conjugated to a very small ligand

  15. Antibody sensed protein surface conformation

    Directory of Open Access Journals (Sweden)

    Scott R. Schricker

    2011-12-01

    Full Text Available An antibody-modified atomic force microscope (AFM tip was used to detect conformational changes of fibronectin deposited on a poly(methyl methacrylate/poly(acrylic acid block copolymer compared to PMMA and a random poly(methyl methacrylate/poly(acrylic acid copolymer with an identical chemical composition. Based on the antibody-protein adhesive force maps and phase imaging, it was found that the nanomorphology of the triblock copolymer induces the desired conformation of fibronectin. This finding demonstrates that block copolymer nanomorphology can be used to regulate protein conformation and potentially cellular response.

  16. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A; Thompson, Vicki S

    2013-02-26

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  17. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A.; Thompson, Vicki S.

    2013-02-26

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  18. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A.; Thompson, Vicki S

    2010-04-13

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  19. Antibody profiling sensitivity through increased reporter antibody layering

    International Nuclear Information System (INIS)

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  20. Use of antinucleoside antibodies to probe the organization of chromosomes denatured by ultraviolet irradiation

    International Nuclear Information System (INIS)

    Ultraviolet irradiation of methanol:acetic acid-fixed human and mouse metaphase chromosomes rendered them capable of binding antibodies specific for purine or pyrimidine bases. Since these antibodies react with single-stranded but not with native DNA, our results indicate that UV irradiation generated single-stranded regions in chromosomal DNA. Using an indirect immunofluorescence technique to detect antibody binding, highly characteristic, nonrandom patterns of antibody binding were observed. Antibodies to adenosine (anti-A) and thymidine (anti-T) produced identical patterns of binding which in most respects matched the chromosome banding patterns produced by quinacrine. However, additional foci of intense fluorescence were seen in the paracentromeric regions of constitutive heterochromatin on chromosomes 1, 9 and 16, regions which had been shown by in situ DNA--RNA hybridization to be the locations of AT-rich human satellite DNA. Antibodies to cytidine were also bound to the same region of chromosome 9. In mouse chromosome preparations, both anti-A and anti-T produced bright fluorescence of the region containing centromeric heterochromatin, which had been shown to be the location of the AT-rich satellite DNA of this species. (U.S.)

  1. Highly sensitive and unbiased approach for elucidating antibody repertoires.

    Science.gov (United States)

    Lin, Sherry G; Ba, Zhaoqing; Du, Zhou; Zhang, Yu; Hu, Jiazhi; Alt, Frederick W

    2016-07-12

    Developing B lymphocytes undergo V(D)J recombination to assemble germ-line V, D, and J gene segments into exons that encode the antigen-binding variable region of Ig heavy (H) and light (L) chains. IgH and IgL chains associate to form the B-cell receptor (BCR), which, upon antigen binding, activates B cells to secrete BCR as an antibody. Each of the huge number of clonally independent B cells expresses a unique set of IgH and IgL variable regions. The ability of V(D)J recombination to generate vast primary B-cell repertoires results from a combinatorial assortment of large numbers of different V, D, and J segments, coupled with diversification of the junctions between them to generate the complementary determining region 3 (CDR3) for antigen contact. Approaches to evaluate in depth the content of primary antibody repertoires and, ultimately, to study how they are further molded by secondary mutation and affinity maturation processes are of great importance to the B-cell development, vaccine, and antibody fields. We now describe an unbiased, sensitive, and readily accessible assay, referred to as high-throughput genome-wide translocation sequencing-adapted repertoire sequencing (HTGTS-Rep-seq), to quantify antibody repertoires. HTGTS-Rep-seq quantitatively identifies the vast majority of IgH and IgL V(D)J exons, including their unique CDR3 sequences, from progenitor and mature mouse B lineage cells via the use of specific J primers. HTGTS-Rep-seq also accurately quantifies DJH intermediates and V(D)J exons in either productive or nonproductive configurations. HTGTS-Rep-seq should be useful for studies of human samples, including clonal B-cell expansions, and also for following antibody affinity maturation processes. PMID:27354528

  2. Structural Characterization of Peptide Antibodies

    DEFF Research Database (Denmark)

    Chailyan, Anna; Marcatili, Paolo

    2015-01-01

    better understand the underlying mechanisms of antibody-antigen interaction here we present a pipeline developed by us to structurally classify immunoglobulin antigen binding sites and to infer key sequence residues and other variables that have a prominent role in each structural class....

  3. A multicenter evaluation of a new antibody test kit for lymphatic filariasis employing recombinant Brugia malayi antigen Bm-14

    OpenAIRE

    Weil, Gary J; Curtis, Kurt C.; Fischer, Peter U.; Kimberly Y Won; Lammie, Patrick J; Joseph, Hayley; Melrose, Wayne D; Brattig, Norbert W.

    2010-01-01

    Antibody tests are useful for mapping the distribution of lymphatic filariasis (LF) in countries and regions and for monitoring progress in elimination programs based on mass drug administration (MDA). Prior antibody tests have suffered from poor sensitivity and/or specificity or from a lack of standardization. We conducted a multicenter evaluation of a new commercial ELISA that detects IgG4 antibodies to the recombinant filarial antigen Bm14. Four laboratories tested a shared panel of coded ...

  4. Alternative affinity tools: more attractive than antibodies?

    NARCIS (Netherlands)

    Ruigrok, V.J.B.; Levisson, M.; Eppink, M.H.M.; Smidt, H.; Oost, van der J.

    2011-01-01

    Antibodies are the most successful affinity tools used today, in both fundamental and applied research (diagnostics, purification and therapeutics). Nonetheless, antibodies do have their limitations, including high production costs and low stability. Alternative affinity tools based on nucleic acids

  5. Detection of Campylobacter species using monoclonal antibodies

    Science.gov (United States)

    Young, Colin R.; Lee, Alice; Stanker, Larry H.

    1999-01-01

    A panel of species specific monoclonal antibodies were raised to Campylobacter coli, Campylobacter jejuni and Campylobacter lari. The isotypes, and cross-reactivity profiles of each monoclonal antibody against an extensive panel of micro- organisms, were determined.

  6. Progranulin antibodies in autoimmune diseases.

    Science.gov (United States)

    Thurner, Lorenz; Preuss, Klaus-Dieter; Fadle, Natalie; Regitz, Evi; Klemm, Philipp; Zaks, Marina; Kemele, Maria; Hasenfus, Andrea; Csernok, Elena; Gross, Wolfgang L; Pasquali, Jean-Louis; Martin, Thierry; Bohle, Rainer Maria; Pfreundschuh, Michael

    2013-05-01

    Systemic vasculitides constitute a heterogeneous group of diseases. Autoimmunity mediated by B lymphocytes and their humoral effector mechanisms play a major role in ANCA-associated vasculitis (AAV) as well as in non-ANCA associated primary systemic vasculitides and in the different types of autoimmune connective tissue disorders and rheumatoid arthritis. In order to detect autoantibodies in systemic vasculitides, we screened protein macroarrays of human cDNA expression libraries with sera from patients with ANCA-associated and ANCA-negative primary systemic vasculitides. This approach led to the identification of antibodies against progranulin, a 88 kDA secreted glycoprotein with strong anti-inflammatory activity in the course of disease of giant-cell arteritis/polymyalgia rheumatica (14/65), Takayasu's arteritis (4/13), classical panarteritis nodosa (4/10), Behcet's disease (2/6) and in the course of disease in granulomatosis with polyangiitis (31/75), Churg-Strauss syndrome (7/23) and in microscopic polyangiitis (7/19). In extended screenings the progranulin antibodies were also detected in other autoimmune diseases such as systemic lupus erythematosus (39/91) and rheumatoid arthritis (16/44). Progranulin antibodies were detected only in 1 of 97 healthy controls. Anti-progranulin positive patients with systemic vasculitides, systemic lupus erythematosus or rheumatoid arthritis had significant lower progranulin plasma levels, indicating a neutralizing effect. In light of the anti-inflammatory effects of progranulin, progranulin antibodies might exert pro-inflammatory effects thus contributing to the pathogenesis of the respective autoimmune diseases and might serve as a marker for disease activity. This hypothesis is supported by the fact that a positive progranulin antibody status was associated with active disease in granulomatosis with polyangiitis. PMID:23149338

  7. Virus Strain Discrimination Using Recombinant Antibodies

    OpenAIRE

    Boonham, N.; Barker, I.

    2002-01-01

    Most routine testing for plant viruses is currently carried out using monoclonal and polyclonal antibodies. Traditional methods of antibody production however can be time consuming and require the use of expensive cell culture facilities. Recombinant antibody technology however is starting to make an impact in this area, enabling the selection of antibody fragments in a few weeks compared with the many months associated with traditional methods and requires only basic microbiological faciliti...

  8. Antibody-dependent cellular cytotoxicity-mediating antibodies from an HIV-1 vaccine efficacy trial target multiple epitopes and preferentially use the VH1 gene family.

    Science.gov (United States)

    Bonsignori, Mattia; Pollara, Justin; Moody, M Anthony; Alpert, Michael D; Chen, Xi; Hwang, Kwan-Ki; Gilbert, Peter B; Huang, Ying; Gurley, Thaddeus C; Kozink, Daniel M; Marshall, Dawn J; Whitesides, John F; Tsao, Chun-Yen; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Pitisuttithum, Punnee; Rerks-Ngarm, Supachai; Kim, Jerome H; Michael, Nelson L; Tomaras, Georgia D; Montefiori, David C; Lewis, George K; DeVico, Anthony; Evans, David T; Ferrari, Guido; Liao, Hua-Xin; Haynes, Barton F

    2012-11-01

    The ALVAC-HIV/AIDSVAX-B/E RV144 vaccine trial showed an estimated efficacy of 31%. RV144 secondary immune correlate analysis demonstrated that the combination of low plasma anti-HIV-1 Env IgA antibodies and high levels of antibody-dependent cellular cytotoxicity (ADCC) inversely correlate with infection risk. One hypothesis is that the observed protection in RV144 is partially due to ADCC-mediating antibodies. We found that the majority (73 to 90%) of a representative group of vaccinees displayed plasma ADCC activity, usually (96.2%) blocked by competition with the C1 region-specific A32 Fab fragment. Using memory B-cell cultures and antigen-specific B-cell sorting, we isolated 23 ADCC-mediating nonclonally related antibodies from 6 vaccine recipients. These antibodies targeted A32-blockable conformational epitopes (n = 19), a non-A32-blockable conformational epitope (n = 1), and the gp120 Env variable loops (n = 3). Fourteen antibodies mediated cross-clade target cell killing. ADCC-mediating antibodies displayed modest levels of V-heavy (VH) chain somatic mutation (0.5 to 1.5%) and also displayed a disproportionate usage of VH1 family genes (74%), a phenomenon recently described for CD4-binding site broadly neutralizing antibodies (bNAbs). Maximal ADCC activity of VH1 antibodies correlated with mutation frequency. The polyclonality and low mutation frequency of these VH1 antibodies reveal fundamental differences in the regulation and maturation of these ADCC-mediating responses compared to VH1 bNAbs. PMID:22896626

  9. Structure-based, targeted deglycosylation of HIV-1 gp120 and effects on neutralization sensitivity and antibody recognition

    International Nuclear Information System (INIS)

    The human immunodeficiency virus (HIV-1) exterior envelope glycoprotein, gp120, mediates receptor binding and is the major target for neutralizing antibodies. Primary HIV-1 isolates are characteristically more resistant to broadly neutralizing antibodies, although the structural basis for this resistance remains obscure. Most broadly neutralizing antibodies are directed against functionally conserved gp120 regions involved in binding to either the primary virus receptor, CD4, or the viral coreceptor molecules that normally function as chemokine receptors. These antibodies are known as CD4 binding site (CD4BS) and CD4-induced (CD4i) antibodies, respectively. Inspection of the gp120 crystal structure reveals that although the receptor-binding regions lack glycosylation, sugar moieties lie proximal to both receptor-binding sites on gp120 and thus in proximity to both the CD4BS and the CD4i epitopes. In this study, guided by the X-ray crystal structure of gp120, we deleted four N-linked glycosylation sites that flank the receptor-binding regions. We examined the effects of selected changes on the sensitivity of two prototypic HIV-1 primary isolates to neutralization by antibodies. Surprisingly, removal of a single N-linked glycosylation site at the base of the gp120 third variable region (V3 loop) increased the sensitivity of the primary viruses to neutralization by CD4BS antibodies. Envelope glycoprotein oligomers on the cell surface derived from the V3 glycan-deficient virus were better recognized by a CD4BS antibody and a V3 loop antibody than were the wild-type glycoproteins. Absence of all four glycosylation sites rendered a primary isolate sensitive to CD4i antibody-mediated neutralization. Thus, carbohydrates that flank receptor-binding regions on gp120 protect primary HIV-1 isolates from antibody-mediated neutralization

  10. Prevalence of antibodies against Neospora spp. and Sarcocystis neurona in donkeys from northeastern Brazil.

    Science.gov (United States)

    Gennari, Solange Maria; Pena, Hilda Fátima de Jesus; Lindsay, David Scott; Lopes, Marcos Gomes; Soares, Herbert Sousa; Cabral, Aline Diniz; Vitaliano, Sérgio Netto; Amaku, Marcos

    2016-01-01

    Sarcocystis neurona and Neospora hughesi are coccidian protozoa that can cause neurological illness in horses in America. In this study we report seroprevalence of Neospora spp. andS. neurona in sera of 333 donkeys from the northeastern region of Brazil. Antibodies to Neospora spp. were detected in 2% (7 donkeys) of 333 sera tested by the indirect fluorescent antibody test (IFAT) with a cut-off dilution of 1:40. Antibodies to S. neurona were found in 3% (10 donkeys) of the samples tested by IFAT (cut-off ≥50) and 21% (69 donkeys) by the direct agglutination test (SAT ≥50). The SAT and IFAT results for S. neurona showed a poor concordance (value of Kappa=0.051). This is the first report of Neospora spp. antibodies in Brazilian donkeys and the first detection of antibodies against S. neurona in this animal species. PMID:26982557

  11. Prevalence of antibodies against Neospora spp. and Sarcocystis neurona in donkeys from northeastern Brazil

    Directory of Open Access Journals (Sweden)

    Solange Maria Gennari

    2016-03-01

    Full Text Available Abstract Sarcocystis neurona and Neospora hughesi are coccidian protozoa that can cause neurological illness in horses in America. In this study we report seroprevalence of Neospora spp. andS. neurona in sera of 333 donkeys from the northeastern region of Brazil. Antibodies to Neospora spp. were detected in 2% (7 donkeys of 333 sera tested by the indirect fluorescent antibody test (IFAT with a cut-off dilution of 1:40. Antibodies to S. neurona were found in 3% (10 donkeys of the samples tested by IFAT (cut-off ≥50 and 21% (69 donkeys by the direct agglutination test (SAT ≥50. The SAT and IFAT results for S. neurona showed a poor concordance (value of Kappa=0.051. This is the first report ofNeospora spp. antibodies in Brazilian donkeys and the first detection of antibodies against S. neurona in this animal species.

  12. Immunoglobulin G4: an odd antibody

    NARCIS (Netherlands)

    R.C. Aalberse; S.O. Stapel; J. Schuurman; T. Rispens

    2009-01-01

    Despite its well-known association with IgE-mediated allergy, IgG4 antibodies still have several poorly understood characteristics. IgG4 is a very dynamic antibody: the antibody is involved in a continuous process of half-molecules (i.e. a heavy and attached light-chain) exchange. This process, also

  13. Neutralizing Antibody Response and Antibody-Dependent Cellular Cytotoxicity in HIV-1-Infected Individuals from Guinea-Bissau and Denmark.

    Science.gov (United States)

    Borggren, Marie; Jensen, Sanne Skov; Heyndrickx, Leo; Palm, Angelica A; Gerstoft, Jan; Kronborg, Gitte; Hønge, Bo Langhoff; Jespersen, Sanne; da Silva, Zacarias José; Karlsson, Ingrid; Fomsgaard, Anders

    2016-05-01

    The development of therapeutic and prophylactic HIV vaccines for African countries is urgently needed, but the question of what immunogens to use needs to be answered. One approach is to include HIV envelope immunogens derived from HIV-positive individuals from a geographically concentrated epidemic with more limited viral genetic diversity for a region-based vaccine. To address if there is a basis for a regional selected antibody vaccine, we have screened two regionally separate cohorts from Guinea-Bissau and Denmark for neutralizing antibody activity and antibody-dependent cellular cytotoxicity (ADCC) against local and nonlocal circulating HIV-1 strains. The neutralizing activity did not demonstrate higher potential against local circulating strains according to geography and subtype determination, but the plasma from Danish individuals demonstrated significantly higher inhibitory activity than that from Guinea-Bissau individuals against both local and nonlocal virus strains. Interestingly, an opposite pattern was observed with ADCC activity, where Guinea-Bissau individual plasma demonstrated higher activity than Danish plasma and was specifically against the local circulating subtype. Thus, on basis of samples from these two cohorts, no local-specific neutralizing activity was detected, but a local ADCC response was identified in the Guinea-Bissau samples, suggesting potential use of regional immunogens for an ADCC-inducing vaccine. PMID:26621287

  14. Production, isolation, and characterization of rabbit anti-idiotypic antibodies directed against human antithyrotrophin receptor antibodies.

    OpenAIRE

    Baker, J. R.; Lukes, Y G; Burman, K. D.

    1984-01-01

    Previous studies have shown that anti-idiotypic antibodies can be developed in vivo through animal immunization with idiotype, and that these antibodies can be isolated from other anti-immunoglobulin antibodies by affinity purification. These techniques have relied on large amounts of idiotype, which were produced either by hyperimmunization or by monoclonal antibodies, to serve as the affinity adsorbent. In the present study, we produced anti-idiotypic antibodies to human anti-thyroid-stimul...

  15. New high affinity monoclonal antibodies recognize non-overlapping epitopes on mesothelin for monitoring and treating mesothelioma.

    Science.gov (United States)

    Zhang, Yi-Fan; Phung, Yen; Gao, Wei; Kawa, Seiji; Hassan, Raffit; Pastan, Ira; Ho, Mitchell

    2015-01-01

    Mesothelin is an emerging cell surface target in mesothelioma and other solid tumors. Most antibody drug candidates recognize highly immunogenic Region I (296-390) on mesothelin. Here, we report a group of high-affinity non-Region I rabbit monoclonal antibodies. These antibodies do not compete for mesothelin binding with the immunotoxin SS1P that binds Region I of mesothelin. One pair of antibodies (YP218 and YP223) is suitable to detect soluble mesothelin in a sandwich ELISA with high sensitivity. The new assay can also be used to measure serum mesothelin concentration in mesothelioma patients, indicating its potential use for monitoring patients treated with current antibody therapies targeting Region I. The antibodies are highly specific and sensitive in immunostaining of mesothelioma. To explore their use in tumor therapy, we have generated the immunotoxins based on the Fv of these antibodies. One immunotoxin (YP218 Fv-PE38) exhibits potent anti-tumor cytotoxicity towards primary mesothelioma cell lines in vitro and an NCI-H226 xenograft tumor in mice. Furthermore, we have engineered a humanized YP218 Fv that retains full binding affinity for mesothelin-expressing cancer cells. In conclusion, with their unique binding properties, these antibodies may be promising candidates for monitoring and treating mesothelioma and other mesothelin-expressing cancers. PMID:25996440

  16. Solid phase double-antibody radioimmunoassay procedure

    International Nuclear Information System (INIS)

    The present invention is concerned with the radioimmunoassay (RIA) procedure for assaying body fluid content of an antigenic substance which may either be an antigen itself or a hapten capable of being converted, such as by means of reaction with a protein, to an antigenic material. The present invention is concerned with a novel and improved modification of a double-antibody RIA technique in which there is a first antibody that is specific to the antigenic substance suspected to be present in a body fluid from which the assay is intended. The second antibody, however, is not specific to the antigenic substance or analyte, but is an antibody against the first antibody

  17. The role of antibodies in myasthenia gravis.

    Science.gov (United States)

    De Baets, M; Stassen, M H W

    2002-10-15

    Myasthenia gravis is an autoimmune disease associated with antibodies directed to the postsynaptic acetylcholine receptor. These antibodies reduce the number of receptors. Autoantibodies against AChR and other muscle antigens can be used for the diagnosis of myasthenia gravis and related disorders. The origin and the role of these antibodies in the disease are discussed. Experimental autoimmune myasthenia gravis, an experimental model closely mimicking the disease, has provided answers to many questions about the role of antibodies, complement macrophages and AChR anchor proteins. Genetically modified anti-AChR antibodies may also be used in the future to treat myasthenia. PMID:12220686

  18. Ocorrência de anticorpos anti-Neospora caninum em cães da microrregião da Serra de Botucatu, Estado de São Paulo, Brasil Occurrence of antibodies anti-Neospora caninum in dogs of Botucatu range Micro region, State of São Paulo, Brazil

    Directory of Open Access Journals (Sweden)

    Carla Cristina G. de Moraes

    2008-03-01

    of bovine abortion and canine neuropathy. Considering the isolation of N. caninum and the frequent serology in bovines of our region, the objectives of the present trial were to evaluate the occurrence of antibodies anti -N. caninum in dogs of the Botucatu range micro region, in the State of São Paulo, and its association with gender, age and origin (urban area - exclusively from the city; rural area - only farms and cottages; and periurban area - access to the urban and rural areas of the dogs studied. A total of 963 dogs of defined breeds or non-defined breeds, of both genders and different ages, showing no clinical symptoms, were analyzed. Animals were randomly selected during the anti-rabies vaccination campaign in the region, from May to September 1998. Serum samples obtained from the animals were evaluated by means of the Indirect Fluorescent Antibody Test (IFAT using as antigen N. caninum, standard strain NC-1. A total of 245 animals were reagent (25.4% positive results, with 161 (27.5% males and 84 (22.3% females. According to the origin - urban, rural and periurban areas - 223 (25.8%, 11(16.9% and 11(33.3% dogs were, respectively, reagent to the IFAT. All of the 11 cities in this region presented seropositive dogs with occurrence rates ranging from 53.5 to 8.9%. The lower positive percentage was observed in dogs below 1 year old (16.2% when compared with animals from 1 to 4 years old and more than 4 years old (28.4 % and 28.0% respectively, which did not present any differences between them. Results obtained characterized the seropositivity for N. caninum in dogs from all Municipalities in the Botucatu micro region showing the wide distribution of the agent in the region.

  19. Two Escape Mechanisms of Influenza A Virus to a Broadly Neutralizing Stalk-Binding Antibody

    Science.gov (United States)

    Chai, Ning; Swem, Lee R.; Reichelt, Mike; Chen-Harris, Haiyin; Luis, Elizabeth; Park, Summer; Fouts, Ashley; Lupardus, Patrick; Wu, Thomas D.; Li, Olga; McBride, Jacqueline; Lawrence, Michael; Xu, Min; Tan, Man-Wah

    2016-01-01

    Broadly neutralizing antibodies targeting the stalk region of influenza A virus (IAV) hemagglutinin (HA) are effective in blocking virus infection both in vitro and in vivo. The highly conserved epitopes recognized by these antibodies are critical for the membrane fusion function of HA and therefore less likely to be permissive for virus mutational escape. Here we report three resistant viruses of the A/Perth/16/2009 strain that were selected in the presence of a broadly neutralizing stalk-binding antibody. The three resistant viruses harbor three different mutations in the HA stalk: (1) Gln387Lys; (2) Asp391Tyr; (3) Asp391Gly. The Gln387Lys mutation completely abolishes binding of the antibody to the HA stalk epitope. The other two mutations, Asp391Tyr and Asp391Gly, do not affect antibody binding at neutral pH and only slightly reduce binding at low pH. Interestingly, they enhance the fusion ability of the HA, representing a novel mechanism that allows productive membrane fusion even in the presence of antibody and hence virus escape from antibody neutralization. Therefore, these mutations illustrate two different resistance mechanisms used by IAV to escape broadly neutralizing stalk-binding antibodies. Compared to the wild type virus, the resistant viruses release fewer progeny viral particles during replication and are more sensitive to Tamiflu, suggesting reduced viral fitness. PMID:27351973

  20. Selection of apoptotic cell specific human antibodies from adult bone marrow.

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    Caroline Grönwall

    Full Text Available Autoreactive antibodies that recognize neo-determinants on apoptotic cells in mice have been proposed to have protective, homeostatic and immunoregulatory properties, although our knowledge about the equivalent antibodies in humans has been much more limited. In the current study, human monoclonal antibodies with binding specificity for apoptotic cells were isolated from the bone marrow of healthy adults using phage display technology. These antibodies were shown to recognize phosphorylcholine (PC-associated neo-determinants. Interestingly, three of the four identified apoptotic cell-specific antibody clones were encoded by VH3 region rearrangements with germline or nearly germline configuration without evidence of somatic hypermutation. Importantly, the different identified antibody clones had diverse heavy chain CDR3 and deduced binding surfaces as suggested by structure modeling. This may suggest a potentially great heterogeneity in human antibodies recognizing PC-related epitopes on apoptotic cells. To re-construct the postulated structural format of the parental anti-PC antibody, the dominant clone was also expressed as a recombinant human polymeric IgM, which revealed a substantially increased binding reactivity, with dose-dependent and antigen-inhibitable binding of apoptotic cells. Our findings may have implication for improved prognostic testing and therapeutic interventions in human inflammatory disease.

  1. Production and characterization of polyclonal antibody against a synthetic peptide from β-actin protein

    Directory of Open Access Journals (Sweden)

    Nazila Amini

    2014-06-01

    Full Text Available Objective(s:Antibodies against actin, as one of the most widely studied structural and multifunctional housekeeping proteins in eukaryotic cells, are used as internal loading controls in western blot analyses. The aim of this study was to produce polyclonal antibody against a synthetic peptide derived from N-terminal region of β-actin protein to be used as a protein loading control in western blot and other assay systems. Materials and Methods: A synthetic peptide derived from β-actin protein was designed and conjugated to Keyhole limpet hemocyanin (KLH (and used to immunize a white New Zealand rabbit. The antibody was purified from serum by affinity chromatography column. The purity of the antibody was determined by SDS-PAGE and its ability to recognize the immunizing peptide was measured by ELISA. The reactivity of the antibody with β-actin protein in a panel of different cell lysates was then evaluated by western blot. In addition, the reactivity of the antibody with the corresponding protein was also evaluated by Immunocytochemistry and Immunohistochemistry in different samples. Results: The antibody could recognize the immunizing peptide in ELISA. It could also recognize            β-actin protein in western blot as well as in immunocytochemistry and immunohistochemistry. Conclusion: Our data suggest that this antibody may be used as an internal control in western blot analyses as well as in other immunological applications such as ELISA,immunocytochemistry and immunohistochemistry.

  2. 9 CFR 113.452 - Erysipelothrix Rhusiopathiae Antibody.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Erysipelothrix Rhusiopathiae Antibody... REQUIREMENTS Antibody Products § 113.452 Erysipelothrix Rhusiopathiae Antibody. Erysipelothrix Rhusiopathiae Antibody is a specific antibody product containing antibodies directed against one or more somatic...

  3. Monoclonal Antibody Therapies against Anthrax

    OpenAIRE

    Zhaochun Chen; Mahtab Moayeri; Robert Purcell

    2011-01-01

    Anthrax is a highly lethal infectious disease caused by the spore-forming bacterium Bacillus anthracis. It not only causes natural infection in humans but also poses a great threat as an emerging bioterror agent. The lethality of anthrax is primarily attributed to the two major virulence factors: toxins and capsule. An extensive effort has been made to generate therapeutically useful monoclonal antibodies to each of the virulence components: protective antigen (PA), lethal factor (LF) and ede...

  4. Antibody Peptide Based Antifungal Immunotherapy

    OpenAIRE

    Magliani, Walter; Conti, Stefania; Giovati, Laura; Zanello, Pier Paolo; Sperindè, Martina; Ciociola, Tecla; Polonelli, Luciano

    2012-01-01

    Fungal infections still represent relevant human illnesses worldwide and some are accompanied by unacceptably high mortality rates. The limited current availability of effective and safe antifungal agents makes the development of new drugs and approaches of antifungal vaccination/immunotherapy every day more needed. Among them, small antibody(Ab)-derived peptides are arousing great expectations as new potential antifungal agents. In this topic, the search path from the study of the yeast kill...

  5. Epigenetics of the antibody response

    OpenAIRE

    Li, Guideng; Zan, Hong; Xu, Zhenming; Casali, Paolo

    2013-01-01

    Epigenetic marks, such as DNA methylation, histone posttranslational modifications and microRNAs, are induced in B cells by the same stimuli that drive the antibody response. They play major roles in regulating somatic hypermutation (SHM), class switch DNA recombination (CSR) and differentiation to plasma cells or long-lived memory B cells. Histone modifications target the CSR and, possibly, SHM machinery to the immunoglobulin locus; they together with DNA methylation and microRNAs modulate t...

  6. Pharmacological selection of antibodies for immunoscintigraphy

    International Nuclear Information System (INIS)

    The recent development of hybridoma technology has resulted in the production of monoclonal antibodies that recognize a variety of tumor antigens. Many antibodies have been developed and some of them are used with different success in clinical practice. A list of criteria is proposed for the selection of antibodies suitable for imaging studies illustrated with the example of two monoclonal antibodies anti-CEA and 19.9 used in colorectal carcinoma imaging. Monoclonal antibodies obtained today are not truly tumor-specific, they are tumor-associated; this suggests that some cross-reactions with normal tissues exist. For immunoscintigraphical use it is important to select antibodies which procedure high tumor cell staining with limited reactivity against normal tissues. Antibodies can be separated into F(ab')2 and Fab fragments which diffuse more easily into the tumor with a rapid clearance from the circulation giving higher tumor to normal tissues ratio at an early time. Antibodies with both high affinity and avidity towards tumor cell receptors produce better imaging results. Antibodies can be labelled directly with iodine or technetium and with indium using chelating agents. In vivo kinetics of radiolabelled antibodies are very different considering the nuclide and the labelling method used. Pharmacokinetics on nude mice grated with human tumors are very useful for selecting the most appropriate nuclide antibody fragment and the most efficient labelling technique for a given application. (author)

  7. Application of Monoclonal Antibodies in Veterinary Parasitology

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    Gupta A.

    2011-08-01

    Full Text Available The discovery of hybridoma technology by Kohler and Milstein in 1975, heralded a new era in antibody research. Mouse hybridomas were the first reliable source of monoclonal antibodies. The generation of monoclonal antibodies from species other than rats and mice, has developed slowly over the last 30 years. The advent of antibody engineering and realization of the advantages of non murine antibodies has increased their relevance recently. However, in the area of veterinary parasitology, monoclonal antibodies are just beginning to fulfill the promises inherent in their great specificity for recognizing and selectively binding to antigens. This review describes the recent advances in the application of monoclonal antibodies for immunodiagnosis / prophylaxis and immunotherapy of parasitic diseases. [Vet. World 2011; 4(4.000: 183-188

  8. Functional analysis of neutralizing antibodies against Clostridium perfringens epsilon-toxin.

    Science.gov (United States)

    McClain, Mark S; Cover, Timothy L

    2007-04-01

    The Clostridium perfringens epsilon-toxin causes a severe, often fatal illness (enterotoxemia) characterized by cardiac, pulmonary, kidney, and brain edema. In this study, we examined the activities of two neutralizing monoclonal antibodies against the C. perfringens epsilon-toxin. Both antibodies inhibited epsilon-toxin cytotoxicity towards cultured MDCK cells and inhibited the ability of the toxin to form pores in the plasma membranes of cells, as shown by staining cells with the membrane-impermeant dye 7-aminoactinomycin D. Using an antibody competition enzyme-linked immunosorbent assay (ELISA), a peptide array, and analysis of mutant toxins, we mapped the epitope recognized by one of the neutralizing monoclonal antibodies to amino acids 134 to 145. The antibody competition ELISA and analysis of mutant toxins suggest that the second neutralizing monoclonal antibody also recognizes an epitope in close proximity to this region. The region comprised of amino acids 134 to 145 overlaps an amphipathic loop corresponding to the putative membrane insertion domain of the toxin. Identifying the epitopes recognized by these neutralizing antibodies constitutes an important first step in the development of therapeutic agents that could be used to counter the effects of the epsilon-toxin. PMID:17261609

  9. Development of antibody against sulfamethazine

    International Nuclear Information System (INIS)

    Sulfamethazine (SMT) is widely used to treat bacterial and protozoan infections in food animals. So its residue has been detected in various food products, and in Europe, the tolerance level for sulfonamides in meat and milk is 100 ng/g. To ensure that residues in animal food products do not exceed this limit, a simple, sensitive, and rapid method to determinate their residues in animal tissues is needed. In this paper the development of polyclonal or monoclonal antibodies against sulfamethazine (SMT) and a simplified method to identify residual sulfamethazine by radio immunoassay (RIA) is presented. Polyclonal antibodies (PcAbs) against sulfamethazine (SMT) were obtained by immunizing rabbits with SMT-conjugated bovine serum albumin (BSA). The association constants (Ka) of the PcAbs were higher than 108 and the cross-reactivities with Sulfadiazine(SD), Sulfaquinoxaline(SQX) which were structurally related compounds were lower than 0.05%(RIA). Simultaneous, six strains of hybridoma cell were prepared which can secrete monoclonal antibodies (McAbs) against SMT . The Ka of the McAbs against SMT were higher than 107 and the cross-reactivities with SD, SQX were lower than 0.1%(RIA). (authors)

  10. Monoclonal antibodies in targeted therapy

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    Beata Powroźnik

    2012-09-01

    Full Text Available Targeted therapy is a new therapeutic method consisting in the inhibition of specific molecular pathways. In modern therapy, the key role is played by monoclonal antibodies, included in the group of biological agents. The success of molecularly targeted therapy is to define the proper “molecular target”, selecting the right drug active against a specific “target” and selecting a group of patients who benefit from treatment. Introduction of targeted therapy resulted in improved results of the treatment of many serious and chronic diseases. In general, targeted molecular therapies have good toxicity profiles, but some patients are exquisitely sensitive to these drugs and can develop particular and severe toxicities. Patient selection and proper monitoring significantly decrease the risk of life-threatening adverse events. Data concerning late side effects are still unavailable because of the short follow-up of molecularly targeted therapy. Currently in the U.S. and Europe there are approximately 31 registered therapeutic monoclonal antibodies, while 160 are subjected to clinical trials. This paper presents an overview of therapeutic monoclonal antibodies currently used in therapy and the present state of knowledge about them. 

  11. Serosurveillance for antibodies to rinderpest and peste de petits ruminants in bovins and small ruminants in Mali

    International Nuclear Information System (INIS)

    567 sera from 58 herds of small ruminants showed a very low prevalence of rinderpest antibodies (2 sera positive). The prevalence of antibodies to peste des petits ruminants (PPR) was very high and in the region of Segou only 7 herds were negative. The study of bovine sera included 436 sera from 15 herds with a low antibody prevalence to rinderpest. The prevalence of antibodies to rinderpest was very low (8 positive sera). These results would indicate that in Mali PPR infection in small ruminants only very occasionally infects cattle and would have a negligible effect on a rinderpest vaccination programme. (author). 3 refs, 9 tabs

  12. Soroprevalência de anticorpos anti-Toxoplasma gondii em bovinos e funcionários de matadouros da microrregião de Pato Branco, Paraná, Brasil Seroprevalence of anti-Toxoplasma gondii antibodies in cattle and slaughterhouse workers in the region of Pato Branco, Paraná, Brazil

    Directory of Open Access Journals (Sweden)

    Heitor Daguer

    2004-08-01

    Full Text Available Visando avaliar a participação da carne bovina na epidemiologia da toxoplasmose, foram coletadas amostras de soro de 348 bovinos e de 64 funcionários em quatro matadouros da microrregião de Pato Branco, Estado do Paraná, Brasil. Os soros dos bovinos foram avaliados pela reação de imunofluorescência indireta (RIFI e apresentaram soropositividade (IgG em 41,4% das amostras examinadas. O título mais freqüentemente encontrado foi o de 64 (92,4%. Nenhum animal apresentou título superior a 1024. Não foram encontradas diferenças significativas em relação ao sexo, idade e procedência dos animais. Os soros humanos, avaliados pelos testes de RIFI e de ELISA (imunoensaio enzimático para IgG, apresentaram 67,2% e 84,4% de positividade, respectivamente. Não foram encontradas diferenças significativas com relação às variáveis idade, sexo, tempo de serviço no abatedouro, contato com gatos e hábito de ingerir carne crua ou mal cozida. Os resultados sugerem que a carne bovina pode desempenhar importante papel na manutenção da toxoplasmose na região.In order to determine the role of bovine meat in the epidemiology of toxoplasmosis, 348 serum samples were collected from cattle and 64 serum samples from slaughterhouse workers at four plants in the region of Pato Branco, Paraná state, southern Brazil. Cattle sera were examined by the indirect fluorescent antibody test (IFAT, showing an IgG-seropositivity of 41.4%. The most frequently titer found was 64 (92.4%. Maximum titer found was 1024. There was no significant difference concerning sex, age and origin of positive sera. Human sera were tested by the IFAT and enzyme-linked immunosorbent assay (ELISA, showing 67.2% and 84.4% of IgG-seropositivity, respectively. No significant difference was observed between prevalences and age, sex, duration of employment at slaughterhouse, contact with cats and eating raw or undercooked meat. Results suggest that bovine meat may be a possible source of

  13. Presence of antibotulinum neurotoxin antibodies in selected wild canids in Israel.

    Science.gov (United States)

    Steinman, Amir; Millet, Neta; Frenkel, Chana; King, Roni; Shpigel, Nahum Y

    2007-07-01

    Serum samples from 35 golden jackals (Canis aureus syriacus), eight wolves (Canis lupus), and four red foxes (Vulpes vulpes) from various regions of Israel were collected during the years 2001-04 and tested for antibodies to Clostridium botulinum neurotoxin (BoNT) types C and D. Antibodies against BoNT types C and D were detected in 10 (29%) and in 3 (9%) of 35 golden jackals, respectively, using enzyme-linked immunosorbent assay. This report describes detection of anti BoNT antibodies in wild canids other than coyotes (Canis latrans) for the first time and demonstrates that C. botulinum type C is prevalent in Israel. PMID:17699099

  14. Prevalence of antibodies against Neospora spp. and Sarcocystis neurona in donkeys from northeastern Brazil

    OpenAIRE

    Solange Maria Gennari; Hilda Fátima de Jesus Pena; David Scott Lindsay; Marcos Gomes Lopes; Herbert Sousa Soares; Aline Diniz Cabral; Sérgio Netto Vitaliano; Marcos Amaku

    2016-01-01

    Abstract Sarcocystis neurona and Neospora hughesi are coccidian protozoa that can cause neurological illness in horses in America. In this study we report seroprevalence of Neospora spp. andS. neurona in sera of 333 donkeys from the northeastern region of Brazil. Antibodies to Neospora spp. were detected in 2% (7 donkeys) of 333 sera tested by the indirect fluorescent antibody test (IFAT) with a cut-off dilution of 1:40. Antibodies to S. neurona were found in 3% (10 donkeys) of the samples te...

  15. Labeling and use of monoclonal antibodies in immunofluorescence: protocols for cytoskeletal and nuclear antigens.

    Science.gov (United States)

    Bauer, Christoph R

    2014-01-01

    Antibodies are widely used to target and label specifically extra- or intracellular antigens within cells and tissues. Most protocols follow an indirect approach implying the successive incubation with primary and secondary antibodies. In these protocols the primary antibodies are specifically targeted against the antigen in question and are normally not labeled. The secondary antibodies come from a different species and are in contrast fluorescently labeled. The idea is that the primary antibodies specifically bind to their targets but cannot be visualized directly. Only binding of the secondary (fluorescent) antibodies to the constant region of the primary antibodies allows consecutively the visualization in a fluorescent microscope.Primary antibodies can be either of monoclonal (normally produced in mouse) or of polyclonal origin (normally produced in rabbit, goat, sheep, or donkey). Using (primary) monoclonal antibodies has the clear advantage that all antibodies used are identical in origin and behavior and should thus give a more clear-cut labeling result. On the other hand the demands towards labeling protocols might be concomitantly higher: Binding of primary antibodies will only occur if fixation and labeling protocols preserve the antigen sufficiently to keep its specific and unique target structure available. One could imagine that for polyclonal antibodies this demand is slightly lower as there is a pool of antibodies with varying specificities against multiple parts of their target antigens. Certain fractions of this pool might thus tolerate a larger variety of conditions, and consequently a larger variety of protocols might still result in successful labeling.Each step in a labeling protocol can be decisive for the outcome of an experiment especially if monoclonal antibodies are used. Especially critical are choice of buffer and fixation and permeabilization parameters of the protocol.In this chapter we discuss and detail proven protocols using

  16. Advances in monoclonal antibody application in myocarditis

    Institute of Scientific and Technical Information of China (English)

    Li-na HAN; Shuang HE; Yu-tang WANG; Li-ming YANG; Si-yu LIU; Ting ZHANG

    2013-01-01

    Monoclonal antibodies have become a part of daily preparation technologies in many laboratories.Attempts have been made to apply monoclonal antibodies to open a new train of thought for clinical treatments of autoimmune diseases,inflammatory diseases,cancer,and other immune-associated diseases.This paper is a prospective review to anticipate that monoclonal antibody application in the treatment of myocarditis,an inflammatory disease of the heart,could be a novel approach in the future.In order to better understand the current state of the art in monoclonal antibody techniques and advance applications in myocarditis,we,through a significant amount of literature research both domestic and abroad,developed a systematic elaboration of monoclonal antibodies,pathogenesis of myocarditis,and application of monoclonal antibodies in myocarditis.This paper presents review of the literature of some therapeutic aspects of monoclonal antibodies in myocarditis and dilated cardiomyopathy to demonstrate the advance of monoclonal antibody application in myocarditis and a strong anticipation that monoclonal antibody application may supply an effective therapeutic approach to relieve the severity of myocarditis in the future.Under conventional therapy,myocarditis is typically associated with congestive heart failure as a progressive outcome,indicating the need for alternative therapeutic strategies to improve long-term results.Reviewing some therapeutic aspects of monoclonal antibodies in myocarditis,we recently found that monoclonal antibodies with high purity and strong specificity can accurately act on target and achieve definite progress in the treatment of viral myocarditis in rat model and may meet the need above.However,several issues remain.The technology on howto make a higher homologous and weak immunogenic humanized or human source antibody and the treatment mechanism of monoclonal antibodies may provide solutions for these open issues.If we are to further stimulate

  17. Antibody to E1 peptide of hepatitis C virus genotype 4 inhibits virus binding and entry to HepG2 cells in vitro

    OpenAIRE

    El-Awady, Mostafa K.; Tabll, Ashraf A.; Atef, Khaled; Yousef, Samar S; Omran, Moataza H; El-Abd, Yasmin; Bader-Eldin, Noha G; Salem, Ahmad M; Zohny, Samir F; El-Garf, Wael T

    2006-01-01

    AIM: To analyze the neutralizing activity of antibodies against E1 region of hepatitis C virus (HCV). Specific polyclonal antibody was raised via immunization of New Zealand rabbits with a synthetic peptide that had been derived from the E1 region of HCV and was shown to be highly conserved among HCV published genotypes.

  18. Generation and Characterization of Novel Human IRAS Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Bo Wang

    2009-01-01

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

  19. The antibody repertoire in evolution: chance, selection, and continuity.

    Science.gov (United States)

    Marchalonis, John J; Adelman, Miranda K; Schluter, Samuel F; Ramsland, Paul A

    2006-01-01

    All jawed vertebrates contain the genetic elements essential for the function of the adaptive/combinatorial immune response, have diverse sets of natural antibodies resulting from segmental gene recombination, express comparable functional repertoires and can produce specific antibodies following appropriate immunization. Profound variability occurs in the third hypervariable (CDR3) segments of light and heavy chains even within antibodies of the same ostensible specificity. Germline VH and VL elements, as well as the joining (J) segments are highly conserved among the distinct vertebrate species. Conservation is particularly noted among the VH3-like sequences of all jawed vertebrates in the FR2 and FR3 segments, as well as in the FGXGT(R or K)L J-segment characteristic of light chains and TCRs and the WGXGT(uncharged)VT JH segments. Human VH3-53 and Vlambda6 family orthologs may be present over the entire range of vertebrates. Models of the three-dimensional structures of shark VH/VL combining sites indicate similarity in framework structure and comparable CDR usage to those of man. Although carcharhine shark VH regions show greater than 50% identity to the human VH germline prototype, searches of lower deuterostome and invertebrate databases fail to detect molecules with significant relatedness. Overall, antibodies of jawed vertebrates show tremendous individual diversity, but are constructed incorporating design features that arose with the evolutionary emergence of the jawed vertebrates and have been conserved through at least 450 million years of evolutionary time. PMID:16083959

  20. Prevalence and incidence of dengue virus and antibody placental transfer during late pregnancy in central Brazil

    OpenAIRE

    Argolo, Angela FLT; Féres, Valéria CR; Silveira, Lucimeire A; Oliveira, Anna Carolina M; Luiz A. Pereira; Júnior, João Bosco Siqueira; Braga, Cynthia; Martelli, Celina MT

    2013-01-01

    Background Maternal dengue antibodies are considered to play a significant role in dengue pathogenesis among infants. Determining the transplacental specific antibody transfer is invaluable for establishing the optimal vaccination age among infants in endemic regions. Methods We conducted a cross-sectional study among pairs of maternal and corresponding umbilical cord blood samples in public hospitals. The prevalence and incidence of dengue infection were determined in 505 pairs of pregnant w...

  1. Differences in the composition of the human antibody repertoire by B cell subsets in the blood

    OpenAIRE

    Eva Szymanska eMroczek; Ippolito, Gregory C.; Tobias eRogosch; Kam Hon eHoi; Tracy A Hwangpo; Marsha G Brand; Yingxin eZhuang; Cun Ren eLiu; Schneider, David A; Michael eZemlin; Brown, Elizabeth E.; George eGeorgiou; Schroeder, Harry W.

    2014-01-01

    The vast initial diversity of the antibody repertoire is generated centrally by means of a complex series of V (D) J gene rearrangement events, variation in the site of gene segment joining, and TdT catalyzed N- region addition. Although the diversity is great, close inspection has revealed distinct and unique characteristics in the antibody repertoires expressed by different B cell developmental subsets. In order to illustrate our approach to repertoire analysis, we present an in-depth com...

  2. Differences in the Composition of the Human Antibody Repertoire by B Cell Subsets in the Blood

    OpenAIRE

    Mroczek, Eva Szymanska; Ippolito, Gregory C.; Rogosch, Tobias; Hoi, Kam Hon; Tracy A Hwangpo; Marsha G Brand; Zhuang, Yingxin; Liu, Cun Ren; Schneider, David A; Zemlin, Michael; Brown, Elizabeth E.; Georgiou, George; Schroeder, Harry W.

    2014-01-01

    The vast initial diversity of the antibody repertoire is generated centrally by means of a complex series of V(D)J gene rearrangement events, variation in the site of gene segment joining, and TdT catalyzed N-region addition. Although the diversity is great, close inspection has revealed distinct and unique characteristics in the antibody repertoires expressed by different B cell developmental subsets. In order to illustrate our approach to repertoire analysis, we present an in-depth comparis...

  3. Production and characterization of polyclonal antibody against a synthetic peptide from β-actin protein

    OpenAIRE

    Nazila Amini; Mohadeseh Naghi Vishteh; Omid Zarei; Reza Hadavi; Negah Ahmadvand; Hodjattallah Rabbani; Mahmood Jeddi-Tehrani

    2014-01-01

    Objective(s):Antibodies against actin, as one of the most widely studied structural and multifunctional housekeeping proteins in eukaryotic cells, are used as internal loading controls in western blot analyses. The aim of this study was to produce polyclonal antibody against a synthetic peptide derived from N-terminal region of β-actin protein to be used as a protein loading control in western blot and other assay systems. Materials and Methods: A synthetic peptide derived from β-actin protei...

  4. Detection of anti-Neospora caninum antibodies in Iranian native cattle

    OpenAIRE

    Jamal Gharekhani; Heidar Heidari

    2014-01-01

    Neospora caninum is an Apicomplexan parasite which may cause abortion in cattle. This study investigated occurrences of antibodies against N. caninum in Iranian native cattle. From September 2010 to September 2011, blood samples (n=768) of native cows were collected randomly from different rural regions of Hamedan (n=400) and Kurdistan provinces (n=368) located to the western part of Iran. All the samples were evaluated for IgG antibodies against N. caninum using Enzyme Linked Immunosorbent A...

  5. scFv-based “grababody” as a general strategy to improve recruitment of immune effector cells to antibody-targeted tumors

    OpenAIRE

    Cai, Zheng; Fu, Ting; Nagai, Yasuhiro; Lam, Lian; Yee, Marla; Zhu, Zhiqiang; Zhang, Hongtao

    2013-01-01

    Recruitment of immune cells to tumor cells targeted by a therapeutic antibody can heighten the antitumor efficacy of the antibody. For example, p185her2/neu-targeting antibodies not only downregulate the p185her2/neu kinase (ERBB2) but also trigger complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) through the antibody Fc region. Here we describe a generalized strategy to improve immune cell recruitment to targeted cancer cells, using a modified scFv a...

  6. BLUETONGUE VIRUS ANTIBODIES DETECTIONS IN SHEEP FROM ARAÇATUBA REGION –SAO PAULO, BRAZIL DETECÇÃO DE ANTICORPOS CONTRA O VÍRUS DA LÍNGUA AZUL EM OVINOS NA REGIÃO DE ARAÇATUBA – SÃO PAULO, BRASIL

    Directory of Open Access Journals (Sweden)

    Adriana Hellmeister de Campos Nogueira

    2009-12-01

    Full Text Available

    Bluetongue (BT is an infectious, insect-born viral disease of ruminants. The causative agent of BT is bluetongue virus (BTV that belongs to the family Reoviridae genus Orbivirus. Insect vectors in the genus Culicoides transmit this virus. BT affects domestic and wild ruminants, however small ruminants are considered the most affected specie. The aim of the study was to detect antibodies against BTV in commercial sheep farms, of the Northeastern region of Sao Paulo State, Brazil. A total of 1002 sera samples collected from adult sheep (above 1 year-old, comprising a total of 31 farms, were screened for the presence of BTV antibodies, by agar gel immunodiffusion test (AGID and ELISA-CFS (Enzyme Linked Immunosorbent Assay – competitive solid phase, both produced by Pan American Center of FMDV. From a total of 1002 samples, 651 (65% were positive by AGID and 742 (74.1%, were positive by ELISA-CFS. These results suggest that the BTV is widespread among farms, probably causing subclinical infections.

    KEY WORDS: AGID, bluetongue virus, ELISA-CFS, seroepidemiological survey.

    A língua azul é uma doença viral, cujo agente etiológico pertence à família Reoviridae, gênero Orbivirus, transmitida por um vetor (artrópode hematófago, do gênero Culicoides. Os animais acometidos são ruminantes domésticos e selvagens, porém os pequenos ruminantes são os mais afetados. O estudo teve como objetivo detectar a presença de anticorpos para língua azul em ovinos da região de Araçatuba, por possuir um rebanho expressivo e condições climáticas favoráveis à multiplicação de insetos. Foram analisadas 1.002 amostras de soros ovinos, provenientes de 31 cabanhas, pelas provas de imunodifusão dupla em gel de ágar (AGID e ELISA (Enzyme Linked immunosorbent Assay de competição da fase sólida (ELISA CFS, provenientes do Centro Panamericano de Febre Aftosa. Desses soros, 651 (65% foram

  7. Frequency of antibodies to Babesia bigemina, B. bovis, Anaplasma marginale, Trypanosoma vivax and Borrelia burdgorferi in cattle from the northeastern region of the state of Pará, Brazil Freqüência de anticorpos para Babesia bigemina, B. bovis, Anaplasma marginale, Trypanosoma vivax e Borrelia burgdorferi em bovinos do nordeste do Estado do Pará, Brasil

    Directory of Open Access Journals (Sweden)

    Daniel S. Guedes Junior

    2008-06-01

    Full Text Available Babesiosis, anaplasmosis, and trypanosomosis are relevant diseases, potentially causing morbidity in cattle, leading to economic losses. Borreliosis is import as a potential zoonosis. The objective of this study was to determine, by indirect enzyme-linked immunosorbent assay (ELISA, the frequency of seropositive cattle to Babesia bigemina, B. bovis, Anaplasma marginale, Trypanosoma vivax and Borrelia burgdorferi in cattle from the Northeastern region of Pará, Brazil. Sera samples from 246 female adult cattle from municipalities of Castanhal and São Miguel do Guamá were used. Crude antigens ELISAs were used to detect antibodies to all agents, except to A. marginale, to which an indirect ELISA with recombinant major surface 1a protein (MSP1a antigen was used. Overall frequencies of seropositive animals were: B. bigemina - 99.2%; B. bovis - 98.8%; A. marginale - 68.3%; T. vivax - 93.1% and B. burgdorferi - 54.9%. The frequencies of seropositive cattle to B. bovis and B. bigemina suggest a high rate of transmission of these organisms by tick in the studied region, which can be classified as enzootically stable to these hemoprotozoans. The low frequency of seropositive cattle to A. marginale may be attributed to a lower sensitivity of the recombinant antigen ELISA utilized or a distinct rate of inoculation of this rickettsia by ticks, as compared with Babesia sp. transmission. The high frequency of seropositive cattle to T. vivax indicates that this hemoprotozoan is prevalent in herds from the Northeastern region of Pará. The rate of animal that showed homologues antibodies to B. burgdorferi indicates the presence of the tickborne spirochaetal agent in the cattle population in the studied region.A babesiose, a anaplasmose e a tripanossomose são enfermidades relevantes, potencialmente causadoras de morbidade em bovinos, levando a perdas econômicas. A borreliose assume importância como zoonose potencial. O objetivo desse estudo foi determinar

  8. Development of monoclonal antibodies against parathyroid hormone: Genetic control of the immune response to human PTH

    International Nuclear Information System (INIS)

    The authors embarked upon a program to develop monoclonal antibodies to the biologically active amino terminal region of PTH. Using the BALB/c mouse for immunization, fully biologically active synthetic human PTH-(1-34) and bovine PTH-(1-84) as immunogens, monoclonal antibody methods and a solid-phase screening assay in which PTH-(1-34) was adhered to polyvinylchloride plates in a manner that preserved immunoreactivity. They generated 17 monoclonal antibodies against the amino-terminal portion of parathyroid hormone. Isotypic analysis of these monoclonal antibodies was performed using affinity purified goat anti-mouse immunoglobins specific for IgG heavy chains, γ/sub 1/, γ/sub 2a/, γ/sub 2b/, γ/sub 3/; α(IgA); and μ(Igm). All antibodies were IgM as evidenced by 40 times greater than background radioactivity when 25,000 cpm of /sup 125/I-labeled goat anti-mouse IgM was used as second antibody in a solid-phase radioimmunoassay. All incubations with iodinated second antibodies to other heavy chain classes of immunoglobins demonstrated background radioactivity. Extensive synthetic work in the laboratory for multiple biologic studies of structure-activity relationships of PTH, as well as analog design, has led to the synthesis of many peptide analogues and fragments from 7 to 34 amino acids in length. Study of the antibody recognition site (region specificity) by two of these monoclonal antibodies, 10A/sub 7/, and 6B/sub 1/, was undertaken with synthetic peptides

  9. Mechanistic insights into the neutralization of cytotoxic abrin by the monoclonal antibody D6F10.

    Directory of Open Access Journals (Sweden)

    Shradha Bagaria

    Full Text Available Abrin, an A/B toxin obtained from the Abrus precatorius plant is extremely toxic and a potential bio-warfare agent. Till date there is no antidote or vaccine available against this toxin. The only known neutralizing monoclonal antibody against abrin, namely D6F10, has been shown to rescue the toxicity of abrin in cells as well as in mice. The present study focuses on mapping the epitopic region to understand the mechanism of neutralization of abrin by the antibody D6F10. Truncation and mutational analysis of abrin A chain revealed that the amino acids 74-123 of abrin A chain contain the core epitope and the residues Thr112, Gly114 and Arg118 are crucial for binding of the antibody. In silico analysis of the position of the mapped epitope indicated that it is present close to the active site cleft of abrin A chain. Thus, binding of the antibody near the active site blocks the enzymatic activity of abrin A chain, thereby rescuing inhibition of protein synthesis by the toxin in vitro. At 1∶10 molar concentration of abrin:antibody, the antibody D6F10 rescued cells from abrin-mediated inhibition of protein synthesis but did not prevent cell attachment of abrin. Further, internalization of the antibody bound to abrin was observed in cells by confocal microscopy. This is a novel finding which suggests that the antibody might function intracellularly and possibly explains the rescue of abrin's toxicity by the antibody in whole cells and animals. To our knowledge, this study is the first report on a neutralizing epitope for abrin and provides mechanistic insights into the poorly understood mode of action of anti-A chain antibodies against several toxins including ricin.

  10. Antibody response to hidden epitope of influenza a hemagglutinin elicited by anti-idiotypic antibodies

    International Nuclear Information System (INIS)

    Monoclonal antibody (MoAb) IIF4 defines an epitope on the HA2 part of influenza hemagglutinin (HA)It was also found this epitope becomes fully accessible after pH 5 treatment of the antigen and is shared by strains of H3 subtype. In this study we found binding of MoAb IIF4 also to some strains belonging to H2, H4, H7, and H10 subtypes. We prepared rabbit polyclonal anti-IIF4 anti-idiotype (anti-Id) antibody. In competitive assays, the inhibition potential of anti-Id was considerably higher than that of native HA. Anti-Id was used for the preparation of mouse Ab3 (anti-anti-IIF4) serum. Reactivity pattern of Ab3 with influenza virus strains differed from Ab1 in in (i) appearance of binding to some strains of H2 and H7 subtype and (ii) decreased dependency of Ab3 binding on the pH forms of antigen. The reactivity of Ab1 and Ab3 with two amantadine-resistant virus mutants indicates that IIF4 epitope (and its related region recognized by Ab3) becomes accessible in consequence of destabilization of trimeric arrangement of HA and it it also correlates with expulsion of N-terminus of HA2. (author)

  11. Affitins as alternative to antibodies

    International Nuclear Information System (INIS)

    Full text of publication follows. We have developed the use of Sac7d archaeal polypeptide and its homologues as a non-antibody scaffold from which artificial affinity proteins (Affitins) can be derived with a number of favorable properties. Affitins show affinity (sub-nanomolar) and specificity that compare well with those of antibodies [Ref.1]. They are thermally (up to 90 C. degrees) and chemically stable (pH 0-12+, denaturants), well expressed in E. coli (up to 200 mg/L), lack disulfide bridge and have a size compatible with chemical synthesis (7 kDa). We have demonstrated their use as reagents for intra-cellular inhibition [Ref.1], affinity purification [Ref.2], immuno-localization [Ref.3], protein chip array [Ref.4] and biosensors [Ref.5]. We have also shown that Affitins are plastic enough to tolerate several mutagenesis schemes while their fold and their favorable properties are conserved [Ref.6]. Compared to Affitins, monoclonal antibodies are 20 times larger, less stable and more complex molecules. Furthermore, the remarkable stability properties of Affitins make them suited for demanding labeling protocols that are usually used for peptides. All together, these results show that Affitins should be well suited for biomedical applications where fine tuning of the affinity reagent properties is needed. References: [Ref.1] Mouratou, B. et al., (2007), Proc Natl Acad Sci U S A 104, 17983-17988; [Ref.2] Krehenbrink, M. et al. (2008), J Mol Biol 383, 1058-1068; [Ref.3] Buddelmeijer, N. et al. (2009), J Bacteriol 191, 161-168; [Ref.4] Cinier, M. et al. (2009), Bioconjug. Chem. 20, 2270-2277; [Ref.5] Miranda, F. F. et al. (2011), Biosens. Bioelectron. 26, 4184-4190; [Ref.6] Behar G.et al. (2013), Protein Eng Des Sel. 26(4):267-75. (authors)

  12. Production and characterization of peptide antibodies

    DEFF Research Database (Denmark)

    Trier, Nicole Hartwig; Hansen, Paul Robert; Houen, Gunnar

    2012-01-01

    Proteins are effective immunogens for generation of antibodies. However, occasionally the native protein is known but not available for antibody production. In such cases synthetic peptides derived from the native protein are good alternatives for antibody production. These peptide antibodies are...... powerful tools in experimental biology and are easily produced to any peptide of choice. A widely used approach for production of peptide antibodies is to immunize animals with a synthetic peptide coupled to a carrier protein. Very important is the selection of the synthetic peptide, where factors such as......, including solid-phase peptide-carrier conjugation and peptide-carrier conjugation in solution. Upon immunization, adjuvants such as Al(OH)(3) are added together with the immunogenic peptide-carrier conjugate, which usually leads to high-titred antisera. Following immunization and peptide antibody...

  13. Oncocytic lesions of the ophthalmic region

    DEFF Research Database (Denmark)

    Østergaard, Jens; Prause, Jan U; Heegaard, Steffen

    2011-01-01

    Purpose: This study aimed to make a nationwide clinicopathological study of oncocytic lesions in the ophthalmic region and to characterize their cytokeratin (CK) expression. Methods: All histologically diagnosed oncocytic lesions in the ophthalmic region registered in Denmark over a 25-year period...... expression. Basal-type oncocytic cells reacted with antibodies against CK 5/6, CK 7, CK 8, CK 13, CK 14, CK 17, CK 18 and CK 19, and suprabasal cells with CK 4, CK 7, CK 8, CK 18 and CK 19. Antibodies against CK 1+10 and CK 20 showed no reaction. Conclusions: Oncocytic lesions of the ophthalmic region most...

  14. Soroprevalência de anticorpos contra o antígeno CagA do Helicobacter pylori em pacientes com úlcera gástrica na região Norte do Brasil Seroprevalence of antibodies against the CagA antigen the Helicobacter pylori in patients with gastric ulcer in the North region of Brazil

    Directory of Open Access Journals (Sweden)

    Luisa Caricio Martins

    2002-08-01

    Full Text Available O Helicobacter pylori é um agente patogênico largamente distribuído no mundo, estando envolvido no desenvolvimento de várias doenças gastrointestinais. Atualmente a infecção pela cepa virulenta (CagA+ do H. pylori é considerado um dos principais fatores etiológicos para o desenvolvimento de ulcerações gástricas. Baseado nessa informação, investigamos a soroprevalência das cepas virulentas entre os pacientes com úlcera gástrica da nossa região, utilizando testes sorológicos para detecção de anticorpos contra o H. pylori e a proteína CagA. Sendo observado que 82% (45/55 dos pacientes estavam infectados pela cepa virulenta, entre esses 89% (40/45 apresentaram grau de inflamação aumentado na mucosa gástrica, com denso infiltrado de leucócitos no tecido, o que provavelmente favoreceu a formação das ulcerações gástricas.Helicobacter pylori is a pathogenic agent with a worldwide distribution and is involved in the development of many gastrointestinal diseases. Nowadays infection with the virulent strain CagA+ of H. pylori is considered one of the main etiological factors in the development of gastric ulcer. Based on this information, we investigated the seroprevalence of virulent strains among patients with gastric ulcer from one region, using serologic tests to detect antibodies against H. pylori and CagA protein. Infection by the virulent strain was found in 82% (40/55 of the patients, and among these, 89% (40/45 presented an increased degree of inflammation in the gastric mucosa, with a dense infiltration of leukocytes in the tissue, which probably favored the formation of gastric ulcer. We concluded that the presence of the virulent strain is related to the development of an increased inflammation in the gastric mucosa.

  15. Antibody-Mediated Lung Transplant Rejection

    OpenAIRE

    Hachem, Ramsey

    2012-01-01

    Antibody-mediated rejection after lung transplantation remains enigmatic. However, emerging evidence over the past several years suggests that humoral immunity plays an important role in allograft rejection. Indeed, the development of donor-specific antibodies after transplantation has been identified as an independent risk factor for acute cellular rejection and bronchiolitis obliterans syndrome. Furthermore, cases of acute antibody-mediated rejection resulting in severe allograft dysfunctio...

  16. Imaging tumors with radiolabelled monoclonal antibodies

    International Nuclear Information System (INIS)

    Using a metallic radionuclide, either directly bound to a monoclonal antibody, or to a chelating agent (such as di-ethylenetriamine-pentaacetic acid (DTPA)) conjugated to the antibody, a tumor can be traced rapidly and with high specificity. The labelled antibody is injected into the host. In some cases, a localization of distant metastases is possible, giving an indication of tumor spreading. Detection occurs by photoscanning. (Auth.)

  17. Haptens, conjugates and antibodies for pyrimethanil fungicide

    OpenAIRE

    Mercader Badia, Josep Vicent; Abad Fuentes, Antonio; Abad Somovilla, Antonio; Agulló, Consuelo

    2012-01-01

    [EN] The invention relates to haptens, conjugates and antibodies for pyrimethanil fungicide. In addition, the invention relates to the use of pyrimethanil conjugates as assay antigens or immunogens in order to obtain antibodies of the aforementioned fungicide, and to the use of the labelled derivatives of pyrimethanil as assay antigens. The invention also relates to a pyrimethanil analysis method using the antibodies obtained, at times together with assay antigens which are conjugates or labe...

  18. Single-domain antibodies for biomedical applications.

    Science.gov (United States)

    Krah, Simon; Schröter, Christian; Zielonka, Stefan; Empting, Martin; Valldorf, Bernhard; Kolmar, Harald

    2016-02-01

    Single-domain antibodies are the smallest antigen-binding units of antibodies, consisting either only of one variable domain or one engineered constant domain that solely facilitates target binding. This class of antibody derivatives comprises naturally occurring variable domains derived from camelids and sharks as well as engineered human variable or constant antibody domains of the heavy or light chain. Because of their high affinity and specificity as well as stability, small size and benefit of multiple re-formatting opportunities, those molecules emerged as promising candidates for biomedical applications and some of these entities have already proven to be successful in clinical development. PMID:26551147

  19. Mouse monoclonal antibodies against estrogen receptor.

    Science.gov (United States)

    De Rosa, Caterina; Rossi, Valentina; Abbondanza, Ciro

    2014-01-01

    The production of monoclonal antibodies, by cloning hybridoma derived from the fusion of myeloma cells and spleen lymphocytes, has allowed to obtain great advances in many fields of biological knowledge. The use of specific antibodies to the estrogen receptor, in fact, has been an invaluable method to bring out its mechanisms of action and its effects, both genomic and extra-genomic. Here we describe, step by step, the production of monoclonal antibodies, starting from protocol for antigen preparation to the selection of antibody-secreting hybridoma. PMID:25182770

  20. Heparin-Induced Thrombocytopenia Antibody Test

    Science.gov (United States)

    ... Thrombocytopenia Platelet Factor 4 Antibody Related tests: Complete Blood Count , Platelet Count , Serotonin Release Assay, Heparin-induced Platelet Aggregation All content on Lab Tests Online has been ...

  1. Monoclonal Antibodies for Lipid Management.

    Science.gov (United States)

    Feinstein, Matthew J; Lloyd-Jones, Donald M

    2016-07-01

    In recent years, biochemical and genetic studies have identified proprotein convertase subtilisin/kexin type 9 (PCSK9) as a major mediator of low-density lipoprotein cholesterol (LDL-c) levels and thereby a potential novel target for reducing risk of coronary heart disease (CHD). These observations led to the development of PCSK9 inhibitors, which lower LDL-c levels more than any other non-invasive lipid-lowering therapy presently available. The PCSK9 inhibitors furthest along in clinical trials are subcutaneously injected monoclonal antibodies. These PCSK9 inhibitors have demonstrated LDL-c-lowering efficacy with acceptable safety in phase III clinical trials and may offer a useful therapy in addition to maximally tolerated HMG-CoA reductase inhibitors (statins) in certain patient groups. Longer-term data are required to ensure sustained efficacy and safety of this new class of medications. This review provides an overview of the biology, genetics, development, and clinical trials of monoclonal antibodies designed to inhibit PCSK9. PMID:27221501

  2. Antiphospholipid Antibodies and Systemic Scleroderma

    Directory of Open Access Journals (Sweden)

    Awa Oumar Touré

    2013-03-01

    Full Text Available Objective: Antiphospholipid antibodies (APLs could be associated with an increased risk of vascular pathologies in systemic scleroderma. The aim of our study was to search for APLs in patients affected by systemic scleroderma and to evaluate their involvement in the clinical manifestations of this disease. Materials and Methods: We conducted a cross-sectional descriptive study, from January 2009 until August 2010, with patients received at the Department of Dermatology (Dakar, Senegal. Blood samples were taken at the hematology laboratory and were analyzed for the presence of APLs. Results: Forty patients were recruited. Various types of either isolated or associated APLs were found in 23 patients, i.e. 57.5% of the study population. The most frequently encountered antibody was IgG anti-β2 GPI (37.5% of the patients, followed by anticardiolipins (17.5% and lupus anticoagulants (5%. No statistically significant association of positive antiphospholipid-related tests to any of the scleroderma complications could be demonstrated. Conclusion: A high proportion of patients showing association of systemic scleroderma and APLs suggests the presence of a morbid correlation between these 2 pathologies. It would be useful to follow a cohort of patients affected by systemic scleroderma in order to monitor vascular complications following confirmation of the presence of antiphospholipid syndrome.

  3. Structures of Adnectin/Protein Complexes Reveal an Expanded Binding Footprint

    Energy Technology Data Exchange (ETDEWEB)

    Ramamurthy, Vidhyashankar; Krystek, Jr., Stanley R.; Bush, Alexander; Wei, Anzhi; Emanuel, Stuart L.; Gupta, Ruchira Das; Janjua, Ahsen; Cheng, Lin; Murdock, Melissa; Abramczyk, Bozena; Cohen, Daniel; Lin, Zheng; Morin, Paul; Davis, Jonathan H.; Dabritz, Michael; McLaughlin, Douglas C.; Russo, Katie A.; Chao, Ginger; Wright, Martin C.; Jenny, Victoria A.; Engle, Linda J.; Furfine, Eric; Sheriff, Steven (BMS)

    2014-10-02

    Adnectins are targeted biologics derived from the tenth type III domain of human fibronectin ({sup 10}Fn3), a member of the immunoglobulin superfamily. Target-specific binders are selected from libraries generated by diversifying the three {sup 10}Fn3 loops that are analogous to the complementarity determining regions of antibodies. The crystal structures of two Adnectins were determined, each in complex with its therapeutic target, EGFR or IL-23. Both Adnectins bind different epitopes than those bound by known monoclonal antibodies. Molecular modeling suggests that some of these epitopes might not be accessible to antibodies because of the size and concave shape of the antibody combining site. In addition to interactions from the Adnectin diversified loops, residues from the N terminus and/or the {beta} strands interact with the target proteins in both complexes. Alanine-scanning mutagenesis confirmed the calculated binding energies of these {beta} strand interactions, indicating that these nonloop residues can expand the available binding footprint.

  4. Polyclonal antibody against conserved sequences of mce1A protein blocks MTB infection in macrophages.

    Science.gov (United States)

    Sivagnanam, Sasikala; Namasivayam, Nalini; Chellam, Rajamanickam

    2012-03-01

    The pathogenesis of Mycobacterium tuberculosis is largely due to its ability to enter and survive within human macrophages. It is suggested that a specific protein namely mammalian cell entry protein is involved in the pathogenesis and the specific gene for this protein mce1A has been identified in several pathogenic organisms such as Rickettsia, Shigella, Escherichia coli, Helicobacter, Streptomyces, Klebsiella, Vibrio, Neisseria, Rhodococcus, Nocardioides, Saccharopolyspora erthyrae, and Pseudomonas. Analysis of mce1 operons in the above mentioned organisms through bioinformatics tools has revealed the presence of unique sequences (conserved regions) suggesting that these sequences may be involved in the process of infection. Presently, the mce1A full-length (1,365 bp) region from Mycobacterium bovis and its conserved regions (303 bp) were cloned in to an expression vector and the purified expressed proteins of molecular weight ~47 and ~11 kDa, respectively, were injected to rabbits to raise the polyclonal antibodies. The purified polyclonal antibodies were checked for their ability to inhibit the Mycobacterium infection in cultured human macrophages. In macrophage invasion assay, when antibody added at high concentration, decrease in viable counts was observed in all cell cultures within the first 5 days after infection, where the intracellular bacterial CFU obtained from the infected MTB increased by the 3rd day at low concentration of antibody. The macrophage invasion assay has indicated that the purified antibodies of mce1A conserved region can inhibit the infection of Mycobacterium. PMID:22159737

  5. 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 not...... 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....

  6. Stratification of Antibody-Positive Subjects by Antibody Level Reveals an Impact of Immunogenicity on Pharmacokinetics

    OpenAIRE

    Zhou, Lei; Hoofring, Sarah A.; Wu, Yu; Vu, Thuy; Ma, Peiming; Swanson, Steven J.; Chirmule, Narendra; Starcevic, Marta

    2012-01-01

    The availability of highly sensitive immunoassays enables the detection of antidrug antibody (ADA) responses of various concentrations and affinities. The analysis of the impact of antibody status on drug pharmacokinetics (PK) is confounded by the presence of low-affinity or low-concentration antibody responses within the dataset. In a phase 2 clinical trial, a large proportion of subjects (45%) developed ADA following weekly dosing with AMG 317, a fully human monoclonal antibody therapeutic....

  7. High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells

    OpenAIRE

    Jäger, Volker; Büssow, Konrad; Wagner, Andreas; Weber, Susanne; Hust, Michael; Frenzel, André; Schirrmann, Thomas

    2013-01-01

    Background The demand of monospecific high affinity binding reagents, particularly monoclonal antibodies, has been steadily increasing over the last years. Enhanced throughput of antibody generation has been addressed by optimizing in vitro selection using phage display which moved the major bottleneck to the production and purification of recombinant antibodies in an end-user friendly format. Single chain (sc)Fv antibody fragments require additional tags for detection and are not as suitable...

  8. Antibodies to human fetal erythroid cells from a nonimmune phage antibody library

    OpenAIRE

    Huie, Michael A.; Cheung, Mei-Chi; Muench, Marcus O.; Becerril, Baltazar; Kan, Yuet W.; Marks, James D.

    2001-01-01

    The ability to isolate fetal nucleated red blood cells (NRBCs) from the maternal circulation makes possible prenatal genetic analysis without the need for diagnostic procedures that are invasive for the fetus. Such isolation requires antibodies specific to fetal NRBCs. To generate a panel of antibodies to antigens present on fetal NRBCs, a new type of nonimmune phage antibody library was generated in which multiple copies of antibody fragments are displayed on each pha...

  9. Passive antibody transfer in chickens to model maternal antibody after avian influenza vaccination

    Science.gov (United States)

    Birds transfer maternal antibodies (MAb) to their offspring through the egg yolk where the antibody is absorbed and enters the circulatory system. These maternal antibodies, depending on the pathogen, can provide early protection from some diseases, but it may also interfere with the vaccination re...

  10. Comparative Analysis of Immune Repertoires between Bactrian Camel's Conventional and Heavy-Chain Antibodies.

    Science.gov (United States)

    Li, Xinyang; Duan, Xiaobo; Yang, Kai; Zhang, Wei; Zhang, Changjiang; Fu, Longfei; Ren, Zhe; Wang, Changxi; Wu, Jinghua; Lu, Ruxue; Ye, Yanrui; He, Mengying; Nie, Chao; Yang, Naibo; Wang, Jian; Yang, Huanming; Liu, Xiao; Tan, Wen

    2016-01-01

    Compared to classical antibodies, camel heavy chain antibodies (HCAbs) are smaller in size due to lack of the light chain and the first constant domain of the heavy chain (CH1 region). The variable regions of HCAbs (VHHs) are more soluble and stable than that of conventional antibodies (VHs). Even with such simple structure, they are still functional in antigen binding. Although HCAbs have been extensively investigated over the past two decades, most efforts have been based upon low throughput sequence analysis, and there are only limited reports trying to analyze and describe the complete immune repertoire (IR) of camel HCAbs. Here we leveraged the high-throughput data generated by Next Generation Sequencing (NGS) of the variable domains of the antibody heavy chains from three Bactrian camels to conduct in-depth comparative analyses of the immunoglobulin repertoire. These include analyses of the complementary determining region 3 (CDR3) length and distribution, mutation rate, antibody characteristic amino acids, the distribution of the cysteine (Cys) codons, and the non-classical VHHs. We found that there is higher diversity in the CDR2 than in the other sub-regions, and there is a higher mutation rate in the VHHs than in the VHs (P Arg AA substitution at the first position of framework 4 for all types of clones. We present, for the first time, a relatively complete picture of the Bactrian camel antibody immune repertoire, including conventional antibody (Ab) and HCAbs, using PCR and in silico analysis based on high-throughput NGS data. PMID:27588755

  11. Specific biodetection of B16 mouse melanoma in vivo by syngeneic monoclonal antibody

    International Nuclear Information System (INIS)

    The specific detection of tumors in vivo using a radiolabeled syngeneic monoclonal antibody made by fusion of P3U1 (BALB/c myeloma cells) and C57BL/6 spleen cells primed with syngeneic B16 melanoma cells was investigated by color imaging, autoradiography, and biodistribution. The radiolabeled antimelanoma antibody specifically accumulated only in the tumor lesions, whereas no radioactivity was observed in normal tissues or organs. The distribution patterns of the radioactive antibody in the tumor lesions depended on the sizes of the tumor. Almost the entire region of the small metastatic tumor in lymph nodes was labeled, whereas the radioactive antibody was irregularly localized mainly in the center of the medium-sized tumor. However, only the peripheral region of the large primary tumor was labeled. The highest uptake of radioactivity (tumor:blood ratio) was observed in the small lymph node metastatic tumor lesions rather than in the large primary tumor. Furthermore, high resolution color imaging of B16 melanoma was also obtained by using 125I-labeled monoclonal antibody. Tumor location was specifically visible without subtraction or enhancement methods 3-5 days after injection of the radiolabeled antibody

  12. Overcoming low yields of plant-made antibodies by a protein engineering approach.

    Science.gov (United States)

    Zischewski, Julia; Sack, Markus; Fischer, Rainer

    2016-01-01

    The commercial development of plant-based antibody production platforms is often limited by low and variable yields, but little is known about the factors that affect antibody accumulation during and after translation. Here, we present a strategy to identify yield-limiting regions in the transcript and protein. We exchanged variable heavy chain (VH) domain sequences between two human antibodies at structurally conserved positions, thus creating ten chimeric VH domains containing sequences from M12 (∼1000 μg/g leaf fresh weight [FW]) and 4E10 (∼100 μg/g FW). After transient expression in Nicotiana benthamiana leaves, we measured mRNA and protein levels by quantitative real-time PCR and surface plasmon resonance spectroscopy, respectively. Transcript levels were similar for all constructs, but antibody levels ranged from ∼250 μg/g to over 2000 μg/g FW. Analysis of the expression levels showed that: i) 4E10 yields were only marginally increased by suppression of post-transcriptional gene silencing; ii) the CDR3 of 4E10 contains a protease site; and iii) a bipartite, yield-limiting region exists in the CDR2/CDR3. Our findings highlight the strong impact of cotranslational and posttranslational events on antibody yields and show that protein engineering is a powerful tool that can be used to overcome the remaining limitations affecting antibody production in plants. PMID:26632507

  13. Subsetting of acetylcholine receptor-reactive antibodies by preparative isoelectric focusing.

    Science.gov (United States)

    Thompson, P A; Krolick, K A

    1991-01-01

    The antibodies produced against most foreign antigens are composed of a family of immunoglobulins, a family composed of members that are of a number that often reflects the size/complexity of the molecule that stimulates their production. In other words, such responses involve the activation of a "polyclonal" B lymphocyte population. The antibody products of the B cells, although all capable of binding the original antigen, bind at various immunogenic sites (epitopes) on that antigen. Such differences in antigen-binding fine specificity is determined by amino acid residues in the antibody variable region domains found associated with the antigen combining site and tend to have a complimentary biochemistry with the molecule for which they are intended to interact. Furthermore, in addition to amino acid differences that dictate the isotypes and allotypes of antibody molecules, differences in the amino acids that compose the variable regions can produce differences in net charge of particular antibody molecules; thus, families of polyclonal antibodies, all reactive with the same antigen but with different fine specificities, can be separated and, as shown below, purified based on their isoelectric points by preparative isoelectric focusing (pIEF). PMID:1780274

  14. 21 CFR 866.3290 - Gonococcal antibody test (GAT).

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Gonococcal antibody test (GAT). 866.3290 Section... antibody test (GAT). (a) Identification. A gonococcal antibody test (GAT) is an in vitro device that..., indirect fluorescent antibody, or radioimmunoassay, antibodies to Neisseria gonorrhoeae in sera...

  15. Synergistic cytotoxic effects of antibodies directed against different cell surface determinants

    International Nuclear Information System (INIS)

    Three antibody populations were raised in rabbits against surface antigens on guinea-pig L2C leukaemic lymphocytes: against idiotypic determinants on the lambda chain of the surface immunoglobulin, against C region determinants on the lambda chain, and against the surface antigens recognised by conventional anti-lymphocyte sera. Complement and K-cell cytotoxicities effected by the antibodies on L2C cells were studied in vitro. In both cytotoxic systems mixtures of the antibodies revealed synergy, in that the titres of the mixtures exceeded predicted additive titres of their components. The synergy was greater when the mixed antibodies were directed to determinants on the same molecule rather than to determinants on different molecules. (author)

  16. Molecular basis of high viscosity in concentrated antibody solutions: Strategies for high concentration drug product development.

    Science.gov (United States)

    Tomar, Dheeraj S; Kumar, Sandeep; Singh, Satish K; Goswami, Sumit; Li, Li

    2016-01-01

    Effective translation of breakthrough discoveries into innovative products in the clinic requires proactive mitigation or elimination of several drug development challenges. These challenges can vary depending upon the type of drug molecule. In the case of therapeutic antibody candidates, a commonly encountered challenge is high viscosity of the concentrated antibody solutions. Concentration-dependent viscosity behaviors of mAbs and other biologic entities may depend on pairwise and higher-order intermolecular interactions, non-native aggregation, and concentration-dependent fluctuations of various antibody regions. This article reviews our current understanding of molecular origins of viscosity behaviors of antibody solutions. We discuss general strategies and guidelines to select low viscosity candidates or optimize lead candidates for lower viscosity at early drug discovery stages. Moreover, strategies for formulation optimization and excipient design are also presented for candidates already in advanced product development stages. Potential future directions for research in this field are also explored. PMID:26736022

  17. Phage Display-Derived Cross-Reactive Neutralizing Antibody against Enterovirus 71 and Coxsackievirus A16.

    Science.gov (United States)

    Zhang, Xiao; Sun, Chunyun; Xiao, Xiangqian; Pang, Lin; Shen, Sisi; Zhang, Jie; Cen, Shan; Yang, Burton B; Huang, Yuming; Sheng, Wang; Zeng, Yi

    2016-01-01

    Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are members of the Picornaviridae family and are considered the main causative agents of hand, foot and mouth disease (HFMD). In recent decades large HFMD outbreaks caused by EV71 and CVA16 have become significant public health concerns in the Asia-Pacific region. Vaccines and antiviral drugs are unavailable to prevent EV71 and CVA16 infection. In the current study, a chimeric antibody targeting a highly conserved peptide in the EV71 VP4 protein was isolated by using a phage display technique. The antibody showed cross-neutralizing capability against EV71 and CVA16 in vitro. The results suggest that this phage display-derived antibody will have great potential as a broad neutralizing antibody against EV71 and CVA16 after affinity maturation and humanization. PMID:26073737

  18. Kinetics of Anti-Phlebotomus perniciosus Saliva Antibodies in Experimentally Bitten Mice and Rabbits.

    Directory of Open Access Journals (Sweden)

    Inés Martín-Martín

    Full Text Available Sand flies are hematophagous arthropods that act as vectors of Leishmania parasites. When hosts are bitten they develop cellular and humoral responses against sand fly saliva. A positive correlation has been observed between the number of bites and antibody levels indicating that anti-saliva antibody response can be used as marker of exposure to sand flies. Little is known about kinetics of antibodies against Phlebotomus perniciosus salivary gland homogenate (SGH or recombinant salivary proteins (rSP. This work focused on the study of anti-P. perniciosus saliva antibodies in sera of mice and rabbits that were experimentally exposed to the bites of uninfected sand flies.Anti-saliva antibodies were evaluated by ELISA and Western blot. In addition, antibody levels against two P. perniciosus rSP, apyrase rSP01B and D7 related protein rSP04 were determined in mice sera. Anti-saliva antibody levels increased along the immunizations and correlated with the number of sand fly bites. Anti-SGH antibody levels were detected in sera of mice five weeks after exposure, and persisted for at least three months. Anti-apyrase rSP01B antibodies followed similar kinetic responses than anti-SGH antibodies while rSP04 showed a delayed response and exhibited a greater variability among sera of immunized mice. In rabbits, anti-saliva antibodies appeared after the second week of exposure and IgG antibodies persisted at high levels, even 7 months post-exposure.Our results contributed to increase the knowledge on the type of immune response P. perniciosus saliva and individual proteins elicited highlighting the use of rSP01B as an epidemiological marker of exposure. Anti-saliva kinetics in sera of experimentally bitten rabbits were studied for the first time. Results with rabbit model provided useful information for a better understanding of the anti-saliva antibody levels found in wild leporids in the human leishmaniasis focus in the Madrid region, Spain.

  19. Photonic crystal fiber based antibody detection

    DEFF Research Database (Denmark)

    Duval, A; Lhoutellier, M; Jensen, J B; Hoiby, P E; Missier, V; Pedersen, L H; Hansen, Theis Peter; Bjarklev, Anders Overgaard; Bang, Ole

    An original approach for detecting labeled antibodies based on strong penetration photonic crystal fibers is introduced. The target antibody is immobilized inside the air-holes of a photonic crystal fiber and the detection is realized by the means of evanescent-wave fluorescence spectroscopy and...

  20. Receptor antibodies as novel therapeutics for diabetes

    DEFF Research Database (Denmark)

    Ussar, Siegfried; Vienberg, Sara Gry; Kahn, C Ronald

    2011-01-01

    Antibodies to receptors can block or mimic hormone action. Taking advantage of receptor isoforms, co-receptors, and other receptor modulating proteins, antibodies and other designer ligands can enhance tissue specificity and provide new approaches to the therapy of diabetes and other diseases....

  1. Monoclonal Antibody Therapy for Advanced Neuroblastoma

    Science.gov (United States)

    NCI is sponsoring two clinical trials of a monoclonal antibody called ch14.18, in combination with other drugs, to see if the antibody may be helpful for children or young adults (up to age 21) with relapsed or refractory neuroblastoma.

  2. Bioconjugation of antibodies to horseradish peroxidase (hrp)

    Science.gov (United States)

    The bioconjugation of an antibody to an enzymatic reporter such as horseradish peroxidase (HRP) affords an effective mechanism by which immunoassay detection of a target antigen can be achieved. The use of heterobifunctional cross—linkers to covalently link antibodies to HRP provides a simple and c...

  3. Thyrotropin receptor antibodies and its clinical application

    International Nuclear Information System (INIS)

    Thyrotropin receptor antibodies (TRAb) are not homogeneous, which are composed by four antibodies at least. TRAb plays very important roles in autoimmune thyroid diseases ad off-thyroid symptoms associated, and other thyroiditis in clinical diagnosis, assessment of curative effects, determination of the time to stop medicine, prognostication of recurrence and inspection of high risk population

  4. Monoclonal antibodies to Leptospira interrogans serovar pomona.

    OpenAIRE

    Ainsworth, A J; Lester, T L; Capley, G

    1985-01-01

    Three monoclonal antibodies produced against Leptospira interrogans serovar pomona have been studied for their diagnostic usefulness. All three monoclonals reacted strongly in the enzyme-linked immunosorbent assay and indirect fluorescent antibody test with serovar pomona and did not react with serovars grippotyphosa, canicola, icterohaemorrhagiae and hardjo.

  5. "Unconventional" Neutralizing Activity of Antibodies Against HIV

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Neutralizing antibodies are recognized to be one of the essential elements of the adaptive immune response that must be induced by an effective vaccine against HIV. However, only a limited number of antibodies have been identified to neutralize a broad range of primary isolates of HIV-1 and attempts to induce such antibodies by immunization were unsuccessful. The difficulties to generate such antibodies are mainly due to intrinsic properties of HIV-1 envelope spikes, such as high sequence diversity, heavy glycosylation, and inducible and transient nature of certain epitopes. In vitro neutralizing antibodies are identified using "conventional" neutralization assay which uses phytohemagglutinin (PHA)-stimulated human PBMCs as target cells. Thus, in essence the assay evaluates HIV-1 replication in CD4+ T cells. Recently, several laboratories including us demonstrated that some monoclonal antibodies and HIV-1-specific polyclonal IgG purified from patient sera, although they do not have neutralizing activity when tested by the "conventional" neutralization assay, do exhibit potent and broad neutralizing activity in "unconventional" ways. The neutralizing activity of these antibodies and IgG fractions is acquired through post-translational modifications, through opsonization of virus particles into macrophages and immature dendritic cells (iDCs), or through expression of antibodies on the surface of HIV-1-susceptible cells. This review will focus on recent findings of this area and point out their potential applications in the development of preventive strategies against HIV.

  6. Determination of Biotin: Antibody Molar Ratio

    International Nuclear Information System (INIS)

    The determination of the biotinylation yield (number of biotin molecules per molecule of antibody) is important to ensure that the MAb has maintained its immunoreactivity. If the biotinylation of the MAb is carried out with a molar ratio of biotin:antibody ~10:1, then the number of biotins per MAb usually ranges between 6 and 8

  7. Anti-influenza M2e antibody

    Energy Technology Data Exchange (ETDEWEB)

    Bradbury, Andrew M.

    2013-04-16

    Humanized recombinant and monoclonal antibodies specific for the ectodomain of the influenza virus M2 ion channel protein are disclosed. The antibodies of the invention have anti-viral activity and may be useful as anti-viral therapeutics and/or prophylactic/vaccine agents for inhibiting influenza virus replication and for treating individuals infected with influenza.

  8. Anti-influenza M2e antibody

    Energy Technology Data Exchange (ETDEWEB)

    Bradbury, Andrew M. (Santa Fe, NM)

    2011-12-20

    Humanized recombinant and monoclonal antibodies specific for the ectodomain of the influenza virus M2 ion channel protein are disclosed. The antibodies of the invention have anti-viral activity and may be useful as anti-viral therapeutics and/or prophylactic/vaccine agents for inhibiting influenza virus replication and for treating individuals infected with influenza.

  9. PREPARATION AND CHARACTERIZATION OF MONOCLONAL ANTIBODY AGAINST HUMAN TELOMERASE REVERSE TRANSCRIPTASE

    Institute of Scientific and Technical Information of China (English)

    王俊梅; 张波; 杨邵敏; 韩继生; 李冰思; 侯琳

    2003-01-01

    Objective. To develop monoclonal antibodies against the catalytic subunit of human telomerase reverse transcriptase (hTERT) for its expression detection of human tumors. Methods. A dominant epitope in hTERT (peptide hTERT7)was automatically synthesized based on Fmoc method, and was used to immunize Balb/c mice. Hybridomas were generated and screened by ELISA for specific monoclonal antibodies, and the characterization was performed by Western blotting and immunohistochemical staining. The heavy chain variable region of antibody was cloned by RT-PCR and sequenced. Results. Antigenic peptide hTERT7 was synthesized and confirmed by MALDI-TOF-MS and HPLC analysis. One hybridoma cell line secreting anti-hTERT7 antibodies designated as M2 was established after primary screening and consequent 3 rounds of limited dilution. M2 was IgG1 in isotyping. The competi tive assay showed that the M2 antibody was hTERT7 -specific, and the affinity constant was about 1×106 mol-1. The antibody reacted with cell extracts from HeLa cancer cells but not with those from normal 2BS cells in ELISA assay. For in situ staining of immunohistochemistry, the positive staining presented in the nuclear compartment of HeLa, while 2BS was negative. The heavy chain variable region from M2 re vealed that the monoclonal antibody was mouse origin. Conclusions. The developed mouse monoclonal antibody is hTERT-specific and able to recognize native cellular hTERT in ELISA and immunohistochemistry, which makes the immuno-detection of telom erase hTERT expression in cancer cells or tissues possible.

  10. Elicitation of broadly neutralizing influenza antibodies in animals with previous influenza exposure.

    Science.gov (United States)

    Wei, Chih-Jen; Yassine, Hadi M; McTamney, Patrick M; Gall, Jason G D; Whittle, James R R; Boyington, Jeffrey C; Nabel, Gary J

    2012-08-15

    The immune system responds to influenza infection by producing neutralizing antibodies to the viral surface protein, hemagglutinin (HA), which regularly changes its antigenic structure. Antibodies that target the highly conserved stem region of HA neutralize diverse influenza viruses and can be elicited through vaccination in animals and humans. Efforts to develop universal influenza vaccines have focused on strategies to elicit such antibodies; however, the concern has been raised that previous influenza immunity may abrogate the induction of such broadly protective antibodies. We show here that prime-boost immunization can induce broadly neutralizing antibody responses in influenza-immune mice and ferrets that were previously infected or vaccinated. HA stem-directed antibodies were elicited in mice primed with a DNA vaccine and boosted with inactivated vaccine from H1N1 A/New Caledonia/20/1999 (1999 NC) HA regardless of preexposure. Similarly, gene-based vaccination with replication-defective adenovirus 28 (rAd28) and 5 (rAd5) vectors encoding 1999 NC HA elicited stem-directed neutralizing antibodies and conferred protection against unmatched 1934 and 2007 H1N1 virus challenge in influenza-immune ferrets. Indeed, previous exposure to certain strains could enhance immunogenicity: The strongest HA stem-directed immune response was observed in ferrets previously infected with a divergent 1934 H1N1 virus. These findings suggest that broadly neutralizing antibodies against the conserved stem region of HA can be elicited through vaccination despite previous influenza exposure, which supports the feasibility of developing stem-directed universal influenza vaccines for humans. PMID:22896678

  11. Antigliadin antibody in sporadic adult ataxia

    Directory of Open Access Journals (Sweden)

    Mahdi Aloosh

    2012-09-01

    Full Text Available Background: The most common neurologic manifestationof gluten sensitivity is ataxia, which accounts for up to 40%of idiopathic sporadic ataxia. Timing of diagnosis of glutenataxia is vital as it is one of the very few treatable causes ofsporadic ataxia and causes irreversible loss of Purkinje cells.Antigliadin antibody (AGA of the IgG type is the bestmarker for neurological manifestations of gluten sensitivity.This study was conducted to measure the prevalence ofgluten ataxia in a group of Iranian patients with idiopathicataxia.Methods: For 30 patients with idiopathic cerebellar ataxia, aquestionnaire about clinical and demographic data wascompleted. Serum AGA (IgA and IgG and antiendomysialantibody (AEA were assessed. Gluten ataxic patientsunderwent duodenal biopsy. Magnetic resonanceimaging was done for all patients to see if cerebellaratrophy is present.Results: Only 2 patients had a positive IgG AGA (6.7%who both had a positive AEA while none of themshowed changes of celiac disease in their duodenalbiopsies. Only presence of gastrointestinal symptomsand pursuit eye movement disorders were higher inpatients with gluten ataxia.Conclusion: Prevalence of gluten ataxia in Iranianpatients with idiopathic ataxia seems to be lower thanmost of other regions. This could be explained by smallsample size, differences in genetics and nutritionalhabits and also effect of serologic tests in clinical versusresearch setting. Further researches with larger samplesize are recommended.

  12. Nano antibody therapy for cancer

    International Nuclear Information System (INIS)

    Nanomedicine, an offshoot of nanotechnology, refers to highly specific medical intervention at the molecular scale for curing disease or repairing damaged tissues, such as bone, muscle, or nerve. Nanotechnology can have an early, paradigm-changing impact on how clinicians will detect cancer in its earliest stages. Exquisitely sensitive devices constructed of nanoscale components-such as nanocantilevers, nanowires and nanochannels-offer the potential for detecting even the rarest molecular signals associated with malignancy. One of the most pressing needs in clinical oncology is for imaging agents that can identify tumors that are far smaller than is possible with today's technology, at a scale of 100,000 cells rather than 1,000,000,000 cells. A new approach in nanotechnology for treating cancer incorporates nano iron particles and attaches them to an antibody that has targets only cancer cells and not healthy cells. The treatment works in two steps. This treatment is an ingenious way to make localized tumor ablation a systemic treatment. The advantages are incredible. There are absolutely no side effects from this treatment. It is not painful or even uncomfortable. The iron particles get flushed harmlessly from the body. It is not a drug and so the cancer cannot build up a resistance to the treatment. It is a systematic treatment; even cancer cells and tumors that are not known about get heated up and ablated. This treatment can even be used to enhance imaging of the cancer because once the cancer cells are coated with the iron particles, they are easy to identify. Everything depends on how reliably the antibodies target cancer cells and not healthy cells. When used in conjunction with other systemic treatments, such as vaccine treatments, we could be looking at a time when even advanced cancers can be brought under control. (author)

  13. Synthetic Antibodies for Reversible Cell Recognition

    Science.gov (United States)

    Zhou, Jing Zhou

    2011-12-01

    Antibody-mediated cell recognition plays a critical role in various biological and biomedical applications. However, strong antibody-cell interactions can lead to the difficulty of separating antibodies from the bound cells in a simple and non-destructive manner, which is often necessary to numerous applications such as cell sorting or separation. Thus, this thesis research is aimed to create an antibody-like nanomaterial with the function of reversible cell recognition It was hypothesized that nucleic acid aptamer and dendrimer could be used as fundamental structural components to develop an antibody-like nanomaterial. The aptamer functions as the binding site of an antibody; the dendrimer is used as a robust, defined nano-scaffold to support the aptamer and to carry small molecules (e.g., fluorophores). To test this hypothesis, a novel method was first developed to discover the essential nucleotides of full-length aptamers to mimic the binding sites of antibodies. The essential nucleotides were further conjugated with a dendrimer to synthesize a monovalent aptamer-dendrimer nanomaterial. The results clearly showed that the essential nucleotides could maintain high affinity and specificity after tethered on dendrimer surface. To further test the hypothesis that antibody-like nanomaterials can be rationally designed to acquire the capability of reversible cell recognition, an aptamer that was selected at 0 °C was used as a model to synthesize a "Y-shaped" nanomaterial by conjugating two aptamers to the same dendrimer. The results showed that the nanomaterial-cell interaction could be affected by the distance between two binding aptamers. In addition, the "Y-shaped" antibody-like nanomaterial could bind target cells more strongly than its monovalent control. Importantly, the strong cell-nanomaterial interaction could be rapidly reversed when the temperature was shifted from 0 °C to 37 °C. In summary, we developed a synthetic antibody that can not only mimic the

  14. Antibodies against chromosomal beta-lactamase

    DEFF Research Database (Denmark)

    Giwercman, B; Rasmussen, J W; Ciofu, Oana; Clemmentsen, I; Schumacher, H; Høiby, N

    1994-01-01

    A murine monoclonal anti-chromosomal beta-lactamase antibody was developed and an immunoblotting technique was used to study the presence of serum and sputum antibodies against Pseudomonas aeruginosa chromosomal group 1 beta-lactamase in patients with cystic fibrosis (CF). The serum antibody...... response was studied with serum samples collected in 1992 from 56 CF patients in a cross-sectional study and with serum samples from 18 CF patients in a longitudinal study. Anti-beta-lactamase immunoglobulin G antibodies were present in all of the serum samples from the patients with chronic...... bronchopulmonary P. aeruginosa infection (CF + P) but in none of the CF patients with no or intermittent P. aeruginosa infection. Anti-beta-lactamase antibodies were present in serum from CF + P patients after six antipseudomonal courses (median) and correlated with infection with a beta-lactam-resistant strain of...

  15. Antibody-Mediated Lung Transplant Rejection

    Science.gov (United States)

    Hachem, Ramsey

    2012-01-01

    Antibody-mediated rejection after lung transplantation remains enigmatic. However, emerging evidence over the past several years suggests that humoral immunity plays an important role in allograft rejection. Indeed, the development of donor-specific antibodies after transplantation has been identified as an independent risk factor for acute cellular rejection and bronchiolitis obliterans syndrome. Furthermore, cases of acute antibody-mediated rejection resulting in severe allograft dysfunction have been reported, and these demonstrate that antibodies can directly injure the allograft. However, the incidence and toll of antibody-mediated rejection are unknown because there is no widely accepted definition and some cases may be unrecognized. Clearly, humoral immunity has become an important area for research and clinical investigation. PMID:23002428

  16. Trends in Malignant Glioma Monoclonal Antibody Therapy

    Science.gov (United States)

    Chekhonin, Ivan; Gurina, Olga

    2015-01-01

    Although new passive and active immunotherapy methods are emerging, unconjugated monoclonal antibodies remain the only kind of biological preparations approved for high-grade glioma therapy in clinical practice. In this review, we combine clinical and experimental data discussion. As antiangiogenic therapy is the standard of care for recurrent glioblastoma multiforme (GBM), we analyze major clinical trials and possible therapeutic combinations of bevacizumab, the most common monoclonal antibody to vascular endothelial growth factor (VEGF). Another humanized antibody to gain recognition in GBM is epidermal growth factor (EGFR) antagonist nimotuzumab. Other antigens (VEGF receptor, platelet-derived growth factor receptor, hepatocyte growth factor and c-Met system) showed significance in gliomas and were used to create monoclonal antibodies applied in different malignant tumors. We assess the role of genetic markers (isocitrate dehydrogenase, O6-methylguanine-DNA methyltransnsferase) in GBM treatment outcome prediction. Besides antibodies studied in clinical trials, we focus on perspective targets and briefly list other means of passive immunotherapy.

  17. Antiphospholipid antibody: laboratory, pathogenesis and clinical manifestations

    Directory of Open Access Journals (Sweden)

    T. Ziglioli

    2011-06-01

    Full Text Available Antiphospholipid antibodies (aPL represent a heterogeneous group of antibodies that recognize various antigenic targets including beta2 glycoprotein I (β2GPI, prothrombin (PT, activated protein C, tissue plasminogen activator, plasmin and annexin A2. The most commonly used tests to detect aPL are: lupus anticoagulant (LAC, a functional coagulation assay, anticardiolipin antibody (aCL and anti-β2GPI antibody (anti-β2GPI, which are enzyme-linked immunoassay (ELISA. Clinically aPL are associated with thrombosis and/or with pregnancy morbidity. Apparently aPL alone are unable to induce thrombotic manifestations, but they increase the risk of vascular events that can occur in the presence of another thrombophilic condition; on the other hand obstetrical manifestations were shown to be associated not only to thrombosis but mainly to a direct antibody effect on the trophoblast.

  18. Isolation of Balamuthia mandrillaris-specific antibody fragments from a bacteriophage antibody display library.

    Science.gov (United States)

    Siddiqui, Ruqaiyyah; Kulsoom, Huma; Lalani, Salima; Khan, Naveed Ahmed

    2016-07-01

    Balamuthia mandrillaris is a protist pathogen that can cause encephalitis with a mortality rate of more than 95%. Early diagnosis followed by aggressive treatment is a pre-requisite for successful prognosis. Current methods for identifying this organism rely on culture and microscopy, antibody-based methods using animals, or involve the use of molecular tools that are expensive. Here, we describe the isolation of antibody fragments that can be used for the unequivocal identification of B. mandrillaris. B. mandrillaris-specific antibody fragments were isolated from a bacteriophage antibody display library. Individual clones were studied by enzyme-linked immunosorbent assay, and immunofluorescence. Four antibody clones showed specific binding to B. mandrillaris. The usefulness of phage antibody display technology as a diagnostic tool for isolating antibody fragments against B. mandrillaris antigens and studying their biological role(s) is discussed further. PMID:27055361

  19. Immobilization of antibodies and enzyme-labeled antibodies by radiation polymerization

    International Nuclear Information System (INIS)

    Immobilization of antibodies and enzyme-labeled antibodies by radiation polymerization at low temperatures was studied. The antibody activity of antibody was not affected by irradiation at an irradiation dose of below 8 MR and low temperatures. Immobilization of peroxidase-labeled anti-rabbit IgG goat IgG, anti-peroxidase, peroxidase, and anti-alpha-fetoprotein was carried out with hydrophilic and hydrophobic monomers. The activity of the immobilized enzyme-labeled antibody membranes varied with the thickness of the membranes and increased with decreasing membrane thickness. The activity of the immobilized antibody particles was varied by particle size. Immobilized anti-alpha-fetoprotein particles and membranes can be used for the assay of alpha-fetoprotein by the antigen-antibody reaction, such as a solid-phase sandwich method with high sensitivity

  20. Vector-Mediated In Vivo Antibody Expression.

    Science.gov (United States)

    Schnepp, Bruce C; Johnson, Philip R

    2014-08-01

    This article focuses on a novel vaccine strategy known as vector-mediated antibody gene transfer, with a particular focus on human immunodeficiency virus (HIV). This strategy provides a solution to the problem of current vaccines that fail to generate neutralizing antibodies to prevent HIV-1 infection and AIDS. Antibody gene transfer allows for predetermination of antibody affinity and specificity prior to "immunization" and avoids the need for an active humoral immune response against the HIV envelope protein. This approach uses recombinant adeno-associated viral (rAAV) vectors, which have been shown to transduce muscle with high efficiency and direct the long-term expression of a variety of transgenes, to deliver the gene encoding a broadly neutralizing antibody into the muscle. Following rAAV vector gene delivery, the broadly neutralizing antibodies are endogenously synthesized in myofibers and passively distributed to the circulatory system. This is an improvement over classical passive immunization strategies that administer antibody proteins to the host to provide protection from infection. Vector-mediated gene transfer studies in mice and monkeys with anti-HIV and simian immunodeficiency virus (SIV)-neutralizing antibodies demonstrated long-lasting neutralizing activity in serum with complete protection against intravenous challenge with virulent HIV and SIV. These results indicate that existing potent anti-HIV antibodies can be rapidly moved into the clinic. However, this methodology need not be confined to HIV. The general strategy of vector-mediated antibody gene transfer can be applied to other difficult vaccine targets such as hepatitis C virus, malaria, respiratory syncytial virus, and tuberculosis. PMID:26104192

  1. Radiohalogenated half-antibodies and maleimide intermediate therefor

    Science.gov (United States)

    Kassis, A.I.; Khawli, L.A.

    1991-02-19

    N-(m-radiohalophenyl) maleimide can be conjugated with a reduced antibody having a mercapto group to provide a radiolabeled half-antibody having immunological specific binding characteristics of whole antibody. No Drawings

  2. Genetically engineered multivalent single chain antibody constructs for cancer therapy

    International Nuclear Information System (INIS)

    increase its tumor: normal tissue ratio for improved therapeutic index, we engineered a variety antibody constructs. These constructs were evaluated using novel approaches like special radionuclides, pretargeting and optimization. Due to the smaller size, the engineered antibody molecules should penetrate better throughout a tumor mass, with less dose heterogeneity, than is the case with intact IgG. Multivalent scFvs with an appropriate radionuclide, therefore, hold promising prospects for cancer therapy and clinical imaging in MAb-based radiopharmaceuticals. In addition, the human anti-mouse antibodies (HAMA) responses in patients against antibody-based therapy are usually directed against the immunoglobulin constant regions; however, anti-idiotypic responses can also be detected. The HAMA responses reduce the efficacy of treatment by removing the circulating antibody molecules, fragments, and possibly scFvs by altering the pharmacokinetic properties of the antibody. HAMA responses against divalent IgG, divalent Ig fragments, and possibly multimeric scFvs could cause immune complex formation with hypersensitivity or allergic reactions that could be harmful to patients. The use of small molecules, such as scFvs (monomeric as well as multimeric), with their shorter biological half-lives and the lack of the constant regions and humanized variable (binding regions) performed in our studies should reduce the development of HAMA. The generation of humanized and fully human scFvs should further reduce the development of HAMA. Specific accomplishments on the project are the production of large amounts of recombinant antibodies as they are required in large amounts for cancer diagnosis and therapy. A variety of single-chain Fv (scFv) constructs were engineered for the desired pharmacokinetic properties. Tetrameric and dimeric scFvs showed a two-fold advantage: (1) there was a considerable gain in avidity as compared to smaller fragments, and (2) the biological half-life was more

  3. Genetically engineered multivalent single chain antibody constructs for cancer therapy

    Energy Technology Data Exchange (ETDEWEB)

    Surinder Batra, Ph D

    2006-02-27

    its tumor: normal tissue ratio for improved therapeutic index, we engineered a variety antibody constructs. These constructs were evaluated using novel approaches like special radionuclides, pretargeting and optimization. Due to the smaller size, the engineered antibody molecules should penetrate better throughout a tumor mass, with less dose heterogeneity, than is the case with intact IgG. Multivalent scFvs with an appropriate radionuclide, therefore, hold promising prospects for cancer therapy and clinical imaging in MAb-based radiopharmaceuticals. In addition, the human anti-mouse antibodies (HAMA) responses in patients against antibody-based therapy are usually directed against the immunoglobulin constant regions; however, anti-idiotypic responses can also be detected. The HAMA responses reduce the efficacy of treatment by removing the circulating antibody molecules, fragments, and possibly scFvs by altering the pharmacokinetic properties of the antibody. HAMA responses against divalent IgG, divalent Ig fragments, and possibly multimeric scFvs could cause immune complex formation with hypersensitivity or allergic reactions that could be harmful to patients. The use of small molecules, such as scFvs (monomeric as well as multimeric), with their shorter biological half-lives and the lack of the constant regions and humanized variable (binding regions) performed in our studies should reduce the development of HAMA. The generation of humanized and fully human scFvs should further reduce the development of HAMA. Specific accomplishments on the project are the production of large amounts of recombinant antibodies as they are required in large amounts for cancer diagnosis and therapy. A variety of single-chain Fv (scFv) constructs were engineered for the desired pharmacokinetic properties. Tetrameric and dimeric scFvs showed a two-fold advantage: (1) there was a considerable gain in avidity as compared to smaller fragments, and (2) the biological half-life was more

  4. Designer genes. Recombinant antibody fragments for biological imaging

    International Nuclear Information System (INIS)

    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-CH3 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

  5. Generation and characterization of heavy chain antibodies derived from Camelids

    OpenAIRE

    Schmidthals, Katrin

    2013-01-01

    Antibodies and antibody fragments are essential tools in basic research, diagnostics and therapy. Conventional antibodies consist of two heavy and two light chains with both chains contributing to the antigen-binding site. In addition to these conventional antibodies, camelids (llamas, alpacas, dromedaries and camels) possess so-called heavy chain antibodies (hcAbs) that lack the light chains. The antigen binding site of these unusual antibodies is formed by one single domain only, the so cal...

  6. Structural Basis of Zika Virus-Specific Antibody Protection.

    Science.gov (United States)

    Zhao, Haiyan; Fernandez, Estefania; Dowd, Kimberly A; Speer, Scott D; Platt, Derek J; Gorman, Matthew J; Govero, Jennifer; Nelson, Christopher A; Pierson, Theodore C; Diamond, Michael S; Fremont, Daved H

    2016-08-11

    Zika virus (ZIKV) infection during pregnancy has emerged as a global public health problem because of its ability to cause severe congenital disease. Here, we developed six mouse monoclonal antibodies (mAbs) against ZIKV including four (ZV-48, ZV-54, ZV-64, and ZV-67) that were ZIKV specific and neutralized infection of African, Asian, and American strains to varying degrees. X-ray crystallographic and competition binding analyses of Fab fragments and scFvs defined three spatially distinct epitopes in DIII of the envelope protein corresponding to the lateral ridge (ZV-54 and ZV-67), C-C' loop (ZV-48 and ZV-64), and ABDE sheet (ZV-2) regions. In vivo passive transfer studies revealed protective activity of DIII-lateral ridge specific neutralizing mAbs in a mouse model of ZIKV infection. Our results suggest that DIII is targeted by multiple type-specific antibodies with distinct neutralizing activity, which provides a path for developing prophylactic antibodies for use in pregnancy or designing epitope-specific vaccines against ZIKV. PMID:27475895

  7. Antibody Engineering & Therapeutics, the annual meeting of The Antibody Society December 7-10, 2015, San Diego, CA, USA.

    Science.gov (United States)

    Pauthner, Matthias; Yeung, Jenny; Ullman, Chris; Bakker, Joost; Wurch, Thierry; Reichert, Janice M; Lund-Johansen, Fridtjof; Bradbury, Andrew R M; Carter, Paul J; Melis, Joost P M

    2016-01-01

    The 26th Antibody Engineering & Therapeutics meeting, the annual meeting of The Antibody Society united over 800 participants from all over the world in San Diego from 6-10 December 2015. The latest innovations and advances in antibody research and development were discussed, covering a myriad of antibody-related topics by more than 100 speakers, who were carefully selected by The Antibody Society. As a prelude, attendees could join the pre-conference training course focusing, among others, on the engineering and enhancement of antibodies and antibody-like scaffolds, bispecific antibody engineering and adaptation to generate chimeric antigen receptor constructs. The main event covered 4 d of scientific sessions that included antibody effector functions, reproducibility of research and diagnostic antibodies, new developments in antibody-drug conjugates (ADCs), preclinical and clinical ADC data, new technologies and applications for bispecific antibodies, antibody therapeutics for non-cancer and orphan indications, antibodies to harness the cellular immune system, building comprehensive IgVH-gene repertoires through discovering, confirming and cataloging new germline IgVH genes, and overcoming resistance to clinical immunotherapy. The Antibody Society's special session focused on "Antibodies to watch" in 2016. Another special session put the spotlight on the limitations of the new definitions for the assignment of antibody international nonproprietary names introduced by the World Health Organization. The convention concluded with workshops on computational antibody design and on the promise and challenges of using next-generation sequencing for antibody discovery and engineering from synthetic and in vivo libraries. PMID:26909869

  8. Expression of recombinant multi-coloured fluorescent antibodies in gor -/trxB- E. coli cytoplasm

    Directory of Open Access Journals (Sweden)

    Markiv Anatoliy

    2011-11-01

    Full Text Available Abstract Background Antibody-fluorophore conjugates are invaluable reagents used in contemporary molecular cell biology for imaging, cell sorting and tracking intracellular events. However they suffer in some cases from batch to batch variation, partial loss of binding and susceptibility to photo-bleaching. In theory, these issues can all be addressed by using recombinant antibody fused directly to genetically encoded fluorescent reporters. However, single-chain fragment variable domains linked by long flexible linkers are themselves prone to disassociation and aggregation, and in some cases with isoelectric points incompatible with use in physiologically relevant milieu. Here we describe a general approach that permits fully functional intracellular production of a range of coloured fluorescent recombinant antibodies with optimally orientated VH/VL interfaces and isoelectric points compatible for use in physiological solutions at pH 7.4 with a binding site to fluorophore stoichiometry of 1:1. Results Here we report the design, assembly, intracellular bacterial production and purification of a panel of novel antibody fluorescent protein fusion constructs. The insertion of monomeric fluorescent protein derived from either Discosoma or Aequorea in-between the variable regions of anti-p185HER2-ECD antibody 4D5-8 resulted in optimal VH/VL interface interactions to create soluble coloured antibodies each with a single binding site, with isoelectric points of 6.5- 6. The fluorescent antibodies used in cell staining studies with SK-BR-3 cells retained the fluorophore properties and antibody specificity functions, whereas the conventional 4D5-8 single chain antibody with a (Gly4Ser3 linker precipitated at physiological pH 7.4. Conclusions This modular monomeric recombinant fluorescent antibody platform may be used to create a range of recombinant coloured antibody molecules for quantitative in situ, in vivo and ex vivo imaging, cell sorting and cell

  9. IBC’s 23rd Annual Antibody Engineering, 10th Annual Antibody Therapeutics International Conferences and the 2012 Annual Meeting of The Antibody Society

    OpenAIRE

    Klöhn, Peter-Christian; Wuellner, Ulrich; Zizlsperger, Nora; Zhou, Yu; Tavares, Daniel; Berger, Sven; Zettlitz, Kirstin A.; Proetzel, Gabriele; Yong, May; Begent, Richard H.J.; Reichert, Janice M

    2013-01-01

    The 23rd Annual Antibody Engineering, 10th Annual Antibody Therapeutics international conferences, and the 2012 Annual Meeting of The Antibody Society, organized by IBC Life Sciences with contributions from The Antibody Society and two Scientific Advisory Boards, were held December 3–6, 2012 in San Diego, CA. The meeting drew over 800 participants who attended sessions on a wide variety of topics relevant to antibody research and development. As a prelude to the main events, a pre-conference ...

  10. Antibodies to the N-terminal block 2 of Plasmodium falciparum merozoite surface protein 1 are associated with protection against clinical malaria

    DEFF Research Database (Denmark)

    Cavanagh, David R; Dodoo, Daniel; Hviid, Lars; Kurtzhals, Jørgen; Theander, Thor G; Akanmori, Bartholomew D; Polley, Spencer; Conway, David J; Koram, Kojo; McBride, Jana S

    2004-01-01

    This longitudinal prospective study shows that antibodies to the N-terminal block 2 region of the Plasmodium falciparum merozoite surface protein 1 (MSP-1) are associated with protection against clinical malaria in an area of stable but seasonal malaria transmission of Ghana. Antibodies to the bl....... falciparum and, thus, a promising new candidate for the development of a malaria vaccine.......This longitudinal prospective study shows that antibodies to the N-terminal block 2 region of the Plasmodium falciparum merozoite surface protein 1 (MSP-1) are associated with protection against clinical malaria in an area of stable but seasonal malaria transmission of Ghana. Antibodies to the...... block 2 region of MSP-1 were measured in a cohort of 280 children before the beginning of the major malaria transmission season. The cohort was then actively monitored for malaria, clinically and parasitologically, over a period of 17 months. Evidence is presented for an association between antibody...

  11. Antibody reactivity to conserved linear epitopes of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1)

    DEFF Research Database (Denmark)

    Staalsø, T; Khalil, E A; Elhassan, I M;

    1998-01-01

    The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family of protein antigens are involved in adhesion of P. falciparum infected erythrocytes to the capillary endothelium of the host. Antibodies to variable regions of these proteins, measured by agglutination, correlates with...... synthetic peptides derived from conserved regions of PfEMP1. The antibody responses to these peptides increased with age and were higher in individuals with asymptomatic P. falciparum infection compared to individuals presenting with fever attributable to falciparum malaria. This indicates that antibodies...... recognising the conserved regions of PfEMP1 arise upon exposure to the parasite, and that these may be involved in the development of protection against malaria. Antibodies to the Pfalhesin peptide of the human aniontransporter, band3, were measured by the same method. The magnitude of this antibody response...

  12. Antiphospholipid Antibodies in Lupus Nephritis.

    Science.gov (United States)

    Parodis, Ioannis; Arnaud, Laurent; Gerhardsson, Jakob; Zickert, Agneta; Sundelin, Birgitta; Malmström, Vivianne; Svenungsson, Elisabet; Gunnarsson, Iva

    2016-01-01

    Lupus nephritis (LN) is a major manifestation of systemic lupus erythematosus (SLE). It remains unclear whether antiphospholipid antibodies (aPL) alter the course of LN. We thus investigated the impact of aPL on short-term and long-term renal outcomes in patients with LN. We assessed levels of aPL cross-sectionally in SLE patients diagnosed with (n = 204) or without (n = 294) LN, and prospectively in 64 patients with active biopsy-proven LN (52 proliferative, 12 membranous), before and after induction treatment (short-term outcomes). Long-term renal outcome in the prospective LN cohort was determined by the estimated glomerular filtration rate (eGFR) and the Chronic Kidney Disease (CKD) stage, after a median follow-up of 11.3 years (range: 3.3-18.8). Cross-sectional analysis revealed no association between LN and IgG/IgM anticardiolipin or anti-β2-glycoprotein I antibodies, or lupus anticoagulant. Both aPL positivity and levels were similar in patients with active LN and non-renal SLE. Following induction treatment for LN, serum IgG/IgM aPL levels decreased in responders (p<0.005 for all), but not in non-responders. Both at active LN and post-treatment, patients with IgG, but not IgM, aPL had higher creatinine levels compared with patients without IgG aPL. Neither aPL positivity nor levels were associated with changes in eGFR from either baseline or post-treatment through long-term follow-up. Moreover, aPL positivity and levels both at baseline and post-treatment were similar in patients with a CKD stage ≥3 versus 1-2 at the last follow-up. In conclusion, neither aPL positivity nor levels were found to be associated with the occurrence of LN in SLE patients. However, IgG aPL positivity in LN patients was associated with a short-term impairment of the renal function while no effect on long-term renal outcome was observed. Furthermore, IgG and IgM aPL levels decreased following induction treatment only in responders, indicating that aPL levels are affected by

  13. Antiphospholipid Antibodies in Lupus Nephritis.

    Directory of Open Access Journals (Sweden)

    Ioannis Parodis

    Full Text Available Lupus nephritis (LN is a major manifestation of systemic lupus erythematosus (SLE. It remains unclear whether antiphospholipid antibodies (aPL alter the course of LN. We thus investigated the impact of aPL on short-term and long-term renal outcomes in patients with LN. We assessed levels of aPL cross-sectionally in SLE patients diagnosed with (n = 204 or without (n = 294 LN, and prospectively in 64 patients with active biopsy-proven LN (52 proliferative, 12 membranous, before and after induction treatment (short-term outcomes. Long-term renal outcome in the prospective LN cohort was determined by the estimated glomerular filtration rate (eGFR and the Chronic Kidney Disease (CKD stage, after a median follow-up of 11.3 years (range: 3.3-18.8. Cross-sectional analysis revealed no association between LN and IgG/IgM anticardiolipin or anti-β2-glycoprotein I antibodies, or lupus anticoagulant. Both aPL positivity and levels were similar in patients with active LN and non-renal SLE. Following induction treatment for LN, serum IgG/IgM aPL levels decreased in responders (p<0.005 for all, but not in non-responders. Both at active LN and post-treatment, patients with IgG, but not IgM, aPL had higher creatinine levels compared with patients without IgG aPL. Neither aPL positivity nor levels were associated with changes in eGFR from either baseline or post-treatment through long-term follow-up. Moreover, aPL positivity and levels both at baseline and post-treatment were similar in patients with a CKD stage ≥3 versus 1-2 at the last follow-up. In conclusion, neither aPL positivity nor levels were found to be associated with the occurrence of LN in SLE patients. However, IgG aPL positivity in LN patients was associated with a short-term impairment of the renal function while no effect on long-term renal outcome was observed. Furthermore, IgG and IgM aPL levels decreased following induction treatment only in responders, indicating that aPL levels are

  14. Studies on Purification of Methamidophos Monoclonal Antibodies and Comoarative Immunoactivity of Purified Antibodies

    Institute of Scientific and Technical Information of China (English)

    SU-QING ZHAO; YUAN-MING SUN; CHUN-YAN ZHANG; XIAO-YU HUANG; HOU-RUI ZHANG; ZHEN-YU ZHU

    2003-01-01

    Objective To purify Methamidophos (Met) monoclonal antibodies with two methods andcompare immune activity of purified antibodies. Method Caprylic acid ammonium sulphateprecipition (CAASP) method and Sepharose protein-A (SPA) affinity chromatography method wereused to purify Met monoclonal antibodies, UV spectrum scanning was used to determine proteincontent and recovery of purified antibodies, sodium dodecylsulphate polyacrylamide gelelectrophoresis (SDS-PAGE) was used to analyze the purity of purified antibodies, and enzyme-linkedimmunosorbent assay (ELISA) was used to determine immune activity of purified antibodies.Results Antibody protein content and recovery rate with CAASP method were 7.62 mg/mL and8.05% respectively, antibody protein content and recovery rate with SPA method were 6.45 mg/mLand 5.52% respectively. Purity of antibodies purified by SPA method was higher than that by CAASPmethod. The half-maximal inhibition concentration (IC50) of antibodies purified by SPA to Met was181.26 μg/mL, and the linear working range and the limit of quantification (LOD) were 2.43-3896.01μg/mL and 1.03 μg/mL, respectively. The IC50 of antibodies purified by CAASP to Met was 352.82μg/mL, and the linear working range and LOD were 10.91-11412.29 ug/mL and 3.42 μg/mL,respectively. Conclusion Antibodies purified by SPA method are better than those by CAASPmethod, and Met monoclonal antibodies purified by SPA method can be used to prepare gold-labelledtesting paper for analyzing Met residue in vegetable and drink water.

  15. Clinical application of a new antimyosin antibody

    International Nuclear Information System (INIS)

    A mouse monoclonal antibody, 3-48 (Rougier Bio-Tech Ltd, Montreal) which recognizes the alpha and beta heavy chains of human atrial and ventricular myosin, and the beta heavy chain of human slow skeletal muscle, has recently been developed. In the rat isoproterenol-induced infarction model and the canine model of selective obstruction of a coronary artery, the antibody was shown to be specifically localized to the necrotic myocardium. A selected group of patients with known infarction was imaged with the 111indium labeled F(ab')2 protion of this antibody in a pre-clinical feasibility study, and the results therefrom are reported in this communication. (orig.)

  16. Antibody catalysis of peptide bond formation.

    OpenAIRE

    Jacobsen, J R; Schultz, P. G.

    1994-01-01

    An antibody generated against a neutral phosphonate diester transition-state (TS not equal to) analog catalyzes the formation of an amide bond between a phenylalanyl amino group and an acyl azide derived from L-alanine. The antibody is selective for L- vs. D-alanine and does not catalyze the hydrolysis of the acyl azide to an appreciable degree. A rate acceleration of 10,000-fold relative to the uncatalyzed reaction is observed. The antibody may achieve its catalytic efficiency both by acting...

  17. The antibody approach of labeling blood cells

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, S.C.

    1991-12-31

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are criticality assessed and evaluated.

  18. The antibody approach of labeling blood cells

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, S.C.

    1992-12-31

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are critically assessed and evaluated.

  19. The antibody approach of labeling blood cells

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, S.C.

    1991-01-01

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are criticality assessed and evaluated.

  20. The antibody approach of labeling blood cells

    International Nuclear Information System (INIS)

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are critically assessed and evaluated

  1. Reshaping Human Antibodies: Grafting an Antilysozyme Activity

    Science.gov (United States)

    Verhoeyen, Martine; Milstein, Cesar; Winter, Greg

    1988-03-01

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

  2. The antibody approach of labeling blood cells

    International Nuclear Information System (INIS)

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are criticality assessed and evaluated

  3. Immunotherapy with GD2 specific monoclonal antibodies

    International Nuclear Information System (INIS)

    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 GD2 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

  4. Class specific antibody response to gonococcal infection.

    OpenAIRE

    Miettinen, A; Hakkarainen, K; Grönroos, P; Heinonen, P.; Teisala, K; Aine, R; Sillantaka, I; Saarenmaa, K; Lehtinen, M; Punnonen, R

    1989-01-01

    An enzyme immunoassay was used to determine IgM, IgG, and IgA antibodies to gonococcal pili in 68 patients with uncomplicated gonorrhoea, 35 women with pelvic inflammatory disease, and in 115 normal controls. A clear difference in response rate in all three antibody classes between patients with gonorrhoea and healthy controls was evident. Among women with gonorrhoea, the magnitude of antibody response was higher than among men with gonorrhoea, especially in the IgM class. No major difference...

  5. Primary structure and functional scFv antibody expression of an antibody against the human protooncogen c-myc.

    Science.gov (United States)

    Fuchs, P; Breitling, F; Little, M; Dübel, S

    1997-06-01

    The immunoglobulin heavy- and light-chain variable region (Vh and Vl) genes were isolated from Myc1-9E10 hybridoma cells, which secreted monoclonal antibody against human oncogen c-myc. The expression vector pOPE52-c-myc was constructed for the recombinant production in E. coli. A 30 kDa single chain fragment (scFv) expression product was found in the periplasmic space by SDS-PAGE and immunoblotting. A significant fraction was processed correctly as demonstrated with an antiserum recognizing the processed aminoterminus only. The specific binding of the scFv fragment to the peptide epitope of the maternal monoclonal antibody was demonstrated and the primary sequence of the variable regions was determined. Sequence comparison with previously published partial Vh and Vl sequences from this hybridoma cell line revealed a genetic heterogeneity for the light chain variable region. The potential use of this scFv as a new tool for detection and purification of tagged proteins, for adding costimulatory signals to the surface of cancer cells as well as for analyzing c-myc function in the living cell by cytoplasmic expression is discussed. PMID:9219032

  6. Antibodies against the Plasmodium falciparum glutamate-rich protein from naturally exposed individuals living in a Brazilian malaria-endemic area can inhibit in vitro parasite growth

    DEFF Research Database (Denmark)

    Pratt-Riccio, Lilian Rose; Bianco, Cesare; Totino, Paulo Renato Rivas;

    2011-01-01

    The glutamate-rich protein (GLURP) is an exoantigen expressed in all stages of the Plasmodium falciparum life cycle in humans. Anti-GLURP antibodies can inhibit parasite growth in the presence of monocytes via antibody-dependent cellular inhibition (ADCI), and a major parasite-inhibitory region h...

  7. Monoclonal antibody against boron carriers of BNCT. Part 1. Preparation and characterization of anti mercaptoundecahydrododecaborate antibody (anti-BSH MAb)

    International Nuclear Information System (INIS)

    The monoclonal antibody against mercaptoundecahydrododecaborate (BSH) was prepared, which recognized specifically the icosahedral boron cluster moiety and named 'anti-BSH MAb'. The dissociation constant of anti-BSH MAb against BSH was determined, and the cross reactivity was also clarified by using the enzyme-linked immunosorbent assay (ELISA). In addition, the amino acid sequences of the antigen-binding site in the variable region of heavy and light chains were partly determined and characterized upon protein database. Furthermore, a highly specific, rapid and practical immunoassay for BSH including quantitative determination of the BSH concentrations in blood by the competitive ELISA system using anti-BSH MAb has been explored. (author)

  8. Solid-phase peptide quantitation assay using labeled monoclonal antibody and glutaraldehyde fixation

    International Nuclear Information System (INIS)

    A solid-phase radioimmunoassay utilizing iodinated peptide-specific monoclonal antibody as a detection system instead of labeled peptide has been developed. Regional specific monoclonal antibodies to either gastrin-releasing peptide or gastrin were used as models to validate the general application of our modified assay. Conditions for radioactive labeling of the monoclonal antibody were determined to minimize oxidant damage, which compromises the sensitivity of other reported peptide quantitation assays. Pretreatment of 96-well polyvinyl chloride test plates with a 5% glutaraldehyde solution resulted in consistent retention of sufficient target peptide on the solid-phase matrix to allow precise quantitation. This quantitative method is completed within 1 h of peptide solid phasing. Pretreatment of assay plates with glutaraldehyde increased binding of target peptide and maximized antibody binding by optimizing antigen presentation. The hypothesis that glutaraldehyde affects both peptide binding to the plate and orientation of the peptide was confirmed by analysis of several peptide analogs. These studies indicate that peptide binding was mediated through a free amino group leaving the carboxy-terminal portion of the target peptide accessible for antibody binding. It was observed that the length of the peptide also affects the amount of monoclonal antibody that will bind. Under the optimal conditions, results from quantitation of gastrin-releasing peptide in relevant samples agree well with those from previously reported techniques. Thus, we report here a modified microplate assay which may be generally applied for the rapid and sensitive quantitation of peptide hormones

  9. Overcoming the Constraints of Anti-HIV/CD89 Bispecific Antibodies That Limit Viral Inhibition

    Directory of Open Access Journals (Sweden)

    Xiaocong Yu

    2016-01-01

    Full Text Available Innovative strategies are necessary to maximize the clinical application of HIV neutralizing antibodies. To this end, bispecific constructs of human antibody F240, reactive with well-conserved gp41 epitope and antibody 14A8, reactive with the IgA receptor (CD89 on effector cells, were constructed. A F240 × 14A8 bispecific single chain variable region (scFv molecule was constructed by linking two scFvs using a conventional GGGGS linker. Despite immunoreactivity with HIV gp41 and neutrophils, this bispecific scFv failed to inhibit HIV infection. This is in sharp contrast to viral inhibition using a chemical conjugate of the Fab of these two antibodies. Therefore, we constructed two novel Fab-like bispecific antibody molecules centered on fusion of the IgG1 CH1 domain or CH1-hinge domain to the C-terminus of F240scFv and fusion of the kappa chain CL domain to the C-terminus of 14A8scFv. Both Bi-Fab antibodies showed significant ADCVI activity for multiple clade B and clade C isolates by arming the neutrophils to inhibit HIV infection. The approach presented in this study is unique for HIV immunotherapy in that the impetus of neutralization is to arm and mobilize PMN to destroy HIV and HIV infected cells.

  10. Maximum-Entropy Models of Sequenced Immune Repertoires Predict Antigen-Antibody Affinity.

    Science.gov (United States)

    Asti, Lorenzo; Uguzzoni, Guido; Marcatili, Paolo; Pagnani, Andrea

    2016-04-01

    The immune system has developed a number of distinct complex mechanisms to shape and control the antibody repertoire. One of these mechanisms, the affinity maturation process, works in an evolutionary-like fashion: after binding to a foreign molecule, the antibody-producing B-cells exhibit a high-frequency mutation rate in the genome region that codes for the antibody active site. Eventually, cells that produce antibodies with higher affinity for their cognate antigen are selected and clonally expanded. Here, we propose a new statistical approach based on maximum entropy modeling in which a scoring function related to the binding affinity of antibodies against a specific antigen is inferred from a sample of sequences of the immune repertoire of an individual. We use our inference strategy to infer a statistical model on a data set obtained by sequencing a fairly large portion of the immune repertoire of an HIV-1 infected patient. The Pearson correlation coefficient between our scoring function and the IC50 neutralization titer measured on 30 different antibodies of known sequence is as high as 0.77 (p-value 10-6), outperforming other sequence- and structure-based models. PMID:27074145

  11. Single domain antibodies: promising experimental and therapeutic tools in infection and immunity.

    Science.gov (United States)

    Wesolowski, Janusz; Alzogaray, Vanina; Reyelt, Jan; Unger, Mandy; Juarez, Karla; Urrutia, Mariela; Cauerhff, Ana; Danquah, Welbeck; Rissiek, Björn; Scheuplein, Felix; Schwarz, Nicole; Adriouch, Sahil; Boyer, Olivier; Seman, Michel; Licea, Alexei; Serreze, David V; Goldbaum, Fernando A; Haag, Friedrich; Koch-Nolte, Friedrich

    2009-08-01

    Antibodies are important tools for experimental research and medical applications. Most antibodies are composed of two heavy and two light chains. Both chains contribute to the antigen-binding site which is usually flat or concave. In addition to these conventional antibodies, llamas, other camelids, and sharks also produce antibodies composed only of heavy chains. The antigen-binding site of these unusual heavy chain antibodies (hcAbs) is formed only by a single domain, designated VHH in camelid hcAbs and VNAR in shark hcAbs. VHH and VNAR are easily produced as recombinant proteins, designated single domain antibodies (sdAbs) or nanobodies. The CDR3 region of these sdAbs possesses the extraordinary capacity to form long fingerlike extensions that can extend into cavities on antigens, e.g., the active site crevice of enzymes. Other advantageous features of nanobodies include their small size, high solubility, thermal stability, refolding capacity, and good tissue penetration in vivo. Here we review the results of several recent proof-of-principle studies that open the exciting perspective of using sdAbs for modulating immune functions and for targeting toxins and microbes. PMID:19529959

  12. Maximum-Entropy Models of Sequenced Immune Repertoires Predict Antigen-Antibody Affinity

    Science.gov (United States)

    Marcatili, Paolo; Pagnani, Andrea

    2016-01-01

    The immune system has developed a number of distinct complex mechanisms to shape and control the antibody repertoire. One of these mechanisms, the affinity maturation process, works in an evolutionary-like fashion: after binding to a foreign molecule, the antibody-producing B-cells exhibit a high-frequency mutation rate in the genome region that codes for the antibody active site. Eventually, cells that produce antibodies with higher affinity for their cognate antigen are selected and clonally expanded. Here, we propose a new statistical approach based on maximum entropy modeling in which a scoring function related to the binding affinity of antibodies against a specific antigen is inferred from a sample of sequences of the immune repertoire of an individual. We use our inference strategy to infer a statistical model on a data set obtained by sequencing a fairly large portion of the immune repertoire of an HIV-1 infected patient. The Pearson correlation coefficient between our scoring function and the IC50 neutralization titer measured on 30 different antibodies of known sequence is as high as 0.77 (p-value 10−6), outperforming other sequence- and structure-based models. PMID:27074145

  13. PREVALENCE OF ANTIBODIES AGAINST INFLUENZA VIRUS IN NON-VACCINATED EQUINES FROM THE BRAZILIAN PANTANAL

    Directory of Open Access Journals (Sweden)

    Lucas Gaíva E Silva

    2014-12-01

    Full Text Available The prevalence of antibodies against Equine Influenza Virus (EIV was determined in 529 equines living on ranches in the municipality of Poconé, Pantanal area of Brazil, by means of the hemagglutination inhibition test, using subtype H3N8 as antigen. The distribution and possible association among positive animal and ranches were evaluated by the chi-square test, spatial autoregressive and multiple linear regression models. The prevalence of antibodies against EIV was estimated at 45.2% (95% CI 30.2 - 61.1% with titers ranging from 20 to 1,280 HAU. Seropositive equines were found on 92.0% of the surveyed ranches. Equine from non-flooded ranches (66.5% and negativity in equine infectious anemia virus (EIAV (61.7% were associated with antibodies against EIV. No spatial correlation was found among the ranches, but the ones located in non-flooded areas were associated with antibodies against EIV. A negative correlation was found between the prevalence of antibodies against EIV and the presence of EIAV positive animals on the ranches. The high prevalence of antibodies against EIV detected in this study suggests that the virus is circulating among the animals, and this statistical analysis indicates that the movement and aggregation of animals are factors associated to the transmission of the virus in the region.

  14. Prevalence of antibodies against influenza virus in non-vaccinated equines from the Brazilian Pantanal.

    Science.gov (United States)

    Gaíva e Silva, Lucas; Borges, Alice Mamede Costa Marques; Villalobos, Eliana Monteforte Cassaro; Lara, Maria do Carmo Custodio Souza Hunold; Cunha, Elenice Maria Siquetin; de Oliveira, Anderson Castro Soares; Braga, Isis Assis; Aguiar, Daniel Moura

    2014-01-01

    The prevalence of antibodies against Equine Influenza Virus (EIV) was determined in 529 equines living on ranches in the municipality of Poconé, Pantanal area of Brazil, by means of the hemagglutination inhibition test, using subtype H3N8 as antigen. The distribution and possible association among positive animal and ranches were evaluated by the chi-square test, spatial autoregressive and multiple linear regression models. The prevalence of antibodies against EIV was estimated at 45.2% (95% CI 30.2 - 61.1%) with titers ranging from 20 to 1,280 HAU. Seropositive equines were found on 92.0% of the surveyed ranches. Equine from non-flooded ranches (66.5%) and negativity in equine infectious anemia virus (EIAV) (61.7%) were associated with antibodies against EIV. No spatial correlation was found among the ranches, but the ones located in non-flooded areas were associated with antibodies against EIV. A negative correlation was found between the prevalence of antibodies against EIV and the presence of EIAV positive animals on the ranches. The high prevalence of antibodies against EIV detected in this study suggests that the virus is circulating among the animals, and this statistical analysis indicates that the movement and aggregation of animals are factors associated to the transmission of the virus in the region. PMID:25351542

  15. Overcoming the Constraints of Anti-HIV/CD89 Bispecific Antibodies That Limit Viral Inhibition.

    Science.gov (United States)

    Yu, Xiaocong; Duval, Mark; Gawron, Melissa; Posner, Marshall R; Cavacini, Lisa A

    2016-01-01

    Innovative strategies are necessary to maximize the clinical application of HIV neutralizing antibodies. To this end, bispecific constructs of human antibody F240, reactive with well-conserved gp41 epitope and antibody 14A8, reactive with the IgA receptor (CD89) on effector cells, were constructed. A F240 × 14A8 bispecific single chain variable region (scFv) molecule was constructed by linking two scFvs using a conventional GGGGS linker. Despite immunoreactivity with HIV gp41 and neutrophils, this bispecific scFv failed to inhibit HIV infection. This is in sharp contrast to viral inhibition using a chemical conjugate of the Fab of these two antibodies. Therefore, we constructed two novel Fab-like bispecific antibody molecules centered on fusion of the IgG1 CH1 domain or CH1-hinge domain to the C-terminus of F240scFv and fusion of the kappa chain CL domain to the C-terminus of 14A8scFv. Both Bi-Fab antibodies showed significant ADCVI activity for multiple clade B and clade C isolates by arming the neutrophils to inhibit HIV infection. The approach presented in this study is unique for HIV immunotherapy in that the impetus of neutralization is to arm and mobilize PMN to destroy HIV and HIV infected cells. PMID:27419146

  16. Maximum-Entropy Models of Sequenced Immune Repertoires Predict Antigen-Antibody Affinity.

    Directory of Open Access Journals (Sweden)

    Lorenzo Asti

    2016-04-01

    Full Text Available The immune system has developed a number of distinct complex mechanisms to shape and control the antibody repertoire. One of these mechanisms, the affinity maturation process, works in an evolutionary-like fashion: after binding to a foreign molecule, the antibody-producing B-cells exhibit a high-frequency mutation rate in the genome region that codes for the antibody active site. Eventually, cells that produce antibodies with higher affinity for their cognate antigen are selected and clonally expanded. Here, we propose a new statistical approach based on maximum entropy modeling in which a scoring function related to the binding affinity of antibodies against a specific antigen is inferred from a sample of sequences of the immune repertoire of an individual. We use our inference strategy to infer a statistical model on a data set obtained by sequencing a fairly large portion of the immune repertoire of an HIV-1 infected patient. The Pearson correlation coefficient between our scoring function and the IC50 neutralization titer measured on 30 different antibodies of known sequence is as high as 0.77 (p-value 10-6, outperforming other sequence- and structure-based models.

  17. [Anti-basal ganglia antibody].

    Science.gov (United States)

    Hayashi, Masaharu

    2013-04-01

    Sydenham's chorea (SC) is a major manifestation of rheumatic fever, and the production of anti-basal ganglia antibodies (ABGA) has been proposed in SC. The pathogenesis is hypothesized as autoimmune targeting of the basal ganglia via molecular mimicry, triggered by streptococcal infection. The spectrum of diseases in which ABGA may be involved has been broadened to include other extrapyramidal movement disorders, such as tics, dystonia, and Parkinsonism, as well as other psychiatric disorders. The autoimmune hypothesis in the presence and absence of ABGA has been suggested in Tourette's syndrome (TS), early onset obsessive-compulsive disorders (OCD), and pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS). Recently, the relationship between ABGA and dopamine neurons in the basal ganglia has been examined, and autoantibodies against dopamine receptors were detected in the sera from patients with basal ganglia encephalitis. In Japan, the occurrence of subacute encephalitis, where patients suffer from episodes of altered behavior and involuntary movements, has increased. Immune-modulating treatments are effective, indicating the involvement of an autoimmune mechanism. We aimed to detect the anti-neuronal autoantibodies in such encephalitis, using immunohistochemical assessment of patient sera. The sera from patients showing involuntary movements had immunoreactivity for basal ganglia neurons. Further epitopes for ABGA will be investigated in basal ganglia disorders other than SC, TS, OCD, and PANDAS. PMID:23568985

  18. Antibody-guided irradiation of hepatic metastases using intrahepatically administered radiolabelled anti-CEA antibodies with simultaneous and reversible hepatic blood flow stasis using biodegradable starch microspheres.

    Science.gov (United States)

    Epenetos, A A; Courtenay-Luck, N; Dhokia, B; Snook, D; Hooker, G; Lavender, J P; Hemmingway, A; Carr, D; Paraharalambous, M; Bosslet, K

    1987-12-01

    Two monoclonal antibodies to carcinoembryonic antigen (CEA) were radiolabelled with 131I and used for the treatment of hepatic metastases in a patient who had a primary colonic carcinoma. Approximately 100 mCi of 131I-labelled antibody were administered via the hepatic artery on two occasions. On the second occasion, radiolabelled antibody was given concurrently with biodegradable starch microspheres in an attempt to enhance tumour uptake of antibody by achieving temporary stasis or delay of hepatic blood flow. The procedure was carried out uneventfully. There was clinical improvement and a fall in circulating CEA levels after each course of treatment. Furthermore, after the second course of therapy the clinical improvement was sustained for a longer period (more than 3 months) and there was evidence of diminution in the size of some of the liver metastases. Regional administration of 131I-labelled anti-CEA antibody concurrently with biodegradable starch microspheres appears to be a promising new method for the treatment of hepatic metastases from colonic carcinoma. PMID:3449789

  19. Impact of variable domain glycosylation on antibody clearance: an LC/MS characterization.

    Science.gov (United States)

    Huang, Lihua; Biolsi, Susan; Bales, Kelly R; Kuchibhotla, Uma

    2006-02-15

    Variable (Fv) domain N-glycosylation sites are found in approximately 20% of human immunoglobulin Gs (IgGs) in addition to the conserved N-glycosylation sites in the C(H)2 domains. The carbohydrate structures of the Fv glycans and their impact on in vivo half-life are not well characterized. Oligosaccharide structures in a humanized anti-Abeta IgG1 monoclonal antibody (Mab) with an N-glycosylation site in the complementary determining region (CDR2) of the heavy chain variable region were elucidated by LC/MS analysis following sequential exoglycosidase treatments of the endoproteinase Lys-C digest. Results showed that the major N-linked oligosaccharide structures in the Fv region have three characteristics (core-fucosylated biantennary oligosaccharides with one or two N-glycolylneuraminic acid [NeuGc] residues, zero or one alpha-linked Gal residue, and zero or one beta-linked GalNAc residue), whereas N-linked oligosaccharides in the Fc region contained typical Fc glycans (core-fucosylated, biantennary oligosaccharides with zero to two Gal residues). To elucidate the contribution of Fv glycans to the half-life of the antibody, a method that allows capture of the Mab and determination of its glycan structures at various time points after administration to mice was developed. Anti-Abeta antibody in mouse serum was immunocaptured by immobilized goat anti-human immunoglobulin Fc(gamma) antibody resin, and the captured material was treated with papain to generate Fab and Fc for LC/MS analysis. Different glycans in the Fc region showed the same clearance rate as demonstrated previously. In contrast to many other non-antibody glycosylated therapeutics, there is no strong correlation between oligosaccharide structures in the Fv region and their clearance rates in vivo. Our data indicated that biantennary oligosaccharides lacking galactosylation had slightly faster clearance rates than other structures in the Fv domain. PMID:16360109

  20. Evidence for a novel human-specific xeno-auto-antibody response against vascular endothelium.

    Science.gov (United States)

    Pham, Tho; Gregg, Christopher J; Karp, Felix; Chow, Renee; Padler-Karavani, Vered; Cao, Hongzhi; Chen, Xi; Witztum, Joseph L; Varki, Nissi M; Varki, Ajit

    2009-12-10

    Humans are genetically unable to synthesize the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc). However, Neu5Gc can be metabolically incorporated and covalently expressed on cultured human cell surfaces. Meanwhile, humans express varying and sometimes high titers of polyclonal anti-Neu5Gc antibodies. Here, a survey of human tissues by immunohistochemistry with both a monospecific chicken anti-Neu5Gc antibody and with affinity-purified human anti-Neu5Gc antibodies demonstrates endothelial expression of Neu5Gc, likely originating from Neu5Gc-rich foods like red meats. We hypothesized that the combination of Neu5Gc incorporation and anti-Neu5Gc antibodies can induce endothelial activation. Indeed, the incubation of high-titer human sera with Neu5Gc-fed endothelial cells led to Neu5Gc-dependent antibody binding, complement deposition, endothelial activation, selectin expression, increased cytokine secretion, and monocyte binding. The proinflammatory cytokine tumor necrosis factor-alpha also selectively enhanced human anti-Neu5Gc antibody reactivity. Anti-Neu5Gc antibodies affinity-purified from human serum also directed Neu5Gc-dependent complement deposition onto cultured endothelial cells. These data indicate a novel human-specific mechanism in which Neu5Gc-rich foods deliver immunogenic Neu5Gc to the endothelium, giving anti-Neu5Gc antibody- and complement-dependent activation, and potentially contributing to human vascular pathologies. In the case of atherosclerosis, Neu5Gc is present both in endothelium overlying plaques and in subendothelial regions, providing multiple pathways for accelerating inflammation in this disease. PMID:19828701

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

    International Nuclear Information System (INIS)

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

  2. The structural analysis of shark IgNAR antibodies reveals evolutionary principles of immunoglobulins.

    Science.gov (United States)

    Feige, Matthias J; Gräwert, Melissa A; Marcinowski, Moritz; Hennig, Janosch; Behnke, Julia; Ausländer, David; Herold, Eva M; Peschek, Jirka; Castro, Caitlin D; Flajnik, Martin; Hendershot, Linda M; Sattler, Michael; Groll, Michael; Buchner, Johannes

    2014-06-01

    Sharks and other cartilaginous fish are the phylogenetically oldest living organisms that rely on antibodies as part of their adaptive immune system. They produce the immunoglobulin new antigen receptor (IgNAR), a homodimeric heavy chain-only antibody, as a major part of their humoral adaptive immune response. Here, we report the atomic resolution structure of the IgNAR constant domains and a structural model of this heavy chain-only antibody. We find that despite low sequence conservation, the basic Ig fold of modern antibodies is already present in the evolutionary ancient shark IgNAR domains, highlighting key structural determinants of the ubiquitous Ig fold. In contrast, structural differences between human and shark antibody domains explain the high stability of several IgNAR domains and allowed us to engineer human antibodies for increased stability and secretion efficiency. We identified two constant domains, C1 and C3, that act as dimerization modules within IgNAR. Together with the individual domain structures and small-angle X-ray scattering, this allowed us to develop a structural model of the complete IgNAR molecule. Its constant region exhibits an elongated shape with flexibility and a characteristic kink in the middle. Despite the lack of a canonical hinge region, the variable domains are spaced appropriately wide for binding to multiple antigens. Thus, the shark IgNAR domains already display the well-known Ig fold, but apart from that, this heavy chain-only antibody employs unique ways for dimerization and positioning of functional modules. PMID:24830426

  3. Antibodies against the calcium-binding protein

    International Nuclear Information System (INIS)

    Plant microsomes contain a protein clearly related to a calcium-binding protein, calsequestrin, originally found in the sarcoplasmic reticulum of muscle cells, responsible for the rapid release and uptake of Ca2+ within the cells. The location and role of calsequestrin in plant cells is unknown. To generate monoclonal antibodies specific to plant calsequestrin, mice were immunized with a microsomal fraction from cultured cells of Streptanthus tortuosus (Brassicaceae). Two clones cross-reacted with one protein band with a molecular weight equal to that of calsequestrin (57 kilodaltons) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. This band is able to bind 45Ca2+ and can be recognized by a polyclonal antibody against the canine cardiac muscle calsequestrin. Rabbit skeletal muscle calsequestrin cross-reacted with the plant monoclonal antibodies. The plant monoclonal antibodies generated here are specific to calsequestrin protein

  4. Polynucleotides encoding anti-sulfotyrosine antibodies

    Science.gov (United States)

    Bertozzi, Carolyn R.; Kehoe, John; Bradbury, Andrew M.

    2011-01-11

    The invention provides anti-sulfotyrosine specific antibodies capable of detecting and isolating polypeptides that are tyrosine-sulfated. The sulfotyrosine antibodies and antibody fragments of the invention may be used to discriminate between the non-sulfated and sulfated forms of such proteins, using any number of immunological assays, such ELISAs, immunoblots, Western Blots, immunoprecipitations, and the like. Using a phage-display system, single chain antibodies (scFvs) were generated and screened against tyrosine-sulfated synthetic peptide antigens, resulting in the isolation of scFvs that specifically recognize sulfotyrosine-containing peptides and/or demonstrate sulfotyrosine-specific binding in tyrosine sulfated proteins. The VH and VL genes from one such sulfotyrosine-specific scFv were employed to generate a full length, sulfotyrosine-specific immunoglobulin.

  5. Patient-Derived Antibody Targets Tumor Cells

    Science.gov (United States)

    An NCI Cancer Currents blog on an antibody derived from patients that killed tumor cells in cell lines of several cancer types and slowed tumor growth in mouse models of brain and lung cancer without evidence of side effects.

  6. Chemical biology: How to minimalize antibodies

    Science.gov (United States)

    Rader, Christoph

    2015-02-01

    The success of antibodies as pharmaceuticals has triggered interest in crafting much smaller mimics. A crucial step forward has been taken with the chemical synthesis of small molecules that recruit immune cells to attack cancer cells.

  7. Immunoglobulin Classification Using the Colored Antibody Graph.

    Science.gov (United States)

    Bonissone, Stefano R; Pevzner, Pavel A

    2016-06-01

    The somatic recombination of V, D, and J gene segments in B-cells introduces a great deal of diversity, and divergence from reference segments. Many recent studies of antibodies focus on the population of antibody transcripts that show which V, D, and J gene segments have been favored for a particular antigen, a repertoire. To properly describe the antibody repertoire, each antibody must be labeled by its constituting V, D, and J gene segment, a task made difficult by somatic recombination and hypermutation events. While previous approaches to repertoire analysis were based on sequential alignments, we describe a new de Bruijn graph-based algorithm to perform VDJ labeling and benchmark its performance. PMID:27149636

  8. Deep sequencing and human antibody repertoire analysis.

    Science.gov (United States)

    Boyd, Scott D; Crowe, James E

    2016-06-01

    In the past decade, high-throughput DNA sequencing (HTS) methods and improved approaches for isolating antigen-specific B cells and their antibody genes have been applied in many areas of human immunology. This work has greatly increased our understanding of human antibody repertoires and the specific clones responsible for protective immunity or immune-mediated pathogenesis. Although the principles underlying selection of individual B cell clones in the intact immune system are still under investigation, the combination of more powerful genetic tracking of antibody lineage development and functional testing of the encoded proteins promises to transform therapeutic antibody discovery and optimization. Here, we highlight recent advances in this fast-moving field. PMID:27065089

  9. Monoclonal antibodies for radioimmunoimaging: Current perspectives

    International Nuclear Information System (INIS)

    The ability to image tumor using radiolabeled monoclonal antibody products has been widely demonstrated. The questions of safety and efficacy remain open and require further experience, but in some clinical situations, radioimmunoimaging has provided clinically useful information. This paper deals with a set of current problems in imaging with radiolabeled monoclonal antibodies and current perspectives on the possible solutions to these problems. The major areas discussed here are the following: (a) The selection process. How might we choose the ''best'' antibody for imaging from among the multitude now available and what form (i.e., which fragments) may be useful? (b) The imaging procedure: What are the basic optimal imaging parameters and how does the data produced by this modality interface with information obtained by more standard methods of imaging? (c) Quantitative techniques: How can noninvasive quantitative techniques provide information useful to the antibody selection process and to the diagnostic and therapeutic applications

  10. Antibody deficiency in Rubinstein-Taybi syndrome.

    Science.gov (United States)

    Herriot, R; Miedzybrodzka, Z

    2016-03-01

    The developmental disorder Rubinstein-Taybi syndrome (RTS) is frequently complicated by recurrent respiratory infections. In many cases this is likely to be the result of microaspiration or gastro-oesophageal reflux but, in a proportion, underlying antibody deficiency is a potentially modifiable susceptibility factor for infection. Relatively subtle, specific defects of pneumococcal antibody production have previously been described in the context of RTS. Here, we report a rare association between the syndrome and an overt, major primary antibody deficiency disorder (common variable immune deficiency) which was successfully managed with immunoglobulin replacement therapy. Early recognition and investigation for antibody deficiency associated with RTS allied to effective and optimized treatment are essential to minimize morbidity and mortality and improve quality and duration of life. PMID:26307339

  11. New haptens and antibodies for ractopamine.

    Science.gov (United States)

    Wang, Zhanhui; Liu, Meixuan; Shi, Weimin; Li, Chenglong; Zhang, Suxia; Shen, Jianzhong

    2015-09-15

    In this work, three unreported immunizing haptens of ractopamine (RAC) were synthesized and used to produce highly sensitive and specific polyclonal antibody. The spacer arms of haptens for coupling to protein carrier were located on different position of RAC with different length. High affinity polyclonal antibodies were obtained and characterized in terms of titer and sensitivity by using enzyme-linked immunosorbent assay (ELISA). The best antibody employed in a heterologous competitive ELISA exhibited an IC50 value as low as 0.12ngmL(-1) and could not recognize other 10 β-agonists including clenbuterol and salbutamol. The heterologous competitive ELISA was preliminary applied to swine urine and the results showed the new antibody was sufficiently sensitive and specific, and potentially used for the detection of RAC at trace level in real samples. PMID:25863617

  12. Localization of tumors by radiolabelled antibodies

    International Nuclear Information System (INIS)

    A method of utilizing radiolabelled antibodies to carcinoembryonic antigens for determining the site of tumors which produce or are associated with carcinoembryonic antigen is disclosed. 3 claims, no drawings

  13. The effect of anti-human plasminogen monoclonal antibodies on Glu-plasminogen activation by plasminogen activators

    Directory of Open Access Journals (Sweden)

    M. Akrami

    2006-07-01

    the A1D12 F(ab region to Glu-plasminogen increases the catalytic efficiency of plasminogen activation by plasminogen activators. Therefore, it may be useful to apply clinically A1D12 for the therapy of thromboembolic events such as myocardial infarction by humanizing the F(ab fragment of the A1D12 antibody. Inhibition pattern of antibody MC2B8 obey the mixed type of enzyme inhibition by binding the antibody probably at, or near, the cleavage site of Glu-plasminogen.

  14. Seroprevalence of Toxoplasma gondii antibodies in captive wild mammals and birds in Brazil.

    Science.gov (United States)

    In this study serum samples of 203 animals from different locations from zoos and breeding facilities from the north and northeast region of Brazil were analyzed for the presence of anti-Toxoplasma gondii antibodies by the modified agglutination test (MAT) with a cutoff of 1:25. Of the sampled anima...

  15. Lack of in Vivo Antibody Dependent Cellular Cytotoxicity with Antibody Containing Gold Nanoparticles

    OpenAIRE

    Ahmed, Marya; Pan, Dorothy W.; Davis, Mark E.

    2015-01-01

    Antibody-dependent cellular cytotoxicity (ADCC) is a cytolytic mechanism that can elicit in vivo antitumor effects and can play a significant role in the efficacy of antibody treatments for cancer. Here, we prepared cetuximab, panitumumab, and rituximab containing gold nanoparticles and investigated their ability to produce an ADCC effect in vivo. Cetuximab treatment of EGFR-expressing H1975 tumor xenografts showed significant tumor regression due to the ADCC activity of the antibody in vivo,...

  16. Antibody-Specific Model of Amino Acid Substitution for Immunological Inferences from Alignments of Antibody Sequences

    OpenAIRE

    Mirsky, Alexander; Kazandjian, Linda; Anisimova, Maria

    2014-01-01

    Antibodies are glycoproteins produced by the immune system as a dynamically adaptive line of defense against invading pathogens. Very elegant and specific mutational mechanisms allow B lymphocytes to produce a large and diversified repertoire of antibodies, which is modified and enhanced throughout all adulthood. One of these mechanisms is somatic hypermutation, which stochastically mutates nucleotides in the antibody genes, forming new sequences with different properties and, eventually, hig...

  17. Quantitative Assessment of Antibody Internalization with Novel Monoclonal Antibodies against Alexa Fluorophores

    OpenAIRE

    Liao-Chan, Sindy; Daine-Matsuoka, Barbara; Heald, Nathan; Wong, Tiffany; Lin, Tracey; Cai, Allen G.; Lai, Michelle; D’Alessio, Joseph A.; Theunissen, Jan-Willem

    2015-01-01

    Antibodies against cell surface antigens may be internalized through their specific interactions with these proteins and in some cases may induce or perturb antigen internalization. The anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen. Numerous techniques, including microscopy and flow cytometry, have been used to identify antibodies with desired cellular uptake rates. To enable quantitative measurements of inte...

  18. Development of Monoclonal Antibodies Suitable for Rabies Virus Antibody and Antigen Detection

    OpenAIRE

    Chander, Vishal; Singh, R.P.; Verma, P. C.

    2012-01-01

    The control of an infectious viral disease as rabies is made easier by rapid and accurate diagnosis. Successful rabies prophylaxis is dependent upon the active immunization with vaccine along with passive administration of rabies virus neutralizing antibodies which together clear the virus before widespread infection of central nervous system occurs. The present study aimed at the development of monoclonal antibodies (MAbs) suitable for rabies virus antibody and antigen detection. For the pro...

  19. A monoclonal thyroid-stimulating antibody

    OpenAIRE

    Ando, Takao; Latif, Rauf; Pritsker, Alla; Moran, Thomas; Nagayama, Yuji; Davies, Terry F.

    2002-01-01

    The thyrotropin receptor, also known as the thyroid-stimulating hormone receptor (TSHR), is the primary antigen of Graves disease. Stimulating TSHR antibodies are the cause of thyroid overstimulation and were originally called long-acting thyroid stimulators due to their prolonged action. Here we report the successful cloning and characterization of a monoclonal antibody (MS-1) with TSHR-stimulating activity. The thyroid-stimulating activity of MS-1 was evident at IgG concentrations as low as...

  20. History and Practice: Antibodies in Infectious Diseases.

    Science.gov (United States)

    Hey, Adam

    2015-04-01

    Antibodies and passive antibody therapy in the treatment of infectious diseases is the story of a treatment concept which dates back more than 120 years, to the 1890s, when the use of serum from immunized animals provided the first effective treatment options against infections with Clostridium tetani and Corynebacterium diphtheriae. However, after the discovery of penicillin by Fleming in 1928, and the subsequent introduction of the much cheaper and safer antibiotics in the 1930s, serum therapy was largely abandoned. However, the broad and general use of antibiotics in human and veterinary medicine has resulted in the development of multi-resistant strains of bacteria with limited to no response to existing treatments and the need for alternative treatment options. The combined specificity and flexibility of antibody-based treatments makes them very valuable tools for designing specific antibody treatments to infectious agents. These attributes have already caused a revolution in new antibody-based treatments in oncology and inflammatory diseases, with many approved products. However, only one monoclonal antibody, palivizumab, for the prevention and treatment of respiratory syncytial virus, is approved for infectious diseases. The high cost of monoclonal antibody therapies, the need for parallel development of diagnostics, and the relatively small markets are major barriers for their development in the presence of cheap antibiotics. It is time to take a new and revised look into the future to find appropriate niches in infectious diseases where new antibody-based treatments or combinations with existing antibiotics, could prove their value and serve as stepping stones for broader acceptance of the potential for and value of these treatments. PMID:26104697

  1. Single-domain antibodies for brain targeting

    OpenAIRE

    Lalatsa, Katerina; Moreira Leite, Diana

    2014-01-01

    Smaller recombinant antibody fragments as single-domain antibodies (sdAbs) are emerging as credible alternatives because of their target specificity, high affinity, and cost-effective recombinant production. sdAbs have been forged into multivalent and multispecif ic therapeutics, or targeting moieties, that are able to shuttle their linked therapeutic cargo (i.e., drugs, nanoparticles, toxins, enzymes, and radionuclides) to the receptor of interest. Their ability to permeate across the blood ...

  2. Influenza-Specific Antibody-Dependent Phagocytosis

    OpenAIRE

    Ana-Sosa-Batiz, Fernanda; Vanderven, Hillary; Jegaskanda, Sinthujan; Johnston, Angus; Rockman, Steven; Laurie, Karen; Barr, Ian; Reading, Patrick; Lichtfuss, Marit; Stephen J Kent

    2016-01-01

    Background Immunity to human influenza A virus (IAV) infection is only partially understood. Broadly non-neutralizing antibodies may assist in reducing disease but have not been well characterized. Methods We measured internalization of opsonized, influenza protein-coated fluorescent beads and live IAV into a monocytic cell line to study antibody-dependent phagocytosis (ADP) against multiple influenza hemagglutinin (HA) subtypes. We analyzed influenza HA-specific ADP in healthy human donors, ...

  3. Clearance of pathological antibodies using biomimetic nanoparticles

    OpenAIRE

    Copp, Jonathan A.; Fang, Ronnie H.; Luk, Brian T.; Hu, Che-Ming J.; Gao, Weiwei; Zhang, Kang; Zhang, Liangfang

    2014-01-01

    The selective depletion of disease-causing antibodies using nanoparticles offers a new model in the management of type II immune hypersensitivity reactions. The demonstration of pathophysiologically inspired nanoengineering serves as a valuable prototype for additional therapeutic improvements with the goal of minimizing therapy-related adverse effects. Through the use of cell membrane-cloaked nanoparticles, nanoscale decoys with strong affinity to pathological antibodies can be administered ...

  4. Monoclonal antibodies to Bacteroides fragilis lipopolysaccharide.

    OpenAIRE

    Linko-Kettunen, L; Arstila, P; Jalkanen, M; Jousimies-Somer, H; Lassila, O; Lehtonen, O P; Weintraub, A; Viljanen, M K

    1984-01-01

    Monoclonal antibodies (MoAbs) to the lipopolysaccharide (LPS) of Bacteroides fragilis were produced by immunizing mice before hybridization with bacterial outer membranes solubilized with Triton X-100. Nineteen stabile clones were established. They all produced antibodies that reacted more strongly with purified B. fragilis LPS than with crude sonicated antigen in an enzyme immunoassay. Four MoAbs were studied by immunoblotting and enzyme immunoassay inhibition. Immunoblotting confirmed that ...

  5. Simultaneous Assessment of Asp Isomerization and Asn Deamidation in Recombinant Antibodies by LC-MS following Incubation at Elevated Temperatures

    OpenAIRE

    Diepold, Katharina; Bomans, Katrin; Wiedmann, Michael; Zimmermann, Boris; Petzold, Andreas; Schlothauer, Tilman; Mueller, Robert; Moritz, Bernd; Stracke, Jan Olaf; Mølhøj, Michael; Reusch, Dietmar; Bulau, Patrick

    2012-01-01

    The degradation of proteins by asparagine deamidation and aspartate isomerization is one of several chemical degradation pathways for recombinant antibodies. In this study, we have identified two solvent accessible degradation sites (light chain aspartate-56 and heavy chain aspartate-99/101) in the complementary-determining regions of a recombinant IgG1 antibody susceptible to isomerization under elevated temperature conditions. For both hot-spots, the degree of isomerization was found to be ...

  6. Antibody-Conjugated Nanoparticles for Biomedical Applications

    Directory of Open Access Journals (Sweden)

    Manuel Arruebo

    2009-01-01

    Full Text Available Nanoscience and Nanotechnology have found their way into the fields of Biotechnology and Medicine. Nanoparticles by themselves offer specific physicochemical properties that they do not exhibit in bulk form, where materials show constant physical properties regardless of size. Antibodies are nanosize biological products that are part of the specific immune system. In addition to their own properties as pathogens or toxin neutralizers, as well as in the recruitment of immune elements (complement, improving phagocytosis, cytotoxicity antibody dependent by natural killer cells, etc., they could carry several elements (toxins, drugs, fluorochroms, or even nanoparticles, etc. and be used in several diagnostic procedures, or even in therapy to destroy a specific target. The conjugation of antibodies to nanoparticles can generate a product that combines the properties of both. For example, they can combine the small size of nanoparticles and their special thermal, imaging, drug carrier, or magnetic characteristics with the abilities of antibodies, such as specific and selective recognition. The hybrid product will show versatility and specificity. In this review, we analyse both antibodies and nanoparticles, focusing especially on the recent developments for antibody-conjugated nanoparticles, offering the researcher an overview of the different applications and possibilities of these hybrid carriers.

  7. Decay of maternal antibodies in broiler chickens.

    Science.gov (United States)

    Gharaibeh, Saad; Mahmoud, Kamel

    2013-09-01

    The objective of this study was to determine the decay rate of maternal antibodies against major broiler chicken pathogens. A total of 30 one-day-old broiler chicks were obtained from a commercial hatchery and reared in isolation. These chicks were retrieved from a parent flock that received a routine vaccination program. Chicks were bled at hatch and sequentially thereafter every 5 d through 30 d of age. Maternal antibody titers were measured by ELISA for avian encephalomyelitis (AEV), avian influenza virus (AIV), chicken anemia virus (CAV), infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS), and reovirus (Reo). Maternal antibody titers for Newcastle disease virus (NDV) were measured using a hemagglutination inhibition test. Half-life estimates of maternal antibody titers were 5.3, 4.2, 7, 5.1, 3.9, 3.8, 4.9, 4.1, 6.3, and 4.7 d for AEV, AIV, CAV, IBDV, IBV, ILTV, MG, MS, NDV, and Reo, respectively. The statistical analysis revealed significant differences among half-lives of maternal antibody titers against certain pathogens. Furthermore, all maternal antibody titers were depleted by 10 d of age except for IBDV. PMID:23960115

  8. Quality control of antibodies for assay development.

    Science.gov (United States)

    Schumacher, Sarah; Seitz, Harald

    2016-09-25

    Antibodies are used as powerful tools in basic research, for example, in biomarker identification, and in various forms for diagnostics, for example, identification of allergies or autoimmune diseases. Due to their robustness and ease of handling, immunoassays are favourite methods for investigation of various biological or medical questions. Nevertheless in many cases, additional analyses such as mass spectrometry are used to validate or confirm the results of immunoassays. To minimize the workload and to increase confidence in immunoassays, there are urgent needs for antibodies which are both highly specific and well validated. Unfortunately many commercially available antibodies are neither well characterized nor fully tested for cross-reactivities. Adequate quality control and validation of an antibody is time-consuming and can be frustrating. Such validation needs to be performed for every assay/application. However, where an antibody validation is successful, a highly specific and stable reagent will be on hand. This article describes the validation processes of antibodies, including some often neglected factors, as well as unspecific binding to other sample compounds in a multiparameter diagnostic assay. The validation consists of different immunological methods, with important assay controls, and is performed in relation to the development of a diagnostic test. PMID:26873787

  9. 21 CFR 866.5110 - Antiparietal antibody immunological test system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Antiparietal antibody immunological test system....5110 Antiparietal antibody immunological test system. (a) Identification. An antiparietal antibody... the specific antibody for gastric parietal cells in serum and other body fluids. Gastric...

  10. 21 CFR 866.5100 - Antinuclear antibody immunological test system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Antinuclear antibody immunological test system....5100 Antinuclear antibody immunological test system. (a) Identification. An antinuclear antibody... the autoimmune antibodies in serum, other body fluids, and tissues that react with cellular...

  11. Avian Diagnostic and Therapeutic Antibodies to Viral Emerging Pathogens

    Energy Technology Data Exchange (ETDEWEB)

    David Bradley

    2011-03-31

    During the current period the following key objectives were achieved: demonstration of high titer antibody production by geese following immunization with inactived H1N1 virus; completion of the epitope mapping of West Nile Virus-specific goose antibodies and initiation of epitope mapping of H1N1 flu-specific goose antibodies; advancement in scalable purification of goose antibodies.

  12. Fast antibody fragment motion: flexible linkers act as entropic spring.

    Science.gov (United States)

    Stingaciu, Laura R; Ivanova, Oxana; Ohl, Michael; Biehl, Ralf; Richter, Dieter

    2016-01-01

    A flexible linker region between three fragments allows antibodies to adjust their binding sites to an antigen or receptor. Using Neutron Spin Echo Spectroscopy we observed fragment motion on a timescale of 7 ns with motional amplitudes of about 1 nm relative to each other. The mechanistic complexity of the linker region can be described by a spring model with Brownian motion of the fragments in a harmonic potential. Displacements, timescale, friction and force constant of the underlying dynamics are accessed. The force constant exhibits a similar strength to an entropic spring, with friction of the fragment matching the unbound state. The observed fast motions are fluctuations in pre-existing equilibrium configurations. The Brownian motion of domains in a harmonic potential is the appropriate model to examine functional hinge motions dependent on the structural topology and highlights the role of internal forces and friction to function. PMID:27020739

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

    International Nuclear Information System (INIS)

    . (J. Mol. Biol. 275:269), and the participation of specific residues in antigen recognition was assessed using site-directed mutagenesis. Three amino acids in the light chain variable region, H39, Y54 and F103, were particularly important in antigen recognition. In a separate series of experiments, a recombinant phage-displayed antibody library has been prepared using RNA isolated from the spleens of sheep and rabbits immunized with specific metal-chelate complexes. Phage-display libraries produced from an immunized source are inclined to include variable genes specific for the immunized antigen(s), many of which are already affinity matured. An antibody fragment specific for the UO22+-DCP complex was isolated from this combined phage display library. While the binding affinity of this antibody fragment for UO22+-DCP was not as high as that of the 12F6 monoclonal antibody, the beauty of antibody phage display technology is that it allows for the potential manipulation and saturation of the antibody's binding affinity, which may drastically improve and ultimately surpass that of monoclonal antibodies.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2005-04-18

    canonical structures method detailed by Morea et al. (J. Mol. Biol. 275:269), and the participation of specific residues in antigen recognition was assessed using site-directed mutagenesis. Three amino acids in the light chain variable region, H39, Y54 and F103, were particularly important in antigen recognition. In a separate series of experiments, a recombinant phage-displayed antibody library has been prepared using RNA isolated from the spleens of sheep and rabbits immunized with specific metal-chelate complexes. Phage-display libraries produced from an immunized source are inclined to include variable genes specific for the immunized antigen(s), many of which are already affinity matured. An antibody fragment specific for the UO{sub 2}{sup 2+}-DCP complex was isolated from this combined phage display library. While the binding affinity of this antibody fragment for UO{sub 2}{sup 2+}-DCP was not as high as that of the 12F6 monoclonal antibody, the beauty of antibody phage display technology is that it allows for the potential manipulation and saturation of the antibody's binding affinity, which may drastically improve and ultimately surpass that of monoclonal antibodies.

  15. A multi-Fc-species system for recombinant antibody production

    OpenAIRE

    Nizak Clément; Vielemeyer Ole; El Marjou Ahmed; Moutel Sandrine; Benaroch Philippe; Dübel Stefan; Perez Franck

    2009-01-01

    Abstract Background Genomic, transcriptomic and proteomic projects often suffer from a lack of functional validation creating a strong demand for specific and versatile antibodies. Antibody phage display represents an attractive approach to select rapidly in vitro the equivalent of monoclonal antibodies, like single chain Fv antibodies, in an inexpensive and animal free way. However, so far, recombinant antibodies have not managed to impose themselves as efficient alternatives to natural anti...

  16. Antigen-Specific Antibody Glycosylation Is Regulated via Vaccination

    OpenAIRE

    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.

    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 (Fc domain) regulate antibody functionality: changes in antibody subclass and changes in a single N-linked glycan located in the CH2 domain of the IgG Fc. Together, these modifications provide a spe...

  17. Ability to develop broadly neutralizing HIV-1 antibodies is not restricted by the germline immunoglobulin gene repertoire1

    Science.gov (United States)

    Scheepers, Cathrine; Shrestha, Ram K.; Lambson, Bronwen E.; Jackson, Katherine J. L.; Wright, Imogen A.; Naicker, Dshanta; Goosen, Mark; Berrie, Leigh; Ismail, Arshad; Garrett, Nigel; Karim, Quarraisha Abdool; Karim, Salim S. Abdool; Moore, Penny L.; Travers, Simon A.; Morris, Lynn

    2015-01-01

    The human immunoglobulin repertoire is vast, producing billions of unique antibodies from a limited number of germline immunoglobulin genes. The immunoglobulin heavy chain variable region (IGHV) is central to antigen binding and is comprised of 48 functional genes. Here we analyzed whether HIV-1 infected individuals who develop broadly neutralizing antibodies show a distinctive germline IGHV profile. Using both 454 and Illumina technologies we sequenced the IGHV repertoire of 28 HIV-infected South African women from the Center for the AIDS Programme of Research in South African (CAPRISA) 002 and 004 cohorts, 13 of whom developed broadly neutralizing antibodies. Of the 259 IGHV alleles identified in this study, approximately half were not found in the International Immunogenetics Database (IMGT). This included 85 entirely novel alleles and 38 alleles that matched rearranged sequences in non-IMGT databases. Analysis of the rearranged H chain V region genes of monoclonal antibodies isolated from 7 of the CAPRISA women and previously isolated broadly neutralizing antibodies from other donors provided evidence that at least 8 novel or non-IMGT alleles contributed to functional antibodies. Importantly, we found that despite a wide range in the number of IGHV alleles in each individual, including alleles used by known broadly neutralizing antibodies, there were no significant differences in germline IGHV repertoires between individuals who do and do not develop broadly neutralizing antibodies. This study reports novel IGHV repertoires and highlights the importance of a fully comprehensive immunoglobulin database for germline gene usage prediction. Furthermore, these data suggest a lack of genetic bias in broadly neutralizing antibody development in HIV-1 infection, with implications for HIV vaccine design. PMID:25825450

  18. Detection of antibodies against Chlamydophila abortus in Costa Rican sheep flocks

    OpenAIRE

    R. Villagra-Blanco; Dolz, G.; Montero-Caballero, D.; Romero-Zúñiga, J.J.

    2015-01-01

    A total of 359 sheep samples from 15 flocks were analyzed for the presence of antibodies against Chlamydophila abortus using a commercial Enzyme linked Immunosorbent Assay (ELISA). Antibodies were detected in 19 (5.29%) sheep from 12 (80%) flocks. Seropositive animals were found in all analyzed regions (Central, Chorotega, Atlantic Huetar, North Huetar and Central Pacific) determining prevalence between 0.28% and 4.4%, and intra-flock positivity between 3.7% and 25.0%. The survey revealed two...

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

    OpenAIRE

    Xuemei Zhang; Gary Guishan Xiao; Ying Gao

    2013-01-01

    In this study, the peptide sized 21 kDa covering P-gp transmembrane region was first prepared for generating a novel mouse monoclonal antibody Fab fragment with biological activity against multiple drug resistance protein P-gp21 by phage display technology. Phage-displayed antibody library prepared from mice spleen tissues was selected against the recombinant protein P-gp21 with five rounds of panning. A number of clones expressing Fab bound to P-gp21, showing neutralized activity in vitro, w...

  20. Simple Method To Prepare Oligonucleotide-Conjugated Antibodies and Its Application in Multiplex Protein Detection in Single Cells.

    Science.gov (United States)

    Gong, Haibiao; Holcomb, Ilona; Ooi, Aik; Wang, Xiaohui; Majonis, Daniel; Unger, Marc A; Ramakrishnan, Ramesh

    2016-01-20

    The diversity of nucleic acid sequences enables genomics studies in a highly multiplexed format. Since multiplex protein detection is still a challenge, it would be useful to use genomics tools for this purpose. This can be accomplished by conjugating specific oligonucleotides to antibodies. Upon binding of the oligonucleotide-conjugated antibodies to their targets, the protein levels can be converted to oligonucleotide levels. In this report we describe a simple method for preparing oligonucleotide-conjugated antibodies and discuss this method's application in oligonucleotide extension reaction (OER) for multiplex protein detection. Conjugation is based on strain-promoted alkyne-azide cycloaddition (the Cu-free click reaction), in which the antibody is activated with a dibenzocyclooctyne (DBCO) moiety and subsequently linked covalently with an azide-modified oligonucleotide. In the functional test, the reaction conditions and purification processes were optimized to achieve maximum yield and best performance. The OER assay employs a pair of antibody binders (two antibodies, each conjugated with its own oligonucleotide) developed for each protein target. The two oligonucleotides contain unique six-base complementary regions at their 3' prime ends to allow annealing and extension by DNA synthesis enzymes to form a DNA template. Following preamplification, the DNA template is detected by qPCR. Distinct oligonucleotide sequences are assigned to different antibody binders to enable multiplex protein detection. When tested using recombinant proteins, some antibody binders, such as those specific to CSTB, MET, EpCAM, and CASP3, had dynamic ranges of 5-6 logs. The antibody binders were also used in a multiplexed format in OER assays, and the binders successfully detected their protein targets in cell lysates, and in single cells in combination with the C1 system. This click reaction-based antibody conjugation procedure is cost-effective, needs minimal hands-on time, and