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Sample records for monoclonal neutralizing antibodies

  1. Recent Progress toward Engineering HIV-1-Specific Neutralizing Monoclonal Antibodies

    OpenAIRE

    Ming Sun; Yue Li; Huiwen Zheng; Yiming Shao

    2016-01-01

    The recent discoveries of broadly potent neutralizing human monoclonal antibodies represent a new generation of antiretrovirals for the treatment and prophylaxis. Antibodies are generally considered more effective and safer and have been proved to provide passive protection against mucosal challenge in humanized mice and macaques. Several neutralizing Abs could protect animals against HIV-1 but are not effective when used in an established infected model for therapy. In order to overcome the ...

  2. [Neutralizing Monoclonal and Chimeric Antibodies to Human IFN-γ].

    Science.gov (United States)

    Larina, M V; Aliev, T K; Solopova, O N; Pozdnyakova, L P; Korobova, S V; Yakimov, S A; Sveshnikov, P G; Dolgikh, D A; Kirpichnikov, M P

    2015-01-01

    Autoiminune disorders are chronic diseases characterized by abnormal immune response directed against self-antigens that leads to tissue damage and violation of its normal functioning. Such diseases often result in disability or even death of patients. Nowadays a number of monoclonal antibodies to pro-inflammatory cytokines and their receptors are successfully used for the targeted treatment of autoimmune diseases. One of the perspective targets in autoimmune disease therapy is interferon gamma, a key cytokine in Th1 cells differentiation, activation of macrophages, and inflammation. In the present work, 5 monoclonal antibodies to human IFN-γ were obtained. For the development of potential therapeutic agent, we have performed neutralizing activity and affinity analysis of the antibodies. Based on the data obtained, the monoclonal antibody F1 was selected. This antibody has a dissociation constant 1.7 x 10(-9) M and IC90 = 8.9 ± 2.0 nM measured upon antibody inhibition of the IFN-γ-induced HLA-DR expression on the surface of U937 cells. We have constructed a bicistronic vector for the production of recombinant chimeric Fab fragment F1 chim in E. coli cells. The recombinant chimeric Fab fragment Fl chim neutralizes IFN-γ activity in vitro and has a dissociation constant 1.8 x 10(-9) M.

  3. Novel neutralizing monoclonal antibodies protect rodents against lethal filovirus challenges

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    Caleb D. Marceau

    2014-01-01

    Full Text Available Filoviruses are the causative agents of lethal hemorrhagic fever in human and non-human primates (NHP. The family of Filoviridae is composed of three genera, Ebolavirus, Marburgvirus and Cuevavirus. There are currently no approved vaccines or antiviral therapeutics for the treatment of filovirus infections in humans. Passive transfer of neutralizing antibodies targeting the Ebola virus (EBOV glycoprotein (GP has proven effective in protecting mice, guinea pigs and NHP from lethal challenges with EBOV. In this study, we generated two neutralizing monoclonal antibodies (MAbs, termed S9 and M4 that recognize the GP of EBOV or multiple strains of Marburg virus (MARV, respectively. We characterized the putative binding site of S9 as a linear epitope on the glycan cap of the GP1 subunit of the EBOV-GP. The M4 antibody recognizes an unknown conformational epitope on MARV-GP. Additionally, we demonstrated the post-exposure protection potential of these antibodies in both the mouse and guinea pig models of filovirus infection. These data indicate that MAbs S9 and M4 would be good candidates for inclusion in an antibody cocktail for the treatment of filovirus infections.

  4. Incomplete Neutralization and Deviation from Sigmoidal Neutralization Curves for HIV Broadly Neutralizing Monoclonal Antibodies.

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    Laura E McCoy

    2015-08-01

    Full Text Available The broadly neutralizing HIV monoclonal antibodies (bnMAbs PG9, PG16, PGT151, and PGT152 have been shown earlier to occasionally display an unusual virus neutralization profile with a non-sigmoidal slope and a plateau at <100% neutralization. In the current study, we were interested in determining the extent of non-sigmoidal slopes and plateaus at <100% for HIV bnMAbs more generally. Using both a 278 panel of pseudoviruses in a CD4 T-cell (U87.CCR5.CXCR4 assay and a panel of 117 viruses in the TZM-bl assay, we found that bnMAbs targeting many neutralizing epitopes of the spike had neutralization profiles for at least one virus that plateaued at <90%. Across both panels the bnMAbs targeting the V2 apex of Env and gp41 were most likely to show neutralization curves that plateaued <100%. Conversely, bnMAbs targeting the high-mannose patch epitopes were less likely to show such behavior. Two CD4 binding site (CD4bs Abs also showed this behavior relatively infrequently. The phenomenon of incomplete neutralization was also observed in a large peripheral blood mononuclear cells (PBMC-grown molecular virus clone panel derived from patient viral swarms. In addition, five bnMAbs were compared against an 18-virus panel of molecular clones produced in 293T cells and PBMCs and assayed in TZM-bl cells. Examples of plateaus <90% were seen with both types of virus production with no consistent patterns observed. In conclusion, incomplete neutralization and non-sigmoidal neutralization curves are possible for all HIV bnMAbs against a wide range of viruses produced and assayed in both cell lines and primary cells with implications for the use of antibodies in therapy and as tools for vaccine design.

  5. Human Monoclonal Antibodies Broadly Neutralizing against Influenza B Virus

    Science.gov (United States)

    Yasugi, Mayo; Kubota-Koketsu, Ritsuko; Yamashita, Akifumi; Kawashita, Norihito; Du, Anariwa; Sasaki, Tadahiro; Nishimura, Mitsuhiro; Misaki, Ryo; Kuhara, Motoki; Boonsathorn, Naphatsawan; Fujiyama, Kazuhito; Okuno, Yoshinobu; Nakaya, Takaaki; Ikuta, Kazuyoshi

    2013-01-01

    Influenza virus has the ability to evade host immune surveillance through rapid viral genetic drift and reassortment; therefore, it remains a continuous public health threat. The development of vaccines producing broadly reactive antibodies, as well as therapeutic strategies using human neutralizing monoclonal antibodies (HuMAbs) with global reactivity, has been gathering great interest recently. Here, three hybridoma clones producing HuMAbs against influenza B virus, designated 5A7, 3A2 and 10C4, were prepared using peripheral lymphocytes from vaccinated volunteers, and were investigated for broad cross-reactive neutralizing activity. Of these HuMAbs, 3A2 and 10C4, which recognize the readily mutable 190-helix region near the receptor binding site in the hemagglutinin (HA) protein, react only with the Yamagata lineage of influenza B virus. By contrast, HuMAb 5A7 broadly neutralizes influenza B strains that were isolated from 1985 to 2006, belonging to both Yamagata and Victoria lineages. Epitope mapping revealed that 5A7 recognizes 316G, 318C and 321W near the C terminal of HA1, a highly conserved region in influenza B virus. Indeed, no mutations in the amino acid residues of the epitope region were induced, even after the virus was passaged ten times in the presence of HuMAb 5A7. Moreover, 5A7 showed significant therapeutic efficacy in mice, even when it was administered 72 hours post-infection. These results indicate that 5A7 is a promising candidate for developing therapeutics, and provide insight for the development of a universal vaccine against influenza B virus. PMID:23408886

  6. Purification and identification of Haemophilus ducreyi cytotoxin by use of a neutralizing monoclonal antibody.

    OpenAIRE

    Purvén, M; Frisk, A.; Lönnroth, I.; Lagergard, T

    1997-01-01

    Haemophilus ducreyi produces a cytotoxin responsible for the killing of cultured human epithelial cells. Cytotoxin-neutralizing antibodies were detected in the majority of sera from patients with culture-proven chancroid, and a significantly higher level of such antibodies in patients than in blood donors was noted both in areas where the disease is endemic and those where it is not. We produced neutralizing monoclonal antibodies (MAbs) in mice with a crude osmotic preparation of the cytotoxi...

  7. Monoclonal antibodies neutralizing the haemolytic activity of box jellyfish (Chironex fleckeri) tentacle extracts.

    Science.gov (United States)

    Collins, S P; Comis, A; Marshall, M; Hartwick, R F; Howden, M E

    1993-09-01

    1. Three monoclonal antibodies have been produced which neutralize in vitro the haemolytic activity present in tentacle extracts of the box jellyfish (Chironex fleckeri). 2. Two of these monoclonal antibodies bound specifically to a component of relative molecular mass 50,000 in tentacle extract on Western blots. 3. This binding only occurred when the extracts were electrophoresed under non-reducing conditions. 4. The third monoclonal antibody did not display binding to Western blots of tentacle extract under any of our experimental conditions.

  8. Induction and characterization of monoclonal anti-idiotypic antibodies reactive with idiotopes of canine parvovirus neutralizing monoclonal antibodies.

    NARCIS (Netherlands)

    G.F. Rimmelzwaan (Guus); J. van Es (Johan); G.A. Drost; F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert)

    1991-01-01

    textabstractMonoclonal anti-idiotypic (anti-Id) antibodies (Ab2) were generated against idiotypes (Id) of canine parvovirus (CPV) specific monoclonal antibodies (MoAbs). The binding of most of these anti-Id antibodies to their corresponding Id could be inhibited by antigen, thus classifying these an

  9. Characterization of a Novel Neutralizing Monoclonal Antibody Against Ebola Virus GP.

    Science.gov (United States)

    Reynard, Olivier; Volchkov, Viktor E

    2015-10-01

    Ebola virus is the etiological agent of a severe hemorrhagic fever with a high mortality rate. As the only protein exposed on the surface of viral particles, the spike glycoprotein GP is the unique target for neutralizing monoclonal antibodies. In this study, we demonstrate the strong neutralization capacity of the monoclonal antibody #3327 and characterize its activity. GP residues that are required for recognition and neutralization were found to be located both in the internal fusion loop and in the receptor-binding domain. Analysis of Ebola virus entry in the presence of #3327 allows us to hypothesize that this antibody binds to the virus particle before internalization and endosomal processing of GP and likely prevents the final viral fusion step. Importantly, #3327 is able to block entry of virions bearing GP that contain the Q508 escape mutation common to a number of virus-neutralizing antibodies, and therefore provides future perspectives for treatment strategies against Ebola virus infection.

  10. Characterization of Two Human Monoclonal Antibodies Neutralizing Influenza A H7N9 Viruses

    Science.gov (United States)

    Wang, Jianmin; Chen, Zhe; Bao, Linlin; Zhang, Weijia; Xue, Ying; Pang, XingHuo; Zhang, Xi

    2015-01-01

    H7N9 was a cause of significant global health concern due to its severe infection and approximately 35% mortality in humans. By screening a Fab antibody phage library derived from patients who recovered from H7N9 infections, we characterized two human monoclonal antibodies (HuMAbs), HNIgGD5 and HNIgGH8. The epitope of these two antibodies was dependent on two residues in the receptor binding site at positions V186 and L226 of the hemagglutinin glycoprotein. Both antibodies possessed high neutralizing activity. PMID:26063436

  11. Characterization of Hemolysin of Moraxella bovis Using a Hemolysis-Neutralizing Monoclonal Antibody

    OpenAIRE

    2000-01-01

    A concentrated bacterial culture supernatant from the hemolytic Moraxella bovis strain UQV 148NF was used to immunize mice and generate monoclonal antibodies (MAbs). One, MAb G3/D7, neutralized the hemolytic activity of M. bovis and recognized a 94-kDa protein by Western blot analysis in hemolytic M. bovis strains representing each of the different fimbrial serogroups. Exposure of corneal epithelial cells to M. bovis concentrated culture supernatants demonstrated a role for an exotoxin in the...

  12. Protection Against Clostridium difficile Infection With Broadly Neutralizing Antitoxin Monoclonal Antibodies

    OpenAIRE

    2012-01-01

    The spore-forming bacterium Clostridium difficile represents the principal cause of hospital-acquired diarrhea and pseudomembranous colitis worldwide. C. difficile infection (CDI) is mediated by 2 bacterial toxins, A and B; neutralizing these toxins with monoclonal antibodies (mAbs) provides a potential nonantibiotic strategy for combating the rising prevalence, severity, and recurrence of CDI. Novel antitoxin mAbs were generated in mice and were humanized. The humanized antitoxin A mAb PA-50...

  13. Production of neutralizing monoclonal antibody against human vascular endothelial growth factor receptor Ⅱ

    Institute of Scientific and Technical Information of China (English)

    Rong LI; Dong-sheng XIONG; Xiao-feng SHAO; Jia LIU; Yuan-fu XU; Yuan-sheng XU; Han-zhi LIU; Zhen-ping ZHU; Chun-zheng YANG

    2004-01-01

    AIM: To prepare neutralizing monoclonal antibody (mAb) against extracellular immunoglobulin (Ig)-like domainⅢ of vascular endothelial growth factor receptor KDR and study its biological activity. METHODS: Soluble KDR Ig domain Ⅲ (KDR-Ⅲ) fusion protein was expressed in E Coli and purified from the bacterial periplasmic extracts via an affinity chromatography. Monoclonal antibodies against KDR-Ⅲ were prepared by hybridoma technique. ELISA and FACS analysis were used to identify its specificity. Immunoprecipitation and [3H]-thymidine incorporation assay were also used to detect the activity of anti-KDR mAb blocking the phosphorylation of KDR tyrosine kinase receptor and the influence on vascular endothelial growth factor-induced mitogenesis of human endothelial ceils.RESULTS: A monoclonal antibody, Ycom1D3 (IgG1), was generated from a mouse immunized with the recombinant KDR-Ⅲ protein. Ycom1D3 bound specifically to both the soluble KDR-Ⅲ and the cell-surface expressed KDR. Ycom1D3 effectively blocked VEGF/KDR interaction and inhibited VEGF-stimulated KDR activation in human endothelial cells. Furthermore, the antibody efficiently neutralized VEGF-induced mitogenesis of human endothelial cells. CONCLUSION: Our results suggest that the anti-KDR mAb, Ycom1D3, has potential applications in the treatment of cancer and other diseases where pathological angiogenesis is involved.

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

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    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.

  15. Stoichiometry of monoclonal antibody neutralization of T-cell line-adapted human immunodeficiency virus type 1

    DEFF Research Database (Denmark)

    Schønning, Kristian; Lund, O; Lund, O S

    1999-01-01

    In order to study the stoichiometry of monoclonal antibody (MAb) neutralization of T-cell line-adapted human immunodeficiency virus type 1 (HIV-1) in antibody excess and under equilibrium conditions, we exploited the ability of HIV-1 to generate mixed oligomers when different env genes are coexpr......In order to study the stoichiometry of monoclonal antibody (MAb) neutralization of T-cell line-adapted human immunodeficiency virus type 1 (HIV-1) in antibody excess and under equilibrium conditions, we exploited the ability of HIV-1 to generate mixed oligomers when different env genes...

  16. Stoichiometry of monoclonal antibody neutralization of T-cell line-adapted human immunodeficiency virus type 1

    DEFF Research Database (Denmark)

    Schønning, Kristian; Lund, O; Lund, O S;

    1999-01-01

    In order to study the stoichiometry of monoclonal antibody (MAb) neutralization of T-cell line-adapted human immunodeficiency virus type 1 (HIV-1) in antibody excess and under equilibrium conditions, we exploited the ability of HIV-1 to generate mixed oligomers when different env genes are coexpr......In order to study the stoichiometry of monoclonal antibody (MAb) neutralization of T-cell line-adapted human immunodeficiency virus type 1 (HIV-1) in antibody excess and under equilibrium conditions, we exploited the ability of HIV-1 to generate mixed oligomers when different env genes...

  17. Monoclonal Antibodies Follow Distinct Aggregation Pathways During Production-Relevant Acidic Incubation and Neutralization

    DEFF Research Database (Denmark)

    Pedersen, Thomas Skamris; Tian, Xinsheng; Thorolfsson, Matthias

    2016-01-01

    PURPOSE: Aggregation aspects of therapeutic monoclonal antibodies (mAbs) are of common concern to the pharmaceutical industry. Low pH treatment is applied during affinity purification and to inactivate endogenous retroviruses, directing interest to the mechanisms of acid-induced antibody...... aggregation. METHODS: We characterized the oligomerization kinetics at pH 3.3, as well as the reversibility upon neutralization, of three model mAbs with identical variable regions, representative of IgG1, IgG2 and IgG4 respectively. We applied size-exclusion high performance liquid chromatography......H IgG1 retained monomeric, whereas IgG2 and IgG4 exhibited two-phase oligomerization processes. After neutralization, IgG2 oligomers partially reverted to the monomeric state, while on the contrary, IgG4 oligomers tended to aggregate. Subclass-specific aggregation-prone motifs on the Fc fragments were...

  18. Structural Basis for Recognition of Human Enterovirus 71 by a Bivalent Broadly Neutralizing Monoclonal Antibody.

    Science.gov (United States)

    Ye, Xiaohua; Fan, Chen; Ku, Zhiqiang; Zuo, Teng; Kong, Liangliang; Zhang, Chao; Shi, Jinping; Liu, Qingwei; Chen, Tan; Zhang, Yingyi; Jiang, Wen; Zhang, Linqi; Huang, Zhong; Cong, Yao

    2016-03-01

    Enterovirus 71 (EV71) is the main pathogen responsible for hand, foot and mouth disease with severe neurological complications and even death in young children. We have recently identified a highly potent anti-EV71 neutralizing monoclonal antibody, termed D5. Here we investigated the structural basis for recognition of EV71 by the antibody D5. Four three-dimensional structures of EV71 particles in complex with IgG or Fab of D5 were reconstructed by cryo-electron microscopy (cryo-EM) single particle analysis all at subnanometer resolutions. The most critical EV71 mature virion-Fab structure was resolved to a resolution of 4.8 Å, which is rare in cryo-EM studies of virus-antibody complex so far. The structures reveal a bivalent binding pattern of D5 antibody across the icosahedral 2-fold axis on mature virion, suggesting that D5 binding may rigidify virions to prevent their conformational changes required for subsequent RNA release. Moreover, we also identified that the complementary determining region 3 (CDR3) of D5 heavy chain directly interacts with the extremely conserved VP1 GH-loop of EV71, which was validated by biochemical and virological assays. We further showed that D5 is indeed able to neutralize a variety of EV71 genotypes and strains. Moreover, D5 could potently confer protection in a mouse model of EV71 infection. Since the conserved VP1 GH-loop is involved in EV71 binding with its uncoating receptor, the scavenger receptor class B, member 2 (SCARB2), the broadly neutralizing ability of D5 might attribute to its inhibition of EV71 from binding SCARB2. Altogether, our results elucidate the structural basis for the binding and neutralization of EV71 by the broadly neutralizing antibody D5, thereby enhancing our understanding of antibody-based protection against EV71 infection.

  19. Structural Basis for Recognition of Human Enterovirus 71 by a Bivalent Broadly Neutralizing Monoclonal Antibody.

    Directory of Open Access Journals (Sweden)

    Xiaohua Ye

    2016-03-01

    Full Text Available Enterovirus 71 (EV71 is the main pathogen responsible for hand, foot and mouth disease with severe neurological complications and even death in young children. We have recently identified a highly potent anti-EV71 neutralizing monoclonal antibody, termed D5. Here we investigated the structural basis for recognition of EV71 by the antibody D5. Four three-dimensional structures of EV71 particles in complex with IgG or Fab of D5 were reconstructed by cryo-electron microscopy (cryo-EM single particle analysis all at subnanometer resolutions. The most critical EV71 mature virion-Fab structure was resolved to a resolution of 4.8 Å, which is rare in cryo-EM studies of virus-antibody complex so far. The structures reveal a bivalent binding pattern of D5 antibody across the icosahedral 2-fold axis on mature virion, suggesting that D5 binding may rigidify virions to prevent their conformational changes required for subsequent RNA release. Moreover, we also identified that the complementary determining region 3 (CDR3 of D5 heavy chain directly interacts with the extremely conserved VP1 GH-loop of EV71, which was validated by biochemical and virological assays. We further showed that D5 is indeed able to neutralize a variety of EV71 genotypes and strains. Moreover, D5 could potently confer protection in a mouse model of EV71 infection. Since the conserved VP1 GH-loop is involved in EV71 binding with its uncoating receptor, the scavenger receptor class B, member 2 (SCARB2, the broadly neutralizing ability of D5 might attribute to its inhibition of EV71 from binding SCARB2. Altogether, our results elucidate the structural basis for the binding and neutralization of EV71 by the broadly neutralizing antibody D5, thereby enhancing our understanding of antibody-based protection against EV71 infection.

  20. Computational prediction of neutralization epitopes targeted by human anti-V3 HIV monoclonal antibodies.

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    Evgeny Shmelkov

    Full Text Available The extreme diversity of HIV-1 strains presents a formidable challenge for HIV-1 vaccine design. Although antibodies (Abs can neutralize HIV-1 and potentially protect against infection, antibodies that target the immunogenic viral surface protein gp120 have widely variable and poorly predictable cross-strain reactivity. Here, we developed a novel computational approach, the Method of Dynamic Epitopes, for identification of neutralization epitopes targeted by anti-HIV-1 monoclonal antibodies (mAbs. Our data demonstrate that this approach, based purely on calculated energetics and 3D structural information, accurately predicts the presence of neutralization epitopes targeted by V3-specific mAbs 2219 and 447-52D in any HIV-1 strain. The method was used to calculate the range of conservation of these specific epitopes across all circulating HIV-1 viruses. Accurately identifying an Ab-targeted neutralization epitope in a virus by computational means enables easy prediction of the breadth of reactivity of specific mAbs across the diversity of thousands of different circulating HIV-1 variants and facilitates rational design and selection of immunogens mimicking specific mAb-targeted epitopes in a multivalent HIV-1 vaccine. The defined epitopes can also be used for the purpose of epitope-specific analyses of breakthrough sequences recorded in vaccine clinical trials. Thus, our study is a prototype for a valuable tool for rational HIV-1 vaccine design.

  1. Use of Monoclonal Antibodies in the Sensitive Detection and Neutralization of Botulinum Neurotoxin Serotype B

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    Luisa W. Cheng

    2015-11-01

    Full Text Available Botulinum neurotoxins (BoNT are some of nature’s most potent toxins. Due to potential food contamination, and bioterrorism concerns, the development of detection reagents, therapeutics and countermeasures are of urgent interest. Recently, we have developed a sensitive electrochemiluminescent (ECL immunoassay for BoNT/B, using monoclonal antibodies (mAbs MCS6-27 and anti-BoNT/B rabbit polyclonal antibodies as the capture and detector. The ECL assay detected as little as 1 pg/mL BoNT/B in the buffer matrix, surpassing the detection sensitivities of the gold standard mouse bioassays. The ECL assay also allowed detection of BoNT/B in sera matrices of up to 100% sera with negligible matrix effects. This highly-sensitive assay allowed the determination of the biological half-lives of BoNT/B holotoxin in vivo. We further tested the toxin neutralization potential of our monoclonal antibodies using the mouse systemic and oral intoxication models. A combination of mAbs protected mice in both pre- and post-exposure models to lethal doses of BoNT/B. MAbs were capable of increasing survival of animals when administered even 10 h post-intoxication in an oral model, suggesting a likely time for BoNT/B complexes to reach the blood stream. More sensitive detection assays and treatments against BoNT intoxication will greatly enhance efforts to combat botulism.

  2. Safety, pharmacokinetics and neutralization of the broadly neutralizing HIV-1 human monoclonal antibody VRC01 in healthy adults.

    Science.gov (United States)

    Ledgerwood, J E; Coates, E E; Yamshchikov, G; Saunders, J G; Holman, L; Enama, M E; DeZure, A; Lynch, R M; Gordon, I; Plummer, S; Hendel, C S; Pegu, A; Conan-Cibotti, M; Sitar, S; Bailer, R T; Narpala, S; McDermott, A; Louder, M; O'Dell, S; Mohan, S; Pandey, J P; Schwartz, R M; Hu, Z; Koup, R A; Capparelli, E; Mascola, J R; Graham, B S

    2015-12-01

    VRC-HIVMAB060-00-AB (VRC01) is a broadly neutralizing HIV-1 monoclonal antibody (mAb) isolated from the B cells of an HIV-infected patient. It is directed against the HIV-1 CD4 binding site and is capable of potently neutralizing the majority of diverse HIV-1 strains. This Phase I dose-escalation study in healthy adults was conducted at the National Institutes of Health (NIH) Clinical Center (Bethesda, MD, USA). Primary objectives were the safety, tolerability and pharmacokinetics (PK) of VRC01 intravenous (i.v.) infusion at 5, 20 or 40 mg/kg, given either once (20 mg/kg) or twice 28 days apart (all doses), and of subcutaneous (s.c.) delivery at 5 mg/kg compared to s.c. placebo given twice, 28 days apart. Cumulatively, 28 subjects received 43 VRC01 and nine received placebo administrations. There were no serious adverse events or dose-limiting toxicities. Mean 28-day serum trough concentrations after the first infusion were 35 and 57 μg/ml for groups infused with 20 mg/kg (n = 8) and 40 mg/kg (n = 5) doses, respectively. Mean 28-day trough concentrations after the second infusion were 56 and 89 μg/ml for the same two doses. Over the 5-40 mg/kg i.v. dose range (n = 18), the clearance was 0.016 l/h and terminal half-life was 15 days. After infusion VRC01 retained expected neutralizing activity in serum, and anti-VRC01 antibody responses were not detected. The human monoclonal antibody (mAb) VRC01 was well tolerated when delivered i.v. or s.c. The mAb demonstrated expected half-life and pharmacokinetics for a human immunoglobulin G. The safety and PK results support and inform VRC01 dosing schedules for planning HIV-1 prevention efficacy studies.

  3. Protective efficacy of neutralizing monoclonal antibodies in a nonhuman primate model of Ebola hemorrhagic fever.

    Science.gov (United States)

    Marzi, Andrea; Yoshida, Reiko; Miyamoto, Hiroko; Ishijima, Mari; Suzuki, Yasuhiko; Higuchi, Megumi; Matsuyama, Yukie; Igarashi, Manabu; Nakayama, Eri; Kuroda, Makoto; Saijo, Masayuki; Feldmann, Friederike; Brining, Douglas; Feldmann, Heinz; Takada, Ayato

    2012-01-01

    Ebola virus (EBOV) is the causative agent of severe hemorrhagic fever in primates, with human case fatality rates up to 90%. Today, there is neither a licensed vaccine nor a treatment available for Ebola hemorrhagic fever (EHF). Single monoclonal antibodies (MAbs) specific for Zaire ebolavirus (ZEBOV) have been successfully used in passive immunization experiments in rodent models, but have failed to protect nonhuman primates from lethal disease. In this study, we used two clones of human-mouse chimeric MAbs (ch133 and ch226) with strong neutralizing activity against ZEBOV and evaluated their protective potential in a rhesus macaque model of EHF. Reduced viral loads and partial protection were observed in animals given MAbs ch133 and ch226 combined intravenously at 24 hours before and 24 and 72 hours after challenge. MAbs circulated in the blood of a surviving animal until virus-induced IgG responses were detected. In contrast, serum MAb concentrations decreased to undetectable levels at terminal stages of disease in animals that succumbed to infection, indicating substantial consumption of these antibodies due to virus replication. Accordingly, the rapid decrease of serum MAbs was clearly associated with increased viremia in non-survivors. Our results indicate that EBOV neutralizing antibodies, particularly in combination with other therapeutic strategies, might be beneficial in reducing viral loads and prolonging disease progression during EHF.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-09-11

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

  5. Neutralizing monoclonal antibodies against hepatitis C virus E2 protein bind discontinuous epitopes and inhibit infection at a postattachment step

    DEFF Research Database (Denmark)

    Sabo, Michelle C; Luca, Vincent C; Prentoe, Jannick

    2011-01-01

    The E2 glycoprotein of hepatitis C virus (HCV) mediates viral attachment and entry into target hepatocytes and elicits neutralizing antibodies in infected patients. To characterize the structural and functional basis of HCV neutralization, we generated a novel panel of 78 monoclonal antibodies...... of the homologous HCV strain in cell culture. Two of these bound E2 proteins from strains representative of HCV genotypes 1 to 6, and one of these MAbs, H77.39, neutralized infection of strains from five of these genotypes. The three most potent neutralizing MAbs in our panel, H77.16, H77.39, and J6.36, inhibited...

  6. Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits

    Science.gov (United States)

    Robinson, James E.; Hastie, Kathryn M.; Cross, Robert W.; Yenni, Rachael E.; Elliott, Deborah H.; Rouelle, Julie A.; Kannadka, Chandrika B.; Smira, Ashley A.; Garry, Courtney E.; Bradley, Benjamin T.; Yu, Haini; Shaffer, Jeffrey G.; Boisen, Matt L.; Hartnett, Jessica N.; Zandonatti, Michelle A.; Rowland, Megan M.; Heinrich, Megan L.; Martínez-Sobrido, Luis; Cheng, Benson; de la Torre, Juan C.; Andersen, Kristian G.; Goba, Augustine; Momoh, Mambu; Fullah, Mohamed; Gbakie, Michael; Kanneh, Lansana; Koroma, Veronica J.; Fonnie, Richard; Jalloh, Simbirie C.; Kargbo, Brima; Vandi, Mohamed A.; Gbetuwa, Momoh; Ikponmwosa, Odia; Asogun, Danny A.; Okokhere, Peter O.; Follarin, Onikepe A.; Schieffelin, John S.; Pitts, Kelly R.; Geisbert, Joan B.; Kulakoski, Peter C.; Wilson, Russell B.; Happi, Christian T.; Sabeti, Pardis C.; Gevao, Sahr M.; Khan, S. Humarr; Grant, Donald S.; Geisbert, Thomas W.; Saphire, Erica Ollmann; Branco, Luis M.; Garry, Robert F.

    2016-01-01

    Lassa fever is a severe multisystem disease that often has haemorrhagic manifestations. The epitopes of the Lassa virus (LASV) surface glycoproteins recognized by naturally infected human hosts have not been identified or characterized. Here we have cloned 113 human monoclonal antibodies (mAbs) specific for LASV glycoproteins from memory B cells of Lassa fever survivors from West Africa. One-half bind the GP2 fusion subunit, one-fourth recognize the GP1 receptor-binding subunit and the remaining fourth are specific for the assembled glycoprotein complex, requiring both GP1 and GP2 subunits for recognition. Notably, of the 16 mAbs that neutralize LASV, 13 require the assembled glycoprotein complex for binding, while the remaining 3 require GP1 only. Compared with non-neutralizing mAbs, neutralizing mAbs have higher binding affinities and greater divergence from germline progenitors. Some mAbs potently neutralize all four LASV lineages. These insights from LASV human mAb characterization will guide strategies for immunotherapeutic development and vaccine design. PMID:27161536

  7. Conformation-Dependent High-Affinity Potent Ricin-Neutralizing Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Wei-Gang Hu

    2013-01-01

    Full Text Available Ricin is a potential biothreat agent with no approved antidote available for ricin poisoning. The aim of this study was to develop potent antibody-based antiricin antidotes. Four strong ricin resistant hybridoma clones secreting antiricin monoclonal antibodies (mAbs were developed. All four mAbs are bound to conformational epitopes of ricin toxin B (RTB with high affinity (KD values from 2.55 to 36.27 nM. RTB not only triggers cellular uptake of ricin, but also facilitates transport of the ricin toxin A (RTA from the endoplasmic reticulum to the cytosol, where RTA exerts its toxic activity. The four mAbs were found to have potent ricin-neutralizing capacities and synergistic effects among them as determined by an in vitro neutralization assay. In vivo protection assay demonstrated that all four mAbs had strong efficacy against ricin challenges. D9 was found to be exceptionally effective. Intraperitoneal (i.p. administration of D9, at a dose of 5 μg, 6 weeks before or 6 hours after an i.p. challenge with 5 × LD50 of ricin was able to protect or rescue 100% of the mice, indicating that mAb D9 is an excellent candidate to be developed as a potent antidote against ricin poisoning for both prophylactic and therapeutic purposes.

  8. Characterization of hemolysin of Moraxella bovis using a hemolysis-neutralizing monoclonal antibody.

    Science.gov (United States)

    Billson, F M; Harbour, C; Michalski, W P; Tennent, J M; Egerton, J R; Hodgson, J L

    2000-06-01

    A concentrated bacterial culture supernatant from the hemolytic Moraxella bovis strain UQV 148NF was used to immunize mice and generate monoclonal antibodies (MAbs). One, MAb G3/D7, neutralized the hemolytic activity of M. bovis and recognized a 94-kDa protein by Western blot analysis in hemolytic M. bovis strains representing each of the different fimbrial serogroups. Exposure of corneal epithelial cells to M. bovis concentrated culture supernatants demonstrated a role for an exotoxin in the pathogenesis of infectious bovine keratoconjunctivitis, while neutralization of hemolytic and cytotoxic activities by MAb G3/D7 implies that these activities are related or have common epitopes. The action of M. bovis hemolysin was further characterized in sheep erythrocyte preparations with a binding step and Ca(2+) required for lysis to proceed, similar to the RTX family of bacterial exotoxins. Neutralization of lytic activity in vitro is evidence for the presence of M. bovis antigens, which may be capable of protecting cattle from the development of infectious bovine keratoconjunctivitis.

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

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    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.

  10. Sub-domains of ricin's B subunit as targets of toxin neutralizing and non-neutralizing monoclonal antibodies.

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    Anastasiya Yermakova

    Full Text Available The B subunit (RTB of ricin toxin is a galactose (Gal-/N-acetylgalactosamine (GalNac-specific lectin that mediates attachment, entry, and intracellular trafficking of ricin in host cells. Structurally, RTB consists of two globular domains with identical folding topologies. Domains 1 and 2 are each comprised of three homologous sub-domains (α, β, γ that likely arose by gene duplication from a primordial carbohydrate recognition domain (CRD, although only sub-domains 1α and 2γ retain functional lectin activity. As part of our ongoing effort to generate a comprehensive B cell epitope map of ricin, we report the characterization of three new RTB-specific monoclonal antibodies (mAbs. All three mAbs, JB4, B/J F9 and C/M A2, were initially identified based on their abilities to neutralize ricin in a Vero cell cytotoxicity assay and to partially (or completely block ricin attachment to cell surfaces. However, only JB4 proved capable of neutralizing ricin in a macrophage apoptosis assay and in imparting passive immunity to mice in a model of systemic intoxication. Using a combination of techniques, including competitive ELISAs, pepscan analysis, differential reactivity by Western blot, as well as affinity enrichment of phage displayed peptides, we tentatively localized the epitopes recognized by the non-neutralizing mAbs B/J F9 and C/M A2 to sub-domains 2α and 2β, respectively. Furthermore, we propose that the epitope recognized by JB4 is within sub-domain 2γ, adjacent to RTB's high affinity Gal/GalNAc CRD. These data suggest that recognition of RTB's sub-domains 1α and 2γ are critical determinants of antibody neutralizing activity and protective immunity to ricin.

  11. A three monoclonal antibody combination potently neutralizes multiple botulinum neurotoxin serotype F subtypes

    Science.gov (United States)

    Fan, Yongfeng; Garcia-Rodriguez, Consuelo; Lou, Jianlong; Wen, Weihua; Conrad, Fraser; Zhai, Wenwu; Smith, Theresa J.; Smith, Leonard A.

    2017-01-01

    Human botulism is primarily caused by botulinum neurotoxin (BoNT) serotypes A, B and E, with around 1% caused by serotype F (BoNT/F). BoNT/F comprises at least seven different subtypes with the amino acid sequence difference between subtypes as high as 36%. The sequence differences present a significant challenge for generating monoclonal antibodies (mAbs) that can bind, detect and neutralize all BoNT/F subtypes. We used repertoire cloning of immune mouse antibody variable (V) regions and yeast display to generate a panel of 33 lead single chain Fv (scFv) mAbs that bound one or more BoNT/F subtypes with a median equilibrium dissociation constant (KD) of 4.06 × 10−9 M. By diversifying the V-regions of the lead mAbs and selecting for cross reactivity we generated five mAbs that bound each of the seven subtypes. Three scFv binding non-overlapping epitopes were converted to IgG that had KD for the different BoNT/F subtypes ranging from 2.2×10−8 M to 1.47×10−12 pM. An equimolar combination of the mAbs was able to potently neutralize BoNT/F1, F2, F4 and F7 in the mouse neutralization assay. The mAbs have potential utility as diagnostics capable of recognizing the known BoNT/F subtypes and could be developed as antitoxins to prevent and treat type F botulism. PMID:28323873

  12. Antigenic characterization of Brazilian bovine viral diarrhea virus isolates by monoclonal antibodies and cross-neutralization

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    Botton S.A.

    1998-01-01

    Full Text Available Nineteen Brazilian isolates of bovine viral diarrhea virus (BVDV were characterized antigenically with a panel of 19 monoclonal antibodies (mAbs (Corapi WV, Donis RO and Dubovi EJ (1990 American Journal of Veterinary Research, 55: 1388-1394. Eight isolates were further characterized by cross-neutralization using sheep monospecific antisera. Analysis of mAb binding to viral antigens by indirect immunofluorescence revealed distinct patterns of reactivity among the native viruses. Local isolates differed from the prototype Singer strain in recognition by up to 14 mAbs. Only two mAbs - one to the non-structural protein NS23/p125 and another to the envelope glycoprotein E0/gp48 - recognized 100% of the isolates. No isolate was recognized by more than 14 mAbs and twelve viruses reacted with 10 or less mAbs. mAbs to the major envelope glycoprotein E2/gp53 revealed a particularly high degree of antigenic variability in this glycoprotein. Nine isolates (47.3% reacted with three or less of 10 E2/gp53 mAbs, and one isolate was not recognized by any of these mAbs. Virus-specific antisera to eight isolates plus three standard BVDV strains raised in lambs had virus-neutralizing titers ranging from 400 to 3200 against the homologous virus. Nonetheless, many antisera showed significantly reduced neutralizing activity when tested against heterologous viruses. Up to 128-fold differences in cross-neutralization titers were observed for some pairs of viruses. When the coefficient of antigenic similarity (R was calculated, 49 of 66 comparisons (74.24% between viruses resulted in R values that antigenically distinguish strains. Moreover, one isolate had R values suggesting that it belongs to a distinct serologic group. The marked antigenic diversity observed among Brazilian BVDV isolates should be considered when planning diagnostic and immunization strategies.

  13. A human monoclonal antibody with neutralizing activity against highly divergent influenza subtypes.

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    Nicola Clementi

    Full Text Available The interest in broad-range anti-influenza A monoclonal antibodies (mAbs has recently been strengthened by the identification of anti-hemagglutinin (HA mAbs endowed with heterosubtypic neutralizing activity to be used in the design of "universal" prophylactic or therapeutic tools. However, the majority of the single mAbs described to date do not bind and neutralize viral isolates belonging to highly divergent subtypes clustering into the two different HA-based influenza phylogenetic groups: the group 1 including, among others, subtypes H1, H2, H5 and H9 and the group 2 including, among others, H3 subtype. Here, we describe a human mAb, named PN-SIA28, capable of binding and neutralizing all tested isolates belonging to phylogenetic group 1, including H1N1, H2N2, H5N1 and H9N2 subtypes and several isolates belonging to group 2, including H3N2 isolates from the first period of the 1968 pandemic. Therefore, PN-SIA28 is capable of neutralizing isolates belonging to subtypes responsible of all the reported pandemics, as well as other subtypes with pandemic potential. The region recognized by PN-SIA28 has been identified on the stem region of HA and includes residues highly conserved among the different influenza subtypes. A deep characterization of PN-SIA28 features may represent a useful help in the improvement of available anti-influenza therapeutic strategies and can provide new tools for the development of universal vaccinal strategies.

  14. Mouse in Vivo Neutralization of Escherichia coli Shiga Toxin 2 with Monoclonal Antibodies

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    Larry H. Stanker

    2013-10-01

    Full Text Available Shiga toxin-producing Escherichia coli (STEC food contaminations pose serious health concerns, and have been the subject of massive food recalls. STEC has been identified as the major cause of the life-threatening complication of hemolytic uremic syndrome (HUS. Besides supportive care, there currently are no therapeutics available. The use of antibiotics for combating pathogenic E. coli is not recommended because they have been shown to stimulate toxin production. Clearing Stx2 from the circulation could potentially lessen disease severity. In this study, we tested the in vivo neutralization of Stx2 in mice using monoclonal antibodies (mAbs. We measured the biologic half-life of Stx2 in mice and determined the distribution phase or t1/2 α to be 3 min and the clearance phase or t1/2 β to be 40 min. Neutralizing mAbs were capable of clearing Stx2 completely from intoxicated mouse blood within minutes. We also examined the persistence of these mAbs over time and showed that complete protection could be passively conferred to mice 4 weeks before exposure to Stx2. The advent of better diagnositic methods and the availability of a greater arsenal of therapeutic mAbs against Stx2 would greatly enhance treatment outcomes of life threatening E. coli infections.

  15. Generation and characterization of neutralizing monoclonal antibodies against baculo-expressed HPV 16 VLPs.

    Science.gov (United States)

    Vidyasagar, P; Sridevi, V N; Rajan, S; Praveen, A; Srikanth, A; Abhinay, G; Siva Kumar, V; Verma, R R; Rajendra, L

    2014-03-01

    Human papillomavirus (HPV) is the well-known second most cause of cervical cancer in women worldwide. According to the WHO survey, 70% of the total cervical cancers are associated with types HPV 16 and 18. Presently used prophylactic vaccine for HPV contains mainly capsid protein of L1 virus like particles (VLPs). Correct folding of VLPs and display of neutralizing epitopes are the major constraint for VLP-based vaccines. Further, monoclonal antibodies (mAbs) play a vital role in developing therapeutics and diagnostics. mAbs are also useful for the demonstration of VLP conformation, virus typing and product process assessment as well. In the present study, we have explored the usefulness of mAbs generated against sf-9 expressed HPV 16 VLPs demonstrated as type-specific and conformational dependent against HPV 16 VLPs by ELISA. High affinity and high pseudovirion neutralization titer of mAbs indicated their potential for the development of prophylactic vaccines for HPV. Also, the type-specific and conformational reactivity of the mAbs to HPV 16 VLPs in sf-9 cells by immunofluorescence assay proved their diagnostic potential.

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

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    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.

  17. In vivo neutralization assays by monoclonal antibodies against white spot syndrome virus in crayfish (Cambarus proclarkii)

    Institute of Scientific and Technical Information of China (English)

    WANG Yinan; ZHAN Wenbin; XING Jing; JIANG Yousheng

    2008-01-01

    The neutralizing activities of eight monoclonal antibodies (MAbs) against white spot syndrome virus (WSSV) (2D2,2B2,1D2,1DS,1C2,4A1,6A4 and 6B4) were analyzed by in vivo experiments.Gills from WSSV-infected shrimp were homogenized and ten-fold serially diluted by PBS,and then incubated with MAbs (hybridoma culture supernatant),respectively.The mixture of WSSV and MAbs were injected into crayfish (Procambarus clarkii).Mter challenge,the death rates of crayfish were counted to determine the neutralizing activities of MAbs.At the same time,the mixture of myeloma culture supernatant and WSSV or PBS was served as positive or negative control,respectively.The results showed that,at each virus dilution,the mean time to death of the crayfish injected with MAb-treated virus was significantly longer than that in the positive control,though they all showed 100% mortality within 25 d,and meanwhile,few crayfish died in the negative control.Among the eight MAbs,2D2,2B2,1D2 and 1DS,especially the former two,delayed the mortality significantly,and 1C2,4Al and 6A4 delayed the mortality as well but not so efficiently,while MAb 6B4 was efficient only when the virus concentration increased.The results indicated that the anti-WSSV MAbs can neutralize WSSV in different virus dilutions.

  18. Isolation and characterization of broadly neutralizing human monoclonal antibodies to the e1 glycoprotein of hepatitis C virus

    DEFF Research Database (Denmark)

    Meunier, Jean-Christophe; Russell, Rodney S.; Goossens, Vera

    2008-01-01

    The relative importance of humoral and cellular immunity in the prevention or clearance of hepatitis C virus (HCV) infection is poorly understood. However, there is considerable evidence that neutralizing antibodies are involved in disease control. Here we describe the detailed analysis of human...... monoclonal antibodies (MAbs) directed against HCV glycoprotein E1, which may have the potential to control HCV infection. We have identified two MAbs that can strongly neutralize HCV-pseudotyped particles (HCVpp) bearing the envelope glycoproteins of genotypes 1a, 1b, 4a, 5a, and 6a and less strongly...... neutralize HCVpp bearing the envelope glycoproteins of genotype 2a. Genotype 3a was not neutralized. The epitopes for both MAbs were mapped to the region encompassing amino acids 313 to 327. In addition, robust neutralization was also observed against cell culture-adapted viruses of genotypes 1a and 2a...

  19. Neutralization and Binding Profile of Monoclonal Antibodies Generated Against Influenza A H1N1 Viruses.

    Science.gov (United States)

    Shembekar, Nachiket; Mallajosyula, Vamsee V Aditya; Malik, Ankita; Saini, Ashok; Varadarajan, Raghavan; Gupta, Satish Kumar

    2016-08-01

    Monoclonal antibodies (MAbs) provide scope for the development of better therapeutics and diagnostic tools. Herein, we describe the binding and neutralization profile(s) for a panel of murine MAbs generated against influenza A H1N1 viruses elicited by immunization with pandemic H1 recombinant hemagglutinin (rHA)/whole virus or seasonal H1 rHA. Neutralizing MAbs, MA-2070 and MA-M, were obtained after pandemic A/California/07/2009 (H1N1) virus/rHA immunization(s). Both MAbs reacted specifically with rHA from A/California/07/2009 and A/England/195/2009 in ELISA. MA-2070 bound rHA of A/California/07/2009 with high affinity (KD = 51.36 ± 9.20 nM) and exhibited potent in vitro neutralization (IC50 = 2.50 μg/mL). MA-2070 bound within the stem domain of HA. MA-M exhibited both hemagglutination inhibition (HI, 1.50 μg/mL) and in vitro neutralization (IC50 = 0.66 μg/mL) activity against the pandemic A/California/07/2009 virus and showed higher binding affinity (KD = 9.80 ± 0.67 nM) than MA-2070. MAb, MA-H generated against the seasonal A/Solomon Islands/03/2006 (H1N1) rHA binds within the head domain and bound the seasonal H1N1 (A/Solomon Islands/03/2006 and A/New Caledonia/20/1990) rHAs with high affinity (KD; 0.72-8.23 nM). MA-H showed high HI (2.50 μg/mL) and in vitro neutralization (IC50 = 2.61 μg/mL) activity against the A/Solomon Islands/03/2006 virus. All 3 MAbs failed to react in ELISA with rHA from various strains of H2N2, H3N2, H5N1, H7N9, and influenza virus B, suggesting their specificity for either pandemic or seasonal H1N1 influenza virus. The MAbs reported here may be useful in developing diagnostic assays.

  20. Protection against Clostridium difficile infection with broadly neutralizing antitoxin monoclonal antibodies.

    Science.gov (United States)

    Marozsan, Andre J; Ma, Dangshe; Nagashima, Kirsten A; Kennedy, Brian J; Kang, Yun Kenneth; Arrigale, Robert R; Donovan, Gerald P; Magargal, Wells W; Maddon, Paul J; Olson, William C

    2012-09-01

    The spore-forming bacterium Clostridium difficile represents the principal cause of hospital-acquired diarrhea and pseudomembranous colitis worldwide. C. difficile infection (CDI) is mediated by 2 bacterial toxins, A and B; neutralizing these toxins with monoclonal antibodies (mAbs) provides a potential nonantibiotic strategy for combating the rising prevalence, severity, and recurrence of CDI. Novel antitoxin mAbs were generated in mice and were humanized. The humanized antitoxin A mAb PA-50 and antitoxin B mAb PA-41 have picomolar potencies in vitro and bind to novel regions of the respective toxins. In a hamster model for CDI, 95% of animals treated with a combination of humanized PA-50 and PA-41 showed long-term survival relative to 0% survival of animals treated with standard antibiotics or comparator mAbs. These humanized mAbs provide insight into C. difficile intoxication and hold promise as potential nonantibiotic agents for improving clinical management of CDI.

  1. Epitope mapping of neutralizing monoclonal antibody in avian influenza A H5N1 virus hemagglutinin.

    Science.gov (United States)

    Ohkura, Takashi; Kikuchi, Yuji; Kono, Naoko; Itamura, Shigeyuki; Komase, Katsuhiro; Momose, Fumitaka; Morikawa, Yuko

    2012-02-03

    The global spread of highly pathogenic avian influenza A H5N1 viruses raises concerns about more widespread infection in the human population. Pre-pandemic vaccine for H5N1 clade 1 influenza viruses has been produced from the A/Viet Nam/1194/2004 strain (VN1194), but recent prevalent avian H5N1 viruses have been categorized into the clade 2 strains, which are antigenically distinct from the pre-pandemic vaccine. To understand the antigenicity of H5N1 hemagglutinin (HA), we produced a neutralizing monoclonal antibody (mAb12-1G6) using the pre-pandemic vaccine. Analysis with chimeric and point mutant HAs revealed that mAb12-1G6 bound to the loop (amino acid positions 140-145) corresponding to an antigenic site A in the H3 HA. mAb12-1G6 failed to bind to the mutant VN1194 HA when only 3 residues were substituted with the corresponding residues of the clade 2.1.3.2 A/Indonesia/5/05 strain (amino acid substitutions at positions Q142L, K144S, and S145P), suggesting that these amino acids are critical for binding of mAb12-1G6. Escape mutants of VN1194 selected with mAb12-1G6 carried a S145P mutation. Interestingly, mAb12-1G6 cross-neutralized clade 1 and clade 2.2.1 but not clade 2.1.3.2 or clade 2.3.4 of the H5N1 virus. We discuss the cross-reactivity, based on the amino acid sequence of the epitope.

  2. Interaction between Shiga Toxin and Monoclonal Antibodies: Binding Characteristics and in Vitro Neutralizing Abilities

    Directory of Open Access Journals (Sweden)

    Letícia B. Rocha

    2012-09-01

    Full Text Available Monoclonal antibodies (MAbs have been employed either for diagnosis or treatment of infections caused by different pathogens. Specifically for Shiga toxin-producing Escherichia coli (STEC, numerous immunoassays have been developed for STEC diagnosis, showing variability in sensitivity and specificity when evaluated by reference laboratories, and no therapy or vaccines are currently approved. Thus, the aim of this work was the characterization of the interaction between MAbs against Stx1 and Stx2 toxins and their neutralizing abilities to enable their use as tools for diagnosis and therapy. The selected clones designated 3E2 (anti-Stx1 and 2E11 (anti-Stx2 were classified as IgG1. 3E2 recognized the B subunit of Stx1 with an affinity constant of 2.5 × 10−10 M, detected as little as 6.2 ng of Stx1 and was stable up to 50 ºC. In contrast, 2E11 recognized the A subunit of Stx2, was stable up to 70 ºC, had a high dissociation constant of  6.1 × 10−10 M, and detected as little as 12.5 ng of Stx2. Neutralization tests showed that 160 ng of 3E2 MAb inhibited 80% of Stx1 activity and 500 µg 2E11 MAb were required for 60% inhibition of Stx2 activity. These MAb amounts reversed 25 to 80% of the cytotoxicity triggered by different STEC isolates. In conclusion, these MAbs show suitable characteristics for their use in STEC diagnosis and encourage future studies to investigate their protective efficacy.

  3. Analysis of memory B cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from HIV-1-infected individuals.

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    Davide Corti

    Full Text Available BACKGROUND: The isolation of human monoclonal antibodies (mAbs that neutralize a broad spectrum of primary HIV-1 isolates and the characterization of the human neutralizing antibody B cell response to HIV-1 infection are important goals that are central to the design of an effective antibody-based vaccine. METHODS AND FINDINGS: We immortalized IgG(+ memory B cells from individuals infected with diverse clades of HIV-1 and selected on the basis of plasma neutralization profiles that were cross-clade and relatively potent. Culture supernatants were screened using various recombinant forms of the envelope glycoproteins (Env in multiple parallel assays. We isolated 58 mAbs that were mapped to different Env surfaces, most of which showed neutralizing activity. One mAb in particular (HJ16 specific for a novel epitope proximal to the CD4 binding site on gp120 selectively neutralized a multi-clade panel of Tier-2 HIV-1 pseudoviruses, and demonstrated reactivity that was comparable in breadth, but distinct in neutralization specificity, to that of the other CD4 binding site-specific neutralizing mAb b12. A second mAb (HGN194 bound a conserved epitope in the V3 crown and neutralized all Tier-1 and a proportion of Tier-2 pseudoviruses tested, irrespective of clade. A third mAb (HK20 with broad neutralizing activity, particularly as a Fab fragment, recognized a highly conserved epitope in the HR-1 region of gp41, but showed striking assay-dependent selectivity in its activity. CONCLUSIONS: This study reveals that by using appropriate screening methods, a large proportion of memory B cells can be isolated that produce mAbs with HIV-1 neutralizing activity. Three of these mAbs show unusual breadth of neutralization and therefore add to the current panel of HIV-1 neutralizing antibodies with potential for passive protection and template-based vaccine design.

  4. Label-free detection and characterization of the binding of hemagglutinin protein and broadly neutralizing monoclonal antibodies using terahertz spectroscopy

    Science.gov (United States)

    Sun, Yiwen; Zhong, Junlan; Zhang, Cunlin; Zuo, Jian; Pickwell-MacPherson, Emma

    2015-03-01

    Hemagglutinin (HA) is the main surface glycoprotein of the influenza A virus. The H9N2 subtype influenza A virus is recognized as the most possible pandemic strain as it has crossed the species barrier, infecting swine and humans. We use terahertz spectroscopy to study the hydration shell formation around H9 subtype influenza A virus's HA protein (H9 HA) as well as the detection of antigen binding of H9 HA with the broadly neutralizing monoclonal antibody. We observe a remarkable concentration dependent nonlinear response of the H9 HA, which reveals the formation process of the hydration shell around H9 HA molecules. Furthermore, we show that terahertz dielectric properties of the H9 HA are strongly affected by the presence of the monoclonal antibody F10 and that the terahertz dielectric loss tangent can be used to detect the antibody binding at lower concentrations than the standard ELISA test.

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

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    Sharad P Adekar

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

  6. Stoichiometry of monoclonal antibody neutralization of T-cell line-adapted human immunodeficiency virus type 1

    DEFF Research Database (Denmark)

    Schønning, Kristian; Lund, O; Lund, O S

    1999-01-01

    In order to study the stoichiometry of monoclonal antibody (MAb) neutralization of T-cell line-adapted human immunodeficiency virus type 1 (HIV-1) in antibody excess and under equilibrium conditions, we exploited the ability of HIV-1 to generate mixed oligomers when different env genes...... neutralization of T-cell line-adapted HIV-1 is incremental rather than all or none and that each MAb binding an Env oligomer reduces the likelihood of infection....... are coexpressed. By the coexpression of Env glycoproteins that either can or cannot bind a neutralizing MAb in an env transcomplementation assay, virions were generated in which the proportion of MAb binding sites could be regulated. As the proportion of MAb binding sites in Env chimeric virus increased, MAb...

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

    Science.gov (United States)

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

    2016-01-01

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

  8. Biochemical and pharmacological characterization of human c-Met neutralizing monoclonal antibody CE-355621

    Science.gov (United States)

    Michaud, Neil R.; Jani, Jitesh P.; Hillerman, Stephen; Tsaparikos, Konstantinos E.; Barbacci-Tobin, Elsa G.; Knauth, Elisabeth; Putz Jr., Henry; Campbell, Mary; Karam, George A.; Chrunyk, Boris; Gebhard, David F.; Green, Larry L.; Xu, Jinghai J.; Dunn, Margaret C.; Coskran, Tim M.; Lapointe, Jean-Martin; Cohen, Bruce D.; Coleman, Kevin G.; Bedian, Vahe; Vincent, Patrick; Kajiji, Shama; Steyn, Stefan J.; Borzillo, Gary V.; Los, Gerrit

    2012-01-01

    The c-Met proto-oncogene is a multifunctional receptor tyrosine kinase that is stimulated by its ligand, hepatocyte growth factor (HGF), to induce cell growth, motility and morphogenesis. Dysregulation of c-Met function, through mutational activation or overexpression, has been observed in many types of cancer and is thought to contribute to tumor growth and metastasis by affecting mitogenesis, invasion, and angiogenesis. We identified human monoclonal antibodies that bind to the extracellular domain of c-Met and inhibit tumor growth by interfering with ligand-dependent c-Met activation. We identified antibodies representing four independent epitope classes that inhibited both ligand binding and ligand-dependent activation of c-Met in A549 cells. In cells, the antibodies antagonized c-Met function by blocking receptor activation and by subsequently inducing downregulation of the receptor, translating to phenotypic effects in soft agar growth and tubular morphogenesis assays. Further characterization of the antibodies in vivo revealed significant inhibition of c-Met activity (≥ 80% lasting for 72–96 h) in excised tumors corresponded to tumor growth inhibition in multiple xenograft tumor models. Several of the antibodies identified inhibited the growth of tumors engineered to overexpress human HGF and human c-Met (S114 NIH 3T3) when grown subcutaneously in athymic mice. Furthermore, lead candidate antibody CE-355621 inhibited the growth of U87MG human glioblastoma and GTL-16 gastric xenografts by up to 98%. The findings support published pre-clinical and clinical data indicating that targeting c-Met with human monoclonal antibodies is a promising therapeutic approach for the treatment of cancer. PMID:23007574

  9. Affinity maturation of a broadly neutralizing human monoclonal antibody that prevents acute hepatitis C virus infection in mice.

    Science.gov (United States)

    Keck, Zhen-Yong; Wang, Yong; Lau, Patrick; Lund, Garry; Rangarajan, Sneha; Fauvelle, Catherine; Liao, Grant C; Holtsberg, Frederick W; Warfield, Kelly L; Aman, M Javad; Pierce, Brian G; Fuerst, Thomas R; Bailey, Justin R; Baumert, Thomas F; Mariuzza, Roy A; Kneteman, Norman M; Foung, Steven K H

    2016-12-01

    Direct-acting antivirals (DAAs) have led to a high cure rate in treated patients with chronic hepatitis C virus (HCV) infection, but this still leaves a large number of treatment failures secondary to the emergence of resistance-associated variants (RAVs). To increase the barrier to resistance, a complementary strategy is to use neutralizing human monoclonal antibodies (HMAbs) to prevent acute infection. However, earlier efforts with the selected antibodies led to RAVs in animal and clinical studies. Therefore, we identified an HMAb that is less likely to elicit RAVs for affinity maturation to increase potency and, more important, breadth of protection. Selected matured antibodies show improved affinity and neutralization against a panel of diverse HCV isolates. Structural and modeling studies reveal that the affinity-matured HMAb mediates virus neutralization, in part, by inducing conformational change to the targeted epitope, and that the maturated light chain is responsible for the improved affinity and breadth of protection. A matured HMAb protected humanized mice when challenged with an infectious HCV human serum inoculum for a prolonged period. However, a single mouse experienced breakthrough infection after 63 days when the serum HMAb concentration dropped by several logs; sequence analysis revealed no viral escape mutation. The findings suggest that a single broadly neutralizing antibody can prevent acute HCV infection without inducing RAVs and may complement DAAs to reduce the emergence of RAVs. (Hepatology 2016;64:1922-1933). © 2016 by the American Association for the Study of Liver Diseases.

  10. 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…

  11. 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…

  12. Neutralizing Monoclonal Antibodies Block Chikungunya Virus Entry and Release by Targeting an Epitope Critical to Viral Pathogenesis.

    Science.gov (United States)

    Jin, Jing; Liss, Nathan M; Chen, Dong-Hua; Liao, Maofu; Fox, Julie M; Shimak, Raeann M; Fong, Rachel H; Chafets, Daniel; Bakkour, Sonia; Keating, Sheila; Fomin, Marina E; Muench, Marcus O; Sherman, Michael B; Doranz, Benjamin J; Diamond, Michael S; Simmons, Graham

    2015-12-22

    We evaluated the mechanism by which neutralizing human monoclonal antibodies inhibit chikungunya virus (CHIKV) infection. Potently neutralizing antibodies (NAbs) blocked infection at multiple steps of the virus life cycle, including entry and release. Cryo-electron microscopy structures of Fab fragments of two human NAbs and chikungunya virus-like particles showed a binding footprint that spanned independent domains on neighboring E2 subunits within one viral spike, suggesting a mechanism for inhibiting low-pH-dependent membrane fusion. Detailed epitope mapping identified amino acid E2-W64 as a critical interaction residue. An escape mutation (E2-W64G) at this residue rendered CHIKV attenuated in mice. Consistent with these data, CHIKV-E2-W64G failed to emerge in vivo under the selection pressure of one of the NAbs, IM-CKV063. As our study suggests that antibodies engaging the residue E2-W64 can potently inhibit CHIKV at multiple stages of infection, antibody-based therapies or immunogens that target this region might have protective value.

  13. Neutralizing Monoclonal Antibodies Block Chikungunya Virus Entry and Release by Targeting an Epitope Critical to Viral Pathogenesis

    Directory of Open Access Journals (Sweden)

    Jing Jin

    2015-12-01

    Full Text Available We evaluated the mechanism by which neutralizing human monoclonal antibodies inhibit chikungunya virus (CHIKV infection. Potently neutralizing antibodies (NAbs blocked infection at multiple steps of the virus life cycle, including entry and release. Cryo-electron microscopy structures of Fab fragments of two human NAbs and chikungunya virus-like particles showed a binding footprint that spanned independent domains on neighboring E2 subunits within one viral spike, suggesting a mechanism for inhibiting low-pH-dependent membrane fusion. Detailed epitope mapping identified amino acid E2-W64 as a critical interaction residue. An escape mutation (E2-W64G at this residue rendered CHIKV attenuated in mice. Consistent with these data, CHIKV-E2-W64G failed to emerge in vivo under the selection pressure of one of the NAbs, IM-CKV063. As our study suggests that antibodies engaging the residue E2-W64 can potently inhibit CHIKV at multiple stages of infection, antibody-based therapies or immunogens that target this region might have protective value.

  14. A neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute nipah virus infection.

    Directory of Open Access Journals (Sweden)

    Katharine N Bossart

    2009-10-01

    Full Text Available Nipah virus is a broadly tropic and highly pathogenic zoonotic paramyxovirus in the genus Henipavirus whose natural reservoirs are several species of Pteropus fruit bats. Nipah virus has repeatedly caused outbreaks over the past decade associated with a severe and often fatal disease in humans and animals. Here, a new ferret model of Nipah virus pathogenesis is described where both respiratory and neurological disease are present in infected animals. Severe disease occurs with viral doses as low as 500 TCID(50 within 6 to 10 days following infection. The underlying pathology seen in the ferret closely resembles that seen in Nipah virus infected humans, characterized as a widespread multisystemic vasculitis, with virus replicating in highly vascular tissues including lung, spleen and brain, with recoverable virus from a variety of tissues. Using this ferret model a cross-reactive neutralizing human monoclonal antibody, m102.4, targeting the henipavirus G glycoprotein was evaluated in vivo as a potential therapeutic agent. All ferrets that received m102.4 ten hours following a high dose oral-nasal Nipah virus challenge were protected from disease while all controls died. This study is the first successful post-exposure passive antibody therapy for Nipah virus using a human monoclonal antibody.

  15. Pharmacokinetics and immunogenicity of broadly neutralizing HIV monoclonal antibodies in macaques.

    Science.gov (United States)

    Rosenberg, Yvonne; Sack, Markus; Montefiori, David; Labranche, Celia; Lewis, Mark; Urban, Lori; Mao, Lingjun; Fischer, Rainer; Jiang, Xiaoming

    2015-01-01

    The identification of highly potent broadly neutralizing antibodies (bnAbs) against HIV-1, and success in preventing SHIV infection following their passive administration, have increased the likelihood that immunotherapeutic strategies can be adopted to prevent and treat HIV-1 infection. However, while broad and potent neutralizing activity is an essential prerequisite, in vivo properties such as good circulatory stability and non-immunogenicity are equally critical for developing a human treatment. In the present study, glycoforms of the bnAbs 10-1074, NIH45-46G54W, 10E8, PGT121, PGT128, PGT145, PGT135, PG9, PG16, VRC01 and b12 were produced by Agrobacterium-mediated transient transfection of Nicotiana benthamiana and assessed following administration in rhesus macaques. The results indicate that (i) N-glycans within the VL domain impair plasma stability of plant-derived bnAbs and (ii) while PGT121 and b12 exhibit no immunogenicity in rhesus macaques after multiple injections, VRC01, 10-1074 and NIH45-46G54W elicit high titer anti-idiotypic antibodies following a second injection. These anti-idiotypic antibodies specifically bind the administered bnAb or a close family member, and inhibit the bnAb in neutralization assays. These findings suggest that specific mutations in certain bnAbs contribute to their immunogenicity and call attention to the prospect that these mutated bnAbs will be immunogenic in humans, potentially compromising their value for prophylaxis and therapy of HIV-1.

  16. Characterization of a Large Panel of Rabbit Monoclonal Antibodies against HIV-1 gp120 and Isolation of Novel Neutralizing Antibodies against the V3 Loop.

    Directory of Open Access Journals (Sweden)

    Yali Qin

    Full Text Available We recently reported the induction of potent, cross-clade neutralizing antibodies (nAbs against Human Immunodeficiency Virus type-1 (HIV-1 in rabbits using gp120 based on an M-group consensus sequence. To better characterize these antibodies, 93 hybridomas were generated, which represent the largest panel of monoclonal antibodies (mAbs ever generated from a vaccinated rabbit. The single most frequently recognized epitope of the isolated mAbs was at the very C-terminal end of the protein (APTKAKRRVVEREKR, followed by the V3 loop. A total of seven anti-V3 loop mAbs were isolated, two of which (10A3 and 10A37 exhibited neutralizing activity. In contrast to 10A3 and most other anti-V3 loop nAbs, 10A37 was atypical with its epitope positioned more towards the C-terminal half of the loop. To our knowledge, 10A37 is the most potent and broadly neutralizing anti-V3 loop mAb induced by vaccination. Interestingly, all seven anti-V3 loop mAbs competed with PGT121, suggesting a possibility that early induction of potent anti-V3 loop antibodies could prevent induction of more broadly neutralizing PGT121-like antibodies that target the conserved base of the V3 loop stem.

  17. Variant-specific monoclonal and group-specific polyclonal human immunodeficiency virus type 1 neutralizing antibodies raised with synthetic peptides from the gp120 third variable domain.

    OpenAIRE

    Laman, J. D.; Schellekens, M M; Abacioglu, Y H; Lewis, G K; Tersmette, M; Fouchier, R A; Langedijk, J. P.; Claassen, E.; Boersma, W J

    1992-01-01

    The third variable (V3) domain of the human immunodeficiency virus type 1 (HIV-1) external membrane glycoprotein gp120 is of crucial importance in eliciting neutralizing antibodies in infected persons. Polyclonal (PAb) and monoclonal (MAb) antibodies directed against selected epitopes in the V3 domain are valuable tools for analysis of the involvement of such sequences in neutralization and for definition of the relation between amino acid variability and immunological cross-reactions. The ai...

  18. Modeling neutralization kinetics of HIV by broadly neutralizing monoclonal antibodies in genital secretions coating the cervicovaginal mucosa.

    Directory of Open Access Journals (Sweden)

    Scott A McKinley

    Full Text Available Eliciting broadly neutralizing antibodies (bnAb in cervicovaginal mucus (CVM represents a promising "first line of defense" strategy to reduce vaginal HIV transmission. However, it remains unclear what levels of bnAb must be present in CVM to effectively reduce infection. We approached this complex question by modeling the dynamic tally of bnAb coverage on HIV. This analysis introduces a critical, timescale-dependent competition: to protect, bnAb must accumulate at sufficient stoichiometry to neutralize HIV faster than virions penetrate CVM and reach target cells. We developed a model that incorporates concentrations and diffusivities of HIV and bnAb in semen and CVM, kinetic rates for binding (kon and unbinding (koff of select bnAb, and physiologically relevant thicknesses of CVM and semen layers. Comprehensive model simulations lead to robust conclusions about neutralization kinetics in CVM. First, due to the limited time virions in semen need to penetrate CVM, substantially greater bnAb concentrations than in vitro estimates must be present in CVM to neutralize HIV. Second, the model predicts that bnAb with more rapid kon, almost independent of koff, should offer greater neutralization potency in vivo. These findings suggest the fastest arriving virions at target cells present the greatest likelihood of infection. It also implies the marked improvements in in vitro neutralization potency of many recently discovered bnAb may not translate to comparable reduction in the bnAb dose needed to confer protection against initial vaginal infections. Our modeling framework offers a valuable tool to gaining quantitative insights into the dynamics of mucosal immunity against HIV and other infectious diseases.

  19. A broadly flavivirus cross-neutralizing monoclonal antibody that recognizes a novel epitope within the fusion loop of E protein.

    Directory of Open Access Journals (Sweden)

    Yong-Qiang Deng

    Full Text Available Flaviviruses are a group of human pathogenic, enveloped RNA viruses that includes dengue (DENV, yellow fever (YFV, West Nile (WNV, and Japanese encephalitis (JEV viruses. Cross-reactive antibodies against Flavivirus have been described, but most of them are generally weakly neutralizing. In this study, a novel monoclonal antibody, designated mAb 2A10G6, was determined to have broad cross-reactivity with DENV 1-4, YFV, WNV, JEV, and TBEV. Phage-display biopanning and structure modeling mapped 2A10G6 to a new epitope within the highly conserved flavivirus fusion loop peptide, the (98DRXW(101 motif. Moreover, in vitro and in vivo experiments demonstrated that 2A10G6 potently neutralizes DENV 1-4, YFV, and WNV and confers protection from lethal challenge with DENV 1-4 and WNV in murine model. Furthermore, functional studies revealed that 2A10G6 blocks infection at a step after viral attachment. These results define a novel broadly flavivirus cross-reactive mAb with highly neutralizing activity that can be further developed as a therapeutic agent against severe flavivirus infections in humans.

  20. Direct administration in the respiratory tract improves efficacy of broadly neutralizing anti-influenza virus monoclonal antibodies.

    Science.gov (United States)

    Leyva-Grado, Victor H; Tan, Gene S; Leon, Paul E; Yondola, Mark; Palese, Peter

    2015-07-01

    The emergence of influenza virus strains resistant to approved neuraminidase inhibitors and the time constrains after infection when these drugs can be effective constitute major drawbacks for this class of drugs. This highlights a critical need to discover new therapeutic agents that can be used for the treatment of influenza virus-infected patients. The use of broadly neutralizing anti-influenza monoclonal antibodies (MAbs) has been sought as an alternative immunotherapy against influenza infection. Here, we tested in mice previously characterized broadly neutralizing anti-hemagglutinin (HA) stalk MAbs prophylactically and therapeutically using different routes of administration. The efficacy of treatment against an influenza H1N1 pandemic virus challenge was compared between two systemic routes of administration, intraperitoneal (i.p.) and intravenous (i.v.), and two local routes, intranasal (i.n.) and aerosol (a.e.). The dose of MAb required for prophylactic protection was reduced by 10-fold in animals treated locally (i.n. or a.e.) compared with those treated systemically (i.p. or i.v.). Improved therapeutic protection was observed in animals treated i.n. on day 5 postinfection (60% survival) compared with those treated via the i.p. route (20% survival). An increase in therapeutic efficacy against other influenza virus subtypes (H5N1) was also observed when a local route of administration was used. Our findings demonstrate that local administration significantly decreases the amount of broadly neutralizing monoclonal antibody required for protection against influenza, which highlights the potential use of MAbs as a therapeutic agent for influenza-associated disease.

  1. Development and characterization of neutralizing monoclonal antibodies against canine distemper virus hemagglutinin protein.

    Science.gov (United States)

    Bi, Zhenwei; Xia, Xingxia; Wang, Yongshan; Mei, Yongjie

    2015-04-01

    Canine distemper virus (CDV) causes a serious multisystemic disease in dogs and other carnivora. Hemagglutinin (H) protein-specific antibodies are mainly responsible for protective immunity against CDV infection. In the present study, six neutralizing MAbs to the H protein of CDV were newly obtained and characterized by immunizing BALB/c mice with a recent Chinese field isolate. Competitive binding inhibition assay revealed that they recognized four distinct antigenic regions of the H protein. Immunofluorescence assay and western blotting showed that all MAbs recognize the conformational rather than the linear epitopes of the H protein. Furthermore, in immunofluorescence and virus neutralization assays, two of the MAbs were found to react only with the recent Chinese field isolate and not with older CDV strains, including vaccine strain Onderstepoort, indicating there are neutralization-related antigenic variations between the recent Chinese field isolate and the older CDV strains examined in this study. The newly established MAbs are useful for differentiating the expanding CDV strains and could be used in immunotherapy and immunodiagnosis against infection with CDV.

  2. Monoclonal antibody "gold rush".

    Science.gov (United States)

    Maggon, Krishan

    2007-01-01

    The market, sales and regulatory approval of new human medicines, during the past few years, indicates increasing number and share of new biologics and emergence of new multibillion dollar molecules. The global sale of monoclonal antibodies in 2006 were $20.6 billion. Remicade had annual sales gain of $1 billion during the past 3 years and five brands had similar increase in 2006. Rituxan with 2006 sales of $4.7 billion was the best selling monoclonal antibody and biological product and the 6th among the top selling medicinal brand. It may be the first biologic and monoclonal antibody to reach $10 billion annual sales in the near future. The strong demand from cancer and arthritis patients has surpassed almost all commercial market research reports and sales forecast. Seven monoclonal antibody brands in 2006 had sales exceeding $1 billion. Humanized or fully human monoclonal antibodies with low immunogenicity, enhanced antigen binding and reduced cellular toxicity provide better clinical efficacy. The higher technical and clinical success rate, overcoming of technical hurdles in large scale manufacturing, low cost of market entry and IND filing, use of fully human and humanized monoclonal antibodies has attracted funds and resources towards R&D. Review of industry research pipeline and sales data during the past 3 years indicate a real paradigm shift in industrial R&D from pharmaceutical to biologics and monoclonal antibodies. The antibody bandwagon has been joined by 200 companies with hundreds of new projects and targets and has attracted billions of dollars in R&D investment, acquisitions and licensing deals leading to the current Monoclonal Antibody Gold Rush.

  3. Heterogeneity of monoclonal antibodies.

    Science.gov (United States)

    Liu, Hongcheng; Gaza-Bulseco, Georgeen; Faldu, Dinesh; Chumsae, Chris; Sun, Joanne

    2008-07-01

    Heterogeneity of monoclonal antibodies is common due to the various modifications introduced over the lifespan of the molecules from the point of synthesis to the point of complete clearance from the subjects. The vast number of modifications presents great challenge to the thorough characterization of the molecules. This article reviews the current knowledge of enzymatic and nonenzymatic modifications of monoclonal antibodies including the common ones such as incomplete disulfide bond formation, glycosylation, N-terminal pyroglutamine cyclization, C-terminal lysine processing, deamidation, isomerization, and oxidation, and less common ones such as modification of the N-terminal amino acids by maleuric acid and amidation of the C-terminal amino acid. In addition, noncovalent associations with other molecules, conformational diversity and aggregation of monoclonal antibodies are also discussed. Through a complete understanding of the heterogeneity of monoclonal antibodies, strategies can be employed to better identify the potential modifications and thoroughly characterize the molecules.

  4. Strategic addition of an N-linked glycan to a monoclonal antibody improves its HIV-1-neutralizing activity.

    Science.gov (United States)

    Song, Ruijiang; Oren, Deena A; Franco, David; Seaman, Michael S; Ho, David D

    2013-11-01

    Ibalizumab is a humanized monoclonal antibody that binds human CD4--a key receptor for HIV--and blocks HIV-1 infection. However, HIV-1 strains with mutations resulting in loss of an N-linked glycan from the V5 loop of the envelope glycoprotein gp120 are resistant to ibalizumab. Previous structural analysis suggests that this glycan fills a void between the gp120 V5 loop and the ibalizumab light chain, perhaps causing steric hindrance that disrupts viral entry. If this void contributes to HIV-1 resistance to ibalizumab, we reasoned that 'refilling' it by engineering an N-linked glycan into the ibalizumab light chain at a position spatially proximal to gp120 V5 may restore susceptibility to ibalizumab. Indeed, one such ibalizumab variant neutralized 100% of 118 diverse HIV-1 strains tested in vitro, including 10 strains resistant to parental ibalizumab. These findings demonstrate that the strategic placement of a glycan in the variable region of a monoclonal antibody can substantially enhance its activity.

  5. Human monoclonal antibodies to a novel cluster of conformational epitopes on HCV E2 with resistance to neutralization escape in a genotype 2a isolate

    DEFF Research Database (Denmark)

    Keck, Zhen-yong; Xia, Jinming; Wang, Yong

    2012-01-01

    The majority of broadly neutralizing antibodies to hepatitis C virus (HCV) are against conformational epitopes on the E2 glycoprotein. Many of them recognize overlapping epitopes in a cluster, designated as antigenic domain B, that contains residues G530 and D535. To gain information on other...... regions that will be relevant for vaccine design, we employed yeast surface display of antibodies that bound to genotype 1a H77C E2 mutant proteins containing a substitution either at Y632A (to avoid selecting non-neutralizing antibodies) or D535A. A panel of nine human monoclonal antibodies (HMAbs......) was isolated and designated as HC-84-related antibodies. Each HMAb neutralized cell culture infectious HCV (HCVcc) with genotypes 1-6 envelope proteins with varying profiles, and each inhibited E2 binding to the viral receptor CD81. Five of these antibodies neutralized representative genotypes 1-6 HCVcc...

  6. 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

    2013-01-01

    polyclonal antibodies to aa 412 to 423 from HCV-infected individuals confirmed broad neutralization, conflicting findings have been reported on polyclonal antibodies to an adjacent region, aa 434 to 446, that may or may not interfere with neutralization by antibodies to aa 412 to 423. To define the interplay......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...... between these antibodies, we isolated human monoclonal antibodies (HMAbs) to aa 412 to 423, designated HC33-related HMAbs (HC33 HMAbs), and characterized their interactions with other HMAbs to aa 434 to 446. A subset of the HC33 HMAbs neutralized genotype 1 to 6 infectious cell culture-derived HCV virions...

  7. Broad-range neutralizing anti-influenza A human monoclonal antibodies: new perspectives in therapy and prophylaxis.

    Science.gov (United States)

    Clementi, Nicola; Criscuolo, Elena; Castelli, Matteo; Clementi, Massimo

    2012-10-01

    Broadly neutralizing monoclonal antibodies (mAbs) directed against different subtypes of influenza A viruses are novel tools for the potential development of effective anti-influenza prophylactic and therapeutic strategies. In both cases, the main candidates for passive transfer and new vaccine development are represented by protective mAbs directed against influenza hemagglutinin (HA). A large number of mAbs directed against influenza HA has been developed to date. However, even if they can be useful and contribute to develop new vaccinal strategies, only few of them can be a good candidate for human administration. In this review, we will describe the most relevant human mAb directed against influenza HA able to recognize highly divergent influenza isolates and possibly useful for human therapy and prophylaxis.

  8. Crystal structure of the Hendra virus attachment G glycoprotein bound to a potent cross-reactive neutralizing human monoclonal antibody.

    Directory of Open Access Journals (Sweden)

    Kai Xu

    Full Text Available The henipaviruses, represented by Hendra (HeV and Nipah (NiV viruses are highly pathogenic zoonotic paramyxoviruses with uniquely broad host tropisms responsible for repeated outbreaks in Australia, Southeast Asia, India and Bangladesh. The high morbidity and mortality rates associated with infection and lack of licensed antiviral therapies make the henipaviruses a potential biological threat to humans and livestock. Henipavirus entry is initiated by the attachment of the G envelope glycoprotein to host cell membrane receptors. Previously, henipavirus-neutralizing human monoclonal antibodies (hmAb have been isolated using the HeV-G glycoprotein and a human naïve antibody library. One cross-reactive and receptor-blocking hmAb (m102.4 was recently demonstrated to be an effective post-exposure therapy in two animal models of NiV and HeV infection, has been used in several people on a compassionate use basis, and is currently in development for use in humans. Here, we report the crystal structure of the complex of HeV-G with m102.3, an m102.4 derivative, and describe NiV and HeV escape mutants. This structure provides detailed insight into the mechanism of HeV and NiV neutralization by m102.4, and serves as a blueprint for further optimization of m102.4 as a therapeutic agent and for the development of entry inhibitors and vaccines.

  9. Crystal structure of the Hendra virus attachment G glycoprotein bound to a potent cross-reactive neutralizing human monoclonal antibody.

    Science.gov (United States)

    Xu, Kai; Rockx, Barry; Xie, Yihu; DeBuysscher, Blair L; Fusco, Deborah L; Zhu, Zhongyu; Chan, Yee-Peng; Xu, Yan; Luu, Truong; Cer, Regina Z; Feldmann, Heinz; Mokashi, Vishwesh; Dimitrov, Dimiter S; Bishop-Lilly, Kimberly A; Broder, Christopher C; Nikolov, Dimitar B

    2013-01-01

    The henipaviruses, represented by Hendra (HeV) and Nipah (NiV) viruses are highly pathogenic zoonotic paramyxoviruses with uniquely broad host tropisms responsible for repeated outbreaks in Australia, Southeast Asia, India and Bangladesh. The high morbidity and mortality rates associated with infection and lack of licensed antiviral therapies make the henipaviruses a potential biological threat to humans and livestock. Henipavirus entry is initiated by the attachment of the G envelope glycoprotein to host cell membrane receptors. Previously, henipavirus-neutralizing human monoclonal antibodies (hmAb) have been isolated using the HeV-G glycoprotein and a human naïve antibody library. One cross-reactive and receptor-blocking hmAb (m102.4) was recently demonstrated to be an effective post-exposure therapy in two animal models of NiV and HeV infection, has been used in several people on a compassionate use basis, and is currently in development for use in humans. Here, we report the crystal structure of the complex of HeV-G with m102.3, an m102.4 derivative, and describe NiV and HeV escape mutants. This structure provides detailed insight into the mechanism of HeV and NiV neutralization by m102.4, and serves as a blueprint for further optimization of m102.4 as a therapeutic agent and for the development of entry inhibitors and vaccines.

  10. Structural basis of clade-specific HIV-1 neutralization by humanized anti-V3 monoclonal antibody KD-247.

    Science.gov (United States)

    Kirby, Karen A; Ong, Yee Tsuey; Hachiya, Atsuko; Laughlin, Thomas G; Chiang, Leslie A; Pan, Yun; Moran, Jennifer L; Marchand, Bruno; Singh, Kamalendra; Gallazzi, Fabio; Quinn, Thomas P; Yoshimura, Kazuhisa; Murakami, Toshio; Matsushita, Shuzo; Sarafianos, Stefan G

    2015-01-01

    Humanized monoclonal antibody KD-247 targets the Gly(312)-Pro(313)-Gly(314)-Arg(315) arch of the third hypervariable (V3) loop of the HIV-1 surface glycoprotein. It potently neutralizes many HIV-1 clade B isolates, but not of other clades. To understand the molecular basis of this specificity, we solved a high-resolution (1.55 Å) crystal structure of the KD-247 antigen binding fragment and examined the potential interactions with various V3 loop targets. Unlike most antibodies, KD-247 appears to interact with its target primarily through light chain residues. Several of these interactions involve Arg(315) of the V3 loop. To evaluate the role of light chain residues in the recognition of the V3 loop, we generated 20 variants of KD-247 single-chain variable fragments with mutations in the antigen-binding site. Purified proteins were assessed for V3 loop binding using AlphaScreen technology and for HIV-1 neutralization. Our data revealed that recognition of the clade-specificity defining residue Arg(315) of the V3 loop is based on a network of interactions that involve Tyr(L32), Tyr(L92), and Asn(L27d) that directly interact with Arg(315), thus elucidating the molecular interactions of KD-247 with its V3 loop target.

  11. Characterization of botulinum neurotoxin type A neutralizing monoclonal antibodies and influence of their half-lives on therapeutic activity.

    Directory of Open Access Journals (Sweden)

    Christelle Mazuet

    Full Text Available Botulinum toxins, i.e. BoNT/A to/G, include the most toxic substances known. Since botulism is a potentially fatal neuroparalytic disease with possible use as a biowarfare weapon (Centers for Disease Control and Prevention category A bioterrorism agent, intensive efforts are being made to develop vaccines or neutralizing antibodies. The use of active fragments from non-human immunoglobulins (F(ab'(2, Fab', scFv, chemically modified or not, may avoid side effects, but also largely modify the in vivo half-life and effectiveness of these reagents. We evaluated the neutralizing activity of several monoclonal anti-BoNT/A antibodies (mAbs. F(ab'(2 fragments, native or treated with polyethyleneglycol (PEG, were prepared from selected mAbs to determine their half-life and neutralizing activity as compared with the initial mAbs. We compared the protective efficiency of the different biochemical forms of anti-toxin mAbs providing the same neutralizing activity. Among fourteen tested mAbs, twelve exhibited neutralizing activity. Fragments from two of the best mAbs (TA12 and TA17, recognizing different epitopes, were produced. These two mAbs neutralized the A1 subtype of the toxin more efficiently than the A2 or A3 subtypes. Since mAb TA12 and its fragments both exhibited the greatest neutralizing activity, they were further evaluated in the therapeutic experiments. These showed that, in a mouse model, a 2- to 4-h interval between toxin and antitoxin injection allows the treatment to remain effective, but also suggested an absence of correlation between the half-life of the antitoxins and the length of time before treatment after botulinum toxin A contamination. These experiments demonstrate that PEG treatment has a strong impact on the half-life of the fragments, without affecting the effectiveness of neutralization, which was maintained after preparation of the fragments. These reagents may be useful for rapid treatment after botulinum toxin A

  12. A neutralizing monoclonal antibody targeting the acid-sensitive region in chikungunya virus E2 protects from disease.

    Directory of Open Access Journals (Sweden)

    Suganya Selvarajah

    Full Text Available The mosquito-borne alphavirus, chikungunya virus (CHIKV, has recently reemerged, producing the largest epidemic ever recorded for this virus, with up to 6.5 million cases of acute and chronic rheumatic disease. There are currently no licensed vaccines for CHIKV and current anti-inflammatory drug treatment is often inadequate. Here we describe the isolation and characterization of two human monoclonal antibodies, C9 and E8, from CHIKV infected and recovered individuals. C9 was determined to be a potent virus neutralizing antibody and a biosensor antibody binding study demonstrated it recognized residues on intact CHIKV VLPs. Shotgun mutagenesis alanine scanning of 98 percent of the residues in the E1 and E2 glycoproteins of CHIKV envelope showed that the epitope bound by C9 included amino-acid 162 in the acid-sensitive region (ASR of the CHIKV E2 glycoprotein. The ASR is critical for the rearrangement of CHIKV E2 during fusion and viral entry into host cells, and we predict that C9 prevents these events from occurring. When used prophylactically in a CHIKV mouse model, C9 completely protected against CHIKV viremia and arthritis. We also observed that when administered therapeutically at 8 or 18 hours post-CHIKV challenge, C9 gave 100% protection in a pathogenic mouse model. Given that targeting this novel neutralizing epitope in E2 can potently protect both in vitro and in vivo, it is likely to be an important region both for future antibody and vaccine-based interventions against CHIKV.

  13. Rapid Generation of Human-Like Neutralizing Monoclonal Antibodies in Urgent Preparedness for Influenza Pandemics and Virulent Infectious Diseases.

    Directory of Open Access Journals (Sweden)

    Weixu Meng

    Full Text Available The outbreaks of emerging infectious diseases caused by pathogens such as SARS coronavirus, H5N1, H1N1, and recently H7N9 influenza viruses, have been associated with significant mortality and morbidity in humans. Neutralizing antibodies from individuals who have recovered from an infection confer therapeutic protection to others infected with the same pathogen. However, survivors may not always be available for providing plasma or for the cloning of monoclonal antibodies (mAbs.The genome and the immunoglobulin genes in rhesus macaques and humans are highly homologous; therefore, we investigated whether neutralizing mAbs that are highly homologous to those of humans (human-like could be generated. Using the H5N1 influenza virus as a model, we first immunized rhesus macaques with recombinant adenoviruses carrying a synthetic gene encoding hemagglutinin (HA. Following screening an antibody phage display library derived from the B cells of immunized monkeys, we cloned selected macaque immunoglobulin heavy chain and light chain variable regions into the human IgG constant region, which generated human-macaque chimeric mAbs exhibiting over 97% homology to human antibodies. Selected mAbs demonstrated potent neutralizing activities against three clades (0, 1, 2 of the H5N1 influenza viruses. The in vivo protection experiments demonstrated that the mAbs effectively protected the mice even when administered up to 3 days after infection with H5N1 influenza virus. In particular, mAb 4E6 demonstrated sub-picomolar binding affinity to HA and superior in vivo protection efficacy without the loss of body weight and obvious lung damage. The analysis of the 4E6 escape mutants demonstrated that the 4E6 antibody bound to a conserved epitope region containing two amino acids on the globular head of HA.Our study demonstrated the generation of neutralizing mAbs for potential application in humans in urgent preparedness against outbreaks of new influenza infections or

  14. Rapid Transient Production of a Monoclonal Antibody Neutralizing the Porcine Epidemic Diarrhea Virus (PEDV) in Nicotiana benthamiana and Lactuca sativa.

    Science.gov (United States)

    Rattanapisit, Kaewta; Srijangwad, Anchalee; Chuanasa, Taksina; Sukrong, Suchada; Tantituvanont, Angkana; Mason, Hugh S; Nilubol, Dachrit; Phoolcharoen, Waranyoo

    2017-06-02

    Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea, vomiting, dehydration, weight loss, and high mortality rate in neonatal piglets. Porcine epidemic diarrhea (PED) has been reported in Europe, America, and Asia including Thailand. The disease causes substantial losses to the swine industry in many countries. Presently, there is no effective PEDV vaccine available. In this study, we developed a plant-produced monoclonal antibody (mAb) 2C10 as a prophylactic candidate to prevent the PEDV infection. Recently, plant expression systems have gained interest as an alternative for the production of antibodies because of many advantages, such as low production cost, lack of human and animal pathogen, large scalability, etc. The 2C10 mAb was transiently expressed in Nicotiana benthamiana and lettuce using geminiviral vector. After purification by protein A affinity chromatography, the antibody was tested for the binding and neutralizing activity against PEDV. Our result showed that the plant produced 2C10 mAb can bind to the virus and also inhibit PEDV infection in vitro. These results show excellent potential for a plant-expressed 2C10 as a PEDV prophylaxis and a diagnostic for PEDV infection. Georg Thieme Verlag KG Stuttgart · New York.

  15. Neutralization of feline infectious peritonitis virus: preparation of monoclonal antibody that shows cell tropism in neutralizing activity after viral absorption into the cells.

    Science.gov (United States)

    Kida, K; Hohdatsu, T; Kashimoto-Tokunaga, J; Koyama, H

    2000-01-01

    Feline infectious peritonitis virus (FIPV) infection of feline macro-phages is enhanced by mouse anti-FIPV monoclonal antibody (MAb). This anti-body-dependent enhancement (ADE) of FIPV infection is dependent on mouse MAb subclass, and MAb of IgG2a subclass has a strong ADE activity. Furthermore, MAb showing strong neutralizing activity in Felis catus whole fetus (fcwf-4) cells and Crandell feline kidney (CrFK) cells shows strong enhancing activity in feline macrophages, indicating that the neutralizing epitope and the enhancing epitope are closely related. In this study, we prepared MAb FK50-4 that showed a strong neutralizing activity in feline macrophages, despite the fact that the MAb belonged to the IgG2a subclass. However, MAb FK50-4 did not exhibit neutralizing activity in CrFK cells or fcwf-4 cells, thus showing a very unusual property. MAb FK50-4 recognized FIPV small integral membrane glycoprotein (M protein). Even when feline macrophages were pretreated with MAb FK50-4 prior to FIPV inoculation, this antibody prevented FIPV infection. This reaction disappeared after treatment of FK50-4 with protein A. The neutralizing activity of FK50-4 was also effective on feline macrophages after the cells were inoculated with FIPV. These findings indicated that the FIPV replication mechanism differs between feline macrophages and CrFK/fcwf-4 cells and that a neutralizing epitope that can prevent FIPV infection of feline macrophages after viral absorption is present on M protein.

  16. A broadly neutralizing human monoclonal antibody is effective against H7N9

    Science.gov (United States)

    Tharakaraman, Kannan; Subramanian, Vidya; Viswanathan, Karthik; Sloan, Susan; Yen, Hui-Ling; Barnard, Dale L.; Leung, Y. H. Connie; Szretter, Kristy J.; Koch, Tyree J.; Delaney, James C.; Babcock, Gregory J.; Wogan, Gerald N.; Sasisekharan, Ram; Shriver, Zachary

    2015-01-01

    Emerging strains of influenza represent a significant public health threat with potential pandemic consequences. Of particular concern are the recently emerged H7N9 strains which cause pneumonia with acute respiratory distress syndrome. Estimates are that nearly 80% of hospitalized patients with H7N9 have received intensive care unit support. VIS410, a human antibody, targets a unique conserved epitope on influenza A. We evaluated the efficacy of VIS410 for neutralization of group 2 influenza strains, including H3N2 and H7N9 strains in vitro and in vivo. VIS410, administered at 50 mg/kg, protected DBA mice infected with A/Anhui/2013 (H7N9), resulting in significant survival benefit upon single-dose (−24 h) or double-dose (−12 h, +48 h) administration (P H7N9) resulted in significant decreased lung viral load (P = 0.002) and decreased lung cytokine responses for nine of the 11 cytokines measured. Based on these results, we find that VIS410 may be effective either as monotherapy or combined with antivirals in treating H7N9 disease, as well as disease from other influenza strains. PMID:26283346

  17. A neutralizing anti-gH/gL monoclonal antibody is protective in the guinea pig model of congenital CMV infection.

    Directory of Open Access Journals (Sweden)

    Marcy R Auerbach

    2014-04-01

    Full Text Available Human cytomegalovirus (HCMV is the most common cause of congenital virus infection. Congenital HCMV infection occurs in 0.2-1% of all births, and causes birth defects and developmental abnormalities, including sensorineural hearing loss and developmental delay. Several key studies have established the guinea pig as a tractable model for the study of congenital HCMV infection and have shown that polyclonal antibodies can be protective. In this study, we demonstrate that an anti-guinea pig CMV (GPCMV glycoprotein H/glycoprotein L neutralizing monoclonal antibody protects against fetal infection and loss in the guinea pig. Furthermore, we have delineated the kinetics of GPCMV congenital infection, from maternal infection (salivary glands, seroconversion, placenta to fetal infection (fetus and amniotic fluid. Our studies support the hypothesis that a neutralizing monoclonal antibody targeting an envelope GPCMV glycoprotein can protect the fetus from infection and may shed light on the therapeutic intervention of HCMV congenital infection in humans.

  18. Fully human broadly neutralizing monoclonal antibodies against influenza A viruses generated from the memory B cells of a 2009 pandemic H1N1 influenza vaccine recipient

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Weibin [Molecular Virus Unit, Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025 (China); Chen, Aizhong [Key Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Miao, Yi [Shanghai Xuhui Central Hospital, Shanghai 200031 (China); Xia, Shengli [Center for Disease Control and Prevention of Henan Province, Zhengzhou 450016 (China); Ling, Zhiyang; Xu, Ke; Wang, Tongyan [Molecular Virus Unit, Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025 (China); Xu, Ying; Cui, Jun; Wu, Hongqiang; Hu, Guiyu; Tian, Lin; Wang, Lingling [Key Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Shu, Yuelong [Chinese Center for Disease Control and Prevention, Beijing 102206 (China); Ma, Xiaowei [Hualan Biological Bacterin Company, Xinxiang 453003 (China); Xu, Bianli; Zhang, Jin [Center for Disease Control and Prevention of Henan Province, Zhengzhou 450016 (China); Lin, Xiaojun, E-mail: linxiaojun@hualan.com [Hualan Biological Bacterin Company, Xinxiang 453003 (China); Bian, Chao, E-mail: cbian@sibs.ac.cn [Key Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Sun, Bing, E-mail: bsun@sibs.ac.cn [Molecular Virus Unit, Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025 (China); Key Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China)

    2013-01-20

    Whether the 2009 pandemic H1N1 influenza vaccine can induce heterosubtypic cross-protective anti-hemagglutinin (HA) neutralizing antibodies is an important issue. We obtained a panel of fully human monoclonal antibodies from the memory B cells of a 2009 pandemic H1N1 influenza vaccine recipient. Most of the monoclonal antibodies targeted the HA protein but not the HA1 fragment. Among the analyzed antibodies, seven mAbs exhibited neutralizing activity against several influenza A viruses of different subtypes. The conserved linear epitope targeted by the neutralizing mAbs (FIEGGWTGMVDGWYGYHH) is part of the fusion peptide on HA2. Our work suggests that a heterosubtypic neutralizing antibody response primarily targeting the HA stem region exists in recipients of the 2009 pandemic H1N1 influenza vaccine. The HA stem region contains various conserved neutralizing epitopes with the fusion peptide as an important one. This work may aid in the design of a universal influenza A virus vaccine.

  19. 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...

  20. Mapping of a region of dengue virus type-2 glycoprotein required for binding by a neutralizing monoclonal antibody.

    Science.gov (United States)

    Trirawatanapong, T; Chandran, B; Putnak, R; Padmanabhan, R

    1992-07-15

    Envelope glycoprotein E of flaviviruses is exposed at the surface of the virion, and is responsible for eliciting a neutralizing antibody (Ab) response, as well as protective immunity in the host. In this report, we describe a method for the fine mapping of a linear sequence of the E protein of dengue virus type-2 (DEN-2), recognized by a type-specific and neutralizing monoclonal Ab (mAb), 3H5. First, an Escherichia coli expression vector containing a heat-inducible lambda pL promoter was used to synthesize several truncated, and near-full length E polypeptides. Reactivities of these polypeptides with polyclonal mouse hyperimmune sera, as well as the 3H5 mAb revealed the location of the 3H5-binding site to be within a region of 166 amino acids (aa) between aa 255 and 422. For fine mapping, a series of targeted deletions were made inframe within this region using the polymerase chain reaction (PCR). The hydrophilicity pattern of this region was used as a guide to systematically delete the regions encoding the various groups of surface aa residues within the context of a near-full-length E polypeptide by using PCR. The 3H5-binding site was thus precisely mapped to a region encoding 12 aa (between aa 386 and 397). A synthetic peptide containing this sequence was able to bind to the 3H5 mAb specifically, as shown by enzyme-linked immunosorbent assay. In addition, we show that rabbit Abs raised against the synthetic peptide of 12 aa were able to bind to the authentic E protein, and to neutralize DEN-2 virus in a plaque reduction assay.

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

    National Research Council Canada - National Science Library

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

    .... There are 3 main isoforms, TGFβ1, 2, and 3. As multiple TGFβ isoforms are involved in disease processes, maximal therapeutic efficacy may require neutralization of 2 or more of the TGFβ isoforms...

  2. Neutralization resistance of hepatitis C virus can be overcome by recombinant human monoclonal antibodies

    DEFF Research Database (Denmark)

    Pedersen, Jannie L; Carlsen, Thomas H R; Prentoe, Jannick

    2013-01-01

    -derived genotype 2a (strain T9), 2b (strains DH8 and DH10), and 2c (strain S83) consensus sequences, were viable in Huh7.5 hepatoma cells without requirement for adaptive mutations, reaching HCV infectivity titers of 3.9-4.5 log10 focus-forming units per milliliter. In in vitro neutralization assays, we...

  3. Human Monoclonal Antibodies against West Nile Virus Induced by Natural Infection Neutralize at a Postattachment Step

    NARCIS (Netherlands)

    Vogt, Matthew R.; Moesker, Bastiaan; Goudsmit, Jaap; Jongeneelen, Mandy; Austin, S. Kyle; Oliphant, Theodore; Nelson, Steevenson; Pierson, Theodore C.; Wilschut, Jan; Throsby, Mark; Diamond, Michael S.

    West Nile virus (WNV) is a neurotropic flavivirus that is now a primary cause of epidemic encephalitis in North America. Studies of mice have demonstrated that the humoral immune response against WNV limits primary infection and protects against a secondary challenge. The most-potent neutralizing

  4. Non-neutralizing monoclonal antibodies to a trypsin-sensitive site on the major glycoprotein of rotavirus which discriminate between virus serotypes.

    Science.gov (United States)

    Coulson, B S; Fowler, K J; White, J R; Cotton, R G

    1987-01-01

    Monoclonal antibodies were derived to a human rotavirus purified from stools. Three of the antibodies immunoprecipitated the rotavirus outer capsid glycoprotein gp 34 and were non-neutralizing. These antibodies reacted by enzyme immunoassay with cultivable rotaviruses showing the "long" RNA electropherotype but were inefficient as detectors of "long" RNA pattern rotaviruses in stools. Treatment of SA 11 rotavirus with 7.5 micrograms/ml porcine trypsin for 30 minutes at 37 degrees C irreversibly reduced binding of the antibodies to SA 11 rotavirus in enzyme immunoassays by 50 per cent. Binding was abolished in the presence of rotavirus-negative faecal extracts. These results indicate that non-neutralizing sites on gp 34 of rotaviruses can vary with RNA electropherotype and serotype, and that levels of trypsin currently in use to assist growth of rotaviruses in cell culture may alter the serological profile of the viruses.

  5. Mechanisms mediating enhanced neutralization efficacy of staphylococcal enterotoxin B by combinations of monoclonal antibodies.

    Science.gov (United States)

    Dutta, Kaushik; Varshney, Avanish K; Franklin, Matthew C; Goger, Michael; Wang, Xiaobo; Fries, Bettina C

    2015-03-13

    Staphylococcal enterotoxin B (SEB) is a superantigen that cross-links the major histocompatibility complex class II and specific V-β chains of the T-cell receptor, thus forming a ternary complex. Developing neutralizing mAb to disrupt the ternary complex and abrogate the resulting toxicity is a major therapeutic challenge because SEB is effective at very low concentrations. We show that combining two SEB-specific mAbs enhances their efficacy, even though one of the two mAbs by itself has no effect on neutralization. Crystallography was employed for fine-mapping conformational epitopes in binary and ternary complexes between SEB and Fab fragments. NMR spectroscopy was used to validate and identify subtle allosteric changes induced by mAbs binding to SEB. The mapping of epitopes established that a combination of different mAbs can enhance efficacy of mAb-mediated protection from SEB induced lethal shock by two different mechanisms: one mAb mixture promoted clearance of the toxin both in vitro and in vivo by FcR-mediated cross-linking and clearance, whereas the other mAb mixture induced subtle allosteric conformational changes in SEB that perturbed formation of the SEB·T-cell receptor·major histocompatibility complex class II trimer. Finally structural information accurately predicted mAb binding to other superantigens that share conformational epitopes with SEB. Fine mapping of conformational epitopes is a powerful tool to establish the mechanism and optimize the action of synergistic mAb combinations.

  6. Structural Analysis of Human and Macaque Monoclonal Antibodies 2909 and 2.5B: Implications for the Configuration of the Quaternary Neutralizing Epitope of HIV-1 gp120

    Energy Technology Data Exchange (ETDEWEB)

    B Spurrier; J Sampson; M Totrov; H Li; T ONeal; C Williams; J Robinson; M Gorny; S Zolla-Pazner; X Kong

    2011-12-31

    The quaternary neutralizing epitope (QNE) of HIV-1 gp120 is preferentially expressed on the trimeric envelope spikes of intact HIV virions, and QNE-specific monoclonal antibodies (mAbs) potently neutralize HIV-1. Here, we present the crystal structures of the Fabs of human mAb 2909 and macaque mAb 2.5B. Both mAbs have long beta hairpin CDR H3 regions >20 {angstrom} in length that are each situated at the center of their respective antigen-binding sites. Computational analysis showed that the paratopes include the whole CDR H3, while additional CDR residues form shallow binding pockets. Structural modeling suggests a way to understand the configuration of QNEs and the antigen-antibody interaction for QNE mAbs. Our data will be useful in designing immunogens that may elicit potent neutralizing QNE Abs.

  7. Escape from neutralization by the respiratory syncytial virus-specific neutralizing monoclonal antibody palivizumab is driven by changes in on-rate of binding to the fusion protein

    Energy Technology Data Exchange (ETDEWEB)

    Bates, John T. [The Vanderbilt Vaccine Center, Departments of Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN (United States); Keefer, Christopher J. [The Vanderbilt Vaccine Center, Departments of Pediatrics, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN (United States); Slaughter, James C. [The Vanderbilt Vaccine Center, Departments of Biostatistics and Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN (United States); Kulp, Daniel W. [IAVI Neutralizing Antibody Center and Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA (United States); Schief, William R. [IAVI Neutralizing Antibody Center and Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA (United States); Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA (United States); Crowe, James E., E-mail: james.crowe@vanderbilt.edu [The Vanderbilt Vaccine Center, Departments of Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN (United States); The Vanderbilt Vaccine Center, Departments of Pediatrics, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN (United States)

    2014-04-15

    The role of binding kinetics in determining neutralizing potency for antiviral antibodies is poorly understood. While it is believed that increased steady-state affinity correlates positively with increased virus-neutralizing activity, the relationship between association or dissociation rate and neutralization potency is unclear. We investigated the effect of naturally-occurring antibody resistance mutations in the RSV F protein on the kinetics of binding to palivizumab. Escape from palivizumab-mediated neutralization of RSV occurred with reduced association rate (K{sub on}) for binding to RSV F protein, while alteration of dissociation rate (K{sub off}) did not significantly affect neutralizing activity. Interestingly, linkage of reduced K{sub on} with reduced potency mirrored the effect of increased K{sub on} found in a high-affinity enhanced potency palivizumab variant (motavizumab). These data suggest that association rate is the dominant factor driving neutralization potency for antibodies to RSV F protein antigenic site A and determines the potency of antibody somatic variants or efficiency of escape of viral glycoprotein variants. - Highlights: • The relationship of affinity to neutralization for virus antibodies is uncertain. • Palivizumab binds to RSV escape mutant fusion proteins, but with reduced affinity. • Association rate (K{sub on}) correlated well with the potency of neutralization.

  8. Drift of the HIV-1 envelope glycoprotein gp120 toward increased neutralization resistance over the course of the epidemic: a comprehensive study using the most potent and broadly neutralizing monoclonal antibodies.

    Science.gov (United States)

    Bouvin-Pley, M; Morgand, M; Meyer, L; Goujard, C; Moreau, A; Mouquet, H; Nussenzweig, M; Pace, C; Ho, D; Bjorkman, P J; Baty, D; Chames, P; Pancera, M; Kwong, P D; Poignard, P; Barin, F; Braibant, M

    2014-12-01

    Extending our previous analyses to the most recently described monoclonal broadly neutralizing antibodies (bNAbs), we confirmed a drift of HIV-1 clade B variants over 2 decades toward higher resistance to bNAbs targeting almost all the identified gp120-neutralizing epitopes. In contrast, the sensitivity to bNAbs targeting the gp41 membrane-proximal external region remained stable, suggesting a selective pressure on gp120 preferentially. Despite this evolution, selected combinations of bNAbs remain capable of neutralizing efficiently most of the circulating variants.

  9. Strategic addition of an N-linked glycan to a monoclonal antibody improves its HIV-1-neutralizing activity

    OpenAIRE

    Song, Ruijiang; Oren, Deena A.; Franco, David; Seaman, Michael S; Ho, David D.

    2013-01-01

    Ibalizumab is a humanized monoclonal antibody that binds human CD4—a key receptor for HIV—and blocks HIV-1 infection. However, HIV-1 strains with mutations resulting in loss of an N-linked glycan from the V5 loop of the envelope protein gp120 are resistant to ibalizumab. Previous structural analysis suggests that this glycan fills a void between the gp120 V5 loop and the ibalizumab L chain, perhaps causing steric hindrance that disrupts viral entry. If this void contributes to HIV-1 resistanc...

  10. Polyclonal and monoclonal antibodies in clinic.

    Science.gov (United States)

    Wootla, Bharath; Denic, Aleksandar; Rodriguez, Moses

    2014-01-01

    Immunoglobulins (Ig) or antibodies are heavy plasma proteins, with sugar chains added to amino-acid residues by N-linked glycosylation and occasionally by O-linked glycosylation. The versatility of antibodies is demonstrated by the various functions that they mediate such as neutralization, agglutination, fixation with activation of complement and activation of effector cells. Naturally occurring antibodies protect the organism against harmful pathogens, viruses and infections. In addition, almost any organic chemical induces antibody production of antibodies that would bind specifically to the chemical. These antibodies are often produced from multiple B cell clones and referred to as polyclonal antibodies. In recent years, scientists have exploited the highly evolved machinery of the immune system to produce structurally and functionally complex molecules such as antibodies from a single B clone, heralding the era of monoclonal antibodies. Most of the antibodies currently in the clinic, target components of the immune system, are not curative and seek to alleviate symptoms rather than cure disease. Our group used a novel strategy to identify reparative human monoclonal antibodies distinct from conventional antibodies. In this chapter, we discuss the therapeutic relevance of both polyclonal and monoclonal antibodies in clinic.

  11. Human monoclonal antibodies derived from a patient infected with 2009 pandemic influenza A virus broadly cross-neutralize group 1 influenza viruses

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Yang [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Sasaki, Tadahiro [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Kubota-Koketsu, Ritsuko [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Kanonji Institute, The Research Foundation for Microbial Diseases of Osaka University, Kanonji, Kagawa (Japan); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Inoue, Yuji [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Yasugi, Mayo [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka (Japan); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Yamashita, Akifumi; Ramadhany, Ririn; Arai, Yasuha [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Du, Anariwa [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Boonsathorn, Naphatsawan [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Department of Medical Sciences, Ministry of Public Health, Muang, Nonthaburi (Thailand); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Ibrahim, Madiha S. [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Department of Microbiology and Immunology, Faculty of Veterinary Medicine, Damanhour University, Damanhour (Egypt); and others

    2014-07-18

    Highlights: • Influenza infection can elicit heterosubtypic antibodies to group 1 influenza virus. • Three human monoclonal antibodies were generated from an H1N1-infected patient. • The antibodies predominantly recognized α-helical stem of viral hemagglutinin (HA). • The antibodies inhibited HA structural activation during the fusion process. • The antibodies are potential candidates for future antibody therapy to influenza. - Abstract: Influenza viruses are a continuous threat to human public health because of their ability to evolve rapidly through genetic drift and reassortment. Three human monoclonal antibodies (HuMAbs) were generated in this study, 1H11, 2H5 and 5G2, and they cross-neutralize a diverse range of group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H5N1 and H9N2. The three HuMAbs were prepared by fusing peripheral blood lymphocytes from an H1N1pdm-infected patient with a newly developed fusion partner cell line, SPYMEG. All the HuMAbs had little hemagglutination inhibition activity but had strong membrane-fusion inhibition activity against influenza viruses. A protease digestion assay showed the HuMAbs targeted commonly a short α-helix region in the stalk of the hemagglutinin. Furthermore, Ile45Phe and Glu47Gly double substitutions in the α-helix region made the HA unrecognizable by the HuMAbs. These two amino acid residues are highly conserved in the HAs of H1N1, H5N1 and H9N2 viruses. The HuMAbs reported here may be potential candidates for the development of therapeutic antibodies against group 1 influenza viruses.

  12. Variant-specific monoclonal and group-specific polyclonal human immunodeficiency virus type 1 neutralizing antibodies raised with synthetic peptides from the gp120 third variable domain.

    Science.gov (United States)

    Laman, J D; Schellekens, M M; Abacioglu, Y H; Lewis, G K; Tersmette, M; Fouchier, R A; Langedijk, J P; Claassen, E; Boersma, W J

    1992-03-01

    The third variable (V3) domain of the human immunodeficiency virus type 1 (HIV-1) external membrane glycoprotein gp120 is of crucial importance in eliciting neutralizing antibodies in infected persons. Polyclonal (PAb) and monoclonal (MAb) antibodies directed against selected epitopes in the V3 domain are valuable tools for analysis of the involvement of such sequences in neutralization and for definition of the relation between amino acid variability and immunological cross-reactions. The aim of this study was to obtain such site-specific antibodies. By using synthetic peptides derived from the V3 domain, a group-specific neutralizing PAb, two high-affinity HIV-1 IIIB neutralizing MAb, and two nonneutralizing MAb were raised. A 15-amino-acid peptide overlapping the tip of the V3 domain of HIV-1 MN was used to produce a rabbit PAb (W0/07). This PAb inhibited syncytium formation induced by HIV-1 IIIB and four field isolates. A similar IIIB-derived peptide was used to generate two murine immunoglobulin G1 (IgG1) MAb (IIIB-V3-13 and IIIB-V3-34). Pepscan analysis mapped the binding site of IIIB-V3-34 to the sequence IRIQRGPGR. The Kds of IIIB-V3-13 and IIIB-V3-34 for gp120 were 6.8 x 10(-11) and 1.6 x 10(-10) M, respectively. These MAb neutralized IIIB but not MN and inhibited syncytium formation induced by IIIB. They are applicable in enzyme-linked immunosorbent assays, immunocytochemistry, and flow cytometry. A peptide covering the left base of the V3 domain was used to generate two murine IgG1 MAb (IIIB-V3-21 and IIIB-V3-26). The binding site of IIIB-V3-21 was mapped to the sequence INCTRPN. These MAb did not neutralize HIV-1 and did not inhibit syncytium formation. This study supports the notion that HIV-1 neutralizing antibodies suitable for multiassay performance can be obtained with synthetic peptides and that high-affinity MAb can be generated. Such site-specific antibodies are useful reagents in the analysis of HIV-1 neutralization. In addition, the cross-neutralization

  13. Cross-neutralizing activity of human anti-V3 monoclonal antibodies derived from non-B clade HIV-1 infected individuals.

    Science.gov (United States)

    Andrabi, Raiees; Williams, Constance; Wang, Xiao-Hong; Li, Liuzhe; Choudhary, Alok K; Wig, Naveet; Biswas, Ashutosh; Luthra, Kalpana; Nadas, Arthur; Seaman, Michael S; Nyambi, Phillipe; Zolla-Pazner, Susan; Gorny, Miroslaw K

    2013-05-10

    One approach to the development of an HIV vaccine is to design a protein template which can present gp120 epitopes inducing cross-neutralizing antibodies. To select a V3 sequence for immunogen design, we compared the neutralizing activities of 18 anti-V3 monoclonal antibodies (mAbs) derived from Cameroonian and Indian individuals infected with clade AG and C, respectively. It was found that V3 mAbs from the Cameroonian patients were significantly more cross-neutralizing than those from India. Interestingly, superior neutralizing activity of Cameroonian mAbs was also observed among the nine VH5-51/VL lambda genes encoding V3 mAbs which mediate a similar mode of recognition. This correlated with higher relative binding affinity to a variety of gp120s and increased mutation rates in V3 mAbs from Cameroon. These results suggest that clade C V3 is probably weakly immunogenic and that the V3 sequence of CRF02_AG viruses can serve as a plausible template for vaccine immunogen design.

  14. Monoclonal neutralizing antibodies against EV71 screened from mice immunized with yeast-produced virus-like particles

    Institute of Scientific and Technical Information of China (English)

    Tao; Lin; Lingzhi; Xianyu; Songya; Lyu

    2015-01-01

    Periodic outbreaks of hand, foot and mouth disease(HFMD) occur in children under 5 years old, and can cause death in some cases. The C4 strain of enterovirus 71(EV71) is the main pathogen that causes HFMD in China. Although no drugs against EV71 are available, some studies have shown that candidate vaccines or viral capsid proteins can produce anti-EV71 immunity. In this study, female BABL/c mice(6–8 weeks old) were immunized with virus-like particles(VLPs) of EV71 produced in yeast to screen for anti-EV71 antibodies. Two hybridomas that could produce neutralizing antibodies against EV71 were obtained. Both neutralizing m Abs(D4 and G12) were confirmed to bind the VP1 capsid protein of EV71, and could protect > 95% cells from 100 TCID50 EV71 infection at 25 μg/m L solution(lowest concentration). Those two neutralizing m Abs identified in the study may be promising candidates in development for m Abs to treat EV71 infection, and utilized as suitable reagents for use in diagnostic tests and biological studies.

  15. A humanized monoclonal antibody neutralizes yellow fever virus strain 17D-204 in vitro but does not protect a mouse model from disease.

    Science.gov (United States)

    Calvert, Amanda E; Dixon, Kandice L; Piper, Joseph; Bennett, Susan L; Thibodeaux, Brett A; Barrett, Alan D T; Roehrig, John T; Blair, Carol D

    2016-07-01

    The yellow fever virus (YFV) vaccine 17D-204 is considered safe and effective, yet rare severe adverse events (SAEs), some resulting in death, have been documented following vaccination. Individuals exhibiting post-vaccinal SAEs are ideal candidates for antiviral monoclonal antibody (MAb) therapy; the time until appearance of clinical signs post-exposure is usually short and patients are quickly hospitalized. We previously developed a murine-human chimeric monoclonal antibody (cMAb), 2C9-cIgG, reactive with both virulent YFV and 17D-204, and demonstrated its ability to prevent and treat YF disease in both AG129 mouse and hamster models of infection. To counteract possible selection of 17D-204 variants that escape neutralization by treatment with a single MAb (2C9-cIgG), we developed a second cMAb, 864-cIgG, for use in combination with 2C9-cIgG in post-vaccinal therapy. MAb 864-cIgG recognizes/neutralizes only YFV 17D-204 vaccine substrain and binds to domain III (DIII) of the viral envelope protein, which is different from the YFV type-specific binding site of 2C9-cIgG in DII. Although it neutralized 17D-204 in vitro, administration of 864-cIgG had no protective capacity in the interferon receptor-deficient AG129 mouse model of 17D-204 infection. The data presented here show that although DIII-specific 864-cIgG neutralizes virus infectivity in vitro, it does not have the ability to abrogate disease in vivo. Therefore, combination of 864-cIgG with 2C9-cIgG for treatment of YF vaccination SAEs does not appear to provide an improvement on 2C9-cIgG therapy alone.

  16. Localization of epitopes recognized by monoclonal antibodies that neutralized the H3N2 influenza viruses in man

    Science.gov (United States)

    Okada, Jun; Ohshima, Nobuko; Kubota-Koketsu, Ritsuko; Iba, Yoshitaka; Ota, Sayuri; Takase, Wakana; Yoshikawa, Tetsushi; Ishikawa, Toyokazu; Asano, Yoshizo; Okuno, Yoshinobu; Kurosawa, Yoshikazu

    2011-01-01

    Through extensive isolation of neutralizing mAbs against H3N2 influenza viruses representing the in vivo repertoire in a human donor, we examined the relationships between antigenic drift of influenza virus and protective antibodies generated in an infected individual. The majority of mAbs isolated from a donor born in 1960 were divided into three major groups with distinct strain specificity: 1968–1973, 1977–1993 and 1997–2003. In the present study, we developed a new method that allowed us to comprehensively determine the location of epitopes recognized by many mAbs. Original haemagglutinins (HAs) of several strains and chimaeric variants, in which one of the seven sites (A, B1, B2, C1, C2, D or E) was replaced by some other strain-derived sequence, were artificially expressed on the cell surface. The binding activity of mAbs to the HAs was examined by flow cytometry. By using this method, we determined the location of epitopes recognized by 98 different mAbs. Clones that neutralize the 1968–1973 strains bind to site B2/D, A or A/B1. While sites C, E and B were recognized by clones that neutralized the 1977–1993 strains, the majority of these clones bind to site C. Clones that neutralize the 1997–2003 strains bind to site B, A/B1, A/B2 or E/C2. PMID:21068214

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

    Directory of Open Access Journals (Sweden)

    Charles Sabin

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

  18. Cloning of the first human anti-JCPyV/VP1 neutralizing monoclonal antibody: epitope definition and implications in risk stratification of patients under natalizumab therapy.

    Science.gov (United States)

    Diotti, Roberta Antonia; Mancini, Nicasio; Clementi, Nicola; Sautto, Giuseppe; Moreno, Guisella Janett; Criscuolo, Elena; Cappelletti, Francesca; Man, Petr; Forest, Eric; Remy, Louise; Giannecchini, Simone; Clementi, Massimo; Burioni, Roberto

    2014-08-01

    JC virus (JCPyV) has gained novel clinical importance as cause of progressive multifocal leukoencephalopathy (PML), a rare demyelinating disease recently associated to immunomodulatory drugs, such as natalizumab used in multiple sclerosis (MS) cases. Little is known about the mechanisms leading to PML, and this makes the need of PML risk stratification among natalizumab-treated patients very compelling. Clinical and laboratory-based risk-stratification markers have been proposed, one of these is represented by the JCPyV-seropositive status, which includes about 54% of MS patients. We recently proposed to investigate the possible protective role of neutralizing humoral immune response in preventing JCPyV reactivation. In this proof-of-concept study, by cloning the first human monoclonal antibody (GRE1) directed against a neutralizing epitope on JCPyV/VP1, we optimized a robust anti-JCPyV neutralization assay. This allowed us to evaluate the neutralizing activity in JCPyV-positive sera from MS patients, demonstrating the lack of correlation between the level of anti-JCPyV antibody and anti-JCPyV neutralizing activity. Relevant consequences may derive from future clinical studies induced by these findings; indeed the study of the serum anti-JCPyV neutralizing activity could allow not only a better risk stratification of the patients during natalizumab treatment, but also a better understanding of the pathophysiological mechanisms leading to PML, highlighting the contribution of peripheral versus central nervous system JCPyV reactivation. Noteworthy, the availability of GRE1 could allow the design of novel immunoprophylactic strategies during the immunomodulatory treatment. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Fragmentation of monoclonal antibodies

    Science.gov (United States)

    Vlasak, Josef

    2011-01-01

    Fragmentation is a degradation pathway ubiquitously observed in proteins despite the remarkable stability of peptide bond; proteins differ only by how much and where cleavage occurs. The goal of this review is to summarize reports regarding the non-enzymatic fragmentation of the peptide backbone of monoclonal antibodies (mAbs). The sites in the polypeptide chain susceptible to fragmentation are determined by a multitude of factors. Insights are provided on the intimate chemical mechanisms that can make some bonds prone to cleavage due to the presence of specific side-chains. In addition to primary structure, the secondary, tertiary and quaternary structures have a significant impact in modulating the distribution of cleavage sites by altering local flexibility, accessibility to solvent or bringing in close proximity side chains that are remote in sequence. This review focuses on cleavage sites observed in the constant regions of mAbs, with special emphasis on hinge fragmentation. The mechanisms responsible for backbone cleavage are strongly dependent on pH and can be catalyzed by metals or radicals. The distribution of cleavage sites are different under acidic compared to basic conditions, with fragmentation rates exhibiting a minimum in the pH range 5–6; therefore, the overall fragmentation pattern observed for a mAb is a complex result of structural and solvent conditions. A critical review of the techniques used to monitor fragmentation is also presented; usually a compromise has to be made between a highly sensitive method with good fragment separation and the capability to identify the cleavage site. The effect of fragmentation on the function of a mAb must be evaluated on a case-by-case basis depending on whether cleavage sites are observed in the variable or constant regions, and on the mechanism of action of the molecule. PMID:21487244

  20. Isolation of a high affinity neutralizing monoclonal antibody against 2009 pandemic H1N1 virus that binds at the 'Sa' antigenic site.

    Directory of Open Access Journals (Sweden)

    Nachiket Shembekar

    Full Text Available Influenza virus evades host immunity through antigenic drift and shift, and continues to circulate in the human population causing periodic outbreaks including the recent 2009 pandemic. A large segment of the population was potentially susceptible to this novel strain of virus. Historically, monoclonal antibodies (MAbs have been fundamental tools for diagnosis and epitope mapping of influenza viruses and their importance as an alternate treatment option is also being realized. The current study describes isolation of a high affinity (K(D = 2.1±0.4 pM murine MAb, MA2077 that binds specifically to the hemagglutinin (HA surface glycoprotein of the pandemic virus. The antibody neutralized the 2009 pandemic H1N1 virus in an in vitro microneutralization assay (IC(50 = 0.08 µg/ml. MA2077 also showed hemagglutination inhibition activity (HI titre of 0.50 µg/ml against the pandemic virus. In a competition ELISA, MA2077 competed with the binding site of the human MAb, 2D1 (isolated from a survivor of the 1918 Spanish flu pandemic on pandemic H1N1 HA. Epitope mapping studies using yeast cell-surface display of a stable HA1 fragment, wherein 'Sa' and 'Sb' sites were independently mutated, localized the binding site of MA2077 within the 'Sa' antigenic site. These studies will facilitate our understanding of antigen antibody interaction in the context of neutralization of the pandemic influenza virus.

  1. Plasma pharmacokinetics and biological activity of a human immunodeficiency virus type 1 neutralizing human monoclonal antibody, F105, in cynomolgus monkeys.

    Science.gov (United States)

    Cavacini, L A; Power, J; Emes, C L; Mace, K; Treacy, G; Posner, M R

    1994-05-01

    The IgG1 kappa human monoclonal antibody (HMab), F105 reacts with a discontinuous epitope on the CD4 binding site (CD4BS) of human immunodeficiency virus type 1 (HIV-1)/gp120 and has broad neutralizing activity. F105 HMab (60 mg/kg bolus) was administered intravenously to four monkeys and serum was collected at intervals to determine pharmacokinetics in a primate model. Average serum F105 concentrations, as determined by enzyme-linked immunosorbent assay, were analyzed with MINSQ software using a two-compartment, first-order model. The half-life for the alpha phase of the distribution curve is 6.7 h and for the beta elimination phase, 9.6 days. The volume of distribution is 0.65 L/kg and the rate of clearance 2 ml/kg/h. Serum levels of 1.3-1.6 mg/ml of F105 were maintained for 24 h. When monkey serum from day 15 postdose was tested, total serum F105 was 230 +/- 79 micrograms/ml and was immunoreactive with cells infected with the MN and IIIB strains of HIV-1 as determined by flow cytometry. Binding activity was identical to that obtained with stock F105 HMab. Identical neutralizing activity between the injected and uninjected antibody was also observed. Thus, serum neutralizing titers (90%) of 1:2000 at peak and 1:30 at day 15 postdose for MN virus were observed. These data indicate that high in vivo levels of HMab F105 can be attained by single bolus administration with full retention of biological activity. Of importance, levels of antibody necessary for effective neutralization can be achieved and maintained.

  2. Applying antibody-sensitive hypervariable region 1-deleted hepatitis C virus to the study of escape pathways of neutralizing human monoclonal antibody AR5A

    DEFF Research Database (Denmark)

    Velazquez-Moctezuma, Rodrigo; Law, Mansun; Bukh, Jens

    2017-01-01

    Hepatitis C virus (HCV) is a major cause of end-stage liver diseases. With 3–4 million new HCV infections yearly, a vaccine is urgently needed. A better understanding of virus escape from neutralizing antibodies and their corresponding epitopes are important for this effort. However, for viral is...

  3. The N276 glycosylation site is required for HIV-1 neutralization by the CD4 binding site specific HJ16 monoclonal antibody.

    Directory of Open Access Journals (Sweden)

    Sunita S Balla-Jhagjhoorsingh

    Full Text Available Immunogen design for HIV-1 vaccines could be based on epitope identification of naturally occurring neutralizing antibodies in infected patients. A tier 2 neutralizing monoclonal antibody (mAb, HJ16 recognizes a new epitope in the CD4 binding site (CD4bs region that only partially overlaps with the b12 epitope. We aimed to identify the critical binding site by resistance induction in a sensitive primary CRF02_AG strain. In four independent dose-escalation studies, the N276D mutation was consistently the only alteration found and it was confirmed to be responsible for resistance to HJ16 by site-directed mutagenesis in envelopes (envs of the homologous CRF02_AG, as well as of a subtype A and a subtype C primary isolate. This mutation removes an N-linked glycosylation site. The effect of N276D was very selective, as it failed to confer resistance to a range of other entry inhibitors. Remarkably, sensitivity to the CD4bs VRC01 and VRC03 mAbs was increased in the N276D mutated viruses. These data indicate that binding of the CD4bs specific HJ16 mAb critically depends on the interaction with the N276-glycan, thus indicating that HJ16 is the first glycan dependent CD4bs-specific mAb.

  4. Neutralization of West Nile virus by cross-linking of its surface proteins with Fab fragments of the human monoclonal antibody CR4354

    Energy Technology Data Exchange (ETDEWEB)

    Kaufmann, Bärbel; Vogt, Matthew R.; Goudsmit, Jaap; Holdaway, Heather A.; Aksyuk, Anastasia A.; Chipman, Paul R.; Kuhn, Richard J.; Diamond, Michael S.; Rossmann, Michael G. (Purdue); (WU-MED); (Crucell)

    2010-11-15

    Many flaviviruses are significant human pathogens, with the humoral immune response playing an essential role in restricting infection and disease. CR4354, a human monoclonal antibody isolated from a patient, neutralizes West Nile virus (WNV) infection at a postattachment stage in the viral life-cycle. Here, we determined the structure of WNV complexed with Fab fragments of CR4354 using cryoelectron microscopy. The outer glycoprotein shell of a mature WNV particle is formed by 30 rafts of three homodimers of the viral surface protein E. CR4354 binds to a discontinuous epitope formed by protein segments from two neighboring E molecules, but does not cause any detectable structural disturbance on the viral surface. The epitope occurs at two independent positions within an icosahedral asymmetric unit, resulting in 120 binding sites on the viral surface. The cross-linking of the six E monomers within one raft by four CR4354 Fab fragments suggests that the antibody neutralizes WNV by blocking the pH-induced rearrangement of the E protein required for virus fusion with the endosomal membrane.

  5. Role of neutralizing anti-murine interleukin-17A monoclonal antibody on chronic ozone-induced airway inflammation in mice.

    Science.gov (United States)

    Zhang, Min; Fei, Xia; Zhang, Guo-Qing; Zhang, Peng-Yu; Li, Feng; Bao, Wu-Ping; Zhang, Ying-Ying; Zhou, Xin

    2016-10-01

    Exposure to ozone has led to airway inflammation and airway hyperresponsiveness, which potential mechanisms relate to ozone-induced oxidative stress. IL-17 is a growing target for autoimmune and inflammatory diseases. The aim of the study was to examine the inhibitory effects of anti-murine interleukin-17A monoclonal antibody (IL-17mAb) on adverse effects of ozone which are noted above. After C57/BL6 mice were exposed to ozone (2.5ppm; 3h) for 12 times over 6 weeks, IL-17mAb, PBS was intraperitoneally injected into mice 1h after ozone or air exposure for 6 weeks and mice were studied 24h after final exposure, monitoring bronchial responsiveness, airway inflammatory cells, lung histology, levels of neutrophil-related chemokine and proinflammatory cytokines in bronchoalveolar lavage (BAL) fluid and serum, the expression of IL-17A mRNA and protein, glucocorticoid receptors (GR), and the phosphorylation of p38MAPK in lung tissues. The administration of IL-17mAb reduced the ozone-induced increases in total cells, especially neutrophils; decreased levels of cytokines, including IL-8 in BAL fluid, IL-8 and IL-17A in serum; mitigated the severity of airway hyperresponsiveness; attenuated lung inflammation scores and histologic analysis confirmed the suppression of lung inflammation, compared with the administration of a control PBS. Exposure to ozone results in increases in IL-17A production rate, mRNA and protein levels of IL-17A and the protein level of GR. These effects were halted and reversed by IL-17mAb treatment. Furthermore, IL-17mAb also reduced the phosphorylation of p38MAPK. Therefore, we conclude that IL-17mAb may be a useful therapy in ozone-related diseases, including COPD.

  6. Potent and synergistic neutralization of human immunodeficiency virus (HIV) type 1 primary isolates by hyperimmune anti-HIV immunoglobulin combined with monoclonal antibodies 2F5 and 2G12.

    Science.gov (United States)

    Mascola, J R; Louder, M K; VanCott, T C; Sapan, C V; Lambert, J S; Muenz, L R; Bunow, B; Birx, D L; Robb, M L

    1997-10-01

    Three antibody reagents that neutralize primary human immunodeficiency virus type 1 (HIV-1) isolates were tested for magnitude and breadth of neutralization when used alone or in double or triple combinations. Hyperimmune anti-HIV immunoglobulin (HIVIG) is derived from the plasma of HIV-1-infected donors, and monoclonal antibodies (MAbs) 2F5 and 2G12 bind to distinct regions of the HIV-1 envelope glycoprotein. The antibodies were initially tested against a panel of 15 clade B HIV-1 isolates, using a single concentration that is achievable in vivo (HIVIG, 2,500 microg/ml; MAbs, 25 microg/ml). Individual antibody reagents neutralized many of the viruses tested, but antibody potency varied substantially among the viruses. The virus neutralization produced by double combinations of HIVIG plus 2F5 or 2G12, the two MAbs together, or the triple combination of HIVIG, 2F5, and 2G12 was generally equal to or greater than that predicted by the effect of individual antibodies. Overall, the triple combination displayed the greatest magnitude and breadth of neutralization. Synergistic neutralization was evaluated by analyzing data from dose-response curves of each individual antibody reagent compared to the triple combination and was demonstrated against each of four viruses tested. Therefore, combinations of polyclonal and monoclonal anti-HIV antibodies can produce additive or synergistic neutralization of primary HIV-1 isolates. Passive immunotherapy for treatment or prophylaxis of HIV-1 should consider mixtures of potent neutralizing antibody reagents to expand the magnitude and breadth of virus neutralization.

  7. 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.

  8. Five birds, one stone: neutralization of α-hemolysin and 4 bi-component leukocidins of Staphylococcus aureus with a single human monoclonal antibody.

    Science.gov (United States)

    Rouha, Harald; Badarau, Adriana; Visram, Zehra C; Battles, Michael B; Prinz, Bianka; Magyarics, Zoltán; Nagy, Gábor; Mirkina, Irina; Stulik, Lukas; Zerbs, Manuel; Jägerhofer, Michaela; Maierhofer, Barbara; Teubenbacher, Astrid; Dolezilkova, Ivana; Gross, Karin; Banerjee, Srijib; Zauner, Gerhild; Malafa, Stefan; Zmajkovic, Jakub; Maier, Sabine; Mabry, Robert; Krauland, Eric; Wittrup, K Dane; Gerngross, Tillman U; Nagy, Eszter

    2015-01-01

    Staphylococcus aureus is a major human pathogen associated with high mortality. The emergence of antibiotic resistance and the inability of antibiotics to counteract bacterial cytotoxins involved in the pathogenesis of S. aureus call for novel therapeutic approaches, such as passive immunization with monoclonal antibodies (mAbs). The complexity of staphylococcal pathogenesis and past failures with single mAb products represent considerable barriers for antibody-based therapeutics. Over the past few years, efforts have focused on neutralizing α-hemolysin. Recent findings suggest that the concerted actions of several cytotoxins, including the bi-component leukocidins play important roles in staphylococcal pathogenesis. Therefore, we aimed to isolate mAbs that bind to multiple cytolysins by employing high diversity human IgG1 libraries presented on the surface of yeast cells. Here we describe cross-reactive antibodies with picomolar affinity for α-hemolysin and 4 different bi-component leukocidins that share only ∼26% overall amino acid sequence identity. The molecular basis of cross-reactivity is the recognition of a conformational epitope shared by α-hemolysin and F-components of gamma-hemolysin (HlgAB and HlgCB), LukED and LukSF (Panton-Valentine Leukocidin). The amino acids predicted to form the epitope are conserved and known to be important for cytotoxic activity. We found that a single cross-reactive antibody prevented lysis of human phagocytes, epithelial and red blood cells induced by α-hemolysin and leukocidins in vitro, and therefore had superior effectiveness compared to α-hemolysin specific antibodies to protect from the combined cytolytic effect of secreted S. aureus toxins. Such mAb afforded high levels of protection in murine models of pneumonia and sepsis.

  9. 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.

  10. Staphylococcus aureus Alpha-Toxin Is Conserved among Diverse Hospital Respiratory Isolates Collected from a Global Surveillance Study and Is Neutralized by Monoclonal Antibody MEDI4893.

    Science.gov (United States)

    Tabor, David E; Yu, Li; Mok, Hoyin; Tkaczyk, Christine; Sellman, Bret R; Wu, Yuling; Oganesyan, Vaheh; Slidel, Tim; Jafri, Hasan; McCarthy, Michael; Bradford, Patricia; Esser, Mark T

    2016-09-01

    Staphylococcus aureus infections lead to an array of illnesses ranging from mild skin infections to serious diseases, such endocarditis, osteomyelitis, and pneumonia. Alpha-toxin (Hla) is a pore-forming toxin, encoded by the hla gene, that is thought to play a key role in S. aureus pathogenesis. A monoclonal antibody targeting Hla, MEDI4893, is in clinical development for the prevention of S. aureus ventilator-associated pneumonia (VAP). The presence of the hla gene and Hla protein in 994 respiratory isolates collected from patients in 34 countries in Asia, Europe, the United States, Latin America, the Middle East, Africa, and Australia was determined. Hla levels were correlated with the geographic location, age of the subject, and length of stay in the hospital. hla gene sequence analysis was performed, and mutations were mapped to the Hla crystal structure. S. aureus supernatants containing Hla variants were tested for susceptibility or resistance to MEDI4893. The hla gene was present and Hla was expressed in 99.0% and 83.2% of the isolates, respectively, regardless of geographic region, hospital locale, or age of the subject. More methicillin-susceptible than methicillin-resistant isolates expressed Hla (86.9% versus 78.8%; P = 0.0007), and S. aureus isolates from pediatric patients expressed the largest amounts of Hla. Fifty-seven different Hla subtypes were identified, and 91% of the isolates encoded an Hla subtype that was neutralized by MED4893. This study demonstrates that Hla is conserved in diverse S. aureus isolates from around the world and is an attractive target for prophylactic monoclonal antibody (MAb) or vaccine development.

  11. Three amino acid residues in the envelope of human immunodeficiency virus type 1 CRF07_BC regulate viral neutralization susceptibility to the human monoclonal neutralizing antibody IgG1b12

    Institute of Scientific and Technical Information of China (English)

    Jianhui; Nie; Juan; Zhao; Qingqing; Chen; Weijin; Huang; Youchun; Wang

    2014-01-01

    The CD4 binding site(CD4bs) of envelope glycoprotein(Env) is an important conserved target for anti-human immunodeficiency virus type 1(HIV-1) neutralizing antibodies. Neutralizing monoclonal antibodies IgG1 b12(b12) could recognize conformational epitopes that overlap the CD4 bs of Env. Different virus strains, even derived from the same individual, showed distinct neutralization susceptibility to b12. We examined the key amino acid residues affecting b12 neutralization susceptibility using single genome amplification and pseudovirus neutralization assay. Eleven amino acid residues were identified that affect the sensitivity of Env to b12. Through site-directed mutagenesis, an amino acid substitution at position 182 in the V2 region of Env was confirmed to play a key role in regulating the b12 neutralization susceptibility. The introduction of V182 L to a resistant strain enhanced its sensitivity to b12 more than twofold. Correspondingly, the introduction of L182 V to a sensitive strain reduced its sensitivity to b12 more than tenfold. Amino acid substitution at positions 267 and 346 could both enhance the sensitivity to b12 more than twofold. However, no additive effect was observed when the three site mutageneses were introduced into the same strain, and the sensitivity was equivalent to the single V182 L mutation. CRF07_BC is a major circulating recombinant form of HIV-1 prevalent in China. Our data may provide important information for understanding the molecular mechanism regulating the neutralization susceptibility of CRF07_BC viruses to b12 and may be helpful for a vaccine design targeting the CD4 bs epitopes.

  12. A non-VH1-69 heterosubtypic neutralizing human monoclonal antibody protects mice against H1N1 and H5N1 viruses.

    Directory of Open Access Journals (Sweden)

    Donata De Marco

    Full Text Available Influenza viruses are among the most important human pathogens and are responsible for annual epidemics and sporadic, potentially devastating pandemics. The humoral immune response plays an important role in the defense against these viruses, providing protection mainly by producing antibodies directed against the hemagglutinin (HA glycoprotein. However, their high genetic variability allows the virus to evade the host immune response and the potential protection offered by seasonal vaccines. The emergence of resistance to antiviral drugs in recent years further limits the options available for the control of influenza. The development of alternative strategies for influenza prophylaxis and therapy is therefore urgently needed. In this study, we describe a human monoclonal antibody (PN-SIA49 that recognizes a highly conserved epitope located on the stem region of the HA and able to neutralize a broad spectrum of influenza viruses belonging to different subtypes (H1, H2 and H5. Furthermore, we describe its protective activity in mice after lethal challenge with H1N1 and H5N1 viruses suggesting a potential application in the treatment of influenza virus infections.

  13. Human monoclonal antibodies derived from a patient infected with 2009 pandemic influenza A virus broadly cross-neutralize group 1 influenza viruses.

    Science.gov (United States)

    Pan, Yang; Sasaki, Tadahiro; Kubota-Koketsu, Ritsuko; Inoue, Yuji; Yasugi, Mayo; Yamashita, Akifumi; Ramadhany, Ririn; Arai, Yasuha; Du, Anariwa; Boonsathorn, Naphatsawan; Ibrahim, Madiha S; Daidoji, Tomo; Nakaya, Takaaki; Ono, Ken-ichiro; Okuno, Yoshinobu; Ikuta, Kazuyoshi; Watanabe, Yohei

    2014-07-18

    Influenza viruses are a continuous threat to human public health because of their ability to evolve rapidly through genetic drift and reassortment. Three human monoclonal antibodies (HuMAbs) were generated in this study, 1H11, 2H5 and 5G2, and they cross-neutralize a diverse range of group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H5N1 and H9N2. The three HuMAbs were prepared by fusing peripheral blood lymphocytes from an H1N1pdm-infected patient with a newly developed fusion partner cell line, SPYMEG. All the HuMAbs had little hemagglutination inhibition activity but had strong membrane-fusion inhibition activity against influenza viruses. A protease digestion assay showed the HuMAbs targeted commonly a short α-helix region in the stalk of the hemagglutinin. Furthermore, Ile45Phe and Glu47Gly double substitutions in the α-helix region made the HA unrecognizable by the HuMAbs. These two amino acid residues are highly conserved in the HAs of H1N1, H5N1 and H9N2 viruses. The HuMAbs reported here may be potential candidates for the development of therapeutic antibodies against group 1 influenza viruses. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Broadly Neutralizing Antibodies for HIV Eradication.

    Science.gov (United States)

    Stephenson, Kathryn E; Barouch, Dan H

    2016-02-01

    Passive transfer of antibodies has long been considered a potential treatment modality for infectious diseases, including HIV. Early efforts to use antibodies to suppress HIV replication, however, were largely unsuccessful, as the antibodies that were studied neutralized only a relatively narrow spectrum of viral strains and were not very potent. Recent advances have led to the discovery of a large portfolio of human monoclonal antibodies that are broadly neutralizing across many HIV-1 subtypes and are also substantially more potent. These antibodies target multiple different epitopes on the HIV envelope, thus allowing for the development of antibody combinations. In this review, we discuss the application of broadly neutralizing antibodies (bNAbs) for HIV treatment and HIV eradication strategies. We highlight bNAbs that target key epitopes, such as the CD4 binding site and the V2/V3-glycan-dependent sites, and we discuss several bNAbs that are currently in the clinical development pipeline.

  15. 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.

  16. The epitope and neutralization mechanism of AVFluIgG01, a broad-reactive human monoclonal antibody against H5N1 influenza virus.

    Directory of Open Access Journals (Sweden)

    Zhiliang Cao

    Full Text Available The continued spread of highly pathogenic avian influenza (HPAI H5N1 virus underscores the importance of effective antiviral approaches. AVFluIgG01 is a potent and broad-reactive H5N1-neutralizing human monoclonal antibody (mAb showing great potential for use either for therapeutic purposes or as a basis of vaccine development, but its antigenic epitope and neutralization mechanism have not been finely characterized. In this study, we first demonstrated that AVFluIgG01 targets a novel conformation-dependent epitope in the globular head region of H5N1 hemagglutinin (HA. By selecting mimotopes from a random peptide library in combination with computational algorithms and site-directed mutagenesis, the epitope was mapped to three conserved discontinuous sites (I-III that are located closely at the three-dimensional structure of HA. Further, we found that this HA1-specific human mAb can efficiently block both virus-receptor binding and post-attachment steps, while its Fab fragment exerts the post-attachment inhibition only. Consistently, AVFluIgG01 could inhibit HA-mediated cell-cell membrane fusion at a dose-dependent manner and block the acquisition of pH-induced protease sensitivity. These results suggest a neutralization mechanism of AVFluIgG01 by simultaneously blocking viral attachment to the receptors on host cells and interfering with HA conformational rearrangements associated with membrane fusion. The presented data provide critical information for developing novel antiviral therapeutics and vaccines against HPAI H5N1 virus.

  17. Substitution at Aspartic Acid 1128 in the SARS Coronavirus Spike Glycoprotein Mediates Escape from a S2 Domain-Targeting Neutralizing Monoclonal Antibody

    Science.gov (United States)

    Ng, Oi-Wing; Keng, Choong-Tat; Leung, Cynthia Sau-Wai; Peiris, J. S. Malik; Poon, Leo Lit Man; Tan, Yee-Joo

    2014-01-01

    The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is the etiological agent for the infectious disease, SARS, which first emerged 10 years ago. SARS-CoV is a zoonotic virus that has crossed the species barriers to infect humans. Bats, which harbour a diverse pool of SARS-like CoVs (SL-CoVs), are believed to be the natural reservoir. The SARS-CoV surface Spike (S) protein is a major antigenic determinant in eliciting neutralizing antibody production during SARS-CoV infection. In our previous work, we showed that a panel of murine monoclonal antibodies (mAbs) that target the S2 subunit of the S protein are capable of neutralizing SARS-CoV infection in vitro (Lip KM et al, J Virol. 2006 Jan; 80(2): 941–50). In this study, we report our findings on the characterization of one of these mAbs, known as 1A9, which binds to the S protein at a novel epitope within the S2 subunit at amino acids 1111–1130. MAb 1A9 is a broadly neutralizing mAb that prevents viral entry mediated by the S proteins of human and civet SARS-CoVs as well as bat SL-CoVs. By generating mutant SARS-CoV that escapes the neutralization by mAb 1A9, the residue D1128 in S was found to be crucial for its interaction with mAb 1A9. S protein containing the substitution of D1128 with alanine (D1128A) exhibited a significant decrease in binding capability to mAb 1A9 compared to wild-type S protein. By using a pseudotyped viral entry assay, it was shown that the D1128A substitution in the escape virus allows it to overcome the viral entry blockage by mAb 1A9. In addition, the D1128A mutation was found to exert no effects on the S protein cell surface expression and incorporation into virion particles, suggesting that the escape virus retains the same viral entry property as the wild-type virus. PMID:25019613

  18. Substitution at aspartic acid 1128 in the SARS coronavirus spike glycoprotein mediates escape from a S2 domain-targeting neutralizing monoclonal antibody.

    Directory of Open Access Journals (Sweden)

    Oi-Wing Ng

    Full Text Available The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV is the etiological agent for the infectious disease, SARS, which first emerged 10 years ago. SARS-CoV is a zoonotic virus that has crossed the species barriers to infect humans. Bats, which harbour a diverse pool of SARS-like CoVs (SL-CoVs, are believed to be the natural reservoir. The SARS-CoV surface Spike (S protein is a major antigenic determinant in eliciting neutralizing antibody production during SARS-CoV infection. In our previous work, we showed that a panel of murine monoclonal antibodies (mAbs that target the S2 subunit of the S protein are capable of neutralizing SARS-CoV infection in vitro (Lip KM et al, J Virol. 2006 Jan; 80(2: 941-50. In this study, we report our findings on the characterization of one of these mAbs, known as 1A9, which binds to the S protein at a novel epitope within the S2 subunit at amino acids 1111-1130. MAb 1A9 is a broadly neutralizing mAb that prevents viral entry mediated by the S proteins of human and civet SARS-CoVs as well as bat SL-CoVs. By generating mutant SARS-CoV that escapes the neutralization by mAb 1A9, the residue D1128 in S was found to be crucial for its interaction with mAb 1A9. S protein containing the substitution of D1128 with alanine (D1128A exhibited a significant decrease in binding capability to mAb 1A9 compared to wild-type S protein. By using a pseudotyped viral entry assay, it was shown that the D1128A substitution in the escape virus allows it to overcome the viral entry blockage by mAb 1A9. In addition, the D1128A mutation was found to exert no effects on the S protein cell surface expression and incorporation into virion particles, suggesting that the escape virus retains the same viral entry property as the wild-type virus.

  19. Substitution at aspartic acid 1128 in the SARS coronavirus spike glycoprotein mediates escape from a S2 domain-targeting neutralizing monoclonal antibody.

    Science.gov (United States)

    Ng, Oi-Wing; Keng, Choong-Tat; Leung, Cynthia Sau-Wai; Peiris, J S Malik; Poon, Leo Lit Man; Tan, Yee-Joo

    2014-01-01

    The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is the etiological agent for the infectious disease, SARS, which first emerged 10 years ago. SARS-CoV is a zoonotic virus that has crossed the species barriers to infect humans. Bats, which harbour a diverse pool of SARS-like CoVs (SL-CoVs), are believed to be the natural reservoir. The SARS-CoV surface Spike (S) protein is a major antigenic determinant in eliciting neutralizing antibody production during SARS-CoV infection. In our previous work, we showed that a panel of murine monoclonal antibodies (mAbs) that target the S2 subunit of the S protein are capable of neutralizing SARS-CoV infection in vitro (Lip KM et al, J Virol. 2006 Jan; 80(2): 941-50). In this study, we report our findings on the characterization of one of these mAbs, known as 1A9, which binds to the S protein at a novel epitope within the S2 subunit at amino acids 1111-1130. MAb 1A9 is a broadly neutralizing mAb that prevents viral entry mediated by the S proteins of human and civet SARS-CoVs as well as bat SL-CoVs. By generating mutant SARS-CoV that escapes the neutralization by mAb 1A9, the residue D1128 in S was found to be crucial for its interaction with mAb 1A9. S protein containing the substitution of D1128 with alanine (D1128A) exhibited a significant decrease in binding capability to mAb 1A9 compared to wild-type S protein. By using a pseudotyped viral entry assay, it was shown that the D1128A substitution in the escape virus allows it to overcome the viral entry blockage by mAb 1A9. In addition, the D1128A mutation was found to exert no effects on the S protein cell surface expression and incorporation into virion particles, suggesting that the escape virus retains the same viral entry property as the wild-type virus.

  20. "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.

  1. 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

  2. In vivo time-related evaluation of a therapeutic neutralization monoclonal antibody against lethal enterovirus 71 infection in a mouse model.

    Directory of Open Access Journals (Sweden)

    Zhiqun Li

    Full Text Available Enterovirus 71 (EV71 is a neurotropic virus capable of inducing severe neurological symptoms and death. No direct targeting antivirals are useful in the treatment of severe EV71 infection. Because of low toxicity and good specificity, monoclonal antibodies (MAb are a potential candidate for the treatment of viral infections. Therefore, we developed an EV71-specific conformational MAb with high in vitro cross-neutralization activity to heterologous EV71 subgenotypes. The in vivo treatment experiment at different days post-infection indicated that a single treatment of MAb CT11F9 within day 3 post-infection fully protected mice from morbidity and mortality (0% PBS vs. 100% at 10 µg/g per body weight ***P<0.0001. Immunohistochemical and histological analysis confirmed that CT11F9 significantly prohibited EV71 VP1 expression in various tissues and prevented EV71-induced myonecrosis. Moreover, thrice-treatment at day 4, 5, 6 post-infection was associated with an increased survival rate (18.2% single vs. 50% thrice at 20 µg/g per body weight, and the mice recovered from limb paralysis. Competitive ELISA also confirmed that CT11F9-recognized epitopes were immunodominant in humans. In conclusion, MAb CT11F9 is an ideal candidate to be humanized and used in severe EV71 infection.

  3. Motavizumab, A Neutralizing Anti-Respiratory Syncytial Virus (Rsv Monoclonal Antibody Significantly Modifies The Local And Systemic Cytokine Responses Induced By Rsv In The Mouse Model

    Directory of Open Access Journals (Sweden)

    Jafri Hasan S

    2007-10-01

    Full Text Available Abstract Motavizumab (MEDI-524 is a monoclonal antibody with enhanced neutralizing activity against RSV. In mice, motavizumab suppressed RSV replication which resulted in significant reduction of clinical parameters of disease severity. We evaluated the effect of motavizumab on the local and systemic immune response induced by RSV in the mouse model. Balb/c mice were intranasally inoculated with 106.5 PFU RSV A2 or medium. Motavizumab was given once intraperitoneally (1.25 mg/mouse as prophylaxis, 24 h before virus inoculation. Bronchoalveolar lavage (BAL and serum samples were obtained at days 1, 5 (acute and 28 (long-term post inoculation and analyzed with a multiplex assay (Beadlyte Upstate, NY for simultaneous quantitation of 18 cytokines: IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, KC (similar to human IL-8, IL-10, IL-12p40, IL-12p70, IL-13, IL-17, TNF-α, MCP-1, RANTES, IFN-γ and GM-CSF. Overall, cytokine concentrations were lower in serum than in BAL samples. By day 28, only KC was detected in BAL specimens at low concentrations in all groups. Administration of motavizumab significantly reduced (p

  4. Breadth of neutralization and synergy of clinically relevant human monoclonal antibodies against HCV genotypes 1a, 1b, 2a, 2b, 2c, and 3a

    DEFF Research Database (Denmark)

    Carlsen, Thomas H R; Pedersen, Jannie; Prentoe, Jannick C

    2014-01-01

    panel was determined by dose-response neutralization assays in Huh7.5 cells with antibody concentrations ranging from 0.0012 to 100 μg/mL. Interestingly, IC50 values against the different HCVcc's exhibited large variations among the HMAbs, and only three HMAbs (HC-1AM, HC84.24, and AR4A) neutralized all...

  5. Pharmacokinetics interactions of monoclonal antibodies.

    Science.gov (United States)

    Ferri, Nicola; Bellosta, Stefano; Baldessin, Ludovico; Boccia, Donatella; Racagni, Giorgi; Corsini, Alberto

    2016-09-01

    The clearance of therapeutic monoclonal antibodies (mAbs) typically does not involve cytochrome P450 (CYP450)-mediated metabolism or interaction with cell membrane transporters, therefore the pharmacokinetics interactions of mAbs and small molecule drugs are limited. However, a drug may affect the clearance of mAbs through the modulation of immune response (e.g., methotrexate reduces the clearance of infliximab, adalimumab, and golimumab, possibly due to methotrexate's inhibitory effect on the formation of antibodies against the mAbs). In addition, mAbs that are cytokine modulators may modify the metabolism of drugs through their effects on P450 enzymes expression. For example, cytokine modulators such as tocilizumab (anti-IL-6 receptor antibody) may reverse the "inhibitory" effect of IL-6 on CYP substrates, resulting in a "normalization" of CYP activities. Finally, a drug may alter the clearance of mAbs by either increasing or reducing the levels of expression of targets of mAbs on the cell surface. For instance, statins and fibrates induce PCSK9 expression and therefore increase cellular uptake and clearance of alirocumab and evolocumab, anti-PCSK9 antibodies. In the present review, we will provide an overview on the pharmacokinetics properties of mAbs as related to the most relevant examples of mAbs-small molecule drug interaction.

  6. Production and Screening of Monoclonal Peptide Antibodies.

    Science.gov (United States)

    Trier, Nicole Hartwig; Mortensen, Anne; Schiolborg, Annette; Friis, Tina

    2015-01-01

    Hybridoma technology is a remarkable and indispensable tool for generating high-quality monoclonal antibodies. Hybridoma-derived monoclonal antibodies not only serve as powerful research and diagnostic reagents, but have also emerged as the most rapidly expanding class of therapeutic biologicals. In this chapter, an overview of hybridoma technology and the laboratory procedures used routinely for hybridoma production and antibody screening are presented, including characterization of peptide antibodies.

  7. Inhibition of human immunodeficiency virus (HIV) infection in vitro by anticarbohydrate monoclonal antibodies: peripheral glycosylation of HIV envelope glycoprotein gp120 may be a target for virus neutralization

    DEFF Research Database (Denmark)

    Hansen, J E; Clausen, H; Nielsen, C

    1990-01-01

    Carbohydrate structures are often involved in the initial adhesion of pathogens to target cells. In the present study, a panel of anticarbohydrate monoclonal antibodies (MAbs) was tested for their ability to inhibit in vitro human immunodeficiency virus infectivity. MAbs against three different N...... carbohydrate structures expressed by the viral envelope glycoprotein gp120, indicating that glycans of the viral envelope are possible targets for immunotherapy or vaccine development or both....

  8. Monoclonal antibodies in chronic lymphocytic leukemia.

    Science.gov (United States)

    Ferrajoli, Alessandra; Faderl, Stefan; Keating, Michael J

    2006-09-01

    Multiple options are now available for the treatment of chronic lymphocytic leukemia. Over the last 10 years, monoclonal antibodies have become an integral part of the management of this disease. Alemtuzumab has received approval for use in patients with fludarabine-refractory chronic lymphocytic leukemia. Rituximab has been investigated extensively in chronic lymphocytic leukemia both as a single agent and in combination with chemotherapy and other monoclonal antibodies. Epratuzumab and lumiliximab are newer monoclonal antibodies in the early phase of clinical development. This article will review the monoclonal antibodies more commonly used to treat chronic lymphocytic leukemia, the results obtained with monoclonal antibodies as single agents and in combination with chemotherapy, and other biological agents and newer compounds undergoing clinical trials.

  9. A novel rabbit anti-hepatocyte growth factor monoclonal neutralizing antibody inhibits tumor growth in prostate cancer cells and mouse xenografts

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Yanlan; Chen, Yicheng; Ding, Guoqing; Wang, Mingchao; Wu, Haiyang; Xu, Liwei; Rui, Xuefang; Zhang, Zhigen, E-mail: srrshurology@163.com

    2015-08-14

    The hepatocyte growth factor and its receptor c-Met are correlated with castration-resistance in prostate cancer. Although HGF has been considered as an attractive target for therapeutic antibodies, the lack of cross-reactivity of monoclonal antibodies with human/mouse HGFs is a major obstacle in preclinical developments. We generated a panel of anti-HGF RabMAbs either blocking HGF/c-Met interaction or inhibiting c-Met phosphorylation. We selected one RabMAb with mouse cross-reactivity and demonstrated that it blocked HGF-stimulated downstream activation in PC-3 and DU145 cells. Anti-HGF RabMAb inhibited not only the growth of PC-3 cells but also HGF-dependent proliferation in HUVECs. We further demonstrated the efficacy and potency of the anti-HGF RabMAb in tumor xenograft mice models. Through these in vitro and in vivo experiments, we explored a novel therapeutic antibody for advanced prostate cancer. - Highlights: • HGF is an attractive target for castration-refractory prostate cancer. • We generated and characterized a panel of anti-HGF rabbit monoclonal antibodies. • More than half of these anti-HGF RabMAbs was cross-reactive with mouse HGF. • Anti-HGF RabMAb blocks HGF-stimulated phosphorylation and cell growth in vitro. • Anti-HGF RabMAb inhibits tumor growth and angiogenesis in xenograft mice.

  10. Novel Clostridium difficile Anti-Toxin (TcdA and TcdB Humanized Monoclonal Antibodies Demonstrate In Vitro Neutralization across a Broad Spectrum of Clinical Strains and In Vivo Potency in a Hamster Spore Challenge Model.

    Directory of Open Access Journals (Sweden)

    Hongyu Qiu

    Full Text Available Clostridium difficile (C. difficile infection (CDI is the main cause of nosocomial antibiotic-associated colitis and increased incidence of community-associated diarrhea in industrialized countries. At present, the primary treatment of CDI is antibiotic administration, which is effective but often associated with recurrence, especially in the elderly. Pathogenic strains produce enterotoxin, toxin A (TcdA, and cytotoxin, toxin B (TcdB, which are necessary for C. difficile induced diarrhea and gut pathological changes. Administration of anti-toxin antibodies provides an alternative approach to treat CDI, and has shown promising results in preclinical and clinical studies. In the current study, several humanized anti-TcdA and anti-TcdB monoclonal antibodies were generated and their protective potency was characterized in a hamster infection model. The humanized anti-TcdA (CANmAbA4 and anti-TcdB (CANmAbB4 and CANmAbB1 antibodies showed broad spectrum in vitro neutralization of toxins from clinical strains and neutralization in a mouse toxin challenge model. Moreover, co-administration of humanized antibodies (CANmAbA4 and CANmAbB4 cocktail provided a high level of protection in a dose dependent manner (85% versus 57% survival at day 22 for 50 mg/kg and 20 mg/kg doses, respectively in a hamster gastrointestinal infection (GI model. This study describes the protective effects conferred by novel neutralizing anti-toxin monoclonal antibodies against C. difficile toxins and their potential as therapeutic agents in treating CDI.

  11. Monoclonal Antibodies Against Xenopus Greatwall Kinase

    Science.gov (United States)

    Wang, Ling; Fisher, Laura A.; Wahl, James K.

    2011-01-01

    Mitosis is known to be regulated by protein kinases, including MPF, Plk1, Aurora kinases, and so on, which become active in M-phase and phosphorylate a wide range of substrates to control multiple aspects of mitotic entry, progression, and exit. Mechanistic investigations of these kinases not only provide key insights into cell cycle regulation, but also hold great promise for cancer therapy. Recent studies, largely in Xenopus, characterized a new mitotic kinase named Greatwall (Gwl) that plays essential roles in both mitotic entry and maintenance. In this study, we generated a panel of mouse monoclonal antibodies (MAbs) specific for Xenopus Gwl and characterized these antibodies for their utility in immunoblotting, immunoprecipitation, and immunodepletion in Xenopus egg extracts. Importantly, we generated an MAb that is capable of neutralizing endogenous Gwl. The addition of this antibody into M-phase extracts results in loss of mitotic phosphorylation of Gwl, Plk1, and Cdk1 substrates. These results illustrate a new tool to study loss-of-function of Gwl, and support its essential role in mitosis. Finally, we demonstrated the usefulness of the MAb against human Gwl/MASTL. PMID:22008075

  12. Development and characterization of mouse monoclonal antibodies specific for chicken interleukin 18

    Science.gov (United States)

    Four mouse monoclonal antibodies (mAbs) which are specific for chicken interleukin 18 (chIL18) were produced and characterized by enzyme-linked immunosorbent assay (ELISA), Western blotting, quantitative real-time PCR and neutralization assays. Monoclonal antibodies specific for chIL18 identified a ...

  13. Production and Characterization of Monoclonal Antibodies of Shrimp White Spot Syndrome Virus Envelope Protein VP28

    Institute of Scientific and Technical Information of China (English)

    Wan-gang GU; Jun-fa YUAN; Ge-lin XU; Li-juan LI; Ni LIU; Cong ZHANG; Jian-hong ZHANG; Zheng-li SHI

    2007-01-01

    BALB/c mice were immunized with purified White spot syndrome virus (WSSV).Six monoclonal antibody cell lines were selected by ELISA with VP28 protein expressed in E.coll in vitro neutralization experiments showed that 4 of them could inhibit the virus infection in crayfish.Westernblot suggested that all these monoclonal antibodies were against the conformational structure of VP28.The monoclonal antibody 7B4 was labeled with colloidal gold particles and used to locate the VP28 on virus envelope by immunogold labeling.These monoclonal antibodies could be used to develop immunological diagnosis methods for WSSV infection.

  14. Crystallization of the receptor-binding domain of parathyroid hormone-related protein in complex with a neutralizing monoclonal antibody Fab fragment

    Energy Technology Data Exchange (ETDEWEB)

    McKinstry, William J.; Polekhina, Galina; Diefenbach-Jagger, Hannelore; Sato, Koh; Onuma, Etsuro; Gillespie, Matthew T.; Martin, Thomas J.; Parker, Michael W.; (SVIMR-A); (Chugai); (Melbourne)

    2009-04-01

    Parathyroid hormone-related protein (PTHrP) plays an important role in regulating embryonic skeletal development and is abnormally regulated in the pathogenesis of skeletal complications observed with many cancers and osteoporosis. It exerts its action through binding to a G-protein-coupled seven-transmembrane cell-surface receptor (GPCR). Structurally, GPCRs are very difficult to study by X-ray crystallography. In this study, a monoclonal antibody Fab fragment which recognizes the same region of PTHrP as its receptor, PTH1R, was used to aid in the crystallization of PTHrP. The resultant protein complex was crystallized using the hanging-drop vapour-diffusion method with polyethylene glycol as a precipitant. The crystals belonged to the orthorhombic space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 72.6, b = 96.3, c = 88.5 {angstrom}, and diffracted to 2.0 {angstrom} resolution using synchrotron radiation. The crystal structure will shed light on the nature of the key residues of PTHrP that interact with the antibody and will provide insights into how the antibody is able to discriminate between PTHrP and the related molecule parathyroid homone.

  15. Crystallization of the receptor-binding domain of parathyroid hormone-related protein in complex with a neutralizing monoclonal antibody Fab fragment.

    Science.gov (United States)

    McKinstry, William J; Polekhina, Galina; Diefenbach-Jagger, Hannelore; Sato, Koh; Onuma, Etsuro; Gillespie, Matthew T; Martin, Thomas J; Parker, Michael W

    2009-04-01

    Parathyroid hormone-related protein (PTHrP) plays an important role in regulating embryonic skeletal development and is abnormally regulated in the pathogenesis of skeletal complications observed with many cancers and osteoporosis. It exerts its action through binding to a G-protein-coupled seven-transmembrane cell-surface receptor (GPCR). Structurally, GPCRs are very difficult to study by X-ray crystallography. In this study, a monoclonal antibody Fab fragment which recognizes the same region of PTHrP as its receptor, PTH1R, was used to aid in the crystallization of PTHrP. The resultant protein complex was crystallized using the hanging-drop vapour-diffusion method with polyethylene glycol as a precipitant. The crystals belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 72.6, b = 96.3, c = 88.5 A, and diffracted to 2.0 A resolution using synchrotron radiation. The crystal structure will shed light on the nature of the key residues of PTHrP that interact with the antibody and will provide insights into how the antibody is able to discriminate between PTHrP and the related molecule parathyroid homone.

  16. Monoclonal antibodies reactive with hairy cell leukemia

    NARCIS (Netherlands)

    Visser, L; Shaw, A; Slupsky, J; Vos, H; Poppema, S

    Monoclonal antibodies reactive with hairy cell leukemia were developed to aid in the diagnosis of this subtype of B cell chronic lymphocytic leukemia and to gain better insight into the origin of hairy cells. Three antibodies were found to be of value in the diagnosis of hairy cell leukemia.

  17. 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.

  18. Monoclonal antibodies reactive with hairy cell leukemia

    NARCIS (Netherlands)

    Visser, L; Shaw, A; Slupsky, J; Vos, H; Poppema, S

    1989-01-01

    Monoclonal antibodies reactive with hairy cell leukemia were developed to aid in the diagnosis of this subtype of B cell chronic lymphocytic leukemia and to gain better insight into the origin of hairy cells. Three antibodies were found to be of value in the diagnosis of hairy cell leukemia. Antibod

  19. Non-random escape pathways from a broadly neutralizing human monoclonal antibody map to a highly conserved region on the hepatitis C virus E2 glycoprotein encompassing amino acids 412-423.

    Directory of Open Access Journals (Sweden)

    Zhen-yong Keck

    2014-08-01

    Full Text Available A challenge for hepatitis C virus (HCV vaccine development is to define epitopes that are able to elicit protective antibodies against this highly diverse virus. The E2 glycoprotein region located at residues 412-423 is conserved and antibodies to 412-423 have broadly neutralizing activities. However, an adaptive mutation, N417S, is associated with a glycan shift in a variant that cannot be neutralized by a murine but by human monoclonal antibodies (HMAbs against 412-423. To determine whether HCV escapes from these antibodies, we analyzed variants that emerged when cell culture infectious HCV virions (HCVcc were passaged under increasing concentrations of a specific HMAb, HC33.1. Multiple nonrandom escape pathways were identified. Two pathways occurred in the context of an N-glycan shift mutation at N417T. At low antibody concentrations, substitutions of two residues outside of the epitope, N434D and K610R, led to variants having improved in vitro viral fitness and reduced sensitivity to HC33.1 binding and neutralization. At moderate concentrations, a S419N mutation occurred within 412-423 in escape variants that have greatly reduced sensitivity to HC33.1 but compromised viral fitness. Importantly, the variants generated from these pathways differed in their stability. N434D and K610R-associated variants were stable and became dominant as the virions were passaged. The S419N mutation reverted back to N419S when immune pressure was reduced by removing HC33.1. At high antibody concentrations, a mutation at L413I was observed in variants that were resistant to HC33.1 neutralization. Collectively, the combination of multiple escape pathways enabled the virus to persist under a wide range of antibody concentrations. Moreover, these findings pose a different challenge to vaccine development beyond the identification of highly conserved epitopes. It will be necessary for a vaccine to induce high potency antibodies that prevent the formation of escape

  20. Why Does the Molecular Structure of Broadly Neutralizing Monoclonal Antibodies Isolated from Individuals Infected with HIV-1 not Inform the Rational Design of an HIV-1 Vaccine?

    Directory of Open Access Journals (Sweden)

    Marc H V Van Regenmortel

    2015-05-01

    Full Text Available It is commonly assumed that neutralizing Mabs that bind to the HIV-1 Env glycoprotein are more specific reagents than anti-HIV-1 polyclonal antisera and that knowledge of the structure of these Mabs facilitates the rational design of effective HIV-1 vaccine immunogens. However, after more than ten years of unsuccessful experimentation using the structure-based reverse vaccinology approach, it is now evident that it is not possible to infer from the structure of neutralizing Mabs which HIV immunogens induced their formation nor which vaccine immunogens will elicit similar Abs in an immunized host. The use of Mabs for developing an HIV-1 vaccine was counterproductive because it overlooked the fact that the apparent specificity of a Mab very much depends on the selection procedure used to obtain it and also did not take into account that an antibody is never monospecific for a single epitope but is always polyspecific for many epitopes. When the rationale of the proponents of the unsuccessful rational design strategy is analyzed, it appears that investigators who claim they are designing a vaccine immunogen are only improving the binding reactivity of a single epitope-paratope pair and are not actually designing an immunogen able to generate protective antibodies. The task of a designer consists in imagining what type of immunogen is likely to elicit a protective immune response but in the absence of knowledge regarding which features of the immune system are responsible for producing a functional neutralizing activity in antibodies, it is not feasible to intentionally optimize a potential immunogen candidate in order to obtain the desired outcome. The only available option is actually to test possible solutions by trial-and-error experiments until the preset goal is perhaps attained. Rational design and empirical approaches in HIV vaccine research should thus not be opposed as alternative options since empirical testing is an integral part of a so

  1. 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.

  2. Monoclonal antibody technologies and rapid detection assays

    Science.gov (United States)

    Novel methodologies and screening strategies will be outlined on the use of hybridoma technology for the selection of antigen specific monoclonal antibodies. The development of immunoassays used for diagnostic detection of prions and bacterial toxins will be discussed and examples provided demonstr...

  3. Validation of cross-genotype neutralization by hepatitis B virus-specific monoclonal antibodies by in vitro and in vivo infection.

    Science.gov (United States)

    Hamada-Tsutsumi, Susumu; Iio, Etsuko; Watanabe, Tsunamasa; Murakami, Shuko; Isogawa, Masanori; Iijima, Sayuki; Inoue, Takako; Matsunami, Kayoko; Tajiri, Kazuto; Ozawa, Tatsuhiko; Kishi, Hiroyuki; Muraguchi, Atsushi; Joh, Takashi; Tanaka, Yasuhito

    2015-01-01

    Vaccines based on hepatitis B virus (HBV) genotype A have been used worldwide for immunoprophylaxis and are thought to prevent infections by non-A HBV strains effectively, whereas, vaccines generated from genotype C have been used in several Asian countries, including Japan and Korea, where HBV genotype C is prevalent. However, acute hepatitis B caused by HBV genotype A infection has been increasing in Japan and little is known about the efficacy of immunization with genotype C-based vaccines against non-C infection. We have isolated human monoclonal antibodies (mAbs) from individuals who were immunized with the genotype C-based vaccine. In this study, the efficacies of these two mAbs, HB0116 and HB0478, were analyzed using in vivo and in vitro models of HBV infection. Intravenous inoculation of HBV genotype C into chimeric mice with human hepatocytes resulted in the establishment of HBV infection after five weeks, whereas preincubation of the inocula with HB0116 or HB0478 protected chimeric mice from genotype C infection completely. Interestingly, both HB0116 and HB0478 were found to block completely genotype A infection. Moreover, infection by a genotype C strain with an immune escape substitution of amino acid 145 in the hepatitis B surface protein was also completely inhibited by incubation with HB0478. Finally, in vitro analysis of dose dependency revealed that the amounts of HB0478 required for complete protection against genotype C and genotype A infection were 5.5 mIU and 55 mIU, respectively. These results suggested that genotype C-based vaccines have ability to induce cross-genotype immunity against HBV infection.

  4. Monoclonal antibodies to Treponema Pallidum.

    NARCIS (Netherlands)

    H.J.M. van de Donk; J.D.A. van Embden; M.F. van Olderen; A.D.M.E. Osterhaus (Albert); J.C. de Jong (Jan)

    1984-01-01

    textabstractThree successive fusions of mouse myeloma cells and spleen lymphocytes of a mouse immunized with Treponema Pallidum resulted in one hybridoma producing anti T. pallidum antibodies for each fusion. The mice were immunized with live pallidum cells respectively 1, 3 and 5 months before fusi

  5. Human Monoclonal Antibodies as a Countermeasure Against Botulinum Toxins

    Science.gov (United States)

    2012-11-30

    REPORT Human monoclonal antibodies as a countermeasure against Botulinum toxins 14. ABSTRACT 16. SECURITY CLASSIFICATION OF: In this report, we...Prescribed by ANSI Std. Z39.18 - 31-Aug-2012 Human monoclonal antibodies as a countermeasure against Botulinum toxins Report Title ABSTRACT In this report...DTRA Final Report: Human monoclonal antibodies as a countermeasure against Botulinum toxins   Page 1 of 22 DTRA Final Report: Human monoclonal

  6. Monoclonal Antibodies to Plant Growth Regulators

    Science.gov (United States)

    Eberle, Joachim; Arnscheidt, Angelika; Klix, Dieter; Weiler, Elmar W.

    1986-01-01

    Four high affinity monoclonal antibodies, which recognize two plant growth regulators from the cytokinin group, namely trans-zeatin riboside and dihydrozeatin riboside and their derivatives are reported. Six hybridomas were produced from three independent fusions of Balb/c spleen cells with P3-NS1-Ag 4-1 (abbreviated NS1) or X63-Ag 8.653 (X63) myeloma cells. The mice had been hyperimmunized with zeatin riboside-bovine serum albumin conjugate or dihydrozeatin riboside-bovine serum albumin conjugate for 3 months. The hybridomas secrete antibodies of the IgG 1 or IgG 2b subclass and allow the detection of femtomole amounts of the free cytokinins, their ribosides, and ribotides in plant extracts. The use of these monoclonals in radio- and enzyme-linked immunosorbent assay is also discussed. PMID:16664848

  7. Recent developments in monoclonal antibody radiolabeling techniques

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, S.C.; Mease, R.C.

    1989-01-01

    Monoclonal antibodies (MAbs) have shown the potential to serve as selective carriers of radionuclides to specific in vivo antigens. Accordingly, there has been an intense surge of research activity in an effort to develop and evaluate MAb-based radiopharmaceuticals for tumor imaging (radioimmunoscintigraphy) and therapy (radioimmunotherapy), as well as for diagnosing nonmalignant diseases. A number of problems have recently been identified, related to the MAbs themselves and to radiolabeling techniques, that comprise both the selectivity and the specificity of the in vivo distribution of radiolabeled MAbs. This paper will address some of these issues and primarily discuss recent developments in the techniques for radiolabeling monoclonal antibodies that may help resolve problems related to the poor in vivo stability of the radiolabel and may thus produce improved biodistribution. Even though many issues are identical with therapeutic radionuclides, the discussion will focus mainly on radioimmunoscintigraphic labels. 78 refs., 6 tabs.

  8. Use of synthetic peptides to represent surface-exposed epitopes defined by neutralizing dengue complex- and flavivirus group-reactive monoclonal antibodies on the native dengue type-2 virus envelope glycoprotein.

    Science.gov (United States)

    Falconar, Andrew K I

    2008-07-01

    The reactions of neutralizing monoclonal antibodies (mAbs) that defined dengue virus (DENV) complex, flavivirus subgroup or group neutralizing epitopes were tested against synthetic peptide sequences from domains I, II and III of the envelope (E) glycoproteins of different DENV-2 genotypes/strains. The DENV complex-reactive mAb identified the surface-exposed 304-GKFKV/IVKEIA-313 peptides and the DENV complex-conserved 393-KKGSSIGQ/KM-401 peptides in domain III, which were located adjacently in the native glycoprotein. Both flavivirus group-reactive mAbs reacted most strongly with fusion sequence peptides from domain II when they contained a cysteine (C) by glycine (G) substitution (underlined) (101-WGNGGGLFG-109) to represent the native rotated C side chain. The 393-401 sequence represents a newly identified epitope, present as a highly flexible coil located between the 385 and 393 cell-binding sequence and the 401 and 413 sequence involved in the E glycoprotein homo-trimer formation. The 101-109 sequence containing 105-C by G substitution and the 393-401 sequence are good candidates for diagnostic assays and cross-protection experiments.

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

    DEFF Research Database (Denmark)

    Skjødt, K; Schou, C; Koch, C

    1984-01-01

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

  10. Therapeutic monoclonal antibody for Sporotrichosis

    Directory of Open Access Journals (Sweden)

    Sandro eAlmeida

    2012-11-01

    Full Text Available Sporotrichosis is a chronic subcutaneous mycosis that affects either humans or animals and occurs worldwide. This subcutaneous mycosis had been attributed to a single etiological agent, Sporothrix schenckii. S. schenckii exhibits a considerable genetic variability, where recently, was suggesting that this taxon consists of a complex of species. Sporotrichosis is caused by traumatic inoculation of the fungus, which is a ubiquitous environmental saprophyte that can be isolated from soil and plant debris. The infection is limited to the cutaneous forms but, recently, occurrences of more severe clinical forms of this mycosis were described, especially among immunocompromized individuals. The immunological mechanisms involved in prevention and control of sporotrichosis are still not very well understood. Some works suggest that cell-mediated immunity plays an important role in protecting the host against S. schenckii. In contrast, the role of the humoral immune response in protection against this fungus have not been studied in detail. In a previous study, we showed that antigens secreted by S. schenckii induce a specific humoral response in infected animals, mainly against the 70-kDa molecules, indicating a possible participation of specific antibodies to this molecule in infection control. In an other work of the our group, we produced a mAb against a 70-kDa glycoprotein of S. schenckii in order to better understand the effect of passive immunization of mice infected with S. schenckii. Results showed a significant reduction in the number of CFU in organs of mice when the mAb was injected before and during S. schenckii infection. Similar results were observed when T-cell deficient mice were used. Drugs of choice in the treatment of sporothrichosis require long periods and frequently relapses are observed, mainly in immunocompromized patients. The strong protection induced by mAb against a 70-kDa glycoprotein makes it a strong candidate for a

  11. Monoclonal Antibody-Based Therapeutics for Melioidosis and Glanders

    Directory of Open Access Journals (Sweden)

    Hyung-Yong Kim

    2011-01-01

    Full Text Available Problem statement: Burkholderia Pseudomallei (BP and B. Mallei (BM were two closely related pathogenic gram-negative bacteria. They were the causative agents of melioidosis and glanders, respectively and are recognized by CDC as category B select agents. Significant efforts had been devoted to developing the diagnostic and therapeutic measures against these two pathogens. Monoclonal antibody-based therapeutic was a promising targeted therapy to fight against melioidosis and glanders. Valuable findings have been reported by different groups in their attempt to identify vaccine targets against these two pathogens. Approach: Our group has generated neutralizing Monoclonal Antibodies (MAbs against BP and BM and characterized them by both in vitro and in vivo experiments. We present an overview of the MAb-based therapeutic approaches against BP and BM and demonstrate some of our efforts for developing chimeric and fully human MAbs using antibody engineering. Results: Throughout conventional mouse hybridoma technique and antibody engineering (chimerization and in vitro antibody library techniques, we generated 10 chimeric MAbs (3 stable MAbs and 7 transient MAbs and one fully human MAb against BP and BM. In addition, we present the reactive antigen profiles of these MAbs. Our approaches had potentials to accelerate the development of therapeutics for melioidosis and glanders in humans. Conclusion: Our experience and findings presented here will be valuable for choosing the best antigenic targets and ultimately for the production of effective vaccines for these two pathogens.

  12. Characteristics of Monoclonal Antibody Against Infectious Bursal Disease Virus

    Institute of Scientific and Technical Information of China (English)

    LiYan-Fei; WangWei; 等

    1999-01-01

    Thirteen strains of monoclonal antibodies(McAbs) against infections bursal disease virus(IBDV) were obtained by using hydridoma technique and their characteristics were studied by double immunodiffusion,enzyme-linked immunosorbent assay(ELISA),virus neutralization test(VNT) and Western-blotting assay (WBA).The result showed that nine of the thirteen McAbs belonged to IgG class and four of them belonged to IgM class.No crossreactions were detected betwween the McAbs and Newscastle disease virus (NDV),infectious bronchitis virus(IBV) and Marek's disease virus(MDV).All of McAbs were positively specific reactive with IBDV and five of them can neutralize viral infectivity.Their recognized epitopes of the neutralizing McAbs were all presented on VP2 of the IBDV.

  13. Characteristics of Monoclonal Antibody Against Infectious Bursal Disease Virus

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    Thirteen strains of monoclonal antibodies (McAbs) against infectious bursal disease virus (IBDV) were obtained by using hybridoma technique and their characteristics were studied by double immunodiffusion,en- zyme- linked immunosorbent assay (ELISA), virus neutralization test (VNT) and Western- blotting assay (WBA). The result showed that nine of the thirteen McAbs belonged to IgG class and four of them belonged to IgM class. No crossreactions were detected betwween the McAbs and Newscastle disease virus (NDV) ,in- fectious bronchitis virus(IBV) and Marek's disease virus(MDV). All of McAbs were positively specific reac- tive with IBDV and five of them can neutralize viral infectivity. Their recognized epitopes of the neutralizing McAbs were all presented on VP2 of the IBDV.

  14. Aggregates in monoclonal antibody manufacturing processes.

    Science.gov (United States)

    Vázquez-Rey, María; Lang, Dietmar A

    2011-07-01

    Monoclonal antibodies have proved to be a highly successful class of therapeutic products. Large-scale manufacturing of pharmaceutical antibodies is a complex activity that requires considerable effort in both process and analytical development. If a therapeutic protein cannot be stabilized adequately, it will lose partially or totally its therapeutic properties or even cause immunogenic reactions thus potentially further endangering the patients' health. The phenomenon of protein aggregation is a common issue that compromises the quality, safety, and efficacy of antibodies and can happen at different steps of the manufacturing process, including fermentation, purification, final formulation, and storage. Aggregate levels in drug substance and final drug product are a key factor when assessing quality attributes of the molecule, since aggregation might impact biological activity of the biopharmaceutical. In this review it is analyzed how aggregates are formed during monoclonal antibody industrial production, why they have to be removed and the manufacturing process steps that are designed to either minimize or remove aggregates in the final product. Copyright © 2011 Wiley Periodicals, Inc.

  15. ON THE NOTION OF SYNERGY OF MONOCLONAL ANTIBODIES AS DRUGS

    Directory of Open Access Journals (Sweden)

    Michael Sela

    2013-08-01

    Full Text Available History of developing synergy between monoclonal antibodies, anti-tumor activity of monoclonal antibodies against tyrosine-kinases receptors EGFR/ErbB-1 and HER2/ErbB-2 as well as growth factor VEGF in various combinations are considered in the article. There were proposed hypotheses about potential molecular mechanisms underlay synergy between monoclonal antibodies (for homo- and hetero combinations of antibodies appropriately specific for antigenic determinants on the same or different receptors. Future trends in researches necessary to deeper understanding causes of this phenomenon and perspectives for practical application of monoclonal antibodies acted synergistically as immunotherapeutic drugs for human tumors treatment are reviewed.

  16. Characterization of monoclonal and polyclonal antibodies to bovine enteric coronavirus: Establishment of an efficient ELISA for antigen detection in feces

    OpenAIRE

    Czerny, C. P.; Eichhorn, Werner

    1989-01-01

    Monoclonal antibodies to bovine enteric coronavirus (BEC) were produced. Additionally, polyclonal antibodies were made in rabbits and guinea pigs and extracted from the yolk of immunized hens. The antibodies were characterized by neutralization test, hemagglutination inhibition test, enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Neutralizing antibody titers of polyclonal antisera ranged from 1:1280 to 1:40 000. Only one out of 908 hybridoma colonies tested secreted antibodies ...

  17. Monoclonal antibody-based candidate therapeutics against HIV type 1.

    Science.gov (United States)

    Chen, Weizao; Dimitrov, Dimiter S

    2012-05-01

    Treatment of HIV-1 infection has been highly successful with small molecule drugs. However, resistance still develops. In addition, long-term use can lead to toxicity with unpredictable effects on health. Finally, current drugs do not lead to HIV-1 eradication. The presence of the virus leads to chronic inflammation, which can result in increased morbidity and mortality after prolonged periods of infection. Monoclonal antibodies (mAbs) have been highly successful during the past two decades for therapy of many diseases, primarily cancers and immune disorders. They are relatively safe, especially human mAbs that have evolved in humans at high concentrations to fight diseases and long-term use may not lead to toxicities. Several broadly neutralizing mAbs (bnmAbs) against HIV-1 can protect animals but are not effective when used for therapy of an established infection. We have hypothesized that HIV-1 has evolved strategies to effectively escape neutralization by full-size antibodies in natural infections but not by smaller antibody fragments. Therefore, a promising direction of research is to discover and exploit antibody fragments as potential candidate therapeutics against HIV-1. Here we review several bnmAbs and engineered antibody domains (eAds), their in vitro and in vivo antiviral efficacy, mechanisms used by HIV-1 to escape them, and strategies that could be effective to develop more powerful mAb-based HIV-1 therapeutics.

  18. Emerging monoclonal antibodies against Clostridium difficile infection.

    Science.gov (United States)

    Péchiné, Séverine; Janoir, Claire; Collignon, Anne

    2017-04-01

    Clostridium difficile infections are characterized by a high recurrence rate despite antibiotic treatments and there is an urgent need to develop new treatments such as fecal transplantation and immonotherapy. Besides active immunotherapy with vaccines, passive immunotherapy has shown promise, especially with monoclonal antibodies. Areas covered: Herein, the authors review the different assays performed with monoclonal antibodies against C. difficile toxins and surface proteins to treat or prevent primary or recurrent episodes of C. difficile infection in animal models and in clinical trials as well. Notably, the authors lay emphasis on the phase III clinical trial (MODIFY II), which allowed bezlotoxumab to be approved by the Food and Drug Administration and the European Medicines Agency. They also review new strategies for producing single domain antibodies and nanobodies against C. difficile and new approaches to deliver them in the digestive tract. Expert opinion: Only two human Mabs against TcdA and TcdB have been tested alone or in combination in clinical trials. However, many animal model studies have provided rationale for the use of Mabs and nanobodies in C. difficile infection and pave the way for further clinical investigation.

  19. A Potential of an Anti-HTLV-I gp46 Neutralizing Monoclonal Antibody (LAT-27 for Passive Immunization against Both Horizontal and Mother-to-Child Vertical Infection with Human T Cell Leukemia Virus Type-I

    Directory of Open Access Journals (Sweden)

    Hideki Fujii

    2016-02-01

    Full Text Available Although the number of human T-cell leukemia virus type-I (HTLV-I-infected individuals in the world has been estimated at over 10 million, no prophylaxis vaccines against HTLV-I infection are available. In this study, we took a new approach for establishing the basis of protective vaccines against HTLV-I. We show here the potential of a passively administered HTLV-I neutralizing monoclonal antibody of rat origin (LAT-27 that recognizes epitopes consisting of the HTLV-I gp46 amino acids 191–196. LAT-27 completely blocked HTLV-I infection in vitro at a minimum concentration of 5 μg/mL. Neonatal rats born to mother rats pre-infused with LAT-27 were shown to have acquired a large quantity of LAT-27, and these newborns showed complete resistance against intraperitoneal infection with HTLV-I. On the other hand, when humanized immunodeficient mice were pre-infused intravenously with humanized LAT-27 (hu-LAT-27, all the mice completely resisted HTLV-I infection. These results indicate that hu-LAT-27 may have a potential for passive immunization against both horizontal and mother-to-child vertical infection with HTLV-I.

  20. Anaphylaxis to chemotherapy and monoclonal antibodies.

    Science.gov (United States)

    Castells, Mariana C

    2015-05-01

    Hypersensitivity reactions are increasingly prevalent, although underrecognized and underreported. Platins induce immunoglobulin E-mediated sensitization; taxenes and some monoclonal antibodies can induce reactions at first exposure. Severe hypersensitivity can preclude first-line therapy. Tryptase level at the time of a reaction is a useful diagnostic tool. Skin testing provides a specific diagnosis. Newer tests are promising diagnostic tools to help identify patients at risk before first exposure. Safe management includes rapid drug desensitization. This review provides information regarding the scope of hypersensitivity and anaphylactic reactions induced by chemotherapy and biological drugs, as well as diagnosis, management, and treatment options.

  1. The Role of Monoclonal Antibodies in the Management of Leukemia

    Science.gov (United States)

    Al-Ameri, Ali; Cherry, Mohamad; Al-Kali, Aref; Ferrajoli, Alessandra

    2010-01-01

    This article will review the monoclonal antibodies more commonly used in leukemias. In the last three decades, scientists have made considerable progress understanding the structure and the functions of various surface antigens, such as CD20, CD33. The introduction of rituximab, an anti CD20 monoclonal antibody, had a great impact in the treatment of lymphoproliferative disorders. Gemtuzumab, an anti CD 33 conjugated monoclonal antibody has activity in acute mylegenous leukemia (AML). As this field is undergoing a rapid growth, the years will see an increasing use of monoclonal antibodies in hematological malignancies.

  2. The Role of Monoclonal Antibodies in the Management of Leukemia

    Directory of Open Access Journals (Sweden)

    Mohamad Cherry

    2010-10-01

    Full Text Available This article will review the monoclonal antibodies more commonly used in leukemias. In the last three decades, scientists have made considerable progress understanding the structure and the functions of various surface antigens, such as CD20, CD33. The introduction of rituximab, an anti CD20 monoclonal antibody, had a great impact in the treatment of lymphoproliferative disorders. Gemtuzumab, an anti CD 33 conjugated monoclonal antibody has activity in acute mylegenous leukemia (AML. As this field is undergoing a rapid growth, the years will see an increasing use of monoclonal antibodies in hematological malignancies.

  3. Monoclonal antibody disulfide reduction during manufacturing

    Science.gov (United States)

    Hutterer, Katariina M.; Hong, Robert W.; Lull, Jonathon; Zhao, Xiaoyang; Wang, Tian; Pei, Rex; Le, M. Eleanor; Borisov, Oleg; Piper, Rob; Liu, Yaoqing Diana; Petty, Krista; Apostol, Izydor; Flynn, Gregory C.

    2013-01-01

    Manufacturing-induced disulfide reduction has recently been reported for monoclonal human immunoglobulin gamma (IgG) antibodies, a widely used modality in the biopharmaceutical industry. This effect has been tied to components of the intracellular thioredoxin reduction system that are released upon cell breakage. Here, we describe the effect of process parameters and intrinsic molecule properties on the extent of reduction. Material taken from cell cultures at the end of production displayed large variations in the extent of antibody reduction between different products, including no reduction, when subjected to the same reduction-promoting harvest conditions. Additionally, in a reconstituted model in which process variables could be isolated from product properties, we found that antibody reduction was dependent on the cell line (clone) and cell culture process. A bench-scale model using a thioredoxin/thioredoxin reductase regeneration system revealed that reduction susceptibility depended on not only antibody class but also light chain type; the model further demonstrates that the trend in reducibility was identical to DTT reduction sensitivity following the order IgG1λ > IgG1κ > IgG2λ > IgG2κ. Thus, both product attributes and process parameters contribute to the extent of antibody reduction during production. PMID:23751615

  4. Generation of monoclonal antibodies to native active human glycosyltransferases

    DEFF Research Database (Denmark)

    Vester-Christensen, Malene Bech; Bennett, Eric Paul; Clausen, Henrik;

    2013-01-01

    using monoclonal antibodies therefore provides an excellent strategy to analyze the glycosylation process in cells. A major drawback has been difficulties in generating antibodies to glycosyltransferases and validating their specificities. Here we describe a simple strategy for generating...

  5. Epitope Mapping of Dengue-Virus-Enhancing Monoclonal-Antibody Using Phage Display Peptide Library

    OpenAIRE

    Chung-I Rai; Huan-Yao Lei; Yee-Shin Lin; Hsiao-Sheng Liu; Shun-Hua Chen; Lien-Cheng Chen; Trai-Ming Yeh

    2008-01-01

    The Antibody-Dependent Enhancement (ADE) hypothesis has been proposed to explain why more severe manifestations of Dengue Hemorrhagic Fever and Dengue Shock Syndrome (DHF/DSS) occur predominantly during secondary infections of Dengue Virus (DV) with different serotypes. However, the epitopes recognized by these enhancing antibodies are unclear. Recently, anti-pre-M monoclonal antibody (mAb 70-21), which recognized all DV serotypes without neutralizing activity, were generated and demonstrated...

  6. A monoclonal antibody toolkit for C. elegans.

    Directory of Open Access Journals (Sweden)

    Gayla Hadwiger

    Full Text Available BACKGROUND: Antibodies are critical tools in many avenues of biological research. Though antibodies can be produced in the research laboratory setting, most research labs working with vertebrates avail themselves of the wide array of commercially available reagents. By contrast, few such reagents are available for work with model organisms. METHODOLOGY/PRINCIPAL FINDINGS: We report the production of monoclonal antibodies directed against a wide range of proteins that label specific subcellular and cellular components, and macromolecular complexes. Antibodies were made to synaptobrevin (SNB-1, a component of synaptic vesicles; to Rim (UNC-10, a protein localized to synaptic active zones; to transforming acidic coiled-coil protein (TAC-1, a component of centrosomes; to CENP-C (HCP-4, which in worms labels the entire length of their holocentric chromosomes; to ORC2 (ORC-2, a subunit of the DNA origin replication complex; to the nucleolar phosphoprotein NOPP140 (DAO-5; to the nuclear envelope protein lamin (LMN-1; to EHD1 (RME-1 a marker for recycling endosomes; to caveolin (CAV-1, a marker for caveolae; to the cytochrome P450 (CYP-33E1, a resident of the endoplasmic reticulum; to beta-1,3-glucuronyltransferase (SQV-8 that labels the Golgi; to a chaperonin (HSP-60 targeted to mitochondria; to LAMP (LMP-1, a resident protein of lysosomes; to the alpha subunit of the 20S subcomplex (PAS-7 of the 26S proteasome; to dynamin (DYN-1 and to the alpha-subunit of the adaptor complex 2 (APA-2 as markers for sites of clathrin-mediated endocytosis; to the MAGUK, protein disks large (DLG-1 and cadherin (HMR-1, both of which label adherens junctions; to a cytoskeletal linker of the ezrin-radixin-moesin family (ERM-1, which localized to apical membranes; to an ERBIN family protein (LET-413 which localizes to the basolateral membrane of epithelial cells and to an adhesion molecule (SAX-7 which localizes to the plasma membrane at cell-cell contacts. In addition to

  7. Lactogenic immunity in transgenic mice producing recombinant antibodies neutralizing coronavirus.

    Science.gov (United States)

    Castilla, J; Sola, I; Pintado, B; Sánchez-Morgado, J M; Enjuanes, L

    1998-01-01

    Protection against coronavirus infections can be provided by the oral administration of virus neutralizing antibodies. To provide lactogenic immunity, eighteen lines of transgenic mice secreting a recombinant IgG1 monoclonal antibody (rIgG1) and ten lines of transgenic mice secreting recombinant IgA monoclonal antibodies (rIgA) neutralizing transmissible gastroenteritis coronavirus (TGEV) into the milk were generated. Genes encoding the light and heavy chains of monoclonal antibody (MAb) 6A.C3 were expressed under the control of regulatory sequences derived from the mouse genomic DNA encoding the whey acidic protein (WAP) and beta-lactoglobulin (BLG), which are highly abundant milk proteins. The MAb 6A.C3 binds to a highly conserved epitope present in coronaviruses of several species. This MAb does not allow the selection of neutralization escaping virus mutants. The antibody was expressed in the milk of transgenic mice with titers of one million as determined by RIA, and neutralized TGEV infectivity by one million fold corresponding to immunoglobulin concentrations of 5 to 6 mg per ml. Matrix attachment regions (MAR) sequences were not essential for rIgG1 transgene expression, but co-microinjection of MAR and antibody genes led to a twenty to ten thousand-fold increase in the antibody titer in 50% of the rIgG1 transgenic animals generated. Co-microinjection of the genomic BLG gene with rIgA light and heavy chain genes led to the generation of transgenic mice carrying the three transgenes. The highest antibody titers were produced by transgenic mice that had integrated the antibody and BLG genes, although the number of transgenic animals generated does not allow a definitive conclusion on the enhancing effect of BLG co-integration. Antibody expression levels were transgene copy number independent and integration site dependent. The generation of transgenic animals producing virus neutralizing antibodies in the milk could be a general approach to provide protection

  8. Immunochemical Characterization of Anti-Acetylcholinesterase Inhibitory Monoclonal Antibodies

    Science.gov (United States)

    1993-01-01

    formation. This conformation was first proposed using studies with monoclonal antibodies against a synthetic peptide mimicking the sequence of the...distinct antigenic determinants on dengue -2 virus using monoclonal antibodies, Am. J. Trop. Med. Hyg., 31 (1982) 548-555. 7 D. De la Hoz, B.P. Doctor

  9. Monoclonal Antibodies Specific for Hippurate Hydrolase of Campylobacter jejuni

    OpenAIRE

    Steele, Marina; Gyles, Carlton; Chan, Voon Loong; Odumeru, Joseph

    2002-01-01

    Eleven monoclonal antibodies raised against recombinant Campylobacter jejuni hippurate hydrolase were tested for binding to lysates from 19 C. jejuni strains, 12 other Campylobacter strains, and 21 non-Campylobacter strains. Several monoclonal antibodies bound to C. jejuni but not to other Campylobacter species and may be useful in a species-specific immunoassay.

  10. Development of syngeneic monoclonal anti-idiotype antibodies to mouse monoclonal anti-asialoglycoprotein receptor antibody.

    Directory of Open Access Journals (Sweden)

    Hirai M

    2002-06-01

    Full Text Available Anti-idiotype antibodies (Ab2 play an important role in the homeostasis of immune responses and are related to the development and the disease activity of certain autoimmune diseases. The asialoglycoprotein receptor (ASGPR is considered one of the target antigens in the pathogenesis of autoimmune chronic active hepatitis (AIH. We previously developed a mouse monoclonal antibody (clone 8D7 which recognizes rat and human ASGPR. In this study, to help investigate the anti-ASGPR antibody-anti-idiotype antibody network in patients with AIH, we developed a syngeneic mouse monoclonal Ab2 to the 8D7 anti-ASGPR antibody (Ab1. One clone, designated as 3C8, tested positive for specific reactivity to 8D7-Ab1 and did not bind to other irrelevant immunoglobulins. By competitive inhibition assays, the binding of 8D7-Ab1 to liver membrane extracts, i.e., the crude antigen preparation, was inhibited by 3C8-Ab2 in a dose-dependent manner, and the binding of 8D7-Ab1 to 3C8-Ab2 was inhibited by the liver membrane extracts. In the immunohistochemical analysis, 3C8-Ab2 blocked the specific staining of sinusoidal margins of rat hepatocytes by 8D7-Ab1. These results suggest that 3C8 anti-idiotype antibody recognizes the specific idiotypic determinants within the antigen-binding site of 8D7-Ab1.

  11. Monoclonal antibodies based on hybridoma technology.

    Science.gov (United States)

    Yagami, Hisanori; Kato, Hiroshi; Tsumoto, Kanta; Tomita, Masahiro

    2013-03-01

    Based on the size and scope of the present global market for medicine, monoclonal antibodies (mAbs) have a very promising future, with applications for cancers through autoimmune ailments to infectious disease. Since mAbs recognize only their target antigens and not other unrelated proteins, pinpoint medical treatment is possible. Global demand is dramatically expanding. Hybridoma technology, which allows production of mAbs directed against antigens of interest is therefore privileged. However, there are some pivotal points for further development to generate therapeutic antibodies. One is selective generation of human mAbs. Employment of transgenic mice producing human antibodies would overcome this problem. Another focus is recognition sites and conformational epitopes in antigens may be just as important as linear epitopes, especially when membrane proteins such as receptors are targeted. Recognition of intact structures is of critical importance for medical purposes. In this review, we describe patent related information for therapeutic mAbs based on hybridoma technology and also discuss new advances in hybridoma technology that facilitate selective production of stereospecific mAbs.

  12. Arenavirus Glycan Shield Promotes Neutralizing Antibody Evasion and Protracted Infection.

    Directory of Open Access Journals (Sweden)

    Rami Sommerstein

    2015-11-01

    Full Text Available Arenaviruses such as Lassa virus (LASV can cause severe hemorrhagic fever in humans. As a major impediment to vaccine development, delayed and weak neutralizing antibody (nAb responses represent a unifying characteristic of both natural infection and all vaccine candidates tested to date. To investigate the mechanisms underlying arenavirus nAb evasion we engineered several arenavirus envelope-chimeric viruses and glycan-deficient variants thereof. We performed neutralization tests with sera from experimentally infected mice and from LASV-convalescent human patients. NAb response kinetics in mice correlated inversely with the N-linked glycan density in the arenavirus envelope protein's globular head. Additionally and most intriguingly, infection with fully glycosylated viruses elicited antibodies, which neutralized predominantly their glycan-deficient variants, both in mice and humans. Binding studies with monoclonal antibodies indicated that envelope glycans reduced nAb on-rate, occupancy and thereby counteracted virus neutralization. In infected mice, the envelope glycan shield promoted protracted viral infection by preventing its timely elimination by the ensuing antibody response. Thus, arenavirus envelope glycosylation impairs the protective efficacy rather than the induction of nAbs, and thereby prevents efficient antibody-mediated virus control. This immune evasion mechanism imposes limitations on antibody-based vaccination and convalescent serum therapy.

  13. Arenavirus Glycan Shield Promotes Neutralizing Antibody Evasion and Protracted Infection.

    Science.gov (United States)

    Sommerstein, Rami; Flatz, Lukas; Remy, Melissa M; Malinge, Pauline; Magistrelli, Giovanni; Fischer, Nicolas; Sahin, Mehmet; Bergthaler, Andreas; Igonet, Sebastien; Ter Meulen, Jan; Rigo, Dorothée; Meda, Paolo; Rabah, Nadia; Coutard, Bruno; Bowden, Thomas A; Lambert, Paul-Henri; Siegrist, Claire-Anne; Pinschewer, Daniel D

    2015-11-01

    Arenaviruses such as Lassa virus (LASV) can cause severe hemorrhagic fever in humans. As a major impediment to vaccine development, delayed and weak neutralizing antibody (nAb) responses represent a unifying characteristic of both natural infection and all vaccine candidates tested to date. To investigate the mechanisms underlying arenavirus nAb evasion we engineered several arenavirus envelope-chimeric viruses and glycan-deficient variants thereof. We performed neutralization tests with sera from experimentally infected mice and from LASV-convalescent human patients. NAb response kinetics in mice correlated inversely with the N-linked glycan density in the arenavirus envelope protein's globular head. Additionally and most intriguingly, infection with fully glycosylated viruses elicited antibodies, which neutralized predominantly their glycan-deficient variants, both in mice and humans. Binding studies with monoclonal antibodies indicated that envelope glycans reduced nAb on-rate, occupancy and thereby counteracted virus neutralization. In infected mice, the envelope glycan shield promoted protracted viral infection by preventing its timely elimination by the ensuing antibody response. Thus, arenavirus envelope glycosylation impairs the protective efficacy rather than the induction of nAbs, and thereby prevents efficient antibody-mediated virus control. This immune evasion mechanism imposes limitations on antibody-based vaccination and convalescent serum therapy.

  14. A Strategy for Screening Monoclonal Antibodies for Arabidopsis Flowers

    Science.gov (United States)

    Shi, Qian; Zhou, Lian; Wang, Yingxiang; Ma, Hong

    2017-01-01

    The flower is one of the most complex structures of angiosperms and is essential for sexual reproduction. Current studies using molecular genetic tools have made great advances in understanding flower development. Due to the lack of available antibodies, studies investigating the localization of proteins required for flower development have been restricted to use commercial antibodies against known antigens such as GFP, YFP, and FLAG. Thus, knowledge about cellular structures in the floral organs is limited due to the scarcity of antibodies that can label cellular components. To generate monoclonal antibodies that can facilitate molecular studies of the flower, we constructed a library of monoclonal antibodies against antigenic proteins from Arabidopsis inflorescences and identified 61 monoclonal antibodies. Twenty-four of these monoclonal antibodies displayed a unique band in a western blot assay in at least one of the examined tissues. Distinct cellular distribution patterns of epitopes were detected by these 24 antibodies by immunofluorescence microscopy in a flower section. Subsequently, a combination of immunoprecipitation and mass spectrometry analysis identified potential targets for three of these antibodies. These results provide evidence for the generation of an antibody library using the total plant proteins as antigens. Using this method, the present study identified 61 monoclonal antibodies and 24 of them were efficiently detecting epitopes in both western blot experiments and immunofluorescence microscopy. These antibodies can be applied as informative cellular markers to study the biological mechanisms underlying floral development in plants. PMID:28293248

  15. 登革病毒EDⅢ特异性单抗的体内外中和活性观察%A murine monoclone antibody against envelope protein domain Ⅲ neutralizes dengue virus four serotypes in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    黄艳芬; 邓永强; 丘立文; 刘利东; 秦鄂德; 车小燕; 秦成峰

    2013-01-01

    目的 鉴定1株能同时识别登革病毒Ⅰ~Ⅳ型(dengue virus serotypesⅠ-Ⅳ,DENV 1~4)的EDⅢ特异性单抗2D73的体内外中和活性.方法 采用间接酶联免疫吸附实验(ELISA)和间接免疫荧光法(IFA)对单抗2D73的特性进行鉴定,并用基于酶联免疫斑点法的微中和实验(ELISPOT-MNT)和乳鼠保护实验观察单抗2D73对DENV 1~4的体内外中和活性.结果 单抗2D73对4型DENV均具有较强的中和活性,其50%抑制浓度(IC50)分别为0.28、0.16、0.18和18.82 μg/ml.乳鼠保护实验显示,该抗体对DENV 1~4同样具有较好的体内保护效果,与对照组相比,实验组小鼠发病时间明显延迟,生存率显著提高(P<0.05).结论 获得1株同时针对DENV 1~4且具有体内外中和活性的单抗,该单抗可进一步用于DENV致病和免疫机制的研究以及抗病毒药物的研制.%Objective To evaluate the neutralizing activities of a murine monoclonal antibody ( mAb) 2D73 against dengue virus serotypes Ⅰ-Ⅳ( DENV1-4) in vitro and in vivo. Methods Indirect ELISA and immuno fluorescence assay (IFA) were applied to identify specificity of mAb 2D73. Enzyme-linked immunospot micro -neutralizing test (ELISPOT-MNT) was performed to identify its neutralizing activities in vitro, and protective capacity was evaluated in a stringent in -tracranial challenge suckling mice model. Results All the four DENV serotypes were strongly neutralized by mAb 2D73 with 50% inhibitory concentrations (IC50) of 0. 28 , 0. 16 , 0. 18 and 18. 82 μg/ml to DENV1-4 , respectively. The onset of pathogenesis was delayed and the survival rate was improved obviously (P < 0. 05 ) in experimental group compared with control group. Conclusion MAb 2D73 against all the four DENV serotypes has strong neutralizing activities in vitro and protective efficacy in vivo, and might be used for the study of the pathogenic and immunologic mechanism of DENV and for the development of antiviral therapy.

  16. Purification of infectious canine parvovirus from cell culture by affinity chromatography with monoclonal antibodies.

    NARCIS (Netherlands)

    J. Groen (Jan); N. Juntti; J.S. Teppema; F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert); G.F. Rimmelzwaan (Guus)

    1987-01-01

    textabstractImmuno affinity chromatography with virus neutralizing monoclonal antibodies, directed to the haemagglutinating protein of canine parvovirus (CPV) was used to purify and concentrate CPV from infected cell culture. The procedure was monitored by testing the respective fractions in an infe

  17. Treatment with anti-interferon-δ monoclonal antibodies modifies experimental autoimmune encephalomyelitis in interferon-δ receptor knockout mice

    DEFF Research Database (Denmark)

    Espejo, C.; Penkowa, Milena; Saez-Torres, I.

    2001-01-01

    Neuroinflammation, neuronal degeneration, regeneration, monoclonal antibodies, multiple schlerosis......Neuroinflammation, neuronal degeneration, regeneration, monoclonal antibodies, multiple schlerosis...

  18. Drug Development of Therapeutic Monoclonal Antibodies.

    Science.gov (United States)

    Mould, Diane R; Meibohm, Bernd

    2016-08-01

    Monoclonal antibodies (MAbs) have become a substantial part of many pharmaceutical company portfolios. However, the development process of MAbs for clinical use is quite different than for small-molecule drugs. MAb development programs require careful interdisciplinary evaluations to ensure the pharmacology of both the MAb and the target antigen are well-understood. Selection of appropriate preclinical species must be carefully considered and the potential development of anti-drug antibodies (ADA) during these early studies can limit the value and complicate the performance and possible duration of preclinical studies. In human studies, many of the typical pharmacology studies such as renal or hepatic impairment evaluations may not be needed but the pharmacokinetics and pharmacodynamics of these agents is complex, often necessitating more comprehensive evaluation of clinical data and more complex bioanalytical assays than might be used for small molecules. This paper outlines concerns and strategies for development of MAbs from the early in vitro assessments needed through preclinical and clinical development. This review focuses on how to develop, submit, and comply with regulatory requirements for MAb therapeutics.

  19. Neutralizing antibody fails to impact the course of Ebola virus infection in monkeys.

    Directory of Open Access Journals (Sweden)

    Wendelien B Oswald

    2007-01-01

    Full Text Available Prophylaxis with high doses of neutralizing antibody typically offers protection against challenge with viruses producing acute infections. In this study, we have investigated the ability of the neutralizing human monoclonal antibody, KZ52, to protect against Ebola virus in rhesus macaques. This antibody was previously shown to fully protect guinea pigs from infection. Four rhesus macaques were given 50 mg/kg of neutralizing human monoclonal antibody KZ52 intravenously 1 d before challenge with 1,000 plaque-forming units of Ebola virus, followed by a second dose of 50 mg/kg antibody 4 d after challenge. A control animal was exposed to virus in the absence of antibody treatment. Passive transfer of the neutralizing human monoclonal antibody not only failed to protect macaques against challenge with Ebola virus but also had a minimal effect on the explosive viral replication following infection. We show that the inability of antibody to impact infection was not due to neutralization escape. It appears that Ebola virus has a mechanism of infection propagation in vivo in macaques that is uniquely insensitive even to high concentrations of neutralizing antibody.

  20. Monoclonal antibodies against naturally occurring bioactive compounds.

    Science.gov (United States)

    Shoyama, Y; Tanaka, H; Fukuda, N

    1999-09-01

    The ratio of hapten to bovine serum albumin (BSA) in an antigen conjugate was determined by matrix-assisted laser desorption/ionization (MALDI) tof mass spectrometry. A hybridoma secreting monoclonal antibody (MAb) was produced by fusing splenocytes immunized with an antigen-BSA conjugate with HAT-sensitive mouse myeloma cells. The cross-reaction of anti-forskolin antibodies with 7-deacetyl forskolin was 5.6%. A very small cross-reaction appeared with other derivatives. The full measuring range of the assay extends from 5 ng to 5 mug/ml of forskolin. Immunoaffinity column chromatography using anti-forskolin MAbs appears to be far superior to previously published separation methods. The capacity of the immunoaffinity column as determined by ELISA is 9 mug/ml. Forskolin has been isolated directly from the crude extracts of tuberous roots and the callus culture of Coleus forskohlii. A MAb against tetrahydrocannabinolic acid (THCA) was produced. The cross-reaction of anti-THCA antibody against other cannabinoids was very wide. Many cannabinoids and a spiro-compound were reactive, but did not react with other phenolics. It became evident that this ELISA was able to be applied to the biotransformation experiments of cannabinoids in plant tissue culture system. Anti-ginsenoside Rb1 MAbs were produced. New western blotting method of determination for ginsenosides was established. Ginsenosides separated by silica gel TLC were transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was treated with NaIO(4) solution followed by BSA, resulting in a ginsenoside-BSA conjugate. Immunostaining of ginsenosides was more sensitive compared to other staining. Immunostaining of ginsenosides in the fresh ginseng root was succeeded using anti-ginsenoside Rb1 (GRb1) MAb after blotting to PVDF membrane.

  1. Sub-Nanogram Detection of RDX Explosive by Monoclonal Antibodies.

    Science.gov (United States)

    Ulaeto, David O; Hutchinson, Alistair P; Nicklin, Stephen

    2015-08-01

    Polyclonal and monoclonal antibodies were raised to protein carrier molecules haptenized with RDX, a major component of many plastic explosives including Semtex. Sera from immunized mice detected RDX protein conjugates in standard ELISA. Clonally purified monoclonal antibodies had detection limits in the sub-ng/mL range for underivatized RDX in competition ELISA. The monoclonal antibodies are not dependent on the presence of taggants added during the manufacturing process, and are likely to have utility in the detection of any explosive containing RDX, or RDX contamination of environmental sites.

  2. Monoclonal antibodies and Fc fragments for treating solid tumors

    Directory of Open Access Journals (Sweden)

    Eisenbeis AM

    2012-01-01

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

  3. Antibodies in infectious diseases: polyclonals, monoclonals and niche biotechnology.

    Science.gov (United States)

    Berry, Jody D; Gaudet, Ryan G

    2011-09-01

    Antibody preparations have a long history of providing protection from infectious diseases. Although antibodies remain the only natural host-derived defense mechanism capable of completely preventing infection, as products, they compete against inexpensive therapeutics such as antibiotics, small molecule inhibitors and active vaccines. The continued discovery in the monoclonal antibody (mAb) field of leads with broadened cross neutralization of viruses and demonstrable synergy of antibody with antibiotics for bacterial diseases, clearly show that innovation remains. The commercial success of mAbs in chronic disease has not been paralleled in infectious diseases for several reasons. Infectious disease immunotherapeutics are limited in scope as endemic diseases necessitate active vaccine development. Also, the complexity of these small markets draws the interest of niche companies rather than big pharmaceutical corporations. Lastly, the cost of goods for mAb therapeutics is inherently high for infectious agents due to the need for antibody cocktails, which better mimic polyclonal immunoglobulin preparations and prevent antigenic escape. In cases where vaccine or convalescent populations are available, current polyclonal hyperimmune immunoglobulin preparations (pIgG), with modern and highly efficient purification technology and standardized assays for potency, can make economic sense. Recent innovations to broaden the potency of mAb therapies, while reducing cost of production, are discussed herein. On the basis of centuries of effective use of Ab treatments, and with growing immunocompromised populations, the question is not whether antibodies have a bright future for infectious agents, but rather what formats are cost effective and generate safe and efficacious treatments to satisfy regulatory approval. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. Production and characterization of monoclonal antibodies against mink leukocytes

    DEFF Research Database (Denmark)

    Chen, W.S.; Pedersen, Mikael; Gram-Nielsen, S.

    1997-01-01

    Three monoclonal antibodies (mAbs) were generated against mink leukocytes. One antibody reacted with all T lymphocytes, one with all monocytes and one had platelet reactivity. Under reducing conditions, the T lymphocyte reactive antibody immunoprecipitated 18 kDa, 23 kDa, 25 kDa and 32-40 kDa pol...

  5. Syngeneic anti-idiotypic monoclonal antibodies to an anti-NeuGc-containing ganglioside monoclonal antibody.

    Science.gov (United States)

    Vázquez, A M; Pérez, A; Hernández, A M; Macías, A; Alfonso, M; Bombino, G; Pérez, R

    1998-12-01

    An IgM monoclonal antibody (MAb), named P3, has the characteristic to react specifically with a broad battery of N-glycolyl containing-gangliosides and with antigens expressed on breast tumors. When this MAb was administered alone in syngeneic mice, an specific IgG anti-idiotypic antibody (Ab2) response was induced, this Ab2 response was increased when P3 MAb was injected coupled to a carrier protein and in the presence of Freund's adjuvant. Spleen cells from these mice were used in somatic-cell hybridization experiments, using the murine myeloma cell line P3-X63-Ag8.653 as fusion partner. Five Ab2 MAbs specific to P3 MAb were selected. These IgG1 Ab2 MAbs were able to block the binding of P3 MAb to GM3(NeuGc) ganglioside and to a human breast carcinoma cell line. Cross-blocking experiments demonstrated that these Ab2 MAbs are recognizing the same or very close sites on the Abl MAb. The five Ab2 MAbs were injected into syngeneic mice and four of them produced strong anti-anti-idiotypic antibody (Ab3) response. While these Ab2 MAbs were unable to generate Ab3 antibodies with the same antigenic specificity than P3 MAb, three of them induced antibodies bearing P3 MAb idiotopes (Ag-Id+ Ab3). These results demonstrated that these Ab2 MAbs are not "internal image" antibodies, but they could define "regulatory idiotopes."

  6. A Therapeutic Antibody against West Nile Virus Neutralizes Infection by Blocking Fusion within Endosomes

    NARCIS (Netherlands)

    Thompson, Bruce S.; Moesker, Bastiaan; Smit, Jolanda M.; Wilschut, Jan; Diamond, Michael S.; Fremont, Daved H.

    2009-01-01

    Defining the precise cellular mechanisms of neutralization by potently inhibitory antibodies is important for understanding how the immune system successfully limits viral infections. We recently described a potently inhibitory monoclonal antibody (MAb E16) against the envelope (E) protein of West N

  7. A Therapeutic Antibody against West Nile Virus Neutralizes Infection by Blocking Fusion within Endosomes

    NARCIS (Netherlands)

    Thompson, Bruce S.; Moesker, Bastiaan; Smit, Jolanda M.; Wilschut, Jan; Diamond, Michael S.; Fremont, Daved H.

    Defining the precise cellular mechanisms of neutralization by potently inhibitory antibodies is important for understanding how the immune system successfully limits viral infections. We recently described a potently inhibitory monoclonal antibody (MAb E16) against the envelope (E) protein of West

  8. A monoclonal antibody against human MUDENG protein.

    Science.gov (United States)

    Wagley, Yadav; Choi, Jun-Ha; Wickramanayake, Dimuthu Dhammika; Choi, Geun-Yeol; Kim, Chang-Kyu; Kim, Tae-Hyoung; Oh, Jae-Wook

    2013-08-01

    MUDENG (mu-2-related death-inducing gene, MuD) encodes a predicted ∼54-kDa protein in humans, considered to be involved in trafficking proteins from endosomes toward other membranous compartments as well as in inducing cell death. Here we report on the generation of a mouse monoclonal antibody (MAb) against the middle domain of human (h) MuD. This IgG sub 1 MAb, named M3H9, recognizes residues 244-326 in the middle domain of the MuD protein. Thus, the MuD proteins expressed in an astroglioma cell line and primary astrocytes can be detected by the M3H9 MAb. We showed that M3H9 MAb can be useful in enzyme-linked immunosorbent assay (ELISA) and immunoblot experiments. In addition, M3H9 MAb can detect the expression of the MuD protein in formalin-fixed, paraffin-embedded mouse ovary and uterus tissues. These results indicate that the MuD MAb M3H9 could be useful as a new biomarker of hereditary spastic paraplegia and other related diseases.

  9. Neutralizing antibody blocks adenovirus infection by arresting microtubule-dependent cytoplasmic transport.

    Science.gov (United States)

    Smith, Jason G; Cassany, Aurelia; Gerace, Larry; Ralston, Robert; Nemerow, Glen R

    2008-07-01

    Neutralizing antibodies are commonly elicited by viral infection. Most antibodies that have been characterized block early stages of virus entry that occur before membrane penetration, whereas inhibition of late stages in entry that occurs after membrane penetration has been poorly characterized. Here we provide evidence that the neutralizing antihexon monoclonal antibody 9C12 inhibits adenovirus infection by blocking microtubule-dependent translocation of the virus to the microtubule-organizing center following endosome penetration. These studies identify a previously undescribed mechanism by which neutralizing antibodies block virus infection, a situation that may be relevant for other nonenveloped viruses that use microtubule-dependent transport during cell entry.

  10. Neutralizing Antibody Blocks Adenovirus Infection by Arresting Microtubule-Dependent Cytoplasmic Transport▿

    Science.gov (United States)

    Smith, Jason G.; Cassany, Aurelia; Gerace, Larry; Ralston, Robert; Nemerow, Glen R.

    2008-01-01

    Neutralizing antibodies are commonly elicited by viral infection. Most antibodies that have been characterized block early stages of virus entry that occur before membrane penetration, whereas inhibition of late stages in entry that occurs after membrane penetration has been poorly characterized. Here we provide evidence that the neutralizing antihexon monoclonal antibody 9C12 inhibits adenovirus infection by blocking microtubule-dependent translocation of the virus to the microtubule-organizing center following endosome penetration. These studies identify a previously undescribed mechanism by which neutralizing antibodies block virus infection, a situation that may be relevant for other nonenveloped viruses that use microtubule-dependent transport during cell entry. PMID:18448546

  11. Dengue Virus Envelope Dimer Epitope Monoclonal Antibodies Isolated from Dengue Patients Are Protective against Zika Virus

    Directory of Open Access Journals (Sweden)

    J. A. Swanstrom

    2016-07-01

    Full Text Available Zika virus (ZIKV is a mosquito-borne flavivirus responsible for thousands of cases of severe fetal malformations and neurological disease since its introduction to Brazil in 2013. Antibodies to flaviviruses can be protective, resulting in lifelong immunity to reinfection by homologous virus. However, cross-reactive antibodies can complicate flavivirus diagnostics and promote more severe disease, as noted after serial dengue virus (DENV infections. The endemic circulation of DENV in South America and elsewhere raises concerns that preexisting flavivirus immunity may modulate ZIKV disease and transmission potential. Here, we report on the ability of human monoclonal antibodies and immune sera derived from dengue patients to neutralize contemporary epidemic ZIKV strains. We demonstrate that a class of human monoclonal antibodies isolated from DENV patients neutralizes ZIKV in cell culture and is protective in a lethal murine model. We also tested a large panel of convalescent-phase immune sera from humans exposed to primary and repeat DENV infection. Although ZIKV is most closely related to DENV compared to other human-pathogenic flaviviruses, most DENV immune sera (73% failed to neutralize ZIKV, while others had low (50% effective concentration [EC50], 1:100 serum dilution; 9% levels of cross-neutralizing antibodies. Our results establish that ZIKV and DENV share epitopes that are targeted by neutralizing, protective human antibodies. The availability of potently neutralizing human monoclonal antibodies provides an immunotherapeutic approach to control life-threatening ZIKV infection and also points to the possibility of repurposing DENV vaccines to induce cross-protective immunity to ZIKV.

  12. Generation and characterization of monoclonal antibodies specific to Coenzyme A

    Directory of Open Access Journals (Sweden)

    Malanchuk O. M.

    2015-06-01

    Full Text Available Aim. Generation of monoclonal antibodies specific to Coenzyme A. Methods. Hybridoma technique. KLH carrier protein conjugated with CoA was used for immunization. Screening of positive clones was performed with BSA conjugated to CoA. Results. Monoclonal antibody that specifically recognizes CoA and CoA derivatives, but not its precursors ATP and cysteine has been generated. Conclusion. In this study, we describe for the first time the production and characterization of monoclonal antibodies against CoA. The monoclonal antibody 1F10 was shown to recognize specifically CoA in Western blotting, ELISA and immunoprecipitation. These properties make this antiboby a particularly valuable reagent for elucidating CoA function in health and disease.

  13. Sensitive neutralization test for rubella antibody.

    Science.gov (United States)

    Sato, H; Albrecht, P; Krugman, S; Ennis, F A

    1979-01-01

    A modified rubella virus plaque neutralization test for measuring rubella antibody was developed based on the potentiation of the virus-antibody complex by heterologous anti-immunoglobulin. The test is highly sensitive, yielding titers on the average 50 to 100 times higher than the haemagglutination inhibition test or the conventional plaque neutralization test. The sensitivity of this enhanced neutralization test is somewhat limited by the existence of a prozone phenomenon which precludes testing of low-titered sera below a dilution of 1:16. No prozone effect was observed with cerebrospinal fluids. The specificity of the enhanced neutralization test was determined by seroconversion of individuals receiving rubella vaccine. Although the rubella hemagglutination inhibition test remains the test of choice in routine diagnostic and surveillance work, the enhanced rubella neutralization test is particularly useful in monitoring low-level antibody in the cerebrospinal fluid in patients with neurological disorders and in certain instances of vaccine failure. PMID:107192

  14. Purification of Murine Monoclonal IgM Antibody

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    This paper presents the purification of a monoclonal IgM antibody against human tumor associated antigen Lewis-Y by ion exchange chromatography and gel filtration.Enzyme-linked immunosorbent assay (ELISA) and SDS-polyacrylamide gel electrophoresis (PAGE) were used to identify purified IgM antibody.In flow cytometry analysis, the purified IgM antibody recognizes human breast tumor cell line MCF-7 which expresses Lewis-Y antigen.This work presents a new way for the purification of murine monoclonal IgM antibody.

  15. Neutralizing antibodies in hepatitis C virus infection

    Institute of Scientific and Technical Information of China (English)

    Mirjam B Zeisel; Samira Fafi-Kremer; Isabel Fofana; Heidi Barth; Fran(c)oise Stoll-Keller; Michel Doffo(e)l; Thomas F Baumert

    2007-01-01

    Hepatitis C virus (HCV) is a major cause of hepatitis world-wide. The majority of infected individuals develop chronic hepatitis which can then progress to liver cirrhosis and hepatocellular carcinoma. Spontaneous viral clearance occurs in about 20%-30% of acutely infected individuals and results in resolution of infection without sequaelae. Both viral and host factors appear to play an important role for resolution of acute infection. A large body of evidence suggests that a strong, multispecific and long-lasting cellular immune response appears to be important for control of viral infection in acute hepatitis C. Due too the lack of convenient neutralization assays,the impact of neutralizing responses for control of viral infection had been less defined. In recent years, the development of robust tissue culture model systems for HCV entry and infection has finally allowed study of antibody-mediated neutralization and to gain further insights into viral targets of host neutralizing responses.In addition, detailed analysis of antibody-mediated neutralization in individual patients as well as cohorts with well defined viral isolates has enabled the study of neutralizing responses in the course of HCV infection and characterization of the impact of neutralizing antibodies for control of viral infection. This review will summarize recent progress in the understanding of the molecular mechanisms of antibody-mediated neutralization and its impact for HCV pathogenesis.(C) 2007 The WJG Press. All rights reserved.

  16. ELISA Detection of Francisella tularensis using Polyclonaland Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Miroslav Pohanka

    2008-09-01

    Full Text Available The mouse monoclonal and polyclonal antibodies were produced for the detection of intracellular pathogenand potential warfare agent Francisella tularensis. Antibody titers obtained were 1:640 for polyclonal antibodiesand 1:320 for monoclonal antibodies. Both antibodies were used in the indirect enzyme-linked immunosorbentassay (ELISA found to detect F. tularensis whole cells. The limit of detection was 5.4×106 CFU/ml for polyclonalantibodies and 6.9×106 CFU/ml for monoclonal antibodies. The value sample could  be distinguished from anyconcentration of another gram-negative bacterium: Escherichia coli.Defence Science Journal, 2008, 58(5, pp.698-702, DOI:http://dx.doi.org/10.14429/dsj.58.1693

  17. Molecular characterization of the variable heavy and light chain regions of five HIV-1 specific human monoclonal antibodies.

    NARCIS (Netherlands)

    E.M.M. van der Donk; M. Schutten (Martin); A.D.M.E. Osterhaus (Albert); R.W.J. van der Heijden (Roger)

    1994-01-01

    textabstractWe have reported the generation and characterization of four HIV-1 neutralizing human monoclonal antibodies. Three antibodies recognize a conformational epitope within the CD4-binding site of HIV-1 gp120 and one recognizes a linear epitope located within the hypervariable V3 domain of gp

  18. Maturation Pathways of Cross-Reactive HIV-1 Neutralizing Antibodies

    Directory of Open Access Journals (Sweden)

    Dimiter S. Dimitrov

    2009-11-01

    Full Text Available Several human monoclonal antibodies (hmAbs and antibody fragments, including the best characterized in terms of structure-function b12 and Fab X5, exhibit relatively potent and broad HIV-1 neutralizing activity. However, the elicitation of b12 or b12-like antibodies in vivo by vaccine immunogens based on the HIV-1 envelope glycoprotein (Env has not been successful. B12 is highly divergent from the closest corresponding germline antibody while X5 is less divergent. We have hypothesized that the relatively high degree of specific somatic hypermutations may preclude binding of the HIV-1 envelope glycoprotein (Env to closest germline antibodies, and that identifying antibodies that are intermediates in the pathways to maturation could help design novel vaccine immunogens to guide the immune system for their enhanced elicitation. In support of this hypothesis we have previously found that a germline-like b12 (monovalent and bivalent scFv as an Fc fusion protein or IgG lacks measurable binding to an Env as measured by ELISA with a sensitivity in the μM range [1]; here we present evidence confirming and expanding these findings for a panel of Envs. In contrast, a germline-like scFv X5 bound Env with high (nM affinity. To begin to explore the maturation pathways of these antibodies we identified several possible b12 intermediate antibodies and tested their neutralizing activity. These intermediate antibodies neutralized only some HIV-1 isolates and with relatively weak potency. In contrast, germline-like scFv X5 neutralized a subset of the tested HIV-1 isolates with comparable efficiencies to that of the mature X5. These results could help explain the relatively high immunogenicity of the coreceptor binding site on gp120 and the abundance of CD4-induced (CD4i antibodies in HIV-1-infected patients (X5 is a CD4i antibody as well as the maturation pathway of X5. They also can help identify antigens that can bind specifically to b12 germline and

  19. Apolipoprotein E Mediates Evasion From Hepatitis C Virus Neutralizing Antibodies.

    Science.gov (United States)

    Fauvelle, Catherine; Felmlee, Daniel J; Crouchet, Emilie; Lee, JiYoung; Heydmann, Laura; Lefèvre, Mathieu; Magri, Andrea; Hiet, Marie-Sophie; Fofana, Isabel; Habersetzer, François; Foung, Steven K H; Milne, Ross; Patel, Arvind H; Vercauteren, Koen; Meuleman, Philip; Zeisel, Mirjam B; Bartenschlager, Ralf; Schuster, Catherine; Baumert, Thomas F

    2016-01-01

    Efforts to develop an effective vaccine against hepatitis C virus (HCV) have been hindered by the propensity of the virus to evade host immune responses. HCV particles in serum and in cell culture associate with lipoproteins, which contribute to viral entry. Lipoprotein association has also been proposed to mediate viral evasion of the humoral immune response, though the mechanisms are poorly defined. We used small interfering RNAs to reduce levels of apolipoprotein E (apoE) in cell culture-derived HCV-producing Huh7.5-derived hepatoma cells and confirmed its depletion by immunoblot analyses of purified viral particles. Before infection of naïve hepatoma cells, we exposed cell culture-derived HCV strains of different genotypes, subtypes, and variants to serum and polyclonal and monoclonal antibodies isolated from patients with chronic HCV infection. We analyzed the interaction of apoE with viral envelope glycoprotein E2 and HCV virions by immunoprecipitation. Through loss-of-function studies on patient-derived HCV variants of several genotypes and subtypes, we found that the HCV particle apoE allows the virus to avoid neutralization by patient-derived antibodies. Functional studies with human monoclonal antiviral antibodies showed that conformational epitopes of envelope glycoprotein E2 domains B and C were exposed after depletion of apoE. The level and conformation of virion-associated apoE affected the ability of the virus to escape neutralization by antibodies. In cell-infection studies, we found that HCV-associated apoE helps the virus avoid neutralization by antibodies against HCV isolated from chronically infected patients. This method of immune evasion poses a challenge for the development of HCV vaccines. Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.

  20. Broadly Neutralizing Anti-Influenza Virus Antibodies: Enhancement of Neutralizing Potency in Polyclonal Mixtures and IgA Backbones

    Science.gov (United States)

    He, Wenqian; Mullarkey, Caitlin E.; Duty, J. Andrew; Moran, Thomas M.; Palese, Peter

    2015-01-01

    ABSTRACT Current influenza virus vaccines rely upon the accurate prediction of circulating virus strains months in advance of the actual influenza season in order to allow time for vaccine manufacture. Unfortunately, mismatches occur frequently, and even when perfect matches are achieved, suboptimal vaccine efficacy leaves several high-risk populations vulnerable to infection. However, the recent discovery of broadly neutralizing antibodies that target the hemagglutinin (HA) stalk domain has renewed hope that the development of “universal” influenza virus vaccines may be within reach. Here, we examine the functions of influenza A virus hemagglutinin stalk-binding antibodies in an endogenous setting, i.e., as polyclonal preparations isolated from human sera. Relative to monoclonal antibodies that bind to the HA head domain, the neutralization potency of monoclonal stalk-binding antibodies was vastly inferior in vitro but was enhanced by several orders of magnitude in the polyclonal context. Furthermore, we demonstrated a surprising enhancement in IgA-mediated HA stalk neutralization relative to that achieved by antibodies of IgG isotypes. Mechanistically, this could be explained in two ways. Identical variable regions consistently neutralized virus more potently when in an IgA backbone compared to an IgG backbone. In addition, HA-specific memory B cells isolated from human peripheral blood were more likely to be stalk specific when secreting antibodies of IgA isotypes compared to those secreting IgG. Taken together, our data provide strong evidence that HA stalk-binding antibodies perform optimally when in a polyclonal context and that the targeted elicitation of HA stalk-specific IgA should be an important consideration during “universal” influenza virus vaccine design. IMPORTANCE Influenza viruses remain one of the most worrisome global public health threats due to their capacity to cause pandemics. While seasonal vaccines fail to protect against the

  1. Development of monoclonal antibodies that recognize Treponema pallidum.

    OpenAIRE

    Saunders, J M; Folds, J D

    1983-01-01

    We developed a panel of monoclonal antibodies to Treponema pallidum (Nichols) antigens, some of which recognize treponemal antigens on T. pallidum (Nichols), T. pallidum strain 14, and Treponema phagedenis biotype Reiter. The antibodies were detected by either an enzyme-linked immunosorbent assay or a radioimmunoassay.

  2. Monoclonal antibodies for the control of influenza virus vaccines.

    NARCIS (Netherlands)

    H.J.M. van de Donk; M.F. van Olderen; A.D.M.E. Osterhaus (Albert); J.C. de Jong (Jan)

    1984-01-01

    textabstractHybridomas producing haemagglutination inhibiting monoclonal antibodies against influenza A/Texas/1/77 H3N2 were developed. One hybridoma producing antibodies reacting with Victoria/3/75, Texas/1/77 Bangkok/1/79 and England/496/80 was selected to determine the potency of influenza virusv

  3. MONOCLONAL ANTIBODIES TO IDENTIFY TOMATO MOSAIC TOBAMOVIRUS (TOMV

    Directory of Open Access Journals (Sweden)

    Duarte Keila M.R.

    2001-01-01

    Full Text Available Monoclonal antibodies were obtained against Tomato mosaic tobamovirus (ToMV isolated in Brazil. One antibody (8G7G2 isotyped as IgG2b (kappa light chain showed strong specificity and very low cross reaction with the Tobacco mosaic virus (TMV. It can be used in identification of tomato mosaic virus (ToMV.

  4. Serological comparison of tospovirus isolates using polyclonal and monoclonal antibodies.

    NARCIS (Netherlands)

    Adam, G.; Peters, D.; Goldbach, R.W.

    1996-01-01

    A test was conducted to compare tospovirus isolates using different poly- and monoclonal antibodies. All isolates and antibodies were compared under identical conditions. From 130 tospovirus isolates, which were obtained from all over the world and included well-characterized isolates from all four

  5. Serological comparison of tospovirus isolates using polyclonal and monoclonal antibodies.

    NARCIS (Netherlands)

    Adam, G.; Peters, D.; Goldbach, R.W.

    1996-01-01

    A test was conducted to compare tospovirus isolates using different poly- and monoclonal antibodies. All isolates and antibodies were compared under identical conditions. From 130 tospovirus isolates, which were obtained from all over the world and included well-characterized isolates from all four

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

    DEFF Research Database (Denmark)

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

    2005-01-01

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

  7. Immunoblotting with monoclonal antibodies: importance of the blocking solution.

    Science.gov (United States)

    Hauri, H P; Bucher, K

    1986-12-01

    Four commonly used blocking agents, i.e., fetal calf serum, mammalian gelatin-Nonidet-P40, fish gelatin-Nonidet-P40, and defatted powdered milk were compared with respect to their efficiency to block the nonspecific background and to promote maximal immunoreactivity of monoclonal antibodies against human intestinal sucrase-isomaltase during immunoblotting. Two of five monoclonal antibodies were found to react with the electroblotted enzyme. However, one of the reacting antibodies gave optimal results with fish gelatin-Nonidet-P40 and the other with defatted powdered milk, while fetal calf serum lead to unacceptably high backgrounds. The results suggest that some of the difficulties encountered with monoclonal antibodies in immunoblotting may be due to inappropriate blocking conditions.

  8. In vivo emergence of HIV-1 highly sensitive to neutralizing antibodies.

    Directory of Open Access Journals (Sweden)

    Marlén M I Aasa-Chapman

    Full Text Available BACKGROUND: The rapid and continual viral escape from neutralizing antibodies is well documented in HIV-1 infection. Here we report in vivo emergence of viruses with heightened sensitivity to neutralizing antibodies, sometimes paralleling the development of neutralization escape. METHODOLOGY/PRINCIPAL FINDINGS: Sequential viral envs were amplified from seven HIV-1 infected men monitored from seroconversion up to 5 years after infection. Env-recombinant infectious molecular clones were generated and tested for coreceptor use, macrophage tropism and neutralization sensitivity to homologous and heterologous serum, soluble CD4 and monoclonal antibodies IgG1b12, 2G12 and 17b. We found that HIV-1 evolves sensitivity to contemporaneous neutralizing antibodies during infection. Neutralization sensitive viruses grow out even when potent autologous neutralizing antibodies are present in patient serum. Increased sensitivity to neutralization was associated with susceptibility of the CD4 binding site or epitopes induced after CD4 binding, and mediated by complex envelope determinants including V3 and V4 residues. The development of neutralization sensitive viruses occurred without clinical progression, coreceptor switch or change in tropism for primary macrophages. CONCLUSIONS: We propose that an interplay of selective forces for greater virus replication efficiency without the need to resist neutralizing antibodies in a compartment protected from immune surveillance may explain the temporal course described here for the in vivo emergence of HIV-1 isolates with high sensitivity to neutralizing antibodies.

  9. Broadly-Reactive Neutralizing and Non-neutralizing Antibodies Directed against the H7 Influenza Virus Hemagglutinin Reveal Divergent Mechanisms of Protection.

    Science.gov (United States)

    Tan, Gene S; Leon, Paul E; Albrecht, Randy A; Margine, Irina; Hirsh, Ariana; Bahl, Justin; Krammer, Florian

    2016-04-01

    In the early spring of 2013, Chinese health authorities reported several cases of H7N9 influenza virus infections in humans. Since then the virus has established itself at the human-animal interface in Eastern China and continues to cause several hundred infections annually. In order to characterize the antibody response to the H7N9 virus we generated several mouse monoclonal antibodies against the hemagglutinin of the A/Shanghai/1/13 (H7N9) virus. Of particular note are two monoclonal antibodies, 1B2 and 1H5, that show broad reactivity to divergent H7 hemagglutinins. Monoclonal antibody 1B2 binds to viruses of the Eurasian and North American H7 lineages and monoclonal antibody 1H5 reacts broadly to virus isolates of the Eurasian lineage. Interestingly, 1B2 shows broad hemagglutination inhibiting and neutralizing activity, while 1H5 fails to inhibit hemagglutination and demonstrates no neutralizing activity in vitro. However, both monoclonal antibodies were highly protective in an in vivo passive transfer challenge model in mice, even at low doses. Experiments using mutant antibodies that lack the ability for Fc/Fc-receptor and Fc/complement interactions suggest that the protection provided by mAb 1H5 is, at least in part, mediated by the Fc-fragment of the mAb. These findings highlight that a protective response to a pathogen may not only be due to neutralizing antibodies, but can also be the result of highly efficacious non-neutralizing antibodies not readily detected by classical in vitro neutralization or hemagglutination inhibition assays. This is of interest because H7 influenza virus vaccines induce only low hemagglutination inhibiting antibody titers while eliciting robust antibody titers as measured by ELISA. Our data suggest that these binding but non-neutralizing antibodies contribute to protection in vivo.

  10. Broadly-Reactive Neutralizing and Non-neutralizing Antibodies Directed against the H7 Influenza Virus Hemagglutinin Reveal Divergent Mechanisms of Protection.

    Directory of Open Access Journals (Sweden)

    Gene S Tan

    2016-04-01

    Full Text Available In the early spring of 2013, Chinese health authorities reported several cases of H7N9 influenza virus infections in humans. Since then the virus has established itself at the human-animal interface in Eastern China and continues to cause several hundred infections annually. In order to characterize the antibody response to the H7N9 virus we generated several mouse monoclonal antibodies against the hemagglutinin of the A/Shanghai/1/13 (H7N9 virus. Of particular note are two monoclonal antibodies, 1B2 and 1H5, that show broad reactivity to divergent H7 hemagglutinins. Monoclonal antibody 1B2 binds to viruses of the Eurasian and North American H7 lineages and monoclonal antibody 1H5 reacts broadly to virus isolates of the Eurasian lineage. Interestingly, 1B2 shows broad hemagglutination inhibiting and neutralizing activity, while 1H5 fails to inhibit hemagglutination and demonstrates no neutralizing activity in vitro. However, both monoclonal antibodies were highly protective in an in vivo passive transfer challenge model in mice, even at low doses. Experiments using mutant antibodies that lack the ability for Fc/Fc-receptor and Fc/complement interactions suggest that the protection provided by mAb 1H5 is, at least in part, mediated by the Fc-fragment of the mAb. These findings highlight that a protective response to a pathogen may not only be due to neutralizing antibodies, but can also be the result of highly efficacious non-neutralizing antibodies not readily detected by classical in vitro neutralization or hemagglutination inhibition assays. This is of interest because H7 influenza virus vaccines induce only low hemagglutination inhibiting antibody titers while eliciting robust antibody titers as measured by ELISA. Our data suggest that these binding but non-neutralizing antibodies contribute to protection in vivo.

  11. Monoclonal antibodies:Principles and applications of immmunodiagnosis and immunotherapy for hepatitis C virus

    Institute of Scientific and Technical Information of China (English)

    Ashraf; Tabll; Aymn; T; Abbas; Sherif; El-Kafrawy; Ahmed; Wahid

    2015-01-01

    Hepatitis C virus(HCV) is a major health problem worldwide. Early detection of the infection will help better management of the infected cases. The monoclonal antibodies(m Ab) of mice are predominantly used for the immunodiagnosis of several viral,bacterial,and parasitic antigens. Serological detection of HCV antigens and antibodies provide simple and rapid methods of detection but lack sensitivity specially in the window phase between the infection and antibody development. Human mA b are used in the immunotherapy of several blood malignancies,such as lymphoma and leukemia,as well as for autoimmune diseases. In this review article,we will discuss methods of mouse and human monoclonal antibody production. We will demonstrate the role of mouse mA b in the detection of HCV antigens as rapid and sensitive immunodiagnostic assays for the detection of HCV,which is a major health problem throughout the world,particularly in Egypt. We will discuss the value of HCV-neutralizing antibodies and their roles in the immunotherapy of HCV infections and in HCV vaccine development. We will also discuss the different mechanisms by which the virus escape the effect of neutralizing mA b. Finally,we will discuss available and new trends to produce antibodies,such as egg yolk-based antibodies(Ig Y),production in transgenic plants,and the synthetic antibody mimics approach.

  12. Coarse grained modeling of transport properties in monoclonal antibody solution

    Science.gov (United States)

    Swan, James; Wang, Gang

    Monoclonal antibodies and their derivatives represent the fastest growing segment of the bio pharmaceutical industry. For many applications such as novel cancer therapies, high concentration, sub-cutaneous injections of these protein solutions are desired. However, depending on the peptide sequence within the antibody, such high concentration formulations can be too viscous to inject via human derived force alone. Understanding how heterogenous charge distribution and hydrophobicity within the antibodies leads to high viscosities is crucial to their future application. In this talk, we explore a coarse grained computational model of therapeutically relevant monoclonal antibodies that accounts for electrostatic, dispersion and hydrodynamic interactions between suspended antibodies to predict assembly and transport properties in concentrated antibody solutions. We explain the high viscosities observed in many experimental studies of the same biologics.

  13. Neutralizing antibodies to Haemophilus ducreyi cytotoxin.

    OpenAIRE

    Lagergård, T; Purvén, M

    1993-01-01

    Neutralizing antibodies against cytotoxin produced by Haemophilus ducreyi bacteria were studied in rabbits by an assay employing HEp-2 cells and diluted crude cytotoxin preparations from the organism. Antisera to 12 different H. ducreyi strains were prepared by immunization of rabbits with bacterial sonicates combined with Freund's adjuvant. The antibody response during infection with H. ducreyi was studied in two groups of rabbits which were infected with five live strains by either single o...

  14. The Case for Adjunctive Monoclonal Antibody Immunotherapy in Schizophrenia.

    Science.gov (United States)

    Miller, Brian J; Buckley, Peter F

    2016-06-01

    This article presents the case in favor of clinical trials of adjunctive monoclonal antibody immunotherapy in schizophrenia. Evidence for prenatal and premorbid immune risk factors for the development of schizophrenia in the offspring is highlighted. Then key evidence for immune dysfunction in patients with schizophrenia is considered. Next, previous trials of adjunctive anti-inflammatory or other immunotherapy in schizophrenia are discussed. Then evidence for psychosis as a side effect of immunotherapy for other disorders is discussed. Also presented is preliminary evidence for adjunctive monoclonal antibody immunotherapy in psychiatric disorders. Finally, important considerations in the design and implementation of clinical trials of adjunctive monoclonal antibody immunotherapy in schizophrenia are discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Use of monoclonal antibodies as an effective strategy for treatment of ciguatera poisoning.

    Science.gov (United States)

    Inoue, Masayuki; Lee, Nayoung; Tsumuraya, Takeshi; Fujii, Ikuo; Hirama, Masahiro

    2009-06-01

    Ciguatera is a global food poisoning caused by the consumption of fish that have accumulated sodium channel activator toxins, ciguatoxins. At present, most diagnosed cases of ciguatera are treated with symptomatic and supportive remedies, and no specific therapy has been devised. Here we report that ciguatoxin CTX3C can be effectively neutralized in vitro and in vivo by simultaneous use of two anti-ciguatoxin monoclonal antibodies, providing the first rational approach toward directly preventing and treating ciguatera.

  16. Preparation and Identification of Anti-rabies Virus Monoclonal Antibodies

    Institute of Scientific and Technical Information of China (English)

    Wen-juan Wang; Xiong Li; Li-hua Wang; Hu Shan; Lei Cao; Peng-cheng Yu; Qing Tang; Guo-dong Liang

    2012-01-01

    To provide a foundation for the development of rapid and specific methods for the diagnosis of rabies virus infection,anti-rabies virus monoclonal antibodies were prepared and rabies virus nucleoprotein and human rabies virus vaccine strain (PV strain) were used as immunogens to immunize 6-8 week old female BALB/c mice.Spleen cells and SP2/0 myeloma cells were fused according to conventional methods:the monoclonal cell strains obtained were selected using the indirect immunofluorescence test; this was followed by preparation of monoclonal antibody ascitic fluid; and finally,systematic identification of subclass,specificity and sensitivity was carried out.Two high potency and specific monoclonal antibodies against rabies virus were obtained and named 3B12 and 4A12,with ascitic fluid titers of 1∶8000 and 1∶10000,respectively.Both belonged to the IgG2a subclass.These strains secrete potent,stable and specific anti-rabies virus monoclonal antibodies,which makes them well suited for the development of rabies diagnosis reagents.

  17. Structure of a human rhinovirus-bivalently bound antibody complex: implications for viral neutralization and antibody flexibility.

    OpenAIRE

    1993-01-01

    The structure of a neutralizing immunoglobulin (monoclonal antibody mAb17-IA), bound to human rhinovirus 14 (HRV14), has been determined by cryo-electron microscopy and image reconstruction. The antibody bound bivalently across icosahedral twofold axes of the virus, and there were no detectable conformational changes in the capsid. Thus, bivalently bound IgGs do not appear to cause gross deformations in the capsid. Differences between the electron density of the constant domains of the bound ...

  18. Automated pipeline for rapid production and screening of HIV-specific monoclonal antibodies using pichia pastoris.

    Science.gov (United States)

    Shah, Kartik A; Clark, John J; Goods, Brittany A; Politano, Timothy J; Mozdzierz, Nicholas J; Zimnisky, Ross M; Leeson, Rachel L; Love, J Christopher; Love, Kerry R

    2015-12-01

    Monoclonal antibodies (mAbs) that bind and neutralize human pathogens have great therapeutic potential. Advances in automated screening and liquid handling have resulted in the ability to discover antigen-specific antibodies either directly from human blood or from various combinatorial libraries (phage, bacteria, or yeast). There remain, however, bottlenecks in the cloning, expression and evaluation of such lead antibodies identified in primary screens that hinder high-throughput screening. As such, "hit-to-lead identification" remains both expensive and time-consuming. By combining the advantages of overlap extension PCR (OE-PCR) and a genetically stable yet easily manipulatable microbial expression host Pichia pastoris, we have developed an automated pipeline for the rapid production and screening of full-length antigen-specific mAbs. Here, we demonstrate the speed, feasibility and cost-effectiveness of our approach by generating several broadly neutralizing antibodies against human immunodeficiency virus (HIV).

  19. Therapeutic monoclonal antibodies in human breast milk: a case study.

    Science.gov (United States)

    Ross, Elle; Robinson, Steven E; Amato, Carol; McMillan, Colette; Westcott, Jay; Wolf, Tiffany; Robinson, William A

    2014-04-01

    Recently, therapeutic monoclonal antibodies have been introduced for the treatment of advanced melanoma and other diseases. It remains unclear whether these drugs can be safely administered to women who are breast feeding because of the potential hazardous side effects for nursing infants. One such therapy for metastatic melanoma is ipilimumab, a human monoclonal antibody that blocks cytotoxic T-lymphocyte-antigen-4, and is the preferred treatment for patients with metastatic melanoma when other molecular therapies are not viable. This study measured ipilimumab levels in the breast milk of a patient undergoing treatment that were enough to raise concerns for a nursing infant exposed to ipilimumab.

  20. ERBB oncogene proteins as targets for monoclonal antibodies.

    Science.gov (United States)

    Polanovski, O L; Lebedenko, E N; Deyev, S M

    2012-03-01

    General properties of the family of tyrosine kinase ERBB receptors are considered in connection with their role in the generation of cascades of signal transduction in normal and tumor cells. Causes of acquisition of oncogene features by genes encoding these receptors and their role in tumorigenesis are analyzed. Anti-ERBB monoclonal antibodies approved for therapy are described in detail, and mechanisms of their antitumor activity and development of resistance to them are reviewed. The existing and the most promising strategies for creating and using monoclonal antibodies and their derivatives for therapy of cancer are discussed.

  1. Quantitative analysis of monoclonal antibodies by cation-exchange chromatofocusing.

    Science.gov (United States)

    Rozhkova, Anna

    2009-08-07

    A robust cation-exchange chromatofocusing method was developed for the routine analysis of a recombinant humanized monoclonal IgG antibody. We compare the chromatofocusing method to the conventional cation-exchange chromatography (CEX) employing a salt gradient and show clear advantages of chromatofocusing over CEX. We demonstrate the suitability of the present chromatofocusing method for its intended purpose by testing the validation characteristics. To our knowledge, this is the first chromatofocusing method developed for the routine analysis of monoclonal antibody charge species.

  2. Efficient Methods To Isolate Human Monoclonal Antibodies from Memory B Cells and Plasma Cells.

    Science.gov (United States)

    Corti, Davide; Lanzavecchia, Antonio

    2014-10-01

    In this article, we highlight the advantages of isolating human monoclonal antibodies from the human memory B cells and plasma cell repertoires by using high-throughput cellular screens. Memory B cells are immortalized with high efficiency using Epstein-Barr virus (EBV) in the presence of a toll-like receptor (TLR) agonist, while plasma cells are maintained in single-cell cultures by using interleukin 6 (IL-6) or stromal cells. In both cases, multiple parallel assays, including functional assays, can be used to identify rare cells that produce antibodies with unique properties. Using these methods, we have isolated potent and broadly neutralizing antibodies against a variety of viruses, in particular, a pan-influenza-A-neutralizing antibody and an antibody that neutralizes four different paramyxoviruses. Given the high throughput and the possibility of directly screening for function (rather than just binding), these methods are instrumental to implement a target-agnostic approach to identify the most effective antibodies and, consequently, the most promising targets for vaccine design. This approach is exemplified by the identification of unusually potent cytomegalovirus-neutralizing antibodies that led to the identification of the target, a pentameric complex that we are developing as a candidate vaccine.

  3. Surface activity of a monoclonal antibody.

    Science.gov (United States)

    Mahler, Hanns-Christian; Senner, Frank; Maeder, Karsten; Mueller, Robert

    2009-12-01

    The development of high concentration antibody formulations presents a major challenge for the formulation scientist, as physical characteristics and stability behavior change compared to low concentration protein formulations. The aim of this study was to investigate the potential correlation between surface activity and shaking stress stability of a model antibody-polysorbate 20 formulation. The surface activities of pure antibody and polysorbate 20 were compared, followed by a study on the influence of a model antibody on the apparent critical micelle concentration (CMC) of polysorbate 20 over a protein concentration range from 10 to 150 mg/mL. In a shaking stress experiment, the stability of 10, 75, and 150 mg/mL antibody formulations was investigated containing different concentrations of polysorbate 20, both below and above the CMC. The antibody increased significantly the apparent CMC of antibody-polysorbate 20 mixtures in comparison to the protein-free buffer. However, the concentration of polysorbate required for stabilization of the model antibody in a shaking stress experiment did not show dependence on the CMC. A polysorbate 20 level of 0.005% was found sufficient to stabilize both at low and high antibody concentration against antibody aggregation and precipitation.

  4. Anti-MrkA Monoclonal Antibodies Reveal Distinct Structural and Antigenic Features of MrkA

    Science.gov (United States)

    Wang, Qun; Chen, Yan; Cvitkovic, Romana; Pennini, Meghan E.; Chang, Chew shun; Pelletier, Mark; Bonnell, Jessica; Wu, Herren; Dall’Acqua, William F.; Stover, C. Kendall; Xiao, Xiaodong

    2017-01-01

    Antibody therapy against antibiotics resistant Klebsiella pneumoniae infections represents a promising strategy, the success of which depends critically on the ability to identify appropriate antibody targets. Using a target-agnostic strategy, we recently discovered MrkA as a potential antibody target and vaccine antigen. Interestingly, the anti-MrkA monoclonal antibodies isolated through phage display and hybridoma platforms all recognize an overlapping epitope, which opens up important questions including whether monoclonal antibodies targeting different MrkA epitopes can be generated and if they possess different protective profiles. In this study we generated four anti-MrkA antibodies targeting different epitopes through phage library panning against recombinant MrkA protein. These anti-MrkA antibodies elicited strong in vitro and in vivo protections against a multi-drug resistant Klebsiella pneumoniae strain. Furthermore, mutational and epitope analysis suggest that the two cysteine residues may play essential roles in maintaining a MrkA structure that is highly compacted and exposes limited antibody binding/neutralizing epitopes. These results suggest the need for further in-depth understandings of the structure of MrkA, the role of MrkA in the pathogenesis of Klebsiella pneumoniae and the protective mechanism adopted by anti-MrkA antibodies to fully explore the potential of MrkA as an efficient therapeutic target and vaccine antigen. PMID:28107434

  5. Discovery and Characterization of Phage Display-Derived Human Monoclonal Antibodies against RSV F Glycoprotein.

    Directory of Open Access Journals (Sweden)

    Zhifeng Chen

    Full Text Available Respiratory syncytial virus (RSV is a leading cause of lower respiratory tract infection in infants, the elderly and in immunosuppressed populations. The vast majority of neutralizing antibodies isolated from human subjects target the RSV fusion (F glycoprotein, making it an attractive target for the development of vaccines and therapeutic antibodies. Currently, Synagis® (palivizumab is the only FDA approved antibody drug for the prevention of RSV infection, and there is a great need for more effective vaccines and therapeutics. Phage display is a powerful tool in antibody discovery with the advantage that it does not require samples from immunized subjects. In this study, Morphosys HuCAL GOLD® phage libraries were used for panning against RSV prefusion and postfusion F proteins. Panels of human monoclonal antibodies (mAbs against RSV F protein were discovered following phage library panning and characterized. Antibodies binding specifically to prefusion or postfusion F proteins and those binding both conformations were identified. 3B1 is a prototypic postfusion F specific antibody while 2E1 is a prototypic prefusion F specific antibody. 2E1 is a potent broadly neutralizing antibody against both RSV A and B strains. Epitope mapping experiments identified a conformational epitope spanning across three discontinuous sections of the RSV F protein, as well as critical residues for antibody interaction.

  6. Broadly Neutralizing Alphavirus Antibodies Bind an Epitope on E2 and Inhibit Entry and Egress

    NARCIS (Netherlands)

    Fox, Julie M.; Long, Feng; Edeling, Melissa A.; Lin, Hueylie; van Duijl-Richter, Mareike K. S.; Fong, Rachel H.; Kahle, Kristen M.; Smit, Jolanda M.; Jin, Jing; Simmons, Graham; Doranz, Benjamin J.; Crowe, James E.; Fremont, Daved H.; Rossmann, Michael G.; Diamond, Michael S.

    2015-01-01

    We screened a panel of mouse and human monoclonal antibodies (MAbs) against chikungunya virus and identified several with inhibitory activity against multiple alphaviruses. Passive transfer of broadly neutralizing MAbs protected mice against infection by chikungunya, Mayaro, and O'nyong'nyong alphav

  7. Broadly Neutralizing Alphavirus Antibodies Bind an Epitope on E2 and Inhibit Entry and Egress

    NARCIS (Netherlands)

    Fox, Julie M.; Long, Feng; Edeling, Melissa A.; Lin, Hueylie; van Duijl-Richter, Mareike K. S.; Fong, Rachel H.; Kahle, Kristen M.; Smit, Jolanda M.; Jin, Jing; Simmons, Graham; Doranz, Benjamin J.; Crowe, James E.; Fremont, Daved H.; Rossmann, Michael G.; Diamond, Michael S.

    2015-01-01

    We screened a panel of mouse and human monoclonal antibodies (MAbs) against chikungunya virus and identified several with inhibitory activity against multiple alphaviruses. Passive transfer of broadly neutralizing MAbs protected mice against infection by chikungunya, Mayaro, and O'nyong'nyong alphav

  8. Characterization of monoclonal antibodies that strongly inhibit Electrophorus electricus acetylcholinesterase.

    Science.gov (United States)

    Remy, M H; Frobert, Y; Grassi, J

    1995-08-01

    In this study, we describe three different monoclonal antibodies (mAbs Elec-403, Elec-408, and Elec-410) directed against Electrophorus electricus acetylcholinesterase (AChE) which were selected as inhibitors for this enzyme. Two of these antibodies (Elec-403 and Elec-410), recognized overlapping but different epitopes, competed with snake venom toxin fasciculin for binding to the enzyme, and thus apparently recognized the peripheral site of AChE. In addition, the binding of Elec-403 was antagonized by 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide (BW284C51) and propidium, indicating that the corresponding epitope encompassed the anionic site involved in the binding of these low-molecular-mass inhibitors. The third mAb (Elec-408), was clearly bound to another site on the AChE molecule, and its inhibitory effect was cumulative with those of Elec-403, Elec-410, and fasciculin. All mAbs bound AChE with high affinity and were as strong inhibitors with an apparent Ki values less than 0.1 nM. Elec-403 was particularly efficient with an inhibitory activity similar to that of fasciculin. Inhibition was observed with both charged (acetylthiocholine) and neutral substrates (o-nitrophenyl acetate) and had the characteristics of a non-competitive process. Elec-403 and Elec-410 probably exert their effect by triggering allosteric transitions from the peripheral site to the active site. The epitope recognized by mAb Elec-408 has not been localized, but it may correspond to a new regulatory site on AChE.

  9. Characterization of monoclonal antibodies against waterfowl parvoviruses VP3 protein

    Directory of Open Access Journals (Sweden)

    Yin Xiuchen

    2012-11-01

    Full Text Available Abstract Background The VP3 protein of goose parvovirus (GPV or Muscovy duck parvovirus (MDPV, a major structural protein, can induce neutralizing antibodies in geese and ducks, but monoclonal antibodies (MAbs against VP3 protein has never been characterized. Results Three hybridoma cell lines secreting anti-GPV VP3 MAbs were obtained and designated 4A8, 4E2, and 2D5. Immunoglobulin subclass tests differentiated them as IgG2b (4A8 and 4E2 and IgG2a (2D5. Dot blotting assays showed that three MAbs reacted with His-VP3 protein in a conformation-independent manner. A competitive binding assay indicated that the MAbs delineated two epitopes, A and B of VP3. Immunofluorescence assay showed that MAbs 4A8, 4E2, and 2D5 could specifically bind to goose embryo fibroblast cells (GEF or duck fibroblast cells (DEF infected with GPV and MDPV. Dot blotting also showed that the MAbs recognized both nature GPV and MDPV antigen. Western blotting confirmed that the MAbs recognized VP3 proteins derived from purified GPV and MDPV particles. The MAbs 4A8 and 2D5 had universal reactivity to heterologous GPV and MDPV tested in an antigen-capture enzyme-linked immunosorbent assay. Conclusions Preparation and characterization of these the MAbs suggests that they may be useful for the development of a MAb-capture ELISA for rapid detection of both GPV and MDPV. Virus isolation and PCR are reliable for detecting GPV and MDPV infection, but these procedures are laborious, time-consuming, and requiring instruments. These diagnosis problems highlight the ongoing demand for rapid, reproducible, and automatic methods for the sensitive detection of both GPV and MDPV infection.

  10. 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.

  11. A monoclonal antibody for G protein-coupled receptor crystallography.

    Science.gov (United States)

    Day, Peter W; Rasmussen, Søren G F; Parnot, Charles; Fung, Juan José; Masood, Asna; Kobilka, Tong Sun; Yao, Xiao-Jie; Choi, Hee-Jung; Weis, William I; Rohrer, Daniel K; Kobilka, Brian K

    2007-11-01

    G protein-coupled receptors (GPCRs) constitute the largest family of signaling proteins in mammals, mediating responses to hormones, neurotransmitters, and senses of sight, smell and taste. Mechanistic insight into GPCR signal transduction is limited by a paucity of high-resolution structural information. We describe the generation of a monoclonal antibody that recognizes the third intracellular loop (IL3) of the native human beta(2) adrenergic (beta(2)AR) receptor; this antibody was critical for acquiring diffraction-quality crystals.

  12. Heterogeneity of monoclonal antibodies revealed by charge-sensitive methods.

    Science.gov (United States)

    Vlasak, J; Ionescu, R

    2008-12-01

    The expanding field of monoclonal antibody-based pharmaceuticals has triggered increased interest in analytical characterization of these large proteins and in understanding of their heterogeneity and degradation pathways. As a result, a large number of enzymatic modifications as well as chemical and physical degradations have been reported in monoclonal antibodies in recent years. Most heterogeneity is related to changes in the surface charge of the antibody, either directly, as a change in the number of charged residues, or indirectly as a chemical or physical alteration that changes surface-charge distribution. This review presents an overview of the sources of charge-related heterogeneity in monoclonal antibodies and the methods used for their detection. A detailed section is dedicated to deamidation of asparagine and isomerization of aspartic acid residues, two ubiquitous degradation pathways detected in antibodies and other proteins as well. Finally, kinetic modeling of the accumulation of antibody variants is presented as a tool to determine the expected fraction of molecules that have undergone one or more degradation reactions.

  13. Characterization of Binding Epitopes of CA125 Monoclonal Antibodies

    DEFF Research Database (Denmark)

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

    2014-01-01

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

  14. Production and potential use of monoclonal antibodies against polio viruses.

    NARCIS (Netherlands)

    A.D.M.E. Osterhaus (Albert); A.L. van Wezel; G. van Steenis (Bert); A.G. Hazendonk

    1982-01-01

    textabstractLymphocyte hybridomas secreting monoclonal antibodies against different strains of polio virus type 1, 2, or 3 have been produced. For this purpose Balb/C mice were immunized with purified and inactivated virus suspensions and their splenocytes were fused with P3X63Ag8 mouse myeloma cell

  15. Immunohistochemical diagnosis of systemic bovine zygomycosis by murine monoclonal antibodies

    DEFF Research Database (Denmark)

    Jensen, H.E.; Aalbaek, B.; Lind, Peter

    1996-01-01

    Murine monoclonal antibodies (Mabs) against water-soluble somatic antigens (WSSA) and the wall fraction (WF) from Rhizopus arrhizus (Rhizopus oryzae) were produced in vitro by fusion of splenocytes from immunized BALB/c mice with mouse myeloma X63-Ag 8.653 cells. Supernatants reacting only with h...... for the in situ diagnosis of systemic bovine zygomycosis....

  16. A mouse monoclonal antibody against Alexa Fluor 647.

    Science.gov (United States)

    Wuethrich, Irene; Guillen, Eduardo; Ploegh, Hidde L

    2014-04-01

    Fluorophores are essential tools in molecular and cell biology. However, their application is mostly confined to the singular exploitation of their fluorescent properties. To enhance the versatility and expand the use of the fluorophore Alexa Fluor 647 (AF647), we generated a mouse monoclonal antibody against it. We demonstrate its use of AF647 for immunoblot, immunoprecipitation, and cytofluorimetry.

  17. Monoclonal antibodies for the detection of Puccinia striiformis urediniospores

    DEFF Research Database (Denmark)

    Skottrup, Peter Durand; Frøkiær, Hanne; Hearty, Stephen

    2007-01-01

    The fungal pathogen Pst causes yellow rust disease in wheat plants leading to crop losses. The organism spreads by releasing wind-dispersed urediniospores from infected plants. In this study a library of novel monoclonal antibodies (mAbs) was developed against Pst urediniospores. Nine mAb-produci...

  18. Monoclonal Antibodies to Prevent Use of Mycotoxins as Biological Weapons

    Science.gov (United States)

    2007-07-01

    Mycotoxins as Biological Weapons PRINCIPAL INVESTIGATOR: Marta Feldmesser, M.D. CONTRACTING ORGANIZATION: Albert Einstein College of...Monoclonal Antibodies to Prevent Use of Mycotoxins as Biological Weapons 5b. GRANT NUMBER W81XWH-06-1-0085 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR

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

    NARCIS (Netherlands)

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

    1995-01-01

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

  20. 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.

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

    NARCIS (Netherlands)

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

    1995-01-01

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

  2. Characterization of monoclonal and polyclonal antibodies to bovine enteric coronavirus: establishment of an efficient ELISA for antigen detection in feces.

    Science.gov (United States)

    Czerny, C P; Eichhorn, W

    1989-06-01

    Monoclonal antibodies to bovine enteric coronavirus (BEC) were produced. Additionally, polyclonal antibodies were made in rabbits and guinea pigs and extracted from the yolk of immunized hens. The antibodies were characterized by neutralization test, hemagglutination inhibition test, enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Neutralizing antibody titers of polyclonal antisera ranged from 1:1280 to 1:40,000. Only one out of 908 hybridoma colonies tested secreted antibodies with neutralizing activity. By ELISA, polyclonal sera exhibited high background reactions that could be significantly reduced by treatment with kaolin in the case of rabbit sera. Attempts to establish an ELISA for BEC antigen detection based on polyclonal sera failed due to low sensitivity and specificity. Optimal results were achieved when a mixture of two monoclonal antibodies was coated onto microplates for antigen capture, while rabbit hyperimmune serum served as detecting antibodies in an indirect assay. The combination of the two monoclonal antibodies did not increase sensitivity synergistically, but in a compensatory fashion, probably because of epitope differences between BEC field strains.

  3. Structure and specificity of lamprey monoclonal antibodies

    OpenAIRE

    Herrin, Brantley R.; Alder, Matthew N; Roux, Kenneth H.; Sina, Christina; Ehrhardt, Götz R. A.; Boydston, Jeremy A.; Turnbough, Charles L.; Cooper, Max D.

    2008-01-01

    Adaptive immunity in jawless vertebrates (lamprey and hagfish) is mediated by lymphocytes that undergo combinatorial assembly of leucine-rich repeat (LRR) gene segments to create a diverse repertoire of variable lymphocyte receptor (VLR) genes. Immunization with particulate antigens induces VLR-B-bearing lymphocytes to secrete antigen-specific VLR-B antibodies. Here, we describe the production of recombinant VLR-B antibodies specific for BclA, a major coat protein of Bacillus anthracis spores...

  4. Development of Biodegradable Nanocarriers Loaded with a Monoclonal Antibody

    Directory of Open Access Journals (Sweden)

    Andrew Gdowski

    2015-02-01

    Full Text Available Treatments utilizing monoclonal antibody therapeutics against intracellular protein-protein interactions in cancer cells have been hampered by several factors, including poor intracellular uptake and rapid lysosomal degradation. Our current work examines the feasibility of encapsulating monoclonal antibodies within poly(lactic-co-glycolic acid (PLGA nanoparticles using a water/oil/water double emulsion solvent evaporation technique. This method can be used to prepare protective polymeric nanoparticles for transporting functional antibodies to the cytoplasmic compartment of cancer cells. Nanoparticles were formulated and then characterized using a number of physical and biological parameters. The average nanoparticle size ranged from 221 to 252 nm with a low polydispersity index. Encapsulation efficiency of 16%–22% and antibody loading of 0.3%–1.12% were observed. The antibody molecules were released from the nanoparticles in a sustained manner and upon release maintained functionality. Our studies achieved successful formulation of antibody loaded polymeric nanoparticles, thus indicating that a PLGA-based antibody nanoformulation is a promising intracellular delivery vehicle for a large number of new intracellular antibody targets in cancer cells.

  5. Epitopes on the peplomer protein of infectious bronchitis virus strain M41 as defined by monoclonal antibodies.

    NARCIS (Netherlands)

    N.M.C. Bleumink-Pluym; A.D.M.E. Osterhaus (Albert); M.C. Horzinek; B.A.M. van der Zeijst (Ben); H.G.M. Niesters (Bert)

    1987-01-01

    textabstractSixteen monoclonal antibodies (Mcabs) were prepared against infectious bronchitis virus strain M41, all of them reacting with the peplomer protein. One of them, Mcab 13, was able to neutralize the virus and to inhibit hemagglutination. Competition binding assays allowed the definition of

  6. The use of combinations of monoclonal antibodies in clinical oncology.

    Science.gov (United States)

    Henricks, Linda M; Schellens, Jan H M; Huitema, Alwin D R; Beijnen, Jos H

    2015-12-01

    Treatment with monoclonal antibodies is becoming increasingly important in clinical oncology. These antibodies specifically inhibit signaling pathways in tumor growth and/or induce immunological responses against tumor cells. By combining monoclonal antibodies several pathways may be targeted simultaneously, potentially leading to additive or synergistic effects. Theoretically, antibodies are very suitable for use in combination therapy, because of limited overlapping toxicity and lack of pharmacokinetic interactions. In this article an overview is given of preclinical and clinical data on twenty-five different combinations of antibodies in oncology. Some of these combinations have proven clinical benefit, for example the combination of trastuzumab and pertuzumab in HER2-positive breast cancer, which exemplifies an additive or synergistic effect on antitumor activity in clinical studies and the combination of nivolumab and ipilimumab, which results in significant increases in progression-free and overall survival in patients with advanced melanoma. However, other combinations may lead to unfavorable results, such as bevacizumab with cetuximab or panitumumab in advanced colorectal cancer. These combinations result in shorter progression-free survival and increased toxicity compared to therapy with a single antibody. In summary, the different published studies showed widely varying results, depending on the combination of antibodies, indication and patient population. More preclinical and clinical studies are necessary to unravel the mechanisms behind synergistic or antagonistic effects of combining monoclonal antibodies. Most research on combination therapies is still in an early stage, but it is expected that for several tumor types the use of combination therapy of antibodies will become standard of care in the near future.

  7. Enzyme immunoassay of mumps virus in cell culture with peroxidase-labelled virus specific monoclonal antibodies and its application for determination of antibodies

    NARCIS (Netherlands)

    Tiel, F.H. van; Kraaijeveld, C.A.; Baller, J.; Harmsen, T.; Oosterlaken, T.A.M.; Snippe, H.

    1988-01-01

    Mumps neutralizing monoclonal antibodies (MAs) were purified and labelled with horseradish peroxidase and used to detect virus-infected Vero cells, which were seeded as monolayers in wells of 96-well plates. This direct enzyme immunoassay (EIA) in cell culture proved to be a sensitive method for

  8. Enzyme immunoassay of mumps virus in cell culture with peroxidase-labelled virus specific monoclonal antibodies and its application for determination of antibodies

    NARCIS (Netherlands)

    Tiel, F.H. van; Kraaijeveld, C.A.; Baller, J.; Harmsen, T.; Oosterlaken, T.A.M.; Snippe, H.

    1988-01-01

    Mumps neutralizing monoclonal antibodies (MAs) were purified and labelled with horseradish peroxidase and used to detect virus-infected Vero cells, which were seeded as monolayers in wells of 96-well plates. This direct enzyme immunoassay (EIA) in cell culture proved to be a sensitive method for det

  9. [Single B cell monoclonal antibody technologies and applications].

    Science.gov (United States)

    Chi, Xiangyang; Yu, Changming; Chen, Wei

    2012-06-01

    Monoclonal antibodies (mAbs) contribute a lot to the development of numerous fields in life science as a pivotal tool in modern biological research. Development of the PCR methods and maturation of antibody production have made it possible to generate mAbs from single human B cells by single cell RT-PCR with successional cloning and expression in vitro. Compared to traditional monoclonal antibody technologies, single B cell technologies require relatively fewer cells, which are highly efficient in obtaining specific mAbs in a rapid way with preservation of the natural heavy and light chain pairing. With so many advantages, single B cell technologies have been proved to be an attractive approach for retrieval of naive and antigen-experienced antibody repertoires generated in vivo, design of rationale structure-based vaccine, evaluation and development of basic B cell biology concepts in health and autoimmunity, and prevention of infectious diseases by passive immunization and therapy for disorders. Accordingly, this review introduced recent progresses in the single B cell technologies for generating monoclonal antibodies and applications.

  10. Library of monoclonal antibodies against brush border membrane epithelial antigens

    Energy Technology Data Exchange (ETDEWEB)

    Behar, M.; Katz, A.; Silverman, M.

    1986-03-01

    A purified fraction of proximal tubule brush border membranes (BBM) was prepared from dog kidney and used to immunize mice. The standard technique of hybridoma production was followed as described by Kohler and Milstein. Production of antibodies was detected by indirect immunofluorescence on dog kidney slices and by immunodot against the purified fraction on nitrocellulose. Five hybrids exhibited anti BBM activity. These were cloned twice and yielded stable cell lines producing IgG type monoclonal antibodies against BBM. They were designated A/sub 1/, C/sub 7/, D/sub 3/, D/sub 7/ and H/sub 4/. As a family these five monoclonals have broad tissue specificity, i.e. positive staining of the surface mucosa of intestinal kidney proximal tubules. D/sub 3/ exhibits even broader specificity for epithelium reacting with bile canaliculi and choroid plexus. The authors have verified that at least 4/5 antibodies are directed against BBM protein as revealed by immunoprecipitation of solubilized BBM and detected by Coomassie blue staining or autoradiography of lactoperoxidase labelled BBM. Most interestingly all antibodies bind to the surface of LL CPK/sub 1/ cells, a continuous pig kidney cell line of undefined origin but exhibiting many characteristics of proximal tubule cells. The library of monoclonal antibodies obtained provide important probes with which to study membrane biogenesis and polarization in epithelial cells.

  11. Adsorption of monoclonal antibodies to glass microparticles.

    Science.gov (United States)

    Hoehne, Matthew; Samuel, Fauna; Dong, Aichun; Wurth, Christine; Mahler, Hanns-Christian; Carpenter, John F; Randolph, Theodore W

    2011-01-01

    Microparticulate glass represents a potential contamination to protein formulations that may occur as a result of processing conditions or glass types. The effect of added microparticulate glass to formulations of three humanized antibodies was tested. Under the three formulation conditions tested, all three antibodies adsorbed irreversibly at near monolayer surface coverages to the glass microparticles. Analysis of the secondary structure of the adsorbed antibodies by infrared spectroscopy reveal only minor perturbations as a result of adsorption. Likewise, front-face fluorescence quenching measurements reflected minimal tertiary structural changes upon adsorption. In contrast to the minimal effects on protein structure, adsorption of protein to suspensions of glass microparticles induced significant colloidal destabilization and flocculation of the suspension.

  12. Monoclonal IgA Antibodies for Aflatoxin Immunoassays

    OpenAIRE

    2016-01-01

    Antibody based techniques are widely used for the detection of aflatoxins which are potent toxins with a high rate of occurrence in many crops. We developed a murine monoclonal antibody of immunoglobulin A (IgA) isotype with a strong binding affinity to aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2) and aflatoxin M1 (AFM1). The antibody was effectively used in immunoaffinity column (IAC) and ELISA kit development. The performance of the IACs was compatible ...

  13. Cross-reactive broadly neutralizing antibodies: timing is everything

    OpenAIRE

    Euler, Zelda; Schuitemaker, Hanneke

    2012-01-01

    The recent surge of research into new broadly neutralizing antibodies in HIV-1 infection has recharged the field of HIV-1 vaccinology. In this review we discuss the currently known broadly neutralizing antibodies and focus on factors that may shape these antibodies in natural infection. We further discuss the role of these antibodies in the clinical course of the infection and consider immunological obstacles in inducing broadly neutralizing antibodies with a vaccine.

  14. Cross-reactive broadly neutralizing antibodies: timing is everything.

    Science.gov (United States)

    Euler, Zelda; Schuitemaker, Hanneke

    2012-01-01

    The recent surge of research into new broadly neutralizing antibodies in HIV-1 infection has recharged the field of HIV-1 vaccinology. In this review we discuss the currently known broadly neutralizing antibodies and focus on factors that may shape these antibodies in natural infection. We further discuss the role of these antibodies in the clinical course of the infection and consider immunological obstacles in inducing broadly neutralizing antibodies with a vaccine.

  15. Vaccine Elicitation of High Mannose-Dependent Neutralizing Antibodies against the V3-Glycan Broadly Neutralizing Epitope in Nonhuman Primates.

    Science.gov (United States)

    Saunders, Kevin O; Nicely, Nathan I; Wiehe, Kevin; Bonsignori, Mattia; Meyerhoff, R Ryan; Parks, Robert; Walkowicz, William E; Aussedat, Baptiste; Wu, Nelson R; Cai, Fangping; Vohra, Yusuf; Park, Peter K; Eaton, Amanda; Go, Eden P; Sutherland, Laura L; Scearce, Richard M; Barouch, Dan H; Zhang, Ruijun; Von Holle, Tarra; Overman, R Glenn; Anasti, Kara; Sanders, Rogier W; Moody, M Anthony; Kepler, Thomas B; Korber, Bette; Desaire, Heather; Santra, Sampa; Letvin, Norman L; Nabel, Gary J; Montefiori, David C; Tomaras, Georgia D; Liao, Hua-Xin; Alam, S Munir; Danishefsky, Samuel J; Haynes, Barton F

    2017-02-28

    Induction of broadly neutralizing antibodies (bnAbs) that target HIV-1 envelope (Env) is a goal of HIV-1 vaccine development. A bnAb target is the Env third variable loop (V3)-glycan site. To determine whether immunization could induce antibodies to the V3-glycan bnAb binding site, we repetitively immunized macaques over a 4-year period with an Env expressing V3-high mannose glycans. Env immunizations elicited plasma antibodies that neutralized HIV-1 expressing only high-mannose glycans-a characteristic shared by early bnAb B cell lineage members. A rhesus recombinant monoclonal antibody from a vaccinated macaque bound to the V3-glycan site at the same amino acids as broadly neutralizing antibodies. A structure of the antibody bound to glycan revealed that the three variable heavy-chain complementarity-determining regions formed a cavity into which glycan could insert and neutralized multiple HIV-1 isolates with high-mannose glycans. Thus, HIV-1 Env vaccination induced mannose-dependent antibodies with characteristics of V3-glycan bnAb precursors.

  16. Sperm-immobilizing monoclonal antibody to human seminal plasma antigens.

    Science.gov (United States)

    Shigeta, M; Watanabe, T; Maruyama, S; Koyama, K; Isojima, S

    1980-01-01

    Rat spleen cells immunized to human azoospermic semen (a mixture of seminal plasma components) and mouse myeloma cells (P3/X63 Ag8U1; P3U1) (Marguilies et al., 1976) were successfully fused with polyethylene glycol (PEG 1500) and 19 of 89 fused cell cultures were found to produce sperm-immobilizing antibody. The cells that produced antibody indicating the highest sperm-immobilizing activity were distributed into wells for further recloning and 10 clones producing sperm-immobilizing antibody were established. The clone (1C4) producing the highest antibody titre was found to produce a large amount of IgG in culture supernatants and to contain a mixture of rat and mouse chromosomes. It was proved by immunodiffusion test that the monoclonal antibody was produced to the human seminal plasma antigen No. 7 which is common to human milk protein. Using this hybridoma which produced a large amount of monoclonal sperm-immobilizing antibody, a new method could be developed for purifying human seminal plasma antigen by immunoaffinity chromatography with bound antibody from the hybridoma. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:6783353

  17. Generation of monoclonal antibodies against highly conserved antigens.

    Directory of Open Access Journals (Sweden)

    Hongzhe Zhou

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

  18. Monoclonal IgA Antibodies for Aflatoxin Immunoassays

    Directory of Open Access Journals (Sweden)

    Özlem Ertekin

    2016-05-01

    Full Text Available Antibody based techniques are widely used for the detection of aflatoxins which are potent toxins with a high rate of occurrence in many crops. We developed a murine monoclonal antibody of immunoglobulin A (IgA isotype with a strong binding affinity to aflatoxin B1 (AFB1, aflatoxin B2 (AFB2, aflatoxin G1 (AFG1, aflatoxin G2 (AFG2 and aflatoxin M1 (AFM1. The antibody was effectively used in immunoaffinity column (IAC and ELISA kit development. The performance of the IACs was compatible with AOAC performance standards for affinity columns (Test Method: AOAC 991.31. The total binding capacity of the IACs containing our antibody was 111 ng, 70 ng, 114 ng and 73 ng for AFB1, AFB2, and AFG1 andAFG2, respectively. Furthermore, the recovery rates of 5 ng of each AF derivative loaded to the IACs were determined as 104.9%, 82.4%, 85.5% and 70.7% for AFB1, AFB2, AFG1 and AFG2, respectively. As for the ELISA kit developed using non-oriented, purified IgA antibody, we observed a detection range of 2–50 µg/L with 40 min total test time. The monoclonal antibody developed in this research is hitherto the first presentation of quadruple antigen binding IgA monoclonal antibodies in mycotoxin analysis and also the first study of their utilization in ELISA and IACs. IgA antibodies are valuable alternatives for immunoassay development, in terms of both sensitivity and ease of preparation, since they do not require any orientation effort.

  19. Monoclonal IgA Antibodies for Aflatoxin Immunoassays

    Science.gov (United States)

    Ertekin, Özlem; Pirinçci, Şerife Şeyda; Öztürk, Selma

    2016-01-01

    Antibody based techniques are widely used for the detection of aflatoxins which are potent toxins with a high rate of occurrence in many crops. We developed a murine monoclonal antibody of immunoglobulin A (IgA) isotype with a strong binding affinity to aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2) and aflatoxin M1 (AFM1). The antibody was effectively used in immunoaffinity column (IAC) and ELISA kit development. The performance of the IACs was compatible with AOAC performance standards for affinity columns (Test Method: AOAC 991.31). The total binding capacity of the IACs containing our antibody was 111 ng, 70 ng, 114 ng and 73 ng for AFB1, AFB2, and AFG1 andAFG2, respectively. Furthermore, the recovery rates of 5 ng of each AF derivative loaded to the IACs were determined as 104.9%, 82.4%, 85.5% and 70.7% for AFB1, AFB2, AFG1 and AFG2, respectively. As for the ELISA kit developed using non-oriented, purified IgA antibody, we observed a detection range of 2–50 µg/L with 40 min total test time. The monoclonal antibody developed in this research is hitherto the first presentation of quadruple antigen binding IgA monoclonal antibodies in mycotoxin analysis and also the first study of their utilization in ELISA and IACs. IgA antibodies are valuable alternatives for immunoassay development, in terms of both sensitivity and ease of preparation, since they do not require any orientation effort. PMID:27187470

  20. Monoclonal IgA Antibodies for Aflatoxin Immunoassays.

    Science.gov (United States)

    Ertekin, Özlem; Pirinçci, Şerife Şeyda; Öztürk, Selma

    2016-05-12

    Antibody based techniques are widely used for the detection of aflatoxins which are potent toxins with a high rate of occurrence in many crops. We developed a murine monoclonal antibody of immunoglobulin A (IgA) isotype with a strong binding affinity to aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2) and aflatoxin M1 (AFM1). The antibody was effectively used in immunoaffinity column (IAC) and ELISA kit development. The performance of the IACs was compatible with AOAC performance standards for affinity columns (Test Method: AOAC 991.31). The total binding capacity of the IACs containing our antibody was 111 ng, 70 ng, 114 ng and 73 ng for AFB1, AFB2, and AFG1 andAFG2, respectively. Furthermore, the recovery rates of 5 ng of each AF derivative loaded to the IACs were determined as 104.9%, 82.4%, 85.5% and 70.7% for AFB1, AFB2, AFG1 and AFG2, respectively. As for the ELISA kit developed using non-oriented, purified IgA antibody, we observed a detection range of 2-50 µg/L with 40 min total test time. The monoclonal antibody developed in this research is hitherto the first presentation of quadruple antigen binding IgA monoclonal antibodies in mycotoxin analysis and also the first study of their utilization in ELISA and IACs. IgA antibodies are valuable alternatives for immunoassay development, in terms of both sensitivity and ease of preparation, since they do not require any orientation effort.

  1. Signature biochemical properties of broadly cross-reactive HIV-1 neutralizing antibodies in human plasma.

    Science.gov (United States)

    Sajadi, Mohammad M; Lewis, George K; Seaman, Michael S; Guan, Yongjun; Redfield, Robert R; DeVico, Anthony L

    2012-05-01

    The common properties of broadly cross-reactive HIV-1 neutralization antibodies found in certain HIV-1-infected individuals holds significant value for understanding natural and vaccine-mediated anti-HIV immunity. Recent efforts have addressed this question by deriving neutralizing monoclonal anti-envelope antibodies from memory B cell pools of selected subjects. However, it has been more difficult to identify whether broadly neutralizing antibodies circulating in plasma possess shared characteristics among individuals. To address this question, we used affinity chromatography and isoelectric focusing to fractionate plasma immunoglobulin from 10 HIV-1-infected subjects (5 subjects with broad HIV-1 neutralizing activity and 5 controls). We find that plasma neutralizing activity typically partitions into at least two subsets of antibodies. Antibodies with restricted neutralization breadth have relatively neutral isoelectric points and preferentially bind to envelope monomers and trimers versus core antigens from which variable loops and other domains have been deleted. In comparison, broadly neutralizing antibodies account for a minor fraction of the total anti-envelope response. They are consistently distinguished by more basic isoelectric points and specificity for epitopes shared by monomeric gp120, gp120 core, or CD4-induced structures. Such biochemical properties might be exploited to reliably predict or produce broad anti-HIV immunity.

  2. Generation of HER2 monoclonal antibodies using epitopes of a rabbit polyclonal antibody.

    Science.gov (United States)

    Hu, Francis Jingxin; Uhlen, Mathias; Rockberg, Johan

    2014-01-25

    One of the issues in using polyclonal antibodies is the limited amount of reagent available from an immunisation, leading to batch-to-batch variation and difficulties in obtaining the same antibody performance when the same antigen is re-immunised into several separate animals. This led to the development of hybridoma technology allowing, at least theoretically, for an unlimited production of a specific binder. Nevertheless, polyclonal antibodies are widely used in research and diagnostics and there exists a need for robust methods to convert a polyclonal antibody with good binding performance into a renewable monoclonal with identical or similar binding specificity. Here we have used precise information regarding the functional recognition sequence (epitope) of a rabbit polyclonal antibody with attractive binding characteristics as the basis for generation of a renewable mouse monoclonal antibody. First, the original protein fragment antigen was used for immunisation and generation of mouse hybridoma, without obtaining binders to the same epitope region. Instead a peptide designed using the functional epitope and structural information was synthesised and used for hybridoma production. Several of the monoclonal antibodies generated were found to have similar binding characteristics to those of the original polyclonal antibody. These monoclonal antibodies detected native HER2 on cell lines and were also able to stain HER2 in immunohistochemistry using xenografted mice, as well as human normal and cancer tissues.

  3. A Neutralizing Antibody Assay Based on a Reporter of Antibody-Dependent Cell-Mediated Cytotoxicity.

    Science.gov (United States)

    Wu, Yuling; Li, Jia J; Kim, Hyun Jun; Liu, Xu; Liu, Weiyi; Akhgar, Ahmad; Bowen, Michael A; Spitz, Susan; Jiang, Xu-Rong; Roskos, Lorin K; White, Wendy I

    2015-11-01

    Benralizumab is a humanized anti-IL5 receptor α (IL5Rα) monoclonal antibody (mAb) with enhanced (afucosylation) antibody-dependent cell-mediated cytotoxicity (ADCC) function. An ADCC reporter cell-based neutralizing antibody (NAb) assay was developed and characterized to detect NAb against benralizumab in human serum to support the clinical development of benralizumab. The optimal ratio of target cells to effector cells was 3:1. Neither parental benralizumab (fucosylated) nor benralizumab Fab resulted in ADCC activity, confirming the requirement for ADCC activity in the NAb assay. The serum tolerance of the cells was determined to be 2.5%. The cut point derived from normal and asthma serum samples was comparable. The effective range of benralizumab was determined, and 35 ng/mL [80% maximal effective concentration (EC80)] was chosen as the standard concentration to run in the assessment of NAb. An affinity purified goat anti-benralizumab polyclonal idiotype antibody preparation was shown to have NAb since it inhibited ADCC activity in a dose-dependent fashion. The low endogenous concentrations of IL5 and soluble IL5 receptor (sIL5R) did not demonstrate to interfere with the assay. The estimated assay sensitivities at the cut point were 1.02 and 1.10 μg/mL as determined by the surrogate neutralizing goat polyclonal and mouse monoclonal anti-drug antibody (ADA) controls, respectively. The assay can detect NAb (at 2.5 μg/mL) in the presence of 0.78 μg/mL benralizumab. The assay was not susceptible to non-specific matrix effects. This study provides an approach and feasibility of developing an ADCC cell-based NAb assay to support biopharmaceuticals with an ADCC function.

  4. Interactions between Lipids and Human Anti-HIV Antibody 4E10 Can Be Reduced without Ablating Neutralizing Activity

    NARCIS (Netherlands)

    Xu, Hengyu; Song, Likai; Kim, Mikyung; Holmes, Margaret A.; Kraft, Zane; Sellhorn, George; Reinherz, Ellis L.; Stamatatos, Leonidas; Strong, Roland K.

    2010-01-01

    Human 4E10 is one of the broadest-specificity, HIV-1-neutralizing monoclonal antibodies known, recognizing a membrane-proximal linear epitope on gp41. The lipid cross-reactivity of 4E10 has been alternately suggested either to contribute to the apparent rarity of 4E10-like antibody responses in HIV

  5. Immunohistochemical identification of mast cells in formaldehyde-fixed tissue using monoclonal antibodies specific for tryptase.

    Science.gov (United States)

    Walls, A F; Jones, D B; Williams, J H; Church, M K; Holgate, S T

    1990-10-01

    An avidin-biotin enhanced immunoperoxidase procedure using monoclonal antibodies (AA1, AA3, and AA5) prepared against human mast cell tryptase resulted in intense staining of mast cells in paraffin-embedded tissue. The distribution of mast cells observed was similar to that seen when adjacent serial sections were stained using a standard procedure with toluidine blue, though the immunoperoxidase technique permitted the identification of significantly more mast cells. With monoclonal antibody AA1, immunostaining was entirely specific for mast cell granules, and there was negligible background staining in a range of tissues including lung, tonsil, colon, gastric mucosa, skin, and pituitary. There was no staining of antibody on basophils or on any other normal blood leukocyte. The technique was effective with tissue fixed in either Carnoy's or neutral buffered formalin, though the internal mast cell structure was better preserved with formaldehyde fixation. The immunoperoxidase staining procedure with monoclonal antibody AA1 is a highly specific and sensitive means for the detection of mast cells in routinely processed tissues.

  6. The Use of Monoclonal Antibodies in Human Prion Disease

    Science.gov (United States)

    Bodemer, Walter

    Detection of PrP and its pathological isoform(s) is the key to understanding the etiology and pathogenesis of transmissible spongiform encephalopathy. There is ample evidence that PrP isoforms constitute a major component of an unknown and perhaps unconventional infectious agent. An etiological relationship between human and zoonotic transmissible spongiform encephalopathies may be revealed with monoclonal antibodies. Knowledge of the conformational transition rendering a nonpathogenic, almost ubiquitous cellular protein into a pathogenic one is crucial to defining pathomechanisms. The stepwise or even continuous formation of pathogenic molecules can be monitored. Any improvement in the early diagnosis could help to conceive new therapeutic measures which are not currently available. Determination of PrP isoforms in tissue, cells, or body fluids may be of prognostic value. Many experimental approaches in molecular medicine and molecular biology of the prion protein already rely on monoclonal antibodies. Recombinant antibodies such as the single-chain Fv may soon replace traditional hybridoma techniques. Binding affinity can easily be manipulated by a number of techniques, including in vitro mutagenesis - a step which could never be carried out using the traditional hybridoma technology. Monoclonal antibodies are and will remain an essential support for ongoing research on the prion protein in general and on the unconventional infectious prions.

  7. Challenges and opportunities for monoclonal antibody therapy in veterinary oncology.

    Science.gov (United States)

    Beirão, Breno C B; Raposo, Teresa; Jain, Saurabh; Hupp, Ted; Argyle, David J

    2016-12-01

    Monoclonal antibodies (mAbs) have come to dominate the biologics market in human cancer therapy. Nevertheless, in veterinary medicine, very few clinical trials have been initiated using this form of therapy. Some of the advantages of mAb therapeutics over conventional drugs are high specificity, precise mode of action and long half-life, which favour infrequent dosing of the antibody. Further advancement in the field of biomedical sciences has led to the production of different forms of antibodies, such as single chain antibody fragment, Fab, bi-specific antibodies and drug conjugates for use in diagnostic and therapeutic purposes. This review describes the potential for mAbs in veterinary oncology in supporting both diagnosis and therapy of cancer. The technical and financial hurdles to facilitate clinical acceptance of mAbs are explored and insights into novel technologies and targets that could support more rapid clinical development are offered. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    NARCIS (Netherlands)

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

    1999-01-01

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

  9. The neutralization of interferons by antibody. I. Quantitative and theoretical analyses of the neutralization reaction in different bioassay systems.

    Science.gov (United States)

    Grossberg, S E; Kawade, Y; Kohase, M; Yokoyama, H; Finter, N

    2001-09-01

    The highly specific ability of antibodies to inhibit the biologic activity of cytokines or other therapeutic proteins is widely used in research and a subject of increasing clinical importance. The need exists for a standardized approach to the reporting of neutralizing antibody potency soundly based on theoretical and practical considerations and tested by experimental data. Pursuant to the original studies of Kawade on the theoretical and functional aspects of neutralization of interferons (IFN), experimental data were obtained by different laboratories employing varied methodology to address two hypotheses concerning the nature of IFN neutralization reactions, based on a derived formula that allows expression of neutralizing power as the reduction of 10 laboratory units (LU)/ml to 1 LU/ml, the end point of most bioassays. Two hypotheses are posed: (1) antibody acts to neutralize a fixed amount of biologically active IFN molecules, or (2) antibody reduces IFN activity in a set ratio of added/residual biologically active IFN. The first, or fixed amount, hypothesis relates to the reactivity of high-affinity antibodies neutralizing equimolar amounts of antigen, whereas the second, or constant proportion, hypothesis postulates a reduction in the ratio of total added IFN to residual active IFN molecules, such as a low-affinity antibody might exhibit. Analyses of data of the neutralization of IFN-alpha and IFN-beta are presented, employing human polyclonal antibodies and murine monoclonal antibodies (mAb). The theoretical constructs of Kawade are extended in the Appendix and correlated with new experimental data in the text. The data clearly indicate that the low-antibody affinity, constant proportion hypothesis, rather than the high-antibody affinity, fixed amount hypothesis, is applicable, if the bioassay is sensitive to IFN. The findings presented here and in the following paper (pp. 743-755, this issue) taken together provide the basis for a standardized method of

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

    Science.gov (United States)

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

    1993-04-01

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

  11. Structural basis for the antibody neutralization of Herpes simplex virus

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Cheng-Chung; Lin, Li-Ling [Academia Sinica, Taipei 115, Taiwan (China); Academia Sinica, Taipei 115, Taiwan (China); Chan, Woan-Eng [Development Center for Biotechnology, New Taipei City 221, Taiwan (China); Ko, Tzu-Ping [Academia Sinica, Taipei 115, Taiwan (China); Academia Sinica, Taipei 115, Taiwan (China); Lai, Jiann-Shiun [Development Center for Biotechnology, New Taipei City 221, Taiwan (China); Ministry of Economic Affairs, Taipei 100, Taiwan (China); Wang, Andrew H.-J., E-mail: ahjwang@gate.sinica.edu.tw [Academia Sinica, Taipei 115, Taiwan (China); Academia Sinica, Taipei 115, Taiwan (China); Taipei Medical University, Taipei 110, Taiwan (China)

    2013-10-01

    The gD–E317-Fab complex crystal revealed the conformational epitope of human mAb E317 on HSV gD, providing a molecular basis for understanding the viral neutralization mechanism. Glycoprotein D (gD) of Herpes simplex virus (HSV) binds to a host cell surface receptor, which is required to trigger membrane fusion for virion entry into the host cell. gD has become a validated anti-HSV target for therapeutic antibody development. The highly inhibitory human monoclonal antibody E317 (mAb E317) was previously raised against HSV gD for viral neutralization. To understand the structural basis of antibody neutralization, crystals of the gD ectodomain bound to the E317 Fab domain were obtained. The structure of the complex reveals that E317 interacts with gD mainly through the heavy chain, which covers a large area for epitope recognition on gD, with a flexible N-terminal and C-terminal conformation. The epitope core structure maps to the external surface of gD, corresponding to the binding sites of two receptors, herpesvirus entry mediator (HVEM) and nectin-1, which mediate HSV infection. E317 directly recognizes the gD–nectin-1 interface and occludes the HVEM contact site of gD to block its binding to either receptor. The binding of E317 to gD also prohibits the formation of the N-terminal hairpin of gD for HVEM recognition. The major E317-binding site on gD overlaps with either the nectin-1-binding residues or the neutralizing antigenic sites identified thus far (Tyr38, Asp215, Arg222 and Phe223). The epitopes of gD for E317 binding are highly conserved between two types of human herpesvirus (HSV-1 and HSV-2). This study enables the virus-neutralizing epitopes to be correlated with the receptor-binding regions. The results further strengthen the previously demonstrated therapeutic and diagnostic potential of the E317 antibody.

  12. Autologous HIV-1 neutralizing antibodies: emergence of neutralization-resistant escape virus and subsequent development of escape virus neutralizing antibodies

    DEFF Research Database (Denmark)

    Arendrup, M; Nielsen, C; Hansen, J E;

    1992-01-01

    The capacity of consecutive human sera to neutralize sequentially obtained autologous virus isolates was studied. HIV-1 was isolated three times over a 48-164-week period from three individuals immediately after seroconversion and from two individuals in later stages of infection. Development of ...... escape virus may be part of the explanation of the apparent failure of the immune system to control HIV infection.......The capacity of consecutive human sera to neutralize sequentially obtained autologous virus isolates was studied. HIV-1 was isolated three times over a 48-164-week period from three individuals immediately after seroconversion and from two individuals in later stages of infection. Development...... of neutralizing antibodies to the primary virus isolates was detected 13-45 weeks after seroconversion. Emergence of escape virus with reduced sensitivity to neutralization by autologous sera was demonstrated. The patients subsequently developed neutralizing antibodies against the escape virus but after a delay...

  13. Production of monoclonal antibodies to human glomerular basement membrane.

    Directory of Open Access Journals (Sweden)

    Mino,Yasuaki

    1984-10-01

    Full Text Available Using the technique of somatic cell fusion, we produced monoclonal antibodies to collagenase-digested human glomerular basement membrane (GBM. Fourteen monoclonal antibodies which reacted with normal human kidney in indirect immunofluorescence (IIF studies were produced. An analysis of the binding patterns indicated that the antigens recognized could be divided into six broad groups. Monoclonal antibody B3-H10 (Group 1 reacted with only GBM in a fine granular pattern. A5-B12 and B5-C2 (Group 2 reacted with GBM and peritubular capillary in a linear pattern. B2-A12 (Group 3 reacted with only epithelial cells. Al-C9 and A4-E2 (Group 4 showed a mesangial pattern in glomerulus and a lineal pattern in tubular basement membrane (TBM, Bowman's capsule and peritubular capillary. A1-E1, A1-E11, A2-E6, A3-B6, A4-F8 and B5-H2 (Group 5 recognized determinants common to GBM, TBM, Bowman's capsule and/or peritubular capillary. A3-F1 and B5-E10 (Group 6 reacted with TBM and Bowman's capsule. The staining pattern of B3-H10 (Group 1 was characteristic because it was not linear, but finely granular along the GBM. The staining pattern of B2-A12 (Group 3 was also characteristic because only epithelial cells were stained, and processes of epithelial cells were observed as fine fibrils. To the best of our knowledge, these two types of monoclonal antibodies have not been reported previously.

  14. Strategies for Treating Autoimmune Disease With Monoclonal Antibodies

    OpenAIRE

    Wofsy, David

    1985-01-01

    There is no safe and reliable therapy for most serious autoimmune diseases, such as systemic lupus erythematosus. Severe cases usually require treatment with corticosteroids or cytotoxic drugs or both, which frequently provide inadequate disease control and can cause serious complications. These therapies are not restricted in their effects to cells of the immune system, but rather have a broad range of toxic effects on cells throughout the body. The development of monoclonal antibodies has l...

  15. Quantification of Moraxella bovis haemagglutinating adhesins with monoclonal antibodies.

    Science.gov (United States)

    Gil-Turnes, C; Aleixo, J A

    1991-08-01

    Six monoclonal antibodies (MAbs) against Moraxella bovis GF 9 were used to quantify haemagglutinating adhesins of 16 strains of this organism. The amount of each MAb necessary to inhibit one haemagglutinating unit of each strain varied between 4 and 0.007 times that required by strain GF 9. Five strains reacted with six MAbs, one with five, two with four, one with three, two with two and three with none. The procedures used enabled to detect dominant strains candidates for vaccines.

  16. Monoclonal antibody to native P39 protein from Borrelia burgdorferi.

    OpenAIRE

    Sullivan, T J; Hechemy, K E; Harris, H L; Rudofsky, U H; Samsonoff, W A; Peterson, A J; Evans, B. D.; Balaban, S L

    1994-01-01

    We have produced, by using a sonicate of Borrelia burgdorferi, a monoclonal antibody (MAb), NYSP39H, that is specific for the P39 protein band. This MAb reacted with 13 isolates of B. burgdorferi but not with eight different spirochetes (four borrelias, two leptospiras, and two treponemas). Surface labeling of B. burgdorferi with biotin and subsequent treatment with Nonidet P-40 showed that P39 was not biotinylated but was extracted with Nonidet P-40, indicating that it is present within the ...

  17. Monoclonal Antibodies to the Thyrotropin Receptor

    Directory of Open Access Journals (Sweden)

    Takao Ando

    2005-01-01

    Full Text Available The thyrotropin receptor (TSHR is a seven transmembrane G-protein linked glycoprotein expressed on the thyroid cell surface and which, under the regulation of TSH, controls the production and secretion of thyroid hormone from the thyroid gland. This membrane protein is also a major target antigen in the autoimmune thyroid diseases. In Graves' disease, autoantibodies to the TSHR (TSHR-Abs stimulate the TSHR to produce thyroid hormone excessively. In autoimmune thyroid failure, some patients exhibit TSHR-Abs which block TSH action on the receptor. There have been many attempts to generate human stimulating TSHR-mAbs, but to date, only one pathologically relevant human stimulating TSHR-mAb has been isolated. Most mAbs to the TSHR have been derived from rodents immunized with TSHR antigen from bacteria or insect cells. These antigens lacked the native conformation of the TSHR and the resulting mAbs were exclusively blocking or neutral TSHR-mAbs. However, mAbs raised against intact native TSHR antigen have included stimulating mAbs. One such stimulating mAb has demonstrated a number of differences in its regulation of TSHR post-translational processing. These differences are likely to be reflective of TSHR-Abs seen in Graves' disease.

  18. Monoclonal antibodies in animal production; their use in diagnostics and passive immunization.

    NARCIS (Netherlands)

    Booman, P.

    1989-01-01

    One of the landmarks in immunology was the invention and development of monoclonal antibody-secreting hybridomas by Milstein and his coworkers. The enormous promise of monoclonal antibody technology, which became apparent soon after its discovery, may explain the unusual speed with which monoclonal

  19. De Novo Sequencing and Resurrection of a Human Astrovirus-Neutralizing Antibody.

    Science.gov (United States)

    Bogdanoff, Walter A; Morgenstern, David; Bern, Marshall; Ueberheide, Beatrix M; Sanchez-Fauquier, Alicia; DuBois, Rebecca M

    2016-05-13

    Monoclonal antibody (mAb) therapeutics targeting cancer, autoimmune diseases, inflammatory diseases, and infectious diseases are growing exponentially. Although numerous panels of mAbs targeting infectious disease agents have been developed, their progression into clinically useful mAbs is often hindered by the lack of sequence information and/or loss of hybridoma cells that produce them. Here we combine the power of crystallography and mass spectrometry to determine the amino acid sequence and glycosylation modification of the Fab fragment of a potent human astrovirus-neutralizing mAb. We used this information to engineer a recombinant antibody single-chain variable fragment that has the same specificity as the parent monoclonal antibody to bind to the astrovirus capsid protein. This antibody can now potentially be developed as a therapeutic and diagnostic agent.

  20. Antibody function in neutralization and protection against HIV-1

    NARCIS (Netherlands)

    Hessell, A.J.

    2009-01-01

    The ability to induce neutralizing antibodies is generally thought to be of great importance for vaccine efficacy. In HIV-1 research this quality has been elusive as the HIV-1 virus has evolved multiple mechanisms to evade neutralizing antibodies. This thesis traces studies with four broadly neutral

  1. Identification and Characterization of a Broadly Cross-Reactive HIV-1 Human Monoclonal Antibody That Binds to Both gp120 and gp41

    NARCIS (Netherlands)

    Zhang, Mei-Yun; Yuan, Tingting; Li, Jingjing; Borges, Andrew Rosa; Watkins, Jennifer D.; Guenaga, Javier; Yang, Zheng; Wang, Yanping; Wilson, Richard; Li, Yuxing; Polonis, Victoria R.; Pincus, Seth H.; Ruprecht, Ruth M.; Dimitrov, Dimiter S.

    2012-01-01

    Identification of broadly cross-reactive HIV-1-neutralizing antibodies (bnAbs) may assist vaccine immunogen design. Here we report a novel human monoclonal antibody (mAb), designated m43, which co-targets the gp120 and gp41 subunits of the HIV-1 envelope glycoprotein (Env). M43 bound to recombinant

  2. Ofatumumab: a novel monoclonal anti-CD20 antibody

    Directory of Open Access Journals (Sweden)

    Thomas S Lin

    2010-05-01

    Full Text Available Thomas S LinGlaxoSmithKline Oncology R&D, Collegeville, PA, USAAbstract: Ofatumumab, a novel humanized monoclonal anti-CD20 antibody, was recently approved by the FDA for the treatment of fludarabine and alemtuzumab refractory chronic lymphocytic leukemia (CLL. Ofatumumab effectively induces complement-dependent cytotoxicity (CDC in vitro, and recent studies demonstrated that ofatumumab also effectively mediates antibody-dependent cellular cytotoxicity (ADCC. Pharmacokinetic studies indicated that increased exposure to the antibody correlated with improved clinical outcome in CLL. Thus, pharmacogenomics may be important in identifying which patients are more likely to respond to ofatumumab therapy, although such studies have not yet been performed. Patients with the high-affinity FCGR3a 158 V/V polymorphism may be more likely to respond to therapy, if ADCC is the primary in vivo mechanism of action of ofatumumab. Patients with increased expression of the complement defense proteins CD55 and CD59 may be less likely to respond if ofatumumab works in vivo primarily via CDC. Patients with increased metabolism and clearance of ofatumumab may have lower exposure and be less likely to respond clinically. Thus, pharmacogenomics may determine the responsiveness of patients to ofatumumab therapy.Keywords: monoclonal antibody, CD20, CLL, NHL, lymphoma

  3. Monoclonal Antibody Production against Human Spermatozoal Surface Antigens

    Directory of Open Access Journals (Sweden)

    M Jedi-Tehrani

    2005-10-01

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

  4. Prediction and Reduction of the Aggregation of Monoclonal Antibodies.

    Science.gov (United States)

    van der Kant, Rob; Karow-Zwick, Anne R; Van Durme, Joost; Blech, Michaela; Gallardo, Rodrigo; Seeliger, Daniel; Aßfalg, Kerstin; Baatsen, Pieter; Compernolle, Griet; Gils, Ann; Studts, Joey M; Schulz, Patrick; Garidel, Patrick; Schymkowitz, Joost; Rousseau, Frederic

    2017-04-21

    Protein aggregation remains a major area of focus in the production of monoclonal antibodies. Improving the intrinsic properties of antibodies can improve manufacturability, attrition rates, safety, formulation, titers, immunogenicity, and solubility. Here, we explore the potential of predicting and reducing the aggregation propensity of monoclonal antibodies, based on the identification of aggregation-prone regions and their contribution to the thermodynamic stability of the protein. Although aggregation-prone regions are thought to occur in the antigen binding region to drive hydrophobic binding with antigen, we were able to rationally design variants that display a marked decrease in aggregation propensity while retaining antigen binding through the introduction of artificial aggregation gatekeeper residues. The reduction in aggregation propensity was accompanied by an increase in expression titer, showing that reducing protein aggregation is beneficial throughout the development process. The data presented show that this approach can significantly reduce liabilities in novel therapeutic antibodies and proteins, leading to a more efficient path to clinical studies. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  5. Efficient generation of human IgA monoclonal antibodies.

    Science.gov (United States)

    Lorin, Valérie; Mouquet, Hugo

    2015-07-01

    Immunoglobulin A (IgA) is the most abundant antibody isotype produced in humans. IgA antibodies primarily ensure immune protection of mucosal surfaces against invading pathogens, but also circulate and are present in large quantities in blood. IgAs are heterogeneous at a molecular level, with two IgA subtypes and the capacity to form multimers by interacting with the joining (J) chain. Here, we have developed an efficient strategy to rapidly generate human IgA1 and IgA2 monoclonal antibodies in their monomeric and dimeric forms. Recombinant monomeric and dimeric IgA1/IgA2 counterparts of a prototypical IgG1 monoclonal antibody, 10-1074, targeting the HIV-1 envelope protein, were produced in large amounts after expression cloning and transient transfection of 293-F cells. 10-1074 IgAs were FPLC-purified using a novel affinity-based resin engrafted with anti-IgA chimeric Fabs, followed by a monomers/multimers separation using size exclusion-based FPLC. ELISA binding experiments confirmed that the artificial IgA class switching of 10-1074 did not alter its antigen recognition. In summary, our technical approach allows the very efficient production of various forms of purified recombinant human IgA molecules, which are precious tools in dissecting IgA B-cell responses in physiological and pathophysiological conditions, and studying the biology, function and therapeutic potential of IgAs.

  6. Radiolabelled peptides and monoclonal antibodies for therapy decision making in inflammatory diseases

    NARCIS (Netherlands)

    Malviya, G.; Signore, A.; Lagana, B.; Dierckx, R. A.

    2008-01-01

    Radiolabelled peptides and monoclonal antibodies are an emerging class of radiopharmaceuticals for imaging inflammation with clinical implications for several chronic inflammatory disorders for diagnosis, therapy decision making and follow up. In the last decades, a number of novel monoclonal antibo

  7. 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.

  8. Molecular Characterization of the Monoclonal Antibodies Composing ZMAb: A Protective Cocktail Against Ebola Virus

    Science.gov (United States)

    Audet, Jonathan; Wong, Gary; Wang, Han; Lu, Guangwen; Gao, George F.; Kobinger, Gary; Qiu, Xiangguo

    2014-01-01

    Ebola virus (EBOV) causes severe viral hemorrhagic fever in humans and non-human primates, with a case fatality rate of up to 88% in human outbreaks. Over the past 3 years, monoclonal antibody (mAb) cocktails have demonstrated high efficacy as treatments against EBOV infection. One such cocktail is ZMAb, which consists of three mouse antibodies, 1H3, 2G4, and 4G7. Here, we present the epitope binding properties of mAbs 1H3, 2G4, and 4G7. We showed that these antibodies have different variable region sequences, suggesting that the individual mAbs are not clonally related. All three antibodies were found to neutralize EBOV variant Mayinga. Additionally, 2G4 and 4G7 were shown to cross-inhibit each other in vitro and select for an escape mutation at the same position on the EBOV glycoprotein (GP), at amino acid 508. 1H3 selects an escape mutant at amino acid 273 on EBOV GP. Surface plasmon resonance studies showed that all three antibodies have dissociation constants on the order of 10−7. In combination with previous studies evaluating the binding sites of other protective antibodies, our results suggest that antibodies targeting the GP1-GP2 interface and the glycan cap are often selected as efficacious antibodies for post-exposure interventions against EBOV. PMID:25375093

  9. Modulation of desmin intermediate filament assembly by a monoclonal antibody

    Science.gov (United States)

    1988-01-01

    We have used a monoclonal antibody against desmin to examine the assembly of intermediate filaments (IF) from their building blocks, the tetrameric protofilaments. The antibody, designated D76, does not cross react with any other IF proteins (Danto, S.I., and D.A. Fischman. 1984. J. Cell Biol. 98:2179-2191). It binds to a region amino-terminal to cys- 324 of avian desmin that is resistant to chymotrypsin and trypsin digestion, and in the electron microscope appears to bind to the ends of tetrameric protofilaments. In combination, these findings suggest that the epitope of the antibody resides at the amino-terminal end of the alpha-helical rod domain. Preincubation of desmin protofilaments with an excess of D76 antibodies blocks their subsequent assembly into IF. In the presence of sub-stoichiometric amounts of antibodies, IF are assembled from protofilaments but they are morphologically aberrant in that (a) they are capped by IgG molecules at one or both ends; (b) they are unraveled to varying degree, revealing a characteristic right- handed helical arrangement of sub-filamentous strands of different diameters. The antibody binds only to the ends but not along the length of desmin IF. The most straightforward explanation for this is that the epitope resides in a part of the desmin molecule that becomes buried within the core of the filament upon polymerization and is therefore inaccessible to the antibody. PMID:2450097

  10. Serrumab: a novel human single chain-fragment antibody with multiple scorpion toxin-neutralizing capacities.

    Science.gov (United States)

    Pucca, Manuela Berto; Cerni, Felipe Augusto; Peigneur, Steve; Arantes, Eliane Candiani; Tytgat, Jan; Barbosa, José Elpidio

    2014-01-01

    In Brazil, scorpion envenomation is an important public health problem. The yellow scorpion, Tityus serrulatus (Ts), is considered the most dangerous species in the country, being responsible for the most severe clinical cases of envenomation. Currently, the administration of serum produced in horses is recognized and used as a treatment for accidents with scorpions. However, horse herds' maintenance is costly and the antibodies are heterologous, which can cause anaphylaxis and Serum Sickness. In the present work, a human monoclonal fragment antibody, Serrumab, has been analysed. Toxin neutralizing effects of Serrumab were evaluated using a two-electrode voltage-clamp technique. The results show that Serrumab presented a high neutralizing effect against Ts β-toxins (Ts1, 43.2% and Ts2, 68.8%) and none or low neutralizing effect against α-toxins (Ts3, 0% and Ts5, 10%). Additional experiments demonstrated that Serrumab was also able to neutralize the action of toxins from other scorpion genus (Css II, 45.96% and Lqh III, 100%/β- and α-toxins, respectively). This work indicated that Serrumab is able to neutralize many toxins in Ts venom, and could being considered as a neutralizing antibody for formulating a human anti-scorpion serum in Brazil. Additionally, this work demonstrated that Serrumab could neutralize different toxins from distinct scorpion genus. All these results reinforce the idea that Serrumab is a scFv antibody with multiple neutralizing capacities and a promising candidate for inclusion in scorpion anti-venoms against different genera.

  11. Monitoring monoclonal antibody delivery in oncology: the example of bevacizumab.

    Directory of Open Access Journals (Sweden)

    Guillaume Nugue

    Full Text Available Developing therapeutic monoclonal antibodies paves the way for new strategies in oncology using targeted therapy which should improve specificity. However, due to a lack of biomarkers, a personalized therapy scheme cannot always be applied with monoclonal antibodies. As a consequence, the efficacy or side effects associated with this type of treatment often appear to be sporadic. Bevacizumab is a therapeutic monoclonal antibody targeting Vascular Endothelial Growth Factor (VEGF. It is used to limit tumor vascularization. No prognosis or response biomarker is associated with this antibody, we therefore assessed whether the administration protocol could be a possible cause of heterogeneous responses (or variable efficacy. To do this, we developed a bevacizumab assay with a broad sensitivity range to measure blood bevacizumab concentrations. We then analyzed bevacizumab concentrations in 17 patients throughout the first quarter of treatment. In line with previously published data, average blood concentrations were 88+/-27 mg/L following the first dose administered, and 213+/-105 mg/L after the last (6(th dose administered. However, the individual values were scattered, with a mean 4-fold difference between the lowest and the highest concentration for each dose administered. We demonstrated that the bevacizumab administration schedule results in a high inter-individual variability in terms of blood concentrations. Comparison of assay data with clinical data indicates that blood concentrations above the median are associated with side effects, whereas values below the median favor inefficacy. In conclusion, bevacizumab-based therapy could benefit from a personalized administration schedule including follow-up and adjustment of circulating bevacizumab concentrations.

  12. Mechanism of human antibody-mediated neutralization of Marburg virus.

    Science.gov (United States)

    Flyak, Andrew I; Ilinykh, Philipp A; Murin, Charles D; Garron, Tania; Shen, Xiaoli; Fusco, Marnie L; Hashiguchi, Takao; Bornholdt, Zachary A; Slaughter, James C; Sapparapu, Gopal; Klages, Curtis; Ksiazek, Thomas G; Ward, Andrew B; Saphire, Erica Ollmann; Bukreyev, Alexander; Crowe, James E

    2015-02-26

    The mechanisms by which neutralizing antibodies inhibit Marburg virus (MARV) are not known. We isolated a panel of neutralizing antibodies from a human MARV survivor that bind to MARV glycoprotein (GP) and compete for binding to a single major antigenic site. Remarkably, several of the antibodies also bind to Ebola virus (EBOV) GP. Single-particle EM structures of antibody-GP complexes reveal that all of the neutralizing antibodies bind to MARV GP at or near the predicted region of the receptor-binding site. The presence of the glycan cap or mucin-like domain blocks binding of neutralizing antibodies to EBOV GP, but not to MARV GP. The data suggest that MARV-neutralizing antibodies inhibit virus by binding to infectious virions at the exposed MARV receptor-binding site, revealing a mechanism of filovirus inhibition.

  13. Membrane adsorbers as purification tools for monoclonal antibody purification.

    Science.gov (United States)

    Boi, Cristiana

    2007-03-15

    Downstream purification processes for monoclonal antibody production typically involve multiple steps; some of them are conventionally performed by bead-based column chromatography. Affinity chromatography with Protein A is the most selective method for protein purification and is conventionally used for the initial capturing step to facilitate rapid volume reduction as well as separation of the antibody. However, conventional affinity chromatography has some limitations that are inherent with the method, it exhibits slow intraparticle diffusion and high pressure drop within the column. Membrane-based separation processes can be used in order to overcome these mass transfer limitations. The ligand is immobilized in the membrane pores and the convective flow brings the solute molecules very close to the ligand and hence minimizes the diffusional limitations associated with the beads. Nonetheless, the adoption of this technology has been slow because membrane chromatography has been limited by a lower binding capacity than that of conventional columns, even though the high flux advantages provided by membrane adsorbers would lead to higher productivity. This review considers the use of membrane adsorbers as an alternative technology for capture and polishing steps for the purification of monoclonal antibodies. Promising industrial applications as well as new trends in research will be addressed.

  14. Monoclonal Antibody Therapy and Renal Transplantation: Focus on Adverse Effects

    Directory of Open Access Journals (Sweden)

    Gianluigi Zaza

    2014-02-01

    Full Text Available A series of monoclonal antibodies (mAbs are commonly utilized in renal transplantation as induction therapy (a period of intense immunosuppression immediately before and following the implant of the allograft, to treat steroid-resistant acute rejections, to decrease the incidence and mitigate effects of delayed graft function, and to allow immunosuppressive minimization. Additionally, in the last few years, their use has been proposed for the treatment of chronic antibody-mediated rejection, a major cause of late renal allograft loss. Although the exact mechanism of immunosuppression and allograft tolerance with any of the currently used induction agents is not completely defined, the majority of these medications are targeted against specific CD proteins on the T or B cells surface (e.g., CD3, CD25, CD52. Moreover, some of them have different mechanisms of action. In particular, eculizumab, interrupting the complement pathway, is a new promising treatment tool for acute graft complications and for post-transplant hemolytic uremic syndrome. While it is clear their utility in renal transplantation, it is also unquestionable that by using these highly potent immunosuppressive agents, the body loses much of its innate ability to mount an adequate immune response, thereby increasing the risk of severe adverse effects (e.g., infections, malignancies, haematological complications. Therefore, it is extremely important for clinicians involved in renal transplantation to know the potential side effects of monoclonal antibodies in order to plan a correct therapeutic strategy minimizing/avoiding the onset and development of severe clinical complications.

  15. Monoclonal anti-elastin antibody labelled with technetium-99m

    Energy Technology Data Exchange (ETDEWEB)

    Oliveira, Marcia B.N. de; Silva, Claudia R. da; Araujo, Adriano C. de; Bernardo Filho, Mario [Universidade do Estado, Rio de Janeiro, RJ (Brazil). Instituto de Biologia Roberto Alcantara Gomes. Lab. de Radiofarmacia; Porto, Luis Cristovao M.S.; Gutfilen, Bianca [Universidade do Estado, Rio de Janeiro, RJ (Brazil). Instituto de Biologia Roberto Alcantara Gomes; Souza, J.E.Q. [Instituto Nacional do Cancer, Rio de Janeiro, RJ (Brazil). Centro de Pesquisa Basica; Frier, Malcolm [University Hospital, Nottingham (United Kingdom). Dept. of Medical Physics

    1999-11-01

    Technetium-99m ({sup 99m} Tc) is widely employed in nuclear medicine due to its desirable physical, chemical and biological properties. Moreover, it is easily available and normally is inexpensive. A reducing agent is necessary to label cells and molecules with {sup 99m} Tc and stannous chloride (Sn C L{sub 2}) is usually employed. Elastin is the functional protein component of the elastic fiber and it is related with some diseases such as arteriosclerosis, pulmonary emphysema and others. The present study refers to the preparation of the {sup 99m} Tc labeled monoclonal anti-elastin antibody. The monoclonal antibody was incubated with an excess of 2-iminothiolane. The free thiol groups created, were capable of binding with the reduced technetium. Labeling was an exchange reaction with {sup 99m} Tc-glucoheptonate. The labeled preparation was left at 4 deg C for one hour. Then, it was passed through a Sephadex G50 column. Various fractions were collected and counted. A peak corresponding to the radiolabeled antibody was obtained. Stability studies of the labelled anti-elastin were performed at 0,3 6, 24 hours, at both 4 deg C or room temperature. The biodistribution pattern of the {sup 99m} Tc-anti-elastin was studied in healthy male Swiss mice. The immunoreactivity was also determined. An useful labeled-anti-elastin was obtained to future immunoscintigraphic investigations. (author) 4 refs., 7 figs., 6 tabs.

  16. Characterization of oxidative carbonylation on recombinant monoclonal antibodies.

    Science.gov (United States)

    Yang, Yi; Stella, Cinzia; Wang, Weiru; Schöneich, Christian; Gennaro, Lynn

    2014-05-20

    In the biotechnology industry, oxidative carbonylation as a post-translational modification of protein pharmaceuticals has not been studied in detail. Using Quality by Design (QbD) principles, understanding the impact of oxidative carbonylation on product quality of protein pharmaceuticals, particularly from a site-specific perspective, is critical. However, comprehensive identification of carbonylation sites has so far remained a very difficult analytical challenge for the industry. In this paper, we report for the first time the identification of specific carbonylation sites on recombinant monoclonal antibodies with a new analytical approach via derivatization with Girard's Reagent T (GRT) and subsequent peptide mapping with high-resolution mass spectrometry. Enhanced ionization efficiency and high quality MS(2) data resulted from GRT derivatization were observed as key benefits of this approach, which enabled direct identification of carbonylation sites without any fractionation or affinity enrichment steps. A simple data filtering process was also incorporated to significantly reduce false positive assignments. Sensitivity and efficiency of this approach were demonstrated by identification of carbonylation sites on both unstressed and oxidized antibody bulk drug substances. The applicability of this approach was further demonstrated by identification of 14 common carbonylation sites on three highly similar IgG1s. Our approach represents a significant improvement to the existing analytical methodologies and facilitates extended characterization of oxidative carbonylation on recombinant monoclonal antibodies and potentially other protein pharmaceuticals in the biotechnology industry.

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

    Science.gov (United States)

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

    2014-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Laura E McCoy

    2014-12-01

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

  19. Molecular Evolution of Broadly Neutralizing Llama Antibodies to the CD4-Binding Site of HIV-1

    Science.gov (United States)

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

    2014-01-01

    To date, no immunization of humans or animals has elicited broadly neutralizing sera able to prevent HIV-1 transmission; however, elicitation of broad and potent heavy chain only antibodies (HCAb) has previously been reported in llamas. In this study, the anti-HIV immune responses in immunized llamas were studied via deep sequencing analysis using broadly neutralizing monoclonal HCAbs as a guides. Distinct neutralizing antibody lineages were identified in each animal, including two defined by novel antibodies (as variable regions called VHH) identified by robotic screening of over 6000 clones. The combined application of five VHH against viruses from clades A, B, C and CRF_AG resulted in neutralization as potent as any of the VHH individually and a predicted 100% coverage with a median IC50 of 0.17 µg/ml for the panel of 60 viruses tested. Molecular analysis of the VHH repertoires of two sets of immunized animals showed that each neutralizing lineage was only observed following immunization, demonstrating that they were elicited de novo. Our results show that immunization can induce potent and broadly neutralizing antibodies in llamas with features similar to human antibodies and provide a framework to analyze the effectiveness of immunization protocols. PMID:25522326

  20. Characterization of two anti-dengue human monoclonal antibodies prepared from PBMCs of patients with dengue illness in Thailand.

    Science.gov (United States)

    Li, Z-Y; Yamashita, A; Kawashita, N; Sasaki, T; Pan, Y; Ono, K-I; Ikuta, K; Li, Y-G

    2016-06-01

    The global spread of the four dengue virus (DENV) serotypes (dengue-1 to -4) has made this virus a major and growing public health concern. Generally, pre-existing neutralizing antibodies derived from primary infection play a significant role in protecting against subsequent infection with the same serotype. By contrast, these pre-existing antibodies are believed to mediate a non-protective response to subsequent heterotypic DENV infections, leading to the onset of dengue illness. In this study, two monoclonal antibodies prepared by using peripheral blood mononuclear cells (PBMCs) from patients with dengue fever were characterized. Epitope mapping revealed that amino acid residues 254-278 in domain II of the viral envelope protein E were the target region of these antibodies. A database search revealed that certain sequences in this epitope region showed high conservation among the four serotypes of DENV. These two human monoclonal antibodies could neutralize DENV-2,-4 more effectively than DENV-1,-3. The amino acid sequences could not explain this difference in neutralizing activity. However, the 3D structure results showed that amino acid 274 could be the critical residue for the difference in neutralization. These results may provide basic information for the development of a dengue vaccine.

  1. Improved detection of Pneumocystis carinii by an immunofluorescence technique using monoclonal antibodies

    DEFF Research Database (Denmark)

    Orholm, M; Holten-Andersen, W; Lundgren, Jens Dilling

    1990-01-01

    To assess whether a recently developed indirect immunofluorescent stain using monoclonal antibodies was more sensitive in detecting Pneumocystis carinii than the combination of Giemsa and methenamine silver nitrate stains which has routinely been used in the laboratory, 88 lavage fluid specimens...... and 34 induced sputum specimens were examined. All specimens were stained by five techniques: immunofluorescence using a combination of three monoclonal antibodies (from the National Institutes of Health, USA), immunofluorescence using a single monoclonal antibody (from Dakopatts), Giemsa, methenamine...

  2. High-efficiency screening of monoclonal antibodies for membrane protein crystallography.

    Directory of Open Access Journals (Sweden)

    Hyun-Ho Lim

    Full Text Available Determination of crystal structures of membrane proteins is often limited by difficulties obtaining crystals diffracting to high resolution. Co-crystallization with Fab fragments of monoclonal antibodies has been reported to improve diffraction of membrane proteins crystals. However, it is not simple to generate useful monoclonal antibodies for membrane protein crystallography. In this report, we present an optimized process for efficient screening from immunization to final validation of monoclonal antibody for membrane protein crystallography.

  3. Lack of Durable Cross-Neutralizing Antibodies Against Zika Virus from Dengue Virus Infection.

    Science.gov (United States)

    Collins, Matthew H; McGowan, Eileen; Jadi, Ramesh; Young, Ellen; Lopez, Cesar A; Baric, Ralph S; Lazear, Helen M; de Silva, Aravinda M

    2017-05-01

    Cross-reactive antibodies elicited by dengue virus (DENV) infection might affect Zika virus infection and confound serologic tests. Recent data demonstrate neutralization of Zika virus by monoclonal antibodies or human serum collected early after DENV infection. Whether this finding is true in late DENV convalescence (>6 months after infection) is unknown. We studied late convalescent serum samples from persons with prior DENV or Zika virus exposure. Despite extensive cross-reactivity in IgG binding, Zika virus neutralization was not observed among primary DENV infections. We observed low-frequency (23%) Zika virus cross-neutralization in repeat DENV infections. DENV-immune persons who had Zika virus as a secondary infection had distinct populations of antibodies that neutralized DENVs and Zika virus, as shown by DENV-reactive antibody depletion experiments. These data suggest that most DENV infections do not induce durable, high-level Zika virus cross-neutralizing antibodies. Zika virus-specific antibody populations develop after Zika virus infection irrespective of prior DENV immunity.

  4. Selection of peptide mimics of HIV-1 epitope recognized by neutralizing antibody VRC01.

    Directory of Open Access Journals (Sweden)

    Anton N Chikaev

    Full Text Available The ability to induce anti-HIV-1 antibodies that can neutralize a broad spectrum of viral isolates from different subtypes seems to be a key requirement for development of an effective HIV-1 vaccine. The epitopes recognized by the most potent broadly neutralizing antibodies that have been characterized are largely discontinuous. Mimetics of such conformational epitopes could be potentially used as components of a synthetic immunogen that can elicit neutralizing antibodies. Here we used phage display technology to identify peptide motifs that mimic the epitope recognized by monoclonal antibody VRC01, which is able to neutralize up to 91% of circulating primary isolates. Three rounds of biopanning were performed against 2 different phage peptide libraries for this purpose. The binding specificity of selected phage clones to monoclonal antibody VRC01 was estimated using dot blot analysis. The putative peptide mimics exposed on the surface of selected phages were analyzed for conformational and linear homology to the surface of HIV-1 gp120 fragment using computational analysis. Corresponding peptides were synthesized and checked for their ability to interfere with neutralization activity of VRC01 in a competitive inhibition assay. One of the most common peptides selected from 12-mer phage library was found to partially mimic a CD4-binding loop fragment, whereas none of the circular C7C-mer peptides was able to mimic any HIV-1 domains. However, peptides identified from both the 12-mer and C7C-mer peptide libraries showed rescue of HIV-1 infectivity in the competitive inhibition assay. The identification of epitope mimics may lead to novel immunogens capable of inducing broadly reactive neutralizing antibodies.

  5. Alemtuzumab and Natalizumab: The Monoclonal Antibody Story Continues

    Directory of Open Access Journals (Sweden)

    BL Johnston

    2006-01-01

    Full Text Available In the July/August 2006 issue of this journal, the infectious complications associated with the use of infliximab, etanercept and adalimumab were reviewed (1. These represent only three of the many monoclonal antibodies either licensed or in clinical trials for therapeutic use in cancer and autoimmune disease or to prevent rejection in both solid organ and hematopoietic stem cell transplantation. While most of these agents have not been associated with increased infection rates, alemtuzumab and natalizumab have gained particular attention related to either the frequency or type of infection seen in some individuals who have received them.

  6. Rapid analysis of small samples containing forskolin using monoclonal antibodies.

    Science.gov (United States)

    Yanagihara, H; Sakata, R; Shoyama, Y; Murakami, H

    1996-04-01

    The effective range of the competitive ELISA test for detection of forskolin content in clonally propagated plant organs of Coleus forskohlii using monoclonal antibodies extends from 5ng to 5 micrograms. A correlation between the forskolin accumulation and the growth rate was investigated using the clonally propagated shoots. An increase of forskolin content was noted, beginning at week 6. Flowers, rachises, leaves, stems, tuberous roots, and roots were analyzed. Tuberous roots and the stem base contained higher amounts of forskolin than other organs. The forskolin content in the stem decreased gradually towards the top of the shoot.

  7. Fluorescence polarization immunoassay for salinomycin based on monoclonal antibodies

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    A fluorescence polarization immunoassay(FPIA) for the determination of salinomycin(SAL) was developed by using anti-SAL monoclonal antibodies(mAb).Fluorescein labeled SAL(tracer) was synthesized by the N-hydroxysuccinimide active ester method and purified using thin layer chromatography(TLC).The developed FPIA for SAL had a dynamic range from 0.60 to 2193 ng/mL with an IC50 value of 33.2 ng/mL and a detection limit(LOD) of 0.08 ng/mL.No significant cross-reactivities were observed with other drugs but 67.6% with narasin.

  8. Monoclonal antibody therapy in the treatment of Reye's syndrome.

    Science.gov (United States)

    Treon, S P; Broitman, S A

    1992-11-01

    A role for lipopolysaccharides (endotoxins, LPS) in 7 the pathogenesis of Reye's syndrome (RS) has previously been suggested. Impairment of hepatic LPS clearance can lead to systemic endotoxemia as previous studies by this and other laboratories have suggested for several hepatic disorders including RS. Systemic LPS may mediate many of the clinical findings associated with RS by eliciting monokines such as tumor necrosis factor-alpha, interleukin-1, interleukin-6, and interleukin-8. Monoclonal antibody therapy directed at LPS, and monokines may represent a novel approach to the treatment of RS.

  9. Large-scale production of monoclonal antibodies in suspension culture.

    Science.gov (United States)

    Backer, M P; Metzger, L S; Slaber, P L; Nevitt, K L; Boder, G B

    1988-10-01

    Monoclonal antibodies are being manufactured for clinical trials in suspension culture at the 1300-L scale. Suspension culture offers some advantages relative to high-density mammalian cell culture methods; in particular, the ability to closely monitor the behavior of cells in a homogeneous environment. Computer control and on-line mass spectrography of exit gases provide instantaneous information about the culture metabolic activity. Air sparging and agitation by marine impeller provide aeration sufficient to maintain a constant dissolved oxygen tension at cell concentrations up to 5.0 x 10(6) cells/mL without causing apparent cell damage.

  10. Monoclonal Antibodies as Prophylactic and Therapeutic Agents Against Chikungunya Virus.

    Science.gov (United States)

    Clayton, April M

    2016-12-15

    Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that is responsible for considerable epidemics worldwide and recently emerged in the Americas in 2013. CHIKV may cause long-lasting arthralgia after acute infection. With currently no licensed vaccines or antivirals, the design of effective therapies to prevent or treat CHIKV infection is of utmost importance and will be facilitated by increased understanding of the dynamics of chikungunya. In this article, monoclonal antibodies against CHIKV as viable prophylactic and therapeutic agents will be discussed. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  11. Isolation of potent neutralizing antibodies from a survivor of the 2014 Ebola virus outbreak.

    Science.gov (United States)

    Bornholdt, Zachary A; Turner, Hannah L; Murin, Charles D; Li, Wen; Sok, Devin; Souders, Colby A; Piper, Ashley E; Goff, Arthur; Shamblin, Joshua D; Wollen, Suzanne E; Sprague, Thomas R; Fusco, Marnie L; Pommert, Kathleen B J; Cavacini, Lisa A; Smith, Heidi L; Klempner, Mark; Reimann, Keith A; Krauland, Eric; Gerngross, Tillman U; Wittrup, Karl D; Saphire, Erica Ollmann; Burton, Dennis R; Glass, Pamela J; Ward, Andrew B; Walker, Laura M

    2016-03-01

    Antibodies targeting the Ebola virus surface glycoprotein (EBOV GP) are implicated in protection against lethal disease, but the characteristics of the human antibody response to EBOV GP remain poorly understood. We isolated and characterized 349 GP-specific monoclonal antibodies (mAbs) from the peripheral B cells of a convalescent donor who survived the 2014 EBOV Zaire outbreak. Remarkably, 77% of the mAbs neutralize live EBOV, and several mAbs exhibit unprecedented potency. Structures of selected mAbs in complex with GP reveal a site of vulnerability located in the GP stalk region proximal to the viral membrane. Neutralizing antibodies targeting this site show potent therapeutic efficacy against lethal EBOV challenge in mice. The results provide a framework for the design of new EBOV vaccine candidates and immunotherapies.

  12. Monoclonal antibodies to intermediate filament proteins of human cells: unique and cross-reacting antibodies.

    Science.gov (United States)

    Gown, A M; Vogel, A M

    1982-11-01

    Monoclonal antibodies were generated against the intermediate filament proteins of different human cells. The reactivity of these antibodies with the different classes of intermediate filament proteins was determined by indirect immunofluorescence on cultured cells, immunologic indentification on SDS polyacrylamide gels ("wester blot" experiments), and immunoperoxidase assays on intact tissues. The following four antibodies are described: (a) an antivimentin antibody generated against human fibroblast cytoskeleton; (b), (c) two antibodies that recognize a 54-kdalton protein in human hepatocellular carcinoma cells; and (d) an antikeratin antibody made to stratum corneum that recognizes proteins of molecular weight 66 kdaltons and 57 kdaltons. The antivimentin antibody reacts with vimentin (58 kdaltons), glial fibrillary acidic protein (GFAP), and keratins from stratum corneum, but does not recognize hepatoma intermediate filaments. In immunofluorescence assays, the antibody reacts with mesenchymal cells and cultured epithelial cells that express vimentin. This antibody decorates the media of blood vessels in tissue sections. One antihepatoma filament antibody reacts only with the 54 kdalton protein of these cells and, in immunofluorescence and immunoperoxidase assays, only recognizes epithelial cells. It reacts with almost all nonsquamous epithelium. The other antihepatoma filament antibody is much less selective, reacting with vimentin, GFAP, and keratin from stratum corneum. This antibody decorates intermediate filaments of both mesenchymal and epithelial cells. The antikeratin antibody recognizes 66-kdalton and 57-kdalton proteins in extracts of stratum corneum and also identifies proteins of similar molecular weights in all cells tested. However, by immunofluorescence, this antibody decorates only the intermediate filaments of epidermoid carcinoma cells. When assayed on tissue sections, the antibody reacts with squamous epithelium and some, but not all

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

    Directory of Open Access Journals (Sweden)

    LT Coswig

    2007-12-01

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

  14. Moving through three-dimensional phase diagrams of monoclonal antibodies.

    Science.gov (United States)

    Rakel, Natalie; Baum, Miriam; Hubbuch, Jürgen

    2014-01-01

    Protein phase behavior characterization is a multivariate problem due to the high amount of influencing parameters and the diversity of the proteins. Single influences on the protein are not understood and fundamental knowledge remains to be obtained. For this purpose, a systematic screening method was developed to characterize the influence of fluid phase conditions on the phase behavior of proteins in three-dimensional phase diagrams. This approach was applied to three monoclonal antibodies to investigate influences of pH, protein and salt concentrations, with five different salts being tested. Although differences exist between the antibodies, this extensive study confirmed the general applicability of the Hofmeister series over the broad parameter range analyzed. The influence of the different salts on the aggregation (crystallization and precipitation) probability was described qualitatively using this Hofmeister series, with a differentiation between crystallization and precipitation being impossible, however.

  15. Belimumab: anti-BLyS human monoclonal antibody, anti-BLyS monoclonal antibody, BmAb, human monoclonal antibody to B-lymphocyte stimulator.

    Science.gov (United States)

    2008-01-01

    Belimumab is a fully human monoclonal antibody that specifically recognizes and inhibits the biological activity of B-lymphocyte stimulator, or BLyS. Belimumab is in phase III trials for the treatment of systemic lupus erythematosus (SLE) and has completed a phase II trial in rheumatoid arthritis (RA); the product may also have potential in the treatment of other autoimmune disorders. In May 2001, Cambridge Antibody Technology (now MedImmune) completed its discovery programme and Human Genome Sciences identified belimumab as a candidate for clinical development. More than 1000 distinct human antibodies specific to BLyS were characterized by the collaboration.B-lymphocyte stimulator is a naturally occurring protein discovered by Human Genome Sciences that stimulates B-lymphocytes to develop into mature B cells. Laboratory studies have indicated that higher than normal levels of B-lymphocyte stimulator may contribute to the pathogenesis of autoimmune diseases, such as SLE and RA. Human Genome Sciences (HGS) and Cambridge Antibody Technology signed a collaborative agreement in August 1999 to study the B-lymphocyte stimulator as a human protein target. HGS is also developing other BLyS products. In March 2000, HGS and Cambridge Antibody Technology expanded their agreement into a 10-year collaboration and product development alliance, providing Human Genome Sciences with the right to use the antibody technology of Cambridge Antibody Technology to fully develop human antibodies for therapeutic and diagnostic purposes. Cambridge Antibody Technology will receive royalty payments on product sales from HGS, as well as the development and milestone payments it has already received. Belimumab will be manufactured in Human Genome Sciences' manufacturing facility, located in Rockville, MD, USA. HGS holds commercial rights to the drug. In July 2005, GlaxoSmithKline (GSK) exercised its co-development and co-promotion option to belimumab. In an agreement made in June 1996, HGS had

  16. Monoclonal antibodies directed to fucoidan preparations from brown algae.

    Science.gov (United States)

    Torode, Thomas A; Marcus, Susan E; Jam, Murielle; Tonon, Thierry; Blackburn, Richard S; Hervé, Cécile; Knox, J Paul

    2015-01-01

    Cell walls of the brown algae contain a diverse range of polysaccharides with useful bioactivities. The precise structures of the sulfated fucan/fucoidan group of polysaccharides and their roles in generating cell wall architectures and cell properties are not known in detail. Four rat monoclonal antibodies, BAM1 to BAM4, directed to sulfated fucan preparations, have been generated and used to dissect the heterogeneity of brown algal cell wall polysaccharides. BAM1 and BAM4, respectively, bind to a non-sulfated epitope and a sulfated epitope present in the sulfated fucan preparations. BAM2 and BAM3 identified additional distinct epitopes present in the fucoidan preparations. All four epitopes, not yet fully characterised, occur widely within the major brown algal taxonomic groups and show divergent distribution patterns in tissues. The analysis of cell wall extractions and fluorescence imaging reveal differences in the occurrence of the BAM1 to BAM4 epitopes in various tissues of Fucus vesiculosus. In Ectocarpus subulatus, a species closely related to the brown algal model Ectocarpus siliculosus, the BAM4 sulfated epitope was modulated in relation to salinity levels. This new set of monoclonal antibodies will be useful for the dissection of the highly complex and yet poorly resolved sulfated polysaccharides in the brown algae in relation to their ecological and economic significance.

  17. Monoclonal antibodies directed to fucoidan preparations from brown algae.

    Directory of Open Access Journals (Sweden)

    Thomas A Torode

    Full Text Available Cell walls of the brown algae contain a diverse range of polysaccharides with useful bioactivities. The precise structures of the sulfated fucan/fucoidan group of polysaccharides and their roles in generating cell wall architectures and cell properties are not known in detail. Four rat monoclonal antibodies, BAM1 to BAM4, directed to sulfated fucan preparations, have been generated and used to dissect the heterogeneity of brown algal cell wall polysaccharides. BAM1 and BAM4, respectively, bind to a non-sulfated epitope and a sulfated epitope present in the sulfated fucan preparations. BAM2 and BAM3 identified additional distinct epitopes present in the fucoidan preparations. All four epitopes, not yet fully characterised, occur widely within the major brown algal taxonomic groups and show divergent distribution patterns in tissues. The analysis of cell wall extractions and fluorescence imaging reveal differences in the occurrence of the BAM1 to BAM4 epitopes in various tissues of Fucus vesiculosus. In Ectocarpus subulatus, a species closely related to the brown algal model Ectocarpus siliculosus, the BAM4 sulfated epitope was modulated in relation to salinity levels. This new set of monoclonal antibodies will be useful for the dissection of the highly complex and yet poorly resolved sulfated polysaccharides in the brown algae in relation to their ecological and economic significance.

  18. Fingerprinting of Natural Product by Eastern Blotting Using Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Hiroyuki Tanaka

    2012-01-01

    Full Text Available We succeeded in developing the fingerprint of natural product by eastern blotting using monoclonal antibodies. After developing and separating them on a TLC plate, solasodine glycosides are oxidized by NaIO4 and reacted with a protein to give conjugates which are recognized with anti-solamargine monoclonal antibody (MAb. Anti-solamargine MAb having wide cross-reactivity can stain and detect all solasodine glycosides by fingerprint. Different sensitivity between solamargine and solasonine was observed. The detection limit was 1.6 ng of solasonine. The hydrolysed products of solamargine were determined by fingerprint of eastern blotting compared to their Rf values depending on the sugar number. Fingerprint by eastern blotting using anti-ginsenoside Rb1 MAb distinguished the formula containing ginseng prescribed in traditional Chinese medicine. By double-staining of ginsenosides it is possible to suggest that the staining color shows the pharmacological activity, such as the purple bands indicate ginsenosides having stimulation activity, and the blue color indicated compound like ginsenosides possessed the depression affect for the central nervous system (CNS, respectively.

  19. [Obtaining monoclonal antibodies against outer membrane glycoproteins of Entamoeba histolytica].

    Science.gov (United States)

    Agundis, C; Isibasi, A; Ortíz, V; Reyes, J L; Paniagua, J; Ramírez, A; Kumate, J

    1990-01-01

    The goal of this paper was the production of monoclonal antibodies capable of detecting relevant antigens from the surface of Entamoeba histolytica trophozoites, with the purpose of using them as a diagnostic test. The cellular fusion for obtaining the monoclonal antibodies (mAb) was done with spleen cells from BALB/c mice, previously immunized with glycoproteins from the membrane, as well as Sp2/0 cells. The hybridoma supernatants were tested with ELISA, using glycoproteins and lipopeptide phosphoglycans (LPPG) as antigens. Seven hybridomas producing mAb against the glycoproteins were found. Among these, three recognize LPPG. The ability of reacting with the mAb against two molecules disappeared for all the LPPG positive ones when were treated with meta-periodate, and only three reacted against the glycoproteins. All of the mAb were of the Ig M isotypes. They were characterized by Dot blot and Western blot assays. From the results, one may deduce that some mAb recognize as epitopes the polysaccharide portion, and thus infer that they are directed of against the surface and therefore, in the future, could be used with a diagnostic purpose.

  20. Immunotherapy of hepatoma with a monoclonal antibody against murine endoglin

    Institute of Scientific and Technical Information of China (English)

    Guang-Hong Tan; Feng-Ying Huang; Hua Wang; Yong-Hao Huang; Ying-Ying Lin; Yue-Nan Li

    2007-01-01

    AIM: To explore the capability of a monoclonal antibody(mAb) against murine endoglin to inhibit tumor angiogenesis and suppression of hepatoma growth in murine models.METHODS: A monoclonal antibody against murine endoglin was purified by affinity chromatography and passively transfused through tail veins in two murine hepatoma models. Tumor volume and survival time were observed at three-day intervals for 48 d. Microvessels in tumor tissues were detected by immunohistochemistry against CD31, and angiogenesis in vivo was determined by alginate encapsulated assay. In addition, tumor cell apoptosis was detected by TUNEL assay.RESULTS: Passive immunotherapy with anti-endoglin mAb could effectively suppress tumor growth, and prolonged the survival time of hepatoma-bearing mice.Angiogenesis was apparently inhibited within the tumor tissues, and the vascularization of alginate beads was also reduced in the mice passively transfused with antiendoglin mAb. In addition, increased apoptotic cells were observed within the tumor tissues from the mice passively transfused with anti-endoglin mAb.CONCLUSION: Passive immunotherapy with antiendoglin mAb effectively inhibits tumor growth via inhibiting tumor angiogenesis and increasing tumor cell apoptosis, which may be highly correlated with the blockage of endoglin-related signal pathway induced by anti-endoglin mAb.

  1. A High Throughput Protein Microarray Approach to Classify HIV Monoclonal Antibodies and Variant Antigens.

    Directory of Open Access Journals (Sweden)

    Emmanuel Y Dotsey

    Full Text Available In recent years, high throughput discovery of human recombinant monoclonal antibodies (mAbs has been applied to greatly advance our understanding of the specificity, and functional activity of antibodies against HIV. Thousands of antibodies have been generated and screened in functional neutralization assays, and antibodies associated with cross-strain neutralization and passive protection in primates, have been identified. To facilitate this type of discovery, a high throughput-screening tool is needed to accurately classify mAbs, and their antigen targets. In this study, we analyzed and evaluated a prototype microarray chip comprised of the HIV-1 recombinant proteins gp140, gp120, gp41, and several membrane proximal external region peptides. The protein microarray analysis of 11 HIV-1 envelope-specific mAbs revealed diverse binding affinities and specificities across clades. Half maximal effective concentrations, generated by our chip analysis, correlated significantly (P<0.0001 with concentrations from ELISA binding measurements. Polyclonal immune responses in plasma samples from HIV-1 infected subjects exhibited different binding patterns, and reactivity against printed proteins. Examining the totality of the specificity of the humoral response in this way reveals the exquisite diversity, and specificity of the humoral response to HIV.

  2. Complement-Dependent Lysis of Influenza A Virus-Infected Cells by Broadly Cross-Reactive Human Monoclonal Antibodies

    Science.gov (United States)

    Terajima, Masanori; Cruz, John; Co, Mary Dawn T.; Lee, Jane-Hwei; Kaur, Kaval; Wilson, Patrick C.; Ennis, Francis A.

    2011-01-01

    We characterized human monoclonal antibodies (MAbs) cloned from influenza virus-infected patients and from influenza vaccine recipients by complement-dependent lysis (CDL) assay. Most MAbs active in CDL were neutralizing, but not all neutralizing MAbs can mediate CDL. Two of the three stalk-specific neutralizing MAbs tested were able to mediate CDL and were more cross-reactive to temporally distant H1N1 strains than the conventional hemagglutination-inhibiting and neutralizing MAbs. One of the stalk-specific MAbs was subtype cross-reactive to H1 and H2 hemagglutinins, suggesting a role for stalk-specific antibodies in protection against influenza illness, especially by a novel viral subtype which can cause pandemics. PMID:21994454

  3. Broadly neutralizing antibodies targeted to mucin-type carbohydrate epitopes of human immunodeficiency virus

    DEFF Research Database (Denmark)

    Hansen, J E; Nielsen, C; Arendrup, M

    1991-01-01

    The cancer-related mucin-type carbohydrate neoantigen Tn was found on gp160 and gp120 of human immunodeficiency virus (HIV). Immunoglobulin G (IgG) and IgM monoclonal antibodies (MAbs) against Tn neutralized infection with cell-free virus and blocked fusion between HIV-infected and uninfected cells......; this binding was inhibitable by pure Tn antigen, and indications were found that this inhibition occurred at a pre-entry step. Boosting the naturally occurring low-titer anti-Tn activity may be of prophylactic value, as suggested by the in vitro neutralization found in this study....

  4. NCI Requests Targets for Monoclonal Antibody Production and Characterization - Office of Cancer Clinical Proteomics Research

    Science.gov (United States)

    In an effort to provide well-characterized monoclonal antibodies to the scientific community, NCI's Antibody Characterization Program requests cancer-related protein targets for affinity production and distribution.

  5. [Production and characteristics of monoclonal antibodies to the diphtheria toxin].

    Science.gov (United States)

    Valiakina, T I; Lakhtina, O E; Komaleva, R L; Simonova, M A; Samokhvalova, L V; Shoshina, N S; Kalinina, N A; Rubina, A Iu; Filippova, M A; Vertiev, Iu V; Grishin, E V

    2009-01-01

    Monoclonal antibodies to the diphtheria toxin were produced without cross reactivity with the thermolabile toxin (LT) from Escherichia coli; ricin; choleraic toxin; the SeA, SeB, SeE, SeI, and SeG toxins of staphylococcus; the lethal factor of the anthrax toxin; and the protective antigen of the anthrax toxin. A pair of antibodies for the quantitative determination of the diphtheria toxin in the sandwich variation of enzyme-linked immunosorbent assay (ELISA) was chosen. The determination limit of the toxin was 0.7 ng/ml in plate and 1.6 ng/ml in microchip ELISA. The presence of a secretion from the nasopharynx lavage did not decrease the sensitivity of the toxin determination by sandwich ELISA. The immunization of mice with the diphtheria toxin and with a conjugate of the diphtheria toxin with polystyrene microspheres demonstrated that the conjugate immunization resulted in the formation of hybridoma clones which produced antibodies only to the epitopes of the A fragment of the diphtheria toxin. The immunization with the native toxin caused the production of hybridoma clones which predominantly produced antibodies to the epitopes of the B fragment.

  6. [Monoclonal antibodies against PCSK9: from bench to clinic].

    Science.gov (United States)

    Guijarro Herraiz, Carlos

    2016-05-01

    Antibodies are glycoproteins with high specificity binding to multiple antigens due to the large number of structural conformations of the variable chains. Hybridoma technology (fusion of myeloma cells with immunoglobulin-producing lymphocytes) has allowed the synthesis of large quantities of unique antibodies (monoclonal [mAb]). mAbs were initially murine. Subsequently, chimeric mAbs were developed, followed by humanized mAbs and finally human mAbs. The high selectivity and good tolerance of human mAbs allows their therapeutic administration to block specific exogenous or endogenous molecules. Selective human mAbs to the catalytic domain of PCSK9 have recently been developed. These antibodies block PCSK9, favour low-density lipoprotein receptor recycling and markedly reduce circulating cholesterol. Preliminary studies indicate that lowering cholesterol through anti-PCSK9 antibodies may significantly reduce the cardiovascular complications of arteriosclerosis. Copyright © 2016 Elsevier España, S.L.U. y Sociedad Española de Arteriosclerosis. All rights reserved.

  7. Characterization and evaluation of monoclonal antibodies developed for typing influenza A and influenza B viruses.

    OpenAIRE

    Walls, H H; Harmon, M W; Slagle, J J; Stocksdale, C; Kendal, A P

    1986-01-01

    Monoclonal antibodies that are broadly reactive with influenza A or influenza B viruses were produced as stable reagents for typing influenza viruses. Monoclonal antibodies to influenza A were specific for either matrix protein or nucleoprotein. The antibodies to influenza B were specific for nucleoprotein or hemagglutinin protein. In an enzyme immunoassay procedure, influenza A antibodies detected H1N1, H2N2, and H3N2 influenza A virus strains collected between 1934 and 1984. Each of the inf...

  8. New monoclonal antibodies directed against human renin. Powerful tools for the investigation of the renin system.

    OpenAIRE

    Galen, F X; Devaux, C.; Atlas, S; Guyenne, T; Menard, J; Corvol, P; Simon, D.; Cazaubon, C; Richer, P; Badouaille, G

    1984-01-01

    Monoclonal antibodies directed against human renin were obtained by the fusing of myeloma cells with spleen cells from Balb/c or high-responder Biozzi mice injected with pure tumoral or highly purified renal renin. These procedures resulted in the production of seven stable monoclonal antibodies to human renin. Antibodies in the hybridoma culture medium were screened by binding to pure iodinated renin or insolubilized renin in a solid phase assay. The concentration of purified antibodies that...

  9. Hepatitis C virus (HCV infection may elicit neutralizing antibodies targeting epitopes conserved in all viral genotypes.

    Directory of Open Access Journals (Sweden)

    Nicasio Mancini

    Full Text Available Anti-hepatitis C virus (HCV cross-neutralizing human monoclonal antibodies, directed against conserved epitopes on surface E2 glycoprotein, are central tools for understanding virus-host interplay, and for planning strategies for prevention and treatment of this infection. Recently, we developed a research aimed at identifying these antibody specificities. The characteristics of one of these antibodies (Fab e20 were addressed in this study. Firstly, using immunofluorescence and FACS analysis of cells expressing envelope HCV glycoproteins, Fab e20 was able to recognize all HCV genotypes. Secondly, competition assays with a panel of mouse and rat monoclonals, and alanine scanning mutagenesis analyses located the e20 epitope within the CD81 binding site, documenting that three highly conserved HCV/E2 residues (W529, G530 and D535 are critical for e20 binding. Finally, a strong neutralizing activity against HCV pseudoparticles (HCVpp incorporating envelope glycoproteins of genotypes 1a, 1b, 2a, 2b and 4, and against the cell culture-grown (HCVcc JFH1 strain, was observed. The data highlight that neutralizing antibodies against HCV epitopes present in all HCV genotypes are elicited during natural infection. Their availability may open new avenues to the understanding of HCV persistence and to the development of strategies for the immune control of this infection.

  10. Harnessing the protective potential of HIV-1 neutralizing antibodies [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    S Abigail Smith

    2016-01-01

    Full Text Available Recent biological, structural, and technical advances are converging within the HIV-1 vaccine field to harness the power of antibodies for prevention and therapy. Numerous monoclonal antibodies with broad neutralizing activity against diverse HIV-1 isolates have now been identified, revealing at least five sites of vulnerability on the envelope (Env glycoproteins. While there are practical and technological barriers blocking a clear path from broadly neutralizing antibodies (bNAb to a protective vaccine, this is not a dead end. Scientists are revisiting old approaches with new technology, cutting new trails through unexplored territory, and paving new roads in the hopes of preventing HIV-1 infection. Other promising avenues to capitalize on the power of bNAbs are also being pursued, such as passive antibody immunotherapy and gene therapy approaches. Moreover, non-neutralizing antibodies have inhibitory activities that could have protective potential, alone or in combination with bNAbs. With a new generation of bNAbs, and a clinical trial that associated antibodies with reduced acquisition, the field is closer than ever to developing strategies to use antibodies against HIV-1.

  11. Preparation of monoclonal antibody to P53 and its clinical application

    Institute of Scientific and Technical Information of China (English)

    Wenqing Wei; Junhua Wu; Jing Liu; Yuxia Wang

    2013-01-01

    Objective:The aim of this study was to prepare monoclonal antibody against P53, a kind of tumor suppressor protein,and use the antibody initial y in clinical immunoassay. Methods:Monoclonal antibody was prepared and identified via the classic protocol of monoclonal antibody preparation. Identified monoclonal antibodies were purified by af inity chro-matography. Antibody titer was determined by enzyme linked immunosorbent assay (ELISA). The specific binding activity of antibody was detected by Western blotting and immunohistochemistry. Results:Three strains of monoclonal antibodies named 1P15, 2P37 and 3P40 were obtained and purified by af inity chromatography. The purity of antibodies was higher than 90%. The titers of antibodies were more than 1:6000. Western blot and immunohistochemistry assay showed that the specific antibody can combine with endogenous P53 protein in the tumor celllines and determine the expression of P53 in tumor tis-sue. Conclusion:Three strains of monoclonal antibodies with high af inity to P53 were successful y established, which can be used for detecting the expression of P53 in tumor cells or tissue.

  12. Monoclonal antibodies against a synthetic peptide from human immunodeficiency virus type 1 Nef protein

    DEFF Research Database (Denmark)

    Steinaa, L; Wulff, A M; Saermark, T

    1994-01-01

    Monoclonal antibodies against a synthetic peptide (aa 138-152) from HIV-1 Nef protein were produced and characterized. Three hybridoma lines producing monoclonal antibodies (MAbs) against the synthetic peptide were generated by fusion between P3-X63 Ag8.653 myeloma cells and BALB/c splenocytes fr...

  13. Preparation of Europium Induced Conformation—specific anti—calmodulin Monoclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    WeiGuoLI; ChaoQI; 等

    2002-01-01

    Monoclonal antibody technique was employed to detect the conformational difference of CaM induced by metal ions. A trivalent europium ion induced conformation-specific anti-calmodulin monoclonal antibody was successfully prepared with europium-saturated calmodulin as antigen.

  14. Preparation of Europium Induced Conformation-specific anti-calmodulin Monoclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Monoclonal antibody technique was employed to detect the conformational difference of CaM induced by metal ions. A trivalent europium ion induced conformation-specific anti-calmodulin monoclonal antibody was successfully prepared with europium-saturated calmodulin as antigen.

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

    DEFF Research Database (Denmark)

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

    1988-01-01

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

  16. Discovery of functional monoclonal antibodies targeting G-protein-coupled receptors and ion channels.

    Science.gov (United States)

    Wilkinson, Trevor C I

    2016-06-15

    The development of recombinant antibody therapeutics is a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Despite this growth, however, certain classes of important molecular targets have remained intractable to therapeutic antibodies due to complexity of the target molecules. These complex target molecules include G-protein-coupled receptors and ion channels which represent a large potential target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these target proteins. Given this opportunity, substantial effort has been applied to address the technical challenges of targeting these complex membrane proteins with monoclonal antibodies. In this review recent progress made in the strategies for discovery of functional monoclonal antibodies for these challenging membrane protein targets is addressed.

  17. Broad and potent HIV-1 neutralization by a human antibody that binds the gp41-120 interface

    OpenAIRE

    Huang, Jinghe; Kang, Byong H; Pancera, Marie; Lee, Jeong Hyun; Tong, Tommy; Feng, Yu; Georgiev, Ivelin S.; Chuang, Gwo-Yu; Druz, Aliaksandr; Doria-Rose, Nicole A.; Laub, Leo; Sliepen, Kwinten; van Gils, Marit J.; de la Peña, Alba Torrents; Derking, Ronald

    2014-01-01

    The isolation of human monoclonal antibodies (mAbs) is providing important insights regarding the specificities that underlie broad neutralization of HIV-1 (reviewed in 1 ). Here we report a broad and extremely potent HIV-specific mAb, termed 35O22, which binds novel HIV-1 envelope glycoprotein (Env) epitope. 35O22 neutralized 62% of 181 pseudoviruses with an IC50

  18. Interference of coronavirus infection by expression of immunoglobulin G (IgG) or IgA virus-neutralizing antibodies.

    OpenAIRE

    Castilla, J; Sola, I.; Enjuanes, L

    1997-01-01

    Immunoglobulin gene fragments encoding the variable modules of the heavy and light chains of a transmissible gastroenteritis coronavirus (TGEV)-neutralizing monoclonal antibody (MAb) have been cloned and sequenced. The selected MAb recognizes a highly conserved viral epitope and does not lead to the selection of neutralization escape mutants. The sequences of MAb 6A.C3 kappa and gamma 1 modules were identified as subgroup V and subgroup IIIC, respectively. The chimeric immunoglobulin genes en...

  19. Use of commercially available rabbit monoclonal antibodies for immunofluorescence double staining

    DEFF Research Database (Denmark)

    Bzorek, M.; Stamp, I.M.; Frederiksen, L.

    2008-01-01

    Immunohistochemistry, that is, the use of polyclonal and monoclonal antibodies to detect cell and tissue antigens at a microscopical level is a powerful tool for both research and diagnostic purposes. Especially in the field of hematologic disease, there is often a need to detect several antigens...... synchronously, and we report here a fast and easy technique for demonstrating more than 1 antigen in 1 slide using immunofluorescence. We have used commercially available rabbit monoclonal antibodies (Cyclin D1, CD3, CD5, CD23, etc.) paired with mouse monoclonal antibodies (CD7, CD20, CD79a, Pax-5, etc.......) for double immunofluorescence labeling on paraffin-embedded tissue sections. Commercially available rabbit monoclonal antibodies in combination with mouse monoclonal antibodies proved useful in double immunofluorescence labeling on paraffin-embedded tissue, and all combinations used yielded excellent results...

  20. Monoclonal antibodies against human granulocytes and myeloid differentiation antigens.

    Science.gov (United States)

    Mannoni, P; Janowska-Wieczorek, A; Turner, A R; McGann, L; Turc, J M

    1982-12-01

    Monoclonal antibodies (MCA) were obtained by immunizing BALB/c mice with 99% pure granulocytes from normal donors or with a whole leukocyte suspension obtained from a chronic myelogenous leukemia (CML) patient, and then fusing the mouse spleen cells with a 315-43 myeloma cell clone. Four MCA were selected and studied using ELISA, immunofluorescence, cytotoxicity assays, and FACS analysis. Antibodies 80H.1, 80H.3, and 80H.5 (from normals) and 81H.1 (from CML) detected antigens expressed on neutrophils. Antibodies 80H.1 and 80H.3 (IgG) also reacted with monocytes but not with other blood cell subsets. Antibodies 80H.5 and 81H.1 (IgM) were cytotoxic and reacted strongly with most of the cells of the neutrophil maturation sequence, i.e., myeloblasts, promyelocytes, myelocytes, and mature granulocytes. Antibodies 80H.5 and 81H.1 also inhibited CFU-GM growth stimulated by leukocyte feeder layers or placental conditioned media, but did not inhibit BFU-E and CFU-E. Antigens recognized by 80H.3, 80H.5, and 81H.1 were expressed both on a proportion of cells from HL.60, KG.1, ML.1, and K562 myeloid cell lines, and on a proportion of blast cells isolated from patients with acute myelogenous leukemia. They were not found on lymphoid cell lines or lymphoid leukemia cells. These MCA recognize either late differentiation antigens expressed on mature neutrophils and monocytes (80H.1 and 80H.3) or early differentiation antigens (80H.5 and 81H.1) specific to the granulocytic lineage. They may be useful for a better definition of those antigens specific to hematopoietic stem cells and their relationship with normal or neoplastic hematopoiesis.

  1. [ICO-10 monoclonal antibodies to the Thy-1 antigen].

    Science.gov (United States)

    Korotkova, O V; Baryshnikov, A Iu; Tupitsyn, N N; Chimishkian, K L; Kostrykina, V N

    1989-01-01

    Mouse monoclonal antibodies (MAB) ICO-10 to Thy-1 antigen were obtained. MAB ICO-10 reacted in indirect immunofluorescence test with 5.7 +/- 0.8% human thymocytes. Antibodies did not react with granulocytes, monocytes, T- and non-T cells from peripheral blood, and with marrow cells of healthy donors. MAB ICO-10 reacted with blast cells from 25 of 53 patients with T-cell acute lymphoblastic leukemia (ALL), from 2 of 5 patients with B-cell ALL. This antigen was absent on blood and marrow cells from some patients with ALL, 80 patients with chronic lymphoid leukemia, 54 patients with chronic granulocytic leukemia at the stage of blastic crisis, 128 patients with acute nonlymphoblastic leukemia. Antibodies are specifically bound to thymocytes and spleen cells of Thy 1.1 and Thy 1.2 mice. MAB ICO-10 detect Thy-1 antigen expressed on human hematopoietic cells. MAB ICO-10 may be applied for human leukemia and lymphoma immune diagnosis.

  2. Molecular mechanisms of resistance to the EGFR monoclonal antibody cetuximab.

    Science.gov (United States)

    Brand, Toni M; Iida, Mari; Wheeler, Deric L

    2011-05-01

    The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase belonging to the HER family of receptor tyrosine kinases. Receptor activation upon ligand binding leads to down stream activation of the PI3K/AKT, RAS/RAF/MEK/ERK and PLCγ/PKC pathways that influence cell proliferation, survival and the metastatic potential of tumor cells. Increased activation by gene amplification, protein overexpression or mutations of the EGFR has been identified as an etiological factor in a number of human epithelial cancers (e.g., NSCLC, CRC, glioblastoma and breast cancer). Therefore, targeting the EGFR has been intensely pursued as a cancer treatment strategy over the last two decades. To date, five EGFR inhibitors, including three small molecule tyrosine kinase inhibitors (TKIs) and two monoclonal antibodies have gained FDA approval for use in oncology. Both approaches to targeting the EGFR have shown clinical promise and the anti-EGFR antibody cetuximab is used to treat HNSCC and CRC. Despite clinical gains arising from use of cetuximab, both intrinsic resistance and the development of acquired resistance are now well recognized. In this review we focus on the biology of the EGFR, the role of EGFR in human cancer, the development of antibody-based anti-EGFR therapies and a summary of their clinical successes. Further, we provide an in depth discussion of described molecular mechanisms of resistance to cetuximab and potential strategies to circumvent this resistance.

  3. ANTITUMOR EFFECTS OF MONOCLONAL ANTIBODY FAB′ FRAGMENT CONTAINING IMMUNOCONJUGATES

    Institute of Scientific and Technical Information of China (English)

    刘小云; 甄永苏

    2002-01-01

    Objective.Using monoclonal antibody (mAb) Fab′ fragment to develop mAb immunoconjugates for cancer. Methods.Fab′ fragment of mAb 3A5 was prepared by digestion of the antibody with pepsin and then reduced by dithiothreitol (DTT),while Fab′ fragment of mAb 3D6 was obtained by digestion of the antibody with ficin and subsequently reduced by β mercaptoethanol.The conjugation between Fab′ fragment and pingyangmycin (PYM),an antitumor antibiotic,was mediated by dextran T 40.Immunoreactivity of Fab′ PYM conjugates with cancer cells was determined by ELISA,and the cytotoxicity of those conjugates to cancer cells was determined by clonogenic assay.Antitumor effects of the Fab′ PYM conjugates were evaluated by subcutaneously transplanted tumors in mice. Results.The molecular weight of Fab′ fragment was approximately 53 kD,while the average molecular weight of Fab′ PYM conjugate was 170 kD.The Fab′ PYM conjugates showed immunoreactivity with antigen relevant cancer cells and selective cytotoxicity against target cells.Administered intravenously,Fab′ PYM conjugates were more effective against the growth of tumors in mice than free PYM and PYM conjugated with intact mAb. Conclusion.Fab′ PYM conjugate may be capable of targeting cancer cells and effectively inhibiting tumor growth,suggesting its therapeutic potential in cancer treatment.

  4. Predefined GPGRAFY-Epitope-Specific Monoclonal Antibodies with Different Activities for Recognizing Native HIV-1 gp120

    Institute of Scientific and Technical Information of China (English)

    蓝灿辉; 田海军; 陈应华

    2004-01-01

    A seven-amino acid epitope GPGRAFY at the tip of the V3 loop in HIV-1 gp120 is the principal neutralizing epitope,and a subset of anti-V3 antibodies specific for this epitope shows a broad range of neutralizing activity.GPGRAFY-epitope-specific neutralizing antibodies were produced using predefined GPGRAFY-epitope-specific peptides instead of a natural or recombinant gp120 bearing this epitope.All six monoclonal antibodies (mAbs) could recognize the GPGRAFY-epitope on peptides and two of the antibodies,9D8 and 2D7,could recognize recombinant gp120 in enzymelinked immunosorkentassy (ELISA) assays.In the flow cytometry analysis,the mAbs 9D8 and 2D7 could bind to HIV-Env+ CHO-WT cells and the specific bindings could be inhibited by the GPGRAFY-epitope peptide,which suggests that these two mAbs could recognize the native envelope protein gp120 expressed on the cell membrane.However,in syncytium assays,none of the mAbs was capable of inhibiting HIV-Env-mediated cell membrane fusion.The different activities for recognizing native HIV-1 gp120 might be associated with different antibody affinities against the epitopes.The development of conformational mimics of the neutralization epitope in the gp120 V3 loop could elicit neutralizing mAbs with high affinity.

  5. Characterization of golimumab, a human monoclonal antibody specific for human tumor necrosis factor α.

    Science.gov (United States)

    Shealy, David J; Cai, Ann; Staquet, Kim; Baker, Audrey; Lacy, Eilyn R; Johns, Laura; Vafa, Omid; Gunn, George; Tam, Susan; Sague, Sarah; Wang, Dana; Brigham-Burke, Mike; Dalmonte, Paul; Emmell, Eva; Pikounis, Bill; Bugelski, Peter J; Zhou, Honghui; Scallon, Bernard J; Giles-Komar, Jill

    2010-01-01

    We prepared and characterized golimumab (CNTO148), a human IgG1 tumor necrosis factor alpha (TNFα) antagonist monoclonal antibody chosen for clinical development based on its molecular properties. Golimumab was compared with infliximab, adalimumab and etanercept for affinity and in vitro TNFα neutralization. The affinity of golimumab for soluble human TNFα, as determined by surface plasmon resonance, was similar to that of etanercept (18 pM versus 11 pM), greater than that of infliximab (44 pM) and significantly greater than that of adalimumab (127 pM, p=0.018).  The concentration of golimumab necessary to neutralize TNFα-induced E-selectin expression on human endothelial cells by 50% was significantly less than those for infliximab (3.2 fold; p=0.017) and adalimumab (3.3-fold; p=0.008) and comparable to that for etanercept. The conformational stability of golimumab was greater than that of infliximab (primary melting temperature [Tm] 74.8 °C vs. 69.5 °C) as assessed by differential scanning calorimetry.  In addition, golimumab showed minimal aggregation over the intended shelf life when formulated as a high concentration liquid product (100 mg/mL) for subcutaneous administration.  In vivo, golimumab at doses of 1 and 10 mg/kg significantly delayed disease progression in a mouse model of human TNFα-induced arthritis when compared with untreated mice, while infliximab was effective only at 10 mg/kg. Golimumab also significantly reduced histological scores for arthritis severity and cartilage damage, as well as serum levels of pro-inflammatory cytokines and chemokines associated with arthritis. Thus, we have demonstrated that golimumab is a highly stable human monoclonal antibody with high affinity and capacity to neutralize human TNFα in vitro and in vivo.

  6. Effect of polyol sugars on the stabilization of monoclonal antibodies.

    Science.gov (United States)

    Nicoud, Lucrèce; Cohrs, Nicholas; Arosio, Paolo; Norrant, Edith; Morbidelli, Massimo

    2015-02-01

    We investigate the impact of sugars and polyols on the heat-induced aggregation of a model monoclonal antibody whose monomer depletion is rate-limited by protein unfolding. We follow the kinetics of monomer consumption by size exclusion chromatography, and we interpret the results in the frame of two mechanistic schemes describing the enhanced protein stability in the presence of polyols. It is found that the stabilization effect increases with increasing polyol concentration with a comparable trend for all of the tested polyols. However, the stabilization effect at a given polyol concentration is polyol specific. In particular, the stabilization effect increases as a function of polyol size until a plateau is reached above a critical polyol size corresponding to six carbon atoms. Our results show that the stabilization by polyols does not depend solely on the volume fraction filled by the polyol molecules, but is also affected by the polyol chemistry.

  7. Preparation and Identification of Monoclonal Antibodies Against Vibrio anguillarum

    Institute of Scientific and Technical Information of China (English)

    Chen Shiyong(陈师勇); Zhang Peijun; Mo Zhaolan; Zhang Zhendong; Zou Yuxia; Xu Yongli

    2004-01-01

    Monoclonal antibodies (Mabs) against V.anguillarum strain M3 are prepared, and their isotypes are also characterized. Among them, C1C5 is the only Mab which does not crossreact with other eleven non-V.anguillarum strains. The proteinase K digestion test shows that the epitopes recognized by C1C5, C6C3 and C6C32 Mabs contained protein. The periodate oxidation test showed that the epitopes recognized by Mabs except C1C5 are glycosylated. In addition, results of additivity test indicate that the epitopes recognized by C6C3 and C6C32 Mabs are similar, and quite different from those recognized by Mab C1C5.

  8. [Monoclonal antibodies for the treatment of multiple sclerosis].

    Science.gov (United States)

    Sánchez-Seco, Victoria Galán; Casanova Peño, Ignacio; Arroyo González, Rafael

    2014-12-01

    Until the mid 1990s, with the appearance of interferon beta and glatiramer acetate, there was no treatment for multiple sclerosis (MS). However, due to their moderate therapeutic potential in some patients, a broad search was continued to find new and more effective treatment strategies, largely concentrated on monoclonal antibodies (MOAB). Natalizumab, the first MOAB for the treatment of MS, was approved at the end of 2004, representing a major advance in the field of neuroimmunology. Today, there is broad experience with natalizumab and other MOAB (alemtuzumab, daclizumab, rituximab, ocrelizumab, ofatumumab and anti-lingo-1) that are pending commercialization or are under phase II or III of development with promising results. The present review analyzes the efficacy and safety results of all these drugs. Copyright © 2014 Elsevier España, S.L.U. All rights reserved.

  9. Pharmacokinetic, pharmacodynamic and immunogenicity comparability assessment strategies for monoclonal antibodies.

    Science.gov (United States)

    Putnam, Wendy S; Prabhu, Saileta; Zheng, Yanan; Subramanyam, Meena; Wang, Yow-Ming C

    2010-10-01

    Regulatory guidance stipulates that comparability assessment is required to support manufacturing process changes during the development of a biological product or post-approval. However, strategies for assessing the comparability of pre- and post-change materials are still evolving. A hierarchical risk-based approach is recommended, starting with analytical testing to ensure quality, followed by biological characterization and, if needed, in vivo pharmacokinetic (PK), PK-pharmacodynamic (PD), safety and/or efficacy studies. The need for an in vivo study and the type of study required depend on the magnitude and the potential impact of the changes and the timing in the development process. This review discusses factors affecting the PK, PD and immunogenicity of monoclonal antibodies, and provides guidance for determining non-clinical and clinical comparability assessment strategies. Copyright © 2010 Elsevier Ltd. All rights reserved.

  10. Selection of Ceratitis capitata (Diptera: Tephritidae) specific recombinant monoclonal phage display antibodies for prey detection analysis.

    Science.gov (United States)

    Monzó, César; Urbaneja, Alberto; Ximénez-Embún, Miguel; García-Fernández, Julia; García, José Luis; Castañera, Pedro

    2012-01-01

    Several recombinant antibodies against the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), one of the most important pests in agriculture worldwide, were selected for the first time from a commercial phage display library of human scFv antibodies. The specificity and sensitivity of the selected recombinant antibodies were compared with that of a rabbit polyclonal serum raised in parallel using a wide range of arthropod species as controls. The selected recombinant monoclonal antibodies had a similar or greater specificity when compared with classical monoclonal antibodies. The selected recombinant antibodies were successfully used to detect the target antigen in the gut of predators and the scFv antibodies were sequenced and compared. These results demonstrate the potential for recombinant scFv antibodies to be used as an alternative to the classical monoclonal antibodies or even molecular probes in the post-mortem analysis studies of generalist predators.

  11. PCSK9 Inhibition With Monoclonal Antibodies: Modern Management of Hypercholesterolemia.

    Science.gov (United States)

    Ito, Matthew K; Santos, Raul D

    2017-01-01

    Current guidelines for hypercholesterolemia treatment emphasize lifestyle modification and lipid-modifying therapy to reduce the risk for cardiovascular disease. Statins are the primary class of agents used for the treatment of hypercholesterolemia. Although statins are effective for many patients, they fail to achieve optimal reduction in lipids for some patients, including those who have or are at high risk for cardiovascular disease. The PCSK9 gene was identified in the past decade as a potential therapeutic target for the management of patients with hypercholesterolemia. Pharmacologic interventions to decrease PCSK9 levels are in development, with the most promising approach using monoclonal antibodies that bind to PCSK9 in the plasma. Two monoclonal antibodies, alirocumab and evolocumab, have recently been approved for the treatment of hypercholesterolemia, and a third one, bococizumab, is in phase 3 clinical development. All 3 agents achieve significant reductions in levels of low-density lipoprotein cholesterol, as well as reductions in non-high-density lipoprotein cholesterol, apolipoprotein B, and lipoprotein(a). Long-term outcome trials are under way to determine the sustained efficacy, safety, and tolerability of PCSK9 inhibitors and whether this novel class of agents decreases the risk for major cardiovascular events in patients on lipid-modifying therapy. Available data suggest that PCSK9 inhibitors provide a robust reduction in atherogenic cholesterol levels with a good safety profile, especially for patients who fail to obtain an optimal clinical response to statin therapy, those who are statin intolerant or have contraindications to statin therapy, and those with familial hypercholesterolemia. © 2016, The Authors. The Journal of Clinical Pharmacology Published by Wiley Periodicals, Inc. on behalf of American College of Clinical Pharmacology.

  12. HIV therapy by a combination of broadly neutralizing antibodies in humanized mice

    Science.gov (United States)

    Klein, Florian; Gruell, Henning; Scheid, Johannes F.; Bournazos, Stylianos; Mouquet, Hugo; Spatz, Linda A.; Diskin, Ron; Abadir, Alexander; Zang, Trinity; Dorner, Marcus; Billerbeck, Eva; Labitt, Rachael N.; Gaebler, Christian; Marcovecchio, Paola; Incesu, Reha-Baris; Eisenreich, Thomas R.; Bieniasz, Paul D.; Seaman, Michael S.; Bjorkman, Pamela J.; Ravetch, Jeffrey V.; Ploss, Alexander; Nussenzweig, Michel C.

    2013-01-01

    Summary Human antibodies to HIV-1 can neutralize a broad range of viral isolates in vitro and protect non-human primates against infection1,2. Previous work showed that antibodies exert selective pressure on the virus but escape variants emerge within a short period of time3,4. However, these experiments were performed before the recent discovery of more potent anti-HIV-1 antibodies and their improvement by structure-based design5-9. Here we re-examine passive antibody transfer as a therapeutic modality in HIV-1-infected humanized mice (hu-mice). Although HIV-1 can escape from antibody monotherapy, combinations of broadly neutralizing antibodies (bNAbs) can effectively control HIV-1 infection and suppress viral load to levels below detection. Moreover, in contrast to antiretroviral therapy (ART)10-12, the longer half-life of antibodies led to viremic control for an average of 60 days after cessation of therapy. Thus, combinations of potent monoclonal antibodies can effectively control HIV-1 replication in hu-mice, and should be re-examined as a therapeutic modality in HIV-1-infected individuals. PMID:23103874

  13. Human monoclonal antibody HCV1 effectively prevents and treats HCV infection in chimpanzees.

    Directory of Open Access Journals (Sweden)

    Trevor J Morin

    Full Text Available Hepatitis C virus (HCV infection is a leading cause of liver transplantation and there is an urgent need to develop therapies to reduce rates of HCV infection of transplanted livers. Approved therapeutics for HCV are poorly tolerated and are of limited efficacy in this patient population. Human monoclonal antibody HCV1 recognizes a highly-conserved linear epitope of the HCV E2 envelope glycoprotein (amino acids 412-423 and neutralizes a broad range of HCV genotypes. In a chimpanzee model, a single dose of 250 mg/kg HCV1 delivered 30 minutes prior to infusion with genotype 1a H77 HCV provided complete protection from HCV infection, whereas a dose of 50 mg/kg HCV1 did not protect. In addition, an acutely-infected chimpanzee given 250 mg/kg HCV1 42 days following exposure to virus had a rapid reduction in viral load to below the limit of detection before rebounding 14 days later. The emergent virus displayed an E2 mutation (N415K/D conferring resistance to HCV1 neutralization. Finally, three chronically HCV-infected chimpanzees were treated with a single dose of 40 mg/kg HCV1 and viral load was reduced to below the limit of detection for 21 days in one chimpanzee with rebounding virus displaying a resistance mutation (N417S. The other two chimpanzees had 0.5-1.0 log(10 reductions in viral load without evidence of viral resistance to HCV1. In vitro testing using HCV pseudovirus (HCVpp demonstrated that the sera from the poorly-responding chimpanzees inhibited the ability of HCV1 to neutralize HCVpp. Measurement of antibody responses in the chronically-infected chimpanzees implicated endogenous antibody to E2 and interference with HCV1 neutralization although other factors may also be responsible. These data suggest that human monoclonal antibody HCV1 may be an effective therapeutic for the prevention of graft infection in HCV-infected patients undergoing liver transplantation.

  14. Engineering broadly neutralizing antibodies for HIV prevention and therapy.

    Science.gov (United States)

    Hua, Casey K; Ackerman, Margaret E

    2016-08-01

    A combination of advances spanning from isolation to delivery of potent HIV-specific antibodies has begun to revolutionize understandings of antibody-mediated antiviral activity. As a result, the set of broadly neutralizing and highly protective antibodies has grown in number, diversity, potency, and breadth of viral recognition and neutralization. These antibodies are now being further enhanced by rational engineering of their anti-HIV activities and coupled to cutting edge gene delivery and strategies to optimize their pharmacokinetics and biodistribution. As a result, the prospects for clinical use of HIV-specific antibodies to treat, clear, and prevent HIV infection are gaining momentum. Here we discuss the diverse methods whereby antibodies are being optimized for neutralization potency and breadth, biodistribution, pharmacokinetics, and effector function with the aim of revolutionizing HIV treatment and prevention options.

  15. Antibodies to poliovirus detected by immunoradiometric assay with a monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

    Spitz, M.; Fossati, C.A.; Schild, G.C.; Spitz, L.; Brasher, M. (National Inst. for Biological Standards and Control, London (UK))

    1982-10-01

    An immunoradiometric assay (IRMA) for the assay of antibodies to poliovirus antigens is described. Dilutions of the test sera or whole (finger prick) blood samples were incubated with the poliovirus antigen bound to a solid phase and the specific antibody was detected by the addition of a mouse anti-human IgG monoclonal antibody (McAb), which was itself revealed by iodinated sheep IgG antimouse F(ab). The authors have shown that this technique is suitable for the estimation of IgG anti-poliovirus antibodies induced in children following polio vaccine. The present study shows that SPRIA provides a simple and inexpensive method for serological studies with poliovirus particularly for use in large-scale surveys.

  16. Serologic analysis of human rotavirus serotypes P1A and P2 by using monoclonal antibodies.

    Science.gov (United States)

    Padilla-Noriega, L; Werner-Eckert, R; Mackow, E R; Gorziglia, M; Larralde, G; Taniguchi, K; Greenberg, H B

    1993-01-01

    Three human rotavirus (HRV) VP4 serotypes and one subtype have been described on the basis of a fourfold or an eightfold-or-greater difference in neutralization titer when tested with hyperimmune antisera to recombinant VP4 or VP8* (serotypes P1A, P1B, P2, and P3). To start to analyze the antigenic basis underlying serotype specificity, we produced a library of 13 VP4-specific neutralizing monoclonal antibodies (NMAbs) to two HRVs, the serotype P1A strain Wa and the serotype P2 strain ST3, and characterized the reactivity of these NMAbs with a panel of serotypically diverse HRV strains by neutralization assay and enzyme-linked immunosorbent assay (ELISA). We characterized the serotypic specificity of the NMAbs by using a fourfold or an eightfold-or-greater difference in titer against the homologous (i.e., immunogen) and heterologous strains as a criterion for serotype. Some ST3-derived NMAbs reacted specifically with serotype P2 HRVs by ELISA and/or neutralization assay, while some Wa-derived NMAbs reacted specifically by ELISA and/or neutralization assay with some or all serotype P1A HRVs. Other Wa- and ST3-derived NMAbs reacted with some or all serotype P1A and P2 HRV strains by neutralization assay and ELISA. Most NMAbs did not react with serotype P1B or P3 strains. In previous studies, three distinct operationally defined epitopes have been identified on VP4 by examining the reactivity patterns of selected antigenic variants of HRV strain KU. At least one of the NMAbs described here recognizes an epitope unrelated to these previously identified epitopes, since it neutralized both KU and its variants. Images PMID:7681440

  17. Pre- and Postexposure Use of Human Monoclonal Antibody against H5N1 and H1N1 Influenza Virus in Mice: Viable Alternative to Oseltamivir

    NARCIS (Netherlands)

    Koudstaal, W.; Koldijk, M.H.; Brakenhoff, J.P.J.; Cornelissen, A.H.M.; Weverling, G.J.; Friesen, R.H.E.; Goudsmit, J.

    2009-01-01

    New strategies to prevent and treat influenza virus infections are urgently needed. A recently discovered class of monoclonal antibodies (mAbs) neutralizing an unprecedented spectrum of influenza virus subtypes may have the potential for future use in humans. Here, we assess the efficacies of CR6261

  18. Safety and pharmacokinetics of an intramuscular monoclonal antibody (SB209763) against respiratory syncytial virus (RSV) in infants and young children at risk for severe RSV disease

    NARCIS (Netherlands)

    Meissner, HC; Groothuis, [No Value; Rodriguez, WJ; Welliver, RC; Hogg, G; Gray, PH; Loh, R; Simoes, EAF; Sly, P; Miller, AK; Nichols, AI; Jorkasky, DK; Everitt, DE; Thompson, KA

    1999-01-01

    We conducted a multicenter, double-blind, placebo-controlled, randomized trial of a humanized monoclonal antibody against a respiratory syncytial virus (RSV) fusion protein (SE 209763) to evaluate its safety, pharmacokinetics, and fusion inhibition and neutralization titers. Forty-three infants who

  19. Preparation and Biological Evaluation of 188Re Labeled Monoclonal Antibody TGLA

    Institute of Scientific and Technical Information of China (English)

    WEN; Kai; ZHANG; Jun-li; CHEN; Bao-jun; CUI; Hai-ping

    2012-01-01

    <正>Monoclonal antibody TGLA is a specific targeting CD20 chimeric antibody. It can kill tumor cells and inhibit tumor cells’ growth effectively, which has been applied to clinical therapy of lymphoma cell B. 188 Re is easy to get, and emits both β and γ rays. 188Re labeled monoclonal antibody TGLA can be used for the study of lymphoma therapy and imaging. This work got the product 188Re-TGLA by direct labeling

  20. A MONOCLONAL-ANTIBODY AGAINST HUMAN BETA-GLUCURONIDASE FOR APPLICATION IN ANTIBODY-DIRECTED ENZYME PRODRUG THERAPY

    NARCIS (Netherlands)

    Haisma, Hidde; VANMUIJEN, M; SCHEFFER, G; SCHEPER, RJ; PINEDO, HM; BOVEN, E

    1995-01-01

    The selectivity of anticancer agents may be improved by antibody-directed enzyme prodrug therapy (ADEPT), The immunogenicity of antibody-enzyme conjugates and the low tumor to normal tissue ratio calls for the use of a human enzyme and the development of a monoclonal antibody (MAb) against that enzy

  1. Profiling formulated monoclonal antibodies by (1)H NMR spectroscopy.

    Science.gov (United States)

    Poppe, Leszek; Jordan, John B; Lawson, Ken; Jerums, Matthew; Apostol, Izydor; Schnier, Paul D

    2013-10-15

    Nuclear magnetic resonance (NMR) is arguably the most direct methodology for characterizing the higher-order structure of proteins in solution. Structural characterization of proteins by NMR typically utilizes heteronuclear experiments. However, for formulated monoclonal antibody (mAb) therapeutics, the use of these approaches is not currently tenable due to the requirements of isotope labeling, the large size of the proteins, and the restraints imposed by various formulations. Here, we present a new strategy to characterize formulated mAbs using (1)H NMR. This method, based on the pulsed field gradient stimulated echo (PGSTE) experiment, facilitates the use of (1)H NMR to generate highly resolved spectra of intact mAbs in their formulation buffers. This method of data acquisition, along with postacquisition signal processing, allows the generation of structural and hydrodynamic profiles of antibodies. We demonstrate how variation of the PGSTE pulse sequence parameters allows proton relaxation rates and relative diffusion coefficients to be obtained in a simple fashion. This new methodology can be used as a robust way to compare and characterize mAb therapeutics.

  2. Analysis of viral clearance unit operations for monoclonal antibodies.

    Science.gov (United States)

    Miesegaes, George; Lute, Scott; Brorson, Kurt

    2010-06-01

    Demonstration of viral clearance is a critical step in assuring the safety of biotechnology products. We generated a viral clearance database that contains product information, unit operation process parameters, and viral clearance data from monoclonal antibody and antibody-related regulatory submissions to FDA. Here we present a broad overview of the database and resulting analyses. We report that the diversity of model viruses tested expands as products transition to late-phase. We also present averages and ranges of viral clearance results by Protein A and ion exchange chromatography steps, low pH chemical inactivation, and virus filtration, focusing on retro- and parvoviruses. For most unit operations, an average log reduction value (LRV, a measure of clearance power) for retrovirus of >4 log(10) were measured. Cases where clearance data fell outside of the anticipated range (i.e., outliers) were rationally explained. Lastly, a historical analysis did not find evidence of any improvement trend in viral clearance over time. The data collectively suggest that many unit operations in general can reliably clear viruses.

  3. Monoclonal antibodies: pharmacokinetics as a basis for new dosage regimens?

    Science.gov (United States)

    Azanza, J-R; Sádaba, B; Gómez-Guiu, A

    2015-10-01

    Complete monoclonal IgG antibodies which are in use in clinical practice share some pharmacological properties resulting in high concentrations in plasma. This fact is reflected in their low volumes of distribution, which can also be correlated with a high molecular weight and water solubility. This feature allows a novel approach to be applied to the dosing schedule for this group of drugs with fixed doses being used instead of the initially developed weight- or body surface-adjusted dosing schedules. In addition, the development of a new formulation containing hyaluronidase allows a subcutaneous route of administration to be used, because hyaluronidase creates a space in the subcutaneous tissue that helps antibody absorption. This method requires higher doses, but has allowed testing the feasibility of administering a fixed dose, with no individual dose adjustments based on weight or body surface. Moreover, loading doses are not needed, because the first dose results, within 3 weeks, in minimum concentrations that are higher than effective concentrations.

  4. Anti-bacterial monoclonal antibodies: back to the future?

    Science.gov (United States)

    Oleksiewicz, Martin B; Nagy, Gábor; Nagy, Eszter

    2012-10-15

    Today's medicine has to deal with the emergence of multi-drug resistant bacteria, and is beginning to be confronted with pan-resistant microbes. This worsening inadequacy of the antibiotics concept, which has ruled infectious medicine in the last six decades creates an increasing unmet medical need that can be addressed by passive immunization. While past experience from the pre-antibiotic era with serum therapy was in many cases encouraging, antibacterial monoclonal antibodies have so far suffered high attrition rates in the clinic, generally from lack of efficacy. Yet, we believe that recent developments in a number of areas such as infectious disease pathogenesis research, translational medicine, mAb engineering, mAb manufacturing and rapid bedside diagnostics are converging to make the medium-term future permissive for antibacterial mAb development. Here, we review antibacterial mAb-based approaches that are or were in clinical development, and may potentially act as paradigms with regards to molecular targets, antibody formats and mode-of-action, pre-clinical validation and selection of most relevant patient populations, in order to increase the likelihood of successful product development in this field.

  5. Defining process design space for monoclonal antibody cell culture.

    Science.gov (United States)

    Abu-Absi, Susan Fugett; Yang, LiYing; Thompson, Patrick; Jiang, Canping; Kandula, Sunitha; Schilling, Bernhard; Shukla, Abhinav A

    2010-08-15

    The concept of design space has been taking root as a foundation of in-process control strategies for biopharmaceutical manufacturing processes. During mapping of the process design space, the multidimensional combination of operational variables is studied to quantify the impact on process performance in terms of productivity and product quality. An efficient methodology to map the design space for a monoclonal antibody cell culture process is described. A failure modes and effects analysis (FMEA) was used as the basis for the process characterization exercise. This was followed by an integrated study of the inoculum stage of the process which includes progressive shake flask and seed bioreactor steps. The operating conditions for the seed bioreactor were studied in an integrated fashion with the production bioreactor using a two stage design of experiments (DOE) methodology to enable optimization of operating conditions. A two level Resolution IV design was followed by a central composite design (CCD). These experiments enabled identification of the edge of failure and classification of the operational parameters as non-key, key or critical. In addition, the models generated from the data provide further insight into balancing productivity of the cell culture process with product quality considerations. Finally, process and product-related impurity clearance was evaluated by studies linking the upstream process with downstream purification. Production bioreactor parameters that directly influence antibody charge variants and glycosylation in CHO systems were identified.

  6. Purification of human seminal plasma no. 7 antigen by immunoaffinity chromatography on bound monoclonal antibody.

    Science.gov (United States)

    Isojima, S; Koyama, K; Fujiwara, N

    1982-01-01

    Human seminal plasma (HSP) No. 7 antigen was purified by immunoaffinity chromatography on bound 1C4 monoclonal antibody (Moab) (Shigeta et al., 1980b). The pooled HSP protein was applied to a CNBr-activated Sepharose 4B column of bound 1C4 Moab gamma globulin and the antibody bound fraction (fr) eluted was further purified by rechromatography in the same way. The purified antigen in the antibody bound fr obtained by rechromatography gave a single band on SDS-PAGE in a position corresponding to a molecular weight of 15,000 daltons. This preparation was 196.2 times more effective than the original HSP protein in neutralizing the sperm immobilizing activity of 1C4 Moab. The purified HSP No. 7 antigen contained iron, but was different from lactoferrin and transferrin. It did not show any enzymatic activities, such as those of acid phosphatase, LDH or trypsin inhibitor, and shared antigenicity with human milk protein. It was present in seminal plasma as a molecule with a higher molecular weight but seemed to be cleaved to a monomer of 15,000 daltons during purification procedures. This antigen is present on spermatozoa as sperm-coating antigen and the corresponding antibody can immobilize spermatozoa with complement. Images Fig. 3 PMID:7127911

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-11-02

    ABSTRACT

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

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

  8. Generation and applications of monoclonal antibodies for livestock production.

    Science.gov (United States)

    Van Der Lende, T

    1994-01-01

    Monoclonal antibodies (MCAs) have found widespread applications in livestock production. Although the generation of murine MCAs is at present a routine, the production of homologous MCAs, especially important for in vivo applications, is still hampered by the lack of efficient homologous fusion partners for immortalization of antibody producing lymphocytes of livestock species. At present, MCAs are used in immunodiagnostic tests e.g. to monitor livestock reproduction and quality of livestock products. In the future MCAs will also be used in immunosensors for real-time and on-site applications in the same areas. The commercial application of MCAs for the immunomodulation of (pharmacologically induced) physiological processes underlying important (re)production traits is at present limited to the use of anti-PMSG MCAs in PMSG-induced superovulation. However, many potentially interesting applications are under investigation (e.g. immunopotentiation of growth hormone to enhance growth; immunocytolysis of adipocytes to increase lean meat production; immunoneutralization of GnRH for immunocastration; immunoimitation of hormone activity with anti-idiotype antibodies). Attempts to use specific MCAs for the sexing of embryos have been disappointing, mainly because of the relatively low accuracy. In the future, MCAs against membrane proteins which are specific for X- or Y-chromosome bearing spermatozoa might be used for bulk separation of livestock sperm. In general, it is expected that engineered (homologous) recombinant MCAs will largely contribute to the development of a new generation of rapid immunodiagnostic tests and effective immunomodulation applications. They will further increase the use of MCAs in livestock production.

  9. Kinetic analysis of the multistep aggregation mechanism of monoclonal antibodies.

    Science.gov (United States)

    Nicoud, Lucrèce; Arosio, Paolo; Sozo, Margaux; Yates, Andrew; Norrant, Edith; Morbidelli, Massimo

    2014-09-11

    We investigate by kinetic analysis the aggregation mechanism of two monoclonal antibodies belonging to the IgG1 and IgG2 subclass under thermal stress. For each IgG, we apply a combination of size exclusion chromatography and light scattering techniques to resolve the time evolution of the monomer, dimer, and trimer concentrations, as well as the average molecular weight and the average hydrodynamic radius of the aggregate distribution. By combining the detailed experimental characterization with a theoretical kinetic model based on population balance equations, we extract relevant information on the contribution of the individual elementary steps on the global aggregation process. The analysis shows that the two molecules follow different aggregation pathways under the same operating conditions. In particular, while the monomer depletion of the IgG1 is found to be rate-limited by monomeric conformational changes, bimolecular collision is identified as the rate-limiting step in the IgG2 aggregation process. The measurement of the microscopic rate constants by kinetic analysis allows the quantification of the protein-protein interaction potentials expressed in terms of the Fuchs stability ratio (W). It is found that the antibody solutions exhibit large W values, which are several orders of magnitude larger than the values computed in the frame of the DLVO theory. This indicates that, besides net electrostatic repulsion, additional effects delay the aggregation kinetics of the antibody solutions with respect to diffusion-limited conditions. These effects likely include the limited efficiency of the collision events due to the presence of a limited number of specific aggregation-prone patches on the heterogeneous protein surface, and the contribution of additional repulsive non-DLVO forces to the protein-protein interaction potential, such as hydration forces.

  10. Application of monoclonal antibodies to quality control of foot-and-mouth disease vaccines.

    Science.gov (United States)

    Alonso, A; Darsie, G C; Teixeira, A C; Reis, J L; Mesquita, J A

    1994-06-01

    Panels of monoclonal antibodies (mAbs) produced against foot-and-mouth disease (FMD) virus types O, A and C were selected for cell culture neutralization titre (NT), mouse protection index (MPI), trypsin sensitivity (TS) and avidity to different epitopes. The selected sets were used to assay the antigen concentration and the fit between FMDV vaccine and challenge strains. It was observed that FMD vaccines protect more than 75% of vaccinated cattle when manufactured with antigens characterized by (1) a high degree of fit with the potency control virus, and (2) mean ELISA 50% titres (T50) > 28 for O, > 18 for A and > 75 for C types, respectively, using the corresponding mAb set.

  11. Monoclonal antibody against the active site of caeruloplasmin and the ELISA system detecting active caeruloplasmin.

    Science.gov (United States)

    Hiyamuta, S; Ito, K

    1994-04-01

    Serum caeruloplasmin deficiency is a characteristic biochemical abnormality found in patients with Wilson's disease, but the mechanism of this disease is unknown. Although the phenylenediamine oxidase activity of serum caeruloplasmin is markedly low in patients with Wilson's disease, mRNA of caeruloplasmin exists to some extent. To investigate the deficiency of caeruloplasmin oxidase activity in Wilson's disease, we generated 14 monoclonal antibodies (MAbs) and selected ID1, which had the strongest reactivity, and ID2, which had neutralizing ability. We also established a system to measure active caeruloplasmin specifically using these MAbs. These MAbs and the system will be useful tools in analyzing the active site of caeruloplasmin in patients with Wilson's disease.

  12. Potent dengue virus neutralization by a therapeutic antibody with low monovalent affinity requires bivalent engagement.

    Directory of Open Access Journals (Sweden)

    Melissa A Edeling

    2014-04-01

    Full Text Available We recently described our most potently neutralizing monoclonal antibody, E106, which protected against lethal Dengue virus type 1 (DENV-1 infection in mice. To further understand its functional properties, we determined the crystal structure of E106 Fab in complex with domain III (DIII of DENV-1 envelope (E protein to 2.45 Å resolution. Analysis of the complex revealed a small antibody-antigen interface with the epitope on DIII composed of nine residues along the lateral ridge and A-strand regions. Despite strong virus neutralizing activity of E106 IgG at picomolar concentrations, E106 Fab exhibited a ∼20,000-fold decrease in virus neutralization and bound isolated DIII, E, or viral particles with only a micromolar monovalent affinity. In comparison, E106 IgG bound DENV-1 virions with nanomolar avidity. The E106 epitope appears readily accessible on virions, as neutralization was largely temperature-independent. Collectively, our data suggest that E106 neutralizes DENV-1 infection through bivalent engagement of adjacent DIII subunits on a single virion. The isolation of anti-flavivirus antibodies that require bivalent binding to inhibit infection efficiently may be a rare event due to the unique icosahedral arrangement of envelope proteins on the virion surface.

  13. Antiviral Therapy by HIV-1 Broadly Neutralizing and Inhibitory Antibodies

    Directory of Open Access Journals (Sweden)

    Zhiqing Zhang

    2016-11-01

    Full Text Available Human immunodeficiency virus type 1 (HIV-1 infection causes acquired immune deficiency syndrome (AIDS, a global epidemic for more than three decades. HIV-1 replication is primarily controlled through antiretroviral therapy (ART but this treatment does not cure HIV-1 infection. Furthermore, there is increasing viral resistance to ART, and side effects associated with long-term therapy. Consequently, there is a need of alternative candidates for HIV-1 prevention and therapy. Recent advances have discovered multiple broadly neutralizing antibodies against HIV-1. In this review, we describe the key epitopes on the HIV-1 Env protein and the reciprocal broadly neutralizing antibodies, and discuss the ongoing clinical trials of broadly neutralizing and inhibitory antibody therapy as well as antibody combinations, bispecific antibodies, and methods that improve therapeutic efficacy by combining broadly neutralizing antibodies (bNAbs with latency reversing agents. Compared with ART, HIV-1 therapeutics that incorporate these broadly neutralizing and inhibitory antibodies offer the advantage of decreasing virus load and clearing infected cells, which is a promising prospect in HIV-1 prevention and treatment.

  14. Antiviral Therapy by HIV-1 Broadly Neutralizing and Inhibitory Antibodies.

    Science.gov (United States)

    Zhang, Zhiqing; Li, Shaowei; Gu, Ying; Xia, Ningshao

    2016-11-18

    Human immunodeficiency virus type 1 (HIV-1) infection causes acquired immune deficiency syndrome (AIDS), a global epidemic for more than three decades. HIV-1 replication is primarily controlled through antiretroviral therapy (ART) but this treatment does not cure HIV-1 infection. Furthermore, there is increasing viral resistance to ART, and side effects associated with long-term therapy. Consequently, there is a need of alternative candidates for HIV-1 prevention and therapy. Recent advances have discovered multiple broadly neutralizing antibodies against HIV-1. In this review, we describe the key epitopes on the HIV-1 Env protein and the reciprocal broadly neutralizing antibodies, and discuss the ongoing clinical trials of broadly neutralizing and inhibitory antibody therapy as well as antibody combinations, bispecific antibodies, and methods that improve therapeutic efficacy by combining broadly neutralizing antibodies (bNAbs) with latency reversing agents. Compared with ART, HIV-1 therapeutics that incorporate these broadly neutralizing and inhibitory antibodies offer the advantage of decreasing virus load and clearing infected cells, which is a promising prospect in HIV-1 prevention and treatment.

  15. Immunolocation of antisperm monoclonal antibody 6B10 and corresponding antigen

    Institute of Scientific and Technical Information of China (English)

    高绍荣; 胡国俊; 段崇文; 刘辉; 韩之明; 宋祥芬; 陈大元

    1999-01-01

    An antisperm monoclonal antibody 6B10 was produced by hybridoma technique of the isotype IgG. The monoclonal antibody was purified by means of ammonium sulfate precipitation and protein A-Sepharose Cl-4B affinity chromatography. SDS polyacrylamide gel electrophoresis was used to evaluate the purity of the antibody. Evaluation of the sperm acrosomal status was determined by chlortetracycline (CTC) staining. It was found that monoclonal antibody 6B10 can inhibit the sperm acrosome reaction induced by progesterone. The corresponding antigen recognized by monoclonal antibody 6B10 was located on the plasma membrane of the sperm acrosome by indirect immunofluorescent microscopy and immunoelectronmicroscopy. Sperm protein was extracted by 1% Triton X-100. The molecular weight of the antigen is 50 ku, detected by Western blot. The antigen is a key protein in the sperm acrosome reaction and may be the receptor of progesterone on the sperm acrosome. It may either be developed as a candidate contraceptive vaccine

  16. Immunogenicity assessment of monoclonal antibody products: A simulated case study correlating antibody induction with clinical outcomes.

    Science.gov (United States)

    Knezevic, Ivana; Kang, Hye-Na; Thorpe, Robin

    2015-09-01

    Monoclonal antibodies are large molecules with complex structure and functions. They have a wide application for treatment of a broad range of chronic diseases and represent the largest class of biotherapeutic products. Given that biotherapeutic products may induce unwanted humoral and/or cellular immune responses in recipients, it is essential to investigate the immunogenicity of a product prior to licensure. The immune response is influenced by many factors and data generated in the pre-licensure studies are usually somewhat difficult for regulatory review. The knowledge and expertise required for this requires a thorough understanding of animal and human immunology as well as specific product characteristics, including mechanism of action, antibody assays and assessment of results in a given clinical context. The appropriate interpretation of immunogenicity data is of critical importance for defining the safety profile of a monoclonal antibody. Two case studies described in this paper were prepared to mimic a real situation in which regulators need to evaluate immunogenicity studies conducted by manufacturers of monoclonal antibody products. The specific objective of the case studies was to illustrate assessment of unwanted immunogenicity and the important factors that need to be considered in this context. Regulators and manufacturers who attended the World Health Organization (WHO) implementation workshop on Evaluation of Biotherapeutic Products, held in Seoul, Republic of Korea, in May 2014, participated in the case studies and provided valuable input. This article outlines the main aspects of immunogenicity discussed in these case studies and a summary of the lessons learned at this occasion. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  17. Generation and characterization of the human neutralizing antibody fragment Fab091 against rabies virus

    Institute of Scientific and Technical Information of China (English)

    Chen LI; Feng ZHANG; Hong LIN; Zhong-can WANG; Xin-jian LIU; Zhen-qing FENG; Jin ZHU; Xiao-hong GUAN

    2011-01-01

    Aim: To transform the human anti-rabies virus glycoprotein (anti-RABVG) single-chain variable fragment (scFv) into a Fab fragment and to analyze its immunological activity.Methods: The Fab gene was amplified using overlap PCR and inserted into the vector pComb3XSS. The recombinant vector was then transformed into E coli Top10F' for expression and purification. The purified Fab was characterized using SDS-PAGE, Western blotting,indirect ELISA, competitive ELISA, and the fluorescent antibody virus neutralization test (FAVN), respectively, and examined in a Kunming mouse challenge model in vivo.Results: A recombinant vector was constructed. The Fab was expressed in soluble form In E coll Top10F'. Specific binding of the Fab to rabies virus was confirmed by indirect ELISA and immunoprecipitation (IP). The neutralizing antibody titer of Fab was 10.26 IU/mL.The mouse group treated with both vaccine and human rabies immunoglobulin (HRIG)/Fab091 (32 IU/kg) showed protection against rabies, compared with the control group (P<0.05, Logrank test).Conclusion: The antibody fragment Fab was shown to be a neutralizing antibody against RABVG. It can be used together with other monoclonal antibodies for post-exposure prophylaxis of rabies virus in future studies.

  18. Broadly Neutralizing Antibodies Display Potential for Prevention of HIV-1 Infection of Mucosal Tissue Superior to That of Nonneutralizing Antibodies.

    Science.gov (United States)

    Cheeseman, Hannah M; Olejniczak, Natalia J; Rogers, Paul M; Evans, Abbey B; King, Deborah F L; Ziprin, Paul; Liao, Hua-Xin; Haynes, Barton F; Shattock, Robin J

    2017-01-01

    Definition of the key parameters mediating effective antibody blocking of HIV-1 acquisition within mucosal tissue may prove critical to effective vaccine development and the prophylactic use of monoclonal antibodies. Although direct antibody-mediated neutralization is highly effective against cell-free virus, antibodies targeting different sites of envelope vulnerability may display differential activity against mucosal infection. Nonneutralizing antibodies (nnAbs) may also impact mucosal transmission events through Fc-gamma receptor (FcγR)-mediated inhibition. In this study, a panel of broadly neutralizing antibodies (bnAbs) and nnAbs, including those associated with protection in the RV144 vaccine trial, were screened for the ability to block HIV-1 acquisition and replication across a range of cellular and mucosal tissue models. Neutralization potency, as determined by the TZM-bl infection assay, did not fully predict activity in mucosal tissue. CD4-binding site (CD4bs)-specific bnAbs, in particular VRC01, were consistent in blocking HIV-1 infection across all cellular and tissue models. Membrane-proximal external region (MPER) (2F5) and outer domain glycan (2G12) bnAbs were also efficient in preventing infection of mucosal tissues, while the protective efficacy of bnAbs targeting V1-V2 glycans (PG9 and PG16) was more variable. In contrast, nnAbs alone and in combinations, while active in a range of cellular assays, were poorly protective against HIV-1 infection of mucosal tissues. These data suggest that tissue resident effector cell numbers and low FcγR expression may limit the potential of nnAbs to prevent establishment of the initial foci of infection. The solid protection provided by specific bnAbs clearly demonstrates their superior potential over that of nonneutralizing antibodies for preventing HIV-1 infection at the mucosal portals of infection.

  19. Radiolabeled monoclonal antibodies for imaging and therapy: Potential, problems, and prospects: Scientific highlights

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, S.C.; Buraggi, G.L.

    1986-01-01

    This meeting focused on areas of research on radiolabeled monoclonal antibodies. Topics covered included the production, purification, and fragmentation of monoclonal antibodies and immunochemistry of hybridomas; the production and the chemistry of radionuclides; the radiohalogenation and radiometal labeling techniques; the in-vivo pharmacokinetics of radiolabeled antibodies; the considerations of immunoreactivity of radiolabeled preparations; the instrumentation and imaging techniques as applied to radioimmunodetection; the radiation dosimetry in diagnostic and therapeutic use of labeled antibodies; the radioimmunoscintigraphy and radioimmunotherapy studies; and perspectives and directions for future research. Tutorial as well as scientific lectures describing the latest research data on the above topics were presented. Three workshop panels were convened on ''Methods for Determining Immunoreactivity of Radiolabeled Monoclonal Antibodies - Problems and Pitfalls,'' Radiobiological and Dosimetric Considerations for Immunotherapy with Labeled Antibodies,'' and ''The Human Anti-Mouse Antibody Response in Patients.''

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

    Directory of Open Access Journals (Sweden)

    Ouverney E.P.

    2005-01-01

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

  1. Enhanced neutralization potency of botulinum neurotoxin antibodies using a red blood cell-targeting fusion protein.

    Directory of Open Access Journals (Sweden)

    Sharad P Adekar

    Full Text Available Botulinum neurotoxin (BoNT potently inhibits cholinergic signaling at the neuromuscular junction. The ideal countermeasures for BoNT exposure are monoclonal antibodies or BoNT antisera, which form BoNT-containing immune complexes that are rapidly cleared from the general circulation. Clearance of opsonized toxins may involve complement receptor-mediated immunoadherence to red blood cells (RBC in primates or to platelets in rodents. Methods of enhancing immunoadherence of BoNT-specific antibodies may increase their potency in vivo. We designed a novel fusion protein (FP to link biotinylated molecules to glycophorin A (GPA on the RBC surface. The FP consists of an scFv specific for murine GPA fused to streptavidin. FP:mAb:BoNT complexes bound specifically to the RBC surface in vitro. In a mouse model of BoNT neutralization, the FP increased the potency of single and double antibody combinations in BoNT neutralization. A combination of two antibodies with the FP gave complete neutralization of 5,000 LD50 BoNT in mice. Neutralization in vivo was dependent on biotinylation of both antibodies and correlated with a reduction of plasma BoNT levels. In a post-exposure model of intoxication, FP:mAb complexes gave complete protection from a lethal BoNT/A1 dose when administered within 2 hours of toxin exposure. In a pre-exposure prophylaxis model, mice were fully protected for 72 hours following administration of the FP:mAb complex. These results demonstrate that RBC-targeted immunoadherence through the FP is a potent enhancer of BoNT neutralization by antibodies in vivo.

  2. Application of broad-spectrum humanized neutralizing monoclonal antibodies to influenza A virus in prevention and treatment of influenza%广谱全人源化甲型流感病毒中和抗体在流感预防与治疗中的应用

    Institute of Scientific and Technical Information of China (English)

    高灵茜; 杨利

    2013-01-01

    针对多个亚型甲型流感病毒的广谱中和性单克隆抗体(monoclonalantibody,mAb)是极具潜力的新型而有效的流感防治手段.抗流感病毒血凝素(hemagglutinin,HA)的保护性mAb,无论通过主动免疫还是被动免疫,均具有很好的效果.迄今,已有不少甲型流感病毒广谱中和性mAb被开发出来.尽管这些mAb具备临床应用潜能以及作为研制新型疫苗(抗独特型抗体疫苗)的可能,但仅有几种HA mAb对人类流感防治真正有效.此文主要对能高效识别多个亚型HA的人源化甲型流感病毒mAb做一综述.%Broad-spectrum neutralizing monoclonal antibodies (mAbs) against different subtypes of influenza A viruses are potential tools to prevent and treat influenza.Protective mAbs to influenza hemagglutinin (HA) have good results by passive and active immunization.So far,a number of mAbs against influenza HA have been developed.But only few of them are effective for influenza prevention and treatment.In this article,the human mAbs which can recognize divergent influenza isolates are reviewed.

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

    Science.gov (United States)

    Minkoff, Marjorie Sue

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

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

    Science.gov (United States)

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

    2016-04-22

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

  5. Epitope Mapping of Dengue-Virus-Enhancing Monoclonal-Antibody Using Phage Display Peptide Library

    Directory of Open Access Journals (Sweden)

    Chung-I Rai

    2008-01-01

    Full Text Available The Antibody-Dependent Enhancement (ADE hypothesis has been proposed to explain why more severe manifestations of Dengue Hemorrhagic Fever and Dengue Shock Syndrome (DHF/DSS occur predominantly during secondary infections of Dengue Virus (DV with different serotypes. However, the epitopes recognized by these enhancing antibodies are unclear. Recently, anti-pre-M monoclonal antibody (mAb 70-21, which recognized all DV serotypes without neutralizing activity, were generated and demonstrated as an enhancing antibody for DV infection. In the present study, the epitope recognized by mAb 70-21 was identified using a phage-displayed random-peptide library. After three rounds of biopanning, ELISA showed that immunopositive phage clones specifically bound to mAb 70-21 but not to serum or purified IgG from naive mice. DNA sequencing of these phage clones showed a consensus sequence, QNNLGPR. Like mAb70-21, these phage-induced antisera also enhanced the DV infection of cells. In addition, indirect fluorescent assays showed phage-induced antisera bound to human rhabdomyosarcoma or Vero cells. Western blotting and immunoprecipitation analysis showed that phage-induced antisera recognized hsp 60 in BHK cell lysate. Moreover, the sera levels of antibodies against the synthetic peptide QNNLGPR correlated with the disease severity of dengue patients. Taken together, these results suggest that antibodies which recognized epitopes shared by pre-M of DV and hsp 60 of host cells may enhance DV infection and be involved in the development of DHF or DSS.

  6. A monoclonal antibody that recognizes an antigenic determinant shared by HLA A2 and B17.

    Science.gov (United States)

    McMichael, A J; Parham, P; Rust, N; Brodsky, F

    1980-09-01

    A hybridoma monoclonal anti-HLA antibody has been produced by the technique of Kohler and Milstein [1]. This antibody recognizes a new specificity common to HLA A2 and B17. It was shown to be a single antibody by isoelectric focusing and absorption experiments.

  7. EFFECT OF POLYCLONAL AND MONOCLONAL-ANTIBODIES ON SURFACE-PROPERTIES OF STREPTOCOCCUS-SOBRINUS

    NARCIS (Netherlands)

    VANRAAMSDONK, M; VANDERMEI, HC; DESOET, JJ; BUSSCHER, HJ; DEGRAAFF, J

    1995-01-01

    In this study, the effect of antibody adsorption on physicochemical properties of Streptococcus sobrinus was studied. Bacteria were preincubated with polyclonal antibodies or with OMVU10, a monoclonal antibody (MAb) reactive with S. sobrinus. The zeta potentials and the hydrophobicity as determined

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

    Science.gov (United States)

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

    2013-11-01

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

  9. Honing a harder-hitting hammerhead improves broadly neutralizing antibody breadth and potency.

    Science.gov (United States)

    Lewis, George K

    2015-06-01

    While current HIV-1 therapies have greatly improved the quality and duration of life for infected individuals, a vaccine to prevent transmission of the virus is lacking. Broadly neutralizing monoclonal antibodies (bnmAbs) with the capacity to neutralize multiple HIV-1 variants have been isolated from HIV-1-infected individuals, and there has been a great effort to investigate how these bnmAbs arise, due their potential for HIV-1 vaccination. In this issue of the JCI, Willis and colleagues apply a computational approach to design variants of the bnmAb PG9 in an attempt to enhance potency and neutralization breadth. One of these variants was able to target multiple PG9-resistant strains, as the result of stabilization of the long heavy chain complementarity determining region 3 (HCDR3). The results of this study provide important insight and a unique approach to optimizing HIV-1 bnmABs.

  10. A focus reduction neutralization assay for hepatitis C virus neutralizing antibodies

    Directory of Open Access Journals (Sweden)

    Wychowski Czeslaw

    2007-03-01

    Full Text Available Abstract Background/Aim The role of humoral immunity in hepatitis C virus (HCV infection is poorly understood. Nevertheless, there is increasing interest in characterizing the neutralizing antibodies in the serum of HCV-infected patients. Focus reduction assays have been widely used to evaluate neutralizing antibody responses against a range of non-cytopathic viruses. Based on the recent development of a HCV cell culture system using the genotype 2 JFH-1-strain, we developed a focus reduction assay for HCV-neutralizing antibodies. Methods The focus reduction assay was based on a standard microneutralization assay in which immunostained foci on tissue culture plates are counted. The neutralizing anti-HCV antibodies titers of purified serum immunoglobulin samples from seventy-seven individuals were determined using a 50% focus reduction neutralization assay. Each titer was determined as the log value of the reciprocal antibody dilution that reduced the number of viral foci by 50%. IgG antibodies were first purified from each serum in order to avoid the facilitating effect of HDL on HCV entry. Results The assay's cut-off using an ELISA and RNA HCV-negative samples was found to be 1.25 log, corresponding to a dilution of 1:18. The assay was compared with a commercial HCV ELISA and exhibited specificity and sensitivity values of 100% and 96.5%, respectively, and good reproducibility (with intra-assay and inter-assay coefficients of variation of 6.7% and 12.6%, respectively. The assay did not show any cross-reactivity with anti-HIV, anti-HBs or heterophile antibody-positive samples. The neutralizing antibodies titers were 2.13 log (1:134 for homologous samples from HCV genotype 2 infected patients harboring the same genotype as JFH-1 and 1.93 log (1:85 for heterologous samples from patients infected by genotypes other than type 2. These results confirm the presence of broadly cross-neutralizing antibodies already reported using the HCV pseudoparticles

  11. Higher Throughput Quantification of Neutralizing Antibody to Herpes Simplex Viruses.

    Directory of Open Access Journals (Sweden)

    Tamara P Blevins

    Full Text Available We report a rapid, higher throughput method for measuring neutralizing antibody to herpes simplex virus (HSV in human sera. Clinical isolates and sera from the Herpevac Trial for Women were used in a colorimetric assay in which infection of tissue culture (lack of neutralization was indicated by substrate metabolism by beta-galactosidase induced in the ELVIS cell line. The neutralization assay was optimized by addition of guinea pig complement, which particularly enhanced neutralizing antibody titers to HSV-2. Higher neutralizing antibody titers were also achieved using virus particles isolated from the supernatant of infected cells rather than lysate of infected cells as the source of virus. The effect of assay incubation time and incubation time with substrate were also optimized. We found that incubating with substrate until a standard optical density of 1.0 was reached permitted a better comparison among virus isolates, and achieved reliable measurement of neutralizing antibody activity. Interestingly, in contrast to results in the absence of complement, addition of complement allowed sera from HSV-2 gD-vaccinated subjects to neutralize HSV-1 and HSV-2 clinical and laboratory isolates with equal potency.

  12. Use of radiolabeled monoclonal antibodies for diagnostic imaging

    Energy Technology Data Exchange (ETDEWEB)

    Endo, Keigo (Kyoto Univ. (Japan). Faculty of Medicine)

    1990-08-01

    Monoclonal antibodies (MoAbs) are expected to carry radionuclides selectively to target tissues and to offer antigen-specific diagnosis. Indium (In)-111 has many favorable nuclear properties and is efficiently labeled with MoAbs using DAPA as a bifunctional chelating agent. In-111 labeled MoAbs are clinically employed for the diagnosis of malignant melanoma, colorectal cancer and acute myocardial infarction in Japan. Although non-specific deposit of In-111 was seen in liver and bone-marrow, scintigraphy using In-111 labeled MoAbs was encouraging, since it detected about 80% of tumors, tumors missed by conventional diagnostic methods such as CT, and tumors in patients with normal serum CEA values, and acute myocarditis as well as acute myocardial infarction was positive with In-111 labeled Fab fraction of anti-myosin Ab. Acute or subacute toxicity was not observed. Human anti-murine antibody (HAMA) was detected in 53 of 64 (82.8%) patients who were intravenously administered with 20 to 42 mg of anti-melanoma or anti-CEA MoAbs (whole IgG). In contrast, only 5 of 406 (1.2%) patients had detectable levels of HAMA in their serum after receiving 0.5 mg of Fab fraction of MoAb. Recently mouse-human chimeric Ab has been produced by recombinant DNA techniques, which localized well in xenografted tumors and seems to be promising for clinical use. Investigations are under way to increase the tumor to non-tumor ratio by modifying chelating agents for coupling MoAbs with radionuclides. (author).

  13. Characterization of monoclonal antibodies to avian Escherichia coli Iss.

    Science.gov (United States)

    Lynne, Aaron M; Foley, Steven L; Nolan, Lisa K

    2006-09-01

    Colibacillosis accounts for annual multimillion dollar losses in the poultry industry, and control of this disease is hampered by limited understanding of the virulence mechanisms used by avian pathogenic Escherichia coli (APEC). Previous work in our laboratory has found that the presence of the increased serum survival gene (iss) is strongly associated with APEC but not commensal E. coli, making iss and the protein it encodes (Iss) candidate targets of colibacillosis-control procedures. Previously, we produced monoclonal antibodies (MAbs) against Iss to be used as a reagent in studies of APEC virulence and colibacillosis pathogenesis. Unfortunately, the utility of these MAbs was limited because these MAbs exhibited nonspecific binding. It was thought that the lack of specificity might be related to the fact that these MAbs were of the immunoglobulin M (IgM) isotype. In the present study, new MAbs were produced using a different immunization strategy in an effort to generate MAbs of a different isotype. Also, because Iss bears strong similarity to Bor, a lambda-derived protein that occurs commonly among E. coli, MAbs were assessed for their ability to distinguish Iss and Bor. For these studies, the bor gene from an APEC isolate was cloned into an expression vector. The fusion protein expressed from this construct was used to assess the potential of the anti-Iss MAbs produced in the past and present studies to distinguish Bor and Iss. The MAbs produced in this study were of the IgG1 isotype, which appeared to bind more specifically to Iss than previously generated antibodies in certain immunologic procedures. These results suggested that the MAbs generated in this study might prove superior to the previous MAbs as a reagent for study of APEC. However, both MAbs recognized recombinant Iss and Bor, suggesting that any results obtained using anti-Iss MAbs would need to be interpreted with this cross-reactivity in mind.

  14. Broadly neutralizing antibodies targeted to mucin-type carbohydrate epitopes of human immunodeficiency virus

    DEFF Research Database (Denmark)

    Hansen, J E; Nielsen, C; Arendrup, M;

    1991-01-01

    . This inhibition was found in infection of both lymphocytic cells and monocytoid cells. Viruses tested included six HIV-1 and five HIV-2 isolates propagated in different cells, as well as infectious plasma from AIDS patients. The antiviral effect of anti-Tn MAbs occurred by specific binding of the MAb to the virus......The cancer-related mucin-type carbohydrate neoantigen Tn was found on gp160 and gp120 of human immunodeficiency virus (HIV). Immunoglobulin G (IgG) and IgM monoclonal antibodies (MAbs) against Tn neutralized infection with cell-free virus and blocked fusion between HIV-infected and uninfected cells...

  15. [ICO-166 monoclonal antibodies against the CD45RA antigen].

    Science.gov (United States)

    Frolova, E A; Baryshnikov, A Iu; Novikov, V V; Syrkin, A B

    1993-07-01

    Monoclonal antibodies (MCA) ICO-166 against CD45RA antigen were generated and characterized. In the indirect IFA, MCA ICO-166 reacted with 54.1 +/- 1.9% lymphocytes of human peripheral blood and 15.2 +/- 2.3% monocytes but not with granulocytes or thrombocytes. The method of double labelling of cells demonstrated that MCA ICO-166 detected all B-lymphocytes, all NK-cells and 31% of mature T-lymphocytes but only 55% of CD8 suppressor cells and only 21% of CDA helper cells carried this antigen on the surface. Experiments were carried out to block binding of FITC-labeled MCA ALB11 against CD45RA antigen with human lymphocytes by pretreatment of cells with different concentrations of MCA ICO-166. Treatment of cells with MCA ALB11 blocked binding of MCA ALB11-FITC by 85% on the average. MCA ICO-166 blocked binding of MCA ALB11-FITC by 66% on the average. When different dilutions of MCA ICO-166 were used, the dose-dependent effect of blocking of MCA ALB11-FITC binding was observed. MCA ICO-166 immunoprecipitated a protein band of molecular weight 220 kDa from lysates of mononuclear cells of the human peripheral blood.

  16. Role of cosolutes in the aggregation kinetics of monoclonal antibodies.

    Science.gov (United States)

    Nicoud, Lucrèce; Sozo, Margaux; Arosio, Paolo; Yates, Andrew; Norrant, Edith; Morbidelli, Massimo

    2014-10-16

    We propose a general strategy based on kinetic analysis to investigate how cosolutes affect the aggregation behavior of therapeutic proteins. We apply this approach to study the impact of NaCl and sorbitol on the aggregation kinetics of two monoclonal antibodies, an IgG1 and an IgG2. By using a combination of size exclusion chromatography and light scattering techniques, we study the impact of the cosolutes on the monomer depletion, as well as on the formation of dimers, trimers, and larger aggregates. We analyze these macroscopic effects in the frame of a kinetic model based on Smoluchowski's population balance equations modified to account for nucleation events. By comparing experimental data with model simulations, we discriminate the effect of cosolutes on the elementary steps which contribute to the global aggregation process. In the case of the IgG1, it is found that NaCl accelerates the kinetics of aggregation by promoting specifically aggregation events, while sorbitol delays the kinetics of aggregation by specifically inhibiting protein unfolding. In the case of the IgG2, whose monomer depletion kinetics is limited by dimer formation, NaCl and sorbitol are found respectively to accelerate and inhibit conformational changes and aggregation events to the same extent.

  17. Kinetics of Monoclonal Antibody Aggregation from Dilute toward Concentrated Conditions.

    Science.gov (United States)

    Nicoud, Lucrèce; Jagielski, Jakub; Pfister, David; Lazzari, Stefano; Massant, Jan; Lattuada, Marco; Morbidelli, Massimo

    2016-04-07

    Gaining understanding on the aggregation behavior of proteins under concentrated conditions is of both fundamental and industrial relevance. Here, we study the aggregation kinetics of a model monoclonal antibody (mAb) under thermal stress over a wide range of protein concentrations in various buffer solutions. We follow experimentally the monomer depletion and the aggregate growth by size exclusion chromatography with inline light scattering. We describe the experimental results in the frame of a kinetic model based on population balance equations, which allows one to discriminate the contributions of the conformational and of the colloidal stabilities to the global aggregation rate. Finally, we propose an expression for the aggregation rate constant, which accounts for solution viscosity, protein-protein interactions, as well as aggregate compactness. All these effects can be quantified by light scattering techniques. It is found that the model describes well the experimental data under dilute conditions. Under concentrated conditions, good model predictions are obtained when the solution pH is far below the isoelectric point (pI) of the mAb. However, peculiar effects arise when the solution pH is increased toward the mAb pI, and possible explanations are discussed.

  18. DNA immunization as a technology platform for monoclonal antibody induction.

    Science.gov (United States)

    Liu, Shuying; Wang, Shixia; Lu, Shan

    2016-04-06

    To combat the threat of many emerging infectious diseases, DNA immunization offers a unique and powerful approach to the production of high-quality monoclonal antibodies (mAbs) against various pathogens. Compared with traditional protein-based immunization approaches, DNA immunization is efficient for testing novel immunogen designs, does not require the production or purification of proteins from a pathogen or the use of recombinant protein technology and is effective at generating mAbs against conformation-sensitive targets. Although significant progress in the use of DNA immunization to generate mAbs has been made over the last two decades, the literature does not contain an updated summary of this experience. The current review provides a comprehensive analysis of the literature, including our own work, describing the use of DNA immunization to produce highly functional mAbs, in particular, those against emerging infectious diseases. Critical factors such as immunogen design, delivery approach, immunization schedule, use of immune modulators and the role of final boost immunization are discussed in detail.

  19. Characterization of Endotrypanum Parasites Using Specific Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Ramos Franco Antonia Maria

    1997-01-01

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

  20. Development and Evaluation of Monoclonal Antibodies for Paxilline

    Directory of Open Access Journals (Sweden)

    Chris M. Maragos

    2015-09-01

    Full Text Available Paxilline (PAX is a tremorgenic mycotoxin that has been found in perennial ryegrass infected with Acremonium lolii. To facilitate screening for this toxin, four murine monoclonal antibodies (mAbs were developed. In competitive indirect enzyme-linked immunosorbent assays (CI-ELISAs the concentrations of PAX required to inhibit signal development by 50% (IC50s ranged from 1.2 to 2.5 ng/mL. One mAb (2-9 was applied to the detection of PAX in maize silage. The assay was sensitive to the effects of solvents, with 5% acetonitrile or 20% methanol causing a two-fold or greater increase in IC50. For analysis of silage samples, extracts were cleaned up by adsorbing potential matrix interferences onto a solid phase extraction column. The non-retained extract was then diluted with buffer to reduce solvent content prior to assay. Using this method, the limit of detection for PAX in dried silage was 15 µg/kg and the limit of quantification was 90 µg/kg. Recovery from samples spiked over the range of 100 to 1000 µg/kg averaged 106% ± 18%. The assay was applied to 86 maize silage samples, with many having detectable, but none having quantifiable, levels of PAX. The results suggest the CI-ELISA can be applied as a sensitive technique for the screening of PAX in maize silage.

  1. Monoclonal antibody probe for assessing beer foam stabilizing proteins.

    Science.gov (United States)

    Onishi, A; Proudlove, M O; Dickie, K; Mills, E N; Kauffman, J A; Morgan, M R

    1999-08-01

    A monoclonal antibody (Mab; IFRN 1625) has been produced, which is specific for the most hydrophobic polypeptides responsible for foam stabilization. The binding characteristics of the Mab suggest that it is the conformation of certain hydrophobic polypeptides which is important for foam stabilization. An enzyme-linked immunosorbent assay (ELISA) for assessing the foam-positive form of the foam-stabilizing polypeptides in beer was developed using IFRN 1625. A good correlation was obtained between ELISA determination of foam-stabilizing polypeptides and an empirical means of determining foaming, that is, the Rudin head retention values, for a collection of beers of various foam qualities. Application of the ELISA to different stages of the brewing process showed that the amounts of foam-positive polypeptides increased during barley germination. During the brewing process the proportion of foam-positive polypeptides present after fermentation increased slightly, although a large amount was lost along with other beer proteins during subsequent steps, such as filtering. The present study demonstrates that the amounts of beer polypeptide present in a foam-positive form have a direct relationship with the foaming potential of beer, that their levels are altered by processing, and that there is potential for greater quality control.

  2. Monoclonal antibodies against NS1 protein of Goose parvovirus.

    Science.gov (United States)

    Qiu, Zheng; Tian, Wei; Yu, Tianfei; Li, Li; Ma, Bo; Wang, Junwei

    2012-04-01

    In the present study, monoclonal antibodies (MAbs) against NS1 protein of Goose parvovirus (GPV) were generated. The secreted MAbs were obtained by fusing mouse myeloma cells and spleen cells of BALB/c mice, which were immunized with the plasmid pcDNA3.1-GPV-NS1 and recombinant protein of GPV-NS1. With indirect ELISA, six hybridoma cell lines against GPV-NS1 were screened. The subtypes of the two MAbs were IgG2a; the others were IgM. The light chain was κ. Western blot analysis showed that six MAbs reacted with recombinant protein GPV-NS1. GPV-NS1 was dissected into 15 overlapping epitopes, which were used to react with MAbs in Western blot. Results showed that six MAbs recognized NS1 protein linear B-cell epitopes located at the C-terminus 453-514 aa, 485-542 aa, and 533-598 aa.

  3. Immunomodulatory Monoclonal Antibodies in Combined Immunotherapy Trials for Cutaneous Melanoma

    Directory of Open Access Journals (Sweden)

    Mariana Aris

    2017-08-01

    Full Text Available In the last few years, there has been a twist in cancer treatment toward immunotherapy thanks to the impressive results seen in advanced patients from several tumor pathologies. Cutaneous melanoma is a highly mutated and immunogenic tumor that has been a test field for the development of immunotherapy. However, there is still a way on the road to achieving complete and long-lasting responses in most patients. It is desirable that immunotherapeutic strategies induce diverse immune reactivity specific to tumor antigens, including the so-called neoantigens, as well as the blockade of immunosuppressive mechanisms. In this review, we will go through the role of promising monoclonal antibodies in cancer immunotherapy with immunomodulatory function, especially blocking of the inhibitory immune checkpoints CTLA-4 and PD-1, in combination with different immunotherapeutic strategies such as vaccines. We will discuss the rational basis for these combinatorial approaches as well as different schemes currently under study for cutaneous melanoma in the clinical trials arena. In this way, the combination of “push and release” immunomodulatory therapies can contribute to achieving a more robust and durable antitumor immune response in patients.

  4. An update on newer monoclonal antibodies in lymphoma therapy

    Directory of Open Access Journals (Sweden)

    Subhashini Archana Kadavakolan

    2016-01-01

    Full Text Available In 2014, an estimated 9.4% of all new cancers in the US were accounted to hematological cancers. Most of these cancers have a B-cell origin and on the cell surface express antigen CD20-known to restrict B-cells. Considering the intrinsic immune status of the patients receiving chemotherapy, monoclonal antibodies (mAbs are designed to provide active or passive immunotherapy. Clinical success of rituximab-anti-CD20 mAb in the treatment of lymphoma has led to the development of newer generations of mAb to increase the anti-tumor activity. Hence, recent advances in lymphoma therapy are being built on the conventional prototype of anti-CD20 mAb-rituximab. Our review is an update on the advances in lymphoma therapy using mAb against CD20 including the second generation-ofatumumab, veltuzumab, ocrelizumab, and the third-generation mAbs-ocaratuzumab and obinutuzumab.

  5. Establishment of a novel monoclonal antibody against LGR5.

    Science.gov (United States)

    Sasaki, Yuka; Kosaka, Hiromichi; Usami, Katsuaki; Toki, Hiroe; Kawai, Hironori; Shiraishi, Norihiko; Ota, Toshio; Nakamura, Kazuyasu; Furuya, Akiko; Satoh, Mitsuo; Hasegawa, Kazumasa; Masuda, Kazuhiro

    2010-04-09

    LGR5 is an orphan G-protein-coupled receptor (GPCR) that is expressed on the cell surface membrane. LGR5 is reported to be overexpressed in colon, liver, and ovary tumor compared to normal tissue. However, a specific ligand for LGR5 has not yet been determined, and the function is still not clear. An LGR5-specific monoclonal antibody (mAb) is needed as a tool for detection and analysis of LGR5 biological function and cancer therapy. To date, no mAb against LGR5 that retains high affinity and specificity has been reported. Here, we report successful establishment and characterization of a mAb (KM4056) that specifically recognizes the extracellular N-terminal domain of human LGR5, but not LGR4 or LGR6. This mAb has potent complement-dependent cytotoxicity (CDC) activity in vitro and shows strong anti-tumor activity in vivo against xenograft model by transplanting LGR5 expressing CHO transfectants into SCID mice. Thus, KM4056 can be a useful tool for detection of LGR5 positive cells and analysis of LGR5 biological function.

  6. Production and Characterization of Monoclonal Antibody Against Recombinant Human Erythropoietin

    Institute of Scientific and Technical Information of China (English)

    JIE-BO MI; JIN YAN; XIAO-JIE DING; ZHEN-QUAN GUO; MEI-PING ZHAO; WEN-BAO CHANG

    2007-01-01

    Objective To produce specific monoclonal antibody(mAb)against recombinant human erythropoietin(rHuEPO)for development of higmy efficient methods for erythropoietin detection in biological fluids.Methods rHuEPO was covalently coupled with bovine serum albumin(BSA)and the conjugate was used to immunize mice to produce specific mAb against rHuEPO based on hybridoma technology.The obtained F3-mAb was characterized by enzyme-linked immunosorbent assay (ELISA),SDS-PAGE and Western blot.Results The isotype of F3-mAb Was found to be IgM with an affinity constant of 2.1x108 L/mol.The competitive ELISA using the obtained IgM showed a broader linear range and lower detection limit compared with previous work.Conclusions The modification of rHuEPO was proved to be successful in generating required specific mAb with high avidity to rHuEPO.

  7. Downstream processing of monoclonal antibodies--application of platform approaches.

    Science.gov (United States)

    Shukla, Abhinav A; Hubbard, Brian; Tressel, Tim; Guhan, Sam; Low, Duncan

    2007-03-15

    This paper presents an overview of large-scale downstream processing of monoclonal antibodies and Fc fusion proteins (mAbs). This therapeutic modality has become increasingly important with the recent approval of several drugs from this product class for a range of critical illnesses. Taking advantage of the biochemical similarities in this product class, several templated purification schemes have emerged in the literature. In our experience, significant biochemical differences and the variety of challenges to downstream purification make the use of a completely generic downstream process impractical. Here, we describe the key elements of a flexible, generic downstream process platform for mAbs that we have adopted at Amgen. This platform consists of a well-defined sequence of unit operations with most operating parameters being pre-defined and a small subset of parameters requiring development effort. The platform hinges on the successful use of Protein A chromatography as a highly selective capture step for the process. Key elements of each type of unit operation are discussed along with data from 14 mAbs that have undergone process development. Aspects that can be readily templated as well as those that require focused development effort are identified for each unit operation. A brief description of process characterization and validation activities for these molecules is also provided. Finally, future directions in mAb processing are summarized.

  8. Pharmacokinetics of biotech drugs: peptides, proteins and monoclonal antibodies.

    Science.gov (United States)

    Lin, Jiunn H

    2009-09-01

    With the advances in recombinant DNA biotechnology, molecular biology and immunology, the number of biotech drugs, including peptides, proteins and monoclonal antibodies, available for clinical use has dramatically increased in recent years. Although pharmacokinetic principles are equally applicable to the large molecule drugs and conventional small molecule drugs, the underlying mechanisms for the processes of absorption, distribution, metabolism and excretion (ADME) of large molecule drugs are often very different from that of small molecule drugs. Therefore, a good understanding of the ADME processes of large molecule drugs is essential in support of the development of therapeutic biologics. The purpose of this article is to review the current knowledge of the ADME processes that govern the pharmacokinetics of biotech drugs. The challenges encountered by orally administered peptide and protein drugs, and the nature of lymphatic absorption after subcutaneous administration will be discussed. In addition, molecular mechanisms of biodistribution, metabolism and renal excretion of biotech drugs will also be discussed. Finally, approaches used for prediction of human pharmacokinetics of protein drugs will be briefly discussed.

  9. Protective monotherapy against lethal Ebola virus infection by a potently neutralizing antibody.

    Science.gov (United States)

    Corti, Davide; Misasi, John; Mulangu, Sabue; Stanley, Daphne A; Kanekiyo, Masaru; Wollen, Suzanne; Ploquin, Aurélie; Doria-Rose, Nicole A; Staupe, Ryan P; Bailey, Michael; Shi, Wei; Choe, Misook; Marcus, Hadar; Thompson, Emily A; Cagigi, Alberto; Silacci, Chiara; Fernandez-Rodriguez, Blanca; Perez, Laurent; Sallusto, Federica; Vanzetta, Fabrizia; Agatic, Gloria; Cameroni, Elisabetta; Kisalu, Neville; Gordon, Ingelise; Ledgerwood, Julie E; Mascola, John R; Graham, Barney S; Muyembe-Tamfun, Jean-Jacques; Trefry, John C; Lanzavecchia, Antonio; Sullivan, Nancy J

    2016-03-18

    Ebola virus disease in humans is highly lethal, with case fatality rates ranging from 25 to 90%. There is no licensed treatment or vaccine against the virus, underscoring the need for efficacious countermeasures. We ascertained that a human survivor of the 1995 Kikwit Ebola virus disease outbreak maintained circulating antibodies against the Ebola virus surface glycoprotein for more than a decade after infection. From this survivor we isolated monoclonal antibodies (mAbs) that neutralize recent and previous outbreak variants of Ebola virus and mediate antibody-dependent cell-mediated cytotoxicity in vitro. Strikingly, monotherapy with mAb114 protected macaques when given as late as 5 days after challenge. Treatment with a single human mAb suggests that a simplified therapeutic strategy for human Ebola infection may be possible.

  10. Lack of protection following passive transfer of polyclonal highly functional low-dose non-neutralizing antibodies.

    Science.gov (United States)

    Dugast, Anne-Sophie; Chan, Ying; Hoffner, Michelle; Licht, Anna; Nkolola, Joseph; Li, Hualin; Streeck, Hendrik; Suscovich, Todd J; Ghebremichael, Musie; Ackerman, Margaret E; Barouch, Dan H; Alter, Galit

    2014-01-01

    Recent immune correlates analysis from the RV144 vaccine trial has renewed interest in the role of non-neutralizing antibodies in mediating protection from infection. While neutralizing antibodies have proven difficult to induce through vaccination, extra-neutralizing antibodies, such as those that mediate antibody-dependent cellular cytotoxicity (ADCC), are associated with long-term control of infection. However, while several non-neutralizing monoclonal antibodies have been tested for their protective efficacy in vivo, no studies to date have tested the protective activity of naturally produced polyclonal antibodies from individuals harboring potent ADCC activity. Because ADCC-inducing antibodies are highly enriched in elite controllers (EC), we passively transferred highly functional non-neutralizing polyclonal antibodies, purified from an EC, to assess the potential impact of polyclonal non-neutralizing antibodies on a stringent SHIV-SF162P3 challenge in rhesus monkeys. Passive transfer of a low-dose of ADCC inducing antibodies did not protect from infection following SHIV-SF162P3 challenge. Passively administered antibody titers and gp120-specific, but not gp41-specific, ADCC and antibody induced phagocytosis (ADCP) were detected in the majority of the monkeys, but did not correlate with post infection viral control. Thus these data raise the possibility that gp120-specific ADCC activity alone may not be sufficient to control viremia post infection but that other specificities or Fc-effector profiles, alone or in combination, may have an impact on viral control and should be tested in future passive transfer experiments.

  11. THE MECHANISM OF ANTI-IMPLANTATION EFFECT OF PROGESTERONE MONOCLONAL ANTIBODIES IN MICE

    Institute of Scientific and Technical Information of China (English)

    WANGMin-Yi; HEZhi-Ying; WANGHan-Zheng

    1989-01-01

    The purpose of this study is to investigate the mechanism by which antiprogcsterone monoclonal antibodies block early pregnancy in mice. The mechanism of passive immunization is a complex issue as indicated below:

  12. Purification of a Mycoplasma pneumoniae adhesin by monoclonal antibody affinity chromatography.

    OpenAIRE

    Leith, D K; Baseman, J B

    1984-01-01

    A 165,000-dalton surface protein of Mycoplasma pneumoniae, designated protein P1, appears to be the major attachment ligand of the pathogen. We employed monoclonal antibody affinity chromatography to obtain purified protein P1.

  13. The role of radiolabeled anti-TNFα monoclonal antibodies for diagnostic purposes and therapy evaluation

    NARCIS (Netherlands)

    Glaudemans, A. W.J.M.; Dierckx, R. A.J.O.; Kallenberg, C. G.M.; Anzola Fuentes, K. L.

    2010-01-01

    Radiolabelled cytokines and monoclonal antibodies are an emerging class of radiopharmaceuticals for imaging inflammation. These radiopharmaceuticals bind to their targets with high affinity and specificity and therefore have excellent diagnostic potential for imaging of patients with chronic inflamm

  14. CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST BOTH PIG AND RABBIT ZONA PELLUCIDA

    Institute of Scientific and Technical Information of China (English)

    OURU-QIANG

    1989-01-01

    Three monoclonal antibodies (MAbs) were raised against both pig and rabbit zona pellucida with a dual immunization protocol employing heat soluble pig zona (HSPZ) and heat soluble rabbit zona (HSRZ), Of the 140 wells screencd, 12 wells were positive to

  15. Real-time kinetic analysis applied to the production of bispecific monoclonal antibodies for radioimmunodetection of cancer.

    Science.gov (United States)

    Horenstein, A L; Poiesi, C; DeMonte, L; Camagna, M; Mariani, M; Albertini, A; Malavasi, F

    1993-01-01

    An automated biosensor system designed for measuring molecular interactions in real-time and without labelling of the reactants has been used to evaluate the association/dissociation rate and affinity constants of bivalent monoclonal antibodies and a monovalent bispecific monoclonal antibody. Observed differences in affinity between parental and bispecific antibody produced were related to the association rate constants, since the dissociation rate constants were in the same range. Values were also closely related to radioimmunochemical data. These results indicate that the biosensor system, besides presenting several advantages for characterizing antigen-antibody interaction, is valuable for selecting monoclonal antibodies with properties which might be useful in the development of bispecific monoclonal antibodies.

  16. CD4 binding site broadly neutralizing antibody selection of HIV-1 escape mutants.

    Science.gov (United States)

    Dreja, Hanna; Pade, Corinna; Chen, Lei; McKnight, Áine

    2015-07-01

    All human immunodeficiency virus type-1 (HIV-1) viruses use CD4 to enter cells. Consequently, the viral envelope CD4-binding site (CD4bs) is relatively conserved, making it a logical neutralizing antibody target. It is important to understand how CD4-binding site variation allows for escape from neutralizing antibodies. Alanine scanning mutagenesis identifies residues in antigenic sites, whereas escape mutant selection identifies viable mutants. We selected HIV-1 to escape CD4bs neutralizing mAbs b12, A12 and HJ16. Viruses that escape from A12 and b12 remained susceptible to HJ16, VRC01 and J3, whilst six different viruses that escape HJ16 remained sensitive to A12, b12 and J3. In contrast, their sensitivity to VRC01 was variable. Triple HJ16/A12/b12-resistant virus proved that HIV-1 could escape multiple broadly neutralizing monoclonal antibodies, but still retain sensitivity to VRC01 and the llama-derived J3 nanobody. This antigenic variability may reflect that occurring in circulating viruses, so studies like this can predict immunologically relevant antigenic forms of the CD4bs for inclusion in HIV-1 vaccines.

  17. Production and immunoanalytical application of 32 monoclonal antibodies against metacestode somatic antigens of Echinococcus multilocularis.

    Science.gov (United States)

    Wang, Xin; Lu, Rui; Liu, Qiao-Feng; Chen, Jian-Ping; Deng, Qiang; Zhang, Ya-Lou; Zhang, Bing-Hua; Xu, Jia-Nan; Sun, Lei; Niu, Qin-Wang; Liang, Quan-Zeng

    2010-06-01

    Alveolar echinococcosis is a rare but potentially fatal disease. Immunodiagnosis based on antibodies or antigens plays an important role in its diagnosis. In this study, metacestode somatic antigens of Echinococcus multilocularis were used to immunize BALB/c mice, and hybridomas were formed by cell fusion. Making use of the inherent effect of monoclonal antibody techniques to isolate different epitopes, we obtained a repertoire of 32 monoclonal antibodies against the metacestode somatic antigens. These monoclonal antibodies were used to investigate the specificity and localization of the metacestode antigens by enzyme-linked immunosorbent assay and immunohistochemistry, respectively. Nine antibodies specifically reacted with E. multilocularis, while 14 and ten cross-reacted with Echinococcus granulosus and Taenia saginata, respectively. Twenty-five antibodies stained the laminated layer. Eight reacted with the tegument of the protoscolex. Fourteen antibodies recognized the germinal layer. Most of the monoclonal antibodies can react with the antigen Em2. One antibody can react with antigen Em2 and Em10. One antibody that cross-reacted with T. saginata stained the germinal layer and protoscolex, especially its hooklets and suckers, but could not react with Em2 and Em10 antigens. It detected protein bands at 26 and 52 kDa. Two E. multilocularis-specific monoclonal antibodies stained both the germinal and laminated layers and could be used not only to purify specific antigens but also for immunohistochemical studies of E. multilocularis. In summary, these 32 monoclonal antibodies could have potential applications as useful tools in further studies of E. multilocularis antigen profiles.

  18. Identification of Haemophilus influenzae type b by a monoclonal antibody coagglutination assay.

    OpenAIRE

    Hamel, J.; Brodeur, B R; Belmaaza, A; Montplaisir, S; Musser, J M; Selander, R K

    1987-01-01

    A coagglutination assay using monoclonal antibody is described for the identification of Haemophilus influenzae type b. An immunoglobulin G2a monoclonal antibody, Hb-2, directed against a serotype-specific outer membrane protein of H. influenzae type b was adsorbed to Staphylococcus aureus Cowan 1 cells. In a dot enzyme immunoassay, Hb-2 reacted with 453 of 455 H. influenzae type b isolates and did not react with H. influenzae of other serotypes, untypeable H. influenzae strains, or other bac...

  19. Inhibition of lipoxygenase activity in lentil protoplasts by monoclonal antibodies introduced into the cells via electroporation

    OpenAIRE

    J. F. G. Vliegenthart; Maccarrone, M.; Veldink, G.A.

    1992-01-01

    The isolation of lentil protoplasts and the transfer of anti-lipoxygenase monoclonal antibodies into plant protoplasts by electroporation is reported. The dependence of the efficiency of monoclonal antibody incorporation on the field strength is shown as well. The transferred immunoglobulins retained their functional and structural integrity and were able to inhibit the intracellular target enzyme, with a linear relationship between inhibition of lipoxygenase activity and amount of incorporat...

  20. Preexisting human antibodies neutralize recently emerged H7N9 influenza strains

    Science.gov (United States)

    Henry Dunand, Carole J.; Leon, Paul E.; Kaur, Kaval; Tan, Gene S.; Zheng, Nai-Ying; Andrews, Sarah; Huang, Min; Qu, Xinyan; Huang, Yunping; Salgado-Ferrer, Marlene; Ho, Irvin Y.; Taylor, William; Hai, Rong; Wrammert, Jens; Ahmed, Rafi; García-Sastre, Adolfo; Palese, Peter; Krammer, Florian; Wilson, Patrick C.

    2015-01-01

    The emergence and seasonal persistence of pathogenic H7N9 influenza viruses in China have raised concerns about the pandemic potential of this strain, which, if realized, would have a substantial effect on global health and economies. H7N9 viruses are able to bind to human sialic acid receptors and are also able to develop resistance to neuraminidase inhibitors without a loss in fitness. It is not clear whether prior exposure to circulating human influenza viruses or influenza vaccination confers immunity to H7N9 strains. Here, we demonstrate that 3 of 83 H3 HA-reactive monoclonal antibodies generated by individuals that had previously undergone influenza A virus vaccination were able to neutralize H7N9 viruses and protect mice against homologous challenge. The H7N9-neutralizing antibodies bound to the HA stalk domain but exhibited a difference in their breadth of reactivity to different H7 influenza subtypes. Mapping viral escape mutations suggested that these antibodies bind at least two different epitopes on the stalk region. Together, these results indicate that these broadly neutralizing antibodies may contribute to the development of therapies against H7N9 strains and may also be effective against pathogenic H7 strains that emerge in the future. PMID:25689254

  1. Quantitative assessment of antibody internalization with novel monoclonal antibodies against Alexa fluorophores.

    Directory of Open Access Journals (Sweden)

    Sindy Liao-Chan

    Full Text Available 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 internalization of labeled antibodies, an assay based on internalized and quenched fluorescence was developed. For this approach, we generated novel anti-Alexa Fluor monoclonal antibodies (mAbs that effectively and specifically quench cell surface-bound Alexa Fluor 488 or Alexa Fluor 594 fluorescence. Utilizing Alexa Fluor-labeled mAbs against the EphA2 receptor tyrosine kinase, we showed that the anti-Alexa Fluor reagents could be used to monitor internalization quantitatively over time. The anti-Alexa Fluor mAbs were also validated in a proof of concept dual-label internalization assay with simultaneous exposure of cells to two different mAbs. Importantly, the unique anti-Alexa Fluor mAbs described here may also enable other single- and dual-label experiments, including label detection and signal enhancement in macromolecules, trafficking of proteins and microorganisms, and cell migration and morphology.

  2. Structural comparison of four different antibodies interacting with human papillomavirus 16 and mechanisms of neutralization

    Energy Technology Data Exchange (ETDEWEB)

    Guan, Jian [Department of Medicine, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033 (United States); Bywaters, Stephanie M.; Brendle, Sarah A. [Department of Pathology, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033 (United States); Lee, Hyunwook; Ashley, Robert E. [Department of Medicine, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033 (United States); Makhov, Alexander M.; Conway, James F. [Department of Structural Biology, University of Pittsburgh School of Medicine, 3501 5th Ave, Pittsburgh, PA 15260 (United States); Christensen, Neil D. [Department of Pathology, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033 (United States); Hafenstein, Susan, E-mail: shafenstein@hmc.psu.edu [Department of Medicine, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033 (United States)

    2015-09-15

    Cryo-electron microscopy (cryo-EM) was used to solve the structures of human papillomavirus type 16 (HPV16) complexed with fragments of antibody (Fab) from three different neutralizing monoclonals (mAbs): H16.1A, H16.14J, and H263.A2. The structure-function analysis revealed predominantly monovalent binding of each Fab with capsid interactions that involved multiple loops from symmetry related copies of the major capsid protein. The residues identified in each Fab-virus interface map to a conformational groove on the surface of the capsomer. In addition to the known involvement of the FG and HI loops, the DE loop was also found to constitute the core of each epitope. Surprisingly, the epitope mapping also identified minor contributions by EF and BC loops. Complementary immunological assays included mAb and Fab neutralization. The specific binding characteristics of mAbs correlated with different neutralizing behaviors in pre- and post-attachment neutralization assays. - Highlights: • We present HPV16-Fab complexes from neutralizing mAbs: H16.1A, H16.14J, and H263.A2. • The structure-function analysis revealed predominantly monovalent binding of each mAb. • Capsid–Fab interactions involved multiple loops from symmetry related L1 proteins. • Besides the known FG and HI loops, epitope mapping also identified DE, EF, and BC loops. • Neutralizing assays complement the structures to show multiple neutralization mechanisms.

  3. Neutralizing Antibodies and Pathogenesis of Hepatitis C Virus Infection

    Directory of Open Access Journals (Sweden)

    Françoise Stoll-Keller

    2012-10-01

    Full Text Available Hepatitis C virus (HCV infection is a major cause of chronic liver disease worldwide. The interplay between the virus and host innate and adaptive immune responses determines the outcome of infection. There is increasing evidence that host neutralizing responses play a relevant role in the resulting pathogenesis. Furthermore, viral evasion from host neutralizing antibodies has been revealed to be an important contributor in leading both to viral persistence in acute liver graft infection following liver transplantation, and to chronic viral infection. The development of novel model systems to study HCV entry and neutralization has allowed a detailed understanding of the molecular mechanisms of virus-host interactions during antibody-mediated neutralization. The understanding of these mechanisms will ultimately contribute to the development of novel antiviral preventive strategies for liver graft infection and an urgently needed vaccine. This review summarizes recent concepts of the role of neutralizing antibodies in viral clearance and protection, and highlights consequences of viral escape from neutralizing antibodies in the pathogenesis of HCV infection.

  4. Novel method for the high-throughput production of phosphorylation site-specific monoclonal antibodies

    Science.gov (United States)

    Kurosawa, Nobuyuki; Wakata, Yuka; Inobe, Tomonao; Kitamura, Haruki; Yoshioka, Megumi; Matsuzawa, Shun; Kishi, Yoshihiro; Isobe, Masaharu

    2016-01-01

    Threonine phosphorylation accounts for 10% of all phosphorylation sites compared with 0.05% for tyrosine and 90% for serine. Although monoclonal antibody generation for phospho-serine and -tyrosine proteins is progressing, there has been limited success regarding the production of monoclonal antibodies against phospho-threonine proteins. We developed a novel strategy for generating phosphorylation site-specific monoclonal antibodies by cloning immunoglobulin genes from single plasma cells that were fixed, intracellularly stained with fluorescently labeled peptides and sorted without causing RNA degradation. Our high-throughput fluorescence activated cell sorting-based strategy, which targets abundant intracellular immunoglobulin as a tag for fluorescently labeled antigens, greatly increases the sensitivity and specificity of antigen-specific plasma cell isolation, enabling the high-efficiency production of monoclonal antibodies with desired antigen specificity. This approach yielded yet-undescribed guinea pig monoclonal antibodies against threonine 18-phosphorylated p53 and threonine 68-phosphorylated CHK2 with high affinity and specificity. Our method has the potential to allow the generation of monoclonal antibodies against a variety of phosphorylated proteins. PMID:27125496

  5. Monoclonal antibody:the corner stone of modern biotherapeutics%Monoclonal antibody: the corner stone of modern biotherapeutics

    Institute of Scientific and Technical Information of China (English)

    XIA Zhi-nan; CAI Xue-ting; CAO Peng

    2012-01-01

    Worldwide sales of biologic drugs exceeded 100 billion USD in 2011.About 32% is from therapeutic monoclonal antibody (mAb).With many blockbuster biopharmaceutical patents expiring over the next decade,there is a great opportunity for biosimilar to enter the worldwide especially emerging market.Both European Medicines Agency (EMA) and Food and Drug Administration (FDA) have introduced regulatory frameworks for the potential approval of biosimilar mAb therapeutics.Rather than providing a highly abbreviated path,as in the case for small molecule chemical drug,approval for biosimilar mAb will require clinical trial and the details will be very much on a case-by-case basis.Since mAb is the dominant category of biologic drugs,mAb will be the focus of this review.First,the United States (US) and European Union (EU) approved mAb and those in phase 3 trials will be reviewed,then strategies on how to win biosimilar competition will be reviewed.

  6. ANTIBODIES DEFINING RAT ENDOTHELIAL-CELLS - RECA-1, A PAN-ENDOTHELIAL CELL-SPECIFIC MONOCLONAL-ANTIBODY

    NARCIS (Netherlands)

    DUIJVESTIJN, AM; VANGOOR, H; KLATTER, F; MAJOOR, GD; VANBUSSEL, E; VRIESMAN, PJCV

    1992-01-01

    We have been searching for antibodies reactive with rat endothelial cells. Two monoclonal antibodies (mAb), named RECA-1 and RECA-2 were produced and tested in immunoperoxidase staining on frozen sections of various rat tissues. Staining patterns were compared to those obtained with the mAbs OX-2, O

  7. Development of novel monoclonal antibodies against starch and ulvan - Implications for antibody production against polysaccharides with limited immunogenicity

    DEFF Research Database (Denmark)

    Rydahl, Maja Gro; Kračun, Stjepan K.; Fangel, Jonatan U.

    2017-01-01

    Monoclonal antibodies (mAbs) are widely used and powerful research tools, but the generation of mAbs against glycan epitopes is generally more problematic than against proteins. This is especially significant for research on polysaccharide-rich land plants and algae (Viridiplantae). Most antibody...

  8. ANTIBODIES DEFINING RAT ENDOTHELIAL-CELLS - RECA-1, A PAN-ENDOTHELIAL CELL-SPECIFIC MONOCLONAL-ANTIBODY

    NARCIS (Netherlands)

    DUIJVESTIJN, AM; VANGOOR, H; KLATTER, F; MAJOOR, GD; VANBUSSEL, E; VRIESMAN, PJCV

    1992-01-01

    We have been searching for antibodies reactive with rat endothelial cells. Two monoclonal antibodies (mAb), named RECA-1 and RECA-2 were produced and tested in immunoperoxidase staining on frozen sections of various rat tissues. Staining patterns were compared to those obtained with the mAbs OX-2, O

  9. 单克隆抗体S2C4对2型志贺毒素及其亚型毒性的中和作用%Monoclonal Antibody S2C4 Neutralizes The Toxicity of Shiga Toxin 2 and Its Variants

    Institute of Scientific and Technical Information of China (English)

    焦永军; 曾晓燕; 郭喜玲; 史智扬; 冯振卿; 汪华

    2009-01-01

    纯化的2型志贺毒素(Shiga toxin 2,Stx2)经福尔马林脱毒后免疫BALB/c小鼠制备Stx2单克隆抗体,用体外中和试验对具有中和活性的阳性抗体克隆进行初筛,对所获得的中和抗体的重、轻链同种型及结合特异性进行鉴定,其中和保护作用通过体内、体外中和试验加以验证,最后,中和抗体对Stx2亚型Stx2c和Stx2vha的中和谱用体内中和试验验证.结果显示,12株抗Stx2的阳性抗体克隆中,只有1株具有中和活性,命名为S2C4,其重、轻链同种犁为Gi/κ,其靶分子为Stx2的A亚单位,与Stx2的B亚单位或Stx1不结合.在体外中和试验中S2C4可有效中和Stx2对Vero细胞的杀伤作用,同样,S2C4可中和致死量的Stx2及其亚型Stx2e和Stx2vha对小鼠的毒性作用.该抗体有望成为治疗产志贺毒素火肠杆菌感染的候选分子.%Shiga toxin 2 (Stx2) toxoid produced by formaldehyde treatment of purified toxin was used to immunize BALB/c mice for monoclonal antibody (MAb) production.The neutralizing activities of positive clones against Stx2 were screened by in vitro cytotoxicity assay.The isotype and specificity of resultant clone was determined,and its efficacy to neutralize the activity of purified Stx2 was evaluated by in vitro and in vivo toxicity model.Lastly,its spectrum of activity against Stx2 variants was also accessed by mouse toxicity model.It was demonstrated that one of the 12 positive MAb clones against Stx2,designating $2C4 had neutralizing activity.S2C4 belongs to the immunoglobulin G1 subclass and has a K light chain,and it reacts with the A subunit of Stx2 and does not bind to Stx2 B subunit or to Stx1.S2CA could efficiently neutralize the cytotoxicity of Stx2 to Veto cells and mice.It also protected mice against lethal doses of Stx2 variants challenge including Stx2c and Stx2vha.S2C4 is a promising candidate molecule in preventing the progression of hemolytic-uremic syndrome (HUS) mediated mainly by Stx2 in Stx

  10. Monoclonal antibody mapping of the envelope glycoprotein of the dengue 2 virus, Jamaica.

    Science.gov (United States)

    Roehrig, J T; Bolin, R A; Kelly, R G

    1998-07-05

    Although dengue (DEN) virus is the etiologic agent of dengue fever, the most prevalent vector-borne viral disease in the world, precise information on the antigenic structure of the dengue virion is limited. We have prepared a set of murine monoclonal antibodies (MAbs) specific for the envelope (E) glycoprotein of DEN 2 virus and used these antibodies in a comprehensive biological and biochemical analysis to identify 16 epitopes. Following domain nomenclature developed for the related flavivirus, tick-borne encephalitis, three functional domains were identified. Five epitopes associated with domain A were arranged in three spatially independent regions. These A-domain epitopes were destroyed by reduction, and antibodies reactive with these epitopes were able to block virus hemagglutination, neutralize virus infectivity, and block virus-mediated cell membrane fusion. Domain-A epitopes were present on the full-length E glycoprotein, a 45-kDa tryptic peptide representing its first 400 amino acids (aa) and a 22-kDa tryptic peptide representing at least aa 1-120. Four epitopes mapped into domain B, as determined by their partial resistance to reduction and the localization of these epitopes on a 9-kDa tryptic or chymotryptic peptide fragment (aa 300-400). One domain-B-reactive MAb was also capable of binding to a DEN 2 synthetic peptide corresponding to aa 333-351 of the E glycoprotein, confirming the location of this domain. Domain-B epitopes elicited MAbs that were potent neutralizers of virus infectivity and blocked hemagglutination, but they did not block virus-mediated cell-membrane fusion. Domains A and B were spatially associated. As with tick-borne encephalitis virus, determination of domain C was more problematic; however, at least four epitopes had biochemical characteristics consistent with C-domain epitopes.

  11. 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.

  12. Antigen-specific monoclonal antibodies isolated from B cells expressing constitutively active STAT5.

    Directory of Open Access Journals (Sweden)

    Ferenc A Scheeren

    Full Text Available BACKGROUND: Fully human monoclonal antibodies directed against specific pathogens have a high therapeutic potential, but are difficult to generate. METHODOLOGY/PRINCIPAL FINDINGS: Memory B cells were immortalized by expressing an inducible active mutant of the transcription factor Signal Transducer and Activator of Transcription 5 (STAT5. Active STAT5 inhibits the differentiation of B cells while increasing their replicative life span. We obtained cloned B cell lines, which produced antibodies in the presence of interleukin 21 after turning off STAT5. We used this method to obtain monoclonal antibodies against the model antigen tetanus toxin. CONCLUSIONS/SIGNIFICANCE: Here we describe a novel and relatively simple method of immortalizing antigen-specific human B cells for isolation of human monoclonal antibodies. These results show that STAT5 overexpression can be employed to isolate antigen specific antibodies from human memory B cells.

  13. [Generation and characterization of monoclonal antibodies against chicken interleukin 4].

    Science.gov (United States)

    Guan, Xiaoyu; Xu, Zhichao; Wang, Yongqiang; Li, Xiaoqi; Cao, Hong; Zheng, Shijun

    2017-01-25

    To develop monoclonal antibodies (McAbs) against chicken interleukin 4 (chIL-4), we subcloned the mature chIL-4 gene into prokaryotic expression vectors pET-28a and pGEX-6P-1, then expressed and purified the recombinant proteins. We immunized BALB/c mice with the purified His-chIL-4 protein and fused the murine splenocytes with SP2/0 after 4 times of immunization. We used the GST-chIL-4 protein as a coating antigen to establish an indirect ELISA to screen positive clones. After screening and 3 rounds of cloning process, we obtained 3 hybridomas that stably secreted McAbs against chIL-4, and named 1G11-3B, 2E5-3D, and 1G11-5H. The isotypes of these McAbs were all IgG1 and the dissociation constant (Kd) of these McAbs were 1.79×10⁻⁹, 1.61×10⁻⁹, and 2.36×10⁻⁹, respectively. These McAbs specifically bound to chIL-4 expressed by either prokaryotic or eukaryotic system as determined by Western blotting and indirect immunofluorescence assay. The binding domains of chIL-4 recognized by 1G11-3B, 2E5-3D, and 1G11-5H were located between aa 1-40, 80-112, and 40-80, respectively, as determined by Western blotting. These McAbs would help to detect chIL-4 and to elucidate the biological roles of chIL-4 in immune responses.

  14. Differences in Glycoprotein Complex Receptor Binding Site Accessibility Prompt Poor Cross-Reactivity of Neutralizing Antibodies between Closely Related Arenaviruses.

    Science.gov (United States)

    Brouillette, Rachel B; Phillips, Elisabeth K; Ayithan, Natarajan; Maury, Wendy

    2017-04-01

    The glycoprotein complex (GPC) of arenaviruses, composed of stable signal peptide, GP1, and GP2, is the only antigen correlated with antibody-mediated neutralization. However, despite strong cross-reactivity of convalescent antisera between related arenavirus species, weak or no cross-neutralization occurs. Two closely related clade B viruses, Machupo virus (MACV) and Junín virus (JUNV), have nearly identical overall GPC architecture and share a host receptor, transferrin receptor 1 (TfR1). Given structural and functional similarities of the GP1 receptor binding site (RBS) of these viruses and the recent demonstration that the RBS is an important target for neutralizing antibodies, it is not clear how these viruses avoid cross-neutralization. To address this, MACV/JUNV chimeric GPCs were assessed for interaction with a group of α-JUNV GPC monoclonal antibodies (MAbs) and mouse antisera against JUNV or MACV GPC. All six MAbs targeted GP1, with those that neutralized JUNV GPC-pseudovirions competing with each other for RBS binding. However, these MAbs were unable to bind to a chimeric GPC composed of JUNV GP1 containing a small disulfide bonded loop (loop 10) unique to MACV GPC, suggesting that this loop may block MAbs interaction with the GP1 RBS. Consistent with this loop causing interference, mouse anti-JUNV GPC antisera that solely neutralized pseudovirions bearing autologous GP1 provided enhanced neutralization of MACV GPC when this loop was removed. Our studies provide evidence that loop 10, which is unique to MACV GP1, is an important impediment to binding of neutralizing antibodies and contributes to the poor cross-neutralization of α-JUNV antisera against MACV.IMPORTANCE Multiple New World arenaviruses can cause severe disease in humans, and some geographic overlap exists among these viruses. A vaccine that protects against a broad range of New World arenaviruses is desirable for purposes of simplicity, cost, and broad protection against multiple National

  15. Identification and specificity of broadly neutralizing antibodies against HIV

    OpenAIRE

    McCoy, Laura E.; Burton, Dennis R.

    2017-01-01

    Summary Beginning in 2009, studies of the humoral responses of HIV‐positive individuals have led to the identification of scores, if not hundreds, of antibodies that are both broadly reactive and potently neutralizing. This development has provided renewed impetus toward an HIV vaccine and led directly to the development of novel immunogens. Advances in identification of donors with the most potent and broad anti‐HIV serum neutralizing responses were crucial in this effort. Equally, developme...

  16. Application of GP5 Protein to Develop Monoclonal Antibody against Porcine Reproductive and Respiratory Syndrome Virus

    Institute of Scientific and Technical Information of China (English)

    Hong Tian; Yan Cheng; Jin-yang Wu; Jian-hui He; You-jun Shang; Xiang-tao Liu

    2011-01-01

    In this study,a panel of monoclonal antibodies (mAbs) against Porcine reproductive and respiratory syndrome virus(PRRSV),named as 8C9 and4B4,were produced by fusing SP2/0 myeloma cells and spleen cells of BALB/c mice immunized with the PRRSV (TCID50=5.5),screened by the indirect ELISA and subjected to several limiting dilutions.mAbs were then identified by biological characterization.Among the two fusion cell strains,8C9 belonged to the IgG1 subclass and 4B4 belonged to the IgG2a subclass.The titers in cell culture supernatant and abdomen liquor reached to 1:104and 1:105,respectively.The specificity test indicated that the two cells had specific reactions for the PRRSV and GP5 protein respectively,and no reaction with Classical swine fever virus (CSFV) or Swine vesicular disease virus (SVDV).The molecular weights of the heavy chain and light chain were about 45.0 kDa and 25.0 kDa,respectively.In neutralization activity tests,the results showed that the prepared mAb 4B4 can protect 50% of cells with no CPE in dilution up to 1:512,but mAB 8C9 has no neutralization activities to PRRSV.

  17. Natural strain variation and antibody neutralization of dengue serotype 3 viruses.

    Directory of Open Access Journals (Sweden)

    Wahala M P B Wahala

    2010-03-01

    Full Text Available Dengue viruses (DENVs are emerging, mosquito-borne flaviviruses which cause dengue fever and dengue hemorrhagic fever. The DENV complex consists of 4 serotypes designated DENV1-DENV4. Following natural infection with DENV, individuals develop serotype specific, neutralizing antibody responses. Monoclonal antibodies (MAbs have been used to map neutralizing epitopes on dengue and other flaviviruses. Most serotype-specific, neutralizing MAbs bind to the lateral ridge of domain III of E protein (EDIII. It has been widely assumed that the EDIII lateral ridge epitope is conserved within each DENV serotype and a good target for vaccines. Using phylogenetic methods, we compared the amino acid sequence of 175 E proteins representing the different genotypes of DENV3 and identified a panel of surface exposed amino acids, including residues in EDIII, that are highly variant across the four DENV3 genotypes. The variable amino acids include six residues at the lateral ridge of EDIII. We used a panel of DENV3 mouse MAbs to assess the functional significance of naturally occurring amino acid variation. From the panel of antibodies, we identified three neutralizing MAbs that bound to EDIII of DENV3. Recombinant proteins and naturally occurring variant viruses were used to map the binding sites of the three MAbs. The three MAbs bound to overlapping but distinct epitopes on EDIII. Our empirical studies clearly demonstrate that the antibody binding and neutralization capacity of two MAbs was strongly influenced by naturally occurring mutations in DENV3. Our data demonstrate that the lateral ridge "type specific" epitope is not conserved between strains of DENV3. This variability should be considered when designing and evaluating DENV vaccines, especially those targeting EDIII.

  18. Generation of Monoclonal Antibodies against Dengue Virus Type 4 and Identification of Enhancing Epitopes on Envelope Protein.

    Directory of Open Access Journals (Sweden)

    Chung-Tao Tang

    Full Text Available The four serotypes of dengue virus (DENV1-4 pose a serious threat to global health. Cross-reactive and non-neutralizing antibodies enhance viral infection, thereby exacerbating the disease via antibody-dependent enhancement (ADE. Studying the epitopes targeted by these enhancing antibodies would improve the immune responses against DENV infection. In order to investigate the roles of antibodies in the pathogenesis of dengue, we generated a panel of 16 new monoclonal antibodies (mAbs against DENV4. Using plaque reduction neutralization test (PRNT, we examined the neutralizing activity of these mAbs. Furthermore, we used the in vitro and in vivo ADE assay to evaluate the enhancement of DENV infection by mAbs. The results indicate that the cross-reactive and poorly neutralizing mAbs, DD11-4 and DD18-5, strongly enhance DENV1-4 infection of K562 cells and increase mortality in AG129 mice. The epitope residues of these enhancing mAbs were identified using virus-like particle (VLP mutants. W212 and E26 are the epitope residues of DD11-4 and DD18-5, respectively. In conclusion, we generated and characterized 16 new mAbs against DENV4. DD11-4 and D18-5 possessed non-neutralizing activities and enhanced viral infection. Moreover, we identified the epitope residues of enhancing mAbs on envelope protein. These results may provide useful information for development of safe dengue vaccine.

  19. Cross-Reactive Human IgM-Derived Monoclonal Antibodies that Bind to HIV-1 Envelope Glycoproteins

    Directory of Open Access Journals (Sweden)

    Barton F. Haynes

    2010-02-01

    Full Text Available Elicitation of antibodies with potent and broad neutralizing activity against HIV by immunization remains a challenge. Several monoclonal antibodies (mAbs isolated from humans with HIV-1 infection exhibit such activity but vaccine immunogens based on structures containing their epitopes have not been successful for their elicitation. All known broadly neutralizing mAbs (bnmAbs are immunoglobulin (Ig Gs (IgGs and highly somatically hypermutated which could impede their elicitation. Ig Ms (IgMs are on average significantly less divergent from germline antibodies and are relevant for the development of vaccine immunogens but are underexplored compared to IgGs. Here we describe the identification and characterization of several human IgM-derived mAbs against HIV-1 which were selected from a large phage-displayed naive human antibody library constructed from blood, lymph nodes and spleens of 59 healthy donors. These antibodies bound with high affinity to recombinant envelope glycoproteins (gp140s, Envs of HIV-1 isolates from different clades. They enhanced or did not neutralize infection by some of the HIV-1 primary isolates using CCR5 as a coreceptor but neutralized all CXCR4 isolates tested although weakly. One of these antibodies with relatively low degree of somatic hypermutation was more extensively characterized. It bound to a highly conserved region partially overlapping with the coreceptor binding site and close to but not overlapping with the CD4 binding site. These results suggest the existence of conserved structures that could direct the immune response to non-neutralizing or even enhancing antibodies which may represent a strategy used by the virus to escape neutralizing immune responses. Further studies will show whether such a strategy plays a role in HIV infection of humans, how important that role could be, and what the mechanisms of infection enhancement are. The newly identified mAbs could be used as reagents to further

  20. Functional Transplant of a Dengue Virus Serotype 3 (DENV3)-Specific Human Monoclonal Antibody Epitope into DENV1

    Science.gov (United States)

    Messer, William B.; Yount, Boyd L.; Royal, Scott R.; de Alwis, Ruklanthi; Widman, Douglas G.; Smith, Scott A.; Crowe, James E.; Pfaff, Jennifer M.; Kahle, Kristen M.; Doranz, Benjamin J.; Ibarra, Kristie D.; Harris, Eva

    2016-01-01

    ABSTRACT The four dengue virus (DENV) serotypes, DENV1 through 4, are endemic throughout tropical and subtropical regions of the world. While first infection confers long-term protective immunity against viruses of the infecting serotype, a second infection with virus of a different serotype carries a greater risk of severe dengue disease, including dengue hemorrhagic fever and dengue shock syndrome. Recent studies demonstrate that humans exposed to DENV infections develop neutralizing antibodies that bind to quaternary epitopes formed by the viral envelope (E) protein dimers or higher-order assemblies required for the formation of the icosahedral viral envelope. Here we show that the quaternary epitope target of the human DENV3-specific neutralizing monoclonal antibody (MAb) 5J7 can be partially transplanted into a DENV1 strain by changing the core residues of the epitope contained within a single monomeric E molecule. MAb 5J7 neutralized the recombinant DENV1/3 strain in cell culture and was protective in a mouse model of infection with the DENV1/3 strain. However, the 5J7 epitope was only partially recreated by transplantation of the core residues because MAb 5J7 bound and neutralized wild-type (WT) DENV3 better than the DENV1/3 recombinant. Our studies demonstrate that it is possible to transplant a large number of discontinuous residues between DENV serotypes and partially recreate a complex antibody epitope, while retaining virus viability. Further refinement of this approach may lead to new tools for measuring epitope-specific antibody responses and new vaccine platforms. IMPORTANCE Dengue virus is the most important mosquito-borne pathogen of humans worldwide, with approximately one-half the world's population living in regions where dengue is endemic. Dengue immunity following infection is robust and thought to be conferred by antibodies raised against the infecting virus. However, the specific viral components that these antibodies recognize and how they

  1. Therapeutic effect of anti-feline TNF-alpha monoclonal antibody for feline infectious peritonitis.

    Science.gov (United States)

    Doki, Tomoyoshi; Takano, Tomomi; Kawagoe, Kohei; Kito, Akihiko; Hohdatsu, Tsutomu

    2016-02-01

    Feline infectious peritonitis virus (FIPV) replication in macrophages/monocytes induced tumor necrosis factor (TNF)-alpha production, and that the TNF-alpha produced was involved in aggravating the pathology of FIP. We previously reported the preparation of a feline TNF-alpha (fTNF-alpha)-neutralizing mouse monoclonal antibody (anti-fTNF-alpha mAb). This anti-fTNF-alpha mAb 2-4 was confirmed to inhibit the following fTNF-alpha-induced conditions in vitro. In the present study, we investigated whether mAb 2-4 improved the FIP symptoms and survival rate of experimentally FIPV-inoculated SPF cats. Progression to FIP was prevented in 2 out of 3 cats treated with mAb 2-4, whereas all 3 cats developed FIP in the placebo control group. Plasma alpha1-glycoprotein and vascular endothelial growth factor levels were improved by the administration of mAb 2-4, and the peripheral lymphocyte count also recovered. These results strongly suggested that the anti-fTNF-alpha antibody is effective for the treatment of FIP.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-10-15

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

  3. 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.

  4. A Spectrum of Monoclonal Antibodies Reactive with Human Mammary Tumor Cells

    Science.gov (United States)

    Colcher, D.; Horan Hand, P.; Nuti, M.; Schlom, J.

    1981-05-01

    Splenic lymphocytes of mice, immunized with membrane-enriched fractions of metastatic human mammary carcinoma tissues, were fused with the NS-1 non-immunoglobulin-secreting murine myeloma cell line. This resulted in the generation of hybridoma cultures secreting immunoglobulins reactive in solid-phase radioimmunoassays with extracts of metastatic mammary carcinoma cells from involved livers, but not with extracts of apparently normal human liver. As a result of further screening of immunoglobulin reactivities and double cloning of cultures, 11 monoclonal antibodies were chosen that demonstrated reactivities with human mammary tumor cells and not with apparently normal human tissues. These monoclonal antibodies could be placed into at least five major groups on the basis of their differential binding to the surface of various live human mammary tumor cells in culture, to extracts of mammary tumor tissues, or to tissue sections of mammary tumor cells studied by the immunoperoxidase technique. Whereas a spectrum of reactivities to mammary tumors was observed with the 11 monoclonal antibodies, no reactivity was observed to apparently normal cells of the following human tissues: breast, lymph node, lung, skin, testis, kidney, thymus, bone marrow, spleen, uterus, thyroid, intestine, liver, bladder, tonsils, stomach, prostate, and salivary gland. Several of the antibodies also demonstrated a ``pancarcinoma'' reactivity, showing binding to selected non-breast carcinomas. None of the monoclonal antibodies showed binding to purified ferritin or carcinoembryonic antigen. Monoclonal antibodies of all five major groups, however, demonstrated binding to human metastatic mammary carcinoma cells both in axillary lymph nodes and at distal sites.

  5. Partial analysis of the flagellar antigenic determinant recognized by a monoclonal antibody to Clostridium tyrobutyricum.

    Science.gov (United States)

    Bédouet, L; Arnold, F; Robreau, G; Batina, P; Talbot, F; Malcoste, R

    1998-01-01

    In order to count Clostridium tyrobutyricum spores in milk after membrane filtration, murine 21E7-B12 monoclonal antibody was produced. Elution of the monoclonal antibody from this antigen, the flagellar filament protein, by carbohydrate ligands was used to study the epitope structure. A competitive elution of an anti-dextran monoclonal antibody by carbohydrate ligands served as a control in order to validate the immunological tool applied to flagellin epitope study. The carbohydrate moiety of flagellin contained D-glucose and N-acetyl-glucosamine in a molar ration of 11:1 as determined by gas-liquid chromatography and 2 low-abundancy unidentified compounds. In ELISA, D-glucose and N-acetyl-glucosamine did not dissociate the antibody-flagellin complex contrary to maltose, maltotriose, maltotetraose and maltopentaose. The efficiency of elution increased from the dimer to the pentamer and became nil for maltohexaose and maltoheptaose. The fact that the hexamer and heptamer could not react with the 21E7-B12 monoclonal antibody could be explained by a drastic conformational change. The over-all stretched maltopentaose switch to a helical-shaped maltoheptaose which could not fit the 21E7-B12 monoclonal antibody antigen-combining site. Thus, flagellin epitope may contain alpha (1-->4) linked glucose residues plus either N-actyl-glucosamine or an unidentified compound that maintain it in an extended shape.

  6. The generation of monoclonal antibodies and their use in rapid diagnostic tests

    Science.gov (United States)

    Antibodies are the most important component of an immunoassay. In these proceedings we outline novel methods used to generate and select monoclonal antibodies that meet performance criteria for use in rapid lateral flow and microfluidic immunoassay tests for the detection of agricultural pathogens ...

  7. Human monoclonal HLA antibodies reveal interspecies crossreactive swine MHC class I epitopes relevant for xenotransplantation.

    NARCIS (Netherlands)

    Mulder, A.; Kardol, M.J.; Arn, J.S.; Eijsink, C.; Franke, M.E.; Schreuder, G.M.; Haasnoot, G.W.; Doxiadis, I.I.; Sachs, D.H.; Smith, D.M.; Claas, F.H.

    2010-01-01

    Crossreactivity of anti-HLA antibodies with SLA alleles may limit the use of pig xenografts in some highly sensitized patients. An understanding of the molecular basis for this crossreactivity may allow better selection of xenograft donors. We have tested 68 human monoclonal HLA class I antibodies (

  8. The Synthesis of N-Morphine Hapten and Production of Monoclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Those antibodies elicited by different tether site for attachment to carrier protein have different specificity. Herein we reported that a monoclonal antibody against morphine with high specificity and affinity was successfully produced by using different linkers to couple to different carrier proteins.

  9. Synthetic methyl hexagalacturonate hapten inhibitors of antihomogalacturonan monoclonal antibodies LM7, JIM5 and JIM7

    DEFF Research Database (Denmark)

    Clausen, Mads Hartvig; Willats, William George Tycho; Knox, J. Paul

    2003-01-01

    A range of synthetic methyl hexagalacturonates were used as potential hapten inhibitors in competitive-inhibition enzyme-linked immunosorbent assays (ELISAs) with anti-homogalacturonan monoclonal antibodies LM7, JIM5 and JIM7. The selective inhibition of these antibodies by different haptens prov...

  10. B lymphocyte depletion with the monoclonal antibody rituximab in Graves' disease: a controlled pilot study

    DEFF Research Database (Denmark)

    El Fassi, Daniel; Nielsen, Claus H; Bonnema, Steen J

    2007-01-01

    Graves' disease (GD) is a common TSH receptor autoantibody (TRAb)-mediated disorder. Because B lymphocytes are important self-antigen presenting cells and precursors for antibody-secreting plasma cells, temporary B-lymphocyte depletion with the monoclonal antibody rituximab (RTX) might be of bene...

  11. Development of a PBPK model for monoclonal antibodies and simulation of human and mice PBPK of a radiolabelled monoclonal antibody.

    Science.gov (United States)

    Heiskanen, Tomi; Heiskanen, Tomas; Kairemo, Kalevi

    2009-01-01

    Physiology based pharmacokinetic (PBPK) modeling and simulation is a useful method for prediction of biodistribution of both macromolecules and small molecules. It can enhance our understanding of the underlying mechanisms of biodistribution and hence may help in rational design of macromolecules used as diagnostic and therapeutic agents. In this review we discuss PBPK modeling and simulation of a radiolabelled Monoclonal Antibody ((111)In-DOTA-hAFP31 IgG) ("MAB") in mice without tumor and in a human with tumor. This study is part of Xemet Co.'s effort to develop a more accurate and reliable PBPK model and simulation platform, which is applicable both for small molecules and macromolecules. The simulated results were fitted to experimental time series data by varying parameters which were not fixed a priori. It was demonstrated that the PBPK model describes the main features of the pharmacokinetics of the studied systems. It was also shown that simulation can be used for evaluating the parameters of the system and scaling up the pharmacokinetics of MAB from mice to man. We identified several areas of improvement and further development needed to improve the accuracy of PBPK simulation for MAB and other macromolecules. It was concluded that the transvascular permeabilities are the most important parameters and more research is needed to enable prediction of permeabilities from molecular characteristics of macromolecules. It would also be necessary to understand better and describe with a more detailed model the microstructure of the tumor and to measure or predict the antigen concentration in tumor. Non-specific, non-saturable binding in other organs/tissues should be understood better and the kinetic constants of the binding should be measured experimentally. Although the metabolism and clearance were neglected in this study they need to be included in more detailed studies. Also the intracellular trafficking of macromolecules, which was not included in this study

  12. Secretory leukocyte protease inhibitor (SLPI) might contaminate murine monoclonal antibodies after purification on protein G.

    Science.gov (United States)

    Schenk, Jörg A; Fettke, Joerg; Lenz, Christine; Albers, Katharina; Mallwitz, Frank; Gajovic-Eichelmann, Nenad; Ehrentreich-Förster, Eva; Kusch, Emely; Sellrie, Frank

    2012-03-31

    The large scale production of a monoclonal anti-progesterone antibody in serum free medium followed by affinity chromatography on protein G lead to a contamination of the antibody sample with a protein of about 14 kDa. This protein was identified by mass spectrometry as secretory leukocyte protease inhibitor (SLPI). This SLPI contamination lead to a failure of the fiber-optic based competitive fluorescence assay to detect progesterone in milk. Purification of the monoclonal antibody using protein A columns circumvented this problem.

  13. Rescue and expression of human immunoglobulin genes to generate functional human monoclonal antibodies.

    Science.gov (United States)

    Lewis, A P; Parry, N; Peakman, T C; Crowe, J S

    1992-07-01

    Human monoclonal antibody production has been hampered for many years by the instability of cell lines and low levels of expression of the antibodies. We describe here the rescue of human immunoglobulin genes utilizing micro-mRNA preparation from a small number of human hybridoma cells and conventional cDNA cloning. This allows cloning and immediate high-level expression from full-length human heavy and light chain cDNA molecules and provides a mechanism to rescue whole human monoclonal antibodies of proven efficacy.

  14. Broadly Neutralizing Antibodies Display Potential for Prevention of HIV-1 Infection of Mucosal Tissue Superior to That of Nonneutralizing Antibodies

    Science.gov (United States)

    Cheeseman, Hannah M.; Olejniczak, Natalia J.; Rogers, Paul M.; Evans, Abbey B.; King, Deborah F. L.; Ziprin, Paul; Liao, Hua-Xin; Haynes, Barton F.

    2016-01-01

    ABSTRACT Definition of the key parameters mediating effective antibody blocking of HIV-1 acquisition within mucosal tissue may prove critical to effective vaccine development and the prophylactic use of monoclonal antibodies. Although direct antibody-mediated neutralization is highly effective against cell-free virus, antibodies targeting different sites of envelope vulnerability may display differential activity against mucosal infection. Nonneutralizing antibodies (nnAbs) may also impact mucosal transmission events through Fc-gamma receptor (FcγR)-mediated inhibition. In this study, a panel of broadly neutralizing antibodies (bnAbs) and nnAbs, including those associated with protection in the RV144 vaccine trial, were screened for the ability to block HIV-1 acquisition and replication across a range of cellular and mucosal tissue models. Neutralization potency, as determined by the TZM-bl infection assay, did not fully predict activity in mucosal tissue. CD4-binding site (CD4bs)-specific bnAbs, in particular VRC01, were consistent in blocking HIV-1 infection across all cellular and tissue models. Membrane-proximal external region (MPER) (2F5) and outer domain glycan (2G12) bnAbs were also efficient in preventing infection of mucosal tissues, while the protective efficacy of bnAbs targeting V1-V2 glycans (PG9 and PG16) was more variable. In contrast, nnAbs alone and in combinations, while active in a range of cellular assays, were poorly protective against HIV-1 infection of mucosal tissues. These data suggest that tissue resident effector cell numbers and low FcγR expression may limit the potential of nnAbs to prevent establishment of the initial foci of infection. The solid protection provided by specific bnAbs clearly demonstrates their superior potential over that of nonneutralizing antibodies for preventing HIV-1 infection at the mucosal portals of infection. IMPORTANCE Key parameters mediating effective antibody blocking of HIV-1 acquisition within mucosal

  15. Mammalian tissue distribution of a large heparan sulfate proteoglycan detected by monoclonal antibodies

    DEFF Research Database (Denmark)

    Couchman, J R; Ljubimov, A V

    1989-01-01

    A panel of nine monoclonal antibodies has been characterized, all of which have reactivity with the core protein of a large heparan sulfate proteoglycan derived from the murine EHS tumor matrix. These rat monoclonal antibodies stained mouse basement membranes intensely, including those of all...... muscle, endothelia, peripheral nerve fibers and epithelia so far examined. In addition, two of the monoclonal antibodies show cross-species reactivity, staining bovine and human basement membranes, and immunoprecipitating proteoglycans from human endothelial cell cultures. These antibodies do not......, however, cross-react with avian tissues. These results show the ubiquitous distribution of a heparan sulfate proteoglycan in mammalian tissues, which will be useful in vitro and in vivo for studies on the biology of basement membrane proteoglycans and investigations of possible roles of these molecules...

  16. Structural and molecular basis for Ebola virus neutralization by protective human antibodies.

    Science.gov (United States)

    Misasi, John; Gilman, Morgan S A; Kanekiyo, Masaru; Gui, Miao; Cagigi, Alberto; Mulangu, Sabue; Corti, Davide; Ledgerwood, Julie E; Lanzavecchia, Antonio; Cunningham, James; Muyembe-Tamfun, Jean Jacques; Baxa, Ulrich; Graham, Barney S; Xiang, Ye; Sullivan, Nancy J; McLellan, Jason S

    2016-03-18

    Ebola virus causes hemorrhagic fever with a high case fatality rate for which there is no approved therapy. Two human monoclonal antibodies, mAb100 and mAb114, in combination, protect nonhuman primates against all signs of Ebola virus disease, including viremia. Here, we demonstrate that mAb100 recognizes the base of the Ebola virus glycoprotein (GP) trimer, occludes access to the cathepsin-cleavage loop, and prevents the proteolytic cleavage of GP that is required for virus entry. We show that mAb114 interacts with the glycan cap and inner chalice of GP, remains associated after proteolytic removal of the glycan cap, and inhibits binding of cleaved GP to its receptor. These results define the basis of neutralization for two protective antibodies and may facilitate development of therapies and vaccines.

  17. Binding of a neutralizing antibody to dengue virus alters the arrangement of surface glycoproteins

    Energy Technology Data Exchange (ETDEWEB)

    Lok, Shee-Mei; Kostyuchenko, Victor; Nybakken, Grant E.; Holdaway, Heather A.; Battisti, Anthony J.; Sukupolvi-Petty, Soila; Sedlak, Dagmar; Fremont, Daved H.; Chipman, Paul R.; Roehrig, John T.; Diamond, Michael S.; Kuhn, Richard J.; Rossmann, Michael G. (Purdue); (WU-MED); (CDC)

    2008-04-02

    The monoclonal antibody 1A1D-2 has been shown to strongly neutralize dengue virus serotypes 1, 2 and 3, primarily by inhibiting attachment to host cells. A crystal structure of its antigen binding fragment (Fab) complexed with domain III of the viral envelope glycoprotein, E, showed that the epitope would be partially occluded in the known structure of the mature dengue virus. Nevertheless, antibody could bind to the virus at 37 degrees C, suggesting that the virus is in dynamic motion making hidden epitopes briefly available. A cryo-electron microscope image reconstruction of the virus:Fab complex showed large changes in the organization of the E protein that exposed the epitopes on two of the three E molecules in each of the 60 icosahedral asymmetric units of the virus. The changes in the structure of the viral surface are presumably responsible for inhibiting attachment to cells.

  18. Epidemiologic investigation by macrorestriction analysis and by using monoclonal antibodies of nosocomial pneumonia caused by Legionella pneumophila serogroup 10.

    OpenAIRE

    Lück, P. C.; Helbig, J H; Günter, U; Assmann, M.; Blau, R; Koch, H.; Klepp, M.

    1994-01-01

    A 67-year-old woman was hospitalized with an acute pneumonia of the left lower lobe. Legionella pneumophila serogroup 10 was cultured from two sputum specimens taken on days 18 and 20 and was also detected by direct immunofluorescence assay by using a commercially available species-specific monoclonal antibody as well as serogroup 10-specific monoclonal antibodies. Antigenuria was detected in enzyme-linked immunosorbent assays by using serogroup 10-specific polyclonal and monoclonal antibodie...

  19. HIV-1 resistance to neutralizing antibodies: Determination of antibody concentrations leading to escape mutant evolution.

    Science.gov (United States)

    Magnus, Carsten; Reh, Lucia; Trkola, Alexandra

    2016-06-15

    Broadly neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) are considered vital components of novel therapeutics and blueprints for vaccine research. Yet escape to even the most potent of these antibodies is imminent in natural infection. Measures to define antibody efficacy and prevent mutant selection are thus urgently needed. Here, we derive a mathematical framework to predict the concentration ranges for which antibody escape variants can outcompete their viral ancestors, referred to as mutant selection window (MSW). When determining the MSW, we focus on the differential efficacy of neutralizing antibodies against HIV-1 in two canonical infection routes, free-virus infection and cell-cell transmission. The latter has proven highly effective in vitro suggesting its importance for both in vivo spread as well as for escaping targeted intervention strategies. We observed a range of MSW patterns that highlight the potential of mutants to arise in both transmission pathways and over wide concentration ranges. Most importantly, we found that only when the arising mutant has both, residual sensitivity to the neutralizing antibody and reduced infectivity compared to the parental virus, antibody dosing outside of the MSW to restrict mutant selection is possible. Emergence of mutants that provide complete escape and have no considerable fitness loss cannot be prevented by adjusting antibody doses. The latter may in part explain the ubiquitous resistance to neutralizing antibodies observed in natural infection and antibody treatment. Based on our findings, combinations of antibodies targeting different epitopes should be favored for antibody-based interventions as this may render complete resistance less likely to occur and also increase chances that multiple escapes result in severe fitness loss of the virus making longer-term antibody treatment more feasible. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  20. Short Communication: Neutralizing Antibodies in HIV-1-Infected Brazilian Individuals

    Science.gov (United States)

    Morgado, Mariza Gonçalvez; Côrtes, Fernanda Heloise; Guimarães, Monick Lindermeyer; Mendonça-Lima, Leila; Pilotto, Jose Henrique; Grinsztejn, Beatriz; Veloso, Valdiléa Gonçalves; Bongertz, Vera

    2013-01-01

    Abstract Tests for the detection of the humoral immune response to HIV-1 have to be standardized and established, demanding regional efforts. For this purpose the neutralizing antibody (NAb) assay for HIV-1 in TZM-bl cells was introduced in Brazil. Twenty plasma samples from HIV-1-infected individuals were assayed: 10 progressors and 10 long-term nonprogressors. These were tested against eight env-pseudotyped viruses (psVs) in the TZM-bl NAb assay and against HIV-1 strain HTLV/IIIB (HIV-1 IIIB) in primary lymphocytes. Forty-four percent of the samples showed neutralizing titers for psVs and 55% for HIV-1 IIIB. Plasma from progressors showed a broader neutralization and a higher potency. The introduction of these reference reagents encourages the participation of Brazil in future comparative assessments of anti-HIV-1 antibodies. PMID:23145941

  1. Generation and characterization of monoclonal antibodies against the transcription factor Nkx6.1.

    Science.gov (United States)

    Pedersen, Inger L; Klinck, Rasmus; Hecksher-Sorensen, Jacob; Zahn, Stefan; Madsen, Ole D; Serup, Palle; Jorgensen, Mette C

    2006-05-01

    We present the generation of a panel of monoclonal antibodies (F55A10, F55A12, F64A6B4, and F65A2) against the homeodomain transcription factor Nkx6.1, one of the essential transcription factors that regulates the multistep differentiation process of precursor cells into endocrine beta-cells in the pancreas. Expression of Nkx6.1 can be detected in developing pancreatic epithelium and in adult insulin-producing beta-cells, making this transcription factor a unique beta-cell marker. For production of monoclonal antibodies, RBF mice were immunized with a GST-Nkx6.1 fusion protein containing a 66-amino acid C-terminal fragment of rat Nkx6.1. Four clones were established as stable hybridoma cell lines and the produced antibodies were of the mouse IgG1/kappa subtype. When applied for immunohistochemistry on frozen sections of adult mouse pancreas, monoclonal antibodies stain specifically the beta-cells in the endocrine islets of Langerhans with patterns comparable to that of a previously produced polyclonal rabbit serum. Monoclonal antibodies can be divided into two groups that appear to recognize different epitopes, as determined by competition ELISA. The presented antibodies are useful tools for the further characterization of the role and function of Nkx6.1 in pancreatic development, especially for use in double-labeling experiments with existing polyclonal rabbit antibodies.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1985-09-01

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

  3. Overloading ion-exchange membranes as a purification step for monoclonal antibodies

    OpenAIRE

    Brown, Arick; Bill, Jerome; Tully, Timothy; Radhamohan, Asha; Dowd, Chris

    2010-01-01

    The present study examined the overloading of ion-exchange membrane adsorbers, a form of frontal chromatography, as the final purification step in the production of mAbs (monoclonal antibodies) produced from CHO (Chinese-hamster ovary) cells. Preferential binding of impurities over antibody product was exploited using commercially available cation- and anion-exchange membranes. Three different antibody feedstreams previously purified over Protein A and ion-exchange column chromatography were ...

  4. Toward Effective HIV Vaccination INDUCTION OF BINARY EPITOPE REACTIVE ANTIBODIES WITH BROAD HIV NEUTRALIZING ACTIVITY

    Energy Technology Data Exchange (ETDEWEB)

    Nishiyama, Yasuhiro; Planque, Stephanie; Mitsuda, Yukie; Nitti, Giovanni; Taguchi, Hiroaki; Jin, Lei; Symersky, Jindrich; Boivin, Stephane; Sienczyk, Marcin; Salas, Maria; Hanson, Carl V.; Paul, Sudhir; (Texas-MED); (Viral Rickettsial)

    2009-11-23

    We describe murine monoclonal antibodies (mAbs) raised by immunization with an electrophilic gp120 analog (E-gp120) expressing the rare ability to neutralize genetically heterologous human immunodeficiency virus (HIV) strains. Unlike gp120, E-gp120 formed covalent oligomers. The reactivity of gp120 and E-gp120 with mAbs to reference neutralizing epitopes was markedly different, indicating their divergent structures. Epitope mapping with synthetic peptides and electrophilic peptide analogs indicated binary recognition of two distinct gp120 regions by anti-E-gp120 mAbs, the 421-433 and 288-306 peptide regions. Univalent Fab and single chain Fv fragments expressed the ability to recognize both peptides. X-ray crystallography of an anti-E-gp120 Fab fragment revealed two neighboring cavities, the typical antigen-binding cavity formed by the complementarity determining regions (CDRs) and another cavity dominated by antibody heavy chain variable (VH) domain framework (FR) residues. Substitution of the FR cavity VH Lys-19 residue by an Ala residue resulted in attenuated binding of the 421-433 region peptide probe. The CDRs and VH FR replacement/silent mutation ratios exceeded the ratio for a random mutation process, suggesting adaptive development of both putative binding sites. All mAbs studied were derived from VH1 family genes, suggesting biased recruitment of the V gene germ line repertoire by E-gp120. The conserved 421-433 region of gp120 is essential for HIV binding to host CD4 receptors. This region is recognized weakly by the FR of antibodies produced without exposure to HIV, but it usually fails to induce adaptive synthesis of neutralizing antibodies. We present models accounting for improved CD4-binding site recognition and broad HIV neutralizing activity of the mAbs, long sought goals in HIV vaccine development.

  5. Specific ion and buffer effects on protein-protein interactions of a monoclonal antibody.

    Science.gov (United States)

    Roberts, D; Keeling, R; Tracka, M; van der Walle, C F; Uddin, S; Warwicker, J; Curtis, R

    2015-01-05

    Better predictive ability of salt and buffer effects on protein-protein interactions requires separating out contributions due to ionic screening, protein charge neutralization by ion binding, and salting-in(out) behavior. We have carried out a systematic study by measuring protein-protein interactions for a monoclonal antibody over an ionic strength range of 25 to 525 mM at 4 pH values (5, 6.5, 8, and 9) in solutions containing sodium chloride, calcium chloride, sodium sulfate, or sodium thiocyante. The salt ions are chosen so as to represent a range of affinities for protein charged and noncharged groups. The results are compared to effects of various buffers including acetate, citrate, phosphate, histidine, succinate, or tris. In low ionic strength solutions, anion binding affinity is reflected by the ability to reduce protein-protein repulsion, which follows the order thiocyanate > sulfate > chloride. The sulfate specific effect is screened at the same ionic strength required to screen the pH dependence of protein-protein interactions indicating sulfate binding only neutralizes protein charged groups. Thiocyanate specific effects occur over a larger ionic strength range reflecting adsorption to charged and noncharged regions of the protein. The latter leads to salting-in behavior and, at low pH, a nonmonotonic interaction profile with respect to sodium thiocyanate concentration. The effects of thiocyanate can not be rationalized in terms of only neutralizing double layer forces indicating the presence of an additional short-ranged protein-protein attraction at moderate ionic strength. Conversely, buffer specific effects can be explained through a charge neutralization mechanism, where buffers with greater valency are more effective at reducing double layer forces at low pH. Citrate binding at pH 6.5 leads to protein charge inversion and the formation of attractive electrostatic interactions. Throughout the report, we highlight similarities in the measured

  6. Intracellular reprogramming of expression, glycosylation, and function of a plant-derived antiviral therapeutic monoclonal antibody.

    Directory of Open Access Journals (Sweden)

    Jeong-Hwan Lee

    Full Text Available Plant genetic engineering, which has led to the production of plant-derived monoclonal antibodies (mAb(Ps, provides a safe and economically effective alternative to conventional antibody expression methods. In this study, the expression levels and biological properties of the anti-rabies virus mAb(P SO57 with or without an endoplasmic reticulum (ER-retention peptide signal (Lys-Asp-Glu-Leu; KDEL in transgenic tobacco plants (Nicotiana tabacum were analyzed. The expression levels of mAb(P SO57 with KDEL (mAb(PK were significantly higher than those of mAb(P SO57 without KDEL (mAb(P regardless of the transcription level. The Fc domains of both purified mAb(P and mAb(PK and hybridoma-derived mAb (mAb(H had similar levels of binding activity to the FcγRI receptor (CD64. The mAb(PK had glycan profiles of both oligomannose (OM type (91.7% and Golgi type (8.3%, whereas the mAb(P had mainly Golgi type glycans (96.8% similar to those seen with mAb(H. Confocal analysis showed that the mAb(PK was co-localized to ER-tracker signal and cellular areas surrounding the nucleus indicating accumulation of the mAb(P with KDEL in the ER. Both mAb(P and mAb(PK disappeared with similar trends to mAb(H in BALB/c mice. In addition, mAb(PK was as effective as mAb(H at neutralizing the activity of the rabies virus CVS-11. These results suggest that the ER localization of the recombinant mAb(P by KDEL reprograms OM glycosylation and enhances the production of the functional antivirus therapeutic antibody in the plant.

  7. Generation and diagnostic application of monoclonal antibodies against Seneca Valley virus.

    Science.gov (United States)

    Yang, Ming; van Bruggen, Rebekah; Xu, Wanhong

    2012-01-01

    Seneca Valley virus (SVV), a member of the Picornaviridae family, was implicated in a suspicious vesicular disease discovered in pigs from Canada in 2007. Because any outbreak of vesicular disease in pigs is assumed to be foot-and-mouth disease (FMD) until confirmed otherwise, a test for diagnosing the presence of SVV would be a very useful tool. To develop the diagnostic tests for SVV infection, 5 monoclonal antibodies (mAbs) were produced from mice immunized with binary ethylenimine (BEI)-inactivated SVV. Using a dot blot assay, the reactivity of the mAbs was confirmed to be specific for SVV, not reacting with any of the other vesicular disease viruses tested. The mAbs demonstrated reactivity with SVV antigen in infected cells by an immunohistochemistry assay. An SVV-specific competitive enzyme-linked immunosorbent assay (cELISA) was developed using BEI-inactivated SVV antigen and a mAb for serodiagnosis. The cELISA results were compared to the indirect isotype (immunoglobulin [Ig]M and IgG) ELISA and the virus neutralization test. All SVV experimentally inoculated pigs exhibited a positive SVV-specific antibody response at 6 days postinoculation, and the sera remained positive until the end of the experiment on day 57 (>40% inhibition) using the cELISA. The cELISA reflected the profile of the indirect ELISA for both IgM and IgG. This panel of SVV-specific mAbs is valuable for the identification of SVV antigen and the serological detection of SVV-specific antibodies.

  8. New Stx2e Monoclonal Antibodies for Immunological Detection and Distinction of Stx2 Subtypes.

    Directory of Open Access Journals (Sweden)

    Craig Skinner

    Full Text Available Stx2e is a primary virulence factor in STEC strains that cause edema disease in neonatal piglets. Though Stx2a and Stx2e are similar, many antibody-based Stx detection kits are designed to detect Stx2a and do not recognize the Stx2e subtype.Four monoclonal antibodies against Stx2e were developed and characterized. Two of these mAbs recognize the B subunit of Stx2e, Stx2f, and to a lesser extent, Stx2b, Stx2c, and Stx2d. The other two mAbs recognize the A subunit of Stx2e, and cross-react with all Stx2 subtypes except Stx2f. The most sensitive sandwich ELISA using these mAbs has a limit of detection for Stx2e of 11.8 pg/mL. The ability of the neutralizing antibody Stx2e-2 to block Stx2e-receptor binding in Vero cells was visualized using immunofluorescence. Combinations of these and previously developed mAbs permit ELISA-based differentiation between closely related Stx2a, Stx2c, and Stx2d (using mAbs Stx2-5/2-1, Stx2-5/2e-2, and Stx2e-3/2e-2, respectively.The sensitive immunoassays developed in this study should augment our capacity to detect Stx2e in porcine environments and biological samples. Moreover, immunoassays that can distinguish between the closely related Stx2a, Stx2c, and Stx2d subtypes can be useful in quickly analyzing Stx subtypes in samples containing more than one strain of STEC.

  9. Production and characterization of human anti-V3 monoclonal antibodies from the cells of HIV-1 infected Indian donors

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    Andrabi Raiees

    2012-09-01

    Full Text Available Abstract Background Analysis of human monoclonal antibodies (mAbs developed from HIV-1 infected donors have enormously contributed to the identification of neutralization sensitive epitopes on the HIV-1 envelope glycoprotein. The third variable region (V3 is a crucial target on gp120, primarily due to its involvement in co-receptor (CXCR4 or CCR5 binding and presence of epitopes recognized by broadly neutralizing antibodies. Methods Thirty-three HIV-1 seropositive drug naive patients (18 males and 15 females within the age range of 20–57 years (median = 33 years were recruited in this study for mAb production. The mAbs were selected from EBV transformed cultures with conformationally constrained Cholera-toxin-B containing V3C (V3C-CTB fusion protein. We tested the mAbs for their binding with HIV-1 derived proteins and peptides by ELISA and for neutralization against HIV-1 viruses by TZM-bl assays. Results We isolated three anti-V3 mAbs, 277, 903 and 904 from the cells of different individuals. The ELISA binding revealed a subtype-C and subtype-A specific binding of antibody 277 and 903 while mAb 904 exhibited cross reactivity also with subtype-B V3. Epitope mapping of mAbs with overlapping V3 peptides showed exclusive binding to V3 crown. The antibodies displayed high and low neutralizing activity against 2/5 tier 1 and 1/6 tier 2 viruses respectively. Overall, we observed a resistance of the tier 2 viruses to neutralization by the anti-V3 mAbs, despite the exposure of the epitopes recognized by these antibodies on two representative native viruses (Du156.12 and JRFL, suggesting that the affinity of mAb might equally be crucial for neutralization, as the epitope recognition. Conclusions Our study suggests that the anti-V3 antibodies derived from subtype-C infected Indian patients display neutralization potential against tier 1 viruses while such activity may be limited against more resistant tier 2 viruses. Defining the fine epitope

  10. Limited cross-reactivity of mouse monoclonal antibodies against Dengue virus capsid protein among four serotypes

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    Noda M

    2012-11-01

    Full Text Available Megumi Noda,1 Promsin Masrinoul,1 Chaweewan Punkum,1 Chonlatip Pipattanaboon,2,3 Pongrama Ramasoota,2,4 Chayanee Setthapramote,2,3 Tadahiro Sasaki,6 Mikiko Sasayama,1 Akifumi Yamashita,1,5 Takeshi Kurosu,6 Kazuyoshi Ikuta,6 Tamaki Okabayashi11Mahidol-Osaka Center for Infectious Diseases, 2Center of Excellence for Antibody Research, 3Department of Microbiology and Immunology, 4Department of Social and Environmental Medicine, Faculty of Tropical Medicine, Mahidol University, Ratchathewi, Bangkok, Thailand; 5Graduate School of Life Science, Tohoku University, Sendai, Miyagi, 6Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, JapanBackground: Dengue illness is one of the important mosquito-borne viral diseases in tropical and subtropical regions. Four serotypes of dengue virus (DENV-1, DENV-2, DENV-3, and DENV-4 are classified in the Flavivirus genus of the family Flaviviridae. We prepared monoclonal antibodies against DENV capsid protein from mice immunized with DENV-2 and determined the cross-reactivity with each serotype of DENV and Japanese encephalitis virus.Methods and results: To clarify the relationship between the cross-reactivity of monoclonal antibodies and the diversity of these viruses, we examined the situations of flaviviruses by analyses of phylogenetic trees. Among a total of 60 prepared monoclonal antibodies specific for DENV, five monoclonal antibodies stained the nuclei of infected cells and were found to be specific to the capsid protein. Three were specific to DENV-2, while the other two were cross-reactive with DENV-2 and DENV-4. No monoclonal antibodies were cross-reactive with all four serotypes. Phylogenetic analysis of DENV amino acid sequences of the capsid protein revealed that DENV-2 and DENV-4 were clustered in the same branch, while DENV-1 and DENV-3 were clustered in the other branch. However, these classifications of the capsid protein were different from those of the

  11. Boronated monoclonal antibody 225. 28S for potential use in neutron capture therapy of malignant melanoma

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    Tamat, S.R.; Moore, D.E.; Patwardhan, A.; Hersey, P. (Univ. of Sydney (Australia))

    1989-07-01

    The concept of conjugating boron cluster compounds to monoclonal antibodies has been examined by several groups of research workers in boron neutron capture therapy (BNCT). The procedures reported to date for boronation of monoclonal antibodies resulted in either an inadequate level of boron incorporation, the precipitation of the conjugates, or a loss of immunological activity. The present report describes the conjugation of dicesium-mercapto-undecahydrododecaborate (Cs2B12H11SH) to 225.28S monoclonal antibody directed against high molecular weight melanoma-associated antigens (HMW-MAA), using poly-L-ornithine as a bridge to increase the carrying capacity of the antibody and to minimize change in the conformational structure of antibody. The method produces a boron content of 1,300 to 1,700 B atoms per molecule 225.28S while retaining the immunoreactivity. Characterization in terms of the homogeneity of the conjugation of the boron-monoclonal antibody conjugates has been studied by gel electrophoresis and ion-exchange HPLC.

  12. Production and Purification of Monoclonal Antibody Against Tumor Marker of TPA

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    Seyyed Amir Abbas Ghodrat

    2016-05-01

    Full Text Available Considering the invasive nature of cancer cells, one of the most important and best indicator of them is the markers inside them. One of the most important markers that observed in some types of cancer cells in various parts of the body is the Cytokeratin. Tissue plasminogen activator antigen (TPA is a Cytokeratin composed of molecules with various molecular weights. The level of TPA serum as associated with cellular growth level and tumorization of cells. In this research, the hybrid of spleen cells in BALB/c female mouse with myeloma cells was conducted with a ratio of 10:1. The resulting monoclonal antibodies were confirmed by SDS-PAGE and western blot. Protein G chromatography was utilized to purify monoclonal antibodies. The results for determining isotypes showed IgM and IgG classes. The titer of the antibody obtained from various clones was capable of identifying Cytokeratin antigen with a dilution of 1/10000. The resulting antibodies were finally confirmed by western blot and all the 5 resulting monoclonal antibodies were capable of identifying a 48 kDa protein. The results indicate that with the help of TPA marker and the monoclonal antibodies produced against them, this marker can be recognized quickly with great accuracy in suspicious cases of cancer. Thus, appropriate measures will be taken to prevent and fight off its probable side effects. This factor can be further used to build a diagonal kit with high sensitivity.

  13. Recognition determinants of broadly neutralizing human antibodies against dengue viruses.

    Science.gov (United States)

    Rouvinski, Alexander; Guardado-Calvo, Pablo; Barba-Spaeth, Giovanna; Duquerroy, Stéphane; Vaney, Marie-Christine; Kikuti, Carlos M; Navarro Sanchez, M Erika; Dejnirattisai, Wanwisa; Wongwiwat, Wiyada; Haouz, Ahmed; Girard-Blanc, Christine; Petres, Stéphane; Shepard, William E; Desprès, Philippe; Arenzana-Seisdedos, Fernando; Dussart, Philippe; Mongkolsapaya, Juthathip; Screaton, Gavin R; Rey, Félix A

    2015-04-02

    Dengue disease is caused by four different flavivirus serotypes, which infect 390 million people yearly with 25% symptomatic cases and for which no licensed vaccine is available. Recent phase III vaccine trials showed partial protection, and in particular no protection for dengue virus serotype 2 (refs 3, 4). Structural studies so far have characterized only epitopes recognized by serotype-specific human antibodies. We recently isolated human antibodies potently neutralizing all four dengue virus serotypes. Here we describe the X-ray structures of four of these broadly neutralizing antibodies in complex with the envelope glycoprotein E from dengue virus serotype 2, revealing that the recognition determinants are at a serotype-invariant site at the E-dimer interface, including the exposed main chain of the E fusion loop and the two conserved glycan chains. This 'E-dimer-dependent epitope' is also the binding site for the viral glycoprotein prM during virus maturation in the secretory pathway of the infected cell, explaining its conservation across serotypes and highlighting an Achilles' heel of the virus with respect to antibody neutralization. These findings will be instrumental for devising novel immunogens to protect simultaneously against all four serotypes of dengue virus.

  14. Hepatitis C virus resistance to broadly neutralizing antibodies measured using replication-competent virus and pseudoparticles.

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    Wasilewski, Lisa N; Ray, Stuart C; Bailey, Justin R

    2016-11-01

    A better understanding of natural variation in neutralization resistance and fitness of diverse hepatitis C virus (HCV) envelope (E1E2) variants will be critical to guide rational development of an HCV vaccine. This work has been hindered by inadequate genetic diversity in viral panels and by a lack of standardization of HCV entry assays. Neutralization assays generally use lentiviral pseudoparticles expressing HCV envelope proteins (HCVpp) or chimeric full-length viruses that are replication competent in cell culture (HCVcc). There have been few systematic comparisons of specific infectivities of E1E2-matched HCVcc and HCVpp, and to our knowledge, neutralization of E1E2-matched HCVpp and HCVcc has never been compared using a diverse panel of human broadly neutralizing monoclonal antibodies (bNAbs) targeting distinct epitopes. Here, we describe an efficient method for introduction of naturally occurring E1E2 genes into a full-length HCV genome, producing replication-competent chimeric HCVcc. We generated diverse panels of E1E2-matched HCVcc and HCVpp and measured the entry-mediating fitness of E1E2 variants using the two systems. We also compared neutralization of E1E2-matched HCVcc and HCVpp by a diverse panel of human bNAbs targeting epitopes across E1E2. We found no correlation between specific infectivities of E1E2-matched HCVcc versus HCVpp, but found a very strong positive correlation between relative neutralization resistance of these same E1E2-matched HCVcc and HCVpp variants. These results suggest that quantitative comparisons of neutralization resistance of E1E2 variants can be made with confidence using either HCVcc or HCVpp, allowing the use of either or both systems to maximize diversity of neutralization panels.

  15. Production and characterization of monoclonal antibodies to the edta extract of Leptospira interrogans, serovar icterohaemorrhagiae

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    Lilian Terezinha de Queiroz Leite

    1996-10-01

    Full Text Available Monoclonal antibodies (MABs ivere produced against an etbylenediaminetetraacetate (EDTA extract of Leptospira interrogans serovar icterohaemorrhagiae being characterized by gel precipitation as IgM and IgG (IgGl and IgG2b. The EDTA extract was detected as several bands by silver staining in SDS-PAGE. In the Western blot the bands around 20 KDa reacted with a monoclonal antibody, 47B4D6, and was oxidized by periodate and was not digested by pronase, suggesting that the determinant is of carbohydrate nature, lmmunocytochemistry, using colloidal gold labeling, showed that an EDTA extract determinant recognized by monoclonal antibody 47B4D6, is localized under the outer envelope of serovar icterohaemorrhagiae. Hoe AIAB raised against the EDTA extract was not able to protect hamsters from lethal challenge with virulent homologous leptospires.

  16. Simultaneous Raising of Rabbit Monoclonal Antibodies to Fluoroquinolones with Diverse Recognition Functionalities via Single Mixture Immunization.

    Science.gov (United States)

    Liu, Na; Zhao, Zhiyong; Tan, Yanglan; Lu, Lei; Wang, Lin; Liao, Yucai; Beloglazova, Natalia; De Saeger, Sarah; Zheng, Xiaodong; Wu, Aibo

    2016-01-19

    Highly specific monoclonal and polyclonal antibodies are the key components in a diverse set of immunoassay applications, from research work to routine monitoring and analysis. In the current manuscript, combinatorial strategies for a single mixture immunization, screening and rabbit hybridoma cell technology were described. Fluoroquinolones (FQs) drugs were chosen as representative analytes. Six FQs were conjugated with bovine serum albumin and used as immunogens for subsequent immunization, while a mixture of all was injected for coimmunization. The hybridomas obtained against the individual and multiple FQs were used for the production of diverse varieties of rabbit monoclonal antibodies (RabMAbs) against the target analytes. As was proven by indirect competitive ELISA and quantitative lateral flow immunoassay, this approach opens a new way for simultaneously obtaining functional monoclonal antibodies which are capable of recognizing both individual and multiple analytes in a single preparation circle. This addresses various needs of different monitoring regulations as analytical methodology advances.

  17. Viraemia suppressed in HIV-1-infected humans by broadly neutralizing antibody 3BNC117.

    Science.gov (United States)

    Caskey, Marina; Klein, Florian; Lorenzi, Julio C C; Seaman, Michael S; West, Anthony P; Buckley, Noreen; Kremer, Gisela; Nogueira, Lilian; Braunschweig, Malte; Scheid, Johannes F; Horwitz, Joshua A; Shimeliovich, Irina; Ben-Avraham, Sivan; Witmer-Pack, Maggi; Platten, Martin; Lehmann, Clara; Burke, Leah A; Hawthorne, Thomas; Gorelick, Robert J; Walker, Bruce D; Keler, Tibor; Gulick, Roy M; Fätkenheuer, Gerd; Schlesinger, Sarah J; Nussenzweig, Michel C

    2015-06-25

    HIV-1 immunotherapy with a combination of first generation monoclonal antibodies was largely ineffective in pre-clinical and clinical settings and was therefore abandoned. However, recently developed single-cell-based antibody cloning methods have uncovered a new generation of far more potent broadly neutralizing antibodies to HIV-1 (refs 4, 5). These antibodies can prevent infection and suppress viraemia in humanized mice and nonhuman primates, but their potential for human HIV-1 immunotherapy has not been evaluated. Here we report the results of a first-in-man dose escalation phase 1 clinical trial of 3BNC117, a potent human CD4 binding site antibody, in uninfected and HIV-1-infected individuals. 3BNC117 infusion was well tolerated and demonstrated favourable pharmacokinetics. A single 30 mg kg(-1) infusion of 3BNC117 reduced the viral load in HIV-1-infected individuals by 0.8-2.5 log10 and viraemia remained significantly reduced for 28 days. Emergence of resistant viral strains was variable, with some individuals remaining sensitive to 3BNC117 for a period of 28 days. We conclude that, as a single agent, 3BNC117 is safe and effective in reducing HIV-1 viraemia, and that immunotherapy should be explored as a new modality for HIV-1 prevention, therapy and cure.

  18. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library.

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    Wang, Han; Yu, Rui; Fang, Ting; Yu, Ting; Chi, Xiangyang; Zhang, Xiaopeng; Liu, Shuling; Fu, Ling; Yu, Changming; Chen, Wei

    2016-09-11

    Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective.

  19. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library

    Science.gov (United States)

    Wang, Han; Yu, Rui; Fang, Ting; Yu, Ting; Chi, Xiangyang; Zhang, Xiaopeng; Liu, Shuling; Fu, Ling; Yu, Changming; Chen, Wei

    2016-01-01

    Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective. PMID:27626445

  20. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library

    Directory of Open Access Journals (Sweden)

    Han Wang

    2016-09-01

    Full Text Available Tetanus neurotoxin (TeNT produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective.

  1. Structural basis of hepatitis C virus neutralization by broadly neutralizing antibody HCV1

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    Kong, Leopold; Giang, Erick; Robbins, Justin B.; Stanfield, Robyn L.; Burton, Dennis R.; Wilson, Ian A.; Law, Mansun (Scripps)

    2012-10-29

    Hepatitis C virus (HCV) infects more than 2% of the global population and is a leading cause of liver cirrhosis, hepatocellular carcinoma, and end-stage liver diseases. Circulating HCV is genetically diverse, and therefore a broadly effective vaccine must target conserved T- and B-cell epitopes of the virus. Human mAb HCV1 has broad neutralizing activity against HCV isolates from at least four major genotypes and protects in the chimpanzee model from primary HCV challenge. The antibody targets a conserved antigenic site (residues 412-423) on the virus E2 envelope glycoprotein. Two crystal structures of HCV1 Fab in complex with an epitope peptide at 1.8-{angstrom} resolution reveal that the epitope is a {beta}-hairpin displaying a hydrophilic face and a hydrophobic face on opposing sides of the hairpin. The antibody predominantly interacts with E2 residues Leu{sup 413} and Trp{sup 420} on the hydrophobic face of the epitope, thus providing an explanation for how HCV isolates bearing mutations at Asn{sup 415} on the same binding face escape neutralization by this antibody. The results provide structural information for a neutralizing epitope on the HCV E2 glycoprotein and should help guide rational design of HCV immunogens to elicit similar broadly neutralizing antibodies through vaccination.

  2. Holes in the Glycan Shield of the Native HIV Envelope Are a Target of Trimer-Elicited Neutralizing Antibodies

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    Laura E. McCoy

    2016-08-01

    Full Text Available A major advance in the search for an HIV vaccine has been the development of a near-native Envelope trimer (BG505 SOSIP.664 that can induce robust autologous Tier 2 neutralization. Here, potently neutralizing monoclonal antibodies (nAbs from rabbits immunized with BG505 SOSIP.664 are shown to recognize an immunodominant region of gp120 centered on residue 241. Residue 241 occupies a hole in the glycan defenses of the BG505 isolate, with fewer than 3% of global isolates lacking a glycan site at this position. However, at least one conserved glycan site is missing in 89% of viruses, suggesting the presence of glycan holes in most HIV isolates. Serum evidence is consistent with targeting of holes in natural infection. The immunogenic nature of breaches in the glycan shield has been under-appreciated in previous attempts to understand autologous neutralizing antibody responses and has important potential consequences for HIV vaccine design.

  3. JC polyomavirus mutants escape antibody-mediated neutralization.

    Science.gov (United States)

    Ray, Upasana; Cinque, Paola; Gerevini, Simonetta; Longo, Valeria; Lazzarin, Adriano; Schippling, Sven; Martin, Roland; Buck, Christopher B; Pastrana, Diana V

    2015-09-23

    JC polyomavirus (JCV) persistently infects the urinary tract of most adults. Under conditions of immune impairment, JCV causes an opportunistic brain disease, progressive multifocal leukoencephalopathy (PML). JCV strains found in the cerebrospinal fluid of PML patients contain distinctive mutations in surface loops of the major capsid protein, VP1. We hypothesized that VP1 mutations might allow the virus to evade antibody-mediated neutralization. Consistent with this hypothesis, neutralization serology revealed that plasma samples from PML patients neutralized wild-type JCV strains but failed to neutralize patient-cognate PML-mutant JCV strains. This contrasted with serological results for healthy individuals, most of whom robustly cross-neutralized all tested JCV variants. Mice administered a JCV virus-like particle (VLP) vaccine initially showed neutralizing "blind spots" (akin to those observed in PML patients) that closed after booster immunization. A PML patient administered an experimental JCV VLP vaccine likewise showed markedly increased neutralizing titer against her cognate PML-mutant JCV. The results indicate that deficient humoral immunity is a common aspect of PML pathogenesis and that vaccination may overcome this humoral deficiency. Thus, vaccination with JCV VLPs might prevent the development of PML.

  4. Precipitating and non-precipitating monoclonal antibodies against chicken avidin. Significance of epitope density.

    Science.gov (United States)

    Krohn, K; Ashorn, R; Ashorn, P; Kulomaa, M

    1987-01-01

    1. Monoclonal antibodies, generated against chicken avidin, were characterized in Ouchterlony's immunodiffusion. 2. Of the nine antibodies three were non-precipitable but six could form clear visible precipitation lines with egg-white avidin in agarose gel. 3. The latter six antibodies could be divided into two groups according to their reactive pattern in immunodiffusion. 4. Antibodies belonging to the first group precipitated both dimeric as well as tetrameric avidin molecules, while those of the second group precipitated only the tetrameric avidin molecules. 5. The relevance of these results to the structure of avidin as well as possibilities to use monoclonal antibodies and the immunodiffusion technique to compare the structure of avidin induced by different factors are discussed.

  5. Rapid high-resolution characterization of functionally important monoclonal antibody N-glycans by capillary electrophoresis.

    Science.gov (United States)

    Szabo, Zoltan; Guttman, András; Bones, Jonathan; Karger, Barry L

    2011-07-01

    Characterization of the N-glycosylation present in the Fc region of therapeutic monoclonal antibodies requires rapid, high-resolution separation methods to guarantee product safety and efficacy during all stages of process development. Determination of fucosylated oligosaccharides is particularly important during clone selection, product characterization, and lot release as fucose has been shown to adversely affect the ability of mAbs to induce antibody dependent cellular cytotoxicity (ADCC). Here, we apply a general capillary electrophoresis optimization strategy to separate functionally relevant fucosylated and afucosylated glycans on mononclonal antibody products in the presence of several high mannose oligosaccharides. The N-glycans chosen represent those most commonly reported on CHO cell derived therapeutic antibodies. A rapid (processing for automated 96 well plate-based glycosylation analyses of two nonproprietary therapeutic monoclonal antibodies, demonstrating ruggedness and suitability for high-throughput process and product monitoring applications.

  6. Structure-guided alterations of the gp41-directed HIV-1 broadly neutralizing antibody 2F5 reveal new properties regarding its neutralizing function.

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    Javier Guenaga

    Full Text Available The broadly neutralizing HIV-1 antibody 2F5 recognizes an epitope in the gp41 membrane proximal external region (MPER. The MPER adopts a helical conformation as free peptide, as post-fusogenic forms of gp41, and when bound to the 4E10 monoclonal antibody (Mab. However, when bound to 2F5, the epitope is an extended-loop. The antibody-peptide structure reveals binding between the heavy and light chains with most the long, hydrophobic CDRH3 not contacting peptide. However, mutagenesis identifies this loop as critical for binding, neutralization and for putative hydrophobic membrane interactions. Here, we examined length requirements of the 2F5 CDRH3 and plasticity regarding binding and neutralization. We generated 2F5 variants possessing either longer or shorter CDRH3s and assessed function. The CDRH3 tolerated elongations and reductions up to four residues, displaying a range of binding affinities and retaining some neutralizing capacity. 2F5 antibody variants selective recognition of conformationally distinctive MPER probes suggests a new role for the CDRH3 loop in destabilizing the helical MPER. Binding and neutralization were enhanced by targeted tryptophan substitutions recapitulating fully the activities of the wild-type 2F5 antibody in a shorter CDRH3 variant. MPER alanine scanning revealed binding contacts of this variant downstream of the 2F5 core epitope, into the 4E10 epitope region. This variant displayed increased reactivity to cardiolipin-beta-2-glycoprotein. Tyrosine replacements maintained neutralization while eliminating cardiolipin-beta-2-glycoprotein interaction. The data suggest a new mechanism of action, important for vaccine design, in which the 2F5 CDRH3 contacts and destabilizes the MPER helix downstream of its core epitope to allow induction of the extended-loop conformation.

  7. Generation and molecular characterization of a monoclonal antibody reactive with conserved epitope in sphingomyelinases D from Loxosceles spider venoms.

    Science.gov (United States)

    Dias-Lopes, C; Felicori, L; Rubrecht, L; Cobo, S; Molina, L; Nguyen, C; Galéa, P; Granier, C; Molina, F; Chávez-Olortegui, C

    2014-04-11

    We report the production of a neutralizing monoclonal antibody able to recognize the venoms of three major medically important species of Loxosceles spiders in Brazil. The mAb was produced by immunization of mice with a toxic recombinant L. intermedia sphingomyelinase D {SMases D isoform (rLiD1)} [1] and screened by enzyme-linked immunosorbent assay (ELISA) using L. intermedia, L. laeta and L. gaucho venoms as antigens. One clone (LiD1mAb16) out of seventeen anti-rLiD1 hybridomas was cross-reactive with the three whole Loxosceles venoms. 2D Western blot analysis indicated that LiD1mAb16 was capable of interacting with 34 proteins of 29-36kDa in L. intermedia, 33 in L. gaucho and 27 in L. laeta venoms. The results of immunoassays with cellulose-bound peptides revealed that the LiD1mAb16 recognizes a highly conserved linear epitope localized in the catalytic region of SMases D toxins. The selected mAb displayed in vivo protective activity in rabbits after challenge with rLiD1. These results show the potential usefulness of monoclonal antibodies for future therapeutic approaches and also opens up the perspective of utilization of these antibodies for immunodiagnostic assays in loxoscelism.

  8. Broadly Neutralizing Alphavirus Antibodies Bind an Epitope on E2 and Inhibit Ent