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Sample records for protective monoclonal antibodies

  1. Monoclonal Antibodies.

    ERIC Educational Resources Information Center

    Killington, R. A.; Powell, K. L.

    1984-01-01

    Monoclonal antibodies have provided an exciting addition to the "armory" of the molecular biologist and immunologist. This article discusses briefly the concept of, techniques available for, production of, and possible uses of monoclonal antibodies. (Author)

  2. Monoclonal Antibodies.

    ERIC Educational Resources Information Center

    Killington, R. A.; Powell, K. L.

    1984-01-01

    Monoclonal antibodies have provided an exciting addition to the "armory" of the molecular biologist and immunologist. This article discusses briefly the concept of, techniques available for, production of, and possible uses of monoclonal antibodies. (Author)

  3. Monoclonal Antibodies.

    PubMed

    Geskin, Larisa J

    2015-10-01

    Use of monoclonal antibodies (mAbs) has revolutionized cancer therapy. Approaches targeting specific cellular targets on the malignant cells and in tumor microenvironment have been proved to be successful in hematologic malignancies, including cutaneous lymphomas. mAb-based therapy for cutaneous T-cell lymphoma has demonstrated high response rates and a favorable toxicity profile in clinical trials. Several antibodies and antibody-based conjugates are approved for use in clinical practice, and many more are in ongoing and planned clinical trials. In addition, these safe and effective drugs can be used as pillars for sequential therapies in a rational stepwise manner.

  4. A Fungicidal Monoclonal Antibody Protects against Murine Invasive Candidiasis

    PubMed Central

    Sevilla, María J.; Robledo, Beatriz; Rementeria, Aitor; Moragues, María D.; Pontón, José

    2006-01-01

    Mice infected by Candida albicans and treated with monoclonal antibody C7 survived longer than saline-treated animals. A prozone-like effect was observed. The in vitro candidacidal activity of macrophages was strongly enhanced when C. albicans was opsonized by C7 and complete murine serum was present. PMID:16622248

  5. Production and Characterization of Monoclonal Antibodies Against the Protective Antigen Component of Bacillus anthracis Toxin

    DTIC Science & Technology

    1988-01-21

    F. Jaquet, P. Luethy, R. Huetter, and D. G. Braun. 1986. Characterization of mcnoclonal antibodies to a crystal protein of Bacillus thuringiensis ...AD-A192 855 UT FILE COPY Production and Characterization of Monoclonal Antibodies Against the Protective Antigen Component of Bacillus anthracis...Author Tel. No. 301-663-7341 1--ac",- 88 3 14 05 6 Krhirty-six monoclonal antibodies to the protective antigen protein of Bacillus anthracis exotoxin

  6. Production, characterization, and protective effect of monoclonal antibodies to Clostridium chauvoei flagella.

    PubMed

    Tanaka, M; Hirayama, N; Tamura, Y

    1987-08-01

    Monoclonal antibodies to flagella of Clostridium chauvoei were obtained by the fusion of murine myeloma cells (P3-X63-Ag8-U1) and spleen cells from BALB/c mice immunized with partially purified flagella of strain Okinawa. Enzyme-linked immunosorbent assay and Western blot analysis with partially purified flagella, flagellated cells, and nonflagellated mutants were used to show that five monoclonal antibodies are specific for the flagella. In the Western blot analysis, all five antiflagellar antibodies reacted strongly with the 56,000-molecular-weight protein, which corresponds to the flagellin. By using the ELISA-derived reactivity of monoclonal antibodies to the various clostridia and the competitive binding assay, we showed that the flagella of C. chauvoei had at least three epitopes. The three antiflagellar monoclonal antibodies (one immunoglobulin G and two immunoglobulin M) demonstrated passive protective effects in mice. These results strongly suggest that the flagella of C. chauvoei are important for protective immunity in mice.

  7. Antigenic analysis of Clostridium chauvoei flagella with protective and non-protective monoclonal antibodies.

    PubMed

    Tamura, Y; Kijima, M; Ohishi, K; Takahashi, T; Suzuki, S; Nakamura, M

    1992-03-01

    Five monoclonal antibodies (mAbs) directed against the flagellin of Clostridium chauvoei were used to analyse the structural and antigenic characteristics on the bacterial flagellar surface. Immune electron microscopy showed that three protective mAbs recognized the surfaced-exposed epitopes on the flagellar filament of this bacteria. In contrast, two non-protective mAbs recognized internal epitopes of the flagellar filament. These findings have been confirmed by ELISA using mAbs absorbed with whole cells of C. chauvoei possessing flagella. Competitive binding assays showed that protective mAbs indicated reciprocal competition, while each of the non-protective mAbs had topographically distinct epitopes. Moreover, immunoblotting analysis with cyanogen-bromide-cleaved flagellin showed that protective mAbs may preferentially recognize conformational epitopes, whilst one of the non-protective mAbs may recognize a linear and conformation-independent epitope in the flagellin of C. chauvoei.

  8. Protection of mice against Clostridium chauvoei infection by anti-idiotype antibody to a monoclonal antibody to flagella.

    PubMed

    Kijima-Tanaka, M; Nakamura, M; Nagamine, N; Takahashi, T; Aoki, A; Tamura, Y

    1994-03-01

    Polyclonal rabbit anti-idiotypic antibody (anti-Id) against the protective monoclonal antibody specific to the flagella of Clostridium chauvoei was produced, purified, and characterized. Anti-Id inhibited the binding of its related monoclonal antibody to the flagellar antigen, suggesting that the anti-Id bore an internal image of the flagellar antigen. When mice were immunized with anti-Id intraperitoneally, the survival rate increased significantly, compared with mice immunized with normal rabbit IgG (P < 0.01), and specific anti-flagellar antibodies were induced.

  9. Enhanced activity of immobilized dimethylmaleic anhydride-protected poly- and monoclonal antibodies.

    PubMed

    Hadas, E; Koppel, R; Schwartz, F; Raviv, O; Fleminger, G

    1990-06-27

    The effect of reversible protection of the free amino groups of poly- and monoclonal antibodies by dimethylmaleic anhydride on their binding activity following immobilization onto various carriers was studied. The treatment with dimethylmaleic anhydride resulted in a 1.6-1.8-fold increase in the activity of immobilized goat anti-mouse immunoglobulin antibody immobilized onto different epoxy containing carriers and a 3-10.7-fold increase in the activity of immobilized monoclonal antibodies specific for carboxypeptidase A. The increase in activity was most pronounced at low antigen to carrier loads and over a wide range of modifier to protein ratios. The application of reversible protection of antibodies may permit the development of highly active immobilized antibody preparations for use in immunoaffinity purification.

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

    PubMed Central

    Swanstrom, J. A.; Plante, J. A.; Plante, K. S.; Young, E. F.; McGowan, E.; Gallichotte, E. N.; Widman, D. G.; Heise, M. T.; de Silva, A. M.

    2016-01-01

    ABSTRACT 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; 18%) or moderate to high (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. PMID:27435464

  11. Humanized Monoclonal Antibody That Passively Protects Mice against Systemic and Intranasal Ricin Toxin Challenge

    PubMed Central

    Sully, Erin K.; Bohorova, Natasha; Bohorov, Ognian; Kim, Do; Pauly, Michael H.; Whaley, Kevin J.

    2016-01-01

    PB10 is a murine monoclonal antibody against an immunodominant epitope on ricin toxin's enzymatic subunit. Here, we characterize a fully humanized version of PB10 IgG1 (hPB10) and demonstrate that it has potent in vitro and in vivo toxin-neutralizing activities. We also report the minimum serum concentrations of hPB10 required to protect mice against 10 times the 50% lethal dose of ricin when delivered by injection and inhalation. PMID:27466351

  12. Pan-ebolavirus and Pan-filovirus Mouse Monoclonal Antibodies: Protection against Ebola and Sudan Viruses

    PubMed Central

    Holtsberg, Frederick W.; Shulenin, Sergey; Vu, Hong; Howell, Katie A.; Patel, Sonal J.; Gunn, Bronwyn; Karim, Marcus; Lai, Jonathan R.; Frei, Julia C.; Nyakatura, Elisabeth K.; Zeitlin, Larry; Douglas, Robin; Fusco, Marnie L.; Froude, Jeffrey W.; Saphire, Erica Ollmann; Herbert, Andrew S.; Wirchnianski, Ariel S.; Lear-Rooney, Calli M.; Alter, Galit; Dye, John M.; Glass, Pamela J.; Warfield, Kelly L.

    2015-01-01

    ABSTRACT The unprecedented 2014-2015 Ebola virus disease (EVD) outbreak in West Africa has highlighted the need for effective therapeutics against filoviruses. Monoclonal antibody (MAb) cocktails have shown great potential as EVD therapeutics; however, the existing protective MAbs are virus species specific. Here we report the development of pan-ebolavirus and pan-filovirus antibodies generated by repeated immunization of mice with filovirus glycoproteins engineered to drive the B cell responses toward conserved epitopes. Multiple pan-ebolavirus antibodies were identified that react to the Ebola, Sudan, Bundibugyo, and Reston viruses. A pan-filovirus antibody that was reactive to the receptor binding regions of all filovirus glycoproteins was also identified. Significant postexposure efficacy of several MAbs, including a novel antibody cocktail, was demonstrated. For the first time, we report cross-neutralization and in vivo protection against two highly divergent filovirus species, i.e., Ebola virus and Sudan virus, with a single antibody. Competition studies indicate that this antibody targets a previously unrecognized conserved neutralizing epitope that involves the glycan cap. Mechanistic studies indicated that, besides neutralization, innate immune cell effector functions may play a role in the antiviral activity of the antibodies. Our findings further suggest critical novel epitopes that can be utilized to design effective cocktails for broad protection against multiple filovirus species. IMPORTANCE Filoviruses represent a major public health threat in Africa and an emerging global concern. Largely driven by the U.S. biodefense funding programs and reinforced by the 2014 outbreaks, current immunotherapeutics are primarily focused on a single filovirus species called Ebola virus (EBOV) (formerly Zaire Ebola virus). However, other filoviruses including Sudan, Bundibugyo, and Marburg viruses have caused human outbreaks with mortality rates as high as 90%. Thus

  13. Pan-ebolavirus and Pan-filovirus Mouse Monoclonal Antibodies: Protection against Ebola and Sudan Viruses.

    PubMed

    Holtsberg, Frederick W; Shulenin, Sergey; Vu, Hong; Howell, Katie A; Patel, Sonal J; Gunn, Bronwyn; Karim, Marcus; Lai, Jonathan R; Frei, Julia C; Nyakatura, Elisabeth K; Zeitlin, Larry; Douglas, Robin; Fusco, Marnie L; Froude, Jeffrey W; Saphire, Erica Ollmann; Herbert, Andrew S; Wirchnianski, Ariel S; Lear-Rooney, Calli M; Alter, Galit; Dye, John M; Glass, Pamela J; Warfield, Kelly L; Aman, M Javad

    2015-10-14

    The unprecedented 2014-2015 Ebola virus disease (EVD) outbreak in West Africa has highlighted the need for effective therapeutics against filoviruses. Monoclonal antibody (MAb) cocktails have shown great potential as EVD therapeutics; however, the existing protective MAbs are virus species specific. Here we report the development of pan-ebolavirus and pan-filovirus antibodies generated by repeated immunization of mice with filovirus glycoproteins engineered to drive the B cell responses toward conserved epitopes. Multiple pan-ebolavirus antibodies were identified that react to the Ebola, Sudan, Bundibugyo, and Reston viruses. A pan-filovirus antibody that was reactive to the receptor binding regions of all filovirus glycoproteins was also identified. Significant postexposure efficacy of several MAbs, including a novel antibody cocktail, was demonstrated. For the first time, we report cross-neutralization and in vivo protection against two highly divergent filovirus species, i.e., Ebola virus and Sudan virus, with a single antibody. Competition studies indicate that this antibody targets a previously unrecognized conserved neutralizing epitope that involves the glycan cap. Mechanistic studies indicated that, besides neutralization, innate immune cell effector functions may play a role in the antiviral activity of the antibodies. Our findings further suggest critical novel epitopes that can be utilized to design effective cocktails for broad protection against multiple filovirus species. Filoviruses represent a major public health threat in Africa and an emerging global concern. Largely driven by the U.S. biodefense funding programs and reinforced by the 2014 outbreaks, current immunotherapeutics are primarily focused on a single filovirus species called Ebola virus (EBOV) (formerly Zaire Ebola virus). However, other filoviruses including Sudan, Bundibugyo, and Marburg viruses have caused human outbreaks with mortality rates as high as 90%. Thus, cross-protective

  14. Therapeutic Recombinant Monoclonal Antibodies

    ERIC Educational Resources Information Center

    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…

  15. Therapeutic Recombinant Monoclonal Antibodies

    ERIC Educational Resources Information Center

    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…

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

    PubMed Central

    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

  17. Molecular characterization of the monoclonal antibodies composing ZMAb: a protective cocktail against Ebola virus.

    PubMed

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

    2014-11-06

    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.

  18. Human Anti-Plague Monoclonal Antibodies Protect Mice from Yersinia pestis in a Bubonic Plague Model

    PubMed Central

    Xiao, Xiaodong; Zhu, Zhongyu; Dankmeyer, Jennifer L.; Wormald, Michael M.; Fast, Randy L.; Worsham, Patricia L.; Cote, Christopher K.; Amemiya, Kei; Dimitrov, Dimiter S.

    2010-01-01

    Yersinia pestis is the etiologic agent of plague that has killed more than 200 million people throughout the recorded history of mankind. Antibiotics may provide little immediate relief to patients who have a high bacteremia or to patients infected with an antibiotic resistant strain of plague. Two virulent factors of Y. pestis are the capsid F1 protein and the low-calcium response (Lcr) V-protein or V-antigen that have been proven to be the targets for both active and passive immunization. There are mouse monoclonal antibodies (mAbs) against the F1- and V-antigens that can passively protect mice in a murine model of plague; however, there are no anti-Yersinia pestis monoclonal antibodies available for prophylactic or therapeutic treatment in humans. We identified one anti-F1-specific human mAb (m252) and two anti-V-specific human mAb (m253, m254) by panning a naïve phage-displayed Fab library against the F1- and V-antigens. The Fabs were converted to IgG1s and their binding and protective activities were evaluated. M252 bound weakly to peptides located at the F1 N-terminus where a protective mouse anti-F1 mAb also binds. M253 bound strongly to a V-antigen peptide indicating a linear epitope; m254 did not bind to any peptide from a panel of 53 peptides suggesting that its epitope may be conformational. M252 showed better protection than m253 and m254 against a Y, pestis challenge in a plague mouse model. A synergistic effect was observed when the three antibodies were combined. Incomplete to complete protection was achieved when m252 was given at different times post-challenge. These antibodies can be further studied to determine their potential as therapeutics or prophylactics in Y. pestis infection in humans. PMID:20976274

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

    PubMed

    Xiao, Xiaodong; Zhu, Zhongyu; Dankmeyer, Jennifer L; Wormald, Michael M; Fast, Randy L; Worsham, Patricia L; Cote, Christopher K; Amemiya, Kei; Dimitrov, Dimiter S

    2010-10-13

    Yersinia pestis is the etiologic agent of plague that has killed more than 200 million people throughout the recorded history of mankind. Antibiotics may provide little immediate relief to patients who have a high bacteremia or to patients infected with an antibiotic resistant strain of plague. Two virulent factors of Y. pestis are the capsid F1 protein and the low-calcium response (Lcr) V-protein or V-antigen that have been proven to be the targets for both active and passive immunization. There are mouse monoclonal antibodies (mAbs) against the F1- and V-antigens that can passively protect mice in a murine model of plague; however, there are no anti-Yersinia pestis monoclonal antibodies available for prophylactic or therapeutic treatment in humans. We identified one anti-F1-specific human mAb (m252) and two anti-V-specific human mAb (m253, m254) by panning a naïve phage-displayed Fab library against the F1- and V-antigens. The Fabs were converted to IgG1s and their binding and protective activities were evaluated. M252 bound weakly to peptides located at the F1 N-terminus where a protective mouse anti-F1 mAb also binds. M253 bound strongly to a V-antigen peptide indicating a linear epitope; m254 did not bind to any peptide from a panel of 53 peptides suggesting that its epitope may be conformational. M252 showed better protection than m253 and m254 against a Y, pestis challenge in a plague mouse model. A synergistic effect was observed when the three antibodies were combined. Incomplete to complete protection was achieved when m252 was given at different times post-challenge. These antibodies can be further studied to determine their potential as therapeutics or prophylactics in Y. pestis infection in humans.

  20. A natural anti-T-cell receptor monoclonal antibody protects against experimental autoimmune encephalomyelitis.

    PubMed

    Fernández-Malavé, Edgar; Stark-Aroeira, Luiz

    2011-05-01

    The therapeutic potential of natural anti-T-cell receptor (TCR) antibodies is largely unknown. We investigated whether passive administration of C1-19, a novel natural anti-TCRVβ8 monoclonal antibody, could interfere with the development of EAE. Treatment with C1-19 prevented myelin basic protein (MBP)-induced EAE in Vβ8-sufficient B10.PL but not in Vβ8-deficient SJL mice. Furthermore, C1-19 reduced disease severity when administrated shortly after disease onset. These protective effects of C1-19 correlated with a Th2 bias of the cytokine response, in the absence of T-cell deletion or anergy. Together, these findings indicate that natural anti-TCR antibodies could function as therapeutic tools in autoimmune inflammatory diseases.

  1. Monoclonal antibody "gold rush".

    PubMed

    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.

  2. Heterogeneity of monoclonal antibodies.

    PubMed

    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.

  3. Monoclonal antibodies and cancer therapy

    SciTech Connect

    Reisfeld, R.A.; Sell, S.

    1985-01-01

    These proceedings collect papers on the subject of monoclonal antibodies. Topics include: Monoclonal antibody, biochemical effects and cancer therapeutic potential of tunicamycin, use of monoclonal antibodies for detection of lymph node metastases, active specific immunotherapy, and applications of monoclonal antibodies to investigations of growth factors.

  4. A cross-reactive monoclonal antibody to nematode haemoglobin enhances protective immune responses to Nippostrongylus brasiliensis.

    PubMed

    Nieuwenhuizen, Natalie E; Meter, Jeanne M; Horsnell, William G; Hoving, J Claire; Fick, Lizette; Sharp, Michael F; Darby, Matthew G; Parihar, Suraj P; Brombacher, Frank; Lopata, Andreas L

    2013-01-01

    Nematode secreted haemoglobins have unusually high affinity for oxygen and possess nitric oxide deoxygenase, and catalase activity thought to be important in protection against host immune responses to infection. In this study, we generated a monoclonal antibody (48Eg) against haemoglobin of the nematode Anisakis pegreffii, and aimed to characterize cross-reactivity of 4E8g against haemoglobins of different nematodes and its potential to mediate protective immunity against a murine hookworm infection. Immunoprecipitation was used to isolate the 4E8g-binding antigen in Anisakis and Ascaris extracts, which were identified as haemoglobins by peptide mass fingerprinting and MS/MS. Immunological cross-reactivity was also demonstrated with haemoglobin of the rodent hookworm N. brasiliensis. Immunogenicity of nematode haemoglobin in mice and humans was tested by immunoblotting. Anisakis haemoglobin was recognized by IgG and IgE antibodies of Anisakis-infected mice, while Ascaris haemoglobin was recognized by IgG but not IgE antibodies in mouse and human sera. Sequencing of Anisakis haemoglobin revealed high similarity to haemoglobin of a related marine nematode, Psuedoterranova decipiens, which lacks the four -HKEE repeats of Ascaris haemoglobin important in octamer assembly. The localization of haemoglobin in the different parasites was examined by immunohistochemistry and associated with the excretory-secretary ducts in Anisakis, Ascaris and N. brasiliensis. Anisakis haemoglobin was strongly expressed in the L3 stage, unlike Ascaris haemoglobin, which is reportedly mainly expressed in adult worms. Passive immunization of mice with 4E8g prior to infection with N. brasiliensis enhanced protective Th2 immunity and led to a significant decrease in worm burdens. The monoclonal antibody 4E8g targets haemoglobin in broadly equivalent anatomical locations in parasitic nematodes and enhances host immunity to a hookworm infection.

  5. A Cross-Reactive Monoclonal Antibody to Nematode Haemoglobin Enhances Protective Immune Responses to Nippostrongylus brasiliensis

    PubMed Central

    Nieuwenhuizen, Natalie E.; Meter, Jeanne M.; Horsnell, William G.; Hoving, J. Claire; Fick, Lizette; Sharp, Michael F.; Darby, Matthew G.; Parihar, Suraj P.; Brombacher, Frank; Lopata, Andreas L.

    2013-01-01

    Background Nematode secreted haemoglobins have unusually high affinity for oxygen and possess nitric oxide deoxygenase, and catalase activity thought to be important in protection against host immune responses to infection. In this study, we generated a monoclonal antibody (48Eg) against haemoglobin of the nematode Anisakis pegreffii, and aimed to characterize cross-reactivity of 4E8g against haemoglobins of different nematodes and its potential to mediate protective immunity against a murine hookworm infection. Methodology/Principal Findings Immunoprecipitation was used to isolate the 4E8g-binding antigen in Anisakis and Ascaris extracts, which were identified as haemoglobins by peptide mass fingerprinting and MS/MS. Immunological cross-reactivity was also demonstrated with haemoglobin of the rodent hookworm N. brasiliensis. Immunogenicity of nematode haemoglobin in mice and humans was tested by immunoblotting. Anisakis haemoglobin was recognized by IgG and IgE antibodies of Anisakis-infected mice, while Ascaris haemoglobin was recognized by IgG but not IgE antibodies in mouse and human sera. Sequencing of Anisakis haemoglobin revealed high similarity to haemoglobin of a related marine nematode, Psuedoterranova decipiens, which lacks the four –HKEE repeats of Ascaris haemoglobin important in octamer assembly. The localization of haemoglobin in the different parasites was examined by immunohistochemistry and associated with the excretory-secretary ducts in Anisakis, Ascaris and N. brasiliensis. Anisakis haemoglobin was strongly expressed in the L3 stage, unlike Ascaris haemoglobin, which is reportedly mainly expressed in adult worms. Passive immunization of mice with 4E8g prior to infection with N. brasiliensis enhanced protective Th2 immunity and led to a significant decrease in worm burdens. Conclusion The monoclonal antibody 4E8g targets haemoglobin in broadly equivalent anatomical locations in parasitic nematodes and enhances host immunity to a hookworm

  6. Protective effect of polyclonal and monoclonal antibodies against abortion in mice infected by Chlamydia psittaci.

    PubMed

    Buzoni-Gatel, D; Bernard, F; Andersen, A; Rodolakis, A

    1990-08-01

    The role of antibody in preventing placental and fetal infection by Chlamydia psittaci was studied in mice. Pregnant mice were passively immunized with polyclonal sera or monoclonal antibodies (mAbs) at day 11 of gestation. The mice were intravenously challenged the following day with the virulent AB7 ovine abortion strain of C. psittaci. Mice were either killed on day 16 of gestation to determine placental and fetal chlamydial infection levels or were permitted to have and raise their young until 8 days old for comparison of survival rates. Immune sera produced a decrease in both placental and fetal infection and reduced the number of young dying in utero or shortly after birth. Polyclonal sera to the highly invasive AB7 and AB4 strains or to the invasive 1B strain were more effective than serum to the invasive AB13 strain. The B577/F3 and B577/A11 monoclonal antibodies gave almost complete protection, with only low levels of placental infection and no detectable fetal infection or decrease in survival rate. The study demonstrates that immune sera and type-specific mAbs can passively transfer resistance to placental and fetal colonization and to abortion and fetal loss in mice intravenously challenged with P. psittaci.

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

    PubMed

    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.

  8. Synthetic human monoclonal antibodies toward staphylococcal enterotoxin B (SEB) protective against toxic shock syndrome.

    PubMed

    Karauzum, Hatice; Chen, Gang; Abaandou, Laura; Mahmoudieh, Mahta; Boroun, Atefeh R; Shulenin, Sergey; Devi, V Sathya; Stavale, Eric; Warfield, Kelly L; Zeitlin, Larry; Roy, Chad J; Sidhu, Sachdev S; Aman, M Javad

    2012-07-20

    Staphylococcal enterotoxin B (SEB) is a potent toxin that can cause toxic shock syndrome and act as a lethal and incapacitating agent when used as a bioweapon. There are currently no vaccines or immunotherapeutics available against this toxin. Using phage display technology, human antigen-binding fragments (Fabs) were selected against SEB, and proteins were produced in Escherichia coli cells and characterized for their binding affinity and their toxin neutralizing activity in vitro and in vivo. Highly protective Fabs were converted into full-length IgGs and produced in mammalian cells. Additionally, the production of anti-SEB antibodies was explored in the Nicotiana benthamiana plant expression system. Affinity maturation was performed to produce optimized lead anti-SEB antibody candidates with subnanomolar affinities. IgGs produced in N. benthamiana showed characteristics comparable with those of counterparts produced in mammalian cells. IgGs were tested for their therapeutic efficacy in the mouse toxic shock model using different challenge doses of SEB and a treatment with 200 μg of IgGs 1 h after SEB challenge. The lead candidates displayed full protection from lethal challenge over a wide range of SEB challenge doses. Furthermore, mice that were treated with anti-SEB IgG had significantly lower IFNγ and IL-2 levels in serum compared with mock-treated mice. In summary, these anti-SEB monoclonal antibodies represent excellent therapeutic candidates for further preclinical and clinical development.

  9. Synthetic Human Monoclonal Antibodies toward Staphylococcal Enterotoxin B (SEB) Protective against Toxic Shock Syndrome*

    PubMed Central

    Karauzum, Hatice; Chen, Gang; Abaandou, Laura; Mahmoudieh, Mahta; Boroun, Atefeh R.; Shulenin, Sergey; Devi, V. Sathya; Stavale, Eric; Warfield, Kelly L.; Zeitlin, Larry; Roy, Chad J.; Sidhu, Sachdev S.; Aman, M. Javad

    2012-01-01

    Staphylococcal enterotoxin B (SEB) is a potent toxin that can cause toxic shock syndrome and act as a lethal and incapacitating agent when used as a bioweapon. There are currently no vaccines or immunotherapeutics available against this toxin. Using phage display technology, human antigen-binding fragments (Fabs) were selected against SEB, and proteins were produced in Escherichia coli cells and characterized for their binding affinity and their toxin neutralizing activity in vitro and in vivo. Highly protective Fabs were converted into full-length IgGs and produced in mammalian cells. Additionally, the production of anti-SEB antibodies was explored in the Nicotiana benthamiana plant expression system. Affinity maturation was performed to produce optimized lead anti-SEB antibody candidates with subnanomolar affinities. IgGs produced in N. benthamiana showed characteristics comparable with those of counterparts produced in mammalian cells. IgGs were tested for their therapeutic efficacy in the mouse toxic shock model using different challenge doses of SEB and a treatment with 200 μg of IgGs 1 h after SEB challenge. The lead candidates displayed full protection from lethal challenge over a wide range of SEB challenge doses. Furthermore, mice that were treated with anti-SEB IgG had significantly lower IFNγ and IL-2 levels in serum compared with mock-treated mice. In summary, these anti-SEB monoclonal antibodies represent excellent therapeutic candidates for further preclinical and clinical development. PMID:22645125

  10. Blocking by anti-idiotypic antibodies of monoclonal antibody-mediated protection against lethal Semliki Forest virus in mice.

    PubMed

    Oosterlaken, T A; Harmsen, M; Kraaijeveld, C A; Snippe, H

    1990-02-01

    Semliki Forest virus-(SEV) neutralizing monoclonal antibodies (MoAbs), produced after fusion of spleen cells from BALB/c mice and myeloma cell line P3-X63-AG8. 653 or SP2/0, were used for anti-idiotypic immunization of female BALB/c mice. Two intracutaneous immunizations (2 x 40 micrograms per animal), 3 weeks apart, with keyhole limpet haemocyanin-conjugated MoAbs mixed with the saponin Quil A were sufficient to induce high levels of anti-idiotypic antibodies in the circulation of these mice with the capacity to block specifically in vitro MoAb-mediated virus neutralization. Anti-idiotypic antibodies against SFV-neutralizing MoAbs, either passively transferred or actively acquired by immunization, are also able to abrogate (specifically) passive immunity, mediated by critical protective doses of MoAb, in mice against infection with a lethal strain of SFV. Furthermore we confirmed by intervention with anti-idiotypic serum in vivo that an SFV-neutralizing MoAb exerts its greatest protective effect during the first 2 days of infection.

  11. Monoclonal Antibodies against Pectin

    PubMed Central

    Liners, Françoise; Letesson, Jean-Jacques; Didembourg, Christian; Van Cutsem, Pierre

    1989-01-01

    Monoclonal antibodies have been produced that recognize a conformation of homopolygalacturonic acid (pectic acid) induced by an optimum concentration of calcium and sodium of about 1 and 150 millinormal, respectively. The epitope recognized is probably part of the dimers of pectin chains associated according to the `egg box' model. Images Figure 2 PMID:16667195

  12. Monoclonal antibodies against bacteria.

    PubMed

    Macario, A J; Conway de Macario, E

    1988-01-01

    Highlights are presented of most recent work in which monoclonal antibodies have been instrumental in the study of bacteria and their products. Topics summarized pertain to human and veterinary medicines, dentistry, phytopathology, ichthyology, and bacterial ecophysiology, differentiation, evolution and methanogenic biotechnology.

  13. Neutralizing monoclonal antibodies to the E2 protein of chikungunya virus protects against disease in a mouse model.

    PubMed

    Goh, Lucas Y H; Hobson-Peters, Jody; Prow, Natalie A; Gardner, Joy; Bielefeldt-Ohmann, Helle; Pyke, Alyssa T; Suhrbier, Andreas; Hall, Roy A

    2013-12-01

    Chikungunya virus (CHIKV) recently caused the largest epidemic ever recorded for this virus involving an estimated 1.4-6.5million cases, with imported cased reported in over 40 countries. The number of monoclonal antibodies specific for this re-emerging alphavirus is currently limited. Herein we describe the generation and characterisation of five monoclonal antibodies specific for the E2 glycoprotein of CHIKV. The antibodies detected a range of CHIKV isolates in several assays including ELISA, Western blot, immunofluorescence assay (IFA) and immunohistochemistry (IHC) without evidence of cross-reactivity with other alphaviruses. Four antibodies also neutralised CHIKV in vitro, two of which provided complete protection against arthritis in a CHIKV mouse model when administered prior to infection. Given the current shortage of widely available reagents for CHIKV, these specific antibodies will be useful not only in research, but may also provide the basis for new diagnostics and treatments.

  14. Isolation of protective antigens from the gut of Boophilus microplus using monoclonal antibodies.

    PubMed Central

    Lee, R P; Opdebeeck, J P

    1991-01-01

    Monoclonal antibodies (mAb) were produced against midgut membrane (GM) antigens of Boophilus microplus. The isotypes of these mAb were established and their specificity characterized using double diffusion and Western blotting. GM antigens solubilized by Triton X-100 were precipitated by mAb QU13, and the precipitate was then injected into cattle to test for the presence of protective antigens. Vaccinated cattle challenged with 10-day-old larval ticks showed evidence of protection with a 62% reduction in eggs produced by ticks from vaccinated cattle compared to tick eggs from control cattle. In a second vaccine-challenge experiment, the dose of precipitate was increased and greater than 99% protection was provided to these vaccinated cattle following challenge (calculated from tick egg weights compared to the control group). The solubilized antigen(s) precipitated by QU13 were subjected to SDS-PAGE separation and the calculated sizes of these molecules were greater than 200,000, 80,000, 74,000, 62,000 57,000 and less than 30,000 MW. Images Figure 1 Figure 2 PMID:1997395

  15. Generation and Characterization of Human Monoclonal Antibodies Targeting Anthrax Protective Antigen following Vaccination with a Recombinant Protective Antigen Vaccine.

    PubMed

    Chi, Xiangyang; Li, Jianmin; Liu, Weicen; Wang, Xiaolin; Yin, Kexin; Liu, Ju; Zai, Xiaodong; Li, Liangliang; Song, Xiaohong; Zhang, Jun; Zhang, Xiaopeng; Yin, Ying; Fu, Ling; Xu, Junjie; Yu, Changming; Chen, Wei

    2015-05-01

    The anthrax protective antigen (PA) is the central component of the three-part anthrax toxin, and it is the primary immunogenic component in the approved AVA anthrax vaccine and the "next-generation" recombinant PA (rPA) anthrax vaccines. Animal models have indicated that PA-specific antibodies (AB) are sufficient to protect against infection with Bacillus anthracis. In this study, we investigated the PA domain specificity, affinity, mechanisms of neutralization, and synergistic effects of PA-specific antibodies from a single donor following vaccination with the rPA vaccine. Antibody-secreting cells were isolated 7 days after the donor received a boost vaccination, and 34 fully human monoclonal antibodies (hMAb) were identified. Clones 8H6, 4A3, and 22F1 were able to neutralize lethal toxin (LeTx) both in vitro and in vivo. Clone 8H6 neutralized LeTx by preventing furin cleavage of PA in a dose-dependent manner. Clone 4A3 enhanced degradation of nicked PA, thereby interfering with PA oligomerization. The mechanism of 22F1 is still unclear. A fourth clone, 2A6, that was protective only in vitro was found to be neutralizing in vivo in combination with a toxin-enhancing antibody, 8A7, which binds to domain 3 of PA and PA oligomers. These results provide novel insights into the antibody response elicited by the rPA vaccine and may be useful for PA-based vaccine and immunotherapeutic cocktail design.

  16. Recombinant monoclonal antibody technology.

    PubMed

    Siegel, D L

    2002-01-01

    With the development of murine hybridoma technology over a quarter century ago, the ability to produce large quantities of well-characterized monoclonal antibody preparations revolutionized diagnostic and therapeutic medicine. For many applications in transfusion medicine, however, the production of serological reagents in mice has certain biological limitations relating to the difficulty in obtaining murine monoclonal antibodies specific for many human blood group antigens. Furthermore, for therapeutic purposes, the efficacy of murine-derived immunoglobulin preparations is limited by the induction of anti-mouse immune responses. Technical difficulties inherent in human hybridoma formation have led to novel molecular approaches that facilitate the isolation and production of human antibodies without the need for B-cell transformation, tissue culture, or even immunized individuals. These technologies, referred to as 'repertoire cloning' or 'Fab/phage display', involve the rapid cloning of immunoglobulin gene segments to create immune libraries from which antibodies with desired specificities can be selected. The use of such recombinant methods in transfusion medicine is anticipated to play an important role in the development and production of renewable supplies of low-cost reagents for diagnostic and therapeutic applications.

  17. Protection against pneumococcal pneumonia in mice by monoclonal antibodies to pneumolysin.

    PubMed

    García-Suárez, María del Mar; Cima-Cabal, María Dolores; Flórez, Noelia; García, Pilar; Cernuda-Cernuda, Rafael; Astudillo, Aurora; Vázquez, Fernando; De los Toyos, Juan R; Méndez, F Javier

    2004-08-01

    Pneumolysin (PLY) is an important virulence factor of Streptococcus pneumoniae. We examined the ability of three murine monoclonal antibodies (MAbs) to PLY (PLY-4, PLY-5, and PLY-7) to affect the course of pneumococcal pneumonia in mice. The intravenous administration of antibodies PLY-4 and PLY-7 protected the mice from the lethal effect of the purified toxin. Mice treated with PLY-4 before intranasal inoculation of S. pneumoniae type 2 survived longer (median survival time, 100 h) than did untreated animals (median survival time, 60 h) (P < 0.0001). The median survival time for mice treated with a combination of PLY-4 and PLY-7 was 130 h, significantly longer than that for mice given isotype-matched indifferent MAbs (P = 0.0288) or nontreated mice (P = 0.0002). The median survival time for mice treated with a combination of three MAbs was significantly longer (>480 h) than that for mice treated with PLY-5 (48 h; P < 0.0001), PLY-7 (78 h; P = 0.0007), or PLY-4 (100 h; P = 0.0443) alone. Similarly, the survival rate for mice treated with three MAbs (10 of 20 mice) was significantly higher than the survival rate obtained with PLY-5 (1 of 20; P = 0.0033), PLY-4 (2 of 20; P = 0.0138), or PLY-7 (3 of 20; P = 0.0407) alone. These results suggest that anti-PLY MAbs act with a synergistic effect. Furthermore, MAb administration was associated with a significant decrease in bacterial lung colonization and lower frequencies of bacteremia and tissue injury with respect to the results for the control groups.

  18. Monoclonal antibodies to gonadotropin subunits

    SciTech Connect

    Ehrlich, P.H.; Moyle, W.R.; Canfield, R.E.

    1985-01-01

    The production of monoclonal antibodies to peptide hormones, with their unifocal binding sites, can provide tools for understanding hormone structure and function. The paper focuses on techniques that are important for the study of monoclonal antibodies to chorionic gonadotropin (hCG), including hybridoma production, methods of screening for desired clones, properties of the monoclonal antibodies, effect of antibodies on hormone-receptor interaction, inhibition of binding of radiolabeled hCG, inhibition of hCG induced steroidogenesis, determination of relative orientation of epitopes, and synergistic actions of monoclonal antibodies to hCG.

  19. Human-monoclonal-antibody therapy protects nonhuman primates against advanced Lassa fever.

    PubMed

    Mire, Chad E; Cross, Robert W; Geisbert, Joan B; Borisevich, Viktoriya; Agans, Krystle N; Deer, Daniel J; Heinrich, Megan L; Rowland, Megan M; Goba, Augustine; Momoh, Mambu; Boisen, Mathew L; Grant, Donald S; Fullah, Mohamed; Khan, Sheik Humarr; Fenton, Karla A; Robinson, James E; Branco, Luis M; Garry, Robert F; Geisbert, Thomas W

    2017-09-04

    There are no approved treatments for Lassa fever, which is endemic to the same regions of West Africa that were recently devastated by Ebola. Here we show that a combination of human monoclonal antibodies that cross-react with the glycoproteins of all four clades of Lassa virus is able to rescue 100% of cynomolgus macaques when treatment is initiated at advanced stages of disease, including up to 8 d after challenge.

  20. Monoclonal antibodies to Actinobacillus actinomycetemcomitans.

    PubMed Central

    Place, D A; Scidmore, N C; McArthur, W P

    1988-01-01

    Murine hybridoma cell lines were developed which synthesized monoclonal antibodies against Actinobacillus actinomycetemcomitans-associated antigens. Monoclonal antibodies specific for an antigen(s) common to all A. actinomycetemcomitans isolates tested but not detected on other gram-negative oral plaque microorganisms or other Actinobacillus species were identified. Monoclonal antibodies specific for each serotype group of A. actinomycetemcomitans which did not bind to other Actinobacillus species or oral plaque microorganisms were also identified. PMID:3356470

  1. A neutralizing human monoclonal antibody protects African Green monkeys from Hendra virus challenge

    PubMed Central

    Bossart, Katharine N.; Geisbert, Thomas W.; Feldmann, Heinz; Zhu, Zhongyu; Feldmann, Friederike; Geisbert, Joan B.; Yan, Lianying; Feng, Yan-Ru; Brining, Doug; Scott, Dana; Wang, Yanping; Dimitrov, Antony S.; Callison, Julie; Chan, Yee-Peng; Hickey, Andrew C.; Dimitrov, Dimiter S.; Broder, Christopher C.; Rockx, Barry

    2012-01-01

    Hendra virus (HeV) is a recently emerged zoonotic paramyxovirus that can cause a severe and often fatal disease in horses and humans. HeV is categorized as a biosafety level 4 agent, which has made the development of animal models and testing of potential therapeutics and vaccines challenging. Infection of African Green monkeys (AGMs) with HeV was recently demonstrated and disease mirrored fatal HeV infection in humans, manifesting as a multisystemic vasculitis with widespread virus replication in vascular tissues and severe pathologic manifestations in the lung, spleen and brain. Here, we demonstrate that m102.4, a potent HeV neutralizing human monoclonal antibody (hmAb), can protect AGMs from disease post infection (p.i.) with HeV. Fourteen AGMs were challenged intratracheally with a lethal dose of HeV and twelve subjects were infused twice with a 100 mg dose of m102.4 beginning at either 10 hr, 24 hr or 72 hr p.i. and again approximately 48 hrs later. The presence of viral RNA, infectious virus and HeV-specific immune responses demonstrated that all subjects were infected following challenge. All twelve AGMs that received m102.4 survived infection; whereas the untreated control subjects succumbed to disease on day 8 p.i.. Animals in the 72 hr treatment group exhibited neurological signs of disease but all animals started to recover by day 16 p.i.. These results represent successful post-exposure in vivo efficacy by an investigational drug against HeV and highlight the potential impact a hmAb can have on human disease. PMID:22013123

  2. [The pharmacokinetics of monoclonal antibodies].

    PubMed

    Keizer, R J; Huitema, A D R; Damen, C W N; Schellens, J H M; Beijnen, J H

    2007-03-24

    Monoclonal antibodies (MOABs) are, due to their specificity, increasingly being deployed for therapeutic purposes. MOABs are derived from immunoglobulins and are fully or partially of murine or human origin. They are administered parenterally: mostly intravenously, but subcutaneous or intramuscular administration is also possible, in which case absorption probably occurs through the lymphatic system. The distribution of MOABs from the bloodstream into the tissues is slow and is hampered by the high molecular size of the MOABs, which is a lesser problem for fragments of antibodies (Fab fragments). MOABs are metabolised to peptides and amino acids. This process takes place in many tissues of the body, but probably predominantly in epithelial cells. As a consequence of the saturable binding of the MOAB to its target, a dose-dependent (non-linear) elimination is often observed. Immune reactions can accelerate the elimination of antibodies, partially depending on the degree ofhumanisation of the antibody. Antibodies and endogenous immunoglobulins are protected from elimination by binding to protective receptors (neonatal Fc-receptor; FcRn), which explains their long half-lives (up to 4 weeks). Metabolic pharmacokinetic interactions with other drugs have not been reported and are not expected. It is expected that in the years to come, new MOABs directed towards new targets will appear on the market, as well as existing antibodies with improved pharmacokinetic properties.

  3. X-linked immunodeficient mice as a model for testing the protective efficacy of monoclonal antibodies against Pseudomonas aeruginosa.

    PubMed Central

    Zweerink, H J; Gammon, M C; Hutchison, C F; Jackson, J J; Pier, G B; Puckett, J M; Sewell, T J; Sigal, N H

    1988-01-01

    (DBA/N[female] X CBA/2[male])F1 males have been reported to be deficient in producing antibodies against a number of antigens, including carbohydrates (I. Scher, Adv. Immunol. 35:1-71, 1982). We show that F1 male mice, in contrast to females, made less lipopolysaccharide (LPS)-specific antibodies after immunization with heat-inactivated Pseudomonas aeruginosa and had significantly less naturally occurring LPS-specific antibodies. Furthermore, neutropenic males were 50 to 1,000 times more sensitive to challenge with representative isolates belonging to the seven Fisher immunotypes. Administration to neutropenic F1 males of a human monoclonal antibody specific for the O carbohydrates of P. aeruginosa immunotype 2 LPS or administration of serum from rabbits immunized with heat-inactivated P. aeruginosa immunotype 1 raised the level of resistance to bacterial challenge close to that of females. The results show that the X-linked immunodeficient mouse is an excellent model with which to test the protective efficacy of P. aeruginosa-specific monoclonal antibodies. PMID:3128480

  4. Identification of a novel linear epitope in tetanus toxin recognized by a protective monoclonal antibody: implications for vaccine design.

    PubMed

    Luo, Ping; Qin, Liyan; Mao, Xuhu; Chen, Li; Yu, Shu; Li, Qian; Liu, Wei; Zhang, Weijun; Gu, Jiang; Zou, Quanming

    2012-10-05

    Tetanus, a severe infectious disease, is caused by tetanus toxin (TT) from Clostridium tetani, which remains one of the most critical unsolved health problems despite preventive strategies. The carboxyl terminal of TT (TTC) is responsible for the binding of TT to neurons and for its toxicity and has been proven to be immunogenic and protective in various forms. It would therefore be extremely interesting to identify the epitope on TTC at a molecular level. In this study, we generated a neutralizing monoclonal antibody, 5C4, which inhibited TT binding to its receptor and was efficiently protective at 73.7 IU/mg. Moreover, 5C4 recognized a novel linear epitope on TT, namely TC((1155-1171)), which spans from Lys1155 to Val1171. In addition, TC((1155-1171)) was shown to elicit the production of a serum IgG that protected mice against a challenge with TT. These results suggested that TC((1155-1171)) and the monoclonal antibody 5C4 are good candidates for the development of epitope-based vaccines and therapeutic antibodies against tetanus. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Monoclonal antibodies and neuroblastoma

    SciTech Connect

    Miraldi, F. )

    1989-10-01

    Several antineuroblastoma monoclonal antibodies (MoAbs) have been described and two have been used in radioimmunoimaging and radioimmunotherapy in patients. MoAb 3F8 is a murine IgG3 antibody specific for the ganglioside GD2. Radioiodine-labeled 3F8 has been shown to specifically target human neuroblastoma in patients, and radioimmunoimaging with this agent has provided consistently high uptakes with tumor-to-background ratios of greater than or equal to 10:1. Radioimmunotherapy has been attempted with both MoAb 3F8 and MoAb UJ13A, and although encouraging results have been obtained, dosimetry data and tissue dose response information for these agents is lacking, which impedes the development of such therapy. 124I, a positron emitter, can be used with 3F8 in positron emission tomography (PET) scanning to provide dosimetry information for radioimmunotherapy. The tumor radiation dose response from radiolabeled MoAb also can be followed with PET images with fluorodeoxyglucose (FDG) scanning of neuroblastoma tumors. Results to date indicate that radioimmunoimaging has clinical use in the diagnosis of neuroblastoma and the potential for radioimmunotherapy for this cancer remains high.48 references.

  6. High-Sensitivity Monoclonal Antibodies Specific for Homoserine Lactones Protect Mice from Lethal Pseudomonas aeruginosa Infections

    PubMed Central

    Downham, Christina; Broadbent, Ian; Charlton, Keith; Porter, Andrew J.

    2014-01-01

    A number of bacteria, including pathogens like Pseudomonas aeruginosa, utilize homoserine lactones (HSLs) as quorum sensing (QS) signaling compounds and engage in cell-to-cell communication to coordinate their behavior. Blocking this bacterial communication may be an attractive strategy for infection control as QS takes a central role in P. aeruginosa biology. In this study, immunomodulation of HSL molecules by monoclonal antibodies (MAbs) was used as a novel approach to prevent P. aeruginosa infections and as tools to detect HSLs in bodily fluids as a possible first clue to an undiagnosed Gram-negative infection. Using sheep immunization and recombinant antibody technology, a panel of sheep-mouse chimeric MAbs were generated which recognized HSL compounds with high sensitivity (nanomolar range) and cross-reactivity. These MAbs retained their nanomolar sensitivity in complex matrices and were able to recognize HSLs in P. aeruginosa cultures grown in the presence of urine. In a nematode slow-killing assay, HSL MAbs significantly increased the survival of worms fed on the antibiotic-resistant strain PA058. The therapeutic benefit of these MAbs was further studied using a mouse model of Pseudomonas infection in which groups of mice treated with HSL-2 and HSL-4 MAbs survived, 7 days after pathogen challenge, in significantly greater numbers (83 and 67%, respectively) compared with the control groups. This body of work has provided early proof-of-concept data to demonstrate the potential of HSL-specific, monoclonal antibodies as theranostic clinical leads suitable for the diagnosis, prevention, and treatment of life-threatening bacterial infections. PMID:24185854

  7. Monoclonal Antibody Targeting Staphylococcus aureus Surface Protein A (SasA) Protect Against Staphylococcus aureus Sepsis and Peritonitis in Mice.

    PubMed

    Yang, Yilong; Qian, Mengying; Yi, Shaoqiong; Liu, Shuling; Li, Bing; Yu, Rui; Guo, Qiang; Zhang, Xiaopeng; Yu, Changming; Li, Jianmin; Xu, Junjie; Chen, Wei

    2016-01-01

    Epidemic methicillin-resistant Staphylococcus aureus (MRSA) imposes an increasing impact on public health. Due to multi-antibiotics resistance in MRSA strains, there is an urgent need to develop novel therapeutics such as effective monoclonal antibodies (mAbs) against MRSA infections. Staphylococcus aureus surface protein A (SasA), a large surface-located protein (~240 kDa), is one of MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) and a potential target for immunotherapeutic approaches against S. aureus infections. In the present study, we analyzed the sequence of SasA with bioinformatics tools and generated a protective monoclonal antibody (2H7) targeting the conserved domain of SasA. 2H7 was shown to recognize wild-type S. aureus and promote opsonophagocytic killing of S. aureus. In both sepsis and peritoneal infection models, prophylactic administration of 2H7 improved the survival of BALB/c mice challenged by S. aureus strain USA300 and ST239 (prevalent MRSA clones in North America and Asian countries, respectively) and enhanced bacterial clearance in kidneys. Additionally, 2H7 prophylaxis prevented the formation of intraperitoneal abscess in a murine model of peritoneal infection and therapeutic administration of 2H7 showed protective efficacy in a murine sepsis model. Our results presented here provide supporting evidences that an anti-SasA mAb might be a potential component in an antibody-based immunotherapeutic treatment of MRSA infections.

  8. Monoclonal Antibody Targeting Staphylococcus aureus Surface Protein A (SasA) Protect Against Staphylococcus aureus Sepsis and Peritonitis in Mice

    PubMed Central

    Yang, Yilong; Qian, Mengying; Yi, Shaoqiong; Liu, Shuling; Li, Bing; Yu, Rui; Guo, Qiang; Zhang, Xiaopeng; Yu, Changming; Li, Jianmin; Xu, Junjie; Chen, Wei

    2016-01-01

    Epidemic methicillin-resistant Staphylococcus aureus (MRSA) imposes an increasing impact on public health. Due to multi-antibiotics resistance in MRSA strains, there is an urgent need to develop novel therapeutics such as effective monoclonal antibodies (mAbs) against MRSA infections. Staphylococcus aureus surface protein A (SasA), a large surface-located protein (~240 kDa), is one of MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) and a potential target for immunotherapeutic approaches against S. aureus infections. In the present study, we analyzed the sequence of SasA with bioinformatics tools and generated a protective monoclonal antibody (2H7) targeting the conserved domain of SasA. 2H7 was shown to recognize wild-type S. aureus and promote opsonophagocytic killing of S. aureus. In both sepsis and peritoneal infection models, prophylactic administration of 2H7 improved the survival of BALB/c mice challenged by S. aureus strain USA300 and ST239 (prevalent MRSA clones in North America and Asian countries, respectively) and enhanced bacterial clearance in kidneys. Additionally, 2H7 prophylaxis prevented the formation of intraperitoneal abscess in a murine model of peritoneal infection and therapeutic administration of 2H7 showed protective efficacy in a murine sepsis model. Our results presented here provide supporting evidences that an anti-SasA mAb might be a potential component in an antibody-based immunotherapeutic treatment of MRSA infections. PMID:26926145

  9. Delayed Treatment of Ebola Virus Infection with Plant-Derived Monoclonal Antibodies Provides Protection in Rhesus Macaques

    DTIC Science & Technology

    2012-10-15

    Delayed treatment of Ebola virus infection with plant -derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger, Jr.a...Michael Paulyb, Kevin J. Whaleyb, Calli M. Leara, Julia E. Bigginsa, Corinne Scullya, Lisa Hensleya,3, and Larry Zeitlinb,2 aDivision of Virology , United...three anti-Ebola virus mouse/human chimeric mAbs (c13C6, h-13F6, and c6D8) were produced in Chinese hamster ovary and in whole plant

  10. Hemagglutinin Stalk- and Neuraminidase-Specific Monoclonal Antibodies Protect against Lethal H10N8 Influenza Virus Infection in Mice.

    PubMed

    Wohlbold, Teddy John; Chromikova, Veronika; Tan, Gene S; Meade, Philip; Amanat, Fatima; Comella, Phillip; Hirsh, Ariana; Krammer, Florian

    2015-10-28

    Between November 2013 and February 2014, China reported three human cases of H10N8 influenza virus infection in the Jiangxi province, two of which were fatal. Using hybridoma technology, we isolated a panel of H10- and N8-directed monoclonal antibodies (MAbs) and further characterized the binding reactivity of these antibodies (via enzyme-linked immunosorbent assay) to a range of purified virus and recombinant protein substrates. The H10-directed MAbs displayed functional hemagglutination inhibition (HI) and neutralization activity, and the N8-directed antibodies displayed functional neuraminidase inhibition (NI) activity against H10N8. Surprisingly, the HI-reactive H10 antibodies, as well as a previously generated, group 2 hemagglutinin (HA) stalk-reactive antibody, demonstrated NI activity against H10N8 and an H10N7 strain; this phenomenon was absent when virus was treated with detergent, suggesting the anti-HA antibodies inhibited neuraminidase enzymatic activity through steric hindrance. We tested the prophylactic efficacy of one representative H10-reactive, N8-reactive, and group 2 HA stalk-reactive antibody in vivo using a BALB/c challenge model. All three antibodies were protective at a high dose (5 mg/kg). At a low dose (0.5 mg/kg), only the anti-N8 antibody prevented weight loss. Together, these data suggest that antibody targets other than the globular head domain of the HA may be efficacious in preventing influenza virus-induced morbidity and mortality. Avian H10N8 and H10N7 viruses have recently crossed the species barrier, causing morbidity and mortality in humans and other mammals. Although these reports are likely isolated incidents, it is possible that more cases may emerge in future winter seasons, similar to H7N9. Furthermore, regular transmission of avian influenza viruses to humans increases the risk of adaptive mutations and reassortment events, which may result in a novel virus with pandemic potential. Currently, no specific therapeutics or

  11. Polyclonal and monoclonal antibodies in clinic.

    PubMed

    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.

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

    PubMed

    Bossart, Katharine N; Zhu, Zhongyu; Middleton, Deborah; Klippel, Jessica; Crameri, Gary; Bingham, John; McEachern, Jennifer A; Green, Diane; Hancock, Timothy J; Chan, Yee-Peng; Hickey, Andrew C; Dimitrov, Dimiter S; Wang, Lin-Fa; Broder, Christopher C

    2009-10-01

    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.

  13. Anti–IL-20 monoclonal antibody inhibits the differentiation of osteoclasts and protects against osteoporotic bone loss

    PubMed Central

    Hsu, Yu-Hsiang; Chen, Wei-Yu; Chan, Chien-Hui; Wu, Chih-Hsing; Sun, Zih-Jie

    2011-01-01

    IL-20 is a proinflammatory cytokine of the IL-10 family that is involved in psoriasis, rheumatoid arthritis, atherosclerosis, and stroke. However, little is known about the role of IL-20 in bone destruction. We explored the function of IL-20 in osteoclastogenesis and the therapeutic potential of anti–IL-20 monoclonal antibody 7E for treating osteoporosis. Higher serum IL-20 levels were detected in patients with osteopenia and osteoporosis and in ovariectomized (OVX) mice. IL-20 mediates osteoclastogenesis by up-regulating the receptor activator of NF-κB (RANK) expression in osteoclast precursor cells and RANK ligand (RANKL) in osteoblasts. 7E treatment completely inhibited osteoclast differentiation induced by macrophage colony-stimulating factor (M-CSF) and RANKL in vitro and protected mice from OVX-induced bone loss in vivo. Furthermore, IL-20R1–deficient mice had significantly higher bone mineral density (BMD) than did wild-type controls. IL-20R1 deficiency also abolished IL-20–induced osteoclastogenesis and increased BMD in OVX mice. We have identified a pivotal role of IL-20 in osteoclast differentiation, and we conclude that anti–IL-20 monoclonal antibody is a potential therapeutic for protecting against osteoporotic bone loss. PMID:21844205

  14. Partial passive protection with two monoclonal antibodies and frequency of feeding of hyperimmune anti-transmissible gastroenteritis virus (TGEV) serum for protection of three-day-old piglets from a TGEV challenge infection.

    PubMed

    Wesley, R D; Woods, R D

    2001-07-01

    Passive protection experiments were conducted to determine the frequency and amounts of hyperimmune antiserum needed to block a transmissible gastroenteritis virus (TGEV) challenge infection and to identify monoclonal antibodies that are partially protective against TGEV. Hyperimmune antiserum or monoclonal antibodies were added to milk at each feeding or at selected feedings when the amount of antiserum was reduced. Three-day-old piglets were challenged with virulent virus that had been preincubated with antiserum or monoclonal antibodies. The results indicated that supplementing antiserum every other day was not efficacious for protection. Supplementing even small quantities of hyperimmune antiserum (0.5 ml) at least once a day in most cases was sufficient for piglet survival but did not prevent morbidity. Increasing the amount (>2 ml) and providing antiserum 3 times/day completely blocked the TGEV challenge infection. Two monoclonal antibodies were discovered that also provided passive protection for baby pigs. One monoclonal antibody, 5G1, had a high neutralizing titer, and the other, 6C4, was more effective in neutralizing and binding to virulent TGEV than to attenuated TGEVs. Both of these monoclonal antibodies were partially effective as supplements in milk for passive protection. Furthermore, these monoclonal antibodies were useful for boosting the efficacy of TGEV-neutralizing colostrum, which by itself was ineffective. These results show that other antigenic sites, different from the 4-well characterized epitopes on the S glycoprotein of TGEV, also are important for passive protection.

  15. Detection of anthrax protective antigen (PA) using europium labeled anti-PA monoclonal antibody and time-resolved fluorescence ◊

    PubMed Central

    Stoddard, Robyn A.; Quinn, Conrad P.; Schiffer, Jarad M.; Boyer, Anne E.; Goldstein, Jason; Bagarozzi, Dennis A.; Soroka, Stephen D.; Dauphin, Leslie A.; Hoffmaster, Alex R.

    2015-01-01

    Inhalation anthrax is a rare but acute infectious disease following adsorption of Bacillus anthracis spores through the lungs. The disease has a high fatality rate if untreated, but early and correct diagnosis has a significant impact on case patient recovery. The early symptoms of inhalation anthrax are, however, non-specific and current anthrax diagnostics are primarily dependent upon culture and confirmatory real-time PCR. Consequently, there may be a significant delay in diagnosis and targeted treatment. Rapid, culture-independent diagnostic tests are therefore needed, particularly in the context of a large scale emergency response. The aim of this study was to evaluate the ability of monoclonal antibodies to detect anthrax toxin proteins that are secreted early in the course of B. anthracis infection using a time-resolved fluorescence (TRF) immunoassay. We selected monoclonal antibodies that could detect protective antigen (PA), as PA83 and also PA63 and LF in the lethal toxin complex. The assay reliable detection limit (RDL) was 6.63 × 10−6 μM (0.551 ng/ml) for PA83 and 2.51 × 10−5 μM (1.58 ng/ml) for PA63. Despite variable precision and accuracy of the assay, PA was detected in 9 out of 10 sera samples from anthrax confirmed case patients with cutaneous (n=7), inhalation (n=2), and gastrointestinal (n=1) disease. Anthrax Immune Globulin (AIG), which has been used in treatment of clinical anthrax, interfered with detection of PA. This study demonstrates a culture-independent method of diagnosing anthrax through use of monoclonal antibodies to detect PA and LF in the lethal toxin complex. PMID:24857756

  16. Detection of anthrax protective antigen (PA) using europium labeled anti-PA monoclonal antibody and time-resolved fluorescence.

    PubMed

    Stoddard, Robyn A; Quinn, Conrad P; Schiffer, Jarad M; Boyer, Anne E; Goldstein, Jason; Bagarozzi, Dennis A; Soroka, Stephen D; Dauphin, Leslie A; Hoffmaster, Alex R

    2014-06-01

    Inhalation anthrax is a rare but acute infectious disease following adsorption of Bacillus anthracis spores through the lungs. The disease has a high fatality rate if untreated, but early and correct diagnosis has a significant impact on case patient recovery. The early symptoms of inhalation anthrax are, however, non-specific and current anthrax diagnostics are primarily dependent upon culture and confirmatory real-time PCR. Consequently, there may be a significant delay in diagnosis and targeted treatment. Rapid, culture-independent diagnostic tests are therefore needed, particularly in the context of a large scale emergency response. The aim of this study was to evaluate the ability of monoclonal antibodies to detect anthrax toxin proteins that are secreted early in the course of B. anthracis infection using a time-resolved fluorescence (TRF) immunoassay. We selected monoclonal antibodies that could detect protective antigen (PA), as PA83 and also PA63 and LF in the lethal toxin complex. The assay reliable detection limit (RDL) was 6.63×10(-6)μM (0.551ng/ml) for PA83 and 2.51×10(-5)μM (1.58ng/ml) for PA63. Despite variable precision and accuracy of the assay, PA was detected in 9 out of 10 sera samples from anthrax confirmed case patients with cutaneous (n=7), inhalation (n=2), and gastrointestinal (n=1) disease. Anthrax Immune Globulin (AIG), which has been used in treatment of clinical anthrax, interfered with detection of PA. This study demonstrates a culture-independent method of diagnosing anthrax through the use of monoclonal antibodies to detect PA and LF in the lethal toxin complex. Published by Elsevier B.V.

  17. Fragmentation of monoclonal antibodies

    PubMed Central

    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

  18. Monoclonal Anti-HMGB1 (High Mobility Group Box Chromosomal Protein 1) Antibody Protection in Two Experimental Arthritis Models

    PubMed Central

    Schierbeck, Hanna; Lundbäck, Peter; Palmblad, Karin; Klevenvall, Lena; Erlandsson-Harris, Helena; Andersson, Ulf; Ottosson, Lars

    2011-01-01

    High mobility group box chromosomal protein 1 (HMGB1) is a DNA-binding nuclear protein that can be released from dying cells and activated myeloid cells. Extracellularly, HMGB1 promotes inflammation. Experimental studies demonstrate HMGB1 to be a pathogenic factor in many inflammatory conditions including arthritis. HMGB1-blocking therapies in arthritis models alleviate disease and confer significant protection against cartilage and bone destruction. So far, the most successful HMGB1-targeted therapies have been demonstrated with HMGB1-specific polyclonal antibodies and with recombinant A box protein, a fragment of HMGB1. The present study is the first to evaluate the potential of a monoclonal anti-HMGB1 antibody (2G7, mouse IgG2b) to ameliorate arthritis. Effects of repeated injections of this antibody have now been studied in two conceptually different models of arthritis: collagen type II–induced arthritis (CIA) in DBA/1 mice and in a spontaneous arthritis disease in mice with combined deficiencies for genes encoding for the enzyme DNase type II and interferon type I receptors. These mice are unable to degrade phagocytozed DNA in macrophages and develop chronic, destructive polyarthritis. Therapeutic intervention in CIA and prophylactic administration of anti-HMGB1 monoclonal antibody (mAb) in the spontaneous arthritis model significantly ameliorated the clinical courses. Anti-HMGB1 mAb therapy also partially prevented joint destruction, as demonstrated by histological examination. The beneficial antiarthritic effects by the anti-HMGB1 mAb in two diverse models of arthritis represent additional proof-of-concept, indicating that HMGB1 may be a valid target molecule to consider for development of future clinical therapy. PMID:21666956

  19. Protection Against H7 Subtype Influenza Virus Infection in Mice by Passive Transfer of Neutralizing Monoclonal Antibody.

    PubMed

    Zhang, Zhuo; Liu, Ming; Zheng, Shimin

    2015-10-01

    H7 subtype influenza viruses pose serious threats to both the poultry industry and public health. Recent human infections of avian H7N9 influenza viruses with substantial morbidity and mortality have raised concerns about this virus becoming a potential pandemic pathogen. Neutralizing antibodies have been proven to be highly effective in blocking influenza virus infections. In this study, in order to develop an antibody-based immunoprophylaxis against H7 subtype influenza virus, we first generated a neutralizing monoclonal antibody (MAb) by using a pseudotyped lentiviral vector carrying the hemagglutinin protein of H7 subtype influenza virus. In vitro studies demonstrated that this neutralizing MAb completely inhibited the infection of an H7 subtype influenza virus to cells. The protective efficacy of this MAb was then further tested in a mouse model. It was shown that passive immunization of this MAb protected mice from local virus challenge. Results of the current study lay a foundation for the development of neutralizing MAb-mediated prophylactic strategies to combat human H7 influenza virus infections.

  20. Cross-protection of newly emerging HPAI H5 viruses by neutralizing human monoclonal antibodies: A viable alternative to oseltamivir

    PubMed Central

    Ren, Huanhuan; Wang, Guiqin; Wang, Shuangshuang; Chen, Honglin; Chen, Zhiwei; Hu, Hongxing; Cheng, Genhong; Zhou, Paul

    2016-01-01

    ABSTRACT Newly emerging highly pathogenic avian influenza (HPAI) H5N2, H5N3, H5N5, H5N6, H5N8 and H5N9 viruses have been spreading in poultry and wild birds. The H5N6 viruses have also caused 10 human infections with 4 fatal cases in China. Here, we assessed the cross-neutralization and cross-protection of human and mouse monoclonal antibodies against 2 viruses: a HPAI H5N8 virus, A/chicken/Netherlands/14015526/2014 (NE14) and a HPAI H5N6 virus, A/Sichuan/26221/2014 (SC14). The former was isolated from an infected chicken in Netherlands in 2014 and the latter was isolated from an infected human patient in Sichuan, China. We show that antibodies FLA5.10, FLD21.140, 100F4 and 65C6, but not AVFluIgG01, AVFluIgG03, S139/1 and the VRC01 control, potently cross-neutralize the H5N8 NE14 and H5N6 SC14 viruses. Furthermore, we show that a single injection of >1 mg/kg of antibody 100F4 at 4 hours before, or 20 mg/kg antibody 100F4 at 72 hours after, a lethal dose of H5N8 NE14 enables mice to withstand the infection. Finally, we show that a single injection of 0.5 or 1 mg/kg antibody 100F4 prophylactically or 10 mg/kg 100F4 therapeutically outperforms a 5-day course of 10 mg/kg/day oseltamivir treatment against lethal H5N8 NE14 or H5N6 SC14 infection in mice. Our results suggest that further preclinical evaluation of human monoclonal antibodies against newly emerging H5 viruses is warranted. PMID:27167234

  1. Uses of monoclonal antibody 8H9

    DOEpatents

    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.

  2. Uses of monoclonal antibody 8H9

    DOEpatents

    Cheung, Nai-Kong V.

    2010-06-22

    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.

  3. Monoclonal antibodies for treating cancer

    SciTech Connect

    Dillman, R.O. )

    1989-10-01

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

  4. Detection of Campylobacter species using monoclonal antibodies

    NASA Astrophysics Data System (ADS)

    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.

  5. A Neutralizing Anti-gH/gL Monoclonal Antibody Is Protective in the Guinea Pig Model of Congenital CMV Infection

    PubMed Central

    Auerbach, Marcy R.; Yan, Donghong; Vij, Rajesh; Hongo, Jo-Anne; Nakamura, Gerald; Vernes, Jean-Michel; Meng, Y. Gloria; Lein, Samantha; Chan, Pamela; Ross, Jed; Carano, Richard; Deng, Rong; Lewin-Koh, Nicholas; Xu, Min; Feierbach, Becket

    2014-01-01

    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 [1]–[3]. 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. PMID:24722349

  6. Monoclonal Antibodies in Diagnosis and Therapy

    NASA Astrophysics Data System (ADS)

    Waldmann, Thomas A.

    1991-06-01

    Monoclonal antibodies have been applied clinically to the diagnosis and therapy of an array of human disorders, including cancer and infectious diseases, and have been used for the modulation of immune responses. Effective therapy using unmodified monoclonal antibodies has, however, been elusive. Recently, monoclonal antibody-mediated therapy has been revolutionized by advances such as the definition of cell-surface structures on abnormal cells as targets for effective monoclonal antibody action, genetic engineering to create less immunogenic and more effective monoclonal antibodies, and the arming of such antibodies with toxins or radionuclides to enhance their effector function.

  7. Application of monoclonal antibodies in tumor pathology

    SciTech Connect

    Ruiter, D.J. ); Fleuren, G.J.; Warnaar, S.O. )

    1987-01-01

    This book contains papers under the following three section headings: Basic and technical aspects; Tumor associated antigens; and Practical application and case presentations. Some of the paper titles are: Monoclonal antibodies to oncofetal antigens; Monoclonal antibodies against ovarian cancer; and Tumor associated antigens and oncogene products defined by monoclonal antibodies.

  8. In Vitro Neutralization of Low Dose Inocula at Physiological Concentrations of a Monoclonal Antibody Which Protects Macaques against SHIV Challenge

    PubMed Central

    Davis, David; Koornstra, Wim; Fagrouch, Zahra; Verschoor, Ernst J.; Heeney, Jonathan L.; Bogers, Willy M. J. M.

    2013-01-01

    Background Passive transfer of antibodies can be protective in the simian human immunodeficiency virus (SHIV) – rhesus macaque challenge model. The human monoclonal antibody IgG1 b12 neutralizes human immunodeficiency type 1 (HIV-1) in vitro and protects against challenge by SHIV. Our hypothesis is that neutralizing antibodies can only completely inactivate a relatively small number of infectious virus. Methods And Findings We have used GHOST cell assays to quantify individual infectious events with HIV-1SF162 and its SHIV derivatives: the relatively neutralization sensitive SHIVSF162P4 isolate and the more resistant SHIVSF162P3. A plot of the number of fluorescent GHOST cells with increasing HIV-1SF162 dose is not linear. It is likely that with high-dose inocula, infection with multiple virus produces additive fluorescence in individual cells. In studies of the neutralization kinetics of IgG1 b12 against these isolates, events during the absorption phase of the assay, as well as the incubation phase, determine the level of neutralization. It is possible that complete inactivation of a virus is limited to the time it is exposed on the cell surface. Assays can be modified so that neutralization of these very low doses of virus can be quantified. A higher concentration of antibody is required to neutralize the same dose of resistant SHIVSF162P3 than the sensitive SHIVSF162P4. In the absence of selection during passage, the density of the CCR5 co-receptor on the GHOST cell surface is reduced. Changes in the CD4 : CCR5 density ratio influence neutralization. Conclusions Low concentrations of IgG1 b12 completely inactivate small doses of the neutralization resistant SHIV SF162P3. Assays need to be modified to quantify this effect. Results from modified assays may predict protection following repeated low-dose shiv challenges in rhesus macaques. It should be possible to induce this level of antibody by vaccination so that modified assays could predict the outcome of

  9. In vitro neutralization of low dose inocula at physiological concentrations of a monoclonal antibody which protects macaques against SHIV challenge.

    PubMed

    Davis, David; Koornstra, Wim; Fagrouch, Zahra; Verschoor, Ernst J; Heeney, Jonathan L; Bogers, Willy M J M

    2013-01-01

    Passive transfer of antibodies can be protective in the simian human immunodeficiency virus (SHIV)--rhesus macaque challenge model. The human monoclonal antibody IgG1 b12 neutralizes human immunodeficiency type 1 (HIV-1) in vitro and protects against challenge by SHIV. Our hypothesis is that neutralizing antibodies can only completely inactivate a relatively small number of infectious virus. We have used GHOST cell assays to quantify individual infectious events with HIV-1SF162 and its SHIV derivatives: the relatively neutralization sensitive SHIV(SF162P4) isolate and the more resistant SHIV(SF162P3). A plot of the number of fluorescent GHOST cells with increasing HIV-1SF162 dose is not linear. It is likely that with high-dose inocula, infection with multiple virus produces additive fluorescence in individual cells. In studies of the neutralization kinetics of IgG1 b12 against these isolates, events during the absorption phase of the assay, as well as the incubation phase, determine the level of neutralization. It is possible that complete inactivation of a virus is limited to the time it is exposed on the cell surface. Assays can be modified so that neutralization of these very low doses of virus can be quantified. A higher concentration of antibody is required to neutralize the same dose of resistant SHIV(SF162P3) than the sensitive SHIV(SF162P4). In the absence of selection during passage, the density of the CCR5 co-receptor on the GHOST cell surface is reduced. Changes in the CD4 : CCR5 density ratio influence neutralization. Low concentrations of IgG1 b12 completely inactivate small doses of the neutralization resistant SHIV(SF162P3). Assays need to be modified to quantify this effect. Results from modified assays may predict protection following repeated low-dose shiv challenges in rhesus macaques. It should be possible to induce this level of antibody by vaccination so that modified assays could predict the outcome of human trials.

  10. Therapeutic Administration of a Monoclonal Anti-Il-1β Antibody Protects Against Experimental Melioidosis

    PubMed Central

    Weehuizen, Tassili A. F.; Lankelma, Jacqueline M.; De Jong, Hanna K.; De Boer, Onno J.; Roelofs, Joris J. T. H.; Day, Nicholas P.; Gram, Hermann; De Vos, Alex F.; Wiersinga, W. Joost

    2016-01-01

    ABSTRACT Background: Melioidosis, caused by the gram-negative bacterium Burkholderia pseudomallei, is a common cause of community-acquired sepsis in Southeast Asia and Northern Australia. The NLRP3 inflammasome and its downstream product interleukin-1 beta (IL-1β) have been proposed to play crucial roles in melioidosis. In this study, we characterized the role of IL-1β more closely and we assessed its therapeutic potential. Methods: mRNA expression of inflammasome components was determined in isolated leukocytes of 32 healthy controls and 34 patients with sepsis caused by B pseudomallei. Wild-type (WT), NLRP3-deficient (Nlrp3−/−), and Asc−/− mice were infected with B pseudomallei. In additional experiments, infected WT mice were treated with an anti-IL-1β antibody. After 24, 48, and 72 hours (h) mice were sacrificed and organs were harvested. Furthermore, survival studies were performed. Results: Patients with melioidosis exhibited lower mRNA levels of caspase-1, NLRP3, and ASC. Bacterial dissemination and organ damage were increased in B pseudomallei-infected Nlrp3−/− and Asc−/− mice, together with a reduced pulmonary cell influx. Anti-IL-1β treatment of B pseudomallei challenged mice resulted in strongly reduced bacterial counts, organ damage, and pulmonary granulocyte influx together with reduced mortality. Postponement of anti-IL-1β treatment for 24 h postinfection still protected mice during melioidosis. Conclusion: Expression of caspase-1, NLRP3, and ASC is altered in melioidosis patients. In mice, both NLRP3 and ASC contribute to the host defense against melioidosis. Anti-IL-1β treatment protects mice against B pseudomallei infection and might be a novel treatment strategy in melioidosis. PMID:27219859

  11. A monoclonal antibody against leptin.

    PubMed

    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.

  12. Combinations of polyclonal or monoclonal antibodies to proteins of the outer membranes of the two infectious forms of vaccinia virus protect mice against a lethal respiratory challenge.

    PubMed

    Lustig, Shlomo; Fogg, Christiana; Whitbeck, J Charles; Eisenberg, Roselyn J; Cohen, Gary H; Moss, Bernard

    2005-11-01

    Previous studies demonstrated that antibodies to live vaccinia virus infection are needed for optimal protection against orthopoxvirus infection. The present report is the first to compare the protective abilities of individual and combinations of specific polyclonal and monoclonal antibodies that target proteins of the intracellular (IMV) and extracellular (EV) forms of vaccinia virus. The antibodies were directed to one IMV membrane protein, L1, and to two outer EV membrane proteins, A33 and B5. In vitro studies showed that the antibodies to L1 neutralized IMV and that the antibodies to A33 and B5 prevented the spread of EV in liquid medium. Prophylactic administration of individual antibodies to BALB/c mice partially protected them against disease following intranasal challenge with lethal doses of vaccinia virus. Combinations of antibodies, particularly anti-L1 and -A33 or -L1 and -B5, provided enhanced protection when administered 1 day before or 2 days after challenge. Furthermore, the protection was superior to that achieved with pooled immune gamma globulin from human volunteers inoculated with live vaccinia virus. In addition, single injections of anti-L1 plus anti-A33 antibodies greatly delayed the deaths of severe combined immunodeficiency mice challenged with vaccinia virus. These studies suggest that antibodies to two or three viral membrane proteins optimally derived from the outer membranes of IMV and EV, may be beneficial for prophylaxis or therapy of orthopoxvirus infections.

  13. Monoclonal antibodies of three different immunoglobulin G isotypes produced by immunization with a synthetic peptide or native protein protect mice against challenge with Plasmodium yoelii sporozoites.

    PubMed

    Ak, M; Bower, J H; Hoffman, S L; Sedegah, M; Lees, A; Carter, M; Beaudoin, R L; Charoenvit, Y

    1993-06-01

    Passive transfer of monoclonal antibodies (MAbs) against malaria circumsporozoite (CS) proteins protects animals against malaria. Active immunization with synthetic or recombinant peptides induces a level of polyclonal antibodies to sporozoites comparable to those found after passive immunization but does not provide comparable protection. In the Plasmodium yoelii system, synthetic or recombinant peptide-induced antibodies have never been shown to protect. The current studies were designed to determine whether immunogen structure (native protein versus synthetic peptide) or immunoglobulin G (IgG) subclass of antibodies was responsible for the absolute differences between protective, passively transferred MAbs and nonprotective, actively induced polyclonal antibodies. In this study we produced two MAbs, QGP-S1 (IgG1) and QGP-S2 (IgG2b), by immunization with a synthetic peptide based on the P. yoelii CS major repeat, (QGPGAP)4, conjugated to keyhole limpet hemocyanin. These MAbs were compared tp NYS1 (IgG3), an anti-CS protein MAb previously produced by immunization with irradiated P. yoelii sporozoites, which recognizes (QGP GAP)2. QGP-S1 and QGP-S2 passively transferred protection. However, when compared with NYS1, there was a hierarchy of protection, NYS1 > QGP-S1 > QGP-S2. There was no correlation between antibody level at challenge as determined by immunofluorescent antibody test against sporozoites or enzyme-linked immunosorbent assay against (QGPGAP)2 or apparent antibody avidity for (QGPGAP)2 by sodium thiocyanate elution assay. The data demonstrate that a synthetic peptide can induce protective antibodies and that a specific antibody subclass is not required for protection. Work to determine whether antibody affinity or fine specificity can explain the hierarchy of protection among the MAbs is under way.

  14. [Evolution of monoclonal antibodies in cancer treatment].

    PubMed

    Kubczak, Małgorzata; Rogalińska, Małgorzata

    2016-01-01

    Since late 90s of last century the new age of directed therapy began using mainly biological constructs produced in rodents called monoclonal antibodies. The side effects of monoclonal antibodies were a challenge for pharmaceutical companies to improve the biological properties of these biological drugs. The humanization of monoclonal constructs was an idea to improve monoclonal antibodies next generation activity cancer cell reduction in humans. Moreover for some other patients sensitive for monoclonal antibodies therapy could also potentially induce immunological differences that might imply on human health. The new idea related to monoclonal antibodies was to design a small molecule constructs of nanoantibodies with ability to enter into cells. Such small molecules could find their targets inside human cells, even in nuclei leading to differences in cancer cells expression. The existing knowledge on monoclonal antibodies as well as directed activity of nanoantibodies could improve anticancer treatment efficancy of diseases.

  15. Monoclonal anti-envelope antibody AP33 protects humanized mice against a patient-derived hepatitis C virus challenge.

    PubMed

    Desombere, Isabelle; Fafi-Kremer, Samira; Van Houtte, Freya; Pessaux, Patrick; Farhoudi, Ali; Heydmann, Laura; Verhoye, Lieven; Cole, Sarah; McKeating, Jane A; Leroux-Roels, Geert; Baumert, Thomas F; Patel, Arvind H; Meuleman, Philip

    2016-04-01

    End-stage liver disease (ESLD) caused by hepatitis C virus (HCV) infection is a major indication for liver transplantation. However, immediately after transplantation, the liver graft of viremic patients universally becomes infected by circulating virus, resulting in accelerated liver disease progression. Currently available direct-acting antiviral therapies have reduced efficacy in patients with ESLD and prophylactic strategies to prevent HCV recurrence are still highly needed. In this study, we compared the ability of two broadly reactive monoclonal antibodies (mAbs), designated 3/11 and AP33, recognizing a distinct, but overlapping, epitope in the viral E2 glycoprotein to protect humanized mice from a patient-derived HCV challenge. Their neutralizing activity was assessed using the HCV pseudoparticles and cell-culture-derived HCV systems expressing multiple patient-derived envelopes and a human-liver chimeric mouse model. HCV RNA was readily detected in all control mice challenged with a patient-derived HCV genotype 1b isolate, whereas 3 of 4 AP33-treated mice were completely protected. In contrast, only one of four 3/11-treated mice remained HCV-RNA negative throughout the observation period, whereas the other 3 had a viral load that was indistinguishable from that in the control group. The increased in vivo efficacy of AP33 was in line with its higher affinity and neutralizing capacity observed in vitro. Although mAbs AP33 and 3/11 target the same region in E2, only mAb AP33 can efficiently protect from challenge with a heterologous HCV population in vivo. Given that mAb AP33 efficiently neutralizes viral variants that escaped the humoral immune response and reinfected the liver graft of transplant patients, it may be a valuable candidate to prevent HCV recurrence. In addition, our data are valuable for the design of a prophylactic vaccine. © 2015 by the American Association for the Study of Liver Diseases.

  16. A human monoclonal antibody targeting the conserved staphylococcal antigen IsaA protects mice against Staphylococcus aureus bacteremia.

    PubMed

    van den Berg, Sanne; Bonarius, Hendrik P J; van Kessel, Kok P M; Elsinga, Goffe S; Kooi, Neeltje; Westra, Hans; Bosma, Tjibbe; van der Kooi-Pol, Magdalena M; Koedijk, Danny G A M; Groen, Herman; van Dijl, Jan Maarten; Buist, Girbe; Bakker-Woudenberg, Irma A J M

    2015-01-01

    Due to substantial therapy failure and the emergence of antibiotic-resistant Staphylococcus aureus strains, alternatives for antibiotic treatment of S. aureus infections are urgently needed. Passive immunization using S. aureus-specific monoclonal antibodies (mAb) could be such an alternative to prevent and treat severe S. aureus infections. The invariantly expressed immunodominant staphylococcal antigen A (IsaA) is a promising target for passive immunization. Here we report the development of the human anti-IsaA IgG1 mAb 1D9, which was shown to bind to all 26 S. aureus isolates tested. These included both methicillin-susceptible and methicillin-resistant S. aureus (MSSA and MRSA, respectively). Immune complexes consisting of IsaA and 1D9 stimulated human as well as murine neutrophils to generate an oxidative burst. In a murine bacteremia model, the prophylactic treatment with a single dose of 5 mg/kg 1D9 improved the survival of mice challenged with S. aureus isolate P (MSSA) significantly, while therapeutic treatment with the same dose did not influence animal survival. Neither prophylactic nor therapeutic treatment with 5 mg/kg 1D9 resulted in improved survival of mice with S. aureus USA300 (MRSA) bacteremia. Importantly, our studies show that healthy S. aureus carriers elicit an immune response which is sufficient to generate protective mAbs against invariant staphylococcal surface antigens. Human mAb 1D9, possibly conjugated to for example another antibody, antibiotics, cytokines or chemokines, may be valuable to fight S. aureus infections in patients.

  17. Monoclonal Antibodies for Dengue Virus prM Glycoprotein Protect Mice against Lethal Dengue Infection

    DTIC Science & Technology

    1989-09-15

    Nile virus and a prelysozomal endosome prM glycoprotein of dengue virus can also be required for viral replication . PrM Mabs 2H2 protective against...tech- bodies can prevent lethal alphavirus encepha- niques to preserve immunogenicity, to deter- litis. Nature 297: 70-72. UI:82173237 mine whether

  18. Monoclonal Antibodies Passively Protect BALB/c Mice Against Burkholderia mallei Aerosol Challenge

    DTIC Science & Technology

    2006-03-01

    Infectious Diseases, 1425 Porter St., Fort Detrick, Frederick, MD 21702-5001. Phone: (301) 619-4893. Fax: (301) 619-2152. E -mail: david.waag@det.amedd.army.mil...negative control MAb ( E ) 18 h before an aerosol challenge with 20 LD50 of B. mallei strain China 7 (ATCC 23344) or challenged and then given per mouse 1... E ., S. Wong, D. E . Woods, D. A. B. Dance, and W. Chaowagul. 1994. Passive protection of diabetic rats with antisera specific for the poly

  19. A monoclonal antibody targeting a highly conserved epitope in influenza B neuraminidase provides protection against drug resistant strains.

    PubMed

    Doyle, Tracey M; Li, Changgui; Bucher, Doris J; Hashem, Anwar M; Van Domselaar, Gary; Wang, Junzhi; Farnsworth, Aaron; She, Yi-Min; Cyr, Terry; He, Runtao; Brown, Earl G; Hurt, Aeron C; Li, Xuguang

    2013-11-08

    All influenza viral neuraminidases (NA) of both type A and B viruses have only one universally conserved sequence located between amino acids 222-230. A monoclonal antibody against this region has been previously reported to provide broad inhibition against all nine subtypes of influenza A NA; yet its inhibitory effect against influenza B viral NA remained unknown. Here, we report that the monoclonal antibody provides a broad inhibition against various strains of influenza B viruses of both Victoria and Yamagata genetic lineage. Moreover, the growth and NA enzymatic activity of two drug resistant influenza B strains (E117D and D197E) are also inhibited by the antibody even though these two mutations are conformationally proximal to the universal epitope. Collectively, these data suggest that this unique, highly-conserved linear sequence in viral NA is exposed sufficiently to allow access by inhibitory antibody during the course of infection; it could represent a potential target for antiviral agents and vaccine-induced immune responses against diverse strains of type B influenza virus.

  20. A non-glycosylated, plant-produced human monoclonal antibody against anthrax protective antigen protects mice and non-human primates from B. anthracis spore challenge.

    PubMed

    Mett, Vadim; Chichester, Jessica A; Stewart, Michelle L; Musiychuk, Konstantin; Bi, Hong; Reifsnyder, Carolyn J; Hull, Anna K; Albrecht, Mark T; Goldman, Stanley; Baillie, Les W J; Yusibov, Vidadi

    2011-01-01

    The health and economic burden of infectious diseases in general and bioterrorism in particular necessitate the development of medical countermeasures. One proven approach to reduce the disease burden and spread of pathogen is treatment with monoclonal antibodies (mAb). mAbs can prevent or reduce severity of the disease by variety of mechanisms, including neutralizing pathogen growth, limiting its spread from infected to adjacent cells, or by inhibiting biological activity of toxins, such as anthrax lethal toxin. Here, we report the production of glycosylated (pp-mAb (PA) ) and non-glycosylated (pp-mAb (PANG) ) versions of a plant-derived mAb directed against protective antigen (PA) of Bacillus anthracis in Nicotiana benthamiana plants using agroinfiltration. Both forms of the antibody were able to neutralize anthrax lethal toxin activity in vitro and protect mice against an intraperitoneal challenge with spores of B. anthracis Sterne strain. A single 180 µg intraperitoneal dose of pp-mAb (PA) or pp-mAb (PANG) provided 90% and 100% survival, respectively. When tested in non-human primates, pp-mAb (PANG) was demonstrated to be superior to pp-mAb (PA) in that it had a significantly longer terminal half-life and conferred 100% protection against a lethal dose of aerosolized anthrax spore challenge after a single 5 mg/kg intravenous dose compared to a 40% survival rate conferred by pp-mAb (PA) . This study demonstrates the potential of a plant-produced non-glycosylated antibody as a useful tool for the treatment of inhalation anthrax.

  1. Monoclonal Antibodies for Lipid Management.

    PubMed

    Feinstein, Matthew J; Lloyd-Jones, Donald M

    2016-07-01

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

  2. Uses of monoclonal antibody 8H9

    DOEpatents

    Cheung, Nai-Kong V

    2013-08-06

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

  3. Uses of monoclonal antibody 8H9

    DOEpatents

    Cheung, Nai-Kong V.

    2010-06-15

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

  4. Monoclonal antibodies as catalysts for cyanide removal

    SciTech Connect

    Cook, C.E.; Whisnant, C.C.; Miller, D.B.; Allen, D.A.; Basta, P.V.

    1993-05-13

    We have shown that hydrogen cyanide reacts with alpha, beta-unsaturated ketones to form stable compounds under physiological conditions (temperature, pH). Although spontaneous reaction is too slow for protection against cyanide intoxication, rate enhancement in the presence of a suitable catalyst would permit the use of alpha, beta-unsaturated ketones (enones) as prophylactics for cyanide exposure. Based on the accepted mechanism for this 1,4-addition reaction, we have designed and synthesized sized a transition state analog (TSA), conjugated it to protein and used the conjugate to produce more than 300 monoclonal antibodies which bind the TSA. Approximately 10% of these antibodies have been purified from ascites and tested for catalysis of the addition reaction of cyanide to enone. Product formation was measured by HPLC. Four antibodies have been found which significantly enhance the initial velocity of the reaction. The TSA markedly diminishes the reaction velocity, indicating the involvement of the antibody binding site in the observed enhancement. Preliminary kinetic analysis on one antibody gave values of K sub (enone) and K sub KCN 51 uM and 9.6 mM, respectively. The value of k sub (cat) was 2.33 hr-1. The data suggest a rate enhancement of 2 x 10 to the 4th power for the encounter of the enone with the antibody-cyanide complex, whereas the rate enhancement for encounter of cyanide with the antibody-enone complex is 70. To utilize the potential of genetic engineering for modifying the proper-lies of anti-TSA monoclonal antibodies, we are cloning heavy and light chain genes for sequencing and subsequent site-specific mutagenesis.

  5. Monoclonal antibodies in chronic lymphocytic leukemia.

    PubMed

    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.

  6. Improved monoclonal antibodies to halodeoxyuridine

    DOEpatents

    Vanderlaan, M.; Dolbeare, F.A.; Gray, J.W.; Thomas, C.B.

    1983-10-18

    The development, method of production, characterization and methods of use of two hybridomas, CIdU-1 (ATCC Accession No. HB-8321) and CIdU-2 (ATCC Accession No. HB-8320), are described. These secrete IgG/sub 1/(K) immunoglobulins that react with halodeoxyuridine (HdU or halodU) such as bromo, chloro, fluoro and iodo deoxyuridine (BrdU, CldU, FdU and IdU), whether these are free in solution or incorporated into single stranded DNA in whole cells. The antibodies do not react with naturally occurring free nucleic acids or with deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) polymers. These antibodies are suitable for use in enzyme immunoassays for free CldU, FdU, IdU and BrdU and for detecting cells with these nucleotides incorporated into them. The monoclonal antibodies are useful in the detection of the sensitivity of tumor cells to specific chemotherapeutic agents, in the measurement of the rate of cellular DNA synthesis, in the measurement of the rate of proliferation of normal and malignant cells and in the detection of HPRT deficiency in cells. 1 tab.

  7. Guardians at the gate: patent protection of therapeutic monoclonal antibodies through product life cycle management--Part 3.

    PubMed

    McCabe, Kevin W; Calvo, Paul A

    2009-01-01

    Product life cycle management, which necessarily utilizes a multi-disciplinary approach, is an essential tool for companies that develop or market therapeutic monoclonal antibodies (mAbs). Too little attention to such a plan, or use of the wrong resources, could substantially curtail a product's life span. The most difficult part of the therapeutic antibody business is the development of high-quality, safe and effective products. Great care should thus be taken to ensure that products with these characteristics are positioned in a marketplace that is competition-free for as long as possible. In an era of mAbs with billion dollar markets, the loss of even a single day of sales could cost companies millions of dollars in lost revenue.

  8. A potent broad-spectrum protective human monoclonal antibody crosslinking two haemagglutinin monomers of influenza A virus

    PubMed Central

    Wu, Ying; Cho, MyungSam; Shore, David; Song, Manki; Choi, JungAh; Jiang, Tao; Deng, Yong-Qiang; Bourgeois, Melissa; Almli, Lynn; Yang, Hua; Chen, Li-Mei; Shi, Yi; Qi, Jianxu; Li, An; Yi, Kye Sook; Chang, MinSeok; Bae, Jin Soo; Lee, HyunJoo; Shin, JiYoung; Stevens, James; Hong, SeoungSuh; Qin, Cheng-Feng; Gao, George F.; Chang, Shin Jae; Donis, Ruben O.

    2015-01-01

    Effective annual influenza vaccination requires frequent changes in vaccine composition due to both antigenic shift for different subtype hemagglutinins (HAs) and antigenic drift in a particular HA. Here we present a broadly neutralizing human monoclonal antibody with an unusual binding modality. The antibody, designated CT149, was isolated from convalescent patients infected with pandemic H1N1 in 2009. CT149 is found to neutralize all tested group 2 and some group 1 influenza A viruses by inhibiting low pH-induced, HA-mediated membrane fusion. It promotes killing of infected cells by Fc-mediated antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. X-ray crystallographic data reveal that CT149 binds primarily to the fusion domain in HA2, and the light chain is also largely involved in binding. The epitope recognized by this antibody comprises amino-acid residues from two adjacent protomers of HA. This binding characteristic of CT149 will provide more information to support the design of more potent influenza vaccines. PMID:26196962

  9. Tumor detection using radiolabeled monoclonal antibodies

    SciTech Connect

    Moldofsky, P.J.; Powe, J.; Hammond, N.D.

    1987-01-01

    Radioisotope conjugated to monoclonal antibody products has been used for imaging tumors targeted by the antibody. As imaging progresses, new sets of procedural and technical questions arise. In this chapter, we discuss several current problems in imaging tumor with radiolabeled monoclonal antibody. These include (1) methods for selection of specific antibody and, once the particular antibody is selected, which fragment form is to be used; (2) imaging procedures: what are the optimum imaging parameters, such as optimum time for imaging after administration of tracer and considerations regarding background subtraction; and (3) noninvasive quantitative techniques: quantitation of localization of antibody indirectly from quantitative information in the images.100 references.

  10. Adverse cardiac events to monoclonal antibodies used for cancer therapy

    PubMed Central

    Kounis, Nicholas G; Soufras, George D; Tsigkas, Grigorios; Hahalis, George

    2014-01-01

    Monoclonal antibodies are currently used in the treatment of neoplastic, hematological, or inflammatory diseases, a practice that is occasionally associated with a variety of systemic and cutaneous adverse events. Cardiac adverse events include cardiomyopathy, ventricular dysfunction, arrhythmias, arrests, and acute coronary syndromes, such as acute myocardial infarction and vasospastic angina pectoris. These events generally follow hypersensitivity reactions including cutaneous erythema, pruritus chills, and precordial pain. Recently, IgE specific for therapeutic monoclonal antibodies have been detected, pointing to the existence of hypersensitivity and Kounis hypersensitivity-associated syndrome. Therefore, the careful monitoring of cardiovascular events is of paramount importance in the course of monoclonal antibody-based therapies. Moreover, further studies are needed to elucidate the pathophysiology of cardiovascular adverse events elicited by monoclonal antibodies and to identify preventive, protective, and therapeutic measures. PMID:25340003

  11. A histoplasma capsulatum-specific IgG1 isotype monoclonal antibody, H1C, to a 70-kilodalton cell surface protein is not protective in murine histoplasmosis.

    PubMed

    Lopes, Livia Cristina Liporagi; Guimarães, Allan J; de Cerqueira, Mariana Duarte; Gómez, Beatriz L; Nosanchuk, Joshua D

    2010-07-01

    Monoclonal antibodies to Histoplasma capsulatum can modify pathogenesis. We now show that monoclonal antibody H1C to a 70-kDa antigen increases intracellular fungal growth and reduces macrophage nitric oxide release but has no effect on fungal burden or survival in murine infection. This further demonstrates the complexities of host-pathogen interactions.

  12. Serum-neutralizing, non-precipitating, partially protective monoclonal IgA antibody against VSV-NJ

    SciTech Connect

    Englen, M.D.; Ristow, S.; Yilma, T.

    1986-03-05

    A BALB/c mouse hybridoma that secretes an isotype IgA kappa-antibody specific for the New Jersey serotype of vesicular stomatitis virus (VSV-NJ) has been cloned. This antibody is serum-neutralizing yet fails to immunoprecipitate /sup 35/S-labeled G glycoprotein from virus-infected cell lysates. To determine if the antibody could protect mice from a lethal challenge dose of VSV-NJ, passive transfer experiments were performed with ascites fluid containing the IgA antibody. Mice given antibody IP or IV 24 hr before challenge all succumbed to progressive encephalitis, as did control mice. The only mice to survive were those given a virus preparation that had been preincubated with the ascites fluid for 1 hr at 37/sup 0/C. To characterize this antibody physically, Coomassie-stained polyacrylamide gels were prepared in which samples of affinity-purified antibody were prepared under reducing and non-reducing conditions. The results indicate that the molecule is composed of a disulfide bonded light-chain dimer and a disulfide bonded heavy-chain dimer.

  13. Monoclonal antibodies in the treatment of cancer

    SciTech Connect

    Dillman, R.O.

    1984-01-01

    Potential uses of monoclonal antibodies in anti-cancer treatment include passive serotherapy, radioisotope conjugates, toxin-linked conjugates, and chemotherapy-monoclonal antibody conjugates. The bases for these applications have been founded in research with heterologous antisera, and in some cases with monoclonal antibodies in animal tumor models. Human trials with passive serotherapy have already begun in both hematopoietic and solid tumor malignancies. Promising results have been reported in cutaneous T cell lymphoma with anti-T cell monoclonal antibody, and in nodular lymphoma with anti-idiotype monoclonal antibody. Radioisotope conjugate work appears promising for imaging in both animals and humans, and this work will lay the foundation for possible therapeutic application of radio-immunotherapy. Toxin-linked conjugates are promising in vitro and may have application in autologous bone marrow transplantation. Research with chemotherapy conjugates is also underway. Preliminary results suggest that murine monoclonal antibodies will be well tolerated clinically except in the setting of circulating cells which bear the target antigen, where rapid infusions may be associated with intolerable side effects. In certain diseases, production of endogenous anti-mouse antibodies may also limit application. Advances in the technology for human-human hybridoma production may help solve some of these problems. 132 references.

  14. Monoclonal Antibodies for Multiple Sclerosis Treatment.

    PubMed

    Palavra, Filipe

    2015-01-01

    Since their introduction in medical therapy, in the last quarter of the 20th century, monoclonal antibodies have gained an increasing importance in the treatment of various diseases. Neurology has been one of the medical specialties benefiting of the therapeutic potential of these monoclonal antibodies and certain neurological conditions may now contain such drugs in their therapeutic algorithms. Multiple sclerosis is one of these diseases and, in addition to the monoclonal antibodies already licensed for clinical use, several others are in development for future utilization in this specific area. The future will certainly pass through this kind of drugs and, in this article, a review of the most relevant data related to monoclonal antibodies already in use and also in clinical development for multiple sclerosis treatment will be performed.

  15. Preparation of astatine-labeled monoclonal antibodies

    SciTech Connect

    Milesz, S.; Norseev, Yu.V.; Szucs, Z. |

    1995-07-01

    In the cationic state astatine forms a stable complex with diethylenetriaminepentaacetic acid. Thanks to this complex, astatine can be bound to monoclonal antibodies of the RYa{sub 1} type. The most favorable conditions for preparing astatine-labeled antibodies are established. The chromatographic analysis and electromigration experiments showed that astatine is firmly linked to a biomolecule in vitro and it did not escape from labeled monoclonal antibodies even under treatment with such highly effective astatine-complexing agent as thiourea. The immune activity of astatine-labeled antibodies did not change even after 20 h.

  16. Monoclonal Antibody That Defines Human Myoepithelium

    NASA Astrophysics Data System (ADS)

    Dairkee, Shahnaz Hashmi; Blayney, Carlene; Smith, Helene S.; Hackett, Adeline J.

    1985-11-01

    We have isolated a mouse monoclonal antibody that, upon immunohistochemical localization in frozen sections, displays specificity for human myoepithelial cells in the resting mammary gland, sweat glands, and salivary glands. Furthermore, this antibody was strongly and homogeneously reactive with frozen sections of 3 of 60 breast carcinoma specimens. Using immunolocalization techniques in conjunction with polyacrylamide gel electrophoresis, we have determined that the reactivity of this monoclonal antibody is directed toward a 51,000-dalton keratin polypeptide. The potential uses of this antibody in the prognosis of human mammary carcinoma and in understanding the role of the myoepithelium in development and differentiation are discussed.

  17. Monoclonal Antibodies against the Drosophila Nervous System

    NASA Astrophysics Data System (ADS)

    Fujita, Shinobu C.; Zipursky, Stephen L.; Benzer, Seymour; Ferrus, Alberto; Shotwell, Sandra L.

    1982-12-01

    A panel of 148 monoclonal antibodies directed against Drosophila neural antigens has been prepared by using mice immunized with homogenates of Drosophila tissue. Antibodies were screened immunohistochemically on cryostat sections of fly heads. A large diversity of staining patterns was observed. Some antigens were broadly distributed among tissues; others were highly specific to nerve fibers, neuropil, muscle, the tracheal system, cell nuclei, photoreceptors, or other structures. The antigens for many of the antibodies have been identified on immunoblots. Monoclonal antibodies that identify specific molecules within the nervous system should prove useful in the study of the molecular genetics of neural development.

  18. From rabbit antibody repertoires to rabbit monoclonal antibodies

    PubMed Central

    Weber, Justus; Peng, Haiyong; Rader, Christoph

    2017-01-01

    In this review, we explain why and how rabbit monoclonal antibodies have become outstanding reagents for laboratory research and increasingly for diagnostic and therapeutic applications. Starting with the unique ontogeny of rabbit B cells that affords highly distinctive antibody repertoires rich in in vivo pruned binders of high diversity, affinity and specificity, we describe the generation of rabbit monoclonal antibodies by hybridoma technology, phage display and alternative methods, along with an account of successful humanization strategies. PMID:28336958

  19. Trends in Malignant Glioma Monoclonal Antibody Therapy

    PubMed Central

    Chekhonin, Ivan; Gurina, Olga

    2015-01-01

    Although new passive and active immunotherapy methods are emerging, unconjugated monoclonal antibodies remain the only kind of biological preparations approved for high-grade glioma therapy in clinical practice. In this review, we combine clinical and experimental data discussion. As antiangiogenic therapy is the standard of care for recurrent glioblastoma multiforme (GBM), we analyze major clinical trials and possible therapeutic combinations of bevacizumab, the most common monoclonal antibody to vascular endothelial growth factor (VEGF). Another humanized antibody to gain recognition in GBM is epidermal growth factor (EGFR) antagonist nimotuzumab. Other antigens (VEGF receptor, platelet-derived growth factor receptor, hepatocyte growth factor and c-Met system) showed significance in gliomas and were used to create monoclonal antibodies applied in different malignant tumors. We assess the role of genetic markers (isocitrate dehydrogenase, O6-methylguanine-DNA methyltransnsferase) in GBM treatment outcome prediction. Besides antibodies studied in clinical trials, we focus on perspective targets and briefly list other means of passive immunotherapy.

  20. Passive Transfer of A Germline-like Neutralizing Human Monoclonal Antibody Protects Transgenic Mice Against Lethal Middle East Respiratory Syndrome Coronavirus Infection

    PubMed Central

    Agrawal, Anurodh Shankar; Ying, Tianlei; Tao, Xinrong; Garron, Tania; Algaissi, Abdullah; Wang, Yanping; Wang, Lili; Peng, Bi-Hung; Jiang, Shibo; Dimitrov, Dimiter S.; Tseng, Chien-Te K.

    2016-01-01

    Middle East Respiratory Syndrome coronavirus (MERS-CoV) has repeatedly caused outbreaks in the Arabian Peninsula. To date, no approved medical countermeasures (MCM) are available to combat MERS-CoV infections. Several neutralizing human monoclonal antibodies (mAbs), including m336, a germline-like human mAb, have been chosen as promising MCM for MERS-CoV. However, their clinical development has been hindered by the lack of a robust animal model that recapitulate the morbidity and mortality of human infections. We assessed the prophylactic and therapeutic efficacy of m336 by using well-characterized transgenic mice shown to be highly sensitive to MERS-CoV infection and disease. We found that mice treated with m336 prior to or post lethal MERS-CoV challenging were fully protected, compared to control mice which sufferered from profound weight loss and uniform death within days after infection. Taken together, these results support further development of m336 and other human monoclonal antibodies as potential therapeutics for MERS-CoV infection. PMID:27538452

  1. Protection against Invasive Amebiasis by a Single Monoclonal Antibody Directed against a Lipophosphoglycan Antigen Localized on the Surface of Entamoeba histolytica

    PubMed Central

    Marinets, Alexandra; Zhang, Tonghai; Guillén, Nancy; Gounon, Pierre; Bohle, Barbara; Vollmann, Ute; Scheiner, Otto; Wiedermann, Gerhard; Stanley, Samuel L.; Duchêne, Michael

    1997-01-01

    A panel of monoclonal antibodies was raised from mice immunized with a membrane preparation from Entamoeba histolytica, the pathogenic species causing invasive amebiasis in humans. Antibody EH5 gave a polydisperse band in immunoblots from membrane preparations from different E. histolytica strains, and a much weaker signal from two strains of the nonpathogenic species Entamoeba dispar. Although the exact chemical structure of the EH5 antigen is not yet known, the ability of the antigen to be metabolically radiolabeled with [32P]phosphate or [3H]glucose, its sensitivity to digestion by mild acid and phosphatidylinositol-specific phospholipase C, and its specific extraction from E. histolytica trophozoites by a method used to prepare lipophosphoglycans from Leishmania showed that it could be classified as an amebal lipophosphoglycan. Confocal immunofluorescence and immunogold labeling of trophozoites localized the antigen on the outer face of the plasma membrane and on the inner face of internal vesicle membranes. Antibody EH5 strongly agglutinated amebas in a similar way to concanavalin A (Con A), and Con A bound to immunoaffinity-purified EH5 antigen. Therefore, surface lipophosphoglycans may play an important role in the preferential agglutination of pathogenic amebas by Con A. The protective ability of antibody EH5 was tested in a passive immunization experiment in a severe combined immunodeficient (SCID) mouse model. Intrahepatic challenge of animals after administration of an isotype-matched control antibody or without treatment led to the development of a liver abscess in all cases, whereas 11 out of 12 animals immunized with the EH5 antibody developed no liver abscess. Our results demonstrate the importance and, for the first time, the protective capacity of glycan antigens on the surface of the amebas. PMID:9348313

  2. Monoclonal antibodies to leukotoxin of Actinobacillus actinomycetemcomitans.

    PubMed Central

    DiRienzo, J M; Tsai, C C; Shenker, B J; Taichman, N S; Lally, E T

    1985-01-01

    Hybridoma cell lines which produce monoclonal antibodies to a leukotoxin from Actinobacillus actinomycetemcomitans were prepared. The monoclonal antibodies were selected for their ability to neutralize the cytotoxic activity of the leukotoxin and recognize the toxin on nitrocellulose blots. The antibodies belonged to either the immunoglobulin G1 (IgG1) or IgG2 subclass and differed in their ability to bind to the leukotoxin on nitrocellulose blots. However, only slight differences in neutralization titers were observed. Use of the monoclonal antibodies revealed that polymyxin B-extracted or osmotic shock-released leukotoxin could be separated into several high-molecular-weight polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblot analysis with the monoclonal antibodies also demonstrated that the leukotoxin was present in eight oral strains of A. actinomycetemcomitans that had been previously classified by a biological assay as leukotoxic. The availability of these monoclonal antibodies should facilitate and expand studies concerning the role of the leukotoxin in the pathogenicity of A. actinomycetemcomitans. Images PMID:3965404

  3. Production of monoclonal antibodies to plant pathogens.

    PubMed

    Thornton, Christopher R

    2009-01-01

    The use of monoclonal antibodies in plant pathology has improved the quality and specificity of detection methods for diseases. Hybridoma technology allows the limitless production of highly specific antibodies which can be used to identify pathogens to the species or even sub-species level.

  4. Monoclonal Antibody Therapy for Advanced Neuroblastoma

    Cancer.gov

    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.

  5. Prevention of diabetes in nonobese diabetic mice by anti-I-A monoclonal antibodies: transfer of protection by splenic T cells.

    PubMed Central

    Boitard, C; Bendelac, A; Richard, M F; Carnaud, C; Bach, J F

    1988-01-01

    The nonobese diabetic (NOD) mouse has been developed as a model for insulin-dependent diabetes. One gene required for the development of diabetes is associated with the major histocompatibility complex. This gene possibly could be linked to class II genes, which show a unique pattern in NOD mice. To evaluate the role of the I-A class II antigen expressed in NOD mice, we studied the effect of anti-I-A monoclonal antibodies on disease onset in vivo. Long-term treatment with anti-class II IgG2a antibodies specific for NOD I-A antigen prevented the spontaneous development of diabetes, as opposed to control antibodies shown not to react with NOD I-A antigen. Anti-class II antibodies apparently elicited active immune suppression, requiring a fully immunocompetent host, rather than passive blockade of class II antigen. Treatment with anti-class II antibody effectively prevented the adoptive transfer of diabetes produced by splenocytes from diabetic NOD mice into newborn mice but failed to prevent adoptive transfer into irradiated adult NOD recipients. Direct evidence for the induction of suppressor cells was obtained from the passive transfer of spleen cells from anti-class II antibody-treated NOD donors. The injection of anti-class II antibody-treated spleen cells collected from NOD donors prevented the development of diabetes, which normally follows transfer of diabetogenic spleen cells into irradiated 8-week-old male NOD recipients. Depletion experiments indicate that CD4+ cells are responsible for anti-class II-induced protection transferred by spleen cells. PMID:3264405

  6. A humanised murine monoclonal antibody protects mice from Venezuelan equine encephalitis virus, Everglades virus and Mucambo virus when administered up to 48 h after airborne challenge

    SciTech Connect

    O'Brien, Lyn M. Goodchild, Sarah A.; Phillpotts, Robert J.; Perkins, Stuart D.

    2012-05-10

    Currently there are no licensed antiviral treatments for the Alphaviruses Venezuelan equine encephalitis virus (VEEV), Everglades virus and Mucambo virus. We previously developed a humanised version of the mouse monoclonal antibody 1A3B-7 (Hu1A3B-7) which exhibited a wide range of reactivity in vitro and was able to protect mice from infection with VEEV. Continued work with the humanised antibody has now demonstrated that it has the potential to be a new human therapeutic. Hu1A3B-7 successfully protected mice from infection with multiple Alphaviruses. The effectiveness of the humanisation process was determined by assessing proliferation responses in human T-cells to peptides derived from the murine and humanised versions of the V{sub H} and V{sub L} domains. This analysis showed that the number of human T-cell epitopes within the humanised antibody had been substantially reduced, indicating that Hu1A3B-7 may have reduced immunogenicity in vivo.

  7. A humanised murine monoclonal antibody protects mice from Venezuelan equine encephalitis virus, Everglades virus and Mucambo virus when administered up to 48 h after airborne challenge.

    PubMed

    O'Brien, Lyn M; Goodchild, Sarah A; Phillpotts, Robert J; Perkins, Stuart D

    2012-05-10

    Currently there are no licensed antiviral treatments for the Alphaviruses Venezuelan equine encephalitis virus (VEEV), Everglades virus and Mucambo virus. We previously developed a humanised version of the mouse monoclonal antibody 1A3B-7 (Hu1A3B-7) which exhibited a wide range of reactivity in vitro and was able to protect mice from infection with VEEV. Continued work with the humanised antibody has now demonstrated that it has the potential to be a new human therapeutic. Hu1A3B-7 successfully protected mice from infection with multiple Alphaviruses. The effectiveness of the humanisation process was determined by assessing proliferation responses in human T-cells to peptides derived from the murine and humanised versions of the V(H) and V(L) domains. This analysis showed that the number of human T-cell epitopes within the humanised antibody had been substantially reduced, indicating that Hu1A3B-7 may have reduced immunogenicity in vivo. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

  8. Fixed Dosing of Monoclonal Antibodies in Oncology.

    PubMed

    Hendrikx, Jeroen J M A; Haanen, John B A G; Voest, Emile E; Schellens, Jan H M; Huitema, Alwin D R; Beijnen, Jos H

    2017-07-28

    Most monoclonal antibodies in oncology are administered in body-size-based dosing schedules. This is believed to correct for variability in both drug distribution and elimination between patients. However, monoclonal antibodies typically distribute to the blood plasma and extracellular fluids only, which increase less than proportionally with the increase in body weight. Elimination takes place via proteolytic catabolism, a nonspecific immunoglobulin G elimination pathway, and intracellular degradation after binding to the target. The latter is the primary route of elimination and is related to target expression levels rather than body size. Taken together, the minor effects of body size on distribution and elimination of monoclonal antibodies and their usually wide therapeutic window do not support body-size-based dosing. We evaluated effects of body weight on volume of distribution and clearance of monoclonal antibodies in oncology and show that a fixed dose for most of these drugs is justified based on pharmacokinetics. A survey of the savings after fixed dosing of monoclonal antibodies at our hospital showed that fixed dosing can reduce costs of health care, especially when pooling of preparations is not possible (which is often the case in smaller hospitals). In conclusion, based on pharmacokinetic parameters of monoclonal antibodies, there is a rationale for fixed dosing of these drugs in oncology. Therefore, we believe that fixed dosing is justified and can improve efficiency of the compounding. Moreover, drug spillage can be reduced and medication errors may become less likely. The currently available knowledge of elimination of monoclonal antibodies combined with the publicly available data from clinical trials and extensive population pharmacokinetic (PopPK) modeling justifies fixed dosing. Interpatient variation in exposure is comparable after body weight and fixed dosing and most monoclonal antibodies show relatively flat dose-response relationships

  9. Clinical pharmacokinetics of therapeutic monoclonal antibodies.

    PubMed

    Keizer, Ron J; Huitema, Alwin D R; Schellens, Jan H M; Beijnen, Jos H

    2010-08-01

    Monoclonal antibodies (mAbs) have been used in the treatment of various diseases for over 20 years and combine high specificity with generally low toxicity. Their pharmacokinetic properties differ markedly from those of non-antibody-type drugs, and these properties can have important clinical implications. mAbs are administered intravenously, intramuscularly or subcutaneously. Oral administration is precluded by the molecular size, hydrophilicity and gastric degradation of mAbs. Distribution into tissue is slow because of the molecular size of mAbs, and volumes of distribution are generally low. mAbs are metabolized to peptides and amino acids in several tissues, by circulating phagocytic cells or by their target antigen-containing cells. Antibodies and endogenous immunoglobulins are protected from degradation by binding to protective receptors (the neonatal Fc-receptor [FcRn]), which explains their long elimination half-lives (up to 4 weeks). Population pharmacokinetic analyses have been applied in assessing covariates in the disposition of mAbs. Both linear and nonlinear elimination have been reported for mAbs, which is probably caused by target-mediated disposition. Possible factors influencing elimination of mAbs include the amount of the target antigen, immune reactions to the antibody and patient demographics. Bodyweight and/or body surface area are generally related to clearance of mAbs, but clinical relevance is often low. Metabolic drug-drug interactions are rare for mAbs. Exposure-response relationships have been described for some mAbs. In conclusion, the parenteral administration, slow tissue distribution and long elimination half-life are the most pronounced clinical pharmacokinetic characteristics of mAbs.

  10. Monoclonal antibodies as diagnostics; an appraisal.

    PubMed

    Siddiqui, M Z

    2010-01-01

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

  11. Therapeutic monoclonal antibody for sporotrichosis

    PubMed Central

    Almeida, Sandro R.

    2012-01-01

    Sporotrichosis is a chronic subcutaneous mycosis that affects both humans and animals worldwide. This subcutaneous mycosis had been attributed to a single etiological agent, Sporothrix schenckii. S. schenckii exhibits considerable genetic variability, and recently, it was suggested 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 cutaneous forms, but recently, more severe clinical forms of this mycosis have been described, especially among immunocompromised individuals. The immunological mechanisms involved in the prevention and control of sporotrichosis are not well understood. Some studies 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 has not been studied in detail. In a previous study, we showed that antigens secreted by S. schenckii induced a specific humoral response in infected animals, primarily against a 70-kDa molecule, indicating a possible role of specific antibodies against this molecule in infection control. In another study by our group, we produced a mAb against a 70-kDa glycoprotein of S. schenckii to better understand the effect of the passive immunization of mice infected with S. schenckii. The results showed a significant reduction in the number of CFUs in various mice organs when the mAb was injected before or during S. schenckii infection. Similar results were observed when T-cell-deficient mice were used. The drugs of choice in the treatment of sporotrichosis require long periods, and relapses are frequently observed, primarily in immunocompromised patients. The strong protection induced by the mAb against a 70-kDa glycoprotein makes it a strong candidate as a therapeutic vaccine against sporotrichosis. PMID

  12. Production of Monoclonal Antibody against Human Nestin.

    PubMed

    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.

  13. Production of Monoclonal Antibody against Human Nestin

    PubMed Central

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

    2010-01-01

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

  14. A Non-VH1-69 Heterosubtypic Neutralizing Human Monoclonal Antibody Protects Mice against H1N1 and H5N1 Viruses

    PubMed Central

    Solforosi, Laura; Moreno, Guisella J.; Sun, Xiangjie; Tumpey, Terrence M.; Gubareva, Larisa V.; Mishin, Vasiliy; Clementi, Massimo; Burioni, Roberto

    2012-01-01

    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. PMID:22496802

  15. Pre-exposure Prophylaxis With OspA-Specific Human Monoclonal Antibodies Protects Mice Against Tick Transmission of Lyme Disease Spirochetes.

    PubMed

    Wang, Yang; Kern, Aurélie; Boatright, Naomi K; Schiller, Zachary A; Sadowski, Andrew; Ejemel, Monir; Souders, Colby A; Reimann, Keith A; Hu, Linden; Thomas, William D; Klempner, Mark S

    2016-07-15

    Tick transmission of Borrelia spirochetes to humans results in significant morbidity from Lyme disease worldwide. Serum concentrations of antibodies against outer surface protein A (OspA) were shown to correlate with protection from infection with Borrelia burgdorferi, the primary cause of Lyme disease in the United States. Mice transgenic for human immunoglobulin genes were immunized with OspA from B. burgdorferi to generate human monoclonal antibodies (HuMabs) against OspA. HuMabs were generated and tested in in vitro borreliacidal assays and animal protection assays. Nearly 100 unique OspA-specific HuMabs were generated, and 4 HuMabs (221-7, 857-2, 319-44, and 212-55) were selected as lead candidates on the basis of borreliacidal activity. HuMabs 319-44, 857-2, and 212-55 were borreliacidal against 1 or 2 Borrelia genospecies, whereas 221-7 was borreliacidal (half maximal inhibitory concentration, < 1 nM) against B. burgdorferi, Borrelia afzelii, and Borrelia garinii, the 3 main genospecies endemic in the United States, Europe, and Asia. All 4 HuMabs completely protected mice from infection at 10 mg/kg in a murine model of tick-mediated transmission of B. burgdorferi  Our study indicates that OspA-specific HuMabs can prevent the transmission of Borrelia and that administration of these antibodies could be employed as preexposure prophylaxis for Lyme disease. © 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.

  16. Structures of synthetic O-antigen fragments from serotype 2a Shigella flexneri in complex with a protective monoclonal antibody

    PubMed Central

    Vulliez-Le Normand, B.; Saul, F. A.; Phalipon, A.; Bélot, F.; Guerreiro, C.; Mulard, L. A.; Bentley, G. A.

    2008-01-01

    The anti-LPS IgG mAb F22-4, raised against Shigella flexneri serotype 2a bacteria, protects against homologous, but not heterologous, challenge in an experimental animal model. We report the crystal structures of complexes formed between Fab F22-4 and two synthetic oligosaccharides, a decasaccharide and a pentadecasaccharide that were previously shown to be both immunogenic and antigenic mimics of the S. flexneri serotype 2a O-antigen. F22-4 binds to an epitope contained within two consecutive 2a serotype pentasaccharide repeat units (RU). Six sugar residues from a contiguous nine-residue segment make direct contacts with the antibody, including the nonreducing rhamnose and both branching glucosyl residues from the two RUs. The glucosyl residue, whose position of attachment to the tetrasaccharide backbone of the RU defines the serotype 2a O-antigen, is critical for recognition by F22-4. Although the complete decasaccharide is visible in the electron density maps, the last four pentadecasaccharide residues from the reducing end, which do not contact the antibody, could not be traced. Although considerable mobility in the free oligosaccharides can thus be expected, the conformational similarity between the individual RUs, both within and between the two complexes, suggests that short-range transient ordering to a helical conformation might occur in solution. Although the observed epitope includes the terminal nonreducing residue, binding to internal epitopes within the polysaccharide chain is not precluded. Our results have implications for vaccine development because they suggest that a minimum of two RUs of synthetic serotype 2a oligosaccharide is required for optimal mimicry of O-Ag epitopes. PMID:18621718

  17. Monoclonal antibody technologies and rapid detection assays

    USDA-ARS?s Scientific Manuscript database

    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...

  18. Discovery of a Prefusion Respiratory Syncytial Virus F-Specific Monoclonal Antibody That Provides Greater In Vivo Protection than the Murine Precursor of Palivizumab.

    PubMed

    Zhao, Min; Zheng, Zi-Zheng; Chen, Man; Modjarrad, Kayvon; Zhang, Wei; Zhan, Lu-Ting; Cao, Jian-Li; Sun, Yong-Peng; McLellan, Jason S; Graham, Barney S; Xia, Ning-Shao

    2017-08-01

    Palivizumab, a humanized murine monoclonal antibody that recognizes antigenic site II on both the prefusion (pre-F) and postfusion (post-F) conformations of the respiratory syncytial virus (RSV) F glycoprotein, is the only prophylactic agent approved for use for the treatment of RSV infection. However, its relatively low neutralizing potency and high cost have limited its use to a restricted population of infants at high risk of severe disease. Previously, we isolated a high-potency neutralizing antibody, 5C4, that specifically recognizes antigenic site Ø at the apex of the pre-F protein trimer. We compared in vitro and in vivo the potency and protective efficacy of 5C4 and the murine precursor of palivizumab, antibody 1129. Both antibodies were synthesized on identical murine backbones as either an IgG1 or IgG2a subclass and evaluated for binding to multiple F protein conformations, in vitro inhibition of RSV infection and propagation, and protective efficacy in mice. Although 1129 and 5C4 had similar pre-F protein binding affinities, the 5C4 neutralizing activity was nearly 50-fold greater than that of 1129 in vitro In BALB/c mice, 5C4 reduced the peak titers of RSV 1,000-fold more than 1129 did in both the upper and lower respiratory tracts. These data indicate that antibodies specific for antigenic site Ø are more efficacious at preventing RSV infection than antibodies specific for antigenic site II. Our data also suggest that site Ø-specific antibodies may be useful for the prevention or treatment of RSV infection and support the use of the pre-F protein as a vaccine antigen.IMPORTANCE There is no vaccine yet available to prevent RSV infection. The use of the licensed antibody palivizumab, which recognizes site II on both the pre-F and post-F proteins, is restricted to prophylaxis in neonates at high risk of severe RSV disease. Recommendations for using passive immunization in the general population or for therapy in immunocompromised persons with

  19. Anti-CD25 monoclonal antibody enhances the protective efficacy of Schistosoma japonicum GST vaccine via inhibition of CD4(+)CD25(+)Foxp3(+) regulatory T cells.

    PubMed

    Tang, Chun-Lian; Yang, Jin; Cheng, Liang-Yu; Cheng, Lan-Fang; Liu, Zhi-Ming

    2017-08-21

    The effect of anti-CD25 monoclonal antibody (anti-CD25 mAb) on the protection efficacy of Schistosoma japonicum 26 kDa GST (glutathione-S-transferase) vaccine was evaluated. Mice were immunized with GST before infection with S. japonicum cercariae and then injected with anti-CD25 mAb. The worm reduction rate was promoted from 24.18% in mice with GST immunization to 47.09% in mice with GST plus anti-CD25 mAb. Compared with the control group, the percentages of splenic CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) were significantly lower after administration of anti-CD25 mAb; meanwhile, elevated levels of IFN-γ and IL-2 were secreted by splenocytes. These results indicate that the poor protective efficacy of the GST vaccine against S. japonicum results from the presence of CD4(+)CD25(+)Foxp3(+) Tregs, while anti-CD25 mAb can partially block CD4(+)CD25(+)Foxp3(+) Tregs and thus enhance the protective efficacy of the GST vaccine.

  20. Model for In Vivo Assessment of Humoral Protection against Malaria Sporozoite Challenge by Passive Transfer of Monoclonal Antibodies and Immune Serum

    PubMed Central

    Sack, Brandon K.; Miller, Jessica L.; Vaughan, Ashley M.; Douglass, Alyse; Kaushansky, Alexis; Mikolajczak, Sebastian; Coppi, Alida; Gonzalez-Aseguinolaza, Gloria; Tsuji, Moriya; Zavala, Fidel; Sinnis, Photini

    2014-01-01

    Evidence from clinical trials of malaria vaccine candidates suggests that both cell-mediated and humoral immunity to pre-erythrocytic parasite stages can provide protection against infection. Novel pre-erythrocytic antibody (Ab) targets could be key to improving vaccine formulations, which are currently based on targeting antigens such as the circumsporozoite protein (CSP). However, methods to assess the effects of sporozoite-specific Abs on pre-erythrocytic infection in vivo remain underdeveloped. Here, we combined passive transfer of monoclonal Abs (MAbs) or immune serum with a luciferase-expressing Plasmodium yoelii sporozoite challenge to assess Ab-mediated inhibition of liver infection in mice. Passive transfer of a P. yoelii CSP MAb showed inhibition of liver infection when mice were challenged with sporozoites either intravenously or by infectious mosquito bite. However, inhibition was most potent for the mosquito bite challenge, leading to a more significant reduction of liver-stage burden and even a lack of progression to blood-stage parasitemia. This suggests that Abs provide effective protection against a natural infection. Inhibition of liver infection was also achieved by passive transfer of immune serum from whole-parasite-immunized mice. Furthermore, we demonstrated that passive transfer of a MAb against P. falciparum CSP inhibited liver-stage infection in a humanized mouse/P. falciparum challenge model. Together, these models constitute unique and sensitive in vivo methods to assess serum-transferable protection against Plasmodium sporozoite challenge. PMID:24478094

  1. Characterization of chimpanzee/human monoclonal antibodies to vaccinia virus A33 glycoprotein and its variola virus homolog in vitro and in a vaccinia virus mouse protection model.

    PubMed

    Chen, Zhaochun; Earl, Patricia; Americo, Jeffrey; Damon, Inger; Smith, Scott K; Yu, Fujuan; Sebrell, Andrew; Emerson, Suzanne; Cohen, Gary; Eisenberg, Roselyn J; Gorshkova, Inna; Schuck, Peter; Satterfield, William; Moss, Bernard; Purcell, Robert

    2007-09-01

    Three distinct chimpanzee Fabs against the A33 envelope glycoprotein of vaccinia virus were isolated and converted into complete monoclonal antibodies (MAbs) with human gamma 1 heavy-chain constant regions. The three MAbs (6C, 12C, and 12F) displayed high binding affinities to A33 (K(d) of 0.14 nM to 20 nM) and may recognize the same epitope, which was determined to be conformational and located within amino acid residues 99 to 185 at the C terminus of A33. One or more of the MAbs were shown to reduce the spread of vaccinia virus as well as variola virus (the causative agent of smallpox) in vitro and to more effectively protect mice when administered before or 2 days after intranasal challenge with virulent vaccinia virus than a previously isolated mouse anti-A33 MAb (1G10) or vaccinia virus immunoglobulin. The protective efficacy afforded by anti-A33 MAb was comparable to that of a previously isolated chimpanzee/human anti-B5 MAb. The combination of anti-A33 MAb and anti-B5 MAb did not synergize the protective efficacy. These chimpanzee/human anti-A33 MAbs may be useful in the prevention and treatment of vaccinia virus-induced complications of vaccination against smallpox and may also be effective in the immunoprophylaxis and immunotherapy of smallpox and other orthopoxvirus diseases.

  2. Generation of a monoclonal antibody against Mycoplasma spp. following accidental contamination during production of a monoclonal antibody against Lawsonia intracellularis.

    PubMed

    Hwang, Jeong-Min; Lee, Ji-Hye; Yeh, Jung-Yong

    2012-03-01

    This report describes Mycoplasma contamination of Lawsonia intracellularis cultures that led to the unintended acquisition of a monoclonal antibody against Mycoplasma spp. during the attempted generation of a monoclonal antibody against L. intracellularis.

  3. Phase Separation in Solutions of Monoclonal Antibodies

    NASA Astrophysics Data System (ADS)

    Benedek, George; Wang, Ying; Lomakin, Aleksey; Latypov, Ramil

    2012-02-01

    We report the observation of liquid-liquid phase separation (LLPS) in a solution of humanized monoclonal antibodies, IgG2, and the effects of human serum albumin, a major blood protein, on this phase separation. We find a significant reduction of phase separation temperature in the presence of albumin, and a preferential partitioning of the albumin into the antibody-rich phase. We provide a general thermodynamic analysis of the antibody-albumin mixture phase diagram and relate its features to the magnitude of the effective inter-protein interactions. Our analysis suggests that additives (HSA in this report), which have moderate attraction with antibody molecules, may be used to forestall undesirable protein condensation in antibody solutions. Our findings are relevant to understanding the stability of pharmaceutical solutions of antibodies and the mechanisms of cryoglobulinemia.

  4. Chemoenzymatic glyco-engineering of monoclonal antibodies

    PubMed Central

    Giddens, John P.; Wang, Lai-Xi

    2016-01-01

    Summary Monoclonal antibodies (mAbs) are an important class of therapeutic glycoproteins widely used for the treatment of cancer, inflammation, and infectious diseases. Compelling data have shown that the presence and fine structures of the conserved N-glycans at the Fc domain can profoundly affect the effector functions of antibodies. However, mAbs are usually produced as mixtures of Fc glycoforms and the control of glycosylation to a favorable, homogeneous status in various host expression systems is still a challenging task. In this chapter, we describe a detailed procedure of chemoenzymatic glyco-engineering of monoclonal antibodies, using rituximab (a therapeutic monoclonal antibody) as a model system. The protocol includes the deglycosylation of a mAb by an endoglycosidase (such as wild type EndoS) to remove the heterogeneous Fc N-glycans, leaving only the innermost GlcNAc or the core-fucosylated GlcNAc at the glycosylation site. Then the deglycosylated IgG serves as an acceptor for an endoglycosidase-catalyzed transglycosylation to add a desired N-glycan to the GlcNAc acceptor to reconstitute a defined, homogeneous natural glycoform of IgG, using a glycosynthase mutant as the enzyme and activated glycan oxazoline as the donor substrate. A semi-synthesis of sialylated and asialylated biantennary N-glycan oxazolines is also described. This detailed procedure can be used for the Fc glycosylation remodeling of other mAbs to provide homogeneous Fc glycoforms for various applications. PMID:26082235

  5. [Preparation of monoclonal antibody against phosphinothricin acetyltransferase].

    PubMed

    Gao, Xudong; Wang, Yongzhi; Shi, Shengfeng; Li, Zhongpeng; Li, Xiaoyu; Xu, Wenjing; Zhang, Zhengkun; Lu, Yang; Zhang, Jiashi; Li, Qiyun; Wang, Jingang

    2013-05-01

    To express phosphinothricin acetyltransferase (PAT) with biological activity and prepare monoclonal antibodies against PAT. The full length bar gene was cloned by PCR and inserted into prokaryotic expression vector pET28a⁺. The recombinant plasmid pET28-bar was transformed into E.coli BL21(DE3), and under the induction of IPTG, PAT was expressed. The expressed protein was purified by Ni⁺; affinity chromatography to analyze its activity. The purified PAT was used to immunize BALB/c mice, and then the spleen cells from the immunized mice were fused with Sp2/0 cells. The hybridoma clones secreting antibodies against PAT were isolated by indirect ELISA and then subcloned. Soluble PAT was expressed in E.coli. The purified PAT had the activity of acetyltransferase. We totally prepared 9 hybridoma cell lines which secreted specific anti-PAT monoclonal antibodies. The expressed recombinant PAT can be used for biological reagent to prevent and relieve herbicide damage. Monoclonal antibodies against PAT may be used to detect the transgenic products.

  6. Monoclonal antibodies reacting with murine teratocarcinoma cells.

    PubMed Central

    Goodfellow, P N; Levinson, J R; Williams, V E; McDevitt, H O

    1979-01-01

    Monoclonal antibodies were produced in vitro by fusing mouse myeloma cells with spleen cells from a rat immunized with the C3H mouse teratocarcinoma C86-S1. After the fusion two clones were chosen for further analysis. The first clone, 3C4-10, produced an antibody recognizing an antigen with a distribution restricted to teratocarcinoma cell lines, an endoderm cell line, and a neuroblastoma. The second clone, 4A1-9, produced an antibody that reacted with all cultured murine cells tested and adult brain. Neither antibody reacted with preimplantation embryos. The 3C4-10 antibody recognized an antigen associated with proteins. The apparent molecular weight of the 3C4-10 antigen was greater than 100,000. PMID:284353

  7. Immunoglobulin VH determinants defined by monoclonal antibodies.

    PubMed

    Kubagawa, H; Mayumi, M; Kearney, J F; Cooper, M D

    1982-10-01

    Hybridoma clones secreting antibodies against common VH determinants were readily produced by fusion of cells from mice immunized with isolated V mu fragments of human immunoglobulins (Ig), but not with intact Ig molecules or isolated heavy chains. Four monoclonal antibodies to the V mu fragments of different IgM paraproteins were selected for analysis: MH-44 (mu kappa), GB-24 (mu kappa), NF-11 (gamma 1 kappa), and SA-44 (gamma 1 kappa). Each antibody reacted with the homologous V mu fragment, homologous mu chain, and normal gamma chains, but not with the intact IgM molecules, intact IgG, or isolated light chains, as determined by radioimmunoassay. The VH reaction spectra with a panel of myeloma heavy chains showed overlapping but distinctive patterns for the four antibodies. Each of the four monoclonal anti-VH antibodies appeared to react with a different "hidden" VH determinant that is not exposed on undenatured, intact Ig molecules and differs from conventional VH subgroup determinants. In immunofluorescence studies, the monoclonal anti-VH antibodies did not bind to surface Ig on viable B lymphocytes, but visibly stained subpopulations of fixed B lymphocytes, pre-B cells, and normal plasma cells. The mean frequencies of VH+ plasma cells were 30% (MH-44), 17% (GB-24), 13% (NF-11), and 3% (SA-44), and similar frequencies were obtained for the VH+ B cell subpopulations. While subpopulations of B cells could be identified at all stages in differentiation by immunofluorescence with the anti-VH antibodies, neither resting nor activated T cells expressed these VH determinants in detectable amounts.

  8. Immunoglobulin VH determinants defined by monoclonal antibodies

    PubMed Central

    1982-01-01

    Hybridoma clones secreting antibodies against common VH determinants were readily produced by fusion of cells from mice immunized with isolated V mu fragments of human immunoglobulins (Ig), but not with intact Ig molecules or isolated heavy chains. Four monoclonal antibodies to the V mu fragments of different IgM paraproteins were selected for analysis: MH-44 (mu kappa), GB-24 (mu kappa), NF-11 (gamma 1 kappa), and SA-44 (gamma 1 kappa). Each antibody reacted with the homologous V mu fragment, homologous mu chain, and normal gamma chains, but not with the intact IgM molecules, intact IgG, or isolated light chains, as determined by radioimmunoassay. The VH reaction spectra with a panel of myeloma heavy chains showed overlapping but distinctive patterns for the four antibodies. Each of the four monoclonal anti-VH antibodies appeared to react with a different "hidden" VH determinant that is not exposed on undenatured, intact Ig molecules and differs from conventional VH subgroup determinants. In immunofluorescence studies, the monoclonal anti-VH antibodies did not bind to surface Ig on viable B lymphocytes, but visibly stained subpopulations of fixed B lymphocytes, pre-B cells, and normal plasma cells. The mean frequencies of VH+ plasma cells were 30% (MH-44), 17% (GB-24), 13% (NF-11), and 3% (SA-44), and similar frequencies were obtained for the VH+ B cell subpopulations. While subpopulations of B cells could be identified at all stages in differentiation by immunofluorescence with the anti-VH antibodies, neither resting nor activated T cells expressed these VH determinants in detectable amounts. PMID:6185604

  9. Recent developments in monoclonal antibody radiolabeling techniques

    SciTech Connect

    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.

  10. Next generation and biosimilar monoclonal antibodies

    PubMed Central

    2011-01-01

    The Next Generation and Biosimilar Monoclonal Antibodies: Essential Considerations Towards Regulatory Acceptance in Europe workshop, organized by the European Centre of Regulatory Affairs Freiburg (EUCRAF), was held February 3–4, 2011 in Freiburg, Germany. The workshop attracted over 100 attendees from 15 countries, including regulators from 11 agencies, who interacted over the course of two days. The speakers presented their authoritative views on monoclonal antibodies (mAbs) as attractive targets for development, the experience to date with the regulatory process for biosimilar medicinal products, the European Medicines Agency draft guideline on biosimilar mAbs, as well as key elements in the development of mAbs. Participants engaged in many lively discussions, and much speculation on the nature of the quality, non-clinical and clinical requirements for authorization of biosimilar mAbs. PMID:21487235

  11. Innovative Monoclonal Antibody Therapies in Multiple Sclerosis

    PubMed Central

    Kieseier, Bernd C.

    2008-01-01

    The recent years have witnessed great efforts in establishing new therapeutic options for multiple sclerosis (MS), especially for relapsing–remitting disease courses. In particular, the application of monoclonal antibodies provide innovative approaches allowing for blocking or depleting specific molecular targets, which are of interest in the pathogenesis of MS. While natalizumab received approval by the US Food and Drug Administration and the European Medicines Agency in 2006 as the first monoclonal antibody in MS therapy, rituximab, alemtuzumab, and daclizumab were successfully tested for relapsing-remitting MS in small cohorts in the meantime. Here, we review the data available from these recent phase II trials and at the same time critically discuss possible pitfalls which may be relevant for clinical practice. The results of these studies may not only broaden our therapeutic options in the near future, but also provide new insights into disease pathogenesis. PMID:21180564

  12. Monoclonal antibodies as blood grouping reagents.

    PubMed

    Voak, D

    1990-04-01

    The large volume requirements for high quality ABO and Rh(D) typing reagents can now be supplied by selected monoclonal antibodies. Superior anti-A and anti-B monoclonal reagents can be prepared, from blends of at least two antibodies, to optimize the intensity of agglutination for slide tests and the potency for the detection of the weaker sub-groups, including Ax and Bw, by tube techniques. New quality control steps have been described for some highly sensitive anti-A/anti-B antibodies to avoid the detection of traces of A on B cells or traces of B on A1 cells, which results from the non-specific activity of A and B transferases. Excellent anti-A,B reagents may also be made by blends of at least two antibodies to optimize both A and B reactions, but the need for their continued use is now debatable. The development of high titre IgM monoclonal anti-D reagents offers simple rapid saline Rh(D) typing of both patients and donors, but they cannot reliably detect weak D (Du) and some D variants, e.g. the epitopes on D category VI cells. However, this can be achieved by blending an IgM anti-D with IgG (polyclonal) anti-D which can detect these types after conversion of negative saline tests to an antiglobulin phase. In addition, high grade Du, D categories and variants can be reliably detected (for typing donors) by selected monoclonal IgM and IgG anti-Ds by use of suitably enhanced tests without the use of an antiglobulin test.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Monoclonal antibodies against metallothioneins and metalloproteins.

    PubMed

    Talbot, B G; Bilodeau, G; Thirion, J P

    1986-10-01

    Monoclonal antibodies against rabbit metallothioneins (MT) were prepared by in vitro immunization of mouse lymphocytes with a mixture of the two forms of metallothionein MT1 and MT2. Six IgM antibodies (TN1,3,4,5,6,7) which bind to metallothionein were characterized. Antibody TN3 is specific for rabbit MT1 and does not react with any other MT's tested. TN5 is specific for both rabbit MT1 and MT2. TN7 is specific for rabbit MT2 but not MT1 and cross-reacts also with Chinese hamster, mouse and rat metallothioneins. The antibodies TN1, TN4 and TN6 bind not only to rabbit MT1 and MT2 but also to other metal binding proteins like alcohol dehydrogenase and carbonic anhydrase.

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

    PubMed Central

    Sun, Ming; Li, Yue; Zheng, Huiwen; Shao, Yiming

    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 limitation of antiviral activities, multiple antibody-engineering technologies have been explored to generate “the better” neutralizing antibodies against HIV-1 since bNAbs attack viral entry by various mechanisms. Thus, a promising direction of research is to discover and exploit rational antibody combination or engineered antibodies (eAbs) as potential candidate therapeutics against HIV-1. It has been reported that inclusion of fusion-neutralizing antibodies in a set of bNAbs could improve their overall activities and neutralizing spectrum. Here, we review several routes for engineering bNAbs, such as design and generation of bispecific antibodies, specific glycosylation of antibodies to enhance antiviral activity, and variable region-specific modification guided by structure and computer, as well as reviewing antibody-delivery technologies by non-viral vector, viral vector, and human hematopoietic stem/progenitor cells transduced with a lentiviral construct. We also discuss the optimized antiviral activities and benefits of these strategy and potential mechanisms. PMID:27746780

  15. Probing myosin head structure with monoclonal antibodies.

    PubMed

    Winkelmann, D A; Lowey, S

    1986-04-20

    Monoclonal antibodies that react with defined regions of the heavy and light chains of chicken skeletal muscle myosin have been used to provide a correlation between the primary and the tertiary structures of the head. Electron microscopy of rotary shadowed antibody-myosin complexes shows that the sites for three epitopes in the 25,000 Mr tryptic fragment (25k) of subfragment-1, including one within 4000 Mr of the amino terminus of the myosin heavy chain, are clustered 145(+/- 20) A from the head-rod junction. An epitope in the 50,000 Mr fragment maps even further out on the head. These antibodies bind to the head in several orientations, suggesting that each of the heads can rotate can rotate 180 degrees about the head-rod junction. The epitopes are accessible on subfragment-1 bound to actin when they were probed with Fab fragments; therefore, none of these heavy chain sites is is on the contact surface between the head and actin. Two of the anti-25k antibodies affect the K+-EDTA-and Ca2+-ATPase activities of myosin in a manner that mimics the effect on activity of the modification of the reactive thiol, SH-1. These two antibodies also inhibit the actin-activated ATPase non-competitively with respect to actin. None of the other eight antibodies tested had any marked effect on activity. A monoclonal antibody that reacts with an epitope in the amino-terminal third of myosin light chain 2 maps close to the head-rod junction. A polyclonal antibody specific for the amino terminus of light chain 3 binds further up in the "neck region" of the head, indicating that these portions of the two classes of light chains are located at different sites.

  16. Role of outer membrane protein H (OmpH)- and OmpA-specific monoclonal antibodies from hybridoma tumors in protection of mice against Pasteurella multocida.

    PubMed Central

    Vasfi Marandi, M; Mittal, K R

    1997-01-01

    Two major outer membrane proteins of Pasteurella multocida, designated OmpH and OmpA, were characterized and shown to be related to the families of porin and heat-modifiable proteins, respectively. The backpack hybridoma tumor system in BALB/c mice was used to continuously deliver immunoglobulin G2b (IgG2b) monoclonal antibodies (MAbs) specific for OmpH (MAb MT1) and OmpA (MAb MT4.1). MAbs were detected in serum and peritoneal lavage samples of mice bearing hybridoma tumors by an enzyme-linked immunosorbent assay and an immunoblot assay. Highly significant protection was observed in mice bearing MT1 hybridoma tumors against both intraperitoneal and intranasal challenge infections with homologous nontoxigenic P. multocida strains possessing MAb MT1-reacting epitopes, whereas the mice bearing MT4.1 hybridoma tumors were not protected. The numbers of P. multocida organisms in the lungs of mice bearing MT1 hybridoma tumors were significantly less than those in lungs of mice bearing MT4.1 hybridoma tumors at 48 h postchallenge. These results indicate that the OmpH-specific MAb inhibited proliferation of P. multocida in the lungs. MAb MT1 was unable to kill P. multocida in vitro in the presence of complement. However, an enhanced phagocytosis by polymorphonuclear cells (PMNs) was observed in mice bearing MT1 hybridoma tumors. P. multocida induced a more extensive and rapid influx of PMNs into the peritoneal cavity of mice bearing MT1 hybridoma tumors than of mice bearing MT4.1 hybridoma tumors. The results of this study demonstrate for the first time that IgG MAbs against OmpH of P. multocida are involved in the protection of mice against lethal challenge infection by means of opsonization and inhibition of proliferation of P. multocida as a result of increased influx of PMNs into the infection site. PMID:9353026

  17. Polyester-Based Nanoparticles for the Encapsulation of Monoclonal Antibodies.

    PubMed

    Sousa, Flávia; Fonte, Pedro; Cruz, Andreia; Kennedy, Patrick J; Pinto, Inês Mendes; Sarmento, Bruno

    2018-01-01

    Aliphatic polyesters have been widely explored for biomedical applications (e.g., drug delivery systems, biomedical devices, and tissue engineering). Recently, polyesters have been used in nanoparticle formulations for the controlled release of monoclonal antibodies (mAbs) for the enhanced efficacy of antibody-based therapy. Polyester-based nanoparticles for mAb delivery provide decreased antibody dosage, increased antibody stability and protection and longer therapeutic action, ultimately translating to an increased therapeutic index. Additionally, nanoencapsulation holds the potential for the selective cellular recognition and internalization of mAbs, in the disease context when intracellular organelles and molecules (e.g., enzymes, transcription factors and oncogenic proteins) are the preferred target. We present here a detailed method to prepare mAb-loaded polyester-based nanoparticles and the various techniques to characterize the resulting nanoparticles and mAb structure. Finally, we highlight different biological approaches to assess the in vitro bioactivity of the antibody upon nanoparticle release.

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

    PubMed Central

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

    2014-01-01

    Monoclonal antibodies (mAbs) and antibody fragments represent the most important biopharmaceutical products today. Because full length antibodies are glycosylated, mammalian cells, which allow human-like N-glycosylation, are currently used for their production. However, mammalian cells have several drawbacks when it comes to bioprocessing and scale-up, resulting in long processing times and elevated costs. By contrast, antibody fragments, that are not glycosylated but still exhibit antigen binding properties, can be produced in microbial organisms, which are easy to manipulate and cultivate. In this review, we summarize recent advances in the expression systems, strain engineering, and production processes for the three main microbials used in antibody and antibody fragment production, namely Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli. PMID:24183828

  19. Cross-Protective Potential of a Novel Monoclonal Antibody Directed against Antigenic Site B of the Hemagglutinin of Influenza A Viruses

    PubMed Central

    Yoshida, Reiko; Igarashi, Manabu; Ozaki, Hiroichi; Kishida, Noriko; Tomabechi, Daisuke; Kida, Hiroshi; Ito, Kimihito; Takada, Ayato

    2009-01-01

    The hemagglutinin (HA) of influenza A viruses has been classified into sixteen distinct subtypes (H1–H16) to date. The HA subtypes of influenza A viruses are principally defined as serotypes determined by neutralization or hemagglutination inhibition tests using polyclonal antisera to the respective HA subtypes, which have little cross-reactivity to the other HA subtypes. Thus, it is generally believed that the neutralizing antibodies are not broadly cross-reactive among HA subtypes. In this study, we generated a novel monoclonal antibody (MAb) specific to HA, designated MAb S139/1, which showed heterosubtypic cross-reactive neutralization and hemagglutination inhibition of influenza A viruses. This MAb was found to have broad reactivity to many other viruses (H1, H2, H3, H5, H9, and H13 subtypes) in enzyme-linked immunosorbent assays. We further found that MAb S139/1 showed neutralization and hemagglutination-inhibition activities against particular strains of H1, H2, H3, and H13 subtypes of influenza A viruses. Mutant viruses that escaped neutralization by MAb S139/1 were selected from the A/Aichi/2/68 (H3N2), A/Adachi/2/57 (H2N2), and A/WSN/33 (H1N1) strains, and sequence analysis of the HA genes of these escape mutants revealed amino acid substitutions at positions 156, 158, and 193 (H3 numbering). A molecular modeling study showed that these amino acids were located on the globular head of the HA and formed a novel conformational epitope adjacent to the receptor-binding domain of HA. Furthermore, passive immunization of mice with MAb S139/1 provided heterosubtypic protection. These results demonstrate that MAb S139/1 binds to a common antigenic site shared among a variety of HA subtypes and neutralizes viral infectivity in vitro and in vivo by affecting viral attachment to cells. The present study supports the notion that cross-reactive antibodies play some roles in heterosubtypic immunity against influenza A virus infection, and underscores the potential

  20. Cross-protective potential of a novel monoclonal antibody directed against antigenic site B of the hemagglutinin of influenza A viruses.

    PubMed

    Yoshida, Reiko; Igarashi, Manabu; Ozaki, Hiroichi; Kishida, Noriko; Tomabechi, Daisuke; Kida, Hiroshi; Ito, Kimihito; Takada, Ayato

    2009-03-01

    The hemagglutinin (HA) of influenza A viruses has been classified into sixteen distinct subtypes (H1-H16) to date. The HA subtypes of influenza A viruses are principally defined as serotypes determined by neutralization or hemagglutination inhibition tests using polyclonal antisera to the respective HA subtypes, which have little cross-reactivity to the other HA subtypes. Thus, it is generally believed that the neutralizing antibodies are not broadly cross-reactive among HA subtypes. In this study, we generated a novel monoclonal antibody (MAb) specific to HA, designated MAb S139/1, which showed heterosubtypic cross-reactive neutralization and hemagglutination inhibition of influenza A viruses. This MAb was found to have broad reactivity to many other viruses (H1, H2, H3, H5, H9, and H13 subtypes) in enzyme-linked immunosorbent assays. We further found that MAb S139/1 showed neutralization and hemagglutination-inhibition activities against particular strains of H1, H2, H3, and H13 subtypes of influenza A viruses. Mutant viruses that escaped neutralization by MAb S139/1 were selected from the A/Aichi/2/68 (H3N2), A/Adachi/2/57 (H2N2), and A/WSN/33 (H1N1) strains, and sequence analysis of the HA genes of these escape mutants revealed amino acid substitutions at positions 156, 158, and 193 (H3 numbering). A molecular modeling study showed that these amino acids were located on the globular head of the HA and formed a novel conformational epitope adjacent to the receptor-binding domain of HA. Furthermore, passive immunization of mice with MAb S139/1 provided heterosubtypic protection. These results demonstrate that MAb S139/1 binds to a common antigenic site shared among a variety of HA subtypes and neutralizes viral infectivity in vitro and in vivo by affecting viral attachment to cells. The present study supports the notion that cross-reactive antibodies play some roles in heterosubtypic immunity against influenza A virus infection, and underscores the potential

  1. Antibody immunoprophylaxis and immunotherapy for influenza virus infection: utilization of monoclonal or polyclonal antibodies?

    PubMed

    Berry, Cassandra M

    2017-08-30

    Control programs for emerging influenza are in urgent need of novel therapeutic strategies to mitigate potentially devastating threats from pathogenic strains with pandemic potential. Current vaccines and antivirals have inherent limitations in efficacy, especially with rapid evolutionary changes of influenza viruses. Antibody-based antiviral protection harnesses the natural power of the immune system. Antibodies present prophylactic and therapeutic intervention options for prevention and control of influenza, especially for at-risk populations. Specific monoclonal antibodies are well defined in purity and initial efficacy but polyclonal antibodies are easier to scale-up and cost-effective with long-term efficacy, using batches with broadly neutralizing properties against influenza variants. This review presents the pros and cons of monoclonal versus polyclonal antibody therapy for influenza.

  2. Aggregates in monoclonal antibody manufacturing processes.

    PubMed

    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.

  3. Monoclonal antibodies specific for sickle cell hemoglobin

    SciTech Connect

    Jensen, R.H.; Vanderlaan, M.; Grabske, R.J.; Branscomb, E.W.; Bigbee, W.L.; Stanker, L.H.

    1985-01-01

    Two mouse hybridoma cell lines were isolated which produce monoclonal antibodies that bind hemoglobin S. The mice were immunized with peptide-protein conjugates to stimulate a response to the amino terminal peptide of the beta chain of hemoglobin S, where the single amino acid difference between A and S occurs. Immunocharacterization of the antibodies shows that they bind specifically to the immunogen peptide and to hemoglobin S. The specificity for S is high enough that one AS cell in a mixture with a million AA cells is labeled by antibody, and such cells can be analyzed by flow cytometry. Immunoblotting of electrophoretic gels allows definitive identification of hemoglobin S as compared with other hemoglobins with similar electrophoretic mobility. 12 references, 4 figures.

  4. Cross-protection against Vibrio cholerae infection by monoclonal antibodies against Vibrio vulnificus RtxA1/MARTXVv.

    PubMed

    Lee, Tae Hee; Cha, Sun-Shin; Lee, Chang-Seop; Rhee, Joon Haeng; Woo, Hye Ryun; Chung, Kyung Min

    2016-11-01

    Gram-negative Vibrio species secrete multifunctional autoprocessing repeats-in-toxin (MARTX) toxins associated with bacterial pathogenesis. Here, the cross-reactivity and cross-protectivity of mAbs against V. vulnificus RtxA1/MARTXVv was evaluated. Passive administration of any of these mAbs (21RA, 24RA, 46RA, 47RA and 50RA) provided strong protection against lethal V. cholerae infection. Interestingly, 24RA and 46RA, which map to the cysteine protease domain of V. cholerae MARTXVc , inhibited CPD autocleavage in vitro; this process is involved in V. cholerae pathogenesis. These results generate new insight into the development of broadly protective mAbs and/or vaccines against Vibrio species with MARTX toxins. © 2016 The Societies and John Wiley & Sons Australia, Ltd.

  5. Monoclonal Antibodies to Shigella Lipopolysaccharide Are Useful for Vaccine Production.

    PubMed

    Lin, Jisheng; Smith, Mark A; Benjamin, William H; Kaminski, Robert W; Wenzel, Heather; Nahm, Moon H

    2016-08-01

    There is a significant need for an effective multivalent Shigella vaccine that targets the most prevalent serotypes. Most Shigella vaccines under development utilize serotype-specific lipopolysaccharides (LPSs) as a major component based on protection and epidemiological data. As vaccine formulations advance from monovalent to multivalent, assays and reagents need to be developed to accurately and reproducibly quantitate the amount of LPSs from multiple serotypes in the final product. To facilitate this effort, we produced 36 hybridomas that secrete monoclonal antibodies (MAbs) against the O antigen on the LPS from Shigella flexneri 2a, Shigella flexneri 3a, and Shigella sonnei We used six of these monoclonal antibodies for an inhibition enzyme-linked immunosorbent assay (iELISA), measuring LPSs with high sensitivity and specificity. It was also demonstrated that the Shigella serotype-specific MAbs were useful for bacterial surface staining detected by flow cytometry. These MAbs are also useful for standardizing the serum bactericidal assay (SBA) for Shigella Functional assays, such as the in vitro bactericidal assay, are necessary for vaccine evaluation and may serve as immunological correlates of immunity. An S. flexneri 2a-specific monoclonal antibody killed S. flexneri 2b isolates, suggesting that S. flexneri 2a LPS may induce cross-protection against S. flexneri 2b. Overall, the Shigella LPS-specific MAbs described have potential utility to the vaccine development community for assessing multivalent vaccine composition and as a reliable control for multiple immunoassays used to assess vaccine potency.

  6. Monoclonal Antibodies to Shigella Lipopolysaccharide Are Useful for Vaccine Production

    PubMed Central

    Lin, Jisheng; Smith, Mark A.; Benjamin, William H.; Kaminski, Robert W.; Wenzel, Heather

    2016-01-01

    There is a significant need for an effective multivalent Shigella vaccine that targets the most prevalent serotypes. Most Shigella vaccines under development utilize serotype-specific lipopolysaccharides (LPSs) as a major component based on protection and epidemiological data. As vaccine formulations advance from monovalent to multivalent, assays and reagents need to be developed to accurately and reproducibly quantitate the amount of LPSs from multiple serotypes in the final product. To facilitate this effort, we produced 36 hybridomas that secrete monoclonal antibodies (MAbs) against the O antigen on the LPS from Shigella flexneri 2a, Shigella flexneri 3a, and Shigella sonnei. We used six of these monoclonal antibodies for an inhibition enzyme-linked immunosorbent assay (iELISA), measuring LPSs with high sensitivity and specificity. It was also demonstrated that the Shigella serotype-specific MAbs were useful for bacterial surface staining detected by flow cytometry. These MAbs are also useful for standardizing the serum bactericidal assay (SBA) for Shigella. Functional assays, such as the in vitro bactericidal assay, are necessary for vaccine evaluation and may serve as immunological correlates of immunity. An S. flexneri 2a-specific monoclonal antibody killed S. flexneri 2b isolates, suggesting that S. flexneri 2a LPS may induce cross-protection against S. flexneri 2b. Overall, the Shigella LPS-specific MAbs described have potential utility to the vaccine development community for assessing multivalent vaccine composition and as a reliable control for multiple immunoassays used to assess vaccine potency. PMID:27280622

  7. Monoclonal antibodies and method for detecting dioxins and dibenzofurans

    DOEpatents

    Vanderlaan, Martin; Stanker, Larry H.; Watkins, Bruce E.; Bailey, Nina R.

    1989-01-01

    Compositions of matter are described which include five monoclonal antibodies that react with dioxins and dibenzofurans, and the five hybridomas that produce these monoclonal antibodies. In addition, a method for the use of these antibodies in a sensitive immunoassay for dioxins and dibenzofurans is given, which permits detection of these pollutants in samples at concentrations in the range of a few parts per billion.

  8. Labeling of monoclonal antibodies with radionuclides

    SciTech Connect

    Bhargava, K.K.; Acharya, S.A. )

    1989-07-01

    Antibodies, specifically monoclonal antibodies, are potentially very useful and powerful carriers of therapeutic agents to target tissues and diagnostic agents. The loading or charging of antibodies with agents, especially radiotracers, is reviewed here. The choice of radioisotope for immunodetection and/or immunotherapy is based on its availability, half-life, nature of the radiation emitted, and the metabolic pathways of the radionuclide in the body. Most important of all are the derivatization techniques available for labeling the antibody with the given radionuclide. Isotopes of iodine and divalent metal ions are the most commonly used radionuclides. Antibodies labeled with iodine at tyrosine residues are metabolized rapidly in vivo. This leads to the incorporation of metabolized radioactive iodine into various tissues, mainly the thyroid gland and stomach, and to the accumulation of high levels of circulating iodine in the blood, which masks tumor uptake considerably. To overcome these limitations, the use of iodohippurate as an iodine-anchoring molecule to the protein should be considered. When divalent or multivalent metal ions are used as the preferred radionuclide, bifunctional chelating reagents such as EDTA or DTPA are first coupled to the protein or antibody. These chelating molecules are attached to the protein by formation of an isopeptide linkage between the carboxylate of the chelating reagent and the amino group of the protein. Several procedures are available to generate the isopeptide linkage. When the anchoring of the chelating agent through isopeptide linkage results in the inactivation of the antibody, periodate oxidation of the carbohydrate moiety of the antibody, followed by reductive coupling of chelator, could be considered as an alternative. There is still a need for better, simpler, and more direct methods for labeling antibodies with radionuclides. 78 references.

  9. Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques.

    PubMed

    Olinger, Gene Garrard; Pettitt, James; Kim, Do; Working, Cara; Bohorov, Ognian; Bratcher, Barry; Hiatt, Ernie; Hume, Steven D; Johnson, Ashley K; Morton, Josh; Pauly, Michael; Whaley, Kevin J; Lear, Calli M; Biggins, Julia E; Scully, Corinne; Hensley, Lisa; Zeitlin, Larry

    2012-10-30

    Filovirus infections can cause a severe and often fatal disease in humans and nonhuman primates, including great apes. Here, three anti-Ebola virus mouse/human chimeric mAbs (c13C6, h-13F6, and c6D8) were produced in Chinese hamster ovary and in whole plant (Nicotiana benthamiana) cells. In pilot experiments testing a mixture of the three mAbs (MB-003), we found that MB-003 produced in both manufacturing systems protected rhesus macaques from lethal challenge when administered 1 h postinfection. In a pivotal follow-up experiment, we found significant protection (P < 0.05) when MB-003 treatment began 24 or 48 h postinfection (four of six survived vs. zero of two controls). In all experiments, surviving animals that received MB-003 experienced little to no viremia and had few, if any, of the clinical symptoms observed in the controls. The results represent successful postexposure in vivo efficacy by a mAb mixture and suggest that this immunoprotectant should be further pursued as a postexposure and potential therapeutic for Ebola virus exposure.

  10. Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques

    PubMed Central

    Olinger, Gene Garrard; Pettitt, James; Kim, Do; Working, Cara; Bohorov, Ognian; Bratcher, Barry; Hiatt, Ernie; Hume, Steven D.; Johnson, Ashley K.; Morton, Josh; Pauly, Michael; Whaley, Kevin J.; Lear, Calli M.; Biggins, Julia E.; Scully, Corinne; Hensley, Lisa; Zeitlin, Larry

    2012-01-01

    Filovirus infections can cause a severe and often fatal disease in humans and nonhuman primates, including great apes. Here, three anti-Ebola virus mouse/human chimeric mAbs (c13C6, h-13F6, and c6D8) were produced in Chinese hamster ovary and in whole plant (Nicotiana benthamiana) cells. In pilot experiments testing a mixture of the three mAbs (MB-003), we found that MB-003 produced in both manufacturing systems protected rhesus macaques from lethal challenge when administered 1 h postinfection. In a pivotal follow-up experiment, we found significant protection (P < 0.05) when MB-003 treatment began 24 or 48 h postinfection (four of six survived vs. zero of two controls). In all experiments, surviving animals that received MB-003 experienced little to no viremia and had few, if any, of the clinical symptoms observed in the controls. The results represent successful postexposure in vivo efficacy by a mAb mixture and suggest that this immunoprotectant should be further pursued as a postexposure and potential therapeutic for Ebola virus exposure. PMID:23071322

  11. Dissecting monoclonal antibody mega-deals

    PubMed Central

    Villiger, Ralph

    2009-01-01

    Monoclonal antibody (mAb) deals notable in size have made headlines recently. While deals with over US$ 1 bio in milestones were highly unusual in the past, even preclinical agreements, including some with very attractive co-promotion or profit-sharing clauses for the licensor, now reach this mark. This article presents an analysis of the structure of high-value mAb deals and the impact of increasingly sophisticated terms. Strategies of licensees and the impact of strategy on a deal's size and structure are also examined. PMID:20061828

  12. Anti-IL-20 monoclonal antibody inhibited inflammation and protected against cartilage destruction in murine models of osteoarthritis

    PubMed Central

    Hsu, Yu-Hsiang; Yang, Ya-Yu; Huwang, Man-Hsiang; Weng, Yun-Han; Jou, I-Ming; Wu, Po-Tin; Lin, Tain-Yu; Wu, Li-Wha; Chang, Ming-Shi

    2017-01-01

    Osteoarthritis (OA) is a degenerative joint disease characterized by progressive destruction of articular cartilage. Interleukin (IL)-20 is a proinflammatory cytokine involved in the pathogenesis of rheumatoid arthritis. We investigated the role of IL-20 in OA and evaluated whether anti-IL-20 antibody (7E) treatment attenuates disease severity in murine models of surgery-induced OA. Immunohistochemical staining was used to detect IL-20 and its receptors expression in synovial tissue and cartilage from OA patients, and in OA synovial fibroblasts (OASFs) and chondrocytes (OACCs) from rodents with surgery-induced OA. RTQ-PCR and western blotting were used to determine IL-20-regulated OA-associated gene expression in OASFs and OACCs. OA rats and OA mice were treated with 7E. Arthritis severity was determined based on the degree of cartilage damage and the arthritis severity score. We found that IL-20 and its receptors were expressed in OASFs and OACCs. IL-20 induced TNF-α, IL-1β, MMP-1, and MMP-13 expression by activating ERK-1/2 and JNK signals in OASFs. IL-20 not only upregulated MCP-1, IL-6, MMP-1, and MMP-13 expression, but also downregulated aggrecan, type 2 collagen, TGF-β, and BMP-2 expression in OACCs. Arthritis severity was significantly lower in 7E-treated OA rats, and 7E- or MSC-treated OA mice. Therefore, we concluded that IL-20 was involved in the progression and development of OA through inducing proinflammatory cytokines and OA-associated gene expression in OASFs and OACCs. 7E reduced the severity of arthritis in murine models of surgery-induced OA. Our findings provide evidence that IL-20 is a novel target and that 7E is a potential therapeutic agent for OA. PMID:28426699

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

    PubMed Central

    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.

  14. Macaque Monoclonal Antibodies Targeting Novel Conserved Epitopes within Filovirus Glycoprotein

    PubMed Central

    Keck, Zhen-Yong; Enterlein, Sven G.; Howell, Katie A.; Vu, Hong; Shulenin, Sergey; Warfield, Kelly L.; Froude, Jeffrey W.; Araghi, Nazli; Douglas, Robin; Biggins, Julia; Lear-Rooney, Calli M.; Wirchnianski, Ariel S.; Lau, Patrick; Wang, Yong; Herbert, Andrew S.; Dye, John M.; Glass, Pamela J.; Holtsberg, Frederick W.; Foung, Steven K. H.

    2015-01-01

    ABSTRACT Filoviruses cause highly lethal viral hemorrhagic fever in humans and nonhuman primates. Current immunotherapeutic options for filoviruses are mostly specific to Ebola virus (EBOV), although other members of Filoviridae such as Sudan virus (SUDV), Bundibugyo virus (BDBV), and Marburg virus (MARV) have also caused sizeable human outbreaks. Here we report a set of pan-ebolavirus and pan-filovirus monoclonal antibodies (MAbs) derived from cynomolgus macaques immunized repeatedly with a mixture of engineered glycoproteins (GPs) and virus-like particles (VLPs) for three different filovirus species. The antibodies recognize novel neutralizing and nonneutralizing epitopes on the filovirus glycoprotein, including conserved conformational epitopes within the core regions of the GP1 subunit and a novel linear epitope within the glycan cap. We further report the first filovirus antibody binding to a highly conserved epitope within the fusion loop of ebolavirus and marburgvirus species. One of the antibodies binding to the core GP1 region of all ebolavirus species and with lower affinity to MARV GP cross neutralized both SUDV and EBOV, the most divergent ebolavirus species. In a mouse model of EBOV infection, this antibody provided 100% protection when administered in two doses and partial, but significant, protection when given once at the peak of viremia 3 days postinfection. Furthermore, we describe novel cocktails of antibodies with enhanced protective efficacy compared to individual MAbs. In summary, the present work describes multiple novel, cross-reactive filovirus epitopes and innovative combination concepts that challenge the current therapeutic models. IMPORTANCE Filoviruses are among the most deadly human pathogens. The 2014-2015 outbreak of Ebola virus disease (EVD) led to more than 27,000 cases and 11,000 fatalities. While there are five species of Ebolavirus and several strains of marburgvirus, the current immunotherapeutics primarily target Ebola virus

  15. Systemic administration of an anti-tumor necrosis factor-alpha monoclonal antibody protects against endotoxin-induced uveitis in rats

    PubMed Central

    Ge, Qingman; Wang, Shaocheng; Zheng, Yuezhong

    2016-01-01

    Objective: This study was to evaluate the effect of systemic injection of an anti-tumor necrosis factor alpha (TNF-α) monoclonal antibody (mAb) on endotoxin-induced uveitis (EIU). Materials and Methods: Fifty-six male Wistar rats (6–8 weeks old) were randomly divided into three groups: EIU, anti-TNF-α mAb + EIU, and control. EIU was induced by injecting Escherichia coli O55:B5 lipopolysaccharide (LPS) into the hind footpad of the rats (150 μg/rat). The anti-TNF-α mAb (1 μg/kg) was administrated 30 min before LPS injection through one-time intravenous injection. The onset time and peak time of EIU were recorded. The serum and aqueous humor (AH) TNF-α, interleukin (IL)-6, and IL-10 levels were measured by ELISA at 4, 24, and 72 h post-LPS injection. Clinical manifestations of EIU and eye histopathology were scored. Results: Compared with the EIU rats, anti-TNF-α mAb + EIU rats showed significantly delayed onset of uveitis (t = 7.41, P < 0.001), lower clinical scores and histopathological grades (t = 3.18/2.22, P < 0.001), reduced levels of TNF-α (F = 15.06/59.43, P < 0.001) and IL-6 (F = 99.63/14.92, P < 0.001), and increased levels of IL-10 (F = 24.94/8.99, P < 0.001) in the serum and AH. AH TNF-α, serum IL-6, and AH IL-6 levels are positively correlated, whereas serum IL-10 levels were negatively correlated with EIU activity. Conclusion: Antagonizing TNF-α by system injection of the anti-TNF-α mAb protects against EIU in rats. Blocking TNF-α signaling could be a useful strategy for managing uveitis. PMID:28112125

  16. Monoclonal antibody disulfide reduction during manufacturing

    PubMed Central

    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

  17. Structure and specificity of lamprey monoclonal antibodies.

    PubMed

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

    2008-02-12

    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. The recombinant VLR-B antibodies possess 8-10 uniform subunits that collectively bind antigen with high avidity. Sequence analysis, mutagenesis, and modeling studies show that antigen binding involves residues in the beta-sheets lining the VLR-B concave surface. EM visualization reveals tetrameric and pentameric molecules having a central core and highly flexible pairs of stalk-region "arms" with antigen-binding "hands." Remarkable antigen-binding specificity, avidity, and stability predict that these unusual LRR-based monoclonal antibodies will find many biomedical uses.

  18. Recombinant genetic libraries and human monoclonal antibodies.

    PubMed

    Adams, Jarrett J; Nelson, Bryce; Sidhu, Sachdev S

    2014-01-01

    In order to comprehensively manipulate the human proteome we require a vast repertoire of pharmacological reagents. To address these needs we have developed repertoires of synthetic antibodies by phage display, where diversified oligonucleotides are used to modify the complementarity-determining regions (CDRs) of a human antigen-binding fragment (Fab) scaffold. As diversity is produced outside the confines of the mammalian immune system, synthetic antibody libraries allow us to bypass several limitations of hybridoma technology while improving the experimental parameters under which pharmacological reagents are produced. Here we describe the methodologies used to produce synthetic antibody libraries from a single human framework with diversity restricted to four CDRs. These synthetic repertoires can be extremely functional as they produce highly selective, high affinity Fabs to the majority of soluble human antigens. Finally we describe selection methodologies that allow us to overcome immuno-dominance in our selections to target a variety of epitopes per antigen. Together these methodologies allow us to produce human monoclonal antibodies to manipulate the human proteome.

  19. A Monoclonal Antibody Toolkit for C. elegans

    PubMed Central

    Hadwiger, Gayla; Dour, Scott; Arur, Swathi; Fox, Paul; Nonet, Michael L.

    2010-01-01

    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 β-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 α-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 working

  20. The Cys-Rich Region of Hepatitis A Virus Cellular Receptor 1 Is Required for Binding of Hepatitis A Virus and Protective Monoclonal Antibody 190/4

    PubMed Central

    Thompson, Peter; Lu, Jinhua; Kaplan, Gerardo G.

    1998-01-01

    The hepatitis A virus cellular receptor 1 (HAVcr-1) cDNA codes for a class I integral membrane glycoprotein, termed havcr-1, of unknown natural function which serves as an African green monkey kidney (AGMK) cell receptor for HAV. The extracellular domain of havcr-1 has an N-terminal Cys-rich region that displays homology with sequences of members of the immunoglobulin superfamily, followed by a Thr/Ser/Pro (TSP)-rich region characteristic of mucin-like O-glycosylated proteins. The havcr-1 glycoprotein contains four putative N-glycosylation sites, two in the Cys-rich region and two in the TSP-rich region. To characterize havcr-1 and define region(s) involved in HAV receptor function, we expressed the TSP-rich region in Escherichia coli fused to glutathione S-transferase and generated antibodies (Ab) in rabbits (anti-GST2 Ab). Western blot analysis with anti-GST2 Ab detected 62- and 65-kDa bands in AGMK cells and 59-, 62-, and 65-kDa bands in dog cells transfected with the HAVcr-1 cDNA (cr5 cells) but not in dog cells transfected with the vector alone (DR2 cells). Treatment of AGMK and cr5 cell extracts with peptide-N-glycosidase F resulted in the collapse of the havcr-1-specific bands into a single band of 56 kDa, which indicated that different N-glycosylated forms of havcr-1 were expressed in these cells. Treatment of AGMK and cr5 cells with tunicamycin reduced binding of protective monoclonal Ab (MAb) 190/4, which suggested that N-glycans are required for binding of MAb 190/4 to havcr-1. To test this hypothesis, havcr-1 mutants lacking the N-glycosylation motif at the first site (mut1), second site (mut2), and both (mut3) sites were constructed and transfected into dog cells. Binding of MAb 190/4 and HAV to mut1 and mut3 cells was highly reduced, while binding to mut2 cells was not affected and binding to dog cells expressing an havcr-1 construct containing a deletion of the Cys-rich region (d1− cells) was undetectable. HAV-infected cr5 and mut2 cells but not

  1. The Cys-rich region of hepatitis A virus cellular receptor 1 is required for binding of hepatitis A virus and protective monoclonal antibody 190/4.

    PubMed

    Thompson, P; Lu, J; Kaplan, G G

    1998-05-01

    The hepatitis A virus cellular receptor 1 (HAVcr-1) cDNA codes for a class I integral membrane glycoprotein, termed havcr-1, of unknown natural function which serves as an African green monkey kidney (AGMK) cell receptor for HAV. The extracellular domain of havcr-1 has an N-terminal Cys-rich region that displays homology with sequences of members of the immunoglobulin superfamily, followed by a Thr/Ser/Pro (TSP)-rich region characteristic of mucin-like O-glycosylated proteins. The havcr-1 glycoprotein contains four putative N-glycosylation sites, two in the Cys-rich region and two in the TSP-rich region. To characterize havcr-1 and define region(s) involved in HAV receptor function, we expressed the TSP-rich region in Escherichia coli fused to glutathione S-transferase and generated antibodies (Ab) in rabbits (anti-GST2 Ab). Western blot analysis with anti-GST2 Ab detected 62- and 65-kDa bands in AGMK cells and 59-, 62-, and 65-kDa bands in dog cells transfected with the HAVcr-1 cDNA (cr5 cells) but not in dog cells transfected with the vector alone (DR2 cells). Treatment of AGMK and cr5 cell extracts with peptide-N-glycosidase F resulted in the collapse of the havcr-1-specific bands into a single band of 56 kDa, which indicated that different N-glycosylated forms of havcr-1 were expressed in these cells. Treatment of AGMK and cr5 cells with tunicamycin reduced binding of protective monoclonal Ab (MAb) 190/4, which suggested that N-glycans are required for binding of MAb 190/4 to havcr-1. To test this hypothesis, havcr-1 mutants lacking the N-glycosylation motif at the first site (mut1), second site (mut2), and both (mut3) sites were constructed and transfected into dog cells. Binding of MAb 190/4 and HAV to mut1 and mut3 cells was highly reduced, while binding to mut2 cells was not affected and binding to dog cells expressing an havcr-1 construct containing a deletion of the Cys-rich region (d1- cells) was undetectable. HAV-infected cr5 and mut2 cells but not mut1

  2. [Progress in monoclonal antibody-based immunotherapy for cancer treatment].

    PubMed

    Guo, Yajun

    2015-06-01

    More than 100 years ago, Paul Ehrlich first proposed the "magic bullets" concept in which antibody targeting disease related antigen can fight against human disease. Since then, with the development of hybridoma technology for monoclonal antibody production and cancer serum therapy, immunotherapy based monoclonal antibody bas been used in chinical practice to treat hematological and solid tumor. Up to now, more than 20 recombinant antibody drugs were approved for cancer treatment worldwide. In recent years, the next-generation antibody drug, including immune checkpoint antagonists, bi-specific antibody, and antibody drug conjugates have successfully cured various malignant tumor. This review recalled the history of monoclonal antibody as potent immunotherapy of cancer firstly, and focused on the next-generation antibody drug's mechanism of action, construction strategies, and the side effects in clinic. Lastly, the future trend of anti-tumor antibody drug was also discussed.

  3. Immunochemical Characterization of Anti-Acetylcholinesterase Inhibitory Monoclonal Antibodies

    DTIC Science & Technology

    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

  4. Efficient generation of human IgA monoclonal antibodies.

    PubMed

    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.

  5. Monoclonal Antibodies Against Xenopus Greatwall Kinase

    PubMed Central

    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

  6. Monoclonal antibodies based on hybridoma technology.

    PubMed

    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.

  7. Monoclonal antibodies to human urinary thrombopoietin

    SciTech Connect

    McDonald, T.P.; Clift, R.; Cottrell, M.

    1986-01-01

    Monoclonal antibodies (MA) to a thrombocytopoiesis-stimulating factor (TSF or thrombopoietin) were obtained from hybridomas derived from the fusion of P3 x 63/Ag 8 cells and spleen cells from TSF-immunized BALB/c mice. Media from several hybrid cultures were tested in a microantibody detection technique that measured the binding of MA to a /sup 125/I-purified TSF preparation from human embryonic kidney (HEK) cells. Hybridized cells were injected into pristane-primed mice and the antibodies produced in the ascites fluid were also shown to bind the /sup 125/I-TSF. Compared to the results of normal mouse serum, ascites fluid containing MA was shown to bind the unlabeled TSF from HEK cells. The TSF activity was significantly reduced in the supernatant fluid after precipitating the TSF-anti-TSF immune complex by a second antibody when tested in an immunothrombocythemic mouse assay. After SDS-PAGE, the precipitate from this TSF-Ma conjugate showed that the antiserum bound a single 32,000 mol wt component, indicating the monospecificity of the MA. MA directed toward human TSF will allow studies that were not previously possible.

  8. Antibacterial monoclonal antibodies: the next generation?

    PubMed

    DiGiandomenico, Antonio; Sellman, Bret R

    2015-10-01

    There is a clear need for renewed efforts to combat the increasing incidence of antibiotic resistance. While the antibiotic resistance epidemic is due in part to the misuse of antibiotics, even proper empiric antibiotic therapy increases the selective pressure and potential for drug-resistance and spread of resistance mechanisms between bacteria. Antibiotic resistance coupled with the detrimental effects of broad-spectrum antibiotics on the healthy microbiome, have led the field to explore pathogen specific antibacterials such as monoclonal antibodies (mAbs). Medical need along with advances in mAb discovery, engineering, and production have driven significant effort developing mAb-based antibacterials. If successful, they will provide physicians with precision weapons to combat bacterial infections and can help prevent a return to a pre-antibiotic era.

  9. Monoclonal antibodies in treatment of multiple sclerosis

    PubMed Central

    Rommer, P S; Dudesek, A; Stüve, O; Zettl, UK

    2014-01-01

    Monoclonal antibodies (mAbs) are used as therapeutics in a number of disciplines in medicine, such as oncology, rheumatology, gastroenterology, dermatology and transplant rejection prevention. Since the introduction and reintroduction of the anti-alpha4-integrin mAb natalizumab in 2004 and 2006, mAbs have gained relevance in the treatment of multiple sclerosis (MS). At present, numerous mAbs have been tested in clinical trials in relapsing–remitting MS, and in progressive forms of MS. One of the agents that might soon be approved for very active forms of relapsing–remitting MS is alemtuzumab, a humanized mAb against CD52. This review provides insights into clinical studies with the mAbs natalizumab, alemtuzumab, daclizumab, rituximab, ocrelizumab and ofatumumab. PMID:24001305

  10. SPECT assay of radiolabeled monoclonal antibodies

    SciTech Connect

    Jaszczak, R.J.

    1992-02-01

    The long-term goal of this research project is to develop methods to improve the utility of single photon emission computed tomography (SPECI) to quantify the biodistribution of monoclonal antibodies (MoAbs) labeled with clinically relevant radionuclides ({sup 123}I, {sup 131}I, and {sup 111}In) and with another radionuclide,{sup 211}At, recently used in therapy. We describe here our progress in developing quantitative SPECT methodology for {sup 111}In and {sup 123}I. We have focused our recent research thrusts on the following aspects of SPECT: (1) The development of improved SPECT hardware, such as improved acquisition geometries. (2) The development of better reconstruction methods that provide accurate compensation for the physical factors that affect SPECT quantification. (3) The application of carefully designed simulations and experiments to validate our hardware and software approaches.

  11. Building better monoclonal antibody-based therapeutics

    PubMed Central

    Weiner, George J.

    2015-01-01

    For 20 years, monoclonal antibodies (mAbs) have been a standard component of cancer therapy, yet there is still much room for improvement. Efforts continue to build better cancer therapeutics based on mAbs. Anti-cancer mAbs function via a variety of mechanisms including directly targeting the malignant cells, modifying the host response to the malignant cells, delivering cytotoxic moieties to the malignant cells or retargeting cellular immunity towards the malignant cells. Characteristics of mAbs that affect their efficacy include antigen specificity, overall structure, affinity for the target antigen and how a mAb component is incorporated into a construct that can trigger target cell death. This article reviews the various approaches to using mAb-based therapeutics to treat cancer, the strategies used to take advantage of the unique potential of each approach, and provides examples of current mAb-based treatments. PMID:25998715

  12. Monitoring therapeutic monoclonal antibodies in brain tumor

    PubMed Central

    Ait-Belkacem, Rima; Berenguer, Caroline; Villard, Claude; Ouafik, L’Houcine; Figarella-Branger, Dominique; Beck, Alain; Chinot, Olivier; Lafitte, Daniel

    2014-01-01

    Bevacizumab induces normalization of abnormal blood vessels, making them less leaky. By binding to vascular endothelial growth factor, it indirectly attacks the vascular tumor mass. The optimal delivery of targeted therapies including monoclonal antibodies or anti-angiogenesis drugs to the target tissue highly depends on the blood-brain barrier permeability. It is therefore critical to investigate how drugs effectively reach the tumor. In situ investigation of drug distribution could provide a better understanding of pharmacological agent action and optimize chemotherapies for solid tumors. We developed an imaging method coupled to protein identification using matrix-assisted laser desorption/ionization mass spectrometry. This approach monitored bevacizumab distribution within the brain structures, and especially within the tumor, without any labeling. PMID:25484065

  13. Monoclonal antibodies against methionyl recombinant human prolactin.

    PubMed

    Paris, N; Robert, R; Mercier, L

    1993-02-01

    Hybridoma cell lines producing monoclonal antibody (Mab) against recombinant human prolactin (rhPrl) were established from fusion between X63-Ag8 myeloma cells and Balb/c mice splenocytes. Four Mabs numbered I to IV were selected by ELISA, purified and characterized. All these Mabs were of the Ig1 kappa isotype and able to recognize oxidized as well as reduced rhPrl. As shown by a competitive inhibition assay, Mab IV did not compete with any of the three others. Moreover, both rhPrl and hPrl extracted from human pituitaries, were recognized equally by this Mab. Properties displayed by Mab IV make it very attractive for the evaluation of prolactin levels by sandwich immunoassays.

  14. The birth pangs of monoclonal antibody therapeutics

    PubMed Central

    2012-01-01

    This paper examines the development and termination of nebacumab (Centoxin®), a human IgM monoclonal antibody (mAb) drug frequently cited as one of the notable failures of the early biopharmaceutical industry. The non-approval of Centoxin in the United States in 1992 generated major concerns at the time about the future viability of any mAb therapeutics. For Centocor, the biotechnology company that developed Centoxin, the drug posed formidable challenges in terms of safety, clinical efficacy, patient selection, the overall economic costs of health care, as well as financial backing. Indeed, Centocor's development of the drug brought it to the brink of bankruptcy. This article shows how many of the experiences learned with Centoxin paved the way for the current successes in therapeutic mAb development. PMID:22531443

  15. A Strategy for Screening Monoclonal Antibodies for Arabidopsis Flowers

    PubMed Central

    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

  16. Invasion of erythrocytes in vitro by Plasmodium falciparum can be inhibited by monoclonal antibody directed against an S antigen.

    PubMed

    Saul, A; Cooper, J; Ingram, L; Anders, R F; Brown, G V

    1985-11-01

    A monoclonal antibody has been produced which binds to the heat stable S antigen present in the FCQ-27/PNG isolate of Plasmodium falciparum. This monoclonal antibody also inhibits the invasion in vitro of erythrocytes by malarial merozoites thus demonstrating that the S antigens of Plasmodium falciparum may be a target of protective immune responses.

  17. Drug Development of Therapeutic Monoclonal Antibodies.

    PubMed

    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.

  18. Clinical laboratory applications of monoclonal antibodies.

    PubMed Central

    Payne, W J; Marshall, D L; Shockley, R K; Martin, W J

    1988-01-01

    Monoclonal antibody (MAb) technology is well recognized as a significant development for producing specific serologic reagents to a wide variety of antigens in unlimited amounts. These reagents have provided the means for developing a number of highly specific and reproducible immunological assays for rapid and accurate diagnosis of an extensive list of diseases, including infectious diseases. The impact that MAbs have had in characterizing infectious disease pathogens, as well as their current and future applications for use in clinical microbiology laboratories, is reviewed. In addition, the advantages (and disadvantages) of the use of MAbs in a number of immunoassays, such as particle agglutination, radioimmunoassays, enzyme-linked immunosorbent assays, immunofluorescent-antibody assays, and immunohistology, are explored, including the use of these reagents in novel test system assays. Also, nucleic acid probe technology is compared with the use of MAbs from the perspective of their respective applications in the diagnosis of infectious disease agents. There is no question that hybridoma technology has the potential to alter significantly the methods currently used in most clinical microbiology laboratories. PMID:3058298

  19. Monoclonal antibody treatments for rheumatoid arthritis.

    PubMed

    Bossaller, Lukas; Rothe, Achim

    2013-09-01

    Rheumatoid arthritis (RA) is a systemic disease and the most prevalent of all autoimmune disorders. Here we review recent advances in the development and availability of biologic agents with a focus on monoclonal antibody or smaller formats of targeted engineered therapeutics including novel, non-antibody-based therapeutics. Today an array of biologics blocking either proinflammatory cytokines or lymphocyte activation/survival are available that enable a substantial improvement over conventional disease-modifying antirheumatic drugs (DMARDs). We review the engineering process of antibody-based biologics, their preclinical and clinical application, and current efforts to treat RA by interfering with B-cell function (notable targets covered are CD20, CD38, B-cell activating factor, transmembrane activator and calcium-modulating and cyclophilin interactor), with T-cell function (CD3, CD4, CD28), with bone erosion (RANKL), and with cytokines or growth factors (tumor necrosis factor, interleukin-1 [IL-1], IL-6, IL-17, VEGF). Future treatment choices might encompass the blockade or modulation of danger-associated molecular patterns such as HMGB1, pattern recognition receptors, messenger RNAs or noncoding RNAs, histone acetylation, and inflammasome components. Although current therapies can reduce the signs and symptoms of RA for many patients, the quest for a cure (or a more complete blockade of the structural damage) in RA is still ongoing and will need treatment approaches, which are not exclusively confined to blocking a particular cytokine, receptor, or autoreactive B or T cell involved in disease progression. To this end exciting treatment alternatives and drug targets are on the horizon that may become available to patients in the future.

  20. Monoclonal antibody that inhibits infection of HeLa and rhabdomyosarcoma cells by selected enteroviruses through receptor blockade

    SciTech Connect

    Crowell, R.L.; Field, A.K.; Schleif, W.A.; Long, W.L.; Colonno, R.J.; Mapoles, J.E.; Emini, E. A.

    1986-02-01

    BALB/c mice were immunized with HeLa cells, and their spleen cells were fused with myeloma cells to produce hybridomas. Initial screening of culture fluids from 800 fusion products in a cell protection assay against coxsackievirus B3 (CB3) and the CB3-RD virus variant yielded five presumptive monoclonal antibodies with three specificities: (i) protection against CB3 on HeLa, (ii) protection against CB3-RD on rhabdomyosarcoma (RD) cells, and (iii) protection against both viruses on the respective cells. Only one of the monoclonal antibodies (with dual specificity) survived two subclonings and was studied in detail. The antibody was determined to have an immunoglobulin G2a isotype and protected cells by blockade of cellular receptors, since attachment of (/sup 35/S)methionine-labeled CB3 was inhibited by greater than 90%. The monoclonal antibody protected HeLa cells against infection by CB1, CB3, CB5, echovirus 6, and coxsackievirus A21 and RD cells against CB1-RD, CB3-RD, and CB5-Rd virus variants. The monoclonal antibody did not protect either cell type against 16 other immunotypes of picornaviruses. The monoclonal antibody produced only positive fluorescence on those cells which were protected against infection, and /sup 125/I-labeled antibody confirmed the specific binding to HeLa and RD cells. The results suggest that this monoclonal antibody possesses some of the receptor specificity of the group B coxsackieviruses.

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

    PubMed Central

    Hutchinson, Alistair P.; Nicklin, Stephen

    2015-01-01

    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. PMID:26252765

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

    PubMed

    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.

  3. Localisation of malignant glioma by a radiolabelled human monoclonal antibody.

    PubMed Central

    Phillips, J; Alderson, T; Sikora, K; Watson, J

    1983-01-01

    Human monoclonal antibodies were produced by fusing intratumoral lymphocytes from patients with malignant gliomas with a human myeloma line. One antibody was selected for further study after screening for binding activity to glioma cell lines. The patient from whom it was derived developed recurrent glioma. 1 mg of antibody was purified, radiolabelled with 131I, and administered intravenously. The distribution of antibody was determined in the blood, CSF and tumour cyst fluid and compared with that of a control human monoclonal immunoglobulin. Antibody localisation in the tumour was observed and confirmed by external scintiscanning. Images PMID:6101173

  4. Monoclonal Antibody-Based Candidate Therapeutics Against HIV Type 1

    PubMed Central

    Dimitrov, Dimiter S.

    2012-01-01

    Abstract 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. PMID:21827278

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

    PubMed

    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.

  6. Monoclonal antibody specific for a pigmentation associated antigen

    SciTech Connect

    Thomson, T.M.; Mattes, M.J.; Old, L.J.; Lloyd, K.O

    1989-01-17

    Monoclonal antibody TA99, which specifically binds to a pigmentation associated antigen present on melanoma cells is described. Additionally, the hybridoma cell line deposited with the ATCC under Accession Number HB 8704 from which the antibody is derived, as well as methods for using the antibody are described.

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

    PubMed

    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."

  8. Anti-schistosome monoclonal antibodies of different isotypes--correlation with cytotoxicity.

    PubMed Central

    Horowitz, S; Tarrab-Hazdai, R; Eshhar, Z; Arnon, R

    1983-01-01

    Five monoclonal antibodies specific towards Schistosoma mansoni antigens were prepared by fusion of spleen cells of infected and immunized mouse with the murine myeloma NS-1 cells. Three of the five antibodies belonged to the IgG1 class, one was an IgM and the fifth one was an IgE. The IgE monoclonal antibody designated 54.10, induced antigen-specific degranulation of rat basophilic cell line, a property which served as the basis for the screening assay. Its biological function was demonstrated by a specific macrophage activation that led to killing of schistosomula; no such killing was obtained with anti-schistosome antibodies of other classes or with IgE of different antigenic specificity. The second monoclonal antibody of biological significance was an IgG1, designated 27.21 which is reactive in the immunofluorescence staining of surface antigens on intact schistosomula. All three monoclonal antibodies that belonged to the IgG1 class were effective in mediating killing of schistosomula by complement, with the highest effect exerted by 27.21. It is thus apparent that the 27.21 monoclonal antibody is directed against a densely distributed surface antigen on the schistosomula membrane which is possibly involved in the protective immunity. Preliminary data showed that immunoprecipitation with the 27.21 antibodies results in the isolation of three major protein bands, of 60 kd, 50 kd, 19 kd, respectively. Images Fig. 2. Fig. 4. PMID:11894925

  9. Cation-exchange chromatography of monoclonal antibodies

    PubMed Central

    Urmann, Marina; Graalfs, Heiner; Joehnck, Matthias; Jacob, Lothar R

    2010-01-01

    A novel cation-exchange resin, Eshmuno™ S, was compared to Fractogel® SO3− (M) and Toyopearl GigaCap S-650M. The stationary phases have different base matrices and carry specific types of polymeric surface modifications. Three monoclonal antibodies (mAbs) were used as model proteins to characterize these chromatographic resins. Results from gradient elutions, stirred batch adsorptions and confocal laser scanning microscopic investigations were used to elucidate binding behavior of mAbs onto Eshmuno™ S and Fractogel® SO3− and the corresponding transport mechanisms on these two resins. The number of charges involved in mAb binding for Eshmuno™ S is lower than for Fractogel® SO3−, indicating a slightly weaker electrostatic interaction. Kinetics from batch uptake experiments are compared to kinetic data obtained from confocal laser scanning microscopy images. Both experimental approaches show an accelerated protein adsorption for the novel stationary phase. The influence of pH, salt concentrations and residence times on dynamic binding capacities was determined. A higher dynamic binding capacity for Eshmuno™ S over a wider range of pH values and residence times was found compared to Fractogel® SO3− and Toyopearl GigaCap S-650M. The capture of antibodies from cell culture supernatant, as well as post-protein A eluates, were analyzed with respect to their host cell protein (hcp) removal capabilities. Comparable or even better hcp clearance was observed at much higher protein loading for Eshmuno™ S than Fractogel® SO3− or Toyopearl GigaCap S-650M. PMID:20559022

  10. Monoclonal antibodies for use in man: current regulatory situation in the Federal Republic of Germany.

    PubMed

    Haase, M

    1990-01-01

    The article addresses the requirement to be met for approval of monoclonal antibodies with special emphasis on products coupled with radionuclides and on principles for the conduct of clinical trials. According to the German Drug Law monoclonal antibodies are considered as being sera. Therefore, the Paul-Ehrlich-Institut, Federal Office for Sera and Vaccines, is responsible for marketing authorizations. Sera and vaccines need a special manufacturing licence which is given by the competent authority of the Federal State. Batches of monoclonal antibodies can only be marketed if they have been released by the Paul-Ehrlich-Institut; in connexion with batch control the importance of reference preparations is stressed. The standard requirements for the data to be submitted with the applications for marketing authorizations are in accordance with the EEC Council Directives and Notes for Guidance. For the testing of radioactive monoclonal antibodies in clinical trials, compliance with both the Drug Law and The German Radiation Protection Ordinance must be ensured. In addition to the authorizations required for non-labelled monoclonal antibody products, the use of radioactive substances in diagnosis and therapy requires an authorization by the competent Federal State authority. The main purpose of the planning and performance of clinical trials with new monoclonal antibody in diagnosis and therapy must be the comparison with established diagnostic tools and/or established medicinal products of known effect.

  11. Generation and Improvement of Effector Function of a Novel Broadly Reactive and Protective Monoclonal Antibody against Pneumococcal Surface Protein A of Streptococcus pneumoniae

    PubMed Central

    Cho, Rebecca; Groff, Brian C.; Kubota, Tsuguo; Destito, Giuseppe; Laudenslager, John; Koriazova, Lilia; Tahara, Tomoyuki; Kanda, Yutaka

    2016-01-01

    A proof-of-concept study evaluating the potential of Streptococcus pneumoniae Pneumococcal Surface Protein A (PspA) as a passive immunization target was conducted. We describe the generation and isolation of several broadly reactive mouse anti-PspA monoclonal antibodies (mAbs). MAb 140H1 displayed (i) 98% strain coverage, (ii) activity in complement deposition and opsonophagocytic killing (OPK) assays, which are thought to predict the in vivo efficacy of anti-pneumococcal mAbs, (iii) efficacy in mouse sepsis models both alone and in combination with standard-of-care antibiotics, and (iv) therapeutic activity in a mouse pneumonia model. Moreover, we demonstrate that antibody engineering can significantly enhance anti-PspA mAb effector function. We believe that PspA has promising potential as a target for the therapy of invasive pneumococcal disease by mAbs, which could be used alone or in conjunction with standard-of-care antibiotics. PMID:27171010

  12. Passive Immunization with Anti-Glucosaminidase Monoclonal Antibodies Protects Mice from Implant-Associated Osteomyelitis by Mediating Opsonophagocytosis of Staphylococcus aureus Megaclusters

    PubMed Central

    Varrone, John J.; de Mesy Bentley, Karen L.; Bello-Irizarry, Sheila N.; Nishitani, Kohei; Mack, Sarah; Hunter, Joshua G.; Kates, Stephen L.; Daiss, John L.; Schwarz, Edward M.

    2014-01-01

    Towards development of a methicillin-resistant S. aureus (MRSA) vaccine we evaluated a neutralizing anti-glucosaminidase (Gmd) monoclonal antibody (1C11) in a murine model of implant-associated osteomyelitis, and compared its effects on LAC USA300 MRSA versus placebo (alpha-T2m) and a Gmd-deficient isogenic strain (delta-Gmd). 1C11 significantly reduced infection severity, as determined by bioluminescent imaging of bacteria, micro-CT assessment of osteolysis and histomorphometry of abscess numbers (p<0.05). Histology also revealed infiltrating macrophages, and the complete lack of staphylococcal abscess communities (SAC), in marrow abscesses of 1C11 treated mice. In vitro, 1C11 had no direct effects on proliferation, but electron microscopy demonstrated that 1C11 treatment phenocopies delta-Gmd defects in binary fission. Moreover, addition of 1C11 to MRSA cultures induced the formation of large bacterial aggregates (megaclusters) that sedimented out of solution, which was not observed in delta-Gmd cultures or 1C11 treated cultures of a protein A-deficient strain (delta-Spa), suggesting that the combined effects of Gmd inhibition and antibody-mediated agglutination are required. Finally, we demonstrated that macrophage opsonophagocytosis of MRSA and megaclusters is significantly increased by 1C11 (p<0.01). Collectively, these results suggest that the primary mechanism of anti-Gmd humoral immunity against MRSA osteomyelitis is macrophage invasion of SAC and opsonophagocytosis of megaclusters. PMID:24992290

  13. Mice are actively immunized after passive monoclonal antibody prophylaxis and ricin toxin challenge. (Reannouncement with new availability information)

    SciTech Connect

    Lemley, P.V.; Wright, D.C.

    1992-12-31

    Mice passively immunized by a protective, anti-ricin A-chain monoclonal antibody, then challenged intravenously with ricin, were protected from a subsequent ricin challenge, and were actively immunized. Two significant advantages accrued from this experiment: the monoclonal antibody neutralized the toxicity of the ricin immunogen, and active immunization was achieved with very low antigen load (approx. 0.5 micrograms/mouse). We ruled out the possibility that residual monoclonal antibody provided the protection by using three independent criteria. There was significant (four orders of magnitude) enhancement of the immune response in the presence of the monoclonal antibody; control immunizations of mice with ricin A-chain, ricin B-chain or either chain with the monoclonal antibody did not induce active immunity; and the active immunization could not be replicated when protective goat polyclonal antibody was substituted for the monoclonal antibody. Because high titers were achieved rapidly without any adjuvant, we are currently investigating haptenized ricin to determine if anti-hapten monoclonal antibodies can be produced by this refined procedure.

  14. Monoclonal Antibodies Identify Novel Neural Antigens

    NASA Astrophysics Data System (ADS)

    Hawkes, Richard; Niday, Evelyn; Matus, Andrew

    1982-04-01

    Monoclonal antibodies (Mabs) were raised against synaptic plasma membranes from rat cerebellum. The hybridomas were screened with a solid-phase immunoassay, the positive lines were characterized by their immunoperoxidase staining pattern on cerebellum, and the specific polypeptide antigens were identified on protein blots. Among the Mabs described are some that stain only neurons or only glia and others that react with specific parts of cells, such as axons, dendrites, and synapses. Many Mabs reveal novel relationships between antigens and the cells in which they occur. For example, a Mab designated 7D5 reacts with a family of > 30 proteins but stains only glial cells. Several Mabs stain punctate sites of synaptic size and distribution in the cerebellar cortex but each reacts with a different subset of polypeptides. One of the most restricted cytological staining patterns is given by 12D5, which stains punctate sites in the granular layer of the cerebellar cortex and reacts with a single polypeptide band of apparent Mr 270,000. These results illustrate the feasibility of raising Mabs that can be used to follow the expression of specific gene products during brain development.

  15. Monoclonal antibodies against plant cell wall polysaccharides

    SciTech Connect

    Hahn, M.G.; Bucheli, E.; Darvill, A.; Albersheim, P. )

    1989-04-01

    Monoclonal antibodies (McAbs) are useful tools to probe the structure of plant cell wall polysaccharides and to localize these polysaccharides in plant cells and tissues. Murine McAbs were generated against the pectic polysaccharide, rhamnogalacturonan I (RG-I), isolated from suspension-cultured sycamore cells. The McAbs that were obtained were grouped into three classes based upon their reactivities with a variety of plant polysaccharides and membrane glycoproteins. Eleven McAbs (Class I) recognize epitope(s) that appear to be immunodominant and are found in RG-I from sycamore and maize, citrus pectin, polygalacturonic acid, and membrane glycoproteins from suspension-cultured cells of sycamore, maize, tobacco, parsley, and soybean. A second group of five McAbs (Class II) recognize epitope(s) present in sycamore RG-I, but do not bind to any of the other polysaccharides or glycoproteins recognized by Class I. Lastly, one McAb (Class III) reacts with sycamore RG-I, sycamore and tamarind xyloglucan, and sycamore and rice glucuronoarabinoxylan, but does not bind to maize RG-I, polygalacturonic acid or the plant membrane glycoproteins recognized by Class I. McAbs in Classes II and III are likely to be useful in studies of the structure, biosynthesis and localization of plant cell wall polysaccharides.

  16. A monoclonal antibody against human MUDENG protein.

    PubMed

    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.

  17. Viral Epitopes and Monoclonal Antibodies: Isolation of Blocking Antibodies that Inhibit Virus Neutralization

    NASA Astrophysics Data System (ADS)

    Massey, Richard J.; Schochetman, Gerald

    1981-07-01

    The inability of pathogenic animal viruses to be completely neutralized by antibodies can lead to chronic viral infections in which infectious virus persists even in the presence of excess neutralizing antibody. A mechanism that results in this nonneutralized fraction of virus was defined by the topographical relationships of viral epitopes identified with monoclonal antibodies wherein monoclonal antibodies bind to virus and sterically block the binding of neutralizing antibodies.

  18. Monoclonal Antibodies Targeting Tumor Growth | NCI Technology Transfer Center | TTC

    Cancer.gov

    The NCI Nanobiology Program, Protein Interaction Group is seeking parties to license or co-develop, evaluate, or commercialize monoclonal antibodies against the insulin-like growth factor for the treatment of cancer.

  19. DEVELOPMENT OF MONOCLONAL ANTIBODIES AGAINST FATHEAD MINNOW (PIMEPHALES PROMELAS) VITELLOGENIN

    EPA Science Inventory

    We have obtained a panel of monoclonal antibodies directed against fathead minnow vitellogenin (Vtg) for use in sensitive ELISAs to quantify the response of exposure in vivo to estrogen or estrogen mimics.

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

    NASA Astrophysics Data System (ADS)

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

    1995-07-01

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

  1. Use of Human Hybridoma Technology To Isolate Human Monoclonal Antibodies.

    PubMed

    Smith, Scott A; Crowe, James E

    2015-02-01

    The human hybridoma technique offers an important approach for isolation of human monoclonal antibodies. A diversity of approaches can be used with varying success. Recent technical advances in expanding the starting number of human antigen-specific B cells, improving fusion efficiency, and isolating new myeloma partners and new cell cloning methods have enabled the development of protocols that make the isolation of human monoclonal antibodies from blood samples feasible. Undoubtedly, additional innovations that could improve efficiency are possible.

  2. Specific immunofluorescent staining of pathogenic treponemes with a monoclonal antibody.

    PubMed Central

    Ito, F; Hunter, E F; George, R W; Pope, V; Larsen, S A

    1992-01-01

    Two hybrid cell lines which produced mouse monoclonal antibody to the DAL-1 street strain of Treponema pallidum subsp. pallidum were established. These monoclonal antibodies strongly reacted with T. pallidum subsp. pallidum (Nichols strain, DAL-1, and two other street strains, strains MN-1 and MN-3) and T. pallidum subsp. pertenue by indirect microimmunofluorescent antibody and enzyme-linked immunosorbent assay techniques, but they did not react with normal rabbit testicular tissue. These monoclonal antibodies did not react with nonpathogenic treponemes, such as T. phagedenis Reiter, T. denticola MRB, T. refringens Noguchi, or other spirochetes, such as Borrelia burgdorferi and Leptospira interrogans serovar pomona in microimmunofluorescent antibody smear slides or in Western blots (immunoblots). While unlabeled antibodies are useful for investigating the antigenic structures of T. pallidum, we labeled these monoclonal antibodies with fluorescein isothiocyanate and used them for diagnosing syphilis by direct staining of lesion exudate or T. pallidum subsp. pallidum in formalin-fixed tissues from patients suspected of having syphilis. Both monoclonal antibodies were directed against antigens of T. pallidum subsp. pallidum with a molecular weight of 37,000 as determined by the Western blotting technique. Images PMID:1374079

  3. Monoclonal antibodies: new agents for cancer detection and targeted therapy

    SciTech Connect

    Baldwin, R.W.; Byers, V.S. )

    1991-01-01

    Antibodies directed against markers on cancer cells are gaining in importance for the purpose of targeting diagnostic and therapeutic agents. In the past, this approach has had very limited success principally because the classical methods for producing antibodies from blood serum of animals immunized with cancer cells or extracts were unsatisfactory. The situation has changed dramatically since 1975 following the design of procedures for 'immortalizing' antibody-producing cells (lymphocytes) by fusing them with cultured myeloma cells to form hybridomas which continuously secrete antibodies. Since these hybridomas produce antibodies coded for by a single antibody-producing cell, the antibodies are called monoclonal. Building on these advances in biomedical research, it is now possible to reproducibly manufacture monoclonal antibodies on a scale suitable for use in cancer detection and therapy.

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

    PubMed

    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.

  5. [Comparative studies on monoclonal antibody KM10 and anti-CEA monoclonal antibodies].

    PubMed

    Soyama, N; Yamamoto, M; Ohyanagi, H; Saitoh, Y

    1989-11-01

    The specificity of KM10 was evaluated in comparison with newly developed anti-CEA monoclonal antibodies (A10, B9, JA4, AH3). Both KM10 and all anti-CEA monoclonal antibodies reacted with CEA in ELISA system, and with adenocarcinoma of the stomach, colon, and pancreas in the immunohistochemical assay. B9, JA4, and AH3 were suggested to react with CEA related antigens, such as NCA and BGPI, whereas KM10 and A10 were suggested to recognize the distinctive part of CEA. The antigenic determinant of CEA reactive with KM10 and A10 was revealed to be protein moiety after enzyme treatment. The competitive binding inhibition assay, however, indicated that epitopes of KM10 and A10 were different each other. Enzyme immunoassay using both KM10 and A10 could detect CEA. These findings showed the possible use of both KM10 and A10 for clinical diagnosis and treatment by means of targeting for the distinctive part of CEA.

  6. Characterization and utilization of a monoclonal antibody against pancreatic carcinoma

    SciTech Connect

    Kurtzman, S.H.; Sindelar, W.F.; Atcher, R.W.; Mitchell, J.B.; DeGraff, W.G.; Gamson, J.; Russo, A.; Friedman, A.M.; Hines, J.J.

    1994-10-01

    A monoclonal antibody was produced against a human pancreatic adenocarcinoma line and was found to react with several different human carcinomas by immunoperoxidase staining of fixed tissues. The original cells used to generate the monoclonal antibody were treated with detergent to lyse the cell membrane. A membrane associated protein of molecular weight 35kD was isolated from this detergent lysed preparation and found to be recognized by the monoclonal antibody. The binding constant of the antigen antibody reaction on the cells is 5 x 10{sup {minus}5}. It was further determined that there are 700,000 binding sites per cell. Kinetics of the antigen-antibody reaction under several conditions were also explored.

  7. Characterization of monoclonal antibodies against Gnathostoma nipponicum.

    PubMed

    Ikadai, H; Fujii, T; Nagai, T; Yoshioka, K; Nagasao, J; Kudo, N; Oyamada, T

    2003-02-01

    Monoclonal antibodies (mAbs) were produced against the proteins of advanced third-stage larvae (AdL3) of Gnathostoma nipponicum. Six mAbs (Gn2C3, Gn2H3, Gn4C3, Gn4E9, GnSH1, and Gn10B7) were obtained as determined by enzyme-linked immunosorbent assay (ELISA). Gn4E9 and GnSH1 seemed to be genus-specific, as they did not cross-react with Anisakis sp., Dirofilaria immitis, Gongylonema pulchrum, Toxocara canis, Trichinella sp., Trichuris vulpis, Metagonimus sp., or Spirometra erinaceieuropaei by ELISA. Immunohistochemistry showed that Gn2C3, Gn4E9, and Gn5H1 reacted strongly with the central esophagus; Gn2H3 reacted with cuticle,muscle, intestine, and the cervical sac; and Gn4C3 and Gn10B7 reacted with cuticle, muscle, esophagus, intestine, and the cervical sac of AdL3. In Western blotting analysis, Gn2C3, Gn4E9, and Gn5H1 reacted to 60-, 53-, 46-, and 41-kDa proteins; Gn4C3 reacted to the AdL3 protein of G. nipponicum (>42 kDa). Moreover, proteins purified using a mAb Gn4E9 immunoprecipitation method (sizes 60-, 53-, 46-, and 41-kDa) were used as antigens in ELISAs. A significant difference (P < 0.01) was shown between mouse sera infected with G. nipponicum and sera infected with Trichnella sp. or not infected. These results provide a rationale for evaluating esophageal proteins for the development of diagnostic methods for detecting G. nipponicum or Gnathostoma sp. infections.

  8. Palladium-109 labeled anti-melanoma monoclonal antibodies

    DOEpatents

    Srivastava, S.C.; Fawwaz, R.A.; Ferrone, S.

    1984-04-30

    The invention consists of new monoclonal antibodies labelled with Palladium 109, a beta-emitting radionuclide, the method of preparing this material, and its use in the radiotherapy of melanoma. The antibodies are chelate-conjugated and demonstrate a high uptake in melanomas. (ACR)

  9. Prophylaxis and therapy of influenza pneumonia in mice by intratracheal instillation of monoclonal antibody

    SciTech Connect

    Ratcliffe, D.R.

    1985-01-01

    This study on passive immunity dealt principally with the following topics: pathogenesis of the pneumonia produced by influenza virus (PR8) in CF-1 mice; the distribution and retention of monoclonal antibody instilled intratracheally (IT) into the lung; and prophylaxis and therapy of influenza pneumonia using specific monoclonal antibody (IgG 2a/k anti-HA). The fate of a single 50 ul bolus of antibody instilled IT was determined by monitoring the activity of /sup 125/I-labelled monoclonal IgG in the lungs and by lavage recovery of functional antibody.Antibody was demonstrated in high concentrations for the first 3 days and was present in the lungs for a period of 7 days. For prophylaxis several trials indicated that monoclonal antibody provided significant protection from lethal effects of the virus. Antibody given to clinically ill mice on day 3 produced a highly significant reduction in mortality (P < 0.001) when compared to control mice. The treatment reversed the weight loss and apparently arrested the development of lesions in most of the mice within 2 days following antibody administration.

  10. A perspective of monoclonal antibodies: Past, present, and future

    SciTech Connect

    DeLand, F.H. )

    1989-07-01

    In 1975, the development of the technique to produce monoclonal antibodies revolutionized the approach to cancer detection and therapy. Hundreds of monoclonal antibodies to the epitopes of tumor cells have been produced, providing more specific tools for probing the cellular elements of cancer. At the same time, these tools have disclosed greater complexity in the character of these cells and stimulated further investigation. Although there are antibodies to specific epitopes of neoplastic cells, this purity has not provided the improved detection and therapy of cancer first expected. Technical manipulations have provided limited improvement in results, but more sophisticated techniques, such as biologic response modifiers, may be required to attain clinical results that can be universally applied. The intense research in monoclonal antibodies and their application does offer promise that the goal of improved cancer detection and therapy will be forthcoming. 58 references.

  11. Complete De Novo Assembly of Monoclonal Antibody Sequences

    PubMed Central

    Tran, Ngoc Hieu; Rahman, M. Ziaur; He, Lin; Xin, Lei; Shan, Baozhen; Li, Ming

    2016-01-01

    De novo protein sequencing is one of the key problems in mass spectrometry-based proteomics, especially for novel proteins such as monoclonal antibodies for which genome information is often limited or not available. However, due to limitations in peptides fragmentation and coverage, as well as ambiguities in spectra interpretation, complete de novo assembly of unknown protein sequences still remains challenging. To address this problem, we propose an integrated system, ALPS, which for the first time can automatically assemble full-length monoclonal antibody sequences. Our system integrates de novo sequencing peptides, their quality scores and error-correction information from databases into a weighted de Bruijn graph to assemble protein sequences. We evaluated ALPS performance on two antibody data sets, each including a heavy chain and a light chain. The results show that ALPS was able to assemble three complete monoclonal antibody sequences of length 216–441 AA, at 100% coverage, and 96.64–100% accuracy. PMID:27562653

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

    PubMed

    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.

  13. Neutralizing human monoclonal antibodies prevent Zika virus infection in macaques.

    PubMed

    Magnani, Diogo M; Rogers, Thomas F; Beutler, Nathan; Ricciardi, Michael J; Bailey, Varian K; Gonzalez-Nieto, Lucas; Briney, Bryan; Sok, Devin; Le, Khoa; Strubel, Alexander; Gutman, Martin J; Pedreño-Lopez, Núria; Grubaugh, Nathan D; Silveira, Cassia G T; Maxwell, Helen S; Domingues, Aline; Martins, Mauricio A; Lee, David E; Okwuazi, Erica E; Jean, Sherrie; Strobert, Elizabeth A; Chahroudi, Ann; Silvestri, Guido; Vanderford, Thomas H; Kallas, Esper G; Desrosiers, Ronald C; Bonaldo, Myrna C; Whitehead, Stephen S; Burton, Dennis R; Watkins, David I

    2017-10-04

    Therapies to prevent maternal Zika virus (ZIKV) infection and its subsequent fetal developmental complications are urgently required. We isolated three potent ZIKV-neutralizing monoclonal antibodies (nmAbs) from the plasmablasts of a ZIKV-infected patient-SMZAb1, SMZAb2, and SMZAb5-directed against two different domains of the virus. We engineered these nmAbs with Fc LALA mutations that abrogate Fcγ receptor binding, thus eliminating potential therapy-mediated antibody-dependent enhancement. We administered a cocktail of these three nmAbs to nonhuman primates 1 day before challenge with ZIKV and demonstrated that the nmAbs completely prevented viremia in serum after challenge. Given that numerous antibodies have exceptional safety profiles in humans, the cocktail described here could be rapidly developed to protect uninfected pregnant women and their fetuses. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

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

    NASA Astrophysics Data System (ADS)

    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.

  15. [Preparation and Assessment of IL1RAP Monoclonal Antibody].

    PubMed

    Yin, Ling-Ling; Ruan, Su-Hong; Tian, Yu; Xu, Kai Lin; Zhao, Kai

    2015-08-01

    To prepare and identify human monoclonal antibody against IL-1 receptor accessory protein (IL1RAP), which is a new identified surface marker for leukemia stem cells (LSC). BALB/c mice were inoculated intraperitoneally with hybridoma cells (3H6E10, 10D8A7) and their ascites were collected. The monoclonal antibody against hu-IL1RAP specifically was purified from ascites, the nondenaturing-PAGE, ELISA and Western blot were used to detect the purity, titer and sensitivity of antibody. Two purified antibodies were obtained and named as 3H6E10 McAb and 10D8A7 McAb, whose purity was 95% and 94% respectively. The titer of two purified monoclonal antibodies was 1 : 81000 and specific conjugation of IL1RAP purified protein and endogenous protein from normal people and leukemia patients with purified antibodies were confirmed. The purified monoclonal antibodies which can specifically bind to hu-IL1RAP are successfully prepared, thus providing novel way to effectively clear LSC in the future.

  16. Functional Characterization and Evaluation of In Vitro Protective Efficacy of Murine Monoclonal Antibodies BURK24 and BURK37 against Burkholderia pseudomallei

    PubMed Central

    Peddayelachagiri, Bhavani V.; Paul, Soumya; Makam, Shivakiran S.; Urs, Radhika M.; Kingston, Joseph J.; Tuteja, Urmil; Sripathy, Murali H.; Batra, Harsh V.

    2014-01-01

    Burkholderia pseudomallei, the causative agent of melioidosis has been recognized by CDC as a category B select agent. Although substantial efforts have been made for development of vaccine molecules against the pathogen, significant hurdles still remain. With no licensed vaccines available and high relapse rate of the disease, there is a pressing need for development of alternate protection strategies. Antibody-mediated passive protection is promising in this regard and our primary interest was to unravel this frontier of specific mAbs against Burkholderia pseudomallei infections, as functional characterization of antibodies is a pre-requisite to demonstrate them as protective molecules. To achieve this, we designed our study on in vitro-based approach and assessed two mAbs, namely BURK24 and BURK37, reactive with outer membrane proteins and lipopolysaccharide of the pathogen respectively, for their ability to manifest inhibitory effects on the pathogenesis mechanisms of B. pseudomallei including biofilm formation, invasion and induction of apoptosis. The experiments were performed using B. pseudomallei standard strain NCTC 10274 and a clinical isolate, B. pseudomallei 621 recovered from a septicemia patient with diabetic ailment. The growth kinetic studies of the pathogen in presence of various concentrations of each individual mAb revealed their anti-bacterial properties. Minimal inhibitory concentration and minimal bactericidal concentration of both the mAbs were determined by using standards of Clinical and Laboratory Standards Institute (CLSI) and experiments were performed using individual mAbs at their respective bacteriostatic concentration. As an outcome, both mAbs exhibited significant anti-Burkholderia pseudomallei properties. They limited the formation of biofilm by the bacterium and completely crippled its invasion into human alveolar adenocarcinoma epithelial cells. Also, the mAbs were appreciably successful in preventing the bacterium to induce

  17. Two novel anti-von Willebrand factor monoclonal antibodies.

    PubMed

    Spadafora-Ferreira, M; Lopes, A A; Coelho, V; Guilherme, L; Kalil, J

    2000-01-15

    Von Willebrand Factor is a multimer produced by endothelial cells and megakaryocytes, being stored in intracellular organelles, such as the Weibel-Palade bodies and alpha-granules in endothelial cells and platelets, respectively. This molecule acts as a carrier protein for factor VIIIc, involved in the intrinsic pathway of blood coagulation maintaining its stability in circulation. Von Willebrand Factor also plays an important role in platelet aggregation and adhesion to injured vessel wall. It interacts with platelets through two distinct glycoproteins, GPIb and GPIIb/IIIa. We raised two monoclonal antibodies, ECA-3 and ECA-4, against human umbilical vascular endothelial cells that recognize and immunoprecipitate von Willebrand Factor. Interestingly, ECA-4 monoclonal antibody is able to completely inhibit platelet agglutination induced by ristocetin, suggesting that it binds to von Willebrand Factor close to platelet GPIb binding site. The use of monoclonal antibodies to identify von Willebrand Factor binding regions to factor VIII or platelets has been reported by others. In pulmonary hypertension, abnormalities have been detected on the multimeric structure of the molecule as well as on its proteolytic fragments, by using monoclonal antibodies. Moreover, monoclonal antibodies raised against specific regions of von Willebrand Factor molecule may allow studies of functional abnormalities of this protein in inherited and acquired disorders like subtypes of von Willebrand's disease.

  18. New monoclonal antibodies on the horizon in multiple myeloma

    PubMed Central

    O’Donnell, Elizabeth K.; Raje, Noopur S.

    2016-01-01

    Across all cancers, monoclonal antibodies have emerged as a potential strategy for cancer therapy. Monoclonal antibodies target antigens expressed on the surface of cancer cells and accessory cells. This targeted approach uses the host’s immune system to promote the killing of cancer cells. Multiple myeloma (MM) is the second most common hematologic malignancy that remains incurable in the majority of patients. The treatment of MM has evolved dramatically over the past decade and continues to evolve with the approval of four new drugs in 2015. Most recently the United States Food and Drug Administration (US FDA) approved two monoclonal antibodies for the treatment of this disease. Monoclonal antibodies are generally well-tolerated and offer a novel method of action for treated relapsed and refractory disease and are now being studied in the upfront setting. In this article, we review the evidence for the existing approved monoclonal antibodies and discuss promising targeted therapies and innovative strategies for the treatment of MM. PMID:28203341

  19. Breast cancer immunotherapy: monoclonal antibodies and peptide-based vaccines.

    PubMed

    Mohit, Elham; Hashemi, Atieh; Allahyari, Mojgan

    2014-07-01

    Recently, immunotherapy has emerged as a treatment strategy in the adjuvant setting of breast cancer. In this review, monoclonal antibodies in passive and peptide-based vaccines, as one of the most commonly studied in active immunotherapy approaches, are discussed. Trastuzumab, a monoclonal antibody against HER-2/neu, has demonstrated considerable efficacy. However, resistance to trastuzumab has led to development of many targeted therapies which have been examined in clinical trials. Monoclonal antibodies against immune-checkpoint molecules that are dysregulated by tumors as an immune resistance mechanism are also explained in this review. Additionally, monoclonal antibodies with the ability to target breast cancer stem cells that play a role in cancer recurrence are mentioned. Here, clinical trials of HER-2/neu B and T cells, MUC1 and hTERT cancer peptide vaccines are also presented. In addition, various strategies for enhancing vaccine efficacy including combination with monoclonal antibodies and using different delivery systems for peptide/protein-based vaccine are described.

  20. Reversal of intracellular toxicity of the trichothecene mycotoxin T-2 with monoclonal antibody.

    PubMed

    Hunter, K W; Brimfield, A A; Knower, A T; Powell, J A; Feuerstein, G Z

    1990-12-01

    The trichothecene mycotoxin T-2 is a potent inhibitor of intracellular protein synthesis. We have previously shown that a mouse immunoglobulin G1 monoclonal antibody (15H6) specific for T-2 toxin can neutralize the in vitro protein synthesis inhibitory effect of the toxin in human B-lymphoblastoid cultures, and protect rats from lethal toxemia. We now report that these monoclonal antibodies can induce the net efflux of [3H]-T-2 toxin from poisoned human B-lymphoblastoid cells in vitro, and restore protein synthesis. Administration of the monoclonal antibodies (250 mg/kg) 30 min before infusion of a lethal dose (1 mg/kg) of T-2 toxin causes the sequestration of the toxin in the plasma compartment. When administered 35 min after T-2 toxin, a time when the bulk of toxin is in the tissues, the monoclonal antibodies facilitate the migration of toxin back into the plasma compartment. These data demonstrate that monoclonal antibodies can be of therapeutic value against an intracellular toxin.

  1. Novel monoclonal antibodies to study tissue regeneration in planarians.

    PubMed

    Ross, Kelly G; Omuro, Kerilyn C; Taylor, Matthew R; Munday, Roma K; Hubert, Amy; King, Ryan S; Zayas, Ricardo M

    2015-01-21

    Planarians are an attractive model organism for studying stem cell-based regeneration due to their ability to replace all of their tissues from a population of adult stem cells. The molecular toolkit for planarian studies currently includes the ability to study gene function using RNA interference (RNAi) and observe gene expression via in situ hybridizations. However, there are few antibodies available to visualize protein expression, which would greatly enhance analysis of RNAi experiments as well as allow further characterization of planarian cell populations using immunocytochemistry and other immunological techniques. Thus, additional, easy-to-use, and widely available monoclonal antibodies would be advantageous to study regeneration in planarians. We have created seven monoclonal antibodies by inoculating mice with formaldehyde-fixed cells isolated from dissociated 3-day regeneration blastemas. These monoclonal antibodies can be used to label muscle fibers, axonal projections in the central and peripheral nervous systems, two populations of intestinal cells, ciliated cells, a subset of neoblast progeny, and discrete cells within the central nervous system as well as the regeneration blastema. We have tested these antibodies using eight variations of a formaldehyde-based fixation protocol and determined reliable protocols for immunolabeling whole planarians with each antibody. We found that labeling efficiency for each antibody varies greatly depending on the addition or removal of tissue processing steps that are used for in situ hybridization or immunolabeling techniques. Our experiments show that a subset of the antibodies can be used alongside markers commonly used in planarian research, including anti-SYNAPSIN and anti-SMEDWI, or following whole-mount in situ hybridization experiments. The monoclonal antibodies described in this paper will be a valuable resource for planarian research. These antibodies have the potential to be used to better understand

  2. Impact of cell culture on recombinant monoclonal antibody product heterogeneity.

    PubMed

    Liu, Hongcheng; Nowak, Christine; Shao, Mei; Ponniah, Gomathinayagam; Neill, Alyssa

    2016-09-01

    Recombinant monoclonal antibodies are commonly expressed in mammalian cell culture and purified by several steps of filtration and chromatography. The resulting high purity bulk drug substance still contains product variants differing in properties such as charge and size. Posttranslational modifications and degradations occurring during cell culture are the major sources of heterogeneity in bulk drug substance of recombinant monoclonal antibodies. The focus of the current review is the impact of cell culture conditions on the types and levels of various modifications and degradations of recombinant monoclonal antibodies. Understanding the relationship between cell culture and product variants can help to make consistently safe and efficacious products. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1103-1112, 2016. © 2016 American Institute of Chemical Engineers.

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

    PubMed

    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.

  4. Monoclonal antibodies specific for African swine fever virus proteins.

    PubMed Central

    Sanz, A; García-Barreno, B; Nogal, M L; Viñuela, E; Enjuanes, L

    1985-01-01

    We have obtained 60 stable hybridomas which produced immunoglobulins that recognized 12 proteins from African swine fever virus particles and African swine fever virus-infected cells. Most of the monoclonal antibodies were specific for the three major structural proteins p150, p72, and p12. The specificity of some monoclonal antibodies for the structural proteins p150 and p37 and the nonstructural proteins p220 and p60 indicated that proteins p150 and p220 are antigenically related to proteins p37 and p60. The association of some viral antigens to specific subcellular components was determined by immunofluorescence and analysis of the binding of monoclonal antibodies to infected cells. A host protein (p24) seemed to be associated with the virus particles. Images PMID:3882998

  5. Choriocarcinoma: blocking factor and monoclonal antibody iodine 131 imaging

    SciTech Connect

    Pattillo, R.A.; Khazaeli, M.B.; Ruckert, A.C.; Hussa, R.O.; Collier, B.D.; Beierwaltes, W.; Mattingly, R.F.

    1984-04-01

    Postoperative iodine 131 monoclonal antibody localization in metastatic choriocarcinoma was accomplished in this study. The monoclonal antibody was prepared to male choriocarcinoma which cross reacted with gestational choriocarcinoma. The antibody was raised against whole choriocarcinoma cells and human chorionic gonadotropin (hCG) cross reactivity was excluded. The purified antibody was iodinated with /sup 131/I and successfully imaged BeWo choriocarcinoma transplanted in nude mice; however, imaging of choriocarcinoma in a patient was verified only after resection. It is our belief that failure to sufficiently concentrate the antibody in the tumor before operation was due to blocking factor in the serum of the patient. Blocking factor and hCG dropped postoperatively. Blocking factor activity in 15 patients with metastatic trophoblastic disease was monitored and, like hCG, was found to be a sensitive indicator of the presence of disease. Its efficacy may be in the small number of patients without hCG but with persistent disease.

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

    PubMed Central

    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

  7. The clinical application of monoclonal antibody therapies in renal transplantation.

    PubMed

    Dhanireddy, Kiran K; Xu, He; Mannon, Roslyn B; Hale, Douglas A; Kirk, Allan D

    2004-05-01

    Monoclonal antibodies have become valuable tools for the precise clinical manipulation of the immune system. These highly specific proteins have proven their usefulness in both the treatment and prevention of organ transplant rejection. Indeed, they are the centrepieces of many evolving regimens designed to reduce or eliminate the need for chronic immunosuppression. This manuscript will review the monoclonal antibodies that have made their way into the clinic either as experimental therapies or approved drugs. It will provide a general overview of this class of agents and their mechanisms of action. Standard therapies and potential new applications will be described.

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

    PubMed

    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.

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

    PubMed

    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.

  10. ERBB oncogene proteins as targets for monoclonal antibodies.

    PubMed

    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.

  11. Monoclonal Antibodies Against Human Cardiac Troponin I for Immunoassays II.

    PubMed

    Lee, Gregory; Liu, Suefay

    2015-06-01

    Human cardiac troponin I (cTnI) is one of the most specific biomarkers for detection of acute myocardial infarction (AMI). To formulate immunoassay kits for rapid immunodiagnosis of AMI, monoclonal antibodies with high affinity and specificity were generated against cTnI and subsequently tested through a series of experiments. C57BL/6 mice were immunized with cTnI as the immunogen and cell fusions with myeloma cells of BALB/c origin were performed to generate hybridomas. The supernatants of the hybridoma cell culture were routinely screened for antibody secretions against intact cTnI and synthetic peptides from the N-terminal half of cTnI (amino acid residues N1-30, N24-40, N59-79, and N80-95). Monoclonal antibodies specific to different epitope regions were then determined and selected, according to their respective affinity and specificity, for formulation of enzyme immunoassay kits. The results of this study found that most of the selected antibodies revealed comparable binding affinity to cTnI and to the corresponding synthetic peptides. Optimal sandwich enzyme immunoassays with high sensitivity could be achieved through proper combinations of the epitope-distinct monoclonal antibodies in different capture-detection pairs; signal enhancements were frequently observed when a mixture of epitope-distinct anti-cTnI monoclonal antibodies was used for coating. This indicates that a combination of epitope-distinct anti-cTnI monoclonal antibodies recognizing the N-terminal half of cTnI yield reliable detection and greater sensitivity for cTnI in AMI patients.

  12. Characterization of monoclonal antibodies against human apolipoprotein E.

    PubMed Central

    Milne, R W; Douste-Blazy, P; Marcel, Y L; Retegui, L

    1981-01-01

    From a single cell fusion, five stable hybridomas secreting antiapolipoprotein E (apo E) were obtained. The immunoglobulin (Ig)G subclasses containing the respective monoclonal antibodies were isolated and were used as the antibody component in a solid-phase radioimmunoassay. The binding of 125I-apo E to the insolubilized antibody was inhibited by unlabeled apo E but not by unlabeled apoproteins A-I, A-II, C-II, and C-III, or by low density lipoprotein immunodepleted of endogenous apo E. Competition curves were obtained with lipoprotein subfractions that had the same shape as those obtained with purified apo E. Apo E levels in normal and hyperlipidemic plasma were well correlated when measured by the five monoclonal antibodies and polyclonal anti-apo E, although differences in absolute values were observed. In normal subjects 34, 10, 20, and 36% of apo E was recovered in the very low density lipoprotein, low density lipoprotein, high density lipoprotein, and the d greater than 1.21-gl/ml fractions, respectively, whereas these values were 34, 7, 12, and 47%, respectively, in type III patients. All antibodies indicated the same subfraction distribution of apo E. The monoclonal antibodies reacted with all of the isomorphs of apo E. One of the antibodies could be clearly distinguished by its reactivity with chemically modified very low density lipoprotein. Images PMID:6788802

  13. Recent Advances in Monoclonal Antibody Therapies for Multiple Sclerosis.

    PubMed

    Wootla, Bharath; Watzlawik, Jens O; Stavropoulos, Nikolaos; Wittenberg, Nathan J; Dasari, Harika; Abdelrahim, Murtada A; Henley, John R; Oh, Sang-Hyun; Warrington, Arthur E; Rodriguez, Moses

    2016-06-01

    Multiple sclerosis (MS) is the most common chronic inflammatory, demyelinating disease of the CNS and results in neurological disability. Existing immunomodulatory and immunosuppressive approaches lower the number of relapses but do not cure or reverse existing deficits nor improve long-term disability in MS patients. Monogenic antibodies were described as treatment options for MS, however the immunogenicity of mouse antibodies hampered the efficacy of potential therapeutics in humans. Availability of improved antibody production technologies resulted in a paradigm shift in MS treatment strategies. In this review, an overview of immunotherapies for MS that use conventional monoclonal antibodies reactive to immune system and their properties and mechanisms of action will be discussed, including recent advances in MS therapeutics and highlight natural autoantibodies (NAbs) that directly target CNS cells. Recent challenges for MS therapy are the identification of relevant molecular and cellular targets, time frame of treatment, and antibody toxicity profiles to identify safe treatment options for MS patients. The application of monoclonal antibody therapies with better biological efficacy associated with minimum side effects possesses huge clinical potential. Advances in monoclonal antibody technologies that directly target cells of nervous system may promote the CNS regeneration field from bench to bedside.

  14. Recent Advances in Monoclonal Antibody Therapies for Multiple Sclerosis

    PubMed Central

    Stavropoulos, Nikolaos; Wittenberg, Nathan J.; Dasari, Harika; Abdelrahim, Murtada A.; Henley, John R.; Oh, Sang-Hyun; Warrington, Arthur E.; Rodriguez, Moses

    2016-01-01

    Introduction Multiple sclerosis (MS) is the most common chronic inflammatory, demyelinating disease of the CNS and results in neurological disability. Existing immunomodulatory and immunosuppressive approaches lower the number of relapses but do not cure or reverse existing deficits nor improve long-term disability in MS patients. Areas Covered Monogenic antibodies were described as treatment options for MS, however the immunogenicity of mouse antibodies hampered the efficacy of potential therapeutics in humans. Availability of improved antibody production technologies resulted in a paradigm shift in MS treatment strategies. In this review, an overview of immunotherapies for MS that use conventional monoclonal antibodies reactive to immune system and their properties and mechanisms of action will be discussed, including recent advances in MS therapeutics and highlight natural autoantibodies (NAbs) that directly target CNS cells. Expert Opinion Recent challenges for MS therapy are the identification of relevant molecular and cellular targets, time frame of treatment, and antibody toxicity profiles to identify safe treatment options for MS patients. The application of monoclonal antibody therapies with better biological efficacy associated with minimum side effects possesses huge clinical potential. Advances in monoclonal antibody technologies that directly target cells of nervous system may promote the CNS regeneration field from bench to bedside. PMID:26914737

  15. Identification of mutant monoclonal antibodies with increased antigen binding.

    PubMed

    Pollock, R R; French, D L; Gefter, M L; Scharff, M D

    1988-04-01

    Sib selection and an ELISA have been used to isolate hybridoma subclones producing mutant antibodies that bind antigen better than the parental monoclonal antibody. Such mutants arise spontaneously in culture at frequencies of 2.5-5 X 10(-5). The sequences of the heavy and light chain variable regions of the mutant antibodies are identical to that of the parent and the Ka values of the mutants and the parent are the same. The increase in binding is associated with abnormalities of the constant region polypeptide and probably reflect changes in avidity of these antibodies.

  16. Prediction and reduction of the aggregation of monoclonal antibodies.

    PubMed

    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

    2017-03-17

    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 (APRs) and their contribution to the thermodynamic stability of the protein. Although APRs 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.

  17. Monoclonal antibodies to endogenous galactose-specific tumor cell lectins.

    PubMed Central

    Raz, A; Meromsky, L; Carmi, P; Karakash, R; Lotan, D; Lotan, R

    1984-01-01

    A monoclonal antibody, 5D7, was obtained after immunization of syngeneic mice with B16 melanoma cell extracts enriched for endogenous lectin activity and screening for inhibition of lectin-mediated hemagglutination. Binding of this antibody to affinity-purified B16 melanoma galactoside-specific lectin was revealed by solid-phase radioimmunoassay and binding to the surface of viable B16 cells was demonstrated by indirect immunofluorescence. Inhibition of lectin activity and cell surface labeling by 5D7 antibody were also found with several types of cultured human and murine cells including melanoma, sarcoma and carcinoma. This monoclonal antibody should be useful for evaluating the role of tumor cell surface lectins in intercellular interactions and metastasis. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:6084591

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

    PubMed

    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.

  19. Monoclonal Antibodies Attached to Carbon Nanotube Transistors for Paclitaxel Detection

    NASA Astrophysics Data System (ADS)

    Lee, Wonbae; Lau, Calvin; Richardson, Mark; Rajapakse, Arith; Weiss, Gregory; Collins, Philip; UCI, Molecular Biology; Biochemistry Collaboration; UCI, Departments of Physics; Astronomy Collaboration

    Paclitaxel is a naturally-occurring pharmaceutical used in numerous cancer treatments, despite its toxic side effects. Partial inhibition of this toxicity has been demonstrated using weakly interacting monoclonal antibodies (3C6 and 8A10), but accurate monitoring of antibody and paclitaxel concentrations remains challenging. Here, single-molecule studies of the kinetics of antibody-paclitaxel interactions have been performed using single-walled carbon nanotube field-effect transistors. The devices were sensitized with single antibody attachments to record the single-molecule binding dynamics of paclitaxel. This label-free technique recorded a range of dynamic interactions between the antibody and paclitaxel, and it provided sensitive paclitaxel detection for pM to nM concentrations. Measurements with two different antibodies suggest ways of extending this working range and uncovering the mechanistic differences among different antibodies.

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

    DTIC Science & Technology

    2007-07-01

    conjugate to immunize rats. We found that vaccination by either the intraperitoneal or subcutaneous route induced very high aflatoxin B1 -binding antibody...titers that were completely competed by free aflatoxin BB1 in solution. We will make and characterize monoclonal antibodies to aflatoxins B1 and G1...their potential for use in the event of bioterrorism has been highlighted (1). Immunization with an aflatoxin B1 (AF- B1 )-BSA conjugate previously

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

    PubMed

    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.

  2. Cooperative Immunoassays: Ultrasensitive Assays with Mixed Monoclonal Antibodies

    NASA Astrophysics Data System (ADS)

    Ehrlich, Paul H.; Moyle, William R.

    1983-07-01

    Mixtures of certain monoclonal antibodies appear to bind human chorionic gonadotropin in a ``cooperative'' fashion because they form circular complexes with the hormone. Experiments illustrate how this property might be exploited to develop very sensitive immunoassays for human chorionic gonadotropin or any other antigen. Since the assays are not based on competitive inhibition between radiolabeled and unlabeled antigen, they are much more sensitive than a traditional radioimmunoassay in which either one of the same antibodies is used alone.

  3. Generation of monoclonal antibodies to recombinant vascular endothelial growth factor.

    PubMed

    Shein, S A; Gurina, O I; Leopol'd, A V; Baklaushev, V P; Korchagina, A A; Grinenko, N F; Ivanova, N V; Volgina, N E; Ryabukhin, I A; Chekhonin, V P

    2012-05-01

    Female BALB/c mice were subcutaneously immunized with recombinant VEGF-164. After 3 immunization cycles, splenic B cells from immunized mouse were fused with immortalized myeloma culture SP2/0-Ag14 cells. Screening of hybrid cells producing anti-VEGF antibodies was performed by ELISA and immunocytochemical analysis on cultured C6 glioma cells. Subsequent cloning yielded hybridoma stably expressing monoclonal anti-VEGF antibodies recognizing recombinant and native VEGF.

  4. Targeted therapeutics for severe refractory asthma: monoclonal antibodies.

    PubMed

    Grainge, Christopher L; Maltby, Steven; Gibson, Peter G; Wark, Peter A B; McDonald, Vanessa M

    2016-07-01

    Severe asthma is a complex multifactorial disease that requires specialist multidisciplinary input for optimal clinical outcomes. Following multidimensional assessment for optimisation of current therapy, self-management skills and comorbidities, all patients should be accurately phenotyped. Only after this assessment has been completed should new monoclonal antibody therapies be considered. In this review, we summarise the new antibody approaches targeting identified pathological pathways in severe refractory asthma.

  5. Mechanisms of monoclonal antibody stabilization and release from silk biomaterials

    PubMed Central

    Guziewicz, Nicholas A.; Massetti, Andrew J.; Perez-Ramirez, Bernardo J.; Kaplan, David L.

    2013-01-01

    The availability of stabilization and sustained delivery systems for antibody therapeutics remains a major clinical challenge, despite the growing development of antibodies for a wide range of therapeutic applications due to their specificity and efficacy. A mechanistic understanding of protein-matrix interactions is critical for the development of such systems and is currently lacking as a mode to guide the field. We report mechanistic insight to address this need by using well-defined matrices based on silk gels, in combination with a monoclonal antibody. Variables including antibody loading, matrix density, charge interactions, hydrophobicity and water access were assessed to clarify mechanisms involved in the release of antibody from the biomaterial matrix. The results indicate that antibody release is primarily governed by hydrophobic interactions and hydration resistance, which are controlled by silk matrix chemistry, peptide domain distribution and protein density. Secondary ionic repulsions are also critical in antibody stabilization and release. Matrix modification by free methionine incorporation was found to be an effective strategy for mitigating encapsulation induced antibody oxidation. Additionally, these studies highlight a characterization approach to improve the understanding and development of other protein sustained delivery systems, with broad applicability to the rapidly developing monoclonal antibody field. PMID:23859659

  6. The interaction between pertussis toxin and 10 monoclonal antibodies.

    PubMed

    Schou, C; Au-Jensen, M; Heron, I

    1987-10-01

    Data on the epitope specificity of 10 monoclonal hybridoma antibodies (Mabs) that showed positive reaction in enzyme-linked immunosorbent assay (ELISA) towards pertussins toxin (Ptx) are presented. The relative functional affinity of the Mabs was determined in a catching ELISA system. The Mabs were tested for their ability to inhibit the biological activities of this toxin in two in vitro systems, viz. haemagglutination (HA) and Chinese Hamster Ovary cell (CHO) test, and in three in vivo assays: histamine sensitization (HS), leucocytosis-promoting activity (LP) and protection against intra-cerebral challenge (i.c.) with virulent B. pertussis organisms. Four Mabs were found inhibiting HA and three inhibited the effect on CHO cells. Two Mabs showed demonstrable protective effect on mice in i.c. test. The same two Mabs were also able to inhibit HS and LP activity of Ptx. Five of the ten Mabs reacted with Ptx subjected to blotting after separation of the toxin subunits in sodium-dodecyl sulphate polyacrylamide gel electrophoresis. The five Mabs all bound to more than one subunit. The epitopes defined by several of the Mabs might be useful in the context of a third-generation whooping cough vaccine.

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

    PubMed

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

    2017-10-01

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

  8. Identification of two antigenic determinants in pseudomurein by monoclonal antibodies

    SciTech Connect

    Conway de Macario, E.; Macario, A.J.L.; Kandler, O.; Wolin, M.J.

    1982-01-01

    Pseudomurein is a unique peptidoglycan found only in the wall of methanogenic bacteria (MB) of the family Methanobacteriaceae. Although its chemical composition has recently been determined, its immunologic properties have not been elucidated. Methanobacteriaceae elicit antibodies in rabbits and mice. The authors have produced monoclonal antibodies against the bacteria. Antigenic determinants on the MB's surface were resolved with the monoclonal antibodies by means of inhibition-blocking procedures combined with immunoenzymatic assays devised for the structural analysis of bacterial antigens. One monoclonal antibody against Methanobrevibacter arboriphilus DHl recognized a determinant involving the ..gamma..-Glu-Ala end of the pseudomurein peptide. A second antibody did not react with the above determinant but with another involving N-acetylglucosamine. The latter antibody reacted with the immunizing MB, i.e. Methanobacterium thermoautotrophicum ..delta..H and with another strain of this species, GGl, but it did not react with the rest of the pseudomurein-containing bacteria. The data show that pseudomurein possess at least two different determinants, one in the C-terminus of the peptide moiety and the other in the backbone structure and indicate that the spatial arrangement of the peptidoglycan components is distinctive for the species examined and plays a role in antigenicity.

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

    PubMed

    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.

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

    PubMed

    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.

  11. Pulmonary monoclonal antibody delivery via a portable microfluidic nebulization platform

    PubMed Central

    Cortez-Jugo, Christina; Qi, Aisha; Rajapaksa, Anushi; Friend, James R.

    2015-01-01

    Nebulizers have considerable advantages over conventional inhalers for pulmonary drug administration, particularly because they do not require coordinated breath actuation to generate and deliver the aerosols. Nevertheless, besides being less amenable to miniaturization and hence portability, some nebulizers are prone to denature macromolecular drugs due to the large forces generated during aerosolization. Here, we demonstrate a novel portable acoustomicrofluidic device capable of nebulizing epidermal growth factor receptor (EGFR) monoclonal antibodies into a fine aerosol mist with a mass median aerodynamic diameter of approximately 1.1 μm, optimal for deep lung deposition via inhalation. The nebulized monoclonal antibodies were tested for their stability, immunoactivity, and pharmacological properties, which confirmed that nebulization did not cause significant degradation of the antibody. In particular, flow cytometry demonstrated that the antigen binding capability of the antibody is retained and able to reduce phosphorylation in cells overexpressing the EGFR, indicating that the aerosols generated by the device were loaded with stable and active monoclonal antibodies. The delivery of antibodies via inhalation, particularly for the treatment of lung cancer, is thus expected to enhance the efficacy of this protein therapeutic by increasing the local concentration where they are needed. PMID:25945147

  12. Bacterial surface antigens defined by monoclonal antibodies: the methanogens

    SciTech Connect

    Conway de Macario, E.; Macario, A.J.L.; Magarinos, M.C.; Jovell, R.J.; Kandler, O.

    1982-01-01

    The methanogens (MB) are unique microbes of great evolutionary interest with applications in biotechnology-bioengineerings and are important in digestive processes. Their cell-wall composition is distinctively different from that of Eubacteria, e.g. the Methanobacteriaceae possess the peptidoglycan pseudomurein rather than murein. The range of cell-wall compositions among MB and their evolutionary and functional significance is not well known. The authors undertook a systematic study of the MB's surface structure using monoclonal antibodies through the following steps: (1) generation of hybridomas that produce antibody to several MB from 3 of their 4 families; (2) development of immunoenzymatic assays for MB's antigens and antibodies; (3) determination of the fine specificity of monoclonal antibodies by inhibition-blocking tests using cell-wall extracts and compounds of known structure; thus a set of monoclonal probes of predetermined specificity was assembled; and (4) resolution of surface determinants of MB representative of the Methanobacteriaceae using the monoclonal probes. Specific markers of MB strains were characterized. Two epitopes were identified within the pseudomurein molecule.

  13. Indium-111 labeled anti-melanoma monoclonal antibodies

    DOEpatents

    Srivastava, S.C.; Fawwaz, R.A.; Ferrone, S.

    1984-04-30

    A monoclonal antibody to a high molecular weight melanoma-associated antigen was chelated and radiolabeled with indium-111. This material shows high affinity for melanoma and thus can be used in the detection, localization and imaging of melanoma. 1 figure.

  14. Binding properties of monoclonal antibodies to rabies virus.

    PubMed

    Cusi, M G; Valensin, P E; Tollis, M; Bracci, L; Petreni, S; Soldani, P

    1991-07-01

    The monoclonal antibodies (mAbs) obtained by immunizing mice with a tetradecapeptide corresponding to the 190-203 region of rabies virus glycoprotein, involved in binding to the acetylcholine receptor (AchR), displayed different specificities to different rabies virus strains. These mAbs, when used in immunofluorescence tests, allowed differentiation of wild rabies viruses from the attenuated ones.

  15. Development and evaluation of monoclonal antibodies for paxilline

    USDA-ARS?s Scientific Manuscript database

    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 concentrati...

  16. A mouse monoclonal antibody against Alexa Fluor 647.

    PubMed

    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. Anti-glycoprotein D monoclonal antibody protects against herpes simplex virus type 1-induced diseases in mice functionally depleted of selected T-cell subsets or asialo GM1+ cells.

    PubMed Central

    Staats, H F; Oakes, J E; Lausch, R N

    1991-01-01

    Passive transfer of a monoclonal antibody (MAb) specific for glycoprotein D (gD) is highly effective in preventing the development of herpes simplex virus type 1-induced stromal keratitis. In the present study, we investigated whether animals which had been functionally depleted of T-cell subsets or asialo GM1+ cells would continue to be responsive to MAb therapy. BALB/c mice were depleted of CD4+, CD8+, or asialo GM1+ cells by treatment with anti-L3T4, anti-Lyt 2.2, or anti-asialo GM1 antibodies, respectively. Functional depletion of CD4+ cells was documented by the loss of delayed-type hypersensitivity responsiveness, while CD8+ cell depletion was accompanied by abrogation of cytotoxic lymphocyte activity. Anti-asialo GM1 treatment led to the loss of natural killer cell lytic activity. Mice depleted of the desired cell population and infected on the scarified cornea with herpes simplex virus type 1 uniformly developed necrotizing stromal keratitis by 3 weeks postinfection. A single inoculation of anti-gD MAb (55 micrograms) given intraperitoneally 24 h postinfection strongly protected hosts depleted of CD4+ cells against stromal keratitis. Likewise, antibody treatment in CD8+ or asialo GM1+ cell-depleted hosts was as therapeutically effective as that seen in non-cell-depleted mice. We also observed that in cell-depleted mice, the virus spread into the central nervous system and caused encephalitis. The CD4+ cell-depleted mice were the most severely affected, as 100% developed fatal disease. Anti-gD MAb treatment successfully protected all (32 of 32) CD4+-, CD8+-, or asialo GM1(+)-depleted hosts against encephalitis. We therefore conclude that antibody-mediated prevention of stromal keratitis and encephalitis does not require the obligatory participation of CD4+, CD8+, or asialo GM1+ cells. However, when mice were simultaneously depleted of both CD4+ and CD8+ T-cell subsets, antibody treatment could not prevent fatal encephalitis. Thus, antibody can compensate for

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

    PubMed

    Rossmann, Friederike S; Laverde, Diana; Kropec, Andrea; Romero-Saavedra, Felipe; Meyer-Buehn, Melanie; Huebner, Johannes

    2015-01-01

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

  19. Isolation of Highly Active Monoclonal Antibodies against Multiresistant Gram-Positive Bacteria

    PubMed Central

    Rossmann, Friederike S.; Laverde, Diana; Kropec, Andrea; Romero-Saavedra, Felipe; Meyer-Buehn, Melanie; Huebner, Johannes

    2015-01-01

    Multiresistant nosocomial pathogens often cause life-threatening infections that are sometimes untreatable with currently available antibiotics. Staphylococci and enterococci are the predominant Gram-positive species associated with hospital-acquired infections. These infections often lead to extended hospital stay and excess mortality. In this study, a panel of fully human monoclonal antibodies was isolated from a healthy individual by selection of B-cells producing antibodies with high opsonic killing against E. faecalis 12030. Variable domains (VH and VL) of these immunoglobulin genes were amplified by PCR and cloned into an eukaryotic expression vector containing the constant domains of a human IgG1 molecule and the human lambda constant domain. These constructs were transfected into CHO cells and culture supernatants were collected and tested by opsonophagocytic assay against E. faecalis and S. aureus strains (including MRSA). At concentrations of 600 pg/ml, opsonic killing was between 40% and 70% against all strains tested. Monoclonal antibodies were also evaluated in a mouse sepsis model (using S. aureus LAC and E. faecium), a mouse peritonitis model (using S. aureus Newman and LAC) and a rat endocarditis model (using E. faecalis 12030) and were shown to provide protection in all models at a concentration of 4 μg/kg per animal. Here we present a method to produce fully human IgG1 monoclonal antibodies that are opsonic in vitro and protective in vivo against several multiresistant Gram-positive bacteria. The monoclonal antibodies presented in this study are significantly more effective compared to another monoclonal antibody currently in clinical trials. PMID:25706415

  20. A Mixture of Functionally Oligoclonal Humanized Monoclonal Antibodies That Neutralize Clostridium difficile TcdA and TcdB with High Levels of In Vitro Potency Shows In Vivo Protection in a Hamster Infection Model

    PubMed Central

    Davies, Nicola L.; Compson, Joanne E.; MacKenzie, Brendon; O'Dowd, Victoria L.; Oxbrow, Amanda K. F.; Heads, James T.; Turner, Alison; Sarkar, Kaushik; Dugdale, Sarah L.; Jairaj, Mark; Christodoulou, Louis; Knight, David E. O.; Cross, Amanda S.; Hervé, Karine J. M.; Tyson, Kerry L.; Hailu, Hanna; Doyle, Carl B.; Ellis, Mark; Kriek, Marco; Cox, Matthew; Page, Matthew J. T.; Moore, Adrian R.; Lightwood, Daniel J.

    2013-01-01

    Clostridium difficile infections are a major cause of antibiotic-associated diarrhea in hospital and care facility patients. In spite of the availability of effective antibiotic treatments, C. difficile infection (CDI) is still a major cause of patient suffering, death, and substantial health care costs. Clostridium difficile exerts its major pathological effects through the actions of two protein exotoxins, TcdA and TcdB, which bind to and disrupt gut tissue. Antibiotics target the infecting bacteria but not the exotoxins. Administering neutralizing antibodies against TcdA and TcdB to patients receiving antibiotic treatment might modulate the effects of the exotoxins directly. We have developed a mixture of three humanized IgG1 monoclonal antibodies (MAbs) which neutralize TcdA and TcdB to address three clinical needs: reduction of the severity and duration of diarrhea, reduction of death rates, and reduction of the rate of recurrence. The UCB MAb mixture showed higher potency in a variety of in vitro binding and neutralization assays (∼10-fold improvements), higher levels of protection in a hamster model of CDI (82% versus 18% at 28 days), and higher valencies of toxin binding (12 versus 2 for TcdA and 3 versus 2 for TcdB) than other agents in clinical development. Comparisons of the MAb properties also offered some insight into the potential relative importance of TcdA and TcdB in the disease process. PMID:23324518

  1. Natalizumab: AN 100226, anti-4alpha integrin monoclonal antibody.

    PubMed

    2004-01-01

    Natalizumab [AN 100226, anti-alpha4 integrin monoclonal antibody, Antegren] is a humanised monoclonal antibody that blocks alpha4beta1 integrin-mediated leukocyte migration. Natalizumab is in phase III trials for the treatment of multiple sclerosis in North America and the UK, and for the treatment of Crohn's disease also in the UK. It may have potential in the treatment of other immune-related inflammatory disease. Elan Corporation intends to examine the potential of natalizumab in rheumatoid arthritis and ulcerative colitis. 4beta1 integrin on circulating leukocytes binds to vascular cell adhesion molecule-1, which is expressed at high levels in the blood vessels in the CNS during exacerbations of multiple sclerosis. This allows leukocytes expressing alpha4beta1 integrin (very late antigen-4) to move from the peripheral blood into the CNS. Inflammatory proteins and other factors released from lymphocytes in the brain lead to the progression of symptoms. A limitation of natalizumab is that it must be injected and cannot be administered orally. Scientists have transformed the large anti-alpha4 monoclonal antibody into much smaller, drug-like molecules suitable for oral administration. Protein Design Labs has granted a worldwide nonexclusive licence under its antibody humanisation patents to Elan Pharmaceuticals for natalizumab. Biogen Inc. has entered into an agreement with Elan for a worldwide exclusive collaboration to develop, manufacture and commercialise natalizumab for multiple sclerosis and Crohn's disease and rheumatoid arthritis. Development of natalizumab is also being funded, in part, by Axogen (acquired by Elan in 1999). In November 2003, Biogen and IDEC Pharmaceuticals merged to form Biogen Idec. Elan repurchased royalty rights on a package of products, including natalizumab, from Autoimmune Disease Research Company. Elan and Genzyme Transgenics Corporation signed an agreement to produce natalizumab in GTC's genetically engineered goats, which will

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

    PubMed

    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.

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

    PubMed

    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.

  4. Therapeutic monoclonal antibodies and multiple sclerosis: The essentials.

    PubMed

    Heliopoulos, Ioannis; Patousi, Athanasia

    2017-09-06

    Monoclonal antibodies (mAbs) are now established as targeted therapies for malignancies, transplant rejection, autoimmune and infectious diseases. Two monoclonal antibodies are available for treatment and other antibodies are currently being tested in multiple sclerosis (MS) patients. The purpose of the present review paper is to outline the antibody engineering technologies, the immunologic and pharmacologic concepts of mΑbs and the current status of treatment in MS with emphasis on clinical efficacy and safety. We conducted a thorough review of the scientific literature published until 31 December 2014 (print and electronic publications) concerning the production, applications and side effects of the use of Mabs. Sixty five articles were used in total (both original research and review papers). With the introduction of mAbs the treatment of MS has entered a new era, both with respect to efficacy and target specificity. However, administration of mAbs carries the risk of immune reactions such as acute anaphylaxis, serum sickness a, infection and other autoimmune diseases. In addition, unexpected consequences arise from our incomplete knowledge of the immune system. For example, natalizumab, a monoclonal antibody targeting α4-integrin on leukocytes increases the risk of developing progressive multifocal leukoencephalopathy, without causing notable immunosuppression. Further study on the use of mabs is required, both in vitro and in the clinical field, in order to increase our knowledge upon these new revolutionary therapeutic agents. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

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

    SciTech Connect

    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.

  6. Adsorption of monoclonal antibodies to glass microparticles.

    PubMed

    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.

  7. N-Glycosylation Design and Control of Therapeutic Monoclonal Antibodies.

    PubMed

    Sha, Sha; Agarabi, Cyrus; Brorson, Kurt; Lee, Dong-Yup; Yoon, Seongkyu

    2016-10-01

    The N-linked glycan profiles on recombinant monoclonal antibody therapeutics significantly affect antibody biological functions and are largely determined by host cell genotypes and culture conditions. A key step in bioprocess development for monoclonal antibodies (mAbs) involves optimization and control of N-glycan profiles. With pressure from pricing and biosimilars looming, more efficient and effective approaches are sought in the field of glycoengineering. Metabolic studies and mathematical modeling are two such approaches that optimize bioprocesses by better understanding and predicting glycosylation. In this review, we summarize a group of strategies currently used for glycan profile modulation and control. Metabolic analysis and mathematical modeling are then explored with an emphasis on how these two techniques can be utilized to advance glycoengineering.

  8. [Production of human monoclonal antibody reactive with gastrointestinal carcinoma].

    PubMed

    Soyama, N; Ohyanagi, H; Saitoh, Y

    1990-12-01

    Lymphocytes obtained from regional lymph nodes and spleen in the patients with gastrointestinal carcinoma were fused with the human B lymphoblastoid cell line GC01 and human hybridomas producing human monoclonal antibody (MoAb) were derived. Human MoAb No. 235 (IgM) derived from spleen cell of a gastric cancer patient reacted with adenocarcinoma of stomach, colon, and pancreas in the new immunohistochemical assay, modified direct immunoperoxidase method, and reacted with KATO III cells in cultured cell lines. The antigenic determinant of this antibody was suspected to be protein moiety after enzyme treatment. The competitive binding inhibition assay indicated that its epitope was different from anti-CEA monoclonal antibodies (KM10, A10, B9, AH3, JA4) and KM01. These findings suggested the possible use of human MoAb No. 235 for clinical application of targeting cancer chemotherapy in the future.

  9. Process economics of industrial monoclonal antibody manufacture.

    PubMed

    Farid, Suzanne S

    2007-03-15

    Pressures for cost-effective manufacture of antibodies are growing given their high doses and increasing market potential that have resulted in significant increases in total site capacities of up to 200,000 L. This paper focuses on the process economic issues associated with manufacturing antibodies and reviews the cost studies published in the literature; many of the issues highlighted are not only specific to antibodies but also apply to recombinant proteins. Data collated at UCL suggest current benchmark investment costs of $660-$1580/ft2 ($7130-$17,000/m2) and $1765-$4220/L for antibody manufacturing facilities with total site capacities in the range of 20,000-200,000 L; the limitations of the data are highlighted. The complications with deriving benchmark cost of goods per gram (COG/g) values are discussed, stressing the importance of stating the annual production rate and either titre or fermentation capacity with the cost so as to allow comparisons. The uses and limitations of the methods for cost analysis and the available software tools for process economics are presented. Specific examples found in the literature of process economic studies related to antibody manufacture for different expression systems are reviewed. The key economic drivers are identified; factors such as fermentation titre and overall yield are critical determinants of economic success. Future trends in antibody manufacture that are driven by economic pressures are discussed, such as the use of alternative expression systems (e.g. transgenics, E. coli and yeast), disposables, and improvements to downstream technology. The hidden costs and the challenges in each case are highlighted.

  10. Initial Characterization of Monoclonal Antibodies against Human Monocytes

    NASA Astrophysics Data System (ADS)

    Ugolini, Valentina; Nunez, Gabriel; Smith, R. Graham; Stastny, Peter; Capra, J. Donald

    1980-11-01

    Three monoclonal antibodies against human monocytes have been produced by somatic cell fusion. Extensive specificity analysis suggests that these antibodies react with most if not all human peripheral blood monocytes and not with highly purified T or B cells. Initial chemical characterization of the monocyte antigen recognized by two of these antibodies is presented. The molecule is a single polypeptide chain with an apparent molecular weight of 200,000. These reagents should prove useful in the clinical definition of disorders of monocyte differentiation, in studies of monocyte function, and in the elucidation of the genetics and structure of monocyte cell surface antigens.

  11. Idiotypic analysis of a monoclonal anti-Sm antibody.

    PubMed

    Pisetsky, D S; Lerner, E A

    1982-10-01

    Among murine models of autoimmunity, MRL mice are unique in their expression of antibodies to the nuclear antigen Sm. To assess genetic mechanisms in the control of this response, the idiotypes borne by a monoclonal anti-Sm antibody of MRL-Ipr/Ipr origin were investigated. Rabbit antisera were prepared against Y2, a hybridoma product with anti-Sm activity, and were rendered specific for idiotype by extensive absorption with normal globulins from BALB/c mice. In assays of idiotype by an inhibition ELISA, Y2 was shown to share idiotypes with Y12, another monoclonal anti-Sm derived from the same fusion as Y2; other monoclonal autoantibodies of MRL origin but different antigenic specificity failed to display idiotype activity in this assay. The presence of other anti-idiotypic specificities was revealed by absorption and elution of the anti-idiotype from an MRL globulin column; sera from both anti-Sm-positive and negative mice demonstrated these idiotypes. These results suggest that the predominant specificities detected by the anti-idiotype were unique to the monoclonal antibodies of the same animal, although there was also activity to idiotypes not related to anti-Sm binding molecules.

  12. Development of murine monoclonal antibodies to methamphetamine and methamphetamine analogues.

    PubMed

    Danger, Yannic; Gadjou, Caroline; Devys, Anne; Galons, Hervé; Blanchard, Dominique; Folléa, Gilles

    2006-02-20

    Methamphetamine and ecstasy are addictive drugs that cause major health problems in young people. Here we report on the development of high-affinity monoclonal antibodies to methamphetamine and its analogues, which may constitute powerful tools for antibody-based therapy. Six haptens, methamphetamine and ecstasy analogues, were synthesized, linked to a carrier protein and injected into mice. Several specific monoclonal antibodies were subsequently obtained following fusion of splenocytes from the immunized animals, with Sp2/O cells. Antibody specificity was fully investigated by competition ELISA, using a series of analogues, to identify specific amphetamine and/or ecstasy-specific antibodies. Antibody affinity was estimated to be in the range of 10(8) M(-1) with an enantiomeric hapten. Finally, two characteristic hybridoma clones (DAS-M243-6H5 and DAS-M278-4B12), secreting specific and potent mAbs were isolated. The development of drug-specific antibodies as in this study may provide promising therapeutic insight into how to neutralize methamphetamine in vivo during acute intoxication.

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

    PubMed Central

    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

  14. Monoclonal IgA Antibodies for Aflatoxin Immunoassays

    PubMed Central

    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

  15. Potent neutralizing monoclonal antibodies against Ebola virus infection

    PubMed Central

    Zhang, Qi; Gui, Miao; Niu, Xuefeng; He, Shihua; Wang, Ruoke; Feng, Yupeng; Kroeker, Andrea; Zuo, Yanan; Wang, Hua; Wang, Ying; Li, Jiade; Li, Chufang; Shi, Yi; Shi, Xuanling; Gao, George F.; Xiang, Ye; Qiu, Xiangguo; Chen, Ling; Zhang, Linqi

    2016-01-01

    Ebola virus infections cause a deadly hemorrhagic disease for which no vaccines or therapeutics has received regulatory approval. Here we show isolation of three (Q206, Q314 and Q411) neutralizing monoclonal antibodies (mAbs) against the surface glycoprotein (GP) of Ebola virus identified in West Africa in 2014 through sequential immunization of Chinese rhesus macaques and antigen-specific single B cell sorting. These mAbs demonstrated potent neutralizing activities against both pseudo and live Ebola virus independent of complement. Biochemical, single particle EM, and mutagenesis analysis suggested Q206 and Q411 recognized novel epitopes in the head while Q314 targeted the glycan cap in the GP1 subunit. Q206 and Q411 appeared to influence GP binding to its receptor NPC1. Treatment with these mAbs provided partial but significant protection against disease in a mouse model of Ebola virus infection. These novel mAbs could serve as promising candidates for prophylactic and therapeutic interventions against Ebola virus infection. PMID:27181584

  16. Development of a monoclonal antibody specific to cooked mammalian meats.

    PubMed

    Hsieh, Y H; Sheu, S C; Bridgman, R C

    1998-04-01

    Detection of species adulteration in ground meat products is important for consumer protection and food-labeling law enforcement. This study was conducted to develop monoclonal antibodies (MAbs) that can be used in an enzyme-linked immunosorbent assay (ELISA) for rapid detection of any cooked mammalian meats in cooked poultry products. Soluble muscle proteins extracted from cooked pork (heated at 100 degrees C for 15 min) were used as the antigen to immunized mice for developing the MAb. One that was developed, MAb 2F8 (IgG2b class), strongly reacted with cooked meat of five mammalian species (beef cattle, hogs, sheep, horse, and deer) but did not react with any cooked poultry (chicken, turkey, and duck) or raw meats. At least 0.5% by weight of pork, beef, lamb, and horse meats in a chicken-based mixture could not detect using the indirect ELISA with MAb 2F8. The MAb 2F8 is useful in a single initial screening test to detect the presence of five nonpoultry meat adulterants in cooked poultry products.

  17. The therapeutic potential of anti-interleukin-20 monoclonal antibody.

    PubMed

    Hsu, Yu-Hsiang; Chang, Ming-Shi

    2014-01-01

    Interleukin (IL)-20, a member of the IL-10 family of cytokines, was discovered in 2001. IL-20 acts on multiple cell types by activating on a heterodimer receptor complex of either IL-20R1-IL-20R2 or IL-22R1-IL-20R2. Recent evidence indicates that IL-20's interaction with its receptors might have proinflammatory effects on chronic inflammatory diseases, particularly rheumatoid arthritis (RA), osteoporosis, and breast cancer. Updated information about IL-20, such as its identification, expression, receptors, signaling, and biological activities, is illustrated in this review based on our research and the data available in the literature. IL-20 is a pleiotropic cytokine, which promotes inflammation, angiogenesis, and chemotaxis. IL-20 also regulates osteoclast differentiation by altering the receptor activator of NF-κB (RANK) and RANK ligand (RANKL) axis. Inflammation, angiogenesis, and osteoclastogenesis are critical for the pathogenesis of RA, osteoporosis, and breast cancer-induced osteolysis. Based on the in vitro and in vivo data and clinical samples, we demonstrated that IL-20 plays pivotal roles in these three diseases. In experimental models, anti-IL-20 monoclonal antibody ameliorates arthritis severity, protects against ovariectomized-induced bone loss, and inhibits breast tumor-induced osteolysis. This review presents the clinical implications of IL-20, which will lead to a better understanding of the biological functions of IL-20 in these diseases and provide new therapeutic options in the future.

  18. Monoclonal antibodies identify a group of nuclear pore complex glycoproteins

    PubMed Central

    1987-01-01

    Using monoclonal antibodies we identified a group of eight polypeptides of rat liver nuclear envelopes that have common epitopes. Most or all of these proteins are structurally distinct, as shown by tryptic peptide mapping and analysis with polyclonal antibodies. While these polypeptides are relatively tightly bound to nuclear membranes, only one is an integral membrane protein. The eight antigens cofractionate with the nuclear pore complex under various conditions of ionic strength and detergent. It can be seen by immunofluorescence microscopy that the monoclonal antibodies reacting with these antigens stain the nuclear surface of interphase cells in a finely punctate pattern. When the nuclear envelope is disassembled and subsequently reformed during mitosis, the proteins are reversibly dispersed throughout the cytoplasm in the form of minute foci. By EM immunogold localization on isolated nuclear envelopes, the monoclonal antibodies label exclusively the nuclear pore complex, at both its nucleoplasmic and cytoplasmic margins. Considered together, our biochemical and localization data indicate that the eight nuclear envelope polypeptides are pore complex components. As shown in the accompanying paper (Holt, G. D., C. M. Snow, A. Senior, R. S. Haltiwanger, L. Gerace, and G. W. Hart, J. Cell Biol., 104:1157-1164) these eight polypeptides contain a novel form of glycosylation, O-linked N-acetylglucosamine. The relative abundance and disposition of these O-linked glycoproteins in the pore complex are consistent with their having a role in nucleocytoplasmic transport. PMID:2437126

  19. Murine monoclonal antibodies generated against mouse/rat hemokinin-1.

    PubMed

    Jin, Liang; Jin, Bo-quan; Song, Chao-jun; Zhang, Yu

    2009-08-01

    The mouse/rat hemokinin-1 (m/rHK-1) was discovered nearly 9 years ago. This molecule is a peptide comprising 11 amino acids. The m/rHK-1 was found to be mainly expressed in central immune organs like bone marrow, and was proven to have lymphopoietic roles in B and T lymphocyte development. m/rHK-1was also reported to have analgesic roles in rat spinal cord, in addition to other functions such as relaxing activity on coronary artery. Unlike its analogues SP, NKA, and NKB, m/rHK-1 does not express in the nervous system. To further study the distribution and function of m/rHK-1, we carried out conventional immunization and cell fusion procedures to acquire the hybridomas secreting specific monoclonal antibodies to m/rHK-1. In the 17 positive clones obtained, three antibodies named 1B12, 2B4, and 4G5 were shown representative in cross-reactivity against m/rHK-1 and its analogues by indirect ELISA, competitive indirect ELISA, and immunofluorescence assays. Among the three clones, the 2B4 monoclonal antibody appeared to be the high-titered and specific clone to m/rHK-1. Monoclonal antibodies to m/rHK-1 will function as good tools in the physiological study of m/rHK-1 in the near future.

  20. The Use of Monoclonal Antibodies in Human Prion Disease

    NASA Astrophysics Data System (ADS)

    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.

  1. Stimuli-responsive magnetic nanoparticles for monoclonal antibody purification.

    PubMed

    Borlido, Luís; Moura, Leila; Azevedo, Ana M; Roque, Ana C A; Aires-Barros, Maria R; Farinha, José Paulo S

    2013-06-01

    Monoclonal antibodies (mAbs) are important therapeutic proteins. One of the challenges facing large-scale production of monoclonal antibodies is the capacity bottleneck in downstream processing, which can be circumvented by using magnetic stimuli-responsive polymer nanoparticles. In this work, stimuli-responsive magnetic particles composed of a magnetic poly(methyl methacrylate) core with a poly(N-isopropylacrylamide-co-acrylic acid) (P(NIPAM-co-AA)) shell cross-linked with N, N'-methylenebisacrylamide were prepared by miniemulsion polymerization. The particles were shown to have an average hydrodynamic diameter of 317 nm at 18°C, which decreased to 277 nm at 41°C due to the collapse of the thermo-responsive shell. The particles were superparamagnetic in behavior and exhibited a saturation magnetization of 12.6 emu/g. Subsequently, we evaluated the potential of these negatively charged stimuli-responsive magnetic particles in the purification of a monoclonal antibody from a diafiltered CHO cell culture supernatant by cation exchange. The adsorption of antibodies onto P(NIPAM-co-AA)-coated nanoparticles was highly selective and allowed for the recovery of approximately 94% of the mAb. Different elution strategies were employed providing highly pure mAb fractions with host cell protein (HCP) removal greater than 98%. By exploring the stimuli-responsive properties of the particles, shorter magnetic separation times were possible without significant differences in product yield and purity.

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

    PubMed

    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.

  3. Cysteinylation of a monoclonal antibody leads to its inactivation

    PubMed Central

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

    2016-01-01

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

  4. Escape From Monoclonal Antibody Neutralization Affects Henipavirus Fitness In Vitro and In Vivo

    PubMed Central

    Borisevich, Viktoriya; Lee, Benhur; Hickey, Andrew; DeBuysscher, Blair; Broder, Christopher C.; Feldmann, Heinz; Rockx, Barry

    2016-01-01

    Henipaviruses are zoonotic viruses that can cause severe and acute respiratory diseases and encephalitis in humans. To date, no vaccine or treatments are approved for human use. The presence of neutralizing antibodies is a strong correlate of protection against lethal disease in animals. However, since RNA viruses are prone to high mutation rates, the possibility that these viruses will escape neutralization remains a potential concern. In the present study, we generated neutralization-escape mutants, using 6 different monoclonal antibodies, and studied the effect of these neutralization-escape mutations on in vitro and in vivo fitness. These data provide a mechanism for overcoming neutralization escape by use of cocktails of cross-neutralizing monoclonal antibodies that recognize residues within the glycoprotein that are important for virus replication and virulence. PMID:26357909

  5. Escape From Monoclonal Antibody Neutralization Affects Henipavirus Fitness In Vitro and In Vivo.

    PubMed

    Borisevich, Viktoriya; Lee, Benhur; Hickey, Andrew; DeBuysscher, Blair; Broder, Christopher C; Feldmann, Heinz; Rockx, Barry

    2016-02-01

    Henipaviruses are zoonotic viruses that can cause severe and acute respiratory diseases and encephalitis in humans. To date, no vaccine or treatments are approved for human use. The presence of neutralizing antibodies is a strong correlate of protection against lethal disease in animals. However, since RNA viruses are prone to high mutation rates, the possibility that these viruses will escape neutralization remains a potential concern. In the present study, we generated neutralization-escape mutants, using 6 different monoclonal antibodies, and studied the effect of these neutralization-escape mutations on in vitro and in vivo fitness. These data provide a mechanism for overcoming neutralization escape by use of cocktails of cross-neutralizing monoclonal antibodies that recognize residues within the glycoprotein that are important for virus replication and virulence.

  6. Monoclonal antibodies that target pathological assemblies of Abeta.

    PubMed

    Lambert, Mary P; Velasco, Pauline T; Chang, Lei; Viola, Kirsten L; Fernandez, Sara; Lacor, Pascale N; Khuon, Daliya; Gong, Yuesong; Bigio, Eileen H; Shaw, Pamela; De Felice, Fernanda G; Krafft, Grant A; Klein, William L

    2007-01-01

    Amyloid beta (Abeta) immunotherapy for Alzheimer's disease has shown initial success in mouse models of Alzheimer's disease and in human patients. However, because of meningoencephalitis in clinical trials of active vaccination, approaches using therapeutic antibodies may be preferred. As a novel antigen to generate monoclonal antibodies, the current study has used Abeta oligomers (amyloid beta-derived diffusible ligands, ADDLs), pathological assemblies known to accumulate in Alzheimer's disease brain. Clones were selected for the ability to discriminate Alzheimer's disease from control brains in extracts and tissue sections. These antibodies recognized Abeta oligomers and fibrils but not the physiologically prevalent Abeta monomer. Discrimination derived from an epitope found in assemblies of Abeta1-28 and ADDLs but not in other sequences, including Abeta1-40. Immunoneutralization experiments showed that toxicity and attachment of ADDLs to synapses in culture could be prevented. ADDL-induced reactive oxygen species (ROS) generation was also inhibited, establishing this response to be oligomer-dependent. Inhibition occurred whether ADDLs were prepared in vitro or obtained from Alzheimer's disease brain. As conformationally sensitive monoclonal antibodies that selectively immunoneutralize binding and function of pathological Abeta assemblies, these antibodies provide tools by which pathological Abeta assemblies from Alzheimer's disease brain might be isolated and evaluated, as well as offering a valuable prototype for new antibodies useful for Alzheimer's disease therapeutics.

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

    PubMed

    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

  8. Quantitative SPECT of uptake of monoclonal antibodies

    SciTech Connect

    DeNardo, G.L.; Macey, D.J.; DeNardo, S.J.; Zhang, C.G.; Custer, T.R.

    1989-01-01

    Absolute quantitation of the distribution of radiolabeled antibodies is important to the efficient conduct of research with these agents and their ultimate use for imaging and treatment, but is formidable because of the unrestricted nature of their distribution within the patient. Planar imaging methods have been developed and provide an adequate approximation of the distribution of radionuclide for many purposes, particularly when there is considerable specificity of targeting. This is not currently the case for antibodies and is unlikely in the future. Single photon emission computed tomography (SPECT) provides potential for greater accuracy because it reduces problems caused by superimposition of tissues and non-target contributions to target counts. SPECT measurement of radionuclide content requires: (1) accurate determination of camera sensitivity; (2) accurate determination of the number of counts in a defined region of interest; (3) correction for attenuation; (4) correction for scatter and septal penetration; (5) accurate measurement of the administered dose; (6) adequate statistics; and (7) accurate definition of tissue mass or volume. The major impediment to each of these requirements is scatter of many types. The magnitude of this problem can be diminished by improvements in tomographic camera design, computer algorithms, and methodological approaches. 34 references.

  9. Production and characterization of monoclonal antibodies against aflatoxin B1.

    PubMed

    Soukhtanloo, Mohammad; Talebian, Elham; Golchin, Mehdi; Mohammadi, Mojgan; Amirheidari, Bagher

    2014-01-01

    In this article, we embarked on production of mouse monoclonal antibodies against aflatoxin B1 which is the most commonly occurring fungal toxin in food and feed products. After immunization and fusion with myloma cells, two stable clones (A218 and B319) were selected. Isotyping showed that these monoclonal antibodies (mAbs) were IgG2b with kappa light chains. The affinity of A218 and B319 clons were 5×10(11) M(-1) and 6×10(9) M(-1), respectively. Competitive indirect ELISA results indicated these mAbs had complete (100%) cross-reaction with four major types of aflatoxins: B1, B2, G1, and G2. These mAbs could be used for immunoassay measurement of aflatoxins with high affinity and low detection limits.

  10. Biosimilar monoclonal antibodies in lymphoma: a critical appraisal.

    PubMed

    Rioufol, Catherine; Salles, Gilles

    2015-05-01

    Rituximab, an anti-CD20 monoclonal antibody, revolutionized the treatment of lymphoma. Although newer generation anti-CD20 monoclonal antibodies are being examined, patent expiries and patient demand have fueled the development of rituximab biosimilars. The development of such agents is both an important and difficult undertaking. By definition, although they aim to have safety and efficacy comparable with their reference agents, biosimilars are not exact replicas of those agents, and small changes in nonclinical and preclinical properties may ultimately affect in vivo activity. Consideration must be given to the complex mechanisms of action, sensitive patient populations that may be treated, and appropriate clinical trial endpoints. Furthermore, extrapolation of indications is multifaceted, deserving close examination. This review represents a critical look at biosimilars in lymphoma and their safety, efficacy and long-term effects on patient outcomes.

  11. Adverse Events of Monoclonal Antibodies Used for Cancer Therapy

    PubMed Central

    Guan, Mei; Zhou, Yan-Ping; Sun, Jin-Lu; Chen, Shu-Chang

    2015-01-01

    In 1997, the first monoclonal antibody (MoAb), the chimeric anti-CD20 molecule rituximab, was approved by the US Food and Drug administration for use in cancer patients. Since then, the panel of MoAbs that are approved by international regulatory agencies for the treatment of hematopoietic and solid malignancies has continued to expand, currently encompassing a stunning amount of 20 distinct molecules for 11 targets. We provide a brief scientific background on the use of MoAbs in cancer therapy, review all types of monoclonal antibodies-related adverse events (e.g., allergy, immune-related adverse events, cardiovascular adverse events, and pulmonary adverse events), and discuss the mechanism and treatment of adverse events. PMID:26075239

  12. Anaphylaxis: implications of monoclonal antibody use in oncology.

    PubMed

    Gleich, Gerald J; Leiferman, Kristin M

    2009-02-01

    Anaphylaxis is currently classified as an immunologically triggered response with reactions that are IgE-mediated and reactions that are not IgE-mediated. This immunologically mediated phenomenon can result in various clinical manifestations, including decreased blood pressure, generalized skin inflammation, such as hives and pruritus, and respiratory symptoms, such as wheezing or bronchospasm. The severity of anaphylaxis can range from a mild allergic reaction to a potentially fatal anaphylactic shock. Numerous causative agents trigger anaphylactic reactions, and some of the best described include food and bee sting allergens. Monoclonal antibodies, which are increasingly used in the treatment of various malignancies, also can cause anaphylaxis. In this review, the mechanisms governing anaphylaxis along with treatment strategies are reviewed. Diagnostic aids for anaphylaxis are also discussed. Increased awareness of the mechanisms, symptoms, and treatment of anaphylaxis can aid caregivers to make informed decisions when new agents, such as monoclonal antibodies, are introduced into the clinic.

  13. Monoclonal antibodies for the detection of trace chemicals

    SciTech Connect

    Vanderlaan, M.; Van Emon, J.; Watkins, B.; Stanker, L.

    1986-08-15

    Problems in analytical chemistry may limit monitoring for trace organic residues by traditional chromatographic methods. For example, the cost and analysis time per sample may preclude adequate sampling, making the development of alternative technologies desirable. Immunoassays are one such alternative, with the potential for cost reduction by automation and parallel sample processing. A particularly significant advance in the past decade has been the development of monoclonal antibodies, which offer greater selectivity and reproducibility than conventional antisera. Immunoassays can be developed that use simple, field-portable instrumentation, give rapid results, and have detection limits of less than a part-per-billion. This paper reviews the general technology for developing monoclonal antibodies to small organic molecules using the immunoassay of 2,3,7,8-tetrachlorodibenzodioxin (2,3,7,8-TCDD) as an example. 18 refs., 2 figs., 1 tab.

  14. Immunohistochemical identification of cytotoxic lymphocytes using human perforin monoclonal antibody.

    PubMed Central

    Hameed, A.; Olsen, K. J.; Cheng, L.; Fox, W. M.; Hruban, R. H.; Podack, E. R.

    1992-01-01

    Perforin is a potent cytolytic pore-forming protein expressed in cytoplasmic granules of cytotoxic T lymphocytes and natural killer cells. A new monoclonal antibody raised against human perforin was used to detect both in vitro and in vivo perforin expression in cytotoxic cells. Immunohistochemical analysis of human peripheral blood mononuclear cells cultured in recombinant interleukin-2 (rIL-2) showed strong granular cytoplasmic staining of the IL-2 activated cytotoxic cells. Fresh-frozen tissue sections from patients with heart allograft rejection were also stained. Strong granular cytoplasmic staining of the mononuclear inflammatory infiltrate characteristic for perforin in cardiac allograft rejection was observed. The detection and quantitative analysis of perforin-associated cytotoxic cells by the human anti-perforin monoclonal antibody will help to evaluate the significance of these functionally distinct cytotoxic cells in human tissue. Images Figure 1 PMID:1374586

  15. Cellular Mechanisms of CNS Repair by Natural Autoreactive Monoclonal Antibodies

    PubMed Central

    Wright, Brent R.; Warrington, Arthur E.; Edberg, Dale E.; Rodriguez, Moses

    2009-01-01

    Natural autoreactive monoclonal IgMs have demonstrated potential as therapeutic agents for CNS disease. These antibodies bind surface antigens on specific CNS cells activating intracellular repair-promoting signals. IgMs that bind to surface antigens on oligodendrocytes enhanced remyelination in animal models of multiple sclerosis. IgMs that bind to neurons stimulate neurite outgrowth and prevent neuron apoptosis. The neuron-binding IgMs may have utility in CNS axon- or neuron-damaging diseases such as amyotrophic lateral sclerosis, stroke, spinal cord injury or secondary progressive multiple sclerosis. Recombinant remyelination-promoting IgMs have been generated for formal toxicology studies and, after FDA approval, a Phase I clinical trial. Natural autoreactive monoclonal antibodies directed against CNS cells represent novel therapeutic molecules to induce repair of the nervous system. PMID:20008649

  16. Rhenium-186-labeled monoclonal antibodies for radioimmunotherapy: preparation and evaluation.

    PubMed

    John, E; Thakur, M L; DeFulvio, J; McDevitt, M R; Damjanov, I

    1993-02-01

    Rhenium-186 has been determined to be a leading radionuclide for radioimmunotherapy. However, the use of 186Re has been limited due to the lack of a convenient and efficient method by which the radionuclide can be bound to monoclonal antibodies. We have developed a simple technique to label IgM, IgG, fragmented antibodies and tumor necrosis factor-alpha with 186Re. This technique uses ascorbic acid (AA) for controlled reduction of antibody disulfide groups to sulfhydryls and SnCl2 in citric acid for the reduction of 186ReO4-. The labeling yields as determined by instant thin-layer chromatography, molecular filtration and gel filtration were greater than 95% and the colloid formation was less than 5%. The labeled antibodies were stable when challenged with 100 and 250 molar excess of DTPA and HSA for 24 hr at 37 degrees C. SDS-PAGE analysis and autoradiography of labeled IgM, IgG and F(ab')2 monoclonal antibodies indicated uniform labeling and that no fragmentation of the monoclonal antibodies had taken place during the labeling procedure. Immunospecificity of 186Re-labeled human neutrophil specific IgM, as determined by in vitro antigen excess assay, was comparable to that of indium-111-labeled c-DTPA-IgM and technetium-99m-labeled-IgM. A nuclear histone specific 186Re-TNT-1-F(ab')2 was evaluated in mice bearing experimental tumors. The tumor/muscle ratios at 4 and 24 hr were 5.9 +/- 0.21 and 13.8 +/- 6.7, respectively compared to that of 2.4 +/- 0.3 at 4 hr p.i. with a nonspecific protein. The labeling technique is simple, reliable and has already been adapted to a single-vial kit preparation.

  17. Monoclonal antibodies directed against surface molecules of multicell spheroids

    NASA Technical Reports Server (NTRS)

    Martinez, Andrew O.

    1993-01-01

    The objective of this project is to generate a library of monoclonal antibodies (MAbs) to surface molecules of mammalian tumor and transformed cells grown as multicell spheroids (MCS). These MCS are highly organized, three dimensional multicellular structures which exhibit many characteristics of in vivo organized tissues not found in conventional monolayer or suspension culture; therefore, MCS make better in vitro model systems to study the interactions of mammalian cells. Additionally, they provide a functional assay for surface adhesion molecules.

  18. Positron emission tomographic imaging of tumors using monoclonal antibodies

    SciTech Connect

    Zalutsky, M.R.

    1992-08-01

    This research project is developing methods for utilizing positron emission tomography (PET) to increase the clinical potential of radiolabeled monoclonal antibodies (MAbs). This report describes the development of methods for labeling MAbs and their fragments with positron-emitting halogen nuclides, fluorine-18 and iodine-124. These nulides were selected because of the widespread availability of F-18 and because of our extensive experience in the development of new protein radiohalogenation methods.

  19. Recovery and purification process development for monoclonal antibody production

    PubMed Central

    Ma, Junfen; Winter, Charles; Bayer, Robert

    2010-01-01

    Hundreds of therapeutic monoclonal antibodies (mAbs) are currently in development, and many companies have multiple antibodies in their pipelines. Current methodology used in recovery processes for these molecules are reviewed here. Basic unit operations such as harvest, Protein A affinity chromatography and additional polishing steps are surveyed. Alternative processes such as flocculation, precipitation and membrane chromatography are discussed. We also cover platform approaches to purification methods development, use of high throughput screening methods, and offer a view on future developments in purification methodology as applied to mAbs. PMID:20647768

  20. Neutralizing Monoclonal Antibodies against Biological Toxins. Phase 1

    DTIC Science & Technology

    1993-08-14

    the original copy. AD-B’i76 29`8 CONTRACT NO: DAMD17-93-C-3083 TITLE: NEUTRALIZING MONOCLONAL ANTIBODIES AGAINST BIOLOGIC4JL TOXINS PRINCIPAL...Biological ’ Toxins DAMDl7-93-C-308.3 6. AUTHOR($) 65502A 30665502MB02. S4 .274 Mark C. Glassy, Ph.D. WUDA336206 7. PERFORMING ORGANIZATION NAMI(S...NUMBER OF PAGES RA I, SBIR, Antibody, Toxins , BL2, BD 16. PRICE COUE 17. SEC.URIly (LASSIFICATION 18. SECURITY CLASSIMICATION 19. SECURITY

  1. Production of Monoclonal Antibodies in Plants for Cancer Immunotherapy

    PubMed Central

    Moussavou, Ghislain; Ko, Kisung; Lee, Jeong-Hwan; Choo, Young-Kug

    2015-01-01

    Plants are considered as an alternative platform for recombinant monoclonal antibody (mAb) production due to the improvement and diversification of transgenic techniques. The diversity of plant species offers a multitude of possibilities for the valorization of genetic resources. Moreover, plants can be propagated indefinitely, providing cheap biomass production on a large scale in controlled conditions. Thus, recent studies have shown the successful development of plant systems for the production of mAbs for cancer immunotherapy. However, their several limitations have to be resolved for efficient antibody production in plants. PMID:26550566

  2. Monoclonal antibodies in the treatment of pancreatic cancer

    PubMed Central

    Huang, Zhi-Qiang; Buchsbaum, Donald J

    2009-01-01

    Human pancreatic cancer is a malignant disease with almost equal incidence and mortality. Effective diagnostic and therapeutic strategies are still urgently needed to improve its survival rate. With advances in structural and functional genomics, recent work has focused on targeted molecular therapy using monoclonal antibodies. This review summarizes the target molecules on the tumor cell surface and normal tissue stroma, which are related to pancreatic cancer oncogenesis, tumor growth or resistance to chemotherapy, as well as molecules involved in regulating inflammation and host immunoresponses. Targeted molecules include cell-surface receptors, such as the EGF receptor, HER2, death receptor 5 and IGF-1 receptor. Effects of monoclonal antibodies against these target molecules alone or in combination with chemotherapy, small-molecule signal transduction inhibitors, or radiation therapy are also discussed. Also discussed are the use of toxin or radioisotope conjugates, and information relating to the use of these targeting agents in pancreatic cancer clinical trials. Although targeted molecular therapy with monoclonal antibodies has made some progress in pancreatic cancer treatment, especially in preclinical studies, its clinical application to improve the survival rate of pancreatic cancer patients requires further investigation. PMID:20046965

  3. Investigation of a panel of monoclonal antibodies and polyclonal sera against anthrax toxins resulted in identification of an anti-lethal factor antibody with disease-enhancing characteristics.

    PubMed

    Kulshreshtha, Parul; Tiwari, Ashutosh; Priyanka; Joon, Shikha; Sinha, Subrata; Bhatnagar, Rakesh

    2015-12-01

    Hybridomas were created using spleen of mice that were actively immunized with rLFn (recombinant N-terminal domain of lethal factor). Later on, separate group of mice were immunized with rLFn to obtain a polyclonal control for passive immunization studies of monoclonal antibodies. This led to the identification of one cohort of rLFn-immnized mice that harboured disease-enhancing polyclonal antibodies. At the same time, the monoclonal antibodies secreted by all the hybridomas were being tested. Two hybridomas secreted monoclonal antibodies (H10 and H8) that were cross-reactive with EF (edema factor) and LF (lethal factor), while the other two hybridomas secreted LF-specific antibodies (H7 and H11). Single chain variable fragment (LETscFv) was derived from H10 hybridoma. H11 was found to have disease-enhancing property. Combination of H11 with protective monoclonal antibodies (H8 and H10) reduced its disease enhancing nature. This in vitro abrogation of disease-enhancement provides the proof of concept that in polyclonal sera the disease enhancing character of a fraction of antibodies is overshadowed by the protective nature of the rest of the antibodies generated on active immunization. Copyright © 2015. Published by Elsevier Ltd.

  4. Mitosis-specific monoclonal antibodies block cleavage in amphibian embryos.

    PubMed

    Davis, F M; Wright, D A; Penkala, J E; Rao, P N

    1989-04-01

    By microinjecting monoclonal antibodies that bind specifically to mitotic and meiotic cells of a variety of species, we studied the biological activity of antigens recognized by these antibodies. The antibodies recognize a family of phosphoprotein antigens that are found throughout the cytoplasm of mitotic cells and particularly at microtubule organizing centers, including centrosomes and kinetochores. Their binding is dependent on phosphorylation of the polypeptides. Immunoglobulins were introduced into Xenopus laevis and Rana pipiens oocytes or cleaving embryos using glass micropipettes. The ability of the antibody-injected oocytes to undergo mitosis or meiosis was compared with those injected with control mouse immunoglobulins. The antibodies failed to block chromosome condensation and germinal vesicle breakdown in progesterone-treated oocytes. However, functional mitotic spindles were not assembled in cleavage stage frog embryos injected with antibodies. In vitro, the binding of the antibodies to the antigens inhibited the dephosphorylation of the antigens by alkaline phosphatase. The antibody binding to the activated microtubule organizing centers (MTOC) seems to block not only the nucleation of microtubules and the organization of the mitotic spindle, but also the dephosphorylation of proteins associated with the MTOC that normally occurs at the mitosis-G1 transition.

  5. Intravesical administration of radiolabeled antitumor monoclonal antibody in bladder carcinoma

    SciTech Connect

    Bamias, A.; Keane, P.; Krausz, T.; Williams, G.; Epenetos, A.A. )

    1991-01-15

    Tumor associated AUA1 monoclonal antibody and 11.4.1. nonspecific monoclonal antibody, which does not react with human tissues, were radiolabeled with 111In and administered intravesically to 23 patients undergoing cystoscopy for bladder carcinoma. The antibody solution remained in the bladder for 1 h and then was washed out prior to cystoscopy. Tumor and nontumor samples were obtained during cystoscopy and were counted in a gamma counter. Conventional and immunoperoxidase staining with both antibodies were also performed. AUA1 reacted with all bladder carcinomas while 11.4.1. was negative in all cases. The mean uptake of AUA1 at 2, 24, and 48 h after the instillation (expressed as 10(3) x percentage of injected dose/g of tissue) was: 6.12 +/- 5.50 (SD), 1.70 +/- 2.57, 0.30 +/- 0.17 in the tumors and 0.32 +/- 0.50, 0.22 +/- 0.30, 0 in the nontumor areas, and for 11.4.1. it was: 0.075 +/- 0.075, 0.025 +/- 0.025 in the tumors and 0.30 +/- 0.42, 0.15 +/- 0.26 in the nontumor areas. The uptake of AUA1 by the tumors correlated with the tumor grade. There was no radioactivity in the blood at 2 h, and at 1, 2, and 3 days after the instillation. Our results indicate that intravesical administration of radiolabeled monoclonal antibody AUA1 targets selectively to tumor tissue without any significant normal tissue uptake. This finding might allow the development of a nontoxic and specific therapeutic approach for superficial bladder carcinoma.

  6. Antibody-mediated immune suppression is improved when blends of anti-RBC monoclonal antibodies are used in mice.

    PubMed

    Bernardo, Lidice; Amash, Alaa; Marjoram, Danielle; Lazarus, Alan H

    2016-08-25

    Although the prevention of hemolytic disease of the fetus and newborn is highly effective using polyclonal anti-D, a recombinant alternative is long overdue. Unfortunately, anti-D monoclonal antibodies have been, at best, disappointing. To determine the primary attribute defining an optimal antibody, we assessed suppression of murine red blood cell (RBC) immunization by single-monoclonal antibodies vs defined blends of subtype-matched antibodies. Allogeneic RBCs expressing the HOD antigen (hen egg lysozyme [HEL]-ovalbumin-human transmembrane Duffy(b)) were transfused into naïve mice alone or together with selected combinations of HEL-specific antibodies, and the resulting suppressive effect was assessed by evaluating the antibody response. Polyclonal HEL antibodies dramatically inhibited the antibody response to the HOD antigen, whereas single-monoclonal HEL antibodies were less effective despite the use of saturating doses. A blend of monoclonal HEL-specific antibodies reactive with different HEL epitopes significantly increased the suppressive effect, whereas a blend of monoclonal antibodies that block each other's binding to the HEL protein did not increase suppression. In conclusion, these data show that polyclonal antibodies are superior to monoclonal antibodies at suppressing the immune response to the HOD cells, a feature that can be completely recapitulated using monoclonal antibodies to different epitopes.

  7. Monoclonal antibodies and toxins--a perspective on function and isotype.

    PubMed

    Chow, Siu-Kei; Casadevall, Arturo

    2012-06-01

    Antibody therapy remains the only effective treatment for toxin-mediated diseases. The development of hybridoma technology has allowed the isolation of monoclonal antibodies (mAbs) with high specificity and defined properties, and numerous mAbs have been purified and characterized for their protective efficacy against different toxins. This review summarizes the mAb studies for 6 toxins--Shiga toxin, pertussis toxin, anthrax toxin, ricin toxin, botulinum toxin, and Staphylococcal enterotoxin B (SEB)--and analyzes the prevalence of mAb functions and their isotypes. Here we show that most toxin-binding mAbs resulted from immunization are non-protective and that mAbs with potential therapeutic use are preferably characterized. Various common practices and caveats of protection studies are discussed, with the goal of providing insights for the design of future research on antibody-toxin interactions.

  8. Monoclonal Antibodies and Toxins—A Perspective on Function and Isotype

    PubMed Central

    Chow, Siu-Kei; Casadevall, Arturo

    2012-01-01

    Antibody therapy remains the only effective treatment for toxin-mediated diseases. The development of hybridoma technology has allowed the isolation of monoclonal antibodies (mAbs) with high specificity and defined properties, and numerous mAbs have been purified and characterized for their protective efficacy against different toxins. This review summarizes the mAb studies for 6 toxins—Shiga toxin, pertussis toxin, anthrax toxin, ricin toxin, botulinum toxin, and Staphylococcal enterotoxin B (SEB)—and analyzes the prevalence of mAb functions and their isotypes. Here we show that most toxin-binding mAbs resulted from immunization are non-protective and that mAbs with potential therapeutic use are preferably characterized. Various common practices and caveats of protection studies are discussed, with the goal of providing insights for the design of future research on antibody-toxin interactions. PMID:22822456

  9. Polymorphisms of the Hepatitis A Virus Cellular Receptor 1 in African Green Monkey Kidney Cells Result in Antigenic Variants That Do Not React with Protective Monoclonal Antibody 190/4

    PubMed Central

    Feigelstock, Dino; Thompson, Peter; Mattoo, Pravina; Kaplan, Gerardo G.

    1998-01-01

    Monoclonal antibody (MAb) 190/4 blocks binding of hepatitis A virus (HAV) to the HAV cellular receptor 1 (havcr-1) and protects African green monkey kidney (AGMK) clone GL37 cells (GL37 cells) against HAV infection. BS-C-1 and CV-1 cells, two widely used AGMK cell lines, did not react with MAb 190/4 but expressed havcr-1, as judged by Western blot analysis. The cDNA coding for havcr-1 was amplified from BS-C-1 and CV-1 total cellular RNA by reverse transcription-PCR. Alignment of the amino acid sequences inferred from the cDNA nucleotide sequences showed that BS-C-1 and CV-1 havcr-1 differed from GL37 havcr-1 by having two substitutions in the Cys-rich region, N48H and K108Q, and 10 to 11 additional substitutions plus the insertion of 18 to 22 amino acids in the mucin-like region. Studies with chimeras of GL37 havcr-1 and BS-C-1 havcr-1 showed that the K108Q substitution was responsible for the lack of reaction of MAb 190/4 with BS-C-1 and CV-1 cells. Binding studies indicated that HAV bound to dog cell transfectants expressing the BS-C-1 havcr-1 as well as the GL37/BS-C-1 havcr-1 chimeras. These results indicate that antigenic variants of havcr-1 are expressed in AGMK cells and that binding of HAV to these havcr-1 variants tolerates changes in protective epitope 190/4. PMID:9621093

  10. Polymorphisms of the hepatitis A virus cellular receptor 1 in African green monkey kidney cells result in antigenic variants that do not react with protective monoclonal antibody 190/4.

    PubMed

    Feigelstock, D; Thompson, P; Mattoo, P; Kaplan, G G

    1998-07-01

    Monoclonal antibody (MAb) 190/4 blocks binding of hepatitis A virus (HAV) to the HAV cellular receptor 1 (havcr-1) and protects African green monkey kidney (AGMK) clone GL37 cells (GL37 cells) against HAV infection. BS-C-1 and CV-1 cells, two widely used AGMK cell lines, did not react with MAb 190/4 but expressed havcr-1, as judged by Western blot analysis. The cDNA coding for havcr-1 was amplified from BS-C-1 and CV-1 total cellular RNA by reverse transcription-PCR. Alignment of the amino acid sequences inferred from the cDNA nucleotide sequences showed that BS-C-1 and CV-1 havcr-1 differed from GL37 havcr-1 by having two substitutions in the Cys-rich region, N48H and K108Q, and 10 to 11 additional substitutions plus the insertion of 18 to 22 amino acids in the mucin-like region. Studies with chimeras of GL37 havcr-1 and BS-C-1 havcr-1 showed that the K108Q substitution was responsible for the lack of reaction of MAb 190/4 with BS-C-1 and CV-1 cells. Binding studies indicated that HAV bound to dog cell transfectants expressing the BS-C-1 havcr-1 as well as the GL37/BS-C-1 havcr-1 chimeras. These results indicate that antigenic variants of havcr-1 are expressed in AGMK cells and that binding of HAV to these havcr-1 variants tolerates changes in protective epitope 190/4.

  11. Transformation-Related Antigens Identified by Monoclonal Antibodies

    NASA Astrophysics Data System (ADS)

    Strand, Mette

    1980-06-01

    Tumor-cell proteins that were antigenic in a syngeneic animal were identified by immunoprecipitation with monoclonal antibodies. Spleen cells of BALB/c mice immunized with plasma membranes of Kirsten RNA sarcoma virus-transformed BALB/3T3 cells were fused with NS-l myeloma cells. Antibodies secreted into the culture fluid from these hybridomas were distinguished by their reactivity against proteins of different target cells. A total of 191 cultures were established; 143 produced antibodies that bound to BALB/3T3 cells transformed by the RNA sarcoma virus, of which antibodies from 82 bound to BALB/3T3 transformed with simian virus 40, and antibodies from 56 bound to BALB/3T3 cells. Thus, more than 50% of the cultures produced antibodies that possibly were specific to antigens of the transformed cell. Twenty different hybridomas have been cloned, and antibodies from eight of these were found to immunoprecipitate five different proteins. A protein of approximately 32,000 daltons was precipitated from BALB/3T3 cells transformed by the RNA sarcoma virus, simian virus 40, or methylcholanthrene but not from untransformed BALB/3T3 cells. A protein of about 300,000 daltons was precipitated from all four cell lines; precipitation was enhanced in the viral transformed cells. Proteins of approximately 57,000, 54,000, and 8500 daltons were immunoprecipitated from all four cell lines.

  12. A human monoclonal antibody that binds serotype A botulinum neurotoxin.

    PubMed

    Adekar, Sharad P; Jones, R Mark; Elias, M D; Al-Saleem, Fetweh H; Root, Michael J; Simpson, Lance L; Dessain, Scott K

    2008-02-01

    Monoclonal antibodies have demonstrated significant potential as therapeutics for botulinum neurotoxin exposures. We previously described a hybridoma method for cloning native human antibodies that uses a murine myeloma cell line that ectopically expresses the human telomerase catalytic subunit gene (hTERT) and the murine interleukin-6 gene (mIL-6). Here we describe a heterohybridoma cell line that ectopically expresses mIL-6 and hTERT and has improved stability of hTERT expression. We fused this cell line to human peripheral blood B cells from a subject who had received the botulinum toxoid vaccine, cloning a high-affinity antibody (13A) specific for serotype A botulinum neurotoxin (BoNT/A). The 13A antibody is an affinity-matured, post-germinal center IgG(1) lambda antibody that has partial neutralization activity in vivo. 13A binds an epitope on BoNT/A that overlaps the binding epitope of an IgG antibody previously shown to fully neutralize a lethal dose of BoNT/A in vivo. The 13A antibody may be useful for diagnostic testing or for incorporation into an oligoclonal therapeutic to counteract BoNT/A exposure.

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

    PubMed Central

    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

  14. Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies.

    PubMed

    Leon, Paul E; Wohlbold, Teddy John; He, Wenqian; Bailey, Mark J; Henry, Carole J; Wilson, Patrick C; Krammer, Florian; Tan, Gene S

    2017-08-29

    Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

  15. Improved monoclonal antibody tumor/background ratios with exchange transfusions.

    PubMed

    Henry, C A; Clavo, A C; Wahl, R L

    1991-01-01

    Blood exchange transfusions were performed in nude rats with subcutaneous HTB77 human ovarian carcinoma xenografts in an attempt to improve specific monoclonal antibody (MoAb) tumor/non-tumor uptake ratios. Animals were injected intravenously with both 131I-5G6.4 specific and 125I-UPC-10 non-specific MoAb. Twenty-four hours later 65-80% of the original blood was exchanged with normal heparinized rat blood and then these rodents were sacrificed. Exchange transfusion significantly (P less than 0.05) decreased normal tissue activities of 131I (except for muscle) by 63-85%, while tumor activity decreased only 5%. Tumor to background ratios increased from 0.1-0.8 to 2.3-6.3. Exchange transfusions substantially enhance tumor/normal tissue antibody uptake ratios and, along with plasmapheresis, may be useful in enhancing antibody localization in vivo, particularly for therapy.

  16. Monoclonal antibody aggregation intermediates visualized by atomic force microscopy.

    PubMed

    Lee, Hanjoo; Kirchmeier, Marc; Mach, Henryk

    2011-02-01

    Ubiquitous but highly variable processes of therapeutic protein aggregation remain poorly characterized, especially in the context of common infusion reactions and clinical immunogenicity. Among the numerous challenges is the characterization of intermediate steps that lead to the appearance of precipitates. Although the biophysical methods for elucidation of secondary and tertiary structures as well as overall size distribution are typically well established in the development laboratories, the use of molecular scale imaging techniques is still relatively rare due to low throughput and technical complexity. In this work, we present the use of atomic force microscopy to examine morphology of monoclonal antibody aggregates. Despite varying in primary structure as a result of different complementarity defining regions, most antibodies studied exhibited a similar aggregation intermediate consisting of several monomers. However, the manner of subsequent condensation of these oligomers appeared to differ between the antibodies, suggesting stability-dependent mechanisms.

  17. Daratumumab: monoclonal antibody therapy to treat multiple myeloma.

    PubMed

    Xia, C; Ribeiro, M; Scott, S; Lonial, S

    2016-10-01

    Daratumumab (Darzalex[TM]) is a human monoclonal antibody (MAb) that targets CD38; a surface protein highly expressed across multiple myeloma (MM) cells. Preclinical studies have shown daratumumab induces MM cell death through several mechanisms, including complement-dependent cytotoxicity (CDC) antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), apoptosis upon secondary crosslinking and immunomodulatory effects via a decrease in immune suppressive cells. Daratumumab has a favorable toxicity profile and encouraging clinical activity as a single agent and in combination with lenalidomide in heavily pretreated, relapsed patients in whom other novel agents (such as bortezomib, thalidomide and lenalidomide) and stem cell transplant have already failed. Given the encouraging efficacy and acceptable safety profile, daratumumab has emerged as a novel treatment option for MM both as a monotherapy and in combination with conventional and novel anti-MM agents. This review will focus on preclinical pharmacology, pharmacokinetics, safety and clinical development of daratumumab in MM.

  18. [Anti Shiga-like toxin II(SLT-II) humanized monoclonal antibody].

    PubMed

    Matsumoto, Yoh-ichi

    2002-03-01

    Anti-Shiga-Like Toxin II(SLT-II) Humanized Monoclonal Antibody(TMA-15) was constructed from Mouse Monoclonal Antibody(MuVTm1.1) recognizing the same antigen using recombinant and CDR grafting technology. TMA-15 had almost the same affinity to SLT-II as MuVTm1.1 and showed the good protective activity of mice challenged either with SLT-II or with SLT-II secreting Shiga-like Toxin producing E. coli(STEC). TMA-15 showed no acute toxicity to monkeys and no cross-reactivity to human tissues in pre-clinical safety studies. From the preliminary results of Phase 1 clinical trial using healthy adult volunteers, doses up to planned maximum dose were well tolerated and TMA-15 showed long half life in blood almost comparable to gamma globulin preparations. Therefore, TMA-15 is expected to show clinical efficacy in coming clinical trial using pediatric STEC patients.

  19. Efficient generation of monoclonal antibodies from single rhesus macaque antibody secreting cells.

    PubMed

    Meng, Weixu; Li, Leike; Xiong, Wei; Fan, Xuejun; Deng, Hui; Bett, Andrew J; Chen, Zhifeng; Tang, Aimin; Cox, Kara S; Joyce, Joseph G; Freed, Daniel C; Thoryk, Elizabeth; Fu, Tong-Ming; Casimiro, Danilo R; Zhang, Ningyan; A Vora, Kalpit; An, Zhiqiang

    2015-01-01

    Nonhuman primates (NHPs) are used as a preclinical model for vaccine development, and the antibody profiles to experimental vaccines in NHPs can provide critical information for both vaccine design and translation to clinical efficacy. However, an efficient protocol for generating monoclonal antibodies from single antibody secreting cells of NHPs is currently lacking. In this study we established a robust protocol for cloning immunoglobulin (IG) variable domain genes from single rhesus macaque (Macaca mulatta) antibody secreting cells. A sorting strategy was developed using a panel of molecular markers (CD3, CD19, CD20, surface IgG, intracellular IgG, CD27, Ki67 and CD38) to identify the kinetics of B cell response after vaccination. Specific primers for the rhesus macaque IG genes were designed and validated using cDNA isolated from macaque peripheral blood mononuclear cells. Cloning efficiency was averaged at 90% for variable heavy (VH) and light (VL) domains, and 78.5% of the clones (n = 335) were matched VH and VL pairs. Sequence analysis revealed that diverse IGHV subgroups (for VH) and IGKV and IGLV subgroups (for VL) were represented in the cloned antibodies. The protocol was tested in a study using an experimental dengue vaccine candidate. About 26.6% of the monoclonal antibodies cloned from the vaccinated rhesus macaques react with the dengue vaccine antigens. These results validate the protocol for cloning monoclonal antibodies in response to vaccination from single macaque antibody secreting cells, which have general applicability for determining monoclonal antibody profiles in response to other immunogens or vaccine studies of interest in NHPs.

  20. Analysis of acetylcholine receptor phosphorylation sites using antibodies to synthetic peptides and monoclonal antibodies.

    PubMed Central

    Safran, A; Neumann, D; Fuchs, S

    1986-01-01

    Three peptides corresponding to residues 354-367, 364-374, 373-387 of the acetylcholine receptor (AChR) delta subunit were synthesized. These peptides represent the proposed phosphorylation sites of the cAMP-dependent protein kinase, the tyrosine-specific protein kinase and the calcium/phospholipid-dependent protein kinase respectively. Using these peptides as substrates for phosphorylation by the catalytic subunit of cAMP-dependent protein kinase it was shown that only peptides 354-367 was phosphorylated whereas the other two were not. These results verify the location of the cAMP-dependent protein kinase phosphorylation site within the AChR delta subunit. Antibodies elicited against these peptides reacted with the delta subunit. The antipeptide antibodies and two monoclonal antibodies (7F2, 5.46) specific for the delta subunit were tested for their binding to non-phosphorylated receptor and to receptor phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. Antibodies to peptide 354-367 were found to react preferentially with non-phosphorylated receptor whereas the two other anti-peptide antibodies bound equally to phosphorylated and non-phosphorylated receptors. Monoclonal antibody 7F2 reacted preferentially with the phosphorylated form of the receptor whereas monoclonal antibody 5.46 did not distinguish between the two forms. Images Fig. 2. Fig. 4. Fig. 5. PMID:3816758

  1. [Progress in preparation of small monoclonal antibodies of knock out technique].

    PubMed

    Liu, Jing; Mao, Xin-min; Li, Lin-lin; Li, Xin-xia; Wang, Ye; Lan, Yi

    2015-10-01

    With the application of monoclonal antibody technology more and more widely, its production technology is becoming more and more perfect. Small molecule monoclonal antibody technology is becoming a hot research topic for people. The application of traditional Chinese medicine small molecule monoclonal antibody technology has been more and more widely, the technology for effective Chinese medicine component knockout provide strong technical support. The preparation of monoclonal antibodies and small molecule knockout technology are reviewed in this paper. The preparation of several steps, such as: in the process of preparation of antigen, hapten carrier coupling, coupling ratio determination and identification of artificial antigen and establishment of animal immunization and hybridoma cell lines of monoclonal antibody, the large-scale preparation; small molecule monoclonal antibody on Immune in affinity chromatography column method is discussed in detail. The author believes that this technology will make the traditional Chinese medicine research on a higher level, and improve the level of internationalization of Chinese medicine research.

  2. STRUCTURAL ELUCIDATION OF CRITICAL RESIDUES INVOLVED IN BINDING OF HUMAN MONOCLONAL ANTIBODIES TO HEPATITIS C VIRUS E2 ENVELOPE GLYCOPROTEIN

    PubMed Central

    Iacob, Roxana E.; Keck, Zhenyong; Olson, Oakley; Foung, Steven K.H.; Tomer, Kenneth B.

    2008-01-01

    Human monoclonal antibodies derived from B cells of HCV infected individuals provide information on the immune response to native HCV envelope proteins as they are recognized during infection. Monoclonal antibodies have been useful in the determination of the function and structure of specific immunogenic domains of proteins and should also be useful for the structure/function characterization of HCV E1 and E2 envelope glycoproteins. The HCV E2 envelope glycoprotein has at least three immunodistinctive conformation domains, designated A, B, and C. Conformational epitopes within domain B and C are neutralizing antibody targets on HCV pseudoparticles as well as from infectious cell culture virus. In this study, a combination of differential surface modification and mass spectrometric limited proteolysis followed by alanine mutagenesis was used to provide insight into potential conformational changes within the E2 protein upon antibody binding. The arginine guanidine groups in the E2 protein were modified with CHD in both the affinity bound and free states followed by mass spectrometric analysis, and the regions showing protection upon antibody binding were identified. This protection can arise by direct contact between the residues and the monoclonal antibody, or by antibody-induced conformational changes. Based on the mass spectrometric data, site-directed mutagenesis experiments were performed which clearly identified additional amino acids residues on E2 distant from the site of antibody interaction, whose change to alanine inhibited antibody recognition by inducing conformational changes within the E2 protein. PMID:18230369

  3. Monoclonal antibodies against type II rat brain protein kinase

    SciTech Connect

    Nakabayashi, C.H.; Huang, K.P.

    1987-05-01

    Three monoclonal antibodies (8/1, 10/10, and 25/3) against rat brain type II protein kinase C (PKC) were used to carry out the immunochemical characterization of this kinase. These antibodies immunoprecipitated the type II PKC in a dose-dependent manner but did neither to type I nor type III isozyme. Purified type II PKC has a molecular weight of 82,000 and consists of heterogeneous isoelectric point species, all of which are cross reactive with these antibodies. Immunoblot analysis of the tryptic fragments from PKC revealed that all three antibodies recognized the 33-38-KDa fragments, the phospholipid/phorbol ester-binding domain, but not the 45-48-KDa fragments, the kinase catalytic domain. The immune complexes of the kinase and the antibodies retained the kinase activity which was dependent on Ca/sup 2 +/ and phosphatidylserine (PS) and further activated by diacylglycerol. With antibody 8/1, the apparent Km values of the kinase for Ca/sup 2 +/ and PS were not influenced. The initial rate and final extent of autophosphorylation were reduced. The concentration of PS required for half-maximal (/sup 3/H)phorbol 12,13-dibutyrate (PDBu) binding was increased and the total PDBu binding was reduced. In the presence of optimum concentrations of Ca/sup 2 +/ and PS, the Kd of PDBu was unaffected by the antibody but the total binding was reduced. These results demonstrate that the PS/PDBu-binding domain contains the major epitope for the antibodies and the antibody mainly influences the PS/PDBu binding to the kinase.

  4. Practical considerations for nonclinical safety evaluation of therapeutic monoclonal antibodies

    PubMed Central

    Lynch, Carmel M; Hart, Bruce W

    2009-01-01

    Monoclonal antibodies (mAbs) are a well established class of therapeutics as evidenced by a large number of FDA approved mAbs for the treatment of cancers and autoimmune diseases. Monoclonal antibodies that are molecularly engineered for enhanced functions and pharmacokinetic properties are routinely being considered for development by many biotechnology companies. Safety evaluation of current generation of mAbs poses new challenges due to the highly complex nature of engineering aspects and variability induced by the diverse recombinant cell systems to generate them. This review provides a basic outline for nonclinical safety evaluation of therapeutic antibodies. Important considerations for planning a preclinical program, the types of nonclinical safety studies, and a general timeline for their conduct in relation to clinical trials are described. A list of relevant regulatory documents issued by government agencies is also provided. Adoption of these principles will greatly enhance the quality and relevance of the nonclinical safety data generated and will facilitate future development of mAb therapeutics. PMID:20046568

  5. Characterization of oxidative carbonylation on recombinant monoclonal antibodies.

    PubMed

    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.

  6. Trends in capacity utilization for therapeutic monoclonal antibody production.

    PubMed

    Langer, Eric S

    2009-01-01

    The administration of high doses of therapeutic antibodies requires large-scale, efficient, cost effective manufacturing processes. An understanding of how the industry is using its available production capacity is important for production planning, and facility expansion analysis. Inaccurate production planning for therapeutic antibodies can have serious financial ramifications. In the recent 5(th) Annual Report and Survey of Biopharmaceutical Manufacturing Capacity and Production, 434 qualified respondents from 39 countries were asked to indicate, among other manufacturing issues, their current trends and future predictions with respect to the production capacity utilization of monoclonal antibodies in mammalian cell culture systems. While overall production of monoclonals has expanded dramatically since 2003, the average capacity utilization for mammalian cell culture systems, has decreased each year since 2003. Biomanufacturers aggressively attempt to avoid unanticipated high production demands that can create a capacity crunch. We summarize trends associated with capacity utilization and capacity constraints which indicate that biopharmaceutical manufacturers are doing a better job planning for capacity. The results have been a smoothing of capacity use shifts and an improved ability to forecast capacity and outsourcing needs. Despite these data, today, the instability and financial constraints caused by the current global economic crisis are likely to create unforeseen shifts in our capacity utilization and capacity expansion trends. These shifts will need to be measured in subsequent studies.

  7. Immunolocalization of neuroblastoma using radiolabeled monoclonal antibody UJ13A

    SciTech Connect

    Goldman, A.; Vivian, G.; Gordon, I.; Pritchard, J.; Kemshead, J.

    1984-08-01

    The monoclonal antibody UJ13A, raised after immunization of mice with human fetal brain, recognized an antigen expressed on human neuroblastoma cell lines and fresh tumors. Antibody was purified and radiolabeled with iodine isotopes using chloramine-T. In preclinical studies, 125I-labeled UJ13A was injected intravenously into nude mice bearing xenografts of human neuroblastoma. Radiolabeled UJ13A uptake by the tumors was four to 23 times greater than that by blood. In control animals, injected with a similar quantity of a monoclonal antibody known not to bind to neuroblastoma cells in vitro (FD44), there was no selective tumor uptake. Nine patients with histologically confirmed neuroblastoma each received 100 to 300 micrograms UJ13A radiolabeled with 1 to 2.8 mCi 123I or 131I. Sixteen positive sites were visible on gamma scans 1 to 7 days after injection: 15 were primary or secondary tumor sites, and one was a false positive; there were two false negatives. In two of the 15 positive sites, tumor had not been demonstrated by other imaging techniques; these were later confirmed as areas of malignant infiltration. No toxicity was encountered.

  8. Generation of human monoclonal antibodies reactive with cellular antigens.

    PubMed Central

    Cote, R J; Morrissey, D M; Houghton, A N; Beattie, E J; Oettgen, H F; Old, L J

    1983-01-01

    Human lymphocytes from lymph node, peripheral blood, spleen, and tumor specimens have been fused with the LICR-LON-HMy2 (LICR-2) or SKO-007 human cell lines or the NS-1 mouse myeloma line. Over 75 fusions with the three myeloma-lymphoblastoid lines have been performed. Several factors appeared to improve the fusion outcome, including maintenance of the myeloma-lymphoblastoid lines in logarithmic phase growth at greater than or equal to 95% viability, a delay of 24 hr in the introduction of aminopterin to the fused cells, and preselection of the fetal calf serum used in the medium. For a given number of lymphocytes, fusions with NS-1 produced 5-20 times more clones than fusions with LICR-2 or SKO-007, and LICR-2 produced 4 times as many clones as SKO-007. The percentage of clones secreting human immunoglobulin, the range of immunoglobulin production, and the proportion of IgM, IgA, and IgG secretors were comparable for clones derived from the three myeloma-lymphoblastoid lines. Stable Ig-secreting clones were isolated with approximately equal frequency from LICR-2 and NS-1 fusions. A number of stable clones producing human monoclonal antibodies reacting with cell-surface, cytoplasmic, or nuclear antigens have been isolated from tumor-bearing patients and normal individuals. A surface antigenic system present on normal and malignant cells has been defined with a human monoclonal antibody derived from a patient with breast cancer. Techniques for producing human monoclonal antibody now appear to be sufficiently advanced to initiate a serological dissection of the humoral immune response to cancer. Images PMID:6572959

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

    PubMed

    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.

  10. Monoclonal Antibody Shows Promise as Potential Therapeutic for MERS | Poster

    Cancer.gov

    A monoclonal antibody has proven effective in preventing Middle Eastern Respiratory Syndrome (MERS) in lab animals, suggesting further development as a potential intervention for the deadly disease in humans, according to new research. MERS is a newly emerged coronavirus first detected in humans in 2012. Most cases have occurred in the Middle East, but the disease has appeared elsewhere. In all, MERS has infected more than 1,700 individuals and killed more than 600, according to the World Health Organization. No vaccines or antiviral therapies currently exist. Several candidate vaccines are being developed, and some have been tested in animal models, a prerequisite to human clinical trials.

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

    PubMed

    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.

  12. Monoclonal antibody against mouse CAR following genetic immunization.

    PubMed

    Carson, Steven D; Switzer, Barbara L; Tracy, Steven M; Chapman, Nora M

    2004-02-01

    To broaden our repertoire of monoclonal antibodies against CAR (coxsackievirus and adenovirus receptor), we inoculated mice with an expression vector containing the cDNA encoding human CAR extracellular and transmembrane sequence, and boosted the response by inoculation with soluble human CAR protein produced in E. coli. Of the hybridomas obtained following this immunization protocol, one secreted IgG with exceptional reactivity against mouse CAR. Since CAR has been shown to form dimers, expression of human CAR in cells that express mouse CAR may have stimulated the host immune system to recognize endogenous CAR in heterodimers.

  13. Monoclonal Antibody Shows Promise as Potential Therapeutic for MERS | Poster

    Cancer.gov

    A monoclonal antibody has proven effective in preventing Middle Eastern Respiratory Syndrome (MERS) in lab animals, suggesting further development as a potential intervention for the deadly disease in humans, according to new research. MERS is a newly emerged coronavirus first detected in humans in 2012. Most cases have occurred in the Middle East, but the disease has appeared elsewhere. In all, MERS has infected more than 1,700 individuals and killed more than 600, according to the World Health Organization. No vaccines or antiviral therapies currently exist. Several candidate vaccines are being developed, and some have been tested in animal models, a prerequisite to human clinical trials.

  14. Generation of cell lines for monoclonal antibody production.

    PubMed

    Alvin, Krista; Ye, Jianxin

    2014-01-01

    Monoclonal antibodies (mAbs) represent the largest group of therapeutic proteins with 30 products approved in the USA and hundreds of therapies currently undergoing clinical trials. The complex nature of mAbs makes their development as therapeutic agents constrained by numerous criteria such as quality, safety, regulation, and quantity. Identification of a clonal cell line expressing high levels of mAb with adequate quality attributes and generated in compliance with regulatory standards is a necessary step prior to a program moving to large-scale production for clinical material. This chapter outlines the stable transfection technology that generates clonal cell lines for commercial manufacturing processes.

  15. Monoclonal antibodies directed against surface molecules of multicell spheroids

    NASA Technical Reports Server (NTRS)

    Martinez, Andrew O.

    1993-01-01

    The objective of this project is to generate a library of monoclonal antibodies (MAb's) to surface molecules involved in the cell-cell interactions of mammalian cells grown as multicell spheroids (MCS). MCS are highly organized 3-dimensional multicellular structures which exhibit many characteristics in vivo tissues not found in conventional monolayer or suspension culture. They also provide a functional assay for surface adhesion molecules. In brief, MCS combine the relevance of organized tissues with the accuracy of in vitro methodology. Further, one can manipulate these MCS experimentally to discern important information about their biology.

  16. Development of Monoclonal Antibodies in China: Overview and Prospects

    PubMed Central

    Zhang, Mao-Yu; Lu, Jin-Jian; Wang, Liang; Gao, Zi-Chao; Ung, Carolina Oi Lam; Wang, Yi-Tao

    2015-01-01

    Monoclonal antibodies (mAbs) have become increasingly important as human therapeutic agents. Yet, current research concentrates on technology itself and pays attention to developed countries. This paper aims to provide a comprehensive review of mAbs development in China through systematic analysis of drug registry, patent applications, clinical trials, academic publication, and ongoing R&D projects. The trends in therapeutic areas and industrialization process are also highlighted. Development and research trends of mAbs are analyzed to provide a future perspective of mAbs as therapeutic agents in China. PMID:25811022

  17. Anti-interleukin-10R1 monoclonal antibody in combination with bacillus Calmette–Guérin is protective against bladder cancer metastasis in a murine orthotopic tumour model and demonstrates systemic specific anti-tumour immunity

    PubMed Central

    Newton, M R; Askeland, E J; Andresen, E D; Chehval, V A; Wang, X; Askeland, R W; O'Donnell, M A; Luo, Y

    2014-01-01

    Effective treatment of bladder cancer with bacillus Calmette–Guérin (BCG) depends on the induction of a T helper type (Th) 1 immune response. Interleukin (IL)-10 down-regulates the Th1 response and is associated with BCG failure. In this study, we investigated whether blocking IL-10 signalling could enhance the BCG-induced Th1 response and anti-tumour immunity in a murine orthotopic tumour model. Treatment with BCG and anti-IL-10 receptor 1 monoclonal antibody (anti-IL-10R1 mAb) increased the interferon (IFN)-γ to IL-10 ratio in both splenocyte cultures and urine. Mice bearing luciferase-expressing MB49 (MB49-Luc) tumours were treated and followed for tumour growth by bioluminescent imaging, bladder weight and histology. Mice treated with phosphate-buffered saline (PBS) (group 1), BCG plus control immunoglobulin (Ig)G1 (group 2) or BCG plus anti-IL-10R1 mAb (group 3) showed 0, 6 and 22% tumour regression, respectively. The mean bladder weight of group 3 mice was substantially lower than those of groups 1 and 2 mice. Remarkably, 36% of group 1 and 53% of group 2 mice but no group 3 mice developed lung metastasis (P = 0·02). To investigate the mechanisms underlying the effect of combination therapy, splenocytes were stimulated with S12 peptide (serine mutation at codon 12 of the K-ras oncogene) known to be expressed in MB49-Luc cells. Induction of ras mutation-specific IFN-γ and cytotoxicity was observed in mice treated with combination therapy. These observations indicate that BCG, in combination with anti-IL-10R1 mAb, induces enhanced anti-tumour immunity that is protective against lung metastasis. Anti-IL-10R1 mAb demonstrates systemic effects and may prove useful in clinical practice for treating bladder cancer in high-risk patients. PMID:24593764

  18. Anti-interleukin-10R1 monoclonal antibody in combination with bacillus Calmette--Guérin is protective against bladder cancer metastasis in a murine orthotopic tumour model and demonstrates systemic specific anti-tumour immunity.

    PubMed

    Newton, M R; Askeland, E J; Andresen, E D; Chehval, V A; Wang, X; Askeland, R W; O'Donnell, M A; Luo, Y

    2014-07-01

    Effective treatment of bladder cancer with bacillus Calmette-Guérin (BCG) depends on the induction of a T helper type (Th) 1 immune response. Interleukin (IL)-10 down-regulates the Th1 response and is associated with BCG failure. In this study, we investigated whether blocking IL-10 signalling could enhance the BCG-induced Th1 response and anti-tumour immunity in a murine orthotopic tumour model. Treatment with BCG and anti-IL-10 receptor 1 monoclonal antibody (anti-IL-10R1 mAb) increased the interferon (IFN)-γ to IL-10 ratio in both splenocyte cultures and urine. Mice bearing luciferase-expressing MB49 (MB49-Luc) tumours were treated and followed for tumour growth by bioluminescent imaging, bladder weight and histology. Mice treated with phosphate-buffered saline (PBS) (group 1), BCG plus control immunoglobulin (Ig)G1 (group 2) or BCG plus anti-IL-10R1 mAb (group 3) showed 0, 6 and 22% tumour regression, respectively. The mean bladder weight of group 3 mice was substantially lower than those of groups 1 and 2 mice. Remarkably, 36% of group 1 and 53% of group 2 mice but no group 3 mice developed lung metastasis (P = 0·02). To investigate the mechanisms underlying the effect of combination therapy, splenocytes were stimulated with S12 peptide (serine mutation at codon 12 of the K-ras oncogene) known to be expressed in MB49-Luc cells. Induction of ras mutation-specific IFN-γ and cytotoxicity was observed in mice treated with combination therapy. These observations indicate that BCG, in combination with anti-IL-10R1 mAb, induces enhanced anti-tumour immunity that is protective against lung metastasis. Anti-IL-10R1 mAb demonstrates systemic effects and may prove useful in clinical practice for treating bladder cancer in high-risk patients.

  19. A Combination of Three Fully Human Toxin A- and Toxin B-Specific Monoclonal Antibodies Protects against Challenge with Highly Virulent Epidemic Strains of Clostridium difficile in the Hamster Model

    PubMed Central

    Cole, Leah E.; Li, Lu; Zhang, Jinrong; Brown, Anna M.; Mundle, Sophia; Zhang, Jianxin; Ray, Satyajit; Ma, Fuqin; Garrone, Pierre; Bertraminelli, Nicola; Kleanthous, Harry; Anderson, Stephen F.

    2015-01-01

    Clostridium difficile infection (CDI) is the principal cause of nosocomial diarrhea and pseudomembranous colitis associated with antibiotic therapy. Recent increases in the number of outbreaks attributed to highly virulent antibiotic-resistant strains underscore the importance of identifying efficacious alternatives to antibiotics to control this infection. CDI is mediated by two large exotoxins, toxins A and B. Strong humoral toxin-specific immune responses are associated with recovery and a lack of disease recurrence, whereas insufficient humoral responses are associated with recurrent CDI. Multiple approaches targeting these toxins, including intravenous immunoglobulin, neutralizing polymers, active vaccines, and, most recently, monoclonal antibodies (MAbs), have been explored, with various degrees of success. In this study, we describe the characterization of the first MAbs isolated from healthy human donors using a high-throughput B-cell cloning strategy. The MAbs were selected based on their ability to inhibit the actions of toxins A and B in vitro and because of their in vivo efficacy in a hamster challenge model. A potent 2-MAb cocktail was identified and then further potentiated by the addition of a second anti-toxin B MAb. This 3-MAb combination protected animals against mortality and also reduced the severity and duration of diarrhea associated with challenge with highly virulent strains of C. difficile toxinotypes 0 and III. This highly efficacious cocktail consists of one MAb specific to the receptor binding domain of toxin A and two MAbs specific to nonoverlapping regions of the glucosyltransferase domain of toxin B. This MAb combination offers great potential as a nonantibiotic treatment for the prevention of recurrent CDI. PMID:25924765

  20. A Combination of Three Fully Human Toxin A- and Toxin B-Specific Monoclonal Antibodies Protects against Challenge with Highly Virulent Epidemic Strains of Clostridium difficile in the Hamster Model.

    PubMed

    Anosova, Natalie G; Cole, Leah E; Li, Lu; Zhang, Jinrong; Brown, Anna M; Mundle, Sophia; Zhang, Jianxin; Ray, Satyajit; Ma, Fuqin; Garrone, Pierre; Bertraminelli, Nicola; Kleanthous, Harry; Anderson, Stephen F

    2015-07-01

    Clostridium difficile infection (CDI) is the principal cause of nosocomial diarrhea and pseudomembranous colitis associated with antibiotic therapy. Recent increases in the number of outbreaks attributed to highly virulent antibiotic-resistant strains underscore the importance of identifying efficacious alternatives to antibiotics to control this infection. CDI is mediated by two large exotoxins, toxins A and B. Strong humoral toxin-specific immune responses are associated with recovery and a lack of disease recurrence, whereas insufficient humoral responses are associated with recurrent CDI. Multiple approaches targeting these toxins, including intravenous immunoglobulin, neutralizing polymers, active vaccines, and, most recently, monoclonal antibodies (MAbs), have been explored, with various degrees of success. In this study, we describe the characterization of the first MAbs isolated from healthy human donors using a high-throughput B-cell cloning strategy. The MAbs were selected based on their ability to inhibit the actions of toxins A and B in vitro and because of their in vivo efficacy in a hamster challenge model. A potent 2-MAb cocktail was identified and then further potentiated by the addition of a second anti-toxin B MAb. This 3-MAb combination protected animals against mortality and also reduced the severity and duration of diarrhea associated with challenge with highly virulent strains of C. difficile toxinotypes 0 and III. This highly efficacious cocktail consists of one MAb specific to the receptor binding domain of toxin A and two MAbs specific to nonoverlapping regions of the glucosyltransferase domain of toxin B. This MAb combination offers great potential as a nonantibiotic treatment for the prevention of recurrent CDI.

  1. Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats.

    PubMed

    Byrnes-Blake, Kelly A; Laurenzana, Elizabeth M; Carroll, F Ivy; Abraham, Philip; Gentry, W Brooks; Landes, Reid D; Owens, S Michael

    2003-02-14

    Our studies examined pharmacokinetic mechanisms involved in high-affinity (K(d) approximately 11 nM) monoclonal antibody-based antagonism of (+)-methamphetamine-induced locomotor effects. Male rats received (+)-methamphetamine (0.3, 1, or 3 mg/kg i.v.) followed 30 min later by saline or anti-(+)-methamphetamine monoclonal antibody. All groups received a constant dose of monoclonal antibody that was equimolar in binding sites to the body burden of a 1 mg/kg i.v. (+)-methamphetamine dose 30 min after administration. The monoclonal antibody antagonized locomotor effects due to 0.3 and 1 mg/kg (+)-methamphetamine. In contrast, monoclonal antibody treatment increased locomotor activity due to 3 mg/kg (+)-methamphetamine. We also investigated the serum and brain pharmacokinetics of (+)-methamphetamine without and with the monoclonal antibody. Rats received (+)-methamphetamine (1 mg/kg i.v.) followed by saline or monoclonal antibody treatment at 30 min. The monoclonal antibody significantly increased serum methamphetamine concentrations and significantly decreased brain methamphetamine concentrations. These data indicate that anti-(+)-methamphetamine monoclonal antibody-induced pharmacodynamics are complex, but are related to time-dependent changes in (+)-methamphetamine brain distribution. Copyright 2003 Elsevier Science B.V.

  2. Monoclonal antibodies to pancreatic stone protein. Radioimmunoassay and immunological comparison with trypsin 1.

    PubMed

    Provansal-Cheylan, M; Lusher, M; De Caro, A; Multigner, L; Montalto, G; Sarles, H; Delaage, M

    1986-09-01

    Monoclonal antibodies were prepared against pancreatic stone protein, a protein which inhibits calcium carbonate precipitation. Two monoclonal antibodies designated D4 and 2E7 were characterized. Immunoadsorbant columns, obtained by linkage of these monoclonal antibodies to Affigel 10, have been used to isolate immunoreactive forms of pancreatic stone protein from nonactivated human pancreatic juice. These monoclonal antibodies permitted us to test the possible immunological relationship between pancreatic stone protein and human trypsin 1. No immunological similarity was found, in agreement with our previous results, and it was established that pancreatic stone protein is a novel protein and not a degradation product of human trypsin(ogen) 1.

  3. Binding specificity of a monoclonal anti-DNA antibody.

    PubMed

    Pisetsky, D S; Caster, S A

    1982-05-01

    To investigate the interaction of DNA and anti-DNA antibodies in the immune complex disease of systemic lupus erythematosus, the fine specificity of binding of a monoclonal anti-DNA antibody was determined. This antibody, termed Cll, was derived from the fusion of spleen cells from an autoimmune MRL-lpr/lpr mouse with the myeloma cell line M45. In a solid-phase ELISA assay to measure anti-DNA activity, Cll showed preference for single stranded compared to double stranded DNA of animal origin. The Cll antibody also bound some deoxyribohomopolymers as well as ribohomopolymers, but failed to bind synthetic DNA duplexes. Defined size oligonucleotides with a size range of 2-(12-18) failed to inhibit the binding of Cll to single stranded DNA. This pattern of binding is consistent with the recognition of a unique structural determinant that can be represented by a variety of nucleic acids. The absence of antigenic activity among the oligonucleotides suggests that an extended polynucleotide structure is required for antibody binding, possibly because of a bivalent or 'monogamous' mode of interaction. The binding properties of Cll further suggest that its ability to participate in immune complex formation may be limited by the nature of the available DNA antigen.

  4. Monoclonal antibodies with specificity for hairy cell leukemia cells.

    PubMed Central

    Posnett, D N; Chiorazzi, N; Kunkel, H G

    1982-01-01

    Hairy cell leukemia is a well described clinical entity, but the cell of origin for this leukemic cell and its function are still unknown. There are no totally specific markers for this cell, although tartrate-resistant acid phosphatase staining has been used extensively as a diagnostic test. This study describes three monoclonal murine antibodies with variable specificity for hairy cells. Antibody 1 was highly specific for hairy cells and was not found to react with normal or leukemic cells in this limited study. It did not react with the cells of all patients. It also did not react with all of the hairy cells of some of the positive cases. Antibodies 2 and 3 reacted with virtually all hairy cells but not with normal peripheral blood cells. However, reactions were obtained with certain leukemic myelomonoblasts and some activated B cells. The most obvious use for these three antibodies is for diagnostic purposes. They should also be helpful reagents to investigate the origin of the leukemic hairy cell. The possibility that antibody 1 detects a tumor-specific antigen is discussed. Images PMID:7047565

  5. Monoclonal antibodies to human plasma low-density lipoproteins. I. Enhanced binding of 125I-labeled low-density lipoproteins by combined use of two monoclonal antibodies.

    PubMed

    Mao, S J; Patton, J G; Badimon, J J; Kottke, B A; Alley, M C; Cardin, A D

    1983-11-01

    Four monoclonal antibodies (IgG2b) to human plasma low-density lipoproteins (LDL) have been characterized. The binding affinities of each monoclonal antibody to 125I-labeled LDL were moderately high, ranging from 10(8) to 10(10) L/mol at 4 degrees C, but were reduced by at least 50-70% at 37 degrees C. The maximum binding of each monoclonal antibody was unique, ranging from 20 to 95% of total 125I-labeled LDL, suggesting that LDL particles were immunochemically heterogeneous. One antibody, LP-34, had both high and low binding affinities to LDL. Another, LP-47, exhibited high affinity for isolated LDL, yet reacted poorly with native LDL in plasma, indicating that the conformation of isolated LDL differs from that of native LDL in plasma. Unlike polyclonal serum antibodies, a mixture of four monoclonal antibodies failed to precipitate LDL, but did show a drastic increase in binding to LDL. We found that only two of our monoclonal antibodies were necessary for such synergistic enhancement. We propose that one of the monoclonal antibodies may serve as a catalytic reagent, and discuss the clinical significance of this finding.

  6. Monoclonal antibodies to the extracellular glucosyltransferases from Streptococcus sobrinus 6715.

    PubMed Central

    McCabe, M M; Alberts, M; Stein, J

    1987-01-01

    Murine monoclonal antibodies (MAbs) were raised against the glucosyltransferases (GTFs) of Streptococcus sobrinus 6715. The antibody panels included MAbs raised against the primer-independent, soluble product enzyme (GTF-Si) which did not cross-react with other GTFs, as well as MAbs raised against the primer-dependent, soluble product enzyme (GTF-Sd) which recognized both GTF-Si and GTF-Sd, thus indicating that these catalytically distinct enzymes share epitopes. MAbs raised against GTF-I recognized several forms of GTF-I and did not cross-react with the GTF-S enzymes. None of the MAbs recognized the major glucan-binding protein of S. sobrinus. Two MAbs inhibited glucan synthesis, one blocking primer synthesis by GTF-Si by 89% and the second inhibiting that by GTF-I by 92%. Images PMID:2956196

  7. Discovery and characterization of hydroxylysine in recombinant monoclonal antibodies

    PubMed Central

    Xie, Qing; Moore, Benjamin; Beardsley, Richard L.

    2016-01-01

    ABSTRACT Tryptic peptide mapping analysis of a Chinese hamster ovary (CHO)-expressed, recombinant IgG1 monoclonal antibody revealed a previously unreported +16 Da modification. Through a combination of MSn experiments, and preparation and analysis of known synthetic peptides, the possibility of a sequence variant (Ala to Ser) was ruled out and the presence of hydroxylysine was confirmed. Post-translational hydroxylation of lysine was found in a consensus sequence (XKG) known to be the site of modification in other proteins such as collagen, and was therefore presumed to result from the activity of the CHO homolog of the lysyl hydroxylase complex. Although this consensus sequence was present in several locations in the antibody sequence, only a single site on the heavy-chain Fab was found to be modified. PMID:26651858

  8. The application of monoclonal antibodies in cancer diagnosis.

    PubMed

    Zhang, Xuemei; Soori, Gamini; Dobleman, Thomas J; Xiao, Gary G

    2014-01-01

    Cancer becomes the second leading cause of death in the world. An effective strategy for early diagnosis of the disease is key to reduce the mortality and morbidity. Development of effective monoclonal antibody (mAb)-based assays or diagnostic imaging techniques for detection of antigens and small molecules that are released from cancerous cells will enhance modern diagnostic medicine of cancer significantly. Although mAb technology is still under development, recent advances in preparation of recombinant antigen and antibody engineering techniques have dramatically enhanced the applications of this technology in cancer diagnosis. Compared with other methods, mAb-based assays may provide spatial, temporal, accurate and quantitative measurement for diagnosis of the disease. This review summarizes the progress of the mAb-based assays in the field of molecular diagnosis of cancer.

  9. Internal radiation dosimetry for clinical testing of radiolabeled monoclonal antibodies

    SciTech Connect

    Fisher, D.R.; Durham, J.S.; Hui, T.E.; Hill, R.L.

    1990-11-01

    In gauging the efficacy of radiolabeled monoclonal antibodies in cancer treatment, it is important to know the amount of radiation energy absorbed by tumors and normal tissue per unit administered activity. This paper describes methods for estimating absorbed doses to human tumors and normal tissues, including intraperitoneal tissue surfaces, red marrow, and the intestinal tract from incorporated radionuclides. These methods use the Medical Internal Radiation Dose (MIRD) scheme; however, they also incorporate enhancements designed to solve specific dosimetry problems encountered during clinical studies, such as patient-specific organ masses obtained from computerized tomography (CT) volumetrics, estimates of the dose to tumor masses within normal organs, and multicellular dosimetry for studying dose inhomogeneities in solid tumors. Realistic estimates of absorbed dose are provided within the short time requirements of physicians so that decisions can be made with regard to patient treatment and procurement of radiolabeled antibodies. Some areas in which further research could improve dose assessment are also discussed. 16 refs., 3 figs.

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

    PubMed

    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

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

    PubMed

    De Benedictis, Paola; Minola, Andrea; Rota Nodari, Elena; Aiello, Roberta; Zecchin, Barbara; Salomoni, Angela; Foglierini, Mathilde; Agatic, Gloria; Vanzetta, Fabrizia; Lavenir, Rachel; Lepelletier, Anthony; Bentley, Emma; Weiss, Robin; Cattoli, Giovanni; Capua, Ilaria; Sallusto, Federica; Wright, Edward; Lanzavecchia, Antonio; Bourhy, Hervé; Corti, Davide

    2016-04-01

    Currently available rabies post-exposure prophylaxis (PEP) for use in humans includes equine or human rabies immunoglobulins (RIG). The replacement of RIG with an equally or more potent and safer product is strongly encouraged due to the high costs and limited availability of existing RIG. In this study, we identified two broadly neutralizing human monoclonal antibodies that represent a valid and affordable alternative to RIG in rabies PEP. Memory B cells from four selected vaccinated donors were immortalized and monoclonal antibodies were tested for neutralizing activity and epitope specificity. Two antibodies, identified as RVC20 and RVC58 (binding to antigenic site I and III, respectively), were selected for their potency and broad-spectrum reactivity. In vitro, RVC20 and RVC58 were able to neutralize all 35 rabies virus (RABV) and 25 non-RABV lyssaviruses. They showed higher potency and breath compared to antibodies under clinical development (namely CR57, CR4098, and RAB1) and commercially available human RIG. In vivo, the RVC20-RVC58 cocktail protected Syrian hamsters from a lethal RABV challenge and did not affect the endogenous hamster post-vaccination antibody response. © 2016 Humabs BioMed SA Published under the terms of the CC BY 4.0 license.

  12. Biosimilar monoclonal antibodies (mAbs) in oncology.

    PubMed

    Moore, Caroline

    2017-09-07

    Biological medicines are derived from living cells and organisms. Monoclonal antibodies (mAbs) are biological agents that are widely used to treat malignancies including non-Hodgkin's lymphomas and chronic lymphocytic leukaemia. They are effective but expensive. The patents for many mAbs are expiring, so biosimilar medicines, which contain a version of the active ingredient of the original drug, are being developed. Biological medicines cannot be assessed in the same way as standard generic medications because they are difficult to copy and can change over time. A pathway regulates how biosimilars are assessed and compared with the original drug to ensure they are highly similar and have no clinically meaningful differences in terms of structure, function, pharmacodynamics and mechanism of action, pharmacokinetic properties, clinical efficacy and safety. Truxima(® ▾)(rituximab), the first biosimilar monoclonal antibody to be approved for use in the UK in an oncology setting, is biosimilar to intravenous (IV) rituximab; rituximab improves the effectiveness of standard chemotherapy for lymphoma. The two drugs are comparable in efficacy and safety and have the same indications, dosing regimen and storage procedures.

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

    PubMed

    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.

  14. Generation of a rat monoclonal antibody specific for hsp72.

    PubMed

    Tanaka, Masako; Shiota, Masayuki; Okada, Seiji; Harada, Akihito; Odawara, Jun; Mun, Saya; Iwao, Hiroshi; Ohkawa, Yasuyuki

    2011-08-01

    The heat shock protein 70 (Hsp70) family members function as ATP-dependent molecular chaperones that assist in the folding of newly synthesized polypeptides and in the refolding of misfolded/aggregated proteins. These heat shock proteins comprise at least eight sets of molecular groups that share high homology, but differ from each other in their expression level and subcellular localization. Hsp72, which is also known as Hsp70 and Hsp70-1, is localized mainly in the cytoplasm but is also found in the nucleus. Stress-induced Hsp72 functions as a chaperone enabling the cells to cope with harmful aggregations of denatured proteins during and following stress. The difference in the function of Hsp72 from that of other Hsp70 members, however, remains unclear. We report the establishment of a monoclonal antibody specific for Hsp72 using the rat medial iliac lymph node method. Immunoblot analysis revealed that our monoclonal antibody against Hsp72 specifically identified the 65 kDa protein. Immunocytochemical staining also revealed that Hsp72 localized in the cytoplasm and nucleus, and aggregated in the nucleus in response to heat stress. This MAb against Hsp72 will allow for further studies to elucidate the mechanism by which Hsp72 is localized in the cell in response to stress stimuli, and aid in the identification of specific interacting molecules.

  15. Bothropic antivenom based on monoclonal antibodies, is it possible?

    PubMed

    Frauches, Thiago S; Petretski, Jorge H; Arnholdt, Andrea C V; Lasunskaia, Elena B; de Carvalho, Eulógio C Q; Kipnis, Thereza L; da Silva, Wilmar D; Kanashiro, Milton M

    2013-09-01

    Neutralizing monoclonal antibodies against three major toxic components of Bothrops atrox venom were produced and tested. The mAbs against phospholipase A2, hemorrhagic metalloprotease, and thrombin-like enzymes were produced in large amounts and purified with caprylic acid followed by ammonium sulfate precipitation. Purified mAbs were analyzed by SDS-PAGE and their ability to neutralize the respective toxins was tested. Five Swiss mice were injected i.p. with 13.5 mg of pooled mAbs and challenged via s.c. route with venom. Survival rate was recorded for the next 48 h. All mice treated and challenged with venom survived, whereas only one mouse in the control group survived. Bleeding time in mice treated with mAbs was similar to that observed in control mice. Our results show that monoclonal antibodies neutralized the lethal toxicity of Bothrops venom and indicate that there is a reasonable possibility of developing antivenoms based on humanized mAbs to treat victims of venomous animals in the future.

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

    PubMed

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

    2016-05-01

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

  17. Monoclonal Antibodies Directed to Fucoidan Preparations from Brown Algae

    PubMed Central

    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. PMID:25692870

  18. Effects of the orientation of anti-BMP2 monoclonal antibody immobilized on scaffold in antibody-mediated osseous regeneration

    PubMed Central

    Ansari, Sahar; Freire, Marcelo; Choi, Moon G; Tavari, Azadeh; Almohaimeed, Mohammad; Moshaverinia, Alireza; Zadeh, Homayoun H

    2015-01-01

    Recently, we have shown that anti-BMP2 monoclonal antibodies (mAbs) can trap endogenous osteogenic BMP ligands, which can in turn mediate osteodifferentiation of progenitor cells. The effectiveness of this strategy requires the availability of the anti-BMP-2 monoclonal antibodies antigen-binding sites for anti-BMP-2 monoclonal antibodies to bind to the scaffold through a domain that will leave its antigen-binding region exposed and available for binding to an osteogenic ligand. We examined whether antibodies bound to a scaffold by passive adsorption versus through Protein G as a linker will exhibit differences in mediating bone formation. In vitro anti-BMP-2 monoclonal antibodies was immobilized on absorbable collagen sponge (ACS) with Protein G as a linker to bind the antibody through its Fc region and implanted into rat calvarial defects. The biomechanical strength of bone regenerated by absorbable collagen sponge/Protein G/anti-BMP-2 monoclonal antibodies immune complex was compared to ACS/anti-BMP-2 monoclonal antibodies or ACS/Protein G/isotype mAb control group. Results demonstrated higher binding of anti-BMP-2 monoclonal antibodies/BMPs to C2C12 cells, when the mAb was initially attached to recombinant Protein G or Protein G-coupled microbeads. After eight weeks, micro-CT and histomorphometric analyses revealed increased bone formation within defects implanted with absorbable collagen sponge/Protein G/anti-BMP-2 monoclonal antibodies compared with defects implanted with absorbable collagen sponge/anti-BMP-2 monoclonal antibodies (p < 0.05). Confocal laser scanning microscopy (CLSM) confirmed increased BMP-2, -4, and -7 detection in sites implanted with absorbable collagen sponge/Protein G/anti-BMP-2 monoclonal antibodies in vivo. Biomechanical analysis revealed the regenerated bone in sites with Protein G/anti-BMP-2 monoclonal antibodies had higher mechanical strength in comparison to anti-BMP-2 monoclonal antibodies. The negative control group, Protein G

  19. Neutralizing determinants defined by monoclonal antibodies on polypeptides specified by bovine herpesvirus 1.

    PubMed Central

    Collins, J K; Butcher, A C; Riegel, C A; McGrane, V; Blair, C D; Teramoto, Y A; Winston, S

    1984-01-01

    Monoclonal antibodies were used to study neutralizing determinants on polypeptides of bovine herpesvirus 1. Two of three monoclonal antibodies which recognized nonoverlapping epitopes on a glycoprotein of 82,000 daltons were found to neutralize. A second group of monoclonal antibodies that individually precipitated five viral glycopolypeptides ranging in size from 102,000 to 55,000 daltons also neutralized. Two monoclonal antibodies which were the most efficient in neutralization recognized a non-glycosylated protein of 115,000 daltons which was the major polypeptide on the virus. A fourth group of monoclonal antibodies precipitated a non-glycosylated polypeptide of 91,000 daltons and several smaller polypeptides, but these antibodies demonstrated only limited neutralizing activity. Images PMID:6208375

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

    Cancer.gov

    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. Submissions will be accepted through July 9, 2012.

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

    Cancer.gov

    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. Submissions will be accepted through July 11, 2014.

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

    Cancer.gov

    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.

  3. Screening individual hybridomas by microengraving to discover monoclonal antibodies

    PubMed Central

    Ogunniyi, Adebola O; Story, Craig M; Papa, Eliseo; Guillen, Eduardo; Love, J Christopher

    2014-01-01

    The demand for monoclonal antibodies (mAbs) in biomedical research is significant, but the current methodologies used to discover them are both lengthy and costly. Consequently, the diversity of antibodies available for any particular antigen remains limited. Microengraving is a soft lithographic technique that provides a rapid and efficient alternative for discovering new mAbs. This protocol describes how to use microengraving to screen mouse hybridomas to establish new cell lines producing unique mAbs. Single cells from a polyclonal population are isolated into an array of microscale wells (~105 cells per screen). The array is then used to print a protein microarray, where each element contains the antibodies captured from individual wells. The antibodies on the microarray are screened with antigens of interest, and mapped to the corresponding cells, which are then recovered from their microwells by micromanipulation. Screening and retrieval require approximately 1–3 d (9–12 d including the steps for preparing arrays of microwells). PMID:19528952

  4. Intracavitary use of two radiolabeled tumor-associated monoclonal antibodies

    SciTech Connect

    Malamitsi, J.; Skarlos, D.; Fotiou, S.; Papakostas, P.; Aravantinos, G.; Vassilarou, D.; Taylor-Papadimitriou, J.; Koutoulidis, K.; Hooker, G.; Snook, D.

    1988-12-01

    Six patients with metastatic breast cancer and malignant pleural effusions and 13 patients with known or suspected ovarian cancer, underwent immunoscintigraphy after intracavitary (intrapleural or intraperitoneal) administration of iodine-131-(131I) or indium-111-(111In) labeled tumor associated monoclonal antibodies HMFG2 and H17E2. This method proved to be sensitive and specific with a true-positive result in 13 out of 14 patients with tumor and a true-negative result in five out of five patients without tumor. At any one time, 65%-80% of the whole-body radioactivity was closely associated with the cavity into which the radiolabeled antibody was administered while the radioactivity in the blood was always low, (approximately 4 X 10(-3) of administered dose/ml of blood). Concentrations of radiolabeled antibody (per gram of tumor tissue) ranged from 0.02%-0.1% of the injected dose in intracavitary tumors, but only 0.002% in a retroperitoneal metastasis. The specificity of this approach was documented in four control patients with benign ovarian cysts and in two patients who were imaged using both specific and nonspecific radiolabeled antibody. We conclude that the intracavitary administration of 131I- or 111In-labeled HMFG2 and H17E2 is a favorable route of administration and offers significant advantages over previously reported intravenous administration for the localization of breast or ovarian metastases confined to the pleural or peritoneal cavities.

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

    PubMed

    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.

  6. Generation and characterization of monoclonal antibodies against FABP4.

    PubMed

    Gorbenko, Olena; Filonenko, Valeriy; Gout, Ivan

    2006-04-01

    Fatty acid binding protein 4 (FABP4) is a key mediator of intracellular transport and metabolism of fatty acids in adipose tissues. FABP4 binds fatty acids with high affinity and transports them to various compartments in the cell. When in complex with fatty acids, FABP4 interacts with and modulates the activity of two important regulators of metabolism: hormone-sensitive lipase and peroxisome proliferator-activated receptor gamma. Genetic studies in mice clearly indicated that deregulation of FABP4 function may lead to the development of severe diseases such as diabetes II type and atherosclerosis. In this study, we report the production and detailed characterization of monoclonal antibodies (MAbs) against FABP4. Recombinant glutathione S-transferase (GST)-FABP4 or His-FABP4 was expressed in bacteria, affinity purified, and used for immunization of mice, enzyme-linked immunosorbent assay (ELISA) screening, and characterization of selected clones. We have isolated two hybridoma clones that produced antibodies specific for recombinant and native FABP4, as shown by Western blotting and immunoprecipitation. The specificity of generated antibodies was further tested in a cell-based model of adipogenesis. In this analysis, the accumulation of FABP4 during NIH 3T3-L1 differentiation into adipocytes was detected by generated antibodies, which correlates well with previously published data. Taken together, we produced MAbs that will be useful for the scientific community working on fatty acid-binding proteins and lipid metabolism.

  7. Structural Characterization of a Monoclonal Antibody-Maytansinoid Immunoconjugate.

    PubMed

    Luo, Quanzhou; Chung, Hyo Helen; Borths, Christopher; Janson, Matthew; Wen, Jie; Joubert, Marisa K; Wypych, Jette

    2016-01-05

    Structural characterization was performed on an antibody-drug conjugate (ADC), composed of an IgG1 monoclonal antibody (mAb), mertansine drug (DM1), and a noncleavable linker. The DM1 molecules were conjugated through nonspecific modification of the mAb at solvent-exposed lysine residues. Due to the nature of the lysine conjugation process, the ADC molecules are heterogeneous, containing a range of species that differ with respect to the number of DM1 per antibody molecule. The DM1 distribution profile of the ADC was characterized by electrospray ionization mass spectrometry (ESI-MS) and capillary isoelectric focusing (cIEF), which showed that 0-8 DM1s were conjugated to an antibody molecule. By taking advantage of the high-quality MS/MS spectra and the accurate mass detection of diagnostic DM1 fragment ions generated from the higher-energy collisional dissociation (HCD) approach, we were able to identify 76 conjugation sites in the ADC, which covered approximately 83% of all the putative conjugation sites. The diagnostic DM1 fragment ions discovered in this study can be readily used for the characterization of other ADCs with maytansinoid derivatives as payload. Differential scanning calorimetric (DSC) analysis of the ADC indicated that the conjugation of DM1 destabilized the C(H)2 domain of the molecule, which is likely due to conjugation of DM1 on lysine residues in the C(H)2 domain. As a result, methionine at position 258 of the heavy chain, which is located in the C(H)2 domain of the antibody, is more susceptible to oxidation in thermally stressed ADC samples when compared to that of the naked antibody.

  8. From the discovery of monoclonal antibodies to their therapeutic application: an historical reappraisal.

    PubMed

    Ribatti, Domenico

    2014-09-01

    Vertebrate make billions of different antibodies, each with a binding site that recognizes a specific region of a macromolecule. The hybridoma technique allows monoclonal antibodies, highly specific antibodies produced in the laboratory by a variety of methods. In the last 35 years since the first process for creating monoclonal antibodies was introduced, their application have improved the growing biotechnology industry, but the most important application concerns the therapy of human malignancies.

  9. Potent monoclonal antibodies against Clostridium difficile toxin A elicited by DNA immunization.

    PubMed

    Zhang, Chunhua; Jin, Ke; Xiao, Yanling; Cheng, Ying; Huang, Zuhu; Wang, Shixia; Lu, Shan

    2013-10-01

    Recent studies have demonstrated that DNA immunization is effective in eliciting antigen-specific antibody responses against a wide range of infectious disease targets. The polyclonal antibodies elicited by DNA vaccination exhibit high sensitivity to conformational epitopes and high avidity. However, there have been limited reports in literature on the production of monoclonal antibodies (mAb) by DNA immunization. Here, by using Clostridium difficile (C. diff) toxin A as a model antigen, we demonstrated that DNA immunization was effective in producing a panel of mAb that are protective against toxin A challenge and can also be used as sensitive reagents to detect toxin A from various testing samples. The immunoglobulin (Ig) gene usage for such mAb was also investigated. Further studies should be conducted to fully establish DNA immunization as a unique platform to produce mAb in various hosts.

  10. Using monoclonal antibodies as an international standard for the measurement of anti-adalimumab antibodies.

    PubMed

    van Schouwenburg, Pauline A; Kruithof, Simone; Wolbink, Gertjan; Wouters, Diana; Rispens, Theo

    2016-02-20

    Comparing studies investigating anti-drug antibody (ADA) formation is hampered by the lack of comparability between study protocols, assay formats, and standardized reference materials. In this respect, the use of an international standard would mean a major step forward. Here we compared 11 fully human monoclonal antibodies against adalimumab in two assays commonly used for ADA measurement; the bridging ELISA and the antigen binding test (ABT). Our results show non-parallel titration of the monoclonal antibodies in both assays, which we also find for polyclonal ADA sources. Moreover, we observed that the output of the bridging ELISA depends to a large degree on the affinity of the monoclonal antibody. For the ABT, results reflect a combination of affinity and avidity. This suggests that rather than reporting ADA values in nanogram per milliliter, arbitrary units may be more appropriate. Together our data highlight the difficulty of ADA standardization by identifying several pitfalls that should be taken into account when selecting a standard for ADA testing.

  11. Method of rapid production of hybridomas expressing monoclonal antibodies on the cell surface

    DOEpatents

    Meagher, Richard B.; Laterza, Vince

    2006-12-12

    The present invention relates to genetically altered hybridomas, myelomas and B cells. The invention also relates to utilizing genetically altered hybridomas, myelomas and B cells in methods of making monoclonal antibodies. The present invention also provides populations of hybridomas and B cells that can be utilized to make a monoclonal antibody of interest.

  12. Monoclonal antibody typing of Chlamydia psittaci strains derived from avian and mammalian species.

    PubMed Central

    Fukushi, H; Nojiri, K; Hirai, K

    1987-01-01

    A total of 77 Chlamydia psittaci strains of avian, human, and mammalian origin were grouped into four serovars with 11 monoclonal antibodies recognizing the lipopolysaccharide and the major outer membrane protein antigens. The avian and human strains, which were closely related to each other, were distinct from the mammalian strains. Immunological typing of C. psittaci with monoclonal antibodies seems practical. PMID:3667918

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  14. Monoclonal antibodies to cyclodiene insecticides and method for detecting the same

    DOEpatents

    Stanker, L.H.; Vanderlaan, M.; Watkins, B.E.

    1994-08-02

    Methods are described for making specific monoclonal antibodies useful for detection of cyclodienes in foods and environmental samples. Monoclonal antibodies specifically reactive with cyclodienes can detect accumulated pesticides in food, tissue or environmental samples. Extraction and preparation of organic samples for immunoassay in a polar-nonpolar reaction medium permits detection of halogenated organic ring structures at concentrations in samples. 13 figs.

  15. Monoclonal antibodies to cyclodiene insecticides and method for detecting the same

    DOEpatents

    Stanker, Larry H.; Vanderlaan, Martin; Watkins, Bruce E.

    1994-01-01

    Methods are described for making specific monoclonal antibodies useful for detection of cyclodienes in foods and environmental samples. Monoclonal antibodies specifically reactive with cyclodienes can detect accumulated pesticides in food, tissue or environmental samples. Extraction and preparation of organic samples for immunoassay in a polar-nonpolar reaction medium permits detection of halogenated organic ring structures at concentrations in samples.

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

    PubMed

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

    PubMed

    Bzorek, Michael; Stamp, Inger Merete; Petersen, Bodil Laub; Frederiksen, Lisbeth

    2008-07-01

    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.

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

    PubMed

    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.

  19. [Identification and production of monoclonal antibody of Siberian tiger's immunoglobulin].

    PubMed

    Zhang, Yaonglong; Zhang, Duanling; Zhou, Ming; Xue, Yuan; Hua, Yuping; Ma, Jianzhang

    2010-03-01

    To purify immunoglobulin (Ig) of Siberian Tiger and prepare monoclonal antibody (mAb) against the Ig,which can be used to develop immunological diagnostic kits for diagnosing infectious disease in Siberian Tiger. The Ig of Siberian tigers was purified with saturated ammonium sulfate combined with recombinant Protein G. The C57BL/6 mice were immunized with the purified Ig. Spleno-cytes of the mice immunized were collected and fused with the mouse myeloma cell line (Sp2/0-Ag14). The positive hybridoma clones were selected by ELISA and were identified by western blot. The sandwich ELISA was used to detect immunocompetence of the purified Ig and the mAb. We obtained three mouse hybridoma clones that produced mAbs against Ig of Siberian Tiger. The derived McAbs could recognize Ig heavy chain of Siberian Tiger specifically. The biological activity of the Ig and obtained McAbs also could be identified by detecting the antibody induced by panleukopenia virus (FPV-HLJ) vaccine in Siberian Tiger. The antibody also would be useful for assess the vaccine efficacy against the infectious disease on the Siberian Tiger. Protein G can be used in Ig purification of Siberian Tiger. The obtained McAbs from the hybridoma ADT11 in this study owned strong ability to bind Ig of Siberian Tiger and have a stable immunocompetence. They can be used to develop diagnostic methods for detecting infectious disease in Siberian Tiger and vaccine research.

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

    PubMed

    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.

  1. A monoclonal antibody recognizing a differentiation marker on rat gonocytes.

    PubMed

    van Dissel-Emiliani, F M; van Kooten, P J; de Boer-Brouwer, M; de Rooij, D G; van der Donk, J A

    1993-01-01

    Monoclonal antibodies (MAb) were raised against a testicular membrane fraction from 18-day post coitum (p.c.) rat testes. One antibody, designated 4B6.3E10 (mu, kappa), was obtained which specifically reacted with gonocytes in the fetal testis. No significant cross-reactivity with other tissues from the 18-day p.c. embryo was found. MAb 4B6.3E10 was reactive with rat gonocytes from 17-day p.c. until the day of birth. Germ cells at later stages of testis development did not show any labelling. The epitope recognized by 4B6.3E10 is a carbohydrate as periodate treatment leads to a loss of reactivity of the antibody. By SDS-PAGE and Western blotting of proteins extracted from a testicular membrane fraction from 18-day p.c. testes, MAb 4B6.3E10 was found to recognize at least 3 protein moieties with apparent molecular weights in the ranges of 80-100, 120, 160-180 kDa (either under reducing- or non-reducing conditions). The results suggest that MAb 4B6.3E10 recognizes a specific differentiation marker for fetal rat gonocytes.

  2. Removal of drugs from the circulation using immobilized monoclonal antibodies

    SciTech Connect

    Brizgys, M.V.

    1987-01-01

    High-affinity monoclonal antidigoxin antibodies (dig-Ab) were immobilized to a pellicular microbead and characterized in terms of antibody affinity, specificity for other glycosides, and binding capacity. Determination of digoxin binding revealed that the binding capacity decreased to 25% of theoretical capacity. Attempts to improve the binding capacity were ineffective. A guinea pig animal model was developed to determine the efficacy of removing digoxin in vivo from the circulation using an antibody column. Male guinea pigs were hemoperfused with either a dig-Ab or bovine Y-globulin control column 16 h after a single i.v. injection of digoxin. Pre- and postcolumn plasma concentrations were obtained to evaluate the extraction efficiency. Hemoperfusion continued for 3 h at flow rates of 1.0-2.0 mL/min. Bound digoxin was eluted as described earlier and concentrations determined by (/sup 125/I) digoxin RIA. Amounts of digoxin removed represented less than 1% of the total body content. After several studies with the same column, the dig-Ab had lost most of its activity. A freshly prepared dig-Ab column removed approximately 20% of the total body content. Most of the measured constituents of the blood were unaffected by the procedure.

  3. Properties of technetium-99m labeled monoclonal antibodies

    SciTech Connect

    Rhodes, B.A.; Zamora, P.O.; Newell, K.D.; Reed, K.A.

    1984-01-01

    This study was designed to determine the chemical and immunochemical properties of monoclonal antibodies or fragments which have been labeled with Tc-99m using the pretinning method. The labeled proteins were evaluated using: Sephadex G-25 gel column scanning to determine percentage radiolabeled protein; HPLC to determine the molecular weight and purity of the proteins; reactivity with solid phase antigens to determine immunoreactivity under a variety of storage conditions; and the Tc-99m transchelation method of a previous study to determine the strength of the bonding. Percentage labeled protein ranges from 65 to 95%. Under certain labeling conditions small fractions of the F(ab')2 protein can be converted to aggregates of Fab fragments. Immunoreactivity depends on the purity and immunoreactivity of the original protein and is not changed by the labeling procedure. Transchelation is minimal (less than 5% at 4000 molar excess of EDTA). It is concluded that the pretinning method can be used to produce an extremely stable, immunoreactive, Tc-99m labeled antibody or antibody fragments. The labeled proteins retain their biologic activity during storage or during incubation with human plasma.

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

    PubMed Central

    Brand, Toni M; Iida, Mari

    2011-01-01

    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. PMID:21293176

  5. Human liver catalase: cloning, expression and characterization of monoclonal antibodies.

    PubMed

    Jin, Li Hua; Kim, Dae Won; Eum, Won Sik; Yoon, Chang Sik; Jang, Sang Ho; Choi, Hee Soon; Choi, Soo Hyun; Kim, Young Hoon; Kim, So Young; Jung, Mi Ryoung; Kang, Tae-Cheon; Won, Moo Ho; Lee, Hyeon Yong; Kang, Jung Hoon; Kwon, Oh-Shin; Cho, Sung-Woo; Lee, Kil Soo; Park, Jinseu; Choi, Soo Young

    2003-06-30

    We isolated a cDNA encoding liver catalase from a human liver cDNA library. The cDNA had a high degree of sequence similarity to the corresponding enzyme from other sources. It was expressed in E. coli using the pET15b vector. The protein produced was enzymatically active after purification, and its kinetic parameters closely resembled those of other mammalian catalases. Monoclonal antibodies were generated against the purified catalase; six antibodies recognizing different epitopes were obtained, one of which inhibited the enzyme. The cross reactions of the antibodies with brain catalases from human and other mammalian tissues were investigated, and all the immunoreactive bands obtained on Western blots had molecular masses of about 58 kDa. Similarly fractionated extracts of several mammalian cell lines all gave a single band of molecular mass 58 kDa. These results indicate that mammalian livers and human cell lines contain only one major type of immunologically reactive catalase, even though some of catalases have been previously reported to differ in certain properties.

  6. Antiviral Monoclonal Antibodies: Can They Be More Than Simple Neutralizing Agents?

    PubMed

    Pelegrin, Mireia; Naranjo-Gomez, Mar; Piechaczyk, Marc

    2015-10-01

    Monoclonal antibodies (mAbs) are increasingly being considered as agents to fight severe viral diseases. So far, they have essentially been selected and used on the basis of their virus-neutralizing activity and/or cell-killing activity to blunt viral propagation via direct mechanisms. There is, however, accumulating evidence that they can also induce long-lasting protective antiviral immunity by recruiting the endogenous immune system of infected individuals during the period of immunotherapy. Exploiting this property may revolutionize antiviral mAb-based immunotherapies, with benefits for both patients and healthcare systems.

  7. Monoclonal antibodies to mouse cell-surface antigens.

    PubMed

    Alaverdi, Noosheen

    2002-05-01

    The cluster of differentiation (CD) nomenclature, originally devised by the International Workshop on Human Leukocyte Differentiation Antigens (HLDA) to classify leukocyte surface antigens, has become more accepted by researchers in nonhuman animal models. The CD homologues from other species are usually identified by multiple studies, including biochemical, functional analysis, cloning, and immunological assays. This unit compiles a complete (as of August, 2001) list of monoclonal antibodies (mAb) to the mouse CD antigens and several non-CD surface antigens that are commonly used as phenotypic and functional markers in the mouse system. There are many reagents recognizing polymorphic epitopes of the mouse MHC and different subunits of TCR; for simplicity, only a few mAbs to the framework determinants of these molecules are listed here.

  8. Monoclonal antibodies and the transformation of blood typing.

    PubMed

    Marks, Lara

    2014-01-01

    Today, when monoclonal antibodies (mAbs) have become one of the most important classes of therapeutic drugs, it is easy to forget how much they have transformed our healthcare in other ways. One of the first clinical areas, as this paper shows, where mAbs made their mark was in the field of blood typing. The adoption of mAbs for this purpose was done with little public fanfare or funding. Nonetheless, it radically transformed the accuracy and cost of blood typing and shifted the procedure away from a dependence on reagents made from human blood donated by volunteers. This paper argues that the development of mAbs as reagents for blood typing laid the foundation for the first large-scale production of mAbs thereby paving the way to the advent of mAb diagnostics and therapeutics.

  9. A Monoclonal Antibody Specific to Surface Antigen on Candida krusei

    PubMed Central

    Robert, Raymond; Faure, Odile; Carloti, Arnaud; Lebeau, Bernadette; Bernard, Christian; Marot-Leblond, Agnès; Grillot, Renée; Senet, Jean-Marcel

    1998-01-01

    A monoclonal antibody (MAb; MAb 6B3) which reacts specifically with a cell wall antigen found in all strains or isolates of Candida krusei was developed. MAb 6B3 was extensively tested by immunofluorescence assay for cross-reaction with many Candida, Cryptococcus, Saccharomyces, Trichosporon, and Rhodotorula species and was found to react only with the species C. krusei. The specific epitope is expressed on the surface of fungal cells and appears to reside on a protein moiety. Taking into account the increasing importance of fluconazole-resistant strains in nosocomial fungal infections, the very high degree of specificity of this MAb for C. krusei could be useful for the routine detection of C. krusei in culture or in tissue samples. PMID:9455893

  10. High-level iodination of monoclonal antibody fragments for radiotherapy

    SciTech Connect

    Ferens, J.M.; Krohn, K.A.; Beaumier, P.L.; Brown, J.P.; Hellstroem, I.; Hellstroem, K.E.; Carrasquillo, J.A.; Larson, S.M.

    1984-03-01

    Two different murine monoclonal antibody Fab fragments specific for p97, a melanoma-associated antigen, were labeled with I-131 at high activity levels without excessive chemical damage. Up to 20 mg of Fab were labeled with up to 300 mCi of I-131 using the chloramine-T method and large working volumes at room temperature. As much as 90% of the initial activity was recovered as labeled product. The labeled Fabs varied in their sensitivity to radioiodination damage, as measured by an in vitro cell-binding assay. Radioiodination was performed safely using a remote iodination apparatus. The final product was of radiopharmaceutical quality suitable for clinical diagnosis and experimental radiotherapy in humans.

  11. Positron emission tomographic imaging of tumors using monoclonal antibodies

    SciTech Connect

    Zalutsky, M.R.

    1990-12-01

    The overall objective for this research project is to develop methods for utilizing Positron Emission Tomography (PET) to increase the clinical potential of radiolabelled monoclonal antibodies (MAbs). By labeling MAbs with positron-emitting nuclides, it should be possible to quantitate the dynamics of their three-dimensional distribution in vivo. Our long term goals are to apply this approach to investigate the following: normal tissue toxicity; radiation dose to the tumor; and early tumor imaging. The research plans of this proposal include the following specific aims: optimize labeling of MAbs with fluorine 18, bromine 76 and bromine 75; label MAb Mel-14 (reactive against human gliomas and melanomas) and its Fab and F(ab{prime}){sub 2} fragments while retaining immunoreactivity; determine the distribution of Mel-14 in athymic mice bearing human gliomas; determine pharmacokinetics of Mel-14 in nonhuman primates. Experiments with another MAb, TP-1, and iodine 124 and 131 are also planned. 8 figs.

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

    PubMed

    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.

  13. Target Therapy in Hematological Malignances: New Monoclonal Antibodies

    PubMed Central

    Szymczyk, Agnieszka; Pawlowski, Johannes

    2014-01-01

    Apart from radio- and chemotherapy, monoclonal antibodies (MoAbs) represent a new, more selective tool in the treatment of hematological malignancies. MoAbs bind with the specific antigens of the tumors. This interaction is a basis for targeted therapies which exhibit few side effects and significant antitumor activity. This review provides an overview of the functional characteristics of MoAbs, with some examples of their clinical application. The promising results in the treatment of hematological malignancies have led to the more frequent usage of MoAbs in the therapy. Development of MoAbs is a subject of extensive research. They are a promising method of cancer treatment in the future. PMID:27433507

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

    PubMed

    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.

  15. Identification of Eimeria acervulina conoid antigen using chicken monoclonal antibody.

    PubMed

    Matsubayashi, Makoto; Minoura, Chisa; Kimura, Shintaro; Tani, Hiroyuki; Furuya, Masaru; Lillehoj, Hyun S; Matsuda, Haruo; Takenaka, Shigeo; Hatta, Takeshi; Tsuji, Naotoshi; Sasai, Kazumi

    2016-11-01

    In the poultry industry, Eimeria spp. is one of the important pathogens which cause significant economic losses. We have previously generated a chicken monoclonal antibody (mAb), 6D-12-G10, with specificity for an antigen located in the apical cytoskeleton of Eimeria acervulina and with cross-reactive among Apicomplexan parasites, including other Eimeria spp., Toxoplasma, Neospora, and Cryptosporidium spp. Furthermore, the protein of Cryptosporidium parvum recognized by the 6D-12-G10 has been identified as elongation factor-1α (EF-1α). In the present study, to identify the target molecule of E. acervulina by the mAb, we performed two-dimensional Western blotting analysis. Finally, we found two positive molecules which are identified as EF-1α and a related protein. Our previous finding using C. parvum and the results in this study suggest that EF-1α could be associated with the invasion facilitated by the cytoskeleton at the apical region of zoites.

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

    PubMed

    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.

  17. Monoclonal antibodies and the transformation of blood typing

    PubMed Central

    Marks, Lara

    2014-01-01

    Today, when monoclonal antibodies (mAbs) have become one of the most important classes of therapeutic drugs, it is easy to forget how much they have transformed our healthcare in other ways. One of the first clinical areas, as this paper shows, where mAbs made their mark was in the field of blood typing. The adoption of mAbs for this purpose was done with little public fanfare or funding. Nonetheless, it radically transformed the accuracy and cost of blood typing and shifted the procedure away from a dependence on reagents made from human blood donated by volunteers. This paper argues that the development of mAbs as reagents for blood typing laid the foundation for the first large-scale production of mAbs thereby paving the way to the advent of mAb diagnostics and therapeutics. PMID:25484059

  18. Selection of Ceratitis capitata (Diptera: Tephritidae) Specific Recombinant Monoclonal Phage Display Antibodies for Prey Detection Analysis

    PubMed Central

    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. PMID:23272105

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

    PubMed

    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.

  20. Purification and identification of cell surface antigens using lamprey monoclonal antibodies

    PubMed Central

    Yu, Cuiling; Ali, Shabab; St. Germain, Jonathan; Liu, Yanling; Yu, Xuecong; Jaye, David L.; Moran, Michael F.; Cooper, Max D.; Ehrhardt, Götz R.A.

    2013-01-01

    Variable lymphocyte receptor (VLR) B antibodies of the evolutionary distant sea lamprey are structurally distinct from conventional mammalian antibodies. The different protein architecture and large evolutionary distance of jawless vertebrates suggest that VLR antibodies may represent promising tools for biomarker discovery. Here we report the generation of panels of monoclonal VLR antibodies from lamprey larvae immunized with human T cells and the use of a recombinant monoclonal VLR antibody for antigen purification and mass spectrometric identification. We demonstrate that despite predicted low affinity of individual VLR antigen binding units to the antigen, the high avidity resulting from decameric assembly of secreted VLR antibodies allows for efficient antigen capture and subsequent identification by mass spectometry. We show that VLR antibodies detect their antigens with high specificity and can be used in various standard laboratory application techniques. The lamprey antibodies are novel reagents that can complement conventional monoclonal antibodies in multiple scientific research disciplines. PMID:22964555

  1. Brachytherapy attains abscopal effects when combined with immunostimulatory monoclonal antibodies.

    PubMed

    Rodriguez-Ruiz, María E; Rodriguez, Inmaculada; Barbes, Benigno; Mayorga, Lina; Sanchez-Paulete, Alfonso Rodriguez; Ponz-Sarvise, Mariano; Pérez-Gracia, José Luis; Melero, Ignacio

    2017-08-21

    Preclinical and clinical evidence indicate that the proimmune effects of radiotherapy can be synergistically augmented with immunostimulatory monoclonal antibodies (mAb) to act both on irradiated tumor lesions and on tumors at distant, nonirradiated sites. We have recently reported that external beam radiotherapy achieves abscopal effects when combined with antagonist anti-PD1 mAbs and agonist anti-CD137 (4-1BB) mAbs. The goal of this work is to study the abscopal effects of radiotherapy instigated by brachytherapy techniques. Mice bearing a subcutaneous colorectal carcinoma, MC38 (colorectal cancer), in both flanks were randomly assigned to receive brachytherapy or not (8 Gy × three fractions) to only one of the two grafted tumors, in combination with intraperitoneal immunostimulatory monoclonal antibodies (anti-PD1, anti-CD137, and/or their respective isotype controls). To study the abscopal effects of brachytherapy, we established an experimental set up that permits irradiation of mouse tumors sparing a distant site resembling metastasis. Such second nonirradiated tumor was used as indicator of abscopal effect. Tumor size was monitored every 2 days. Abscopal effects on distant nonirradiated subcutaneous tumor lesions of transplanted MC38-derived tumors only took place when brachytherapy was combined with immunostimulatory anti-PD1 and/or anti-CD137 mAbs. Our results demonstrate that immunotherapy-potentiated abscopal effects can be attained by brachytherapy. Accordingly, immunotherapy plus brachytherapy combinations are suitable for clinical translation. Copyright © 2017 American Brachytherapy Society. Published by Elsevier Inc. All rights reserved.

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

    PubMed Central

    Santos, Raul D.

    2016-01-01

    Abstract 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. PMID:27195910

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

    PubMed

    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.

  4. Potential of palladium-109-labeled antimelanoma monoclonal antibody for tumor therapy

    SciTech Connect

    Fawwaz, R.A.; Wang, T.S.T.; Srivastava, S.C.; Rosen, J.M.; Ferrone, S.; Hardy, M.A.; Alderson, P.O.

    1984-07-01

    Palladium-109, a beta-emitting radionuclide, was chelated to the monoclonal antibody 225.28S to the high molecular weight antigen associated with human melanoma. Injection of the radiolabeled monoclonal antibody into nude mice bearing human melanoma resulted in significant accumulation of the radiolabel in the tumors: 19% injected dose/g; 38:1 and 61:1 tumor-to-blood ratios at 24 and 48 hr, respectively. The localization of the radiolabeled antibody in liver and kidney also was high, but appreciably lower than that achieved in tumor. These results suggest Pd-109-labeled monoclonal antibody to tumor-associated antigens may have potential applications in tumor immunotherapy.

  5. Kinetic epitope mapping of monoclonal antibodies raised against the Yersinia pestis virulence factor LcrV.

    PubMed

    Read, Thomas; Olkhov, Rouslan V; Williamson, E Diane; Shaw, Andrew M

    2015-03-15

    Five monoclonal antibodies, mAb7.3, mAb29.3, mAb46.3, mAb12.3 and mAb36.3, raised to the LcrV virulence factor from Yersinia pestis were characterised for their Fab affinity against the purified protein and their Fc affinity to Protein A/G as a proxy for the FcγR receptor. Kinetic measurements were performed label-free in a localised particle plasmon array reader. The Fc-ProteinA/G complex first-order half-life was determined for each antibody and fell in the range of 0.8-3.8h. The Fab first-order half-lives had ranged from 3.4 to 9.2h although two antibodies, mAb12.3 and mAb36.3, showed low affinity interactions. Competitive binding studies of mixtures of the Fab-active antibodies were performed to measure the relative binding efficiency of one antibody in the presence of the other. A geometric relative positioning of the epitopes of mAb7.3, mAb29.3 and mAb46.3 was determined based on the footprint locus of the antibody and the percentage of competitive binding. The two known protective antibodies mAb7.3 and mAb29.3 showed greater interference, indicating epitopes close to one another compared to the non-protective mAb46.3 antibody. The Fab-Fc complex half-life screen and epitope mapping are potentially useful tools in the screening of therapeutic antibodies or vaccine candidates. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Probing Functional Changes in Exocyst Configuration with Monoclonal Antibodies

    PubMed Central

    Inamdar, Shivangi M.; Hsu, Shu-Chan; Yeaman, Charles

    2016-01-01

    Spatial regulation of exocytosis relies on the exocyst, a hetero-octameric protein complex that tethers vesicles to fusion sites at the plasma membrane. Nevertheless, our understanding of mechanisms regulating exocyst assembly/disassembly, localization, and function are incomplete. Here, we have exploited a panel of anti-Sec6 monoclonal antibodies (mAbs) to probe possible configurational changes accompanying transitions in exocyst function in epithelial MDCK cells. Sec6 is quantitatively associated with Sec8 in high molecular weight complexes, as shown by gel filtration and co-immunoprecipitation studies. We mapped epitopes recognized by more than 20 distinct mAbs to one of six Sec6 segments. Surprisingly, mAbs that bound epitopes in each segment labeled distinct subcellular structures. In general, antibodies to epitopes in N-terminal domains labeled Sec6 in either cytosolic or nuclear pools, whereas those that bound epitopes in C-terminal domains labeled membrane-associated Sec6. In this latter group, we identified antibodies that labeled distinct Sec6 populations at the apical junctional complex, desmosomes, endoplasmic reticulum and vimentin-type intermediate filaments. That each antibody was specific was verified by both Sec6 RNAi and competition with fusion proteins containing each domain. Comparison of non-polarized and polarized cells revealed that many Sec6 epitopes either redistribute or become concealed during epithelial polarization. Transitions in exocyst configurations may be regulated in part by the actions of Ral GTPases, because the exposure of Sec6 C-terminal domain epitopes at the plasma membrane is significantly reduced upon RalA RNAi. To determine whether spatio-temporal changes in epitope accessibility was correlated with differential stability of interactions between Sec6 and other exocyst subunits, we quantified relative amounts of each subunit that co-immunoprecipitated with Sec6 when antibodies to N-terminal or C-terminal epitopes were used

  7. Production of Monoclonal Antibodies Directed against the Microsporidium Enterocytozoon bieneusi

    PubMed Central

    Accoceberry, Isabelle; Thellier, Marc; Desportes-Livage, Isabelle; Achbarou, Abderrahim; Biligui, Sylvestre; Danis, Martin; Datry, Annick

    1999-01-01

    Several hybridomas producing antibodies detected by indirect immunofluorescence antibody test (IFAT) were established by fusion of mouse myeloma SP2/O with spleen cells from BALB/c mice immunized against whole spores (protocol 1) or chitinase-treated spores (protocol 2) of Enterocytozoon bieneusi and were cloned twice by limiting dilutions. Two monoclonal antibodies (MAbs), 3B82H2 from protocol 1, isotyped as immunoglobulin M (IgM), and 6E52D9 from protocol 2, isotyped as IgG, were expanded in both ascites and culture. IFAT with the MAbs showed that both MAbs reacted exclusively with the walls of the spores of E. bieneusi, strongly staining the surface of mature spores, and produced titers of greater than 4,096. Immunogold electron microscopy confirmed the specific reactivities of both antibodies. No cross-reaction, either with the spores of the other intestinal microsporidium species Encephalitozoon intestinalis or with yeast cells, bacteria, or any other intestinal parasites, was observed. The MAbs were used to identify E. bieneusi spores in fecal specimens from patients suspected of having intestinal microsporidiosis. The IFAT was validated against standard staining methods (Chromotrope 2R and Uvitex 2B) and PCR. We report here the first description and characterization of two MAbs specific for the spore wall of E. bieneusi. These MAbs have great potential for the demonstration and species determination of E. bieneusi, and their application in immunofluorescence identification of E. bieneusi in stool samples could offer a new diagnostic tool for clinical laboratories. PMID:10565939

  8. Monoclonal antibody therapeutics with up to five specificities

    PubMed Central

    LaFleur, David W.; Abramyan, Donara; Kanakaraj, Palanisamy; Smith, Rodger G.; Shah, Rutul R.; Wang, Geping; Yao, Xiao-Tao; Kankanala, Spandana; Boyd, Ernie; Zaritskaya, Liubov; Nam, Viktoriya; Puffer, Bridget A.; Buasen, Pete; Kaithamana, Shashi; Burnette, Andrew F.; Krishnamurthy, Rajesh; Patel, Dimki; Roschke, Viktor V.; Kiener, Peter A.; Hilbert, David M.; Barbas III, Carlos F.

    2013-01-01

    The recognition that few human diseases are thoroughly addressed by mono-specific, monoclonal antibodies (mAbs) continues to drive the development of antibody therapeutics with additional specificities and enhanced activity. Historically, efforts to engineer additional antigen recognition into molecules have relied predominantly on the reformatting of immunoglobulin domains. In this report we describe a series of fully functional mAbs to which additional specificities have been imparted through the recombinant fusion of relatively short polypeptides sequences. The sequences are selected for binding to a particular target from combinatorial libraries that express linear, disulfide-constrained, or domain-based structures. The potential for fusion of peptides to the N- and C- termini of both the heavy and light chains affords the bivalent expression of up to four different peptides. The resulting molecules, called zybodies, can gain up to four additional specificities, while retaining the original functionality and specificity of the scaffold antibody. We explore the use of two clinically significant oncology antibodies, trastuzumab and cetuximab, as zybody scaffolds and demonstrate functional enhancements in each case. The affect of fusion position on both peptide and scaffold function is explored, and penta-specific zybodies are demonstrated to simultaneously engage five targets (ErbB2, EGFR, IGF-1R, Ang2 and integrin αvβ3). Bispecific, trastuzumab-based zybodies targeting ErbB2 and Ang2 are shown to exhibit superior efficacy to trastuzumab in an angiogenesis-dependent xenograft tumor model. A cetuximab-based bispecific zybody that targeting EGFR and ErbB3 simultaneously disrupted multiple intracellular signaling pathways; inhibited tumor cell proliferation; and showed efficacy superior to that of cetuximab in a xenograft tumor model. PMID:23575268

  9. Generation of monoclonal antibody targeting fibroblast growth factor receptor 3.

    PubMed

    Gorbenko, Olena; Ovcharenko, Galyna; Klymenko, Tetyana; Zhyvoloup, Olexandr; Gaman, Nadia; Volkova, Darija; Gout, Ivan; Filonenko, Valeriy

    2009-08-01

    Fibroblast growth factor receptor 3 (FGFR3) is a member of the FGFR family of receptor tyrosine kinases, whose function has been implicated in diverse biological processes, including cell proliferation, differentiation, survival, and tumorigenesis. Deregulation of FGFR3 signaling has been implicated with human pathologies, including cancer. Activating mutations in FGFR3 gene are frequently detected in bladder cancer, multiple myeloma, and noninvasive papillary urothelial cell carcinomas, while the overexpression of the receptor is observed in thyroid lymphoma and bladder cancer. The main aim of this study was to generate hybridoma clones producing antibody that could specifically recognize FGFR3/S249C mutant, but not the wild-type FGFR. To achieve this, we used for immunization bacterially expressed fragment of FGFR3 corresponding to loops II-III of the extracellular domain (GST-His/FGFR3/S249C-LII-III), which possesses oncogenic mutation at Ser249 detected in at least 50% of bladder cancers. Primary ELISA screening allowed us to isolate several hybridoma clones that showed specificity towards FGFR3/S249C, but not FGFR3wt protein. Unfortunately, these clones were not stable during single-cell cloning and expansion and lost the ability to recognize specifically FGFR3/S249C. However, this study allowed us to generate several monoclonal antibodies specific towards both FGFR3wt and FGFR3/S249C recombinant proteins. Produced hybridomas secreted MAbs that were specific in Western blotting towards bacterially expressed FGFR3wt and FGFR3/S249C, as well as the full-length receptors ectopically expressed in Sf21 and HEK293 cells. Moreover, transiently expressed wild-type and oncogenic forms of FGFR were efficiently immunoprecipitated with selected antibodies from the lysates of infected Sf21 and transiently transfected HEK293. In summary, generated antibodies should be useful as tools for examining the expression pattern and biological functions of FGFR3 in normal and

  10. Defining process design space for monoclonal antibody cell culture.

    PubMed

    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.

  11. Monoclonal antibodies: pharmacokinetics as a basis for new dosage regimens?

    PubMed

    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.

  12. Hierarchical Cluster Formation in Concentrated Monoclonal Antibody Formulations

    NASA Astrophysics Data System (ADS)

    Godfrin, P. Douglas; Zarzar, Jonathan; Zarraga, Isidro Dan; Porcar, Lionel; Falus, Peter; Wagner, Norman; Liu, Yun

    Reversible cluster formation has been identified as an underlying cause of large solution viscosities observed in some concentrated monoclonal antibody (mAb) formulations. As high solution viscosity prevents the use of subcutaneous injection as a delivery method for some mAbs, a fundamental understanding of the interactions responsible for high viscosities in concentrated mAb solutions is of significant relevance to mAb applications in human health care as well as of intellectual interest. Here, we present a detailed investigation of a well-studied IgG1 based mAb to relate the short time dynamics and microstructure to significant viscosity changes over a range of pharmaceutically relevant physiochemical conditions. Using a combination of experimental techniques, it is found that upon adding Na2SO4, these antibodies dimerize in solution. Proteins form strongly bounded reversible dimers at dilute concentrations that, when concentrated, interact with each other to form loosely bounded, large, transient clusters. The combined effect of forming strongly bounded dimers and a large transient network is a significant increase in the solution viscosity. Strongly bounded, reversible dimers may exist in many IgG1 based mAb systems such that these results contribute to a more comprehensive understanding of the physical mechanisms producing high viscosities in concentrated protein solutions.

  13. Analysis of viral clearance unit operations for monoclonal antibodies.

    PubMed

    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.

  14. Generation and characterization of monoclonal antibodies to equine CD16.

    PubMed

    Noronha, Leela E; Harman, Rebecca M; Wagner, Bettina; Antczak, Douglas F

    2012-04-15

    The low-affinity Fc receptor CD16 plays a central role in the inflammatory and innate immune responses of many species, but has not yet been investigated in the horse. Using the predicted extracellular region of equine CD16 expressed as a recombinant fusion protein with equine IL-4 (rIL-4/CD16), we generated a panel of mouse monoclonal antibodies (mAbs) that recognize equine CD16. Nine mAbs were chosen for characterization based upon recognition of CD16, but not IL-4, in ELISA. All nine mAbs recognized full-length, cell-surface CD16 expressed as a GFP fusion protein by CHO cells, but not the closely related Fc receptor CD32 expressed in the same system. In flow cytometric analysis with equine peripheral leukocytes, the mAbs labeled cells in the granulocyte, monocyte, and lymphocyte populations in a pattern consistent with other species. Monocytes that were strongly labeled with CD16 mAb 9G5 were also positive for the LPS receptor CD14. Cytospins made with peripheral leukocytes were immunohistochemically labeled and showed mAb recognition of primarily mononuclear cells. ELISA revealed that the nine mAbs can be grouped into three patterns of epitope recognition. These new antibodies will serve as useful tools in the investigation of equine immune responses and inflammatory processes.

  15. Production of human anti-HLA monoclonal antibodies

    SciTech Connect

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

    1986-03-01

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

  16. Monoclonal Antibodies for the Diagnosis of Borrelia crocidurae.

    PubMed

    Fotso Fotso, Aurélien; Mediannikov, Oleg; Nappez, Claude; Azza, Saïd; Raoult, Didier; Drancourt, Michel

    2016-01-01

    Relapsing fever borreliae, produced by ectoparasite-borne Borrelia species, cause mild to deadly bacteremia and miscarriage. In the perspective of developing inexpensive assays for the rapid detection of relapsing fever borreliae, we produced 12 monoclonal antibodies (MAbs) against Borrelia crocidurae and characterized the two exhibiting the highest titers. P3A10 MAb reacts with the 35.6-kDa flagellin B (flaB) of B. crocidurae while P6D9 MAb recognizes a 35.1-kDa variable-like protein (Vlp) in B. crocidurae and a 35.2-kDa Vlp in Borrelia duttonii. Indirect immunofluorescence assay incorporating relapsing fever and Lyme group borreliae and 11 blood-borne organisms responsible for fever in West Africa confirmed the reactivity of these two MAbs. Combining these two MAbs in indirect immunofluorescence assays detected relapsing fever borreliae including B. crocidurae in ticks and the blood of febrile Senegalese patients. Both antibodies could be incorporated into inexpensive and stable formats suited for the rapid point-of-care diagnosis of relapsing fever. These first-ever MAbs directed against African relapsing fever borreliae are available for the scientific community to promote research in this neglected field.

  17. Profiling formulated monoclonal antibodies by (1)H NMR spectroscopy.

    PubMed

    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.

  18. Direct detection of idiotypic determinants on blotted monoclonal antibodies.

    PubMed

    Petit, C; Sauron, M E; Gilbert, M; Thèze, J

    1982-01-01

    The protein-blotting technique has been tested as a mean to study the expression of idiotypic determinants. A monoclonal BALB/c antipoly (Glu60-Ala30-Tyr10) GAT antibody (G5) was caused to migrate on SDS gel and transferred to a nitrocellulose filter. To facilitate the renaturation of the idiotypic determinants, the blotted proteins were incubated in NP40 buffer, immediately after the transfer. The ability of two anti-idiotypic sera to detect two defined idiotypic specificities of the blotted G5 molecules was investigated. When G5 was electrophoresed on SDS gel under non-reducing conditions, a specific detection of two idiotypic specificities of the G5-blotted molecules was obtained. On the other hand, when G5 was migrated under reducing conditions, none of the two antiidiotypic sera gave a staining of the heavy and the light chains. This result indicates that molecules expressing conformational idiotypic determinants can be detected by protein-blotting technique after migration on SDS gel. Moreover, this suggests the possible interest of this technique to analyse non-antibody molecules bearing idiotypic determinants.

  19. Monoclonal antibody-based therapies for microbial diseases

    PubMed Central

    Saylor, Carolyn; Dadachova, Ekaterina; Casadevall, Arturo

    2009-01-01

    The monoclonal antibody (mAb) revolution that currently provides many new options for the treatment of neoplastic and inflammatory diseases has largely bypassed the field of infectious diseases. Only one mAb is licensed for use against an infectious disease, although there are many in various stages of development. This situation is peculiar given that serum therapy was one of the first effective treatments for microbial diseases and that specific antibodies have numerous antimicrobial properties. The underdevelopment and underutilization of mAb therapies for microbial diseases has various complex explanations that include the current availability of antimicrobial drugs, small markets, high costs and microbial antigenic variation. However, there are signs that the climate for mAb therapeutics in infectious diseases is changing given increasing antibiotic drug resistance, the emergence of new pathogenic microbes for which no therapy is available, and development of mAb cocktail formulations. Currently, the major hurdle for the widespread introduction of mAb therapies for microbial diseases is economic, given the high costs of immunoglobulin preparations and relatively small markets. Despite these obstacles there are numerous opportunities for mAb development against microbial diseases and the development of radioimmunotherapy provides new options for enhancing the magic bullet. Hence, there is cautious optimism that the years ahead will see more mAbs in clinical use against microbial diseases. PMID:20006139

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

    PubMed

    Cheng, Luisa W; Henderson, Thomas D; Lam, Tina I; Stanker, Larry H

    2015-11-27

    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.

  1. Monoclonal antibodies against rabbit mammary prolactin receptors. Specific antibodies to the hormone binding domain

    SciTech Connect

    Katoh, M.; Djiane, J.; Kelly, P.A.

    1985-09-25

    Three monoclonal antibodies (M110, A82, and A917) were obtained by fusing myeloma cells and spleen cells from mice immunized with partially purified rabbit mammary gland prolactin (PRL) receptors. All 3 antibodies were capable of complete inhibition of SVI-ovine prolactin (oPRL) binding to rabbit mammary PRL receptors in either particulate or soluble form. M110 showed slightly greater potency than oPRL in competing for SVI-oPRL binding. These antibodies also inhibited PRL binding to microsomal fractions from rabbit liver, kidney, adrenal, ovary, and pig mammary gland, although A82 showed poor inhibition in pig mammary gland. There was no cross-reaction of any of the 3 monoclonal antibodies (mAbs) for the other species tested: human (T-47D breast cancer cells) and rat (liver, ovary). In order to confirm that these antibodies are specific to the binding domain, antibodies were purified, iodinated, and binding characteristics were investigated. SVI-M110 and SVI-A82 binding was completely inhibited by lactogenic hormones, whereas nonlactogenic hormones did not cross-react. Competition of 125I-M110 by oPRL was comparable to that of SVI-oPRL by unlabeled oPRL, while SVI-A917 binding was only partially competed (30-60%) by lactogenic hormones. Tissue and species specificity of labeled antibody binding paralleled results of binding inhibition experiments using 125I-oPRL. In addition, A82 and A917 completely inhibited 125I-M110 binding. In contrast, 125I-A82 binding was stimulated by A917 and 125I-A917 binding was stimulated by A82.

  2. Isolation and characterization of broad and ultrapotent human monoclonal antibodies with therapeutic activity against chikungunya virus

    PubMed Central

    Smith, Scott A.; Silva, Laurie A.; Fox, Julie M.; Flyak, Andrew; Kose, Nurgun; Sapparapu, Gopal; Khomadiak, Solomiia; Ashbrook, Alison W.; Kahle, Kristen M.; Fong, Rachel H.; Swayne, Sherri; Doranz, Benjamin J.; McGee, Charles E.; Heise, Mark T.; Pal, Pankaj; Brien, James D.; Austin, S. Kyle; Diamond, Michael S.; Dermody, Terence S.; Crowe, James E.

    2015-01-01

    SUMMARY Chikungunya virus (CHIKV) is a mosquito-transmitted RNA virus that causes acute febrile infection associated with polyarthralgia in humans. Mechanisms of protective immunity against CHIKV are poorly understood, and no effective therapeutics or vaccines are available. We isolated and characterized human monoclonal antibodies (mAbs) that neutralize CHIKV infectivity. Among the 30 mAbs isolated, 13 had broad and ultrapotent neutralizing activity (IC50 < 10 ng/mL), and all of these mapped to domain A of the E2 envelope protein. Potent inhibitory mAbs blocked post-attachment steps required for CHIKV membrane fusion, and several were protective in a lethal challenge model in immunocompromised mice, even when administered at late time points after infection. These highly protective mAbs could be considered for prevention or treatment of CHIKV infection, and their epitope location in domain A of E2 could be targeted for rational structure-based vaccine development. PMID:26159721

  3. Selectivity verification of cardiac troponin monoclonal antibodies for cardiac troponin detection by using conventional ELISA

    NASA Astrophysics Data System (ADS)

    Fathil, M. F. M.; Arshad, M. K. Md; Gopinath, Subash C. B.; Adzhri, R.; Ruslinda, A. R.; Hashim, U.

    2017-03-01

    This paper presents preparation and characterization of conventional enzyme-linked immunosorbent assay (ELISA) for cardiac troponin detection to determine the selectivity of the cardiac troponin monoclonal antibodies. Monoclonal antibodies, used to capture and bind the targets in this experiment, are cTnI monoclonal antibody (MAb-cTnI) and cTnT monoclonal antibody (MAb-cTnT), while both cardiac troponin I (cTnI) and T (cTnT) are used as targets. ELISA is performed inside two microtiter plates for MAb-cTnI and MAb-cTnT. For each plate, monoclonal antibodies are tested by various concentrations of cTnI and cTnT ranging from 0-6400 µg/l. The binding selectivity and level of detection between monoclonal antibodies and antigen are determined through visual observation based on the color change inside each well on the plate. ELISA reader is further used to quantitatively measured the optical density of the color changes, thus produced more accurate reading. The results from this experiment are utilized to justify the use of these monoclonal antibodies as bio-receptors for cardiac troponin detection by using field-effect transistor (FET)-based biosensors coupled with substrate-gate in the future.

  4. The history of monoclonal antibody development – Progress, remaining challenges and future innovations

    PubMed Central

    Liu, Justin K.H.

    2014-01-01

    As medicine progresses into a new era of personalised therapy, the use of monoclonal antibodies to treat a wide range of diseases lies at the heart of this new forefront. Since the licencing of the first monoclonal antibody for clinical use 30 years ago, the monoclonal antibody industry has expanded exponentially and is now valued at billions of dollars. With major advances in genetic sequencing and biomedical research, much research into monoclonal antibodies now focuses on identifying new targets for development and maximising their efficacy for use in clinical practice. However, a balance has to be struck with regards to reducing numbers of side-effects and overall economic cost, which arguably somewhat blighted their early clinical and commercial successes. Nowadays, there are approximately 30 monoclonal antibodies that have been approved for use in clinical practice with many more currently being tested in clinical trials. Some of the current major limitations include: the use of inefficient models for generation, a lack of efficacy and issues of cost-effectiveness. Some of the current research focuses on ways to improve the efficacy of existing monoclonal antibodies through optimising their effects and the addition of beneficial modifications. This review will focus on the history of monoclonal antibody development – how it has increasingly moved away from using laborious animal models to a more effective phage display system, some of the major drawbacks from a clinical and economical point of view and future innovations that are currently being researched to maximise their effectiveness for future clinical use. PMID:25568796

  5. Antipneumococcal effects of C-reactive protein and monoclonal antibodies to pneumococcal cell wall and capsular antigens.

    PubMed Central

    Briles, D E; Forman, C; Horowitz, J C; Volanakis, J E; Benjamin, W H; McDaniel, L S; Eldridge, J; Brooks, J

    1989-01-01

    Antibodies to pneumococcal capsular polysaccharides are well known for their ability to protect against pneumococcal infection. Recent studies indicate that antibodies to cell wall antigens, including pneumococcal surface protein A and the phosphocholine (PC) determinant of teichoic acids as well as human C-reactive protein (which also binds to PC), can protect mice against pneumococcal infection. In the present study we compared the protective effects of these agents as measured by mouse protection, the blood bactericidal assay, and clearance of pneumococci from the blood and peritoneal cavity. Our findings extend previous results indicating that human C-reactive protein and antibodies to noncapsular antigens are generally less protective than anticapsular antibodies. The new results obtained indicate the following: (i) mouse protection studies with intraperitoneal and intravenous infections provide very similar results; (ii) monoclonal immunoglobulin G2a (IgG2a) antibodies to PC, like IgG1, IgG2b, and IgG3 antibodies to PC, are highly protective against pneumococcal infection in mice; (iii) human antibody to PC is able to protect against pneumococcal infection in mice; (iv) antibodies to PspA are effective at mediating blood and peritoneal clearance of pneumococci; (v) complement is required for the in vivo protective effects of both IgG and IgM antibodies to PC; (vi) IgG1, IgG2b, and IgG3 anti-PC antibodies all mediate complement-dependent lysis of PC-conjugated erythrocytes; and (vii) antibodies and human C-reactive proteins that are reactive with capsular antigens but not cell wall antigens are able to mediate significant antibacterial activity in the blood bactericidal assay. PMID:2707854

  6. Kinetic analysis of the multistep aggregation mechanism of monoclonal antibodies.

    PubMed

    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.

  7. Development of new staining technology "eastern blotting" using monoclonal antibody.

    PubMed

    Morinaga, Osamu; Shoyama, Yukihiro

    2011-03-01

    Ginsenosides contained in Panax species were separated by silica gel TLC blotted to a polyvinylidene difluoride (PVDF) membrane which was dipped in a sodium periodide (NaIO(4)) solution and reacted with protein, preparing a ginsenoside-protein conjugate for binding a ginsenoside on a PVDF membrane. The blotted spots were stained by anti-ginsenoside-Rb1 monoclonal antibody (MAb) and anti-ginsenoside-Rg1MAb, respectively. The newly established immunostaining method, eastern blotting was applied for the determination of ginsenosides possessing protopanaxadiol and/or protopanaxatriol. Double staining of eastern blotting for ginsenosides using anti-ginsenoside-Rb1 MAb and anti-ginsenoside-Rg1 MAb promoted complete identification of ginsenosides in Panax species. This technique has been devised for the chromatographic separation and identification of ginsenosides using polyethersulfone (PES) membrane. It caused an acceptable separation of ginsenoside-Rb1, -Rc and -Rd in various ginseng extracts. Newly developed technique is quite simple and applies for immunoassay system. Ginsenosides separated using a PES membrane were directly treated with a NaIO(4) solution and then reacted with bovine serum albumin (BSA) for making a ginsenoside-protein conjugate. After the blocking, anti-ginsenoside-Rb1 MAb recognized a ginsenoside on a PES membrane and then a sec-ond antibody labeled with enzyme reacted to the first antibody. Finally a substrate was oxidized with the enzyme and de-veloped the staining of ginsenosides. The staining spots of ginsenosides on membrane were quantitatively evaluated by NIH Image indicating at least 62.5 ng of each ginsenoside-Rb1, -Rc and -Rd were detected with clarity. The determination range of three ginsenosides was from 0.125 to 2.0 µg of direct amount on PES membrane.

  8. High-throughput epitope binning of therapeutic monoclonal antibodies: why you need to bin the fridge.

    PubMed

    Brooks, Benjamin D; Miles, Adam R; Abdiche, Yasmina N

    2014-08-01

    Analytical tools are evolving to meet the need for the higher-throughput characterization of therapeutic monoclonal antibodies. An antibody's epitope is arguably its most important property because it underpins its functional activity but, because epitope selection is innate, it remains an empirical process. Here, we focus on the emergence of label-free biosensors with throughput capabilities orders of magnitude higher than the previous state-of-the-art, which can facilitate large assays such as epitope binning so that they can be incorporated alongside functional activity screens, enabling the rapid identification of leads that exhibit unique and functional epitopes. In addition to streamlining the drug development process by saving time and cost, the information from epitope binning assays could provide the basis for intellectual property protection. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Genetic characterization of mouse immunoglobulin allotypic determinants (allotopes) defined by monoclonal antibodies.

    PubMed

    Huang, C M; Parsons, M; Oi, V T; Huang, H J; Herzenberg, L A

    1983-01-01

    We have generated a new series of monoclonal antibodies recognizing allotypic determinants on mouse IgG1, IgG2a, and IgG2b. In this communication we describe their reactivities with immunoglobulins of the inbred mouse strains. Comparison with serology charts indicates that many of these monoclonal antibodies detect allotypic specificities previously defined by conventional antisera; others define previously undescribed specificities. Strain and isotype distribution allows us to assign five new allotypic specificities to Igh-1 and three new specificities to Igh-3. In addition, on the basis of reactivity with the monoclonal antibodies, we have defined a new Igh haplotype in SWR/J mice, Ighp.

  10. The Use of Humanized Monoclonal Antibodies for the Prevention of Respiratory Syncytial Virus Infection

    PubMed Central

    Arcuri, Santo; Galletti, Silvia; Faldella, Giacomo

    2013-01-01

    Monoclonal antibodies are widely used both in infants and in adults for several indications. Humanized monoclonal antibodies (palivizumab) have been used for many years for the prevention of respiratory syncytial virus infection in pediatric populations (preterm infants, infants with chronic lung disease or congenital heart disease) at high risk of severe and potentially lethal course of the infection. This drug was reported to be safe, well tolerated and effective to decrease the hospitalization rate and mortality in these groups of infants by several clinical trials. In the present paper we report the development and the current use of monoclonal antibodies for prophylaxis against respiratory syncytial virus. PMID:23840240

  11. A broadly reactive monoclonal antibody detects multiple genotypes of hepatitis B virus X protein.

    PubMed

    Wei, Lili; Shen, Zhongliang; Zhao, Xue; Wu, Yanxin; Liu, Wei; Zhang, Junqi; Xie, Youhua; Liu, Jing

    2014-10-01

    A highly specific and broadly reactive monoclonal antibody against hepatitis B virus X (HBx) protein was developed that detected, in both immunoblotting and immunofluorescence, HBx proteins of seven of the eight currently known genotypes of HBV, which were overexpressed in cultured cells. Evaluation of HBx expression levels in cultured hepatocytes using this monoclonal antibody showed that cells transiently and stably transfected with HBV genomes expressed far less HBx protein than cells transiently transfected with an HBx overexpression plasmid routinely used for studying HBx functions. The availability of such sensitive and broadly reactive monoclonal antibodies against HBx will enable more-quantitative studies of HBx functions.

  12. Radiolabeled monoclonal antibodies for imaging and therapy: Potential, problems, and prospects: Scientific highlights

    SciTech Connect

    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.''

  13. Structure of solid tumors and their vasculature: Implications for therapy with monoclonal antibodies

    SciTech Connect

    Dvorak, H.F.; Nagy, J.A.; Dvorak, A.M. )

    1991-03-01

    Delivery of monoclonal antibodies to solid tumors is a vexing problem that must be solved if these antibodies are to realize their promise in therapy. Such success as has been achieved with monoclonal antibodies is attributable to the local hyperpermeability of the tumor vasculature, a property that favors antibody extravasation at tumor sites and that is mediated by a tumor-secreted vascular permeability factor. However, leaky tumor blood vessels are generally some distance removed from target tumor cells, separated by stroma and by other tumor cells that together represent significant barriers to penetration by extravasated monoclonal antibodies. For this reason, alternative approaches may be attractive. These include the use of antibody-linked cytotoxins, which are able to kill tumor cells without immediate contact, and direction of antibodies against nontumor cell targets, for example, antigens unique to the tumor vascular endothelium or to tumor stroma. 50 refs.

  14. Production and characterization of monoclonal antibodies against dog immunoglobulin isotypes.

    PubMed

    Arce, C; Moreno, A; Millán, Y; Martín de las Mulas, J; Llanes, D

    2002-09-06

    A panel of six monoclonal antibodies (mAbs) recognizing antigenic determinants on canine immunoglobulin (Ig) heavy or light chains was produced and characterized. All monoclonals recognized the IgG(2) subclass, although only two were subclass-specific (CA3H1 and CA4F1). The CA3B8 mAb was found to be specific for an epitope on canine immunoglobulin G heavy chain, (IgG(1) and IgG(2) subclasses). Two mAbs (CA2E9 and CA5B2) reacted with an epitope on the heavy chain of canine IgG and IgM and another, CA4E7, bound to canine IgA, IgG and IgM isotypes; CA4E7 recognized an epitope on canine immunoglobulin light chain. CA4E7, CA4F1 and CA5B2 recognized an epitope in the Fab region. Three mAbs, CA3B8, CA4E7 and CA5B2, showed much lower reactivity with canine IgG by ELISA when IgG was periodate-treated, suggesting that they recognized a carbohydrate determinant. Cross-reactivity analysis of these mAbs with sera from horse, goat, cow, sheep, pig, cat, rabbit, hamster, rat, mouse and human indicated that two mAbs, CA3B8 and CA5B2, recognized a canine IgG-specific epitope; two others, CA3H1 and CA4E7, recognized an epitope also present in rabbit and sheep immunoglobulin respectively; and the remaining two (CA2E9 and CA4F1) recognized an epitope broadly present on the Igs of the species analyzed. This panel of antibodies will be a useful tool for future canine immunodiagnosis tests. With the exception of CA2E9, all mAbs were able to recognize plasma cells on paraffin-embedded tissues, and will thus be useful for immunohistochemical assays.

  15. Antibody specificity and antigen characterization of rat monoclonal antibodies against Streptococcus mutans cell wall-associated protein antigens.

    PubMed Central

    Ackermans, F; Klein, J P; Cormont, F; Bazin, H; Ogier, J A; Frank, R M; Vreven, J

    1985-01-01

    Monoclonal antibodies to Streptococcus mutans OMZ175 (serotype f) cell wall-associated antigens (wall-extracted antigens [WEA]) were derived from the fusion of Lou C plasmocytoma rat cells (IR 983 F) and spleen cells from Wistar R inbred rats immunized with WEA. Four cell lines producing monoclonal antibodies directed against a component of S. mutans WEA have been established. All four monoclonal antibodies reacted only with two antigens of WEA from S. mutans OMZ175 by Western blotting and immunoprecipitation techniques, enzyme-linked immunosorbent assay (ELISA), and competitive ELISA. Western blot analysis of WEA showed that the four monoclonal antibodies recognized two related cell wall-associated proteins with apparent molecular weights of 125,000 and 76,000. Immunoprecipitation of whole cells with the monoclonal antibodies confirmed the surface localization of the two antigens. The ELISA and competitive ELISA were used to analyze the distribution of the epitopes on seven S. mutans serotypes. All S. mutans serotypes were found to express the recognized epitopes; however, different reactivity patterns could be distinguished among the various strains tested, and the four monoclonal antibodies reacted only weakly with S. mutans serotypes d and g. Images PMID:2410364

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

    NASA Astrophysics Data System (ADS)

    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

  17. Prevention of Herpes Simplex Virus Induced Stromal Keratitis by a Glycoprotein B-Specific Monoclonal Antibody

    PubMed Central

    Krawczyk, Adalbert; Dirks, Miriam; Kasper, Maren; Buch, Anna; Dittmer, Ulf; Giebel, Bernd; Wildschütz, Lena; Busch, Martin; Goergens, Andre; Schneweis, Karl E.; Eis-Hübinger, Anna M.; Sodeik, Beate; Heiligenhaus, Arnd; Roggendorf, Michael; Bauer, Dirk

    2015-01-01

    The increasing incidence of acyclovir (ACV) and multidrug-resistant strains in patients with corneal HSV-1 infections leading to Herpetic Stromal Keratitis (HSK) is a major health problem in industrialized countries and often results in blindness. To overcome this obstacle, we have previously developed an HSV-gB-specific monoclonal antibody (mAb 2c) that proved to be highly protective in immunodeficient NOD/SCID-mice towards genital infections. In the present study, we examined the effectivity of mAb 2c in preventing the immunopathological disease HSK in the HSK BALB/c mouse model. Therefore, mice were inoculated with HSV-1 strain KOS on the scarified cornea to induce HSK and subsequently either systemically or topically treated with mAb 2c. Systemic treatment was performed by intravenous administration of mAb 2c 24 h prior to infection (pre-exposure prophylaxis) or 24, 40, and 56 hours after infection (post-exposure immunotherapy). Topical treatment was performed by periodical inoculations (5 times per day) of antibody-containing eye drops as control, starting at 24 h post infection. Systemic antibody treatment markedly reduced viral loads at the site of infection and completely protected mice from developing HSK. The administration of the antiviral antibody prior or post infection was equally effective. Topical treatment had no improving effect on the severity of HSK. In conclusion, our data demonstrate that mAb 2c proved to be an excellent drug for the treatment of corneal HSV-infections and for prevention of HSK and blindness. Moreover, the humanized counterpart (mAb hu2c) was equally effective in protecting mice from HSV-induced HSK when compared to the parental mouse antibody. These results warrant the future development of this antibody as a novel approach for the treatment of corneal HSV-infections in humans. PMID:25587898

  18. Antibodies: Protective, destructive and regulatory role

    SciTech Connect

    Milgrom, F.; Abeyounis, C.J.; Albini, B.

    1985-01-01

    This book contains papers under 10 subject headings. The headings are: Production and Function of Antibodies, Protective Role of Antibodies, Antibodies to Foreign and Neoplastic Cells, Autoantibodies, Regulatory Mechanisms, Allergy, Immune Complexes, Antibodies in Pregnancy and Aging, Administration of Antibodies for Prevention and Therapy, and Abstracts of Poster Presentations.

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

    PubMed

    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.

  20. A monoclonal antibody specific for 6-monoacetylmorphine reduces acute heroin effects in mice.

    PubMed

    Bogen, Inger Lise; Boix, Fernando; Nerem, Elisabeth; Mørland, Jørg; Andersen, Jannike Mørch

    2014-06-01

    Immunotherapy against drugs of abuse is being studied as an alternative treatment option in addiction medicine and is based on antibodies sequestering the drug in the bloodstream and blocking its entry into the brain. Producing an efficient vaccine against heroin has been considered particularly challenging because of the rapid metabolism of heroin to multiple psychoactive molecules. We have previously reported that heroin's first metabolite, 6-monoacetylmorphine (6-MAM), is the predominant mediator for heroin's acute behavioral effects and that heroin is metabolized to 6-MAM primarily prior to brain entry. On this basis, we hypothesized that antibody sequestration of 6-MAM is sufficient to impair heroin-induced effects and therefore examined the effects of a monoclonal antibody (mAb) specific for 6-MAM. In vitro experiments in human and rat blood revealed that the antibody was able to bind 6-MAM and block the metabolism to morphine almost completely, whereas the conversion of heroin to 6-MAM remained unaffected. Mice pretreated with the mAb toward 6-MAM displayed a reduction in heroin-induced locomotor activity that corresponded closely to the reduction in brain 6-MAM levels. Intraperitoneal and intravenous administration of the anti-6-MAM mAb gave equivalent protection against heroin effects, and the mAb was estimated to have a functional half-life of 8 to 9 days in mice. Our study implies that an antibody against 6-MAM is effective in counteracting heroin effects.

  1. Monoclonal antibodies against the native urease of Helicobacter pylori: synergistic inhibition of urease activity by monoclonal antibody combinations.

    PubMed Central

    Nagata, K; Mizuta, T; Tonokatu, Y; Fukuda, Y; Okamura, H; Hayashi, T; Shimoyama, T; Tamura, T

    1992-01-01

    Monoclonal antibodies (MAbs) against the native urease of Helicobacter pylori NCTC 11637 were found to clearly inhibit the urease activity. Interestingly, synergistic inhibition by two MAbs recognizing different subunits was also observed. Ten MAbs were produced and classified as two isotypes of the immunoglobulin G (IgG) subclass, IgG1, and IgG2a. Western blot (immunoblot) analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that five MAbs recognized the large subunit and the other five recognized the small subunit of the urease. Among the MAbs, L2 and S2, which recognized the large and the small subunits, respectively, were also able to inhibit the urease activity of clinical isolates from H. pylori-infected patients. The combination of L2 and S2 led to augmented synergistic inhibition. L2, but not S2, could also inhibit the urease activity from Helicobacter mustelae; enzyme-linked immunosorbent assay and Western blot analysis showed that L2 cross-reacted with this urease. These results suggested that the epitope recognized by L2 had a structure common to both Helicobacter species and may be involved in the active site of the urease. In contrast to the MAbs, a polyclonal antibody in sera from mice immunized with H. pylori urease did not have the ability to inhibit H. pylori urease activity. However, the polyclonal antibody retained the ability to abolish the inhibitory action of these MAbs. Moreover, other MAbs which could not inhibit H. pylori urease activity also abolished the inhibitory action. Images PMID:1383158

  2. Reagent Target Request for Monoclonal Antibody Production and Characterization | Office of Cancer Clinical Proteomics Research

    Cancer.gov

    NCI's Antibody Characterization Program provides reagents and other critical resources to support protein/peptide measurements and analysis. In an effort to produce and distribute well-characterized monoclonal antibodies to the scientific community, the program is seeking cancer related protein targets for antibody production and characterization for distribution to the research community. Submission Period: May 20, 2011 - July 1, 2011.

  3. Antibody response that protects against disseminated candidiasis.

    PubMed Central

    Han, Y; Cutler, J E

    1995-01-01

    We previously showed that surface mannans of Candida albicans function as adhesins during yeast cell attachment to mouse splenic marginal zone macrophages. The mannan adhesin fraction was encapsulated into liposomes and used to vaccinate mice over a 5- to 6-week period. Circulating agglutinins specific for the fraction correlated with increased resistance to disseminated candidiasis. Antiserum from vaccinated animals protected naive BALB/cByJ mice against C. albicans serotype A and B strains and Candida tropicalis. Antiserum also protected SCID mice against disseminated disease. The serum protective ability was stable at 56 degrees C, but this ability was adsorbed by C. albicans cells. The antiserum was divided into three fractions after separation by high-performance liquid chromatography. One fraction contained all of the agglutinin activity and transferred resistance to naive mice. A second fraction also transferred resistance. Two monoclonal antibodies (MAbs) specific for candidal surface determinants were obtained. MAb B6.1 is specific for a mannan epitope in the adhesin fraction, and MAb B6 is specific for a different epitope in the fraction. Both MAbs are immunoglobulin M, and both strongly agglutinate candidal cells, but only MAb B6.1 protected both normal and SCID mice against disseminated candidiasis. In one experiment, 10 normal mice were given MAb B6.1 and challenged with yeast cells. Six mice survived the 67-day observation period; 4 of the survivors were cured as evidenced by the lack of CFU in the kidney and spleen. Our studies show that antibodies against certain cell surface antigens of C. albicans help the host resist disseminated candidiasis. PMID:7790089

  4. Heterobifunctional reagents: A new approach to radiolabeling of monoclonal antibodies

    SciTech Connect

    Wang, T.S.T.; Ng, A.K.; Fawwaz, R.A.; Liu, Z.; Alderson, P.O.

    1985-05-01

    The use of bifunctional chelate such as the cyclic anhydride of DTPA for radiolabeling antibodies (Abs) may lead to homopolymerization, and intra- or intermolecular cross-linking, with resulting denaturation and decrease immunoreactivity of Abs. The authors, therefore, investigated the use of heterobifunctional reagents, whereby one group selectively couples to the amino group of the Ab and the other group to the radiometal for Ab labeling. One such reagent, 2,6-Dioxo-N-(carboxymethyl)morphine (DCM) was synthesized by reacting nitrilotriacetic acid with acetic anhydride. The other agent tested was commercially available N-Succinimidyl-3-(2-pyridyldithio) propionate (SPDP). These agents were evaluated independently for their ability to label a monoclonal antibody (MoAb) to a melanoma associated antigen (Ag). Labeling proceeded at a 2mg/ml concentration of the Ab, at HEPES pH 8.2, and 7.0, respectively, at room temperature for 30 min. The conjugate subsequently was labeled with Tc-99m or In-111. For comparison, the same labeled Abs also were prepared by using the cyclic anhydride of DTPA. Binding of the Ab to melanoma cells and control cells then was assayed. The results of cell binding experiments (N=3 per agent) in the region of Ag excess (X+-SD) were as follows: 62.6 +- 2.83% for Tc-99m-DCM-MoAb and 41.3+-1.84% for Tc-99m-SPDP-MoAb vs. 28.6 +- 1.16% for Tc-99m-DTPA-MoAb (p<0.01); 56.2 +- 2.97% for In-111-DCM-MoAb vs. 28.6 +- 1.16% for In-111-DTPA-M0Ab. Binding of all agents to the control lymphoid cell line was less than 3%. These results suggest that heterobifunctional reagents can reduce the loss of immunoreactivity of labeled MoAbs.

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

    PubMed

    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.

  6. Human Monoclonal Antibodies Broadly Neutralizing against Influenza B Virus

    PubMed Central

    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

  7. Characterization of monoclonal antibodies to avian Escherichia coli Iss.

    PubMed

    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.

  8. Monoclonal antibodies recognizing single amino acid substitutions in hemoglobin

    SciTech Connect

    Stanker, L.H.; Branscomb, E.; Vanderlaan, M.; Jensen, R.H.

    1986-06-01

    Four monoclonal antibodies (mAb) to non-human primate hemoglobin referred to as Cap-4, Cap-5, Rh-2, and Rh-4, and two mAb to human hemoglobin, referred to as H-1 and H-3 were isolated and were partially characterized. Binding studies with these mAb on a panel of hemoglobins and isolated ..cap alpha.. and ..beta.. globin chains revealed a unique reactivity pattern for each mAb. Amino acid sequence analysis of the antigens used to generate the binding data suggests that the specific recognition of certain hemoglobin antigens by each mAb is controlled by the presence of a particular amino acid at a specific position within the epitope. The use of synthetic peptides as antigens confirmed this observation for five of the mAb. No synthetic peptides were tested with the sixth mAb, Rh-2. The amino acids required for binding of mAb Cap-4, Cap-5, Rh-4, and Rh-2 to hemoglobin are alanine at ..beta..5, threonine at ..beta..13, glutamine at ..beta..125, and leucine at ..cap alpha..68. The non-human primate hemoglobin antibodies require a specific amino acid that is not present in human hemoglobin. The amino acid required for binding of Cap-4, Cap-5, and Rh-4 could arise by a single base change in the ..beta.. globin gene, whereas the amino acid required for Rh-2 binding could only occur if two base changes occurred. Thus these mAb are candidate probes for a somatic cell mutation assay on the basis of the detection of peripheral blood red cells that possess single amino acid substituted hemoglobin as a result of single base substitutions in the globin genes of precursor cells.

  9. Monoclonal antibodies to human apolipoproteins: application to the study of high density lipoprotein subpopulations.

    PubMed

    Bustos, P; Ulloa, N; Calvo, C; Muller, D; Durán, D; Martínez, J; Salazar, L; Quiroga, A

    2000-09-01

    We produced, selected and cloned hybridomas that secrete monoclonal antibodies against human apolipoprotein (apo) A-I. All of the antibodies corresponded to the IgG(1) subclass and were named 1C11, 2B4, 2C10, 7C5, 8A4 and 8A5. The antibodies were characterized by their reactivity with whole lipoproteins, apolipoproteins, synthetic peptides and fragments generated by cleavage of the apo A-I. Three of the monoclonal antibodies studied (2B4, 2C10 and 7C5) were similarly inhibited by an amino-terminal peptide (amino acid sequence 1-20) of apo A-I, whereas antibodies 1C11, 8A4 and 8A5 had no reaction. Other results show that monoclonal antibody 1C11 recognizes an epitope located between amino acids 135-148. We evaluated the monoclonal antibody 8A4 against different HDL subpopulations by competitive displacement analysis and it showed a similar reactivity with the HDL particles: LpA-I and LpA-I:A-II. This antibody was used to standardize a sandwich ELISA to quantitate LpA-I in plasma. We conclude that these monoclonal antibodies are relevant for the study of apo A-I epitope expression and for quantitating apo A-I containing lipoparticles.

  10. Localization of a tumor cell adhesion domain of laminin by a monoclonal antibody

    SciTech Connect

    Skubitz, A.P.N.; Charonis, A.S.; Tsilibary, E.C.; Furcht, L.T. )

    1987-12-01

    Monoclonal antibodies were prepared to localize the domain(s) of laminin to which tumor cells adhere. Rat Y3-Ag 1.2.3 myeloma cells were fused with spleen cells from a rat immunized with a purified 440-kDa fragment of chymotrypsin-digested laminin. Three monoclonal antibodies (AL-1 to AL-3) that bound to intact laminin in a solid-phase radioimmunoassay were chosen for further analysis. The epitopes recognized by these antibodies were characterized by radioimmunoassays, immunoblotting, radioimmunoprecipitation, and immunoaffinity chromatography. In cell adhesion assays, monoclonal antibody AL-2 inhibited the highly metastatic melanoma cell line, K-1735-M4, to both intact laminin and the 440-kDa fragment of laminin. Electron microscopic examination of laminin-monoclonal antibody interactions showed that monoclonal antibody AL-2 reacted with the long arm of laminin directly below the cross-region. Two monoclonal antibodies that failed to inhibit tumor cell adhesion to laminin reacted with epitopes on the lateral short arms or cross-region of laminin as seen by electron microscopy. These results suggest that a new tumor cell binding domain of laminin may be located close to the cross-region on the long arm of laminin.

  11. An immunogen synthesis strategy for the development of specific anti-deoxynivalenol monoclonal antibodies.

    PubMed

    Sanders, Melanie; Guo, Yirong; Iyer, Abhishek; García, Yara Ruiz; Galvita, Anastasia; Heyerick, Arne; Deforce, Dieter; Risseeuw, Martijn D P; Van Calenbergh, Serge; Bracke, Marc; Eremin, Sergei; Madder, Annemieke; De Saeger, Sarah

    2014-01-01

    An immunogen synthesis strategy was designed to develop anti-deoxynivalenol (DON) monoclonal antibodies with low cross-reactivity against structurally similar trichothecenes. A total of eight different DON immunogens were synthesised, differing in the type and position of the linker on the DON molecule. After immunisation, antisera from mice immunised with different DON immunogens were checked for the presence of relevant antibodies. Then, both homologous and heterologous enzyme-linked immunosorbent assays (ELISAs) were performed for hybridoma screening. Finally, three monoclonal antibodies against DON and its analogues were generated. In addition, monoclonal antibody 13H1 could recognise DON and its analogues in the order of HT-2 toxin > 15-acetyldeoxynivalenol (15-ADON) > DON, with IC₅₀ ranging from 1.14 to 2.13 µg ml⁻¹. Another monoclonal antibody 10H10 manifested relatively close sensitivities to DON, 3-acetyldeoxynivalenol (3-ADON) and 15-ADON, with IC₅₀ values of 22, 15 and 34 ng ml⁻¹, respectively. Using an indirect ELISA format decreases the 10H10 sensitivity to 15-ADON with 92%. A third monoclonal antibody 2A9 showed to be very specific and sensitive to 3-ADON, with IC₅₀ of 0.38 ng ml⁻¹. Using both 2A9 and 10H10 monoclonal antibodies allows determining sole DON contamination.

  12. Monoclonal antibodies against soman: Characterization of soman stereoisomers. (Reannouncement with new availability information)

    SciTech Connect

    Lenz, D.E.; Yourick, J.J.; Dawson, J.S.; Scott, J.

    1992-12-31

    Hybridomas were produced which expressed monoclonal anti-soman antibodies as determined by microtiter enzyme-linked-antibody immunoassay (EIA). Each of these antibodies was titrated using a competitive inhibition enzyme immunoassay (CIEIA) with a variety of test ligands. The ligands used included soman (a racemic mixture), sarin, tabun, and each of the four stereoisomers of soman(C+P+, C+P-, C-P+ and C-P-). In all cases the antibodies tested exhibited IC50 values of 10 - 4 - 5 X 10 - 6 M for soman. When sarin or tabun was used as a ligand, the antibodies exhibited no cross reactivity. All of the antibodies cross reacted with the four soman stereoisomers. A second group of hybridomas were produced which expressed monoclonal antibodies against CsPs-soman. These antibodies were used to make preliminary absolute chiral assignments to the four soman stereoisomers. Soman; Antibodies; Stereoisomers; Absolute configuration.

  13. Trial Watch: Tumor-targeting monoclonal antibodies in cancer therapy.

    PubMed

    Vacchelli, Erika; Aranda, Fernando; Eggermont, Alexander; Galon, Jérôme; Sautès-Fridman, Catherine; Zitvogel, Laurence; Kroemer, Guido; Galluzzi, Lorenzo

    2014-01-01

    In 1997, for the first time in history, a monoclonal antibody (mAb), i.e., the chimeric anti-CD20 molecule rituximab, was approved by the US Food and Drug Administration for use in cancer patients. Since then, the panel of mAbs that are approved by international regulatory agencies for the treatment of hematopoietic and solid malignancies has not stopped to expand, nowadays encompassing a stunning amount of 15 distinct molecules. This therapeutic armamentarium includes mAbs that target tumor-associated antigens, as well as molecules that interfere with tumor-stroma interactions or exert direct immunostimulatory effects. These three classes of mAbs exert antineoplastic activity via distinct mechanisms, which may or may not involve immune effectors other than the mAbs themselves. In previous issues of OncoImmunology, we provided a brief scientific background to the use of mAbs, all types confounded, in cancer therapy, and discussed the results of recent clinical trials investigating the safety and efficacy of this approach. Here, we focus on mAbs that primarily target malignant cells or their interactions with stromal components, as opposed to mAbs that mediate antineoplastic effects by activating the immune system. In particular, we discuss relevant clinical findings that have been published during the last 13 months as well as clinical trials that have been launched in the same period to investigate the therapeutic profile of hitherto investigational tumor-targeting mAbs.

  14. Reversible cluster formation in concentrated monoclonal antibody solutions

    NASA Astrophysics Data System (ADS)

    Godfrin, P. Douglas; Porcar, Lionel; Falus, Peter; Zarraga, Isidro; Wagner, Norm; Liu, Yun

    2015-03-01

    Protein cluster formation in solution is of fundamental interest for both academic research and industrial applications. Recently, industrial scientists are also exploring the effect of reversible cluster formation on biopharmaceutical processing and delivery. However, despite of its importance, the understanding of protein clusters at concentrated solutions remains scientifically very challenging. Using the neutron spin echo technique to study the short time dynamics of proteins in solutions, we have recently systematically studied cluster formation in a few monoclonal antibody (mAb) solutions and their relation with solution viscosity. We show that the existence of anisotropic attraction can cause the formation of finite sized clusters, which increases the solution viscosity. Interestingly, once clusters form at relatively low concentrations, the average size of clusters in solutions remains almost constant over a wide range of concentrations similar to that of micelle formation. For a different mAb we have also investigated, the attraction is mostly induced by hydrophobic patches. As a result, these mAbs form large clusters with loosely linked proteins. In both cases, the formation of clusters all increases the solution viscosity substantially. However, due to different physics origins of cluster formation, solutions viscosities for these two different types of mAbs need to be controlled by different ways.

  15. [ICO-166 monoclonal antibodies against the CD45RA antigen].

    PubMed

    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. Monoclonal Antibodies for Systemic Lupus Erythematosus (SLE) †

    PubMed Central

    Ponticelli, Claudio; Moroni, Gabriella

    2010-01-01

    A number of monoclonal antibodies (mAb) are now under investigation in clinical trials to assess their potential role in Systemic Lupus Erythematosus (SLE). The most frequently used mAb is rituximab, which is directed against CD20, a membrane protein expressed on B lymphocytes. Uncontrolled trials reported an improvement of SLE activity in non-renal patients and other studies even reported an improvement of severe lupus nephritis unresponsive to conventional treatments. However two randomized trials failed to show the superiority of rituximab over conventional treatment in non renal SLE and in lupus nephritis. Preliminary trials reported promising results with epratuzumab, a humanized mAb directed against CD22, and with belimumab, a human mAb that specifically recognizes and inhibits the biological activity of BLyS a cytokine of the tumor-necrosis-factor (TNF) ligand superfamily. Other clinical trials with mAb directed against TNF-alpha, interleukin-10 (Il-10), Il-6, CD154, CD40 ligand, IL-18 or complement component C5 are under way. At present, however, in spite of good results reported by some studies, no firm conclusion on the risk-benefit profile of these mAbs in patients with SLE can be drawn from the available studies. PMID:27713252

  17. Pharmacokinetics of biotech drugs: peptides, proteins and monoclonal antibodies.

    PubMed

    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.

  18. Guidelines to cell engineering for monoclonal antibody production.

    PubMed

    Rita Costa, A; Elisa Rodrigues, M; Henriques, Mariana; Azeredo, Joana; Oliveira, Rosário

    2010-02-01

    Monoclonal antibodies (mAbs) are currently used for many diagnostic and therapeutic applications. The high demand for these biopharmaceuticals has led to the development of large-scale manufacturing processes, with productivity improvements being mainly achieved by optimization of bioreactor systems. However, more recently, the early steps of production, previous to bioreactor culture, have been presented as alternative areas where productivity enhancements can be achieved. Thus, this review describes the progress made for the improvement of productivity in mammalian expression systems for the high production of mAbs. Advances in the development of mAb-producing cell lines are being made, particularly regarding expression vector design and methods used for transfection, with the intent to create a reproducible methodology. Selection of the most suitable clones is also a critical step that can be improved, by including variables other than the expression level, which is still the common practice. Furthermore, strategies of cell engineering, although still mostly based on trial-and-error experimentation and not in standard protocols, hold great interest to improve cell growth and productivity, as well as product quality in the future. Improvements of the initial steps of the production process would not only result in cells with higher expression ability, but would also speed-up the process development.

  19. Anti-CD20 monoclonal antibodies in multiple sclerosis.

    PubMed

    Moreno Torres, Irene; García-Merino, Antonio

    2017-04-01

    The therapeutic utility of the anti-CD20 monoclonal antibodies (mAbs) is currently being evaluated in multiple sclerosis (MS) in line with the better understanding of the role of B lymphocytes in MS pathogenesis. Area covered: We conducted a literature search using Medline/Pub Med database of basic research and available controlled trials about anti-CD20 mAbs in MS. Additionally, ongoing studies were identified in the ClinicalTrials.gov database. B cells exert multiple inflammatory and regulatory functions playing an important role in MS pathogenesis as is demonstrated by the production of autoantibodies, infiltration of B cells in MS lesions and the formation of ectopic B cell follicle-like structures in meninges, among others. B-cell depletion by anti-CD20 mAbs has been shown to have an impact on these pathogenic mechanisms. The efficacy of three of them, rituximab, ocrelizumab and ofatumumab in MS has been confirmed by placebo-controlled clinical trials demonstrating a significant reduction of the annualized relapsing rate (ARR), new gadolinium-enhancing (GdE) and T2 lesions. There have been no significant safety problems so far but the overall benefit to risk profile is still to be determined. Expert commentary: After recent good results of these agents in MS therapy, questions related to maintenance therapy, markers of response and control of B cells values remain unanswered.

  20. Monoclonal antibodies directed against surface molecules of multicell spheroids

    NASA Technical Reports Server (NTRS)

    Martinez, Andrew O.

    1994-01-01

    The objective of this project is to generate a library of monoclonal antibodies (MAbs) directed against surface molecules of tumor and transformed cells grown as multicell spheroids (MCS). These MCS are highly organized, 3-dimensional multicellular structures which exhibit many characteristics of in vivo organized tissues not found in conventional monolayer or suspension culture. Therefore MCS make better in vitro model systems to study the interactions of mammalian cells, and provide a functional assay for surface adhesion molecules. This project also involves investigations of cell-cell interactions in a gravity-based environment. It will provide a base of scientific information necessary to expand the focus of the project in future years to microgravity and hypergravity-based environments. This project also has the potential to yield important materials (e.g., cellular products) which may prove useful in the diagnosis and/or treatment of certain human diseases. Moreover, this project supports the training of both undergraduate and graduate students; thus, it will assist in developing a pool of future scientists with research experience in an area (gravitational biology) of interest to NASA.

  1. DNA immunization as a technology platform for monoclonal antibody induction.

    PubMed

    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.

  2. Establishment of a novel monoclonal antibody against LGR5.

    PubMed

    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.

  3. Downstream processing of monoclonal antibodies--application of platform approaches.

    PubMed

    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.

  4. Elotuzumab: the first approved monoclonal antibody for multiple myeloma treatment

    PubMed Central

    Magen, Hila; Muchtar, Eli

    2016-01-01

    Elotuzumab is a monoclonal antibody directed against the SLAMF7 receptor, expressed on normal and malignant plasma cells with a lower expression on other lymphoid cells such as natural killer (NK) cells. Elotuzumab has no significant antimyeloma activity when given as a single agent to patients with relapsed or refractory multiple myeloma (RRMM). However, when combined with other antimyeloma agents, it results in improved response and outcome. Owing to the results from the landmark ELOQUENT-2 phase III clinical trial, which compared lenalidomide and dexamethasone with or without elotuzumab in patients with RRMM, elotuzumab in combination with lenalidomide and dexamethasone was approved by the American Food and Drug Administration (FDA) in November 2015 for multiple myeloma (MM) patients who received one to three prior lines of therapy. This review will give a brief description of the signaling lymphocytic activation molecule (SLAM) family receptors, the unique SLAMF7 receptor and the mechanism of action of elotuzumab. Thereafter, we will give an overview on its antimyeloma activity in preclinical and clinical trials, including its toxicity profile and management thereof. PMID:27493709

  5. Advective hydrogel membrane chromatography for monoclonal antibody purification in bioprocessing.

    PubMed

    Hou, Ying; Brower, Mark; Pollard, David; Kanani, Dharmesh; Jacquemart, Renaud; Kachuik, Bradley; Stout, James

    2015-01-01

    Protein A chromatography is widely employed for the capture and purification of monoclonal antibodies (mAbs). Because of the high cost of protein A resins, there is a significant economic driving force to seek new downstream processing strategies. Membrane chromatography has emerged as a promising alternative to conventional resin based column chromatography. However, to date, the application has been limited to mostly ion exchange flow through (FT) mode. Recently, significant advances in Natrix hydrogel membrane has resulted in increased dynamic binding capacities for proteins, which makes membrane chromatography much more attractive for bind/elute operations. The dominantly advective mass transport property of the hydrogel membrane has also enabled Natrix membrane to be run at faster volumetric flow rates with high dynamic binding capacities. In this work, the potential of using Natrix weak cation exchange membrane as a mAb capture step is assessed. A series of cycle studies was also performed in the pilot scale device (> 30 cycles) with good reproducibility in terms of yield and product purities, suggesting potential for improved manufacturing flexibility and productivity. In addition, anion exchange (AEX) hydrogel membranes were also evaluated with multiple mAb programs in FT mode. Significantly higher binding capacity for impurities (support mAb loads up to 10Kg/L) and 40X faster processing speed were observed compared with traditional AEX column chromatography. A proposed protein A free mAb purification process platform could meet the demand of a downstream purification process with high purity, yield, and throughput.

  6. Trial watch: Tumor-targeting monoclonal antibodies for oncological indications

    PubMed Central

    Vacchelli, Erika; Pol, Jonathan; Bloy, Norma; Eggermont, Alexander; Cremer, Isabelle; Fridman, Wolf Hervé; Galon, Jérôme; Marabelle, Aurélien; Kohrt, Holbrook; Zitvogel, Laurence; Kroemer, Guido; Galluzzi, Lorenzo

    2015-01-01

    An expanding panel of monoclonal antibodies (mAbs) that specifically target malignant cells or intercept trophic factors delivered by the tumor stroma is now available for cancer therapy. These mAbs can exert direct antiproliferative/cytotoxic effects as they inhibit pro-survival signal transduction cascades or activate lethal receptors at the plasma membrane of cancer cells, they can opsonize neoplastic cells to initiate a tumor-targeting immune response, or they can be harnessed to specifically deliver toxins or radionuclides to transformed cells. As an indication of the success of this immunotherapeutic paradigm, international regulatory agencies approve new tumor-targeting mAbs for use in cancer patients every year. Moreover, the list of indications for previously licensed molecules is frequently expanded to other neoplastic disorders as the results of large, randomized clinical trials become available. Here, we discuss recent advances in the preclinical and clinical development of tumor-targeting mAbs for oncological indications. PMID:25949870

  7. Monoclonal antibody probe for assessing beer foam stabilizing proteins.

    PubMed

    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.

  8. [Study of plant lectins from Viscum album using monoclonal antibodies].

    PubMed

    Tonevitskiĭ, A G; Rakhmanova, V A; Shamshiev, A T; Usachaeva, E A; Agapov, I I; Prokov'ev, S A; Denisenko, O N; Pfuller, U; Eifler, R

    1995-01-01

    Monoclonal antibodies (monAT) against both native (TA5, TB12) and denatured (TB33, TB35) plant toxin ML1 from Viscum album have been obtained. The interaction of monAT against native toxin with its isoforms ML2 and ML3 was investigated. It was shown that monAT TA5 to A-chain of ML1 toxin cross-reacted with ML2 and ML3 isoforms. TA5 did not inhibit enzyme activity of A-chain in cell-free rabbit reticulocyte system. It was shown that monAT TB12 reacted with galactose-binding site of B-subunit. Both monAT had no cross-reactions with plant toxin ricin. The binding constants for TA5 with ML1, ML2, ML3 respectively were 4.3.10(7) M-1, 1.2.10(7) M-1, and 0.3.10(7) M-1. The binding constants for TB12 were 2.10(7) M-1 with ML1 toxin, and more than 10(6) M-1 with ML2 and ML3. The nature of heterogeneity in ML toxin family is discussed. Test-systems for ML1 determination in different V. album extracts are suggested.

  9. Production of a Chaetomium globosum Enolase Monoclonal Antibody

    PubMed Central

    Nayak, Ajay P.; Lemons, Angela R.; Rittenour, William R.; Hettick, Justin M.; Beezhold, Donald H.

    2014-01-01

    Chaetomium globosum is a hydrophilic fungal species and a contaminant of water-damaged building materials in North America. Methods to detect Chaetomium species include subjective identification of ascospores, viable culture, or molecular-based detection methods. In this study, we describe the production and initial characterization of a monoclonal antibody (MAb) for C. globosum enolase. MAb 1C7, a murine IgG1 isotype MAb, was produced and reacted with recombinant C. globosum enolase (rCgEno) in an enzyme-linked immunosorbent assay and with a putative C. globosum enolase in a Western blot. Epitope mapping showed MAb 1C7 specific reactivity to an enolase decapeptide, LTYEELANLY, that is highly conserved within the fungal class Sordariomycetes. Cross-reactivity studies showed MAb 1C7 reactivity to C. atrobrunneum but not C. indicum. MAb 1C7 did not react with enolase from Aspergillus fumigatus, which is divergent in only two amino acids within this epitope. The results of this study suggest potential utility of MAb 1C7 in Western blot applications for the detection of Chaetomium and other Sordariomycetes species. PMID:25495488

  10. Anti-ICOS Monoclonal Antibody Treatment of Canine Chronic GVHD.

    PubMed

    Graves, Scott S; Parker, Maura H; Stone, Diane; Sale, George E; Pillai, Smitha P S; Johnson, Melissa M; Storb, Rainer

    2017-09-25

    In murine model systems, inducible costimulator (ICOS) signaling has been implicated in the formation of chronic graft-versus-host disease (GVHD). Previously, we showed that chronic GVHD can be reproducibly produced in the dog hematopoietic cell transplantation (HCT) model and that ICOS expression is upregulated on T cells in dogs with chronic GVHD. The goal of the present study was to determine whether administration of a short course of anti-canine ICOS monoclonal antibody (mAb) could alter the rapid and progressive course of chronic GVHD. Five dogs underwent HCT from dog leukocyte antigen mismatched unrelated donors following total body irradiation. Post-grafting immunosuppression consisted of methotrexate (days 1, 3, 6, and 11) and cyclosporine (days -1 through 78). Anti-ICOS mAb (3 injections, 72 hours apart) was administered upon diagnosis of GVHD. One dog failed to respond to anti-ICOS mAb therapy and succumbed to chronic GVHD in a time course similar to control untreated dogs. Overall, anti-ICOS-treated dogs experienced a significant prolongation in survival from the time of diagnosis of chronic GVHD compared to control dogs. Within the limitations of the number of study dogs, we suggest that a short course of anti-ICOS mAb may be useful in the treatment of chronic canine GVHD. Copyright © 2017. Published by Elsevier Inc.

  11. Kinetics of Monoclonal Antibody Aggregation from Dilute toward Concentrated Conditions.

    PubMed

    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.

  12. Role of cosolutes in the aggregation kinetics of monoclonal antibodies.

    PubMed

    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.

  13. Monoclonal anti-thrombopoietin antibodies generated by genetic immunization.

    PubMed

    Lim, Nam-Kyu; Kim, Jung-Hwan; Kim, Se-Yeon; Kang, Hyun-Jung; Kim, Keun-Soo; Lee, Sangyoon; Hong, Hyo Jeong; Inn, Kyung-Soo

    2006-04-01

    Thrombopoietin (TPO) is a megakaryocyte growth and differentiation factor that is currently being investigated as a therapeutic for cancer patients undergoing myelosuppressive chemotherapy. We generated monoclonal antibodies (MAbs) specific for human thrombopoietin (hTPO) by genetic immunization using an hTPO expression plasmid and an adjuvant plasmid that encodes mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). All genetically immunized mice exhibited a high humoral immune response. Splenocytes from these mice were used to generate hybridomas. Two MAbs, designated 2B9A10 and 4C16B15 (of IgG1 and IgG3 isotypes, respectively), were subsequently selected and produced. They specifically recognized and precipitated recombinant hTPO produced by mammalian cells and were effective in sandwich enzyme-linked immunosorbent assays (ELISAs) for hTPO quantitation. Our results demonstrate that these MAbs should be useful for purification and quantitation of hTPO in clinical and laboratory settings.

  14. Therapeutic monoclonal antibodies: strategies and challenges for biosimilars development.

    PubMed

    Calvo, Begoña; Zuñiga, Leyre

    2012-01-01

    Biosimilar medicines already on the market may have a primary structure identical to their reference products (e.g., amino acid sequences should be identical). In the case of monoclonal antibodies (mAbs), and due to their more complex structure, a greater level of demand would be in order and identity at other levels (e.g., post-translational modifications within the Fc region of the molecule) should be proved to establish "similarity". These requirements would lead to a greater development in the process and tighter quality controls during the production of biosimilar mAbs. The following issues should be taken into account in the comparability exercise: - The designs of the studies carried out to obtain approval of the reference product are not always adequate to show that safety and efficacy of the biosimilar mAbs are comparable. A similar efficacy does not necessarily imply a similar safety profile between the innovator and biosimilar products. - The design of clinical tests to demonstrate comparability must be flexible and adaptable throughout the development of the product. - The European Medicines Agency (EMA) will consider suitable goals in the evaluation of biosimilar mAbs for their approval (e.g., to specify whether their goal is to check similarity with the reference product or to show that the treatment is effective at a clinical level).

  15. DNA immunization as a technology platform for monoclonal antibody induction

    PubMed Central

    Liu, Shuying; Wang, Shixia; Lu, Shan

    2016-01-01

    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. PMID:27048742

  16. Ontogeny of Rat Thymic Epithelium Defined by Monoclonal Anticytokeratin Antibodies

    PubMed Central

    Jovanović, Suzana; Vasiljevski, Milijana; Dujić, Aleksandar

    1990-01-01

    Ontogenetic study on the expression of cytokeratin (CK) polypeptides within particular subsets of rat thymic epithelial cells (TEC) has been performed by a large panel of anti-CK monoclonal antibodies (mAbs) using the streptavidin-biotin immunoperoxidase method. Simultaneous presence of two or more CK subunits in the same TEC has been demonstrated by double immunoflouorescence labeling. The obtained results showed that the expression of CK polypeptides in fetal and neonatal thymus differed from the adult patterns. The main difference was observed in expression of CK10, 18, and 19 polypeptides. During fetal ontogeny, CK10 and 18 are markers for most medullary TEC or a subset of medullary TEC, respectively, whereas CK19 is mainly a pan-TEC marker. In the adult animals, they are localized in the cortical and a subset of medullary TEC (CK18), subcapsular/perivascular and some medullary TEC (CK19), or in a subset of medullary TEC and Hasall’s corpuscles (HC) (CK10). The switch in their expression in the cortex was observed during the first two weeks of postnatal life. PMID:1726554

  17. Anti-CD20 monoclonal antibodies: beyond B-cells.

    PubMed

    Avivi, Irit; Stroopinsky, Dina; Katz, Tamar

    2013-09-01

    Anti-CD20 monoclonal antibodies (MoAbs), employed in treating CD20⁺ lymphomas and autoimmune diseases, appear to have broader functions than just eradicating malignant B-cells and decreasing autoantibody production. Rituximab-induced T-cell inactivation, reported both in-vitro and in-vivo, may contribute to the increased risk of T-cell-dependent infections, observed in patients receiving this therapy. T-cell polarization into a suppressive phenotype, often observed in patients receiving rituximab for autoimmune disorders, was reported to be associated with prolonged remissions. Elimination of B-cells serving as antigen-presenting cells, thereby causing impaired T-cell activation, could play a significant role in induction of these changes. Direct binding of rituximab to a CD20dim T-cell population, inducing its depletion, may contribute to the decreased T-cell activation following rituximab therapy. Further investigation of the complex network through which rituximab and new anti-CD20 MoAbs act, would advance the employment of these agents in different clinical settings.

  18. Trial Watch: Immunomodulatory monoclonal antibodies for oncological indications

    PubMed Central

    Buqué, Aitziber; Bloy, Norma; Aranda, Fernando; Castoldi, Francesca; Eggermont, Alexander; Cremer, Isabelle; Fridman, Wolf Hervé; Fucikova, Jitka; Galon, Jérôme; Marabelle, Aurélien; Spisek, Radek; Tartour, Eric; Zitvogel, Laurence; Kroemer, Guido; Galluzzi, Lorenzo

    2015-01-01

    Immunomodulatory monoclonal antibodies (mAbs) differ from their tumor-targeting counterparts because they exert therapeutic effects by directly interacting with soluble or (most often) cellular components of the immune system. Besides holding promise for the treatment of autoimmune and inflammatory disorders, immunomodulatory mAbs have recently been shown to constitute a potent therapeutic weapon against neoplastic conditions. One class of immunomodulatory mAbs operates by inhibiting safeguard systems that are frequently harnessed by cancer cells to establish immunological tolerance, the so-called “immune checkpoints.” No less than 3 checkpoint-blocking mAbs have been approved worldwide for use in oncological indications, 2 of which during the past 12 months. These molecules not only mediate single-agent clinical activity in patients affected by specific neoplasms, but also significantly boost the efficacy of several anticancer chemo-, radio- or immunotherapies. Here, we summarize recent advances in the development of checkpoint-blocking mAbs, as well as of immunomodulatory mAbs with distinct mechanisms of action. PMID:26137403

  19. Production and characterization of a monoclonal antibody to Campylobacter jejuni.

    PubMed

    Heo, S A; Nannapaneni, R; Johnson, M G; Park, J S; Seo, K H

    2009-04-01

    Campylobacter species are a group of spiral-shaped bacteria that can cause disease in humans and animals. We developed a high-affinity monoclonal antibody (MAb) probe that recognizes Campylobacter jejuni cells. Cell suspensions grown under microaerobic conditions at 42 degrees C for 20 h on Bolton agar plates with lysed horse blood were used as live and heat-killed preparations, centrifuged at 8,000 x g for 20 min, and resuspended in carbonate buffer (pH 9.6) for coating on the enzyme-linked immunosorbent assay plates. BALB/c mice were immunized with C. jejuni sonicated cells at 10(7) CFU/ml to generate MAb-producing hybridoma clones. Of about 500 initial hybridoma clones, MAb 33D2, which reacted with C. jejuni and Campylobacter coli, was selected for further evaluation. MAb 33D2 is in the immunoglobulin subclass G2a and had relatively weaker reactivity with the C. coli strains tested. MAb 33D2 did not show any cross-reactions with the nine non-Campylobacter bacteria tested in the enzyme-linked immunosorbent assay and had a stronger affinity for C. jejuni as live versus heat-killed cells. In Western blot assays, MAb 33D2 recognized two major antigens of 62 and 43 kDa in extracts from C. jejuni cells but only one antigen of 62 kDa in extracts from C. coli cells.

  20. Adverse events to monoclonal antibodies used for cancer therapy

    PubMed Central

    Baldo, Brian A

    2013-01-01

    Fifteen monoclonal antibodies (mAbs) are currently registered and approved for the treatment of a range of different cancers. These mAbs are specific for a limited number of targets (9 in all). Four of these molecules are indeed directed against the B-lymphocyte antigen CD20; 3 against human epidermal growth factor receptor 2 (HER2 or ErbB2), 2 against the epidermal growth factor receptor (EGFR), and 1 each against epithelial cell adhesion molecule (EpCAM), CD30, CD52, vascular endothelial growth factor (VEGF), tumor necrosis factor (ligand) superfamily, member 11 (TNFSF11, best known as RANKL), and cytotoxic T lymphocyte-associated protein 4 (CTLA4). Collectively, the mAbs provoke a wide variety of systemic and cutaneous adverse events including the full range of true hypersensitivities: Type I immediate reactions (anaphylaxis, urticaria); Type II reactions (immune thrombocytopenia, neutopenia, hemolytic anemia); Type III responses (vasculitis, serum sickness; some pulmonary adverse events); and Type IV delayed mucocutaneous reactions as well as infusion reactions/cytokine release syndrome (IRs/CRS), tumor lysis syndrome (TLS), progressive multifocal leukoencephalopathy (PML) and cardiac events. Although the term “hypersensitivity” is widely used, no common definition has been adopted within and between disciplines and the requirement of an immunological basis for a true hypersensitivity reaction is sometimes overlooked. Consequently, some drug-induced adverse events are sometimes incorrectly described as “hypersensitivities” while others that should be described are not. PMID:24251081

  1. Kinase inhibitors and monoclonal antibodies in oncology: clinical implications.

    PubMed

    Gharwan, Helen; Groninger, Hunter

    2016-04-01

    Molecularly targeted cancer therapies, such as small-molecule kinase inhibitors and monoclonal antibodies, constitute a rapidly growing and an important part of the oncology armamentarium. Unlike conventional (cytotoxic) chemotherapeutics, targeted therapies were designed to disrupt cancer cell pathogenesis at specific biological points essential for the development and progression of the tumour. These agents were developed to disrupt specific targets with the aim of minimizing treatment burden compared with conventional chemotherapy. Nevertheless the increasingly common use of targeted therapies has revealed some unanticipated, often clinically significant toxic effects, as well as compromising effective palliative and end-of-life management approaches. Although patients and clinicians welcome improvements in cancer prognosis, these changes can also impact patient quality-of-life. Therefore, as demand for oncology expertise increases, physicians need to apprise themselves of targeted therapies and their clinical implications, including drug-specific side effects, impact on quality of life, and cost issues, especially in relation to end-of-life care. This Review provides a useful summary and guide for professionals treating patients with malignant diseases.

  2. Development and Evaluation of Monoclonal Antibodies for Paxilline

    PubMed Central

    Maragos, Chris M.

    2015-01-01

    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. PMID:26426046

  3. Characterization of epitopes recognized by monoclonal antibodies: experimental approaches supported by freely accessible bioinformatic tools.

    PubMed

    Clementi, Nicola; Mancini, Nicasio; Castelli, Matteo; Clementi, Massimo; Burioni, Roberto

    2013-05-01

    Monoclonal antibodies (mAbs) have been used successfully both in research and for clinical purposes. The possible use of protective mAbs directed against different microbial pathogens is currently being considered. The fine definition of the epitope recognized by a protective mAb is an important aspect to be considered for possible development in epitope-based vaccinology. The most accurate approach to this is the X-ray resolution of mAb/antigen crystal complex. Unfortunately, this approach is not always feasible. Under this perspective, several surrogate epitope mapping strategies based on the use of bioinformatics have been developed. In this article, we review the most common, freely accessible, bioinformatic tools used for epitope characterization and provide some basic examples of molecular visualization, editing and computational analysis.

  4. Application of monoclonal antibodies to quality control of foot-and-mouth disease vaccines.

    PubMed

    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.

  5. Enzymic oxidation of monoclonal antibodies by soluble and immobilized bifunctional enzyme complexes.

    PubMed

    Solomon, B; Koppel, R; Schwartz, F; Fleminger, G

    1990-06-27

    Site-specific modification of monoclonal antibodies was achieved by oxidation of the carbohydrate moieties of antibodies which are located remote from the antigen binding sites. Sialic acid and galactose are terminal sugars of these carbohydrate chains. Concomitant treatment of the antibodies with neuraminidase and galactose oxidase generated aldehyde groups in the oligosaccharide moieties of immunoglobulins which reacted selectively with amino or hydrazide groups of the matrix. Subsequent immobilization of neuraminidase and galactose oxidase on Eupergit C-adipic dihydrazide proved to be an efficient and selective system for the enzymic oxidation of the monoclonal antibodies without impairing their immunological activity. Oriented immobilization of enzymically oxidized monoclonal antibodies on hydrazide or amino Eupergit C derivatives thus leads to the formation of antibody matrix conjugates which possess high antigen-binding activities.

  6. Comparative testing of monoclonal antibodies against Plasmodium falciparum sporozoites for ELISA development*

    PubMed Central

    Wirtz, R. A.; Zavala, F.; Charoenvit, Y.; Campbell, G. H.; Burkot, T. R.; Schneider, I.; Esser, K. M.; Beaudoin, R. L.; Andre, R. G.

    1987-01-01

    Ten monoclonal antibodies developed against Plasmodium falciparum sporozoites at four institutions were evaluated for use in an enzyme-linked immunosorbent assay (ELISA). Four of the antibodies were eliminated because of their low sensitivity or requirement for high concentrations of capture antibody, while an additional four were rejected because they exhibited cross-reactivity with P. berghei sporozoites. Of the two remaining monoclonal antibodies, that designated 2A10 had the highest sensitivity, a requirement for lower concentrations of capture antibody, and had been tested successfully against sporozoites from a wider range of geographical areas than the others. Use of this monoclonal antibody in a standardized ELISA method gave a test ten times more sensitive than previously reported for P. falciparum sporozoites and its detection limit was less than 100 sporozoites per mosquito. PMID:3555879

  7. Anti-Mesothelin Monoclonal Antibodies for the Treatment of Cancer | NCI Technology Transfer Center | TTC

    Cancer.gov

    The National Cancer Institute, Laboratory of Molecular Biology is seeking parties interested in collaborative research to further co-develop monoclonal antibodies for the treatment of mesothelin-expressing cancers.

  8. Demonstration of a surface antigen of Clostridium tyrobutyricum by use of immunoblotting with a monoclonal antibody.

    PubMed

    Gueguen, F; Robreau, G; Talbot, F; Malcoste, R

    1990-01-01

    A monoclonal antibody, prepared against whole cells of Clostridium tyrobutyricum, recognized a surface antigen extracted by heat treatment or by hot phenol-water treatment. This antigen, after analysis by polyacrylamide gel electrophoresis and immunoblotting, has been shown to present a regularly-spaced ladder pattern similar to those shown by the lipopolysaccharide of many gram-negative bacteria. The proteinase K has been sho