Pub Date : 2000-12-01DOI: 10.1089/027245700750053968
P. Leo, P. Ucelli, E. Augusto, M. S. Oliveira, W. Tamashiro
The aim of this study was to produce anti-TNP monoclonal antibodies (MAbs) that could be conjugated and used for the detection of antigen-antibody reactions, in which the antigen specific-antibody had been previously bound to trinitrophenyl (TNP). For hybridoma production, SP2/0-Ag14 cells were fused with spleen cells from mice previously immunized with TNP-ovalbumin (TNP-OVA). After 10 days, enzyme-linked immunoadsorbent assay (ELISA) was used to detect anti-TNP antibodies in the supernatants, and five cultures were found to be strictly positive for TNP. Three of these were subsequently cloned by limiting dilution, and 15 clones were chosen for expansion based on the criterion of high reactivity against TNP. Anti-TNP MAbs produced by those clones were isotyped as IgG1, and purified by Sepharose-protein G affinity cromatography from ascites developed in BALB/c mice. Two purified MAbs (1B2.1B6 and 1B2.1E12) were coupled to horseradish peroxidase (HRPO). The resulting conjugates were evaluated in ELISA tests for interferon-gamma and interleukin-4 detection, in which the secondary anti-cytokine antibodies were coupled either to TNP or biotin. The performance of anti-TNP conjugates in these assays were compared with a biotin-streptavidin/peroxidase system. Both types of conjugates were similarly able to detect cytokines with r2 (linear correlation coefficient) close to unity value. Growth studies of one of those hybridomas (1B2.1B6) yielded a specific growth rate of 0.042 h(-1) and a doubling time of 16.5 h. Data discussed here show that at least two MAbs against TNP raised in this work can be used as a reagent for enzyme immunoassays.
{"title":"Anti-TNP monoclonal antibodies as reagents for enzyme immunoassay (ELISA).","authors":"P. Leo, P. Ucelli, E. Augusto, M. S. Oliveira, W. Tamashiro","doi":"10.1089/027245700750053968","DOIUrl":"https://doi.org/10.1089/027245700750053968","url":null,"abstract":"The aim of this study was to produce anti-TNP monoclonal antibodies (MAbs) that could be conjugated and used for the detection of antigen-antibody reactions, in which the antigen specific-antibody had been previously bound to trinitrophenyl (TNP). For hybridoma production, SP2/0-Ag14 cells were fused with spleen cells from mice previously immunized with TNP-ovalbumin (TNP-OVA). After 10 days, enzyme-linked immunoadsorbent assay (ELISA) was used to detect anti-TNP antibodies in the supernatants, and five cultures were found to be strictly positive for TNP. Three of these were subsequently cloned by limiting dilution, and 15 clones were chosen for expansion based on the criterion of high reactivity against TNP. Anti-TNP MAbs produced by those clones were isotyped as IgG1, and purified by Sepharose-protein G affinity cromatography from ascites developed in BALB/c mice. Two purified MAbs (1B2.1B6 and 1B2.1E12) were coupled to horseradish peroxidase (HRPO). The resulting conjugates were evaluated in ELISA tests for interferon-gamma and interleukin-4 detection, in which the secondary anti-cytokine antibodies were coupled either to TNP or biotin. The performance of anti-TNP conjugates in these assays were compared with a biotin-streptavidin/peroxidase system. Both types of conjugates were similarly able to detect cytokines with r2 (linear correlation coefficient) close to unity value. Growth studies of one of those hybridomas (1B2.1B6) yielded a specific growth rate of 0.042 h(-1) and a doubling time of 16.5 h. Data discussed here show that at least two MAbs against TNP raised in this work can be used as a reagent for enzyme immunoassays.","PeriodicalId":55044,"journal":{"name":"Hybridoma","volume":"19 6 1","pages":"473-9"},"PeriodicalIF":0.0,"publicationDate":"2000-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/027245700750053968","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60497201","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2000-12-01DOI: 10.1089/027245700750053940
N. Sato, N. Kiyokawa, K. Takada, M. Itagaki, M. Saito, T. Sekino, T. Suzuki, T. Taguchi, K. Mimori, F. Lanza, J. Fujimoto
The mouse- and rat-platelet-specific hamster monoclonal antibody (MAb) 1C2, previously found to react with a thrombin-sensitive 74-kD glycoprotein, was now shown to recognize platelet glycoprotein V (GPV, CD42d). 1C2 reacted with NIH-3T3 cells in which recombinant mouse or rat GPV was expressed. Both 1C2 and 4A5, another mouse-platelet-specific rat MAb, immunoprecipitated GVP, although they recognized different epitopes. Side-by-side comparison confirmed that 1C2 as well as RPM.9, a MAb against rat GPV, recognized the same rat platelet molecule. In a mouse bone marrow culture, 1C2+ megakaryocytes emerged from CD41 (GPIIb)+1C2- megakaryocytes. Because 1C2+ megakaryocytes exhibited higher DNA ploidy distribution than CD41+ cells, GPV likely appears in the late stage of megakaryocyte maturation. This study established 1C2 as a MAb against mouse and rat GPV, namely CD42d, and as useful tool to study rodent megakaryopoiesis.
{"title":"Characterization of monoclonal antibodies against mouse and rat platelet glycoprotein V (CD42d).","authors":"N. Sato, N. Kiyokawa, K. Takada, M. Itagaki, M. Saito, T. Sekino, T. Suzuki, T. Taguchi, K. Mimori, F. Lanza, J. Fujimoto","doi":"10.1089/027245700750053940","DOIUrl":"https://doi.org/10.1089/027245700750053940","url":null,"abstract":"The mouse- and rat-platelet-specific hamster monoclonal antibody (MAb) 1C2, previously found to react with a thrombin-sensitive 74-kD glycoprotein, was now shown to recognize platelet glycoprotein V (GPV, CD42d). 1C2 reacted with NIH-3T3 cells in which recombinant mouse or rat GPV was expressed. Both 1C2 and 4A5, another mouse-platelet-specific rat MAb, immunoprecipitated GVP, although they recognized different epitopes. Side-by-side comparison confirmed that 1C2 as well as RPM.9, a MAb against rat GPV, recognized the same rat platelet molecule. In a mouse bone marrow culture, 1C2+ megakaryocytes emerged from CD41 (GPIIb)+1C2- megakaryocytes. Because 1C2+ megakaryocytes exhibited higher DNA ploidy distribution than CD41+ cells, GPV likely appears in the late stage of megakaryocyte maturation. This study established 1C2 as a MAb against mouse and rat GPV, namely CD42d, and as useful tool to study rodent megakaryopoiesis.","PeriodicalId":55044,"journal":{"name":"Hybridoma","volume":"162 1","pages":"455-61"},"PeriodicalIF":0.0,"publicationDate":"2000-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/027245700750053940","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60497127","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2000-12-01DOI: 10.1089/027245700750053913
M. Nakamura, M. Terada, H. Sasaki, M. Kamada, T. Ohno
Antibody humanization by transplanting the complimentarity determining region (CDR) to a human framework aims to reduce the response of the human immune system against a foreign molecule during passive immunization. We transferred the CDR from the murine monoclonal antibody (MAb) NM-01 to a human IgG frame. The humanized NM-01 (hNM-01) recognizes the same epitope on Human Immunodeficiency Virus type 1 (HIV-1) envelope as its murine progenitor, but with greater efficiency, and shows enhanced neutralization of HIV-1. We have shown that this increase in reactivity may be attributed to residue 4 of the humanized kappa chain, where the presence of a methionine residue rather than the murine leucine appears to promote a more advantageous conformation of the antigen-binding site, perhaps via packing interactions with the V(kappa) CDR1. The capacity of humanized NM-01 to neutralize direct clinical isolates was also examined with the expectation that hNM-01 will prove suitable for development as a therapeutic agent. This reshaped antibody reacted with several clinical isolates of HIV-1 tested. Moreover, we proved the ability of this antibody of its activation of complement by flow cytometry and electron microscopy analysis. Although hNM-01 alone was capable of neutralizing HIV-1, the presence of complement enhanced neutralization. The enhancement of complement activation was also observed in hNM-01 than murine progenitor. This finding supports a potential role for antibody-dependent complement-mediated virolysis and more effective neutralization in HIV-1 therapy.
{"title":"Virolysis and in vitro neutralization of HIV-1 by humanized monoclonal antibody hNM-01.","authors":"M. Nakamura, M. Terada, H. Sasaki, M. Kamada, T. Ohno","doi":"10.1089/027245700750053913","DOIUrl":"https://doi.org/10.1089/027245700750053913","url":null,"abstract":"Antibody humanization by transplanting the complimentarity determining region (CDR) to a human framework aims to reduce the response of the human immune system against a foreign molecule during passive immunization. We transferred the CDR from the murine monoclonal antibody (MAb) NM-01 to a human IgG frame. The humanized NM-01 (hNM-01) recognizes the same epitope on Human Immunodeficiency Virus type 1 (HIV-1) envelope as its murine progenitor, but with greater efficiency, and shows enhanced neutralization of HIV-1. We have shown that this increase in reactivity may be attributed to residue 4 of the humanized kappa chain, where the presence of a methionine residue rather than the murine leucine appears to promote a more advantageous conformation of the antigen-binding site, perhaps via packing interactions with the V(kappa) CDR1. The capacity of humanized NM-01 to neutralize direct clinical isolates was also examined with the expectation that hNM-01 will prove suitable for development as a therapeutic agent. This reshaped antibody reacted with several clinical isolates of HIV-1 tested. Moreover, we proved the ability of this antibody of its activation of complement by flow cytometry and electron microscopy analysis. Although hNM-01 alone was capable of neutralizing HIV-1, the presence of complement enhanced neutralization. The enhancement of complement activation was also observed in hNM-01 than murine progenitor. This finding supports a potential role for antibody-dependent complement-mediated virolysis and more effective neutralization in HIV-1 therapy.","PeriodicalId":55044,"journal":{"name":"Hybridoma","volume":"19 6 1","pages":"427-34"},"PeriodicalIF":0.0,"publicationDate":"2000-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/027245700750053913","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60497105","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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