Human antibodies and hybridomas最新文献_第8页

Serum preparation and methods for the large-scale production of IgG monoclonal antibody. 血清制备及大规模生产IgG单克隆抗体的方法。

Human antibodies and hybridomas Pub Date : 1994-01-01

Q S Zeng, H Takeyama, S Kanda, R F Irie

{"title":"Serum preparation and methods for the large-scale production of IgG monoclonal antibody.","authors":"Q S Zeng, H Takeyama, S Kanda, R F Irie","doi":"","DOIUrl":"","url":null,"abstract":"In this study we demonstrated that fetal calf serum (FCS) depleted of IgG by protein G affinity chromatography (G-FCS) is superior to whole FCS or serum-free culture media as a culture supplement for the production of purified IgG monoclonal antibodies (MAb). One hundred ml FCS was applied to a 25 ml protein G Sepharose 4 Fast Flow Column, which was shaken gently for 2 days at 4 degrees C. The procedure was repeated using a protein G column for an additional day. G-FCS was used at a concentration of 5% in RPMI 1640 medium to grow the mouse myeloma cell line P3X63.Ag8.653, which secretes an IgG-1 mouse-human chimeric monoclonal antibody (TVE-1). Cell density, viability, doubling time, and antibody production were used as indices to compare the efficacy of this medium with that of whole FCS medium, AIM-V (Gibco, USA) and other serum-free media. The results demonstrate that cell growth and antibody production in -GFCS medium did not differ significantly from that in FCS medium, but were significantly better than in the serum-free media (p < 0.001). TVE-1 antibody in the spent tissue culture media was purified by 50% ammonium sulfate precipitation and Protein A affinity chromatography. An antibody that was more than 99% pure was obtained. Endotoxin analysis revealed that the IgG depletion process does not generate a significant level of endotoxin in FCS (< 0.06 EU/ml).(ABSTRACT TRUNCATED AT 250 WORDS)","PeriodicalId":77166,"journal":{"name":"Human antibodies and hybridomas","volume":"5 1-2","pages":"75-80"},"PeriodicalIF":0.0,"publicationDate":"1994-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18855967","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}

引用次数: 0

Human monoclonal antibodies recognize early and late viral proteins of human cytomegalovirus. 人单克隆抗体识别人巨细胞病毒的早期和晚期病毒蛋白。

Human antibodies and hybridomas Pub Date : 1994-01-01

H Alexander, J Harpprecht, H G Podzuweit, P Rautenberg, W Müller-Ruchholtz

{"title":"Human monoclonal antibodies recognize early and late viral proteins of human cytomegalovirus.","authors":"H Alexander, J Harpprecht, H G Podzuweit, P Rautenberg, W Müller-Ruchholtz","doi":"","DOIUrl":"","url":null,"abstract":"Human monoclonal antibodies directed against human cytomegalovirus were generated by fusion of in vitro stimulated human spleen lymphocytes from an HCMV-seropositive 53-year-old organ donor with the mouse myeloma cell line Ag8.653. Fourteen human/mouse hybridomas producing anti-cytomegalovirus IgG were screened by an ELISA technique and four selected clones have been established since March 1988, generating about 5-40 micrograms/24 h IgG per ml culture supernatant. Reference and local cytomegalovirus strains were stained by the antibodies without showing cross-reactivity to other herpes viruses. Three monoclonal antibodies, A4B4 (IgG11), A6B3 (IgG1k) and A6A2 (IgG1k), immunoprecipitated a 68 kDa early viral protein which appears during the infectious cycle, first in the nucleus (18-24 h) and then also in the cytoplasm (24-96 h) of infected cells. Inhibition of DNA replication restricted the detection of the 68 kDa viral protein to the nucleus of infected cells. Staining of unfixed infected cells showed that two of the antibodies bound at the surface of a few cells. The fourth monoclonal antibody A3C5 (IgG11) immunoprecipitated a 34/38 kDa late viral protein which appears in the nucleus (48-72 h) of infected cells. These antibodies enable us to study the human host response to human cytomegalovirus and to elucidate the functions of human antibodies especially in their interaction with the T-cell response.","PeriodicalId":77166,"journal":{"name":"Human antibodies and hybridomas","volume":"5 1-2","pages":"81-90"},"PeriodicalIF":0.0,"publicationDate":"1994-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18855968","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}

引用次数: 0

Galactosylation of human IgG monoclonal anti-D produced by EBV-transformed B-lymphoblastoid cell lines is dependent on culture method and affects Fc receptor-mediated functional activity. ebv转化的b淋巴母细胞样细胞系产生的人IgG单克隆抗d的半乳糖基化依赖于培养方法，并影响Fc受体介导的功能活性。

Human antibodies and hybridomas Pub Date : 1994-01-01

B M Kumpel, T W Rademacher, G A Rook, P J Williams, I B Wilson

{"title":"Galactosylation of human IgG monoclonal anti-D produced by EBV-transformed B-lymphoblastoid cell lines is dependent on culture method and affects Fc receptor-mediated functional activity.","authors":"B M Kumpel, T W Rademacher, G A Rook, P J Williams, I B Wilson","doi":"","DOIUrl":"","url":null,"abstract":"Human monoclonal antibodies to the Rh D blood group antigen were produced by EBV-transformed B cell lines grown in serum-free medium in low density (LD) static cultures or high density (HD) hollow fiber bioreactors. Glycosylation analysis of the purified IgG was determined by the binding of anti-GlcNAc monoclonal antibody (GN7) and by analysis of oligosaccharides released by hydrazinolysis. The LD MAbs had only trace levels of agalactosyl oligosaccharides (G0), the major species (> 70%) being digalactosyl structures (G2). The HD MAbs, by contrast, contained about 10% G0 and relatively high levels (over 50%) of monogalactosyl (G1) oligosaccharides. beta 1-4 galactosyltransferase activity in the LD cell lines was similar to that found previously for other EBV-transformed B cell lines. The predominant oligosaccharides of an IgG3 anti-D, BRAD-3, contained bisecting N-acetylglucosamine. In functional assays with Fc receptor (Fc gamma R) positive effector cells, the highly galactosylated LD form of BRAD-3 was more active than the HD form in monocyte (Fc gamma RI) and K cell (Fc gamma RIII) mediated lysis of erythrocytes in ADCC assays, although these preparations showed no difference in Fc gamma RI-mediated rosette formation with U937 cells. One MAb, JAC10, was over 10-fold less active than two other IgG1 MAbs, 2B6 and BRAD-5, at mediating lysis of erythrocytes by Fc gamma RIII+ K cells; differences in sialylation may have contributed to this heterogeneity.(ABSTRACT TRUNCATED AT 250 WORDS)","PeriodicalId":77166,"journal":{"name":"Human antibodies and hybridomas","volume":"5 3-4","pages":"143-51"},"PeriodicalIF":0.0,"publicationDate":"1994-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18759964","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}

引用次数: 0

Antibody production of a human EBV-transformed B cell line and its heterohybridoma and trioma cell line descendants in different culture systems. 人ebv转化B细胞系及其异杂交瘤和三瘤细胞系后代在不同培养体系中的抗体产生

Human antibodies and hybridomas Pub Date : 1994-01-01

B Gustafsson, J Hinkula

引用次数: 0

Secretion of human monoclonal antibody after fusion of an immortal T cell line and lymphocytes from the peripheral blood of a patient with colon carcinoma. 大肠癌患者外周血淋巴细胞与不朽T细胞系融合后分泌人单克隆抗体。

Human antibodies and hybridomas Pub Date : 1994-01-01 DOI: 10.3233/HAB-1994-51-205

S. Dillman, P. Aldenderfer, A. Strelkauskas

{"title":"Secretion of human monoclonal antibody after fusion of an immortal T cell line and lymphocytes from the peripheral blood of a patient with colon carcinoma.","authors":"S. Dillman, P. Aldenderfer, A. Strelkauskas","doi":"10.3233/HAB-1994-51-205","DOIUrl":"https://doi.org/10.3233/HAB-1994-51-205","url":null,"abstract":"A series of human hybridomas were derived by the fusion of the immature T cell line, JM, and lymphocytes from the peripheral blood of a colon carcinoma patient in long term remission who was shown to produce anti-tumor antibody. Five of the hybridomas have been selected for further study. These hybridomas express the T cell surface markers CD2, CD3, CD4, and CD8. The human IgG2/kappa antibody produced by these clones has been purified using affinity chromatography and has been used in immunohistiologic staining procedures. Biotinylated monoclonal antibody (MAb) stained colon carcinoma tissue but did not stain normal colon. Positive immunostaining was also seen utilizing colon carcinoma cell lines but not with other cell lines tested. These hybrids were constructed without using the conventional fusion technology which employs 8-azoguanine resistant, HAT sensitive malignant fusion partners. Because the fusion partner was a T cell, we were able to select hybrids on the basis of surface immunoglobulin. This approach was accompanied by stringent selection of patients as lymphocyte donors. Utilizing these unique methods, we have successfully produced hybridomas with T cell surface markers that produce human MAb with reactivity to colon carcinoma.","PeriodicalId":77166,"journal":{"name":"Human antibodies and hybridomas","volume":"5 1-2 1","pages":"32-40"},"PeriodicalIF":0.0,"publicationDate":"1994-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3233/HAB-1994-51-205","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"69906310","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}

引用次数: 0

Humanization of the murine anti-human CD3 monoclonal antibody OKT3. 小鼠抗人CD3单克隆抗体OKT3的人源化。

Human antibodies and hybridomas Pub Date : 1994-01-01 DOI: 10.3233/HAB-1994-51-206

J. Adair, D. Athwal, M. Bodmer, S. Bright, A. Collins, V. Pulito, P. Rao, R. Reedman, A. Rothermel, D. Xu

{"title":"Humanization of the murine anti-human CD3 monoclonal antibody OKT3.","authors":"J. Adair, D. Athwal, M. Bodmer, S. Bright, A. Collins, V. Pulito, P. Rao, R. Reedman, A. Rothermel, D. Xu","doi":"10.3233/HAB-1994-51-206","DOIUrl":"https://doi.org/10.3233/HAB-1994-51-206","url":null,"abstract":"OKT3 is a murine monoclonal antibody which recognizes an epitope on the epsilon-subunit within the human CD3 complex. OKT3 possesses potent immunosuppressive properties in vivo and has been proven effective in the treatment of renal, heart and liver allograft rejection. Despite its efficacy, significant problems remain associated with OKT3 therapy, i.e. T-cell activation and the anti-murine antibody response. To address the problem of the anti-murine antibody response we have constructed humanized versions of OKT3. One of the humanized derivatives, gOKT3-7 incorporating the OKT3 complementarity determining regions plus a small number of alterations to the human framework, has an affinity of 1.4 x 10(9) M-1 compared with 1.2 x 10(9) M-1 for the murine OKT3. A humanized antibody (gOKT3-1) incorporating only the CDRs from OKT3 was found to be functionally inactive, confirming the requirement for nonCDR substitutions. gOKT3-7 retains the ability of mOKT3 to induce T cell proliferation in vitro and appears to be a good candidate for further development for in vivo therapy.","PeriodicalId":77166,"journal":{"name":"Human antibodies and hybridomas","volume":"5 1-2 1","pages":"41-7"},"PeriodicalIF":0.0,"publicationDate":"1994-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3233/HAB-1994-51-206","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"69906319","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}

引用次数: 58

Anti-bacterial specificities in the human fetal B cell repertoire. 人胎儿B细胞库的抗菌特异性。

Human antibodies and hybridomas Pub Date : 1994-01-01

U Settmacher, A Delvig, S Jahn

引用次数: 0

Phage surface presentation and secretion of antibody fragments using an adaptable phagemid vector. 使用适应性噬菌体载体的噬菌体表面呈递和抗体片段的分泌。

Human antibodies and hybridomas Pub Date : 1994-01-01

M Lah, A Goldstraw, J F White, O Dolezal, R Malby, P J Hudson

{"title":"Phage surface presentation and secretion of antibody fragments using an adaptable phagemid vector.","authors":"M Lah, A Goldstraw, J F White, O Dolezal, R Malby, P J Hudson","doi":"","DOIUrl":"","url":null,"abstract":"Phagemid vectors have been developed which promise to supersede hybridoma technology for the selection and production of human antibodies. We have modified an existing phagemid vector to improve the stability of synthesized soluble antibody fragments. The vector allows the antibody fragment to be produced: i) as a soluble protein incorporating a stable carboxyl terminal octapeptide (FLAG) or, ii) on the surface of a bacteriophage fused to a minor coat protein (the gene III protein). The antibody gene encoding the well characterized monoclonal antibody NC10 (an antibody that recognizes the neuraminidase of the influenza strain N9) was inserted as a single chain Fv construct into the phagemid vectors pHFA and pHFA/SacII. Western blotting, ELISA and electron microscopy studies showed that recombinant clones could be manipulated to either synthesize soluble protein into the periplasm or present the protein on the surface of bacteriophage. Cosynthesis of GroEL and GroES chaperonins resulted in complete proteolysis of the scFvNC10-FLAG-gIIIp fusion product and did not improve total phage production. Coexpression of chaperonins should be used with caution for library construction due to the expected selection pressure for protease resistant gene III fusions.","PeriodicalId":77166,"journal":{"name":"Human antibodies and hybridomas","volume":"5 1-2","pages":"48-56"},"PeriodicalIF":0.0,"publicationDate":"1994-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18855965","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}

引用次数: 0

Comparison of expression of a humanized monoclonal antibody in mouse NSO myeloma cells and Chinese hamster ovary cells. 人源化单克隆抗体在小鼠NSO骨髓瘤细胞和中国仓鼠卵巢细胞中的表达比较。

Human antibodies and hybridomas Pub Date : 1994-01-01 DOI: 10.3233/HAB-1994-51-209

T. Peakman, J. Worden, R. Harris, H. Cooper, J. Tite, M. Page, D. Gewert, M. Bartholemew, J. Crowe, S. Brett

{"title":"Comparison of expression of a humanized monoclonal antibody in mouse NSO myeloma cells and Chinese hamster ovary cells.","authors":"T. Peakman, J. Worden, R. Harris, H. Cooper, J. Tite, M. Page, D. Gewert, M. Bartholemew, J. Crowe, S. Brett","doi":"10.3233/HAB-1994-51-209","DOIUrl":"https://doi.org/10.3233/HAB-1994-51-209","url":null,"abstract":"We report a detailed comparison of two commonly used stable, amplifiable mammalian expression systems (Chinese Hamster Ovary cells/dihydrofolate reductase and Mouse NSO myeloma/glutamine synthetase) used to express a humanized IgG1 monoclonal antibody. We compare copy number and steady state mRNA levels of both the selectable marker and heavy chain of the antibody throughout the selection and amplification process. In both cell lines, copy number and steady state levels of heavy chain and selectable marker increased during selection and were further increased during amplification. As expected, an increase in steady state mRNA levels of heavy chain correlated with an increase in expression of antibody whilst an increase in the steady state levels of mRNA of the selectable marker correlated with increased resistance to the selective agent. In NSO and CHO cells producing equivalent amounts of antibody, the copy number of the antibody genes and selectable marker was significantly higher in the CHO cells than in the NSO cells. However, the steady state mRNA levels of the heavy chain of the antibody were virtually identical. Rates of protein secretion in the two cell lines were also compared and found to be very similar. When the antibody purified from both systems was compared in a number of functional assays they behaved identically.","PeriodicalId":77166,"journal":{"name":"Human antibodies and hybridomas","volume":"5 1-2 1","pages":"65-74"},"PeriodicalIF":0.0,"publicationDate":"1994-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3233/HAB-1994-51-209","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"69905927","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}

引用次数: 21

Secretion of human monoclonal antibody after fusion of an immortal T cell line and lymphocytes from the peripheral blood of a patient with colon carcinoma. 大肠癌患者外周血淋巴细胞与不朽T细胞系融合后分泌人单克隆抗体。

Human antibodies and hybridomas Pub Date : 1994-01-01

S L Dillman, P Aldenderfer, A Strelkauskas

{"title":"Secretion of human monoclonal antibody after fusion of an immortal T cell line and lymphocytes from the peripheral blood of a patient with colon carcinoma.","authors":"S L Dillman, P Aldenderfer, A Strelkauskas","doi":"","DOIUrl":"","url":null,"abstract":"A series of human hybridomas were derived by the fusion of the immature T cell line, JM, and lymphocytes from the peripheral blood of a colon carcinoma patient in long term remission who was shown to produce anti-tumor antibody. Five of the hybridomas have been selected for further study. These hybridomas express the T cell surface markers CD2, CD3, CD4, and CD8. The human IgG2/kappa antibody produced by these clones has been purified using affinity chromatography and has been used in immunohistiologic staining procedures. Biotinylated monoclonal antibody (MAb) stained colon carcinoma tissue but did not stain normal colon. Positive immunostaining was also seen utilizing colon carcinoma cell lines but not with other cell lines tested. These hybrids were constructed without using the conventional fusion technology which employs 8-azoguanine resistant, HAT sensitive malignant fusion partners. Because the fusion partner was a T cell, we were able to select hybrids on the basis of surface immunoglobulin. This approach was accompanied by stringent selection of patients as lymphocyte donors. Utilizing these unique methods, we have successfully produced hybridomas with T cell surface markers that produce human MAb with reactivity to colon carcinoma.","PeriodicalId":77166,"journal":{"name":"Human antibodies and hybridomas","volume":"5 1-2","pages":"32-40"},"PeriodicalIF":0.0,"publicationDate":"1994-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18855963","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}

引用次数: 0