Pub Date : 2001-04-01DOI: 10.1089/02724570152057616
M. Malakaneh, M. Rasaee, F. Rahbarizadeh, R. Madani, M. Forozandeh, K. Khabiri, M. H. Alimohammadian
An active ester derivative of neopterin was prepared using 4-(N-maleimidomethyl) cyclohexan 4-carboxilic acid N-hydroxy succinimide ester (MCH-NHS), conjugated to bovine serum albumin (BSA) and injected for antibody production (for both monoclonal and polyclonal antibodies). High titer antibody producing spleen cells were removed and fused with myeloma cells of Sp2/0 origin. Neopterin was conjugated to the enzyme penicillinase by a one-step glutaraldehyde method, which was used as tracer. A novel enzyme immunoassay was developed using this conjugate to screen and characterize the monoclonal antibody (MAb) produced in these experiments. After limiting dilutions, it was found that antibody produced by one clone with a Ka value of 7.6 x 10-7 mol/L was specific for a number of structurally related molecules. This clone was found to be of IgG class and IgG2a subclass. The standard curvewas constructed with a sensitivity of 10 pg/well (100 pg/mL) covering up to 1 ng/mL.
{"title":"Characterization of a monoclonal antibody against neopterin using an enzyme-linked immunosorbent assay with penicillinase as label.","authors":"M. Malakaneh, M. Rasaee, F. Rahbarizadeh, R. Madani, M. Forozandeh, K. Khabiri, M. H. Alimohammadian","doi":"10.1089/02724570152057616","DOIUrl":"https://doi.org/10.1089/02724570152057616","url":null,"abstract":"An active ester derivative of neopterin was prepared using 4-(N-maleimidomethyl) cyclohexan 4-carboxilic acid N-hydroxy succinimide ester (MCH-NHS), conjugated to bovine serum albumin (BSA) and injected for antibody production (for both monoclonal and polyclonal antibodies). High titer antibody producing spleen cells were removed and fused with myeloma cells of Sp2/0 origin. Neopterin was conjugated to the enzyme penicillinase by a one-step glutaraldehyde method, which was used as tracer. A novel enzyme immunoassay was developed using this conjugate to screen and characterize the monoclonal antibody (MAb) produced in these experiments. After limiting dilutions, it was found that antibody produced by one clone with a Ka value of 7.6 x 10-7 mol/L was specific for a number of structurally related molecules. This clone was found to be of IgG class and IgG2a subclass. The standard curvewas constructed with a sensitivity of 10 pg/well (100 pg/mL) covering up to 1 ng/mL.","PeriodicalId":55044,"journal":{"name":"Hybridoma","volume":"20 2 1","pages":"117-21"},"PeriodicalIF":0.0,"publicationDate":"2001-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/02724570152057616","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60499010","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 : 2001-04-01DOI: 10.1089/02724570152057625
Y. Park, C. Shin, W. J. Lee, M. Jo, J. Ryu, E. Choi, K. C. Kim, K. Ko
Airway mucins are high molecular mass (>10(6) dalton) glycoproteins with various types of associated molecules including glycoproteins, lipoproteins, and lipids. The study of mucin-associated proteins is limited largely due to the lack of specific probes. In this study, we produced a monoclonal antibody, MAbHT10, against a 190-kDa mucin associated-protein by immunizing mice with hamster airway mucin purified in nondissociative condition. Using HT10, the 190-kDa mucin-associated protein was characterized immunologically. The 190-kDa mucin-associated protein is glycoprotein and HT10 recognized carbohydrate containing portion of the protein. The association of 190-kDa protein with mucin is strong enough that heat and detergent treatment is required to dissociate it from mucin as evidenced by gel filtration chromatography, Western blot, enzyme-linked immunoadsorbent assay (ELISA), and co-immunoprecipitation. The expression of the 190-kDa protein is increased with the development of hamster tracheal epithelial cells in culture, but showed differences with the pattern of the regulation of mucin expression. Adenosine triphosphate (ATP), a known strong mucin secretagogue, dose-dependently increased mucin release but caused only marginal increase in the release of the 190-kDa protein. The MAb should be useful in the structural and functional analysis of the 190-kDa mucin-associated proteins in physiological and pathological situations such as chronic airway diseases.
{"title":"Immunological characterization of a mucin-associated protein from hamster tracheal epithelial cell culture.","authors":"Y. Park, C. Shin, W. J. Lee, M. Jo, J. Ryu, E. Choi, K. C. Kim, K. Ko","doi":"10.1089/02724570152057625","DOIUrl":"https://doi.org/10.1089/02724570152057625","url":null,"abstract":"Airway mucins are high molecular mass (>10(6) dalton) glycoproteins with various types of associated molecules including glycoproteins, lipoproteins, and lipids. The study of mucin-associated proteins is limited largely due to the lack of specific probes. In this study, we produced a monoclonal antibody, MAbHT10, against a 190-kDa mucin associated-protein by immunizing mice with hamster airway mucin purified in nondissociative condition. Using HT10, the 190-kDa mucin-associated protein was characterized immunologically. The 190-kDa mucin-associated protein is glycoprotein and HT10 recognized carbohydrate containing portion of the protein. The association of 190-kDa protein with mucin is strong enough that heat and detergent treatment is required to dissociate it from mucin as evidenced by gel filtration chromatography, Western blot, enzyme-linked immunoadsorbent assay (ELISA), and co-immunoprecipitation. The expression of the 190-kDa protein is increased with the development of hamster tracheal epithelial cells in culture, but showed differences with the pattern of the regulation of mucin expression. Adenosine triphosphate (ATP), a known strong mucin secretagogue, dose-dependently increased mucin release but caused only marginal increase in the release of the 190-kDa protein. The MAb should be useful in the structural and functional analysis of the 190-kDa mucin-associated proteins in physiological and pathological situations such as chronic airway diseases.","PeriodicalId":55044,"journal":{"name":"Hybridoma","volume":"20 2 1","pages":"123-9"},"PeriodicalIF":0.0,"publicationDate":"2001-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/02724570152057625","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60498758","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 : 2001-04-01DOI: 10.1089/02724570152057562
I. Bank, R. Dardik, V. Lévy, I. Goldstein, J. Shoham
A monoclonal antibody (MAb), CH11, was developed by immunizing mice with CD4+ gammadelta T-cell receptor (TCR)+ cells. It recognized an antigen expressed in the surface membrane of T-cell lines, but not of U937, lymphoblastoid B cells (LBC), K562, Raji or Daudi cells, indicating selectivity for the T-cell lineage. In addition, it labelled 70-80% of normal peripheral blood mononuclear cells (PBMC), with high expression on the erythrocyte rosetting (E+) fraction, and low/absent expression on E- cells. However, CD4+ T cells expressed higher levels of reactivity than CD8+ or gammadelta+ T-cell receptor (TCR)+ lymphocytes in PB. Furthermore, in 7 of 10 individuals tested, 7.34+/-3.88% of unselected PBMC were CH11- CD3+ and were relatively enriched in CD8+ and in gammadelta TCR+-cells. In addition, thymic gammadelta T cells, and gammadelta lymphoproliferations from two patients were nonreactive or weakly reactive with the MAb. Activation of E+ cells with phorbol-12-myristate-13-acetate (PMA) enhanced CH11 expression uniformly, whereas activation with phytohemagglutinin (PHA) selectively down-regulated expression of the antigen on the CD8+ subset. In Western blots performed in nonreducing (NR) conditions, MAb CH11 detected a 100 kDa molecule in PBMC and Jurkat T-cell lysates. Preincubation of T cells with MAb CH11 specifically abrogated their subsequent reactivity with MAb to CD6, suggesting that MAb CH11 is recognizing an epitope of CD6. Given its function as a receptor for ligands on thymic epithelium, activated leukocytes and synoviocytes, this newly defined heterogeneity of expression and regulation of the CD6 molecule on subsets of T cells may help determine their functional repertoire in vivo.
{"title":"Differential expression and regulation of CD6 on T-cell subsets revealed by monoclonal antibody (MAb) CH11.","authors":"I. Bank, R. Dardik, V. Lévy, I. Goldstein, J. Shoham","doi":"10.1089/02724570152057562","DOIUrl":"https://doi.org/10.1089/02724570152057562","url":null,"abstract":"A monoclonal antibody (MAb), CH11, was developed by immunizing mice with CD4+ gammadelta T-cell receptor (TCR)+ cells. It recognized an antigen expressed in the surface membrane of T-cell lines, but not of U937, lymphoblastoid B cells (LBC), K562, Raji or Daudi cells, indicating selectivity for the T-cell lineage. In addition, it labelled 70-80% of normal peripheral blood mononuclear cells (PBMC), with high expression on the erythrocyte rosetting (E+) fraction, and low/absent expression on E- cells. However, CD4+ T cells expressed higher levels of reactivity than CD8+ or gammadelta+ T-cell receptor (TCR)+ lymphocytes in PB. Furthermore, in 7 of 10 individuals tested, 7.34+/-3.88% of unselected PBMC were CH11- CD3+ and were relatively enriched in CD8+ and in gammadelta TCR+-cells. In addition, thymic gammadelta T cells, and gammadelta lymphoproliferations from two patients were nonreactive or weakly reactive with the MAb. Activation of E+ cells with phorbol-12-myristate-13-acetate (PMA) enhanced CH11 expression uniformly, whereas activation with phytohemagglutinin (PHA) selectively down-regulated expression of the antigen on the CD8+ subset. In Western blots performed in nonreducing (NR) conditions, MAb CH11 detected a 100 kDa molecule in PBMC and Jurkat T-cell lysates. Preincubation of T cells with MAb CH11 specifically abrogated their subsequent reactivity with MAb to CD6, suggesting that MAb CH11 is recognizing an epitope of CD6. Given its function as a receptor for ligands on thymic epithelium, activated leukocytes and synoviocytes, this newly defined heterogeneity of expression and regulation of the CD6 molecule on subsets of T cells may help determine their functional repertoire in vivo.","PeriodicalId":55044,"journal":{"name":"Hybridoma","volume":"20 2 1","pages":"75-84"},"PeriodicalIF":0.0,"publicationDate":"2001-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/02724570152057562","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60498750","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 : 2001-02-01DOI: 10.1089/027245701300060427
T. Atsuta, S. Fujimura, H. Moriya, M. Vidal, T. Akasaka, H. Koseki
The Polycomb group (PcG) genes play a role of transcriptional repressor for long-term maintenance of the Hox cluster gene expression. Recently two structurally similar gene products, Ring1A and Ring1B, were identified. Genetic evidence has indicated that Ring1A has Pc-G properties, however, Ring1B functions are still unknown. To gain functional insights for Ring1B, we raised the mouse monoclonal antibodies (MAbs) against murine Ring1B protein. Using these antibodies, we have detected specifically mouse, human and monkey Ring1B gene products from whole cell extracts in immunoblot and immunoprecipitation analyses. Immunofluorescent staining by the antibodies has shown that endogenous Ring1B proteins clearly co-localize with Ring1A at the pattern of diffuse nuclear speckles. Together with their sequence similarity, Ring1B also may function as a Pc-G protein. Finally, we have proposed that the anti-Ring1B would be useful for biochemical and cytological analyses of Ring1B and Pc-G complex.
Polycomb group (PcG)基因在Hox簇基因表达的长期维持中发挥转录抑制因子的作用。最近发现了两个结构相似的基因产物Ring1A和Ring1B。遗传证据表明，Ring1A具有Pc-G特性，然而，Ring1B的功能仍然未知。为了进一步了解Ring1B的功能，我们构建了针对小鼠Ring1B蛋白的小鼠单克隆抗体(mab)。使用这些抗体，我们已经在免疫印迹和免疫沉淀分析中从全细胞提取物中特异性地检测到小鼠、人类和猴子的Ring1B基因产物。抗体的免疫荧光染色显示内源性Ring1B蛋白与Ring1A以弥漫性核斑的模式明显共定位。结合它们序列的相似性，Ring1B也可能具有Pc-G蛋白的功能。最后，我们提出了抗Ring1B可用于Ring1B和Pc-G复合物的生化和细胞学分析。
{"title":"Production of monoclonal antibodies against mammalian Ring1B proteins.","authors":"T. Atsuta, S. Fujimura, H. Moriya, M. Vidal, T. Akasaka, H. Koseki","doi":"10.1089/027245701300060427","DOIUrl":"https://doi.org/10.1089/027245701300060427","url":null,"abstract":"The Polycomb group (PcG) genes play a role of transcriptional repressor for long-term maintenance of the Hox cluster gene expression. Recently two structurally similar gene products, Ring1A and Ring1B, were identified. Genetic evidence has indicated that Ring1A has Pc-G properties, however, Ring1B functions are still unknown. To gain functional insights for Ring1B, we raised the mouse monoclonal antibodies (MAbs) against murine Ring1B protein. Using these antibodies, we have detected specifically mouse, human and monkey Ring1B gene products from whole cell extracts in immunoblot and immunoprecipitation analyses. Immunofluorescent staining by the antibodies has shown that endogenous Ring1B proteins clearly co-localize with Ring1A at the pattern of diffuse nuclear speckles. Together with their sequence similarity, Ring1B also may function as a Pc-G protein. Finally, we have proposed that the anti-Ring1B would be useful for biochemical and cytological analyses of Ring1B and Pc-G complex.","PeriodicalId":55044,"journal":{"name":"Hybridoma","volume":"20 1 1","pages":"43-6"},"PeriodicalIF":0.0,"publicationDate":"2001-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/027245701300060427","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60498510","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 : 2001-02-01DOI: 10.1089/027245701300060463
L. Huang, H. W. Liu, K. Chang
Human arginase was purified from liver and two monoclonal antibodies (MAbs), HA1 and HA2, were produced by fusion of spleen cells from an arginase-immunized BALB/c mouse and the NS-1 myeloma cell line. Both MAbs were of the IgG3 subclass and contained the kappa light chain. HA1 inhibited arginase activity, suggesting that it binds to the arginase catalytic site. HA1 and a horseradish peroxidase-conjugated polyclonal rabbit anti-human arginase antibody were used to develop a sandwich enzyme-linked immunoadsorbent assay (ELISA) for the quantification of human arginase, which can be used in the 1 to 300 ng/mL range. Because of its sensitivity and specificity, this MAb can be successfully applied to the ELISA quantification of arginase in serum and culture supernatants.
{"title":"Development of a sandwich ELISA test for arginase measurement based on monoclonal antibodies.","authors":"L. Huang, H. W. Liu, K. Chang","doi":"10.1089/027245701300060463","DOIUrl":"https://doi.org/10.1089/027245701300060463","url":null,"abstract":"Human arginase was purified from liver and two monoclonal antibodies (MAbs), HA1 and HA2, were produced by fusion of spleen cells from an arginase-immunized BALB/c mouse and the NS-1 myeloma cell line. Both MAbs were of the IgG3 subclass and contained the kappa light chain. HA1 inhibited arginase activity, suggesting that it binds to the arginase catalytic site. HA1 and a horseradish peroxidase-conjugated polyclonal rabbit anti-human arginase antibody were used to develop a sandwich enzyme-linked immunoadsorbent assay (ELISA) for the quantification of human arginase, which can be used in the 1 to 300 ng/mL range. Because of its sensitivity and specificity, this MAb can be successfully applied to the ELISA quantification of arginase in serum and culture supernatants.","PeriodicalId":55044,"journal":{"name":"Hybridoma","volume":"20 1 1","pages":"53-7"},"PeriodicalIF":0.0,"publicationDate":"2001-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/027245701300060463","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60498315","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 : 2001-02-01DOI: 10.1089/027245701300060346
F. Yücel, B. Çirakoğlu, Z. Mustafaeva, M. Mustafaev
Cu2+-mediated complex formation between copolymers of acrylic acid with N-isopropyl-acyrlamide (CP1) and negatively charged covalent conjugate of bovine serum albumin with progesterone (BSA.P) was studied in neutral water in the presence of Cu2+. It was shown that under conditions where CP and BSA.P are negatively charged and incapable of binding to one another, the divalent Cu2+ act as "fasteners" promoting the formation of relatively stable water-soluble ternary polycomplexes. The immunogenic properties of ternary mixtures BSA.P-Cu2+-CP1 and BSA.P+IFA were investigated and the production of monoclonal antibodies (MAbs) against progesterone hormone was analyzed. Fusion following the two different immunization procedures resulted in the growth of comparable numbers of progesterone-specific MAbs with apparently similar antigen affinities. Thus, immunizations using antigens in BSA.P-Cu2+-CP1 appear to provide an efficient alternative to incomplete Freund's adjuvant.
在中性水中研究了Cu2+介导的丙烯酸- n -异丙基丙烯酰胺共聚物(CP1)和带负电荷的牛血清白蛋白-黄体酮共价物(BSA.P)的络合物形成。结果表明，在CP和BSA。P带负电荷，不能相互结合，二价Cu2+作为“紧固件”促进形成相对稳定的水溶性三元多配合物。牛血清白蛋白三元混合物的免疫原性。P-Cu2+-CP1和BSA。对P+IFA进行了研究，并分析了抗孕激素单克隆抗体(mab)的产生。两种不同免疫程序的融合导致具有明显相似抗原亲和力的黄体酮特异性单克隆抗体数量相当。因此，利用牛血清白蛋白抗原进行免疫。P-Cu2+-CP1似乎是不完全弗氏佐剂的有效替代。
{"title":"Immune response to progesterone immobilized on Cu2+-induced amphifilic polyelectrolyte-protein complex: antigen specificity and affinity of hybridoma clones.","authors":"F. Yücel, B. Çirakoğlu, Z. Mustafaeva, M. Mustafaev","doi":"10.1089/027245701300060346","DOIUrl":"https://doi.org/10.1089/027245701300060346","url":null,"abstract":"Cu2+-mediated complex formation between copolymers of acrylic acid with N-isopropyl-acyrlamide (CP1) and negatively charged covalent conjugate of bovine serum albumin with progesterone (BSA.P) was studied in neutral water in the presence of Cu2+. It was shown that under conditions where CP and BSA.P are negatively charged and incapable of binding to one another, the divalent Cu2+ act as \"fasteners\" promoting the formation of relatively stable water-soluble ternary polycomplexes. The immunogenic properties of ternary mixtures BSA.P-Cu2+-CP1 and BSA.P+IFA were investigated and the production of monoclonal antibodies (MAbs) against progesterone hormone was analyzed. Fusion following the two different immunization procedures resulted in the growth of comparable numbers of progesterone-specific MAbs with apparently similar antigen affinities. Thus, immunizations using antigens in BSA.P-Cu2+-CP1 appear to provide an efficient alternative to incomplete Freund's adjuvant.","PeriodicalId":55044,"journal":{"name":"Hybridoma","volume":"20 1 1","pages":"11-5"},"PeriodicalIF":0.0,"publicationDate":"2001-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/027245701300060346","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60498053","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 : 2001-02-01DOI: 10.1089/027245701300060409
M. Pupo-Antúnez, R. Rodríguez, M. Álvarez, N. Amin, H. Rodríguez, A. Otero, G. Guzmán
A mouse monoclonal antibody (MAb, 4B6) was able to recognize dengue virus type 4 envelope (E) protein both as a recombinant protein in Pichia pastoris and when it was present in infected brains of suckling mice. 4B6 was characterized by enzyme-linked immunoadsorbent assay (ELISA), hemaglutination inhibition, neutralization, and immunoblot. The MAb was isotyped as IgG2a. It was serotype 4 specific and it inhibited hemaglutination and neutralized homologous virus. It did not enhance infection of P338D1 cells by dengue type 4 virus strain H-241 strain. This MAb was reactive with recombinant E protein and dengue 4 virus, as revealed by Western blot. In vivo, MAb 4B6 conferred passive protection in mice challenged with homologous virus. Currently, this MAb is being used to purify recombinant E protein for further studies.
{"title":"Development of a monoclonal antibody specific to a recombinant envelope protein from dengue virus type 4 expressed in Pichia pastoris.","authors":"M. Pupo-Antúnez, R. Rodríguez, M. Álvarez, N. Amin, H. Rodríguez, A. Otero, G. Guzmán","doi":"10.1089/027245701300060409","DOIUrl":"https://doi.org/10.1089/027245701300060409","url":null,"abstract":"A mouse monoclonal antibody (MAb, 4B6) was able to recognize dengue virus type 4 envelope (E) protein both as a recombinant protein in Pichia pastoris and when it was present in infected brains of suckling mice. 4B6 was characterized by enzyme-linked immunoadsorbent assay (ELISA), hemaglutination inhibition, neutralization, and immunoblot. The MAb was isotyped as IgG2a. It was serotype 4 specific and it inhibited hemaglutination and neutralized homologous virus. It did not enhance infection of P338D1 cells by dengue type 4 virus strain H-241 strain. This MAb was reactive with recombinant E protein and dengue 4 virus, as revealed by Western blot. In vivo, MAb 4B6 conferred passive protection in mice challenged with homologous virus. Currently, this MAb is being used to purify recombinant E protein for further studies.","PeriodicalId":55044,"journal":{"name":"Hybridoma","volume":"20 1 1","pages":"35-41"},"PeriodicalIF":0.0,"publicationDate":"2001-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/027245701300060409","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60498409","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 : 2001-02-01DOI: 10.1089/027245701300060382
F. Zhang, X. Shao, H. Li, J. G. Robison, B. K. Murray, K. O’Neill
Previous research has shown that thymidine kinase 1 (TK1), a nucleotide salvage pathway enzyme, is an accurate prognostic and diagnostic tumor marker. However, the current radioisotope assay for TK1 is cumbersome and has hampered the clinical application of this diagnostic technique in cancer management. To overcome the problems of the current radioisotope assay, we have produced monoclonal antibodies (MAbs) using purified TK1 from Raji cell extract. Production and confirmation of their specificity was confirmed using Western blot, immunohistochemical staining, TK1 activity inhibition assays, and enzyme-linked immunoadsorbent assay (ELISA) techniques. Thus, in the future, these antibodies may aid in the early detection of cancer and more accurate prognosis, as well as allowing for an increased ability to study the function of TK1 in basic cellular processes.
{"title":"A monoclonal antibody specific for human thymidine kinase 1.","authors":"F. Zhang, X. Shao, H. Li, J. G. Robison, B. K. Murray, K. O’Neill","doi":"10.1089/027245701300060382","DOIUrl":"https://doi.org/10.1089/027245701300060382","url":null,"abstract":"Previous research has shown that thymidine kinase 1 (TK1), a nucleotide salvage pathway enzyme, is an accurate prognostic and diagnostic tumor marker. However, the current radioisotope assay for TK1 is cumbersome and has hampered the clinical application of this diagnostic technique in cancer management. To overcome the problems of the current radioisotope assay, we have produced monoclonal antibodies (MAbs) using purified TK1 from Raji cell extract. Production and confirmation of their specificity was confirmed using Western blot, immunohistochemical staining, TK1 activity inhibition assays, and enzyme-linked immunoadsorbent assay (ELISA) techniques. Thus, in the future, these antibodies may aid in the early detection of cancer and more accurate prognosis, as well as allowing for an increased ability to study the function of TK1 in basic cellular processes.","PeriodicalId":55044,"journal":{"name":"Hybridoma","volume":"20 1 1","pages":"25-34"},"PeriodicalIF":0.0,"publicationDate":"2001-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/027245701300060382","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60498561","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 : 2001-02-01DOI: 10.1089/027245701300060364
J. H. Park, S. Na, H. H. Lee, Y. J. Lee, K. L. Kim
The 15-meric S-tag is a truncated form of the S-peptide, which builds together with the 103 amino acid large S-protein the whole ribonuclease S-protein. Its small size and excessive solubility have made the S-tag an excellent fusion partner in the production of recombinant proteins, and a large variety of applications have been reported using the S-tag as a carrier. While S-tagged proteins were mostly detected and analyzed so far by use of their affinity to S-proteins, monoclonal antibodies (MAbs) for this tag have been not available. The generation of antibodies specific for S-tagged proteins is expected to broaden the range of applications of such S-tag fused recombinant proteins, and in this context, a novel MAb termed ATOM-2 was generated that specifically binds S-tagged proteins, which have been expressed using pET-vectors. Antigen specificity of ATOM-2 was confirmed in Western blot and enzyme-linked immunoadsorbent assay analysis, and using a series of amino acid deletion mutants, the binding epitope of ATOM-2 was precisely mapped. This showed that ATOM-2 recognizes the C-terminal part of the 15-meric S-tag in context with a few residues of vector encoded sequences. The core sequence for ATOM-2 binding epitope is "His-Met-Asp-Ser-Pro-Asp-Leu-Gly-Thr," which is present in all pET-expression vectors encoding S-tag fusion proteins. Because the ATOM-2 binding region does not overlap with the S-protein binding sequence, a convenient tool is provided for the simultaneous or alternative detection, purification, and analysis of recombinant S-tagged proteins to conventional S-proteins.
{"title":"Detection of pET-vector encoded, recombinant S-tagged proteins using the monoclonal antibody ATOM-2.","authors":"J. H. Park, S. Na, H. H. Lee, Y. J. Lee, K. L. Kim","doi":"10.1089/027245701300060364","DOIUrl":"https://doi.org/10.1089/027245701300060364","url":null,"abstract":"The 15-meric S-tag is a truncated form of the S-peptide, which builds together with the 103 amino acid large S-protein the whole ribonuclease S-protein. Its small size and excessive solubility have made the S-tag an excellent fusion partner in the production of recombinant proteins, and a large variety of applications have been reported using the S-tag as a carrier. While S-tagged proteins were mostly detected and analyzed so far by use of their affinity to S-proteins, monoclonal antibodies (MAbs) for this tag have been not available. The generation of antibodies specific for S-tagged proteins is expected to broaden the range of applications of such S-tag fused recombinant proteins, and in this context, a novel MAb termed ATOM-2 was generated that specifically binds S-tagged proteins, which have been expressed using pET-vectors. Antigen specificity of ATOM-2 was confirmed in Western blot and enzyme-linked immunoadsorbent assay analysis, and using a series of amino acid deletion mutants, the binding epitope of ATOM-2 was precisely mapped. This showed that ATOM-2 recognizes the C-terminal part of the 15-meric S-tag in context with a few residues of vector encoded sequences. The core sequence for ATOM-2 binding epitope is \"His-Met-Asp-Ser-Pro-Asp-Leu-Gly-Thr,\" which is present in all pET-expression vectors encoding S-tag fusion proteins. Because the ATOM-2 binding region does not overlap with the S-protein binding sequence, a convenient tool is provided for the simultaneous or alternative detection, purification, and analysis of recombinant S-tagged proteins to conventional S-proteins.","PeriodicalId":55044,"journal":{"name":"Hybridoma","volume":"20 1 1","pages":"17-23"},"PeriodicalIF":0.0,"publicationDate":"2001-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/027245701300060364","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60497910","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 : 2001-02-01DOI: 10.1089/027245701300060328
M. C. Long, Kelly E.J. Marshall, Brian J. Kearney, George V. Ludwig, Jonathan P. Wong, L. Nagata
A novel recombinant single-chain fragment variable (scFv) antibody against western equine encephalitis (WEE) virus has been previously constructed and partially characterized. The RS10B5huFc antibody was made by fusing an anti-WEE scFv to a human heavy-chain IgG1 constant region. The RS10B5huFc antibody was functional in binding to WEE virus in enzyme-linked immunosorbent assays (ELISAs), and the Fc domain of the antibody was capable of effector functions, such as binding to protein G and human complement. In this study, the RS10B5huFc antibody was further characterized by BIAcore analyses and was found to possess a binding affinity to a WEE virus epitope (K[D] = 9.14 x 10(-6) M), 4.5-fold lower than its parental mouse monoclonal antibody (MAb) 10B5 E7E2 (K[D] = 2 x 10(-6) M). No cross-reactivity was found between the RS10B5huFc antibody and three other alphaviruses (Sindbis virus [SIN], Venezuelan equine encephalitis [VEE] virus, and eastern equine encephalitis [EEE] virus). Pharmacokinetics studies showed that the RS10B5huFc antibody (free and encapsulated) was found to be retained in the lungs of mice for greater than 48 h when administered intranasally. In contrast, when administered intramuscularly to mice, the RS10B5huFc antibody was not detected in the lungs and only found in the liver and kidneys.
{"title":"Pharmacokinetics study of a novel chimeric single-chain variable fragment antibody against western equine encephalitis virus.","authors":"M. C. Long, Kelly E.J. Marshall, Brian J. Kearney, George V. Ludwig, Jonathan P. Wong, L. Nagata","doi":"10.1089/027245701300060328","DOIUrl":"https://doi.org/10.1089/027245701300060328","url":null,"abstract":"A novel recombinant single-chain fragment variable (scFv) antibody against western equine encephalitis (WEE) virus has been previously constructed and partially characterized. The RS10B5huFc antibody was made by fusing an anti-WEE scFv to a human heavy-chain IgG1 constant region. The RS10B5huFc antibody was functional in binding to WEE virus in enzyme-linked immunosorbent assays (ELISAs), and the Fc domain of the antibody was capable of effector functions, such as binding to protein G and human complement. In this study, the RS10B5huFc antibody was further characterized by BIAcore analyses and was found to possess a binding affinity to a WEE virus epitope (K[D] = 9.14 x 10(-6) M), 4.5-fold lower than its parental mouse monoclonal antibody (MAb) 10B5 E7E2 (K[D] = 2 x 10(-6) M). No cross-reactivity was found between the RS10B5huFc antibody and three other alphaviruses (Sindbis virus [SIN], Venezuelan equine encephalitis [VEE] virus, and eastern equine encephalitis [EEE] virus). Pharmacokinetics studies showed that the RS10B5huFc antibody (free and encapsulated) was found to be retained in the lungs of mice for greater than 48 h when administered intranasally. In contrast, when administered intramuscularly to mice, the RS10B5huFc antibody was not detected in the lungs and only found in the liver and kidneys.","PeriodicalId":55044,"journal":{"name":"Hybridoma","volume":"20 1 1","pages":"1-10"},"PeriodicalIF":0.0,"publicationDate":"2001-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/027245701300060328","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60497935","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}
京公网安备 11010802042870号
京ICP备2023020795号-1
扫码关注我们
求助内容：
应助结果提醒方式：