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Analysis of TRAIL receptor expression using anti-TRAIL death receptor-5 monoclonal antibodies
作者姓名:Ma Y  Yang D  Chen Y
作者单位:[1]TongjiMedicalCollege,HuazhongUniversityofScienceandTechnology,Wuhan430030,China [2]DepartmentofPathologyandLaboratoryMedicine,SchoolofMedicineUniversityofPennsylvania,USA
基金项目:supported in part by grants HNIFOS 0321001800 and HNIFMSTS 2002-119.
摘    要:Objective To establish hybridomas that produce anti-death receptor-5 (DR5) monoclonal antibodies(mAbs) and check the surface expression of DR5 (sDR5) on cell lines.Methods The cDNA of human DR5 was cloned into pGAPZa. Recombinant Pichia pastoris clones generated via homologous recombination secreted high levels of sDR5. The sDR5 was purified using a nickel ion column. BALB/c mice were immunized with sDR5 and spleen cells were fused with the SP2/0-Ag 14. Monoclonal antibodies were tested by ELISA for their abilities binding to sDR5 and by flow cytometry for the reactivities to surface DR5 of Jurkat cells. Surface expression of the TRAIL receptor was determined by flow cytometric analysis measuring the binding of anti-DR5 mAb.Results Isotypes of mAbs were determined to be IgG1 and IgM, all of which were reactive to sDR5 as observed through ELISA. It was discovered using flow cytometry that only IgG was able to bind to DR5 on the plasma membrane of Jurkat cells, sDR5 was found to completely inhibit anti-DR5 mAb binding to Jurkat cells. Approximately 95% of Jurkat cells, 98% SW480, 99% U937, 100% U87,86% HCT116, 64% HL-60, 47% HeLa and 13% K562 cells express membrane DR5.Conclusions These results demonstrate that anti-DR5 mAb is able to specifically bind to DR5 and that DR5 is expressed at high levels on Jurkat, SW480, U87, U937 and HCT116 cell lines, and at medium levels on HL-60 and HeLa cell lines. The expression of DR5 on K562 cell line is low.

关 键 词:单克隆抗体  TRAIL  受体  细胞凋亡  抗死亡受体-5

Analysis of TRAIL receptor expression using anti-TRAIL death receptor-5 monoclonal antibodies
Ma Y,Yang D,Chen Y.Analysis of TRAIL receptor expression using anti-TRAIL death receptor-5 monoclonal antibodies[J].Chinese Medical Journal,2003,116(6):947-950.
Authors:Ma Yuanfang  Yang Dongliang  Chen Youmei
Institution:1. Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China;Department of Immunology, Medical College, Henan University, Kaifeng 475001, China
2. Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
3. Department of Pathology and Laboratory Medicine, School of Medicine University of Pennsylvania, USA
Abstract:OBJECTIVE: To establish hybridomas that produce anti-death receptor-5 (DR5) monoclonal antibodies (mAbs) and check the surface expression of DR5 (sDR5) on cell lines. METHODS: The cDNA of human DR5 was cloned into pGAPZalpha. Recombinant Pichia pastoris clones generated via homologous recombination secreted high levels of sDR5. The sDR5 was purified using a nickel ion column. BALB/c mice were immunized with sDR5 and spleen cells were fused with the SP2/0-Ag 14. Monoclonal antibodies were tested by ELISA for their abilities binding to sDR5 and by flow cytometry for the reactivities to surface DR5 of Jurkat cells. Surface expression of the TRAIL receptor was determined by flow cytometric analysis measuring the binding of anti-DR5 mAb. RESULTS: Isotypes of mAbs were determined to be IgG(1) and IgM, all of which were reactive to sDR5 as observed through ELISA. It was discovered using flow cytometry that only IgG was able to bind to DR5 on the plasma membrane of Jurkat cells. sDR5 was found to completely inhibit anti-DR5 mAb binding to Jurkat cells. Approximately 95% of Jurkat cells, 98% SW480, 99% U937, 100% U87, 86% HCT116, 64% HL-60, 47% HeLa and 13% K562 cells express membrane DR5. CONCLUSIONS: These results demonstrate that anti-DR5 mAb is able to specifically bind to DR5 and that DR5 is expressed at high levels on Jurkat, SW480, U87, U937 and HCT116 cell lines, and at medium levels on HL-60 and HeLa cell lines. The expression of DR5 on K562 cell line is low.
Keywords:TRAIL  TRAIL-R  apoptosis  death receptor-5  monoclonal antibody
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