ABSTRACT
Objective:Generation and identification of hybridoma to produce monoclonal antibody against MICA/B. Methods:SP2/0 cells were fused with murine splenocyte immunized with recombinant protein rMICA?012 to get hybridoma cell lines. The titer of the monoclonal antibody produced by 9B10 cell line was determined by ELISA and its specificity was tested by Western blot,Luminex mutiplex microsphere-based immunoassay and immunofluorescence assay. Results:Six hybridoma cell lines were selected by ELISA screening test. The minimum reaction concentration of mAb 9B10 was 0. 02 ng/μl,and the specificity of mAb 9B10 was determinated by Western blot,Luminex mutiplex microsphere-based immunoassay and immunofluorescence. Conclusion:The monoclonal antibody 9B10 was available to apply for the detection of MICA and MICB expression.
ABSTRACT
Objective:Generation and identification of hybridoma to produce monoclonal antibody against MICA/B. Methods:SP2/0 cells were fused with murine splenocyte immunized with recombinant protein rMICA?012 to get hybridoma cell lines. The titer of the monoclonal antibody produced by 9B10 cell line was determined by ELISA and its specificity was tested by Western blot,Luminex mutiplex microsphere-based immunoassay and immunofluorescence assay. Results:Six hybridoma cell lines were selected by ELISA screening test. The minimum reaction concentration of mAb 9B10 was 0. 02 ng/μl,and the specificity of mAb 9B10 was determinated by Western blot,Luminex mutiplex microsphere-based immunoassay and immunofluorescence. Conclusion:The monoclonal antibody 9B10 was available to apply for the detection of MICA and MICB expression.
ABSTRACT
Objective To construct a prokaryotic expression system for major histocompatibility complex class Ⅰ chain-related gene B ( MICB) and to establish an ELISA method for the detection of anti-MICB antibodies in patients with kidney transplantation. Methods The MICB cDNA fragments were ob-tained by RT-PCR with a pair of specific primers. The MICB cDNA and the prokaryotic expression vector pET-28a were digested by two restriction enzymes and ligated to construct the recombinant expression plas-mid pET-28a-MICB. The transformed E. coli BL21 DE3 strains carrying recombinant expression plasmid were induced by IPTG to express MICB protein. The expressed recombinant proteins were identified by Western blot assay and purified by Ni-NTA Spin column. The purified proteins were coupled to ELISA for the detection of anti-MICB antibodies in patients with kidney transplantation. Results Three common MICB fragments contained the exons 2 and 3 were obtained. The recombinant proteins were expressed in E. coli BL21 DE3 strains carrying pET-28a-MICB and successfully purified by the Ni-NTA Spin column. Results of the Western blot assay confirmed that the obtained proteins were the target proteins. The ELISA method was successfully established and used for the detection of anti-MICB antibodies in 24 patients with kidney trans-plantation. The absorbance values indicated that the sensitivities of three recombinant MICB proteins were different. Conclusion The expression system for MICB gene was successfully constructed. The established ELISA for the detection of anti-MICB antibodies would pave the way for further investigation on the correla-tions between MICB protein and transplantation immunity.
ABSTRACT
Objective: To analyze the expression of HLA class Ⅰ molecules and MHC classⅠ related chain A and B (MICA/MICB) in human nasopharyngeal carcinoma cell line (CNE2) and multi-drug resistant nasopharyngeal carcinoma cell line (CNE2/ DDP), and to assess their influence on NK cell-mediated lysis.Methods: Expression of HLA classⅠ molecules and MICA/MICB on the surface of CNE2 and CNE2/DDP cell lines was analyzed by flow cytometry. Cytotoxicity of NK cells (isolated from 3 healthy persons) against CNE2 and CNE2/DDP cells were detected by LDH releasing assay at different effect-to-target cell ratios (E∶T). In blocking experiments, anti-MHC class Ⅰ monoclonal antibody (mAb) (W6/32, a pan anti-HLA class Ⅰ antibody) and anti-MHC class I chain related molecules mAb (BAMO-1, specificly against MICA and MICB) were added to the target cells at a E∶T ratio of 10∶1. Results:It was found that the expression of HLA class Ⅰ molecules and MICA/MICB on CNE2 was higher than that on CNE2/DDP(P