| Objective To obtain stable expression of human thrombomodulin in CHO andto prepare monoclonal antibody(McAb) against human thrombomodulin , which helps establish a base for vascular endothelium injury.Methods (1)Recombinant expression plasmid pThr402 was transfected into CHOby lipofectamine and stable expressing hTM clonal cells lines were obtained after selecting with G418 . hTM expression in CHO was identified by flow cytometry and Western blotting. (2) Balb/c mice obtained active immunity by injecting CHO-TM into abdomen and McAb was screened by CELISA after normal cell hybridization, cell specificity, tissue specificity and recognizing antigen specificity of McAb were identified respectively by flow cytometry , immunochemical ABC staining and Western blotting. (3) McAb was applicated on Western blotting analyzing sTM levels of cardiovascular diseases.Results (1) 5 stable expressing hTM clonal cells lines were obtained. (2)Amurine McAb NH-1 was obtained and its titer in ascites was 2×10-5, and its content in ascites was 20mg/ml.McAb was typed by reaction with subclass-specific rabbit anti-mouse antibodies and were found to be IgGl. Flow...
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