Preparation, Charactorization And Preliminary Application Of Monoclonal Antibody Against Human Endothelial Cell Specific Molecule-1

Objective To establish the monoclonal antibodys against human ESM-1, study its characterization and analyze ESM-1 expression of tumor tissues in human. Methods Using hybridoma techique, we prepared human ESM-1 protein as antigen. The hybridoma cells were obtained by fusing spleen cells of BALB,C mice to myeloma cells SP2,0 and culured in the HAT selective medium. The positive clones were screened by indirect ELISA and the limited dilution methods were used to select anti-human ESM-1 McAbs. We finally got the hybridoma strain which secreted antibody against ESM-1. McAb class and subclass were identified by Immunotype?kit. The Westen blotting method was analysed the specificity of McAb. We checked the expression of ESM-1 in endothelial cell by immune fluorescence. The relative affinity of antibody was demonstrated by indirect ELISA. We analysised ESM-1 expression in normal human kidney tissues and the renal cell carcinomas by immunohistochemistry. Results Seven stains of hybridoma cells secreting anti-human ESM-1 mAbs were obtained. The McAb 3A7 secreted by one of them has the highest speciality. Its agglutinaton titer was 1: 6000 and its subclass was IgG2b. The affinity contents of the 3A7 was 1.67 104L,mol. The chromosomes of hybridoma cell were 100 or so. The size of ESM-1 was demonstrated by Westen blot on endothelial cell lysates and supernatants. The location of ESM-1 was major in the cytoplasm. The positive expression of ESM-1 was higher in the renal cell carcinomas. Conclusion The antibodies against human ESM-1 has been prepared successfully. The results obtained provides that hybridoma cell has high sensitivity and high specificity. These antibodies can be used in immunohistochemistry study of the normal and tumor tissues in human, and may be beneficial to cancer diagnosis, prognosis and cancer theraoy.