Preparation And Preliminary Application Of Monoclonal Antibodies Against Denatured Insulin

Objective:To prepare and identify monoclonal antibodies against pure denatured insulin and establish ELISA to measure the denatured insulin level in DM patient.Methods:BALB/c mice were immunized subcutaneously with pure denatured insulin on its back. We use of the chessboard titration method to determined the working concentrations of coated antigen.Indirect ELISA was used to detect antibody titers in the serum of immunized mice, and the mouse with high antibody titer was selected used for monoclonal antibody preparation. The mouse spleen was removed under the sterile condition, and the spleen cells were dispersed through the strainer to get a single cell suspension. Hybridoma cell lines were obtained by fusing immunized mouse spleen cells with myeloma SP2/0cells and cultured selectively with HAT and HT. Each supernatant was screened by its reactivity to denatured insulin.10positive hybridoma with the capability of secreting monoclonal antibody against denatured insulin were screened by indirect ELISA, and were subjected to clone and subclone by limiting dilution method. Incrementation culture was used to preparation of McAbs. The selected hybridoma cells were proliferated and injected into BALB/c mice for the generation of ascites. Each ascites was detected for the titers of the denatured insulin monoclonal antibodies. The antibodies in the ascites were purified through Saturated ammonium sulfate. The obtained monoclonal antibody were analyzed by SDS-PAGE. Ig classes and subclasses of the McAbs were detected by Mouse Monoclonal Antibody Isotyping kit. The titers of McAbs secreted by hybridoma before and after freeze were tested by indirect ELISA. The specificities of McAbs were determined by ELISA and Western blot analysis.Result:10μg/ml was selected as the working concentration of coated antigen. The work concentration of second-antibody was1:5000. we use the HAT medium to screen the hybridoma that can secrete the McAb against denatured insulin. finally, two lines of hybridoma cells were obtained after5rounds of cloning which can stably secrete the McAb against denatured insulin. We named them as INS1G9and INS3D7. Through Saturated ammonium sulfate, we purified the ascites and got pure antibodies. Both McAbs were IgGl. ELISA and Western blot analysis showed that they could recognize a target molecule with relative molecular mass of6KD. INS1G9and INS3D7secreted by two strains of hybridoma cells before and after freeze were1:8000and1:5000. Preliminary establishment double neo-confucianism in serum antibodies to clip detection insulin levels,Conclusion:Two McAbs, NS1G9and INS3D7against denatured insulin are prepared successfully, which provides valuable experiment foundation for measuring the denatured level in DM patient. We detect the denatured insulin level in DM patient and found type2diabetes serum degeneration insulin detection level is high, the difference between the normal group with a statistical significance.