Studies On The Preparation Of Cyclosporin A Monoclonal Antibody

OBJECTIVE: To study the methodology of immunogen synthesis and the monoclonal antibody preparation of Cyclosporine A.METHODS: With the stirring at room temperature and dehydrolyzing agent 1-ethyl-3-(3'-dimethylaminopropyl) carbodiimide (EDCI ), the antigen of cyclosporine A(CsA-BBa-PL) was synthesized through the reaction between the conjugate of cyclosporine A and 4-benzoulbenzoic (CsA-BBa) and poly-L-lysine(PL), the weight proportion was 3.4 to 1. The products were isolated and purified by dialysis, the specification of the dialysis-membrane was 10,000. It was identified by UV spectra within 190 to 300 nm. The specificity of the antigen of CsA was identified by enzyme-linked immunosorbent assay (ELISA), the concentration of antigen used for the identification was 5μg/ml. The female BALB/C mice, seven weeks old, were immunized with the antigen of cyclosporine A, the blood serum potencies were measured by indirect enzyme-linked immunosorbent assay. With the help of fusogenic agent polyethylene glycol (PEG), the concentration of which is 50%, the spleen cells, which had higher blood serum potencies, were fused with SP2/0 myeloma cells (the ratio is ten to one). Then the fused cells were cultured in the cell culture plates which had owned feeder layer cells for 24 hours. The selected solution HAT was used to culture the fusion cells so as to select hybridoma cells. The hybridoma cells was selected and identified by ELISA.RESULTS: The UV spectra peaks of CsA-BBa and PL and the antigen of CsA were 212nm, 203nm, 209.5nm respectively, compared to the materials used to synthesize antigen of cyclosporine A, the UV spectra of the antigen changed obviously, which proved that the conjugate was formed through the reaction. The positive to negative ratio of the specificity identification was 23.22. The blood serum potencies of the immune mice were 3.35, 3.43, 3.37, 3.31, and 3.38 respectively. The blank control and negative control and positive control were 0.080, 0.42 and 3.43 respectively. The potencies of the mice were obviously higher than the blank control and negative control, which were close to the positive control. The selected hybridoma cells were able to secrete monoclonal antibodies of cyclosporine A stably. CONCLUSIONS: The antigen of cyclosporine A was able to synthetized by this reaction. The antigen was able to induce the immunological reaction of mice and made the body form the antibodies accordingly. The hybridoma technique was able to prepare monoclonal antibody of Cyclosporine A successfully.