A Study On The Preparation And Identification Of Monoclonal Antibodies Against Alanine Aminotransferase

BACKGRONND: Alanine aminotransferase is one of important catalytic enzymes in human serum and play a aminotransference role during the process of protein metabolization. Alanine aminotransferase mainly esits in hepatocellular plasma, when hepatic injury is occurred , a great deal of Alanine aminotransferase releases into blood, so ALT is a sensitive marker for diagnosis of hepatic injury. To reduce the incidence of post-transfusion hepatitis, The Ministry of Health of our country has required that serum alanine aminotransferase activity was one of the tests that must be detected to blood donors before donation. In China, approximately 6% to 8% of all blood donations will be discarded owing to an elevated alanine aminotransferase level. Facing these problem, It was essential for us to eastablish a sensitive, accurate and convenient method for detection of serum ALT. Nowadays velocity method and the chemistry dry powder method are widely used during the screening for blood donors . Both of the methods are time-consumming, with a low accuracy and are easily interfered by environment condition. The colloidal gold immunochromatographic assay is a specific, sensitive, simple and rapid assay which don't need a special equipments and easily observe result. For all these reasons, we try to study on the gold immunochromatographic assay for the detection of ALT in human serum. In the study, the preparation of monoclonal antibodies with high specificity was the very important step.AIMS : To obtain the hybridoma secreting ALT monoclonal antibodies ,to prepare monoclonal antibodies against ALT and to identify the ALT monoclonal antibodies. METHODS: Balb/c mice were immunized subcutaneously four times with 20μg of ALT purified from pig heart in 50% Freund's adjuvant at a interval of 2 weeks, and titers in immunized mice sera were determined by indirect ELISA . The splenic lymphocytes from the immunized mouse were fused to myeloma Sp2/0 cells and cultured selectively with HAT and HT. Using human serum with high activity of ALT as detecting antigen, Each supernatant was screened by its reactivity to ALT. One of antibody-producing wells was selected for further use through following successive subclonings by limiting dilution method. The selected hybridoma cells were proliferated and injected into Balb/c mice for generation of ascites. The The titers and affinities of ascites and were determined by indirect ELISA. The chromosome of hybridoma cells was analyzed by the method of Colchicine treatment. The subtype of the antibodies was identified by SBA Clontyping System/HRP. The obtained ALT monoclonal antidodies were analyzed by SDS-PAGE. The specificity of the monoclonal antibody was assessed by western blotting. The application was studied by a Sandwich ELISA which was established after the ALT monoclonal antibodies were labeled with Horseradish peroxidase.RESULTS: One hybridoma cell line(7Dl) secreting monoclonal antibodies against ALT was obtained. The chromosome number of 7D1 hybridoma cells varied from 95 to 106. The subtype of 7D1 monoclonal antibodies is IgG1. The titer of ascites and supernatant are 1:10~4 and 1:10~6 respectively. SDS-PAGE of ALT monoclonal antibodies showed that heavy and light chain with a relative molecular weight of 55KDa and 23KDa respectively. The purified monoclonal antibodies showed good affinity and specificity against ALT in Western blotting. The biochemistry value of ALT was consistent with the result of Sandwich ELISA.CONCLUSION: In present study, one strain hybridoma cell was obtained, which could stably secret ALT monoclonal antibodies. The antibodies that prepared showed good affinity and could recognize ALT specially without cross-reactions with other proteins in human serum, which provide a potential value for establishing the colloidal gold immunochromatography assay .