Preparation and identification of monoclonal antibodies against alanine aminotransferase

Objective To obtain the hybridoma secreting ALT monoclonal antibodies,and to prepare monoclonal antibodies against ALT and to identify the ALT monoclonal antibodies.Methods Five Balb/c mice were immunized subcutaneously four times with 20 μg of ALT 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(5∶1)and cultured selectively with HAT and HT.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 antibodies in the ascites were purified by the method of acetic acid amino sulphate precipitation.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(7D1) secreting monoclonal antibodies against ALT was obtained.The chromosome number of 7D1 hybridoma cells varied from 95 to106.The subtype of 7D1 monoclonal antibodies was IgG1.The titer of ascites and supernatant are 1∶104 and 1∶106,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.