Abstract:The objectiveof this study is to establish direct and indirect competitive enzyme-linked immunoassay for detection of melamine.For this,BALB/c mice were immunized with MEL-BSA,after a fusion of mouse spleen cells with SP2/0 cells,the hybridoma cells secreting monoclonal antibody against MEL were generated.MEL-mAb was produced by ascites in mice.Melamine was conjugated with HRP,and the conjugates activity was identified.MEL-OVA and MEL mAb were coated in wells respectively,standard melamine were used as inhibitors,direct and indirect competitive ELISA were compared in sensitivity,work range and testing time.Four hybridism cell strains were obtained,which could stably secrete mAbs against melamine.One of them,C4 strain,had a titers of 1∶104 at least in ascites by indirect ELISA and a high affinity constant(Ka) with 2.92×108L/mol.IC50 of direct and indirect competitive ELISA methods were 3.12 μg/L and 56.88 μg/L respectively,and their work ranges were 0.1~1000 μg/L and 0.05~10 μg/L,respectively.Both direct and indirect competitive ELISA methods can be used for the detection of melamine,while the latter is more sensitive and suitable for further research.
