Preparation And Identification Of Monoclonal Antibodies Against Mycobaterium Bovis

Two stable hybridoma cell lines secreting monoclonal antibodies (McAbs) against Mycobaterium bovis,were developed by fusion between the murine myeloma cell SP2/0 and spleen cells of BALB/C mice immunized with Mycobaterium bovis . Hybridomas were screened by Enzyme-linked Immunosorbent Assays(ELISA). Hybridomas whose supernatants show stronger positive were subcloned by limited dilution methods 3-4times,until the positive rate come up to 100%.The two McAbs designated as 5D₄ and 4C₃.Preliminary characterizations of the two hybridoma cell lines were identificated.The results of the ELISA showed subtypes of the two McAbs belong to class IgG₁.The number of the chromosome of the two hybridoma cell lines is calculated that average number of the 5D₄ is 103 and the 4C₃ is 10⁵.Indirect enzyme-linked immunosorbent assays(ELISA) were used to screen those hybridomas producing antibodies.The ELISA titers of the ascites were 10-4 to 10-5while the ELISA titers of the 5D₄ supernatants is 1:2 560 and 4C₃ is 1:1 280 . Antigen dots and specific tests of the two McAbs were analysed.The two McAbs are against different epitopes of Mycobaterium bovis .The results of the SDS-PAGE showed molecular weight of heavy chains of the two McAbs is 50KD roughly and the light chain of 5D₄ is 30KD while 4C₃ is 25KD approximately. The OD₄₅₀ values of the two McAbs in 4 times cloning culture were ascending and the ELISA titers of the ascites were 10-4 to 10-5,which showed the two hybridoma cell lines are stable .Interspecies and exterspecies reactivity of the two McAbs were analysed .Cross reactivity of the two McAbs with other antigens were not observed (Escherichia coli,Salmonella,StapHylococcus aureus),except with Mycobacterium tuberculosis. The two McAbs were applied to prepare the rapid test papers by colloidal gold immunochromato- graphy technology which the sensitivity is 10⁶CFU/mL to detect Mycobaterium bovis. The test papers were positive against Mycobaterium bovis and Mycobacterium tuberculosis, negative against Escherichia coli,Salmonella, StapHylococcus aureus and so on.These results showed colloidal gold immunochromatography technology has advantages of stronger spcificity,better stability and easier manipulation .The characterization of the two McAbs and the test papers and their applications to the serodiagnosis of Mycobaterium bovis need further evaluation.