Production and biological characteristics of monoclonal antibodies against recombinant human interleukin-12

Objective To produce hybridoma cell lines of monoclonal antibodies(McAb) against recombinant human interleukin-12(rhIL-12) by purified rhIL-12 and identify the specificity of anti-rhIL-12 McAb,and provide a valuable tool for the diagnose and treatment of tumor and immune associated disease .Methods BALB/C mice were immunized with purified rhIL-12p70 and hybridomas were generated with traditional technique.McAb were screened by ELISA with limited dilution and subcloning approach and purified with clutathione sepharose 4B affinity chromatography column.The subtype and titers in the ascites and specificity of McAb were identified by kit and Western blotting respectively.Results Three cell lines of hybridoma constantly secreted McAbs against rhIL-12p70.The chromosomal number of three strains were about 100 and cell lines of hybridoma had been confirmed.Two of them belonged to IgG1 and one of them belonged to IgG2a,Kappa isotypes.Ascites titers were 1∶5.9×106,1∶2.9×107, and 1∶3.6×107.The affinity were EM5EM6EV9 in proper order.Conclusion Three hybridoma cell strains are constructed successfully which stably secrete high-titer and high-specific McAb against rhIL-12.