[Preparation of mouse monoclonal antibody against human vasorin (VASN) protein by high-efficacy electrofusion-based protocol].

Objective To prepare and identify mouse monoclonal antibodies against human vasorin (VASN) protein using electrofusion method. Methods The mice were immunized with human recombinant protein VASN-His, and then the cells were fused by electrofusion apparatus. Indirect ELISA was used to screen the positive hybridoma cells which could bind natural protein VASN. The titer and affinities of the antibodies were detected by ELISA, and Western blotting was used to determine whether the antibody could recognize VASN protein in HepG2 cells. Results The fusion rate reached 0.31% when the ratio of spleen cells and Sp2/0 myeloma cells was 2:1, the alternating electric field intensity was 50 V, 2 MHz for 20 seconds, and the direct current pulse intensity was 500 V for 0.5 second. Two mouse anti-human VASN monoclonal antibodies (4H1and 8B9) were obtained, with the highest titer of 1:256 000 and the highest affinity constant (Ka) of 4.9×106 L/mol. Western blotting showed that both monoclonal antibodies could specifically recognize VASN in HepG2 cells. Conclusion Two mouse anti-human VASN monoclonal antibodies have been successfully prepared by the cell electrofusion method.