[Preparation and characterization of monoclonal antibodies against human neuropilin 1 b1b2 domain (HuNRP1 b )].

Objective To prepare recombinant protein of human neuropilin 1 b1b2 domain (HuNRP1b ) and monoclonal antibodies (mAbs) against the recombinant HuNRP1b (rHuNRP1b ). Methods The coding sequence of HuNRP1b was amplified and cloned into vector pET22b to construct recombinant plasmid pET-HuNRP1b . After analyzed by restriction enzyme digestion and DNA sequencing, pET-HuNRP1b was transformed into Escherichia coli and induced to express rHuNRP1b with histidine tag (His-HuNRP1b ), which was identified by SDS-PAGE analysis. Then His-HuNRP1b was used as immunogen to immune BALB/c mice. After the fusion of spleen cells and Sp2/0 cells, the positive hybridoma cells were screened by indirect ELISA that was established with recombinant HuNRP1b with a trigger factor tag (TF-HuNRP1b ). The specificity of mAbs against HuNRP1b was identified by ELISA and Western blotting. The ability of mAbs to bind native HuNRP1 was revealed by indirect fluorescence assay (IFA). Results His-HuNRP1b was expressed and obtained. Western blotting showed that mAbs 1A10, 6A2 against rHuNRP1b could bind His-HuNRP1b with the band of 34 kDa and TF-HuNRP1b with 89-kDa band, respectively, but not the proteins from bacteria with empty vectors. IFA revealed that the mAbs could bind native HuNRP1 expressed on breast cancer cell line MDA-MB-231. Conclusion The mAbs specific to rHuNRP1b were generated successfully.