Preparation and identification of mouse anti-human B7-H6 monoclonal antibodies

Abstract

Objective To prepare mouse anti-human monoclonal antibodies against B7-H6 and to identify their biological characteristics. Methods The B7-H6 gene was cloned by RT-PCR from a human lung adenocarcinoma cell line (A549) and then subcloned into the eukaryote expression vector pCMV3 to construct the recombinant vector pCMV3-B7-H6. The recombinant vector pCMV3-B7-H6 that was verified with enzyme digestion and gene sequencing was transfected into NIH/3T3 cells by electroporation. BALB/c mice were immunized with the successfully transfected cells named 2H8 through intraperitoneal injection. The monoclonal antibodies against human B7-H6 with the advantages of high affinity and specificity were prepared by using hybridoma technology. Western blot assay and flow cytometry analysis were used to identify the specificity of prepared monoclonal antibodies. Results The recombinant eukaryotic expression vector encoding B7-H6 was successfully constructed. Two hybridoma clones that stably secreted monoclonal antibodies against B7-H6 were screened out by using flow cytometry analysis and the monoclonal antibodies secreted by them were belonged to IgG2a isotype. Specific reactions between B7-H6 and the secreted monoclonal antibodies were confirmed by Western blot assay and flow cytometry analysis. Conclusion The monoclonal antibodies which recognized B7-H6 specifically were prepared successfully. Key words: B7-H6; Eukaryotic expression; Monoclonal antibody; Specificity