[Preparation and characterization of monoclonal antibodies against CMTM7].

OBJECTIVE:To obtain monoclonal antibodies against CMTM7 (CKLF-like MARVEL transmembrane domain containing 7) for further study of the structure and biological function of CMTM7. METHODS:Three polypeptides were synthesized based on the bioinformatics analysis of the CMTM7 and coupled with keyhole limpet hemocyanin (KLH). Balb/c mice were immunized with these mixed CMTM7 polypeptides. Hybridomas were generated by the fusion of the spleenocytes from these mice with Sp2/0 myeloma cells. Resulting hybridomas producing anti-CMTM7 antibodies were screened by enzymejlinked immunosorbent assay (ELISA). The specificities of these monoclonal antibodies were determined by Western blot, immunofluorescecence and immunocytochemistry (ICC). RESULTS:Two hybridoma cell lines (MC9 and 2C9) stable in secreting anti-CMTM7 monoclonal antibodies (MAbs) were generated. Both of them produced immunoglobulin G1 (IgG1) against CMTM7. 2C9 and MC9 recognized a region between amino acid residues 19-44 and 163-175 of CMTM7, respectively. MC9 antibody could be used for Western blot, immunofluorescecence and immunocytochmistry assay. However, 2C9 antibody could be only used for Western blot. Data obtained from immunofluorescence and ICC indicated that CMTM7 protein expression was upregulated in the early stage of lymphocyte activation treated with phytohemagglutinin (PHA). CONCLUSION:Monoclonal antibodies of high specificity against CMTM7 have been successfully generated, which could be utilized as a useful reagent for the analysis of biochemical, structural, and functional properties of CMTM7.