Expression,purification and preparation of monoclonal antibody of HCA518 protein / 中国免疫学杂志

ABSTRACT

Objective; To express and purify HCA518 protein and prepare its monoclonal antibody ( McAb). Methods: The HCA518 protein was expressed with gene recombinant technique in prokaryotic system and purified with nickel chelate nitrilotriacetic acid(Ni-NTA) affinity chromatography column. Hybridoma cell lines that secreted anti-HCA518 McAb were established by cells fusion and screened by enzyme linked immunosorbent assay( ELISA). The specificity of anti-HCA518 McAb wa3 identified by Western blot assay. The HCA518 protein in tumor cells was stained by immunoflourescence assay. Results: Rcombinant HCA518 protein was expressed with a purity of 98%. Two hybridoma cell lines was selected and anti-HCA518 McAb was purified from mice ascites. The titers of anti HCA518 McAb in ascites were 1?10-4 and 5?10-4 respectively. The antibody belonged to IgG2b subtype and IgM. Anti-HCA518 McAb specifically reacted with recombinant HCA518 protein and tumor cells'nuclear protein (P100). The HCA518 protein was mainly located in cell nucleus. Conclusion: Stable hybridoma cell lines that secreted anti-HCA518 McAb have been established and anti HCA518 McAb was prepared with high specificity. It has important significance for detecting HCA518 protein in tumor tissues and determining malignant proliferation status of tumor cells and predicting its prognosis.