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目的制备和筛选拮抗型人肿瘤坏死因子-α(hTNF-α)单克隆抗体(McAb)并检测其活性,为临床靶向治疗提供理论依据。方法用经典免疫方法获得抗hTNF-αMcAb,经接种于小鼠腹腔后获得腹水,采用硫酸铵沉淀和蛋白G亲和层析法纯化得到纯度>95%的抗hTNF-αMcAb,采用ELISA方法测定效价、抗体亚型和亲和力,MTT法测定抗体阻断hTNF-α对L929细胞的细胞毒作用和筛选拮抗型抗hTNF-αMcAb,流式细胞仪法测定抗体阻断hTNF-α诱导L929细胞凋亡的作用。结果获得1株能稳定分泌抗hTNF-αMcAb的杂交瘤细胞株XB10,分泌的抗体亚型为IgG2b;该抗体能高亲和性与hTNF-α结合,特异性阻断hTNF-α对L929细胞的细胞毒作用和抑制细胞凋亡,并呈一定剂量依赖性。结论成功制备能稳定分泌拮抗型抗hTNF-α的McAb的杂交瘤细胞株,其分泌的抗体对hTNF-α有特异性拮抗作用,为今后研制临床治疗型抗hTNF-α的基因工程抗体奠定了实验基础。
Objective To prepare and screen the McAb of antagonist human tumor necrosis factor-α (hTNF-α) and test its activity to provide a theoretical basis for clinical targeted therapy. Methods The anti-hTNF-αMcAb was obtained by classical immunization. The ascites was obtained after being inoculated into the peritoneal cavity of mice. Anti-hTNF-αMcAb> 95% was purified by ammonium sulfate precipitation and protein G affinity chromatography. Antibody could block the cytotoxicity of hTNF-α on L929 cells and the antagonistic anti-hTNF-αMcAb by MTT assay, and the antibody could block the apoptosis of L929 cells induced by hTNF-α by flow cytometry Role. Results A hybridoma cell line XB10 secreting anti-hTNF-αMcAb was obtained and the secreted antibody subtype was IgG2b. This antibody could bind hTNF-α with high affinity and block the specific binding of hTNF-α to L929 cells Cytotoxicity and inhibition of apoptosis, and in a dose-dependent manner. Conclusion The hybridoma cell line that can stably secrete McAb against antagonistic hTNF-α can be successfully prepared, and the secreted antibody has specific antagonism to hTNF-α, laying a foundation for the future development of genetically engineered anti-hTNF-α antibody Experimental basis.