白癜风相关黑素细胞膜抗原VIT150、VIT90、VIT75纯化及分析

Purification and analysis of melanocyte surface antigens associated with vitiligo by immunoprecipitation and chemiluminescence

目的:纯化和分析白癜风相关的黑素细胞膜抗原,为蛋白质微量测序及筛选、克隆黑素细胞膜抗原打下基础。方法:培养高纯度的正常人黑素细胞,活细胞ELISA法和蛋白免疫印迹法(Westein blot)检测并筛选抗黑素细胞的高滴度IgG抗体血清,生物素标记可溶性黑素细胞膜抗原,加入筛选血清与蛋白A-琼脂糖凝胶(protein A-sepharose)进行免疫共沉淀,沉淀后的抗原抗体复合物行平极电泳及电转印,碱性磷酸酶标记的亲合素进行化学发光法检测及鉴定。结果:活细胞ELISA法和Westein blot检测结果中共筛选了10例白癜风患者高滴度IgG血清,免疫沉淀、免疫印迹、化学发光法检测后发现10例患者均有阳性条带,其抗原相对分子质量为150 000、90 000、75 000、60 000。结论:白癜风患者血清中存在抗黑素细胞膜抗原的自身抗体,抗原相对分子质量主要为150 000、90 000、75 000,通过免疫共沉淀初步纯化了150 000、90 000和75 000抗原。

Objective: To purify and analyze melanocyte surface antigens associated with vitiligo and build the foundation for screening, sequencing and cloning for the protein antigens. Methods: High liter IgG of vitiligo serum to melanocytes was screened by using alive cell enzyme-linked immunoabsorbent assay and immunoblotting and then the biotin labelled soluble melanocyte antigens. Soluble melanocyte antigens were co-precipitated with protein A-sepharose and the screened sera, run on 10% SDS-PAGE, then blotted on the membrane. Streptavidin linked with alkaline phosphatase was added to NC membrane. Finally, the antigens were visualized by the BM chemiluminescence Western blotting Kit. Results: The sera of 10 cases were chosen by immunoblotting and live cell enzyme-linked immunoabsorbent assay from 238 vitiligo patients. The immunoprecipitation and chemiluminescence showed that all sera had positive strips. Autoantibodies could react with several antigens with approximate MWs of 150 000, 90 000, 75 000, 60 000. Conclusions:. Autoantibodies directed to melanocyte surface antigens are present in the sera of patients with vitiligo. The antigens with approximate MWs of 150 000, 90 000, 75 000 located on the surface of melanocyte were primarily purified by immunoprecipitation.