胰岛素瘤相关蛋白-2自身抗体酶联免疫吸附测定方法的建立

Establishment of Enzyme-linked Immunosorbent Assay for Insulinnoma-associated Protein-2 Autoant ibodies.

目的:建立糖尿病患者血清中胰岛素瘤相关蛋白鄄2(IA鄄2)自身抗体的酶联免疫吸附测定(ELISA)方法。方法:以链亲和素预包被的聚苯乙烯微量滴定板为固相载体,原核表达的生物素酰化IA鄄2胞内段融合蛋白为抗原,辣根过氧化物酶标记的山羊抗人IgG为二抗,2,2’鄄连氮鄄双鄄[3鄄乙基苯并噻唑啉磺酸](ABTS)为显色底物建立ELISA。结果:本方法的平均批内变异系数为8.8%,批间变异系数为7.7%。用该方法和Roche公司生产的“DiapletsAnti鄄IA鄄2”IA鄄2自身抗体酶联免疫试剂盒进行对比,共检测218例糖尿病患者,以Roche试剂盒的检测结果为准,本方法的诊断灵敏度为92.1%,特异度为98.9%。结论:本研究建立的IA鄄2自身抗体ELISA法有较好的灵敏度、特异度和精密度,且操作简单方便,有较广阔的应用前景。

Objectiv e: To establish enzyme-linked immunosorbent assay(ELISA)for insulinnoma-associate d protein-2(IA-2)au-toantibodies in serum of patients with diabetes mellitu s.Methods:The E.coli-derived biotinylated intracytoplasmic part of IA-2fusion protein was used as antigen and it was captured on streptavidin pre-coated poly styrene microtiter plates.The horseradish peroxidase(HRP)labeled goat anti-huma n-IgG was used as secondary antibody.2,2'-azino-di-Establishment of Enzyme-linked Immunosorbent Assay for Insulinnoma-associated Protein-2 Autoant ibodies.$$$$ JIA Xiujuan,XIE Xiaoyan,ZHANG Di,et al.Ruijin Hospital,Shanghai Institut e of Endocrinology,Shanghai Second Medical University,Shanghai(200025)Objectiv e: To establish enzyme-linked immunosorbent assay(ELISA)for insulinnoma-associate d protein-2(IA-2)au-toantibodies in serum of patients with diabetes mellitu s.Methods:The E.coli-derived biotinylated intracytoplasmic part of IA-2fusion protein was used as antigen and it was captured on streptavidin pre-coated poly styrene microtiter plates.The horseradish peroxidase(HRP)labeled goat anti-huma n-IgG was used as secondary antibody.2,2'-azino-di-[3-ethylbenzthia-zoline sulfonatel(ABTS)solution was used as substrate for HRP.Results:The overall intr a-assay coefficient of variation(CV)was8.8%and the inter-assay CV was7.7%.Our kit was compared with'Diaplets anti-IA-2'IA-2autoantibodies ELISA kit produ ced by Roche company.A total of218patients with diabetes mellitus were detected. When the Roche kit was used as standard,the diagnostic sensitivity and specifici ty of our kit were92.1%and98.9%respectively.Conclu-sions:The method of ELISA sh ows good sensitivity,specificity and precision in detecting IA-2autoantibody,al so it is con-venient to be served as a routine use.