北京大学学报(医学版) ›› 2014, Vol. 46 ›› Issue (3): 483-487.

• 技术方法 • 上一篇    下一篇

人叶酸受体自身抗体IgG酶联免疫吸附试验检测方法的建立及评价

王琳琳,杨娜,袁悦,任爱国△   


  1. (北京大学公共卫生学院流行病与卫生统计学系,北京100191;北京大学生育健康研究所,卫生部生育健康重点实验室,北京100191)
  • 出版日期:2014-06-18 发布日期:2014-06-18

Establishment and evaluation of enzyme-linked immunosorbent assay for measuring human autoantibody IgG to folate receptor

WANG Lin-lin, YANG Na, YUAN Yue, REN Ai-guo△   

  1. (Department of Epidemiology and Health Statistics, Peking University School of Public Health, Beijing 100191, China; Institute of Reproductive and Child Health, Peking University; Ministry of Health Key Laboratory of Reproductive Health, Beijing 100191, China)
  • Online:2014-06-18 Published:2014-06-18

摘要: 目的:建立血浆叶酸受体自身抗体免疫球蛋白G(immunoglobulin G,IgG)的酶联免疫吸附试验(enzymelinked immunosorbent assay,ELISA)检测方法,并对其进行评价。方法:从正常人的胎盘样品中提取叶酸受体蛋白并将其纯化,以纯化的人源叶酸受体蛋白为包被蛋白,以单克隆抗体为检测二抗,建立血浆叶酸受体自身抗体IgG的间接ELISA检测方法,评价其灵敏度、精密度及稳定性。随机选取24例血浆标本,同时以商品化的牛源叶酸结合蛋白与本方法中提取的人源叶酸受体蛋白作为包被蛋白,采用ELISA方法分别检测血浆中叶酸受体抗体IgG的水平,比较两种方法的检测结果。结果:标准曲线可检测的IgG浓度范围为6.25×10-4~ 8.00×10-2(标准品采用健康人混合血浆,IgG浓度定为1),最低检测限为3.13×10-4,批内差异为2.74%~8.07%,批间差异为4.16%~8.23%。同一样本不同稀释倍数下IgG的检测结果均在可接受范围内,结果较稳定。血浆标本的牛源包被蛋白与人源包被蛋白检测方法对比结果显示两种方法高度相关,相关系数为0.954(P<0.001);人源包被蛋白检测结果比牛源包被蛋白高14%。结论:成功建立以人源叶酸受体蛋白作为包被蛋白、针对血浆叶酸受体自身抗体IgG水平的ELISA检测方法,该方法检测灵敏,重复性好,可用于大样本量的人群检测。

关键词: 叶酸受体, 自身抗体, 酶联免疫吸附试验, 免疫球蛋白G

Abstract: Objective:To establish and evaluate a newly established method of enzyme-linked immunosorbent assay (ELISA) for measuring human autoantibody to folate receptor (FR). Methods: Folate receptor was extracted and purified from healthy woman placenta tissues. The protein was coated on 96-well plates. Goat monoclonal antibody was used as detecting antibody to set up the indirect ELISA procedure. The sensitivity, precision and linearity of the method were evaluated. Further, the method was compared with the ELISA method with commercialized bovine folate binding protein (FBP) by determining autoantibody levels in 24 individuals. Results: The measuring range of the standard curve was from 6.25×10-4 to 8×10-2 (the IgG concentration of pooled plasma from healthy donors was defined as 1). The lowest detectable level was 3.13×10-4. The intra- and inter-assay coefficients of variations were 2.74%-8.07% and 4.16%-8.23%, respectively. Linearity test results were considered within acceptable limits. The data from FBP-ELISA and FR-ELISA were highly correlated (r=0.954, P<0.001); The value from FR-ELISA was higher by 14% than that from FBP-ELISA. Conclusion: The ELISA method for measuring human autoantibody IgG to folate receptor was successfully established using human FR as coating protein. The method is sensitive and repeatable and can be used in large-scale population study.

Key words: Folate receptor, Autoantibodies, Enzyme-linked immunosorbent assay, Immunoglobulin G

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