弓形虫抗体免疫印迹试剂盒的研制

Development of Immunoblot Kit for the Detection of Anti,Toxoplasma Antibodies

目的研制敏感、特异的检测弓形虫IgM和IgG抗体免疫印迹试剂盒。方法收集人工感染RH株弓形虫速殖子昆明系小鼠的腹腔液,提取弓形虫胞质蛋白,采用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离弓形虫可溶性抗原,并经电泳转印至硝酸纤维膜,以无毒灵敏的四甲基联苯胺(TMB)为底物分别检测30份弓形虫IgM和28份IgG阳性血清,40份健康人血清。通过比较抗原制备方法和使用剂量、封闭剂、洗涤和稀释剂、工作浓度、作用时间以及反应带出现率,选择最佳实验条件,以敏感性、特异性、Youden指数以及稳定性作为试剂盒评价标准。结果用免疫印迹试剂盒检测30份弓形虫IgM和28份IgG阳性血清,敏感性分别为90.0%(27/30)和85.7%(24/28),40份健康对照者血清的弓形虫IgM和IgG抗体均为阴性,特异性均为100%,Youden指数分别为0.9和0.86。试剂盒于4℃保存约6个月的检测结果一致。结论该免疫印迹试剂盒敏感性和特异性均较高,且操作简便、快速。

Objective To develop a diagnostic kit for the detection of anti-Toxoplasna antibodies in human sera by immuuobdot technique. Methods The cytoplasm proteins of Toxoplasmna gondii were extracted from collected ascites in Kumning strain mice infected by tachvzoites of Toxoplasma gondii （RH strain）. Toxoplasma gondii soluble antigens were separated by SDS-PAGE electrophoresis and transferred to pyroxylin membrane. Using nonpoisonous tetramethylbenzidine （TMB） as horseradish peroxidase substrate for immunoblot, the optimal experimental conditions were selected through comparison of antigen preparation, reagents for blocking or washing, dilution concentration, reaction time, and frequencies of reaeting band. Sensitivity, specificity, Youden index and stability were evaluated as the standard for the kit. Results In 30 sera with anti-Toxoplasma IgM antibodies and 28 with IgG antibodies, tire sensitivity for IgM and IgG antibody detection was 90.0%（27/30） and 85.7% （24/28） respectively, the specificity was all 100% in examining 40 heahhy control sera, and the Youden index was 0.9 and 0.86 respectively. The kit was stable at 4℃ for 6 months. Conclusion The immunoblot kit shows an easy operation, fast reaction and reliable result, and may be practical.