猪附红细胞体病间接ELISA诊断试剂盒的研制及初步应用

以纯化的猪附红细胞体特异性抗原为包被抗原,在建立猪附红细胞体病间接ELISA检测方法的基础上对各反应参数进行了优化,并初步组装成试剂盒。组装的试剂盒抗原最佳包被质量浓度为30 mg/L,被检血清稀释度为1∶100,酶标二抗的最适工作滴度为1∶4 000,底物TMB最适反应条件为室温15 min;检测血清的判定标准为D450值≥0.13定为阳性反应,D450值≤0.11定为阴性反应,0.11~0.13定为疑似反应;该试剂盒不与猪大肠杆菌病、猪弓形虫病及猪瘟等阳性血清发生交叉反应,具有较好的特异性;在不同时间对阴阳性样品重复检测8次,阴阳性血清的D450值变异系数均未超过10%,说明该试剂盒具有良好的重复性;通过对组装试剂盒中各组分的稳定性和保存时间的测定结果证实,该试剂盒的保质期暂定为6个月;应用保存6个月的试剂盒通过对吉林省延边地区的60份猪血清样本检测,并与间接ELISA比较,其阳性符合率达100%,说明该试剂盒具有较好的稳定性。

Development and validation of indirect ELISA test kit for detecting eperythrozoonosis

In this research,E.suis antigen which was purified and protected as envelope antigen was made.The optimal reaction conditions were determined and the kit was assembled initially.The result showed that the optimal concentration of antigen was 30 mg/L;the optimal concentration of serum dilution was 1∶100;the optimal condition of HRP-labeled rabbit anti-swine IgG was 1∶4 000;the optimal reaction condition of TMB was room temperature for 15 minutes.D450≥0.13 was positive,D450≤0.11 was negative,and 0.11-0.13 was suspected.The method has no cross-reaction with swine E.coli,swine fever and swine toxoplasmosis serum.Coefficient of variation of negative and positive serum at D450 was not exceed 10% after detecting for different 8 times.This showed that the kit had a good repeatability.The kits could be kept for 6 months by the determination of stability.60 samples of serum in Yanbian area were detected by the kit which had saved for 6 months.Compared with indirect ELISA,the result showed the the positive coincidence was 100%.This study provides a new effective way for diagnosis of eperythrozoonosis.