泰勒焦虫重组蛋白TaSP-Tams1-SPAG1间接ELISA检测方法的建立

Development of ELISA with Recombinant Protein Antigens for Theileria annulata

以真核表达的焦虫表面抗原重组蛋白TaSP-Tams1-SPAG1作为ELISA板包被抗原,通过方阵实验确定抗原包被浓度,探索抗原包被时间、温度、一抗及二抗血清稀释度及作用时间,建立牛环形泰勒焦虫表面抗原重组蛋白ELISA方法。结果得出TaSP-Tams1-SPAG1的最佳包被浓度为10μg/mL,血清最佳稀释度为1∶100,ELISA阳性反应的临界值为OD492nm≥0.348,重复试验变异系数小于12%,所建立的ELISA方法不与牛巴贝斯虫及牛瑟氏泰勒虫血清发生反应。TaSP-Tams1-SPAG1为抗原建立的ELISA方法特异性强,敏感度高,重复性好,为牛环形泰勒焦虫病的准确检测及防治提供技术支持。

The objective of this paper was to discuss the antigen coating time,temperature,serum dilution of first antibody and second antibody and action time,and develop antigen recombinant protein ELISA method against Theileria annulata in cattle,to determine antigen coating concentration on the square ex-periment by using antigen recombinant protein TaSP-Tams1-SPAG1 against Theileria annulata expressed by eukarya as ELISA coating antigen.The results indicated that the optimal concentration of coating anti-gen was 10 μg/mL and the optimal blocking dilution of serum sample was 1 ∶100 in the cross assay.The critical value reacted by ELISA positive was an OD 492nm ≥ 0.348.The variation coefficient of intra-batch and the inter-batch in the repeating tests was less than 12%.No cross reactions were found among the piro-plasms.The improved indirect TaSP-Tams1-SPAG1 ELISA Was highly sensitive specific and reproducible, which provided a technical basis for accurate detection and control of Theileria annulata diseases in cattle.