应用旋毛虫GAD和GLS重组抗原建立间接ELISA方法的研究

Study of an indirect ELISA establishment using GAD and GLS recombinant antigens of Trichinella spiralis

为了建立快速检测猪旋毛虫病的血清学方法,分别用旋毛虫GAD、GLS重组蛋白作为包被抗原,建立2种检测旋毛虫血清抗体的间接ELISA方法。对抗原包被条件、血清稀释度、封闭液的选择与封闭时间、酶标二抗的稀释度与孵育时间和TM B作用时间等条件进行了优化。旋毛虫GAD包被抗原间接ELISA方法和GLS包被抗原间接ELISA方法的阴阳性临界值分别为0.848和0.963;这2种间接ELISA方法批内和批间重复试验的最大变异系数均小于10%,且用这2种方法分别对猪蛔虫阳性血清、华支睾吸虫阳性血清和弓形虫阳性血清进行了检测,均无交叉反应,表明这2种方法具有很好的稳定性和特异性。同时用这2种间接ELISA方法和旋毛虫抗体快速检测卡分别对感染旋毛虫不同数量、不同时间的小鼠血清进行检测,结果显示,这2种间接ELISA方法具有较高的敏感性。本研究中建立的这2种间接ELISA方法为旋毛虫病的早期诊断和流行病学调查提供了必要的手段。

In order to establish a rapid serological method for detecting trichinellosis,two detecting Trichinella spiralis serum antibodies indirect ELISA methods were established which used the T.spiralis GAD and GLS proteins recombinant as coating antigens.The conditions inculding antigen encapsulation conditions,serum dilution,blocking buffer selection and sealing time,enzyme-labeled secondary antibody dilution,incubation time and TMB action time were optimized,respectively.The negative and positive critical values determination criterion of indirect ELISA methods of T.spiralis GAD and GLS coated antigens were 0.848 and 0.963,respectively.The maximum variation coefficients of intraassay and inter-assay of the two indirect ELISA methods were all less than 10%.And the positive sera of Ascaris suis,Clonorchis sinensis and Toxoplasma gondii were detected by the two methods,no cross reaction was found.The results showed that the methods established in present study have good stability and high specificity.At the same time,the comparison was also made between the established methods in the present study and the T.spiralis antibody rapid detection cards,the sera of infected T.spiralis mice with different numbers and days were detected which showed that the ELISA methods established in the present study had high sensitivity.The two established indirect ELISA methods in this study provide a necessary tool for the early diagnosis and epidemiological investigation of trichinellosis.