猪弓形虫病阻断ELISA方法的建立及初步应用

Development and Preliminary Application of a Blocking ELISA for Detection of Porcine Toxoplasmosis

本研究利用制备的GRA7单克隆抗体为基础建立猪弓形虫病的阻断ELISA方法。以rGRA7重组蛋白免疫小鼠,取脾细胞进行细胞融合,筛选出4株能够稳定产生单克隆抗体的阳性单克隆细胞株。经对此单克隆抗体进行IFA和Western blot检测确认成功制备GRA7蛋白的单克隆抗体后,以重组蛋白为包被抗原对阻断ELISA方法进行优化后,经过重复性、交叉性、符合性实验验证本方法具有可重复性,与MAT符合性高。初步应用于288份临床样品检测,检出率为11.46%,与MAT检测结果重合率为84.8%。本研究成功制备弓形虫GRA7蛋白的单克隆抗体,并建立了一种猪弓形虫病的阻断ELISA方法,为弓形虫病的检测提出了新的方案。

In this study,a blocking ELISA method for swine toxoplasmosis was developed based on the monoclonal antibody GRA7.Mice were immunized with the recombinant protein rGRA7 and then their spleen cells were taken for cell fusion.The resulting hybridomas were screened and 4 positive cell lines were found to stably produce monoclonal antibodies.The monoclonal antibodies against GRA7 protein was confirmed in IFA and Western blotting and used to develop a blocking ELISA method.The blocking ELISA was optimized for the concentration of the coating antigen and tested for its repeatability,cross-compatibility and conformity.The results showed that this blocking ELISA had good repeatability and high compliance with MAT.Subsequently,this method was used to test 288 clinical samples and the positive rate was 11.46%with 84.9%coincidence with the MAT.Collectively,a monoclonal antibody against GRA7 protein of Toxoplasma gondii was prepared for development of a blocking ELISA method,which might be used for the detection of swine toxoplasmosis.