牛环形泰勒虫GST-Tams1融合蛋白间接ELISA检测方法的建立

Development of an Improved Indirect Tams1 ELISA for Detection of Theileria Annulata in Cattle

【目的与方法】以纯化的牛环形泰勒虫GST-Tams1融合蛋白作为检测抗原,通过优化ELISA反应条件,建立检测牛环形泰勒虫血清特异性抗体的间接ELISA方法。【结果】方阵试验确定的GST-Tams1抗原的最适包被浓度为10μg/mL,血清最佳稀释倍数为80倍,ELISA阳性反应的临界值为OD450≥0.282,批内和批间重复试验的变异系数均小于15%。Tams1间接ELISA方法能排除GST的干扰,与其它梨形虫病无交叉反应,与环形泰勒虫病巢式PCR检测方法的阳性符合率为96%。【结论】建立的Tams1间接ELSIA检测法重复性好、特异性强、灵敏度高。这是国内首次利用重组蛋白建立的牛环形泰勒虫病血清学诊断方法,为大规模地进行牛环形泰勒虫病的流行病学调查和血清学诊断提供有效的技术手段。

【Objective and Method】An improved indirect Tams1 enzyme-linked immunosorbent ELISA assay（indirect Tams1 ELISA） was developed for the detection of specific antibody against Theileria annulata in cattle after optimizing its reacting conditions.【Result】The optimal concentration of coating antigen was 10 μg/mLand the optimal dilution of serum sample was 1：80 in the cross assay.The cutoff was chosen as an OD450≥0.282 for positive response.The variation coefficient of intra-batch and the inter-batch in the repeating tests was less than 15%.The interference of murine GST antiserum could be eliminated and no cross reactions were found among the piroplasms.The coincidences of identified positive serum samples for bovine piroplasmosis in indirect Tams 1 ELISAs were 96%,comparing with that in nested PCR.【Conclusion】The improved indirect Tams1 ELISA was highly sensitive,specific and reproducible.This was the first case to establish an improved indirect Tams1 ELISA for detection of specific antibodies against Theileria annulata in China,which provided a new tool for the large-scale epidemiological surveys and serological diagnosis of bovine theileriosis infected with Theileria annulata.