牛双芽巴贝斯虫GST-HSP20(exon)融合蛋白间接ELISA检测方法的建立

目的与方法]以纯化的牛双芽巴贝斯虫GST-HSP20(exons) 融合蛋白作为检测抗原,通过优化ELISA反应条件,建立了检测牛双芽巴贝斯虫血清特异性抗体的新型间接ELISA方法.结果]方阵试验确定的GST-HSP20抗原的最适包被浓度为5 μg/mL,血清最佳稀释倍数为40倍,ELISA阳性反应的临界值为OD450≥0.292,批内和批间重复试验的变异系数均小于10;.HSP20间接ELISA方法能排除GST的干扰,与其它梨形虫病无交叉反应,与巢式PCR检测方法的阳性符合率为96;.结论]所建立的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双芽巴贝斯病血清学诊断方法,为大规模地进行牛巴贝斯虫病的流行病学调查和血清学诊断提供有效的技术手段.

Development of an improved indirect HSP20(exons) ELISA for detection of Babesia bigemina in cattle

【Objective and Method】An improved indirect HSP20 enzyme-linked immunoborbent assay(indirect HSP20 ELISA) was developed for the detection of specific antibody against Babesia bigemina in cattle after optimizing its reacting conditions.【Result】The optimal concentration of coating recombinant antigen was 5 μg/ml and the optimal dilution of serum sample was 1:40 in the cross assay.The cutoff was chosen as an OD450≥0.292 for positive response.The variation coefficient of intra-batch and the inter-batch in the repeating tests was less than 10%.The interference of murine GST antiserum could be eliminated and no cross reactions were foud among the piroplasms.The coincidences of identified positive serum samples for bovine piroplasmosis in indirect HSP20 ELISAs were 96%,comparing with that in nested PCR.【Conclusion】Indirect HSP20 ELISA was highly sensitive, specific and reproducible.This was the first case to establish an inproved indirect HSP20 ELISA for ditection of specific antibody against Babesia bigemina in China,which was provided a new tool for the large-scale epidemiological svrveys and serological dignosis of bovine babesiosis infected with Babesia bigemina.