马链球菌兽疫亚种类M蛋白抗体间接ELISA检测方法的建立及应用

Development of indirect ELISA for detection of antibodies against M-like protein of Streptococcus equi ssp.zooepidemicus

为建立简便实用的马链球菌兽疫亚种间接ELISA,对马链球菌兽疫亚种ATCC35246菌株的类M蛋白(SzP)进行了原核表达,以纯化的SzP作为包被抗原,建立了马链球菌兽疫亚种血清抗体间接ELISA,并对抗原包被浓度、血清稀释度、封闭液的选择等反应条件进行了优化。结果显示,重组SzP的最佳包被浓度为0.625μg/mL,标准阳性血清的最佳稀释度为1∶40,最适封闭液为含50g/L脱脂奶粉的PBS溶液,待检血清的最佳孵育时间为60min,酶标二抗的最佳作用时间为45min。用建立的马链球菌兽疫亚种血清抗体间接ELISA对采自河南、山东、安徽、上海、江苏等地猪场的1 080份血清进行检测。结果显示,上述五地猪场血清样本马链球菌兽疫亚种抗体的阳性率分别为33.3%、16.5%、21.8%、13.9%和13.5%。上述结果表明,本试验建立的间接ELISA的特异性和重复性良好,具有重要的临床应用价值。

To establish a simple and practical indirect ELISA for detection of Streptococcus equi ssp.zooepidemicus,the M-like protein gene of S.zooepidemicus ATCC35246 strain was expressed in Escherichia coli BL21(DE3).In the optimized indirect ELISA,the recombinant protein was coated at 0.625μg/mL.The optimal dilution of standard positive serum was 1∶40.The optimal blocking buffer was 50g/L skimmed milk powder in PBS.The optimal incubation time of tested serum was 60min.The optimal reaction time of HRP-SPA was 45min.Total 1 080 serum samples from Henan,Shandong,Anhui,Shanghai and Jiangsu provinces were tested for anti-S.zooepidemicus serum antibodies by this indirect ELISA.The results showed that the positive rates in these 5 provinces were 33.3%,16.5%,21.8%,13.9%and 13.5%,respectively.In summary,the indirect ELISA for detection of anti-S.zooepidemicus serum antibodies was successfully established.