山羊化脓隐秘杆菌溶血素抗体间接ELISA检测方法的建立、验证及应用

Development verification and application of indirect ELISA for pyolysin antibody against Trueperella pyogenes in goats

目的建立山羊化脓隐秘杆菌溶血素(pyolysin,PLO)抗体的间接ELISA检测方法,并进行验证及应用。方法通过E.coli表达系统表达重组溶血素(recombinant pyolysin,r PLO),采用Ni-NTA亲和层析进行纯化。以r PLO作为包被抗原,HRP标记的兔抗山羊IgG抗体为二抗,建立检测山羊化脓隐秘杆菌PLO抗体的间接ELISA法。采用方阵滴定法确定最佳抗原包被浓度(3.5、0.7、0.14和0.05μg/mL)及最佳二抗稀释度(1∶10000、1∶20000、1∶40000),同时验证方法的特异性、灵敏性及精密性。分别采用琼脂糖凝胶扩散试验和优化的间接ELISA法检测15份化脓隐秘杆菌自然感染的山羊血清,计算两者符合率;并采用优化的间接ELISA法检测360份临床山羊血清样本。结果纯化的r PLO相对分子质量约34000,纯度达90%。优化后ELISA法最佳抗原包被浓度为0.14μg/mL,二抗稀释度为1∶40000。优化后的ELISA法对E.coli、链球菌和伪结核棒状杆菌的阳性血清均无交叉反应;可检测出1∶800稀释的阳性血清;批内和批间变异系数(CV)均<10%。两种方法检测15份化脓隐秘杆菌自然感染山羊血清阳性检出率均为100%,符合率为100%;采用优化方法检测360份山羊血清的阳性率为24.1%(87/360)。结论成功建立了用于检测山羊化脓隐秘杆菌PLO抗体的间接ELISA法,该方法具有良好的特异性、灵敏性及精密性。

Objective To develop verify and apply an indirect ELISA for pyolysin antibody against Trueperella pyogenes in goats. Methods Recombinant pyoysin(r PLO) was expressed in E. coli and purified by Ni-NTA chromatography. An indirect ELISA was established for detection of anti-PLO IgG antibody against T. pyogenes in goats was developed using r PLO as coating antigen and HRP-labeled rabbit anti-goat IgG as the secondary antibody. The concentration of coating antigen(3. 5,0. 7,0. 14 and 0. 05 μg/mL)and the dilution of secondary antibody(1∶10 000,1∶20 000 and 1∶40 000)were optimized by block titration. The developed method was verified for specificity,sensitivity and reproducibility.Fifteen goat serum samples naturally infected with T. pyogenes were determined by agarose gel diffusion test and the optimized indirect ELISA respectively,and the coincidence rate of results was calculated. A total of 360 clinical serum samples of goats were determined by the optimized indirect ELISA. Results The purified r PLO,with a relative molecular mass of about 34 000,reached a purity of 90%. The optimal concentration of coating antigen was 0. 14 μg/mL,while the optimal dilution of secondary antibody was 1∶40 000. The developed ELISA showed no cross reaction with the sera positive for E. coli Streptococcus,or Corynebacterium pseudotuberculosis. The positive serum samples at a dilution of 1∶800 was detected by the developed method. Both the coefficients of variation of determination result by the developed ELISA in the intra-batch and inter-batch repetitive tests were less than 10%. Both the positive rates of fifteen goat serum samples naturally infected with T. pyogenes determined by two methods were 100%,of which the incidence rate was 100%. However,the positive rate of 360 goat serum samples determined by the optimized ELISA ws 24. 1%(87/360).Conclusion The indirect ELISA for PLO antibody against T. pyogenes in goats was successfully developed,which showed high specificity,sensitivity and precision.