检测犬弓形虫SAG3抗体间接ELISA方法的建立

Establishment of an indirect ELISA technique to detect antibodies against the SAG3 surface antigen of Toxoplasma gondii in dogs

目的以弓形虫重组SAG3蛋白为抗原,建立一种检测犬弓形虫抗体的间接ELISA方法。方法采用棋盘滴定法确定抗原的包被浓度和阳性血清稀释度,再对该方法的封闭剂以及封闭时间、犬弓形虫阳性血清孵育时间、二抗稀释度以及加入显色液孵育时间等各项条件进行优化;通过对弓形虫感染犬血清及犬心丝虫、等孢球虫、蛔虫及巴贝斯虫感染血清的检测评价方法的敏感性和特异性。结果该方法最佳优化条件分别为:抗原包被浓度0.82μg/孔,封闭剂为含10%胎牛血清的PBST,被检血清稀释度1∶20;兔抗犬酶标二抗稀释度1∶8 000。优化的ELISA敏感性为1∶1 280,犬心丝虫、等孢球虫、蛔虫及巴贝斯虫感染血清均未发生交叉阳性反应;批间、批内重复性试验的变异系数均<15%。结论建立的ELISA方法特异性较强,敏感性高,批内、批间重复性较好,可用于犬弓形虫感染的检测。

Objective To develop an indirect ELISA technique using the recombinant protein SAG3 to detect antibodies against the SAG3 surface antigen of Toxoplasma gondii in dogs. Methods Optimal antigen coating conditions and the dilution of serum positive for T.gondii were determined using chessboard titration.A variety of conditions including the type of blocking agent and reaction time,the duration of incubation of serum positive for T.gondii,the dilution of secondary antibodies,and the duration of incubation of a soluble TMB substrate solution were optimized.Sera positive for T.gondii,Dirofilaria immitis,Isospora canis,Toxocara canis,and Babesia canis were used to infect dogs in order to detect the sensitivity and specificity of the indirect ELISA technique. Results The optimal conditions were an antigen coating of 0.82μg/well and a PBST solution of 10%fetal bovine serum as a blocking agent.The optimal dilution of the serum positive for T.gondii was 1：20,and the optimal dilution of secondary antibodies was 1：8 000.The indirect ELISA technique had a sensitivity of 1：1 280.There were no cross-reactions with sera positive for Dirofilaria immitis,Isospora canis,Toxocara canis,or Babesia canis.Intra-and inter-batch coefficients of variation were both less than 15%. Conclusion The indirect ELISA technique developed here had a high level of specificity,a high level of sensitivity,and good inter-and intra-batch reproducibility,and this technique can be used to detect toxoplasmosis in dogs.