检测犬弓形虫感染双抗体夹心ELISA方法的建立

Establishment of a double antibody sandwich ELISA technique for detection of toxoplasmosis infection in dogs

目的用42#抗弓形虫SAG3单抗为捕获抗体、29#抗弓形虫SAG3单抗作为检测抗体建立检测犬弓形虫感染的双抗体夹心ELISA方法。方法利用HRP标记检测29#抗弓形虫SAG3单抗;采用棋盘滴定法确定捕获抗体42#抗弓形虫SAG3单抗的包被浓度和弓形虫阳性血清稀释度;对封闭剂、检测抗体的工作浓度等条件进行优化,并进行敏感性、特异性、重复性以及稳定性测定。用所建立的双抗体夹心ELISA方法对76份犬待检血清进行检测,计算阳性率。结果最佳优化条件为捕获抗体包被浓度为1∶800稀释(0.34μg/孔),弓形虫阳性血清稀释度为1∶12,封闭剂为10%FBS溶液,最佳封闭时间为2h,参考阳性血清最佳作用时间为2h,检测抗体稀释度1∶3 000,TMB底物最佳显色时间为10min。优化的ELISA敏感性为1∶48;与犬心丝虫阳性血清、犬等孢球虫阳性血清、犬蛔虫阳性血清及犬巴贝斯虫阳性血清之间均无交叉反应;批内、批间重复性试验的CV均<15%;保存于4℃中的包被板稳定性>90d。用该方法检测76份犬血清中的弓形虫抗原,阳性率为5.26%。结论建立的双抗体夹心ELISA方法特异、敏感,重复性好,可用于犬弓形虫感染的早期检测。

Objectives To develop a double antibody sandwich ELISA for the detection of toxoplasmosis in dogs using no.42 McAb against Toxoplasma gondii SAG3 as a capture antibody and no.29 McAb against Toxoplasma SAG3 as a detection antibody. Method The detection antibody was labelled using an HRP Labeling Kit,and its optimal dilution was determined using direct ELISA.The coating of the capture antibody and dilution of serum positive for Toxoplasma（created in this laboratory by artificially infecting dogs）were determined using chessboard titration.Various conditions including the blocking agent used and dilution of the detection antibody were optimized.The specificity of the method was determined with serum positive for Dirofilaria immitis,Isospora canis,Toxocara canis,and Babesia canis.Seventy-six serum samples of dogs were tested with this technique. Result Optimal conditions were coating of the capture antibody at a ratio of 1：800（0.34μg/well）and use of 10% fetal bovine serum as the blocking agent.The optimal blocking time and the duration of incubation for serum positive for Toxoplasma were both 2 hours,the optimal dilution of serum positive for Toxoplasma was 1：12,the optimal dilution of the detection antibody was 1：3 000,and the duration of incubation of a TMB substrate solution was 10 min.The sensitivity of the method was 1：1 280.There was no cross-reactivity with serum samples containing D.immitis,I.canis,T.canis,or B.canis.The intra-and inter-batch coefficients of variation were both less than 15%.Sealed plates that were kept at 4℃ were stable for at least 90 d.ELISA detected Toxoplasma in the 76 serum samples from dogs at a rate of 5.26%. Conclusion The double antibody sandwich ELISA technique that was developed here is highly specific,highly sensitive,and highly reproducible,and it can be used for early detection of toxoplasmosis in dogs.