猪弓形虫循环抗原双抗体夹心ELISA检测方法的建立及应用

Development and application of double antibody sandwich ELISA for Toxoplasma gondii circulating antigens in swine

目的建立一种检测猪血清中弓形虫循环抗原(circulating antigen,CAg)的双抗体夹心ELISA法。方法利用抗弓形虫表面抗原3(surface antigen 3,SAG3)的单克隆抗体Anti-A-SAG3-7作为捕获抗体,辣根过氧化物酶(HRP)标记抗弓形虫SAG3的单克隆抗体Anti-A-SAG3-23作为检测抗体,建立双抗体夹心ELISA法,并对方法的捕获抗体(1∶400~1∶6 400倍比稀释)、检测抗体(1∶400~1∶12 800倍比稀释)、血清稀释度(1∶10~1∶80倍比稀释)及封闭液的种类(5%脱脂奶粉、10%胎牛血清、5%BSA、1%BSA)进行优化,同时验证该方法的敏感性、特异性及精密性。采用优化后的方法对广东和吉林地区的各94份猪血清样本进行检测。结果双抗体夹心ELISA法的最佳检测条件为:捕获抗体Anti-A-SAG3-7及检测抗体HRP-Anti-A-SAG3-23稀释度均为1∶3 200,封闭液为1%BSA,血清稀释度为1∶80。该方法检测两份猪囊虫阳性血清无交叉反应,灵敏度达1∶640,批内及批间的变异系数(CV)均<11%。广东和吉林地区各94份样品的阳性率分别为20.2%(19/94)和11.7%(11/94)。结论该方法具有良好的特异性、敏感性及精密性,可用于猪弓形虫CAg的检测。

Objective To develop a double antibody sandwich ELISA method for determination of Toxoplasma gondii circulating antigen（CAg） in swine. Methods A double antibody sandwich ELISA method was developed using monoclonal antibody Anti-A-SAG3-7 against Toxoplasma gondii as capture antibody and HRP-labeled monoclonal antibody Anti-A-SAG3-23 against Toxoplasma gondii as detection antibody. The dilutions of capture antibody（1 ∶ 400 - 1 ∶ 6 400）,detection antibody（1 ∶ 400 - 1 ∶ 12 800）and serum（1 ∶ 10 - 1 ∶ 80）and blocking agents（5% skimmed milk, 10% fetal bovine serum, 5% BSA and 1% BSA） were optimized, and the method was verified for sensitivity, specificity and reproducibility. The 94 swine serum samples from Guangdong and 94 from Jilin regions were determined by the optimized method. Results Both the optimal dilutions of capture antibody Anti-A-SAG3-7 and detection antibody HRP-Anti-ASAG3-23 were 1 ∶ 3 200. The optimal blocking agent was 1% BSA, and the optimal dilution of serum was 1 ∶ 80. No cross reaction with serum of pigs with swine cysticercosis was observed. The sensitivity of the developed method was 1 ∶ 640,while the CVs in intra-and inter-assays were less than 11%. The positive rates of samples from Guangdong and Jilin regions were 20. 2%（19/94）and 11. 7%（11/94）respectively. Conclusion The developed ELISA method showed high specificity, sensitivity and precision, which might be used for determination of T. gondii CAg.