中文摘要：

为检测弓形虫循环抗原,以实现弓形虫急性感染的早期诊断,本研究建立了基于ABC（avidin biotin-peroxidase complex）放大系统的双抗体夹心ELISA方法。通过制备弓形虫排泄分泌抗原（ESA）并免疫动物,经3种抗原的筛选以获得抗循环抗原（CAg）的单抗,以方阵滴定法确定最佳多抗包被浓度和单抗工作浓度,利用夹心法和ABC放大系统建立检测弓形虫循环抗原的夹心ABC-ELISA方法,用该方法对人工感染犬血清和其他阳性血清样本进行检测以确定该方法的检出时间和准确性,并应用于临床样品的检测。结果：获得了3株针对不同抗原表位的单克隆抗体,并对CAg有较高特异性。双抗体夹心ABC-ELISA反应条件：兔多抗包被浓度为3.7μg/mL,生物素标记3A5、3E5和10F5-3单抗在混合工作液中的浓度分别为0.1、0.13和0.12μg/mL。该ELISA方法对ESA最低检测限为11.9 ng/mL,并与隐孢子虫早期感染牛血清、血吸虫尾蚴早期感染牛血清、艾美耳球虫早期感染鸡血清、犬瘟热急性感染早期血清、犬细小病毒急性感染血清无交叉反应。用该ELISA方法检测人工感染的犬,于感染后2 d血清即显示阳性,该方法能明显区分标准阳性血清和阴性血清。用该方法检测68份猪临床血样（其中包括2份标准阳性血清）,检测结果与Nest-PCR结果一致。表明该双抗体夹心ABC-ELISA方法特异性强、敏感性高,可用于弓形虫急性感染的早期或活动期的诊断。

英文摘要：

A double antibody sandwich ELISA method based on ABC（ avidin biotin-peroxidase complex） amplification system was established to detect Toxoplasma gondii circulating antigen in order to realize the early diagnosis of acute infection of T. gondii. In this study,animals were immunized with T. gondii excretory- secretory antigens（ ESA） and anti- CAg monoclonal antibodies were raised after the hybridoma cell supernatant being screened by three kinds of antigens. The optimal concentration of coating antibody and optimal dilution of detection antibody were determined by square titration method. Sera from experimental toxoplasmosis canines and other T. gondii positive serum samples were analysed to determine the detection time and the accuracy of this method. The method developed here was applied to detect CAg in clinical samples. Results showed that three stable hybridoma cell clones（ 3A5,3E5,10F5-3） producing anti-CAg monoclonal antibody were established. The antibodies had high antibody titer against CAg and could also recognize different epitopes. Double antibody sandwich ELISA reaction conditions were as follows： the optimum coating concentration of rabbit polyclonal antibody was 3. 7 μg / mL,the working concentration of biotin-labeled monoclonal antibody was 0. 1 μg / mL,0. 13 μg / mL and 0. 12 μg / mL for 3A5,3E5 and 10F5-3,respectively. The limit detection of ESA was 11. 9 ng / mL by this method. This ELISA method had no cross-reaction with Cryptosporidium,Schistosoma japonicum,Eimeria tenella,Canine distemper and Canine parvovirus. CAg could be detected in canine serum 2 days after inoculation with tachyzoites by double antibody sandwich ELISA method. The method could efficiently distinguish positive acute toxoplasmosis sera from negative. The detection results of the two positive control sera,which were mixed in 68 serum samples,were consistent with that of Nest-PCR.These results indicate that the double antibody sandwich ABC-ELISA developed here is a highly sensitive and specific method for d