犬细粒棘球绦虫粪抗原夹心ELISA检测方法的建立

Establishment of an Antibody Sandwich ELISA for Detection of Echinococcus granulosus Antigen in Canine Feces

为建立特异性和敏感性高的检验犬细粒棘球绦虫感染的方法。用细粒棘球绦虫(简称,Eg)成虫抗原分别免疫兔和绵羊,收集高免血清,纯化的高免抗体。依据抗体夹心ELISA工作原理,以兔抗体包被,检测感染Eg、不同犬带科绦虫的实验犬和空白犬粪样,绵羊抗体扑捉抗原,HRP标记兔抗绵羊IgG(1∶8 000)催化显色,用酶标仪测定OD 405nm吸光度,用以确定其特异性和敏感性。试验结果表明,敏感性为82.69%(43/52),特异性为85.88%(140/163);粪抗原在感染细粒棘球绦虫16d后可检出,最低抗原浓度为9.7ng/mL即犬感染5条成虫时可检测出阳性。该检测方法具有较好的特异性和灵敏性,为进一步研制检测细粒棘球绦虫虫体抗原ELISA检测试剂盒奠定了基础。

The aim of this study is to establish a high specific and sensitive method,which used for detection of Echinococcus granulosus（Eg） infection in dogs.Rabbits and sheep were immuned with the adult worm＇s antigen of Echinococcus granulosus,and purified hyperimmune antibodies from rabbits and sheep were obtained.According to sandwhich ELISA,rabbit antibodies were coated in the wells and attached the fecal antigens of dogs infected with Eg or other tapeworm or control dogs.The sheep＇s antibody was used to capture antigen,HRP labeled rabbit anti-sheep IgG（1∶8 000） reacted,was used.The specificity and sensitivity of this test were 85.88%（140/163）and 82.69%（43/52）.The most early day of detecting fecal antigen of Echinococcus granulosus was the 16th day post-challnged with protoscloses.The lowest does of fecal antigen of Echinococcus granulosus was 9.7ng/mL which means dogs infected with 5 adults can be detected as positive.This study established the method to detect parasite Echinococcus granulosus antigen,which has high specificity and sensitivity,lays a foundation for development of Echinococcus granulosus parasite antigen ELISA test kit.