刘原源;黄书晨;高珺珊;王晓岑;宫鹏涛;张壮志;李建华;杨举;李赫;张西臣;
LIU Yuan-yuan;HUANG Shu-chen;GAO Jun-shan;WANG Xiao-cen;GONG Peng-tao;ZHANG Zhuang-zhi;LI Jian-hua;YANG Ju;LI He;ZHANG Xi-chen;College of Veterinary Medicine, Jilin University;

• 1:吉林大学动物医学学院
• 2:新疆畜牧科学院兽医研究所

摘要(Abstract)：

目的建立简便、特异的检测犬粪便中细粒棘球绦虫抗原的双抗体夹心ELISA方法,并进行验证及初步应用。方法以细粒棘球绦虫EdiagA864蛋白为抗原,免疫日本大耳白兔,制备多克隆抗体;利用HRP标记兔抗细粒棘球绦虫EdiagA864多克隆抗体,通过溶解、超声方法处理犬粪便样品;以抗细粒棘球绦虫单克隆抗体2D12作为捕获抗体,HRP标记的兔抗细粒棘球绦虫EdiagA864多克隆抗体为检测抗体,通过棋盘法确定抗体最佳包被浓度、最佳封闭剂、待检粪样最佳稀释比例及酶标抗体最佳浓度。应用建立的双抗体夹心ELISA法对来自长春地区的64份犬粪便样品及来自新疆的8份犬粪便阳性样品进行检测。结果兔抗EdiagA864多克隆抗体的效价为105。建立的双抗体夹心ELISA法的最佳检测条件为:抗体包被浓度为1∶50,封闭剂为1%BSA,粪液稀释度为1∶5,酶标二抗稀释度为1∶800。建立的双抗体夹心ELISA法与犬贾第虫和犬蛔虫阳性样品均无交叉反应;检测不同稀释度的粪便液,当稀释至1∶20时,P/N仍大于2;检测6份阳性样品与4份阴性样品的批间和批内变异系数均小于8。用建立的方法检测8份阳性样品的结果均为阳性,64份待检样品的结果均为阴性。结论建立的双抗夹心ELISA方法特异性较强,敏感性较高,重复性较好,为细粒棘球绦虫流行病学调查及诊断提供了一种更简便、快速、特异的免疫学检测方法。
Objective To establish, verify and preliminarily apply a simple and specific double antibody sandwich ELISA for determination of Echinococcus granulosus antigen(EdiagA864)in dog feces. Methods Rabbits were immunized with EdiagA864, and the prepared polyclonal antibody was labeled by HRP. Dog feces samples were disposed in ultrasonic processing and digestion. A double antibody sandwich ELISA method was established using monoclonal antibody 2D12 as capture antibody, and HRP-labeled polyclonal antibody against EdiagA864 as detection antibody, of which the dilutions of the capture antibody, blocking agent, feces samples, detection antibody and HRP-labeled polyclonal antibody were optimized by chessboard titration. A total of 64 feces samples from Changchun Region and 8 positive feces samples from Xinjiang were detected by the established ELISA method. Results The titer of polyclonal antibody against EdiagA864 was 105. The optimal antibody concentration for coating was 1 ∶ 50, while the optimal blocking agent was 1% BSA, the optimal dilution of feces samples and HRP-labeled antibody were 1 ∶ 5 and 1 ∶ 800 respectively. The established ELISA method showed no cross reaction with positive feces samples of Giardia canis and Toxocara canis. The P/N value of samples at a dilution of 1 ∶ 20 was more than 2. Both the coefficients of variation(CVs) of determination results of 6positive and 4 negative samples in inter-and intra-assays were less than 8. All the determination results of 8 positive samples were positive, and those of 64 test samples were negative. Conclusion The established double-antibody sandwich ELISA method showed high specificity, sensitivity and reproducibility, which provided a simple, convenient and specific immunological method for diagnosis and epidemiological investigation of Echinococcus granulosus antigen in dog feces.

关键词(KeyWords)： 细粒棘球绦虫;双抗体夹心ELISA;EdiagA864;多克隆抗体
Echinococcus granulosus;Double-antibody sandwich ELISA;EdiagA864;Polyclonal antibody

Abstract:

Keywords:

基金项目(Foundation): 国家公益性行业(农业)科研专项(201303042)

作者(Authors): 刘原源;黄书晨;高珺珊;王晓岑;宫鹏涛;张壮志;李建华;杨举;李赫;张西臣;
LIU Yuan-yuan;HUANG Shu-chen;GAO Jun-shan;WANG Xiao-cen;GONG Peng-tao;ZHANG Zhuang-zhi;LI Jian-hua;YANG Ju;LI He;ZHANG Xi-chen;College of Veterinary Medicine, Jilin University;

DOI: 10.13200/j.cnki.cjb.001619

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