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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 a simple and specific double antibody sandwich ELISA for the detection of Echinococcus granulosus antigen in canine faeces, and to verify and preliminary apply it. Methods The Echinococcus granulosus EdiagA864 protein was used as antigen to immunize the Japanese white rabbits to prepare polyclonal antibody. The rabbit anti-Echinococcus antibody E964 polyclonal antibody was stained with HRP and the canine stool samples were treated by dissolution and sonication. Anti-Echinococcus granulosus monoclonal antibody 2D12 as capture antibody, HRP-labeled rabbit anti-Echinococcus granulosus EdiagA864 polyclonal antibody as the detection antibody, by chessboard method to determine the optimal antibody concentration, the best blocking agent to be seized The best dilution ratio of fecal sample and the optimal concentration of enzyme-labeled antibody. A total of 64 canine stool samples from Changchun area and 8 canine stool samples from Xinjiang were detected by the established double antibody sandwich ELISA. Results The titer of rabbit anti-EdiagA864 polyclonal antibody was 105. The best detection conditions for double antibody sandwich ELISA were as follows: the concentration of antibody was 1:50, the concentration of BSA was 1% BSA, the dilution of exudate was 1: 5, and the dilution of ELISA was 1: 800. The established double-antibody sandwich ELISA did not cross-react with Giardia Iniensis and Ascaris suum positive samples. Different dilutions of fecal fluid were tested. When the dilution was 1:20, P / N was still greater than 2. Six positive samples Inter-batch and intra-batch variation coefficients were less than 8 with 4 negative samples. The positive results of the eight positive samples were positive by the established method, and the results of the 64 samples were negative. Conclusion The established double-antibody sandwich ELISA method is more specific, sensitive and reproducible, providing a simple, rapid and specific immunological detection method for the epidemiological investigation and diagnosis of Echinococcus granulosus .