检测犬源狂犬病毒中和抗体ELISA方法的建立

摘要

为了有效监测犬免疫狂犬病疫苗后的保护效力,以狂犬病毒(Rabies virus,RV)糖蛋白的主要优势抗原表位区G3蛋白(RV G3)作为包被抗原,建立了一种检测狂犬病毒中和抗体效价的间接ELISA方法.通过优化反应条件,确定抗原最佳包被量为8 mg/L,血清的最佳稀释度为1∶100,酶标二抗的稀释度为1∶3000.特异性试验表明,该抗原不与犬瘟热病毒、犬腺病毒、犬细小病毒阳性血清发生交叉反应；批内和批间重复性试验的平均变异系数都小于10％；敏感性达1∶1 280.此方法检测134份血清样品的结果与美国SYNBIOTICS试剂盒相比,总符合率达95.6％.%To monitor the effectiveness of canine rabies vaccination, the indirect ELISA method had been developed to detect the neutralizing antibodies against rabies virus (RV) by using the main antigenic determinant of glycoprotein ( RV G3) as coating antigen. The optimum test conditions were as follows: the optimal concentration of RV G3 coating the ELISA plate was 8 mg/L. The optimal concentration of serum samples and HRP-labeled goat anti-canine IgG was 1: 100 and 1: 3 000 respectively. RV G3 had no reaction with positive serum against canine distemper virus ( CDV ) , canine adenovirus ( CAV) or canine par-vovirus ( CPV) by the specificity test. The coefficients of variation of intro-batch and inter-batch duplica-bility test were fewer than 10%. The sensitivity was 1= 1 280. Compared with America SYNBIOTICS kit, the coincidence rate of indirect ELISA was 95. 6% . Therefore, the indirect ELISA has good specificity, repeatability and sensitivity, which can be a good candidate for routine detection of neutralizing antibodies of canine rabies.