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目的定量检测狂犬病病毒病毒样颗粒糖蛋白含量。方法以狂犬病病毒糖蛋白单克隆抗体作为捕获抗体,狂犬病病毒多克隆抗体作为检测抗体,标准品为以细胞半数感染量(TCID50)准确定量的狂犬病病毒SRV9毒株病毒液,建立检测狂犬病病毒糖蛋白(RABV-GP)含量的双抗体夹心ELISA检测方法,对各反应条件进行优化并分析其特异性、重复性和灵敏度。结果双抗体夹心ELISA定量检测方法最佳工作条件为:狂犬病病毒糖蛋白蛋白单克隆抗体包被浓度为2.5μg/ml,4℃包被过夜;1%BSA,37℃封闭2h;狂犬病病毒多克隆抗体1∶200稀释,37℃孵育1.5h;酶标二抗1∶5 000稀释,37℃孵育1h;TMB室温避光显色30min。结论该方法可特异性定量检测狂犬病病毒,与犬细小病毒、犬瘟热病毒和犬传染性肝炎病毒均不发生反应。该方法线性检测范围为1×104~1.0×107 TCID50/ml,平均批内变异系数小于6%,平均批间变异系数小于8%。
Objective To quantitatively detect rabies virus-like particle glycoprotein content. Methods The rabies virus glycoprotein monoclonal antibody was used as the capture antibody and the rabies virus polyclonal antibody was used as the detection antibody. The standard was the virus of rabies virus SRV9 strain which was accurately quantified with half the number of cells (TCID50), and the rabies virus glycoprotein (RABV-GP) content of the double antibody sandwich ELISA detection method, the reaction conditions were optimized and analyzed for specificity, repeatability and sensitivity. Results The optimal working conditions for double antibody sandwich ELISA assay were as follows: Rabbit virus glycoprotein monoclonal antibody was coated at a concentration of 2.5 μg / ml overnight at 4 ° C; 1% BSA was blocked at 37 ° C for 2 hours; and rabies virus polyclonal Antibody diluted 1: 200, incubated at 37 ℃ 1.5h; enzyme-labeled secondary antibody diluted 1: 5000, incubated at 37 ℃ for 1h; TMB room temperature dark for 30min. Conclusion The method can detect rabies virus specifically and quantitatively, and can not react with canine parvovirus, canine distemper virus and canine infectious hepatitis virus. The linear detection range of this method was 1 × 104 ~ 1.0 × 107 TCID50 / ml, the average intra-assay CV was less than 6% and the mean inter-assay CV was less than 8%.