猪伪狂犬病病毒gE抗体胶体金免疫层析检测方法的建立及应用

Establishement and Application of Colloidal Gold Immunochromatographic Assay for Antibodies of gE of Pseudorabies Virus in Swine

以纯化的抗人红细胞单链抗体(ScFv)-猪伪狂犬病病毒(PRV)gE蛋白双功能融合蛋白为诊断抗原和胶体金标记物,以羊抗猪IgG包被硝酸纤维膜作为质控带,制作检测猪伪狂犬病毒gE抗体的双抗原胶体金试纸条。利用方阵滴定试验筛选出金标抗原最佳工作浓度为17.6μg,检测线诊断抗原最佳标记量为1.76μg,血清最佳稀释度为1∶10,作用时间15 min,与猪瘟病毒(CSFV)、猪细小病毒(PPV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪乙型脑炎病毒(JEV)、猪布鲁菌(Brucella)阳性血清和PRVgE缺失疫苗接种的猪免疫血清检测线均不出现红色条带,与PRV标准阳性血清反应检测线出现红色条带。试纸条操作简单,肉眼于15 min内可判定结果;试纸条在室温保存6个月,其特异性和敏感性没有明显变化;与美国IDEXX和法国LSI gE-ELISA抗体检测诊断试剂盒检测结果比较,1 164份猪血清的符合率均为90.55%。制备的胶体金试纸条具有操作简便、敏感性和特异性较高的特点,可用于PRV野毒感染的快速筛查。

An immunochromatographic strip with double-antigen colloidal gold for detecting the antidody of gE protein of pseudorabies virus in swine was prepared.The purified anti-human erythrocyte single chain fragment variable-pseudorabies virus gE bifunctional fusion protein labeled with colloidal gold used as the diagnostic antigen and a goat-anti-pig antidody was used on the control line of the nitrocellulose membrane while blotting the fusion protein on the test line.Through Phalanx titration,a double antigen was screened out,in which the optimal working concentration of the colloidal gold-labeled antigen was 17.6 μg,the optimal label amount of the diagnostic antigen on the test line was 1.76 μg,and the optimal dilution of serum was 1∶10 with a reaction time of 15 min.No red strips were displayed on the test line while detected with positive sera of classical swine fever virus(CSFV),porcine parvovirus virus(PPV),porcine reproduction and respiratory syndrome virus(PRRSV),Japanese encephalitis virus(JEV),Brucella and serum of swine inoculated with PRV gE deleted vaccines,but the reaction of the double antigen with PRV standard positive serum was positive.The results demonstrated that the operation of this immunochromatographic strip is simple and the results can be judged with naked eyes with in 15 min;storage of the strips at room temperature for six months has not changed their sensitivity and specificity.Compared with the results from the American IDEXX and the French LSI gE ELISA antibody detection kits,the total coincidence rate of the negative and positive reaction reached 90.55%.These results suggested that the established colloidal gold immunochromatography assay can be used to detect the porcine serum antibodies of gE protein field PRV with the advantages of simple operation,high sensibility and specificity.