牛肠道病毒抗体检测方法及病毒试验感染小鼠抗体消长规律

Establishment of an indirect ELISA for detection of antibody against bovine enterovirus and antibody change in experimentally infected mice

应用表达纯化的E种肠道病毒HY12VP2重组蛋白作为包被抗原,建立了检测E种肠道病毒抗体的间接ELISA方法,并对实验感染小鼠抗体消长规律进行了研究。结果表明,VP2抗原最适包被浓度为300ng/孔,抗原包被最佳条件为37℃60min;封闭条件为5%脱脂奶粉37℃封闭60min;HRP酶标二抗最适稀释度为3 000×,最佳感作条件37℃90min。通过测定阴性小鼠血清样品,确定阴性和阳性血清临界值判定标准为0.091 2。与病毒中和试验相比,间接ELISA方法敏感性高,特异性强。统计学分析显示,阳性血清和阴性血清样品板内变异系数分别为3.8%和5.2%;板间阳性和阴性样品检测百分率变异系数分别为4%和4.3%,具有良好的重复性。小鼠感染病毒1周后开始产生抗体,随后抗体滴度逐渐增加,至感染6周时,抗体滴度达到峰值,之后逐渐下降。小鼠实验感染病毒抗体消长规律为本病的免疫机理和疫苗研制打下基础。

An indirect ELISA for detecting antibody against enterovirus E was established using the purified recombinant VP2 protein from enterovirus HY12 isolates as antigen,and employed to determine the antibody change in mice experimentally infected with enterovirus HY12.The optimal condition for antigen-coating is 300ng/100μL with an incubation at 37℃ for 60 min,and blocking reagent is 5% nonfat milk with an incubation at 37℃ for 60 min.Threshold defining positive and negative samples was determined statistically based on BEV-negative mice serum samples,and serum with D_（490）≥0.10 is considered as BEV-positive sample.Compared with the virus neutralization test,indirect ELISA method had a high sensitivity and specificity.Statistical analysis also showed that the coefficients of variation for positive serum and negative serum within the sample plate were 3.8% and 5.2%,respectively,indicating a stability of the ELISA assay.Mice were chosen as animal model and infected with enterovirus HY12 to determine viral immunogenicity.Antibody titers were assayed using indirect ELISA.The antibody was detected as early as 7dpi,then gradually increased and reached peak level 42 dpi before it decreased.The antibody change revealed in experimentally infected mice will lay a solid basis of future investigation on vaccine and immune mechanism for HY12 enteroviruses.