猪轮状病毒间接ELISA检测方法的建立

Establishment of an indirect ELISA for detection of antibodies against porcine rotavirus

以纯化的猪轮状病毒VP6蛋白作为检测抗原,建立检测猪轮状病毒血清抗体的间接ELISA。优化后的反应条件为:抗原包被量为6μg/mL,血清的最佳稀释度为1∶100,酶标二抗的工作浓度为1∶5 000。应用该方法检测阴性血清样本30份,确定的检测临界值为0.143(D490nm≥0.143,则判定为阳性)。特异性试验表明,用建立的间接ELISA对传染性胃肠炎病毒、猪流行性腹泻病毒、猪细小病毒、猪瘟病毒和伪狂犬病病毒阳性血清进行检测时无交叉反应。重复性试验证实,批内和批间变异系数均小于10%。结果表明,本研究建立的用以检测猪轮状病毒抗体的间接ELISA具有较高的特异性和敏感性,可应用于猪轮状病毒的病原学检测。

An indirect ELISA was established for rapid detection of antibodies against porcine rotavirus(PRV)using the purified recombinant VP6protein expressed in pET-30a-VP6-BL21as a coating antigen.The reaction conditions were optimized,including 6μg/mL coating antigen of the purified VP6protein,1∶100dilution of the tested serum and 1∶5 000dilution of HRP conjugated IgG with a cut off-value of0.143(D490nm).The indirect ELISA was specific to detect PRV,and had no cross-reaction with transmissible gastroenteritis virus,porcine epidemic diarrhea virus,porcine parvovirus,classical swine fever virus and porcine pseudorabies virus.Its intra-assay and inter-assay coefficient of variability were within 10%.The results revealed that the indirect ELISA was sensitive and specific,which could be used for laboratory diagnosis for PRV infection.