牛轮状病毒双抗体夹心ELISA检测方法的建立

Development of a Double Antibody Sandwich ELISA for Detection of Bovine Rotavirus

用差速离心法纯化的牛轮状病毒(BRV)分别免疫鼠和兔,制备了鼠抗BRV和兔抗BRV超免疫血清,并用层析方法进行了纯化,建立了检测BRV的双抗体夹心ELISA方法。该双抗体夹心ELISA的最佳反应条件为:兔抗BRV IgG包被浓度为10μg.mL-1,鼠抗BRV IgG工作浓度为5μg.mL-1,样品反应时间60 min,酶标抗体工作浓度为1∶5 000,以OD450 nm≥0.432作为阳性判定标准。该方法的板间、板内重复性变异系数小于10%,最低检测浓度为1.41μg.mL-1,并与牛呼吸道合胞体病毒(BRSV)、牛冠状病毒(BCV)和牛传染性鼻气管炎病毒(IBRV)等病原无交叉反应。用建立的ELISA方法与BRV RT-PCR方法同时检测40份临床粪便样品,符合率达到95%。结果表明,建立的双抗体夹心ELISA方法具有较好的特异性和较高的敏感性,可用于BRV的快速检测。

Two kinds of hyperimmune sera,mouse-anti-rotavirus IgG and rabbit-anti-rotavirus IgG,were prepared by inoculating respectively piglets and rabbits with bovine rotavirus（BRV） purified by differential centrifugation,and purified by affinity chromatography.A double antibody sandwich ELISA for detection of BRV was developed based on the two kinds of IgG.The optimal coating concent ration of rabbit-anti-rotavirus IgG was 10 μg·mL-1,the optimal working concent ration of mouse-anti-rotavirus IgG was 5 μg·mL-1,the reaction time of sample was 60 min,and the optimal working dilution of HRP-labelled goat-anti-mouse IgG was 1：5 000.The positive standard value was 0.432（OD450 nm）.The coefficient of variation of reproducibility was less than 10%,and at least 1.41 μg·mL-1 antigen could be detectable.The ELISA had no cross-reaction with IBRV,BCV,BRSV.Forty clinical fecal samples were detected by the ELISA and RT-PCR with agreement rate of 95%.The results revealed that the ELISA possessed good specificity and reproducibility,and higher sensitivity,indicating a suitable method for rapid detection of BRV.