检测牛轮状病毒抗体的双抗原夹心ELISA方法的建立

Establishment of a double-antigen sandwich ELISA method for detection of bovine rotavirus antibodies

为建立一种检测牛轮状病毒(BRV)抗体的双抗原夹心ELISA方法,本试验根据NCBI公布的BRV参考毒株NCDV序列,设计针对牛轮状病毒VP6基因的引物,用载体pCold-TF表达VP6蛋白,将表达的VP6蛋白作为捕获抗原,HRP标记的VP6蛋白作为检测抗原,建立检测BRV血清抗体的双抗原夹心ELISA方法。特异性试验表明该方法特异性良好;敏感性试验显示,该方法可检测到血清最大稀释倍数为1∶1600;重复性试验表明,批间变异系数在4.21%~7.24%之间,批内变异系数为在1.35%~3.52%之间,重复性良好;临床样品检测表明与微球间接凝集方法检测结果符合率为94.4%。结果表明,建立的检测BRV血清抗体的双抗原夹心ELISA方法适用于临床样本的检测,可为牛轮状病毒的快速诊断提供技术支持。

In order to establish a double antigen sandwich ELISA method for detecting bovine rotavirus antibody,primers for VP6 gene of BRV were designed according to the equence of BRV NCDV strain deposited by NCBI.VP6 protein was expressed using p Cold-TF vector.A double-antigen sandwich ELISA method was established for detecting BRV serum antibodies,using the expressed VP6 protein as the capture antigen and VP6 protein labeled with HRP as the detection antigen.Specificity experiment showed that the method was specific.Sensitivity experiment revealed that the maximum dilution ratio of serum detected was 1∶1600.Repeatability experiment showed good repeatability with inter-assay coefficients of variation ranging from 4.21% to 7.24% and intra-assay coefficients of variation ranging from 1.35% to 3.52%.The results of detecting clinical samples showed that the coincidence rate with indirect agglutination method of microspheres was 94.4%.This indicates that the established double antigen sandwich ELISA method for detecting BRV serum antibodies is suitable for the detection of clinical samples,which can provide technical support for the rapid diagnosis of BRV.