猪伪狂犬病病毒gB抗体检测的胶体金免疫层析试纸条研制

Development of a colloidal gold immunochromatographic test strip for detecting gB antibodies of porcine pseudorabies virus

摘要:

本研究旨在建立一种检测猪伪狂犬病病毒(porcine pseudorabies virus, PRV) gB蛋白抗体胶体金免疫层析试纸条，为PRV gB抗体检测提供一种简单快速的检测技术。本研究通过杂交瘤技术制备gB蛋白单克隆抗体，将gB蛋白与胶体金偶联，再将gB蛋白及其单克隆抗体分别喷涂在硝酸纤维素膜上作为检测线和质控线，经一系列条件的优化，成功制备了检测PRV gB抗体的胶体金免疫层析试纸条，并对该试纸条的特异性、敏感性、稳定性和符合性进行验证。结果发现，该试纸条的敏感性为1:128；与猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus, PRRSV)、猪瘟病毒(classical swine fever virus, CSFV)、塞内卡病毒(senecavirus A, SVA)、口蹄疫病毒(foot-and-mouth disease virus, FMDV)阳性血清均无交叉反应；在37 ℃可耐受至少72 d；通过检测96份临床猪血清样本，发现与商品化ELISA试剂盒检测结果的符合率为92.71%。本研究制备的PRV gB抗体胶体金免疫层析试纸条特异性好、灵敏度高、结果易判定，适用于猪伪狂犬病基层现场快速检测。

Abstract:

This study aims to establish a colloidal gold immunochromatographic test strip for the detection of glycoprotein B (gB) antibodies of porcine pseudorabies virus (PRV), thus providing a simple and rapid technique for the detection of PRV gB antibodies. The monoclonal antibodies to gB were prepared by the hybridoma technology. The gB protein was coupled with colloidal gold, and then gB and its monoclonal antibodies were separately sprayed onto a nitrocellulose membrane as the test line and control line. After optimization of conditions, a colloidal gold immunochromatographic test strip for detecting PRV gB antibodies was successfully developed. The specificity, sensitivity, stability, and concordance of the test strip were subsequently validated. The sensitivity of the test strip was 1:128. There was no cross-reactivity with positive sera of porcine reproductive and respiratory syndrome virus, classical swine fever virus, senecavirus A, and foot-and-mouth disease virus. The test strip could tolerate 37 °C for at least 72 d. In the test of 96 clinical pig serum samples, the concordance rate of the test strip results with the results of the commercial ELISA kit was 92.71%. The colloidal gold immunochromatographic test strip prepared in this study for PRV gB antibodies has good specificity, high sensitivity, and simple result interpretation, being suitable for rapid detection of porcine pseudorabies in the field.