猪瘟病毒抗体胶体金免疫层析试纸条的研制及初步应用

Development and application of colloidal gold immunochromatography strip for detecting classical swine fever virus antibodies

为建立一种简便、快捷、特异、敏感的猪瘟病毒(CSFV)E2抗体的胶体金检测方法,本研究将E2蛋白用胶体金颗粒标记,作为金标抗原,将E2蛋白及其单克隆抗体包被在硝酸纤维素膜(NC)上,分别作为检测线(T线)和质控线(C线),组装成检测猪瘟E2蛋白抗体的胶体金免疫层析试纸条。结果显示,该试纸条可在5~10 min完成检测,检测健康猪血清、猪繁殖与呼吸综合征病毒(PRRSV)、口蹄疫A型病毒(FMDV-A)、口蹄疫O型病毒(FMDV-O)、塞内卡病毒(SVA)、牛病毒性腹泻病毒(BVDV)、非洲猪瘟病毒(ASFV)的阳性血清均无交叉反应;检测CSFV阳性血清的灵敏度可达1∶64(ELISA效价为1∶128);通过对田间101份猪血清样品进行检测,并与商品化猪瘟病毒抗体检测试剂盒的检测结果比较,两者的kappa值为0.92,为高度一致;试纸条在常温(18~25℃)和4℃条件下可分别稳定保存6个月和9个月。上述结果表明,本研究制备的试纸条具有较高的敏感性和特异性,且具有检测快速、操作简便、结果容易判断等特点,因此,对猪瘟现场检测有实际应用价值。

A simple,fast,specific and sensitive method of colloidal gold strip was established to detect the antibodies against classical swine fever virus(CSFV) E2.In this study,the E2 protein was labeled with colloidal gold particles as the gold-labeled antigen.Furthermore,the E2 protein and its monoclonal antibody were coated on the nitrocellulose membrane(NC) as the test(T) line and control(C)line,respectively.The results showed that the test can be completed within 5—10 min.Meanwhile,the established assay was used for detecting healthy pig serum,porcine reproductive and respiratory syndrome virus(PRRSV),foot-and-mouth disease type A virus(FMDV-A),foot-and-mouth disease type O virus(FMDV-O),senecavirus A(SVA),bovine viral diarrhea virus(BVDV),african swine fever virus(ASFV),and the results showed all of tested pathogens were negative,while classical swine fever virus(CSFV) was postive.The sensitivity of the colloidal gold immunochromatography strips(ICS) indicated that the dilution ratio of positive serum of CSFV was up to 1 ∶ 64(ELISA titer:1 ∶ 128).Finally,101 clinical samples were simultaneously detected by ICS and commercial swine fever virus antibody detection kit,the kappa value of both was 0.92. Moreover,The test strips were stably stored at 18—25 ℃ and 4 ℃ for 6 and 9 months,respectively.The above results indicated that the test used in this study had high sensitivity and specificity and were characterized by rapid detection,simple operation,and easy interpretation of results.Therefore,it has the practical application value of classical swine fever on-site detection.