基于非洲猪瘟病毒p72蛋白的双抗体夹心ELISA检测方法的建立

Establishment of a double antibody sandwich ELISA method based on p72 protein of African swine fever virus

为建立非洲猪瘟病毒(African swine fever virus,ASFV)p72蛋白的双抗体夹心ELISA(DAS-ELISA)检测方法,本研究以实验室前期制备的ASFV单克隆抗体32H5作为捕获抗体,以HRP标记的MAb 2B2-1作为检测抗体,经条件优化,建立了基于非洲猪瘟病毒p72蛋白的双抗体夹心ELISA检测方法,并进行了敏感性、特异性和重复性试验,同时初步探索了两种抗体在试纸条方面的应用。结果显示,该夹心ELISA方法的最佳工作条件为:捕获抗体包被浓度为2μg/m L,酶标检测抗体的最适稀释倍数为1∶200;判定标准为:当检测样品D值大于等于0.252时判定为阳性;该方法检测猪伪狂犬病毒、猪繁殖与呼吸综合征病毒和猪瘟病毒均呈阴性反应,具有良好的特异性;最低可检测到50 ng/m L p72重组蛋白;其批间和批内重复性变异系数均小于10%。利用两种抗体制备的蓝乳胶免疫层析试纸条也取得了较好的检测结果。这些结果表明本研究建立的ASFV p72双抗体夹心ELISA检测方法特异性强、灵敏度高、重复性好,为非洲猪瘟的快速诊断、p72蛋白的功能研究及检测提供了技术支持。

In order to establish a double antibody sandwich ELISA method based on p72 protein of african swine fever virus(ASFV),the monoclonal antibody(MAb)32H5 of ASFV prepared in our lab was used as capture antibody,and the HRP-labeled MAb 2B2-1 was used as detection antibody.The reaction conditions were optimized to establish a double-monoclonal antibody sandwich ELISA assay based on p72 protein of ASFV.The sensitivity,specificity and repeatability tests were carried out with the optimized reaction conditions,and the application of immunochromatographic test strip was preliminarily explored.The optimized reaction conditions showed that the optimal coating concentration of MAb 32H5 was 2μg/m L,and the optimal working dilution of HRP-labeled MAb 2B2-1 was 1∶200 with a cut-off value of 0.252(D450).The detection results were negative for pseudorabies virus(PRV),porcine reproductive and respiratory syndrome virus(PRRSV),classical swine fever virus(CSFV),showing a good specificity.The result of sensitivity test showed that at least 50 ng/m L p72 protein of ASFV was able to be detected,displaying the good sensitivity of this assay.The intra-and inter-assay reproducibility coefficient of variation is less than 10%.The blue latex particles immunochromatographic test strip prepared with two antibodies also achieved good detection results.The ELISA assay established in this study exhibited strong specificity,high sensitivity and good reproducibility,which provides technical support for the rapid diagnosis of ASFV,the function research and detection of p72 protein.