检测非洲猪瘟病毒抗原的双抗体夹心ELISA方法的建立

Development of double antibody sandwich ELISA assay for detection of African swine fever virus

利用非洲猪瘟病毒(ASFV)p72蛋白不同抗原表位的2株单克隆抗体,1株作为捕获抗体,另1株用辣根过氧化物酶(HRP)标记后作为检测抗体,采用方阵法对ELISA反应条件进行优化,建立了检测ASFV抗原的双抗体夹心ELISA方法。结果显示,该方法中捕获抗体包被质量浓度为1.25 mg/L,酶标单克隆抗体的最适稀释度为1∶4000。通过试验确定该方法的临界值为0.299,当样品D_(450)值≥0.3,且P/N大于2时,判定为阳性。该双单抗夹心ELISA方法可特异性检测ASFV抗原,其他抗原检测结果均为阴性,特异性强;重复性好,批内和批间变异系数(CV)均小于8%。应用本研究建立的方法与荧光PCR方法同时对225份临床样品进行检测,总符合率为96.89%。结果表明,本研究建立的双抗体夹心ELISA方法可用于ASFV抗原的大量样品检测,为ASF防控提供了技术支持。

Two monoclonal antibodies targeting different antigenic epitopes of African swine fever virus(ASFV)p72 protein were used to develop the sandwich ELISA for ASFV antigen.One McAb was coated on the 96-well plate as the capture antibody,and the other was labeled with HRP as the detection antibody.The square matrix method was used to optimize the ELISA reaction conditions.The results showed that the optimized concentration for coating antibody was 1.25 mg/L,and the optimal dilution of the enzyme-conjugate monoclonal antibody was 1∶4000.The critical value of this method was determined to be 0.299 by test.When the D_(450)value of the sample was greater than or equal to 0.3 and P/N was greater than 2,it could be considered to be positive.The double antibody sandwich ELISA method can specifically detect ASFV antigen,and the results of other antigens were negative.The intra-and inter-assay coefficients of variation of D_(450)values were less than 8%,respectively.The method established in this study and fluorescent PCR method were used to detect 225 clinical samples at the same time,and the total coincidence rate was 96.86%.The ELISA assay established in this study can be used for the detection of ASFV antigen with a large scale of samples,providing technical support for the prevention and control of ASF.