H3亚型猪流感病毒抗原捕捉ELISA检测方法的建立

Development of antigen capture ELISA for detecting H3 subtype swine influenza virus

为快速检测H3亚型猪流感病毒(SIV),本研究将一株H3N2亚型SIV特异性单克隆抗体(MAb)进行纯化,建立了以多抗作为包被抗体,MAb作为检测抗体的H3亚型SIV抗原捕捉ELISA(AC-ELISA)方法。并对该方法的特异性和敏感性进行了分析,其敏感性是血凝试验的4倍,与猪繁殖与综合征病毒、猪圆环病毒、猪瘟病毒、猪细小病毒以及H1、H5、H9亚型SIV均没有明显的交叉性反应,批间和批内重复试验变异系数均小于10%。应用建立的AC-ELISA方法对84份H3亚型SIV实验室感染样品进行检测与鸡胚滴定病毒结果符合率达90.47%。本实验建立的AC-ELISA方法具有良好的特异性和敏感性,可用于H3亚型SIV的临床检测。

To developed the rapid detection method for H3 subtype swine influenza virus （SIV）, an antigen-capture enzyme-linked immunosorbent assay （AC-ELISA） was developed with the monoclonal antibody （MAb） 4E8 against HA protein of H3 SIV as detector antibody, and the polyclonal antibody as capture antibody. The results showed that the AC-ELISA was 4 times sensitive higher than that of hemagglutination assay. In addition, it had no cross-reaction with porcine reproductive syndrome virus, porcine circovirus virus, classical swine fever virus, porcine parvovirus and H1, H5, H9 subtypes of SIV. The coefficients of variation for both intra-assay and inter-assay was less than 10%. Moreover, the agreement rate of AC-ELISA and virus isolation was 90.47% tested on samples from experimentally infected pigs. Our data demonstrated the developed MAb-based AC-ELISA provided a specifically and sensitive diagnostic approach for the clinical detection of H3 SIV infection.