犬瘟热病毒单抗夹心ELISA检测方法的建立

Establishment of a sandwich ELISA for detection of canine distemper virus based on monoclonal antibodies

以2株犬瘟热病毒(CDV)特异性单克隆抗体2H11和1G7为基础建立了检测CDV的单抗夹心ELISA方法。用单抗2H11作为捕获抗体,以辣根过氧化物酶(HRP)标记的单抗1G7为检测抗体,通过方阵试验确定2H11和1G7-HRP的最佳工作浓度分别为200和475ng.mL-1。建立的单抗夹心ELISA方法具有良好的特异性,与纯化的犬细小病毒(CPV)无交叉反应,对纯化的CDV最小检出浓度为0.01μg.mL-1。采集经CDV抗原快速检测试纸条检测为阳性的40份疑似感染犬样品和检测为阴性的18份对照犬样品,分别用单抗夹心ELISA法和套式PCR法(N-PCR)进行同步检测,结果表明:单抗夹心ELISA法敏感性接近于N-PCR法,两种方法的符合率为85%。

A sandwich ELISA based on monoclonal antibodies(mAb) for rapid diagnosis of canine distemper virus(CDV) was established.The ELISA was standardized by pair-matching experiment,and the working concentrations of the mAb 2H11 and HRP-conjugated mAb 1G7 were 200 and 475 ng·mL-1,respectively.There was no cross reactivity with canine parvovirus(CPV) and the minimum detection limit was 0.01 μg·mL-1 of purified CDV antigen by the established ELISA.40 positive samples and 18 negative samples detected by solid phase chromatographic immunoassay were tested with ELISA and nested-PCR(N-PCR).The results showed that the sensitivity of ELISA was similar to N-PCR,with 85% agreement of the two methods.