口蹄疫病毒非结构蛋白单克隆抗体阻断ELISA的建立与效果评价

Development and validation of a monoclonal antibody trapping NSP-3ABC blocking ELISA for differentiation of foot-and-mouth disease virus infected from vaccinated animals

使用组氨酸单克隆抗体作为捕获抗体,口蹄疫病毒非结构蛋白(NSP)3B单克隆抗体作为检测抗体,原核表达的口蹄疫病毒非结构蛋白3ABC作为抗原,建立了一种以区分疫苗免疫动物与康复带毒动物或持续感染动物为目的,检测口蹄疫病毒非结构蛋白3ABC抗体的阻断ELISA(FMDV NSP B-ELISA)。使用本研究建立的ELISA检测了大量牛、羊及猪的血清,并用公式(1-样品D450nm/阴性对照D450nm)来计算血清阻断率,最终确定:血清阴断率≥0.46判为阳性,血清阻断率<0.46判为阴性。根据此标准检测试验感染牛、羊及猪血清,敏感性≥85.4%;检测免疫健康牛、羊及猪血清,特异性≥99.2%。通过比较发现本研究建立的ELISA与Ceditest NS ELISA试剂盒有很高的符合率,对感染动物血清与疫区田间牛血清的阳性检出率均高于同类试剂盒。表明,本研究建立的ELISA可以作为田间疫情监测方法,在口蹄疫灭活疫苗免疫动物群体中使用。

The hi stidine monoclonal antibody（McAb） and McAb against non-structural protein（NSP） 3B of foot-and-mouth disease virus（FMDV） were prepared and used as capture antibody and detection antibody respectively. The purified histidine-tagged 3ABC fusion protein of FMDV, which was expressed in Escherichia coli ,was used as antigen. Then a NSP 3B trapping 3ABC-blocking ELISA for the detection of NSP 3ABC antibody of FMDV was developed. With statistics,the critical value 0.46 was defined as blocking rate after detection of cattle（229） ,sheep（181） and swine（256） sera. According to the criteria,the specificity of the positive serum was ≥99.2% ,and the sensitivity of the negative serum was ≥85.4%. The method has a high coincident rate compared with Ceditest NS ELISA kit,with a higher positive detection rate. The present study provided a promising tool for large-scale serological surveys in field.