检测马传贫病毒跨膜蛋白主要免疫决定区抗体间接ELISA诊断方法的建立

Development of an indirect ELISA for detecting the antibody of immunodominant region of transmembrane envelope protein of EIAV

本试验以纯化的重组马传贫病毒跨膜蛋白主要免疫决定区蛋白作为诊断抗原建立了间接ELISA诊断方法,确定其最佳工作条件为每孔包被重组抗原2μg,兔抗马IgG酶标抗体以1∶4000,血清以1∶40倍稀释,底物作用时间为10min;判定标准为P/N值大于2,且OD值大于0.2的血清为阳性,否则判为阴性。本试验所建立的诊断方法与琼扩试验以及以琼扩抗原作为包被抗原的ELISA试验进行了比较,表明该诊断方法敏感性更高。特异性和重复性试验表明该诊断方法具有良好的特异性和重复性。通过对4匹马传贫驴白细胞弱毒疫苗免疫马血清抗体的检测,发现均在接种2周后可产生抗马传贫病毒跨膜蛋白主要免疫决定区相应抗体。此抗体一直持续存在到本试验检测的免疫后17个月,且效价较高。用此方法检测203份马血清样品,其中161份呈抗体阳性,还检测了马传贫自然感染马血清93份,结果全部为阳性。

An indirect enzyme-linked immuosorbent assay （iELISA） for detecting anti-TMlR antibody has been developed by coating plate with the recombinant TMIR protein. The optimum conditions of iELISA were that the foht of the TMIR protein coated is 2μg, the rabbit anti-horse IgG conjugated with perosidase is 1：4 000, the sera are 1：40 and the reaction time of o-phenylenediamine（OPD） is 10 minutes. A positive/negative（P/N） value higher than 2.0 and OD value over 0.2 was determined as comprehensive positive standard for the ELISA. The iELISA is more sensitive compared with Agar gel immunodiffusion （AGID） and ELISA with AGID antigen. The result showed that the assay was specific and repeatable. Serum antibody of 4 horses inoculated with DLAV were detected with the iELISA. The result indicated that the 4 horses induced the antibody against TMIR 2 weeks after inoculation. The TMIR-specific antibody titer eventually reached a plateau and remained 17 months. 203 serum samples were detected by the diagnostic assay, in which 161 serum samples were positive. In addition,93 serum samples collected from EIAV-infected horses were detected with the iELISA, and the results showed that all samples were positive.