牛地方流行性白血病液相蛋白芯片检测方法的建立

Development of a fluorescent microsphere-based assay for the detection of enzootic bovine leukosis

应用xMAP液相芯片技术原理,以原核表达的重组牛白血病病毒gp51蛋白为抗原,建立了检测牛白血病病毒抗体的LiquiChip液相蛋白芯片检测方法,并进行了特异性、敏感性、重复性与稳定性试验。结果表明,该方法对牛白血病病毒阳性血清检测阳性,对其他常见牛病毒阳性血清检测阴性,表明特异性好;液相蛋白芯片方法对牛白血病病毒抗体的检测敏感性可达119.7ng/μL,对多份牛白血病病毒阳性血清的检测结果显示该方法的检测敏感性与商品化ELISA试剂盒无显著差异;对3份牛白血病病毒阳性血清进行4次重复检测,批内、批间的变异系数均低于10%。所制备的偶联微球芯片保存3个月,其检测结果无显著差异。采用该方法对169份临床牛血清样品进行检测,检出阳性率达57.4%,该检测结果与瑞典进口ELISA试剂盒检测结果的符合率为94.67%。本方法为牛地方流行性白血病的进出境检疫、疫病监测和流行学调查研究提供了一种特异、敏感的新型快速检测技术。

Recombinant gp51 protein of bovine leukemia virus（BLV） was expressed using a prokaryotic expression system, and purified and eovalently coupled to fluorescent xMap microspheres. The purified mysB-gp51 protein was used to establish an xMap method for the detection of antibodies against BLV. And the specificity,sensitivity＇reproducibility and stability of the method were examined. For specificity analysis,no positive reaction with positive sera of bluetongue virus, bovine viral diarrhea virus,infectious bovine rhinotracheitis virus, foot-and-mouth disease virus,or bovine ephemeral fever virus was found. For sensitivity analysis,as low as 119. 7 ng/μL BLV positive serum could be detected and no significant difference with commercial ELISA kit. The variation coefficients of intra- or inter-batch were all less than 10% based on 4 replications of 3 samples. Stability test showed that the coupled-beads could be saved up to 3 months without significant difference. The established method was performed to 169 bovine serum samples with a positive rate of 57. 40%which was 94. 67% insistent with commercial ELISA kit from Sweden. The results suggested that the BLV xMap assay established in the present study was high specific, being good sensitivity and easy to be manipulated, which provided an available teehnioue for the detection and serological suivey of BLV of bovine serum