管式磁微粒非洲猪瘟病毒pp62蛋白化学发光抗体检测方法的建立

A carboxylated magnetic bead-based CLIA method for detecting anti-pp62 antibody of African swine fever virus

摘要:

【背景】非洲猪瘟(African swine fever, ASF)作为高致病性传染病，给我国生猪养殖业造成了严重的经济损失，因此建立快捷、灵敏的诊断方法至关重要。【目的】建立一种管式非洲猪瘟病毒pp62蛋白化学发光抗体检测方法。【方法】以重组pp62蛋白作为包被抗原，羧基磁珠(carboxylic magnetic beads)作为固相载体，碱性磷酸酶(alkaline phosphatase, AP)标记的兔抗猪IgG作为酶标二抗，通过对反应条件进行优化，采用非洲猪瘟国家参考品作为溯源血清绘制出标准曲线，建立基于ASFV pp62蛋白的化学发光抗体检测方法。【结果】用建立的化学发光方法检测277份临床样品血清，通过受试者工作特征(receiver operating characteristic, ROC)曲线分析确定阴性、阳性临界值，并确定判定标准：浓度值>140.02 U时为抗体阳性，浓度值<140.02 U时为抗体阴性。此方法对6种不同的病原血清抗体进行检测均无交叉反应，且批内和批间变异系数均在10%以内，与商品化非洲猪瘟抗体检测试剂盒的符合率达96.7%。【结论】本研究建立的管式非洲猪瘟病毒化学发光抗体检测方法具有良好的特异性、敏感性和重复性，可为非洲猪瘟早期监测及试剂盒研发提供参考。

关键词:非洲猪瘟病毒;重组pp62蛋白;羧基磁珠;化学发光检测方法

Abstract:

[Background] African swine fever (ASF), as a highly pathogenic infectious disease, has caused serious economic losses in China’s pig farming industry, so it is crucial to establish a fast and sensitive diagnostic method. [Objective] To develop a chemiluminescence immunoassay (CLIA) for the detection of the anti-pp62 antibody of African swine fever virus (ASFV). [Methods] The recombinant pp62 protein was used as the coating antigen, carboxylated magnetic beads as the solid-phase carrier, and alkaline phosphatase (AP)-conjugated rabbit anti-swine IgG as the secondary antibody. The reaction conditions were optimized, and the standard curve was established with the national reference product of ASFV as the traceable serum. Finally, a CLIA method for detecting the anti-pp62 antibody of ASFV was established. [Results] The established CLIA method was used to test 277 clinical samples, and the negative and positive thresholds were determined by the receiver operating characteristic (ROC) curve. The concentrations >140.02 U and <140.02 U were determined as antibody positive and negative, respectively. Furthermore, the established method was used to detect the serum antibodies to six different pathogens, which showed no cross-reactivity, the intra- and inter-batch coefficients of variation below 10%, and a compliance rate of 96.7% with the results obtained by commercial ASFV antibody detection kits. [Conclusion] The CLIA method established for the detection of ASFV has good specificity, sensitivity, and reproducibility, which can provide a reference for the early monitoring of ASF and the research and development of the relevant kits.

Key words:African swine fever virus;recombinant pp62 protein;carboxylated magnetic beads;chemiluminescence immunoassay