An indirect enzyme-linked immunosorbent assay using a recombinant truncated capsid protein of Porcine circovirus-2
An indirect enzyme-linked immunosorbent assay using a recombinant truncated capsid protein of Porcine circovirus-2
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DOI：
10.1177/1040638712461251
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发表时间：
2012-11-01
影响因子：
1.5
通讯作者：
Thanawongnuwech, Roongroje
中科院分区：
文献类型：
--
作者：
Jittimanee, Suphattra;Ayudhya, Suparlark Nuntawan Na;Thanawongnuwech, Roongroje
猪圆环病毒2型（PCV-2）血清学常用于PCV-2猪群状态测定和PCV-2疫苗接种计划的最佳时机。本研究的目的是开发一种内部间接酶联免疫吸附试验（ELISA）使用重组核定位信号截短衣壳（rntCap）蛋白在大肠杆菌系统中表达，并确定开发的rntCap间接ELISA的诊断性能与免疫过氧化物酶单层试验（IPMA）相比。基于rntCap间接ELISA（n = 90）的受试者工作特征（ROC）曲线分析，确定最佳截止光密度（OD）为0.330，这导致诊断灵敏度、诊断特异性和准确性分别为98.33%、93.33%和96.67%。用纯化的谷胱甘肽-S-转移酶（GST）蛋白或rntCap蛋白作为包被抗原检测阳性和阴性血清的平均OD值，结果表明rntCap间接ELISA检测的平均OD值与GST单独检测的平均OD值有显著性差异（P < 0.005）。建立的rntCap间接ELISA法与IPMA法测定结果之间的相关性为线性回归（斯皮尔曼相关系数= 0.772，P < 0.005），表明rntCap间接ELISA法测定的OD值可用于预测IPMA的效价水平。需要更多的样本来提高诊断的敏感性、特异性和准确性。总之，建立的rntCap间接ELISA可用作大规模检测猪PCV-2抗体的血清学诊断试验，并有能力商业化生产，用于诊断实验室的常规使用。
Porcine circovirus-2 (PCV-2) serology is commonly used for PCV-2 herd status determination and optimal timing of PCV-2 vaccination programs. The objectives of the current study were to develop an in-house indirect enzyme-linked immunosorbent assay (ELISA) using a recombinant nuclear localization signal truncated capsid (rntCap) protein expressed in an Escherichia coli system and to determine the diagnostic performance of the developed rntCap indirect ELISA in comparison with immunoperoxidase monolayer assays (IPMAs). Based on a receiver operating characteristic (ROC) curve analysis of the rntCap indirect ELISA (n = 90), an optimum cutoff optical density (OD) of 0.330 was determined, which resulted in diagnostic sensitivity, diagnostic specificity, and accuracy of 98.33%, 93.33%, and 96.67%, respectively. Average OD values of the positive (n = 8) and negative sera (n = 8) tested by either purified glutathione-S-transferase (GST) protein or the rntCap protein as the coating antigen revealed that the mean OD values tested by the rntCap indirect ELISA were significantly different from using GST alone (P < 0.005). The correlation between the established rntCap indirect ELISA and the IPMA results demonstrated as the linear regression (Spearman correlation coefficient = 0.772, P < 0.005) indicated that the OD ratio obtained from the rntCap indirect ELISA could be used to predict the levels of the IPMA titers. More samples are needed for enhancing the diagnostic sensitivity, specificity and accuracy. In conclusion, the establishment of the rntCap indirect ELISA could be used as a serodiagnostic assay for large-scale detection of PCV-2 antibodies in swine and has the capability to be produced commercially for routine use in diagnostic laboratories.