Development of an Indirect Enzyme-Linked Immunosorbent Assay Based on the Yeast-Expressed CO-26K-Equivalent Epitope-Containing Antigen for Detection of Serum Antibodies against Porcine Epidemic Diarrhea Virus.
Development of an Indirect Enzyme-Linked Immunosorbent Assay Based on the Yeast-Expressed CO-26K-Equivalent Epitope-Containing Antigen for Detection of Serum Antibodies against Porcine Epidemic Diarrhea Virus.
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DOI：
10.3390/v15040882
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发表时间：
2023-03-29
期刊：
影响因子：
--
通讯作者：
Wang H
中科院分区：
文献类型：
--
作者：
Yang X;Li L;Su X;Li J;Liao J;Yang J;Xiao Z;Wen P;Wang H
猪流行性腹泻（Porcine epidemic diarrhea，PED）是由猪流行性腹泻病毒（Porcine epidemic diarrhea virus，PEDV）引起的一种严重的肠道传染病。本研究通过对PEDV的53个全长spike基因和COE结构域的分析，选择了优势菌株SC 1402的spike蛋白的保守COE片段作为目的蛋白，并在毕赤酵母（Pichia pastoris）中成功表达。此外，还建立了基于重组COE蛋白的间接酶联免疫吸附试验（iELISA），用于检测猪血清中的抗PEDV抗体。结果表明，在优化条件下，COE-iELISA的临界值为0.12。以血清中和试验为标准，COE-iELISA的相对灵敏度为94.4%，特异性为92.6%。同时，该检测试剂盒与其他猪病原体无交叉反应性。批内和批间变异系数均小于7%。对164份免疫血清样品的检测结果表明，COE-iELISA与实际诊断结果的总体符合率达99.4%。与商品化ELISA试剂盒检测结果的一致率为95.08%（Kappa值为0.88），表明表达的COE蛋白是一种有效的血清学检测抗原，所建立的COE-iELISA可用于PEDV感染的监测和疫苗的免疫效果检测。
Porcine epidemic diarrhea (PED) is a severe contagious intestinal disease caused by the porcine epidemic diarrhea virus (PEDV), which leads to high mortality in piglets. In this study, by analyzing a total of 53 full-length spike genes and COE domain regions of PEDVs, the conserved COE fragment of the spike protein from the dominant strain SC1402 was chosen as the target protein and expressed successfully in Pichia pastoris (P. pastoris). Furthermore, an indirect enzyme-linked immunosorbent assay (iELISA) based on the recombinant COE protein was developed for the detection of anti-PEDV antibodies in pig sera. The results showed that under the optimized conditions, the cut-off value of COE-based indirect ELISA (COE-iELISA) was determined to be 0.12. Taking the serum neutralization test as standard, the relative sensitivity of the COE-iELISA was 94.4% and specificity 92.6%. Meanwhile, no cross-reactivity to other porcine pathogens was noted with this assay. The intra-assay and inter-assay coefficients of variation were less than 7%. Moreover, 164 vaccinated serum samples test showed that overall agreement between COE-iELISA and the actual diagnosis result was up to 99.4%. More importantly, the developed iELISA exhibited a 95.08% agreement rate with the commercial ELISA kit (Kappa value = 0.88), which suggested that the expressed COE protein was an effective antigen in serologic tests and the established COE-iELISA is reliable for monitoring PEDV infection in pigs or vaccine effectiveness.
猪流行腹泻病毒Chinju99的尖峰基因的克隆和序列分析。
影响因子：
1.6
作者：
Yeo SG;Hernandez M;Krell PJ;Nagy EE
通讯作者：
Nagy EE
影响因子：
3.3
作者：
Knuchel M;Ackermann M;Müller HK;Kihm U
通讯作者：
Kihm U