A species-independent indirect-ELISA for detection of antibodies to the non-structural protein 2B of foot-and-mouth disease virus

IF 16.4 1区化学 Q1 CHEMISTRY, MULTIDISCIPLINARY

Accounts of Chemical Research Pub Date : 2024-08-01 DOI:10.1016/j.biologicals.2024.101785

Jitendra K. Biswal , Samarendra Das , Jajati K. Mohapatra, Manoranjan Rout, Rajeev Ranjan, Rabindra Prasad Singh

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引用次数: 0

Abstract

Diagnostic assays that are able to detect foot-and-mouth disease (FMD) virus infection in the vaccinated population are essential tools in the progressive control pathway for the FMD. However, testing of serum samples using a single diagnostic assay may not completely substantiate freedom from the virus infection. Therefore, viral non-structural proteins (NSPs)-based various serological assays have been developed for the detection of FMD infection. Nevertheless, the NSPs-based ELISAs have been developed in the indirect-ELISA format, thereby necessitating the use of species-specific conjugated secondary-antibodies for the detection of anti-NSP antibodies in various FMD-susceptible species. Therefore, this study presents a novel recombinant 2B-NSP-based indirect ELISA, employing HRP-conjugated protein-A/G detection system which can detect anti-NSPs antibodies from multiple FMD-susceptible species in a single ELISA platform. Recombinant 2B (r2B) protein was expressed as His-SUMO tagged protein in the E. Coli cells and purified using NI-NTA affinity column chromatography. Using the r2B protein and HRP-conjugated protein A/G, an indirect ELISA was developed and validated for the detection of anti-2B antibodies in serum samples collected from multiple FMD-susceptible animal species with known FMD status. Further, a resampling based statistical technique has been reported for determination of optimal cut-off value for the diagnostic assay. Through this technique, the optimal cut-off of 44 percentage of positivity value was determined for the assay. At this optimal cut-off value, the developed diagnostic assay provided diagnostic sensitivity, specificity, and accuracy, positive and negative predictive values (PPV and NPV) of 92.35 %, 98.41 %, 95.21 %, 98.58 %, and 91.67 %, respectively. The assay was validated further by analyzing random serum samples collected across multi-locations in India. The assay can be used as a single platform for testing serum samples from different species of FMDV-susceptible animals and will be useful for NSP-based serosurveillance of FMDV.

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用于检测口蹄疫病毒非结构蛋白 2B 抗体的物种无关间接-ELISA。

能够在接种疫苗的人群中检测口蹄疫病毒感染情况的诊断测定是口蹄疫渐进控制途径中的重要工具。然而，使用单一诊断检测方法检测血清样本可能无法完全证实未感染病毒。因此，人们开发了基于病毒非结构蛋白（NSPs）的各种血清学检测方法来检测口蹄疫感染。然而，基于 NSPs 的 ELISA 是以间接-ELISA 的形式开发的，因此需要使用物种特异性的共轭二级抗体来检测各种 FMD 易感物种的抗 NSP 抗体。因此，本研究提出了一种基于重组 2B-NSP 的新型间接 ELISA 方法，该方法采用 HRP 结合物蛋白-A/G 检测系统，可在单一 ELISA 平台上检测多种 FMD 易感物种的抗 NSPs 抗体。重组 2B 蛋白（r2B）在大肠杆菌细胞中表达为 His-SUMO 标记蛋白，并使用 NI-NTA 亲和柱色谱法纯化。利用 r2B 蛋白和 HRP 结合物蛋白 A/G 开发并验证了一种间接 ELISA 方法，用于检测多种已知 FMD 状态的 FMD 易感动物血清样本中的抗 2B 抗体。此外，还报告了一种基于重采样的统计技术，用于确定诊断测定的最佳临界值。通过这项技术，确定了该检测法的最佳临界值为阳性值的 44%。在此最佳临界值下，所开发的诊断测定的诊断灵敏度、特异性、准确性、阳性预测值和阴性预测值（PPV 和 NPV）分别为 92.35 %、98.41 %、95.21 %、98.58 % 和 91.67 %。通过分析在印度多个地点采集的随机血清样本，该检测方法得到了进一步验证。该检测方法可作为单一平台用于检测不同种类的 FMDV 易感动物的血清样本，并将用于基于 NSP 的 FMDV 血清监测。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Accounts of Chemical Research 化学-化学综合

CiteScore

31.40

自引率

1.10%

发文量

312

审稿时长

2 months

期刊介绍： Accounts of Chemical Research presents short, concise and critical articles offering easy-to-read overviews of basic research and applications in all areas of chemistry and biochemistry. These short reviews focus on research from the author’s own laboratory and are designed to teach the reader about a research project. In addition, Accounts of Chemical Research publishes commentaries that give an informed opinion on a current research problem. Special Issues online are devoted to a single topic of unusual activity and significance. Accounts of Chemical Research replaces the traditional article abstract with an article "Conspectus." These entries synopsize the research affording the reader a closer look at the content and significance of an article. Through this provision of a more detailed description of the article contents, the Conspectus enhances the article's discoverability by search engines and the exposure for the research.