设计基于同质竞争性生物发光的蜱传脑炎病毒(TBEV)即时检测方法。

IF 3.8 2区 化学 Q1 BIOCHEMICAL RESEARCH METHODS
Alexander N Kudryavtsev, Eugenia E Denisova, Vasilisa V Krasitskaya, Ivan K Baykov, Nina V Tikunova, Ludmila A Frank
{"title":"设计基于同质竞争性生物发光的蜱传脑炎病毒(TBEV)即时检测方法。","authors":"Alexander N Kudryavtsev, Eugenia E Denisova, Vasilisa V Krasitskaya, Ivan K Baykov, Nina V Tikunova, Ludmila A Frank","doi":"10.1007/s00216-025-06090-w","DOIUrl":null,"url":null,"abstract":"<p><p>Tick-borne encephalitis virus (TBEV), a highly pathogenic infectious agent that causes serious damage to the nervous system is mainly transmitted by Ixodidae ticks. The laboratory methods (immunoassay and the PCR-based one) are successfully used to detect the virus in tick samples thereby avoiding unwarranted immunoprophylaxis. However, there is a need to determine the tick infection outside the laboratory conditions. In this study, we have developed a one-stage (of mix-and-read type) method for detecting virus in biological samples based on split NanoLuc complementation assay. Artificial NanoLuc luciferase split fragments NLuc(N-residue), 17.6 kDa, and NLuc(C-residue), 11 a.a., were genetically fused with the protein prED3 (fragment of the TBEV capsid protein E) or mouse anti-TBEV single-chain antibody 14D5a in all possible variants. The corresponding hybrid proteins were synthesized in E. coli recombinant cells, purified and studied. Assembling of the luciferase fragments into a bioluminescent complex proceeded due to antigen-antibody affinity interaction. The most efficient luciferase complementation was observed for the pair 14D5a-NLucCter + prED3-NLucNter: the integral bioluminescence of the complex was 2.4% of that of the intact luciferase. Using this complex, a single-phase competitive enzyme immunoassay of TBEV-associated targets was developed. A large number of native ticks were analyzed and a statistically significant difference was shown between \"healthy\" and virus-carrying ticks. Lyophilized all-in-one reagents, reconstituted upon addition of the sample matrices, were developed and tested in model assay. The results offer a basis for the development of a point-of-need portable device for rapid tick detection.</p>","PeriodicalId":462,"journal":{"name":"Analytical and Bioanalytical Chemistry","volume":" ","pages":""},"PeriodicalIF":3.8000,"publicationDate":"2025-08-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Designing the homogeneous competitive bioluminescence-based assay for tick-borne encephalitis virus (TBEV) point-of-care detection.\",\"authors\":\"Alexander N Kudryavtsev, Eugenia E Denisova, Vasilisa V Krasitskaya, Ivan K Baykov, Nina V Tikunova, Ludmila A Frank\",\"doi\":\"10.1007/s00216-025-06090-w\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Tick-borne encephalitis virus (TBEV), a highly pathogenic infectious agent that causes serious damage to the nervous system is mainly transmitted by Ixodidae ticks. The laboratory methods (immunoassay and the PCR-based one) are successfully used to detect the virus in tick samples thereby avoiding unwarranted immunoprophylaxis. However, there is a need to determine the tick infection outside the laboratory conditions. In this study, we have developed a one-stage (of mix-and-read type) method for detecting virus in biological samples based on split NanoLuc complementation assay. Artificial NanoLuc luciferase split fragments NLuc(N-residue), 17.6 kDa, and NLuc(C-residue), 11 a.a., were genetically fused with the protein prED3 (fragment of the TBEV capsid protein E) or mouse anti-TBEV single-chain antibody 14D5a in all possible variants. The corresponding hybrid proteins were synthesized in E. coli recombinant cells, purified and studied. Assembling of the luciferase fragments into a bioluminescent complex proceeded due to antigen-antibody affinity interaction. The most efficient luciferase complementation was observed for the pair 14D5a-NLucCter + prED3-NLucNter: the integral bioluminescence of the complex was 2.4% of that of the intact luciferase. Using this complex, a single-phase competitive enzyme immunoassay of TBEV-associated targets was developed. A large number of native ticks were analyzed and a statistically significant difference was shown between \\\"healthy\\\" and virus-carrying ticks. Lyophilized all-in-one reagents, reconstituted upon addition of the sample matrices, were developed and tested in model assay. The results offer a basis for the development of a point-of-need portable device for rapid tick detection.</p>\",\"PeriodicalId\":462,\"journal\":{\"name\":\"Analytical and Bioanalytical Chemistry\",\"volume\":\" \",\"pages\":\"\"},\"PeriodicalIF\":3.8000,\"publicationDate\":\"2025-08-27\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Analytical and Bioanalytical Chemistry\",\"FirstCategoryId\":\"92\",\"ListUrlMain\":\"https://doi.org/10.1007/s00216-025-06090-w\",\"RegionNum\":2,\"RegionCategory\":\"化学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"BIOCHEMICAL RESEARCH METHODS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Analytical and Bioanalytical Chemistry","FirstCategoryId":"92","ListUrlMain":"https://doi.org/10.1007/s00216-025-06090-w","RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0

摘要

蜱传脑炎病毒(TBEV)是一种对神经系统造成严重损害的高致病性传染病,主要由蜱传播。实验室方法(免疫测定法和基于聚合酶链反应的方法)成功地用于检测蜱虫样本中的病毒,从而避免了不必要的免疫预防。然而,有必要在实验室以外的条件下确定蜱虫感染。在这项研究中,我们开发了一种基于分裂NanoLuc互补试验的生物样品中一阶段(混合-读取型)病毒检测方法。人工NanoLuc荧光素酶分裂片段NLuc(n -残基),17.6 kDa和NLuc(c -残基),11 a.a,在所有可能的变体中与蛋白prED3 (TBEV衣壳蛋白E片段)或小鼠抗TBEV单链抗体14D5a基因融合。在大肠杆菌重组细胞中合成了相应的杂交蛋白,并进行了纯化和研究。由于抗原-抗体亲和相互作用,荧光素酶片段组装成生物发光复合物。对14D5a-NLucCter + prED3-NLucNter进行了最有效的荧光素酶互补,复合物的整体生物发光量为完整荧光素酶的2.4%。利用该复合物,开发了tbev相关靶标的单相竞争性酶免疫测定。对大量本地蜱虫进行了分析,发现“健康”蜱虫和携带病毒的蜱虫之间存在统计学上的显著差异。冻干的一体化试剂,在添加样品基质后重组,在模型分析中开发和测试。研究结果为开发便携式蜱虫快速检测设备奠定了基础。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Designing the homogeneous competitive bioluminescence-based assay for tick-borne encephalitis virus (TBEV) point-of-care detection.

Tick-borne encephalitis virus (TBEV), a highly pathogenic infectious agent that causes serious damage to the nervous system is mainly transmitted by Ixodidae ticks. The laboratory methods (immunoassay and the PCR-based one) are successfully used to detect the virus in tick samples thereby avoiding unwarranted immunoprophylaxis. However, there is a need to determine the tick infection outside the laboratory conditions. In this study, we have developed a one-stage (of mix-and-read type) method for detecting virus in biological samples based on split NanoLuc complementation assay. Artificial NanoLuc luciferase split fragments NLuc(N-residue), 17.6 kDa, and NLuc(C-residue), 11 a.a., were genetically fused with the protein prED3 (fragment of the TBEV capsid protein E) or mouse anti-TBEV single-chain antibody 14D5a in all possible variants. The corresponding hybrid proteins were synthesized in E. coli recombinant cells, purified and studied. Assembling of the luciferase fragments into a bioluminescent complex proceeded due to antigen-antibody affinity interaction. The most efficient luciferase complementation was observed for the pair 14D5a-NLucCter + prED3-NLucNter: the integral bioluminescence of the complex was 2.4% of that of the intact luciferase. Using this complex, a single-phase competitive enzyme immunoassay of TBEV-associated targets was developed. A large number of native ticks were analyzed and a statistically significant difference was shown between "healthy" and virus-carrying ticks. Lyophilized all-in-one reagents, reconstituted upon addition of the sample matrices, were developed and tested in model assay. The results offer a basis for the development of a point-of-need portable device for rapid tick detection.

求助全文
通过发布文献求助,成功后即可免费获取论文全文。 去求助
来源期刊
CiteScore
8.00
自引率
4.70%
发文量
638
审稿时长
2.1 months
期刊介绍: Analytical and Bioanalytical Chemistry’s mission is the rapid publication of excellent and high-impact research articles on fundamental and applied topics of analytical and bioanalytical measurement science. Its scope is broad, and ranges from novel measurement platforms and their characterization to multidisciplinary approaches that effectively address important scientific problems. The Editors encourage submissions presenting innovative analytical research in concept, instrumentation, methods, and/or applications, including: mass spectrometry, spectroscopy, and electroanalysis; advanced separations; analytical strategies in “-omics” and imaging, bioanalysis, and sampling; miniaturized devices, medical diagnostics, sensors; analytical characterization of nano- and biomaterials; chemometrics and advanced data analysis.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:604180095
Book学术官方微信