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}
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.
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.