Scholarlink

<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&#xa0;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 <i>E. coli</i> 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> Graphical Abstract <p></p>

错误：搜索内容不能为空，请输入英文关键词
错误：关键词超出字数限制，请精简
高级检索

Designing the homogeneous competitive bioluminescence-based assay for tick-borne encephalitis virus (TBEV) point-of-care detection

  • 期刊论文

  • 发表日期：

    2025年8月27日

查看全文
  • Alexander N. Kudryavtsev,
  • Eugenia E. Denisova,
  • Vasilisa V. Krasitskaya,
  • Ivan K. Baykov,
  • Nina V. Tikunova,
  • Ludmila A. Frank

摘要

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.

Graphical Abstract