{"title":"利用酪氨酸酶介导的ha标签标记生成q -小体的新方法的发展。","authors":"Hui-Seon Yun, Hanool Yun, Hee-Jin Jeong","doi":"10.1007/s00216-025-06087-5","DOIUrl":null,"url":null,"abstract":"<p><p>Quenchbodies (Q-bodies) are fluorescent antibodies that respond to antigen binding via a fluorescence quenching and de-quenching mechanism. To enhance the versatility of the generation method and expand the color range, we developed a novel Q-body generation approach using tyrosinase-mediated site-specific conjugation to a tyrosine-rich hemagglutinin (HA)-tag. A single-chain variable fragment (scFv) against programmed cell death-ligand 1 (PDL1) was engineered with an N-terminal HA-tag and expressed in Escherichia coli with high yield and purity. Site-specific conjugation of four hydrazide-functionalized dyes with different emission wavelengths was achieved using recombinant tyrosinase. The resulting Q-bodies were evaluated via fluorescence-linked immunosorbent assay, all of which demonstrated antigen concentration-dependent fluorescence enhancement. Notably, the tetramethylrhodamine (TAMRA)-labeled Q-body showed the highest signal-to-background ratio, limit of detection of 0.51 ± 0.01 μg/mL. This HA-tag-mediated Q-body generation strategy offers a robust and versatile tool for the production of Q-bodies with broad dye compatibility, enabling sensitive and multiplexed fluorescent immunoassays.</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\":\"Development of a novel method for generating Q-bodies using tyrosinase-mediated HA-tag labeling.\",\"authors\":\"Hui-Seon Yun, Hanool Yun, Hee-Jin Jeong\",\"doi\":\"10.1007/s00216-025-06087-5\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Quenchbodies (Q-bodies) are fluorescent antibodies that respond to antigen binding via a fluorescence quenching and de-quenching mechanism. To enhance the versatility of the generation method and expand the color range, we developed a novel Q-body generation approach using tyrosinase-mediated site-specific conjugation to a tyrosine-rich hemagglutinin (HA)-tag. A single-chain variable fragment (scFv) against programmed cell death-ligand 1 (PDL1) was engineered with an N-terminal HA-tag and expressed in Escherichia coli with high yield and purity. Site-specific conjugation of four hydrazide-functionalized dyes with different emission wavelengths was achieved using recombinant tyrosinase. The resulting Q-bodies were evaluated via fluorescence-linked immunosorbent assay, all of which demonstrated antigen concentration-dependent fluorescence enhancement. Notably, the tetramethylrhodamine (TAMRA)-labeled Q-body showed the highest signal-to-background ratio, limit of detection of 0.51 ± 0.01 μg/mL. This HA-tag-mediated Q-body generation strategy offers a robust and versatile tool for the production of Q-bodies with broad dye compatibility, enabling sensitive and multiplexed fluorescent immunoassays.</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/s00216-025-06087-5\",\"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/s00216-025-06087-5","RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
Development of a novel method for generating Q-bodies using tyrosinase-mediated HA-tag labeling.
Quenchbodies (Q-bodies) are fluorescent antibodies that respond to antigen binding via a fluorescence quenching and de-quenching mechanism. To enhance the versatility of the generation method and expand the color range, we developed a novel Q-body generation approach using tyrosinase-mediated site-specific conjugation to a tyrosine-rich hemagglutinin (HA)-tag. A single-chain variable fragment (scFv) against programmed cell death-ligand 1 (PDL1) was engineered with an N-terminal HA-tag and expressed in Escherichia coli with high yield and purity. Site-specific conjugation of four hydrazide-functionalized dyes with different emission wavelengths was achieved using recombinant tyrosinase. The resulting Q-bodies were evaluated via fluorescence-linked immunosorbent assay, all of which demonstrated antigen concentration-dependent fluorescence enhancement. Notably, the tetramethylrhodamine (TAMRA)-labeled Q-body showed the highest signal-to-background ratio, limit of detection of 0.51 ± 0.01 μg/mL. This HA-tag-mediated Q-body generation strategy offers a robust and versatile tool for the production of Q-bodies with broad dye compatibility, enabling sensitive and multiplexed fluorescent immunoassays.
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.