人微粒体环氧化物水解酶双纳米体夹心酶联免疫吸附测定的固定化和信号扩增策略

IF 6.7 1区 化学 Q1 CHEMISTRY, ANALYTICAL
Qiyi He, Bofeng Pan, Mark McCoy, Junkang Pan, Zhihao Xu, Christophe Morisseau, Gang Sun, Dongyang Li* and Bruce D. Hammock*, 
{"title":"人微粒体环氧化物水解酶双纳米体夹心酶联免疫吸附测定的固定化和信号扩增策略","authors":"Qiyi He,&nbsp;Bofeng Pan,&nbsp;Mark McCoy,&nbsp;Junkang Pan,&nbsp;Zhihao Xu,&nbsp;Christophe Morisseau,&nbsp;Gang Sun,&nbsp;Dongyang Li* and Bruce D. Hammock*,&nbsp;","doi":"10.1021/acs.analchem.4c0450510.1021/acs.analchem.4c04505","DOIUrl":null,"url":null,"abstract":"<p >The microsomal epoxide hydrolase (mEH) is important in the detoxification of carcinogens in the liver and other tissues but is also a blood biomarker of hepatitis and liver cancer. Improved analytical methods are needed for the study of its role in the metabolism of xenobiotics and endogenous roles as a blood biomarker of diseases. The development of a double nanobody sandwich ELISA offers significant improvements over traditional polyclonal or monoclonal antibody-based assays, enhancing both the homogeneity and the stability of assay production. This study focuses on selecting and optimizing nanobody pairs for detecting human mEH. Four high-affinity nanobodies were identified and tested for thermal stability. Combinations of these nanobodies were evaluated, revealing that the MQ4–MQ30 pair achieved the best performance with a limit of detection (LOD) of 1 ng/mL. Additionally, polyHRP was also employed for signal amplification, enhancing detection capabilities despite challenges related to the small size and single epitope recognition of the nanobodies. Comparative studies using microplates and NHS@MF membranes were also performed. The superior performance of the NHS@MF membranes highlighted their potential as a promising alternative for point-of-care testing. The assay exhibited high specificity for human mEH and minimal cross-reactivity with related enzymes and effectively addressed matrix effects in plasma and tissue samples. These findings underscore the potential of double nanobody sandwich ELISAs for reliable and sensitive biomarker detection.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"96 49","pages":"19605–19614 19605–19614"},"PeriodicalIF":6.7000,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.analchem.4c04505","citationCount":"0","resultStr":"{\"title\":\"Strategies for the Immobilization and Signal Amplification of a Double Nanobody Sandwich ELISA for Human Microsomal Epoxide Hydrolase\",\"authors\":\"Qiyi He,&nbsp;Bofeng Pan,&nbsp;Mark McCoy,&nbsp;Junkang Pan,&nbsp;Zhihao Xu,&nbsp;Christophe Morisseau,&nbsp;Gang Sun,&nbsp;Dongyang Li* and Bruce D. Hammock*,&nbsp;\",\"doi\":\"10.1021/acs.analchem.4c0450510.1021/acs.analchem.4c04505\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p >The microsomal epoxide hydrolase (mEH) is important in the detoxification of carcinogens in the liver and other tissues but is also a blood biomarker of hepatitis and liver cancer. Improved analytical methods are needed for the study of its role in the metabolism of xenobiotics and endogenous roles as a blood biomarker of diseases. The development of a double nanobody sandwich ELISA offers significant improvements over traditional polyclonal or monoclonal antibody-based assays, enhancing both the homogeneity and the stability of assay production. This study focuses on selecting and optimizing nanobody pairs for detecting human mEH. Four high-affinity nanobodies were identified and tested for thermal stability. Combinations of these nanobodies were evaluated, revealing that the MQ4–MQ30 pair achieved the best performance with a limit of detection (LOD) of 1 ng/mL. Additionally, polyHRP was also employed for signal amplification, enhancing detection capabilities despite challenges related to the small size and single epitope recognition of the nanobodies. Comparative studies using microplates and NHS@MF membranes were also performed. The superior performance of the NHS@MF membranes highlighted their potential as a promising alternative for point-of-care testing. The assay exhibited high specificity for human mEH and minimal cross-reactivity with related enzymes and effectively addressed matrix effects in plasma and tissue samples. These findings underscore the potential of double nanobody sandwich ELISAs for reliable and sensitive biomarker detection.</p>\",\"PeriodicalId\":27,\"journal\":{\"name\":\"Analytical Chemistry\",\"volume\":\"96 49\",\"pages\":\"19605–19614 19605–19614\"},\"PeriodicalIF\":6.7000,\"publicationDate\":\"2024-11-22\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://pubs.acs.org/doi/epdf/10.1021/acs.analchem.4c04505\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Analytical Chemistry\",\"FirstCategoryId\":\"92\",\"ListUrlMain\":\"https://pubs.acs.org/doi/10.1021/acs.analchem.4c04505\",\"RegionNum\":1,\"RegionCategory\":\"化学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"CHEMISTRY, ANALYTICAL\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Analytical Chemistry","FirstCategoryId":"92","ListUrlMain":"https://pubs.acs.org/doi/10.1021/acs.analchem.4c04505","RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"CHEMISTRY, ANALYTICAL","Score":null,"Total":0}
引用次数: 0

摘要

微粒体环氧化物水解酶(mEH)在肝脏和其他组织中的致癌物解毒中很重要,但也是肝炎和肝癌的血液生物标志物。需要改进的分析方法来研究其在外源性代谢中的作用以及作为疾病血液生物标志物的内源性作用。双纳米体夹心酶联免疫吸附试验的开发与传统的基于多克隆或单克隆抗体的酶联免疫吸附试验相比有了显著的改进,提高了酶联免疫吸附试验的均匀性和稳定性。本研究的重点是选择和优化用于人体mEH检测的纳米体对。鉴定了四种高亲和纳米体,并对其热稳定性进行了测试。对这些纳米体的组合进行了评估,结果表明MQ4-MQ30对的检测限(LOD)为1 ng/mL,性能最佳。此外,polyHRP还用于信号扩增,增强了检测能力,尽管存在与纳米体的小尺寸和单表位识别相关的挑战。使用微孔板和NHS@MF膜也进行了比较研究。NHS@MF膜的优越性能突出了它们作为即时检测的有希望的替代方案的潜力。该方法对人mEH具有很高的特异性,与相关酶的交叉反应性最小,并有效地解决了血浆和组织样品中的基质效应。这些发现强调了双纳米体夹心elisa在可靠和敏感的生物标志物检测方面的潜力。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Strategies for the Immobilization and Signal Amplification of a Double Nanobody Sandwich ELISA for Human Microsomal Epoxide Hydrolase

The microsomal epoxide hydrolase (mEH) is important in the detoxification of carcinogens in the liver and other tissues but is also a blood biomarker of hepatitis and liver cancer. Improved analytical methods are needed for the study of its role in the metabolism of xenobiotics and endogenous roles as a blood biomarker of diseases. The development of a double nanobody sandwich ELISA offers significant improvements over traditional polyclonal or monoclonal antibody-based assays, enhancing both the homogeneity and the stability of assay production. This study focuses on selecting and optimizing nanobody pairs for detecting human mEH. Four high-affinity nanobodies were identified and tested for thermal stability. Combinations of these nanobodies were evaluated, revealing that the MQ4–MQ30 pair achieved the best performance with a limit of detection (LOD) of 1 ng/mL. Additionally, polyHRP was also employed for signal amplification, enhancing detection capabilities despite challenges related to the small size and single epitope recognition of the nanobodies. Comparative studies using microplates and NHS@MF membranes were also performed. The superior performance of the NHS@MF membranes highlighted their potential as a promising alternative for point-of-care testing. The assay exhibited high specificity for human mEH and minimal cross-reactivity with related enzymes and effectively addressed matrix effects in plasma and tissue samples. These findings underscore the potential of double nanobody sandwich ELISAs for reliable and sensitive biomarker 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
Analytical Chemistry
Analytical Chemistry 化学-分析化学
CiteScore
12.10
自引率
12.20%
发文量
1949
审稿时长
1.4 months
期刊介绍: Analytical Chemistry, a peer-reviewed research journal, focuses on disseminating new and original knowledge across all branches of analytical chemistry. Fundamental articles may explore general principles of chemical measurement science and need not directly address existing or potential analytical methodology. They can be entirely theoretical or report experimental results. Contributions may cover various phases of analytical operations, including sampling, bioanalysis, electrochemistry, mass spectrometry, microscale and nanoscale systems, environmental analysis, separations, spectroscopy, chemical reactions and selectivity, instrumentation, imaging, surface analysis, and data processing. Papers discussing known analytical methods should present a significant, original application of the method, a notable improvement, or results on an important analyte.
×
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