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, Bofeng Pan, Mark McCoy, Junkang Pan, Zhihao Xu, Christophe Morisseau, Gang Sun, Dongyang Li* and Bruce D. Hammock*, ","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, Bofeng Pan, Mark McCoy, Junkang Pan, Zhihao Xu, Christophe Morisseau, Gang Sun, Dongyang Li* and Bruce D. Hammock*, \",\"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. 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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.
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.