{"title":"<i>Luciola mingrelica</i> firefly luciferase as a marker in bioluminescent immunoassays.","authors":"Galina Yu Lomakina, Natalia N Ugarova","doi":"10.1007/s12551-023-01115-z","DOIUrl":null,"url":null,"abstract":"<p><p>Chemical modification of the enzymes with biospecific macromolecules is used in various fields of biotechnology to impart new functions or improve their properties and is a fast and convenient way to get the final products. The preparation of highly active, stable, and functionally active conjugates of the thermostable luciferase through the NH<sub>2</sub>-groups or free SH-groups of the enzyme with target molecules of different molecular weight (albumin, avidin from chicken eggs, antibodies, and progesterone) is described. The obtained conjugates were successfully tested as a reporter in bioluminescent immunoassay for the detection of the molecules and pathogens. Thus, the luc-albumin (Luc-Alb) and luc-insulin (Luc-Ins) conjugates were used in competitive ELISA for the detection of an analyte (albumin or insulin) in the samples. Luc-progesterone (Luc-Pg) was used in the rapid homogeneous immunoassay of progesterone by the BRET technique with the detection limit of 0.5 ng/ml. Luciferase conjugates with avidin (Luc-Avi) and secondary and primary antibodies (Luc-RAM and Luc-Sal) were used for enzyme immunoassay detection of <i>Salmonella paratyphi A</i> cells with the cell detection limit of 5 × 10<sup>4</sup> CFU/ml. To reduce the detection limit of <i>Salmonella</i> cells, we developed a pseudo-homogeneous bioluminescent enzyme immunoassay of cells using a new matrix for the analyte capture-polystyrene microparticles coated with Pluronic F108, covalently labeled with Sal antibodies. This allowed to achieve efficient trapping of cells from solution, significantly reduced nonspecific sorption and decreased the cell detection limit to 2.7 × 10<sup>3</sup> CFU/ml without prior concentration of the sample. The methodology that was developed in this study can be applied for the development of novel bioanalytical systems based on firefly luciferases.</p>","PeriodicalId":4,"journal":{"name":"ACS Applied Energy Materials","volume":" ","pages":"955-962"},"PeriodicalIF":5.4000,"publicationDate":"2023-08-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10643422/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"ACS Applied Energy Materials","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1007/s12551-023-01115-z","RegionNum":3,"RegionCategory":"材料科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2023/10/1 0:00:00","PubModel":"eCollection","JCR":"Q2","JCRName":"CHEMISTRY, PHYSICAL","Score":null,"Total":0}
引用次数: 0
Abstract
Chemical modification of the enzymes with biospecific macromolecules is used in various fields of biotechnology to impart new functions or improve their properties and is a fast and convenient way to get the final products. The preparation of highly active, stable, and functionally active conjugates of the thermostable luciferase through the NH2-groups or free SH-groups of the enzyme with target molecules of different molecular weight (albumin, avidin from chicken eggs, antibodies, and progesterone) is described. The obtained conjugates were successfully tested as a reporter in bioluminescent immunoassay for the detection of the molecules and pathogens. Thus, the luc-albumin (Luc-Alb) and luc-insulin (Luc-Ins) conjugates were used in competitive ELISA for the detection of an analyte (albumin or insulin) in the samples. Luc-progesterone (Luc-Pg) was used in the rapid homogeneous immunoassay of progesterone by the BRET technique with the detection limit of 0.5 ng/ml. Luciferase conjugates with avidin (Luc-Avi) and secondary and primary antibodies (Luc-RAM and Luc-Sal) were used for enzyme immunoassay detection of Salmonella paratyphi A cells with the cell detection limit of 5 × 104 CFU/ml. To reduce the detection limit of Salmonella cells, we developed a pseudo-homogeneous bioluminescent enzyme immunoassay of cells using a new matrix for the analyte capture-polystyrene microparticles coated with Pluronic F108, covalently labeled with Sal antibodies. This allowed to achieve efficient trapping of cells from solution, significantly reduced nonspecific sorption and decreased the cell detection limit to 2.7 × 103 CFU/ml without prior concentration of the sample. The methodology that was developed in this study can be applied for the development of novel bioanalytical systems based on firefly luciferases.
期刊介绍:
ACS Applied Energy Materials is an interdisciplinary journal publishing original research covering all aspects of materials, engineering, chemistry, physics and biology relevant to energy conversion and storage. The journal is devoted to reports of new and original experimental and theoretical research of an applied nature that integrate knowledge in the areas of materials, engineering, physics, bioscience, and chemistry into important energy applications.