title: Application of ADA1 as a new marker enzyme in sandwich ELISA to study the effect of adenosine on activated monocytes.
abstract: Enzyme-linked immunosorbent assay (ELISA) is a valuable technique to detect antigens in biological fluids. Horse radish peroxidase (HRP) is one of the most common enzymes used for signal amplification in ELISA. Despite new advances in technology, such as a large-scale production of recombinant enzymes and availability of new detection systems, limited research is devoted to finding alternative enzymes and their substrates to amplify the ELISA signals. Here, HRP-avidin was substituted with the human adenosine deaminase (hADA1)-streptavidin complex and adenosine as a detection system in commercial ELISA kits. The hADA1 ELISA was successfully used to demonstrate that adenosine, bound to A1 and A3 adenosine receptors, increases cytokine secretion by LPS activated monocytes. We show that hADA1-based ELISA has the same sensitivity, and also provides identical results, as HRP ELISA. In addition, the sensitivity of hADA1-based ELISA could be easily adjusted by changing the adenosine concentration and the incubation time. Therefore, hADA1 could be used as a detection enzyme with any commercial ELISA kit with a wide range of concentration of antigens.
authors: Turku Centre for Biotechnology, University of Turku, Tykistokatu 6, 20520 Turku, Finland.
authors: Chengqian
authors: C
authors: Liu
authors: Turku Centre for Biotechnology, University of Turku, Tykistokatu 6, 20520 Turku, Finland.
authors: Maksym
authors: M
authors: Skaldin
authors: Tulane National Primate Research Center, 18703 Three River Road, Covington, LA 70433, USA.|Department of Public Health, John A. Burns School of Medicine, University of Hawaii at Manoa, 1960 East West Road, Biomed, Honolulu, HI 96822, USA.
authors: Chengxiang
authors: C
authors: Wu
authors: Department of Public Health, John A. Burns School of Medicine, University of Hawaii at Manoa, 1960 East West Road, Biomed, Honolulu, HI 96822, USA.
authors: Yuanan
authors: Y
authors: Lu
authors: Turku Centre for Biotechnology, University of Turku, Tykistokatu 6, 20520 Turku, Finland.
authors: Andrey V
authors: AV
authors: Zavialov
mesh_terms: D000241:Adenosine / Q000378:metabolism*; D000243:Adenosine Deaminase / Q000737:chemistry / Q000378:metabolism*; D015415:Biomarkers / Q000378:metabolism*; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008070:Lipopolysaccharides / Q000009:adverse effects; D009000:Monocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D012680:Sensitivity and Specificity; D019809:Streptavidin / Q000737:chemistry
chemical_list: D015415:Biomarkers; D008070:Lipopolysaccharides; D019809:Streptavidin; C548482:ADA protein, human; D000243:Adenosine Deaminase; D000241:Adenosine