Enzyme-linked coagulation assay: a clot-based, solid-phase assay for thrombin |
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| Authors: | G J Doellgast H Rothberger |
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| Institution: | 1. Department of Pharmacognosy, School of Pharmaceutical Sciences, Lovely Professional University, Phagwara 144411, Punjab, India;2. Department of Pharmacy, Uttarakhand Technical University, Dehradun 248007, Uttarakhand, India;3. Department of Pharmaceutics, School of Pharmacy, Graphic Era Hill University, Dehradun 248001, Uttarakhand, India;4. Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Lovely Professional University, Phagwara 144411, Punjab, India;5. Shree SK Patel College of Pharmaceutical Education and Research, Ganpat University, Mehsana 384012, Gujarat, India;6. Department of Pharmaceutics, School of Pharmaceutical Sciences, Lovely Professional University, Phagwara 144411, Punjab, India;7. College of Osteopathic Medicine, Lake Erie College of Osteopathic Medicine, Bradenton, FL 34211, USA;1. GEOGLOB, University of Sfax, 3038 Sfax, Tunisia;2. Institute of Environmental Science and Technology (ICTA), Universitat Autònoma de Barcelona (UAB), 08193, Bellaterra, Barcelona, Spain;3. GZN, GeoZentrum Nordbayern, Universität Erlangen-Nürnberg, Loewenichstr. 28, D-91054 Erlangen, Germany;4. ISMAR-CNR, Istituto di Scienze Marine, Consiglio Nazionale delle Ricerche, Via Gobetti 101, 40129 Bologna, Italy;5. ICREA, Catalan Institution for Research and Advanced Studies, Pg. Lluís Companys 23, 08010 Barcelona, Spain;6. School of Earth Sciences and UWA Oceans Institute, University of Western Australia, Crawley, Australia;7. Laboratoire des Sciences du Climat et de l''Environnement, LSCE/IPSL, UMR 8212 CNRS-CEA-UVSQ, 91198 Gif-sur-Yvette, France;8. Oceans Graduate School, UWA Oceans Institute, and ARC Centre of Excellence for Coral Reef Studies, University of Western Australia, Crawley, Australia;9. Biology Department, Woods Hole Oceanographic Institute, 266 Woods Hole Road, MA 02543, USA;10. Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Naples, Italy;11. Department of Evolutionary Biology, Ecology and Environmental Sciences, Institut de Recerca de la Biodiversitat (IRBIO), University of Barcelona, Av. Diagonal 643, 08028 Barcelona, Spain;12. Lamont-Doherty Earth Observatory, Columbia University, 61 Route 9W, Palisades, NY 10964, USA |
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| Abstract: | A new, solid-phase microtiter plate assay for thrombin has been developed, using fibrinogen bound to wells of a microtiter plate and peroxidase-fibrinogen in solution as an indicator system. When small amounts of thrombin are added to the mixture, peroxidase-fibrin and plate-bound fibrin are formed, and the peroxidase-fibrin binds to the plate-bound fibrin. The amount of peroxidase-fibrin binding is proportional to the thrombin concentration and time of incubation. Using this assay, thrombin was measured at concentrations as low as 0.25 ng/ml (0.006 nM) in 150 microliter of sample. In the presence of the specific inhibitors benzamidine and D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone, the thrombin activity is reduced, at relative concentrations of inhibitors consistent with their affinities and mechanisms of action. The enzyme-linked coagulation assay is generally useful as a highly sensitive and convenient alternative to conventional "clot-based" tests of coagulation. |
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