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Enzyme kinetic assays with surface plasmon resonance (BIAcore) based on competition between enzyme and creatinine antibody

Item Type:Article
Title:Enzyme kinetic assays with surface plasmon resonance (BIAcore) based on competition between enzyme and creatinine antibody
Creators Name:Stoecklein, W.F.M. and Behrsing, O. and Scharte, G. and Micheel, B. and Benkert, A. and Schoessler, W. and Warsinke, A. and Scheller, F.W.
Abstract:A procedure is described which allows the characterization of enzyme by a hybrid approach using an enzyme and an antibody. The presented method is related to the affinity determination of antibodies by the 'affinity in solution' procedure for BIAcore. The antibody is used as an indicator for the concentration of substrate, which is also the antigen. A mixture of enzyme, substrate and antibody is incubated, and an aliquot of this solution is injected periodically into a flowcell containing immobilized substrate, which is bound by the antibody, but not cleaved by the enzyme. The chosen initial concentration of substrate inhibits the binding of antibody to the immobilized substrate by 90%. During the enzymatic reaction, increased amounts of antibody bind to the surface, as the substrate concentration is decreased. With this method, the cleavage of creatinine with creatinine iminohydrolase (6 mU/ml) was monitored for up to 11 h. A recently developed monoclonal antibody against creatinine was used as the indicating protein. For the calculation of enzyme activity, the signals were compared with a calibration curve for inhibition of antibody binding to the chip by creatinine in solution. (C) 2000 Elsevier Science S.A.
Keywords:BIAcore, Creatinine, Creatinine Iminohydrolase, Monoclonal Antibody, Surface Plasmon Resonance
Source:Biosensors and Bioelectronics
ISSN:0956-5663
Publisher:Elsevier Advanced technology (England)
Volume:15
Number:7-8
Page Range:377-382
Date:1 October 2000
Official Publication:https://doi.org/10.1016/S0956-5663(00)00094-4
PubMed:View item in PubMed

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