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

A procedure is described which allows the characterization of enzyme by a h ybrid approach using an enzyme and an antibody. The presented method is rel ated to the affinity determination of antibodies by the 'affinity in soluti on' procedure for BIAcore. The antibody is used as an indicator for the con centration of substrate, which is also the antigen. A mixture of enzyme, su bstrate and antibody is incubated, and an aliquot of this solution is injec ted periodically into a flowcell containing immobilized substrate, which is bound by the antibody, but not cleaved by the enzyme. The chosen initial c oncentration of substrate inhibits the binding of antibody to the immobiliz ed substrate by 90%. During the enzymatic reaction, increased amounts of an tibody bind to the surface, as the substrate concentration is decreased. Wi th this method, the cleavage of creatinine with creatinine iminohydrolase ( 6 mU/ml) was monitored for up to 11 h. A recently developed monoclonal anti body against creatinine was used as the indicating protein. For the calcula tion 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. All rights reserved.