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 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 con...

Full description

Saved in:
Bibliographic Details
Published in:Biosensors & bioelectronics 2000-10, Vol.15 (7), p.377-382
Main Authors: Stöcklein, Walter F.M, Behrsing, Olaf, Scharte, Gudrun, Micheel, Burkhard, Benkert, Alexander, Schößler, Werner, Warsinke, Axel, Scheller, Frieder W
Format: Article
Language:English
Subjects:
Aminohydrolases - metabolism
Antibody Affinity
BIAcore
Biological and medical sciences
Biosensors
Biotechnology
Creatinine
Creatinine - immunology
Creatinine - metabolism
Creatinine iminohydrolase
Fundamental and applied biological sciences. Psychology
Kinetics
Methods. Procedures. Technologies
Monoclonal antibody
Surface Plasmon Resonance
Various methods and equipments
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:0956-5663
1873-4235
DOI:10.1016/S0956-5663(00)00094-4