Determination of antigen-antibody affinity by immunocapillary electrophoresis.
The use of affinity capillary electrophoresis for the characterization of antigen-antibody interactions (immunocapillary electrophoresis) is shown using monoclonal antibodies against phosphotyrosine as a model system. The influence of the interaction kinetics on the peak profiles was demonstrated in experiments with addition of phosphotyrosine to the electrophoresis buffer. One of the two antibodies that were tested exhibited peak broadening while the other showed no change in peak shape but had a decreased mobility proportional to the amount of phosphotyrosine present. The migration shifts which were of the order 0.05 to 0.15 min at 439 V/cm were a consequence of the antibody-antigen complexes having a slower mobility than the non-complexed antibody. On the basis of measurement of migration shifts at different antigen concentrations, dissociation constants were estimated and shown to be independent on the applied field strength. Thus, when certain requirements are fulfilled, immuno-capillary electrophoresis is a fast and simple method for establishing binding characteristics of unlabelled antigen and antibody molecules under non-denaturing conditions and consumes minute amounts of sample.
Heegaard
N H
NH
Department of Autoimmunology, Statens Seruminstitut, Copenhagen, Denmark.
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002197:Capillary Action; D004586:Electrophoresis / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007158:Immunologic Techniques; D007700:Kinetics; D010768:Phosphoserine / Q000276:immunology; D019000:Phosphotyrosine; D014443:Tyrosine / Q000031:analogs & derivatives / Q000276:immunology
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D010768:Phosphoserine; D019000:Phosphotyrosine; D014443:Tyrosine