Determination of antigen-antibody affinity by immunocapillary electrophoresis

Journal of Chromatography. a
N H Heegaard

Abstract

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.

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Citations

Mar 17, 1999·Journal of Molecular Recognition : JMR·N H Heegaard
Feb 10, 1998·Electrophoresis·G RippelJ Frank
Feb 10, 1998·Electrophoresis·K L Rundlett, D W Armstrong
Feb 10, 1998·Electrophoresis·V Dolnik
Apr 29, 1998·Electrophoresis·M T Bowser, D D Chen
Apr 10, 2002·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Niels H H Heegaard, Robert T Kennedy
May 23, 2002·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Marie TicháKarel Stulík
Apr 9, 2010·Analytical Chemistry·Ryan T TurgeonMichael T Bowser
Jun 5, 1998·Nucleic Acids Research·I I HamdanR D Waigh
Feb 22, 2014·Monoclonal Antibodies in Immunodiagnosis and Immunotherapy·Yaghoub SafdariHossein Zarei Jaliani
Oct 6, 2000·Methods : a Companion to Methods in Enzymology·J Z XingX C Le
Mar 29, 2002·Electrophoresis·Niels H H HeegaardDavid D Y Chen
Mar 29, 2002·Electrophoresis·Philip Britz-McKibbin, David D Y Chen
Sep 23, 2016·International Journal of Molecular Sciences·Marc H V Van Regenmortel
Nov 14, 2013·Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire·Missag Hagop Parseghian
Mar 11, 2018·Biochemistry. Biokhimii︠a︡·D V SotnikovB B Dzantiev
May 31, 1996·Journal of Chromatography. B, Biomedical Applications·M J SchmerrA L Jenny

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