DETERMINATION OF THE DISSOCIATION-CONSTANT OF PHOSVITIN-ANTI-PHOSPHOSERINE INTERACTION BY AFFINITY CAPILLARY ELECTROPHORESIS

We used affinity capillary electrophoresis (ACE) to study the interact ion of a monoclonal anti-phosphoserine antibody (mAb) to a homopolyval ent antigen (hpAg), phosvitin. A model system, which allows the measur ement of the true dissociation constant (K-d) in Ag excess based on me asurement of migration shifts of mAb-hpAg complexes at different Ag co ncentrations in solution, is presented for the study of the interactio ns between a mAb and an Ag that has identical determinants, The experi mental value of K-d (22.4 X 10(-6) M) obtained by ACE is shown to be i n close agreement with the value (17.8 x 10(-6) M) obtained by the con ventional immunoassay based on indirect competition enzyme-linked immu nosorbent assay (ELISA). Moreover, the K(d)s of mAb-hpAg complexes wer e measured and shown to be independent of the applied electrical field strength, Thus, under conditions where the total Ag concentration is in large excess over the total Ab concentration and when certain requi rements are fulfilled, this method offers the advantage of dealing wit h the determination of K-d for unlabeled mAb and homopolymeric Ag mole cules in free solution rather than at the liquid-solid interface, (C) 1997 Academic Press.