title: Determination of the dissociation constant of phosvitin-anti-phosphoserine interaction by affinity capillary electrophoresis.
abstract: We used affinity capillary electrophoresis (ACE) to study the interaction of a monoclonal anti-phosphoserine antibody (mAb) to a homopolyvalent antigen (hpAg), phosvitin. A model system, which allows the measurement of the true dissociation constant (Kd) in Ag excess based on measurement of migration shifts of mAb-hpAg complexes at different Ag concentrations in solution, is presented for the study of the interactions between a mAb and an Ag that has identical determinants. The experimental value of Kd (22.4 x 10(-6) M) obtained by ACE is shown to be in close agreement with the value (17.8 x 10(-6) M) obtained by the conventional immunoassay based on indirect competition enzyme-linked immunosorbent assay (ELISA). Moreover, the Kds of mAb-hpAg complexes were 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 requirements are fulfilled, this method offers the advantage of dealing with the determination of Kd for unlabeled mAb and homopolymeric Ag molecules in free solution rather than at the liquid-solid interface.
authors.affiliation: Office for Research and Development, National Taiwan University College of Medicine, Taipei, Taiwan.
authors.forename: S
authors.initials: S
authors.lastname: Lin
authors.forename: I Y
authors.initials: IY
authors.lastname: Hsiao
authors.forename: S M
authors.initials: SM
authors.lastname: Hsu
mesh_terms: D000911:Antibodies, Monoclonal / Q000032:analysis / Q000378:metabolism*; D000936:Antigen-Antibody Complex / Q000032:analysis; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D019075:Electrophoresis, Capillary; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D008433:Mathematics; D018448:Models, Immunological; D010768:Phosphoserine / Q000276:immunology*; D010774:Phosvitin / Q000378:metabolism*; D011487:Protein Conformation
chemical_list: D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D010768:Phosphoserine; D010774:Phosvitin