Allowance for antibody bivalence in the determination of association rate constants by kinetic exclusion assay

• Published in: Analytical biochemistry Vol. 441; no. 2; pp. 214 - 217
• Main Author: Winzor, Donald J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15-10-2013
• Subjects: antibodies; Antibodies, Bispecific - immunology; Antibody bivalence; Antigen-Antibody Complex; Antigen-Antibody Reactions; antigens; Antigens - immunology; Antigen–antibody interaction; Association rate constant; Binding Sites, Antibody; dissociation; fluorescence; Kinetic exclusion assay; Kinetics; probability; Reacted site probability; thermodynamics; Antibody bivalence; Kinetic exclusion assay; Association rate constant; Reacted site probability; Antigen–antibody interaction
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2013.06.020

This investigation completes the amendment of theoretical expressions for the characterization of antigen–antibody interactions by kinetic exclusion assay—an endeavor that has been marred by inadequate allowance for the consequences of antibody bivalence in its uptake by the affinity matrix (immobilized antigen) that is used to ascertain the fraction of free antibody sites in a solution with defined total concentrations of antigen and antibody. A simple illustration of reacted site probability considerations in action confirms that the square root of the fluorescence response ratio, RAg/Ro, needs to be taken in order to determine the fraction of unoccupied antibody sites, which is the parameter employed to describe the kinetics of antigen uptake in the mixture of antigen and antibody with defined initial composition. The approximately 2-fold underestimation of the association rate constant (ka) that emanates from the usual practice of omitting the square root factor gives rise to a corresponding overestimate of the equilibrium dissociation constant (Kd)—a situation that is also encountered in the thermodynamic characterization of antigen–antibody interactions by kinetic exclusion assay.