An Overview of ELISA-Based Initial Velocity Methods to Measure the Immunoreactive Fraction, Association Rate, And Equilibrium Constants of Monoclonal Antibodies
Alfredo Toraño, Inmaculada Moreno*, José Antonio Infantes, Mercedes Domínguez
Unidad de Inmunología Microbiana, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid 28220, Spain
Three biochemical parameters—the on-rate (k+1), the dissociation rate (k-1), and the equilibrium (Ka) constants—describe the antibody interaction with a ligand. In addition, the antibody immune reactive fraction (IRF) is important because it affects k+1 and Ka constant values. The on-rate and dissociation constants are kinetic, but the equilibrium constant is a thermodynamic parameter, and the methods for their determination are different. Among the available methods to determine these parameters, only surface plasmon resonance (SPR)-based techniques allow calculation of the three antibody constants in a single experiment. However, conventional immunoassays are also suitable to determine antibody constants. Here, we describe some enzyme immunoassays (ELISA) methods developed in our laboratory to, i) quantitate mAb in culture supernatants from the initial velocity of the progress curve, ii) determine mAb IRF by a two-step ELISA method, iii) calculate mAb on-rate constant by kinetic ELISA based on time-course data of ligand binding to capture mAb, and, iv) describe a competitive-inhibition ELISA to measure the inhibition constant (Ki) of mAb binding to plate-bound ligand, in the presence of competing concentrations of mAb and soluble ligand.