Description of a non-competitive ELISA based on time course analysis of ligand binding at saturation, and a direct method for calculating the affinity of monoclonal antibodies

Abstract

We present a time-course saturation ELISA for measuring the equilibrium constant of the monoclonal antibody (mAb) SIM 28 against horse radish peroxidase (HRP). The curves of HRP binding to a series of fixed mAb dilutions were plotted to completion, and the K_t (= K_s) value (time to occupy 50 % of the mAb paratopes) was determined for each mAb dilution and HRP concentration. Analysis of the kinetic mechanism of the reaction by Lineweaver-Burk and Hanes plots showed that the slope and y-intercept were affected, indicating that mAb ligand saturation follows non-competitive inhibition kinetics in this assay format. In this kinetics, the inhibition constant K_i (= K_d) is the time required to double the slope or halve the V_max of the Lineweaver-Burk plot. The K_t values of the time courses were doubled (2 x K_t) and normalized by dividing by the total reaction time to obtain a unitless factor which, when multiplied by the concentration of HRP, gives the K_i. The affinity constant of mAb SIM 28 was determined from ELISA data (n = 16) by three methods: i) doubling of K_t, ii) Beatty equation (K_aff = (n-1)/2 (n [HRP’]_t - [HRP]_t), and iii) SPR (Biacore) analysis. The calculated affinities (mean ± 95 % confidence limits) were i) 4.6 ± 0.67 × 10⁻⁹ M, ii) K_aff = 0.23 ± 0.03 × 10⁹ M⁻¹ (K_d = 4.8 ± 0.81 × 10⁻⁹ M), and iii) 4.3 ± 0.57 × 10⁻⁹ M, respectively. The similar results obtained with the three different techniques indicate that this time-course saturation ELISA, combined with the double K_t method, is a repeatable and direct approach to mAb affinity determination.