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00925njm 22002777a 4500 dc Toraño, Alfredo author Moreno-Iruela, Inmaculada author Infantes-Lorenzo, Jose Antonio author Dominguez-Rodriguez, Mercedes author 2024-11 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 Kt (= Ks) 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 Ki (= Kd) is the time required to double the slope or halve the Vmax of the Lineweaver-Burk plot. The Kt values of the time courses were doubled (2 x Kt) and normalized by dividing by the total reaction time to obtain a unitless factor which, when multiplied by the concentration of HRP, gives the Ki. The affinity constant of mAb SIM 28 was determined from ELISA data (n = 16) by three methods: i) doubling of Kt, ii) Beatty equation (Kaff = (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) Kaff = 0.23 ± 0.03 × 109 M-1 (Kd = 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 Kt method, is a repeatable and direct approach to mAb affinity determination. Toraño A, Moreno I, Infantes JA, Domínguez M. 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. J Immunol Methods. 2024 Nov;534:113756. 0022-1759 https://hdl.handle.net/20.500.12105/26429 39265885 10.1016/j.jim.2024.113756 1872-7905 Journal of immunological methods Monoclonal antibody affinity determination Noncompetitive inhibition kinetics Solid-phase time-course saturation ELISA 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