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
Year: 2024
Type: article
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 × 10 9 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. • Antibody affinity is a main parameter for assessing immunoglobulin function. • Monoclonal antibody affinity is a key factor in immunotherapy. • Antibody-ligand saturation may follow non-competitive inhibition kinetics.
Source: Journal of Immunological Methods
Language: English
Subfield: Radiology, Nuclear Medicine and Imaging
Field: Medicine
Domain: Health Sciences
Open Access status: hybrid
Estimated APC paid: $2,630
Awards: G1119046657, G2219082655
OpenAlex ID: W4402519733