Monoclonal antibody on-rate constant determined from time-course data of ligand binding by capture ELISA: Evaluation of eight data analysis methods

Abstract

We describe an ELISA method with which to determine monoclonal antibody (mAb) on-rate constants (k+1) based on time-course data of ligand (L) binding to plate-bound mAb. The assay was performed in pseudo-first order kinetic conditions ([L] > > [mAb]) and at various starting ligand concentrations. Time-course initial velocity was analyzed by several methods to derive the pseudo-first order (kobs) and second order (k+1) association rate constants of the antibody; the methods included i) an exponential first order rate equation, ii) reaction half-time from the Michaelis-Menten relationship, iii) the Vmax/Km tangent of the time-course curve, iv) Boeker's extrapolated-vo method, v-vi) modified Hanes-Woolf and Lineweaver-Burk linear plots, vii) a LOS plot, and viii) initial velocity gradient. Due to k+1 value dispersion associated with the methods of analysis, the on-rate constant of mAb SIM 253–19 anti-cholera toxin was estimated as an average value of 1.79 ± 0.11 × 106 M−1 s−1, 95% CL (1.68–1.89) and 5.8 (%CV [coefficient of variation]), which is similar to the k+1 obtained by surface plasmon resonance, 1.60 ± 0.17 × 106 M−1 s −1 (mean ± half range). This kinetic ELISA is a sensitive, quantitative method by which to determine antibody association rate constants.