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.

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 (= K) 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.

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

We describe an ELISA method with which to determine monoclonal antibody (mAb) on-rate constants (k) 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 (k) and second order (k) 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 V/K tangent of the time-course curve, iv) Boeker's extrapolated-v method, v-vi) modified Hanes-Woolf and Lineweaver-Burk linear plots, vii) a LOS plot, and viii) initial velocity gradient.

Development of a competitive inhibition kinetic ELISA to determine the inhibition constant (K) of monoclonal antibodies.

Antibody-antigen interactions are mediated by the same molecular recognition mechanisms as those of an enzyme and its substrate. On this basis, we developed a competitive inhibition kinetic ELISA to measure monoclonal antibody (mAb) inhibition constants. Serially diluted samples of ligand (mAb) and inhibitor (soluble antigen) were incubated to equilibrium in ELISA plates coated with a fixed concentration of antigen (receptor).