A reliable method to estimate the association constant for a monoclonal antibody and a protein antigen by enzyme linked immunosorbent assay, ELISA.

The association constant Ka for mouse monoclonal antibody raised against human angiotensinogen was calculated using a mathematical model, SAM I. K1 represents the equilibrium constant for the binding of antibody to the solid phase with antigen previously absorbed. K2 represents the interaction between antibody and antigen in solution (Ag + Ab = AgAb). K3 represents binding to the antigen absorbed on the solid phase by an antigen-antibody complex. K4 represents the second binding of the antigen to the antigen-antibody complex (AgAb + AgAb = (Ag)2Ab). The model unveils cooperativity for the first (K1 and K2) and second (K3 and K4) binding of antigen to antibody. The model gives the association constant in a high affinity interaction between antigen and antibody.