A simple method for the determination of dissociation constants by displacement ELISA

Abstract

Previously, we have described a linearization procedure for the determination of dissociation constants of antigen–antibody interactions using data from enzyme-linked immunosorbent assays (ELISA) [J. Immunol. Methods 143 (1991) 119]. Here, we present a new linearization procedure-based partly on the former one—to evaluate quantitatively the data of displacement ELISAs. It renders possible the determination of dissociation constants of complexes formed between immobilised antigens and the displacing molecules without knowing the explicit concentration of the antigen and the dissociation constant and stoichiometry of the immunocomplex. In addition, determination of the concentration of specific antibody in the serum is not necessary since the equations use only the ratio of serum concentration and apparent dissociation constant of the immunocomplex (both of which can be expressed as serum dilutions). The method has been applied to determine the strength of immobilised hexokinase–tubulin interactions ( K d=1.2 μM) from a displacement ELISA using antihexokinase serum. The applicability and limitations of the procedure are discussed on the basis of both the experimental and theoretical data.