Determination of dissociation constants of anti-ALP monoclonal antibodies by an ELISA - based method.

Abstract

Dissociation constant (Kd) is of major significance in immunoassay. Since affinity may be influenced by the immunoassay methodology it is important to determine this parameter under the condition of the assay used. To employ a rapid and simple ELISA- based method for measuring dissociation constants of two Alkaline Phosphatase (ALP) specific MAbs (A1G8F7 and A1G9G3) established in our laboratory. A simple and rapid method on enzyme- linked immunosorbent assay (ELISA) was developed for measuring the dissociation constant of antibody – antigen reactions. In this method the ability of increasing amounts of antigen to bind to antibody measured in the fluid phase. Based on the data obtained from this study, the dissociation constants of A1G8F7 and A1G9G3 MAbs were 3.8 × 10−9 and 4.3 × 10−9 M, respectively. A1G8F7 and A1G9G3 MAbs with reasonably high affinity are suggested for used in very immunoassay such as ELISA, immunocytochemistry and immunihistochemistry (APAAP method).