Application of a Sol Particle Immunoassay to the Determination of Affinity Constants of Monoclonal Antibodies

The affinity constants (K a ) of monoclonal antibodies (MAb) for binding to their corresponding antigens (Ag), unlabelled and in buffered solution were determined by the following procedure: 1. Incubation of MAb (fixed concentration) with Ag (concentration dilution series). 2. Rapid bound/free separation by adding immobilized second antibody, followed by centrifugation. 3. Determination of free Ag in the supernatant using a gold sol particle agglutination immunoassay (SPIA) in a microtitration plate format. 4. Calculations and interpretation were based on Scatchard and Sips plots. K a values found by this procedure were found to be similar to those obtained by a radio-immunoassay (RIA) procedure. The present method avoids possible artefacts in K a values introduced by the procedure or chemical modification due to labelling of MAb or Ag. It enables rapid, simultaneous screening of a considerable number of different MAbs under non-specialized (i.e. RIA) laboratory conditions.