Application of a sol particle immunoassay to the determination of affinity constants of monoclonal antibodies.
The affinity constants (Ka) 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. Ka 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 Ka 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.
Clinical Lab Systems Research Unit, Organon Teknika B.V., Boxtel, The Netherlands.
R
R
van Erp
T C
TC
Gribnau
A P
AP
van Sommeren
H P
HP
Bloemers
D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity; D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody; D006063:Chorionic Gonadotropin / Q000276:immunology*; D007118:Immunoassay; D015203:Reproducibility of Results
D000911:Antibodies, Monoclonal; D006063:Chorionic Gonadotropin