Determination of antibody affinity by ELISA with a non-linear regression program. Evaluation of linearized approximations.
ELISA experiments based on competition between immobilized and soluble antibody for soluble antigen, and on the formation of a ternary complex of immobilized antibody, antigen and soluble antibody were used by Hoylaerts et al. (J. Immunol. Methods 126 (1990) 253-261) for the determination of dissociation constants. The dissociation constant was taken from linearized plots according to a theory that required several approximations. The effect of these approximations on the resulting dissociation constants has been investigated using two computer programs, CBEIA-C and CBEIA-S. Since most approximations that have to be made for linearization can be avoided by non-linear regression, the programs provide a more reliable basis for calculation of the dissociation constant. Experiments measuring formation of a ternary complex were found to be unsuitable for affinity determination.
Glaser
R W
RW
Department of Medical Immunology, Humboldt University Berlin, Medical School (Charité), Germany.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity / Q000276:immunology*; D000937:Antigen-Antibody Reactions / Q000276:immunology; D003198:Computer Simulation; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D012044:Regression Analysis; D012984:Software
D000911:Antibodies, Monoclonal