Determination of an antibody-antigen binding constant by enzyme immunoassay and a theory for analysis of competitive binding of two ligands to heterogeneous receptor.
A method for determining antigen-antibody binding constants by using enzyme-labeled antigens has been developed. In the measurement, enzyme-labeled and unlabeled antigens (Ag* and Ag) were allowed to compete in binding to the antibody (Ab) under conditions where Ag* much less than Ab much less than Ag. The data were analyzed according to a new theory developed for the analysis of competitive binding of two ligands to a heterogeneous receptor. The theory indicates that the binding degree of a labeled ligand measured at various concentrations of the receptor can be used to prepare a standard curve relating the binding degree of the labeled ligand and the average of the concentrations of the free receptor components which are in binding equilibrium with another unlabeled ligand. For homogeneous receptors, the method gives usual binding constants for the unlabeled ligand, but for heterogeneous receptors, it gives a new type of average binding constant for the unlabeled ligand in which the contribution of each receptor component is amplified in proportion to its affinity against the labeled ligand. This average binding constant was named the "affinity-average binding constant." A rabbit anti-blasticidin S (BLS) antiserum analyzed by the present method using beta-galactosidase-labeled BLS as the labeled ligand was found to be fairly homogeneous with respect to the affinity and to have a binding constant of 1.48 +/- 0.24 (S.D.) X 10(8) M-1 for unlabeled BLS.
D000818:Animals; D000937:Antigen-Antibody Reactions; D001667:Binding, Competitive; D006146:Guanidines / Q000276:immunology; D007124:Immunoenzyme Techniques; D007700:Kinetics; D008024:Ligands; D008954:Models, Biological; D009705:Nucleosides; D011817:Rabbits; D001616:beta-Galactosidase / Q000378:metabolism
D006146:Guanidines; D008024:Ligands; D009705:Nucleosides; C004500:blasticidin S; D001616:beta-Galactosidase