Modification of an ELISA-based procedure for affinity determination: correction necessary for use with bivalent antibody.
A recently described procedure for the evaluation of the affinity of monoclonal antibodies [Friguet et al., J. Immun. Meth. 77, 305-319 (1985)] uses an ELISA system to determine the quantity of free antibody present in a mixture of antigen and antibody. However, an intact IgG may bind antigen by either of two binding sites, and an IgG can bind to a solid-phase antigen whether one or two of its binding sites are free. Therefore, this procedure does not directly provide the concn of liganded binding sites, the quantity necessary for calculation of the thermodynamic association constant. A binomial probability distribution relates the fraction of liganded binding sites to the concn of unliganded, singly liganded, and doubly liganded IgG assuming that the binding of each Fab to antigen is independent. Simulated experiments were used to compare the apparent binding characteristics of bivalent IgG and monovalent Fab and to calculate apparent association constants in each case. It was found that the affinity of binding sites on intact IgG was underestimated by a factor of at least 2 and that the error was inversely related to the fraction of liganded binding sites. Binding site affinity of an antibody may be underestimated by several orders of magnitude. On the basis of binomial analysis, it is possible to convert apparent concns of bound IgG to actual concns of liganded binding site resulting in the calculation of valid association constants for intact IgG without alteration of the experimental protocol.
Stevens
F J
FJ
Division of Biological and Medical Research, Argonne National Laboratory, IL 60439.
D000915:Antibody Affinity; D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody; D004797:Enzyme-Linked Immunosorbent Assay; D007140:Immunoglobulin Fab Fragments / Q000276:immunology*; D007074:Immunoglobulin G / Q000378:metabolism*; D008954:Models, Biological
D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G