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Influence of solid-phase antigen in competition enzyme-linked immunosorbent assays (ELISAs) on calculated
1Department of Medicine, SUNY/Health Science Center, Brooklyn 11203.
The competition ELISA method described in 1985 by Friguet and colleagues has frequently been used to determine dissociation constants (KD) of antigen-antibody reactions. Subsequently Stevens suggested a correction for the bivalency of IgG. In either case, the KD is assumed to vary only with the composition of competing fluid-phase antigen and consequently should not be affected by the solid-phase antigen. However, during the course of experiments defining epitopes to the V3 loop of human immunodeficiency virus type 1, both the composition and density of solid-phase antigen were capable of significantly influencing the calculated values. With one solid-phase antigen, the calculated KD was a function of antigen density. With a second solid-phase antigen, the calculated KD did not vary with the density. With the latter antigen or with low densities of the former, KDs calculated using the Stevens correction for bivalency were close to the estimate obtained by a radiolabeled peptide precipitation assay. Accordingly, since the density and composition of solid-phase antigen may alter the KD calculated from competition ELISAs, such estimates should be confirmed by a more readily interpretable immunological method.
The competition ELISA method described in 1985 by Friguet and colleagues has frequently been used to determine dissociation constants (KD) of antigen-antibody reactions. Subsequently Stevens suggested a correction for the bivalency of IgG. In either case, the KD is assumed to vary only with the composition of competing fluid-phase antigen and consequently should not be affected by the solid-phase antigen. However, during the course of experiments defining epitopes to the V3 loop of human immunodeficiency virus type 1, both the composition and density of solid-phase antigen were capable of significantly influencing the calculated values. With one solid-phase antigen, the calculated KD was a function of antigen density. With a second solid-phase antigen, the calculated KD did not vary with the density. With the latter antigen or with low densities of the former, KDs calculated using the Stevens correction for bivalency were close to the estimate obtained by a radiolabeled peptide precipitation assay. Accordingly, since the density and composition of solid-phase antigen may alter the KD calculated from competition ELISAs, such estimates should be confirmed by a more readily interpretable immunological method.