Relationships between liquid- and solid-phase antibody association characteristics: implications for the use of competitive ELISA techniques to map the spatial location of idiotopes.
A liquid-phase assay system based on small-zone size-exclusion chromatography was used to examine the binding of a monoclonal anti-idiotopic antibody, F6, to its idiotope on the murine plasmacytoma IgA, TEPC-15. Chromatographic behavior revealed a strong association between T-15 and F6, which was previously characterized by solid-phase immunoassay as recognizing a nonbinding site epitope of the T-15. This chromatographic pattern suggests that the inability of the hapten phosphorylcholine to inhibit the anti-idiotope:idiotope relationship in solid-phase immunoassay might be equally explained by the low affinity of the hapten relative to the high affinity of the anti-idiotope antibody. Bivalent interactions between solid-phase IgA and liquid-phase IgG should enhance the binding of the anti-idiotope to an extent that would prevent the hapten from dissociating the complex. Under these solid-phase assay conditions, observation of hapten inhibition may, in some cases, indicate site specificity, but absence of inhibition cannot be interpreted. Computer simulations of solid-phase hapten inhibition scenarios were used to evaluate the qualitative nature of binding inhibition profiles expected under various conditions of liquid- and solid-phase reactant affinities and concentrations. The apparently unusual inhibition curves previously observed in the T-15:anti-T-15 studies in which the degree of binding inhibition by hapten appeared to be independent of anti-idiotope concentration may be predictable in cases of excess solid-phase epitope; the plateau inhibition value is a function of relative affinity constants and concentrations of solid-phase and inhibitor components. The results additionally suggest that the affinity of a liquid-phase antibody may modulate the effective concentration of solid-phase epitope.
D000915:Antibody Affinity; D001667:Binding, Competitive; D004306:Dose-Response Relationship, Immunologic; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000939:Epitopes; D006241:Haptens; D007070:Immunoglobulin A / Q000276:immunology; D007130:Immunoglobulin Idiotypes / Q000276:immunology*; D012995:Solubility
D000939:Epitopes; D006241:Haptens; D007070:Immunoglobulin A; D007130:Immunoglobulin Idiotypes