The role of pH in modified ELISA procedures used for the estimation of functional antibody affinity

Solid phase assays for the measurement of functional antibody affinity are increasingly being used in both clinical and research settings. The majority of such assays employ a chemical reagent to disturb antibody binding but relatively little is known about the properties of such reagents and the basis of their effect on antigen-antibody binding. We have evaluated the diethylamine (DEA) ELISA procedure for the measurement of functional antibody affinity in two independent assays, one for functional human IgG subclass affinity to an organism, Moraxella catarrhalis, and the other for measuring functional affinity of mouse monoclonals specific for the cat allergen Fel d I. DEA was shown to increase the pH of the buffering solution and it was this rise in pH that affected antibody binding. Alkaline buffer and DEA were equally efficient in the inhibition of binding of both the human IgG subclasses and the two mouse monoclonal antibodies to the solid phase. In contrast, pH was shown to have no role in the chaotropic effect of the ion, thiocyanate.