Solid phase disruption of fluid phase equilibrium in affinity assays with ELISA

Quantitation of antibody affinity for antigen can provide important information in assessment of the human immune response. Enzyme-linked immunosorbent assay (ELISA) methods are sufficiently sensitive to permit affinity measurements of low affinity, low titer antibody. The major assumption in using this method, that the solid phase does not influence the equilibrium of the fluid phase antibody-antigen interaction, is not strictly true. Hence, conditions must be chosen to minimize such interference, which can result in spuriously low values of affinity. The degree of solid phase interference was therefore quantitated for an ELISA that measures the affinity between antibody directed against the capsular polysaccharide of Haemophilus influenzae type b, using a monovalent 3-unit oligosaccharide prepared from polysaccharide. The solid phase antigen density has the greatest effect upon disruption of the fluid phase. The concentration of antibody chosen had a measurable but modest effect on the outcome of ELISA, whereas the time of incubation of the antibody and oligosaccharide had no demonstrable effect. By choosing the conditions of the assay carefully, it was possible to minimize interference from the solid phase, so that the measured affinity of anticapsular polysaccharide antibody for monovalent antigen was similar to that obtained with the more traditional precipitation method.