The effect of divalent and univalent binding on antibody titration curves in solid-phase ELISA

This paper describes the influence of antigen coating concentration, epitope density per antigen molecule and anti-immunoglobulin reagents on antibody titration curves in solid-phase ELISA. Based on results obtained with fluorescein as the hapten and monoclonal anti-fluorescein antibody, which were confirmed in another antigen-antibody system, it is concluded that: (a) Antibody titration curves are independent of antigen-coating concentration in a limited range of concentrations only. (b) The complex between one antibody and two epitopes ('divalent binding') is more stable than the complex between one antibody and one epitope ('univalent binding). The ratio between divalent and univalent binding depends on the epitope density per antigen molecule and on the antigen-coating concentration. (c) The prozone phenomenon can be explained by an increased instability of plate bound antibodies due to a shift from divalent to univalent binding. (d) In solid-phase ELISA a correct evaluation of the antiserum specificity can be performed only if it is ascertained that all target antigens are coated under saturating conditions.