The indirect ELISA for quantitation of specific antibody: analysis of antibody dilution curves.

In the enzyme-linked immunosorbent assay (ELISA), antibody dilution curves of immune sera from different individuals often reach plateaus or an initial flat phase at different absorbance (OD) levels. Two experimental models are presented as possible mechanisms for plateau formation. In both models it is assumed that the immune sera have sufficient quantities of antibody of similar affinity to saturate the antigenic determinants on the solid phase. In the first model specific antisera to two pure antigens, hemoglobin and transferrin, produced dose-response curves with separate plateau levels in ELISA. The comixture of these two antisera produced a plateau level which was equal to the sum of the plateau absorbance for each antigen. In the second model, dose-response curves were generated using immune sera with cross-reacting antibodies to a pure multivalent antigen (bovine serum albumin). Plateau regions at different absorbances were demonstrated which correlated with the degree of cross-reactivity of the antisera and the number of antigenic determinants recognized by antibody specificities in the immune sera. These models suggest that the initial plateau regions of the ELISA dose-response curve represents the summation of antibody specificities in an immune serum to some or all of the antigenic determinants of a complex antigen(s) bound to the solid phase. A new approach to the quantitation of antibody concentrations by ELISA is described.