Immunoassays have been recently developed that measure the avidity of IgG antibodies to complex microbial antigens and are suitable for serologic diagnosis of infectious diseases. In these avidity ELISAs, protein-denaturing agents are applied either as diluents of patient sera to prevent the immune complexing of IgG (diluting principle), or the preformed complexes are treated with the protein denaturants (eluting principle). We compared four protein denaturants previously used in such assays, in a diagnostic avidity ELISA for rubella IgG. Diethylamine, guanidine, thiocyanate, or urea were applied, by either principle at various concentrations, and thiocyanate, or urea were applied, by either principle at various concentrations, and thiocyanate at an optimum pH. Patient sera obtained recently after primary infection were distinguished from sera representing past rubella immunity by any protein denaturant tested by the eluting principle, which was superior to the diluting principle.