The influence of immune complexes, steric effects, and antigen-antigen interactions on the sensitivity of enzyme-linked immunosorbent assays

Abstract

Serum from mice hyperimmunized with 2,4,6-trinitrophenylated bovine gamma globulin (TNP-BGG) or keyhole limpet hemocyanin have been shown to have enhanced binding to several ligands unrelated to the antigen used for immunization. Addition of the immunizing antigen to the serum can result in increased binding to unrelated ligands and to the polyvinyl chloride surface of microtiter wells in solid-phase enzyme-linked immunosorbent assays. In a competitive binding assay using TNP-BGG-hyperimmune serum adsorbed to the microtiter well followed by an alkaline phosphatase conjugate of BGG in the presence or absence of TNP-BSA, substantial inhibition of BGG binding is seen. Steric hindrance appears to be the major cause of such inhibition since addition of hapten alone has little effect on BGG binding. An antigen-antigen interaction between KLH and TNP is also detectable. Immune complex formation, steric effects, and antigen-antigen interactions potentially have substantial influences on ELISA sensitivity and must be considered as possible sources of artifact in these assays.