Epitope mapping of recombinant human gamma interferon using monoclonal antibodies

Abstract Five monoclonal antibodies (MAbs B22, B27, 3–6, 32 and 35) specific for human recombinant IFN-γ were characterized. These MAbs were used to set up quantititative sandwich ELISAs which allowed the detection of 1.25 ng/ml of IFN-γ when diluted in normal human serum. Epitope mapping of the IFN-γ molecule using these MAbs demonstrated that antibodies 3–6 and 32 which did not inhibit the biological activity of IFN-γ recognized an epitope localized on the 15 C-terminal amino acids, suggesting that this portion of the molecule was not implicated in the biological activity of IFN-γ. Sandwich ELISAs were performed using various pairs of MAbs. The level of reactivity obtained when antibodies B22 and B27 were used simultaneously as catcher and tracer was similar to the result obtained with two antibodies recognizing different epitopes. These results confirm that the IFN-γ molecule is a dimer in solution and indicate that the two sites of the IFN-γ dimeric molecule which are associated with the biological activity (epitope B22/B27) are fully exposed. In contrast, the C-terminus is only partially accessible to the antibodies 3-6/32, suggesting that the dimerization of IFN-γ molecule results in the interaction of regions of the monomers that are homologous and adjacent to the C-terminus.