Four monoclonal IgG antibodies to purified, recombinant murine γ-interferon (rIFN-γ) have been produced by fusion of immune hamster splenocytes with HAT-sensitive murine myeloma cells. Specificity was confirmed either with an enzyme-linked immunosorbent assay (ELISA) that used immobilized rIFN-γ or with a radioimmunoassay that employed soluble 125I-rIFN-γ and heat-killed, fixed Staphylococcus aureus-bearing Protein A. Competition binding experiments suggested that the monoclonal antibodies (MoAb) displayed two distinct epitope specificities: one displayed by H1 and H2, and the other displayed by H21 and H22. By using murine-human recombinant IFN-γ hybrid molecules, the H1/H2 epitope was shown to depend on the aminoterminus of IFN-γ, whereas the H21/H22 epitope was formed by the carboxy-terminal amino acid sequence. The MoAb also reacted with natural IFN-γ. When bound to a surface, all four MoAb, but not normal hamster IgG, removed 100 of the antiviral and MAF activities present in supernatants of cultures of the murine 24/G1 T cell hybridoma. In free solution, all four antibodies inhibited IFN-γ dependent antiviral activity, but with different efficiencies. Soluble H21/H22 also blocked all of the 24/G1-derived activity that induces nonspecific tumoricidal activity in macrophages (MAF) while H1/H2 enhanced MAF activity. The differential inhibitory or enhancing activities of H21 or H1 reflected their ability to inhibit or enhance binding of 125I-rIFN-γ to macrophages, respectively. Soluble H21/H22 and solid-phase H1/H2 inhibited 100% of the MAF, microbicidal, and Ia-inducing activities from lymphokine preparations produced by mitogen stimulation of normal murine splenic cells. These results help to establish definitive structure-function relationships for the IFN-γ molecule, and indicate that IFN-γ is the primary lymphokine responsible for inducing nonspecific tumoricidal activity and Ia antigen expression, and for enhancing microbicidal activity in macrophages.
|Number of pages||10|
|Journal||Journal of Immunology|
|State||Published - May 1 1985|