ABSTRACT
The recovery of a predetermined amount of interferon-gamma (IFN-gamma) added to normal serum was studied in two independent sandwich ELISA systems specific for rat and human IFN-gamma. In both assays the ELISA activity was rapidly lost in fresh but not in heat-inactivated (30', 56 degrees C) serum. Ninety percent of the initial activity had disappeared within 30 minutes upon incubation at 37 degrees C. Serum-mediated inhibition was not species-specific as the ELISA activity of rat IFN-gamma diminished equally well in rat and human sera. Inhibition was critically dependent on the isotype of the solid-phase monoclonal antibody (mAb) used in the ELISA systems. IgG1 and IgG2a mAbs efficiently inhibited the ELISA activity of IFN-gamma, whereas an IgA mAb was ineffective. The inhibition was not influenced by a wide variety of anti-proteolytic agents but was effectively blocked by anti-complementary substances or treatments directed to the first (C1) and third (C3) component of complement. Our results indicate that activation of the classical pathway of complement (CPC) and the concomitant covalent binding of C3 to the IFN-gamma molecule play a major role in the inhibitory process. It is concluded that reduction of the ELISA activity is attributable to diminished accessibility of the detector antibody for the IFN-gamma protein as a consequence of C3 binding.
Subject(s)
Complement Pathway, Classical , Enzyme-Linked Immunosorbent Assay , Interferon-gamma/antagonists & inhibitors , Binding Sites , Complement C3b/metabolism , Humans , In Vitro Techniques , Interferon-gamma/blood , KineticsABSTRACT
Eight stable hybridoma cell lines producing monoclonal antibodies reactive with rat interferon-gamma have been generated. The antibodies produced belong to three different immunoglobulin isotypes: IgG1 (4 monoclonal antibodies, designated DB-1, DB-10, DB-12 and DB-13), IgG2a (3 monoclonal antibodies, DB-9, DB-14 and DB-16) and IgA (1 monoclonal antibody, DB-2). The antibodies were characterized in terms of epitope specificity and reactivity with rat, mouse and human interferon-gamma. Three antibodies (DB-1, -2 and -14) show high antiviral neutralizing activity against natural and recombinant DNA derived rat interferon-gamma, whereas the others have low (DB-10, -12, -13 and -16) or no (DB-9) detectable activity. Two antibodies (DB-1 and -2) effectively bind and neutralize mouse interferon-gamma, one antibody (DB-14) exhibits some cross-reactivity and the others show no reactivity towards the mouse lymphokine. None of the antibodies reacts with human interferon-gamma. All antibodies recognize immunoblotted recombinant rat interferon-gamma, although substantial variation in the immunoreactivity existed. Competition binding experiments reveal that the antibodies are directed to three spatially distinct sites on the rat lymphokine. Two non-competing monoclonal antibodies were selected and used for the development of a specific enzyme-linked immunosorbent assay for the detection of rat interferon-gamma in biological fluids.
