Complement-mediated inactivation of interferon-gamma in ELISA systems.
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.
van der Meide
P H
PH
Institute of Applied Radiobiology and Immunology TNO, Rijswijk, The Netherlands.
de Labie
M C
MC
Wubben
J A
JA
Borman
A H
AH
D001665:Binding Sites; D003179:Complement C3b / Q000378:metabolism; D003171:Complement Pathway, Classical; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D066298:In Vitro Techniques; D007371:Interferon-gamma / Q000037:antagonists & inhibitors* / Q000097:blood; D007700:Kinetics
D003179:Complement C3b; D007371:Interferon-gamma