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Complement-mediated inactivation of interferon-gamma in ELISA systems
P H van der Meide1, M C de Labie, J A Wubben
1Institute of Applied Radiobiology and Immunology TNO, Rijswijk, The Netherlands.
Journal of Immunoassay
|January 1, 1991
Summary
Serum rapidly degrades interferon-gamma (IFN-gamma) in ELISA assays, primarily through complement activation. This process involves C3 binding, reducing IFN-gamma accessibility for detection.
Area of Science:
- Immunology
- Biochemistry
Background:
- Interferon-gamma (IFN-gamma) is a crucial cytokine.
- ELISA (Enzyme-Linked Immunosorbent Assay) is widely used for cytokine detection.
- Serum components can interfere with immunoassay results.
Purpose of the Study:
- To investigate the mechanism of serum-mediated inhibition of IFN-gamma in sandwich ELISA systems.
- To identify the specific serum factors and pathways responsible for IFN-gamma degradation.
Main Methods:
- Studied IFN-gamma recovery in rat and human serum using specific sandwich ELISAs.
- Assessed the effect of serum incubation time, temperature, and heat inactivation.
- Investigated the role of complement components (C1, C3) and monoclonal antibody (mAb) isotypes (IgG1, IgG2a, IgA).
Main Results:
- ELISA activity of IFN-gamma was rapidly lost in fresh serum at 37°C.
- Inhibition was not species-specific and was dependent on mAb isotype (IgG1/IgG2a effective, IgA ineffective).
- Complement activation, specifically involving C1 and C3, was identified as the primary inhibitory mechanism.
Conclusions:
- Serum-mediated inhibition of IFN-gamma in ELISA is largely due to the classical complement pathway activation.
- Covalent binding of C3 to IFN-gamma reduces its accessibility to detector antibodies, diminishing ELISA activity.
- Understanding this interaction is vital for accurate IFN-gamma quantification in biological samples.