Ab. Wilson et al., A COMPETITIVE-INHIBITION ELISA FOR THE QUANTIFICATION OF HUMAN INTERFERON-GAMMA, Journal of immunological methods, 162(2), 1993, pp. 247-255
A competitive enzyme immunoassay has been developed for the measuremen
t of human interferon-gamma (IFN-gamma) in cell culture supernatants.
The assay is based on the dose-dependent inhibitory effect of liquid p
hase IFN-gamma on the binding of a specific monoclonal antibody to rec
ombinant IFN-gamma (rIFN-gamma) immobilized on microtitre plate wells.
The extent of monoclonal anti-IFN-gamma inhibition was determined by
the uptake of alkaline phosphatase-conjugated goat anti-mouse IgG and
the subsequent development of enzyme substrate colour. Absorbance read
ings were taken and results for test samples were extrapolated from st
andard rIFN-gamma inhibition curves constructed as logit-log plots. As
say performance was assessed using three different monoclonal antibodi
es (clones 20G7, H-22 and GZ-4). Optimum sensitivity was achieved with
the antibodies of higher affinity, 20G7 and H-22, which gave reliable
quantification of IFN-gamma over a wide range of concentrations from
0.4 ng/ml (3.4 IU/ml), or less, to 250 ng/ml approximately 2000 IU/ml)
. The inhibition assay incorporates the advantages of specificity, rep
roducibility and convenience of performance which are the hallmarks of
monoclonal antibody-based ELISAs. However, compared to the sandwich E
LISAs previously described for human IFN-gamma, it is considerably mor
e economical in its use of monoclonal anti-IFN-gamma, requiring < 50 n
g of a single antibody per 96 well plate. It also uses relatively smal
l volumes of test samples (50 mul/well) which is particularly advantag
eous where limited amounts of cell culture supernatant are available f
or cytokine assays.