Improvement of an ELISA bioassay for the routine titration of murine interferon-gamma.

ABSTRACT

We improved an ELISA bioassay for murine IFN-gamma (MuIFN-gamma) based on measurement of Ia antigen on P388D1, a mouse macrophagic tumour line. Cells were cultured in microtitre plates in medium containing dilutions of IFN-gamma source. They were then washed and stained with a rat anti-mouse I amAb followed by mouse anti-rat peroxidase-labelled antibody. After incubation with substrate, the OD was read directly from microtitre plates. Standard curves obtained with reference NIH MuIFN-gamma showed that this assay allowed for the definition of unit values (giving 50% of the maximal effect) comparable to NIH international units (IU). It detected as low as 0.2 IU/ml of MuIFN-gamma and, in contrast to antiviral assays, was insensitive to IFN-alpha/beta. We used a concanavalin A-conditioned supernatant, which is a mixed source of lymphokines, to assess the specificity of our assay. Indeed, Ia expression induced by ConA-conditioned supernatant was fully inhibited by preincubation with anti-MuIFN-gamma antibodies. Using a stable indicator cell line, the present cell surface assay is easier to perform than other ELISA using bone-marrow-derived macrophages, and does not require cell fixation; its high sensitivity and specificity are comparable to that of immunoradiometric assays. It is thus valuable for routine MuIFN-gamma quantitations in culture supernatant and biological fluids.