A sensitive immunochemical assay for biologically active MuIFN-gamma

Abstract

An immunochemical assay for the detection of mouse gamma interferon (MuIFN-gamma) has been established. Using a purified monoclonal antibody (MAb) that neutralizes the antiviral activity of MuIFN-gamma, and a polyclonal antibody (PAb) prepared against affinity-purified MuIFN-gamma, we have developed a double-sandwich enzyme-linked immunosorbent assay (ELISA). The assay is specific for both natural and recombinant MuIFN-gamma and displays only background activity against MuIFN-alpha + beta, recombinant TNF (rTNF), human gamma interferon (HuIFN-gamma) and crude rat spleen cell supernatants. The assay is more sensitive and reproducible than the measurement of hydrogen peroxide (H2O2) release by macrophages, and is capable of detecting both crude and purified MuIFN-gamma down to 0.03 U/ml of antiviral activity, making this assay 10-100 times more sensitive than the conventional antiviral assay. The ELISA detects only biologically active MuIFN-gamma since treatment of the MuIFN-gamma at high temperature or low pH conditions resulted in abolishment of biological activity, as determined by inhibition of cytopathic effect, coincident with a dramatic decrease in ELISA titer.