Production of antibodies against chicken interferon-γ: demonstration of neutralizing activity and development of a quantitative ELISA

Four monoclonal antibodies (mAbs) specific for chicken interferon-γ (ChIFN-γ) were generated by gene gun immunization and were utilized to develop a mAb-based capture ELISA specific for ChIFN-γ. Each mAb reacted specifically with both baculovirus and Escherichia coli-derived recombinant ChIFN-γ in ELISA and Western Blot analysis or natural ChIFN-γ in immunofluorescence experiments. As determined by competition ELISAs, mAbs 3D5, 4C6 and 3A3 recognized the same or adjacent epitopes on the ChIFN-γ molecule, whereas mAb 1E12 recognized a distant epitope. Moreover, this latter mAb was able to highly neutralize the biological activities of both recombinant and natural ChIFN-γ as measured by inhibition of viral replication and macrophage activation. To improve the detection of ChIFN-γ, a capture ELISA was developed using mAb 1E12 as capture antibody and biotinylated mAb 4C6 as detection antibody. In addition to being more rapid and easier to perform than classical cell-mediated immunity tests, this ELISA has excellent sensitivity and improved specificity. The use of a specific rabbit polyclonal serum as revealing antibody further increased the sensitivity of the detection down to 0.5 ng/ml of ChIFN-γ. This ELISA would provide a sensitive tool to measure the in vitro release of ChIFN-γ by T-cells in response to specific recall antigen.