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

Four monoclonal antibodies (mAbs) specific for chicken interferon-gamma (Ch IFN-gamma) were generated by gene gun immunization and were utilized to dev elop a mAb-based capture ELISA specific for ChIFN-gamma. Each mAb reacted s pecifically with both baculovirus and Escherichia coli-derived recombinant ChIFN-gamma in ELISA and Western Blot analysis or natural ChIFN-gamma in im munofluorescence experiments. As determined by competition ELISAs, mAbs 3D5 , 4C6 and 3A3 recognized the same or adjacent epitopes on the ChIFN-gamma m olecule, whereas mAb 1E12 recognized a distant epitope. Moreover, this latt er mAb was able to highly neutralize the biological activities of both reco mbinant and natural ChIFN-gamma as measured by inhibition of viral replicat ion and macrophage activation. To improve the detection of ChIFN-gamma, a c apture ELISA was developed using mAb 1E12 as capture antibody and biotinyla ted mAb 4C6 as detection antibody. In addition to being more rapid and easi er to perform than classical cell-mediated immunity tests, this ELISA has e xcellent sensitivity and improved specificity. The use of a specific rabbit polyclonal serum as revealing antibody further increased the sensitivity o f the detection down to 0.5 ng/ml of ChIFN-gamma. This ELISA would provide a sensitive tool to measure the in vitro release of ChIFN-gamma by T-cells in response to specific recall antigen. (C) 2000 Elsevier Science B.V. All rights reserved.