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 (ChIFN-gamma) were generated by gene gun immunization and were utilized to develop a mAb-based capture ELISA specific for ChIFN-gamma. Each mAb reacted specifically with both baculovirus and Escherichia coli-derived recombinant ChIFN-gamma in ELISA and Western Blot analysis or natural ChIFN-gamma in immunofluorescence experiments. As determined by competition ELISAs, mAbs 3D5, 4C6 and 3A3 recognized the same or adjacent epitopes on the ChIFN-gamma 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-gamma as measured by inhibition of viral replication and macrophage activation. To improve the detection of ChIFN-gamma, 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 0.5ng/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.
Lambrecht
B
B
Section of Avian Virology and Biotechnology, Veterinary and Agrochemical Research Centre (VAR), Groeselenberg 99, B 1180, Brussels, Belgium. belam@var.fgov.be
Gonze
M
M
Meulemans
G
G
van den Berg
T P
TP
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D001667:Binding, Competitive / Q000276:immunology; D002645:Chickens / Q000276:immunology*; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary*; D007114:Immunization / Q000662:veterinary; D007371:Interferon-gamma / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009500:Neutralization Tests / Q000662:veterinary; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D007371:Interferon-gamma