Development of Fluorescence-linked Immunosorbent Assay for High Throughput Screening of Interferon-

Background: Human interferon-gamma (hIFN-γ) is produced by lymphocytes and has a variety of biological properties. Measurement of hIFN-γ is widely used for various immunological responses for allergic or autoim-mune diseases. Enzyme-linked immunosorbent assay (ELISA) is an established immunoassay used to quan-tify cellular metabolites or cytokines. ELISA requires many incubation and wash steps and is not practically suitable for screening large numbers of samples? Methods: We have developed a fluorescence-linked immunosorbent assay (FLISA) method for the detection of hIFN-γ. We measured the 50 % inhibitory concentration (IC50) value of the hIFN-γ production by interleukin (IL)-18 binding protein and anti-IL-18 monoclonal antibody. The IC50 described by FLISA was compared with that by ELISA. Results: We developed a new system for measuring hIFN-γ using Allophycocyanine (APC) fluorescent protein and compared it with the previous method using Cy5.5. The proposed FLISA had a smaller coefficient of vari-ation than ELISA, and the means of coefficient of variation using the same samples measured by ELISA and FLISA were, respectively, 11.1 % and 3.8%, suggesting that the edge effect often giving non-specific results may be smaller in FLISA than in ELISA? Conclusions: The improved FLISA system proposed is ideally suited for efficient measurements of hIFN-γ. This homogeneous and multiplex method will be a powerful tool for high throughput screening for drug discov-ery research? KEY WORDS enzyme-linked immunosorbent assay, fluorescence-linked immunosorbent assay, fluorometric microvolume assay technology, high-throughput screening, interferon-gamm