Development of fluorescence-linked immunosorbent assay for high throughput screening of interferon-gamma.
BACKGROUND
Human interferon-gamma (hIFN-gamma) is produced by lymphocytes and has a variety of biological properties. Measurement of hIFN-gamma is widely used for various immunological responses for allergic or autoimmune diseases. Enzyme-linked immunosorbent assay (ELISA) is an established immunoassay used to quantify 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-gamma. We measured the 50% inhibitory concentration (IC50) value of the hIFN-gamma 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-gamma using Allophycocyanine (APC) fluorescent protein and compared it with the previous method using Cy5.5. The proposed FLISA had a smaller coefficient of variation 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-gamma. This homogeneous and multiplex method will be a powerful tool for high throughput screening for drug discovery research.
Matsukuma
Eiji
E
Department of Pediatrics, Graduate School of Medicine, Gifu University, Gifu, Japan.
Kato
Zenichiro
Z
Omoya
Kentaro
K
Hashimoto
Kazuyuki
K
Li
Ailian
A
Yamamoto
Yutaka
Y
Ohnishi
Hidenori
H
Hiranuma
Hidenori
H
Komine
Hisakazu
H
Kondo
Naomi
N
D000911:Antibodies, Monoclonal; D004797:Enzyme-Linked Immunosorbent Assay; D015200:Fluoroimmunoassay / Q000379:methods*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000276:immunology; D007371:Interferon-gamma / Q000032:analysis*; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D036341:Intercellular Signaling Peptides and Proteins; C118370:interleukin-18 binding protein; D007371:Interferon-gamma