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Updated: Jul 19, 2026
Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-α
Published on: June 14, 2018
Development of fluorescence-linked immunosorbent assay for high throughput screening of interferon-gamma
Eiji Matsukuma1, Zenichiro Kato, Kentaro Omoya
1Department of Pediatrics, Graduate School of Medicine, Gifu University, Gifu, Japan.
Insights
A new fluorescence-linked immunosorbent assay (FLISA) offers efficient human interferon-gamma (hIFN-gamma) measurement. This method is more precise than ELISA, making it ideal for high-throughput drug discovery screening.
Area of Science:
- Immunology
- Biotechnology
Background:
- Human interferon-gamma (hIFN-gamma) is a cytokine produced by lymphocytes with diverse biological functions.
- Quantifying hIFN-gamma is crucial for diagnosing allergic and autoimmune diseases.
- Enzyme-linked immunosorbent assay (ELISA) is a standard method but is labor-intensive and unsuitable for large-scale screening.
Purpose of the Study:
- To develop and validate a novel fluorescence-linked immunosorbent assay (FLISA) for detecting hIFN-gamma.
- To compare the performance of the developed FLISA with the conventional ELISA method.
Main Methods:
- Development of a FLISA system utilizing Allophycocyanine (APC) fluorescent protein for hIFN-gamma detection.
- Measurement of the 50% inhibitory concentration (IC50) of hIFN-gamma production using IL-18 binding protein and anti-IL-18 monoclonal antibody.
- Comparative analysis of IC50 values obtained from FLISA and ELISA.
Main Results:
- The FLISA system demonstrated a significantly smaller coefficient of variation (3.8%) compared to ELISA (11.1%).
- The improved precision suggests a reduced impact of edge effects in FLISA, leading to more reliable results.
- The new FLISA method using APC showed enhanced performance over previous methods using Cy5.5.
Conclusions:
- The developed FLISA provides an efficient and precise method for measuring hIFN-gamma.
- This homogeneous and multiplex assay is highly suitable for high-throughput screening in drug discovery research.
- FLISA represents a significant advancement for immunological assays requiring large sample analysis.
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.
