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High-throughput miniaturized immunoassay for human interleukin-13 secreted from NK3.3 cells using homogenous
Koji Enomoto1, Akiko Araki, Toshihiro Nakajima
1Developmental Research Laboratories, Shionogi & Co. Ltd., 3-1-1, Futaba-cho, Toyonaka, Osaka, Japan. koji.enomoto@shionogi.co.jp
Insights
A new miniaturized immunoassay for human interleukin-13 (IL-13) offers a highly efficient and economical method for detection. This homogeneous time-resolved fluorescence (HTRF) assay is ideal for high-throughput drug screening.
Area of Science:
- Biochemistry
- Immunology
- Assay Development
Background:
- Human interleukin-13 (IL-13) is a key cytokine involved in immune responses.
- Accurate and efficient detection of IL-13 is crucial for research and drug discovery.
- Existing methods may require significant reagent volumes and multiple steps.
Purpose of the Study:
- To develop a miniaturized immunoassay for quantifying human IL-13.
- To utilize homogeneous time-resolved fluorescence (HTRF) technology for enhanced sensitivity and efficiency.
- To establish an assay suitable for high-throughput screening applications.
Main Methods:
- Development of a homogeneous time-resolved fluorescence (HTRF) immunoassay for IL-13.
- Utilized biotinylated anti-IL-13 antibody, a fluorescently labeled anti-IL-13 antibody, and streptavidin-conjugated acceptor fluorophore.
- Performed detection in a 384-well plate format using NK3.3 cells stimulated with IL-2.
Main Results:
- Achieved a detection limit of less than 600 pg/ml for IL-13.
- Demonstrated high correlation (0.9535) with traditional Enzyme-Linked Immunosorbent Assay (ELISA).
- Reduced reagent consumption by 75% compared to conventional 96-well plate assays.
Conclusions:
- The miniaturized HTRF immunoassay provides an economical and efficient method for IL-13 detection.
- The assay eliminates the need for supernatant transfer and plate washing steps.
- This method is particularly well-suited for high-throughput drug screening and research.
Abstract:
A miniaturized immunoassay for human interleukin-13 (IL-13) using homogeneous time-resolved fluorescence (HTRF) has been developed. In this assay, IL-13 which was secreted from NK3.3 cells stimulated with interleukin-2 (IL-2) was detected by measuring the time-resolved fluorescence after adding a mixture of three reagents, biotinylated anti-IL-13 monoclonal antibody, europium cryptate (fluorescence donor)-labeled different anti-IL-13 monoclonal antibody and crosslinked allophycocyanin (fluorescence acceptor)-conjugated with streptavidin in a 384-well assay plate. The detection limit of IL-13 using this immunoassay was estimated to be less than 600 pg/ml and IL-13 levels measured by this method were very close to those measured by enzyme linked immunosorbent assay (ELISA; the correlation coefficient was 0.9535). The proposed assay requires only a fourth of the quantities of all reagents compared with the assay using a conventional 96-well microtiter plate. Furthermore, there is no need to transfer the culture supernatant to another assay plate and wash the plate. Therefore, this miniaturized immunoassay is economical and efficient and is particularly suitable for high-throughput drug screening.
