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Published on: March 24, 2015
High throughput screening for human interferon-gamma production inhibitor using homogenous time-resolved fluorescence
1Chemical Analysis Laboratories, Shionogi & Co, Ltd, Osaka, Japan. koji.enomoto@shionogi.co.jp
Insights
A new homogeneous time-resolved fluorescence immunoassay accurately detects interferon-gamma (IFN-gamma). This method enables automated, high-throughput screening for IFN-gamma production inhibitors with reduced cost and time.
Area of Science:
- Biochemistry
- Immunology
- Assay Development
Background:
- Interferon-gamma (IFN-gamma) is a crucial cytokine in immune responses.
- Accurate and efficient detection of IFN-gamma is vital for research and drug discovery.
- Existing immunoassay methods can be time-consuming and labor-intensive.
Purpose of the Study:
- To develop a novel homogeneous time-resolved fluorescence (HTRF) immunoassay for IFN-gamma.
- To establish an efficient, high-throughput screening method for IFN-gamma production inhibitors.
- To enable automated, in situ cell-based detection of IFN-gamma.
Main Methods:
- Development of a homogeneous immunoassay utilizing time-resolved fluorescence (HTRF).
- Detection involves a mixture of biotinylated polyclonal antibody, EuK-labeled monoclonal antibody, and XL665-conjugated streptavidin.
- Application of the assay for detecting IFN-gamma secreted by NK3.3 cells and in high-throughput screening.
Main Results:
- The HTRF immunoassay achieved a detection limit of approximately 625 pg/ml for IFN-gamma.
- The method was successfully applied to detect IFN-gamma secreted from NK3.3 cells.
- A streamlined, in situ screening format was developed, eliminating the need for supernatant transfer or plate washing.
Conclusions:
- The developed HTRF immunoassay provides a sensitive and efficient method for IFN-gamma detection.
- The in situ screening format facilitates full automation of cell-based immunoassays.
- This approach significantly reduces experimental time and cost while enhancing accuracy and throughput for drug discovery.
Abstract:
An immunoassay for interferon-gamma (IFN-gamma) using homogeneous time-resolved fluorescence (HTRF) has been developed. In this assay, IFN-gamma can be detected by simply adding a mixture of three reagents-biotinylated polyclonal antibody, europium cryptate (fluorescence donor, EuK)-labeled monoclonal antibody, and crosslinked allophycocyanin (fluorescence acceptor, XL665) conjugated with streptavidin-and then measuring the time-resolved fluorescence. The detection limit of IFN-gamma by the proposed method is about 625 pg/ml. We applied the method to the detection of IFN-gamma secreted from NK3.3 cells and employed it in high throughput screening for IFN-gamma production inhibitors. With this screening format, IFN-gamma can be measured by directly adding the above reagents to microplate wells where NK3.3 cells are being cultured and stimulated with interleukin-12. This "in situ" immunoassay requires only pipetting reagents, with no need to transfer the culture supernatant to another microplate or wash the plate. Therefore, this screening format makes possible full automation of cell-based immunoassay, thus reducing cost and experimental time while increasing accuracy and throughput.
