High throughput screening for human interferon-gamma production inhibitor using homogenous time-resolved fluorescence

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.