A homogeneous 384-well high-throughput binding assay for a TNF receptor using alphascreen technology

Area of Science:

• Biochemistry
• Immunology
• Drug Discovery

Background:

• Developing high-throughput assays for protein-protein interactions is crucial for drug discovery.
• Existing assay technologies face limitations in robustness and scalability for screening modulators of cellular signaling pathways.

Purpose of the Study:

• To develop a high-throughput AlphaScreen assay to monitor ligand binding to the OX40 receptor.
• To establish a sensitive and homogeneous assay in a 384-well format for drug screening.

Main Methods:

• Utilized AlphaScreen technology to create a fusion protein of the OX40 receptor extracellular domain and human IgG constant domains.
• Expressed and purified the fusion protein, and performed binding assays with OX40 ligand.
• Optimized assay conditions for a 384-well format and tested its ability to detect inhibitory peptides.

Main Results:

• The developed AlphaScreen assay accurately measured the binding affinity constant for OX40 receptor-ligand interaction, consistent with reported values.
• The assay demonstrated sensitivity and robustness in a homogeneous, 384-well format.
• The system successfully identified small peptides capable of inhibiting the OX40 receptor and ligand interaction.

Conclusions:

• A novel, high-throughput AlphaScreen assay was successfully developed to monitor OX40 receptor-ligand binding.
• This assay is suitable for large-scale screening campaigns to identify small molecules that inhibit OX40 receptor activation.
• The assay provides a valuable tool for drug discovery targeting the OX40 pathway for immune modulation.