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A Homogeneous 384-Well High-Throughput Binding Assay for a TNF Receptor Using Alphascreen Technology

Serono Pharmaceutical Research Institute, 14 Ch. des Aulx, 1228 Plan-les-Ouates, Geneva, Switzerland

Claudia Pena Rossi

Serono International S.A., 15 Ch. des Mines, 1202 Geneva, Switzerland

Susanna Carboni

Serono Pharmaceutical Research Institute, 14 Ch. des Aulx, 1228 Plan-les-Ouates, Geneva, Switzerland

Christèle Fremaux

Serono Pharmaceutical Research Institute, 14 Ch. des Aulx, 1228 Plan-les-Ouates, Geneva, Switzerland

Dominique Perrin

Serono Pharmaceutical Research Institute, 14 Ch. des Aulx, 1228 Plan-les-Ouates, Geneva, Switzerland

Claudio Soto

Serono International S.A., 15 Ch. des Mines, 1202 Geneva, Switzerland

Marie Kosco-Vilbois

NovImmune SA, 64 Ave de la Roseraie, 1211 Geneva 4, Switzerland

Alexander Scheer

Serono Pharmaceutical Research Institute, 14 Ch. des Aulx, 1228 Plan-les-Ouates, Geneva, Switzerland, alexander.scheer{at}serono.com

To take advantage of the growing knowledge of cellular signaling pathways, modern-day drug discovery faces an increasing challenge to develop assays to screen for compounds that modulate protein-protein interactions. One bottleneck in achieving this goal is a lack of suitable and robust assay technologies amenable to a high-throughput format. In this report, we describe how we utilized Alphascreen^TM technology to develop a high-throughput assay to monitor ligand binding to a member of the tumor necrosis factor receptor superfamily. We expressed a fusion protein consisting of the extracellular domain of the OX40 receptor with the constant domains of human IgG. In the presence of OX40 ligand, we determined a binding affinity constant consistent with reported values and optimized the protocol to develop a simple, homogeneous, and sensitive binding assay in a 384-well format. Finally, we assessed if this system could identify small peptides capable of inhibiting the OX40 receptor and ligand interaction. The results showed that the assay was able to detect such peptides and could be used to launch a high-throughput screening campaign for small molecules able to prevent OX40 receptor activation. (Journal of Biomolecular Screening 2003:522-532)

Key Words: Alphascreen technology • protein-protein interaction • TNF receptor superfamily • high-throughput binding assay

Journal of Biomolecular Screening, Vol. 8, No. 5, 522-532 (2003)
DOI: 10.1177/1087057103257804


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