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Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Published on: January 7, 2019

High-throughput screening in two dimensions: binding intensity and off-rate on a peptide microarray.

Matthew P Greving¹, Paul E Belcher, Conor D Cox

¹The Biodesign Institute at Arizona State University, 1001 S. McAllister Avenue, Tempe, AZ 85287-5201, USA.

Analytical Biochemistry

|March 10, 2010

Summary

We developed a high-throughput microarray assay to analyze thousands of compounds for tumor necrosis factor alpha (TNF-alpha) binding and dissociation. This method accurately identifies compounds with slow off-rates, crucial for drug discovery.

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Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

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Published on: January 7, 2019

Identifying Protein-protein Interaction Sites Using Peptide Arrays

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Published on: November 18, 2014

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Published on: March 25, 2014

Area of Science:

Biochemistry
Molecular Biology
Assay Development

Background:

Tumor necrosis factor alpha (TNF-alpha) is a key inflammatory cytokine.
Developing high-throughput methods to screen for TNF-alpha inhibitors is critical for treating inflammatory diseases.
Accurate measurement of compound binding and dissociation rates is essential for drug candidate evaluation.

Purpose of the Study:

To develop and validate a high-throughput, two-dimensional microarray-based screening method.
To simultaneously analyze target binding intensity and off-rate for thousands of compounds.
To assess the reliability of the microarray method by comparing dissociation rates with Surface Plasmon Resonance (SPR).

Main Methods:

A peptide microarray was used to immobilize TNF-alpha.
Labeled TNF-alpha binding and subsequent dissociation were measured.
Time-resolved dissociation data were fitted to a one-component exponential decay model.
Relative binding intensities and dissociation rates (off-rates) were determined for screened compounds.

Main Results:

The 2D microarray assay successfully analyzed thousands of compounds in a single binding assay.
Relative binding intensities and time-resolved dissociation rates were measured for TNF-alpha.
Most peptides exhibiting slow off-rates on the microarray also showed slow off-rates when validated by SPR.
The method demonstrates high-throughput capability for analyzing compound-target interactions.

Conclusions:

The developed microarray assay is a powerful, high-throughput tool for analyzing compound binding and dissociation kinetics.
This method enables efficient screening of large compound libraries for potential TNF-alpha inhibitors.
The correlation with SPR validates the accuracy and reliability of the microarray-based off-rate determination.