Development of surface plasmon resonance biosensor assays for primary and secondary screening of acetylcholine binding protein ligands |
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Authors: | Kim Retra Matthis Geitmann August B. Smit U. Helena Danielson Hubertus Irth |
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Affiliation: | a Leiden/Amsterdam Center for Drug Research, Division of Biomolecular Analysis, Faculty of Sciences, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands b Beactica, 75237 Uppsala, Sweden c Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands d Leiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Faculty of Sciences, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands e Department of Biochemistry and Organic chemistry, Uppsala University, Box 576, SE-751 23, Uppsala, Sweden |
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Abstract: | Surface plasmon resonance (SPR) biosensors recently gained an important place in drug discovery. Here we present a primary and secondary SPR biosensor screening methodology. The primary screening method is based on a direct binding assay with covalent immobilized drug target proteins. For the secondary screening method, a sequential competition assay has been developed where the captured protein is first exposed to an unknown test compound, followed directly by an exposure to a high-molecular-weight reporter ligand. Using the high-molecular-weight reporter ligand to probe the remaining free binding site on the sensor, a significant signal enhancement is obtained. Furthermore, this assay format allows the validation of the primary direct binding assay format, efficiently revealing false positive data. As a model system, acetylcholine binding protein (AChBP), which is a soluble model protein for neuronal nicotinic acetylcholine receptors, has been used. The secondary assay is lower in throughput than the primary assay; however, the signal-to-noise ratio is two times higher compared with the direct assay, and it has a z′ factor of 0.96. Using both assays, we identified the compound tacrine as a ligand for AChBP. |
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Keywords: | Surface plasmon resonance Biosensor Nicotinic acetylcholine receptor Acetylcholine binding protein Sequential assay Screening |
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