title: Development of surface plasmon resonance biosensor assays for primary and secondary screening of acetylcholine binding protein ligands.
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.
authors.affiliation: Leiden/Amsterdam Center for Drug Research, Division of Biomolecular Analysis, Faculty of Sciences, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands. k.retra@few.vu.nl
authors.forename: Kim
authors.initials: K
authors.lastname: Retra
authors.forename: Matthis
authors.initials: M
authors.lastname: Geitmann
authors.forename: Jeroen
authors.initials: J
authors.lastname: Kool
authors.forename: August B
authors.initials: AB
authors.lastname: Smit
authors.forename: Iwan J P
authors.initials: IJ
authors.lastname: de Esch
authors.forename: U Helena
authors.initials: UH
authors.lastname: Danielson
authors.forename: Hubertus
authors.initials: H
authors.lastname: Irth
mesh_terms: D001665:Binding Sites; D008024:Ligands; D011485:Protein Binding; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000379:methods*; D013619:Tacrine / Q000737:chemistry
chemical_list: D008024:Ligands; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins; D013619:Tacrine