Development of surface plasmon resonance biosensor assays for primary and secondary screening of acetylcholine binding protein ligands
2010 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 407, no 1, 58-64 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2010. Vol. 407, no 1, 58-64 p.
Surface plasmon resonance; Biosensor; Nicotinic acetylcholine receptor; Acetylcholine binding protein; Sequential assay; Screening
Natural Sciences Medical and Health Sciences Pharmaceutical Sciences
IdentifiersURN: urn:nbn:se:uu:diva-129429DOI: 10.1016/j.ab.2010.06.021ISI: 000282500500007PubMedID: 20599657OAI: oai:DiVA.org:uu-129429DiVA: diva2:343707