Development of surface plasmon resonance biosensor assays for primary and secondary screening of acetylcholine binding protein ligands.
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.
Retra
Kim
K
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
Geitmann
Matthis
M
Kool
Jeroen
J
Smit
August B
AB
de Esch
Iwan J P
IJ
Danielson
U Helena
UH
Irth
Hubertus
H
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
D008024:Ligands; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins; D013619:Tacrine