title: Development of a multiplex non-radioactive receptor assay: the benzodiazepine receptor, the serotonin transporter and the beta-adrenergic receptor.
abstract: Binding assays still form a fundamental part of modern drug development. Receptor binding assays are mostly based on radioactivity because of their speed, ease of use and reproducibility. Disadvantages, such as health hazards and production of radioactive waste, have prompted the development of non-radioactive receptor binding assays. This application therefore focuses on measuring receptor-ligand interactions using mass spectrometry. Moreover, the novelty of this approach originates in determining multiple analytes in a single assay (multiplexing). The proof of principle of a non-radioactive multiplex receptor assay is demonstrated using a pool of receptors from rat cortical tissue with flunitrazepam, MADAM and pindolol in one vial with or without their respective displacers. Flunitrazepam, MADAM and pindolol bound specifically at 73%, 30% and 40% to their respective receptors. This corresponds to specific binding sites of 0.61 pmol/mg protein, 0.07 pmol/mg protein and 0.06 pmol/mg protein, respectively. We propose to measure the bound fraction instead of the free fraction in order to reach a significant difference in measured signals (total binding versus non-specific binding). The bound fraction can be obtained after dissociating the ligand from the receptor-ligand complex using 50% methanol in water. The current setup of the assay calls for further improvement with respect to the measurement of binding constants for a multitude of receptors in one assay with sufficient accuracy and precision.
authors.affiliation: Department of Biomonitoring & Sensoring, University Centre for Pharmacy, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands. L.A.A.de.Jong@rug.nl
authors.forename: Lutea A A
authors.initials: LA
authors.lastname: de Jong
authors.forename: C Margot
authors.initials: CM
authors.lastname: Jeronimus-Stratingh
authors.forename: Thomas I F H
authors.initials: TI
authors.lastname: Cremers
mesh_terms: D000319:Adrenergic beta-Antagonists / Q000494:pharmacology; D000818:Animals; D001596:Benzylamines / Q000494:pharmacology; D001667:Binding, Competitive; D001681:Biological Assay / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D004353:Drug Evaluation, Preclinical; D005445:Flunitrazepam / Q000494:pharmacology; D005625:Frontal Lobe / Q000187:drug effects / Q000378:metabolism; D018757:GABA Modulators / Q000494:pharmacology; D008024:Ligands; D010869:Pindolol / Q000494:pharmacology; D051381:Rats; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D011963:Receptors, GABA-A / Q000378:metabolism*; D050486:Serotonin Plasma Membrane Transport Proteins / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D000319:Adrenergic beta-Antagonists; D001596:Benzylamines; D018757:GABA Modulators; D008024:Ligands; C470437:N,N-dimethyl-2-(2-amino-4-methylphenylthio)benzylamine; D011943:Receptors, Adrenergic, beta; D011963:Receptors, GABA-A; D050486:Serotonin Plasma Membrane Transport Proteins; D005445:Flunitrazepam; D010869:Pindolol