A method for radioligand binding assays using a robotic workstation

Radioligand binding assays provide a powerful tool for screening drug candidates at many receptors. We present a method utilizing a robotic workstation, the Biomek® 1000, which automates the tedious and repetitive tasks of these assays. First, the robot handles the serial dilution of up to 8 drugs with 11 concentrations per drug. The sequential addition of diluting buffer, non-radioactively labeled ligand, radioactive ligand, and finally the tissue homogenate or membrane preparation, is fully automated. A novel rack design allows the use of tubes with a maximum capacity of 2 ml, providing a total assay volume of 1 ml. Final filtration on a Brandel cell harvester outfitted with a uniquely designed head allows for processing of 48 samples simultaneously from the rack holder. We have employed this method for the determination of equilibrium dissociation constants (K_ds) for drugs at the 5 human muscarinic acetylcholine receptor subtypes expressed in cultured cells, as well as histamine H₁, dopamine D₂, serotonin 5HT_1A, α₁-and α₂-adrenergic receptors in human brain tissue homogenates. Our results compare favorably with manual methods reported for these receptors, and exhibit a very high degree of reproducibility and throughput, with a minimum of operator input.