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 (Kds) for drugs at the 5 human muscarinic acetylcholine receptor subtypes expressed in cultured cells, as well as histamine H1, dopamine D2, serotonin 5HT1A, alpha 1- and alpha 2-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.
Cusack
B
B
Mayo Clinic, Jacksonville, FL 32224.
Richelson
E
E
D000818:Animals; D001921:Brain / Q000378:metabolism; D016466:CHO Cells; D003201:Computers; D006224:Cricetinae; D004353:Drug Evaluation, Preclinical; D006801:Humans; D066298:In Vitro Techniques; D011869:Radioligand Assay / Q000295:instrumentation / Q000379:methods*; D011976:Receptors, Muscarinic / Q000378:metabolism; D015203:Reproducibility of Results; D012371:Robotics
D011976:Receptors, Muscarinic