MICROTITER PLATE BINDING ASSAY FOR CHOLINERGIC COMPOUNDS UTILIZING THE NICOTINIC ACETYLCHOLINE-RECEPTOR

A receptor-based binding assay for the determination of cholinergic compounds of the nicotinic acetylcholine receptor has been developed. By conducting the assay in a 96-well microtiter plate, the method is suitable for large-scale screening in drug development. Solid-phase extraction of the enzyme label significantly simplifies the assay protocol compared to earlier methods. The assay is based on immobilization of biotin-BSA on the microtiter which takes up avidin-labeled peroxidase due to avidin-biotin interaction. To perform the assay, a ligand (the analyte) and a biotin alpha-bungarotoxin conjugate (alphaBgt-biotin) sequentially bind to a vesicle bound nicotinic acetylcholine receptor. This is done either in a test tube, assay I, or in a biotinylated microtiter well, assay II. Avidin-HRP is then added to this mixture; free alphaBgt-biotin conjugate and immobilized biotin-BSA compete for the avidin sites. After the assay solution has been aspirated off, bound enzyme activity is determined which is directly related to the amount of alphaBgt-biotin added. Dose-response curves of cholinergic compounds and Scatchard plots were generated to evaluate the apparent binding constants. Kinetic studies were conducted for the purpose of optimization. The final assay can be performed in under 4 h with a minimum of sample handling.