Development and validation of a 96-well equilibrium dialysis apparatus for measuring plasma protein binding

Abstract

A 96-well equilibrium dialysis block was designed and constructed that is compatible with most standard 96-well format laboratory supplies and instruments. The unique design of the dialysis apparatus allows one to dispense and aspirate from either or both the sample and dialysate sides from the top of the apparatus, which is not possible with systems currently on the market. This feature permits the investigator to analyze a large number of samples, time points, or replicates in the same experiment. The novel alignment of the dialysis membrane vertically in the well maximizes the surface-to-volume ratio, eliminates problems associated with trapped air pockets, and allows one to add or remove samples independently or all at once. Furthermore, the design of the apparatus allows both the sample and dialysate sides of the dialysis well to be accessible by robotic systems, so assays can be readily automated. Teflon construction is used to minimize nonspecific binding of test samples to the apparatus. The device is reusable, easily assembled, and can be shaken in controlled temperature environments to decrease the time required to reach equilibrium as well as facilitate dissolution of test compounds. Plasma protein binding values obtained for 10 diverse compounds using standard dialysis equipment and the 96-well dialysis block validates this method.