Millipore Technical Publications |
 | TN- TN1021EN00 | |
Automation of PAMPA-Lipid using a MultiScreen-IP PAMPA Filter Plate on a Tecan Freedom EVO® Workstation | ||
| Lit No: | TN1021EN00 | |
| Year: | 2006 | |
| PAMPA-Lipid (Parallel Artificial Membrane Permeation Assay) is a non-cell based assay that utilizes a 96-well filter plate.1, 2 The PAMPA assay is designed to predict passive transcellular permeability of drugs in early drug discovery. The assay is carried out in a 96-well MultiScreen-IP PAMPA filter plate and measures the ability of compounds to diffuse from a donor to an acceptor compartment separated by a PVDF membrane pretreated with a lipid containing solvent. The protocol is robust and easy to automate when using lipids from Avanti Polar Lipids, Inc. or pION Inc. The protocol can be run using either Millipore’s PTFE acceptor plate or the disposable MultiScreen Transport Receiver plate. A lipid artificial membrane is applied to the PVDF membrane in a 96-well filter plate (Donor plate). The Donor plate is filled with buffer solutions containing the compounds to be tested. The Donor plate is placed in a 96-well Acceptor plate (either MultiScreen PTFE or MultiScreen Transport Receiver plate) filled with sufficient buffer to ensure liquid contact between the liquid in the Acceptor plate and the PVDF membrane. The Donor and Acceptor plates are incubated together for 16 hours after which time the Donor plate is removed from the Acceptor plate. Samples from the Acceptor plate are analyzed by LC/MS or transferred to a UV compatible 96 well plate and analyzed immediately in a UV/Vis spectrophotometer. An equilibrium plate (compounds at the theoretical equilibrium, i.e. the resulting concentration if the donor and the acceptor solutions are combined) is also created and analyzed. This equilibrium plate is used to calculate the permeability rate (Log Pe) of the drugs. At the end of the incubation time, the integrity of the artificial membrane layer can be measured using electrical resistance. Processing of the automated PAMPA-Lipid assay on the Freedom EVO workstation takes about 47 minutes (this does not include the 16 hours incubation). This time includes the formation of the artificial membrane, dilution of compounds from a mother plate, addition of compounds to the Donor plate, creating the equilibrium plate for analysis and removal of samples for UV/Vis analysis from the Acceptor plate. |
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