Using the AcroWell™ Filter Plate for Receptor/Ligand Binding

The DELFIA* Europium-labeling system allows researchers to directly detect the binding of labeled ligands to receptors provided the unbound-labeled ligands are removed by washing. Cell-based assays are particularly well suited to the DELFIA system because of its broad detection range and high sensitivity (below 1.0 fmol Europium). This degree of sensitivity can only be achieved when non-specific binding and plate material backgrounds are low. The addition of filtration steps to a binding assay reduces the washing steps and prevents the loss of target that may result from the turbulence created using a pipet plate washer in a microtiter plate.

The most efficient way to remove unlabeled ligand and residual wash solutions, and minimize target loss, is through the use of a filter plate. In the past, the use of filter plates for fluorescent assay systems was limited because of high background fluorescence or phosphorescence emissions from the filtration membrane. Pall's AcroWell 96 filter plate with GHP membrane exhibits extremely low background in fluorescence-based assays. The ability to use a filtration plate for the washing, sample preparation, and detection steps reduces costs and handling for the assay.

1. Prepare cells and label ligand or antibody using manufacturer's protocols.
2. For extra insurance of valuable samples, the AcroWell 96 filter plate can be prescreened. To prescreen simply add 100 µl molecular biology grade water to each well, filter, then read the plate in a VICTOR* multilabel counter or comparable counter. This will insure integrity and that background is within acceptable limits.
3. Membrane fragments can be prepared in 2.5 mM MgCl₂, 50 mM HEPES, pH 7.5 (Liu et al., 1998) and quantitated by the Bradford method using Bovine Serum Albumin (BSA) as standard.
4. Use either a multichannel pipet or robotic station to add cells to a 96-well filter plate for assay incubation. Some assays may be incubated in the AcroWell 96 filter plate if non-specific backgrounds remain low (see Frequently Asked Questions, below).
5. No blocking step is required for the AcroWell 96 filter plate. In fact, blocking may increase the Coefficient of Variation (CV).
6. Filter (or wash) cells to remove buffer or culture media.
7. Cells (< 200,000) or 10 µg membrane protein are resuspended in a final volume of 60 µL binding buffer (25 mM EDTA, 0.2% BSA in Hypotonic Buffer). Saturation binding experiments are frequently carried out using increasing concentrations of labeled ligand in the presence of variable amounts of PEG-3350 and/or DMSO.
8. Allow the ligand to bind for up to two hours in the AcroWell 96 filter plate.
9. After binding, stop the reaction by filtration. Filter either by vacuum (at 25.4 to 38.1 cm Hg) or centrifuge (at 500 to 3,000 x g).
10. Add 200 µL of wash solution, filter, and repeat wash steps up to four times.
11. Add 150 µL of Enhancement Solution per well and incubate according to manufacturer's protocol.
12. Measure time-resolved fluorescence using a VICTOR multilabel counter or comparable counter.

Can I incubate my binding assay in the AcroWell 96 filter plate? Yes, however, sometimes long incubations of ligand or antibody result in higher nonspecific binding. If this occurs, it is recommended that the binding step be done in a separate V-bottom 96-well plate prior to transfer to the AcroWell 96 filter plate for subsequent washes and detection.

Are blocking steps needed for receptor/ligand binding assays? Although most assay protocols recommend a blocking step, the AcroWell 96 filter plate allows you to add the assay mixture directly. In fact, the addition of a blocking step on our low-binding filter plate may result in an increased variability in signal and filtration efficiency.

How can I improve my CV? Because the AcroWell 96 filter plate has a low fluorescence background, a lower number of cells can be used to achieve signal. In addition, because the removal of unbound label using filtration is generally more uniform than plate washing because of the variability and turbulance from needle to needle, it allows lower well-to-well variation.

Your plate has a single membrane coupon. Is there a problem with well-to-well crosstalk? Pall's patented sealing process eliminates the lateral flow of liquids between wells. (For more information, see "The AcroWell 96 Filter Plate Minimizes Crosstalk".)

Can I use the AcroWell 96 filter plate with my robotic fluid handling systems? Yes; the AcroWell 96 filter plate was designed to meet SBS footprint and well location specifications.

Can I use a robotic filtration station? Yes. Tomtec* makes a filtration station that has been tested and works well with the AcroWell 96 filter plate. Because the AcroWell 96 filter plate is designed in accordance to the SBS specifications, it will work will all filtration stations that accept standard multi-well plates.

Does the AcroWell 96 filter plate work on all vacuum manifolds? So far, every manifold tested works with the AcroWell 96 filter plate. The Millipore MultiScreen* Resist manifold contains a metal support grid that is not required for the AcroWell filter plate. Therefore, simply remove the grid before using the AcroWell 96 filter plate as you normally would with any vacuum manifold.

How much vacuum can I apply during filtration? The AcroWell 96 filter plate will reliably filter samples at 25.4 cm Hg (10 in. Hg) vacuum. It may fail if the vacuum pressure applied is greater than 38.1 cm Hg (15 in. Hg).

Can I use the AcroWell 96 filter plate to capture filtrate? Yes, if you use a centrifuge for filtration. Collection of filtrate from vacuum-filtered samples is not recommended.

Is the AcroWell 96 filter plate only used for receptor/ligand binding? No; it can be used in any application where cells or particles are captured for analysis. It can also be used for capture of filtrate in some applications using centrifugal filtration.

Can cells be grown directly in the AcroWell 96 filter plate? The current AcroWell 96 filter plate is non-sterile. It can be sanitized by washing with 70% ethanol or sterilized by gamma irradiation or ethylene oxide.

Can the AcroWell 96 filter plate still filter solutions once the membrane has dried out? Yes; the GHP membrane is intrinsically hydrophilic. The AcroWell 96 filter plate does not contain or shed surfactants and will easily wet-out and filter each time.

Can we filter organic solvents with the AcroWell 96 filter plate? No; the supported Emflon membrane layer is hydrophobic and will wet-out, allowing crosstalk to occur.

1. Appell, K., Chung, T., Solly, K., Chelsky, D. (1998). Biological characterization of neurokinin antagonists discovered through screening a combinatorial library. J Biomol. Screening. 3:19-27.
2. Hemmila, I.A., Webb, S. (1997). Time-resolved fluorometry: An overview of the labels and core technologies for drug screening applications. Drug Discovery Today. 2:373-381.
3. Inglese, J., Samama, P. Patel, S., Burbaum, J., Stroke, I., Appell, K. (1998). Chemokine receptor-ligand interactions measured using time-resolved fluorescence. Biochemistry. 37:2372-2377.
4. Liu, J., Gallagher, M., Horlick, R.A., Robbins, A.K., Webb, M.L. (1988). A Time Resolved Fluorometric Assay for Galanin Receptors. J. Biomol. Screening. 3:199-205.
5. Miller, W., Valenzano, K., Samama, P., Fitzpatrick, D., and Seeley, K. (1999). Development of a fluorescent ligand-binding assay using the AcroWell 96 filter plate. 5th Annual SBS Conference, Edinburgh, Scotland.
6. Rogers, M.V. (1997). Light on high throughput screening: Fluorometric-based assay technology. Drug Discovery Today. 2:156-160.