Section5:Filter Assay Format

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Plate Type	Harvester Instrument	Counting Instrument	Comments
Unifilter GF/C or GF/B	Packard or Brandel	Trilux or TopCount	Filter from an assay plate to the filter plate with washing of the assay plate possible
Multiscreen-FC or Multiscreen-FB	MAP or individual manifold	Trilux or TopCount	Removable bottom plastic piece. Requires solid white adapter for TopCount or clear plastic liner and cassette for Trilux
Multiscreen-GV	MAP or individual manifold	Trilux or TopCount	0.22 mM Durapore membrane. Removable bottom plastic piece. Requires solid white adapter for TopCount or clear plastic liner and cassette for Trilux

The speed of separation is important, particularly for lower affinity interactions (<1 nM), and can be influenced by the filter plate type. Dissociation of bound radioligand from a receptor interaction with an affinity of 1 nM can occur in as little as 1.7 min. Lower affinity interactions can dissociate even quicker, when the separation process disrupts equilibrium.

Order of Addition

The order of addition for reagents may affect assay performance as well as ease of automation. A standard format for order of reagent addition in a filtration method is as follows:

Test compound
Radioligand
Receptor

Experiments may be required to determine the optimum order of addition and if there is any effect by locally high concentrations of DMSO present during the initial additions into the wells.

Non-Specific Binding

Radioligands may bind nonspecifically to components of the assay system such as tubes, pipette tips, assay plates or filters. This may lead to ligand depletion and certain binding assumptions may not be met. To test for nonspecific binding, perform an experiment in the absence of membranes. The amount of activity added can be tracked at each step of the assay to determine where any losses or nonspecific binding is occurring.

Some potential solutions to minimize nonspecific binding include the following:

Pretreatment of tubes (siliconization)
Additions to assay buffers (See table of Agents which Reduce NSB in the Assay Buffer section)
Different filter plate manufacturers (Packard, Millipore, Brandel, Polyfiltronics, etc.)
Different filter plate types (GF/C, GF/B, Durapore, etc.)

Since there may be non-receptor binding (to system components as described above), the use of an unlabeled ligand at a 100-fold excess may not be adequate to fully define all of the nonspecific binding.

Temperature

See SPA section on Temperature.

The filtration format can accommodate temperatures other than room temperature easier than the SPA format. The receptor/ligand/compound can be incubated at the desired temperature and then filtered to capture bound radioactivity. Since the filtration process is rapid, there is not a significant temperature drop during that time. Once the scintillant is added, the filter plates can be counted in the microplate scintillation counter at room temperature.

Plate Treatment Conditions

Filter plates are usually pre-wetted to ensure even distribution of the receptor/ligand reagents. If there is no ligand sticking problems, the pre-wet can be accomplished with Wash Buffer.

Pretreatment of filters with polyethylinimine (PEI) is a common practice to minimize ligand binding to filters:

Presoak 30 to 60 min in 0.1% to 0.5% PEI (in water)
Treat at 4°C to minimize filter degradation
Filter away PEI, then wash with ice-cold buffer prior to filtration of receptor sample

Pretreatment with carrier proteins, serum, or detergents has also been used to minimize binding of ligands to filter plates.

Note of Caution: Millipore Multiscreen glass fiber filter plates have a 0.65 mm Durapore support membrane under the GF filter. Some treatments (including PEI) may change or compromise the stability of this support membrane. Appropriate experiments should be designed to test for stability when using these types of plates.

Vacuum Pressure

The vacuum pressure used for filtration binding assays is a balance between having enough pressure to filter the samples rapidly and prevent ligand dissociation and having too much pressure which can affect filter integrity or the level of membranes retained on the filter. The pressure to be used should be determined experimentally, with a starting guideline of 5 to 10 mm Hg. If necessary, install an appropriate regulator to control consistent vacuum pressure.

Wash Buffer

Several washes of the filters are required to remove as much unbound radioligand as possible and to maximize specific binding. Generally, an ice-cold buffer is used to prevent or reduce dissociation of bound radioligand from the receptor.

Filter Plate Drying Time

If filters are not completely dry prior to the addition of liquid scintillant, the residual water present in the filters can interact with the scintillant to reduce counting efficiency. Dry filters require less liquid scintillant to achieve maximum signal than wetted filters. Drying filters completely may not be practical for medium or high throughput screening applications.

Type and Volume of Scintillant

The type of microplate scintillation counter being used may dictate the type of scintillant required for proper counting conditions.

TopCount - must use a "slow" scintillator such as Microscint-20 or Microscint-40

Trilux - can use virtually any scintillant designed for microplate scintillation counting (Microscint-20/Microscint-40, Optiphase Supermix, Meltilux)

Regular liquid scintillation cocktail such as Ready Pro should not be used, as a rule, for microplate scintillation counting as their load capacities may not be adequate and they may not be compatible with microplate plastics.

General volumes of liquid scintillant used are in the range of 40 to 150 μl. As mentioned above, the volume of scintillant used may depend on the dryness of the filters.

Exposure Time to Scintillant

With filter plates, some of the radioligand may be embedded within the filter and require some time to become accessible to the liquid scintillant for photon generation and signal detection. Therefore, an incubation time may be required following the addition of liquid scintillant and prior to counting. In addition to increasing the maximum signal, the variability of the signal may be reduced following an incubation time as demonstrated in the figure on the following page.

When processing large numbers of plates, it is important that a stable counting signal has been reached, so that all plates from the first counted to the last counted, are comparable.

Over time, more radioactive particles will be removed from the filter and make contact with the liquid scintillant in the well. As the data to the left shows, this can improve signal strength and decrease variability.