Section5:Filtration Assays

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Concept

Filter assays differ from SPA because a separation of free radioligand and radioligand bound to the receptor is required for measurement. However, many of the assay development and optimization steps are the same. Specific information to the filter assay format is included in this section, and reference back to the text under the SPA section is made when there is no significant difference between the two formats. A diagram for a standard filtration assay is shown below.

General Steps for a filtration assay:

1. Add and incubate test compound, radioligand and receptor in a plate (this can be a separate plate or if validated, the filtration plate directly)
2. Apply vacuum to "trap" receptor and bound radioligand onto filter and remove unbound radioligand. Wash several times with an appropriate buffer to minimize nonspecific binding.
3. Allow filters to dry. Add liquid scintillation cocktail or other scintillant (i.e. solid Meltilux).
4. Count filters in microplate scintillation counter. Some time between adding the scintillant and counting may be required.

Advantages		Disadvantages
Less color quenching Traditional, trusted method Higher efficiency than SPA Kinetic experiments easier Association/Dissociation		Separation method (dissociation of ligand) Generated large volumes of liquid waste Variable vacuum across plate Nonspecific binding to filters Accumulation of radioactivity on unit Requires more handling steps