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Development of a histamine H1 receptor binding assay using the LEADseeker Multimodality Imaging System



Costar™ solid white 384-well (Corning, 3705)
non-treated assay plate

Protease-free BSA (Sigma, A3059)

Buffer:
50-mM Tris pH 7.4
5-mM MgCl2
0.2% BSA (w/v)

Graphpad Prism™ software v4.0 (GraphPad Software)


Protocol
Human recombinant Histamine H1 receptor membrane preparation was used in conjunction with [pyridinyl-5-3H]Pyrilamine ligand and WGA YOx SPA Imaging Beads. Non-specific binding (NSB) was determined in the presence of 10-µM pyrilamine. The standard assay format was as follows:

1. Reagents were added in the following order: buffer, unlabeled ligand (NSB wells), labeled ligand, premixed bead and membrane. Total assay volume was 50 µl.

2. Diluted bead and membrane was premixed immediately prior to addition to the wells.

3. Wells contained 10 µl of 28-nM [pyridinyl-53H]Pyrilamine (final concentration 5.6 nM) unless otherwise stated. Wells also contained 5 µg of membrane and 500 µg of WGA YOx SPA Imaging Beads. Bead and membrane were premixed immediately prior to addition to the wells. Premixed bead and membrane was added in a 20-µl volume.

4. NSB wells contained 10 µl of 50-µM pyrilamine (final concentration 10 µM) in addition to the above.

5. Plates were sealed and incubated in darkness for 3 h at room temperature (20–25 ºC) unless otherwise stated.

6. Following incubation, plates were imaged on the LEADseeker Multimodality Imaging System for 5 min using q
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