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The development of a melanocortin MC4 receptor binding assay using the LEADseeker Multimodality Imaging System

cro;l of competing ligand prepared in assay buffer containing 2% (v/v) DMSO was added with [125I] NDP-α-MSH (10 µl, as above), precoupled bead and receptor (20 µl), also giving a total assay volume of 40 µl.

3. NSB wells contained 10 µl of 2-nM [125I] NDP-α-MSH (final concentration 0.5 nM), 31.25 µg of SPA Imaging Bead, 250 ng of receptor preparation added together in a 20 µl volume, and 10 µl of 8-µM unlabeled NDP-α-MSH (final concentration 2 µM).

4. For DMSO tolerance studies, DMSO was diluted in assay buffer to give final concentrations per well as shown in the results.

5. Plates were sealed and incubated overnight at room temperature (20–25 ºC).

6. Following incubation, plates were imaged on the LEADseeker Multimodality Imaging System for 5 min with quasi-coincident averaging and 3 ×3 binning.

7. For background estimation, bead-only wells (31.25 µg of bead/well, 20 µl), plus assay buffer (20 µl) were included in all experiments. Typically backgrounds exhibited from bead-only wells were less than 10 integrated optical density (IOD) units, and were routinely subtracted from total and NSB values during data analyses.

* Diluted membrane and bead were precoupled at room temperature immediately prior to assay addition.


Results
Kd determination

Saturation binding was carried out with dilutions of [125I] NDP-α-MSH to give a range of concentrations from 4.05 pM to 10.43 nM in the assay wells. Figure 1 shows the saturation curve which was fitted using non-linear regression with the data analysis package GraphPad Prism v4.0. A Kd value of 0.931 nM (95% confidence intervals 0.744 to 1.117 nM) was estimated directly from the curve
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