( . P P ...)The development of a melanocortin MC4 receptor binding assay using the LEADseeker Multimodality Imaging System

DMSO tolerance
Dilutions of DMSO were prepared in assay buffer to give a range of concentrations from 0.5 to 32% (v/v) in the assay wells. From the results shown in Figure 2, it can be seen that the assay was tolerant of DMSO up to a final concentration 2% (v/v).

IC₅₀ determination
Competition binding of 0.5-nM (~ 88 000 dpm) [¹²⁵I] NDP-αMSH with unlabeled α- and β-MSH, were assessed and the IC₅₀ values calculated as shown in Figure 3. For the competition assays, the unlabeled ligands were prepared in assay buffer containing 2% (v/v) DMSO over the range of 12.2 nM to 100 µM. The IC₅₀ value for α-MSH was 1.13 µM (95% confidence interval range 0.804 to 1.595 µM), K_i, α-MSH, 0.737 µM (95% confidence interval range 0.524 to 1.04 µM), the IC₅₀ value for α-MSH was 0.567 µM (95% confidence interval range 0.497 to 0.647 µM), K_i, α-MSH 0.369 µM (95% confidence interval range 0.324 to 0.422 µM).

Time course
A time course experiment was carried out using reagent concentrations as described in the protocol. Measurements were made over an assay incubation time of 20.5 h. The assay reached equilibrium after 8-h incubation at room temperature and the signal was stable for at least another 12.5 h (Fig 4).

Z’ analysis
A Z’ analysis was carried out using 48 replicate values for total and NSB wells. Z’ was 0.796 (Fig 5), which confirmed the robustness of the assay (3).

Conclusion
The melanocortin MC₄ receptor binding assay has been successfully miniatur
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