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

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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), Ki, 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), Ki, 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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