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The development of a somatostatin sst4 receptor-binding assay using the LEADseeker Multimodality Imaging System

14(tyr11) (final concentration 0.313 nM), 62.5 µg of SPA Imaging Bead, 0.313 µg of receptor preparation added together in a 20-µl volume, and 10 µl of 8-µM unlabeled somatostatin-28 (final concentration 2 µM).

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

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

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

8. For background estimation, bead-only wells (62.5 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.


Results
Kd determination
Saturation binding was carried out with dilutions of (3-[125I] iodotyrosyl11) Somatostatin-14(tyr11) to give a range of concentrations from 0.0 0347–3.00 nM in the assay wells. The saturation curve (Fig 1) was fitted using non-linear regression with the data analysis package GraphPad Prism v4.0. A Kd value of 0.379 nM (95% confidence intervals 0.298–0.460 nM) was estimated directly from the curve.

DMSO tolerance
Dilutions of DMSO were prepared in assay buffer to give a range of concentrations from 0.125 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 of 2% (v/v). '"/>

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