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

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IC50 determination
Competition binding of 0.313 nM (~ 55 000 dpm) (3-[125I] iodotyrosyl11) Somatostatin-14(tyr11) with unlabeled somatostatin-28 and octreotide were assessed and the IC50 values calculated (Fig 3). For the competition assays, the unlabeled ligands were prepared in assay buffer containing 2% (v/v) DMSO over the following ranges: somatostatin-28 (0.119 pM–1 µM) and octreotide (0.119 pM–8 µM).

For somatostatin-28, the IC50 value was 2.58 nM (95% confidence interval range 2.02 to 3.30 nM) and the Ki value was 1.42 nM (95% confidence interval range 1.11 to 1.81 nM). For octreotide, the IC50 value was 2.63 µM (95% confidence interval range 1.01 to 6.89 µM) and the Ki value was 1.44 µM (95% confidence interval range 0.55 to 3.77 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 6-h incubation at room temperature, and the signal was stable for at least another 14 h (Fig 4).

Z’ analysis
A Z’ analysis was carried out using 48 replicate values for total and NSB wells. Z’ was 0.85 (Fig 5), which confirmed the robustness of the assay (7).


Conclusions
The somatostatin sst4 receptor-binding assay has been successfully miniaturized to a 384-well format on LEADseeker Multimodality Imaging System. The assay is suitable for high-throughput screening and is tolerant up to 2% DMSO (v/v). The assay is robust, exhibiting a Z’ value of 0.85, and has a sta
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