( Key words: ...)The development of a somatostatin sst4 receptor-binding assay using the LEADseeker Multimodality Imaging System

Key words: somatostatin • receptor binding assay • LEADseeker • SPA Imaging Beads

Somatostatin (SS) is a neurotransmitter/hormone with a wide range of biological actions (1–3) and a reduction in cortical SS levels has been reported in Alzheimer’s and Parkinson’s diseases. The SS analog, octreotide (SMS 201–995), is used clinically in the treatment of certain tumors, carcinoid syndrome, and glucagonoma. Somatostatin inhibits cell proliferation and growth hormone secretion through interaction with SS receptors (4). Five SS receptors have been cloned and many studies have investigated the tissue distribution, expression, binding affinity to SS, and down-stream signal transduction pathways of these five receptors (5, 6). It has also been shown that SS receptors are G protein coupled. All five SS receptors have similar binding affinities to their natural ligand SS; however, they bind SS analogs, such as octreotide, at different affinities.

This application note describes a 384-well somatostatin sst4 receptor-binding assay which has been developed on the LEADseeker™ Multimodality Imaging System.

Materials
Reagents and instrumentation
LEADseeker Multimodality Imaging System 18-1140-71

Polyethyleneimine (PEI) PS SPA Imaging Beads RPNQ0098

(3-[¹²⁵I] iodotyrosyl¹¹) Somatostatin-14(tyr¹¹), IM161-50UCI
1.85 MBq, 50 µCi

Other material required
Human recombinant somatostatin sst4 receptor membrane preparation (Euroscreen, ES-524-M)

Somatostatin-28 (Sigma, S-6135)

Octreotide (Sigma, O-1014)

384-well white flat-bottom polystyrene, not-treated microplates, non-sterile (Corning, 3705)

Assay buffer: 25-mM HEPES, pH 7.4, 5-mM MgCl₂, 1-mM CaCl₂, 100-µg/ml bacitracin, and 0.2% (w/v) protease-free BSA.

GraphPad Prism™ software (GraphPad Software).

Protocol
Human recombinant somatostatin sst4 receptor membrane preparation produced in CHO-K1 cells was used in conjunction with (3-[¹²⁵I] iodotyrosyl¹¹) Somatostatin-14(tyr¹¹) and PEI PS SPA Imaging Beads. Non-specific binding (NSB) was determined in the presence of 2-µM somatostatin-28. The standard assay format was as follows.

1. Reagents were added in the following order: assay buffer or buffer containing 2% (v/v) DMSO solution, unlabeled ligand (NSB wells), labeled ligand, and premixed bead and membrane. Total assay volume was 40 µl.

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

3. Wells contained 10 µl (~ 55 000 dpm) of 1.25-nM (3-[¹²⁵I] iodotyrosyl¹¹) Somatostatin-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 assay buffer in the absence of competing ligand. For competition assays, 10 µl of competing ligand prepared in assay buffer containing 2% (v/v) DMSO was added with (3-[¹²⁵I] iodotyrosyl¹¹) Somatostatin-14(tyr¹¹) (10 µl, as above), precoupled bead, and receptor (20 µl), also giving a total assay volume of 40 µl.

4. NSB wells contained 10 µl of 1.25-nM (3-[¹²⁵I] iodotyrosyl¹¹) Somatostatin-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
K_d 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 K_d 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).

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 K_i 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 K_i 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 stable signal of at least 14 h.

References
1. Bell, G.I. and Reisine, T.R., Trends Neurosci. 16, 34–38 (1993).

2. Epelbaum, J., Prog. Neurobiol. 27, 63–100 (1986).

3. Rens-Domiano, S and Reisine, T.R. J., Neurochem. 58, 1987–1996 (1992).

4. Li, M., et al. World J. Surg., 29, 293–296 (2005).

5. Benali, N., et al. Digestion 62, 27–32 (2000).

6. Fisher, W.E., et al. J. Natl. Cancer Inst. 90, 322–324 (1998).

7. Zhang, J., et al. J. Biomol. Screening 4, 67–73 (1999).

back to top
'"/>

Source: