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Utilization of Donkey Anti-Rabbit PS SPA Imaging Beads in the quantitative measurement of cAMP in cells

Key words: SPA Imaging Beads • LEADSeeker • cAMP • receptor binding assay


SPA Imaging Assays are homogeneous immunoassays based on the coupling of one assay component to an SPA Imaging Bead in order to generate a signal. Polystyrene (PS) SPA Imaging Beads coated with donkey anti-rabbit antibody enable the immobilization of rabbit secondary antibodies during the assay. The Donkey Anti-Rabbit PS SPA Imaging Beads bind to the Fc region of the secondary antibody, leaving the antigen binding Fab region unhindered. Application data is demonstrated here for the utilization of Donkey Anti-Rabbit PS SPA Imaging Beads in a cAMP assay.


SPA Immunoassay principle
Binding radiolabeled ligands to antibodies immobilized on the surface of an SPA Imaging Bead brings the radiolabel into close proximity with scintillant incorporated within the bead. The emitted radiation (beta particles for tritium or Auger electrons for iodine-125) then stimulates the scintillant to emit light (Fig 1). Unbound radiolabeled ligand is not in close enough proximity to the scintillant for energy transfer to occur and no signal is generated. Light emitted by stimulated SPA Imaging Beads can be detected by platforms such as the LEADseeker™ Multimodality Imaging System.


cAMP assay
Adrenergic receptors are linked to adenylate cyclase via either inhibitory or stimulatory G proteins. These receptors, when activated by agonist or antagonist occupancy of ligand binding sites, affect the intracellular generation of cAMP. In response to receptor binding, adenylate cyclase converts AMP to cAMP, which exerts its effect by activating a protein kinase capable of phosphorylating specific substrates.

Here, we describe a direct screening 384-well assay that enables the direct extraction and assay of cAMP from cultured cells utilizing Donkey Anti-Rabbit PS SPA Imaging Beads to generate signal in a simple, homogeneous format (Fig 2).


Materials
Products used

LEADseeker Multimodality Imaging System 18-1140-71

Donkey Anti-Rabbit PS SPA Imaging Beads RPNQ0299


Other material required
Assay buffer

cAMP standard

Adenosine 3',5'-cyclic phosphoric acid 2'-0-succinyl-3-[125I] iodotyrosine methyl ester, 147.5 kBq, 4 µCi, lyophilized.

Rabbit anti-succinyl cAMP serum, lyophilized.

Lysis reagent 1

Lysis reagent 2

The above reagents are available in the cAMP – [125I] Direct Biotrak™ SPA Screening Assay (RPA 559).


Method
Chinese hamster ovary (CHO) or A431 cells were seeded into 384-well cluster plates suitable for both cell culture and imaging. Cells were stimulated (20 min 37 °C, 5% CO2, 95% humidity) either with forskolin (1–1000 µM) (Fig 3), one of the β-adrenoreceptor agonists: isoproterenol, epinephrine, salbutamol, or noradrenaline (1.25–1000 nM) (Fig 4), or pre-incubated for 30 min with the non-selective adrenoreceptor antagonist propranolol (0.001–1000 nM), before stimulation with isoproterenol (1000 nM) (Fig 5) (1).


A two-stage method was applied, where the cells were cultured and lyzed in a separate vessel. The culture supernatant was gently aspirated and the cells lyzed with 0.25% (w/v) dodecyltrimethyl ammonium bromide. An aliquot of lysate was transferred to a second plate for assay.

Working standards (0.2–25.6 pmol/well) were pipetted into empty wells of the plate. Equal volumes of antisera (20 µl/well), [125I]cAMP (~ 35 000 cpm/well), and Donkey Anti-Rabbit PS SPA Imaging Beads (20 µl, 0.4 mg/well) were added to the microplate wells containing standards and samples. The plates were sealed and incubated at room temperature for 15–20 h, without agitation. The amount of [125I]cAMP bound to the SPA Imaging Beads was determined by imaging on the LEADseeker Multimodality Imaging System for 5 min (2).


Results
The results in Figure 3 show the cAMP produced by CHO cells in response to stimulation by forskolin, an activator of adenylate cyclase. The cells have responded in a dose dependent manner and an increase in cAMP levels can be detected at 1 pmol forskolin, the lowest concentration used.

Figures 4 and 5 show the measurement of cAMP in A431 cells treated with known B2 adrenergic agonist and antagonists. The EC50 values obtained are comparable to those quoted in the literature (3), showing that this imaging assay in 384-well format can be successfully used for high-throughput assays.


Conclusion
Donkey Anti-Rabbit PS SPA Imaging Beads give a good specific signal and large signal window in the cAMP assay. These beads have been utilized in pharmacological experiments and the results correlate very well with other coated SPA beads and non-SPA formats.

Please refer to the SPA imaging bibliography at www.amershambiosciences.com/LEADseeker for a full list of SPA imaging application references.


References
1. CHIESI, M. et al., Trends in Pharmacol. Sci., 22, (5), 247–254, (2001).

2. PRICE, E. et al., Direct, quantitative, intracellular measurement of cyclic AMP in cells: A LEADseeker assay utilizing Protein A Polystyrene SPA Imaging Beads. Poster presentation, Screening Europe Conference, London (2004).

3. SCHWARTZ, M.W. et al., Nature, 404, 661–671, (2000).



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