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Amersham has developed a commercially available scintillation proximity assay (SPA) kit that can be used for measurement of cAMP. Either whole cells or plasma membranes containing the receptor of interest can be used. The strength of this assay is that laborious separation and washing steps, necessary for traditional measurement methods, are not required resulting in a vast increase in assay throughput. The important components of the SPA cAMP assay system include the use of an anti-cAMP primary antibody and a secondary antibody coupled to SPA beads. The principle of the assay is that trace [125_I] cAMP added to the assay reaction mixture will compete with cAMP generated in the

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assay for binding to the primary antibody. The SPA bead is brought into close proximity of [125I]cAMP through the secondary antibody. As cAMP increases, the signal in the assay decreases.

Figure 15: Receptor-mediated regulation of cellular cAMP levels.

The steps involved in the Amersham cAMP assay are as follows:

1) Célls or membranes, agonist if desired, and test extracts are added to a standard microtiter plate and incubated for a pre-established period at room temperature. 2) Microtiter plates are then centrifuged and an aliquot is removed and placed in another

microtiter plate, along with standards and buffer for blank wells. It may be possible to perform the initial cAMP assay and cAMP determinations in the same plate if low amounts of membranes or cells are used and if the level of cAMP generated in the initial incubation is sufficient to fall within the standard curve. If cells are used, they need to be lysed prior to centrifugation of plates.

3) After transfer of an aliquot of the reaction mixture to a microtiter plate, [125I]cAMP tracer, anti-cAMP antibody, and SPA-anti-rabbit secon dary antibody conjugate are added, and plates are incubated for 15–20 hours at room temperature.

4) Plates are then counted in a microtiter plate scintillation counter. cAMP values in experimental samples are determined by extrapolation from the generated cAMP standard curve. Typically, a standard curve is generated from each assay plate. The components and steps of the SPA-cAMP assay are listed in Figure 16.

Figure 16: Steps involved in the SPA-cAMP assay.

The following equipment would be required for a high throughput SPA cAMP assay: -96 well liquid handling system (optional)

-Microtiter plate scintillation counter (Wallac MicroBeta or Packard Top Count)

-robotics system for plate manipulations (optional)

2.3.3.2 FlashPlate cAMP assay

Dupont-NEN has adapted its FlashPlate™ technology to be used as a high throughput assay method for measurement of cAMP levels. The basic principle of the assay is competition of the trace [125I]cAMP added and cAMP generated during incubations for binding to an anti-cAMP antibody which is affixed to scintillant-coated microtiter plate wells. The primary advantage of this assay method, like the SPA assay, is that separation of bound from free tracer radiolabelled cAMP is not required.

The steps involved in the FlashPlate™ cAMP assay are as follows:

1) Cells or membranes, agonists if desired, and test extacts are added to microtiter plates and incubated for a predetermined period at room temperature.

2) Plates are then centrifuged, an aliquot of the reaction mixture is removed and placed in the FlashPlate. It may be possible to perform the initial cAMP generation assay and the cAMP measurement both in FlashPlates if the amount of membrane added is not too expensive and if the levels of cAMP generated fall within the standard cAMP curve. If cells are used, they need to be lysed prior to centrifugation.

3) cAMP reference standards followed by aliquots of the reaction mixture are added to appropriate wells. Tracer cAMP is then added and the plates are incubated for 18–24 hours at 2–8°C.

4) Plates are then read in a microtiter plate scintillation counter. Concentrations of cAMP are extrapolated from the standard curve. Figure 17 details the components and steps associated with the FlashPlate cAMP assay.

Figure 17: Steps involved in the FlashPlate assay for cAMP.

The following equipment would be required for performing the high troughput FlashPlate cAMP assay:

-96 well liquid handling system (optional)

-Microtiter plate scintillation counter (Packard Top Count only) -robotics system for plate manipulations (optional)