( lation Counter and the level of bound r...)In Vitro Measurement of the Second Messenger cAMP: RIA vs. FlashPlate

Results

Using the RIA method, the agonist 5-HT produced a dose-dependent inhibition of forskolin-stimulated cAMP levels with a pEC₅₀ = 7.5 0.2 and a maximum response of 84 7% inhibition (Fig. 1a). The FlashPlate technique produced a comparable 5-HT inhibition curve with a pEC₅₀ = 7.7 0.1 and a maximum response of 84 4% (Fig. 1b). The two assays produced similar results between other parameters, such as absolute levels of cAMP measured, and intra-assay variation (Table 1). Inter-assay variation using the FlashPlates was minimal, with only 6% error on three separate determinations of the same sample (5.07 0.33 pmol cAMP/ml). However, one obvious difference between the two methods was the time required to assay the same number of samples; the FlashPlate method saved several hours compared to the RIA method.

Discussion

To date, radioimmunoassay has been an effective method to measure cAMP within samples. It is, however, a laborious technique with few options for automation and hence not conducive for use in high throughput screening (HTS) applications. The cAMP [¹²⁵I] FlashPlate Assay has overcome these drawbacks: its 96-well microplate format has proven itself highly efficacious for robotic HTS, permitting cAMP measurements to be fully automated, with improvements realized in both efficiency and accuracy. The assay kit (SMP001) is comprised of fewer components than the RIA kit, and is easier to use. Additionally, the elimination of the separation step in the FlashPlate method is a major time saving advance, and again favors full automation. The results obtained from the FlashPlates are very reproducible and comparable to those obtained from RIA, and so the former can be confidently used as an alternative to the latter. The FlashPlate method is extremely sensitive
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