Effect of assay configuration on determination of cAMPconcentration in the DiscoverX hithunter cAMP II™ assay

Vicki L. Mason,* Christine Williams,† David McLoughlin† and Philip G. Strange* *School of Pharmacy, University of Reading, Whiteknights, Reading, Berkshire †Pfizer Global Research and Development, Ramsgate Road, Sandwich, Kent.

The production of cAMP from ATP on activation of adenylyl cyclase presents a reliable platform for the high-throughput-screening of G-protein coupled receptors (Williams, 2004). To screen for agonist activity at Gi-GPCRs it is necessary to activate adenylyl cyclase with forskolin and measure the agonist-induced inhibition of cAMP production.

Inter-assay comparisons of cAMP levels can be made by performing cAMP standard curves. The DiscoveRx HitHunter cAMP II™ assay is a highly sensitive non-radiometric method for measuring cAMP production (Golla and Seethala, 2002). In this assay, it has been suggested that cAMP standard curves can be performed using either PBS or DMEM as the diluent. However, this study has shown that to ensure accurate quantification of cAMP production, it is important that conditions used for the cAMP standard curve are comparable with those used for forskolin dose response curves.

Assays were performed in triplicate in a 384 well format; total assay volume was 15µµl. cAMP standard curves were obtained by performing 1:3 serial dilutions of the cAMP standard in either DMEM (serum free) or PBS. 15µl cAMP (final concentration 83.33mM – 17.42pM) or 5µl cAMP (DMEM; final concentration 27.78µM – 5.81pM) was placed in to the assay. In the case of the 5µl addition, 10µl PBS was added containing DTT and DMSO (final concentration 100µM and 0.5% respectively). Forskolin dose response curves were produced by performing 1:2 serial dilutions in PBS. Forskolin (5µl, final concentration 250µM – 15.26nM) was placed in the assay with 5µl PBS containing DTT and DMSO (final concentration 100µM and 0.5% respectively), and CHO-K1 cells stably expressing the D2s dopamine receptor (Wilson et al., 2001) (5ml DMEM, 20,000 cells/well). The assay was incubated for 1.5h at 37°C in a humidified atmosphere of 5% CO2.

In the cAMP standard curves potency values for cAMP (pEC50) were not significantly different when using DMEM (pEC50 = 6.91 ± 0.08, n = 3), PBS (pEC50 = 7.08 ± 0.02, n = 3) or assay medium (5µl DMEM, 10µl PBS, 0.5% DMSO, 100µM DTT; pEC50 = 6.88 ± 0.10, n =3) as the diluent (one-way ANOVA, P>0.05). The assay range (relative light units; RLUs) differed significantly depending on the diluent used (DMEM, 6140 ± 365; PBS, 28681 ± 2483; assay, 16496 ± 1163, n = 3; one-way ANOVA, P < 0.05). Only the cAMP standard curve performed in assay medium encompassed the range covered by the forskolin dose response curve, hence it was possible to convert forskolin activity in to units of cAMP only under this condition. No significant difference existed in pEC50 values and Hill slope coefficients (nH) for forskolin dose response curves when considering RLUs or units of cAMP (nM) (RLU pEC50 = 4.33 ± 0.06; RLU nH = 1.82 ± 0.08; cAMP (nM) pEC50 = 4.16 ± 0.09; cAMP (nM) nH = 2.09 ± 0.28; n = 3; two-tailed paired t-test p >0.05).

This study has shown that to ensure accurate conversion of forskolin dose response curve data (RLUs) into units of cAMP (nM) it is extremely important to ensure assay conditions are as comparable as possible. This allows for inter-assay comparisons to be made regarding the sensitivity of assays to cAMP production.


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