Limit of detection: 80% B/B₀: 1 pmol/ml · Sensitivity: 50% B/B₀: 4.6 pmol/ml · The Cayman Chemical cGMP Assay is a competitive EIA that can be used for quantification of cGMP directly obtained from cell lysates, tissue homogenates, plasma, or urine. The EIA typically displays an IC₅₀ (50% B/B₀) of approximately 6 pmol/ml and a detection limit (80% B/B₀) of less than 2 pmol/ml. Since the antibody used in this assay was prepared against a cGMP-carrier protein conjugate, antibody binding is increased if an acetyl group is present on the 2' hydroxyl group of the cGMP. The optional acetylation procedure for both samples and standards increases the sensitivity of the assay approximately 10 fold. A protocol for acetylating both the standards and samples prior to performing the assay is provided. Basal levels of cGMP in cell lysates can often be measured without acetylation, but results will depend on the type and number of cells being utilized. Platelets produce approximately 1.5-2.5 pmol cGMP/10⁹ platelets under basal conditions.^1,2,3 Cells such as NG108-15 cells and monocytes produce considerably more cGMP than platelets (approximately 0.1-1 pmol/10⁶ cells).^4,5

1 Chen, L., Salafranca, M.N., and Mehta, J.L. Cyclooxygenase inhibition decreases nitric oxide synthase activity in human platelets. Am J Physiol 42 H1854-H1859 (1997).

2 Miller, O.V., and Gorman, R.R. Modulation of platelet cyclic nucleotide content by PGE₁ and the prostaglandin endoperoxide PGG₂. J Cyclic Nucleotide Res 2 79-87 (1976).

3 Wu, C., Ko, F., Kuo, S., et al. YC-1 inhibited human platelet aggregation through NO-independent activation of soluble guanylate cyclase. Br J Pharmacol 116 1973-1978 (1995).

4 Arima, T., Ohshima, Y., Mizuno, T., et al. Cyclic GMP elevation by 5-hydroxytryptamine is due to nitric oxide derived from endogenous nitrosothiol in NG108-15 cells. Biochem Biophys Res Commun 227 473-478 (1996).

5 Kolb, J.P., Paul-Eugene, N., Damais, C., et al. Interleukin-4 stimulates cGMP production by IFN-γ-activated human monocytes. Involvement of the nitric oxide synthase pathway. J Biol Chem 269 9811-9816 (1994).

Pradelles, P., Grassi, J., Chabardes, D., et al. Enzyme immunoassays of adenosine cyclic 3',5'-monophosphate and guanosine cyclic 3',5'-monophosphate using acetylcholinesterase. Anal Chem 61 447-452 (1989).

Miller, O.V., and Gorman, R.R. Modulation of platelet cyclic nucleotide content by PGE₁ and the prostaglandin endoperoxide PGG₂. J Cyclic Nucleotide Res 2 79-87 (1976).

Stone, J.R., and Marletta, M.A. Soluble guanylate cyclase from bovine lung: Activation with nitric oxide and carbon monoxide and spectral characterization of the ferrous and ferric states. Biochemistry 33 5636-5640 (1994).

Maxey, K.M., Maddipati, K.R., and Birkmeier, J. Interference in enzyme immunoassays. J Clin Immunoassay 15 116-120 (1992).

Kolb, J.P., Paul-Eugene, N., Damais, C., et al. Interleukin-4 stimulates cGMP production by IFN-γ-activated human monocytes. Involvement of the nitric oxide synthase pathway. J Biol Chem 269 9811-9816 (1994).

Arima, T., Ohshima, Y., Mizuno, T., et al. Cyclic GMP elevation by 5-hydroxytryptamine is due to nitric oxide derived from endogenous nitrosothiol in NG108-15 cells. Biochem Biophys Res Commun 227 473-478 (1996).

Wu, C., Ko, F., Kuo, S., et al. YC-1 inhibited human platelet aggregation through NO-independent activation of soluble guanylate cyclase. Br J Pharmacol 116 1973-1978 (1995).

Ko, F., Wu, C., Kuo, S., et al. YC-1, a novel activator of platelet guanylate cyclase. Blood 84 4226-4233 (1994).

Wedel, B.J., and Garbers, D.L. New insights on the functions of the guanylyl cyclase receptors. FEBS Lett 410 29-33 (1997).

Leitinger, N., Blazek, I., and Sinzinger, H. The influence of isoprostanes on ADP-induced platelet aggregation and cyclic AMP-generation in human platelets. Thromb Res 86 337-342 (1997).

Chen, L., Salafranca, M.N., and Mehta, J.L. Cyclooxygenase inhibition decreases nitric oxide synthase activity in human platelets. Am J Physiol 42 H1854-H1859 (1997).

Schmidt, H.H.H.W., Lohmann, S.M., and Walter, U. The nitric oxide and cGMP signal transduction system: Regulation and mechanism of action. Biochim Biophys Acta 1178 153-175 (1993).

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