How to quantitatively detect cAMP and cGMP

How to quantitatively detect cAMP and cGMP
Background introduction
Sutherland discovered cAMP (cyclic adenosine monophosphate) in 1958 and thus won the Nobel Prize in Physiology and Medicine in 1971 [1]. In 1963, Ashman discovered cGMP (cyclic guanosine monophosphate) [2]. Fifty years have passed, and it has been understood that cAMP and cGMP are second messenger molecules that play important regulatory roles in many physiological processes. Guanylate cyclase catalyzes the production of cGMP by GTP, which can be hydrolyzed to GDP by phosphodiesterase. Activation of guanylate cyclase and inhibition of phosphodiesterase can increase intracellular cGMP levels. Inhibitors of cGMP-specific phosphodiesterase are used to treat certain diseases in humans, such as cGMP-specific phosphodiesterase type 5 inhibitors (Viagra and Cialis) for the treatment of ED (erectile dysfunction); Adenylate cyclase catalyzes the production of cAMP by ATP, which can be hydrolyzed by phosphodiesterase to ADP. Activation of adenylate cyclase and inhibition of phosphodiesterase can increase intracellular cAMP levels. Blockers of adenylate cyclase activating receptors and inhibitors of cAMP-specific phosphodiesterase are used to treat certain diseases in humans, such as blockade of b-adrenergic receptors that raise cAMP levels. The agent is used to treat diseases such as arrhythmia, hypertension, myocardial infarction and heart failure.
In the course of 50 years of research, from the beginning to study the regulation of cAMP, cGMP in various physiological processes, to the related drug development and drug screening in recent years, people have been trying to find a fast, sensitive , specific and reproducible methods for quantitatively detecting cAMP and cGMP levels.
Evolution of detection methods
The molecular weights of cAMP and cGMP are only 329.21 and 345.21, respectively, and the content in the body is extremely low, which is p mol/L level, which cannot be determined by general analytical methods. A series of methods for measuring cAMP and cGMP were invented in the 1970s. The most basic principles are three: competitive protein binding assay [3], radioimmunoassay [4] and thin-layer chromatography [5]. In the 1980s, radioimmunoassay was widely used and improved. Because of its radionuclide labeling, the application range was limited. In order to further improve the sensitivity of detection and the safety of operation, it was invented in the 1990s. A variety of competitive ELISA detection kits for enzymatic labeling and chemiluminescence. The fundamental principle of this type of detection method is based on the immune response of antibody antigens. All the antibodies used in these kits are rabbit antiserum or After specific affinity purification and anti-cAMP or cGMP rabbit polyclonal antibody, there is no doubt that rabbit polyclonal antibody has made great contributions in cAMP and cGMP quantitative detection for about forty years, but it also has obvious defects:
1, rabbit polyclonal antibody is not specific to cAMP, cGMP, and will cross-react with other nucleoside-like molecules, although the crossover can be reduced by specific affinity purification, but the crossover cannot be eliminated;
2. The affinity of rabbit polyclonal antibody for cAMP and cGMP is affected by high concentrations of divalent cations (such as Mg2+ and Ca2+);
3, rabbit polyclonal antibody in the process of preparation there are individual differences and batch-to-batch differences, it is difficult to ensure the repeatability of the test data;
4, rabbit polyclonal antibody kit also needs to be acetylated in the treatment of cAMP, cGMP samples, which is inextricably related to the preparation method of rabbit polyclonal antibody. If it is not acetylated, the sensitivity required for detection will not be achieved.
Therefore, no matter how the labeling method is reformed and the antibody is not improved, the change of cAMP and cGMP quantitative detection cannot be fundamentally ushered in.
Successful development of cAMP and cGMP monoclonal antibodies
After two years of research and development, NewEast Biosciences scientists screened more than 40,000 clones and finally found highly specific, high-affinity cAMP and cGMP monoclonal antibodies. And successfully built the world's only monoclonal antibody-based cAMP and cGMP ELISA test kits. The specific ability of this cAMP(cGMP) mAb to recognize non-acetylated cAMP (cGMP) is 108 times more than its recognition ability for cGMP (cAMP), ATP (GTP) and other nucleoside analogs. The previous multi-anti-reagent kit greatly improved the sensitivity and specificity of cAMP (cGMP) detection, and greatly reduced the batch-to-batch variation of the kit, providing long-term and effective reproducibility detection, and completely eliminating the cumbersome The acetylation process and acetylation reagents that are detrimental to the health of researchers.
Once launched, the kit has been widely recognized by the academic community, and has published more than a dozen papers in recent years.
Published papers:
1. Castro, L. R., J. Schittl, et al. (2010). "Feedback control through cGMP-dependent protein kinase contributes to differential regulation and compartmentation of
cGMP in rat cardiac myocytes." Circ Res 107(10): 1232-1240.
2. Guo, D., J. J. Zhang, et al. (2009). "Stimulation of guanylyl cyclase-D by bicarbonate." Biochemistry 48(20): 4417-4422.
3. Long, Y., Q. Li, et al. (2011). "Molecular analysis, developmental function and heavy
Metal-induced expression of ABCC5 in zebrafish." Comp Biochem Physiol B Biochem Mol Biol 158(1): 46-55.
4. Tseng, K. Y., A. Caballero, et al. (2011). "Inhibition of Striatal Soluble Guanylyl Cyclase-cGMP Signaling Reverses Basal Ganglia Dysfunction and Akinesia in Experimental
Parkinsonism." PLoS One 6(11): e27187.
5. Zhang, Y., Y. Sun, et al. (2011). "Molt-inhibiting hormone from Chinese mitten crab (Eriocheir sinensis): Cloning, tissue
Expression and effects of recombinant peptide on ecdysteroid secretion of YOs." Gen
Comp Endocrinol 173(3): 467-474.
6. Zheng, X., L. Ying, et al. (2011). "Role of sulfhydryl-dependent dimerization of soluble guanylyl cyclase in relaxation of porcine coronary
Artery to nitric oxide." Cardiovasc Res 90(3): 565-572.
7. Tsai, E. J., Y. Liu, et al. (2012). "Pressure-overload-induced subcellular relocalization/oxidation of soluble guanylyl cyclase in the heart
Modulate enzyme stimulation." Circ Res 110(2): 295-303.
8. Andre, L., J. Fauconnier, et al. (2012). "Subendocardial Increase in Reactive Oxygen
Species Production Affects Regional Contractile Function in Ischemic Heart Failure."
Antioxid Redox Signal.
9. Makiya, M., M. Dolan, et al. (2011). "Structural basis of anthrax edema factor
Neutralization by a neutralizing antibody." Biochemical and Biophysical Research
Communications. ???
10. Skrabalova, J., J. Neckar, et al. (2012). "Antiarrhythmic effect of prolonged morphine
Exposure is kissing by altered myocardial adenylyl cyclase signaling in rats."
Pharmacol Rep 64(2): 351-359.
11. Ying, L., X. Xu, et al. (2012). "Heterogeneity in relaxation of different sized porcine
Coronary arteries to nitrovasodilators: role of PKG and MYPT1." Pflugers Arch 463(2): 257-268.

references:
1, Sutherland, et al. Cyclic AMP. Academic Press. Science 174, 392 (1971).
2, Ashman, et al, Biochem Biophys Res Commun, 11, 330 (1963).
3, Honma, M. et al., Biochem. Med., 18, 257 (1977).
4, Goldberg, M. L., Clin. Chem., 23, 576 (1977).
5, Trifilo, R. M. et al., J. Chromatogr., 116, 465 (1976).

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