Sample
Plasma, saliva and serum
Intended Use
For the quantitative determination of cAMP in culture supernatants, serum, and saliva from any species. Cited sample types include cell lysate, tissue, and urine.
Contents of Kit
1. Assay Buffer 2, 27mL, Sodium acetate buffer containing proteins and sodium azide.
2. cyclic AMP Standard, 0.5mL, A solution of 2,000 pmol/mL cAMP.
3. Acetylation Kit, 2 vials
a. Triethylamine, 2 mL
b. Acetic Anhydride, 1 mL
4. Goat anti-Rabbit IgG Microtiter Plate, One plate of 96 wells, A clear plate of break-apart strips coated with a goat anti-rabbit polyclonal antibody.
5. cAMP Antibody, 5mL, A yellow solution of rabbit polyclonal antibody to cAMP.
6. cAMP Conjugate, 5mL, A blue solution of cAMP conjugated to alkaline phosphatase.
7. Wash Buffer Concentrate, 27mL, A 20× solution of tris buffered saline containing detergents.
8. pNpp Substrate, 20 mL, A solution of p-nitrophenyl phosphate.
9. Stop Solution 2, 5 mL, A solution of trisodium phosphate in water.
Storage
All components of this kit, except the Conjugate and Standard, are stable at 4°C until the kit' s expiration date. The Conjugate and Standard should be stored at -20°C upon receipt.
Precision
Intra-assay was determined by assaying 20 replicates of three buffer controls containing cAMP in a single assay.
Inter-assay was determined by measuring buffer controls of varying cAMP concentrations in multiple assays over several days.
Sensitivity
The sensitivity of the assay, defined as the concentration of cAMP measured at 2 standard deviations from the mean of 18 zeros along the standard curve, was determined to be 0.49 pmol/mL in the non-acetylated assay format and 0.027 pmol/mL in the acetylated assay format.
General Description
The cyclic AMP Enzyme-Linked Immunosorbent Assay (ELISA) kit is a competitive immunoassay for the quantitative determination of extracellular cyclic AMP in plasma, saliva, serum, and culture supernatant. The optional acetylated assay format provides a significant increase in sensitivity and is ideal for samples with extremely low levels of cAMP. If expected levels of cAMP are unknown, the investigator may evaluate a few samples in the nonacetylated format in order to determine if higher sensitivity is required.
Adenosine 3', 5'-cyclic monophosphate (cyclic AMP; cAMP) is one of the most important "second messengers" involved as a modulator of physiological processes.cAMP is also involved in regulating neuronal, glandular, cardiovascular, immune and other functions. A number of hormones are known to activate cAMP through the action of the enzyme adenylate cyclase which converts ATP to cAMP. These hormones include a variety of anterior pituitary peptide hormones such as corticotropin (ACTH), glucagon, calcitonin, thyroid stimulating hormone (TSH), and luteinizing hormone (LH). Because cAMP has been shown to be involved in the cardiovascular and nervous systems, immune mechanisms, cell growth and differentiation, and general metabolism, there remains considerable interest in the measurement of intracellular cAMP in tissues and cell cultures. The investigation of cAMP may help to provide a clearer understanding of the physiology and pathology of many disease states.
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