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Monoclonal Anti cAMP Antibody Based Direct cAMP ELISA Kit

Monoclonal Anti-cAMP Antibody Based
Direct cAMP ELISA Kit
(New Non-acetylated Version)
Catalog No. 80203
96 Well Kit

Product Description
Adenosine 3', 5'-cyclic monophosphate (cyclic AMP; cAMP) modulates various physiological functions
such as cardiovascular biology, learning and memory, olfaction, immune response, asthma and kidney
function (1,2). cAMP is produced from ATP by adenylyl cyclases and is degraded by
phosphodiesterases. Stimulation of adenylyl cyclases or inhibition of phosphodiesterases can increase
cellular cAMP concentrations. Blockers of adenylyl cyclase-activating receptors and inhibitors of the
cAMP-specific phosphodiesterases are used for treating human diseases. For example, blocking agents
for cAMP-increasing beta-adrenergic receptors (beta-blockers) are used for
treating abnormal heart rhythms, high blood pressure (hypertension),
myocardial infarction and heart failure. Inhibitors of cAMP specific
phosphodiesterase types 2 and 4 are being tested for cognition
enhancement.
To screen for inhibitors or stimulators of cellular cAMP levels, it is
essential to have a sensitive, selective and reproducible method to measure
the cAMP concentrations. This is especially true for the initial screenings
given the possible weaker effects of larger pools of compounds.
Currently available other ELISA kits measuring cAMP levels are based on
the non-affinity-purified polyclonal anti-cAMP antibody. Despite the
claimed selectivity, these polyclonal anti-cAMP antibodies display certain
cross-reactivity with ATP. Given that ATP is the substrate for the cAMP production, it is very desirable
to have an antibody with high specificity towards cAMP over ATP.
NewEast Biosciences cAMP ELISA kit is based on the unique mouse monoclonal anti-cAMP
antibody. This monoclonal anti-cAMP antibody displays >108 fold of selectivity over ATP, cGMP, and
other nucleoside analogues. NewEast Biosciences cAMP ELISA kit provides significantly improved
sensitivity and selectivity over other kits based on polyclonal anti-cAMP antibodies. Our monoclonal
anti-cAMP antibody-based ELISA kit also avoids the batch-to-batch variations associated with
polyclonal antibody productions from animals, thus providing the reproducibility in the long run.
Furthermore, while polyclonal anti-cAMP antibodies used in other ELISA kits have higher affinity for
acetylated cAMP than non-acetylated cAMP, NewEast Biosciences monoclonal anti-cAMP antibody has
similar affinities to non-acetylated and acetylated cAMP molecules. Therefore, acetylation treatments of
samples and standards are not needed in NewEast Biosciences cAMP ELISA kit. This significantly
reduces the time for the assay. The avoidance of organic reagents used in the acetylation process
provides a safe and healthy work environment.

Principle Outline
NewEast Biosciences cAMP ELISA Kit is a competitive immunoassay to measure the cAMP levels,
either from cell extracts or from in vitro adenylyl cyclase assays. Briefly, multi-well plates are coated
with goat-anti-mouse serum. cAMP in cell extracts or in in vitro adenylyl cyclase assays will
competitively bind to the monoclonal anti-cAMP antibody in the presence of fixed amounts of
cAMP-conjugated horse-radish peroxidase or alkaline phosphatase. Known amounts of cAMP are used
as standards to generate the calculation curve. After a short incubation, the excess reagents are washed
away and substrate is added. The multiwell plates are then read on a microplate reader at 450 nm or 405nm. The intensity of the yellow color is inversely proportional to the concentration of cAMP in samples.
The measured optical density is used to calculate the concentration of cAMP in samples based on the curve
from the cAMP standards.
Background
cAMP is a ubiquitous second messenger mediating cellular responses to various exogenous and
endogenous signaling molecules. cAMP regulates physiological processes by activating protein
kinases, gating specific ion channels, modulating cellular cyclic nucleotide concentrations through
phosphodiesterases, and activating Epac (exchange protein directly activated by cAMP) (3-6). The
conversion of ATP to cAMP is catalyzed by adenylyl cyclases (ACs). The major family of ACs in
mammals is the transmembrane ACs which have nine isoforms and could be activated by G protein Gs
and/or Ca2+/calmodulin (1). There is also one soluble AC which could be modulated by bicarbonate
and/or Ca2+ (7-9).
Materials Supplied
1. Goat anti-Mouse IgG Microtiter Plate, One Plate of 96 Wells, Catalog No. 30101 A
plate using break-apart strips coated with goat antibody specific to mouse IgG.
2. cAMP Direct Conjugate, 6 mL, Catalog No. 30202
A solution of horse radish peroxidase conjugated with cAMP (a 1000 X stock solution and
dilution solution are provided).
Note: For long-term best results, store conjugate at -80℃upon receipt. Add 6μL conjugate into
6mL dilution solution and mix the solution gently before use. Please avoid repeated thawing
and freezing after mixing.
3. cAMP Direct Antibody, 6 mL, Catalog No. 26002-2
A solution of a mouse monoclonal antibody to cAMP(a 1000 X stock solution and dilution
solution are provided).
Note: For long-term best results, store conjugate at -80℃upon receipt. Add 6μL Antibody into
6mL dilution solution and mix the solution gently before use. Please avoid repeated thawing
and freezing after mixing.
4. Neutralizing Reagent, 6 mL, Catalog No. 30103
5. 10X Wash Buffer Concentrate, 15 mL, Catalog No. 30106
Phosphate buffered saline containing detergents.
6. Cyclic AMP Standard, 0.25 mL, Catalog No. 30203
A solution of 5,000 pmol/mL cAMP.
7. Substrate A, 12 mL, Catalog No. 30107
8. Substrate B, 12 mL, Catalog No. 30108
9. Stop Solution, 6 mL, Catalog No. 30110
A solution of sulfuric acid in water. Keep tightly capped. Caution: Caustic.
Storage
All components of this kit are stable at 4°C until the kit's expiration date. For long-term best results,
store 1000 X stock solutions at -80℃upon receipt.
Materials Needed but Not Supplied
1. Deionized or distilled water.
2. Concentrated HCl.
3. Precision pipets for volumes between 5 μL and 1,000 μL.
4. Repeater pipets for dispensing 50 μL and 200 μL.
5. Disposable beakers for diluting buffer concentrates.
6. Graduated cylinders.
7. A microplate shaker.
8. Adsorbent paper for blotting.
9. Microplate reader capable of reading at 450 nm, preferably with correction between 570 and
590 nm.
Sample Handling
NewEast Biosciences EIA is compatible with cAMP samples that have been treated with hydrochloric
acid to stop endogenous phosphodiesterase activity. Samples in this matrix can be measured directly
without evaporation or further treatment.
Tissue samples should be frozen in liquid nitrogen. The tissue should be ground to a fine powder under
liquid nitrogen in a stainless steel mortar. After the liquid nitrogen has evaporated, weigh the frozen tissue
and homogenize in 10 volumes of 0.1M HCl. Centrifuge at > 600 x g at room temperature. The samples
can then be diluted in the 0.1M HCl.
Cells grown in tissue culture media can be treated with 0.1M HCl after first removing the media.
Incubate for 10 minutes and visually inspect the cells to verify cell lysis. If adequate lysis has not
occurred incubate for a further 10 minutes and inspect. Centrifuge at 600 x g at room temperature, then
use the supernatant directly in the assay. Cell or tissue lysis can be enhanced by adding 0.1% to 1% Triton
x-100 to the 0.1M HCl prior to use. When used in this concentration range, the detergent will not interfere
with the binding portion of the assay, however there will be a modest increase in the optical density.
Samples containing Triton should be evaluated against a standard curve diluted in the same for the most
accurate determination. Cyclic AMP in the media can be measured after treating 1 mL of the supernatant
media with 10 μL of concentrated hydrochloric acid. Centrifuge at 600 x g at room temperature. The
supernatants can then be used directly in the assay.
Procedural Notes
1. Allow all reagents to warm to room temperature for at least 30 minutes before opening.
2. Pre-rinse the pipet tip with reagent, use fresh pipet tips for each sample, standard and reagent.
3. Pipet standards and samples to the bottom of the wells.
4. Add the reagents to the side of the well to avoid contamination.
5. This kit uses break-apart microtiter strips, which allow the user to measure as many samples as
desired. Unused wells must be kept desiccated at 4°C in the sealed bag provided. The wells should be used
in the frame provided.
6. Prior to addition of substrate, ensure that there is no residual wash buffer in the wells. Any
remaining wash buffer may cause variation in assay results.
Reagent Preparation
1. cAMP Standard – Non-Acetylated Version
Allow the 5,000 pmol/mL cAMP standard solution to warm to room temperature. Label six (or
more) tubes #1 through #6. Pipet 475 μL 0.1M HCl into tube #1 and 400 μL 0.1M HCl into
tubes #2-6. Add 25 μL of the 5,000 pmol/mL standard to tube #1. Vortex thoroughly. Add
100 μL of tube #1 to tube #2 and vortex thoroughly. Continue this for tubes #3 through #6.
The concentration of cAMP in tubes #1 through #6 will be 250, 50, 10, 2, 0.4,and 0.08
pmol/mL respectively. See Direct cAMP Assay Layout Sheet for dilution details.
Diluted standards should be used within 30 minutes of preparation.
Label one tube as the Zero Standard/NSB tube. Pipet 600L 0.1M HCl into this tube.
2. Wash Buffer
Prepare the Wash Buffer by diluting 15 mL of the supplied concentrate with 135 mL of
deionized water. This can be stored at room temperature until the kit expiration date, or for 3
months, whichever is earlier.
Assay Procedure
Bring all reagents to room temperature for at least 30 minutes prior to opening. All standards and
samples should be run in duplicate.
Add the reagent directly to the samples and vortex for 2 seconds immediately after the addition.
1. Refer to the Assay Layout Sheet to determine the number of wells to be used and put any
remaining wells with the desiccant back into the pouch and seal the ziploc. Store unused
wells at 4 °C.
2. Pipet 50 μL of the Neutralizing Reagent into each well, except the TA (Total Activity) and
Blank wells.
3. Pipet 100 μL of 0.1M HCl into the NSB (None Specific Binding) and the Bo (0 pmol/mL
Standard) wells.
4. Pipet 100 μL of Standards into the appropriate wells.
5. Pipet 100 μL of the Samples into the appropriate wells.
6. Pipet 50 μL of 0.1 M HCl into the NSB wells.
7. Pipet 50 μL of Conjugate into each well except the TA and Blank wells.
8. Pipet 50 μL of Antibody into each well, except the Blank, TA and NSB wells.
9. Incubate the plate at room temperature for 2 hours on a plate shaker at 250~500 rpm.
10. Empty the contents of the wells and wash by adding 400 μL of wash solution to every well.
Repeat the wash 2 more times for a total of 3 washes.
11. After the final wash, empty or aspirate the wells, and firmly tap the plate on a lint free paper
towel to remove any remaining wash buffer.
12. Add 5 μL of the Conjugate to the TA wells.
13. Add 200 μL of the Substrate solution to every well. Incubate at room temperature for 5~30
minutes without shaking. (Substrate A and B should be mixed together in equal volumes
within 15 minutes of use. Protect from light.)
14. Add 50 μL of Stop Solution to every well. This stops the reaction and the plate should be read
immediately.
15. Blank the plate reader against the Blank wells, read the optical density at 450 nm, preferably
with correction between 570 and 590 nm. If the plate reader is not able to be blanked against the
Blank wells, manually subtract the mean optical density of the Blank wells from all readings.
Calculation of Results
Several options are available for the calculation of the concentration of cAMP in the samples. The
X-axis is the concentration of cAMP for the standards. The Y-axis is either the Average Net Optical
Density or the Percent Bound.
1. Calculate the average Net Optical Density (OD) bound for each standard and sample by
subtracting the average NSB OD from the average OD bound:
Average Net OD = Average Bound OD -Average NSB OD
2. Calculate the binding of each pair of standard wells as a percentage of the maximum binding
wells (Bo), using the following formula:
Percent Bound =(Net OD/Net Bo OD)×100
3. Using Logit-Log paper plot Average Net OD or Percent Bound (B/Bo) versus concentration
of cAMP for the standards. The concentration of cAMP in the unknowns can be determined
by interpolation.
Typical Standard Curves
These curves must not be used to calculate cAMP concentrations; each user must run a standard
curve for each assay and version used.

Sensitivity
Sensitivity was calculated by determining the average optical density bound for ten wells run with the Bo,
and comparing to the average optical density for ten wells run with Standard #5. The detection limit was
determined as the concentration of cAMP measured at two standard deviations from the zero along the
standard curve

Publications:
1.  Heterogeneity in relaxation of different sized porcine coronary arteries to nitrovasodilators: role of PKG and MYPT1
    Pflugers Arch. 2012 Feb;463(2):257-68
2.  Antiarrhythmic effect of prolonged morphine exposure is accompanied by altered myocardial adenylyl cyclase signaling in rats
    Pharmacol Rep. 2012;64(2):351-9
3.  Uncoupling of M1 muscarinic receptor/G-protein interaction by amyloid beta(1-42)
    Neuropharmacology. 2013 Apr;67:272-83
4.  Subendocardial Increase in Reactive Oxygen Species Production Affects Regional Contractile Function in Ischemic Heart Failure
    Antioxid Redox Signal. 2013 Mar 20;18(9):1009-20
5.  Heterogeneity in relaxation of different sized porcine coronary arteries to nitrovasodilators: role of PKG and MYPT1
    Pflugers Arch. 2012 Feb;463(2):257-68
6.  Structural basis of anthrax edema factor neutralization by a neutralizing antibody
    Biochemical and Biophysical Research Communications Volume 417, Issue 1, 6 January 2012, Pages 324–329
7.  Transgenic rescue of defective Cd36 enhances myocardial adenylyl cyclase signaling in spontaneously hypertensive rats
    Pflügers Archiv - European Journal of Physiology October 2013, Volume 465, Issue 10, pp 1477-1486
8.  Opposing HDAC4 nuclear fluxes due to phosphorylation by β adrenergic activated PKA or by activity or Epac activated CaMKII in skeletal muscle fibres
    The Journal of Physiology Volume 591, Issue 14, pages 3605–3623, July 2013
9.  Sex is a major determinant of neuronal dysfunction in Neurofibromatosis Type 1
    Annals of Neurology Volume 75, Issue 2, pages 309–316, February 2014
10.  cAMP–PKA inhibition of SK3 channel reduced both Ca2+ entry and cancer cell migration by regulation of SK3–Orai1 complex
    Pflügers Archiv - European Journal of Physiology October 2014, Volume 466, Issue 10, pp 1921-1932
11.  Immunomodulating effects of casein-derived peptide QEPVL and QEPV on lymphocytes in vitro and in vivo
    Food Funct., 2014,5, 2061-2069
12.  Agonist-Dependent And -Independent Dopamine-1-Like Receptor Signalling Differently Regulates Downstream Effectors
    FEBS Journal Volume 281, Issue 21, pages 4792–4804, November 2014
13.  Alteration of vascular reactivity in heart failure: Role of phosphodiesterases type 3 and 4
    British Journal of Pharmacology Volume 171, Issue 23, pages 5361–5375, December 2014
14.  Ischemia/Reperfusion-Induced CHOP Expression Promotes Apoptosis and Impairs Renal Function Recovery: The Role of Acidosis and GPR4
    PLoS One. 2014 Oct 24;9(10):e110944
15.  Comparative study of somatostatin-human serum albumin fusion proteins and natural somatostatin on receptor binding, internalization and activation
    PLoS One. 2014 Feb 27;9(2):e89932

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