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Mouse cyclic adenosine
monophosphate(cAMP)
Elisa Kit
? This immunoassay kit allows for the in vitro quantitative determination of mouse
cAMP concentrations in serum, plasma.
? Expiration date six months from the date of manufacture
? FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
INTRODUCTION
Cyclic adenosine monophosphate (cAMP, cyclic AMP or 3’-5’-cyclic adenosine
monophosphate) is a second messenger important in many biological
processes. cAMP is derived from adenosine triphosphate (ATP) and used for
intracellular signal transduction in many different organisms, conveying the
cAMP-dependent pathway.
cAMP is synthesised from ATP by adenylyl cyclase located on the inner side
of the plasma membrane. Adenylyl cyclase is activated by a range of signaling
molecules through the activation of adenylyl cyclase stimulatory G
(Gs)-protein-coupled receptors and inhibited by agonists of adenylyl cyclase
inhibitory G (Gi)-protein-coupled receptors. Liver adenylyl cyclase responds
more strongly to glucagon, and muscle adenylyl cyclase responds more
strongly to adrenaline.
cAMP decomposition into AMP is catalyzed by the enzyme
phosphodiesterase.
cAMP is a second messenger, used for intracellular signal transduction, such
as transferring the effects of hormones like glucagon and adrenaline, which
cannot pass through the cell membrane. It is involved in the activation of
protein kinases and regulates the effects of adrenaline and glucagon. It also
regulates the passage of Ca2+ through ion channels.
PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with a
goat-anti-mouse IgG. Standards or samples are then added to the appropriate
microtiter plate wells with a HRP-conjugated cAMP and antibody preparation
specific for cAMP and incubated. Then substrate solutions are added to each
well. The enzyme-substrate reaction is terminated by the addition of a
sulphuric acid solution and the color change is measured
spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration
of cAMP in the samples is then determined by comparing the O.D. of the
samples to the standard curve.
DETECTION RANGE
0.08pmol/ml-250 pmol/ml. The standard curve concentrations used for the
ELISA’s were 250 pmol/ml, 50 pmol/ml, 10 pmol/ml, 2 pmol/ml, 0.4 pmol/ml,
0.08 pmol/ml.
SPECIFICITY
This assay recognizes mouse cAMP No significant cross-reactivity or
interference was observed.
MATERIALS PROVIDED
Reagent Quantity
Assay plate 1
Standard
1 x 0.25 ml
(5000pmol/ml)
HRP-conjugate 1(1000 x stock solution)
Antibody 1(1000 x stock solution)
HRP-conjugate Diluent 1 x 6 ml
Antibody Diluent 1 x 6 ml
Neutralizing Reagent 1 x 6 ml
Wash Buffer
1 x 15 ml
(10×concentrate)
Substrate A 1 x 10 ml
Substrate B 1 x 10 ml
Stop Solution 1 x 6 ml
OTHER SUPPLIES REQUIRED
1. Deionized or distilled water.
2. Concentrated HCl.
3. Precision Pipets for volumes between 5μl and 1000μl.
4. Repeater Pipets for dispensing 50μl and 200μl.
5. Disposable breakers 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.
STORAGE
1. For long-term best results, store stocks of the Standard ,Antibody and
HRP-conjugate at -80℃. All other components of this kit are stable at 4°C
until the kit’s expiration date.
SAMPLE COLLECTION AND STORAGE
This ELISA 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. The 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℃
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 resultes.
REAGENT PREPARATION
Bring all reagents to room temperature before use.
1. Wash Buffer If crystals have formed in the concentrate, warm to room
temperature and mix gently until the crystals have completely dissolved.
Dilute 15 ml of Wash Buffer Concentrate into deionized to prepare 150 ml
of Wash Buffer.
2. HRP-conjugate working solution: Centrifuge the vial right before use.
Dilute the HRP-conjugate 1000x stock solution (take 6µl) with the provided
dilution buffers (6 ml).
3. Antibody working solution: Centrifuge the vial right before use. Dilute
the Antibody 1000x stock solution (take 6µl) with the provided dilution
buffers (6 ml).
4. Standards: Centrifuge the vial right before use. Allow the 5,000 pmol/mL
cAMP standard solution to warm to room temperature. Label six 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.
Diluted standards should be used within 30 minutes of preparation.
Label one tube as the Zero Standard/NSB tube. Pipet 600µl 0.1M HCl
into this tube.
ASSAY PROCEDURE
Bring all reagents to room temperature for at least 30 minutes prior opening.ALL
standards and samples should be run in duplicate.
Add the reagen 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℃.
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 HRP-conjugate working solution into each well except
the TA and Blank wells.
8. Pipet 50 µL of Antibody working solution 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 HRP-conjugate working solution 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. A gradient of blue color
should become visible during the incubation period. (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 (for HRP), 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:
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.
Non-Acetylated Version
Mean OD for Bo = 0.685±0.003
Mean OD for Standard #5 = 0.604±0.010
Delta Optical Density(0-0.08pmol/ml) = 0.081
2 SD’s of the Zero Standard = 0.006
Sensitivity =￣0.006/0.081×0.4pmol/ml = 29.6 fmol/mL
Linearity
A sample containing 16.0 pmol/mL cAMP was serially diluted 7 times 1:2 in the
0.1M HCl and measured. The data was plotted graphically as actual cAMP
concentration versus measured cAMP concentration. The line obtained had
a slope of 1.000 with a correlation coefficient of 0.999.
Cross Reactivities
The cross reactivities for a number of related compounds were determined by
competition ELISA assays. Potential cross reactants were dissolved in the kit
Assay Buffer at concentrations from 500,000 to 500 pmol/mL. These samples
were then measured in the cAMP assay, and the measured cAMP
concentration at 50% B/Bo calculated. The % cross reactivity was calculated
by comparison with the actual concentration of cross reactant in the sample
and expressed as a percentage.
Compound Cross Reactivity
cAMP 100%
AMP <0.0001%
ATP <0.0001%
cGMP <0.0001%
GMP <0.0001%
GTP <0.0001%
cUMP <0.0001%
CTP <0.0001%
