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Human cyclic adenosine monophosphate（cAMP） Elisa Kit ? This immunoassay kit allows for the in vitro quantitative determination of human cAMP concentrations in serum, plasma.
? Expiration date six months from the date of manufacture
? FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
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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.
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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 human cAMP No significant cross-reactivity or interference was observed.
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MATERIALS PROVIDED
Reagent
Quantity
Assay plate
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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.
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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
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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.
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1. Wash Buffer If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have compley 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 immediay after the addition.
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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.
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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 immediay.
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.
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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.
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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
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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%

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