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Description
Overview
A non-radioactive competitive immunoassay kit for the rapid quantitative measurement of cAMP. No neutralization, extraction, drying, or reconstitution of samples is required. Samples are treated with 0.1N HCl to lyse the cells, stop endogenous phosphodiesterase activity, and stabilize the released cAMP, which can then be analyzed directly in the microtiter plate.
Catalogue Number
116811
Brand Family
Calbiochem®
Materials Required but Not Delivered
• Deionized or distilled water. • Precision pipets for volumes between 5 µl and 1000 µl. • Repeater pipets for dispensing 50 µl and 200 µl. • Disposable beakers for diluting buffer concentrates. • Graduated cylinders. • A microplate shaker. • Adsorbent paper for blotting. • Microplate reader capable of reading at 405 nm, preferably with correction between 570 and 590 nm.
References
References
Chard, T. In: An Introduction to Radioimmunoassay and Related Techniques. 1990. 4th Ed., Elsevier, Amsterdam. NCCLS Evaluation Protocols, SC1, 1989, NCCLS, Villanova, PA, 19085. Tijssen, P. In: Practice and Theory of Enzyme Immunoassays. 1985. Elsevier, Amsterdam. Chabardes, D., et al. 1980. J. Clin. Invest.65, 439. Grill, V., and Cerasi, E. 1974. J. Biol. Chem.249, 41961. Szentivanyi, A. 1968. J. Allergy42, 203. Haynes, R.C. 1958. J. Biol. Chem.233, 1220.
Product Information
Detection method
Colorimetric
Form
100 Tests
Format
96-well format
Kit contains
One Antibody Coated 96-Well Plate, cAMP Direct Conjugate, Anti-cAMP, cAMP Standard, 0.1 N HCl, Neutralizing Reagent, Wash Buffer, Color Substrate, Stop Solution, Triethylamine, Acetic Anhydride, a Plate Sealer, and a user protocol.
Applications
Application References
Gettys, T.W., et al. 1991. J. Biol. Chem.266, 15949.
Gettys, T.W., et al. 1990. 2nd Messengers & Phosphoprot.13, 37.
Yamamoto, I., and Tsuji, J. 1981. Immunopharm. 3, 53.
Collins, W.P., and Hennam, J.F. 1976. In: Molecular Aspects of Medicine Vol. 1, (Baum, H. and Cergeley, J., eds.) p. 3, Pergamon, England.
Fausto, H., and Butcher, F.R. 1976. Biochim. Biophys. Acta428, 702.
Farmer, R.W., et al. 1975. Anal. Biochem.64, 455.
Steiner, A.L. 1974. Methods Enzymol.38, 96.
Highly flammable. Harmful by inhalation and in contact with skin. Very toxic if swallowed. Contact with acids liberates very toxic gas. Causes severe burns. Very toxic to aquatic organisms. May cause long-term adverse effects in the aquatic environment.
S Phrase
S: 26-3-16-27-28-29-36/37/39-45
In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. Keep in a cool place. Keep away from sources of ignition - No Smoking. Take off immediately all contaminated clothing.
Do not empty into drains. Wear suitable protective clothing, gloves and eye/face protection. In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
Product Usage Statements
Intended use
The Calbiochem® cAMP Direct Immunoassay Kit, Colorimetric is a competitive immunoassay for the quantitative assay of cAMP in cell lysates, tissue extracts, and serum/plasma.
Storage and Shipping Information
Ship Code
Blue Ice Only
Toxicity
Multiple Toxicity Values, refer to MSDS
Hazardous Materials Attention:
Due to the nature of the Hazardous Materials in this shipment, additional shipping charges may be applied to your order. Certain sizes may be exempt from the additional hazardous materials shipping charges. Please contact your local sales office for more information regarding these charges.
Storage
+2°C to +8°C
Storage Conditions
Upon arrival store the cAMP Direct Conjugate and the Cyclic AMP Standard at -20°C and the remaining components of the kit at 4°C.
Do not freeze
Ok to freeze
Packaging Information
Transport Information
Supplemental Information
Kit contains
One Antibody Coated 96-Well Plate, cAMP Direct Conjugate, Anti-cAMP, cAMP Standard, 0.1 N HCl, Neutralizing Reagent, Wash Buffer, Color Substrate, Stop Solution, Triethylamine, Acetic Anhydride, a Plate Sealer, and a user protocol.
Chard, T. In: An Introduction to Radioimmunoassay and Related Techniques. 1990. 4th Ed., Elsevier, Amsterdam. NCCLS Evaluation Protocols, SC1, 1989, NCCLS, Villanova, PA, 19085. Tijssen, P. In: Practice and Theory of Enzyme Immunoassays. 1985. Elsevier, Amsterdam. Chabardes, D., et al. 1980. J. Clin. Invest.65, 439. Grill, V., and Cerasi, E. 1974. J. Biol. Chem.249, 41961. Szentivanyi, A. 1968. J. Allergy42, 203. Haynes, R.C. 1958. J. Biol. Chem.233, 1220.
User Protocol
Revision
26-July-2016 JSW
Form
100 Tests
Format
96-well format
Detection method
Colorimetric
Storage
Upon arrival store the cAMP Direct Conjugate and the Cyclic AMP Standard at -20°C and the remaining components of the kit at 4°C.
Intended use
The Calbiochem® cAMP Direct Immunoassay Kit, Colorimetric is a competitive immunoassay for the quantitative assay of cAMP in cell lysates, tissue extracts, and serum/plasma.
Background
Adenosine 3', 5'-cyclic monophosphate (cyclic AMP) is one of the most important second messengers involved in regulating neuronal, glandular, cardiovascular, immune, and other physiological functions. A number of hormones are known to stimulate the production of cAMP through the action of adenylate cyclase that converts ATP to cAMP.
Principles of the assay
The Calbiochem® cAMP Direct Immunoassay Kit, Colorimetric uses a polyclonal antibody to cAMP that binds to cAMP in samples in a competitive manner. After a simultaneous incubation at room temperature, the excess reagents are washed away and substrate is added. After a short incubation time, the reaction is stopped and the yellow color generated is read on a microplate reader at 405 nm. The intensity of the color is inversely proportional to the concentration of cAMP in standards and samples.
• Deionized or distilled water. • Precision pipets for volumes between 5 µl and 1000 µl. • Repeater pipets for dispensing 50 µl and 200 µl. • Disposable beakers for diluting buffer concentrates. • Graduated cylinders. • A microplate shaker. • Adsorbent paper for blotting. • Microplate reader capable of reading at 405 nm, preferably with correction between 570 and 590 nm.
Precautions and recommendations
• Some kit components contain azide, which may react with lead or copper plumbing. When disposing of reagents always flush with large volumes of water to prevent any azide build-up. • Some solutions supplied in this kit are caustic: care should be taken with their use. • The activity of the alkaline phosphatase conjugate is dependent on the presence of Mg2+ and Zn2+ ions. The activity of the conjugate is affected by concentrations of chelators (>10 mM) such as EDTA and EGTA. • The cyclic AMP Standard provided (Cat. No. KP16707) is supplied in ethanolic buffer at a pH optimized to maintain cAMP integrity. Care should be taken in handling this material because of the known and unknown effects of cAMP. • Do not mix components from different kit lots. • Allow all reagents to warm up to room temperature for at least 30 min before opening. • Standards can be made up in either glass or plastic tubes. • Keep unused plate strips sealed in bag with desiccant. • Pre-rinse the pipet tip with the reagent and use fresh pipet tips for each sample, standard and reagent. • Pipet standards and samples to the bottom of the wells. • Add the reagents to the side of the well to avoid contamination. • This kit uses break-apart strips, which allow the user to measure as many samples as desired. Unused wells must be kept desiccated at 4°C in the sealed foil bag. The wells should be used in the frame provided. • Care must be taken to minimize contamination by endogenous alkaline phosphatase. Contaminating alkaline phosphatase activity, especially in the substrate solution, may lead to high blanks. Care should be taken not to touch pipet tips and other items that are used in the assay with bare hands. • Prior to addition of substrate, ensure that there is no residual wash buffer in the wells. Any wash buffer remaining in the wells will cause variations in results. • All standards and samples should be run in duplicate. • Bring all reagents to room temperature for at least 30 min prior to opening.
Preparation
This kit 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. If samples with very low levels of cAMP are to be measured, we have provided reagents to acetylate samples and standards. Please refer to application references for further methods of extraction of cAMP from samples.
• Serum and Plasma: For serum samples add ~10 µl concentrated hydrochloric acid per 1 ml of serum. The serum should be allowed to stand for 15 min at room temperature, and then centrifuged at 600 x g at room temperature. The supernatants can then be diluted in the 0.1 N HCl provided with the kit. In experiments with serum samples diluted greater than 1:2 in 0.1 N HCl, recoveries of cAMP of greater than 96% were observed. EDTA plasma is not a suitable matrix for the acetylated procedure since it tends to precipitate.• Tissue Extracts: Tissue samples frozen in liquid nitrogen 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 0.1 N HCl. Centrifuge at 600 x g at room temperature. The samples can then be diluted in the 0.1 N HCl provided for the assay.
• Cell Lysates: Cells grown in tissue culture medium can be treated with 0.1 N HCl after first removing the medium. Incubate for 10 min and visually inspect the cells to verify cell lysis. If adequate lysis has not occurred, incubate for an additional 10 min and re-inspect. Centrifuge at ≥600 x g at room temperature. Use the supernatant directly in the assay. Cyclic AMP in the medium can be measured after treating the supernatant with concentrated HCl as described above for plasma. Centrifuge at 600 x g at room temperature. The supernatants can then be used directly in the assay. In experiments with tissue culture media samples diluted greater than 1:2 in 0.1M HCl, recoveries of cAMP of 98% were seen.
Reagent preparation
• cAMP Standard-Non-Acetylated Version: Allow the 2,000 pmol/ml cAMP standard solution to warm to room temperature. Label five 12 x 75 mm glass tubes #1 through #5. Pipet 900 µl of 0.1 N HCl into tube #1 and 750 µl of 0.1 N HCl into tubes #2-5. Add 100 µl of the 2,000 pmol/ml standard to tube #1. Vortex thoroughly. Add 250 µl of tube #1 to tube #2 and vortex thoroughly. Continue this for tubes #3 through #5. The concentration of cAMP in tubes #1 through #5 will be 200, 50, 12.5, 3.12 and 0.78 pmol/ml respectively. Diluted standards should be used within 60 min of preparation.
Table 1: Standard Dilutions (Non-Acetylated)
• Acetylation Reagent: Prepare the Acetylating Reagent by adding 0.5 ml of Acetic Anhydride to 1 ml of Triethylamine. Use the reagent within 60 min of preparation.
• cAMP Standard-Acetylated Version: Allow the 2,000 pmol/ml cAMP standard solution to warm to room temperature. Label five 12 x 75 mm glass tubes #1 through #5. Pipet 990 µl of 0.1M HCl into tube #1 and 750 µl of 0.1M HCl into tubes #2-5. Add 10 µl of the 2,000 pmol/ml standard to tube #1. Vortex thoroughly. Add 250 µl of tube #1 to tube #2 and vortex thoroughly. Continue this for tubes #3 though #5. The concentration of cAMP in tubes #1 through #5 will be 20, 5, 1.25, 0.312, and 0.078 pmol/ml respectively.
Table 2: Standard Dilutions (Acetylated)
Acetylate all standards and samples by adding 10 µl of the Acetylating Reagent for each 200 µl of standard or sample. Add the reagent directly to the samples and vortex for about 2 s. Label one 12 x 17 mm glass tube as the Zero Standard/NSB tube. Pipet 1 ml of 0.1 N HCl into this tube. Add 50 µl of the Acetylating Reagent to the Zero Standard/NSB tube and use in Step 3 under Assay Procedure below. Failure to acetylate the NSB and the Zero Standard will result in inaccurate B/B0 values. Use the acetylated standards or samples within 30 min of preparation.
• Wash Buffer: Prepare the Wash Buffer by diluting 5 ml of the concentrate supplied with 95 ml of deionized water. This can be stored at room temperature for 3 months.
Detailed protocol
Note: If the Acetylated version of the kit is to be run, acetylate all standards and samples by adding 10 µl of the Acetylating Reagent for each 200 µl of standard or sample. Add 50 µl of the Acetylating Reagent to the Zero Standard/NSB tube and use in Steps 3 and 6 below. (Failure to acetylate the NSB and Zero standard will result in inaccurate B/Bo values.) Add the reagent directly to the samples and vortex for 2 s. Use the acetylated standards or samples within 30 min.
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 foil pouch and seal the ziploc. Store unused wells at 4°C.
Table 3: Direct cAMP Plate Layout
2. Pipet 50 µl of the Neutralizing Reagent into each well, except the Total Activity (TA) and Blank wells. 3. Pipet 100 µl of 0.1M HCl into the NSB and the B0 (0 pmol/ml Standard) wells. When assaying acetylated samples add 100 µl 0.1M acetylated HCl in place of nonacetylated 0.1M HCl. 4. Pipet 100 µl of Standards #1 through #5 into the appropriate wells. 5. Pipet 100 µl of the Samples into the appropriate wells. 6. Pipet 50 µl of 0.1M HCl into the NSB wells. Use nonacetylated 0.1M HCl for both acetylated or nonacetylated samples. 7. Pipet 50 µl of blue Conjugate into each well except the TA and Blank wells. 8. Pipet 50 µl of yellow Antibody into each well, except the Blank, TA and NSB wells. NOTE: Every well used should be Green in color except the NSB wells which should be Blue. The Blank and TA wells are empty at this point and have no color. 9. Incubate the plate at room temperature for 2 h on a plate shaker at ~500 rpm. The plate may be covered with the plate sealer provided, if so desired. 10. Empty the contents of the wells and wash by adding 200 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 blue Conjugate to the TA wells. 13. Add 200 µl of the pNpp Substrate solution to every well. Incubate at room temperature for 1 h without shaking. 14. Add 50 µl of Stop Solution to each well. Read the plate immediately. 15. Blank the plate reader against the Blank wells, read the absorbance at 405 nm, preferably with correction between 570 and 590 nm. If the plate reader cannot be set against the Blank wells, manually subtract the mean absorbance of the blank wells from all readings.
Calculations
Several options are available for the calculation of the concentration of cAMP in the samples. We recommend that the data be handled by an immunoassay software package utilizing a weighted 4 parameter logistic curve fitting program such as "AssayZap", sold by Biosoft for Macintosh or IBM-compatible computers running Windows. If this type of data reduction software is not readily available, the concentration of cAMP can be calculated as follows:
Figure 1: Calculations
Assay characteristics and examples
The following parameters for this kit were determined using the guidelines listed in the National Committee for Clinical Laboratory Standards (NCCLS) Evaluation Protocols.
Standard curve
Typical standard curves are shown below. These curves must not be used to calculate cAMP concentrations; each user must run a standard curve for each plate and version used.
Figure 2: Standard Curve
Example data
The results shown below are for illustration only and should not be used to calculate results from another assay.
Sensitivity was calculated by determining the average absorbance bound for sixteen wells run as B₀, and comparing to the average absorbance for sixteen 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.
cAMP concentrations were measured in a variety of different samples including tissue culture media and serum. For all of the samples, cAMP was spiked into the undiluted samples that were diluted with the kit 0.1 N HCl and then assayed in the kit. Recovery values were not obtained with urine samples because the endogenous levels of cAMP are high. The following results were obtained:
Figure 5: Sample Recoveries
Linearity
Non-Acetylated Version
A sample containing 15.44 pmol/ml cAMP was serially diluted 4 times 1:2 in the 0.1 N HCl supplied in the kit and measured in the Correlate-EIA assay. The data was plotted graphically as actual cAMP concentration versus measured cAMP concentration. The line obtained had a slope of 0.98 with a correlation coefficient of 0.988.
Acetylated Version
A sample containing 3.41 pmol/ml cAMP was serially diluted 4 times 1:2 in the 0.1 N HCl supplied in the kit and measured in the Acetylated version of the Correlate-EIA assay. The data was plotted graphically as actual cAMP concentration versus measured cAMP concentration. The line obtained had a slope of 1.226 with a correlation coefficient of 0.999.
Specificity
Cross Reactivities The cross-reactivities for a number of related compounds were determined. Potential cross-reactants were at concentrations from 2,000 to 2 pmol/ml. These samples were then measured in the cAMP assay, and the measured cAMP concentration at 50% B/B₀ calculated. The % cross reactivity was calculated by comparison with the actual concentration of cross reactant in the sample and expressed as a percentage.
Table 4: Cross Reactivities
Protocol Summary
Table 5: Assay Protocol Flow Chart
Application references
Gettys, T.W., et al. 1991. J. Biol. Chem.266, 15949.
Gettys, T.W., et al. 1990. 2nd Messengers & Phosphoprot.13, 37.
Yamamoto, I., and Tsuji, J. 1981. Immunopharm. 3, 53.
Collins, W.P., and Hennam, J.F. 1976. In: Molecular Aspects of Medicine Vol. 1, (Baum, H. and Cergeley, J., eds.) p. 3, Pergamon, England.
Fausto, H., and Butcher, F.R. 1976. Biochim. Biophys. Acta428, 702.
Farmer, R.W., et al. 1975. Anal. Biochem.64, 455.
Steiner, A.L. 1974. Methods Enzymol.38, 96.
Registered Trademarks
Calbiochem® is a registered trademark of EMD Biosciences, Inc. Interactive Pathways™ is a trademark of EMD Biosciences, Inc.
