Cyclic AMP CLIA Kit (High-Sensitivity), Species Independent

Description

The Cyclic AMP (cAMP) CLIA (High-Sensitivity) kit is designed to quantitatively measure cAMP present in lysed cells, EDTA and heparin plasma, urine, saliva and tissue culture media samples. For tissue samples, saliva and urine, where the levels of cAMP are expected to be relatively high, the regular format for the assay can be used. For plasma samples and some dilute cell lysates an optional acetylation protocol can be used. This kit can measure as little as 1 femtomol cAMP per sample. The kit is unique in that all samples and standards are diluted into an acidic Sample Diluent, which contains special additives and stabilizers, for cAMP measurement. This allows plasma, urine and saliva samples to be read in an identical manner to lysed cells. Acidified samples of cAMP are stable and endogenous phosphodiesterases are inactivated in the Sample Diluent. A cAMP standard is provided to generate a standard curve for the assay and all samples should be read off the standard curve. A white microtiter plate coated with an antibody to capture sheep IgG is provided. Prior to the addition of any samples or standards a neutralizing Plate Primer solution is added to all the used wells. Standards or diluted samples, either with or without acetylation, are pipetted into the primed wells. A cAMP-peroxidase conjugate is added to the standards and samples in the wells. The binding reaction is initiated by the addition of a sheep antibody to cAMP to each well. After a 2 hour incubation, the plate is washed and the chemiluminescent substrate is added. The substrate reacts with the bound cAMP-peroxidase conjugate to produce light The generated light is detected in a microtiter plate reader capable of reading luminescence. The concentration of the cAMP in the sample is calculated, after making suitable correction for the dilution of the sample, using software available with most plate readers.

Species Reactivity

Wide Species

Applications

Direct CLIA (Chemiluminescent Immunoassay)

Relevant Diseases

Neurological Disorders

Sensitivity

0.119 pmol/ml

Platform

Microplate

Detection Method

Chemiluminescent Assay

Sample Type

Cell lysates, EDTA Plasma, Heparin Plasma, Saliva, Tissue, Tissue Culture Media, Urine

Assay Type

CLIA Kits

Assay Time

2 Hours

Precision

Intra Assay Precision (Regular): Two human urine samples were diluted with Sample Diluent and run in replicates of 20 in an assay. The mean and precision of the calculated cAMP concentrations were: Sample 1- 11.2 pmol/mL, 3.9% CV Sample 2- 3.6 pmol/mL, 8.3% CV Intra Assay Precision (Acetylated): Two human plasma samples were diluted with Sample Diluent, acetylated and run in replicates of 20 in an assay. The mean and precision of the calculated cAMP concentrations were: Sample 1- 1.32 pmol/mL, 8.4% CV Sample 2- 0.39[pmol 10.9% CV Inter Assay Precision (Regular): Two human urine samples were diluted with Sample Diluent and run in duplicates in fourteen assays run over multiple days by four operators. The mean and precision of the calculated cAMP concentrations were: Sample 1- 6.5 pmol/mL, 8.1% CV Sample 2- 2.1 pmol/mL, 13.0% CV Inter Assay Precision (Acetylated): Two human plasma sample were diluted with Sample Diluent, acetylated and run in duplicates in fourteen assays run over multiple days by four operators. The mean and precision of the calculated cAMP concentrations were: Sample 1- 1.02 pmol/mL, 10.1% CV Sample 2- 0.32 pmol/mL, 16.5% CV

Number Of Samples

38 samples in duplicate

Linear Range

Regular: 0.185 - 15 pmol/ml. Acetylated: 0.039 - 5 pmol/ml

Components

Coated White 96 Well Plates 1 Each
Cyclic AMP Standard 125 µl
StressXpress® Cyclic AMP Antibody 3 ml
StressXpress® Cyclic AMP Conjugate Concentrate 60 µl
Conjugate Diluent 3 ml
Sample Diluent 28 ml
Plate Primer 5 ml
Acetic Anhydride 2 ml
Triethylamine 4 ml
Wash Buffer Concentrate 30 ml
Substrate Solution A 6 ml
Substrate Solution B 6 ml
Plate Sealer 1 Each

Shipping

Blue Ice

Storage

4°C

Research Use Only

For research use only

Official Name

Cyclic AMP

1. Sutherland, E. W. and Rall, T. W. Fractionation and Characterization of a Cyclic Adenine Ribonucleotide Formed by Tissue Particles. J. Biol. Chem., 232:1077, 1958.
2. Marsh, J.M. Biol. Reprod., 14:30-53, 1976.
3. Korenman, S.G. and Krall, J.F. Biol. Reprod., 16:1-17, 1977.
4. Kelley, D.J., Bhattacharyya, A., Lahvis, G.P., Yin, J.C.P., Malter, J., and Davidson, R.J. Neurosci. Biobehav. Rev., 32(8): 1533-1543, 2008.
5. http://www.hmdb.ca/metabolites/HMDB00058
6. Serezani, C.H., Ballinger, M.N., Aronoff, D.M., and Peters-Golden, M. Am. J. Resp. Cell and Mol. Biol., 39 (2): 127, 2008.
7. Hannila, S.S., and Filbin, M.T. Exp. Neurol., 209(2): 321-332, 2008.
8. Shankar, D.B, Cheng, J.C., and Sakamoto, K.M. Cancer, 104(9):1819-24, 2005.
9. Galea E. and Feinstein, D.L. FASEB J., 13:2125-2137, 1999.