NACALAI TESQUE, INC.

Cells to supersensitive results in 2 Hours
High Sensitivity Direct Cyclic AMP Chemiluminescent Immunoassay Kit

 

 

 

 

The new DetectX^® Direct Cyclic AMP (cAMP) Chemiluminescent Immunoassay kit is designed to quantitatively measure cAMP present in lysed cells, tissue plasma, urine, saliva and tissue culture media samples. The kit is unique in that all samples and standards are diluted into an acidic sample Diluent, containing special additives and designed to lyse cells, for measurement. A cAMP standard is provided to generate a standard curve for the assay. A Plate Primer solution is added to all the wells and standards or samples in Diluent are pipetted into the white microtiter plate. cAMP-peroxidase conjugate is added to the standards and samples in the wells. The binding reaction is initiated by the addition of an antibody to cAMP. After 2 hours, the plate is washed and chemiluminescent substrate is added. The substrate reacts with the bound cAMP-peroxidase conjugate. The intensity of the emitted light is detected in a microtiter plate reader capable of measuring luminescence. concentration of cAMP in the sample is calculated, after making suitable correction for the dilutions. An optional acetylation Format allows super low concentrations of cAMP to be measured.

 

 

Features

• Measure to less than 1 fmol cAMP per sample - most sensitive kit available
• Measure cAMP in lysed cells, plasma, saliva, urine or tissue
• Colour-coded, simple protocol
• Liquid 4°C stable components
• High signals in 2 hours

 

 Typical data 

■ Regular format

 

■ Acetylated format

 

Sensitivity

Sensitivity was 0.75fmol/well in the Acetylated Format.

 

Sample preparation

All samples are diluted in the Sample Diluent. Tissue, urine and saliva samples should be run in the Regular Format. Plasma and samples with low expected concentrations of cAMP should be run in the Acetylated Format.
TCM samples should be diluted in TCM and read off a standard curve generated in the same TCM.

 

Assay protocol

・Pipet 50 µl of Plate Primer into all wells used for the assay.
・50 µl of samples or standards into duplicate wells in the microtiter plate.
・Add 25 µl of diluted DetectX^® cAMP-Peroxidase Conjugate, followed by 25 µ of cAMP Antibody and shake at room temperature for 2 hours.
・Wash 4 times with wash buffer.
・Add 100 µl of chemiluminescent substrate to each well and read each well for 0.1 second.

 

Linearity

Linearity was determined by taking two Jurkat cell lysate samples, one with a low cAMP level of 0.2 pmol/ml and one with a higher level of 2.5 pmol/ml and mixing them in the ratios given below.

Intra assay precision

Diluted human samples were run in replicates of 20 in both the Regular and Acetylated Formats. The mean and precision of the calculated Cyclic AMP concentrations were:

Inter assay precision

Diluted human samples were run in duplicates in fourteen assays over multiple days by four operators. The mean and precision of the calculated Cyclic AMP concentrations were:

 

Cross reactivity

Ordering information