DBC cAMP Plasma Elisa

PRINCIPLE OF THE TEST 
The cAMP Plasma ELISA is a competitive immunoassay. Competition occurs between cAMP present in calibrators, controls, specimen samples and an enzyme-labelled antigen (HRP conjugate) for a limited number of anti-cAMP antibody binding sites on the microplate wells. After a washing step that removes unbound materials, the TMB substrate (enzyme substrate) is added which reacts with HRP to form a blue-coloured product that is inversely proportional to the amount of cAMP present. Following an incubation, the enzymatic reaction is terminated by the addition of the stopping solution, converting the colour from blue to yellow. The absorbance is measured on a microplate reader at 450 nm. A set of calibrators is used to plot a calibrator curve from which the amount of cAMP in specimen samples and controls can be directly read.

SPECIMEN COLLECTION, STORAGE AND PRETREATMENT
Approximately 0.05 mL of EDTA plasma is required per duplicate determination.
Collect 4–5 mL of venous blood into an appropriately labelled EDTA plasma tube.
Mix the tube by inverting several times.
Centrifuge the sample at room temperature for 15 minutes at 2000 RCF.
Transfer the plasma sample into a new labelled storage tube.

Plasma samples may be stored at 2-8°C for up to 2 days or at -20°C or lower for up to 3 months. Avoid more than two freeze-thaw cycles.
Consider all human specimens as possible biohazardous materials and take appropriate precautions when handling.

All EDTA plasma specimens must be diluted 1:5 in the provided Sample Diluent before being used in the test. Follow the specimen pre-treatment procedure as stated below for each specimen that is to be tested:
1. Pipette 0.2 mL of the Sample Diluent into a new polypropylene microcentrifuge tube.
2. Pipette 50 μL of the plasma specimen into the tube from step 1 that contains 0.2 mL of Sample Diluent. 
3. Close the tube and label it with specimen identification information. 
4. Mix the contents of the tube by vortexing.

CALCULATIONS 
1. Calculate the mean optical density for each calibrator, control and specimen sample duplicate.
2. Use a 4 -parameter or 5-parameter curve fit with immunoassay software to generate a calibrator curve. 
3. The immunoassay software will calculate the concentrations of the controls and specimen samples using the mean optical density values and the calibrator curve. 
4. The final concentration of the EDTA plasma specimen samples must take into account the 1:5 dilution that was performed during the specimen pre-treatment step. Calculate the final plasma specimen cAMP concentration using the following formula: 
Final plasma specimen cAMP concentration = 
Concentration calculated from calibrator curve x 5 (dilution factor). 
Example: If the plasma sample concentration calculated from the calibrator curve was 5 pmol/mL, then the final concentration of cAMP in the plasma specimen sample = 5 pmol/mL x 5 = 25 pmol/mL. 

Do not perform any calculations to samples that did not undergo the specimen pre-treatment (dilution) step (e.g. kit controls). 

5. If a plasma sample reads more than 100 pmol/mL (500 pmol/mL considering the dilution factor of 1: 5), then dilute the 1:5 diluted sample (normal dilution) up to a 1:5 dilution, using the supplied Sample Diluent. The result obtained must be multiplied by the dilution factor that was used.
Example: If the 1:5 diluted plasma specimen (normal dilution) is further diluted 1:5 and produces a result of 20 pmol/mL, then the final plasma specimen cAMP concentration = 20 pmol/mL x 5 x 5 = 500 pmol/mL.