DBC cAMP Urine Elisa

PRINCIPLE OF THE TEST 
The cAMP Urine 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 0.01 mL of urine is required per duplicate determination. 
Either spontaneous urine or 24-hour urine may be used in this assay.

Urine specimen samples may be stored at room temperature (20-25°C) for up to 3 days, at 2-8°C for up to 7 days or at -20°C or lower for up to 3 months. Avoid more than 3 freeze-thaw cycles. Consider all human specimens as possible biohazardous materials and take appropriate precautions when handling.

After stored urine specimens have been brought to room temperature, inspect each sample to ensure that they are free from precipitates prior to undergoing the specimen pre-treatment. If there are precipitates.
present, vortex and centrifuge the sample. Use the clear supernatant for the pretreatment steps stated below.
All urine specimens must be diluted 1:100 in the provided Assay Buffer 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 0.99 mL of the Assay Buffer into a new polypropylene microcentrifuge tube. 
2. Pipette 10 μL of the urine specimen into the tube from step 1 that contains 0.99 mL of assay buffer. 
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 urine specimen samples must take into account the 1:100 dilution that was performed during the specimen pre-treatment step. Calculate the final urine specimen cAMP concentration using the following formula: