DBC Plasma Renin Activity (PRA) Elisa

PRINCIPLE OF THE TEST 
Prior to testing plasma samples with the PRA ELISA, a specimen pretreatment step is required. First, a protease inhibitor (PMSF) is added to the sample to prevent the degradation of angiotensin-I. Next, the generation buffer is added to bring the pH of the sample to approximately 6.0. The plasma sample is then pipetted into two aliquots. One aliquot is incubated at 0°C (ice bath) and the other is incubated at 37°C. Angiotensin-I will be generated by plasma renin in the fraction incubated at 37°C. 
The PRA ELISA is a competitive immunoassay. In the first incubation step, competition occurs between angiotensin-I present in calibrators, controls, specimen samples and an angiotensin-I-biotin conjugate (biotin conjugate) for a limited number of anti-angiotensin-I antibody binding sites on the microplate wells. During this incubation, protease inhibitors are present to prevent the degradation of angiotensin-I into smaller peptides. In the second incubation step, streptavidin-HRP conjugate is added, which binds specifically to any bound biotin conjugate. Unbound streptavidin HRP conjugate is removed by a washing step. Next, 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 angiotensin-I present. The enzymatic reaction is terminated by the addition of the stopping solution, converting the blue colour to a yellow colour. 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 concentration of angiotensin-I in specimen samples and controls can be directly read. 
The plasma renin activity concentration in the plasma sample is calculated from the angiotensin-I concentration in the 0°C and 37°C aliquots and the generation time used. The plasma renin activity results are expressed in terms of the mass of angiotensin-I generated per volume of human plasma per unit of time (ng/mL/h).

SPECIMEN COLLECTION, STORAGE AND PRETREATMENT
Specimen Collection & Storage A minimum of 0.5 mL of EDTA plasma is required per duplicate determination. Proper sample collection is essential for the accurate determination of angiotensin-I. The in vitro generation and degradation of angiotensin-I can be minimized by following the recommended collection and processing procedure as stated below. 
1. Collect at least 2 mL of venous blood into an appropriately labelled EDTA blood collection tube. 
2. Centrifuge the sample at room temperature for 15 minutes at 2000 g. 
3. Transfer the plasma sample into a new labelled storage tube.
4. If samples are to be assayed immediately, proceed to the Specimen Pre-Treatment section, otherwise store at room temperature for up to 6 hours or freeze at -20°C or lower for up to 30 days. Avoid more than two freeze-thaw cycles.
Consider all human specimens as possible biohazardous materials and take appropriate precautions when handling.
Specimen Pre-Treatment & Storage Prior to being tested, all processed plasma specimens must be pre-treated according to the Angiotensin-I generation procedure as stated below. At the end of this procedure, there will be two pre-treated aliquots per plasma sample, a 0°C aliquot and a 37°C aliquot.

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. Using the obtained concentrations of Angiotensin-I (Ang-I) in the 37°C and 0°C aliquots and the generation time used, calculate the plasma renin activity (PRA) in each sample using the following equation: 

𝑃𝑅𝐴 = ( [𝐴𝑛𝑔­𝐼 (37°𝐶)] − [𝐴𝑛𝑔­𝐼(0°𝐶)]/𝐺𝑒𝑛𝑒𝑟𝑎𝑡𝑖𝑜𝑛 𝑇𝑖𝑚𝑒 (ℎ) ) 𝑥 1.11 

5. If a sample reads more than 60 ng/mL then dilute the sample (that has undergone the angiotensin-I generation procedure) with calibrator A at a dilution of no more than 1:10 and rerun the sample. 
The result obtained must be multiplied by the dilution factor. Note: Samples must be diluted only after they have undergone the angiotensin-I generation procedure; do not dilute any samples before performing the angiotensin-I generation procedure.