Plasma Renin Activity ELISA Kit

Prior to testing plasma samples with the Plasma Renin Activity ELISA kit, a specimen pre-treatment 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 0C (ice bath) and the other is incubated at 37C. Angiotensin-I will be generated by plasma renin in the fraction incubated at 37C.

The Renin Activity 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 colored 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 color to a yellow color. 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 0C and 37C 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).