Development of enzyme-linked immunosorbent assay for estimation of urinary albumin

Yearly estimation of urinary albumin is a prerequisite for predicting renal status in Diabetes Type II patients with negative dipstick results for overt proteinuria. A simple, sensitive, and cost-effective enzyme linked immunosorbent assay (ELISA) for urinary albumin has been developed using human serum albumin antiserum (HSA-antiserum), HSA-biotin, and streptavidin-horseradish peroxidase (SA-HRP) conjugates. To the antibody-coated wells, 100 μL of HSA standards followed by 1:100 diluted urine samples in duplicate were added and then 50 μL of HSA-biotin conjugates was added in all the wells. 100 μL of SA-HRP was added after washing. Bound enzyme activity was measured by adding 100 μL TMB/H2O2. The analytical sensitivity and ED50 of the developed method was found to be 0.01 μg/mL and 0.35 μg/mL, respectively. The percent recovery of the HSA from exogenously spiked urine pools were in the range of 98.13-100.29%. The intra- and inter-assay coefficient of variation (CVs) ranged from 3.38-10.32 % and 4.22-11.01%, respectively. The antibody showed 4.4% and 3.2% cross reactivity with monkey and horse serum albumin, respectively. There was no cross reaction with human β2-microglobulin, γ-globulin, and haemoglobulin.