Development of Enzyme-Linked Immunosorbent Assay for Estimation of Urinary Albumin

Abstract

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.

Colorimetric, Naked-Eye Detection of Lysozyme in Human Urine with Gold Nanoparticles

“…Those interactions, where the adsorption of proteins over the particle leads to the formation of a protein corona, may cause a small degree of protection in the presence of salt, leading to a purple color. In concrete, HSA may appear alongside or instead of lysozyme in some kinds of proteinurias, such as those induced by renal damage or diabetes [48]. Protocols for elimination of those proteins by precipitation have been proposed by Wang et al [18].…”

Homologous ELISA for detection of prednisolone in human serum

“…Coating of antibody to microtitre plates. The 96 well microtitre plates were coated using the immunobridge technique for primary antibody immobilization according to the method of Shrivastav et al (Shrivastav, Chaube, Kariya, Kumari, et al, 2011;Shrivastav, Chaube, Kariya, Singh, et al, 2012;Shrivastav, Kariya, Prasad, Chaube, & Kumar, 2014). Briefly, 250 μL of normal rabbit serum (NRS) diluted (1:250) in water was dispensed into each well and incubated at 37°C overnight.…”

“…It is the ability of a test to recover, or measure, a known incremental amount of an analyte from a sample matrix. The experimental protocol for a study includes adding a known amount of analyte (A) to a base (B) and measuring the concentration (C) (Shrivastav et al, 2014). The percentage of recovery can be calculated by using the formula: (C − B)/A * 100.…”

“…The statistics conventionally used to express the precision profile of an assay are the mean (X ), standard deviation (SD) and the coefficient of variation, CV. The % coefficient of variation is calculated as % CV = (SD/X ) * 100 at a particular analyte level (Shrivastav et al, 2014).…”

“…Sensitivity is the capability to measure the minimum quantity of target analyte under defined standard condition (Shrivastav et al, 2014). The sensitivity of the assay is expressed in terms of its lowest detection dose (LDD) and the effective dose at 50% (ED 50 ).…”