title: 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.
authors.affiliation: a Department of Reproductive Biomedicine , National Institute of Health and Family Welfare , Munirka , New Delhi , India.
authors.forename: Tulsidas G
authors.initials: TG
authors.lastname: Shrivastav
authors.forename: Kiran P
authors.initials: KP
authors.lastname: Kariya
authors.forename: Pramod K V
authors.initials: PK
authors.lastname: Prasad
authors.forename: Shail K
authors.initials: SK
authors.lastname: Chaube
authors.forename: Dinesh
authors.initials: D
authors.lastname: Kumar
mesh_terms: D000293:Adolescent; D000328:Adult; D000419:Albuminuria / Q000150:complications / Q000175:diagnosis* / Q000503:physiopathology; D000818:Animals; D001710:Biotin / Q000737:chemistry; D016022:Case-Control Studies; D003429:Cross Reactions; D003924:Diabetes Mellitus, Type 2 / Q000150:complications / Q000175:diagnosis* / Q000503:physiopathology / Q000652:urine; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D000882:Haplorhini; D006735:Horseradish Peroxidase / Q000737:chemistry; D006736:Horses; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D007106:Immune Sera / Q000737:chemistry; D018796:Immunoconjugates / Q000737:chemistry; D007668:Kidney / Q000378:metabolism / Q000503:physiopathology; D008297:Male; D008875:Middle Aged; D012680:Sensitivity and Specificity; D012709:Serum Albumin / Q000737:chemistry / Q000276:immunology; D019809:Streptavidin / Q000737:chemistry
chemical_list: D007106:Immune Sera; D018796:Immunoconjugates; D012709:Serum Albumin; D001710:Biotin; D019809:Streptavidin; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase