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 ß2-microglobulin, γ-globulin, and haemoglobulin.

Influence of different length spacers containing enzyme conjugate on functional parameters of progesterone ELISA.

In steroid enzyme immunoassay (EIA), there is an increase or decrease of labeled steroid recognition by antibody due to homologous and heterologous combinations of enzyme conjugate with immunogen that affects sensitivity of the assay. We have introduced three to 18 atomic length linkers between enzyme and steroid moieties and studied their effects on functional parameters such as sensitivity, ED(50), and specificity of progesterone enzyme immunoassays. Progesterone-3-carboxymethyloxime-bovine serum albumin (P-3-CMO-BSA) was used as an immunogen to raise the antiserum in New Zealand white rabbits. Five enzyme conjugates were prepared using 17-α-hydroxy-progesterone-3-carboxymethyloxime (17-α-OH-P-3-CMO) as carboxylic derivative of 17-α-hydroxy-progesterone and horseradish peroxidase (HRP) as label. These were 17-α-OH-P-3-CMO-HRP, 17-α-OH-P-3-CMO-urea-HRP (17-α-OH-P-3-CMO-U-HRP), 17-α-OH-P-3-CMO-ehylenediamine-HRP (17-α-OH-P-3-CMO-EDA-HRP), 17-α-OH-P-3-CMO-carbohydrazide-HRP (17-α-OH-P-3-CMO-CH-HRP), and 17-α-OH-P-3-CMO-adipic acid dihydrazide-6-aminocaproic acid-HRP (17-α-OH-P-3-CMO-ADH-6ACA-HRP). The influence of different atomic length linkers on sensitivity, ED(50), and specificity were studied with reference to label without linker. The results of the present investigation revealed that the incorporation of ADH-6ACA spacer in 17-α-hydroxy-progesterone-enzyme conjugate improved the sensitivity in antigen plus bridge heterologous EIA system. The presence of spacer in enzyme conjugate improved the sensitivity and specificity (cross-reactivity) in some antigen plus bridge heterologous assay of progesterone.

Heterologous enzyme linked immunosorbent assay for measurement of testosterone in serum.

In steroid enzyme immunoassay (EIA), homologous and heterologous combinations of enzyme conjugate with immunogen influences labeled steroid recognition by antibodies that affect sensitivity of the assay. To develop testosterone enzyme linked immunosorbent assay (ELISA), antibodies were generated against testosterone-3-carboxymethyloxime-bovine serum albumin (T-3-CMO-BSA), testosterone-11-hemisuccinate-bovine serum albumin (T-11-HS-BSA), testosterone-17-hemisuccinate-bovine serum albumin (T-17-HS-BSA), testosterone-17-glucuronide-bovine serum albumin (T-17-G-BSA), and testosterone-19-carboxymethylether-bovine serum albumin (T-19-CME-BSA). Testosterone horseradish peroxidase (HRP) enzyme conjugate were prepared using carboxyl derivatives of 11-keto-testosterone (11-keto-T) and 1-dehydrotestosterone (1-Dehydro-T). Ten combinations of heterologous assays were evaluated. The data of the present study revealed that the use of the T-11-HS-BSA antibody in antigen plus site heterologous assay that employed 11-keto-testosterone-3-CMO-HRP as the label showed binding and displacement, and led to the development of sensitive and specific assay.