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Shrivastav T.G.,National Institute of Health and Family Welfare | Chaube S.K.,Banaras Hindu University | Kariya K.P.,Vmv Com Jmt Arts And Jjp Science College | Kumar D.,National Institute of Health and Family Welfare | Singh R.,University of Delhi
Journal of Immunoassay and Immunochemistry | Year: 2011

Homologous and heterologous combinations of enzyme conjugate and antibody in steroid enzyme immunoassay (EIA) influences unlabeled steroid recognition by antibody that affects sensitivity of the assay. To develop dehydroepiandrosterone (DHEA) antigen heterologous enzyme linked immunosorbent assay (ELISA), antibodies were generated against DHEA-3-hemisuccinate-bovine serum albumin (DHEA-3-HS-BSA), DHEA-7-carboxymethyloxime-bovine serum albumin (DHEA-7-CMO-BSA), and DHEA-17-carboxymethyloxime-bovine serum albumin (DHEA-17-CMO-BSA). Five horseradish peroxidase (HRP) enzyme conjugates were prepared using five testosterone derivatives [testosterone-3-CMO (T-3-CMO), testosterone-17-HS (T-17-HS), testosterone-17-glucuronoside (T-17-G), testosterone-19-carboxymethylether (T-19-CME), and testosterone-11-HS (T-11-HS)]. Fifteen antigen heterologous combinations of antibody and enzyme conjugates were evaluated in the standard binding assay; only two combinations showed binding. The use of antigen heterologous combination (different antigen in label than the immunogen) resulted in development of a simple, direct, and convenient assay as it permits the direct addition of the serum sample into the assay and it requires only 1.5h to complete. Copyright © Taylor & Francis Group, LLC. Source


Shrivastav T.G.,National Institute of Health and Family Welfare | Chaube S.K.,Banaras Hindu University | Kariya K.P.,Vmv Com Jmt Arts And Jjp Science College | Bhanot S.,National Institute of Health and Family Welfare | And 3 more authors.
Journal of Immunoassay and Immunochemistry | Year: 2010

Homologous and heterologous combinations of enzyme conjugate with immunogen in steroid enzyme immunoassay (EIA) influence labeled steroid recognition by an antibody that affects the sensitivity of the assay. To develop dehydroepiandrosterone (DHEA) enzyme linked immunosorbent assay (ELISA), antibodies were generated against dehydroepiandrosterone-17-carboxymethyloxime- bovine serum albumin (DHEA-17-CMO-BSA) and dehydroepiandrosterone-7- carboxymethyloxime- bovine serum albumin (DHEA-7-CMO-BSA). Two dehydroepiandrosterone (DHEA) horse radish peroxidise (HRP) enzyme conjugates were prepared using two dehydroepiandrosterone derivatives (DHEA-17-CMO and DHEA-7-CMO). Four combinations of homologous and heterologous assays were evaluated. The use of heterologous combination improved the sensitivity of the assay. Copyright © Taylor & Francis Group, LLC. Source


Shrivastav T.G.,National Institute of Health and Family Welfare | Chaube S.K.,Banaras Hindu University | Charu,National Institute of Health and Family Welfare | Charu,Patna University | And 5 more authors.
Journal of Immunoassay and Immunochemistry | Year: 2010

A direct antigen heterologous enzyme linked immunosorbent assay (ELISA) for milk progesterone has been developed using progesterone-3-O-carboxymethyloxime- bovine serum albumin (P-3-O-CMO-BSA) antiserum and 17-α-hydroxy- progesterone-3-O-carboxymethyloxime-horseradish peroxidase (17-α-OH-P-3-O- CMO-HRP) enzyme conjugate. The data of the present study reveal that the homologous assay, which employed P-3-O-CMO-HRP as the label, showed no displacement. On the contrary, replacement of P-3-O-CMO-HRP with 17-α-OH-P-3-O-CMO-HRP as the label showed significant displacement and led to the development of a sensitive and specific assay. The recovery of the exogenously spiked progesterone from milk pools was in the range of 94.3-97.88% for toned milk and 97.6-101% for full-cream milk. The intra-assay and interassay coefficients of variation (CVs) ranged from 4.1-7.8% and 4.4-7.0%, respectively. A high ionic strength buffer was used to obtain released progesterone from binding protein/fat. The progesterone values measured in toned and full-cream milk ranged from 1.198-9.745ng/mL and 6.949-14.923ng/mL, respectively. The milk progesterone values obtained by this method correlated well with those obtained by radioimmunoassay; r=0.95 (n=65). Copyright © Taylor & Francis Group, LLC. Source


Shrivastav T.G.,National Institute of Health and Family Welfare | Kariya K.P.,Vmv Com Jmt Arts And Jjp Science College | Prasad P.K.V.,National Institute of Health and Family Welfare | Chaube S.K.,Banaras Hindu University | Kumar D.,National Institute of Health and Family Welfare
Journal of Immunoassay and Immunochemistry | Year: 2014

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. Copyright © 2014 Taylor & Francis Group, LLC. Source


Shrivastav T.G.,National Institute of Health and Family Welfare | Chaube S.K.,Banaras Hindu University | Prasad P.K.V.,National Institute of Health and Family Welfare | Kariya K.P.,Vmv Com Jmt Arts And Jjp Science College | Kumar D.,National Institute of Health and Family Welfare
Journal of Immunoassay and Immunochemistry | Year: 2012

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. © 2012 Copyright Taylor and Francis Group, LLC. Source

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