Journal
TALANTA
Volume 77, Issue 1, Pages 210-216Publisher
ELSEVIER
DOI: 10.1016/j.talanta.2008.06.009
Keywords
Antigen heterology; Enzyme linked immunosorbent assay; Steroid; Cortisol; Corticosterone; Biotin; Streptavidin-horseradish peroxidase
Categories
Funding
- National Institute of Health and Family Welfare, New Delhi, India
Ask authors/readers for more resources
The present study demonstrates improvement in sensitivity and specificity of hapten assay by using antigen heterology in conjunction with low molecular weight biotin label as compared to high molecular weight horseradish peroxidase (HRP) label. For generation of antiserum, cortisol-3-O-carboxylmethyl-oxime-bovine serum albumin (F-3-CMO-BSA) was used as immunogen whereas, for the preparation of primary label, corticosterone-3-carboxymethyl oxime (B-3-CMO) was coupled with biotinylcaproylhydrazide and HRP by employing N-hydroxysuccinimide mediated carbodiimide reaction. The data of the present study revealed that the antigen heterologous assay which employed high molecular weight HRP label showed 100% cross-reaction with corticosterone. On the contrary, when HRP was replaced with low molecular weight biotin label, less than 0.1% cross-reaction was observed with all analogous C-18, C-19, C-21 and C-27 steroids including corticosterone (0.2%). Moreover, the sensitivity of the later assay was 0.09 mu g/dL, which is appreciable as compared to previously reported enzyme based assays. The recovery of the exogenously spiked serum pools lies in the range of 90.3-104.2%. The intra-assay and inter-assay coefficient of variation (CVs) ranged from 3.3% to 7.8% and 2.3% to 7.7%, respectively. The serum cortisol values obtained by this method correlated well with those obtained by radioimmunoassay; r=0.9 (n=50). The use of much stable biotin label in place of HRP has made the antigen heterologous enzyme linked immunosorbent assay (ELISA) of cortisol assay highly specific and sensitive. (c) 2008 Elsevier B.V. All rights reserved.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available