A novel enzyme-linked immunosorbent assay for cortisol using a long-chain biotinylated cortisol-3-CMO derivative.
An enzyme-linked immunosorbent assay (ELISA) using streptavidin-biotin system as a bridge between antibodies bound antigen and reporter molecule (horseradish peroxidase enzyme) has been described. The cortisol antiserum was generated against cortisol-3-O-carboxylmethyl oxime-bovine serum albumin (F-3-CMO-BSA). We have prepared biotin-labelled cortisol as a primary probe and utilized streptavidin-labelled horseradish peroxidase (SA-HRP) as secondary probe to monitor the antigen-antibody interaction. To the cortisol antibody coated micro wells, 25 microL of standard or samples, along with 100 microL of biotinylated cortisol, were kept for 1 h at room temperature. Thereafter, wells were washed and 100 microL of SA-HRP was added to all wells and kept again for 20 min at room temperature. Bound enzyme activity was measured using tetramethyl benzidine/hydrogen peroxidase (TMB/H2O2) as substrate. The incorporation of streptavidin-biotin system as a bridge between antibody bound antigen and reporter molecule (horseradish peroxidase enzyme) increased sensitivity and specificity of the cortisol assay. The use of low molecular weight primary label (F-3-CMO-biotin) might have facilitated the easy and selective access of the analyte present in serum to compete with the antigen-binding pocket of antibody, thereby detecting as low as 3.42 ng/mL of analyte specifically.
Nara
Seema
S
Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka, New Delhi, India.
Tripathi
Vinay
V
Chaube
Shail K
SK
Rangari
Kiran
K
Singh
Harpal
H
Kariya
Kiran P
KP
Shrivastav
Tulsidas G
TG
D000818:Animals; D000906:Antibodies / Q000276:immunology; D001710:Biotin; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006854:Hydrocortisone / Q000031:analogs & derivatives* / Q000097:blood* / Q000276:immunology; D011817:Rabbits; D011863:Radioimmunoassay; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000276:immunology*; D019809:Streptavidin
D000906:Antibodies; C051191:cortisol 21-acetate-3-(O-carboxymethyl)oxime-bovine serum albumin; D012710:Serum Albumin, Bovine; D001710:Biotin; D019809:Streptavidin; D006854:Hydrocortisone