One step enzyme linked immunosorbent assay for direct estimation of serum testosterone.
One step competitive enzyme linked immunosorbent assay (ELISA) for direct estimation of testosterone in human serum is described. Testosterone-3-O-carboxymethyl-oxime-bovine serum albumin (testosterone-3-O-CMO-BSA), was used as immunogen and testosterone-3-O-carboxymethyl-oxime-adipic-acid dihydrazide-horseradish peroxidase (testosterone-3-O-CMO-ADH-HRP) was used as tracer. To the testosterone antibody coated microtiter wells, standard or serum samples (100 microL), along with testosterone-3-O-CMO-ADH-HRP conjugate (100 microL) were incubated for 1 h at 37 degrees C. Bound enzyme activity was measured by using tetra methyl benzidine/hydrogen peroxide (TMB/H2O2) as a substrate. In this new strategy, charcoal stripped pooled human serum spiked with non-cross reactive C18, C19, C21, and C27 steroids, used for preparing the standards and blocking the sex hormone binding globulin (SHBG)/and other steroid binding globulins (SBG). The sensitivity of the assay was 0.015 ng/mL. The intra-assay and inter-assay coefficients of variation (CVs) were ranged from 7.8 to 11.8 and 4.8 to 10.4, respectively. The serum testosterone values, obtained by this method, were correlated well with those obtained by radioimmunoassay r = .98 (n = 100).
Shrivastav
Tulsidas G
TG
Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka, New Delhi, India. nihfw@delnet.ren.nic.in
Basu
Anupam
A
Kariya
Kiran P
KP
D000272:Adipates / Q000737:chemistry; D000818:Animals; D000918:Antibody Specificity; D001560:Benzidines / Q000737:chemistry; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006041:Goats; D006735:Horseradish Peroxidase / Q000737:chemistry; D006801:Humans; D006854:Hydrocortisone / Q000031:analogs & derivatives* / Q000737:chemistry / Q000276:immunology; D006861:Hydrogen Peroxide / Q000737:chemistry; D011817:Rabbits; D011863:Radioimmunoassay; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000276:immunology; D012738:Sex Hormone-Binding Globulin / Q000037:antagonists & inhibitors; D013739:Testosterone / Q000031:analogs & derivatives* / Q000097:blood* / Q000276:immunology
D000272:Adipates; D001560:Benzidines; D012738:Sex Hormone-Binding Globulin; C045037:testosterone-3-carboxymethyloxime-bovine serum albumin conjugate; D012710:Serum Albumin, Bovine; C021758:3,3',5,5'-tetramethylbenzidine; D013739:Testosterone; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; C007133:hydrocortisone hemisuccinate; C010011:adipic dihydrazide; D006854:Hydrocortisone