One Step Enzyme Linked Immunosorbent Assay for Direct Estimation of Serum Testosterone

Abstract

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).

Critical difference applied to exercise-induced salivary testosterone and cortisol using enzyme-linked immunosorbent assay (ELISA): distinguishing biological from statistical change

“…However, the discrepancy in results (78 % vs. 90%) may be attributed to difference in methodologies; in the present investigation, we categorized CVB as the variation over a period of ~12 h whereas Valero-Politi and Fuentes-Arderiu [20] measured yearly variation at monthly intervals but only within a 90 min daily window. More importantly, the present investigation utilized ELISA whereas Valero-Politi and Fuentes-Arderiu [20] used radioimmunoassay (RIA), known to exhibit smaller CVA [18].…”

Antigen Heterologous Enzyme Linked Immunosorbent Assay for the Measurement of Dhea in Serum

“…Van Weemen and Schuurs [41] observed that hapten and bridge heterology have a lesser effect on sensitivity compared with site-heterologous systems. In later studies, it has been reported that both bridge and hapten heterology improved sensitivity of EIAs for cortisol, [23,35,44] testosterone, [17,45] progesterone, [22,24,46] and DHEA. [34] In these heterologous assays, an EIA becomes specific and sensitive due to the use of structurally different hapten derivatives.…”

“…(i) homologous assay and (ii) heterologous assay. In homologous assay, [17][18][19][20] a steroid derivative is the same for the conjugation with carrier protein and enzyme to generate the antibody and to prepare enzyme conjugate; however, the assay is generally less sensitive. [21] On the other hand, in heterologous assay, [22][23][24][25][26][27][28] certain differences of site, bridge, or hapten exist between the steroid derivatives used for immunogen preparation and for the preparation of enzyme conjugate, while the assay is more sensitive.…”

A study on the effect of age, breed and season on sexual behaviour, testosterone concentrations and scrotal circumference in Egyptian rams

“…The obtained serum was stored until hormone analysis at -20ºC. The concentration of testosterone in serum was measured in all samples by ELISA technique (Shrivastav et al, 2003).…”