ON TESTIS AND EPlDlDYMlS OF RATS - Pondicherry University ...

Mean absorbances for each standard, control and experimental serum samples
were calculated. Using a linear-linear graph paper mean absorbance readings were
plottcd fnr each of the standards along the y-axis versus the testosterone
concentrations in ny/ mL along the x-axis. The best-fitting curve through the mean of
the duplicate points was drawn. The testosterone concentrations of the controls and
experimental serum samples were determined from the standard curve by matching
their mean absorbance readings with the corresponding testosterone concentrations.
3.13.5 Quantitative determination of serum estradiol
The serum levels of estradiol were determined strictly according to the
procedure given along with kit.
binding enzyme immunoassay format.
In principle estradiol ELISA is the competitive
In the assay, standards, controls and
cspcr~~ncntal serum samples are incubated with biotin-labeled estradiol and rabbit
ant]-estradlol antiserum in microtitration wells where the labeled and biotin-labeled
antigens compete for a limited number of anti-estradiol binding sites.
After
~ncubation and washing, the wells are incubated with streptavidin-Horseradish
perosidase (HRPO). which binds to the biotinylated estradiol.
The unbound
atreptavidin-HRPO is washed. followed by incubation with the substrate
tetramethylbenzidine (TMB). An acidic stopping solution is then added and degree of
enzymatic turnover of the substrate is determined by the wavelength absorbance
measurement at 450 nm. Before starting the assay all the reagents and experimental
serum samples were brought to room temperature and were thoroughly mixed. The
assay consists of standards containing 0, 20. 50. 250. 750, 2000 and 6000 pg/ mL (see
Appendix 1.7). controls containing low (level I) and high (Level 11) concentrations of
estradiol. All the assays were performed in duplicate. The microtitration strips were
marked and 50 pL of each standard, controls and experimental serum samples were
addcd to the appropriate wells.
Estradiol-Biotin Conjugate solution (100 PL) was
added to each well. The wells were incubated while shaking at a fast speed on an
orbital microplate shaker for 60 min at room temperatun. The wells were aspkred
and washed five times with the wash solution using an automatic microplate washer.