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

3.13.4 Quantitative determinntiun uf serum testustcrunc
The serum levels of testosterone were determined strictly according to the
procedure given along with kit. In principle testosterone ELlSA follows the basic
principle of enzyme immunoassay where there is competition between an unlabeled
antigen and enzyme-labeled antigen bound to the antibody binding sites. The amount
of enzyme-labeled antigen bound to the antibody is inversely proportional to the
concentration of the unlabeled analyte present. Unbound materials are removed by
decanting and washing the wells. The absorbance measured is inversely proportional
to the concentration of testosterone present in the serwn. A set of testosterone
standards is used to plot a standard curve absorbance versus testosterone
concentration from which the testosterone concentrations in the experimental serum
samples can be calculated.
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, 0.1, 0.5, 2.5, 5, 10 and 25 ng testosterone1 mL (see Appendix
1.6) and controls containing low (Level I) and high (Level 11) concentrations of
testosterone and experimental serum samples. All the assays were performed in
duplicate and internal and external quality controls were maintained. The
microtitration strips were marked and 50 pL of the standard, controls and
experimental serum samples were added to the appropriate wells. Enzyme Conjugate
solution (100 pL) and Testosterone-Antiserum (100 bL) were added to each well.
The wells were covered and incubated while shaking at a fast speed on an orbital
microplate shaker for 60 min at room temperature. The wells were aspirated and
washed five times with the wash solution using an automatic microplate washer. The
microtitre plates were dried by inverting on absorbent material. TMB chromogen
solution (100 &) was added to each well. The wells were incubated while shaking at
a fast speed on an orbital microplate shaker for 10 min at room temperature. Stopping
solution. 0.2 M sulfuric acid. (100 pL) was added to each well and absorhance of the
solution in the wells was read within 30 min using a microplate reader set at 450 nm.