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

lust sped (500 to 700 rpm) on an orbital microplate shakcr Ibr LJO min at room
temperature. The wells were aspirated and washed five times with the wash solution
using an automatic microplate washer.
The microtitration plates were dried by
inverting plate on absorbent material. TMB chromogen solution (100 pL) was added
to each well and incubated. 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 absorbance of the solution in the wells was read within 30 min
using a microplate reader set at 450 nm.
Mean absorbances for standard, control and experimental serum samples were
calculated. Using a linear-linear graph paper mean absorbance readings were plotted
for each of the LH concentrations in mIU/ rnL in x-axis versus along the LH standards
in y-axis. The best fitting curve through the mean of the duplicate points were drawn.
The LH concentrations of the controls and experimental serum samples were
determined from the standard curve by matching their mean absorbance readings with
the corresponding LH concentrations.
3.13.3 Quantitative determination of serum prolactin
Serum levels of prolactin were determined strictly according to the procedure
given along with kit. In principle prolactin ELlSA is an enzymatically amplified
'two-step' sandwich-type immunoassay. In the assay, standards, controls and
experimental serum samples are incubated in microtitration wells, which have been
coated with anti-Prolactin antibody. AAer incubation and washing, the wells are
treated with another anti-Prolactin detection antibody labeled with the enzyme
hotseradish peroxide (HRP). After a second incubation and washing step, the wells
are incubated with the substrate tetrarnethylbcnzidine (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. The absorbance measured is
directly proportional to the concentration of prolactin present in the serum. A set of
prolactin standards is used to plot a standard curve of absorbance versus prolactin