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

concentration from which the prolactin concentrations in the expcrinicntal serum
samples can be calculated. Before starting the assay all the rcagents and cxpcrinicntnl
serum samples were brought to room temperature and were thoroughly mixed. The
assay consists of standards containing 0, 2, 6. 20. 60 and 180 ng prolactin1 mL (see
Appendix 1.5) and controls containing low (Level I) and high (Level 11)
concentrations of prolactin and experimental serum samples. All the assays were
performed in duplicate and internal and external quality controls were maintained.
The microtitmtion strips were marked and 25 pL of the standard, controls and
unknowns were added to the appropriate wells. Added 100 pL of assay buffer and
the wells were incubated while shaking at a fast speed (500 to 700 rpm) 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 the plate on absorbent material. Antibody-
Enzyme 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 temperature. The wells were aspirated and washed five times with the wash
solution using an automatic microplate washer and then dried on absorbent material.
TMB chromogcn solution (100 pL) was added to each well. The wells were
~ncubated 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 absorbance of the solution in the wells was read within 30 niin using a
microplate reader set at 450 nm. Mean absorbances for standard, control and
experimental xrum samples were calculated. Using a linear-linear graph paper. nican
absorbance read~ngs were plotted Ibr each ofthc standards along the y-axis versus the
prolactin concentrations in ng/ mL along the x-axis. The best-fitting curve through
the mean of the duplicate poinu was drawn. The prolactin concentrations of the
controls and experimental serum samples were determined from the standard curve by
matching their mean absorbance readings with the corresponding prolactin
concentrations.