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

anti-FSH antibody. After incubation and washing, the wells are treated with another
anti-FSH detection antibody labeled with the enzyme horseradish peroxidase (HRP).
After a second incubation and washing step, the wells are incubated with the substrate
tetramcthylbenzidine (TMB).
An acidic stopping solution is then added and the
degree of enzymatic turnover of the substrate is determined by absorbance
measurement at 450 nm. The absorbance measured is directly proportional to the
concentration of FSH present in the serum samples. A set of FSH standards is used to
plot a standard curve of absorbance versus FSH concentration from which FSH
concentrations in the experimental serum samples can be calculated.
Before starting the assay all the reagents and serum samples were brought to
room temperature and were thoroughly mixed.
The assay consisted of standards
containing concentrations of approximately 0, 1.5, 4.5, 15.45 and 150 mIU/ mL FSH
(see Appendix 1.3), and controls containing low (Level 1) and high (Level 11)
concentrations of FSH 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 100 pL of the standard. controls
and experimental serum samples were added to the appropriate wells and incubated
while shaking at a fast speed (500 to 700 rpm) on an orbital microplate shaker (P-
Multi~ope ELISA Reader) for 60 min at room temperature.
Antibody-Enzyme
Conjugate solution (100 pL) was added to each well and incubated while shaking at a
fast speed on an orbital microplate shaker for 30 min at room temperature. The wells
were aspirated and washed five times with the wash solution using an automatic
microplate washer (Labsystems Well Wash 4). The microtitration plates were dried
by invening on absorbent material. TMB chromogen solution (100 pL) was added to
each well and incubated while shaking at a fast speed (500 - 700 rpm) on an orbital
microplate shaker for 10 min at room temperature. Stopping solution, 0.2 M sulfuric
acid, (100 a) was added to each well and absorbance of the solution in the wells was
read within 30 min using a microplate reader (P-Multiscope ELISA Reader) set at 450
nm. Mean absorbances for standard, control and experimental serum samples were