ON TESTIS AND EPlDlDYMlS OF RATS - Pondicherry University ...
ON TESTIS AND EPlDlDYMlS OF RATS - Pondicherry University ...
ON TESTIS AND EPlDlDYMlS OF RATS - Pondicherry University ...
You also want an ePaper? Increase the reach of your titles
YUMPU automatically turns print PDFs into web optimized ePapers that Google loves.
calculated. Using a linear-linear gtaph paper mean absorbance readings were plotted<br />
Tor each of the standards along the y-axis versus the FSH concentrations in mllJ1 ml,<br />
along the x-axis. The best fitting curve through the mean of the duplicate points was<br />
drawn. The FSH concentrations of the controls and experimental serum samples were<br />
determined 'om the standard curve by matching their mean absorbance readings with<br />
the corresponding FSH concentrations.<br />
3.13.2 Quantitative determination of serum LH<br />
Serum levels of LH were determined strictly according to the procedure given<br />
along with kit.<br />
sandwich-type immunoassay.<br />
In principle LH ELISA is an enzymatically amplified 'one step'<br />
In the assay, standards, controls and experimental<br />
serum samples are incubated with anti-LH antibody in microtitration wells, which<br />
have been coated with another anti-LH antibody. After incubation and washing, the<br />
wells are incubated with the substrate tetramethylbenzidine (TMB).<br />
An acidic<br />
stopping solution is then added and the degree of enzymatic turnover of the substrate<br />
is determined by wavelength absorbance measurement at 450 nm. The absorbance<br />
measured is directly proportional to the concentration of LH present in the serum. A<br />
set of LH standards is used to plot a standard curve of absorbance versus LH<br />
concentration from which the LH concentrations in the experimental serum sanlples<br />
can be calculated.<br />
Before starting the assay all the reagents and experimental serum samples<br />
were brought to the mom temperature and were thoroughly mixed.<br />
The assay<br />
consisted of standards containing LH concentrations of approximately 1, 3, 10, 30 and<br />
100 mlU1 mL [see Appendix 1.4) and controls containing low (Level 1) and high<br />
(Level 11) concentrations of LH and experimental serum samples. All the assays were<br />
pcrlbr~i~cd in duplicutc and inlcrnal and cx~crn:~l quality controls wcrc mai~ilai~iuil<br />
The microtitration strips were marked and 50 pL of the standard, controls and<br />
experimental serum samples were added to the appropriate wells. Antibody-Enzyme<br />
Conjugate solution (100 pL.) was then added to each well and incubated. shaking at a
Change language