effect of infection of the filarial parasite brugia malayi - Pondicherry ...

Tubes were labeled for calibrators, blank, maximum binding tube, total counts and
samples (tissue homogenate) in duplicate. Two hundred pl of the buffer was taken into the
blank tube and 100 pl ihto the maximum binding tube. Similarly 100 pi of the calibrators
and the sample was taken in the calibrator tubes and sample tubes. Then 100 p1 of the [I2' I]
testosterone was added into all the tubes, followed by 100 p1 of testosterone antiserum to all
the tubes except the blank tubes. The tubes were vortexed and incubated for a minimum of
60 minutes at R.T., which was followed by the addition of 100 pi of goat anti-rabbit gamma
globulin to all the tubes. The contents of the tubes were vortexed and incubated for I hr at
R.T. then 2.0 rnl of cold 4% PEG-saline solution was added and centrifuged at 2,000g for 20
minutes. The supernatant was decanted, the rim of each tube was blotted dry and counted in
a Gamma Counter for 1 minute. Calibration curve was prepared as indicated by the kit
procedure. The testosterone levels were expressed in nanornoles/mg protein.
43.1.2 7-GTP
1-GTP was assayed according to the method of Orlowski and Meister (1965) and
modified by Rosalki and Rau (1972) as described earlier.
43.13 LDH-X
The testes of M.naralemis infected with B.rnalayi were excised on different days of
post inoculation (0,30, 60, 90. 120). washed in chilled potassium phosphate buffer (0.1 M,
pH 8.3) and homogenised in the same buffer. The homogenate was centrifuged at 10,000g
for 30 minutes at 4°C and the supernatant was used for LDH-X isoenzyrne assay.
LDH-X isorymes were visualized on agar gel by following the method of Hanis and
Hopkinson ( 1978).
Reagents for staining
1. 0.05 M Tris-HCl buffer. pH 8.0 : 20 ml
2. Calcium l,actate (pentahydrate)
mixture).
3. NAD
: 100 mg (8 mM final concentration in reaction