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International Journal of Marine Science 2013, Vol.3, No.19, 151-157
http://ijms.sophiapublisher.com
153
stimulation with antigen, the rats were immunized (i.p.)
with SRBC suspended in normal saline (0.15 M).
Approximately 25×10
6
cells/mLwere administered for
the primary and 50×10
6
cells/mL
for the secondary
immunization two weeks after the primary dose. Un-
stimulated mice were treated similarly except
immunization with SRBC antigen. The stimulated rats
were treated with different doses of sponge extract .For
each concentration of sponge extract and control
groups, triplicate experiments were maintained.
2.2.3 Experimental system
Blood samples of stimulated rats were collected on the
first and second weeks following sponge extract treated
by cardiac puncture, after anesthetizing the rats with
chloroform. The serum was separated for each group
separately and kept at
-
20
℃
till analyzed. Sodium
citrate (2.8 g/100mL) was used in collecting whole
blood and leukocyte rich plasma for lymphocyte subset
numeration.
Immunological assays
a) Humoral immune response
I. Antibody titre, II. B cell E rosette assay: to enumerate
B lymphocytes.
b) Cell-mediated immune response (CMI)
I. Delayed type hypersensitivity (DTH), II. T cell E
-rosette assay:
to estimate of T lymphocytes.
2.2.4 Haemagglutination antibody titre (HA)
(SRBC-Antigen)
The rats were divided into five groups consisting of
six animals each Rats in group I (control group)
received vehicle only for 14 days. Groups II received
standard drug Dexamethasone (0.5 mg/kg). On 4
th
and
11
th
day as a single dose. Rats in treatment with group
III were given Immun Aid tab. (immunostimulatory
drug) daily for 12 days. Group IV, V and VI were
given sponge extract (50 mg/kg, 150 mg/kg, 250
mg/kg). On 4
th
and 11
th
day as a single dosing
respectively. On 3
th
and 8
th
day of study, rats from all
the groups (i.e. group I to V were immunized and
challenged respectively, with SRBCs in normal saline
(0.1 mL of 20% SRBCs) intraperitonially. Blood was
withdrawn on 7
th
and 14
th
day from heart puncture
method, by using chloroform to give mild anesthesia
to all rats group. The obtained blood was centrifuged
to raise serum, normal saline was used as a diluents
and SRBCs count was adjusted to (0.1 mL of 20%
SRBCs). Each well of a microtitre plate was filled
initially with 25±l of saline and 25±l of serum was
mixed in the first well of micro titre plate. Subsequently
the 25 ±l diluted serum was removed from first well
and added to the next well to get twofold dilutions of
the antibodies present in the serum. Further twofold
dilutions of this diluted serum were similarly carried
out till the last well of the first row (11
th
well), so that
the antibody concentration of any of the dilutions is
half of the previous dilution. 20±l SRBC (0.1% of
SRBCs) were added to each of these dilutions and the
plates were incubated at 37
℃
for one hour and then
observed for haemoagglutination (Agarwal et al.,
1999; Dhasarathan et al., 2010). The highest dilution
giving haemagglutination was taken as the antibody
titre. The antibody titres were expressed in the graded
manner, the minimum dilution (1/2) being ranked as 1,
and mean ranks of different groups were compared for
statistical significance.
2.2.5 B cell rosette assay
Methodology
Blood is collected from sponge extract treated, control
and standard drug treated rat as mentioned in earlier. B
cell count in the blood was carried out by the following
method.
Five to ten ml of blood was collected and it was
introduced into sterile conical flask/beaker containing
(4-5) sterile glass beads. It was then continuously
swirled until sounds were heard from the beads. This
indicates that all the fibrins have adhered to the beads.
This blood was considered as defibrinated blood. This
defibrinated blood was taken and diluted with equal
volume of physiological saline. 3 mL of the lymph prep
solution was taken in a centrifuge tube. The tube was
kept in slanting position and 9 mL of diluted blood was
slowly added along the sides of the centrifuge tube
using Pasteur pipette. Care was taken so that the
FICON layer of the lymphoprep solution present in the
centrifuge tube was not disturbed. The content of the
centrifuge tube was then centrifuged at 1600 rpm for 20
min. The interphase (containing lymphocytes) was
removed using pipette. The cells were washed with 1
mL saline and excess FICON was removed. The
sample was again washed with 1 mL of saline after
centrifugation the supernatant was decanted by
inverting the tube over a filter paper after all saline was