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Antibody may be produced by inducing
laboratory animals, such as rats, guinea pigs and
rabbits, with an immunogenic antigen. Immune
response induced by antigen after exposure can then
be measured and determined using serological
testing. Serologic tests developed today can be
divided into two categories; primary binding and
secondary binding tests. The primary binding test is a
test that directly measures the antigenic bonding to
antibodies including fluorescence antibody technique
(FAT), radioimmunoassay (RIA) and Enzyme Linked
Immunoassay (ELISA). The secondary binding test is
a test that measures the results of antigen-antibody
interaction in-vitro which includes agar gel
precipitation est (AGPT), serum agglutination test
and complement fixation test (FAT). However, the
primary binding is more sensitive than the secondary
binding test.
The purpose of this study is to produce polyclonal
antibody against the ES product of F. gigantica
Lombok isolate using rabbit and to determine the
antibody response of rabbit to the ES product using
the ELISA test, and Western blotting. The success in
producing specific antibodies against specific
antigens will increase sensitivity and specificity of
immunodiagnostic test to perform early detection of
Fasciolosis in livestock.
2 MATERIALS AND METHODS
2.1 Research Design
Two local rabbits (4 months old) were placed in a
special cage and fed with pellets, fresh vegetables,
and drinking water (ad-libitum). The rabbits were
then immunized with 0.5ml (300μg / ml) of
emulsified ES product with 0.5ml Freund's complete
adjuvant (FCA). Sera were harvested and tested with
the ELISA before initial immunization and after
booster. The ELISA results were then confirmed by
Western blot analysis.
2.2 Immunization and Serum
Collection
Animal Ethics approval (Protocol No.
235/UN18.8/ETIK/2017) had been obtained for
conducting this study. Immunization was carried out
by subcutaneous injection at multiple sites (no more
than 5) behind the neck and was dispersed under the
skin by gentle rubbing. Four weeks after initial
immunization with the ES product emulsified with
the FCA, the rabbits were then immunized twice
subcutaneously with 0.5ml (300 μg / ml) of the ES
product mixed with 0.5ml Freund's incomplete
adjuvant (FICA) at 1-month interval.
Blood sampling was performed by taking blood
from the auricularis vein of the rabbits using a 3ml
syringe. Blood were placed on a sterile tube and were
then incubated at room temperature for 3 hours. After
incubation, the serum can be obtained by
centrifugation at 5000rpm at 4
o
C for 15 min. The sera
were then aliquoted into several eppendorf tubes and
then stored at -20°C until further assays.
2.3 Measurement of Humoral Immune
Response
Humoral immune response of each rabbit to the ES
product was measured by ELISA technique, and then
confirmed by Western blot. ELISA was performed
according to Sriasih’s procedure (Sriasih et al., 2005).
The ELISA plates (96 wells) were coated with 50 μl
of the ES product and incubated for 1 hour at room
temperature. After incubation, the ES product was
removed and the wells were washed 5 times using
phosphate buffer saline (PBS) containing 0.05%
Tween 20. Blocking agent (100 μl PBS containing
bovine serum albumin (BSA) or skim milk) or were
added into each well and were then incubated for 1
hour at room temperature. Following incubation, the
plate was washed again (5 times washing cycling).
Fifty microliters serum that had been diluted a
hundred times were added into each well and then
incubated for 1 hour at room temperature. After 5
times washing, 50μl anti-rabbit IgG horse-radish
peroxidase conjugated were added and then incubated
for 1 hour. One hundred microliters of substrate
(ABTS in 100ml of citrate buffer) were added into the
wells and incubated for 15 min room temperature.
Optical density (OD) was then measured at 405nm
wavelength using an ELISA reader machine.
2.4 Western Blotting Analysis
After electrophoresis, gels were equilibrated in
transfer buffer for at least 10 min. Polyvinylidine
difluoride (PVDF) membrane was pre-incubated in
100% methanol for 1 min then rinsed with several
changes of water. After rinsing, the membrane and
filter papers were also allowed to equilibrate for at
least 15 min in transfer buffer. The transfer of
proteins from the gel to the membrane was carried out
at a constant voltage of 15 Volts for 35-40 minutes
using a Trans-Blot® Semi-Dry (SD) electrophoretic
transfer cell as per the manufacturer’s instructions.
Production of Rabbit Anti-Excretory/Secretory Product of Fasciola gigantica Lombok Isolate Antibody
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