376
Chiang Mai J. Sci. 2013; 40(3)
Chiang Mai J. Sci. 2013; 40(3) : 376-385
http://it.science.cmu.ac.th/ejournal/
Contributed Paper
Production of a Rabbit Polyclonal Anti-Elephant IgG
Antibody for the Elephant Laboratory Investigation
Preeyanat Vongchan
Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University,
Chiang Mai, Thailand.
Author for correspondence; e-mail: [email protected]
Received: 26 March 2012
Accepted: 9 April 2013
ABSTRACT
Blood group phenotyping in elephants is not well researched. Transfusion with
incompatible blood may thus result in blood transfusion reactions and may lead to death of
the animal. Pathologies resulting from blood incompatibility between the mother and fetus
may cause HDN. Several disease investigations also utilized anti-elephant IgG antibody
(anti-EIgG) to identify the antibody titer against particular diseases. Of which, reliable and
rapid diagnostic tools based on antiglobulin test using anti-EIgG should be developed.
This work aimed to purify elephant IgG (EIgG) and produce rabbit anti-EIgG for elephant
laboratory investigation. Sera from elephants were pooled and EIgG was purified.
Rabbits were immunized weekly with purified EIgG for 8 weeks. Investigation of anti-EIgG
rising titer was performed by indirect ELISA. Activity and specificity of rabbit anti-EIgG
was determined by EIgG coated red blood cell (RBC) agglutination. Cross-reactivity to other
animal IgG was verified by agglutination and indirect ELISA. The results showed that EIgG
yield was 21.6 mg/mL serum. Rabbits gradually produced anti-EIgG with a rising titer after
first immunization and reached a plateau during week 6-8. Purified rabbit anti-EIgG was
demonstrated to specifically agglutinate EIgG coated elephant RBC. Taken together, it could
be proposed that the product will be able to serve for elephant blood compatibility testing
as well as investigation for other pathologies.
Keywords: elephant blood compatibility, anti-elephant IgG, elephant immunoglo-
bulin, antiglobulin test, HDN
1. INTRODUCTION
In Thailand, the number of Asian
elephants has dramatically decreased due to
habitat loss and hunting [1, 2]. Asian elephants
therefore are protected by being classified as
endangered (EN) on the International Union
of Conservation for Nature (IUCN) Red List
as well as Appendix I of the Convention for
International Trade in Endangered Species
of Wild Fauna and Flora (CITES). A key
factor for elephant conservation is to increase
the population by controlled breeding.
However, breeding programs can encounter
problems such as embryonic loss, abortion,
preterm birth and elephant calf death [2].
Chiang Mai J. Sci. 2013; 40(3)
During pregnancy, the mother animal’s
transplacental hemorrhage can cause
exposure to fetal RBC and induce the
maternal immune response, which affects
abortion and neonatal hemolytic disease
[3-6]. Studies of elephants during pregnancy
have shown that the anatomy and
morphology of the uterus and placenta
are changed [7]. Moreover, there is evidence
of transplacental IgG in female dogs [6, 8].
Blood incompatibility between mother and
neonate with different blood types due to
transplacental hemorrhage may therefore
lead to termination of the pregnancy. In
addition, according to such pathophysiology,
transfusion with incompatible blood may
thus result in blood transfusion reactions and
may lead to death of the animal.
A key factor for elephant conservation
is to decrease the number of animal death
from disease, of which reliable and rapid
diagnostic tools should be developed.
Anti-elephant IgG played an important role
in these diagnostic kits. Several disease
investigations utilized anti-EIgG to identify
the antibody titer against particular
diseases i.e., tuberculosis [9], or to check the
antibody titer against vaccine e.g. tetanus
toxoid [10], porcine zonapellucida vaccine
[11]. Furthermore, the production of this
EIgG could be served as the serology test
of the lethal Elephant endotheliotropic
herpesvirus (EEHV)[12], which was under
developing procedure. Those mentioned
pathologies can be investigated by direct
and/or indirect antiglobulin tests,
Antiglobulin testing is widely used to
detect free antibodies in the serum or
antibodies bound to RBC [13], It is used
most often in veterinary medicine for
diagnosis of immune-mediated hemolysis and
hemolytic disease of the newborn (HDN)
[14, 15]. In humans, anti-human globulin
reagent, which is composed of anti-human
377
IgG and anti-human complement, is used
for blood compatibility testing and diagnosis
of HDN as well as immune-mediated
hemolysis. Species-specific human or animal
antiglobulin reagents are usually produced
in rabbits and goats. Purified IgG, IgM,
and/or complement from species are injected
into rabbits or goats at various time intervals.
The resulting antiserum is heat-inactivated
to inactivate complement and adsorbed
with normal RBC to diminish hemagglutinin.
In veterinary medicine, canine, equine,
and feline polyspecific reagents and
canine monospecific reagent are available
commercially (VMRD and MP Biomedicals).
However, there is as yet no commercially
available anti-EIgG. In this work, we report
the purification of EIgG. The antibody
raised against EIgG was demonstrated to
specifically induce agglutination of EIgG
bound RBC, with cross reaction only with
cow IgG. We proposed that the product
obtained will be helpful in blood compatibility
testing in elephants, investigation of sensitized
RBC in HDN, as well as breeding program
and the other application for laboratory
investigation for elephant.
2. MATERIALS AND METHODS
2.1 Preparation of Elephant IgG (EIgG)
and Production of Rabbit Anti-EIgG
Antibody
Ten pooled sera used in this study were
kindly provided by Dr. Chatchote Thitaram,
Elephant and Wildlife Clinic, Faculty of
Veterinary Medicine, Chiang Mai University.
These included both normal and abnormal
sera and were ready to be destroyed after
laboratory testing. Pooled serum was
centrifuged to remove any particulate
protein. EIgG was isolated (Melon Gel
Purification kit, PIERCE) according to the
manufacturer’s instructions. Briefly, pooled
serum was diluted (1:10) with 1x Melon Gel
378
purification buffer and dialyzed against the
same buffer. Diluted serum was then mixed
with 20% slurry gel (1:1), incubated with
shaking for 5 min, and then filtered through
0.45 μm filter paper under reduced pressure,
nonspecific proteins were adsorbed to the gel,
and only IgG passed through without elution.
Fractions (1 mL) were collected and pooled.
The resulting purified EIgG was dialyzed
against 3 changes of PBS, pH 7.2. Protein
concentration was determined by Lowry’s
method [16] and analyzed by sodium dodecyl
sulfate-polyacrylamide gel (SDS-PAGE).
Aliquots of purified EIgG were frozen and
stored at -20°C.
Purified EIgG 2 mg/mL was mixed
homogeneously with complete Freund’s
adjuvant (CFA, 1:1, v/v). Rabbit was injected
SC with purified EIgG in CFA (1 mg/dose
1 mL) in the first immunization. For week
2 nd -8 th , EIgG mixed with incomplete
Freund’s adjuvant (IFA, 1:1, v/v) was used to
booster the rabbit weekly. Rabbit blood
was collected prior to each immunization.
Serum was separated and kept frozen for
detection of anti-EIgG antibodies.
The rising titer of rabbit anti-EIgG was
measured using indirect ELISA. Briefly,
purified EIgG was diluted to 10 μg/mL with
0.01M Carbonate/bicarbonate buffer,
pH 9.6 and loaded onto a microtiter plate
(50 μL/well). The plate was incubated at
4°C overnight before being washed three
times with 0.05% Tween-20 in phosphate
buffer saline, pH 7.2 (PBST). Five percent
skimmed milk in PBST was added (200 μL/
well) to block non-specific binding sites.
Plates were incubated at 37°C for 1 h and
washed with PBST. Serial 10-fold dilution
of rabbit post-immunized and preimmunized sera were prepared in PBS and
loaded onto a microtiter plate (50 μL/well).
The reaction was allowed to stand at 37°C
for 1 h. After 3 washes with PBST, horseradish
Chiang Mai J. Sci. 2013; 40(3)
peroxidase (HRP) conjugated swine antirabbit Igs (1:2000, DAKO Cytomation) was
added, 50 μL/well, and incubated for another
1 h at 37°C. The plate was then washed
four times with PBST. Finally, H2O2-TMB
(Sigma) was added, 200 μL/well. The plate
was incubated at room temperature with light
protection for 15 min. H2SO4 (2M) was
added (50 μL/well) to stop the reaction and
the optical density (O.D.) was measured at
450 nm. O.D. of last dilution of tested
sera with 1+ RBC agglutination grading was
determined as titer. Titer of tested sera was
then plotted against week of immunization.
Rabbit serum was incubated at 56°C
for 1 h to inactivate complement. The IgG
fraction of rabbit anti-EIgG antibody was
purified from heat inactivated rabbit serum
(Melon Gel Purification kit, PIERCE).
The procedure followed the same steps as
for EIgG purification as previously described.
Protein concentration was determined by
Lowry’s method and purity was checked by
SDS-PAGE. The product obtained was kept
frozen at -20°C.
2.2 Specificity of Rabbit Anti-EIgG
Antibody
Rabbit anti-EIgG was tested for its
specificity to agglutinate EIgG coated RBC
in vitro. With the advantage of tannic acid [17],
purified EIgG could be coated onto elephant
RBC and was thus used to test for the ability
of rabbit anti-EIgG to agglutinate EIgG
coated RBC. In brief, elephant EDTA
whole blood was collected, RBC was
separated, washed three times with 0.85%
NaCl and suspended to 2% with buffered
saline, BS (1 volume of 0.9%w/v NaCl
mixed with 1 volume of phosphate buffer
[0.15M KH2PO4, 0.15M Na2HPO4, pH 7.2]).
Working tannic acid (1% w/v tannic acid
diluted to 1:30,000 with BS) was prepared
and added to produce a ratio of 1:1 with
Chiang Mai J. Sci. 2013; 40(3)
the elephant red cell suspension. The
mixture was incubated at room
temperature for 10 minutes with end-toend shaking and the solvent was
subsequently discarded. An equal volume
of purified EIgG (2.5-20 mg/mL) diluted
with BS pH 7.2 was added to the tannic
acid treated RBC. The mixture was further
incubated at room temperature for another
10 minutes with shaking. Finally, RBC was
washed three times with 0.85% NaCl and
suspended to 5% with the same buffer. A
direct antiglobulin test was then used to
assay the activity of rabbit anti-EIgG. One
drop of 5% EIgG coated RBC suspension
was transferred to a new clean 12×75 mm
test tube. Cells were washed three times
with 0.85% NaCl and the washing solution
was discarded. One drop of purified rabbit
anti-EIgG was added. The reaction was
mixed and immediately centrifuged at 3000xg
for 15 s. The agglutination of IgG coated RBC
was observed and compared to tannic acid
treated RBC control by a direct antiglobulin
test. The effect of cell concentration on
agglutination was also studied. EIgG (20 mg/
mL) was coated on elephant’s RBC and
various cell concentrations of EIgG coated
RBC (1-5%) were prepared by suspending
washed packed EIgG coated RBC with
0.85% NaCl. Serial dilution of rabbit antiEIgG was prepared and direct antiglobulin
test was performed.
2.3 Cross Reactivity of Rabbit AntiEIgG to Other Animal IgG
To verify the specificity of obtained
anti-EIgG, we checked for cross reaction of
rabbit anti-EIgG to IgG of other species by
indirect ELISA and agglutination assay.
For indirect ELISA, purified IgG (10 μg/mL)
from various animals other than human
(horse, cow, goat, sheep, dog and mouse) and
non-globulin protein (bovine albumin)
379
were separately coated onto microtiter
plates (50 μ L/well). The plates were
incubated at 4°C overnight before being
washed three times with PBST. After
blocking step with 5% skimmed milk in
PBST, purified rabbit anti-EIgG (0.14 μg/
mL) was then added, 50 μ L/well, and
incubated at 37°C for 1 h. The following
steps proceeded as in part 1. The optical
density measured at 450 nm of each well
was used to calculate the percentage by
comparison with the O.D. observed
in control EIgG coated wells. For the
agglutination assay, IgG from various
species (400 μg/mL) were individually
coated on elephant RBC by tannic acid as
described in part 2. Species’ IgG coated
RBC were individually tested with rabbit
anti-EIgG by direct antiglobulin test. Cross
reaction was determined in terms of
agglutination grading compared to control
EIgG coated RBC.
3. RESULTS
3.1 Preparation of Elephant IgG (EIgG)
and Production of Rabbit Anti-EIgG
Antibody
Purification using a Melon Gel
Purification kit yielded 21.6 mg EIgG from
1 mL serum. To verify and confirm the IgG’s
biochemical structure and molecular weight,
SDS-PAGE both in reducing condition and
non-reducing condition was performed.
In the presence of reducing agent, disulfide
bonds will be destroyed resulting in 2 bands
of 25 kD and 50 kD of light chain and heavy
chain, respectively. The result showed that
the purity of obtained elephant’s was
excellent, with a trace amount of other
protein (presumably albumin) as shown by
SDS-PAGE (Figure 1).
Rabbits responded to immunization
with purified EIgG, as indicated by a
rising titer of anti-EIgG from week 1st-6th
380
(Figure 2). Maximum titer was 1×109 and
remained constant until week 8 th of
immunization. Rabbit serum was firstly
heat inactivated at 56°C for 30 minutes to
destroy complement before purification
using a Melon Gel Purification Kit
(Figure 3). According to the benefit of
Chiang Mai J. Sci. 2013; 40(3)
Melon gel, all other proteins including
heterophile antibodies that would cause
false agglutination of RBC were also
eliminated. Two batches of the anti-EIgG
were prepared with protein concentrations
of 6 and 14 mg/mL, respectively.
Figure 1. SDS-PAGE (10%) of purified EIgG. EIgG was purified from elephants’ pooled
sera by Melon Gel Purification Kit as described. Purified EIgG was dialyzed against PBS pH
7.2, protein concentration determined by Lowry’s method prior to electrophoresis. Lane 1:
non-reduced condition, Lane 2: reduced condition.
Figure 2. Titer of rabbit anti-EIgG was increased in time dependent manner. Serial
dilutions of rabbit post immunized sera in each week were reacted with purified EIgG
coated on 96-well microtiter plate. The interaction was determined by indirect ELISA.
Chiang Mai J. Sci. 2013; 40(3)
381
Figure 3. SDS-PAGE (10%) of purified rabbit anti-EIgG antibody. Rabbit serum was
purified by Melon Gel Purification Kit as described. Purified rabbit anti-EIgG was
dialyzed against PBS pH 7.2, protein concentration determined by Lowry’s method
prior to electrophoresis. Lane 1: non-reduced condition, Lane 2: reduced condition.
3.2 Specificity of Rabbit Anti-EIgG
Rabbit anti-EIgG was subjected to
direct antiglobulin test using tannic acid
induced EIgG coated RBC. The titers of
rabbit anti-EIgG to agglutinate EIgG (2.5,
5.0, 10, and 20 mg/mL) coated RBC were
4, 16, 32 and 512, respectively (Table 1).The
effect of cell concentration was determined
and it was found that there was no effect
from cell suspension on cell agglutination
induced by the obtained product (Table 2).
Table 1. Purified rabbit anti-EIgG promoted agglutination of EIgG coated RBC.
Dilution of purified rabbit anti-EIgG
(6 mg/mL)
undiluted
1:2
1:4
1:8
1:16
1:32
1:64
1:128
1:256
1:512
1:1024
Concentration of EIgG used to coat on
elephant RBC (mg/mL)
2.5
5.0
10
20
+
+
+
2
3
3
3+
+
+
+
1
3
3
3+
1+
3+
2+
3+
+
+
neg
2
2
3+
neg
1+
2+
2+
+
neg
neg
1
2+
neg
neg
neg
2+
neg
neg
neg
1+
neg
neg
neg
1+
neg
neg
neg
1+
neg
neg
neg
neg
382
Chiang Mai J. Sci. 2013; 40(3)
Table 2. Concentration of EIgG coated RBC suspension had no effect on rabbit antiEIgG promoting agglutination.
Anti-EIgG
(6 mg/mL)
neat
1:2
1:4
1:8
1:16
1:32
1:64
1:128
1:256
1:512
1:1024
1
3+
3+
3+
2+
2+
2+
1+
1+
1+
neg
neg
Concentration of EIgG coated RBC (%)
3
4
2
5
+
+
+
3
3
3
3+
+
+
+
3
3
3
3+
3+
3+
2+
2+
+
+
+
2
3
2
2+
2+
2+
2+
2+
+
+
+
1
1
2
1+
1+
1+
1+
1+
+
+
+
1
1
1
1+
neg
neg
1+
neg
neg
neg
neg
neg
neg
neg
neg
neg
3.3 Cross Reactivity of Purified Rabbit
Anti-EIgG
In order to apply the obtained product
in serological laboratory testing, cross reactivity
was studied. We performed indirect ELISA
to observe the binding of anti-EIgG to other
animal and human IgG. The absorbance
from each reaction was measured and
calculated as percent of cross reaction based
on O.D. obtained from control EIgG. The
anti-EIgG could react with cow IgG and
mouse IgG. The cross reactivity were
32.8% and 12.8%, respectively (Table 3).
Since the product was aimed for agglutination assays, we then tested for cross reaction
in promoting of IgG coated RBC. IgG
from various species was individually
coated on elephant RBC and agglutination
was observed by a direct antiglobulin test
(Table 4). The results showed that rabbit
anti-EIgG cross reacted with cow IgG at a
titer of 128 compared to EIgG itself (1024).
Table 3. Cross reaction between rabbit anti-EIgG and other species’ IgG by indirect
ELISA.
Anti-EIgG
(0.14 μg/mL, 50 μL/well )
O.D. (450 nm)
% cross reaction**
IgG of other species* or BSA coated on
(10 μg/mL, 50 μL/well)
E
H
C
Hu
G
S
0.70 0.01 0.23 0.02 0.05 0.04
100 1.4 32.8 2.8
7.1
5.6
microtiter plate
D
M BSA
0.03 0.09 0.01
4.2 12.8 1.4
*E: elephant, H: Horse, C: Cow, Hu: Human, G: Goat, S: Sheep, D: Dog, M: Mouse, BSA:
Bovine serum albumin, ** Data was calculated from mean of duplicate in each run and
repeated twice
Chiang Mai J. Sci. 2013; 40(3)
383
Table 4. Cross reactivity between rabbit anti-EIgG and other species’ IgG by direct
antiglobulin test.
Other species’ IgG 400 μg/mL coated RBC*
Dilution of purified
rabbit anti-EIgG
D
S
G
C
(14 mg/mL)
Hu
H
E
neg
neg
neg
3+
undiluted
neg
4+
neg
neg
neg
neg
1:2
neg
neg
4+
3+
+
neg
neg
neg
1:4
neg
neg
4
3+
+
+
neg
neg
neg
1:8
neg
neg
3
3
neg
neg
neg
1:16
neg
neg
3+
2+
+
+
neg
neg
neg
1:32
neg
neg
2
2
neg
neg
neg
1:64
neg
neg
2+
1+
+
+
neg
neg
neg
1:128
neg
neg
2
1
neg
neg
neg
1:256
neg
neg
1+
neg
+
neg
neg
neg
1:512
neg
neg
neg
1
neg
neg
neg
1:1024
neg
neg
neg
1+
neg
neg
neg
1;2048
neg
neg
neg
neg
*E: elephant, H: Horse, C: Cow, Hu: Human, G: Goat, S: Sheep, D: Dog, M: Mouse
4. DISCUSSION
Based on human anti-globulin tests,
anti-IgG may be superior to anti-globulins,
since it can limit the occurrence of positive
results from IgM sensitized red blood cells,
which has no clinical importance [18]. We
therefore purified elephant IgG using a
Melon Gel Purification kit instead of salting
out technique. The EIgG was used as antigen
to immunize rabbit. According to the
method used in purification, the antibody
theoretically produced would be rabbit
anti-EIgG. Practically, many workers prefer
anti-IgG to anti-globulins (polyspecific) in
antibody detection and compatibility
tests because anti-IgG does not react
with complement bound to red cells by
cold-reactive antibodies that are not clinically
significant [19]. Studies of anti-human globulin
tests have indicated that weakly sensitized
red cells failed to agglutinate when neat
antiglobulin reagent was used due to the
prozone phenophenon [20]. Using diluted
antiglobulin reagent, in contrast, could
M
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
result in a positive direct antiglobulin test.
Therefore, before the assays, dilution of
anti-EIgG should be optimized. The
optimal concentration of RBC recommended
by AABB is 5% but can be varied from 25% [19]. The lower concentration may
result in agglutination interpretation. In
our study, we determined the effect of EIgG
coated RBC concentration on agglutination.
We found that all EIgG coated RBC
concentrations tested have no effect on
observation when anti-EIgG was used.
Immunoglobulin genes are conserved in
almost all animal species especially Fc or
constant region. Polyclonal antibodies
specific to one specie, thus may react with
the others at this region. In addition, once
animal was immunized with the whole
molecule of elephant’s IgG, polyclonal
antibodies were produced according to the
polyclonal B cell activation. In order to apply
anti-EIgG for other immunological test,
we also investigated the cross reactivity of
the product to other animals’ IgG. According
to a highly sensitive technique, indirect
384
ELISA, we found that the obtained
anti-EIgG could cross react with cow IgG
(32.8%). The result corresponded to the
report of Kania et al. in 1997 [21]. In this
study, the major aim was to apply the
product for direct and/or indirect
antiglobulin testing. We therefore focused on
cross reactivity in the agglutination technique.
We found that, the titer to agglutinated EIgG
coated RBC was 1024. No cross reactivity
with other animals except cow (titer 128) was
observed.
5. CONCLUSION
Taken together, the present study showed
that elephant IgG could be purified, and
rabbit anti-EIgG was produced. The product
had potency and specificity to agglutinate
EIgG coated RBC. It will be helpful in
elephant crossmatching and investigation
of HDN. Moreover, it can be used to detect
blood compatibility in Asian elephants for
breeding programs. However, before the
assay, protein concentration should be
optimized. Conjugation of the product with
specific enzymes or fluorescent dyes would
be of value for a variety of laboratory tests.
ACKNOWLEDGEMENTS
The authors thanked Assistant Professor
Dr. Chatchote Thitaram, DVM, PhD,
Elephant and Wildlife Clinic, Faculty of
Veterinary Medicine, Chiang Mai University
for his kindly provided elephants’ sera.
This project was generally supported by
Department of Medical Technology,
Faculty of Associated Medical Science,
Chiang Mai University. We also thanked
Thailand Research Fund (TRF, RMU
5080018) and National Research Council of
Thailand (NRCT, Fiscal year 2010) for some
part supporting in this work.
Chiang Mai J. Sci. 2013; 40(3)
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