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CHARACTERIZATION OF SERUM
IMMUNOGLOBULIN IN CHANNA STRIATA
(BLOCH) AND KINETICS OF ITS RESPONSE
TO AEROMONAS HYDROPHILA ANTIGEN
a a a a
P. R. Rauta , J. Mohanty , S. K. Garnayak & P. K. Sahoo
a
Central Institute of Freshwater Aquaculture, Kausalyaganga ,
Bhubaneswar , Odisha , India
Accepted author version posted online: 03 Oct 2012.Published
online: 08 May 2013.

To cite this article: P. R. Rauta , J. Mohanty , S. K. Garnayak & P. K. Sahoo (2013):

CHARACTERIZATION OF SERUM IMMUNOGLOBULIN IN CHANNA STRIATA (BLOCH) AND KINETICS OF ITS
RESPONSE TO AEROMONAS HYDROPHILA ANTIGEN, Journal of Immunoassay and Immunochemistry,
34:3, 283-293

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 Journal of Immunoassay and Immunochemistry, 34:283–293, 2013
Copyright # Taylor & Francis Group, LLC
ISSN: 1532-1819 print/1532-4230 online
DOI: 10.1080/15321819.2012.725445

CHARACTERIZATION OF SERUM IMMUNOGLOBULIN IN

CHANNA STRIATA (BLOCH) AND KINETICS OF ITS RESPONSE
TO AEROMONAS HYDROPHILA ANTIGEN
Downloaded by [University of California, San Diego] at 16:34 13 May 2013

P. R. Rauta, J. Mohanty, S. K. Garnayak, and P. K. Sahoo

Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar,
Odisha, India

& The immunoglobulin (Ig) from the serum of Channa striata was isolated by gel electroelution
and characterized further to understand its nature and subsequent applications in studying the
immune response. The purity of the sample was confirmed with the presence of a single band on
native gradient PAGE and the molecular weight of
897 kDa was determined from the gel. In
SDS-PAGE, C. striata Ig was reduced to produce two bands corresponding to H (heavy)
(
72 kDa) and L (light) (
27 kDa) chain subunits. Polyclonal antiserum against the purified
Ig was raised in a rabbit and adsorbed with 10% liver tissue homogenate of C. striata to enhance
its specificity. By an indirect ELISA standardized using the adsorbed rabbit antiserum, the normal
serum Ig concentration in C. striata was estimated to be 3.48 mg=mL. Further, a kinetic study of
specific immunoglobulin response to formalin-killed Aeromonas hydrophila antigen was under-
taken using another indirect ELISA, which showed a significant increase in serum immunoglob-
ulin titer from day 2 onwards and reached its peak at day 14. Subsequently, the Ig titer was
dropped from day 21 onwards till the completion of the experiment at day 42, although it was
at a significantly higher level than the control.

Keywords Aeromonas hydrophila, Channa striata, ELISA, immunoglobulin, kinetics

INTRODUCTION
Like mammals, humoral immunity in fish in response to pathogens
involves the secretion of specific immunoglobulins directed to neutralise
antigens and to activate complement cascade.[1] The IgM is the most
important and predominant class of immunoglobulin in fish;[2–4] although
other isotypes such as IgD and IgT=IgZ have also been reported.[5] IgM
molecules in general are tetrameric in teleosts and pentameric in elasmo-
branchs.[6,7] Thus, teleost tetrameric IgM consists of 8 light (L) and 8 heavy

Address correspondence to P. K. Sahoo, Fish Health Management Division, Central Institute of

Freshwater Aquaculture, Kausalyaganga, Bhubaneswar-751002, India. E-mail: pksahoo1@hotmail.com
 284 P. R. Rauta et al.

(H) chains and with a molecular weight (MW) of 600–1000 kDa, depending
on the species of the fish.[8,9] The clear understanding of fish immune sys-
tem is very much essential for the study of host pathogen interaction, which
results in effective management of diseases in aquaculture.[10]
Channa striata, commonly known as striped murrel, is a native fresh-
water fish of tropical Africa and Asia.[11] In India, this species is highly
popular as food fish in many parts of the country.[12] Besides the high
quality of their flesh in terms of taste and texture, they also have good
market value due to the low fat, fewer intramuscular spines and medicinal
qualities.[13] By virtue of its preferential habitat in bottom zones of swampy
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waters, where the bacterial population may be 10–20 times higher

than in the water column,[14] it might be at a higher risk of getting
infected during culture. It is also one of the most susceptible species to
epizootic ulcerative syndrome (EUS) showing large scale ulcerations and
mortality.[15]
The bacterium, Aeromonas hydrophila, is of scientific and economic
interest because of its pathogenicity to human and fishes.[16,17] A. hydrophila
infections in fishes have been reported from time to time in many Asian
countries including China, Philippines, Thailand, and India.[18]
In the present article, C. striata immunoglobulin (Ig) was purified and
characterized. Rabbit polyclonal antiserum to channa Ig was developed
and used in an indirect ELISA to quantitate the normal serum immuno-
globulin concentration in C. striata. Further, a kinetic study of specific
immunoglobulin to formalin-killed A. hydrophila antigen was undertaken
using another indirect ELISA that would help in assessing the impact of
vaccination or pathogenic microorganisms on C. striata health.

MATERIALS AND METHODS

Maintenance of Fish and Collection of Serum
Apparently, healthy C. striata (150  50 g; 30 numbers) were obtained
from the farm of the Central Institute of Freshwater Aquaculture, Bhuba-
neswar, India. The fishes were stocked in 500 L ferro-cement tanks with aer-
ated freshwater in a wet laboratory and acclimatized for 15 days before
starting the experiment. They were fed with a standard diet in two divided
doses daily during the experiment. Partial water exchange was carried out
daily to remove waste feed and faecal matter.
Blood was collected from all fishes by bleeding the animals through
caudal vein and allowed to clot at room temperature. Serum samples were
obtained by centrifuging the clotted blood at 3000 rpm for 5 min at 4 C,
pooled, aliquoted and preserved at 20 C for further use.
 Ig Characterization and Response Kinetics in C. striata 285

Purification of Immunoglobulin
The pooled serum was allowed to thaw at room temperature and centri-
fuged (6000 rpm=5 min) to remove any precipitate. The serum was run in a
preparative native gradient PAGE (3–12% polyacrylamide conc.) at 150 V
overnight. Considering Ig as the largest molecular size protein in fish serum,
the upper most band (
900 kDa) was excised from the gel and the protein
was eluted from the gel using an electro eluter (Model 422, Biorad, Hercules,
CA, USA). The protein concentration was determined following Bradford.[19]
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Characterization of Immunoglobulin
Determination of the purity and molecular weight of Channa Ig was
performed by a native gradient PAGE of 2.8–22.5% acrylamide concen-
tration.[20] The gel was run at 200 V in TBE (0.09M Tris, 0.08 M boric acid,
0.0025 M Na2 EDTA, pH 8.4) buffer for 10 h at 4 C. Gels were stained with
0.025% coomassie brilliant blue (R-250). Estimation of molecular weight of
the Ig band in comparison to molecular weight standards was made from
four samples using AlphaEase1FC Imaging Software (Alpha Innotech
Corp., San Leandro, CA, USA) and expressed as mean standard error.
The types of Ig subunits and their molecular weights were determined
by SDS-PAGE.[21] Samples and molecular weight markers were reduced by
boiling in sample buffer containing 5% (v=v) 2-mercaptoethanol and were
loaded into wells of a stacking gel of 5% above the separating gel of 10%
acrylamide. Loaded samples were electrophoresed at 200 V for 45 min.
The gels were stained and the molecular weights of the bands were deter-
mined as described earlier.

Development of Rabbit Polyclonal Antiserum

Polyclonal antiserum to purified immunoglobulin was prepared in an
one-year-old New Zealand white rabbit using standard immunization proto-
cols. C. striata purified Ig (
200 mg) was emulsified in an equal volume of
Freund’s complete adjuvant (FCA) and injected to the rabbit intramuscu-
larly, followed by 2 booster doses (with Freund’s incomplete adjuvant) at
day 14 and 28. After 14 days of the last injection, the 
rabbit was bled.
The separated sera was aliquoted and preserved at 20 C for future use.
The specificity of the rabbit antiserum to Channa Ig was checked in
western blotting.
Channa serum and purified Ig were electrotransferred to PVDF mem-
brane from SDS-PAGE gel. Transfer success was assessed by staining of one
part of PVDF membrane with amido black. The other replicate part of PVDF
 286 P. R. Rauta et al.

membrane was immunostained. The reagents used in immunostaining were

added in the following sequence: 5% skimmed milk powder, rabbit
antiserum, and anti-rabbit conjugate (goat anti-rabbit IgG conjugated with
alkaline phosphatase; Genei, Bangalore, India). The membrane was incu-
bated for one hour in each reagent and washed three times with TBST
(TBS with 0.05% Tween 20) after each incubation step. After final washing,
the PVDF membrane was treated with the substrate, BCIP=NBT (Genei)
for approximately 3 min for the color to develop. The membrane was washed
with distilled water, dried, and preserved for photography. The rabbit anti-
serum was found to cross react with many unrelated bands in channa serum
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and hence, was adsorbed with 10% channa liver tissue homogenate at 1:1 pro-
portion. The specificity of the adsorbed antiserum was checked as above.

Quantification of Normal Serum Immunoglobulin

Concentration in C. striata
An indirect ELISA assay was performed to determine the normal serum
Ig concentration in C. striata following the protocol of Behera et al.[22] The
standard curve was made using purified channa Ig. Briefly, different dilu-
tions of purified channa Ig (two-fold dilution from 2 mg=mL up to 10th
dilution) in 100 mL volume were incubated overnight at 4 C in duplicate
wells of polystyrene ELISA plates. The wells were washed thrice with TBST
(TBS with 0.05% Tween 20). After blocking remaining sites with 5% skim
milk powder for 2 hr, 100 mL of a 1:80,000 dilution of the adsorbed rabbit
antiserum was added to each well for 60 min at 25 C. The wells were care-
fully washed with TBST, and 100 mL of HRP-labeled secondary antibody
(goat anti-rabbit; Genei), diluted 1:15,000 in TBST was added for 60 min.
The wells were washed again with TBST, and the reaction visualized after
adding 100 mL=well of TMB=H2O2 (Genei). The reaction was allowed to
proceed for 10 min, stopped with 100 mL 1 N sulphuric acid, and absor-
bance was read at 450 nm with an automatic plate reader (Imax Microplate
Reader, Biorad). Thirty individual channa serum samples were assayed in
duplicate wells at a pre-determined dilution (1:64,000) and the immuno-
globulin concentrations were determined from the standard curve. The
relative Ig concentration was expressed as mean SE.

Kinetics of Immunoglobulin Response in C. striata to A.

hydrophila Antigen
A kinetic study of specific immunoglobulin titer in C. striata to
formalin-killed A. hydrophila antigen was undertaken using an indirect
ELISA following Li et al.[23] Apparently, healthy C. striata fishes were
 Ig Characterization and Response Kinetics in C. striata 287

injected with formalin-killed A. hydrophila (109 cfu=fish) suspended in PBS

and emulsified with equal volume of Freund’s complete adjuvant (FCA).
The immunized fishes were distributed into 3 replicate groups and blood
samples were collected at different time intervals i.e., 0, 2, 4, 6, 10, 14,
21, 28, 35, and 42 days. The separated sera were used in the ELISA to deter-
mine the specific immunoglobulin titer. For ELISA, the 96-well polystyrene
ELISA plate was coated with formalin-killed A. hydrophila whole cell suspen-
sion (109cells=well) to which C. striata serum (1:20,000) was added. Then
the adsorbed rabbit antiserum to C. striata immunoglobulin (1:40,000)
was added followed by HRP conjugated anti-rabbit antibody (Genei). After
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treatment with the substrate (TMB=H2O2), the reaction was stopped with
H2SO4 and the color reaction was read on an ELISA plate reader at
450 nm. The immunoglobulin titer was expressed as P=N values, where P
represents the OD450 of immunized sera samples of different time periods
and N, the OD450 of control (0 day). A kinetic curve was plotted with time
period vs. P=N value and one-way ANOVA followed by DMRT was per-
formed to find out the significant differences among the immunoglobulin
titers at different time intervals using SPSS18 software.

RESULTS AND DISCUSSION

The C. striata immunoglobulin was purified in the present study by a sim-
ple process of electroelution from electrophoresed native gel considering
IgM as the largest molecular sized protein in fish serum. The purified sample
run in native gradient PAGE, showed only one band that indicated the purity.
Such electroelution process has also been adopted for purification of
immunoglobulin in Paralichthys olivaceus[24] or subunit chains of immuno-
globulin in Acipenser baeri[25] and Trematomus bernacchii.[26] Immunoglobulin
from C. striata has also been purified by different researchers before.[27,28]
The tetrameric serum Ig with native molecular mass ranging from 700
and 1000 kDa has been reported in majority of the teleostean species.[9]
The molecular weight of C. striata Ig molecule was determined in native
gradient PAGE to be 896.93  7.32 kDa (Figure 1a). However, the molecu-
lar weight of C. striata Ig was reported earlier as 820 kDa[28] and
670 kDa.[27] These variations of result might be due to the use of different
techniques for estimation of molecular weight. Such variations in molecu-
lar size of teleostean immunoglobulin have been reported for some fish
species such as Sparus aurata,[29,30] Lates calcarifer,[31,32] and Cyprinus
carpio.[33,34] Based on the high molecular weight, the C. striata Ig could
be considered as IgM type, like in other teleostean species.
In SDS-PAGE, the C. striata Ig was reduced to produce two bands corre-
sponding to Ig H (heavy) and L (light) chain subunits (Figure 1b). The
 288 P. R. Rauta et al.
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FIGURE 1 Analysis of C. striata Ig in (a) native gradient PAGE (Lane 1- channa Ig; Lane 2- molecular
weight marker) and (b) SDS-PAGE (Lane 1- Reduced channa Ig; Lane 2- molecular weight marker).

H chain subunits possessed molecular weight of approximately

72.27  0.25 kDa and light chains of 27.15  0.28 kDa. In the earlier studies
of C. striata Ig, molecular weights of heavy chain and light chain were esti-
mated as 67 kDa and 29 kDa,[27] and 70.4 kDa and 25.3 kDa,[28] respectively.
Similar variations of results for the molecular weights of heavy and light
chains have also been observed for common carp, C. carpio.[34,35]
The successful transfer of protein bands from SDS-PAGE gel could be
assessed by amido black staining of the blot (Figure 2a). In immunostain-
ing of the blot with the rabbit anti-channa Ig serum, both H and L chains
of reduced channa Ig were found to be stained. However, with the reduced
channa serum the rabbit antiserum cross-reacted with several other bands
additionally. So, the rabbit antisera was adsorbed with 10% channa liver
tissue homogenate with an objective to remove the cross reacting anti-
bodies. The adsorbed antiserum was then reacted only with the H chain
band of channa Ig (Figure 2b) with increased specificity. The adsorbed
antiserum in the present investigation was used in our ELISA tests later.
Studying the normal serum immunoglobulin concentration is impor-
tant in understanding fish physiology and pathology.[36] An indirect ELISA
test was standardized with the rabbit anti-channa Ig serum to quantify the
normal serum Ig concentration in C. striata. The parallel sigmoid curves
obtained with both purified channa Ig and normal channa serum indicated
that the test could be used to quantify Ig level in normal channa serum
(Figure 3). ELISA has been used by other workers to measure the serum
Ig concentrations in fish.[22,37]
 Ig Characterization and Response Kinetics in C. striata 289
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FIGURE 2 Analysis of C. striata Ig in western blot. (a) Amido black staining (Lane 1. molecular weight
marker; Lane 2. channa Ig; Lane 3. channa serum); (b) Immunostaining (Lane 1. channa Ig; Lane 2.
channa serum) (color figure available online).

In teleosts, the serum Ig concentration has been shown to vary between

2–7 mg=mL.[38] In the present study, the relative Ig concentration in C.
striata was estimated to be 3.48  0.41 mg=mL. These results were expressed
as the mean of duplicate determinations for each sample to which the
blank values were subtracted. Similar serum Ig concentrations have been
reported in other fish species such as Clarias batrachus (3.5 mg=mL)[20]
and Oncorhynchus gairdneri (3.3 mg=mL).[39]
Vaccination seems to be an important strategy in the control of the dis-
eases caused by A. hydropila among farmed fish.[40] Antibody kinetic study is
very much essential to study the impact of vaccination or the immune
response to infection with pathogenic microorganisms. The kinetics of spe-
cific antibody response has been studied by performing ELISA earlier.[23,41]
In the current study, ELISA test showed a significant increase in immuno-
globulin titer from day 2 onwards and such early rise in titer possibly indi-
cates the previous exposure of the animals to environmental A. hydrophila
or related organisms. The titer reached the peak at day 14 (P=N ¼ 2.69),
which is significantly higher compared to the titers of all other time inter-
vals. Subsequently, the immunoglobulin titer was dropped from day 21 (P=
N ¼ 1.8) until the completion of the experiment at day 42 (P=N ¼ 1.53),
even then it was at significantly higher level than the control on that day
 290 P. R. Rauta et al.
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FIGURE 3 Standard curve used for quantitation of immunoglobulin in C. striata serum by indirect
ELISA (color figure available online).

FIGURE 4 Changes in C. striata Ig titers over time following A. hydrophila immunizations. Mean value
bearing same superscript are not statistically significant, p > 0.05.
 Ig Characterization and Response Kinetics in C. striata 291

(Figure 4). Similarly, the kinetics of antibody production in mucus and

serum of European eel (Anguilla anguilla L.) after vaccination against Vibrio
vulnificus was studied by ELISA earlier.[41] Understanding this immuno-
globulin kinetics would be helpful in designing the vaccination strategy
for C. striata.

CONCLUSION
The snakehead murrel, C. striata (Bloch) Ig, could be isolated to hom-
ogeneity by electroelution and it could be proved that the C. striata pos-
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sesses only one type of high molecular weight tetrameric Ig similar to Ig

of other teleosts. The normal Ig concentration in C. striata was estimated
through an indirect ELISA to be 3.48  0.41 mg=mL. The kinetic study of
specific immunoglobulin response to formalin-killed A. hydrophila antigen
was undertaken using another indirect ELISA that may find applications
in studying the impact of vaccination in C. striata.

ACKNOWLEDGMENTS
The authors are thankful to the Director, CIFA, for providing necessary
facilities during the study. Thanks are also due to Dr. K.M. Shankar, CCPI of
NAIP funded project and Dean, College of Fisheries, Mangalore, India, for
his suggestions and guidance to carry out this work. Funding support from
NAIP (ICAR, New Delhi) is gratefully acknowledged.

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