Fasciola gigantica: production and characterization of a monoclonal antibody against recombinant cathepsin B3.

Abstract

A number of monoclonal antibodies (MoAbs) against a recombinant cathepsin B3 (rCatB3) of Fasciola gigantica were produced in BALB/c mice. Reactivity and specificity of these MoAbs were assessed by indirect ELISA and immunoblotting techniques. Six stable clones, namely 1C4, 1E9, 2E5, 2F9, 5B4, 5D7 were obtained. All MoAbs reacted with rCatB3 at molecular weight (MW) 37 kDa as well as the glycosylated peptide at 55-75 kDa and with the native CatB3 at MW 37 kDa in WB extracts of metacercariae (Met) and newly excysted juveniles (NEJ). It was found to be IgG(1) and λ light chain isotypes. Immunolocalization of CatB3 in metacercariae, NEJ, 4-week-old juvenile and adult F. gigantica performed by immunoperoxidase technique by using these MoAbs as probes indicated that CatB3 was present in high concentration in the caecal epithelium and caecal lumen of the Met and NEJ, but not in the 4-week-old juvenile and adult fluke. The MoAbs show no cross-reactions with antigens of other parasites including Gigantocotyl explanatum, Eurytrema pancreaticum, Paramphistomum cervi, Schistosoma spindale, S. mansoni, Haemonchus placei and Setaria labiato-papillosa. Thus, it is possible that these MoAbs could be a good candidate for immunodiagnosis of fasciolosis.

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Fasciola gigantica: Production and characterization of a monoclonal antibody
against recombinant cathepsin B3
Panat Anuracpreedaa,b, Sineenart Songkoomkrongb, Manussabhorn Sethadavitc,
Charoonroj Chotwiwatthanakunb,d, Yotsawan Tinikulb,d, Prasert Sobhonb,⇑
aAgricultural Science Division, Mahidol University, Kanchanaburi Campus, Saiyok, Kanchanaburi 71150, Thailand
bDepartment of Anatomy, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand
cDepartment of Anatomy, Faculty of Medicine, Chiang Mai University, Chiang Mai 52000, Thailand
dMahidol University, Nakhonsawan Campus, Nakhonsawan 60000, Thailand
a r t i c l e i n f o
Article history:
Received 8 July 2010
Received in revised form 11 August 2010
Accepted 12 August 2010
Available online 22 August 2010
Keywords:
Fasciola gigantica
Juvenile fluke
Cathepsin B3
Recombinant protein
Monoclonal antibody
Immunolocalization
Cross-reaction
a b s t r a c t
Anumberofmonoclonalantibodies(MoAbs)againstarecombinantcathepsinB3(rCatB3)ofFasciolagigan-
tica were produced in BALB/c mice. Reactivity and specificity of these MoAbs were assessed by indirect
ELISA and immunoblotting techniques. Six stable clones, namely 1C4, 1E9, 2E5, 2F9, 5B4, 5D7 were
obtained.AllMoAbsreactedwithrCatB3atmolecularweight(MW)37 kDaaswellastheglycosylatedpep-
tideat55–75 kDaandwiththenativeCatB3atMW37 kDainWBextractsofmetacercariae(Met)andnewly
excysted juveniles (NEJ). It was found to be IgG1and k light chain isotypes. Immunolocalization of CatB3 in
metacercariae, NEJ, 4-week-old juvenile and adult F. gigantica performed by immunoperoxidase technique
by using these MoAbs as probes indicated that CatB3 was present in high concentration in the caecal epi-
theliumandcaecallumenoftheMetandNEJ,butnotinthe4-week-oldjuvenileandadultfluke.TheMoAbs
shownocross-reactionswithantigensofotherparasitesincludingGigantocotylexplanatum,Eurytremapan-
creaticum, Paramphistomum cervi, Schistosoma spindale, S. mansoni, Haemonchus placei and Setaria labiato-
papillosa.Thus,itispossiblethattheseMoAbscouldbeagoodcandidateforimmunodiagnosisoffasciolosis.
? 2010 Elsevier Inc. All rights reserved.
1. Introduction
Fasciolosis is an economically important disease caused by
Fasciolasp.infectioninruminants,andisestimatedtocauseaworld-
wide loss to the livestock industry at more than 3 billion US dollars
per annum (Spithill et al., 1997; Sobhon et al., 1998). Fasciola gigan-
tica is the most common Fasciola species infecting ruminants in the
tropical parts of Asia and Africa. In these liver flukes, cathepsin B
(CatB)isamajorprotease,butitislessstudiedintermsofcharacter-
istics and functions in comparison to other proteases. The cathepsin
BfromF.hepaticawasfirstclonedbyWilsonetal.(1998)andnamed
FhCatB. This enzyme has been shown to be the most abundant pro-
tease secreted by the newly excysted juvenile (NEJ) (Wilson et al.,
1998; Tkalcevic et al., 1995), while the adult parasites expressed
mainly the cathepsin L (Dalton and Heffernan, 1989; Smith et al.,
1993). The excretory/secretory extracts from F. hepatica NEJ have
been shown to cleave the bovine serum albumin and immunoglob-
ulin, but not bovine haemoglobin. Hence, it was suggested that pos-
sible functions of CatB in Fasciola sp. may be in the excystment of
metacercariae,andthemigrationofjuvenileflukesthroughthehost
intestinal wall and tissues (Wilson et al., 1998). In F. gigantica, Mee-
mon et al. (2004) had cloned cathepsin B cDNAs from adult, newly
excysted juvenile (NEJ), and metacercarial stages and named them
FGcatB1,FGcatB2,andFGcatB3.FGcatB1transcriptsweredetected
in all stages, whereas FG catB2 and FG catB3 transcripts were ex-
pressed in metacercariae, NEJ, and juvenile parasites only. The
switching off of the catB2 and catB3 genes during the maturation
of the parasites implicates that these enzymes may be used by the
juvenile stages to digest host tissues during their penetration and
migration to the liver; whereas catB1, which is present in all stages,
may perform general digestive function. The isolation and purifica-
tion of isotypes of the native cathepsin B is very difficult, hence our
group had produced recombinant cathepsin B3 protease (CatB3)
using molecular cloning method (Sethadavit et al., 2009). Since this
proteinisreleasedintothehostfluidinafairlylargeamount,itcould
be a good candidate for immunodiagnosis of fasciolosis by F. gigan-
tica, especially the early phase of infection. In the present study, the
monoclonal antibodies (MoAbs) against recombinant F. gigantica
cathepsin B3 (rFgCatB3) were produced and characterized for their
bindingwith both the recombinant and native CatB3 in various par-
asite’stissuesatvariousstagesofthelifecycle.Thecrossreactivities
with other trematode and nematode parasite antigens were also
tested in order to verify their specificity for possible applications
in immunodiagnosis.
0014-4894/$ - see front matter ? 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.exppara.2010.08.012
⇑Corresponding author. Fax: +66 0 2354 7168.
E-mail addresses: Panat1@yahoo.com, kapanat@mahidol.ac.th (P. Anuracpreeda),
scpso@mahidol.ac.th (P. Sobhon).
Experimental Parasitology 127 (2011) 340–345
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2. Materials and methods
2.1. Collection of parasites
2.1.1. Metacercariae (Met)
The method described by Anuracpreeda et al. (2009a) was used
to obtain metacercariae of F. gigantica. Briefly, the Lymnaea ollula
snails were infected with miracidia and allowed to develop spo-
rocysts and cercariae. On day 45 after infection, the cercariae were
shed from the snails and settled on the 5 ? 5 cm cellophane paper
as metacercariae. The metacercariae were brushed off and col-
lected from the cellophane paper and washed several times with
0.85% NaCl solution and used immediately.
2.1.2. Newly excysted juveniles (NEJ)
Metacercariae were excysted by incubation in distilled water
containing 1% pepsin and 0.4% HCl at 37 ?C for 45 min, and washed
with distilled water. They were subsequently resuspended in
0.02 M sodium dithionite, 0.2% taurocholic acid, 1% NaHCO3and
0.8% NaCl in water. Then, 0.005% HCl was added to generate CO2
gas. Specimens were incubated at 37 ?C for 45 min and washed
with distilled water. The activated metacercariae were transferred
to fresh RPMI-1640 medium containing 10 lg/ml gentamycin, and
10% fetal calf serum, and incubated at 37 ?C overnight. On the fol-
lowing day, the newly excysted juveniles (NEJ) were collected and
washed several times with 0.85% NaCl solution and used immedi-
ately (Anuracpreeda et al., 2009a).
2.1.3. Four-week-juvenile parasites (4 wk)
Juvenile F. gigantica were obtained from infected male Golden
Syrian hamsters according to a method described by Anuracpreeda
et al. (2009a). The juvenile parasites were obtained by sacrificing
the infected animals at 7 weeks after infections, and teasing the li-
ver to collect the parasites. The specimens were washed several
times with 0.85% NaC1 and used immediately.
2.1.4. Adult parasites
Adult F. gigantica were collected from the bile ducts and gall
bladders of infected cattle or water buffaloes killed at slaughter-
houses. For cross-reactivity study, other trematodes (Gigantocotyl
explanatum, Eurytrema pancreaticum, Paramphistomum cervi and
Schistosoma spindale) and nematode parasites (Haemonchus placei
and Setaria labiato-papillosa) were also collected from the same
group of animals. Adult Schistosoma mansoni were obtained by per-
fusing mice, 8 weeks after infection with schistosome cercariae. All
parasite samples were washed several times with 0.85% NaCl solu-
tion to remove the host blood, bile and contaminating microorgan-
isms before being processed for further experiments.
2.2. Preparation of antigens
2.2.1. Whole body (WB) antigens of adult and juvenile (JP) parasites
The method described by Anuracpreeda et al. (2009b) was used
to obtain WB antigens. Briefly, whole parasites (all stages of
F. gigantica and other species) were homogenized in lysis buffer
(10 mM Tris–HCl, pH 7.2, 150 mM NaCl, 0.5% Triton X-100, 1 mM
EDTA and 1 mM PMSF) and rotated at 4 ?C for 1 h. The suspensions
were centrifuged at 5000g, 4 ?C, for 20 min to get rid of the eggs,
and the supernatants were collected. Protein concentration was
determined by Lowry’s method (Lowry et al., 1951). These extracts
were stored at ?70 ?C until use in subsequent experiments.
2.2.2. Tegumental antigens (TA) of adult F. gigantica
TA were obtained by extraction from live adult parasites with
1% Triton X-100 in 0.05 M Tris buffer, pH 8.0 containing 0.01 M
EDTA, 0.15 M NaCl for 20 min at room temperature according to
the method described by Anuracpreeda et al. (2006). The extract-
ing solution was collected and centrifuged at 5000g for 20 min at
4 ?C to remove the contaminating eggs. The supernatant containing
soluble TA was collected and dialyzed against 0.01 M phosphate
buffered saline (PBS) overnight at 4 ?C, using Spectra/Por dialysis
membrane (Spectrum Medical Industries, Los Angeles, California,
USA) with molecular weight cut off at 6–8 kDa. The protein con-
centration was measured by Lowry’s method and stored at
?70 ?C until used in later experiments.
2.2.3. Excretory–secretory (ES) antigens of adult F. gigantica
The method described by Anuracpreeda et al. (2009b) was used
to obtain ES antigens. Briefly, ES antigens were prepared by incu-
bating freshly collected, living adult F. gigantica in RPMI-1640
media for 3 h at 37 ?C. At the end of the incubation, the parasites’
eggs in the culture medium was removed by centrifugation at
5000g for 20 min at 4 ?C. The supernatant was then filtered
through 0.22 lm Millipore filter. The protein concentration was
determined by Lowry’s method and the protein solution was
stored at ?70 ?C until further use.
2.3. Preparation of recombinant FgCatB3
Recombinant F. gigantica cathepsin B3 protease (rFgCatB3) was
cloned by PCR screening of a cDNA library (Sethadavit et al., 2009).
The PCR products from tertiary screen were further subcloned into
pGEM?-T Easy vector (Promega) and the sequence confirmed by
DNA sequence analysis. The proFgCatB3 cDNA was generated and
subcloned into the pPICZaA vector using the primer pairs 50-GC
TGAAGCTGAATTCAAGCCAAACTAC-30and 50-ATCTTGGTAGACGCGG
CCGCAGGTAATCCGGC-30containing the EcoRI and NotI restriction
endonuclease sites (underlined), respectively, using Platinum?Pfx
DNA polymerase (Invitrogen). The sequence was confirmed by
DNA sequence analysis. The pPICZaA containing proFgCatB3 gene
was linearized with SacI then electroporated into Pichia pastoris
GS115. Genomic integration was confirmed by screening genomic
DNA by direct PCR using 5_AOX1 and 3_AOX1 primers (Linder
et al., 1996). Recombinant FgCatB3 (rFgCatB3) was expressed in
Pichia pastoris and purified by Ni2+-NTA affinity chromatography
(QIAGEN).
2.4. Production of monoclonal antibodies (MoAbs) against rFgCatB3
Six inbred female BALB/c mice 6–8-weeks-old were immunized
according to the method described by Anuracpreeda et al. (2006).
Briefly, the animals were primed by subcutaneous injection on
the back region with 25 lg rFgCatB3 in 100 ll PBS solution emul-
sified with an equal volume of complete Freund’s adjuvant (Sigma–
Aldrich Inc., Saint Louis, MO, USA). Three weeks later, the animals
were given a booster injection of 25 lg rFgCatB3 in PBS emulsified
in incomplete Freund’s adjuvant (Sigma–Aldrich Inc.) via the same
route. Three days before blood collection, the final boosting with
25 lg rFgCatB3 was given by the intravenous route without adju-
vant. The mice were bled, and the polyclonal antibody (PoAb) titer
in the antisera was tested by indirect ELISA against rFgCatB3. The
hybridoma clones expressing MoAb against rFgCatB3 were pro-
duced by the fusion of immunized spleen cells and mouse myelo-
ma cells (P3x63-Ag8.653). The hybridoma cells that grew
successfully in culture were cloned by limiting dilution methods.
Only the hybridoma clones that produced high titers of antibodies
against rFgCatB3, as screened by indirect ELISA, were selected. The
antibody isotypes were determined by ELISA using the SBA Clono-
typing™ System/HRP (SouthernBiotech, USA). All animal experi-
ments were approved by the Animal Care and Use Committee,
Faculty of Science, Mahidol University, Thailand.
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2.5. Immunoblotting detection
SDS–PAGE and immunoblotting were performed as described
by Anuracpreeda et al. (2008). Briefly, metacercariae, NEJ, JP, ES,
TA, and WB antigens of F. gigantica were separated using 12.5%
SDS–PAGE and transferred onto nitrocellulose membranes for
immunodetection by the selected MoAb. Cattle infected sera (CIS)
obtained from the pooled sera of the naturally infected animals
were used as positive controls. For negative controls, the myeloma
culture fluid (CF) and normal mouse serum (NMS) were used as
probes. Cattle antibodies that reacted with the antigenic molecules
were detected by peroxidase-conjugated rabbit anti-bovine immu-
noglobulin, while the monoclonal and polyclonal antibody–antigen
complexes were detected by peroxidase-conjugated goat anti-
mouse IgG. The reaction was visualized by further incubation in
a specific substrate 3,30,5,50-tetramethyl benzidine (TMB) until
the bands appeared. Finally, the reaction was stopped by adding
distilled water.
2.6. Immunolocalization of CatB3
Six MoAbs were used for the analysis of the distribution and rel-
ative concentration of CatB3 in the paraffin sections of F. gigantica
at each developmental stages (metacercariae, NEJ, 4-week-juvenile
and adult) by using immunoperoxidase techniques according to
the method described by Anuracpreeda et al. (2009a). Briefly, the
endogenous peroxidase in the tissues was quenched by treatment
with 3% H2O2diluted in tap water for 30 min, and washed three
times with 10 mM PBS containing 0.1% Tween-20 for 5 min each.
Then, non-specific binding was blocked by incubating sections in
0.1% glycine in 100 mM PBS pH 7.4 and 4% BSA in 100 mM PBS
pH 7.4, for 5 min and 2 h, respectively. Following the blocking step,
the sections were incubated for 1 h at room temperature in specific
MoAbs against rFgCatB3 and washed three times for 5 min each.
Subsequently, the sections were incubated with biotinylated goat
anti-mouse IgG (Zymed Laboratory Inc., South San Francisco, CA,
USA), at 1:200 dilution, at room temperature for 30 min. After
washing three times with the same buffer, they were incubated
with HRP-conjugated streptavidin (Zymed Laboratory Inc.), at
1:200 dilution, at room temperature for 30 min, and then washed.
Finally, the sections were immersed in the AEC (3-amino-9-ethylc-
arbazole) substrate solution (Zymed Laboratory Inc.) in a dark-
room. The reaction was stopped by immersing in tap water and
the stained sections were mounted in permount before being ob-
served under a Nikon E600 light microscope equipped with a
DXM 1200F digital camera.
3. Results
3.1. Monoclonal antibodies (MoAbs) against rFgCatB3
Six stable clones of MoAb, designated 1C4, 1E9, 2E5, 2F9, 5B4,
5D7, were selected and expanded in culture flasks to obtain large
volume of MoAb which were then collected for further experi-
ments. All MoAbs exhibited specific binding to the rFgCatB3 bands.
The immunoglobulin classes and sub-isotypes of all MoAb selected
were found to be IgG1and k light chain. Clone 2F9 had the highest
titer (up to 3.25 in ELISA OD reading at 450 nm with the cut off
point at 0.12).
3.2. Immunoblotting detection
The immunoblotting experiment showed that all MoAbs re-
acted with two bands of rFgCatB3 which have molecular weight
(MW) of 37 kDa and 55–75 kDa (Fig. 1). However, when tested
against WB antigens in each developmental stages (metacercariae,
NEJ, 4-week-old juvenile and adult) including TA and ES antigens
from adult F. gigantica, these MoAbs reacted with native CatB3
which appeared as a single band at MW 37 kDa in WB extracts of
metacercariae and NEJ, but no positive band was detected in TA,
ES, and WB extracts of 4-week-old juvenile or of adult parasites
(Fig. 2A). When similar antigenic fractions were analyzed with
PoAbs against native CatB3, the positive band was only observed
at MW 37 kDa in WB extracts of metacercariae as well as at MW
37 kDa and 34 kDa in those of NEJ (Fig. 2B) which confirmed that
the proteins detected by MoAb were CatB3.
In the cross-reactivity study, no positive band was detected in
WB antigens from five trematode parasites (G. explanatum, E. pan-
creaticum, P. cervi, S. spindale and S. mansoni) and from two nema-
tode parasites (Haemonchus placei and S. labiato-papillosa), while
the band was very prominent in WB of F. gigantica NEJ (Fig. 3).
3.3. Immunolocalization
The distribution of cathepsin B3 was examined in each develop-
ment stages of the F. gigantica (i.e., metacercariae, NEJ, 4-week-old
juvenile and adult parasites) by immunoperoxidase staining using
MoAbs. Since all MoAbs exhibited similar immunoperoxidase
staining characteristics, the data obtained from clone 2F9, which
exhibited the strongest reaction, were used as representatives of
the group. The sites and intensities of the reddish reaction products
indicated the location and relative concentration of CatB3 that
were bound to the MoAb.
In metacercariae, specific immunostaining was observed in
both caecal epithelium and in the lumen of the caecum, while
the tegument and parenchymal cells were not stained (Fig. 4C).
In NEJ, the intense immunostaining was also observed in both cae-
cal epithelium and in the lumen of the caecum, while the tegu-
ment, muscle and parenchymal cells were not stained (Figs. 4D
and E). At a higher magnification of a NEJ section, intense reddish
immunostaining was detected in both caecal epithelium and in the
lumen of the caecum (Figs. 4D and F). Consistent with the immu-
oblotting analysis, there was no immunostaining in 4-week-old
juvenile and adult parasite tissues (Figs. 4G and H). The negative
control sections of the parasite tissues stained with myeloma
Fig. 1. Immunoblot analysis of the recombinant F. gigantica cathepsin B3 (rFgCatB3)
reacted with myeloma culture fluid-CF (lane 1), normal mouse serum-NMS (lane 2),
cattle infected serum-CIS (lane 3), MoAb 1C4 (lane 4), 1E9 (lane 5), 2E5 (lane 6), 2F9
(lane 7), 5B4 (lane 8) and 5D7 (lane 9). STD is the lane containing standard protein
molecular weight markers.
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culture fluid, showed no reddish staining in any of the parasite
tissues (Figs. 4A and B).
4. Discussion
The full length F. hepatica cathepsin B (FhCatB) cDNA has re-
cently been cloned and characterized by Law et al. (2003), showing
the open reading frame encoding preprocathepsin B with 339 ami-
no acids. The recombinant FhCatB protease had been expressed in
the yeast Saccharomyces cerevisiae, and the purified protein exhib-
ited a molecular weight (MW) of 38 kDa. Following the self-pro-
cessing of the recombinant proFhCatB, two peptides at MW 32
and 28 kDa could be detected. N-terminal sequencing of these
two proteins indicated that 45 amino acids of the proregion was
lost from the 32 kDa peptide, and eight amino acids of the mature
sequence was lost from the 28 kDa peptide. Antibodies raised
against recombinant FhCatB could detect 36 and 31 kDa native
peptides in the juvenile ES material, which are believed to be
proFhCatB and mature FhCatB, respectively. Therefore, the recom-
binant proFhCatB might be improperly self-cleaved. In F. gigantica,
the full length ORF of the cathepsin B3 cDNA (accession number
AY227675) (Meemon et al., 2004) encodes a pre pro-enzyme of
337 amino acids (with predicted MW of 37.8 kDa), consisting of
15 amino acids signal sequence, 70 amino acids prosequence and
252 amino acids mature sequence. The predicted sizes of the
pro-enzyme and mature enzyme are 36.2 kDa and 27.9 kDa,
respectively (Sethadavit et al., 2009). However, when the recombi-
nant proFgCatB3 with the c-myc epitope and hexahistidine tag at
the C-terminus was expressed in the yeast Pichia pastoris GS115,
using the pPICZaA vector designed to secrete recombinant proteins
into the extracellular culture media, SDS–PAGE analysis of the re-
combinant protein eluted from the Ni2+-NTA column revealed a
minor band at about 38–39 kDa, while the bulk of the protein mi-
grated as a smear ranging from 55–75 kDa. Since Pichia is known to
glycosylate secreted proteins that contain an N-linked sugar glyco-
sylation motif, two of which are present in the mature enzyme do-
main of proFgCatB3, it was rationalized that this higher molecular
weight band was most likely due to glycosylation, as observed ear-
lier with the F. hepatica cathepsin CB2 sequence (Beckham et al.,
2006). In both species of Fasciola, CatB proteases is expressed
mainly in the juvenile parasites and demonstrated to play a role
in the penetration of these parasites through the host’s gut wall,
and in mediating their migration to the liver (Wilson et al., 1998;
Law et al., 2003; Meemon et al., 2004).
In the present study, we could produce the specific MoAbs
against rCatB3 of F. gigantica. The isotype of these MoAbs is IgG1
and k light chain. These MoAbs could react with rCatB3, both the
37 kDa and glycosylated peptide at 55–75 kDa. However, in whole
body extracts of F. gigantica, the MoAb specific to rCatB3 could de-
tect only a single band of 37 kDa in metacercairae and NEJ which
might reflect the existence of only one isotype in these two anti-
genic fractions. This 37 kDa band likely corresponds to the proca-
thepsin B3 based on its close approximation to the theoretical
Fig. 2. Determination of the immuno-reactivity of MoAb (2F9) and PoAb against
native CatB3. (A) Immunoblot analysis of F. gigantica newly excysted juveniles (NEJ)
antigens blotted with myeloma CF (lane 1), NMS (lane 2), CIS (lane 3), while lane 4-
metacercariae (Met), lane 5-NEJ, lane 6–4-week-old juvenile (4 wk), lane 7-whole
body extract (WB), lane 8-tegumental antigen (TA) and lane 9-excretory-secretory
(ES) antigens reacted with MoAb clone 2F9. Other clones of MoAb, showing a
similar pattern, are not shown. (B) Immunoblot analysis of F. gigantica NEJ antigens
reacted with myeloma CF (lane 1), NMS (lane 2), CIS (lane 3), while Met (lane 4), NEJ
(lane 5), 4 wk (lane 6), WB (lane 7), TA (lane 8) and ES (lane 9) antigens blotted with
polyclonal antibodies (PoAb) against native CatB3. STD is the lane containing
standard protein molecular weight markers.
Fig. 3. Immunoblot analysis of the cross reactivities of MoAb clone 2F9 reacted
with WB antigens from F. gigantica NEJ and other trematode parasites. NEJ = newly
excysted juveniles in lane 4, Ge = G. explanatum in lane 5, Ep = E. pancreaticum in
lane 6, Pc = P. cervi in lane 7, Ss = S. spindale in lane 8, Sm = S. mansoni in lane 9,
Hp = H. placei in lane 10 and Sp = S. labiato-papillosa in lane 11. The controls show
WB antigens from F. gigantica NEJ blotted with myeloma CF (lane 1), NMS (lane 2)
and CIS (lane 3). Other clones of MoAb, showing similar pattern, are not shown. STD
is the lane containing standard protein molecular weight markers.
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Fig. 4. Immunoperoxidase staining of paraffin sections of various stages of F.gigantica: A–B, control; C–H, immunoperoxidase stained paraffin sections of metacercaria, NEJ,
4-week-old juvenile and adult of F. gigantica by using MoAb specific to rFgCatB3 as a probe. (A) The negative control of a paraffin section of a metacercaria stained with
myeloma culture fluid, showing unstained caecum (Ca), parenchymal cells (Pc), tegument (Te), outer cyst wall (Cw1) and inner cyst wall (Cw2). (B) The negative control of a
paraffin section of a NEJ stained with myeloma culture fluid, showing unstained caecum (Ca), parenchymal cells (Pc), bladder (Bl), tegument (Te), muscle (Mu), oral sucker
(Os) and ventral sucker (Vs). (C) A high magnification micrograph showing intense staining in the caecal epithelium and in the lumen of the caecum (Ca) of the metacercaria
of F. gigantica. (D) A high magnification micrograph of a NEJ showing intense staining in the caecal epithelium (arrow heads) and in the lumen of the caecum (Ca) (arrows),
whereas parenchymal cells (Pc), muscle (Mu), oral sucker (Os), pharynx (Ph) and tegument (Te) are not stained. Inset is the higher magnification of the lower left part showing
intense staining in the caecal epithelium (arrow heads) and in the lumen of the caecum (Ca) (arrows). (E) A high magnification micrograph of a NEJ showing intense staining
in the caecal epithelium (arrow heads) and in the lumen of the caecum (Ca) (arrows), whereas parenchymal cells (Pc), muscle (Mu), bladder (Bl) and tegument (Te) are not
stained. (F) A higher magnification of the lower half of (E) exhibits intense staining in the caecal epithelium (arrow heads) and in the lumen of the caecum (Ca) (arrows), while
parenchymal cells (Pc), muscle (Mu), bladder (Bl) and tegument (Te) are not stained. (G) A medium magnification micrograph of the middle part of a 4-week-old juvenile
showing unstained caecum (Ca), parenchymal cells (Pc), bladder (Bl), muscle (Mu) and tegument (Te). (H) A low magnification micrograph of the anterior part of an adult
fluke shows no staining in the caecum (Ca), parenchymal cells (Pc), testes (Ti), vitelline gland (Vi), muscle (Mu) and tegument (Te).
344
P. Anuracpreeda et al./Experimental Parasitology 127 (2011) 340–345