Acta Tropica 136 (2014) 1–9

Contents lists available at ScienceDirect

Acta Tropica journal homepage: www.elsevier.com/locate/actatropica

Production and characterization of a monoclonal antibody against recombinant cathepsin L1 of Fasciola gigantica Panat Anuracpreeda a,b,∗ , Thippawan Srirakam a , Sudarat Pandonlan a , Narin Changklungmoa b , Charoonroj Chotwiwatthanakun b,c , Yotsawan Tinikul b,c , Jaruwan Poljaroen b,c , Krai Meemon b , Prasert Sobhon b a

Division of Agricultural Science, Mahidol University, Kanchanaburi Campus, Saiyok, Kanchanaburi 71150, Thailand Department of Anatomy, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand c Mahidol University, Nakhonsawan Campus, Nakhonsawan 60130, Thailand b

a r t i c l e

i n f o

Article history: Received 9 January 2014 Received in revised form 6 April 2014 Accepted 7 April 2014 Available online 13 April 2014 Keywords: Fasciola gigantica Cathepsin L1 Monoclonal antibody Immunolocalization Cross-reaction

a b s t r a c t Monoclonal antibodies (MoAbs) against a recombinant cathepsin L1 of Fasciola gigantica (rFgCatL1) were produced in vitro by fusion of BALB/c mice spleen cells immunized with rFgCatL1 and mouse myeloma cells. Reactivity and specificity of these MoAbs were evaluated by indirect ELISA and immunoblotting techniques. Seven MoAb clones were selected from the stable hybridoma clones, namely 1E10, 1F5, 3D11, 4B10, 4D3, 4E3 and 5E7. Clones 1E10, 1F5 and 3D11 were IgM, whereas clones 4B10, 4D3, 4E3 and 5E7 were IgG1 . All MoAbs had kappa light chain isotypes. All MoAbs reacted with rCatL1 at molecular weight (MW) 30 kDa and with the native CatL1 at MW 27 kDa in whole body (WB) extracts of metacercariae (Met), newly excysted juveniles (NEJ), 1, 3, 5-week-old juveniles (Ju), adult WB and adult excretory–secretory (ES) fractions, but not with adult tegumental antigens (TA). All of these MoAbs showed no cross-reactions with antigens of other parasites commonly found in ruminants and human, including Paramphistomum cervi, Eurytrema pancreaticum, Gigantocotyle explanatum, Schistosoma spindale, Schistosoma mansoni, Moniezia benedeni, Avitellina centripunctata, Trichuris sp., Haemonchus placei and Setaria labiato-papillosa. Localization of CatL1 in each developmental stages of F. gigantica by immunoperoxidase technique, using these MoAbs as probes, indicated that CatL1 was present at high concentration in the caecal epithelium and caecal lumen of metacercariae, NEJ, 1, 3, 5-week-old juveniles and adult fluke. This finding indicated that CatL1 is a copiously expressed parasite protein that is released into the ES, thus CatL1 and its MoAb could be a good candidate for immunodiagnosis of fasciolosis in ruminant and human. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Tropical fasciolosis is an economically important disease caused by infection with the hermaphroditic trematode parasite, Fasciola gigantica, in ruminants throughout Asia and in Africa. The worldwide losses in livestock industry due to fasciolosis are estimated over US $ 3.2 billion dollars per annum (Sobhon et al., 1998; Spithill et al., 1999). Likewise, the economic losses in Thailand are estimated to be at least 350 million bahts (US $10 million) per year (Srihakim and Pholpark, 1991). The prevalent rate of infection in

∗ Corresponding author. Division of Agricultural Science, Mahidol University, Kanchanaburi Campus, Saiyok, Kanchanaburi 71150, Thailand. Tel.: +66 3458 5060/+66 2201 5418; fax: +66 3458 5077/+66 2354 7168. E-mail addresses: [email protected], [email protected] (P. Anuracpreeda). http://dx.doi.org/10.1016/j.actatropica.2014.04.012 0001-706X/© 2014 Elsevier B.V. All rights reserved.

Thailand varies from 4 to 24% in cattle and buffaloes, with highest incidents in the Northeast (up to 85% of cattle and buffaloes) (Phonpark and Srikitjakara, 1989; Srihakim and Pholpark, 1991; Sukhapesna et al., 1994). Furthermore, the disease is recognized by World Health Organization as an emerging human disease, and recent reports estimate that at least 2.4 million people are presently infected worldwide and about 91 million are at risk. Humans are usually infected by the ingestion of water plants contaminated with infective stage of the parasite or metacercariae (Keiser and Utzinger, 2009). Cathepsin L is one of the cysteine proteinase which was expressed in many parasitic helminthes. Cathepsin L is secreted by gastrodermal epithelial cells of immature and adult flukes (Collins et al., 2004). This enzyme makes up over 80% of protein contents of excretory–secretory (ES) materials of the fluke (Dowd et al., 1994). In adult F. hepatica, there are at least three types of cathepsin L which have been identified in the ES by a SDS-PAGE technique: cathepsin L1 exhibits molecular

2

P. Anuracpreeda et al. / Acta Tropica 136 (2014) 1–9

weight (MW) at 27 kDa (Smith et al., 1993), cathepsin L2 appears at MW 29.5 kDa (Dowd et al., 1994) and cathepsin L5 appears at MW 24.4 kDa (Smooker et al., 2000). It has been reported that cathepsin L1 cleaves immunoglobulin at the hinge region and cathepsin L2 cleaves fibrinogen and produces a fibrin clot (Dowd et al., 1995). Unlike cathepsin L1 and all others cathepsins, cathepsin L2 can cleave substrates with the proline residue in the P2 position. In addition, cathepsin L1 and L2 can also degrade collagen, laminin and fibronectin in the extracellular matrix and basement membrane of the hosts’ tissues during the juvenile parasites’ invasion (Berasain et al., 1997). Both enzymes comprise of 326 amino acids, 17 amino acids of the signal peptide, 90 amino acids of the pro region, and 219 amino acids of the mature region (Wijffles et al., 1994; Dalton et al., 2003). In F. gigantica, cathepsin L cysteine proteases are isolated from the ES products of adult worms. On gelatin substrate-PAGE, the major protein bands appear MW at 26–28 kDa, and they display immunoglobulin-degradating activity even after partial purification (Dixit et al., 2003). These enzymes comprise of FgCL1 (Maleewong et al., 1999), FgCL2 (Estuningsih et al., 1997), FgCL3 (Dixit et al., 2002; Yadav et al., 2005) which possess MWs at 27 kDa, 27–28 kDa, and 28 kDa, respectively. Several cathepsin L cDNAs and recombinant cathepsin L cysteine proteinases had been cloned and expressed from adult flukes, namely rFgCL1 (Grams et al., 2001; Tantrawatpan et al., 2005), rFgCL1-D (Raina et al., 2006), and rFgCL2 (Yamasaki et al., 2002). By RNA in situ hybridization, cathepsin L mRNA transcripts were detected in the cytoplasm of the caecal epithelial cells of the digestive tract, whereas the reproductive organs, including testis, Mehlis’ gland, and ovary were not stained (Grams et al., 2001; Meemon et al., 2010). Using immunolocalization, Yamasaki and Aoki (1993) reported that cathepsin Ls were synthesized and packaged in vesicles within the caecal epithelial cells of Fasciola sp. Since this protein is released into the host fluid in a fairly large amount, it could be a good candidate for immunodiagnosis of fasciolosis by F. gigantica, especially the early and late of infection. In this study, monoclonal antibodies (MoAbs) against recombinant F. gigantica cathepsin L1 (rFgCatL1) were produced and characterized for their binding with both the recombinant and native CatL1 in various tissues of each developmental stages of the fluke. Cross reactions with antigens of other trematode and nematode parasites were also examined in order to verify their specificity for possible application in immunodiagnosis. 2. Materials and methods 2.1. Collection of parasite samples 2.1.1. Metacercariae (Met) Metacercariae of F. gigantica were obtained from experimentally infected snail, Lymnaea ollula. These snails were infected with miracidia and allowed to develop sporocysts and cercariae. On day 45, the cercariae were shed from the snails and settled on the 5 × 5 cm cellophane papers and transformed into metacercariae. The metacercariae were brushed off and collected from the cellophane paper and washed several times with Hank’s balance salt (HBS) solution containing 100 U/ml penicillin and 100 mg/ml streptomycin and used immediately (Anuracpreeda et al., 2011, 2013a). 2.1.2. Newly excysted juveniles (NEJ) To obtain NEJ of F. gigantica, the metacercariae were excysted by incubation in distilled water containing 1% (w/v) pepsin (pepsin A from porcine gastric mucosa, P-7000, Sigma–Aldrich Co.) and 0.4% (v/v) HCl at 37 ◦ C for 45 min, and then washed with distilled water. After that, they were resuspened in a solution of 0.02 M sodium dithionite (Fluka Biochemika), 0.2% (w/v) taurocholic acid (T-4009,

Sigma–Aldrich Co.), 1% (w/v) NaHCO3 , 0.8% (w/v) NaCl, and 0.005% (v/v) HCl. Specimens were incubated at 37 ◦ C for 45 min and washed with distilled water. The activated metacercariae were excysted in fresh RPMI-1640 medium (Sigma Chemical Co., St. Louis, MO, USA) containing 10% fetal calf serum, and 10 ␮g/ml gentamycin at 37 ◦ C overnight. On the following day, the newly excysted juveniles (NEJ) were collected and washed several times with Hank’s balance salt (HBS) solution and used immediately (Anuracpreeda et al., 2011, 2013a). 2.1.3. Juvenile parasites (Ju) A method described by Anuracpreeda et al. (2009a, 2011) was used to obtain juveniles of F. gigantica from male Golden Syrian hamsters experimentally infected with 30 metacercariae. The juvenile worms were obtained by sacrificing the infected animals at 1, 3, and 5 weeks after infections, and teasing the liver to collect the parasites. The specimens were washed several times with Hank’s balance salt (HBS) solution and used immediately. 2.1.4. Adult parasites Adult stages of F. gigantica were obtained from the bile ducts and gall bladders of naturally infected cattle or water buffaloes killed at the local abattoirs. Other parasites collected from the same group of animals for the cross-reactivity study included trematodes (Paramphistomum cervi, Eurytrema pancreaticum, Gigantocotyle explanatum, and Schistosoma spindale), cestodes (Moniezia benedeni and Avitellina centripunctata), and nematode parasites (Trichuris sp., Haemonchus placei and Setaria labiato-papillosa). Adult Schistosoma mansoni were obtained by perfusing mice 8 weeks after being infected with schistosome cercariae. All parasite samples were washed several times with Hank’s balance salt (HBS) solution before being used further (Anuracpreeda et al., 2012, 2013a,b). 2.2. Preparations of parasite antigens 2.2.1. Whole body (WB) antigens of adult and juvenile (JP) parasites To obtain WB antigens, whole parasites (metacercariae, NEJ, 1, 3, 5-week-old juveniles, adults of F. gigantica and other species) were homogenized in a lysis buffer containing 10 mM Tris–HCl, pH 7.2, 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA and 1 mM PMSF (P-7626, Sigma–Aldrich Co.) and then sonicated for 5 min in an ice bath with 15-s pulses. After rotation at 4 ◦ C for 1 h, the suspensions were centrifuged at 5000 × g, for 20 min at 4 ◦ C to get rid of the eggs, and the supernatants were collected, lyophilized, and stored at −70 ◦ C until use in later experiments (Anuracpreeda et al., 2013a,b; Panyarachun et al., 2013). 2.2.2. Tegumental antigens (TA) of adult F. gigantica The method described by Anuracpreeda et al. (2006) was used to obtain TA. Briefly, live adult worms were treated with extracting buffer containing 1% Triton X-100 in 0.05 M Tris buffer, pH 8.0, 0.01 M EDTA, 0.15 M NaCl for 20 min at room temperature. The extracting solution was collected and centrifuged at 5000 × g for 20 min at 4 ◦ C to remove the eggs which were released during the extraction. The soluble TA was collected and dialyzed in 0.01 M phosphate buffered saline (PBS), pH 7.2, for 24 h at 4 ◦ C, using Spectra/Por dialysis membrane (Spectrum Medical Industries, Los Angeles, California, USA) with molecular weight cut off at 6–8 kDa. Then it was lyophilized, and kept at −70 ◦ C until further use. 2.2.3. Excretory–secretory (ES) antigens of adult F. gigantica The ES antigens were prepared by incubating freshly collected, live adult worms in RPMI-1640 media for 3 h at 37 ◦ C. Thereafter, the parasites’ eggs in the culture medium were removed by centrifugation at 5000 × g for 20 min at 4 ◦ C. The supernatant was

P. Anuracpreeda et al. / Acta Tropica 136 (2014) 1–9

3

collected and dialyzed in 0.01 M PBS, pH 7.2, for 24 h at 4 ◦ C, using Spectra/Por dialysis membrane before it was lyophilized and stored at −70 ◦ C until use in subsequent experiments (Anuracpreeda et al., 2009b).

Committee (SCMUACUC), Faculty of Science, Mahidol University, Thailand.

2.2.4. Determination of protein extracts Protein concentrations of all parasites’ extracts (WB, TA, ES) were determined by Lowry’s method (Lowry et al., 1951) using bovine serum albumin as a standard. These extracts were stored at −70 ◦ C until use.

2.5.1. Characterization of antibody isotypes of MoAbs The antibody isotypes were determined by indirect ELISA using the SBA ClonotypingTM System/HRP (SouthernBiotech, Birmingham, USA). A 50 ␮l of 1 ␮g/ml rFgCatL1 of F. gigantica diluted in 0.05 M carbonate buffer (15 mM Na2 CO3 , 35 mM NaHCO3 , pH 9.6) was added into each well of a flat bottom F96 microtiter plate (Nunc A/S, Roskilde, Denmark) and incubated for 2 h at 37 ◦ C. The coated plate was then washed three times with distilled water to remove excess antigens and unbound materials. Each time the washing fluid was left in the wells for approximately 1 min at room temperature. Subsequently, the nonspecific binding was blocked by adding 100 ␮l/well of 0.25% bovine serum albumin (BSA), 0.05% Tween 20 (Sigma) in 0.01 M PBS, pH 7.2 for 1 h at 37 ◦ C. Then the coated plate was similarly washed prior to adding 50 ␮l of undiluted hybridoma fluid, and incubated for 2 h at 37 ◦ C. After washing, the plate was incubated with 50 ␮l/well of a panel of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgM, IgG1 , IgG2a , IgG2b , IgG3 and IgA (SouthernBiotech, Birmingham, USA) at 1:6000 dilution in the blocking solution for 1 h at 37 ◦ C, for indentifications of heavy chain, and к, ␭ light chains of immunoglobulins. After washings as previously described, the color development was generated by adding 50 ␮l/well of 2,2’-azinobis [3-ethylbenzothiazoline6-sulfonic acid]-diammonium salt (ABTS) substrate (Southern Biotech, Birmingham, USA). The enzymatic reaction was allowed to take place for 10 min at room temperature. Finally, the optical density (OD) value was measured at 405 nm using a microplate reader (Multiskan Ascent, Labsystems, Helsinki, Finland).

2.3. Preparation of recombinant F. gigantica cathepsin L1 protease (rFgCatL1) The methods described by Grams et al. (2001) and Meemon et al. (2010) were used to obtain rFgCatL1. Briefly, the adult F. gigantica cDNA library was used for amplification of a DNA fragment of the CatL1 gene. A fragment of FgCatL1 was isolated by PCR analysis and subcloned into pGEM® -T Easy vector (Promega, Madison, USA) and the sequence confirmed by DNA sequence analysis (Macrogen, South Korea). The full-length FgCatL1 cDNA was subcloned into the bacterial expression vector, pET30b (Novagen), which was transformed into Escherichia coli BL21 (DE). The rFgCatL1 was expressed at 37 ◦ C by inducing the bacteria with isopropyl-␤-d-thiogalactoside (IPTG). The bacterial cells were collected and resuspended in a lysis buffer containing 50 mM NaH2 PO4 , 300 mM NaCl, 10 mM imidazole, pH 8.0 and sonicated at 200–300 watt for 10 s, with a 10 s burst cycle (using six cycles) on ice. The recombinant FgCatL1 (rFgCatL1) was purified by Ni2+ -NTA affinity chromatography (QIAGEN) at room temperature. The column was washed two times with a washing buffer containing 50 mM NaH2 PO4 , 300 mM NaCl, 20 mM imidazole, pH 8.0,and the protein was eluted by an elution buffer containing 50 mM NaH2 PO4 , 300 mM NaCl, 250 mM imidazole, pH 8.0. The eluate was dialyzed against 0.01 M PBS, pH 7.4, at 4 ◦ C overnight, and concentrated by membrane filtration using an Amicon Ultra centrifugal filter devices 10,000 with nominal molecular weight limit (Millipore, Bedford, MA, USA), and dissolved in 0.01 M PBS, pH 7.4. The rFgCatL1 was stored at −70 ◦ C until use. 2.4. Production and screening of monoclonal antibodies (MoAbs) against rFgCatL1 Inbred eight week-old female BALB/c mice were immunized according to the method described by Anuracpreeda et al. (2013a). Briefly, mice were primed by subcutaneous injection with 25 ␮g rFgCatL1 in 100 ␮l PBS solution thoroughly mixed with an equal volume of complete Freund’s adjuvant (Sigma–Aldrich Inc., St. Louis, MO, USA). The second and third injections were given at 2-week intervals with 25 ␮g rFgCatL1 in PBS emulsified in incomplete Freund’s adjuvant (Sigma–Aldrich Inc.) via the same route. Three days before blood collection, a final boosting with 25 ␮g rFgCatL1 was given by the intravenous route without adjuvant. Serum samples from the immunized mice were collected, and the polyclonal antibody (PoAb) titer in the antisera was estimated by indirect ELISA. The hybridoma clones expressing MoAb against rFgCatL1 were produced by fusion of the spleen cells of immunized BALB/c mouse and mouse myeloma cells (P3 × 63-Ag8.653), using polyethylene glycol (PEG) (Sigma–Aldrich Inc.). The hybridoma cells that grew successfully in culture were cloned by limiting dilution methods using a feeder layer of spleen cells. The MoAbs produced by the hybridoma cells were screened by indirect ELISA. The hybridoma fluid that produced high titers of MoAbs against rFgCatL1 was selected. All animal experiments were approved by the Animal Care and Use

2.5. Characterization and reactivity of MoAbs against rFgCatL1

2.5.2. Reactivity of MoAbs The reactivity of MoAbs against rFgCatL1 and native protein in WB, TA, and ES of adult F. gigantica were determined by indirect ELISA. A 50 ␮l of 100 ␮g/ml of WB, TA, and ES of F. gigantica diluted in 0.05 M carbonate buffer was added into each well of a flat bottom F96 microtiter plate (Nunc A/S, Roskilde, Denmark) and incubated for 2 h at 37 ◦ C. After washing three times with distilled water, the nonspecific binding was blocked by adding 100 ␮l/well of 0.25% BSA, 0.05% Tween 20 (Sigma) in 0.01 M PBS, pH 7.2 for 1 h at 37 ◦ C. Thereafter, the coated plate was similarly washed, and 50 ␮l of undiluted hybridoma fluid (MoAbs clone 4E3) was added and incubated for 2 h at 37 ◦ C. After washing, the plate was incubated with 50 ␮l/well of HRP-conjugated goat anti-mouse immunoglobulin (Sigma–Aldrich Inc.) diluted in blocking solution at 1:6000 for 1 h at 37 ◦ C. After that the plate was washed with distilled water, and 50 ␮l/well of 3,3’,5,5’-tetramethyl benzidine (TMB) substrate (KPL, Gaithersburg, USA) was added and incubated for 10 min at room temperature. Finally, the enzymatic reaction was stopped by the addition of 50 ␮l 1N HCl. The OD value at 450 nm was read in a microplate reader (Multiskan Ascent, Labsystems, Helsinki, Finland). 2.6. Immunoblotting assay Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and was performed as described by Laemmli (1970). Briefly, rFgCatL1, metacercariae, NEJ, JP, ES, TA, and WB antigens of F. gigantica as well as WB antigens from other trematode, cestode and nematode parasites were separated in 12.5% SDSPAGE. After electrophoresis, the resolved polypeptide bands were either revealed by Coomassie blue staining or electrophoretically

4

P. Anuracpreeda et al. / Acta Tropica 136 (2014) 1–9

Fig. 1. The levels of the native CatL1 in WB, ES, and TA fractions of adult F. gigantica was estimated by its reactivity with MoAb 4E3 using indirect ELISA. Significant increase of optical densities (OD) representing CatL1 levels were shown in WB and ES (dark blue column) when compared with the control myeloma fluid (yellow column). No significant difference in the levels of CatL1 was observed in TA fraction when compared to the control. The result was considered significant if p-value is lower than 0.05 (p-value < 0.05) represented in asterisk (* ). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

transferred onto nitrocellulose membranes for immunoblotting (Towbin et al., 1979).

2.6.1. Coomassie blue staining The gel was placed and gently shaken in a Coomassie blue solution (0.025% Coomassie Blue R-250, 7% acetic acid and 40% methanol) for 2 h at room temperature. Thereafter, the background of Coomassie bleu- stained gels was removed using destaining solution (7% acetic acid and 40% methanol) for 24 h at room temperature to allow stained protein bands to be seen clearly.

2.6.2. Immunoblotting Each blotted nitrocellulose membrane was cut into strips. The non specific binding was blocked with a blocking solution (5% skimmed milk in Tris buffered saline (TBS) pH 7.4 containing 0.05% Tween 20) for 2 h at room temperature. After that, the membrane strips were incubated in undiluted hybridoma fluid containing MoAbs or PoAb antiserum against rFgCatL1 for 2 h at room temperature. Cattle-infected sera obtained from the pooled sera of the naturally infected animals were used as positive controls, while myeloma culture fluid and normal mouse serum were used as negative controls. After washing with TBS, the MoAbs and PoAb–antigen complexes were detected by HRP-conjugated goat anti-mouse immunoglobulin (Sigma–Aldrich Inc.), while cattle antibodies that reacted with the antigenic molecules were detected by HRP-conjugated goat anti-bovine immunoglobulin (Sigma–Aldrich Inc.), diluted at 1:4000 with 1% skimmed milk in TBS, containing 0.05% Tween 20, pH 7.4, for 1 h at room temperature. After washing, the color reaction was visualized by further incubation in a specific substrate 3,3’,5,5’-tetramethyl benzidine (TMB) (KPL, Gaithersburg, USA) for 3–5 min at room temperature until positive bands appeared. Finally, the reaction was stopped by adding distilled water.

Fig. 2. Determination of the immuno-reactivity of MoAbs against native and recombinant F. gigantica cathepsin L1 (rFgCatL1): (A) immunoblot patterns of the rFgCatL1 reacted with myeloma culture fluid-CF (lane 1), normal mouse serum-NMS (lane 2), cattle infected serum-CIS (lane 3), MoAb 1E10 (lane 4), 1F5 (lane 5), 3D11 (lane 6), 4B10 (lane 7), 4D3 (lane 8), 4E3 (lane 9) and 5E7 (lane 10). (B) Immunoblot patterns of F. gigantica whole body extract (WB) antigens reacted with myeloma culture fluidCF (lane 1), normal mouse serum-NMS (lane 2), cattle infected serum-CIS (lane 3), MoAb 1E10 (lane 4), 1F5 (lane 5), 3D11 (lane 6), 4B10 (lane 7), 4D3 (lane 8), 4E3 (lane 9) and 5E7 (lane 10). STD is the lane containing standard protein molecular weight markers on the left side.

2.7. Localization of CatL1 in developmental stages of F. gigantica by immunoperoxidase technique The immunoperoxidase technique described by Anuracpreeda et al. (2009a) was used to analyze the distribution and relative concentration of CatL1 in the paraffin sections of F. gigantica at each developmental stages (metacercariae, NEJ, 1, 3, 5-week-juveniles and adults) using seven MoAbs as probes. Briefly, the sections were deparaffinized and rehydrated through xylene and decreasing serial concentrations of ethyl alcohol (from 100% to 50%) for 3 min each before rinsing with tap water. The sections were incubated in 0.01 M citrate buffer, pH 6.0 in a microwave oven at 500–700 kV for 5 min, 3 times. Then the endogenous peroxidase in the tissues

P. Anuracpreeda et al. / Acta Tropica 136 (2014) 1–9

Fig. 3. Determination of the immuno-reactivity of MoAb clone 4E3 and PoAb against native CatL1. (A) Immunoblot analysis of F. gigantica whole body extract (WB) antigens reacted with myeloma CF (lane 1), NMS (lane 2), CIS (lane 3), while metacercariae (Met) in lane 4, NEJ in lane 5, 1-week-old juvenile (1wk) in lane 6, 3-week-old juvenile (3wk) in lane 7, 5-week-old juvenile (5wk) in lane 8, WB in lane 9, excretory–secretory (ES) in lane 10 and tegumental antigen (TA) antigens in lane 11 were blotted with MoAb 4E3. Other clones of MoAb showed similar pattern and were not shown. (B) Immunoblot analysis of F. gigantica WB antigens blotted with myeloma CF (lane 1), NMS (lane 2), CIS (lane 3), while metacercariae (Met) in lane 4, NEJ in lane 5, 1-week-old juvenile (1wk) in lane 6, 3-week-old juvenile (3wk) in lane 7, 5-week-old juvenile (5wk) in lane 8, WB in lane 9, excretory–secretory (ES) in lane 10 and tegumental antigen (TA) antigens in lane 11 were blotted with polyclonal antibodies (PoAb) against native CatL1. STD is the lane containing standard protein molecular weight markers.

was quenched by treatment with 3% H2 O2 diluted in tap water for 30 min, and washed with 0.01 M PBS containing 0.1% Tween20 for 5 min, 3 times. Thereafter, non-specific binding was blocked by incubation in 0.1% (w/v) glycine in 0.01 M PBS pH 7.4 and 4% (w/v) BSA in 0.01 M PBS pH 7.4, for 30 min and 1 h, respectively. The sections were incubated in specific MoAbs against rFgCatL1 for 2 h at room temperature. After washing three times for 5 min each, the sections were incubated with biotinylated goat antimouse IgG (Zymed Laboratory Inc., South San Francisco, CA, USA), diluted to 1:200 in 0.01 M PBS, for 30 min at room temperature. Subsequently, they were incubated with HRP-conjugated streptavidin (Zymed Laboratory Inc.), diluted to 1:200 in 0.01 M PBS, for 30 min at room temperature, and then washed. The color reaction was developed by using AEC (3-amino-9-ethylcarbazole) substrate solution (Zymed Laboratory Inc.) in the dark. Finally, the reactions

5

Fig. 4. SDS-PAGE of whole body (WB) antigens from F. gigantica, other trematode, cestode and nematode parasites showing the protein profiles after Coomassie blue staining, and immunoblot analysis of these proteins using MoAb clone 4E3 as probe. (A) Coomassie blue staining of SDS-PAGE separated WB antigens from F. gigantica, other trematode, cestode and nematode parasites, showing protein bands with a wide range of molecular weights. WB = F. gigantica (lane1), Pc = P. cervi (lane 2), Ep = E. pancreaticum (lane 3), Ge = G. explanatum (lane 4), Ss = S. spindale (lane 5), Sm = S. mansoni (lane 6), Mb = M. benedeni (lane 7), Ac = A. centripunctata (lane 8), Ts = Trichuris sp. (lane 9), Hp = H. placei (lane 10) and Sp = S. labiato-papillosa (lane 11). STD is the lane containing standard protein molecular weight markers. (B) Immunoblot analysis showing the cross-reactivity of MoAb clone 4E3 with WB antigens from F. gigantica (WB) (lane 4), Pc = P. cervi (lane 5), Ep = E. pancreaticum (lane 6), Ge = G. explanatum (lane 7), Ss = S. spindale (lane 8), Sm = S. mansoni (lane 9), Mb = M. benedeni (lane 10), Ac = A. centripunctata (lane 11), Ts = Trichuris sp. (lane 12), Hp = H. placei (lane 13) and Sp = S. labiato-papillosa (lane 14). The controls show WB antigens from F. gigantica adult blotted with myeloma CF (lane 1), NMS (lane 2) and CIS (lane 3). Other clones of MoAb showed similar pattern and were not shown. STD is the lane containing standard protein molecular weight markers.

were stopped by adding tap water and the stained sections were mounted in Vectashield (Vector Laboratories Inc., Burlingame, CA, USA). The sections were observed and photographed under a light microscope (Nikon, Eclipse E600) equipped with a DXM 1200F digital camera. 3. Results 3.1. Monoclonal antibodies (MoAbs) against rFgCatL1 Seven hybridoma clones, namely 1E10, 1F5, 3D11, 4B10, 4D3, 4E3 and 5E7, were selected based on the indirect ELISA and immunoblotting results. These clones produced MoAbs specific to rFgCatL1 of F. gigantica, and they were expanded in culture flasks to obtain large volume of MoAb which were collected for further experiments. Clones 1E10, 1F5 and 3D11 were found to be IgM,

6

P. Anuracpreeda et al. / Acta Tropica 136 (2014) 1–9

P. Anuracpreeda et al. / Acta Tropica 136 (2014) 1–9

whereas clones 4B10, 4D3, 4E3 and 5E7 were IgG1 . All MoAbs had ␬ light chain. The highest titer was obtained from MoAb 4E3 as analyzed by indirect ELISA; hence, it was used in the following experiments. 3.2. Evaluation of the native CatL1 in WB, ES, and TA fractions of adult F. gigantica (FgCatL1) The native FgCatL1 in WB, ES, and TA reacted with MoAb 4E3 and the relative levels of FgCatL1 in each fraction were evaluated by indirect ELISA. The levels of reactivity of FgCatL1 in WB and ES were significantly higher when compared with control myeloma fluid, whereas the levels of FgCatL1 in TA was only slightly positive but with no significant difference from the control (Fig. 1). 3.3. Immunoblotting analysis The immunobotting experiment revealed that all MoAbs reacted with a single band of rFgCatL1 which has a molecular weight (MW) of 30 kDa (Fig. 2). However, when tested against WB antigens in all developmental stages (metacercariae, NEJ, 1, 3, 5-week-old juveniles and adults), and TA and ES antigens from adult F. gigantica, these MoAbs reacted intensely with native CatL1 which appeared as a single band at MW 27 kDa in all WB and ES extracts. In contrast, no positive band was detected in adult TA fraction (Fig. 3A). When similar antigenic fractions were analyzed with PoAbs against native CatL1, the positive band was observed at MW 27 kDa which confirmed that the protein detected by MoAb was CatL1 (Fig. 3B). In addition, there was another positive band at MW 27 kDa in adult TA, 17 kDa and 14 kDa in WB extracts of metacercariae, NEJ, 1, 3, 5-week-old juveniles, adult WB and adult ES (Fig. 3B). For the cross-reactivity study, the SDS-PAGEs of WB antigens from F. gigantica, other trematode, cestode and nematode parasites were shown in Fig. 4A. No positive band was detected when the MoAb reacted with WB antigens from five trematode parasites (P. cervi, E. pancreaticum, G. explanatum, S. spindale and S. mansoni), from two cestode parasites (Moniezia benedeni and Avitellina centripunctata), and from three nematode parasites (Trichuris sp., H. placei and S. labiato-papillosa), while the band was very conspicuous in adult F. gigantica WB antigen (Fig. 4B). 3.4. Localization and distribution of CatL1 The distribution of CatL1 in each development stages of F. gigantica (metacercariae, NEJ, 1, 3, 5-week-old juveniles, adults) was examined by immunoperoxidase staining using the MoAbs as probes. All MoAbs exhibited similar immunoperoxidase staining pattern as represented by MoAb 4E3 (Fig. 5) which showed the strongest reaction. The positions and intensities of the brownish reaction products indicated the location and relative concentration of CatL1 that were bound to the MoAb. The myeloma culture fluid, used as a negative control, showed no brownish staining in any tissues of the parasite (Fig. 5A).

7

Consistent with the immuoblotting analysis, the positive immunostaining was detected in both caecal epithelium and in the lumen of the caecum of metacercariae, NEJ, 1, 3 and 5-week-old juveniles F. gigantica, while the tegument, tegumental cells, and parenchymal cells were not stained (Fig. 5B–F). Similar to juveniles, immunostaining of the adult parasite sections was intense in both the caecal epithelium and in lumen of the caecum (Fig. 5G and H), while the tegument, tegumental cells, vitelline cells, testes, muscle and parenchymal cells showed no staining (Fig. 5G).

4. Discussion Several F. hepatica cathepsin L genes have been identified and isolated (Yamasaki and Aoki, 1993; Heussler and Dobbelaere, 1994). A recombinant cathepsin L1 of F. hepatica (rFhCL1) was also generated by expression of a cDNA from the adult stage (Roche et al., 1997). In this report, the recombinant protein of F. gigantica CatL1 (rFgCatL1) could be expressed in E. coli BL21 (DE) and exhibited at a MW 30 kDa. The size of this recombinant protein was larger than the protein analyzed from the coding sequence because of the addition of hexahistidine tag at the C-terminus. In this study, we could also produce MoAbs reacting with rFgCatL1. These MoAbs were both IgM and IgG1 —isotypes with ␬ light chain. Immunoblotting assay showed that these MoAbs could react with the rFgCatL1 at MW 30 kDa. Futhermore, they could also detect a single band of 27 kDa native protein in whole body extracts of F. gigantica metacercariae, NEJ, 1, 3, 5-week-old juveniles, adult WB and adult ES, but not in adult TA fraction. The MW of 27 kDa of this band is equal to MW of the putative protein deduced from the amino acid sequence of FgCatL1 (GenBank accession number AF112566) (Grams et al., 2001). However, it was recently reported that F. gigantica metacercariae and NEJ expressed catthepsin L1H (FgCatL1H) (GenBank accession number AY428949) in caecal epithelial cells instead of the CatL1 which is the adult isotype. Similarly, in F. hepatica, CatL1 was not expressed in metacercariae and NEJ (Robinson et al., 2008). The deduced amino acid sequence of the FgCatL1H shared 78.5% identity with FgCatL1 (Sansri et al., 2013). Thus the MoAbs might cross-react with the common epitope present in the juvenile-type CatL1H present in metacercariae and NEJ stage. This was reflected by the fact that these MoAbs exhibited less intensity upon binding with the antigens in the whole body extracts of F. gigantica metacercariae, NEJ, juveniles, than with the adult WB and ES, which could be due to binding affinity. Recently, we have produced a MoAb against FgCatL1H and found that it could cross reacted with FgCatL1 (unpublished result). In contrast to the MoAbs, PoAb could detect three bands of FgCatL1 at MW 27, 17 and 14 kDa in WB extracts of metacercariae, NEJ, 1, 3, 5-week-old juveniles, adult WB and ES which might reflect the existence of more than one isotype of these proteins in the F. gigantica which carried different epitopes. A phylogenetic analysis of the Fasciola Cat L family demonstrated that FgCatL1 belonged to a distinct group, clade CL1C, as reported by Morphew et al. (2011). In addition, it

Fig. 5. Light micrographs of paraffin sections of various stages of F. gigantica stained by immunoperoxidase technique using MoAb specific to rFgCatL1 as a probe. (A) A negative control section of an adult parasite stained with myeloma culture fluid, showing unstained caecum (Ca), parenchymal cells (Pc), tegument (Te), spine (Sp), muscle (Mu) and vitelline cells (Vi). (B) A high magnification micrograph of a metacercarial section showing intense staining in the caecal epithelium and in the lumen of the caecum (Ca), while tegument (Te), parenchymal cells (Pc), outer cyst wall (Cw1) and inner cyst wall (Cw2) were not stained. (C) A high magnification micrograph of a NEJ showing intense staining in the caecal epithelium and in the lumen of the caecum (Ca), while no staining was detected in oral sucker (Os), pharynx (Ph), tegument (Te), muscle (Mu), and parenchymal cells (Pc). (D) A high magnification micrograph of a 3-week-old juvenile showing intense staining in the caecal epithelium and in the lumen of the caecum (Ca), while the tegument (Te), parenchymal cells (Pc) and muscle (Mu) were not stained. (E) A medium magnification micrograph of a 5-week-old juvenile showing intense staining in the caecal epithelium and in the lumen of the caecum (Ca), while the oral sucker (Os), ventral sucker (Vs), tegument (Te), muscle (Mu) and parenchymal cells (Pc) were not stained. (F–G) Low and medium magnification micrographs of adult F. gigantica sections showing intense staining only in the caecal epithelium and in the lumen of the caecum (Ca), while no staining was observed in the tegument (Te), spine (Sp), muscle (Mu), parenchymal cells (Pc), testes (Ti) and vitelline cells (Vi). (H) A higher magnification micrograph of an adult F. gigantica section showing strong intense staining the cytoplasm of bifurcation (Bi) of caecal epithelium (Ca) and accumulation of the granules (arrows) in the apical and basal cytoplasm, while the basal lamina (Ba) was not stained.

8

P. Anuracpreeda et al. / Acta Tropica 136 (2014) 1–9

has been shown that there are 14 different isoforms of F. hepatica Cat L proteases in the adult CL1, CL2 and CL5 Cat L protease clades (Morphew et al., 2011). The deduced amino acid sequence of the FgCatL1 shared 67.1–94.4% identity with other F. hepatica CatLs (Sansri et al., 2013). Hence, eventhough the monoclonal antibody 4E3 did not cross reacted with antigens from other trematode and nematode parasites, it could still cross-reacted with a possible common epitope in CatLs of F. hepatica, which needed to be tested in the future. The localization and distribution studies revealed that our MoAbs could detect the native CatL1 or its homologs in epithelium and lumen of the caecum in metacercariae, NEJ, 1, 3 and 5-weekold juveniles and adult F. gigantica with a similar pattern to that described by Meemon et al. (2010) using a mouse polyclonal antiserum against native cathepsin L of adult F. gigantica. This finding suggested that the native cathepsin L1 could be synthesized and stored in secretory vesicles of the gastrodermal epithelial cells in an inactive proenzyme form or procathepsin L in both juvenile and adult stage. Procathepsin L might then be secreted into an acidic environment of the caecal lumen where it was activated to become an active enzyme for the nutrient digestion. In F. hepatica, the CatL proteases were also detected in the caecal lumen and within secretory vesicles of the caecal epithelia of juveniles and adult flukes using polyclonal anti-CatL antibodies (Collins et al., 2004). Therefore, the CatL may function as a protease for nutrition acquisitions in Fasciola sp., during late stages; and the early juveniles may employ its homolog (CatL1H) for nutritional acquisition as well as for invasion of the host’s tissues. The MoAb that we produced is quite specific to FgCatL1 and showed no cross-reactivity with antigens in other trematode, cestode and nematode parasites, including P. cervi, E. pancreaticum, G. explanatum, S. spindale, S. mansoni, M. benedeni, A. centripunctata, Trichuris sp., H. placei and S. labiato-papillosa. This implied that this MoAb might binds only to a common epitope, which is present exclusively in Fasciola cathepsin Ls. Hence, it is possible that this MoAb and its corresponding antigen could be used for immunodiagnosis of fasciolosis in the detection of both early and late infections with high sensitivity and specificity. Alternatively, the detection of other antigen such as tegumental antigen at MW 28.5 kDa and CatB3 by their corresponding MoAbs could also be employed in conjunction with the MoAb against CatL1, since these antigens were proven to be present and released from the adult and juvenile stages of F. gigantica, and their exploits in immunodiagnosis by sandwich ELISA have already been reported by our group (Anuracpreeda et al., 2009b, 2011). Acknowledgments This research was financially supported by Research Grants from The Thailand Research Fund (Senior Research Scholar Fellowship) and Faculty of Science, Mahidol University to Prasert Sobhon, and a Research Grant for New Scholar co-funded by The Thailand Research Fund, Commission on Higher Education and Mahidol University to Panat Anuracpreeda (MRG5580012). References Anuracpreeda, P., Wanichanon, C., Chaithirayanon, K., Preyavichyapugdee, N., Sobhon, P., 2006. Distribution of 28.5 kDa antigen in tegument of adult Fasciola gigantica. Acta Trop. 100, 31–40. Anuracpreeda, P., Wanichanon, C., Sobhon, P., 2009a. Fasciola gigantica: immunolocalization of 28.5 kDa antigen in the tegument of metacercaria and juvenile fluke. Exp. Parasitol. 122, 75–83. Anuracpreeda, P., Wanichanon, C., Chawengkirtikul, R., Chaithirayanon, K., Sobhon, P., 2009b. Fasciola gigantica: immunodiagnosis of fasciolosis by detection of circulating 28.5 kDa tegumental antigen. Exp. Parasitol. 123, 334–340. Anuracpreeda, P., Songkoomkrong, S., Sethadavit, M., Chotwiwatthanakun, C., Tinikul, Y., Sobhon, P., 2011. Fasciola gigantica: production and characterization

of a monoclonal antibody against recombinant cathepsin B3. Exp. Parasitol. 127, 340–345. Anuracpreeda, P., Panyarachun, B., Ngamniyom, A., Tinikul, Y., Chotwiwatthanakun, C., Poljaroen, J., Sobhon, P., 2012. Fischoederius cobboldi: a scanning electron microscopy investigation of surface morphology of adult rumen fluke. Exp. Parasitol. 130, 400–407. Anuracpreeda, P., Chawengkirtikul, R., Tinikul, Y., Poljaroen, J., Chotwiwatthanakun, C., Sobhon, P., 2013a. Diagnosis of Fasciola gigantica infection using a monoclonal antibody-based sandwich ELISA for detection of circulating cathepsin B3 protease. Acta Trop. 127, 38–45. Anuracpreeda, P., Poljaroen, J., Chotwiwatthanakun, C., Tinikul, Y., Sobhon, P., 2013b. Antigenic components, isolation and partial characterization of excretionsecretion fraction of Paramphistomum cervi. Exp. Parasitol. 133, 327–333. Berasain, P., Goni, F., McGonigle, S., Dowd, A.J., Dalton, J.P., Frangione, B., Carmona, C., 1997. Proteinases secreted by Fasciola hepatica degrade extracellular matrix and basement membrane components. J. Parasitol. 83, 1–5. Collins, P.R., Stack, C.N., O’Neill, S.M., Doyle, S., Ryan, T., Brennan, G.P., Mousley, A., Stewart, M., Maule, A.G., Dalton, J.P., Donnelly, S., 2004. Cathepsin L1, the major protease involved in liver fluke (Fasciola hepatica) virulence. J. Biol. Chem. 279, 17038–17046. Dalton, J.P., Neill, S.O., Stack, C., Collins, P., Walshe, A., Sekiya, M., Doyle, S., Mulcahy, G., Hoyle, D., Khaznadji, E., Moiré, N., Brennan, G., Mousley, A., Kreshchenko, N., Maule, A.G., Donnelly, S.M., 2003. Fasciola hepatica cathepsin L-like proteases: biology, function, and potential in the development of first generation liver fluke vaccines. Int. J. Parasitol. 33, 1173–1181. Dixit, A.K., Yadav, S.C., Sharma, R.L., 2002. 28 kDa Fasciola gigantica cysteine proteinase in the diagnosis of prepatent ovine fasciolosis. Vet. Parasitol. 109, 233–247. Dixit, A.K., Yadav, S.C., Saini, M., Sharma, R.L., 2003. Purification and characterization of 28 kDa cysteine proteinase for immunodiagnosis of tropical fasciolosis. J. Vet. Parasitol. 17, 5–9. Dowd, A.J., Smith, A.M., McGonigle, S., Dalton, J.P., 1994. Purification and characterisation of a second cathepsin L proteinase secreted by the parasitic trematode Fasciola hepatica. Eur. J. Biochem. 223, 91–98. Dowd, A.J., McGonigle, S., Dalton, J.P., 1995. Fasciola hepatica cathepsin L cleaves fibrinogen and produces a novel type of fibrinogen clot. Eur. J. Biochem. 232, 241–246. Estuningsih, S.E., Smooker, P.M., Wiedosari, E., Widjajanti, S., Vaiano, S., Partoutomo, S., Spithill, T.W., 1997. Evaluation of antigens of Fasciola gigantica as vaccines against tropical fasciolosis in cattle. Int. J. Parasitol. 27, 1419–1428. Grams, R., Vichasri-Grams, S., Sobhon, P., Upatham, E.S., Viyanant, V., 2001. Molecular cloning and characterisation of cathepsin L encoding genes from Fasciola gigantica. Parasitol. Int. 50, 105–114. Heussler, V.T., Dobbelaere, D.A.E., 1994. Cloning of a protease gene family of Fasciola hepatica by the polymerase chain reaction. Mol. Biochem. Parasitol. 64, 11–23. Keiser, J., Utzinger, J., 2009. Food-borne trematodiases. Clin. Microbiol. Rev. 22, 466–483. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randal, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Maleewong, W., Wongkham, C., Intapan, P.M., Pipitgool, V., 1999. Fasciola giganticaspecific antigens: purification by a continuouselution method and its evaluation for the diagnosis of human fascioliasis. Am. J. Trop. Med. Hyg. 61, 648–651. Meemon, K., Khawsuk, W., Sriburee, S., Meepool, A., Sethadavit, M., Sansri, V., Wanichanon, C., Sobhon, P., 2010. Fasciola gigantica: histology of the digestive tract and the expression of cathepsin L. Exp. Parasitol. 125, 371–379. Morphew, R.M., Wright, H.A., LaCourse, E.J., Porter, J., Barrett, J., Woods, D.J., Brophy, P.M., 2011. Towards delineating functions within the Fasciola secreted cathepsin L protease family by integrating in vivo based sub-proteomics and phylogenetics. PLoS Negl. Trop. Dis. 5, e937. Panyarachun, B., Ngamniyom, A., Sobhon, P., Anuracpreeda, P., 2013. Morphology and histology of the adult Paramphistomum gracile Fischoeder, 1901. J. Vet. Sci. 14, 425–432. Phonpark, M., Srikitjakara, L., 1989. The control of parasitism in swamp buffalo and cattle in northest Thailand. In: International Seminar on Animal Health and Production Service for Village Livestock, Khon Kaen, Thailand, pp. 244–249. Raina, O.K., Yadav, S.C., Sriveny, D., Gupta, S.C., 2006. Immunodiagnosis of bubaline fasciolosis with Fasciola gigantica Cathepsin L and recombinant Cathepsin L 1-d proteases. Acta Trop. 98, 145–151. Robinson, M.W., Dalton, J.P., Donnelly, S., 2008. Helminth pathogen cathepsin proteases: it’s a family affair. Trends Biochem. Sci. 33, 601–608. Roche, L., Dowd, A.J., Tort, J., McGonigle, S., McSweeney, A., Curley, G.P., Ryan, T., Dalton, J.P., 1997. Functional expression of Fasciola hepatica cathepsin L1 in Saccharomyces cerevisiae. Eur. J. Biochem. 245, 373–380. Sansri, V., Changklungmoa, N., Chaichanasak, P., Sobhon, P., Meemon, K., 2013. Molecular cloning, characterization and functional analysis of a novel juvenilespecific cathepsin L of Fasciola gigantica. Acta Trop. 128, 76–84. Smith, A.M., Dowd, A.J., McGonigle, S., Keegan, P.S., Brennan, G., Trudgett, A., Dalton, J.P., 1993. Purification of a cathepsin L-like proteinase secreted by adult Fasciola hepatica. Mol. Biochem. Parasitol. 62, 1–8. Smooker, P.M., Whisstock, J.C., Irving, J.A., Siyaguna, S., Spithill, T.W., Pike, R.N., 2000. A single amino acid substitution affects substrate specificity in cysteine proteinases from Fasciola hepatica. Protein Sci. 9, 2567–2572. Sobhon, P., Anantavara, S., Dangprasert, T., Viyanant, V., Krailas, D., Upatham, E.S., Wanichanon, C., Kusamran, T., 1998. Fasciola gigantica: studies of the tegument

P. Anuracpreeda et al. / Acta Tropica 136 (2014) 1–9 as a basis for the developments of immunodiagnosis and vaccine. Southeast Asian J. Trop. Med. Public Health 29, 387–400. Spithill, T.W., Smooker, P.M., Copeman, D.B., 1999. Fasciola gigantica: epidemiology, control, immunology and molecular biology. In: Dalton, J.P. (Ed.), Fasciolosis. CABI Publishing, Oxon, pp. 465–525. Srihakim, S., Pholpark, M., 1991. Problem of fasciolosis in animal husbandry in Thailand. Southeast Asian J. Trop. Med. Public Health 22, 352–355. Sukhapesna, V., Tantasuvan, D., Sarataphan, N., Imsup, K., 1994. Economic impact of fasciolosis in buffalo production. J. Thai. Vet. Med. 45, 45–52. Tantrawatpan, C., Maleewong, W., Wongkham, C., Wongkham, S., Intapan, P.M., Nakashima, K., 2005. Serodiagnosis of human fascioliasis by a cystatin capture enzyme-linked immunosorbent assay with recombinant Fasciola gigantica cathepsin L antigen. Am. J. Trop. Med. Hyg. 72, 82–86.

9

Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheet: procedure and some applications. Proc. Nat. Acad. Sci. U.S.A. 76, 4350–4354. Wijffles, G.L., Panaccio, M., Salvatore, L., Wilson, L., Walker, I.D., Spithill, T.W., 1994. The secreted cathepsin L-like proteinases of the trematode Fasciola hepatica, contain 3-hydroxyproline residues. Biochem. J. 299, 781–790. Yadav, S.C., Saini, M., Raina, O.K., Nambi, P.A., Jadav, K., Sriveny, D., 2005. Fasciola gigantica cathepsin L cysteine proteinase in the detection of early experimental fasciolosis in ruminants. Parasitol. Res. 97, 527–534. Yamasaki, H., Aoki, T., 1993. Cloning and sequence analysis of the major cysteine protease expressed in the trematode parasite Fasciola spp. Biochem. Mol. Biol. Int. 31, 537–542. Yamasaki, H., Mineki, R., Murayama, F., Ito, A., Aoki, T., 2002. Characterisation and expression of the Fasciola gigantica cathepsin L gene. Int. J. Parasitol. 32, 1031–1042.