ORIGINAL PAPER

Serological diagnosis of clonorchiasis: using a recombinantpropeptide of cathepsin L proteinase from Clonorchis sinensisas a candidate antigen

Yanwen Li & Xuchu Hu & Xiaoquan Liu & Yan Huang &

Jin Xu & Junhong Zhao & Zhongdao Wu & Xinbing Yu

Received: 29 October 2011 /Accepted: 30 November 2011 /Published online: 16 December 2011# Springer-Verlag 2011

Abstract Clonorchiasis is a common zoonosis in southernand northeastern parts of China, especially in Guangdong,Guangxi and Jilin province. Anti-Clonorchis sinensis anti-body detection by enzyme-linked immunosorbent assay(ELISA) has been used for epidemiological surveys of clo-norchiasis for its convenience and celerity, but it is still ameaningful work to screen ideal diagnostic antigen or anti-body subtype for improvement of diagnostic sensitivity andspecificity and for judgement of curative effect. In thepresent study, recombinant CsCatL-propeptide (rCsCatL-propeptide) was highly expressed in form of inclusion bodyin Escherichia coli. Soluble rCsCatL-propeptide with highpurity were obtained after purification in denatured condi-tion by using His Bind Purification kit, and then renatured.The major antibody subtypes responding to rCsCatL-propeptide in sera from clonorchiasis patients were IgG1and IgG4, but the level of IgG4 was more predominant(P<0.05). The sensitivity of specific IgG4 detection (91.7%)

was statistically significantly higher than that of IgG1 (25.0%)with rCsCatL-propeptide (P<0.01). The specificities of IgG1and IgG4 detection with rCsCatL-propeptide were 83.3% and88.5%, respectively, and the difference between them was notstatistically significant (P>0.05). Cross-reactions took placewhen we detected IgG1 of sera from patients infected withSchistosoma japonicum, Paragonimus westermani, hook-worm, Trichuris trichiura and Ascaris lumbricoides withrCsCatL-propeptide, while cross-reactions only took place insera from patients infected with S. japonicum and P. wester-mani when we detected specific IgG4. The positive rate ofIgG4 detection in sera from clonorchiasis patients with<1,000, 1,000–4,999, 5,000–9,999, and ≥10,000 eggs per gramfaeces (EPG) were 76.9%, 89.3%, 95.6%, and 100.0%, respec-tively. The positive rates of serodiagnosis correlated well withthe EPG (r00.93). Overall, rCsCatL-propeptide is a valuablecandidate for specific IgG4 detection in sera from clonorchiasispatients by the method of ELISA for its few cross-reaction andacceptable sensitivity. In addition, specific IgG4 detection canbe used to valuate infected degree and therapeutic effect ofclonorchiasis patients.

Introduction

Clonorchis sinensis, the Chinese liver fluke, is the mostimportant food-borne parasite with the highest prevalencein Southern and Northeastern China. It is estimated to infect35 million people worldwide, among which more than 12million are in China mainland (Lun et al. 2005). Clonorchiasiscaused by C. sinensis can led to severe hepatobiliary compli-cation, and also have a close relationship with cholangiocar-cinoma (Choi et al. 2004; Lim et al. 2006). Based on the abovedescription, clonorchiasis has become an important publichealth problem in China.

Y. Li :X. LiuDepartment of Parasitology, Guangxi Medical University,Nanning 530021, China

Y. Li :X. Hu :Y. Huang : J. Xu : J. Zhao : Z. Wu :X. Yu (*)Department of Parasitology, Zhongshan School of Medicine,Sun Yat-Sen University,Guangzhou 510080, Chinae-mail: yuhxteam@163.com

X. Hu :Y. Huang : J. Xu : J. Zhao : Z. Wu :X. YuKey Laboratory of Tropical Disease Control(Sun Yat-Sen University), Ministry of Education,Guangzhou, China

Y. HuangSchool of Public Health, Sun Yat-Sen University,Guangzhou 510080, China

Parasitol Res (2012) 110:2197–2203DOI 10.1007/s00436-011-2749-x

Since praziquantel has a very good effect in expelling C.sinensis from human body, early diagnosis and standardizedtreatment could contribute to effective control of clonorch-iasis. The methods for its laboratory diagnosis include path-ogenic diagnosis, immunological diagnosis, and molecularbiological diagnosis, which has not been established yet inmany primary hospitals in China. Egg detection in feces isreliable but laborious. Additionally, the cases with slightinfection or early infection usually are missed diagnosiswhen using this method. With the advances in immunolog-ical diagnostic methods, the detection of specific antibody isused routinely for clonorchiasis, especially for epidemiolog-ical surveys. Genetic engineering antigen not only solves thesource of diagnostic antigen but also elevates sensitivity andspecificity (Kang et al. 2001; Lee et al. 2003; Zhao et al. 2004).

Cysteine proteases from parasites play a key role in immuneevasion, excystment/encystment, and host tissue invasion.They are unusually immunogenic and have been exploited asserodiagnostic markers and vaccine candidates (Sajid andMcKerrow 2002). Cathepsin L, one of cysteine proteinases,has been found in many parasites. It is secreted from all stagesof the developing parasites and is highly antigenic to hosts(Sajid and McKerrow 2002). Reportedly, some studies showedthat the diagnostic sensitivity and specificity of human para-gonimiasis (Yun et al. 2000), fascioliasis (Cornelissen et al.2001), schistosomiasis (Dalton et al. 1996) and clonorchiasis(Nagano et al. 2004) were higher when using recombinantcysteine proteinase as an antigen than when using adult crudeantigens. In a previous study, a new cathepsin L gene from C.sinensis (CsCatL) was identified, and the recombinant CsCatLwas expressed and purified; it was confirmed that CsCatLlocalized at adult intestine and was a component of excreto-ry–secretory products (ESPs) from C. sinensis (Li et al. 2009).Moreover, bioinformatic analysis of CsCatL (Hu et al. 2008)showed that it comprised signal peptide, propeptide and maturepeptide, and the homology of the propeptide compared withthat of other species is lower than the mature peptide, and thereare three main linear B cell epitopes in the propeptide. Thesedata showed that the propeptide should be of higher antigenic-ity and specificity. In the present study,CsCatL-propeptide wasexpressed and purified. As a diagnostic antigen, its specificityand sensitivity were first evaluated by enzyme-linked immuno-sorbent assay (ELISA).

Methods

Identification of CsCatL from cDNA libraryof adult C. sinensis

From 1,775 Unigenes obtained from cDNA plasmid libraryby expression sequence tags (EST) strategy, an EST clone(No.cs002d09) encoding a cathepsin L-like protease was

identified by BLASTx. The structure and character ofCsCatL were predicted by bioinformatic analysis (Hu et al.2008). The sequence was submitted to GenBank and wasassigned the accession number DQ909016.

PCR amplification

No. cs002d09 plasmid (100 ng) as template was added to apolymerase chain reaction (PCR) mix (50 μl final volume)containing 2 mM MgCl2, 250 μM each dNTP, 1.5 U Taqpolymerase (Invitrogen) and 0.4 μM specific primers (5′-AAACATATGGATCCGCTGCCTGTCCCA-3′ and 5′-GCCGGATCCCTACAATTCGCTGTTTGT-3′, respectively).After 5 min incubation at 95°C, samples were subjected to 35cycles of 30 s at 94°C, 45 s at 55°C and 30 s at 72°C, followedby an extension period of 10 min at 72°C. Amplicons werevisualized in ethidium bromide-stained 0.8% agarose gels andsized by comparisonwith a 1-kb Plus DNA ladder (Invitrogen).

Expression and purification of recombinant CsCatLpropeptide

The encoding sequence of CsCatL-propeptide amplified byPCR from No. cs002d09 plasmid was purified using GFXPCR DNA and Gel Band Purification kit (SBS GenetechCo. China). After digestion with NdeI and BamHI, therestriction fragments of purified PCR products and pET28a(+) vector (Novagen) were purified and linked with T4ligase. DH5α cells were transformed with the recombinantvector CsCatL-propeptide-pET28a (+), and the correct read-ing frame of the recombinant construct was verified bysequencing. Escherichia coli BL21 (DE3; Promega) cellswere transformed with rCsCatL-propeptide-pET28a(+) andexpression was induced with 1 mM IPTG at 28°C in LBmedium containing 50 μg/ml Kanamycin for 5 h. Cells werecentrifuged and disrupted by ultrasonication in 20 mM Tris–HCl buffer (pH 8.0). The lysate were solubilized completelywith 6 M urea in 20 mM Tris–HCl buffer (pH 8.0), purifiedin denatured condition by His Bind Purification kit (Nova-gen), and eluted with 200 mM imidazole. Renaturation wasfollowed with dialysis through stepwise dilution of urea in20 mM Tris–HCl, 5 mM EDTA buffer (pH 8.0). The puri-fication efficiency was analyzed by SDS-PAGE (12% gel)and stained with Coomassie blue, and the concentration ofrecombinant CsCatL-propeptide was determined using theBradford assay.

Crude extract of C. sinensis

Adult C. sinensis were collected from experimental ratsinfected with the metacercariae, homogenized in PBS (pH7.4) and then centrifuged at 10,000×g for 20 min at 4°C.

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The supernatant containing crude antigens was stored at−80°C.

ELISA

Sera from healthy people and patients infected with differentparasites including C. sinensis, Schistosoma japonicum,Paragonimus westermani, hookworm, Ascaris lumbri-coides, and Trichuris trichiura were collected. They wereconfirmed by microscopic examination of feces. All serawere stored at −20°C.

The wells of a micro-ELISA plate (Corning) were coatedwith 0.5 μg of C. sinensis crude extract and rCsCatL-propeptide in 0.05 M bicarbonate buffer (pH 9.6, 100 μl/well) overnight at 4°C, respectively. After washed with PBScontaining 0.05% Tween 20, the microplates were blockedwith 200 μl of PBS containing 1% BSA for 2 h at 37°C. Thesera (dilution: 1:200 for IgG; 1:5 for IgG1, IgG2a, IgE andIgA) were added to the wells (100 μl/well) as primaryantibody. The plates were incubated for 2 h at 37°C. Afterthe microplates were washed, the secondary antibody, HRP-conjugated anti-human IgG (Boster, 1:25,000) or HRP-conjugated anti-human IgG1, IgG2a, IgG4, IgE, IgA(Southern Biotech; 1:2,000) was added to the wells(100 μl/well) and incubated for 1 h at 37°C. All antibodieswere diluted with PBS containing 0.1% BSA. After washingwith PBS containing 0.05% Tween 20 and adding TMB(BD Biosciences; 100 μl/well) to the wells, the color wasdeveloped for 10 min and the reaction was stopped by 50 μl/well of 2 M H2SO4. Absorbance value of each well wasmeasured at a wavelength of 450 nm on ELISA reader(Labsystems Dragon, Multiskan MK3).

The cutoff point was determined by adding 3 standarddeviations (SD) to the mean absorbance value of the controlgroup. The results were analyzed by statistical soft of SPSSversion 11. A value of P<0.05 was considered statisticallysignificant.

Western blotting analysis

rCsCatL-propeptide (5 μg) were subjected to SDS-PAGE(12% gel), and electrotransferred to PVDF membrane(Qbiogene). The membranes were blocked with PBS (pH7.4)–0.05% Tween 20 (PBS-T) containing 1% BSA over-night at 4°C and then probed with sera (1:200 diluted with0.1% BSA in PBS-T) from patients infected with C. sinen-sis, S. japonicum, P. westermani, hookworm, A. lumbri-coides and T. trichiura, respectively. After they were washedwith PBS-T, membranes were incubated with HRP-conjugated anti-human IgG (dilution: 1:2,000) for 1 h at roomtemperature. After rewashing, the color of membranes wasdeveloped with the diaminobenzidine solution (Boster) assubstrate.

Results

Expression, purification, and renaturationof rCsCatL-propeptide

rCsCatL-propeptide is 130 amino acids in length, and itstheoretical MW is 14.9 kDa (Fig. 1, lane 2). It is highlyexpressed in inclusion body with a 6×His tag at the Nterminus. After purification, rCsCatL-propeptide showed asingle band in SDS-PAGE as expected (Fig. 1, lane 3), andwas soluble in PBS after renaturation.

ELISA examination of specific IgG in sera from patientsinfected with helminthes by using rCsCatL-propeptide

The sensitivity and specificity of crude extract from C. sinen-sis adult worm were 94% and 78.3%, respectively. The cross-reaction existed not only in sera from patients infected withtrematodes but also in sera from patients with nematodeinfection by using crude extract as antigen (Table 1). The poorsensitivity observed when we employed rCsCatL-propeptideto detect specific IgG in sera from patients infected withhelminthes including C. sinensis by ELISA was attributed tothe high background of the IgG in sera from healthy people(Table 1). CsCatL-propeptide could be probed by sera frompatients infected with different helminth with S. japonicum,P. westermani, hookworm and T. trichiura (Fig. 2) usingWestern blotting.

ELISA examination of specific antibody subtyperesponding to rCsCatL-propeptide in serafrom clonorchiasis patients

The level of specific IgG4 (A45000.327±0.146) and IgG1(A45000.186±0.076) responding to rCsCatL-propeptide insera from clonorchiasis patients increased compared withthat of healthy people, while the A450 values of those were

Fig. 1 Analysis of expression and purification of rCsCatL-propeptide by12% SDS-PAGE stained with Coomassie brilliant blue. Lane 1, lysate ofE. coliwith pET28a (+) after IPTG induction; lane 2, lysate of E. coliwithrCsCatLpropeptide-pET28a(+) after IPTG induction; lane 3, purifiedrecombinant rCsCatL-propeptide

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0.074±0.054 and 0.039±0.026 in healthy people. The level ofspecific IgG4 and IgG1, especially the former, showed astatistically significant difference (P<0.01) between clonorch-iasis patients and healthy people, and there was significantdifference (P<0.05) in increased degree between specificIgG4 and IgG1.

The level of specific IgG2a (A45000.023±0.039) and IgA(A45000.025±0.022) responding to rCsCatL-propeptide in-creased slightly in sera from clonorchiasis patients comparedwith that of healthy people, while the A450 values of thosewere 0.005±0.011 and 0.007±0.016 in healthy people. Therewas no significant differences (P>0.05) between the twogroups. The level of specific IgE responding to rCsCatL-propeptide was too low to detect in sera from either clonorch-iasis patients or healthy people (Fig. 3).

Sensitivity and specificity of specific IgG4 and IgG1detections in sera from patients with helminthesinfection by using rCsCatL-propeptide

Sera from patients infected with C. sinensis (n024), S. japo-nicum (n012), P. westermani (n012), hookworm (n012), T.trichiura (n012), A. lumbricoides (n012), and healthy peopleas the control (n012) were employed to analyze the diagnosisvalue of rCsCatL-propeptide.

The sensitivity level of specific IgG1 and IgG4 detectionwith rCsCatL-propeptide was 25.0% (6/24) and 91.7%(22/24), respectively. There was statistically significant(P<0.01) difference between IgG1 detection and IgG4 de-tection. The specificity level of specific IgG1 and IgG4detection with rCsCatL-propeptide was 83.3% (65/78) and88.5% (69/78). The difference was not statistically signifi-cant (P>0.05). Cross-reactions took place when we detectedIgG1 of sera from patients infected with S. japonicum, P.westermani, hookworm, T. trichiura and A. lumbricoideswith rCsCatL-propeptide. Moreover, cross-reactions onlytook place when we detected IgG4 in sera from patientsinfected with S. japonicum and P. westermani. There was nocross-reaction when we detected not only IgG1 but alsoIgG4 of sera from healthy people with rCsCatL-propeptide. Additionally, the A450 value of background ofIgG4 detection was lower than IgG1 detection withrCsCatL-propeptide using ELISA (Fig. 4).

Table 1 ELISA examination of specific IgG in sera from patients infected with helminthes using rCsCatL-propeptide or crude extract from C.sinensis adult worm

Groups No. of samples Adult worm crude extract rCsCatL-propeptide

Mean ± SD Positive rate (%)a Mean ± SD Positive rate (%)b

C. sinensis 120 0.346±0.045 78.3 0.384±0.039 0.8

S. japonicum 30 0.242±0.050 6.7 0.388±0.047 6.7

P. westermani 14 0.257±0.075 14.3 0.402±0.050 14.3

Hookworm 9 0.215±0.069 11.1 0.323±0.023 0.0

T. trichiura 6 0.316±0.009 16.6 0.395±0.028 0.0

A. lumbricoides 6 0.203±0.028 0.0 0.275±0.020 0.0

Healthy people 35 0.192±0.042 0.0 0.360±0.034 0.0

a The cutoff value is 0.318b The cutoff point is 0.462

Fig. 2 Western blotting analysis of rCsCatL-propeptide. Western blot-ting analysis of rCsCatL-propeptide probed with sera from patientsinfected with C. sinensis (lane 1), S. japonicum (lane 2), P. westermani(lane 3), hookworm (lane 4), T. trichiura (lane 5), A. lumbricoides(lane 6), and healthy people (lane 7), respectively

Fig. 3 ELISA analysis of specific antibody subtype responding torCsCatL-propeptide in sera from clonorchiasis patients. The level ofspecific IgG4, IgG1, IgG2a, IgA and IgE in sera from clonorchiasispatients were detected with rCsCatL-propeptide and compared withthat of healthy people

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ELISA examination of specific IgG4 in sera fromclonorchiasis patients with different degrees of infectionby using rCsCatL-propeptide

A total of 101 sera samples from clonorchiasis patients weredivided into four groups according to the EPG of patients(Table 2). The total positive rate was 90.1%. Among thefour groups, the A450 value and positive rate of specific IgG4detection in sera from patients in <1,000 EPG group werethe lowest, and those in sera from patients in ≥10,000 EPGgroup were the highest. There was a statistically significantdifference (P<0.05) between the two groups. The A450 valueand positive rate of serodiagnostic ELISA correlated wellwith the EPG of patients (r00.93) (Fig. 5).

Discussion

In a previous study, researchers filtered assembled ESTsequences from C. sinensis transcriptome (CsAEs) using theABCpred server with default parameters and then removedproteins homologous to mammalian and nuclear proteins.Cathepsin L-like cysteine proteinase was one of 19 CsAEsidentified as putative candidates with good antigenicity afterscreening (Yoo et al. 2011). Gathering the information withthe addition of lower homology of CatL-propeptide comparedwith that of other species than the mature peptide, we analyzedthe diagnostic value of CatL-propeptide in the present study.

Our data showed that the major antibody subtypes in seraof clonorchiasis patients responding to rCsCatL-propeptidewere IgG1 and IgG4, especially the latter. The specificIgG2a and IgA responding to rCsCatL-propeptide increasedslightly in patients’ sera compared with those in healthypeople. Specific IgE had not been detected in sera fromclonorchiasis patients and healthy people. Perhaps therewas no specific IgE induced by CsCatL-propeptide in thepatients, or the concentration of IgE induced by CsCatL-propeptide was too low to detect.

Prominent increase of IgG4 antibodies in serum takesplace in several kinds of human helminthiases, such asschistosomiasis (Naus et al. 2003), ascariasis (Santra et al.2001), hydatid disease (Aceti et al. 1993), and cysticercosis(Huang et al. 2002). IgG is the major responding antibodytype in sera from clonorchiasis patients (Lin et al. 1995; Yenet al. 1992). Polysaccharose and phosphocholine of hel-minthes were mainly responsible for the cross-reaction ofserodiagnosis. The variable region of IgG1, IgG2 and IgG3can bind antigenic determinant consisting of the polysac-charose complex so that there are more cross-reactions ofserodiagnosis when they are used as detected antibodies.

Fig. 4 ELISA examinations ofIgG1 and IgG4 in sera frompatients with helminthesinfection withrCsCatLpropeptide. 1, Healthypeople (n033); 2, C. sinensis(n024); 3, S. japonicum(n012); 4, P. westermani(n012); 5, hookworm (n09);6, T. trichiura (n06); 7, A.lumbricoides (n06)

Table 2 ELISA sensitivity of IgG4 detection in sera from clonorch-iasis patients with different EPG with rCsCatL-propeptide

EPG grade No. ofsamples

Mean ± SD Positiverate (%)a

<1,000 EPG 26 0.343±0.181 76.9*

1,000–4,999 EPG 28 0.383±0.148 89.3

5,000–9,999 EPG 23 0.409±0.183 95.6

≥10,000 EPG 24 0.488±0.114 100.0*

Control 35 0.050±0.039 0.0

*P<0.05a The cutoff value is 0.167

Fig. 5 Specific IgG4 detection in sera from clonorchiasis patients withdifferent EPG with rCsCatL-propeptide. 1, Healthy people; 2 patients with<1,000 EPG; 3, patients with 1,000–4,999 EPG; 4, patients with 5,000–9,999 EPG; 5, patients with ≥10,000 EPG

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The detection specificity of IgG4 is higher than those ofIgG1, IgG2 and IgG3 because the variable region of IgG4cannot recognize polysaccharose and phosphocholine. Inour study, rCsCatL-propeptide without post-translated modi-fication of glycosylation and phosphorylation was expressedand purified in prokaryote. The sensitivity and specificity ofIgG4 detection in sera of clonorchiasis patients with rCsCatL-propeptide are superior to that of IgG1 and IgG, and this mightdue to the recognition capability between the different anti-body and different linear B cell epitopes and has nothing to dowith the recognition between polysaccharose/phosphocholineand antibodies. Considering this premise, we tried to cloneand express a single epitope of CsCatL-propeptide for specificdetection without success. Based on the results of our inves-tigation, IgG1 and IgG2a are responsible for the non-specificimmune reaction and background serological reaction, whichresult in the lower specificity and high background value.

Polysaccharide complex and phosphorylcholine of hel-minthes are responsible for the cross-reaction of serodiagno-sis. The variable regions of IgG1, IgG2 and IgG3 except IgG4can bind with the antigenic determinant of polysaccharosecomplex from worms so that there are more cross-reactionsof serodiagnosis when using them as detected antibodies. Theserodiagnostic specificity of IgG4 is higher. In our study, therecombinant protein was prokaryotic expressed, with no post-translated modifications of glycosylation and phosphoryla-tion. Given that, we inferred that the serodiagnostic sensitivityand specificity of IgG4 detection with rCsCatL-propeptideshould be higher than that of IgG1 and total IgG detection.

Anti-C. sinensis IgG4 antibody levels correlated wellwith the EPG of patients. In addition, IgG4 is a short-range antibody. Its level will reduce significantly within6 months of deworming (Hong 1988; Santra et al. 2001)so that anti-C. sinensis IgG4 antibody detection in sera fromclonorchiasis patients with CsCatL-propeptide using ELISAcan be used for serodiagnosis of clonorchiasis, based on thedegree of infection and valuation of therapeutic effect.

A specific PCR assay for the diagnosis of C. sinensisinfection in humans, cats, fish and snail was establishedusing the sequences of the internal transcribed spacers(ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA) ofC. sinensis as genetic markers (Müller et al. 2007; Huang etal. 2011). However, in many rural areas of China, PCR isdifficult to carry out because of inadequate equipment andless-than-ideal medical conditions. Our findings will behelpful for anti-C. sinensis antibody detection by ELISAfor diagnosis of clonorchiasis in those areas or for epidemi-ological surveys.

Acknowledgements The experimental work was supported by theNational Basic Research Program of China (973 program,2010CB530000), the National S & T Major Program (Grant No.2008ZX10004-011) and the China National Natural Science Founda-tion (No. 30771887).

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