Immunoblot analysis of a 10 kDa antigen in cyst fluid of

Taenia solium metacestodes

H.-J.YANG1, J.-Y.CHUNG2, D.-H.YUN2, Y.KONG2, A.ITO3, L.MA 3, Y.-H.LIU 4, S.-C.LEE5, S.-Y.KANG5 & S.-Y.CHO6

1Biomedical Research Center, Korea Institute of Science and Technology, Seoul 136-791, Korea2Department of Molecular Parasitology, Sungkyunkwan University College of Medicine, Suwon 440-746, Korea3Department of Parasitology, Gifu University School of Medicine, Gifu 500, Japan4Institute of Infectious and Parasitic Diseases, First Affiliated Hospital, Chongqing University of Medical Sciences, Chongqing, China5Department of Parasitology, College of Medicine, Chung-Ang University, Seoul 156-756, Korea6Department of Parasitology, Catholic University of Korea School of Medicine, Seoul, 137-701, Korea

SUMMARY

The diagnostic value of a 10 kDa subunit of 150 kDa proteinin cyst fluid (CF) of Taenia soliummetacestodes wasevaluated. Immunoblot analysis revealed that most serafrom patients with neurocysticercosis recognized the10 kDa subunit strongly (209/217 cases, 84·6%), while afew sera from individuals with other parasitic diseasesincluding alveolar echinococcosis (AE, 2/20, 10%) andcystic echinococcosis (CE, 2/25, 8%) showed faint reac-tions. Sera of cases with other parasitic diseases, especiallyAE and CE, exhibited cross reactions against other bands inCF. Both differential immunoblot and immunoprecipitationanalyses showed that the 10 kDa subunit was the mostspecific to cysticercosis and highly antigenic, whereasother components at 20–40, 64, 95 and 106 kDa in CFwere cross-reactive. IgG subclass ELISA and immunoblotdemonstrated that both IgG4 and IgG1 reactions werepredominant in neurocysticercosis and recognized the10 kDa.

Keywords Taenia solium, cysticercosis, alveolarechinococcosis, cystic echinococcosis, serodiagnosis, cystfluid antigen

INTRODUCTION

Taenia soliumcysticercosis is an important cause of neuro-logic diseases in many developing countries of Asia,Africa and Latin America. It is becoming an emergingdisease in developed countries because of infected immi-grants (Schantzet al. 1992; Craig, Rogan & Allan 1996;Simanjuntak et al. 1997). The availability of effectivechemotherapy and the cost and equivocal nature of CT/MRI findings in neurocysticercosis have increased theneed for serological differentiation from other causes ofintracranial disease (Changet al. 1988).

Antigenic component proteins inT. soliummetacestodes,both in cyst fluid (CF) and in parenchymal extracts, havelong been analysed for the establishment of specific sero-diagnosis (Guerraet al. 1982, Gottstein, Tsang & Schantz1986, Larraldeet al. 1986, Choet al, 1986, Baily et al.1988). Parkhouse & Harrison (1987) and Tsanget al.(1989) purified the glycoproteins (GPs) by lentil-lectinaffinity chromatography and reported that seven bandsaround 15–30 kDa or 13–52 kDa were highly specific toneurocysticercosis. These GPs have widely been used forimmunoblot diagnosis of neurocysticercosis due to theirhigh sensitivity and specificity (Wilsonet al. 1991, reviewedby Schantz 1991).

We previously purified a thermostable 150 kDa proteinfrom CF by immunoaffinity chromatography using a mono-clonal antibody and showed that the 150 kDa protein,composed of three subunits at 7, 10 and 15 kDa, washighly specific and sensitive for serodiagnosis of neuro-cysticercosis by ELISA (Kimet al. 1986, Choet al. 1998).In the present study, we analysed the antigenic specificityof the 10 kDa subunit in a CF component protein ofT.soliummetacestodes to evaluate its reliability for differen-tial serodiagnosis of cysticercosis from other larval cestodeinfections especially from echinococcosis.

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Correspondence to: Seung-Yull Cho, Department of Molecular Para-sitology, Sungkyunkwan University, College of Medicine, Suwon 440-746, KoreaReceived: 17 July 1997Accepted for publication: 30 April 1998

MATERIALS AND METHODS

Antigen

CF of Taenia soliummetacestodes was collected from thenaturally infected pigs (Choet al. 1986). Briefly, measledpork, kept in a refrigerator, was cut into 1 cm thick slices.All the ruptured metacestodes were discarded. Intact meta-cestodes were harvested by washing in physiological saline.Unruptured metacestodes were additionally obtained byremoval from the surrounding wall. About 30% of infectedmetacestodes could be collected unruptured, after whichthey were washed with sterile saline three times and dried onfilter papers. The cysts were punctured individually with asterile lancet, and CF collected in a sterile container. Anaverage of 0·05 ml CF was obtained from an intact meta-cestode; a total of 50–500 ml CF was collected from aninfected pig depending the degree of infection intensity.Crude CF was cleared by centrifugation at 20 000g for one h,and the supernatant was used as a CF antigen, and stored at¹708C until use. All procedures were carried out at 48C.Protein content was measured by the method of Lowryet al.(1951).

Patient sera

Cysticercosis seraTwo hundred forty-seven from patients with confirmedcysticercosis were selected randomly from our sera bank.Patients were diagnosed by positive antibody reactions byELISA, either in sera and/or in cerebrospinal fluid, withconcomitant positive brain CT or MRI (Changet al. 1991).

Other patient seraTwenty sera from cases of alveolar echinococcosis (AE) and25 sera of cystic echinococcosis (CE) from China were used(Ito, Wang & Liu, 1993). In addition, each of 20 sera fromsparganosis, paragonimiosis, clonorchiosis and normal con-trols were subjected to the test. All sera samples were storedat ¹708C until use.

Immunoblot for IgG and subclasses

Sodium dodecyl sulphate polyacrylamide gel electrophore-sis (SDS-PAGE) of CF antigen and transblotting werecarried out using commercially available, precast 4–20%gradient gels (01-022 and 01-026, SDS-PAGE mini,TEFCO, Tokyo, Japan) and polyvinylidene difluoridemicroporous membrane (PVDF, Millipore, Bedford, MA,USA) as described previously (Itoet al. 1993). Blotswere incubated overnight with patient sera, diluted at1 : 200. Peroxidase conjugated anti-human IgG (heavy-and light-chain specific, Cappel, Cochranville, PA, USA)

and monoclonal antibodies against human IgG subclasses(G1, G2, G3 and G4, Zymed, San Francisco, CA, USA)were diluted at 1 : 1,000 and 1 : 500, respectively. The blotswere developed with 0·03% (w/v) 4-chloro-l-naphthol(4C1N) containing 0·03% H2O2, in phosphate buffer(0·1 M, pH 7·4).

Immunoprecipitation

Immunoprecipitation was carried out according to Konget al. (1992). CF antigen (30ml) was incubated with each of20ml serum from eight cysticercosis cases, three AEpatients, three CE patients and normal controls (pooledfrom 10 individuals) for two h at 48C, respectively. Eachof 25ml Pansorbin (Calbiochem, San Diego, CA, USA) wasadded and further incubated for two h at 48C. Immunecomplexes were washed five times with phosphate-bufferedsaline containing 0·05% Tween 20 (PBS/T, pH 7·2) fol-lowed by boiling for five min prior to analysis by 10–17·5%SDS-PAGE. The resolved proteins were transblotted toPVDF membrane and further processed by immunoblottingusing pooled serum from 10 cases of neurocysticercosis asdescribed above.

Differential immunoblot

In order to differentiate specific from cross reactive anti-gens, CF used for immunoblot was incubated after transferto PVDF membranes with either AE or CE sera (each pooledfrom 10 cases) at 1 : 100 dilution for two h and furtherincubated with unlabelled anti-human IgG at 1 : 500 dilution(heavy- and light-chain specific, Vector, CA, USA) fortwo h. Each membrane was cut into strips, and incubatedwith five sera of neurocysticercosis (1 : 100 dilutions) over-night and subsequently with peroxidase conjugated anti-human IgG (Fc specific, 1 : 1,000, Cappel, PA, USA) forover two h. The blots were developed as described above.

ELISA for IgG subclasses

A total of 80 cases of neurocysticercosis whose antibodyabsorbance value in ELISA ranged from 0·2–1·2 wasanalysed. Two hundredml of CF (protein 2·5mg/ml) wereused to coat microtitre plates (Costar, Cambridge, MA,USA). Patient sera (200ml) were diluted at 1 : 100 andmouse anti-human IgG1 (JL512), G2 (GoM1), G3 (ZG4)and G4 (RJ4) (Immunotech, Marseille, France) were dilutedat 1 : 500 and incubated for two h, respectively. Alkalinephosphatase conjugated goat anti-mouse IgG (Fc specific,Sigma, St. Louis, MO, USA), diluted at 1 : 1,000, wasincubated for one h. Substrate (1 mg/mlp-nitrophenyl phos-phate (Sigma, St. Louis, MO, USA) in 1 M diethanolamine,

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0·5 mM MgC12, (pH 9·8) and 30ml of 30% H2O2) wasapplied for 24 min (Wen & Craig, 1994). Absorbance wasmeasured at 405 nm using a Bio-Rad Microplate Reader(M3550, Bio-Rad, Hercules, CA, USA). To detect total IgG,alkaline phosphatase conjugated anti-human IgG (Sigma,St. Louis, MO, USA) was diluted at 1 : 1,000. Other proce-dures were carried out as described above.

RESULTS

Immunoblot analysis

When 247 sera from patients with neurocysticercosis wereexamined by immunoblot, they exhibited a variety of reac-tion patterns. The 7, 10, 15, 20–40, 64, 95 and 160 kDacomponents were recognized by the patient sera. The10 kDa component showed the strongest reaction, 84·6%(209 of 247 cases) being positive. Almost all other antigenicbands appeared to be cross reactive especially with serafrom AE and CE. Figure 1 shows the immunoblot findingsemploying each of 10 sera from cysticercosis (panel A), AE(panel B) and CE (panel C) patients. Sera from patients withother parasitic diseases such as sparganosis, paragonimiosisand clonorchiosis showed positive reactions with highmolecular weight proteins at 64, 95, 106 and 145 kDawhile negligible reactions were observed with low mole-cular weight proteins (data not shown). Table 1 summarizesthe outcome of the immunoblot analysis.

Immunoprecipitation

Figure 2 illustrates the immunoprecipitation results using

sera from patients with neurocysticercosis, AE or CEagainst CF. Only neurocysticercosis sera incubated withCF and Pansorbin precipitated the immunologically specificreactive bands at 10 and 15 kDa (lanes a–h). The 10 kDashowed a stronger reaction than that of 15 kDa. Sera fromAE (lanes j–l), CE (lanes m–o) and normal controls (lane i)did not show positive reactions with the 10 kDa antigen.

Differential immunoblot

As shown in Figure 3, differential immunoblot analysis inwhich AE (panel A) or CE (Panel B) sera were used to maskcross reactive antigenic proteins, demonstrated that the both

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Figure 1 Immunoblot analysis performed withten sera samples each from patients withcysticercosis (panel A), AE (panel B) and CE(panel C). Several bands show positivereactions, of which the 10 kDa (Q) exhibitsstrong and frequent reaction with sera fromcysticercosis cases but not with those from AEor CE.Mr, relative molecular mass in kDa.

Table 1 Positive reactivity of the respective bands in CF ofT. soliummetacestodes out of 247 cases of cysticercosis, 20 sera of alveolarechinococcosis (AE) and 25 sera from cystic echinococcosis (CE)

Cysticercosis AE CE

Bands at kDa No. (%) No. (%) No. (%)

106 196 (79·3) 11 (55) 9 (45)95 201 (81·4) 9 (45) 5 (25)64 183 (74·1) 10 (50) 13 (52)52 136 (47·0) 17 (85) 17 (68)34 132 (53·4) 6 (30) 3 (12)29 182 (73·7) 5 (25) 4 (16)26 186 (75·3) 4 (20) 4 (16)15 165 (66·8) 9 (45) 2 (8)10 209 (84·6) 2 (10) 2 (8)

10 and 15 kDa bands were recognized specifically, whereasother bands ranging from 20–40 kDa became negative orfaint. Recognition of the 15 kDa band was inconsistent.

IgG subclass recognition

Figure 4 shows the typical findings of IgG subclass

responses against CF using 5 sera from cases with neuro-cysticercosis (panel A–E); all reacted mainly with IgG4 andwith IgG1 in a few patients. The 10 kDa was recognizedstrongly by IgG4 (Figure 2). Mean absorbance6 SD ofELISA for IgG subclasses in 80 cases of neurocysticercosiswere; 0·216 0·14 for IgG1, 0·156 0·14 for IgG2,0·146 0·11 for IgG3, 0·506 0·38 for IgG4 and 0·596 0·27for whole IgG (cut-off absorbance for whole IgG; 0·18).

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Figure 2 Immunoprecipitation analysis ofcysticercosis, AE and CE. Only cysticercosissera (lanes a–h), incubated with CF andPansorbin, show the immunoreactive bands at15 (M) and 10 kDa (Q) with the strong reactionat 10 kDa while neither sera from AE (lanesj–l), CE (lanes m–o) nor that from normalcontrol (lane i) exhibit positive reaction at10 kDa.Mr, relative molecular mass in kDa.

Figure 3 Differential immunoblot shows the specificimmunoreactivity at 10 kDa (Q). The blots were pre-treated eitherwith pooled serum of AE (panel A) or CE (panel B) patients, afterwhich each serum from patients with cysticercosis (lanes a–e) asincubated as a primary antibody. While the reactions at the 10 kDaare strong, those against 20–40 kDa bands are diminished or negativein most patients.Mr, relative molecular mass in kDa.

Figure 4 Immunoblot findings for IgG subclasses. The CF antigenwas reacted with sera from cysticercosis patients (panel A–E).Peroxidase conjugated anti-human IgG (lanes G), G1 (lanes 1), G2(lanes 2), G3 (lanes 3), and G4 (lanes 4) were subsequently reacted.Antibody responses are shown against CF antigen mainly with IgG4and weakly with IgG1. The 10 kDa was recognized by IgG4 (Q). Mr,relative molecular mass in kDa.

DISCUSSION

In the present study, the diagnostic reliability of the 10 kDacomponent, one of the three subunits of 150 kDa proteinin CF ofT. soliummetacestodes (Kimet al. 1986, Choet al.1988) was assessed employing sera from cases with neuro-cysticercosis, AE, CE and other helminthic infections.Analyses by immunoblot, differential immunoblot andimmunoprecipitation revealed that the 10 kDa subunitwas recognized most specifically by sera of patients withneurocysticercosis and most strongly with IgG4.

It is well known that sera from AE and CE patients showfrequent cross-reactions against CF ofT. soliummetaces-todes (Al-Yaman & Knobloch 1989, Konget al. 1992,reviewed by Gottstein 1992, Craig, Rogan & Allan 1996).Our main interest was, therefore, to evaluate the sensitivityand specificity of the 10 kDa and other component proteinsin CF comparing them especially with sera of AE and CE.

Antigens for specific and sensitive serodiagnosis of cysti-cercosis have been studied by several workers (Guerraet al.1982, Gottsteinet al. 1986, Choet al. 1986, Bailyet al.1988, Larraldeet al. 1989, Tsanget al. 1989). However, theantigenic components, reported by these groups, seldomcorrelate partly due to differences in immunoblot patternsand variations in molecular mass, and partly due to differ-ences in preparation of antigenic material. Some of thesestudies have concentrated on the antigenic characterizationof T. soliummetacestodes CF (Choet al. 1986, Bailyet al.1988, Larraldeet al. 1986 & 1989).

The present study shows that patient sera gave highlyvariable reaction patterns by immunoblot at 7, 10, 20–40,64, 95, 106 and 145 kDa, of which the 10, 20–40, 64 and95 kDa components appeared to be recognized moststrongly and frequently in comparison with others. Theresponses at 64 and 95 kDa were cross reactive whenexamined with sera of AE and CE, whereas the 10 kDarevealed the least cross reactivity (Figure 1). The 95 kDacomponent has also been reported to cross-react with serafrom other helminthic infections (Olivo, Plancarte & Flisser1988). Positive reactions at 52 kDa and higher molecularmasses were not shown in Figure 1 panel A, and theirfrequencies were not matched with those in Table 1 becausewe selected an immunoblot panel which showed the bestreactions at 10 kDa. The strong antigenicity of the 10 kDaprotein was also observed by immunoprecipitation. Figure 2showed that no proteins at 20–40 kDa were captured speci-fically either by AE or CE sera. Instead, only the sera fromcysticercosis patients exhibited specific reactions against the10 kDa. This result additionally suggests that the immuneresponse against the 10 kDa was strong enough to capturethe protein by immunoprecipitation while other antigenicproteins did not bind the patient sera strongly. Furthermore,

when analysed by differential immunoblot, in which thecross reactive proteins were blocked by treating the CFantigen with AE and CE sera, immunoreactive bands ataround 20–40 kDa became weakened or diminished(Figure 3).

The approach for preparing diagnostic antigens forhuman cysticercosis was based on the presence ofcommon antigenic components in the metacestodes ofdifferent Taeniaspecies. Substitute antigens for diagnosisof bovine T. saginatacysticercosis were found in CF ofT. hydatigena(Rhoads et al. 1985) and T. crassicepsmetacestodes (Kalinna, Geyer & Walter, 1990). A 10 kDaprotein in CF ofT. hydatigenametacestodes was also shownto be specific not only for bovineT. saginatacysticercosis(Rhoadset al. 1985, Kamanga-Sollo, Rhoads & Murrell,1987) but also for human cysticercosis (Kunzet al. 1989,Hayungaet al. 1991). This genus-specific 10 kDa proteinfrom T. hydatigena, T. saginataandT. crassicepshas beencharacterized in detail including its molecular sequence byZarlenga, Rhoads & Al-Yaman (1994). However, the10 kDa in CF ofT. soliummetacestodes has not yet beenclearly described. The present study provides evidence ofT. soliummetacestode antigen, and gives one of the mostspecific components with reliability and effectiveness inserological differentiation of cysticercosis from AE and CE,thus supporting previous findings that this antigen is genusspecific. Further studies to elucidate the biochemical andmolecular biological properties, and usefulness of the10 kDa in CF of T. solium metacestodes for serologicalmonitoring of patients with neurocysticercosis are needed.

ACKNOWLEDGEMENTS

This work was supported by grants from: Korea Science andEngineering Foundation (KOSEF, 90-07-00-10) to S.-Y.Cho, Ministry of Science of Technology, Korea (Interna-tional Collaboration Project, 1995–1996) to Y. Kong, andMinistry of Education, Science, Sports and Culture, Japan(International Collaboration Project on Echinococcosis/Cysticercosis; 07044243 and 09044297) to A. Ito.

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