Zurich Open Repository andArchiveUniversity of ZurichMain LibraryStrickhofstrasse 39CH-8057 Zurichwwwzorauzhch

Year 1991

A monoclonal antibody against Echinococcus multilocularis Em2 antigen

Deplazes P Gottstein B

Abstract A monoclonal antibody (MAb G11) species-specific to the Em2 antigen of Echinococcus mul-tilocularis was generated for (i) further biological characterization of the Em2 antigen (ii) easy affinity-purification of Em2 antigen for immunodiagnostic and immunological investigations and (iii) developmentof a sandwich-ELISA for the detection of Em2 antigen in diagnostic samples and thus species-specificidentification of E multilocularis metacestode material The MAb G11 was used in an antibody sandwich-ELISA to detect soluble Em2 antigen with a methodical sensitivity of 80 ng E multilocularis antigenmlof solution MAb G11 specifically detected Em2 antigen in all of 15 E multilocularis-isolates originatingfrom various geographical areas and in none of other helminth isolates (eg Echinococcus granulosus Evogeli and others) Further biological analysis by FITC-labelled MAb G11 demonstrated unique bindingactivity to the laminated layer of the metacestode Also oncospheres were binding FITC-labelled MAbG11 on an outer layer synthesized during cultivation in vitro for 13 days after hatching Applicationof the MAb G11 antibody sandwich-ELISA for investigation of solubilized oncospheres confirmed the invitro synthesis of Em2 antigen by oncospheres on day 13 pi Adult stages (somatic antigens) and freshlyhatched oncospheres were always MAb G11 negative Solid-phase MAb G11 was used for purificationof the corresponding Em2 antigen by affinity chromatography A preliminary serological evaluation ofthe Em2(G11) antigen by ELISA revealed identical immunodiagnostic characteristics compared to Em2obtained by classical means thus suggesting the presented method for future isolation of large-scale Em2antigen

DOI httpsdoiorg101017s0031182000059278

Posted at the Zurich Open Repository and Archive University of ZurichZORA URL httpsdoiorg105167uzh-154522Journal ArticlePublished Version

Originally published atDeplazes P Gottstein B (1991) A monoclonal antibody against Echinococcus multilocularis Em2antigen Parasitology 103(01)41-49DOI httpsdoiorg101017s0031182000059278

41

A monoclonal antibody against Echinococcus multilocularisEm2 antigen

P DEPLAZES andB GOTTSTEIN

Institute of Parasitology University of Zurich Winterthurerstrasse 266a CH-8057 Zurich Switzerland

(Received 7 November 1990 revised 25 January 1991 accepted 25 January 1991)

SUMMARY

A monoclonal antibody (MAb G i l ) species-specific to the Em2 antigen of Echinococcus multilocularis was generated for(i) further biological characterization of the Em2 antigen (ii) easy affinity-purification of Em2 antigen for immuno-diagnostic and immunological investigations and (iii) development of a sandwich-ELISA for the detection of Em2 antigenin diagnostic samples and thus species-specific identification of is multilocularis metacestode material The MAb Gl 1 wasused in an antibody sandwich-ELISA to detect soluble Em2 antigen with a methodical sensitivity of 80 ng E multilocularisantigenml of solution MAb G i l specifically detected Em2 antigen in all of 15 is multilocularis-isoates originating fromvarious geographical areas and in none of other helminth isolates (eg Echinococcus granulosus E vogeli and others)Further biological analysis by FITC-labelled MAb G i l demonstrated unique binding activity to the laminated layer ofthe metacestode Also oncospheres were binding FITC-labelled MAb G i l on an outer layer synthesized during cul-tivation in vitro for 13 days after hatching Application of the MAb G i l antibody sandwich-ELISA for investigation ofsolubilized oncospheres confirmed the in vitro synthesis of Em2 antigen by oncospheres on day 13 pi Adult stages (somaticantigens) and freshly hatched oncospheres were always MAb G i l negative Solid-phase MAb G i l was used for puri-fication of the corresponding Em2 antigen by affinity chromatography A preliminary serological evaluation of theEm2(Gl 1) antigen by ELISA revealed identical immunodiagnostic characteristics compared to Em2 obtained by classicalmeans thus suggesting the presented method for future isolation of large-scale Em2 antigen

Key words Echinococcus multilocularis Em2 antigen monoclonal antibody affinity chromatography Em2-sandwich-ELISA

INTRODUCTION

Alveolar echinococcosis of humans is caused byinfection with the metacestode (larval stage) ofEchinococcus multilocularis Early serological detec-tion and treatment of persons with alveolar echino-coccosis may reduce mortality (Schantz amp Gottstein1986) By the time the disease becomes clinicallymanifest the lesions have often progressed so thatsurgical complete resection is impossible In aprevious study we showed that a purified antigenicpolypeptide (antigen Em2) from E multilocularisdemonstrated immunodiagnostic characteristics suit-able to detect by ELSA early cases of alveolarechinococcosis among large groups of persons livingin endemic areas (Gottstein Schantz amp Wilson1985 Gottstein et al 1987) This was attributed tothe operating characteristics (diagnostic sensitivity94 deg 0 specificity 100 deg0 for non-Echinococcus re-actions 94 deg0 with respect to Echinococcus granulosusinfections) defining the Em2-ELISA (Gottstein1985) the test furthermore allowed a reliable

Reprint requests to Dr B Gottstein Institute ofParasitology University of Zurich Winterthurerstrasse266a CH-8057 Zurich Switzerland

This publication is dedicated to Professor J Eckert on theoccasion of his 60th birthdav

discrimination of human cases of alveolar form cysticechinococcosis (Gottstein Eckert amp Fey 1983Gottstein et al 1986) Field-application of the Em2-ELISA in the frame of sero-epidemiologicalscreening of human populations resulted in the firstpublished finding of spontaneously died-out or aborted E multilocularis lesions in human patients(Rausch et al 1987) These patients maintained ahigh anti-Em2 antibody concentration despite thefact that only non-viable calcified parasite residuesremained as lesions in the liver Interestingly anti-Em2 antibody concentrations dropped to negativerapidly after surgical resection of the liver lesion inquestion The Em2-ELISA is now being establishedas a WHO reference immunodiagnostic test foralveolar echinococcosis (WHOCDSVPH88-78)The Em2-ELISA was recently also investigated forassessing adult-stage E multilocularis infections indefinitive hosts (foxes) resulting in the hypothesisthat the presence of anti-Em2 immunoglobulinsrather reflected a limited post-oncospheral parasitedevelopment in the definitive host than the actualpresence of intestinal E multilocularis worms thusindicating a metacestode-stage specificity of theantigen (Gottstein et al 1991)

The present report describes the preparation of aspecies-specific anti-Em2 (E multilocularis) mono-clonal antibody for (i) further biological character-

Parasitology (1991) 103 41-49 Printed in Great Britain

httpswwwcambridgeorgcoreterms httpsdoiorg101017S0031182000059278Downloaded from httpswwwcambridgeorgcore University of Basel Library on 30 May 2017 at 174203 subject to the Cambridge Core terms of use available at

P Deplazes and B Gottstein 42

ization of the Em2 antigen (ii) easy purification ofEm2 antigen for immunodiagnostic and immuno-logical investigations and (iii) development andapplication of a sandwich-ELISA for the specificdetection of Em2 antigen (and thus E multilocularis)in diagnostic samples

MATERIALS AND METHODS

Experimental design

Species-specific monoclonal antibodies (MAb) weregenerated against Echinococcus multilocularis withthe following strategy (i) Primary screening withELISA of MAb with antigens reflecting species-specificity and genus-specificity including cross-reactivity and non-specificity Primary selection ofMAb was based on the specific reaction with Em2antigen (Gottstein 1985) (ii) Characterization ofEm2-binding MAb with the following techniquesand aims Sandwich-ELISA using solid-phase MAband a dot-ELISA were performed using differentkinds of parasite and control antigens Both assaysdemonstrated Em2 specificity in relation to stage-and species-specificity Further demonstration ofstage-specificity was by direct immunofluorescencewith native E multilocularis parasite material ofdifferent stages and FITC-labelled MAb (iii) Adirect comparative analysis was made by ELISA ofEm2-antigen purified with solid-phase MAb (anti-gen Em2(Gll)) versus Em2 antigen (Gottstein1985) a purified recombinant II3-10 antigenknown to be not related immunologically to Em2antigen (Muller et al 1989) and a crude Emultilocularis somatic antigen The investigation wasdone with characteristic and defined sera fromhuman patients with alveolar echinococcosis

Antigens

Metacestode tissue of a Swiss Echinococcus multi-locularis isolate (CH24) maintained in jirds (Merionesunguiculatus) by intraperitoneal transplantation(Eckert amp Pohlenz 1976) was homogenized in aPolytron PCU-2 blender and frozenthawed 3 timesusing liquid nitrogen and a +37 degC water-bathSubsequently the material was ultrasonicated (60 s40 W 80 pulse) and sedimented at 10000^ for30 min at + 4 deg C The supernatant fraction con-taining somatic antigens was used to immunize micefor monoclonal antibody production An equiva-lently processed antigen but originating from atransplant in cotton rats (Sigmodon hispidus) wasused for a first step MAb screening and pre-selectionin ELISA Purification of the species-specificE multilocularis Em2 antigen was done exactly asdescribed previously (Gottstein 1985) this Em2antigen was used in parallel for primary MAbscreening Somatic antigens were derived from thefollowing Echinococcus materials for further sero-

logical characterization of MAb (using ELISA dot-ELISA and sandwich-ELISA) (i) E multilocularismetacestodes from other isolates than that listedabove (number of isolates in parentheses) originatingfrom Switzerland (8) France (3) Alaska (1)Germany (1) and Japan (2) (ii) gravid adult stageE multilocularis tapeworms recovered from themucosa of the small intestine from a naturallyinfected Swiss fox washed 3 times with PBS prior tofurther processing (iii) E granulosus protoscolecesfrom hydatid cysts dissected from the lungs ofnaturally infected Swiss cattle (iv) E vogeli meta-cestodes from experimentally infected jirds (giftfrom Dr R L Rausch University of WashingtonSeattle) All Echinococcus somatic antigens wereprepared as described in the paragraph aboveE granulosus hydatid cyst fluid was collected fromfertile lung or liver cysts of the following host species(origin number of isolates) Cattle (Switzerland13) horse (Switzerland 2) sheep (Sardinia 1) pig(Poland 2) camel (Egypt 1) human (Switzerland5) Non- Echinococcus somatic antigens were offollowing origin (origin number of isolates) Taeniasolium cysticerci (Mexico 1 South Africa 1)Cysticerus bovis (T saginata) (Switzerland 2)T crassiceps metacestode (from experimentally in-fected BALBc mice 1) Cysticercus tenuicollis(T hydatigena) (Switzerland 1) Mesocestoides cortimetacestode (from experimentally infected BALBcmice 1) mature adult stages from Toxocara canisFasciola hepatica Dicrocoelium dendriticum controltissues from muscles and liver of human murine andbovine origin All these additional somatic antigenswere prepared exactly as described above Forobtaining excretorysecretory antigens (ES-Ag)from E multilocularis a metacestode (CH 24 isolate)cell suspension (obtained by homogenization ofmetacestode tissue in a Tenbroeks homogenizer)was cultivated for 10 days in DMEM (Gibco) with100 U penicillin G and 100 ig streptomycin per mlwithout foetal calf serum Collected culture mediumwas dialysed and concentrated using an Amiconultrafiltration unit and a YM-10 membrane Purifi-cation of the recombinant E multilocularis II3-10antigen was exactly as described by Muller et al(1989)

All protein concentrations were assessed by theBio-Rad protein assay and with bovine albumin as astandard

Hatching of eggs and activation of oncospheres for invitro cultivation

E multilocularis eggs were isolated under bio-hazardconditions from adult-stage tapeworms recoveredfrom the small intestine of naturally infected necrop-sied foxes Basically the same procedure as describedby Deplazes amp Eckert (1988) for T hydatigena wasemployed for isolation of viable E multilocularis eggs

httpswwwcambridgeorgcoreterms httpsdoiorg101017S0031182000059278Downloaded from httpswwwcambridgeorgcore University of Basel Library on 30 May 2017 at 174203 subject to the Cambridge Core terms of use available at

Monoclonal anti-Eml antibody 43

and subsequent oncosphere activation and in vitrocultivation Embryophore disruption was done in1 sodium hypochlorite solution (1 active chlor-ine concentration pH 11) for 5 min at roomtemperature Oncospheres were washed twice inPBS and once in 0-025 M HC1 (in physiological salinesolution) by sedimentation at 600 pound for 10 minActivation of the oncospheres was performed with asolution of 1 pancreatin (Fluka) 15 dog bileand 1 NaHCO3 in distilled water (sterilized by0-25 jiva pore size filtration) for 15-30 min at + 37 degCwith vigorous shaking once every 5 min Activatingsolution was removed by washing with DMEM(Flow) Activated oncospheres were cultivated in30 ml plastic tissue-culture flasks (Falcon) contain-ing a monolayer of Swiss mouse embryo (3T3) cells(Flow) in 10 ml of tissue-culture medium (DMEMwith 100 U penicillin G and 100 fig streptomycin(KC Biological) per ml with 10 foetal calf serum)The culture medium was replaced on days 5 9 and13 of cultivation The whole process was visuallymonitored in a Leitz Labovert FS microscope

Generation of monoclonal antibody MAb)

Female BALBc mice aged 6 weeks were immunizedby sc injection of 50 firm of somatic metacestodeantigen of E multilocularis (prepared as describedabove) emulsified in complete Freunds adjuvant(Difco) Three weeks later the procedure wasrepeated but with incomplete Freunds adjuvantTen days after the first boost the same amount ofantigen diluted in 200 fi of PBS was injectedintraperitoneally Three to four days after this lastboost spleen cells of mice were fused with AG8 x 63myeloma cells (provided by Dr A Metzler Instituteof Virology University of Zurich) using a 50 polyethylene glycol (PEG) 1500 (Bioproducts) sol-ution in serum free DMEM (Gibco) Fusion andcell-culture procedures were carried out as describedby De St Groth amp Scheidegger (1980)

Ten to 15 days after fusion the supernatantfractions from wells containing hybridomas werescreened by ELISA with somatic E multilocularismetacestode antigen (cotton rat isolate) and Em2antigen Supernatants positive in primary screeningwere subsequently tested in ELISA with somaticE granulosus protoscoleces T hydatigena andT solium metacestode antigen Hybridomas selectedfor species-specific anti-Em2 antibody synthesis(and others) were recloned 3 times by limitingdilution and preamplified in 30 ml plastic tissue-culture flasks (Falcon) for generation of ascites-producing tumours in BALBc mice Pristane(1 mlanimal) was injected ip into the animals 15days and 3 days prior to ip injection of lOxlO6

hybridoma cells Ascites fluid was harvested afternecropsy 2-3 weeks later and stored frozen at- 2 0 degC

Purification and isotyping of MAb

Anti-Em2 (and other) MAb were partially purifiedfrom ascites by precipitation with 50 saturatedammonium sulphate (Fleischmann Pain amp Porter1962)

MAb isotypes were determined by immuno-diffusion (mouse monoclonal typing kit TheBinding Site Ltd)

ELISA for MAb

The ELISA used for screening and selection ofMAb (from supernatants of hybridoma cell-cultures)was carried out as previously described (GottsteinEckert amp Fey 1983) with the following modi-fications The polystyrene surface of MicroELISAplates (Nunc-Immuno Plate MaxiSorp No 4-39454A) was coated with antigen (5 fig proteinantigenml) in 100l of 01 M NaHCO3Na2CO3pH 9-6 ( + 0-02 NaN3) at 4 degC overnight in ahumid chamber The wells were then washed 3 timeswith PBS+ 0-3 Tween 20 (PBSTween) andincubated with the same buffer for 20 min Super-natants were added undiluted to each well andincubated for 2 h at 37 degC After 3 washes (PBSTween) 100d of sheep anti-mouse IgG-IgM-IgA-alkaline phosphatase conjugate (The Binding SiteLtd) was incubated in a 1 500 dilution (PBSTween)for 2 h at 37 degC Further procedures for visualizationof the serological reaction corresponded to thosedescribed previously (Gottstein et al 1983)

Antigen detection by sandwich-ELISA

The polystyrene surface of MicroELISA plates wascoated with 5 ig (well) of partially purified anti-Em2 MAb G i l and control MAb G104 (Giardialamblia-specific (Aggarwal Merritt amp Nash 1989)generously provided by Dr T E Nash LPDNIAID NIH Bethesda) as described aboveSomatic- and ES-antigens of various helminthspecies and control tissues were examined for thepresence of epitopes binding uniquely to MAb G i lat a concentration of 50 figm PBS-TweenMAb G i l coupled to alkaline phosphatase from calfintestine (Grade I Boehringer Ltd) according to astandard method (Engvall amp Perlmann 1972) wasused as conjugate (1200 dilution in PBS-Tween for1 h at room temperature) for detecting primaryantibodymdashantigen reactions The conjugate wasstored as a 50 glycerol dilution at mdash20 degC

Dot-ELISA

Soluble antigen preparations were spotted as 3 fidrops (0-5 mg proteinml PBS) onto strips ofnitrocellulose (BA 85 Schleicher amp Schuell) Strips

httpswwwcambridgeorgcoreterms httpsdoiorg101017S0031182000059278Downloaded from httpswwwcambridgeorgcore University of Basel Library on 30 May 2017 at 174203 subject to the Cambridge Core terms of use available at

P Deplazes and B Gottstein 44

were air-dried at room temperature for 2 h washed3 times with PBS-Tween the last wash solution wasleft for 30 min Strips were then incubated withalkaline phosphatase-conjugated M A b G l l (1500in PBS-Tween) for 2 h at room temperatureVisualization of antigen-MAb G i l (alkalinephosphatase-labelled) complexes was performed asdescribed by Dao (1985)

Direct immunofluorescence assay

The presence of antigens reactive to MAb was alsodetermined using direct immunofluorescence Forthat purpose anti-E multilocularis M A b G l l andcontrol MAb G104 were both conjugated to fluor-escein isothiocyanate according to standard pro-cedures Freshly collected (from naturally or ex-perimentally infected animals) adult-stage E multi-locularis tapeworms E multilocularis metacestodes(protoscoleces and germinal layer including lamin-ated layer) and E multilocularis oncospheres afterhatching and E multilocularis oncospheres after13 days of in vitro cultivation were all washed 3 timesin PBS prior to incubation (at 4 degC) with FITC-labelled M A b G l l or control MAb G104 (120final dilutions) for 30 min After repeated washes(3 times with PBS) the fluorescence was monitoredwith an Olympus BH-2 microscope and photo-graphed onto Kodak Ektachrome EPY-50 film

Affinity purification of antigens

Partially purified ascites (MAb G i l and controlMAb G104) was coupled to CNBr-activated Seph-arose 4B according to the manufacturers instruc-tions (Pharmacia Fine Chemicals) Affinity chrom-atography was performed as described previously byBaumann amp Gottstein (1987) Briefly somatic meta-cestode antigen (05 mg) from E multilocularis(CH10 isolate) was incubated with the antibody-Sepharose (5 ml of slurry gel previously equilibratedwith PBS containing 0-5 M NaCl) for 2 h at roomtemperature by slowly rotating the chromatographycolumn After washing the gel thoroughly with PBS(0-5 M NaCl) the bound protein was eluted with5 M MgCl2 The eluate was dialysed against PBS andconcentrated by ultrafiltration as described abovestorage was at mdash80 degC until use The eluate obtainedfrom solid-phase G i l MAb was designatedEm2(Gll)

Sera

The sera used for analytical and comparative investi-gations of different E multilocularis antigens wereobtained from 30 patients with clinically parasito-logically or histologically proven alveolar echino-coccosis (Echinococcus multilocularis) The group wasstatistically matched by sex and age All sera were

subjectively pre-selected from previous studies ac-cording to their known and quantitatively varyingreactivity with Em2-antigen The data resultingfrom the present study thus do not reflect operatingcharacteristics of antigens such as diagnostic sen-sitivity

ELISA for human serum antibodies

All sera listed above were simultaneously tested inparallel runs with ELISA using E multilocularissomatic antigen (CH10 isolate) Em2 antigen (Gott-stein 1985) Em2(Gll) and recombinant E multi-locularis II3-10 antigen (Muller et al 1989) Thelatter antigen was included as a control E multi-locularis antigen not related to Em2 The ELISAtechnique employed corresponded to that describedpreviously (Muller et al 1989)

RESULTS

Generation of hybridomas and selection of MAb

Five fusions were performed Supernatants (cellculture medium taken from growing hybridomacells) were tested in ELISA with somatic antigenfrom E multilocularis metacestodes (cotton ratisolate) All in all 26 supernatants were consideredantibody-positive (Ai at least gt10 times thenegative control value) Subsequently positivesupernatants were tested in ELISA with Em2antigen (Gottstein 1985) and also with E granulosushydatid cyst fluid (bovine isolate) and somaticantigens derived from T hydatigena and T saginatametacestodes One single cell line out of the 26positive showed a strong reaction with all antigensinvestigated and thus was disposed as a non-specificMAb-producing cell line All other 25 cell linesshowed strong reactivity to Em2 antigen in ELISAand 10 from them were also positive withE granulosus hydatid cyst fluid antigen but negativewith T hydatigena and T saginata metacestodeantigen

One MAb (Gi l IgGj isotype) which reacteduniquely with crude E multilocularis metacestodesand purified Em2-antigen (therefore assumed to beE multilocularis species-specific) was selected forfurther cloning and production of ascites fluid

MAb Gil binding properties in sandwich-ELISAand dot-ELISA

A solid-phase M A b G l l sandwich-ELISA was usedas a first step to establish the operating characteristicssuch as specificity (Fig 1) and methodical sensitivity(Fig 2)

Specificity of the assay was tested by investigating15 isolates of E multilocularis versus 24 isolates ofE granulosus 1 isolate of E vogeli and various non-

httpswwwcambridgeorgcoreterms httpsdoiorg101017S0031182000059278Downloaded from httpswwwcambridgeorgcore University of Basel Library on 30 May 2017 at 174203 subject to the Cambridge Core terms of use available at

Monoclonal anti-Eml antibody 45

1

II

III

IV

V

0

Echinococcus multilocularis(somatic antigens)

E granulosus (cyst fluid)

E vogeli (somatic antigens)

Non- Echinococcus(somatic antigens)

Cut-off point determinedby combining II and IV

^405 nm

0-25 0-5 0-75 10 1-25 1-5 1-75

ndegQ n bdquo bdquo raquo jj deg n

| X + 3 SD

Fig 1 Specificity of MAb G i l sandwich-ELISA with antigens tested at a concentration of 50 ig proteinml Echinococcus multilocularis 15 isolates originating from Switzerland France Alaska Germany Japan and Canada E granulosus hydatid cyst fluids from bovine (n = 13) equine (n = 2) porcine (n = 2) ovine (n = 1) camel (n = 1)and human (n = 5) origin E vogeli somatic metacestode antigen IV Non-Echinococcus somatic antigens werefrom Taenia solium cysticerci (n = 2) T saginata cysticerci (n = 3) T crassiceps metacestodes (n = 1) T hydatigenametacestodes (n = 2) Mesocestoides corti metacestodes (n = 1) Adult-stage somatic antigens were from Toxocara canisFasciola hepatica and Dicrocoelium dendriticum control extracts from muscle and liver of human mice and cattleorigin V The cut-off threshold determining the lower resolving limit was calculated on the basis of yi405nm valuesfrom and IV

1-6-

1-4

1-2-

1-0 bull

0-8

0-6

0-4

0-2

0 mdash bull bull - bull bull - bull bull - bull bull - bull -amdashamdashQmdashp--

800 200 50 12-5 3-1 0-8 0-2 005 001Antigen in test solution (^gml)

Fig 2 Titration of ( bull ) Echinococcus multilocularissomatic antigen ( bull ) E granulosus hydatid cyst fluidantigen and ( bull ) Taenia solium cysticercus somaticantigen by MAb G i l sandwich-ELISA E multilocularissomatic antigen was also controlled by irrelevantMAb G104 sandwich-ELISA (O) The lower resolvinglimit corresponds to that shown in Fig 1

Echinococcus helminth and control antigens (listed indetail in Fig 1) The assay was controlled bysimultaneous testing on solid-phase irrelevantG104 MAb The respective control values (neverexceeding 0-02 Aiob nm for all non-pound multilocularisantigens and 0-20 AM6 nm for all E multilocularis

antigens) were individually subtracted from thevalue obtained with the specific solid-phaseMAb G i l The data show that all E multilocularisisolates were clearly detected by the present sand-wich-ELISA and specificity was 100 due toabsolutely no binding activity with any antigensderived from other Echinococcus species otherhelminths or control tissues All antigen concen-trations had been equivalently adjusted to 50 igproteinml after optimizing test parameters anddetermining methodical sensitivity (see Fig 2)Arbitrarily we selected a positivenegative thresholdcharacterized by a cut-off ^4405nm-value set atJC + 3SD from all non-Zs multilocularis isolates Themethodical sensitivity was determined by plottingtitration values obtained with primary solid-phaseMAb G i l and values from solid-phase irrelevantcontrol MAb G104 including sequentially hom-ologous E multilocularis metacestode antigen andMAb Gil-alkaline phosphatase conjugate in thisreaction (Fig 2) Based on this experiment the finalmethodical sensitivity was depicted as a concen-tration of 80 ng E multilocularis proteinml testsolution For demonstration of insignificant back-ground-reactivity the figure includes also E granu-losus and T solium antigens tested on solid-phaseMAb G i l data of the corresponding control re-actions with G104 are not shown as values neverexceeded 0-02v4405nm Testing titrated E multi-locularis antigen with solid-phase controlMAb G104 showed some non-specific reaction atconcentrations larger than 13 fig proteinml These

httpswwwcambridgeorgcoreterms httpsdoiorg101017S0031182000059278Downloaded from httpswwwcambridgeorgcore University of Basel Library on 30 May 2017 at 174203 subject to the Cambridge Core terms of use available at

P Deplazes and B Gottstein 46

^ V laquo ^ Methods

Antigens ^ ^ ^

Dot-ELISA

1 Echinococcus multilocularis

Metacestode somatic antigen

Metacestode ES-antigen

Adult (gravid) somatic antigen

Oncosphere somatic antigen

Oncosphere (cultivated 13 daysin vitro) somatic antigen

II Purified E multilocularis antigens

Em2

Em2(G11)

Em 113-10

III Control antigens

Mouse liver somatic antigen

Mouse serum

Calf serum

Culture medium

E granulosus protoscolexsomatic antigen

Sandwich ELISA

ControlMAb MAbG11 G104

A405 nm

0-887 0-117

0-811 0023

0002 0004

0002 0004

0-710 0050

0-785 0051

0-516 0008

0002 0002

0002 0007

0006 0006

0001 0002

0004 0016

0002 0006

Fig 3 Specificity of dot-ELISA and sandwich-MAb Gi l - and control MAb G104-ELISA

values reflecting minor non-specificity of E multi-locularis antigen components (they were never ob-served with any other antigens tested) were subse-quently corrected by subtraction similar to thecorrections performed in Fig 1

Analysis of stage-specificity by sandwich-ELISAand dot-ELISA (Fig 3 ) revealed that only antigensderived from the metacestode provided epitodeswith MAb Gil-binding properties No reactivity ofMAb G i l was observed with antigens from adult-stage tapeworms and with oncospheres freshlyhatched from E multilocularis eggs MAb G i l however was able to react with antigen derived fromoncospheres cultivated in vitro for 13 days Forfurther analytical characterization of the antigenwith MAb Gil-binding epitope(s) the previouslypurified Em2 antigen (Gottstein 1985) and therecombinant II3-10-antigen (Muller et al 1989)known to be not related to Em2 were included in the

investigation The respective results shown inFig 3 confirm the Em2 specificity of MAb Gl 1 orin other words proving that the Em2 antigen carriesthe MAb Gil-binding epitope As expected therecombinant 113-10 antigen as a negative controlexhibited no binding activity to MAb G i l Inparallel to the sandwich-ELISA the correspondingimmunological reactions were tested by dot-ELISAwith the same antigens but in solid-phase Sero-logical results were qualitatively identical to thoseobtained in sandwich-ELISA

All control antigens (Fig 3 ) showed no bindingproperties in the MAb G i l - and controlMAb G104-sandwich-ELISA

httpswwwcambridgeorgcoreterms httpsdoiorg101017S0031182000059278Downloaded from httpswwwcambridgeorgcore University of Basel Library on 30 May 2017 at 174203 subject to the Cambridge Core terms of use available at

Monoclonal anti-Enil antibody

A - - bull bull bull - bull -

50 Mm

47

Fig 4 Direct immunofluorescence analysis of the localization of the MAb Gil-binding Em2 antigen (A) MAb Gll-FITC-labelled native Echinococcus multilocularis tissue under normal light (B) The same as in (A) but underfluorescence (C and D) MAb Gl 1-FITC-labelled native oncosphere in vitro cultivated for 13 days under normallight (D) Magnified section of (C) (E) The same as (D) but under fluorescence

Characterization of MAb Gil binding properties byfluorescence microscopy

Fluorescence microscopy analysis showed that fromnative E multilocularis metacestode tissues only thelaminated layer was stained by MAb G i l with arelatively strong surface fluorescence (Fig 4) Nativeprotoscoleces did not appear to bind MAb G i l nordid adult-stage E multilocularis tapeworms or onco-spheres freshly hatched from E multilocularis eggs(data for the latter two not shown) Oncospherescultivated in vitro developed into spherical organismswith a surrounding thin layer This layer demon-strated specific binding of MAb G i l at day 13 of invitro cultivation

Control reactions performed with irrelevantMAb G104 revealed that all parasite tissues investi-

gated did not non-specifically bind irrelevant mouseIgG

Overall the fluorescence microscopy analysis wasin perfect agreement with the results from sandwich-and dot-ELISA

ELISA with Em2(Gll)-antigen

In order to determine the immunodiagnosticcharacteristics of the affinity-purified Em2(Gll)antigen an analysis was performed by ELISA incomparison with other E multilocularis antigenslisted below and in Fig 5 The results of investi-gating sera from human patients with alveolarechinococcosis clearly revealed similar operatingcharacteristics for Em2(Gll) and Em2 (Gottstein1985) antigens demonstrated by the relatively high

httpswwwcambridgeorgcoreterms httpsdoiorg101017S0031182000059278Downloaded from httpswwwcambridgeorgcore University of Basel Library on 30 May 2017 at 174203 subject to the Cambridge Core terms of use available at

P Deplazes and B Gottstein 48

0-6-

0-4 -

0-2 -

n -

r =

bull

GBB

0-87

bull ID

degdeg

P

AD

D

G

lt= 1 - 2 -

O)

1 1-0-o| 0-8 -o

Z 0-6 -EuiE 0-4 -c

O

^ 0 - 2 -

0 -

r=0-23

H bull

D

bull

D

a

Gbull G

D

bull

B

20 -

CDO)

CID

o 1-5 -

0-5 -

0 0-2 0-4 0-6^405 nm Em2(G11) antigen

Fig 5 Direct comparative analysis by ELISA of Em2antigen purified with solid-phase MAb (antigenEm2(Gll)) versus (A) Em2 antigen (Gottstein 1985)(B) a crude Echinococcus multilocularis somatic antigenand (C) a purified recombinant 113-10 antigen knownto be unrelated immunologically to Em2 antigen (Mulleret al 1989) the investigation was done with defined serafrom 30 human patients with alveolar echinococcosis(see Material and Methods section)

correlation coefficient (r = 0-87) Crude E multi-locularis somatic antigen (CH10 isolate) also ex-hibited some minor degree of correlation with theEm2(Gll) antigen (r = 023) whereas the recom-binant II3-10 antigen (Muller et al 1989) demon-strated no statistical correlation with the Em2(Gll)antigen (r = 0-01)

DISCUSSION

In this paper we describe a murine monoclonalantibody (designated MAb Gil) that specificallyreacts with an epitope present on the previously

characterized Em2-antigen of E multilocularis meta-cestodes (Gottstein 1985) The reactivity of thisantibody was investigated by sandwich-ELISA anddot-ELISA with various isolates of E multilocularismetacestodes for demonstrating that the epitopecorresponding to MAb Gil was ubiquitously pre-sented by all isolates This observation confirmeddirectly the demonstration of conservation of theEm2 antigen previously assessed by indirect anti-Em2 antibody detection in human patients withalveolar echinococcosis originating from geographi-cally dispersed endemic areas (Gottstein et al 1986)Direct proof for the localization of the epitoperecognized by MAb Gil on the Em2 antigen wasfound by investigating affinity-purified (according toGottstein (1985)) Em2 antigen in sandwich- anddot-ELISA In both test systems MAb Gil showedbinding activity to the Em2 antigen whereas anegative control antigen (recombinant II3-10 anti-gen known to be not related to the Em2 antigen)remained negative as expected Indirect evidence forEm2 identity was also obtained by a comparativeinvestigation of sera from human patients withalveolar echinococcosis in ELISA with differentsolid-phase antigens Em2(Gll) versus Em2 (Gott-stein 1985) demonstrated statistically a good cor-relation Some degree of correlation occurred alsowith somatic E multilocularis metacestode antigenThis was to be expected because of the relativelyhigh proportion of Em2 antigen observed previouslyin somatic extracts (Gottstein et al 1983 Gottstein1985)

Of considerable interest was the observed meta-cestode stage-specificity of the E multilocularisepitope binding MAb Gil and being localized onthe Em2 antigen According to direct immuno-fluorescence analysis the molecule with MAb Gl l -binding activity appeared to be accumulated in thelaminated layer either adjacent to the germinal layeror to oncospheres developed in vitro for 13 days Thelaminated layer is known to remain for extremelylong times in infected host tissue even afterspontaneous dying-out of the larval parasite(Rausch et al 1987) The remarkably long per-sistence of anti-Em2 antibodies in serum frompatients with such aborted lesions hence becomesexplained as well as the very rapid decrease of anti-Em2 antibody concentrations (finally becomingnegative) observed after complete surgical resectionof such dead lesions (Lanier et al 1987 Gottsteinet al 1989)

Structures of the native germinal layer did ingeneral not bind MAb Gil in the direct immuno-fluorescence analysis although some individual notfurther defined cells appeared fluorescent (data notshown) The acellular nature of the MAb Gil-positive laminated layer still requires clarification of(i) the localization of the site of production of theepitope in question (ii) the nature of the respective

httpswwwcambridgeorgcoreterms httpsdoiorg101017S0031182000059278Downloaded from httpswwwcambridgeorgcore University of Basel Library on 30 May 2017 at 174203 subject to the Cambridge Core terms of use available at

Monoclonal anti-Enil antibody 49

synthesizing parasite cells and (iii) the exact bio-chemical properties of the molecule(s) carrying theMAb Gl 1-binding epitope Such experiments arepresently under investigation

In conclusion the development of a species-specific monoclonal antibody against an epitope ofthe Em2 antigen proved useful for further biologicalcharacterization and purification of the Em2 antigenFurthermore the application of MAb G i l in asandwich-ELISA demonstrated also its potentialvalue for the diagnostic identification of E multi-locularis metacestodes and simultaneously for itsdiscrimination from various other cestode helmin-thic or bacterial cell extracts

We wish to thank Professor J Eckert for the fruitfuldiscussions and suggestions concerning the experimentsand the manuscript The authors would also like to expressgratitude to Dr M Aubert (Nancy France) and Dr PJacquier (Zurich) for providing some of the parasitematerial The work was supported by the Swiss NationalScience Foundation (grant no 3-2965190)

R E F E R E N C E S

AGGARWAL A MERRITT J W amp NASH T E (1989)

Cysteine-rich variant surface proteins of Giardialamblia Molecular and Biochemical Parasitology 2339^8

BAUMANN D amp GOTTSTEIN B (1987) A double-antibodysandwich ELISA for the detection of Entamoebahistolytica antigen in stool samples of humansTropical Medicine and Parasitology 38 81-5

DAO L (1985) An improved method of antigendetection on nitrocellulose in situ staining of alkalinephosphatase conjugated antibody Journal ofImmunological Methods 82 225-31

DE ST GROTH F s amp SCHEIDEGGER D (1980) Productionof monoclonal antibodies strategy and tactics Journalof Immunological Methods 35 1mdash21

DEPLAZES p amp ECKERT j (1988) Massengewinnung undLagerung von Taenia hydatigena-Eiern sowieIsolierung lebensfahiger Onkospharen SchweizerArchiv fur Tierheilkunde 130 307-20

ECKERT j amp POHLENZ j (1976) Zur Wirkung vonMebendazol auf Metazestoden von Mesocestoides cortiund Echinococcus multilocularis Tropical Medicine andParasitology 27 247-62

ENGVALL E amp PERLMANN P (1972) Enzyme-linkedimmunosorbent assay ELISA III Journal ofImmunology 109 120-35

FLEISCHMANN J B PAIN R H amp PORTER R R (1962)

Reduction of y-globulin Archives of Biochemistry and

Biophysics 1 (Suppl) S174-S180GOTTSTEIN B (1985) Purification and characterization of

a specific antigen from Echinococcus multilocularisParasite Immunology 7 201mdash12

GOTTSTEIN B ECKERT J amp FEY H (1983) Serologicaldifferentiation between Echinococcus granulosus andE multilocularis infections in man ParasitologyResearch 69 347-56

GOTTSTEIN B SCHANTZ P M amp WILSON J F (1985)

Serological screening for Echinococcus multilocularisinfections with ELISA Lancet i 1097-8

GOTTSTEIN B SCHANTZ P M TODOROV T SAIMOT A G

amp JACQUIER p (1986) An international study on theserological differential diagnosis of human cystic andalveolar echinococcosis Bulletin of the World HealthOrganization 64 101mdash5

GOTTSTEIN B LENGELER C BACHMANN P HAGEMANN

P KOCHER P BROSSARD M WITASSEK F amp ECKERT J

(1987) Sero-epidemiological survey for alveolarechinococcosis (by Em2-ELISA) of blood donors inan endemic area of Switzerland Transactions of theRoyal Society of Tropical Medicine and Hygiene 81960-4

GOTTSTEIN B TSCHUDI K ECKERT J amp AMMANN R

(1989) Em2-ELISA for the follow-up of alveolarechinococcosis after complete surgical resection ofliver lesions Transactions of the Royal Society ofTropical Medicine and Hygiene 83 389mdash93

GOTTSTEIN B DEPLAZES P J ECKERT J MULLER E

SCHOTT E HELLE O BOUJON P WOLFF K

WANDELER A SCHWIETE U amp MOEGLE H (1991)

Serological (Em2-ELISA) and parasitologicalexaminations of fox populations for Echinococcusmultilocularis infections Journal of VeterinaryMedicine Series B (in the Press)

LANIER A P TRUJILLO D E SCHANTZ P M WILSON

J F GOTTSTEIN B amp MCMAHON B J (1987)

Comparison of serologic tests for the diagnosis andfollow-up of alveolar hydatid disease AmericanJournal of Tropical Medicine and Hygiene 37 609mdash15

MULLER N GOTTSTEIN B VOGEL M FLURY K amp

SEEBECK T (1989) Application of a recombinantEchinococcus multilocularis antigen in an ELISA fordiagnosis of human alveolar echinococcosis Molecularand Biochemical Parasitology 36 151-60

RAUSCH R L WILSON J F SCHANTZ P M amp MCMAHON

B j (1987) Spontaneous death of Echinococcusmultilocularis Cases diagnosed serologically (by Em2-ELISA) and clinical significance American Journal ofTropical Medicine and Hygiene 36 576-85

SCHANTZ P M amp GOTTSTEIN B (1986) Echinococcosis(Hydatidosis) In Immunodiagnosis of ParasiticDiseases vol 1 (ed Walls K W amp Schantz P M)pp 69-107 Orlando Academic Press

httpswwwcambridgeorgcoreterms httpsdoiorg101017S0031182000059278Downloaded from httpswwwcambridgeorgcore University of Basel Library on 30 May 2017 at 174203 subject to the Cambridge Core terms of use available at