Vol. 58, No. 4INFECTION AND IMMUNITY, Apr. 1990, p. 955-9600019-9567/90/040955-06$02.00/0Copyright C) 1990, American Society for Microbiology

Identification of a Pathogenic Isolate-Specific 30,000Mr Antigen ofEntamoeba histolytica by Using a Monoclonal AntibodyHIROSHI TACHIBANA,l* SEIKI KOBAYASHI,2 YASUKO KATO,' KOUICHI NAGAKURA,1

YOSHIMASA KANEDA,' AND TSUTOMU TAKEUCHI2Department of Parasitology, School of Medicine, Tokai University, Bohseidai, Isehara, Kanagawa 259-11,1 and

Department of Parasitology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160,2 Japan

Received 1 November 1989/Accepted 3 January 1990

A monoclonal antibody (MAb) produced against trophozoites of Entamoeba histolytica strain HM-1:IMSS,reacted with all of 42 isolates and 4 clones showing pathogenic zymodeme (Z) patterns, i.e., Z-II, Z-IIa-, Z-II(glucose phosphate isomerase:-y+), Z-VII, Z-VII (glucose phosphate isomerase:aL lack, -y+), Z-XI, Z-XIV, andZ-XIX, regardless of culture conditions, geographical origins, or host symptoms in an indirect fluorescenceantibody test. In contrast, the MAb failed to react with 14 isolates possessing nonpathogenic zymodemes Z-Iand Z-VIII and did not react with other enteric protozoan parasites, such as E. histolytica-like Laredo,Entamoeba hartmanni, Entamoeba coli, Endolimax nana, Dientamoeba fragilis, Trichomonas hominis, andGiardia lamblia. Western immunoblotting analysis showed that the molecular weight of the antigeniccomponent recognized by the MAb was exclusively 30,000 in pathogenic isolates of different zymodemes. Theseresults suggest that the 30,000-molecular-weight antigen is a marker of pathogenic isolates and that the indirectfluorescent-antibody test with the MAb is useful for the accurate discrimination of pathogenic amebae.

Entamoeba histolytica, a protozoan parasite, is the caus-ative agent of human amebiasis. In 1984, it was estimatedthat 500 million people were infected with E. histolytica, ofwhom 40 million had developed disabling colitis or extrain-testinal abscesses, resulting in at least 40,000 deaths (23).However, >90% of infected individuals are usually asymp-tomatic. This is probably due to differences in pathogenicityof amebic strains. Recent studies by Sargeaunt and co-workers have demonstrated that E. histolytica strains iso-lated from patients with dysentery show isoenzymatic pat-terns that differ from those isolated from asymptomaticcarriers (14, 17). More recently, it has been reported thatmonoclonal antibodies (MAbs) and DNA probes also coulddistinguish between pathogenic and nonpathogenic strains(3, 18, 21). However, the immunology of the componentsresponsible for pathogenicity or nonpathogenicity is not wellunderstood.

Ih the present study, we have examined the reactivity of aMAb, produced against a pathogenic strain, to more than 50isolates of E. histolytica. The results show that the MAb isexclusively reactive only with isolates showing pathogenicisozyme patterns. The pathogenic strain-specific componentrecognized by the MAb is also identified.

MATERIALS AND METHODSParasites and culture conditions. The E. histolytica strains

and their culture media were as follows. Trophozoites ofHM-1:IMSS, HK-9, Rahman, H303:NIH and AH-1:KEIOwere axenically grown in BI-S-33 medium (2). These axenicstrains, except for AH-1-KEIO, were obtained from L. S.Diamond in May 1978. The NOT-5 strain was xenicallycultured in Balamuth's medium (1). Strains SAW408,SAW1015, SAW1627, SAW1453, SAW1719, and SAW142,provided in July 1988 by P. G. Sargeaunt, were xenicallycultured in Robinson's medium (12). Trophozoites of strainsNOT-1 to -38 and TNK-1 to -18 were also grown in Robin-son's medium. In addition, some strains were also monoxen-

* Corresponding author.

ically cultured in BI-S-33 medium with epimastigotes ofTrypanosoma cruzi (strain Berenice or Tulahuen). Thesemonoxenic strains and clones were established from cysts infeces, trophozoites xenically cultured with bacteria in Rob-inson's medium, and trophozoites from liver abscesses ofJirds produced experimentally by inoculation of trophozo-ites and bacteria. Clones of xenic strains were isolated by themethod of Sargeaunt (13), except that 96-well microplateswere used instead of glass slides. Trophozoites of E. his-tolytica-like Laredo, Entamoeba hartmanni, Entamoebacoli, Endolimax nana, Dientamoeba fragilis, and Trichomo-nas hominis were also xenically cultivated in Robinson'smedium. Trophozoites of Giardia lamblia Portland I weregrown in modified BI-S-33 medium (4). Most of the E.histolytica strains, such as NOT and TNK, and other speciesof amebae and flagellates were newly isolated from Japaneseand non-Japanese travelers, Indochinese immigrants, insti-tutionalized Japanese children, Japanese homosexual men,and macaque monkeys in zoos between July 1988 andAugust 1989. Amebic species were identified by their mor-phological characteristics and electrophoretic isoenzymepatterns (15). Parasites grown in Robinson's medium wereisolated with 75% Percoll by centrifugation at 400 x g for 5min. All parasites were washed three times with 10 mMphosphate-buffered saline (PBS), pH 7.4, by centrifugationat 400 x g for 2 min at 4°C before use.

Production of MAbs. Eight-week-old female BALB/c micewere inoculated intraperitoneally with 4 x 104 sonicatedtrophozoites of E. histolytica HM-1:IMSS in Freund com-plete adjuvant and again after 2 weeks. After an additional 2weeks, the mice received only sonicated trophozoites with-out adjuvant. Three days later, spleen cells were isolated andfused with X63 Ag8.653 mouse myeloma cells by 50%polyethylene glycol 1500 (5). Hybridomas secreting MAbsagainst E. histolytica were screened by immunoperoxidasemicroscopy (20) or an indirect fluorescent-antibody test(IFA) and subsequently cloned by limiting dilution. Ascitesrich in MAbs were obtained by the intraperitoneal inocula-tion of hybridomas into pristane-primed mice. One of the

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MAbs, 4G6, was used in this study. The MAb was typed asimmunoglobulin G subclass 1 (IgGl) with kappa light chainsby double immunodiffusion in gel. Full details of the hybrid-oma lines obtained will be described elsewhere.

IFA. Trophozoites were fixed with 3% Formalin in PBSfor 30 min at 4°C. After three washes with PBS, the parasiteswere suspended in PBS to a concentration of 106/ml. A 2-p.lportion of suspension was added to the wells of multispotglass slides, which were dried at room temperature. Slideswere stored in sealed containers at -70°C until use. The IFAwas carried out at 23°C in a moist chamber. The wells of themultispot slides were treated with 50 [LI of 3% skim milk(Difco Laboratories, Detroit, Mich.) in PBS (PBS-skim milk)for 15 min and then incubated with 50 ,ul of culture super-natant or diluted mouse ascites (4G6) for 30 min. For thepurpose of titration, ascitic fluid was diluted initially 1:20followed by twofold stepwise dilutions. After being washedthree times with PBS, the wells were treated with 25 RI offluorescein isothiocyanate-labeled goat anti-mouse IgG(Medical & Biological Laboratories, Nagoya, Japan), diluted1:50, in PBS-skim milk for 30 min. The slides were washedwith PBS, mounted with 50% glycerol in PBS, and thenobserved with a Nikon fluorescence microscope. Only whena bright yellow-green fluorescence was observed was thereaction judged positive. As negative and positive controls,MAb TCE04 (IgGl), which is specific for T. cruzi (19), andpooled sera from mice immunized with E. histolytica wereincluded in each assay.

Polyacrylamide gel electrophoresis in sodium dodecyl sul-fate and Western immunoblotting. To determine the proteincontent of the various organisms, the method of Lowry et al.(7) was used. Trophozoites of each parasite were solubilizedwith an equal volume of the sample buffer (6) containing 2mM phenylmethylsulfonyl fluoride, 2 mM N Ot-p-tosyl-L-lysine chloromethyl ketone, 2 mM p-hydroxymercuriphenyl-sulfonic acid, 4 ,uM leupeptin, and 10 mM N-ethylmaleimidefor 5 min at 95°C. The supernatant, obtained followingcentrifugation at 10,000 x g for 3 min, was subjected toelectrophoresis in 10% polyacrylamide gel, using Laemmli'sbuffer system. The electrophoresed protein bands weretransferred onto a sheet of Clear Blot Membrane-p (AttoCorp., Tokyo, Japan) by the procedure of Towbin et al. (22).The electrophoretic transfer was carried out at 200 mA for 1h. Some lanes of the sheet were stained with Coomassiebrilliant blue, while the others were incubated first withPBS-skim milk for 2 h and then with ascitic fluid, diluted1:500 in PBS-skim milk, for an additional 2 h at 23°C. Aftera wash with PBS containing 0.05% Tween 20, the mem-branes were incubated with 1:500 horseradish peroxidase-labeled goat anti-mouse IgG (Medical & Biological Labora-tories, Nagoya, Japan) for 1 h. The sheets were washed anddeveloped in a solution of 0.05% 4-chloro-1-naphthol plus0.015% hydrogen peroxide in PBS for 1 h.

RESULTS

We examined the reactivity ofMAb 4G6 to trophozoites ofvarious species of enteric protozoa by IFA (Table 1). The4G6 reacted strongly with trophozoites of E. histolyticaHM-1:IMSS, and the titer of pooled ascitic fluids was 10,240.Fluorescence was located mainly on the nucleus and faintlyon the cytoplasm (Fig. 1A). On the other hand, MAb 4G6 didnot react with trophozoites of E. histolytica-like Laredo, E.hartmanni, Entamoeba coli, Endolimax nana, D. fragilis,Trichomonas hominis, or G. lamblia at the minimum 1:20dilution of ascitic fluids. The reactivity of 4G6 towards

TABLE 1. Reactivity of MAb 4G6 to trophozoites of variousspecies of enteric protozoa in the IFA'

Species IFA titer

Entamoeba histolytica (HM-1:IMSS) ............................. 10,240E. histolvtica-like Laredo ............................................ <20E. hartmanni ............................................. <20Entamoeba coli............................................. <20Endolimax nana ...................... ....................... <20Dientamoeba fragilis ............................................. <20Trichomonas hominis ............................................. <20Giardia lamblia............................................. <20

" As a source of MAb, pooled ascitic fluids were used, starting at a 1:20dilution.

various isolates and clones of E. histolytica was also exam-ined. The amebae had been isolated in Japan from numeroussources and expressed a number of different zymodemes(Table 2). The 4G6 reacted with all isolates and clonesshowing pathogenic zymodemes (Z), i.e., Z-II, Z-IIax-, Z-II(glucose phosphate isomerase:-y+), Z-VII, Z-VII (glucosephosphate isomerase:(x lack, -y+), Z-XI, Z-XIV, and Z-XIX,regardless of culture conditions (axenic, monoxenic, orxenic) or geographic origin. Fluorescence was observed on

FIG. 1. Immunofluorescent photomicrographs of E. histolyticaHM-1:IMSS (A) and NOT-25 (B) labeled with MAb 4G6. Formalin-fixed trophozoites were treated with 4G6 (1:20-diluted ascitic fluids)followed by fluorescein isothiocyanate-labeled goat anti-mouse IgG.Note fluorescence on nucleus (arrows) and cytoplasm (arrowhead)of the trophozoites. Bar, 10 ,um.

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TABLE 2. Reactivity of MAb 4G6 to various strains and clones of E. histolytica

Isolate Geographic origin Symptom(s)" Culture conditionsb Zymodeme classification IFA'

Korea D

United KingdomMexicoSouth AfricaCambodiaTaiwan

ChinaJapanJapanJapanJapanJapanJapanJapanJapanJapanJapanJapanJapanJapanJapanJapanJapanJapanJapanJapanJapanJapanIndiaCambodiaVietnamJapanJapanIndiaJapanJapan

BangladeshJapanVietnamCambodiaNepalIndiaAustraliaJapanCambodiaPeruMexicoEthiopiaBoliviaPeruParaguayGhanaMexicoIndiaCambodiaJapan

CFUC, DCFCFDDCFDDCFLA, CFDCFCFCFCFCFCFCFCFCFCFCFCFCFCFCFCFCFCFCFLA, DLALA, CFLA, D

LADCFCFCF, UCDCFCFCFCFCFCFCFCFCFCFCFCFCFCF

AxAxAxXeXeAxXeXeMonoxMonoxMonoxXeXeXeXeXeXeXeXe, MonoxXeXeMonoxXe, MonoxXeXe, MonoxXeXeXeXeMonoxXeXeXeXeXeXe, MonoxXe, MonoxMonoxMonoxMonoxXeMonoxMonoxXeXeXeXeXeXeXeXeXeXeXeXeXeXeXeXeXe

IIIIII1111lIlIlIIIIIIIIIIIIIIIIIIIII11II11IIIIIIIIIIIIIIII

IIa

II(GPI:-y+ )d

VIIVII(GPI:a lack,-y+)dXIXIVXIVXIXXIXXIXXIXXIXXIXXIXXIXXIXXIXII

I

I

I

I

I

I

I

I

I

III MI)VIII (ME: more cathodic)d

Vill

a CF, Cysts in feces; D, dysentery; LA, liver abscess; UC, ulcer of colon.b Ax, Cultured axenically in BI-S-33 medium; Monox, cultured monoxenically with epimastigotes of Trypanosoma cruzi in BI-S-33 medium; Xe, cultured

xenically (or monoxenically with Escherichia coli) in Robinson's medium except for NOT-5, which was cultured in Balamuth's medium.I MAb 4G6 (ascitic fluids) was used at a 1:20 dilution.d Variations of typical zymodemes or new zymodemes: GPI, glucose phosphate isomerase (EC 5319); ME, NADP+ oxidoreductase (EC 11140).eMacaque monkeys (Macaca fuscata).f Established clone by Sargeaunt's cloning method (13).

Original zymodeme was type III; presently classified as type I.

HK-9H303:NIHRahmanSAW408SAW1015AH-1:KEIONOT-4NOT-5NOT-6NOT-7NOT-8NOT-14NOT-16TNK-1TNK-2TNK-3TNK-4TNK-5TNK-6TNK-7TNK-8TNK-9TNK-10TNK-11TNK-14TNK-15TNK-16TNK-17TNK-18SAW1627NOT-29NOT-25TNK 12eNOT-13SAW1453NOT-3cdfNOT-1NOT-1cllfNOT-1cl2fNOT-1cl3fNOT-2NOT-12NOT-20NOT-21NOT-26NOT-31SAW1719NOT-10NOT-19NOT-23NOT-27NOT-32NOT-34NOT-35NOT-36NOT-37NOT-38SAW142NOT-33TNK 13e

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L I

FIG. 2. Immunofluorescent (A and C) and phase-contrast (B and D) photomicrographs of E. histolytica NOT-31 (A and B) and NOT-27(C and D) treated with MAb 4G6. Formalin-fixed trophozoites were incubated with 4G6 (1:20-diluted ascitic fluid) followed by fluoresceinisothiocyanate-labeled goat anti-mouse IgG. Bar, 10 p.m.

the nucleus and cytoplasm of trophozoites cultured in Rob-inson's medium (Fig. 1B). However, in some isolates, 4G6failed to react with a small number of trophozoites (Fig. 2Aand B). On the other hand, 4G6 did not react with any

trophozoites of isolates showing nonpathogenic types ofzymodeme (Z-I and Z-VIII) without exception (Table 2; Fig.2C and D). These results clearly demonstrate that 4G6recognizes the pathogenic isolate-specific antigen of E. his-tolytica.To identify the antigen recognized by 4G6, Western immu-

noblotting analysis was carried out. 4G6 showed reactivityagainst a protein band with an apparent molecular weight of30,000 in E. histolytica HM-1:IMSS (Fig. 3). When themolecular size of the antigen recognized by 4G6 in theHM-1:IMSS strain was compared with other isolates of E.histolytica possessing different zymodemes, the resultsshowed that the single band of 30,000 Mr was uniformlyrecognized in pathogenic isolates irrespective of zymodemesand culture conditions (Fig. 4).

DISCUSSION

This study shows that the discrimination of pathogenicfrom nonpathogenic isolates, based on the reactivity of MAb4G6, is entirely in accord with that by isozyme analysis,regardless of zymodeme type or culture conditions. This factindicates that there are immunological differences between

pathogenic and nonpathogenic isolates and that MAbs canbe useful in the detection of pathogenic amebae.One of the advantages of using MAbs in the IFA to

identify the pathogenic isolates is simplicity, as suggested byStrachan et al. (18). Zymodeme analysis and DNA hybrid-ization require large numbers of cells, many chemicals,and/or a complicated procedure as compared with IFA.Examination by IFA saves time and cost. Another advan-tage of IFA is that the reactivity of the MAb with eachorganism can be observed.

It has been reported that mixtures of nonpathogenic andpathogenic zymodemes in any single host have not beenfound, nor were alterations detected in the isozyme pattern,from nonpathogenic to pathogenic or vice versa, in a longi-tudinal culture conducted in the presence of viable bacteriain Robinson's medium (13, 16). In contrast, differences inadhesion, phagocytosis, and virulence of clones isolatedfrom a single strain have been reported (11). Such apparentlycontradictory findings most likely reflected the characteris-tics of major populations only. Namely, if an isolate werecomposed largely of nonpathogenic organisms, then a smallminority of pathogens within the isolate could easily beoverlooked in isozymatic analysis. However, by using themethod described here, IFA with MAbs, it would be possi-ble to detect such minorities. Indeed, 4G6 failed to react witha small number of the organisms in some pathogenic isolates

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FIG. 3. Western immunoblotting analysis of reactivity of MAb4G6 with trophozoites of various enteric protozoa. Lanes: 1, 2, and10, E. histolytica HM-1:IMSS; 3, E. histolytica-like Laredo; 4, E.hartmanni; 5, Entamoeba coli; 6, D. fragilis; 7, G. lamblia; 8,Escherichia coli; 9, Trypanosoma cruzi. Cell lysates (ca. 30 ,ug ofprotein per lane) were subjected to sodium dodecyl sulfate-poly-acrylamide gel electrophoresis and transferred to Clear Blot Mem-brane-p. Protein bands in lane 1 were stained with Coomassiebrilliant blue. Lanes 2 to 9 were treated with 4G6, followed byhorseradish peroxidase-labeled goat anti-mouse IgG. Lane 10 is a

negative control treated with TCE04. Numbers to the left indicatemolecular weight markers. Molecular weight (in parentheses) stan-dards used were phosphorylase b (97,400), bovine serum albumin(66,000), egg albumin (45,000), glyceraldehyde-3-phosphate dehy-drogenase (36,000), carbonic anhydrase (29,000), trypsinogen(24,000), trypsin inhibitor (20,100), and a-lactalbumin (14,200).

(Fig. 2A and B). This observation suggests that an isolatemay consist of subpopulations differing in antigenic compo-sition and pathogenic potential.

In this study, Western immunoblotting analysis showedthat the pathogenic strain-specific antigen recognized byMAb was a component of 30,000 Mr. Recently, Young et al.(24) partially purified a membrane pore-forming protein of30,000 Mr from E. histolytica. This material depolarized J774macrophages and mouse spleen lymphocytes and inducedrapid monovalent cation flux across membranes of lipidvesicles. It was not determined whether this protein was alsosecreted by nonpathogenic isolates. At present, nothingdefinitive is known about a possible correlation of theprotein recognized by MAb 4G6 with the pore-formingprotein.Whether the difference between pathogenic and nonpath-

ogenic isolates is genotypic or phenotypic is important.Recent studies with DNA probes have indicated that patho-genicity is dependent on genes that are specific to thepathogenic isolates (3, 21). On the other hand, it has beenobserved that isozyme conversion from nonpathogenic topathogenic, or the reverse, can occur within a cloned cultureof a strain of E. histolytica during the process of axenizationunder appropriate growth conditions (8-10). Therefore, we

cannot exclude the possibility that the gene which codes forthe 30,000Mr component may also exist in nonpathogenicisolates.

In any event, it should be emphasized that the IFA with

1 2 3 4 5 6 7 8 9101112

30k-

FIG. 4. Western immunoblotting analysis of reactivity of MAb4G6 with trophozoites of various isolates of E. histolytica. Lanes: 1,HK-9 (Z-II, axenic); 2, H303:NIH (Z-1I, axenic); 3, Rahman (Z-II,axenic); 4, TNK-6 (Z-II, xenic); 5, SAW1627 (Z-IIa-, monoxenic);6, TNK-12 [Z-VII (glucose phosphate isomerase:oa lack, -y+), xe-nic]; 7, NOT-13 (Z-XI, xenic); 8, NOT-3cl (Z-XIV, monoxenic); 9,NOT-1 (Z-XIX, monoxenic); 10, NOT-12 (Z-XIX, monoxenic); 11,SAW1719 (Z-I, xenic); 12, TNK-13 (Z-VIII, xenic). Cell lysates (ca.30 ,ug of protein per lane) were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis and transferred to ClearBlot Membrane-p. Membranes were treated with 4G6, followed byhorseradish peroxidase-labeled goat anti-mouse IgG. Number to theleft indicates molecular weight.

MAb 4G6 described herein is a useful strategy for theaccurate detection of pathogenic amebae.

ACKNOWLEDGMENTS

We are grateful to K. Kurihara for technical assistance and to W.Stahl for reviewing the manuscript. We also thank P. G. Sargeauntof the London School of Hygiene and Tropical Medicine for thegenerous supply of six strains of E. histolytica.

This work was supported by grants-in-aid for scientific researchfrom the Ministry of Education, Science and Culture of Japan andby the Tokai University School of Medicine Research Aid.

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