CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY,1071-412X/99/$04.0010

May 1999, p. 383–387 Vol. 6, No. 3

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Preparation of Recombinant Human Monoclonal Antibody FabFragments Specific for Entamoeba histolytica

HIROSHI TACHIBANA,1* XUN-JIA CHENG,1 KATSUOMI WATANABE,1 MASATAKA TAKEKOSHI,2

FUMIKO MAEDA,2 SATOSHI AOTSUKA,3 YOSHIMASA KANEDA,1

TSUTOMU TAKEUCHI,4 AND SEIJI IHARA2

Departments of Infectious Diseases1 and Molecular Life Science,2 Tokai University School of Medicine,Isehara, Kanagawa 259-1193, Tokyo Research Center, Nisshinbo Industries, Inc., Nishiarai Sakaecho,

Adachi-ku, Tokyo 123-8585,3 and Department of Tropical Medicine and Parasitology, Schoolof Medicine, Keio University, Shinanomachi, Shinjuku-ku, Tokyo 160-8582,4 Japan

Received 12 October 1998/Returned for modification 28 December 1998/Accepted 10 February 1999

Genes coding for human antibody Fab fragments specific for Entamoeba histolytica were cloned and expressedin Escherichia coli. Lymphocytes were separated from the peripheral blood of a patient with an amebic liverabscess. Poly(A)1 RNA was isolated from the lymphocytes, and then genes coding for the light chain and Fdregion of the heavy chain were amplified by a reverse transcriptase PCR. The amplified DNA fragments wereligated with a plasmid vector and were introduced into Escherichia coli. Three thousand colonies were screenedfor the production of antibodies to E. histolytica HM-1:IMSS by an indirect fluorescence-antibody (IFA) test.Lysates from five Escherichia coli clones were positive. Analysis of the DNA sequences of the five clones showedthat three of the five heavy-chain sequences and four of the five light-chain sequences differed from each other.When the reactivities of the Escherichia coli lysates to nine reference strains of E. histolytica were examined bythe IFA test, three Fab fragments with different DNA sequences were found to react with all nine strains andanother Fab fragment was found to react with seven strains. None of the four human monoclonal antibody Fabfragments reacted with Entamoeba dispar reference strains or with other enteric protozoan parasites. These re-sults indicate that the bacterial expression system reported here is effective for the production of human mono-clonal antibodies specific for E. histolytica. The recombinant human monoclonal antibody Fab fragments maybe applicable for distinguishing E. histolytica from E. dispar and for use in the serodiagnosis of amebiasis.

Amebiasis is caused by the enteric protozoan Entamoebahistolytica. It has been estimated that 50 million people develophemorrhagic colitis and extraintestinal abscesses, resulting in100,000 deaths annually (36). E. histolytica has recently beenreclassified into two species, E. histolytica Schaudinn, 1903, andEntamoeba dispar Brumpt, 1925, on the basis of biochemical,immunological, and genetic findings (8). The two species aremorphologically inseparable, but only E. histolytica is respon-sible for invasive amebiasis. Therefore, for clinical and epide-miological reasons it is important to distinguish between E. his-tolytica and E. dispar (37).

The use of monoclonal antibodies (MAbs) has been shownto be an important part of a specific and sensitive diagnosticstrategy. To date, MAbs specifically reactive with either E. his-tolytica or E. dispar have been produced by hybridoma tech-nology (10, 19, 20, 25–29, 33). It was reported that some of theMAbs were able to detect E. histolytica antigen in feces andserum by enzyme-linked immunosorbent assay (ELISA) (1, 11,13, 14).

Recently, a new approach for the production of MAbs hasbeen devised on the basis of recombinant DNA technology(2–4, 7, 24). In addition, vectors for the cloning and expressionof immunoglobulin Fab fragment genes have been developed(30, 31). Here we report on the preparation of recombinanthuman MAb Fab fragments specific for E. histolytica. The useof a small amount of peripheral blood from a patient with ame-biasis was effective at producing E. histolytica-specific MAbs.

MATERIALS AND METHODS

Parasites. Trophozoites of several strains of E. histolytica (listed in Table 1)and Entamoeba moshkovskii Laredo were axenically grown in BI-S-33 medium(9). Trophozoites of E. dispar SAW1734RclAR were cultured monoxenicallywith Pseudomonas aeruginosa in BCSI-S medium (32). Trophozoites of E. disparSAW1719 were cultured xenically in Robinson’s medium (22). Trophozoites ofEntamoeba hartmanni, Entamoeba coli, Endolimax nana, Dientamoeba fragilis,and Trichomonas hominis, isolated in our laboratories, were also xenically cul-tured in Robinson’s medium (22). Trophozoites of Giardia intestinalis Portland Iwere grown in modified BI-S-33 medium (15). All of these trophozoites werewashed three times with ice-cold 10 mM phosphate-buffered saline (PBS; pH7.4) before being used.

Amplification and cloning of the genes coding heavy and light chains. Tenmilliliters of peripheral blood was obtained from a patient with amebic liverabscess. Lymphocytes were separated from the blood by centrifugation withFicoll-Paque (Pharmacia, Uppsala, Sweden). Poly(A)1 RNA was isolated fromlymphocytes by using the QuickPrep mRNA Purification Kit (Pharmacia). Re-verse transcriptase PCR was performed with the GeneAmp RNA PCR Kit(Perkin-Elmer Cetus, Norwalk, Conn.) according to the manufacturer’s instruc-tions. For cDNA synthesis, an oligo(dT)16 primer was used. For amplification ofthe genes encoding the k and l light chains and the Fd region of the g heavychain, the primer sets shown in Fig. 1 were used. The primer sequences containthe restriction sites for cloning. PCR amplification was performed in 100-mlreaction mixtures. Both sense and antisense primers were used at 1 mM. Thirty-five cycles of PCR were performed as follows: denaturation at 94°C for 1 min,annealing at 50°C for 2 min, and polymerization at 72°C for 3 min. An initialdenaturation step of 4 min at 94°C and a final polymerization step of 7 min at72°C were also included. The amplified DNA fragments were purified with theQIAquick PCR Purification Kit (QIAGEN GmbH, Hilden, Germany). TheDNA fragments were treated with AscI and NheI or SfiI and NotI and were thenpurified by agarose gel electrophoresis in combination with the QIAEX GelExtraction Kit (QIAGEN). Since the amount of the amplified l light-chain genewas small, only the k light-chain gene was ligated into an expression vector,pFab1-His2 (30), and was then introduced into Escherichia coli JM109. Thebacteria were spread on Luria broth plates containing 50 mg of ampicillin per ml,and the vector with the inserts was selected. Next, the Fd heavy-chain gene wasligated into pFab1-His2, which contained the light-chain gene, and was intro-duced into Escherichia coli.

* Corresponding author. Mailing address: Department of InfectiousDiseases, Tokai University School of Medicine, Bohseidai, Isehara,Kanagawa 259-1193, Japan. Phone: 81 (463) 93-1121. Fax: 81 (463)95-5450. E-mail: htachiba@is.icc.u-tokai.ac.jp.

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Expression in Escherichia coli and screening of clones. Each clone was cul-tured in 2 ml of super broth (30 g of tryptone, 20 g of yeast extract, 10 g of MOPS[morpholinepropanesulfonic acid] per liter [pH 7]) containing ampicillin until anoptical density at 600 nm of 0.6 to 0.8 was achieved. Isopropyl-b-D-thiogalacto-pyranoside (final concentration, 0.1 mM) was added to the bacterial cultures,which were then incubated overnight at 30°C. The bacteria were pelleted bycentrifugation, suspended in 150 ml of PBS containing 1 mM phenylmethylsul-fonyl fluoride, and then sonicated. The lysates were centrifuged at 18,000 3 g for10 min. The resultant supernatant was subjected to screening by an indirect flu-orescent-antibody (IFA) test. Each of the positive clones selected by the screen-ing process was cultured in 1 liter of medium. Twenty milliliters of the resultantsupernatant, prepared as described above, was used in the present study.

DNA sequencing. Cloned DNA fragments coding the heavy and light chainswere recloned into sequencing vectors CV-1 and CV-2, respectively. Cycle se-quencing in both directions was performed with Thermo Sequenase (AmershamLife Science, Cleveland, Ohio) with M13 forward (59-CACGACGTTGTAAAAACGAC-39) and reverse (59-GGATAACAATTTCACACAGG-39) primers. Thereactions were run on a model 4000L automated DNA sequencer (LI-COR, Lin-coln, Nebr.).

IFA test. The IFA test, which was performed with formalin-fixed trophozoiteswhich were smeared on glass slides and air dried, was carried out as describedpreviously (28). Fluorescein isothiocyanate-conjugated goat immunoglobulin G(IgG) to human IgG Fab (Organon Teknica Co., Durham, N.C.) was used as thesecondary antibody. An IFA test with live, intact trophozoites was also per-formed as described previously (29).

Western immunoblot analysis. Western immunoblot analysis was performedas described previously (28) and was based on the procedure of Towbin et al.(34). Trophozoites of E. histolytica HM-1:IMSS suspended in PBS were solubi-lized with an equal volume of the sample buffer (17) containing 2 mM phenyl-methylsulfonyl fluoride, 2 mM N-a-p-tosyl-L-lysine chloromethyl ketone, 2 mMp-hydroxymercuriphenyl sulfonic acid, and 4 mM leupeptin for 5 min at 95°C.After centrifugation, the supernatant was subjected to electrophoresis in a 7.5%running gel under nonreducing conditions with Laemmli’s buffer system andwas then transferred to a polyvinylidene difluoride membrane. Each strip of themembrane was incubated with 500 ml of Escherichia coli extract containing MAbFab fragments. The plasma fraction of peripheral blood from the patient with anamebic liver abscess, obtained during the separation of lymphocytes by Ficoll-Paque centrifugation, served as a positive control. A polyclonal rabbit antibodyto the heavy subunit of galactose (Gal)- and N-acetyl-D-galactosamine (GalNAc)-inhibitable lectin of E. histolytica, provided by W. A. Petri, Jr., of the University

of Virginia, was also used. Horseradish peroxidase (HRP)-conjugated sheepantibodies to human IgG F(ab9)2 and rabbit IgG (Organon Teknica) were usedas secondary antibodies. Development was done with a Konica ImmunostainingHRP-1000 kit (Konica Co., Tokyo, Japan).

Nucleotide sequence accession number. The nucleotide sequence data re-ported here have been submitted to DDBJ and have been assigned accession nos.AB022650 to AB022656.

RESULTS

Cloning and sequencing of the clones. When 3,000 colonieswere screened for human antibody Fab fragments reactive withE. histolytica HM-1:IMSS by the IFA test with formalin-fixedtrophozoites, Escherichia coli lysates from five clones showedfluorescence (Fig. 2).

The genes coding the heavy and k chains of these clones weresequenced. The deduced amino acid sequences are shown inFig. 3. Concerning the heavy-chain genes, the sequences of clonesA235 and A429 and of clones C546 and E244 were identical.The k chain sequences of clones A235 and A429 were alsoidentical. The sequences of the variable (V) regions of theheavy and k chains were compared with sequences in theKabat database. The heavy-chain V gene of A235 (A429) be-longed to VH3; those of B220 and C546 (E244) to the VH1

FIG. 1. Primers used for PCR amplification of human immunoglobulingenes. Primer sequences are aligned from 59 to 39. Restriction sites are under-lined. Degenerate nucleotides are indicated as follows: M 5 A or C, Y 5 C orT, W 5 A or T, R 5 A or G, H 5 A, C, or T, S 5 C or G, and K 5 T or G.

TABLE 1. Reactivity by IFA test of human MAb Fab fragments toreference strains of E. histolytica and various

enteric protozoan parasites

Species Strain Zymo-deme

Reactivity of thefollowing clonea:

A235(A429) B220 C546 E244

Entamoeba histolytica HM-1:IMSS II 11 1 1 1HK-9 II 11 11 1 1200:NIH II 1 1 1 1HB-301:NIH II 11 1 1 11H-302:NIH II 11 1 2 1H-303:NIH II 11 1 1 11DKB II 11 1 1 1C-3-2-1 II 1 1 1 11SAW1627 IIa2 11 11 2 1SAW755CR XIV 11 1 1 11

Entamoeba dispar SAW1734RclAR I 2 2 2 2SAW1719 I 2 2 2 2

Entamoeba coli 2 2 2 2

Entamoeba hartmanni 2 2 2 2

Entamoeba mosh-kovskii

Laredo 2 2 2 2

Endolimax nana 2 2 2 2

Dientamoeba fragilis 2 2 2 2

Trichomonas hominis 2 2 2 2

Giardia intestinalis Portland I 2 2 2 2

a 11, strongly positive; 1, positive; 2, negative.

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group. The kappa-chain V genes of all clones except B220 be-longed to Vk1; that of B220 belonged to Vk4.

Reactivities of Fab fragments. The reactivities of the recom-binant human MAb Fab fragments to nine reference strainsof E. histolytica were examined by the IFA test with formalin-fixed trophozoites (Table 1). Clones A235 (A429), B220, andE244 reacted with all the strains. However, clone C546 failedto react with strains H-302:NIH and SAW1627. When the re-activities of these MAb Fab fragments to various enteric pro-tozoan parasites was examined, none reacted with trophozoitesof E. dispar, E. moshkovskii, E. coli, E. hartmanni, E. nana,D. fragilis, T. hominis, or G. intestinalis. The reactivities of theMAbs to live intact trophozoites of E. histolytica in suspensionwere also examined. However, fluorescence was not observed,indicating that epitopes are not present on the cell surface.

The molecular mass of the antigen(s) recognized by the Fabfragments was examined by Western immunoblot analysis (Fig.

4). Clones A235 (A429) and B220 recognized the 260-kDaband under nonreduced conditions. Similar bands were strong-ly recognized by plasma from the patient with an amebic liverabscess (the donor of the lymphocytes) and by a rabbit poly-clonal antibody to the heavy subunit of Gal- and GalNAc-inhibitable lectin. No positive band was elicited by incubationwith clone C546 or E244.

DISCUSSION

Hybridoma technology (16) has resulted in unlimited sup-plies of specific rodent MAbs. However, it has been much lesssuccessful for the production of human MAbs. The presentstudy demonstrates that the bacterial expression system report-ed here is effective for the production of human MAbs specificfor E. histolytica. To our knowledge, this is the first report ofthe preparation of recombinant human MAb Fab fragmentsspecific for any parasite. Western immunoblot analysis dem-onstrated that the antigen recognized by clones A235 (A429)and B220 is a major antigen of E. histolytica. Therefore, it maybe possible to prepare human MAbs to such antigens by thescreening of several thousand clones.

A possible use of recombinant human MAbs may be ascontrols in serodiagnosis (12). To interpret serologic data,known positive controls must be used. For agglutination tests,antibodies prepared in animals are available. However, forELISA and the IFA test, which use second antibodies, humanantibodies must be used as positive controls. The technologyreported here offers unlimited supplies of human MAbs spe-cific for any given pathogen.

The preparation of rodent MAb by hybridoma technologytakes about 3 months by standard protocols (26, 27, 29). Mostof the technician’s time is spent on the immunization of miceand cloning of the hybridoma cells. By using the peripheralblood of patients with amebiasis, it will be possible to obtainhuman MAbs within 1 month.

The most significant application of human MAbs would beas therapy and passive immunization, just as polyclonal anti-bodies have been used (23). Western immunoblot analysis

FIG. 2. Immunofluorescent photomicrograph of formalin-fixed trophozoitesof E. histolytica HM-1:IMSS treated with recombinant human MAb A429, fol-lowed by fluorescein isothiocyanate-conjugated goat IgG to human IgG Fab. Bar,10 mm.

FIG. 3. Comparison of deduced amino acid sequences of genes coding the variable region of the heavy chain (a) and the k light chain (b). Dots indicate identicalamino acids. Hyphens designate gaps added to permit alignment. FR, framework region; CDR, complementarity-determining region.

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demonstrated that A235 (A429) and B220 are reactive with thesame antigen recognized by a rabbit polyclonal antibody to theheavy subunit of Gal- and GalNAc-inhibitable lectin. It isknown that there are adherence-blocking epitopes in the heavysubunit of the lectin (18, 21). We have recently observed thata recombinant mouse MAb Fab fragment specific for a 150-kDa surface antigen of E. histolytica inhibits amebic adherenceto erythrocytes (6, 30). Therefore, if the human Fab fragmentprepared in the present study can inhibit amebic adherence tomammalian cells, it may be valuable for clinical use. However,in preliminary experiments, the adherence of E. histolytica tro-phozoites to erythrocytes was not affected (data not shown).Since we could not localize the epitope on the cell surface ofintact trophozoites by the IFA test, clones A235 (A429) andB220 may recognize the cytoplasmic domain of the Gal- andGalNAc-inhibitable lectin (35).

It is known that the heavy chain is dominant in determiningantigen binding (5). In the present study, although the heavy-chain sequences of C546 and E244 were identical, the reactivi-ties of these MAbs to several strains of E. histolytica were quitedifferent. This observation suggests that the strength of bindingactivity to antigen is changeable by displacement of the lightchain.

In this study, genes with identical sequences were cloned.The reason why the same genes were cloned is unclear. It seemslikely that a large number of lymphocytes that express the geneexisted. Otherwise, several populations of genes were predom-inantly amplified by PCR. In any event, for the cloning of anti-body genes encoding the minor antigen of E. histolytica, werecommend that high titers of the antibody gene library be

constructed and a phage display system be used for screen-ing (5).

ACKNOWLEDGMENTS

We are grateful to O. Suzuki and W. A. Petri, Jr., for gifts of primersand antibody. We also thank W. Stahl for reviewing the manuscript.

This work was supported by a Grant-in-Aid for Scientific Researchfrom the Ministry of Education, Science and Culture of Japan, a TokaiUniversity School of Medicine Research Aid, a grant from OhyamaHealth Foundation, and a grant from the Ministry of Health and Wel-fare of Japan.

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