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Clinical and Diagnostic Laboratory Immunology, May 1999, p. 383-387, Vol. 6, No. 3
Departments of Infectious
Diseases1 and Molecular Life
Science,
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 expressed in
Escherichia coli. Lymphocytes were separated from the
peripheral blood of a patient with an amebic liver abscess.
Poly(A)+ RNA was isolated from the lymphocytes, and then
genes coding for the light chain and Fd region of the heavy chain were
amplified by a reverse transcriptase PCR. The amplified DNA fragments
were ligated with a plasmid vector and were introduced into
Escherichia coli. Three thousand colonies were screened for
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 showed that 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 by the IFA test, three Fab fragments with different DNA
sequences were found to react with all nine strains and another Fab
fragment was found to react with seven strains. None of the four human
monoclonal antibody Fab fragments reacted with Entamoeba dispar reference strains or with other enteric protozoan
parasites. These results indicate that the bacterial expression
system reported here is effective for the production of human
monoclonal antibodies specific for E. histolytica. The
recombinant human monoclonal antibody Fab fragments may be applicable
for distinguishing E. histolytica from E. dispar and for use in the serodiagnosis of amebiasis.
Amebiasis is caused by the
enteric protozoan Entamoeba histolytica. It
has been estimated that 50 million people develop hemorrhagic colitis
and extraintestinal abscesses, resulting in 100,000 deaths annually
(36). E. histolytica has recently been
reclassified into two species, E. histolytica
Schaudinn, 1903, and Entamoeba dispar Brumpt, 1925, on the basis of biochemical, immunological, and genetic findings
(8). The two species are morphologically inseparable, but
only E. histolytica is responsible for invasive amebiasis. Therefore, for clinical and epidemiological reasons it is
important to distinguish between E. histolytica and
E. dispar (37).
The use of monoclonal antibodies (MAbs) has been shown to be an
important part of a specific and sensitive diagnostic strategy. To
date, MAbs specifically reactive with either E. histolytica or E. dispar have been produced by
hybridoma technology (10, 19, 20, 25-29, 33). It was
reported that some of the MAbs were able to detect E. histolytica antigen in feces and serum by enzyme-linked
immunosorbent assay (ELISA) (1, 11, 13, 14).
Recently, a new approach for the production of MAbs has been devised on
the basis of recombinant DNA technology (2-4, 7, 24). In
addition, vectors for the cloning and expression of immunoglobulin Fab
fragment genes have been developed (30, 31). Here we report
on the preparation of recombinant human MAb Fab fragments specific for
E. histolytica. The use of a small amount of peripheral
blood from a patient with amebiasis was effective at producing
E. histolytica-specific MAbs.
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 monoxenically with
Pseudomonas aeruginosa in BCSI-S medium (32).
Trophozoites of E. dispar SAW1719 were cultured xenically in Robinson's medium (22). Trophozoites of
Entamoeba hartmanni, Entamoeba
coli, Endolimax nana, Dientamoeba fragilis, and Trichomonas hominis, isolated in our laboratories, were
also xenically cultured in Robinson's medium (22).
Trophozoites of Giardia intestinalis Portland I were grown
in modified BI-S-33 medium (15). All of these trophozoites
were washed three times with ice-cold 10 mM phosphate-buffered saline
(PBS; pH 7.4) before being used.
Amplification and cloning of the genes coding heavy and light
chains.
Ten milliliters of peripheral blood was obtained from a
patient with amebic liver abscess. Lymphocytes were separated from the
blood by centrifugation with Ficoll-Paque (Pharmacia, Uppsala, Sweden).
Poly(A)+ RNA was isolated from lymphocytes by using the
QuickPrep mRNA Purification Kit (Pharmacia). Reverse transcriptase PCR
was performed with the GeneAmp RNA PCR Kit (Perkin-Elmer Cetus,
Norwalk, Conn.) according to the manufacturer's instructions. For cDNA
synthesis, an oligo(dT)16 primer was used. For
amplification of the genes encoding the
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Copyright © 1999, American Society for Microbiology. All rights reserved.
Preparation of Recombinant Human Monoclonal
Antibody Fab Fragments Specific for Entamoeba
histolytica
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Reactivity by IFA test of human MAb Fab fragments to
reference strains of E. histolytica and various
enteric protozoan parasites
and 
light chains and
the Fd region of the 
heavy chain, the primer sets shown in Fig.
1 were used. The primer sequences contain
the restriction sites for cloning. PCR amplification was performed in
100-µl reaction mixtures. Both sense and antisense primers were used
at 1 µM. 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 initial denaturation step of 4 min at 94°C and a final polymerization step of 7 min at 72°C were
also included. The amplified DNA fragments were purified with the
QIAquick PCR Purification Kit (QIAGEN GmbH, Hilden, Germany). The DNA
fragments were treated with AscI and NheI or
SfiI and NotI and were then purified by agarose
gel electrophoresis in combination with the QIAEX Gel Extraction Kit
(QIAGEN). Since the amount of the amplified light-chain gene was
small, only the light-chain gene was ligated into an expression
vector, pFab1-His2 (30), and was then introduced into
Escherichia coli JM109. The bacteria were spread on Luria
broth plates containing 50 µg of ampicillin per ml, and the vector
with the inserts was selected. Next, the Fd heavy-chain gene was
ligated into pFab1-His2, which contained the light-chain gene, and was
introduced into Escherichia coli.
View larger version (47K):
[in a new window]
FIG. 1.
Primers used for PCR amplification of human
immunoglobulin genes. Primer sequences are aligned from 5' to 3'.
Restriction sites are underlined. Degenerate nucleotides are indicated
as follows: M = A or C, Y = C or T, W = A or T, R = A or G, H = A, C, or T, S = C or G, and K = T or G.
Expression in Escherichia coli and screening of
clones.
Each clone was cultured 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 an optical density at 600 nm of 0.6 to 0.8 was
achieved. Isopropyl-
-D-thiogalactopyranoside (final
concentration, 0.1 mM) was added to the bacterial cultures, which were
then incubated overnight at 30°C. The bacteria were pelleted by
centrifugation, suspended in 150 µl of PBS containing 1 mM
phenylmethylsulfonyl fluoride, and then sonicated. The lysates were
centrifuged at 18,000 × g for 10 min. The resultant
supernatant was subjected to screening by an indirect
fluorescent-antibody (IFA) test. Each of the positive clones selected
by the screening process was cultured in 1 liter of medium.
Twenty milliliters of the resultant supernatant, prepared as described
above, was used in the present study.
DNA sequencing. Cloned DNA fragments coding the heavy and light chains were recloned into sequencing vectors CV-1 and CV-2, respectively. Cycle sequencing in both directions was performed with Thermo Sequenase (Amersham Life Science, Cleveland, Ohio) with M13 forward (5'-CACGACGTTGTAAAAACGAC-3') and reverse (5'-GGATAACAATTTCACACAGG-3') primers. The reactions were run on a model 4000L automated DNA sequencer (LI-COR, Lincoln, Nebr.).
IFA test. The IFA test, which was performed with formalin-fixed trophozoites which were smeared on glass slides and air dried, was carried out as described previously (28). Fluorescein isothiocyanate-conjugated goat immunoglobulin G (IgG) to human IgG Fab (Organon Teknica Co., Durham, N.C.) was used as the secondary antibody. An IFA test with live, intact trophozoites was also performed as described previously (29).
Western immunoblot analysis.
Western immunoblot analysis was
performed as 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 solubilized with an equal volume of the sample buffer (17)
containing 2 mM phenylmethylsulfonyl fluoride, 2 mM
N-
-p-tosyl-L-lysine chloromethyl
ketone, 2 mM p-hydroxymercuriphenyl sulfonic acid, and 4 µM 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 and
was then transferred to a polyvinylidene difluoride
membrane. Each strip of the membrane was incubated with 500 µl of
Escherichia coli extract containing MAb Fab fragments. The
plasma fraction of peripheral blood from the patient with an amebic
liver abscess, obtained during the separation of lymphocytes by
Ficoll-Paque centrifugation, served as a positive control. A polyclonal
rabbit antibody to 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
sheep antibodies to human IgG F(ab')2 and rabbit IgG
(Organon Teknica) were used as secondary antibodies. Development was
done with a Konica Immunostaining HRP-1000 kit (Konica Co., Tokyo, Japan).
Nucleotide sequence accession number. The nucleotide sequence data reported here have been submitted to DDBJ and have been assigned accession nos. AB022650 to AB022656.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cloning and sequencing of the clones. When 3,000 colonies were screened for human antibody Fab fragments reactive with E. histolytica HM-1:IMSS by the IFA test with formalin-fixed trophozoites, Escherichia coli lysates from five clones showed fluorescence (Fig. 2).
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chains of these clones were
sequenced. The deduced amino acid sequences are shown in
Fig. 3. Concerning the heavy-chain
genes, the sequences of clones A235 and A429 and of clones C546
and E244 were identical. The chain sequences of clones A235
and A429 were also identical. The sequences of the variable (V) regions
of the heavy and chains were compared with sequences in the
Kabat database. The heavy-chain V gene of A235 (A429) belonged to VH3;
those of B220 and C546 (E244) to the VH1 group. The kappa-chain V genes
of all clones except B220 belonged to V1; that of B220 belonged
to V4.
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Reactivities of Fab fragments. The reactivities of the recombinant human MAb Fab fragments to nine reference strains of E. histolytica were examined by the IFA test with formalin-fixed trophozoites (Table 1). Clones A235 (A429), B220, and E244 reacted with all the strains. However, clone C546 failed to react with strains H-302:NIH and SAW1627. When the reactivities of these MAb Fab fragments to various enteric protozoan parasites was examined, none reacted with trophozoites of E. dispar, E. moshkovskii, E. coli, E. hartmanni, E. nana, D. fragilis, T. hominis, or G. intestinalis. The reactivities of the MAbs to live intact trophozoites of E. histolytica in suspension were 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 Fab fragments was examined by Western immunoblot analysis (Fig. 4). Clones A235 (A429) and B220 recognized the 260-kDa band under nonreduced conditions. Similar bands were strongly recognized by plasma from the patient with an amebic liver abscess (the donor of the lymphocytes) and by a rabbit polyclonal antibody to the heavy subunit of Gal- and GalNAc-inhibitable lectin. No positive band was elicited by incubation with clone C546 or E244.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Hybridoma technology (16) has resulted in unlimited supplies of specific rodent MAbs. However, it has been much less successful for the production of human MAbs. The present study demonstrates that the bacterial expression system reported here is effective for the production of human MAbs specific for E. histolytica. To our knowledge, this is the first report of the preparation of recombinant human MAb Fab fragments specific for any parasite. Western immunoblot analysis demonstrated that the antigen recognized by clones A235 (A429) and B220 is a major antigen of E. histolytica. Therefore, it may be possible to prepare human MAbs to such antigens by the screening of several thousand clones.
A possible use of recombinant human MAbs may be as controls in serodiagnosis (12). To interpret serologic data, known positive controls must be used. For agglutination tests, antibodies prepared in animals are available. However, for ELISA and the IFA test, which use second antibodies, human antibodies must be used as positive controls. The technology reported here offers unlimited supplies of human MAbs specific for any given pathogen.
The preparation of rodent MAb by hybridoma technology takes about 3 months by standard protocols (26, 27, 29). Most of the technician's time is spent on the immunization of mice and cloning of the hybridoma cells. By using the peripheral blood of patients with amebiasis, it will be possible to obtain human MAbs within 1 month.
The most significant application of human MAbs would be as therapy and passive immunization, just as polyclonal antibodies have been used (23). Western immunoblot analysis demonstrated that A235 (A429) and B220 are reactive with the same antigen recognized by a rabbit polyclonal antibody to the heavy subunit of Gal- and GalNAc-inhibitable lectin. It is known that there are adherence-blocking epitopes in the heavy subunit of the lectin (18, 21). We have recently observed that a recombinant mouse MAb Fab fragment specific for a 150-kDa surface antigen of E. histolytica inhibits amebic adherence to erythrocytes (6, 30). Therefore, if the human Fab fragment prepared in the present study can inhibit amebic adherence to mammalian cells, it may be valuable for clinical use. However, in preliminary experiments, the adherence of E. histolytica trophozoites to erythrocytes was not affected (data not shown). Since we could not localize the epitope on the cell surface of intact trophozoites by the IFA test, clones A235 (A429) and B220 may recognize the cytoplasmic domain of the Gal- and GalNAc-inhibitable lectin (35).
It is known that the heavy chain is dominant in determining antigen binding (5). In the present study, although the heavy-chain sequences of C546 and E244 were identical, the reactivities of these MAbs to several strains of E. histolytica were quite different. This observation suggests that the strength of binding activity to antigen is changeable by displacement of the light chain.
In this study, genes with identical sequences were cloned. The reason why the same genes were cloned is unclear. It seems likely that a large number of lymphocytes that express the gene existed. Otherwise, several populations of genes were predominantly amplified by PCR. In any event, for the cloning of antibody genes encoding the minor antigen of E. histolytica, we recommend that high titers of the antibody gene library be constructed and a phage display system be used for screening (5).
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ACKNOWLEDGMENTS |
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We are grateful to O. Suzuki and W. A. Petri, Jr., for gifts of primers and antibody. We also thank W. Stahl for reviewing the manuscript.
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, a Tokai University School of Medicine Research Aid, a grant from Ohyama Health Foundation, and a grant from the Ministry of Health and Welfare of Japan.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Infectious Diseases, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa 259-1193, Japan. Phone: 81 (463) 93-1121. Fax: 81 (463) 95-5450. E-mail: htachiba{at}is.icc.u-tokai.ac.jp.
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Discussion
References
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Clinical and Diagnostic Laboratory Immunology, May 1999, p. 383-387, Vol. 6, No. 3
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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