JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1998, p. 3278–3284 Vol. 36, No.

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0095-1137/98/$04.0010
Copyright © 1998, American Society for Microbiology. All Rights Reserved.

Characterization of Monoclonal Antibodies to the 44-Kilodalton

Major Outer Membrane Protein of the Human Granulocytic
Ehrlichiosis Agent
HYUNG-YONG KIM AND YASUKO RIKIHISA*
Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University,
Columbus, Ohio 43210-1093
Received 23 February 1998/Returned for modification 14 May 1998/Accepted 4 August 1998

The major outer membrane proteins (OMPs) of the human granulocytic ehrlichiosis (HGE) agent, with
molecular sizes of 44 to 47 kDa, are immunodominant antigens in human infection. Monoclonal antibodies
(MAbs) to the OMPs were made by immunizing BALB/c mice with the purified HGE agent and then by fusing
spleen cells with myeloma cells. The immunologic specificities of three MAbs (3E65, 5C11, and 5D13) were
examined with five human HGE agent isolates and one tick isolate. By Western blot analysis, all three MAbs
recognized the HGE agent but not Ehrlichia chaffeensis, Ehrlichia sennetsu, Ehrlichia canis, or their host cells.
MAb 3E65 reacted with a 44-kDa protein in the homologous human isolate but not in the remaining five
isolates. The two remaining MAbs recognized proteins with molecular sizes of 44 to 47 kDa in all six isolates.
Western blot results with the OMP fraction of the six isolates were consistent with results with the whole HGE
agent. Immunofluorescent-antibody staining and immunogold labeling with these MAbs showed that these
antigens were primarily present on the membrane of the HGE agent. MAbs 5C11 and 5D13 recognized the
recombinant 44-kDa protein by Western immunoblot analysis, but MAb 3E65 did not. Passive immunization
with MAb 3E65 was more effective in protecting mice from HGE agent infection than with MAbs 5C11 and
5D13. These MAbs would be useful for analyzing the role of the major OMP antigens in HGE agent infection
and for serodiagnosis.

Human granulocytic ehrlichiosis (HGE) is an emerging tick-
borne zoonosis characterized by fever, headache, myalgia, el-
evated liver enzyme (serum aminotransferase) levels, leukope-
nia, anemia, thrombocytopenia, and elevated C-reactive
protein levels (2). The first 12 reported cases were in Wiscon-
sin and Minnesota between 1990 and 1993. Subsequently,
more than 400 cases have been identified in the upper Midwest
and in the northeastern United States (1, 2, 4, 13, 15), and
evidence of HGE has been also reported in Europe (3, 6, 10).
The etiologic agent, currently referred to as the HGE agent, is
an obligate intracellular gram-negative bacterium that repli-
cates in the membrane-bound vacuoles of granulocytes. It is
closely related to the Ehrlichia equi-E. phagocytophila group of
the tribe Ehrlichieae (4). The pathogen is transmitted mainly by
the deer tick, Ixodes scapularis, which is also the vector of
Borrelia burgdorferi, the agent of Lyme disease (9, 13). Human
coinfection with the HGE agent and B. burgdorferi has been
previously reported (8).
Diagnosis is often made by a retrospective immunofluores-
cent antibody (IFA) test with the HGE agent or E. equi antigen
(1–4, 6, 8, 11, 13, 14–17). Recently, we cloned and expressed a
44-kDa major antigen of the HGE agent and demonstrated
that this recombinant antigen (rP44) may be more specific for
serodiagnosis of HGE than whole organism-infected cells due
to the absence of heat shock proteins or other antigenically
cross-reactive proteins (17). PCR amplification of the 16S
rRNA gene fragment of the HGE agent from peripheral blood
has been gaining acceptance as a sensitive test at acute stages
of HGE. Microscopic examination of Romanowsky-stained pe-
* Corresponding author. Mailing address: Department of Veteri-
nary Biosciences, College of Veterinary Medicine, The Ohio State
University, 1925 Coffey Rd., Columbus, OH 43210-1093. Phone: (614)
292-5661. Fax: (614) 292-6473. E-mail: rikihisa.1@osu.edu.
ripheral blood smears may reveal the presence of ehrlichial
morulae in the neutrophils. Using five isolates of HGE agents
and a tick isolate, we reported that the major outer membrane
proteins (OMPs) of the HGE agent, with molecular sizes be-
tween 43 and 49 kDa, are immunodominant antigens in human
infection (16, 17). Western blot analysis also revealed varia-
tions in numbers and molecular sizes of the major antigenic
OMPs of the six isolates. Since polyclonal antisera were used
for that study, it was unclear whether the major OMPs of
similar sizes are common antigens among the six isolates.
Monoclonal antibodies (MAbs) against a 44-kDa OMP of the
HGE agent might be useful for clarifying the relationships of
the OMPs of the HGE agent, for analyzing the antigenic
epitopes, for understanding the immune responses of HGE
agent infection, and for serodiagnosis. In this study, MAbs
against a 44-kDa protein of HGE agent isolate 13 were pro-
duced and characterized by using five HGE agent isolates, a
tick isolate, and rP44.
MATERIALS AND METHODS
Cultures and media. Five isolates of the HGE agent (isolates 13, 2, 11, 3, and
6) (11, 16) and a tick isolate (USG) (5) were propagated in the human promy-
elocytic leukemia cell line HL-60 (American Type Culture Collection [ATCC],
Manassas, Va.) in RPMI 1640 medium supplemented with 5% fetal bovine
serum (FBS) (Atlanta Biologicals, Norcross, Ga.), 1% minimal essential medium
(MEM) nonessential amino acid mixture (GIBCO, Grand Island, N.Y.), 1 mM
MEM sodium pyruvate (GIBCO), and 2 mM L-glutamine (GIBCO) (11, 16).
Ehrlichia chaffeensis in THP-1 cells (ATCC) and Ehrlichia sennetsu in P388D1
cells (ATCC) were maintained in RPMI 1640 medium supplemented with 10%
FBS and 2 mM L-glutamine, while Ehrlichia canis in DH82 cells (12) and my-
eloma cells (SP2/0-Ag14; ATCC) were maintained in Dulbecco’s modified Ea-
gle’s medium (DMEM) (GIBCO) supplemented with 10% FBS and 2 mM
L-glutamine. All cultures were incubated at 37°C in a humidified 5% CO2–95%
air atmosphere. Infectivities of all Ehrlichia spp. were determined by Diff-Quik
(modified Giemsa; Baxter Scientific Products, Obetz, Ohio) staining, as de-
scribed previously (11, 12).

3278
VOL. 36, 1998
TABLE 1. Characteristics of MAbs against the HGE agent
Protein(s) (kDa) recognized in HGE agent isolateb:
MAba Isotype
13 2 11
3E65 IgG1 44, 110 110
5C11 IgG2b 44, 89 41, 46, 47
5D13 IgG2b 44 41, 46, 47
a
Subcloned three times by limiting dilution techniques.
b
Major antigens of the six HGE agent isolates are shown in boldface type.
Preparation of purified ehrlichiae and the outer membrane fraction. Infected
cells were weakly sonicated under predetermined conditions (16) to lyse infected
cells with minimum damage to ehrlichiae. After centrifugation to remove un-
broken cells and nuclei of the host cells, the supernatants containing freed
ehrlichiae were size fractionated by Sephacryl S-1000 (Pharmacia, Uppsala,
Sweden) chromatography, as previously described (12, 16). For sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 25 mg of protein from
each purified organism was aliquoted into 5 ml of 10 mM sodium phosphate
buffer (4 mM NaH2PO4 and 6 mM Na2HPO4, pH 7.4) containing 1 mM phe-
nylmethylsulfonyl fluoride (PMSF) (Sigma, St. Louis, Mo.) and frozen at 285°C.
The outer membrane fractions of the six isolates were prepared by a modified
Sarkosyl differential solubilization method (16). The outer membrane fraction
was washed once with 50 ml of 0.2% Sarkosyl in 10 mM sodium phosphate buffer
by centrifugation at 10,000 3 g for 1 h. The final pellets were resuspended in 10
mM sodium phosphate buffer containing 1 mM PMSF and frozen at 285°C.
Immunization. Eight 6-week-old male BALB/c mice (Harlan Sprague-Dawley,
Indianapolis, Ind.) were injected intraperitoneally with a 0.2-ml mixture of equal
volumes of purified HGE agent 13 (200 mg/mouse) in PBS (2.7 mM KCl, 1.8 mM
KH2PO4, 137 mM NaCl, and 10 mM Na2HPO4 [pH 7.4]) and Freund’s complete
adjuvant (Sigma). Two weeks after the first immunization, each mouse was
boosted subcutaneously with the 0.2-ml mixture of equal volumes of 100 mg of
the purified HGE agent in PBS and Freund’s incomplete adjuvant (Sigma). The
mice were intraperitoneally injected with purified HGE agent in PBS (100
mg/mouse) without adjuvant on the 7th day after the second immunization.
Between 35 and 40 days after the first immunization, 0.5 ml of blood was
collected from the retroorbital venous plexus of immunized mice. Serum was
collected and antibody titers were determined by an IFA test using antigen slides
of HGE agent 13 prepared as previously described (11, 16). Four days before
sacrifice, the purified HGE agent (100 mg/mouse in 0.1 ml of PBS) was intrave-
nously injected.
Hybridoma production. When antibody titers were greater than 1:2,560, mice
were sacrificed and spleen cells were fused with myeloma cells in either 50%
polyethylene glycol 4000 (GIBCO) or 40% polyethylene glycol 4000 containing
7.6% dimethyl sulfoxide (Sigma) in PBS, according to the procedure described
by Goding (7). Two milliliters of the fusion mixture (105 myeloma cells and 3 3
105 to 4 3 105 spleen cells) in complete DMEM (cDMEM) (DMEM containing
20% FBS, 1 mM MEM sodium pyruvate, 1% MEM nonessential amino acid
mixture, 2 mM L-glutamine, and a 1:100 dilution of antibiotic-antimycotic mix-
ture [GIBCO] [penicillin G, 10,000 U/ml; streptomycin sulfate, 10,000 mg/ml;
amphotericin B, 25 mg/ml]) containing HAT supplement (diluted 1:100 from the
stock solution of 10 mM Na-hypoxanthine, 40 mM aminopterin, and 1.6 mM
thymidine [GIBCO]) was aliquoted into each well of 24-well culture plates with
spleen feeder cells. All cultures were incubated at 37°C in a humidified 5%
FIG. 1. Western immunoblot analysis of HGE agent 13, E. chaffeensis, E. canis, E. sennetsu, and their uninfected host cells with three MAbs. (A) Proteins were
separated on a 10% polyacrylamide gel and stained with Coomassie blue. (B) For Western blot analysis, hybridoma (3E65, 5C11, and 5D13) culture supernatants were
used at 1:10 dilutions. Molecular weight (MW) standards were from Bio-Rad.
MAbs TO HGE AGENT 44-kDa PROTEIN 3279
3 6 USG
110 110 110 110
47 47 45 44, 45, 46
47 47 45 44, 45, 46
CO2–95% air atmosphere. When colonies became visible, the medium was grad-
ually replaced with cDMEM containing 100 mM Na-hypoxanthine (GIBCO) and
16 mM thymidine (Sigma). For subcultures of positive clones, cDMEM was used.
Screening of candidate hybridoma clones. To screen clones producing MAbs
against the HGE agent, both the IFA test and enzyme-linked immunosorbent
assay (ELISA) were used. For the IFA test, 10 ml of undiluted supernatant was
added to each well of HGE agent 13 slides prepared as previously described (11,
16) and incubated at 37°C for 90 min. After a wash with 23 PBS (19 mM
K2HPO4, 12 mM KH2PO4, and 300 mM NaCl [pH 7.4]) containing 0.05% Tween
20 (Sigma), 10 ml of fluorescein isothiocyanate-conjugated rabbit anti-mouse
immunoglobulin G (IgG) 1 IgM (Jackson ImmunoResearch Laboratories, West
Grove, Pa.) at a 1:100 dilution was added and incubated at 37°C for 90 min. All
slides were counterstained with 0.1% Evans blue in 23 PBS and examined with
a Nikon (Garden City, N.Y.) Microphot-FX epifluorescence microscope. For
photography of labeled cells, infected cells were cytocentrifuged and fixed with
Diff-Quik fixative containing methanol and incubated with antibodies as de-
scribed above.
For ELISA, 96-well plates were coated with purified HGE agent 13 at 1 mg of
protein/well in 50 mM Na-bicarbonate buffer (pH 9.6) and blocked with 5%
nonfat dry milk in the same buffer. One hundred microliters of undiluted hy-
bridoma supernatant was added to each well, including 100 ml each of both the
positive (polyclonal mouse anti-HGE agent 13) and negative (normal BALB/c
mouse serum) controls at 1:10 dilutions in PBS containing 5% nonfat dry milk.
All plates were incubated at 37°C for 1 h and rinsed three times with PBS
containing 0.05% Tween 20. Peroxidase-conjugated goat anti-mouse immuno-
globulins (IgG 1 IgA 1 IgM) (ICN Pharmaceuticals, Aurora, Ohio) at a 1:500
dilution in PBS containing 5% nonfat dry milk were added and incubated at 37°C
for 1 h. ABTS [0.15% 2,29-azino-di-(3-ethylbenzthiazoline sulfonate)] (Sigma) in
0.1 M sodium citrate buffer (pH 4.2) and 0.03% H2O2 were added and incubated
at room temperature for 15 min. The supernatants which had absorbances at 405
nm of greater than 0.8 were again confirmed by the IFA test. The cutoff value of
0.8 was chosen because negative control wells had absorbances of less than 0.5
and 100% of wells with absorbances of above 1.0 were IFA positive; we chose 0.8
to cover some weakly positive clones. Finally, all hybridomas identified as posi-
tive clones by both tests were subcultured into six-well culture plates. When cells
were confluent, these clones were frozen in 90% FBS and 10% dimethyl sulfox-
ide at 285°C. The wells identified as positive clones by primary screening were
subcloned three times via a limiting dilution technique (7).
Production of ascitic fluids containing MAbs. For rapid production of ascitic
fluids containing MAbs, 3 to 5 days prior to hybridoma injection, 0.5 ml of the
inflammatory agent pristane (Sigma) was intraperitoneally injected into 6-week-
old male BALB/c mice (10 mice per clone). At 12 days after intraperitoneal
injection of actively growing hybridomas (3E65, 5C11, and 5D13) into the mice
3280 KIM AND RIKIHISA
FIG. 2. Effect of PMSF on the molecular size of major antigens recognized
by MAb 5C11. Uninfected HL-60 cells, purified HGE agent 13, 1 mM PMSF-
treated HGE agent, and the OMP fraction of the HGE agent were separated on
a 10% polyacrylamide gel, transferred to a nitrocellulose membrane, and incu-
bated with a 1:50 dilution of MAb 5C11 in ascitic fluid. Molecular weight (MW)
standards were from Bio-Rad.
(2 3 106 to 3 3 106 cells/mouse in 0.2 ml of sterile PBS), ascitic fluids containing
MAbs were harvested and centrifuged at 10,000 3 g for 10 min. After the ascitic
fluid for each clone was pooled and inactivated by incubation at 56°C for 30 min,
antibody titers were determined by the IFA test with an antigen slide of HGE
agent 13. Ascitic fluid containing MAbs was diluted at 1:10, filtered through a
0.45-mm-pore-size filter, and frozen at 220 or 285°C.
Isotyping of MAbs. Using either ascitic fluids or culture supernatants, isotypes
of MAbs were determined by the Mouse Monoclonal Typing Kit (The Binding
Site, Birmingham, United Kingdom), which is based on the Ouchterlony immu-
nodiffusion technique, and by the Mouse MAb ID/SP Kit (Zymed Laboratories,
Inc., South San Francisco, Calif.), which is based on a streptavidin-biotin ampli-
fication system in an HGE agent antigen-dependent ELISA.
SDS-PAGE and Western blot analysis. Twenty-five micrograms of proteins of
purified isolates (isolates 13, 2, 11, 3, 6, and USG) and the OMP fraction of each
isolate; purified E. chaffeensis, E. sennetsu, and E. canis; uninfected host cells
(HL-60, THP-1, and DH82); and 10 mg of rP44, affinity purified as described
elsewhere (17), were dissolved in sample buffer (5% 2-mercaptoethanol, 10%
glycerol, 2% SDS, and 0.08% bromophenol blue in 62.5 mM Tris buffer [pH
6.8]). To examine whether the antigenic epitope is trypsin sensitive, 100 mg of
purified HGE agent was incubated in 0.5 ml of 0.25% trypsin (Sigma) in PBS for
15 min at room temperature. Samples, including 10 ml of diluted (1:20) broad-
range molecular weight standards (Bio-Rad, Hercules, Calif.), were boiled at
FIG. 3. Comparisons of six purified HGE agent isolates with three MAbs on a 10% polyacrylamide gel (A) and by Western blot analysis (B). (A) Proteins from
purified HGE agent isolates (13, 2, 11, 3, 6, and USG) were separated on a 10% polyacrylamide gel and stained with Coomassie blue. (B) For Western blot analysis,
hybridoma (3E65, 5C11, and 5D13) culture supernatants were used at 1:10 dilutions. Molecular weight (MW) standards were from Bio-Rad.
J. CLIN. MICROBIOL.
100°C for 5 min. SDS-PAGE and Western blotting were performed as described
elsewhere (12, 16). Protein blots were immersed in blocking buffer (5% nonfat
dry milk in 23 PBS) at 4°C overnight and incubated with culture supernatants (at
a 1:10 dilution in blocking buffer) or ascitic fluids (at a 1:50 dilution) of hybrid-
oma clones 3E65, 5C11, or 5D13 at room temperature for 2 h. After three rinses
with TNTT buffer (50 mM Tris-HCl, 150 mM NaCl, 0.02% Tween 20, and 0.01%
thimerosal [Sigma] [pH 7.4]), the blots were incubated at room temperature for
2 h with peroxidase-conjugated goat anti-mouse immunoglobulins (IgG 1 IgA 1
IgM) (ICN Pharmaceuticals) at a 1:2,000 dilution in blocking buffer. The blots
were washed three times with TNTT buffer for 5 min each. The peroxidase-
positive bands were detected by immersing the blots in a developing solution (73
mM sodium acetate, pH 6.2) containing 0.3% diaminobenzidine tetrahydrochlo-
ride (Nacalai Tesque, Inc., Kyoto, Japan) and 0.04% H2O2 at room temperature
for 5 min. The enzyme reaction was terminated by washing the blots in 0.1 M
H2SO4.
Immunogold labeling. HGE agent 13-infected cells (2 3 107) were fixed in a
fixative (2.5% paraformaldehyde, 0.5% glutaraldehyde, 0.03% trinitrophenol,
and 0.03% CaCl2 in 0.05 M cacodylate buffer [pH 7.4]) at room temperature for
1 h and en bloc stained with 1% uranyl acetate in 0.1 M maleate buffer (pH 5.2)
at room temperature for 1 h. The specimen was dehydrated in a series of graded
ethanols (50 to 90%) and embedded in LR gold (Polysciences, Inc., Warrington,
Pa.) which had been polymerized at 225°C in a low-temperature UV curing unit
(Polysciences). Ultrathin sections (800 Å) were cut with a diamond knife and
mounted on 300-mesh nickel grids. The grids were incubated for 1 h with each
MAb in ascitic fluid or positive and negative control sera diluted 1:50 with
PBS-GT (0.1% gelatin and 0.01% Tween 20 in PBS, pH 7.4). The grids were
rinsed in PBS-GT and incubated at room temperature for 1 h with goat anti-
mouse IgG 1 IgM conjugated with 10-nm gold particles (Amersham, Arlington
Heights, Ill.) diluted 1:20 with PBS-GT. The grids were gently rinsed in PBS-GT
and distilled water. The sections were stained with 2% uranyl acetate in water
and observed with a Philips 300 transmission electron microscope at 60 kV.
Mouse protection assay. Five groups of three mice each (C3H/HeN, 4-week-
old females; Harlan Sprague-Dawley) were inoculated intraperitoneally with
HGE agent 13-infected HL-60 cells (greater than 90% infected cells, 106 cells/
mouse) which had been preincubated at 37°C for 30 min with 0.3 ml of each MAb
in ascitic fluid or positive or negative control mouse sera. MAbs and the positive
control serum were diluted in PBS so that the final IFA titer against HGE agent
13 was 1:2,000. One day after infection, mice were injected intraperitoneally with
0.3 ml of each MAb or control sera. At 5 days post-HGE agent inoculation, mice
were sacrificed for collection of blood specimens. DNA was extracted from the
blood with the QIAamp blood kit (Qiagen Inc., Chatsworth, Calif.). A pair of
primers, 497-521 (59-TAGGCGGTTCGGTAAGTTAAAG-39) and 747-727 (59-
GCACTCATCGTTTACAGCGTG-39) (13), was used for amplification in a
thermal cycler (model 480; Perkin-Elmer) with 5 min of denaturation at 94°C,
followed by 40 cycles of denaturation at 94°C, annealing at 57°C, and extension
at 72°C for 1 min each. After the last cycle, extension was continued for 7 min at
72°C. Each 50-ml PCR mixture contained 1 mg of template DNA, 5 ml of 103
reaction buffer, 0.2 mM concentrations of deoxynucleoside triphosphates, 2.5
mM MgCl2, 1.25 U of Taq polymerase, and 16 pmol of each primer. PCR
products (10 ml each) were electrophoresed in 1.5% agarose gels containing 0.5
mg of ethidium bromide at 95 V for 1 h and photographed under UV illumina-
tion with a gel video system (Gel Print 2000i; Biophotonics Corporation, Ann
Arbor, Mich.).
VOL. 36, 1998 MAbs TO HGE AGENT 44-kDa PROTEIN 3281

FIG. 4. Western blot analysis of the OMP fractions from six HGE agent isolates with three MAbs. (A) The OMP fractions from the six HGE agent isolates (13,
2, 11, 3, 6, and USG) were separated on a 10% polyacrylamide gel and stained with Coomassie blue. (B) For Western blot analysis, hybridoma (3E65, 5C11, and 5D13)
culture supernatants were used at 1:10 dilutions. Molecular weight (MW) standards were from Bio-Rad.

RESULTS These two proteins appear to have a common epitope in HGE

agent 13, since it is unlikely that, after subcloning three times
Production of hybridomas and MAbs. By both IFA test and by the limiting dilution technique, two hybridomas would be
ELISA, 16.7% of colonies (127 of 761) were positive at the first cocloned in MAb 3E65. Although MAbs 5C11 and 5D13 were
screening. Subsequently, 39 hybridomas with strong antibody derived from different master plates prepared from the same
production were subcloned three times and 20 hybridomas mouse spleen, they reacted in almost identical manners by
were identified as positive by Western blot analysis. Three Western blot analysis. Both recognized proteins that are ap-
clones (3E65, 5C11, and 5D13) which reacted to the approxi- proximately 44 kDa in all six isolates (Fig. 3B). The molecular
mately 44-kDa major antigen of HGE agent 13 were selected sizes and numbers of the major antigens recognized by MAbs
for this study. Ascitic fluid was produced with each MAb, and 5C11 and 5D13 varied slightly, from 44 to 47 kDa, among the
MAb isotypes were determined (Table 1). IFA titers of ascitic six isolates (Fig. 3B and Table 1). MAbs 5C11 and 5D13
fluids and culture supernatants of the three MAbs used were recognized antigens of the same molecular sizes both in whole
1:20,480 and 1:320, respectively. These three clones have been organisms and in the OMP fractions of the six isolates (Fig.
stable for more than 1 year. 4B). Thus, all of these antigenic proteins common within each
Western immunoblotting. All three MAbs recognized the isolate and among the six isolates appear to be present in the
HGE agent but not E. chaffeensis, E. canis, E. sennetsu, or their outer membrane.
uninfected host cells (HL-60, THP-1, and DH82) (Fig. 1B). MAb 3E65 did not recognize the rP44 antigen, while MAbs
Although band intensities and molecular sizes were slightly 5C11 and 5D13 did (Fig. 5). These results show that the
variable among HGE isolates, the major antigens recognized epitope of the 44-kDa protein recognized by MAb 3E65 is
by the three MAbs were around 44 kDa (Fig. 1B and Table 1). different from those recognized by MAbs 5C11 and 5D13.
In our previous study, with polyclonal antibodies, multiple Immunofluorescence and immunogold labeling. Polyclonal
bands of approximately 44 kDa were seen in whole HGE agent anti-HGE agent mouse serum strongly labeled entire HGE
antigen, but fewer bands were seen in the OMP fraction (16).
Since we had used 1 mM PMSF for preparation of the OMP
fraction of HGE agents but not for purification of whole HGE
agent organisms, we examined the effect of 1 mM PMSF on the
molecular size of proteins recognized with MAb 5C11 by West-
ern blot analysis. We found that in the presence of PMSF, the
density of the 47-kDa band was reduced and that of the 44-kDa
band became thicker, suggesting that a 47-kDa protein is the
precursor of the 44-kDa protein and that a protease which
cleaves the 47-kDa protein to a 44-kDa protein might be de-
graded in the absence of PMSF (Fig. 2). Therefore, 1 mM
PMSF was added to all specimens throughout this study. No
difference was detected if PMSF was added at the beginning
or just after purification of the HGE agent, suggesting that
this change primarily occurs after purification of the HGE
agent.
Trypsin treatment of HGE agent 13 completely destroyed
the reactivity of all three MAbs, indicating that these MAbs
recognize trypsin-sensitive epitopes (data not shown).
When the six isolates (13, 2, 11, 3, 6, and USG) were com- FIG. 5. Western blot analysis of rP44 with three MAbs. (A) Purified HGE
agent 13 and affinity-purified rP44 were separated on a 10% polyacrylamide gel
pared, MAb 3E65 reacted strongly with a 44-kDa protein in and stained with Coomassie blue. (B) For Western blot analysis, hybridoma
HGE agent 13 but not in the remaining five isolates. It also (3E65, 5C11, and 5D13) culture supernatants were used at 1:10 dilutions. Mo-
reacted weakly with a 110-kDa protein in all isolates (Fig. 3B). lecular weight (MW) standards were from Bio-Rad.
3282 KIM AND RIKIHISA J. CLIN. MICROBIOL.

FIG. 6. IFA staining of HGE agent 13 in HL-60 cells with three MAbs. Infected HL-60 cells were fixed in Diff-Quik fixative and incubated with MAbs (3E65, 5C11,
and 5D13) in ascitic fluid at 1:100 dilutions and with positive and negative control (NS) mouse sera at 1:50 dilutions. Note the ring-like labeling of the HGE agent with
all three MAbs. Magnification, 31,600.

agent 13 organisms, while the negative control mouse serum gold labeling results show that a 44-kDa OMP antigen is con-
did not label any structure at all in the IFA test. All three centrated on the membrane of the HGE agent; with polyclonal
MAbs labeled both intracellular and extracellular HGE agents antiserum and MAb 3E65, some labeling appeared to be
bound to HL-60 cells in a ring-like pattern (Fig. 6). Immuno- present on inclusion membranes, too (Fig. 7).

FIG. 7. Transmission electron micrograph of HGE agent 13 in HL-60 cells immunogold labeled with MAbs. Infected cells were embedded in LR Gold, and ultrathin
sections on grids were incubated with MAbs (3E65 and 5C11) and positive and negative control mouse sera and with goat anti-mouse IgG 1 IgM conjugated with 10-nm
gold particles (Amersham). Bars, 0.4 mm.
VOL. 36, 1998 MAbs TO HGE AGENT 44-kDa PROTEIN 3283

FIG. 8. PCR detection of the HGE agent 16S rRNA gene in the blood of HGE agent-inoculated mice passively immunized with MAbs. Three mice each were
inoculated with HGE agent 13-infected HL-60 cells and each MAb or positive or negative control mouse serum. Each group of mice was inoculated again on the 2nd
day with the same antibody. DNAs were prepared from the blood of all mice at 5 days post-HGE agent inoculation. One microgram of DNA was used as the template
for PCR. As a positive control, 10 pg of DNA extracted from purified HGE agent 13 was used, while distilled water without template was used as a negative control.
The arrow shows the HGE agent-specific 16S rRNA gene fragment (287 bp) obtained by PCR amplification. N1 to N3, negative control mouse serum-treated mice;
P1 to P3, positive control (polyclonal anti-HGE agent) mouse serum-treated mice; E1 to E3, MAb 3E65-treated mice; C1 to C3, MAb 5C11-treated mice; D1 to D3:
MAb 5D13-treated mice. Numbers on the left indicate molecular sizes of fX174 RF DNA/HaeIII fragments (GIBCO). The experiment was repeated twice.

Mouse protection test. Passive immunization with polyclonal
mouse anti-HGE agent serum and MAb 3E65 consistently
showed superior protection, with 67% (2 of 3 mice) protected
from HGE agent 13 infection; with MAbs 5C11 and 5D13,
33% (1 of 3 mice) were protected (Fig. 8). Although the mice
did not show any clinical signs during the 5-day infection pe-
riod, all negative control mice were positive for HGE agent
DNA by PCR, indicating subclinical infection. The detection
limit of PCR was 0.15 pg of DNA in blood.
DISCUSSION
Our previous study showed that multiple bands between 43
and 49 kDa of HGE agents are recognized in both whole cells
and OMP fractions by polyclonal antisera against the six strains
of the HGE agent. We also found that although these antigenic
epitopes were cross-reactive with antisera to other isolates, the
HGE agent has a strain polymorphism in its major antigenic
proteins (16). The present study, using MAbs against OMPs of
the HGE agent, suggests two major reasons for the presence of
these multiple major antigenic proteins. One is the degrada-
tion of a protease which probably cleaves a 47-kDa protein to
a 44-kDa protein during HGE agent storage or during SDS-
PAGE. One possibility is that this 3-kDa molecular size dif-
ference is due to the cleavage of an N-terminal signal peptide,
termed a leader sequence. Our DNA sequence data of the
44-kDa major antigen gene shows an amino acid sequence
(VRA) recognizable by signal peptidases, and the chemically
determined N-terminal amino acid sequence showed that this
site is actually cleaved in a native mature 44-kDa protein (17).
The bacterial signal peptidase is not inhibited by PMSF (14,
18). Since PMSF treatment of the HGE agent prevented a
degradation-induced artifact of multiple bands of major anti-
gens, we recommend the use of PMSF for HGE agent SDS-
PAGE and Western blot analysis. Although there is no N-
glycosylation site based on the predicted amino acid sequence
from the 44-kDa protein gene (17), whether this protein is O
glycosylated or whether any glycosylation may contribute to
multiple bands is unknown. The second and more significant
reason for the presence of multiple bands may be the presence
of multiple cross-reactive major antigenic proteins of different
molecular sizes, since even in the presence of PMSF these
multiple bands were seen in some isolates. This reason is sup-
ported by our recent study which revealed the presence of
multiple homologous gene copies of the major 44-kDa OMPs
in the HGE agent genome (17).
Our rP44 antigen approximately corresponds to the N-ter-
minal half of the 44-kDa protein and was commonly recog-
nized by all HGE patient sera tested and by anti-E. equi serum
(17). Therefore, MAbs 5C11 and 5D13 appear to recognize an
antigenic epitope present at the N-terminal half of the 44-kDa
protein. Since MAb 3E65 did not recognize rP44, it may be
reacting to the C-terminal half of the 44-kDa protein. If this is
the case, the C-terminal half of the 44-kDa protein of HGE
agent 13 may contain an antigenic epitope in common with the
110-kDa protein, but homologous 44-kDa proteins of the re-
maining isolates may lack this epitope. MAbs 5C11 and 5D13
appear to behave in almost identical manners. An additional
study is required to determine whether the two MAbs recog-
nize the same epitope.
The three MAbs protected mice from HGE agent 13 infec-
tion, suggesting that the 44-kDa protein has a significant role in
establishing HGE agent infection in mice. MAb 3E65 is as
effective as polyclonal antiserum in protecting mice from HGE
agent infection and is more effective than MAbs 5C11 or 5D13,
suggesting that the epitope recognized by MAb 3E65 may be
more accessible to the antibody or may have a more critical
role in HGE agent infection than those recognized by the other
two MAbs. These MAbs may serve as useful reagents in ana-
lyzing the interaction of the HGE agent with host cells and may
also be useful for purifying major OMPs and in HGE serodi-
agnosis.
ACKNOWLEDGMENTS
This research was supported by grant AI40934 from the National
Institutes of Health.
We thank Evelyn Handley, of the EM laboratory of Veterinary
Biosciences, The Ohio State University, for technical assistance. Our
colleagues Norio Ohashi and Ning Zhi are appreciated for helpful
3284 KIM AND RIKIHISA
advice in purifying rP44. We also thank Lawrence Dearth and Ray-
mond Mankoski for editorial assistance.
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