INTRODUCTION

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Human granulocytic ehrlichiosis (HGE) is an emerging tick-borne zoonosis characterized by fever, headache, myalgia, elevated liver enzyme (serum aminotransferase) levels, leukopenia, anemia, thrombocytopenia, and elevated C-reactive protein levels (2). The first 12 reported cases were in Wisconsin 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 replicates 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 immunofluorescent 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 peripheral 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 between 43 and 49 kDa, are immunodominant antigens in human infection (16, 17). Western blot analysis also revealed variations 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 produced and characterized by using five HGE agent isolates, a tick isolate, and rP44.

	MATERIALS AND METHODS

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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 promyelocytic 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 myeloma cells (SP2/0-Ag14; ATCC) were maintained in Dulbecco's modified Eagle'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 described previously (11, 12).

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 unbroken 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 µg of protein from each purified organism was aliquoted into 5 µl of 10 mM sodium phosphate buffer (4 mM NaH₂PO₄ and 6 mM Na₂HPO₄, pH 7.4) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma, St. Louis, Mo.) and frozen at 85°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 µl of 0.2% Sarkosyl in 10 mM sodium phosphate buffer by centrifugation at 10,000 × g for 1 h. The final pellets were resuspended in 10 mM sodium phosphate buffer containing 1 mM PMSF and frozen at 85°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 µg/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 µg 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 µg/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 µg/mouse in 0.1 ml of PBS) was intravenously 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 × 10⁵ to 4 × 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 mixture [GIBCO] [penicillin G, 10,000 U/ml; streptomycin sulfate, 10,000 µg/ml; amphotericin B, 25 µg/ml]) containing HAT supplement (diluted 1:100 from the stock solution of 10 mM Na-hypoxanthine, 40 µM 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% CO₂-95% air atmosphere. When colonies became visible, the medium was gradually replaced with cDMEM containing 100 µM Na-hypoxanthine (GIBCO) and 16 µM 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 µl 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 2× PBS (19 mM K₂HPO₄, 12 mM KH₂PO₄, and 300 mM NaCl [pH 7.4]) containing 0.05% Tween 20 (Sigma), 10 µl of fluorescein isothiocyanate-conjugated rabbit anti-mouse immunoglobulin G (IgG) + 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 2× 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 described above.

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 (2 × 10⁶ to 3 × 10⁶ cells/mouse in 0.2 ml of sterile PBS), ascitic fluids containing MAbs were harvested and centrifuged at 10,000 × 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-µm-pore-size filter, and frozen at 20 or 85°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 immunodiffusion technique, and by the Mouse MAb ID/SP Kit (Zymed Laboratories, Inc., South San Francisco, Calif.), which is based on a streptavidin-biotin amplification 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 µg 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 µg 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 µl of diluted (1:20) broad-range molecular weight standards (Bio-Rad, Hercules, Calif.), were boiled at 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 2× 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 hybridoma 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 + IgA + 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 tetrahydrochloride (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 × 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 25°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 + 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 (5'-TAGGCGGTTCGGTAAGTTAAAG-3') and 747-727 (5'-GCACTCATCGTTTACAGCGTG-3') (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-µl PCR mixture contained 1 µg of template DNA, 5 µl of 10× 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 µl each) were electrophoresed in 1.5% agarose gels containing 0.5 µg of ethidium bromide at 95 V for 1 h and photographed under UV illumination with a gel video system (Gel Print 2000i; Biophotonics Corporation, Ann Arbor, Mich.).

	RESULTS

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Production of hybridomas and MAbs. By both IFA test and ELISA, 16.7% of colonies (127 of 761) were positive at the first screening. Subsequently, 39 hybridomas with strong antibody production were subcloned three times and 20 hybridomas were identified as positive by Western blot analysis. Three clones (3E65, 5C11, and 5D13) which reacted to the approximately 44-kDa major antigen of HGE agent 13 were selected for this study. Ascitic fluid was produced with each MAb, and MAb isotypes were determined (Table 1). IFA titers of ascitic fluids and culture supernatants of the three MAbs used were 1:20,480 and 1:320, respectively. These three clones have been stable for more than 1 year.

Western immunoblotting. All three MAbs recognized the HGE agent but not E. chaffeensis, E. canis, E. sennetsu, or their uninfected host cells (HL-60, THP-1, and DH82) (Fig. 1B). Although band intensities and molecular sizes were slightly variable among HGE isolates, the major antigens recognized by the three MAbs were around 44 kDa (Fig. 1B and Table 1).

View larger version (41K): [in this window] [in a new window]   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.

View larger version (28K): [in this window] [in a new window]   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 incubated with a 1:50 dilution of MAb 5C11 in ascitic fluid. Molecular weight (MW) standards were from Bio-Rad.

View larger version (39K): [in this window] [in a new window]   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.

View larger version (44K): [in this window] [in a new window]   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.

View larger version (45K): [in this window] [in a new window]   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 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.

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

View larger version (38K): [in this window] [in a new window]   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, ×1,600.

View larger version (193K): [in this window] [in a new window]   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 + IgM conjugated with 10-nm gold particles (Amersham). Bars, 0.4 µm.

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 period, 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.

View larger version (34K): [in this window] [in a new window]   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 X174 RF DNA/HaeIII fragments (GIBCO). The experiment was repeated twice.

	DISCUSSION

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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 degradation 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 difference 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 antigens, 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 supported 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-terminal half of the 44-kDa protein and was commonly recognized 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 remaining 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 recognize the same epitope.

The three MAbs protected mice from HGE agent 13 infection, 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 analyzing the interaction of the HGE agent with host cells and may also be useful for purifying major OMPs and in HGE serodiagnosis.