Vol. 58, No. 7
INFECTION AND IMMUNITY, JUlY 1990, p. 2105-2114 0019-9567/90/072105-10$02.00/0 Copyright © 1990, American Society for Microbiology
Production and Characterization of Monoclonal Antibodies to Cell Wall Antigens of Aspergillus fumigatus LINE STE-MARIE, SERGE StNtCHAL, MICHtLE BOUSHIRA, SIMON GARZON, HENRI STRYKOWSKI, LOUISE PEDNEAULT, AND LOUIS DE REPENTIGNY*
Department of Microbiology and Immunology, Faculty of Medicine, University of Montreal, and Ste-Justine Hospital, Montreal, Quebec H3T JC5, Canada Received 27 September 1989/Accepted 20 April 1990
Two murine monoclonal antibodies (MAbs) against AspergUlus fumigatus were produced and characterized. Splenocytes from cell wail-immunized BALB/c mice were fused with SP2/0 myeloma cells. The hybridomas were screened with a cold alkali (CA) extract of mycelium containing protein, mannose, and galactose, and two MAbs of the immunoglobulin M class were purified from ascites fluid. MAbs 1 and 40 were characterized by double immunodiffusion against CA antigen, indirect enzyme immunoassay with mannans of Candida albicans serotypes A or B or Candida tropicalis, indirect immunofluorescence with C. albicans- or A. fumigatus-infected tissues, indirect immunofluorescence with smears of other pathogenic fungi, Western blotting (immunoblotting) with the lectin concanavalin A or BS-1 from the seeds of Bandeiraea simplicifolia, and immunoelectron microscopy. MAb 1 did not cross-react with Candida mannan and recognized a periodate-sensitive, pronaseand heat-resistant epitope in CA antigen and three mannose- and galactose-containing components (80, 62, and 49 kilodaltons) of a mycelial homogenate. Immunoelectron microscopy demonstrated binding of MAb 1 to the inner cell wail and intracellular membranes of hyphae and conidia of A. fumigatus. Circulating antigen was detected in experimental invasive aspergillosis by inhibition enzyme immunoassay with MAb 1 and CA antigen. MAb 40 was a nonprecipitating antibody cross-reactive with Candida species, and competition for an epitope located diffusely in the cell wall of A. fumigatus hyphae was demonstrated by incubating MAb 40 with mannan of C. albicans serotype A. These results suggest that MAb 1 recognizes immunodominant oligogalactoside side chains of A. fumigatus galactomannan, while MAb 40 binds to mannopyranosyl side chains common to A. fumigatus galactomannan and C. albicans mannan.
The galactomannan of Aspergillus fumigatus has gained increased attention because it can be detected in serum (19, 21, 28, 40, 45, 53), bronchoalveolar lavage fluid (1), and urine (21) of aspergillus-infected rabbits and in serum (21, 28, 37, 40, 41, 44, 50, 52, 54), urine (21), bronchoalveolar lavage fluid (2), and peritoneal dialysis fluid (18) of patients with aspergillosis. Barreto-Bergter et al. (7) elucidated the structure of A. fumigatus galactomannan purified from 5-day-old mycelium. It consisted of a main chain of 1,6-linked aD-mannopyranose residues substituted at 0-2 by one to three a-D-mannopyranosyl units that were 1,2-interlinked. P-D-galactofuranosyl units were 1,6-linked to the D-mannan core, being components of side chains with an average length of six units which were 1,5-interlinked. The galactomannans of Aspergillus niger, Aspergillus flavus, Aspergillus terreus, and Aspergillus nidulans contained similar structural features (8). This structure was largely in agreement with the methylation-fragmentation studies of Reiss and Lehmann (37), who also proposed a 1,6-linked mannan backbone with oligogalactoside side chains three units long, terminating in galactofuranose. The presence of mannose as a side-chain component was also inferred. Azuma et al. (4) also found both 1,2- and 1,6linked D-mannose residues but proposed that the 1,6-linked D-mannose residues were located in side chains. The immunodominance of galactofuranosyl groups in A. fumigatus galactomannan has been demonstrated by hapten inhibition studies with antibody raised in rabbits (9) and by loss of precipitin activity after hydrolysis of acid-labile galactofuranosyl residues (42). The production of monoclonal antibodies (MAbs) against pathogenic fungi has been reported for several organisms including B. dermatitidis, Candida albicans, Candida tropi-
Galactomannans are common cell wall polysaccharide antigens widely distributed among leguminous plants and fungi (34). They are generally composed of an a-1,6-linked mannan backbone substituted with mannopyranose oligosaccharides and immunodominant terminal galactofuranose present either singly or linked to oligogalactosides (34). Galactomannans have been isolated from the mycelium or culture filtrate of pathogenic fungi including Aspergillus spp. (4, 7, 8, 37), Penicillium spp. (43), Trichophyton spp. (6, 11, 12, 43), Microsporum spp. (11, 12), Histoplasma capsulatum (3, 38), Histoplasma duboisii (3), Blastomyces dermatitidis (3), Coccidioides immitis (33), Paracoccidioides brasiliensis (3), Sporothrix schenckii (34), Exophiala (Cladosporium) werneckii (29), Fonsecaea (Hormodendrum) pedrosoi (48), Trichosporon spp. (22), and Alternaria spp. (5). The wide distribution and similar chemical structure of the galactomannans have been responsible for serologic cross-reactions. The galactomannans of H. capsulatum, H. duboisii, P. brasiliensis, and B. dermatitidis cross-react immunologically in quantitative precipitation and form lines of identity in double immunodiffusion (3). The galactomannan I of dermatophytes has shared antigenicity with those of H. capsulatum, P. brasiliensis, and B. dermatitidis, while the more heavily galactosylated and serologically distinct galactomannan II of dermatophytes cross-reacts with the galactomannans of Aspergillus, Penicillium, and Cladosporium species (3). Galactomannans from black molds of the genera Phialophora, Wangiella, and Fonsecaea are cross-reactive with galactomannans of A. fumigatus, E. werneckii, and Trichophyton rubrum (48, 49). *
Corresponding author. 2105
STE-MARIE ET AL.
calis, Coccidioides immitis, Cryptococcus neoformans and H. capsulatum (34). These have been used for the characterization (13, 32), ultrastructural localization (14, 15, 26), and purification of antigens (36, 55) and as replacements for polyclonal antibodies in clinical immunoassays (35). This work describes the reactivity and characterization of MAbs to cell wall antigens of A. fumigatus. (This work was presented in part at the 89th Annual Meeting of the American Society for Microbiology, New Orleans, La., 14 to 18 May 1989 [Abstr. Annu. Meet. Am. Soc. Microbiol. 1989, F-30, p. 463].) MATERIALS AND METHODS Microorganisms, culture conditions, and preparation of antigens. A. fumigatus 2085 was obtained from the culture collection of the Division of Mycotic Diseases, Centers for Disease Control, Atlanta, Ga., where it is maintained as strain B2570. The fungus was cultured in a 17-liter glass carboy filled with 13 liters of Czapek Dox medium, and incubation was continued for 96 h at 22°C with submerged aeration and magnetic stirring as previously described (19). A cold alkali (CA) extract of mycelium was prepared by the method described by de Repentigny et al. (19) and Reiss and Lehmann (37) and purified by gel filtration chromatography on Sephacryl S-200 Superfine (Pharmacia, Uppsala, Sweden) to separate galactomannan from glucan (19, 37). The antigen preparation contained 50% protein and 50% carbohydrate, and the monosaccharide composition of an acid hydrolysate, determined by gas-liquid chromatography, was galactose and mannose (1:1.2) (19). Mannans of C. albicans 20A (serotype A), C. albicans 526B (serotype B), and C. tropicalis were kindly supplied by Errol Reiss, Division of Mycotic Diseases, Centers for Disease Control, and were prepared as previously described
Immunization of mice. Cell walls of A. fumigatus 2085 were a gift from Errol Reiss and were prepared as described previously (39). They were selected because a good antibody response was obtained in the production of MAbs to C. tropicalis mannan by immunizing mice with mannan-containing cell walls, while purified mannan is a poor immunogen (35). Five-week-old BALB/c mice were immunized subcutaneously and in the footpads with 50 Fxg of walls in 0.2 ml of incomplete Freund adjuvant (Difco Laboratories, Detroit, Mich.). On days 7, 14, 21, 28, and 35, 50 ,ug of the same antigen in saline was injected intraperitoneally. Animals were bled, and antibodies against CA antigen of A. fumigatus or mannan of C. albicans 20A were quantitated by indirect enzyme immunoassay (EIA). On day 45, 3 days before fusion, a booster dose of 50 ,ug of cell walls in saline was injected intravenously. Fusion procedure. Fusion of the splenocytes of a highresponding BALB/c mouse (anti-CA extract titer, 1/16,000) with the SP2/0 plasmacytoma cell line was performed in a ratio of 2:1 in a solution containing 50% (wt/vol) polyethylene glycol 1450 (Eastman Kodak Co., Rochester, N.Y.) by the method of Brodeur et al. (16) with modifications. Myeloma cells were cultured with 20 puM 6-thioguanidine until passage 2 before fusion. Fused cells at a concentration of 1 x 105 cells per ml were dispensed in 96-well tissue culture plates which already contained a feeder layer of 1 x 104 murine macrophages. Cell suspensions were grown in Dulbecco modified Eagle medium (Flow Laboratories, Mississauga, Ontario, Canada) supplemented with 20% fetal calf serum (GIBCO Laboratories, Grand Island, N.Y.), 2 mM
L-glutamine (GIBCO), and antibiotic mixture (100 U of penicillin and 100 ,ug of streptomycin [GIBCO] per ml) in the presence of hypoxanthine-aminopterin-thymidine selection medium (Flow). The first readings were done on days 5 and 6, and the medium was changed on day 14 with hypoxanthine-thymidine instead of hypoxanthine-aminopterin-thymidine. As soon as possible, usually on day 15, supernatants of wells containing growing clones were tested for MAbs directed against CA antigen. The antibody-producing cells were twice recloned by limiting dilution, and ascitic fluid was produced as described by Brodeur et al. (17). EIA procedure. Screening of hybrid clone culture supernatants for antibodies against CA antigen was performed by indirect EIA (23). Microdilution plates (Immulon 1; Dynatech Industries, Inc., Alexandria, Va.) were coated with 0.2 ml of a 2-,ug/ml solution of antigen in 0.06 M carbonate buffer (pH 9.6). Plates were incubated at 37°C for 3 h and then washed four times with double-strength phosphate-buffered saline (PBS) (0.02 M sodium phosphate buffer [pH 7.2], 0.28 M NaCl), containing 0.5 ml of Tween 20 (Fisher Scientific Co., Fair Lawn, N.J.) per liter (PBS-T). Nonspecific adsorption was blocked by incubating the plates with 1% bovine serum albumin (Sigma Chemical Co., St. Louis, Mo.) and 1% polyvinylpyrrolidone (Sigma) in carbonate buffer for 30 min at 37°C. The plate was washed once with PBS-T and incubated with serial twofold dilutions of supernatant for 1 h at 37°C. After washing four times with PBS-T, 200 ,ul of peroxidase-conjugated heavy-chain-specific goat anti-mouse immunoglobulin G (IgG) or IgM (Cappel Worthington, Malvern, Pa.) diluted 1/800 in PBS-T was added to each well. The plate was incubated for 1 h at 37°C and washed four times with PBS-T. A volume of 200 ,ul of o-phenylenediamine (8 mg in 20 ml of citrate buffer [pH 6.0] containing 40 ,u1 of 3% hydrogen peroxide) was added to each well. The color was developed in the dark for 20 min at 22°C, and the reaction was stopped with 50 pul of 4 M H2SO4. The endpoint was defined as the highest dilution of supematant that had an A490 of 0.200 as measured in a spectrophotometer (MR-600; Dynatech). Determination of immunoglobulin class and purification of MAb. The supernatants were tested for class of immunoglobulins by indirect EIA with peroxidase-conjugated heavychain-specific goat anti-mouse IgG or IgM. The globulins in ascitic fluid were twice precipitated with 45% (NH4)2SO4, and IgM MAb was purified on a column (2.6 by 44 cm) of Sephacryl S-300 (Pharmacia). The IgM was recovered in the void volume and pooled to 0.6 mg/ml. The purified antibody showed only heavy and light immunoglobulin chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 10% acrylamide. Precipitin reactions. Double immunodiffusion was performed in 1% Noble agar (Difco) containing 2% polyethylene glycol 6000 (BDH, Toronto, Ontario, Canada). Undiluted purified MAb (0.6 mg/ml) was tested against CA antigen (1 mg/ml) and CA antigen treated with heat, sodium metaperiodate (36), or pronase (19). Immunodiffusion was continued for 72 h, and the plates were washed with 5% sodium citrate, dried, and stained with 0.16% naphthol blue black (Sigma). CA antigen was treated with heat by boiling for 3 min in a water bath. Sodium metaperiodate (Sigma), 0.5 ml of a 0.1 M solution, was added to 0.5 ml of CA antigen (1 mg/ml) and kept in the dark at 4°C for 18 h. An equimolar amount of glycerol (Fisher) was added to consume the remaining periodate, followed after 15 min by the addition of equimolar sodium borohydride (Sigma), and the solution was kept at 4°C for 1
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h. The solution was then dialyzed against PBS at 4°C, and the original volume was restored by ultrafiltration with a microconcentrator (exclusion limit, 10,000 daltons; Centricon-10; Amicon Corp., Danvers, Mass.). The same experiment was also repeated without sodium metaperiodate. Insoluble protease (pronase) from Streptomyces griseus attached to beaded agarose (Sigma) was dispensed by hypodermic syringe fitted with a 13-mm disk filter holder (Millipore Corp., Bedford, Mass.) containing a filter (pore size, 3.0 I,m). The filter, retaining 1 mg of protease-agarose, was placed in a glass tube containing 250 ,ul of CA antigen (1 mg/ml) and incubated at 37°C for 18 h with magnetic stirring. The mixture was filtered on the disk filter device, and the ifitrate was tested in double immunodiffusion against MAb. The same experiment was also repeated without protease. SDS-PAGE, immunoblotting, and reactivity with lectins. A mycelial homogenate was used for analyses by SDS-PAGE, immunoblotting, and lectin reactivity because it contains a more complete mosaic of A. fumigatus antigens and thus allows the detection of potential reactivity with antigens other than the CA extract. A. fumigatus 2085 was cultured for 96 h at 22°C as for preparation of CA antigen. The washed fungus mat was mechanically disrupted with 0.45-mm glass beads for a total time of 20 min in a Braun MSK cell homogenizer. The homogenate was centrifuged at 37,000 x g for 45 min. The supernatant was filtered through a membrane of 0.22-p.m pore size and concentrated three- to fourfold by polyethylene glycol 20,000. The preparation was again centrifuged at 850 x g for 10 min, and the supernatant was dialyzed against 0.02 M Tris hydrochloride buffer (pH 7.4) containing 0.5 M NaCl (TBS). Protein and carbohydrate contents were 1.3 and 3.0 mg/ml, estimated by the Coomassie blue method (Bio-Rad Laboratories, Richmond, Calif.) and the phenolsulfuric acid method described by Dubois et al. (20), respec-
The antigenic extract was prepared for SDS-PAGE by boiling for 4 min in sample buffer (0.0625 M Tris hydrochloride [pH 6.8], 10% glycerol, 2% SDS, 5% 2-,B-mercaptoethanol, 0.001% bromophenol blue). Electrophoresis was done on 10% polyacrylamide gel at 120 V constant voltage and 3°C in electrode buffer (0.025 M Tris, 0.192 M glycine, 0.1% SDS [pH 8.3]), as described by Laemmli (27). Molecular mass standards (14 to 205 kilodaltons [kDa] [Sigma]) were included in each gel. After electrophoresis, gels were stained with silver nitrate or they were electrophoretically transferred to nitroceilulose as described by Towbin et al. (51). The gels were assembled with nitrocellulose paper, and the electrophoretic transfer was performed in a Transblot cell (Bio-Rad) under a constant voltage of 50 V for 20 to 22 h at 3°C and with a buffer of 0.025 M Tris, 0.192 M glycine, and 20% methanol (pH 8.3). After the transfer, the papers were stained by the Biotin-Blot protein detection method (Bio-Rad), immunochemically with sera from immunized mice or with MAb, and with the lectin concanavalin A (ConA) or BS-1 from the seeds of Bandeiraea simplicifolia. Nitrocellulose papers were sliced vertically into 0.5-cm
strips. These were first washed in Tris-buffered saline (TS) (0.01 M Tris hydrochloride, 0.15 M NaCl [pH 7.4]) and then incubated for 90 min at 37°C in TS containing 0.5% nonfat dry milk (TS-milk). For subsequent steps, TS-milk was used to dilute antibodies and for washing, done at 22°C. After blocking, strips were placed in mouse antiserum or MAb
diluted 1:50 or in buffer alone as a control for 1 h. The blots were washed five times over 30 min and then incubated for 1
h with biotinylated goat anti-mouse IgM (Vector Laborato-
ries, Burlingame, Calif.). After washing, strips were incubated in the Vectastain ABC reagent (a preformed complex between avidin and biotinylated horseradish peroxidase; Vector) for 1 h. The blots were washed in TS only, and chromogenic substrate, a solution containing 4-chloro-1naphthol (Bio-Rad) diluted in TS buffer, was added. Development was stopped by washing the strips several times in distilled water. Reactivity of the lectin ConA with the blotted nitrocellulose papers was determined by the method of Hawkes (24). Binding of B. simplicifolia lectin was determined by washing blotted nitrocellulose papers in 0.01 M PBS (pH 6.8), followed by blocking in PBS-T for 30 min at 22°C. The strips were then incubated in a 30-pug/ml solution of biotinylated BS-1 lectin (Sigma) diluted in PBS-T for 1 h at 22°C and washed in 0.01 M PBS-T (pH 7.2). Papers were incubated in the Vectastain ABC-AP reagent (a preformed complex between avidin and biotinylated alkaline phosphatase; Vector) for 1 h. The blots were washed in 0.01 M Tris hydrochloride (pH 8.2), and chromogenic substrate (Alkaline Phosphatase Substrate Kit; Vector) was added. Development was stopped by washing the strips several times in distilled water. Immunofluorescence. Indirect immunofluorescence was conducted to test the ability of MAb to bind Aspergillus species and other pathogenic fungi. The strains used to study the distribution of the antigen are shown in Table 3. Organisms were grown in liquid Sabouraud dextrose medium (IAF Production, Laval-des-Rapides, Quebec, Canada) for 48 to 96 h at 22°C. The fungal growth was washed with distilled water, and smears of the suspensions were fixed on microscope slides with acetone. The slides were washed in PBS for 10 min, and nonspecific adsorption was blocked by incubating with normal goat serum for 20 min. The slides were incubated with a 1/100 dilution of purified MAb for 30 min. After washing four times with PBS, fluorescein-conjugated heavy-chain-specific goat anti-mouse IgM (Meloy Laboratories, Inc., Springfield, Va.) diluted 1/20 in PBS was added. The slides were incubated for 45 min, washed four times with PBS, and examined in a fluorescence microscope. Indirect immunofluorescence with MAb was also conducted on organs of immunosuppressed rabbits experimentally infected with A. fumigatus 2085 or C. albicans A3181A and on a pulmonary aspergilloma caused by A. fumigatus. Fresh tissues were fixed with acetone, and immunofluorescence reactions were performed by the same procedure as for smears of organisms. Immunoelectron microscopy. A. fumigatus 2085 was cultured in a 1-liter Erlenmeyer flask containing 300 ml of Czapek Dox medium for 48 h at 22°C with magnetic stirring. C. albicans 4454 M, serotype A, originally obtained from the Pasteur Institute, Paris, France, was grown to the exponential phase in tryptose-phosphate broth at 37°C for 8 h with rotation (200 rpm; incubator shaker model G25; New Brunswick Scientific Co., Inc., Edison, N.J.), as previously described (31). After washing, both cultures were fixed in 0.1 M cacodylate-buffered 2.5% glutaraldehyde (pH 6.8) for 18 h at 4°C. A portion of the specimens was postfixed with 1% osmium tetroxide or with 1% osmium tetroxide-1.5% potassium ferrocyanide for 2 h at 22°C. After fixation, samples were washed in the same buffer, dehydrated through a series of graded ethanols, and embedded in Araldite 502. Thin sections (80.0 to 100.0 nm thick) were mounted on 400-mesh naked-nickel grids and processed for the immunocytochemical labeling. The grids were floated for 5 min on a drop of Tris-buffered saline (TBS) (0.05 M, pH 8.0) containing 0.05%
STE-MARIE ET AL.
TABLE 1. Immunization of BALB/c mice with A. fumigatus cell walls No. of
Anti-CA Male:female aemetiter
3 1 1 3 16
0:3 0:1 0:1 0:3 8:8
1/16,000 1/5,000 1/2,000 1/1,000
, ma. a
aw ;- >
its Se I
's - 2 _.f,ew 'j.
d FIG. 4. Reactivity of MAb 1 with an epitope located in the inner cell wall of hyphae (a and b) and conidia (c) of A. fumigatus, with additional binding to intracellular membranes. (d) Binding of MAb 1 to A. fumigatus hyphae was abolished by incubating MAb 1 with CA antigen. (e) Absence of reactivity of MAb 1 with blastoconidia of C. albicans serotype A. Bars = 0.5 ,um. 2110
MONOCLONAL ANTIBODIES TO ASPERGILLUS FUMIGATUS
VOL. 58, 1990
0,";.:, A .
FIG. 5. Reactivity of MAb 40 with an epitope located diffusely in the cell wall of hyphae (a and b) and conidia (c) of A. fumigatus, with less binding to intracellular membranes. (d) Binding of MAb 40 to A. fumigatus hyphae was abolished by incubating MAb 40 with CA antigen. Bars = 0.5 ,um.
STE-MARIE ET AL.
FIG. 6. (a) Binding of MAb 40 to A. fumigatus hyphae was abolished by incubating MAb 40 with mannan of C. albicans serotype A. (b) Reactivity of MAb 40 with an epitope located in the cell walls of blastoconidia of C. albicans serotype A. Bars = 0.5 Fm.
DISCUSSION The galactomannan of A. fumigatus consists of a a1,6-linked mannan backbone and two structurally distinct side chains composed of a-D-mannopyranosyl or P-D-galactofuranosyl units (7). The mannopyranosyl side chains are the probable binding sites to mannose receptors in Kupffer cells in the liver, resulting in endocytosis and rapid clearance of circulating antigen (10). In addition, the similar structure of oligomannoside side chains of A. fumigatus galactomannan and Candida mannan offers a plausible explanation for the observed reactivity of anti-C. albicans antiserum with A. fumigatus galactomannan (47) and the binding of antiserum against A. fumigatus somatic antigen with C. albicans mannan (30). The galactofuranosyl side chains of A. fumigatus galactomannan, however, are immunodominant with antibody raised in rabbits (9) and most likely account for cross-reactivity with the galactomannan II of dermatophytes
In this study, two MAbs, MAb 1 and MAb 40, were produced against a purified CA extract of A. fumigatus. Reactivity of MAb 1 with immunodominant oligogalactoside side chains of A. fumigatus galactomannan is suggested by the following: (i) recognition of a periodate-sensitive, pronase- and heat-resistant epitope in CA antigen which contains mannose and galactose; (ii) binding to two components (80 and 62 kDa) which were also recognized by the aD-galactosyl lectin BS-1 and the a-D-mannopyranosyl or a-D-glucopyranosyl lectin ConA; (iii) failure to recognize C. albicans serotype A or B mannan or C. albicans by indirect immunofluorescence and immunoelectron microscopy; (iv)
reactivity with an epitope located in the cell wall; (v) recognition of circulating antigen in invasive aspergillosis, using CA as the reference antigen. Reactivity of MAb 40 with mannopyranosyl side chains common to A. fumigatus galactomannan and C. albicans mannan is suggested by (i) recognition of C. albicans serotype A or B mannan and C. albicans by indirect immunofluorescence and immunoelectron microscopy; (ii) reactivity with an epitope located in the cell wall of A. fumigatus hyphae, abolished by incubating MAb 40 with mannan of C. albicans serotype A. Reactivity of MAb 40 with the mannans of C. albicans serotype A and B suggests that it recognizes antigenic factor 1, 4, or 5 common to both serotypes (32), rather than the immunodominant mannohexaose side chains of C. albicans serotype A mannan (factor 6), which are absent in serotype B mannan (46). The BS-1 lectin isolated from B. simplicifolia seeds has anomeric specificity for a-D-galactopyranosyl residues (25) and would not be expected to bind to P-D-galactofuranosyl units in side chains of A. fumigatus galactomannan (7). However, structural studies of the galactomannan of the yeast Torulopsis gropengiesseri demonstrated the presence of side chains composed of nonreducing terminal, acid-labile ,-D-galactosyl units (23), and despite this fact, the polysaccharide formed a precipitin band with BS-1 lectin (25). The oligogalactosyl side chains of A. fumigatus galactomannan may also not be entirely p-linked as reported (7) but may be either a mixture of a and ,B or exclusively a. Binding of MAb 1 to A. flavus, A. niger, and the dermatophytes M. canis and T. mentagrophytes but not to the
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systemic dimorphic fungi H. capsulatum and B. dermatitidis is consistent with the known distribution and serologic cross-reactivity of the galactomannan II of dermatophytes among the pathogenic fungi (3). It also supports the view that the shared antigenicity of these galactomannans is mediated by immunodominant oligogalactoside side chains (3, 9). Reactivity of MAbs 1 and 40 with oligogalactoside and oligomannoside side chains, respectively, of A. fumigatus galactomannan will need to be confirmed by inhibition studies with structurally defined monosaccharides (9). These MAbs will be useful as immunoprobes for studying epitopes responsible for the shared antigenicity of fungal mannans and galactomannans and for examining their expression during hyphal and conidial morphogenesis. ACKNOWLEDGMENTS This study was supported by grants from the Fondation de l'H6pital Sainte-Justine, the Fonds de la Recherche en Sante du Quebec, and the Medical Research Council of Canada. We thank Bernard Brodeur and Yolande Larose for many helpful suggestions, Micheline Pelletier for support and advice, and Errol Reiss for supplying the cell walls. We also thank Jeannine Joly and Pierre Auger for preparing fungal cultures and Lucie Martinelli for preparation of the manuscript. LITERATURE CITED 1. Andrews, C. P., and M. H. Weiner. 1981. Immunodiagnosis of invasive pulmonary aspergillosis in rabbits: fungal antigen detected by radioimmunoassay in bronchoalveolar lavage fluid. Am. Rev. Respir. Dis. 124:60-64. 2. Andrews, C. P., and M. H. Weiner. 1982. Aspergillus antigen detection in bronchoalveolar lavage fluid from patients with invasive aspergillosis and aspergillomas. Am. J. Med. 73:372380. 3. Azuma, I., F. Kanetsuna, Y. Tanaka, Y. Yamamura, and L. M. Carboneli. 1974. Chemical and immunological properties of galactomannans obtained from Histoplasma duboisii, Histoplasma capsulatum, Paracoccidioides brasiliensis and Blastomyces dermatitidis. Mycopathol. Mycol. Appl. 54:111-125. 4. Azuma, I., H. Kimura, F. Hirao, E. Tsubura, Y. Yamamura, and A. Misaki. 1971. Biochemical and immunological studies on Aspergillus. III. Chemical and immunological properties of glycopeptide obtained from Aspergillus fumigatus. Jpn. J. Microbiol. 15:237-246. 5. Azuma, I., S. Negoro, F. Kanetsuna, Y. Yamamura, H. Miyaji, and A. Misaki. 1971. Isolation and properties of mannans from Alternaria kikuchiana Tanaka and Alternaria zinniae. Jpn. J. Microbiol. 15:373-376. 6. Barker, S. A., 0. Besarab, and C. N. D. Cruickshank. 1967. Galactomannan peptides of Trichophyton mentagrophytes. Carbohydr. Res. 3:325-332. 7. Barreto-Bergter, E. M., P. A. J. Gorin, and L. R. Travassos. 1981. Cell constituents of mycelia and conidia of Aspergillus fumigatus. Carbohydr. Res. 95:205-218. 8. Barreto-Bergter, E. M., L. R. Travassos, and P. A. J. Gorin. 1980. Chemical structure of the D-galacto-D-mannan component from hyphae of Aspergillus niger and other Aspergillus sp. Carbohydr. Res. 86:273-285. 9. Bennett, J. E., A. K. Bhattacharjee, and C. P. J. Glaudemans. 1985. Galactofuranosyl groups are immunodominant in Aspergillusfumigatus galactomannan. Mol. Immunol. 22:251-254. 10. Bennett, J. E., M. M. Friedman, and B. Dupont. 1987. Receptormediated clearance of Aspergillus galactomannan. J. Infect. Dis. 155:1005-1010. 11. Bishop, C. T., M. B. Perry, and F. Blank. 1966. The watersoluble polysaccharides of dermatophytes. V. Galactomannans II from Trichophyton granulosum, Trichophyton interdigitale, Microsporum quinckeanum, Trichophyton rubrum, and Trichophyton schoenleinii. Can. J. Chem. 44:2291-2298. 12. Bishop, C. T., M. B. Perry, F. Blank, and F. P. Cooper. 1965. The water-soluble polysaccharides of dermatophytes. IV. Ga-
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