CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, Jan. 1994, p. 63-67 Vol. 1, No. 11071-412X/94/$04.00+0Copyright C) 1994, American Society for Microbiology

Monoclonal Antibodies against a 97-Kilodalton Antigen fromAspergillus flavus

SETH V. HETHERINGTON,l.2* SCOFT HENWICK, 2t DAVID M. PARHAM,3'4AND CHRISTIAN C. PATRICK1' 2

Department of Infectious Diseases' and Department of Pathology and Laboratory Medicine,3St. Jude Children's Research Hospital, and Departments of Pediatrics2 and Pathology,4

University of Tennessee, Memphis, Tennessee

Received 25 June 1993/Returned for modification 17 August 1993/Accepted 8 September 1993

We prepared a panel of five monoclonal antibodies (MAbs) directed against Aspergillusflavus that all reactedagainst one 97-kDa antigen by Western blot (immunoblot). Flow cytometry demonstrated that these antibodiesbound (in increasing degrees) to all morphologic stages of A. flavus growth: conidia, swollen conidia, andhyphae. Cross-reactivity among species was examined by enzyme-linked immunosorbent assay of fungalculture filtrates. Four MAbs reacted with 10 of 11 A. flavus isolates, and the fifth one reacted with 9 of them.One MAb also reacted with A.fumigatus, two reacted with A. niger, A. wentii, and A. nidulans, and all five reactedwith A. ochraceus. None reacted with A. terreus, A. glaucus, A. versicolor, or a Penicillium species. Each MAbbound to A. flavus hyphae in formalin-fixed paraffin sections of a muscle biopsy from a confirmed human caseof invasive aspergillosis. In summary, these MAbs identified a 97-kDa antigen found on A. flavus that is bothsurface bound and an exoantigen. Either the same or a cross-reacting antigen is present in A. fumigatus andother AspergiUlus species.

Aspergillus species cause several human diseases, includingallergic bronchopulmonary aspergillosis, hypersensitivity pneu-monitis, aspergilloma, and invasive aspergillosis (3, 9). Physi-cians caring for immunocompromised patients recognize thelast syndrome as one of the most difficult infections to diagnoseand treat. Aspergillus conidia, inhaled by the host, germinate inthe lower airways, and the hyphae aggressively invade pulmo-nary tissues and blood vessels and may disseminate to skin,bone, the brain, and other organs.The diagnosis of invasive aspergillosis presents a formidable

task for the clinician. Detection of circulating or excretedAspergillus antigens would provide a rapid, noninvasivemethod, and toward this end, several systems have beenproposed. Weiner et al. used a polyclonal antiserum raisedagainst Aspergillus fumigatus in a competitive radioimmunoas-say to detect antigen in the sera of immunocompromisedpatients (25). Similarly, George et al. have used a competitiveenzyme-linked immunosorbent assay (ELISA) to quantita-tively assess the effect of treatment of experimental aspergil-losis on the level of circulating antigen (6). Other methodsusing monoclonal antibodies (MAbs) have also been devel-oped, and one such test, the Pastorex Aspergillus, is commer-cially available (24).Although hundreds ofAspergillus species exist abundantly in

the environment, only a handful are responsible for clinicaldiseases. In most medical centers, A. fumigatus is the mostcommon agent of aspergillosis, followed by A. flavus (11, 17).For this reason, almost all research onAspergillus antigens andthe development of MAbs have centered on A. fumigatus (7, 8,12, 18). Only occasionally have studies included antigenic

* Corresponding author. Mailing address: Department of InfectiousDiseases, St. Jude Children's Research Hospital, 332 N. Lauderdale,P.O. Box 318, Memphis, TN 38101-0318. Phone: (901) 522-0485. Fax:(901) 527-6616.

t Present address: Department of Medical Microbiology, William V.McKenzie Hospital, University of Alberta, Edmonton, Alberta, Can-ada P6G 2J2.

comparisons among strains (19). At St. Jude Children's Re-search Hospital, where immunocompromised pediatric pa-tients have been treated for over 30 years, A. flavus accountsfor over 50% of cases of proven Aspergillus pneumonia. A.flavus has been the dominant pathogenic species in one otherinstitution (1). Studies identifyingA. flavus antigens could leadto rapid antigen detection methods for diagnosis that couldpermit earlier diagnosis with greater sensitivity.

Because of the incidence of A. flavus-derived Aspergillusinfection at our institution and the lack of information avail-able on A. flavus, we chose to develop MAbs against thisspecies as potential diagnostic tools. In this report, we describethe production of five MAbs against a previously undescribedantigen of A. flavus that is also present in A. fumigatus andother Aspergillus species.

MATERIALS AND METHODS

AspergiUuls species. We obtained 29 strains of Aspergillusrepresenting nine species: A. flavus (11 strains),A. fumigatus (4strains), A. niger (3 strains), A. ochraceus (2 strains), A. wentii(1 strain), A. nidulans (1 strain), A. glaucus (2 strains), A.terreus (3 strains), and A. versicolor (2 strains). They wereisolated from tissue of infected patients, nasal cultures ofcolonized patients, or material collected in hospital environ-mental surveys; two strains were obtained from the AmericanType Culture Collection (A. fumigatus ATCC 46645 and A.terreus ATCC 46941). For use as a negative control, a singleisolate of a Penicillium sp. was obtained from a routine hospitalenvironmental survey culture.

Preparation of culture filtrate. Since growth conditionsinfluence Aspergillus antigen expression, we chose cultureconditions on the basis of work of others that have resulted inreasonable yields of glycoproteins and diverse antigens (14,26). Conidia from each isolate were seeded onto individualpotato dextrose agar plates and grown for 5 days at roomtemperature. From these starter cultures, conidia were inocu-lated into Czapek Dox broth (106 conidia per 200 ml). Cultures

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64 HETHERINGTON ET AL.

were grown for 4, 6, or 8 weeks at room temperature withoutshaking, at which time they were inactivated by the addition of20 ml of 10% formalin solution and phenylmethylsulfonylfluoride at a final concentration of 1 mM. For some experi-ments, cultures were grown at 37°C for 2 weeks with shaking.The culture filtrates (CFs), recovered by sequential filtrationthrough Whatman no. 1 filter paper and a 0.2-pLm-pore-sizefilter, were concentrated approximately 20-fold by vacuumdialysis and then dialyzed extensively against water. Totalprotein and carbohydrate contents were measured by themethod of Lowry et al. (16) and the phenol-sulfuric acidmethod, respectively. CFs from other Aspergillus spp. wereprepared by inoculating conidia into 10 ml of Czapek Doxbroth and incubating for 7 days at 37°C with tumbling. CFswere not concentrated after dialysis.

Immunization of mice. BALB/c mice were immunized bytwo protocols (2). Mouse 1 received 107 glutaraldehyde-fixedA. flavus conidia in Freund's complete adjuvant intraperitone-ally (i.p.) on day 1. Booster immunizations with glutaralde-hyde-fixed conidia resuspended in phosphate-buffered saline(PBS), pH 7.4, were given on days 15, 34, and 55. Splenocytefusion was performed on day 58. Mouse 2 received 100 p.g ofA. flavus CF protein in complete Freund's adjuvant (i.p.) onday 1, followed by booster immunizations i.p. with 100 p.g ofCF protein in incomplete Freund's adjuvant on days 5, 19, and60. Fusion was performed on day 63.

Production of MAbs. Spleen cells were fused to SP2/0myeloma cells with polyethylene glycol by standard methods(2). Fused cells were cultured in Dulbecco's modified Eagle'smedium supplemented with 10% controlled-process serumreplacement (Sigma Chemical Co., St. Louis, Mo.) and hypo-xanthine, aminopterin, and thymidine. Hybridoma culturesupernatants were screened for immunoglobulin G (IgG)antibodies to A. flavus antigens by ELISA and Western blot(immunoblot) (see below). Positive clones were subclonedtwice by limiting dilution. All subsequent experiments wereperformed with hybridoma culture supernatants.ELISA. All screening for anti-Aspergillus MAbs was per-

formed with a biotin-avidin ELISA. All dilutions of reagentswere made in 10 mM PBS with 0.05% Tween 20, pH 7.4(PBS-T). Prepared A. flavus CF was adsorbed to 96-wellpolystyrene plates in 0.1 M carbonate buffer, pH 9.6, at aconcentration of 40 pLg of protein per ml, overnight at 4°C.After washing, nonspecific binding sites were blocked byincubating the wells with PBS containing 3% bovine serumalbumin (BSA) for 1 h at room temperature. Hybridomaculture supernatants diluted 1:1 were applied and incubatedovernight at 4°C. The plate was washed three times withPBS-T, and then goat anti-mouse IgG-biotin conjugate (SigmaChemical Co.) diluted 1:3,000 was added. Following a 1-hincubation at 37°C and washing, an avidin-alkaline phos-phatase conjugate (Extravidin; Sigma Chemical Co.), diluted1:2,000, was added. After a final 1-h incubation and washing,100 p.l of p-nitrophenyl phosphate substrate (1 mg/ml indiethanolamine buffer, 1.0 M, pH 9.8) was added. Colordevelopment proceeded for 1 h at room temperature and wasterminated by the addition of 75 pL. of 1 N NaOH. The opticaldensities (ODs) were read at 405 nm. Control wells includedpreimmunization mouse serum diluted 1:1,000 or culturesupernatants from non-Aspergillus MAbs. Since backgroundODs were always .0.020, we considered an OD of -0.1positive.SDS-PAGE and immunoblot. Proteins in CFs were sepa-

rated by sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) by the method of Laemmli (15). Sam-ples were boiled for 2 min in reducing buffer and applied to a

12.5% acrylamide gel in a Hoeffer (San Francisco, Calif.)minigel apparatus. The gels were stained with Coomassie blueor the silver stain kit of Bio-Rad (Richmond, Calif.) accordingto the manufacturer's instructions.For immunoblotting, proteins were transferred from gels to

nitrocellulose paper with a Hoeffer semidry apparatus by usingTowbin transfer buffer (23). Molecular weight standards wereidentified by reversible staining with Ponceau S. Nonspecificbinding sites on the nitrocellulose were blocked by overnightincubation in Tris-buffered saline (10 mM; pH 7.4) with 10%evaporated milk at 4°C. All subsequent steps were performedat room temperature, and all dilutions and washes were madewith PBS-T supplemented with 3% BSA. The nitrocellulosepaper was placed in a 10-slot Deca-Probe (Hoeffer); to eachslot we added 0.5 ml of hybridoma culture supernatant. After3 h, the slots were washed and goat anti-mouse IgG-biotin wasadded at a 1:1,000 dilution. After 1 h, the slots were washedagain and 0.5 ml of a 1:1,000 dilution of Extravidin-alkalinephosphatase (Sigma) was added. The immunoblot was incu-bated another 1 h, washed, and finally developed with a fast redstain in 0.1 M Tris buffer, pH 8.2.

Flow cytometry. MAb binding to the three growth phases ofA. flavus was compared by our previously published method offlow cytometry (10). To prepare swollen conidia, restingconidia were inoculated into Czapek Dox broth for 8 h at roomtemperature. During this initial metabolic phase, conidia ap-proximately doubled in diameter but no germ tubes or youngmycelia were evident. These swollen conidia were collected bycentrifugation, washed in PBS, and stored at 4°C. Younghyphae were prepared by allowing conidia to incubate inCzapek Dox broth for 16 h. For flow cytometric analysis,conidia, swollen conidia, and hyphae were incubated in hybri-doma culture supernatant as previously described (10). Lightmicroscopy confirmed the morphologic stages and the absenceof large numbers of hyphal aggregates. Aggregates wereeliminated from analysis by using forward and side scatter toselect singlets. Supernatant from an unrelated MAb culturewas used as a negative control. Samples were then incubatedwith fluorescein-conjugated goat anti-mouse IgG. Mean chan-nel fluorescence was determined as described previously for104 particles of each growth phase (10).Immunoperoxidase. Using the Elite Vectastain kit (Vector

Laboratories, Burlingame, Calif.), we tested the ability of ourA. flavus-positive MAb culture supernatants (diluted 1:10 inPBS) to react with A. flavus in a formalin-fixed paraffin sectionof a muscle biopsy from a patient with disseminated aspergil-losis (20). In control experiments, the anti-Aspergillus MAbswere replaced with a MAb against Haemophilus influenzae typeb (provided by Eric Hanson, University of Texas, Dallas) or aMAb to the common antilymphocytic leukemia antigen.

RESULTS

Production of MAbs. Immunization of mouse I with A.flavus conidia and fusion of spleen cells with myeloma cellsyielded two A. flavus CF-positive MAbs. One was class IgMand will not be discussed further. The second, an IgG MAb,was designated 2G6. Mouse 2, immunized with A. flavus CF,yielded four positives, all IgG, designated 2G3, 2D5, 4B10, and6E4. MAb 2G6 was of IgG3 isotype and subclass, and all otherswere IgGI.SDS-PAGE and immunoblot. All five MAbs reacted with a

broad band of A. flavus CF immunoblots with an apparentmolecular mass of 97 kDa (Fig. 1). Using a different batch ofCF (prepared by the same protocol), we noticed that MAb 6E4also reacted with several bands between 21 and 42 kDa (Fig.

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MONOCLONAL ANTIBODIES AGAINST A. FLAVUS 65

Reduced

97-ll MW RT RT 370

66

Non-Reduced

RT RT 370

43

31

21

Ctrl 2G3 2G6 4B10 2D5 6E4

FIG. 1. Immunoblot of five MAbs against CF from A. flawus. Thecontrol lane (Ctrl) contains a non-Aspergillus MAb. Molecular masses

(in kilodaltons) are indicated on the left.

2). Silver-stained SDS-polyacrylamide gels of the CF showed a

dense band at 97 kDa with the unusual property of negativestaining (not shown), that stained heavily with Coomassie blue(Fig. 3). Since culture conditions are known to influenceantigen expression in other Aspergillus species (14), we testedfor the presence of the A. flavus 97-kDa antigen in CFs aftervarious culture conditions. This antigen was most evident whenCFs were prepared from cultures ofA. flavus grown at 37°C for2 weeks, but it was also present in cultures grown at room

temperature for 6 or 8 weeks.Reactivities of MAbs with Aspergillus spp. MAb 2D5 reacted

on ELISA with CFs from 9 of 11 A. fiavus strains. The otherMAbs reacted with 10 of 11 strains (Table 1). One A. flavusisolate did not react with any MAb. Only MAb 2G3 reactedwith three of four A. fumigatus CFs; MAbs 2G3 and 2G6showed a broader range of reactivities than the other MAbs(positive for six and five species, respectively). The ODs withA. flavus CFs as the antigen were generally >1.0 comparedwith 0.1 to 0.4 with other Aspergillus species CFs. No MAbreacted with A. glaucus, A. terreuls, A. versicolor, or the Penicil-lium sp.MAb binding to Aspergillus growth phases. Flow cytometry

demonstrated that the 97-kDa antigen was present on conidia,

1 2 3 4 5

97.2

66.2 -

~~~~~~~~~~~~~~~~~~~~~. ... ... ....97kd ; _ _

43kd

31kd -

21kd -

Bwk 6wk 2wk 8wk 6wk 2wk

FIG. 3. SDS-PAGE of A. flavus CFs from various culture condi-tions, stained with Coomassie blue. Results for samples run undernonreducing conditions are shown to the right. RT, room temperature;wk, weeks of growth.

swollen conidia, and young hyphae (Fig. 4). The fluorescenceintensity, indicating binding of MAb to A. flavus, increasedwith progressive morphologic development. The net binding ofall MAbs to A. flavus was calculated from the fluorescencecurves, after the fluorescence of A. flavus incubated withoutMAb was subtracted, and was expressed as equivalent solublefluorescent molecules, as previously described (10). Conidiademonstrated a small amount of MAb binding (Table 2). Thequantity of equivalent soluble fluorescent molecules increasedan average of 14-fold (range, 8- to 25-fold) as conidia becameswollen conidia. The increase was not due solely to an increasein surface area, since the swollen conidia, at double thediameter of resting conidia, have only four times the surfacearea. Hyphae had the greatest amount of binding of MAb ofthe three growth stages.

Immunohistochemistry. Finally, we tested the reactions ofeach of the five MAbs to an A. flavus isolate identified byhistologic examination and culture of a muscle biopsy speci-men from an immunocompromised patient with disseminatedaspergillosis. All reacted strongly and specifically with hyphaein formalin-fixed paraffin-embedded clinical tissue sections(Fig. 5). The cell walls and septa were clearly delineated,indicating that the 97-kDa antigen or a cross-reacting epitopeis present. Neither the two control MAbs nor the secondaryantibody reacted with the hyphae (not shown).

42.7

TABLE 1. Reactivities of five MAbs raised against A. flavus to CFof different Aspergillus spp. and isolates

31

21.5

FIG. 2. Immunoblot of MAbs against CF antigen from A. flavus.This was a different preparation of CF from that used for Fig. 1. Lane

1, preimmune mouse serum; lane 2, serum from mouse 2, on the dayof fusion; lane 3, MAb 6E4; lane 4, MAb 4B10; lane 5, MAb 2G6.Molecular masses (in kilodaltons) are indicated on the left.

No. of isolates reacting with MAb

MAb A. fiavus A. furmigattis A. niiger A. ochlraceus A. wetiuii A. nlidulans(Ill) (4) (3) (2) (1) (I)-

2D5 9 0 0 1 0 02G3 10 3 3 1 1 12G6 10 0 3 1 1 14BlO 10 0 1 1 0 06E4 10 0 1 1 0 0

" Numbers in parentheses are the numbers of isolates of each species tested.

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66 HETHERINGTON ET AL.

100

Fluorescence Intensity

FIG. 4. Binding of MAb 2G3 to three morphologic growth phasesof A. fiavus. Each curve shows the number of fungal particles (y axis)with the indicated fluorescence intensity (given in arbitrary units).

DISCUSSION

We have produced five MAbs that react with a 97-kDaantigen present on all three intact morphologic forms of A.flavus. The antigen is produced at an early stage in themetabolic activation of resting conidia, is increasingly ex-pressed through the swollen-conidium and hypha stages, and isshed readily into culture medium at room temperature and at37°C. This suggests that the antigen is not exclusively bound tothe fungal surface but may function as an exoantigen. Althoughwe do not yet know its biological function, the antigen appearsto be a structural protein (or glycoprotein) that may beimportant in host-fungus interactions; it is a major componentof the CF (as determined by SDS-PAGE) and is highlyimmunogenic.Our ELISA experiments demonstrated that all of the anti-A.

flavus MAbs react with more than one Aspergillus species,including A. fumigatus, A. niger, A. ochraceus, A. wentii, and A.nidulans. The results must be interpreted cautiously since thenumber of isolates examined from each species, with theexception of A. flavus, was small. In addition, since we usedculture supernatants as the antibody source, some differencesamong the MAbs with regard to CF reactivity may have beenrelated to antibody concentration. Nevertheless, we believethat our results allow us to place Aspergillus spp. into one oftwo groups: MAb-positive species including those listed above,and MAb nonreactors including A. terreus, A. glaucus, and A.versicolor. The ability to differentiate among species could beimportant for epidemiologic studies. More importantly, the

TABLE 2. Flow cytometric analysis of MAb binding to Aspergillusorganisms at different morphologic stages

Result for':MAb Resting Swollen

conidia conidia Hyphae

2D5 1.2 10.9 43.92G3 5.1 62.6 275.92G6 3.6 46.8 217.54B10 1.5 25.9 183.06E4 0.8 21.8 102.9Non-Aspergillus 0.3 0.6 0.0

'All numbers are mean values for approximately 104 particles and areexpressed as the net (with the background subtracted) equivalent solublefluorescent molecules (in thousands) per particle, compared with a set ofstandardized fluorescent beads (8).

.I. -, ~~~~~~ w A. , , .~~~~~~4{ . ..p_r*+E~~~~~~~~Wa;.

i

FIG. 5. Immunoperoxidase-stained muscle biopsy sample from apatient with disseminated aspergillosis. (A) Magnification of x 43; (B)magnification of x 340. Hyphae are clearly stained with MAb 2G6. Allother MAbs were also positive. The culture grew A. flavus.

ability to distinguish Aspergillus species from other morpholog-ically similar fungi in biopsy specimens could be clinicallyhelpful.Our MAbs identify an antigen different from those previ-

ously identified by reactivity with MAbs raised against A.fumigatus. Stynen et al. (22) described seven MAbs, all IgM,that reacted with galactomannan (GM). Ste-Marie et al. (21)produced two IgM MAbs that also probably reacted with GM.GM is a major cell wall component with a branched structureof mannose, galactofuranose, and galactopyranose. It is clearthat our MAbs do not react with GM because (i) immuno-blotting studies with anti-GM antibodies always yield broadbands between 41 and 90 kDa, smaller than our reactiveantigen; (ii) GM is ubiquitous among Aspergillus spp.; and (iii)the Penicillium species contains an immunologically similar oridentical GM, but none of our MAbs reacted with all nineAspergillus species and none reacted with Penicillium CF. FourIgM MAbs produced by Kurup (13) reacted with multiple 12-to 50-kDa bands, one of which had concanavalin A-bindingactivity. Upon immunoblot, another IgM MAb bound to a pairof bands at 48 and 58 kDa (12). Interestingly, this MAb boundto proteins from A. flavus and A. fumigatus CFs but not tothose from A. niger,A. terreus, Penicillium notatum, or Candidaalbicans, thereby discriminating-as do our MAbs-amongfungi and among certain Aspergillus species.

For diagnostic purposes, IgG MAbs are more useful thanthose of the IgM isotype. MAbs against fungi of the IgG

morly-

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MONOCLONAL ANTIBODIES AGAINST A. FLAVUS 67

isotype are, evidently, more difficult to develop or at least lesscommon. Fratamico and Buckley (4) produced an IgG MAbagainst a 58-kDa major component of A. fumigatus mycelia. Thisantigen was mostly protein in content, but the MAb binding wasdirected against a carbohydrate epitope. Frosco et al. (5)produced a MAb against A. fumigatus elastase (approximately31 kDa) by immunizing animals with denatured antigen.The immunoblots showed that our MAbs reacted with a

broad band, a characteristic ascribed to glycoproteins withvarious degrees of glycosylation. We have not yet determinedwhether the MAb epitope is carbohydrate or protein. Futurestudies in our laboratory will examine the effect of glycosidasesand proteinases on MAb reactivity to CF antigen.We noticed that with some preparations of CF, one MAb

(6E4) reacted with multiple low-molecular-weight bands.These bands may be degradation products of the 97-kDaantigen rather than distinct proteins or glycoproteins withidentical or similar epitopes, since these bands did not appearin all CF preparations from a given strain. In any case, of thefive MAbs, only 6E4 bound to these bands and therefore mustrecognize an epitope different from that recognized by theother MAbs.We have demonstrated the usefulness of these MAbs as a

diagnostic adjuvant in tissue samples. Further studies oncross-reactivity may show that Aspergillus spp. can be groupedby differential reactivity. Finally, to our knowledge these arethe first MAbs produced against A. flavus. In some institutions,such as ours, this species is the most common cause of invasiveaspergillosis and remains a significant cause of mortality in theimmunosuppressed patient.

ACKNOWLEDGMENTS

This work was supported by National Cancer Institute CenterSupport (CORE) grant CA 21765, by Biomedical Research Supportgrant RR 05584, and by the American Lebanese Syrian AssociatedCharities (ALSAC).We thank Hallie Holt for performing the immunohistochemical

tests, Karen Dame for editorial comments, and Jim Houston forperforming the flow cytometry.

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