Vol. 43, No. 3INFECTION AND IMMUNITY, Mar. 1984, p. 1012-10180019-9567/84/031012-07$02.00/0Copyright ©) 1984, American Society for Microbiology

Production and Characterization of Three Monoclonal Antibodies toCandida albicans Proteins

NANCY A. STROCKBINE, MICHAEL T. LARGEN, AND HELEN R. BUCKLEY*

Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140

Received 26 September 1983/Accepted 9 December 1983

Three monoclonal antibodies, designated A2C7, C2C7, and F19, were produced which recognize proteinsfrom Candida albicans. All are of the immunoglobulin GI heavy chain and kappa light chain class. A2C7and C2C7 immunoprecipitated three proteins contained in a partially purified fraction (region A) of amycelial cytoplasmic extract of C. albicans. The apparent molecular weights of these proteins are 120,000(120K) to 135K, 44K to 52K, and 35K to 38K. Monoclonal antibody F19 was reactive with proteins of 42K,43K, and 50K in immunoblotting experiments. F19 was also able to form a precipitin band in agarose gelwith protein(s) contained in region A. Limited proteolytic digestion of the three proteins immunoprecipitat-ed by A2C7 and C2C7 demonstrated that both monoclonal antibodies recognized the same three Candidaproteins and that there exists a significant degree of relatedness in primary structure among the threeproteins. Proteins with apparent molecular weights of 120K to 135K, 44K to 52K, and 35K to 38K that wereimmunoprecipitated by sera from two patients with invasive candidiasis and by the serum from a rabbitimmunized against a 48K (44K to 52K) Candida protein were also analyzed by limited proteolysis. Patternsof peptide fragments generated by enzymatic digestion of these proteins showed that the proteinsrecognized by the monoclonal antibodies are the same proteins recognized by antibodies in the sera ofpatients during an invasive Candida infection and by antibodies in the serum of the immune rabbit.

Monoclonal antibodies have been produced against a vastarray of microorganisms, ranging from viruses and bacteriato fungi and multicellular parasites (1, 2, 10, 11, 15, 17, 18).One application for antibodies with these specificities is inthe development of immunodiagnostic tests for the detectionof antigens associated with specific pathogens or the mea-surement of specific antibodies in the host as a result ofinfection with an organism. Recently, antigens that arerecognized by antibodies in the sera of patients with invasivecandidiasis have been identified (lla). Proteins identified ashaving the greatest serodiagnostic potential were used in thedevelopment of monoclonal antibodies. Several monoclonalantibodies have been produced which react with theseproteins. The production and characterization of three ofthese antibodies are reported here.

MATERIALS AND METHODSOrganism and culture methods. Candida albicans serotype

A (Hasenclever B311, ATCC 32354) was grown on Sabour-aud slants for 18 to 24 h at 25°C. The growth from one slantwas inoculated into 50 ml of liquid synthetic medium (8) andincubated on a gyratory shaker at 150 rpm at 25°C. After 18h, 1-ml samples of this growth were added to 100 ml of liquidsynthetic medium and rotated at 150 rpm for 24 h at 37°C.The cells were harvested by centrifugation and resuspendedwith 0.05 M Tris-hydrochloride (pH 7.4) with 0.02% (wt/vol)NaN3. The cell slurry was stored at -80°C until used for thepreparation of extracts. Cells grown under these conditionswere 90% mycelium and 10% yeast cells.Geheration of monoclonal antibody. (i) Antigen prepara-

tion. The mycelial cytoplasmic extract (MCE) was preparedby mechanical disruption essentially as described by Syver-son et al. (14). Samples were examined microscopically forcell breakage. The reactivity of the MCE prepared from

* Corresponding author.

different batches of cells was tested against sera frompatients with systemic candidiasis by using crossed immuno-affinoelectrophoresis in the presence of concanavalin A(ConA) (13) and was found to be reproducible. The MCEwas then passed through ConA-Sepharose 4B (Pharmacia,Uppsala, Sweden) affinity columns to remove cell wallmannan, glucan, and mannan-glucan complexes. The load-ing and running buffer was 0.05 M Tris-hydrochloride (pH7.4) with 0.02% (wt/vol) NaN3 and 0.001 M each of MnCl2,MgCl2, and CaC12. The mannan-depleted material (MD-MCE) (nonbound fraction) was pooled and dialyzed against0.05 M Tris-hydrochloride (pH 7.8) with 0.02% (wt/vol)NaN3 for ion-exchange chromatography. The removal ofmannan-containing components was monitored by crossedimmunoaffinoelectrophoresis with ConA. The MD-MCEwas fractionated by ion-exchange chromatography through adiethylaminoethyl-Sephacel (Pharmacia) column. Fractionswere eluted with a linear salt gradient of 0 to 0.3 M NaCl in0.05 M Tris-hydrochloride (pH 7.8) with 0.02% (wt/vol)NaN3. Conductivity was measured by using a conductivitybridge (model RC 16B2; Industrial Instruments, CedarGrove, N.J.); the gradient was found to be linear from 0 to0.25 M NaCl (data not shown). Proteins eluting at 0.028 to0.066 M NaCl (region A) of the diethylaminoethyl-Sephacelcolumn or the unfractionated MCE was used for immuniza-tion.

(ii) Immunization. Two groups of BALB/c mice (six miceper group) were immunized with either unfractionated MCEproteins or the proteins from region A. Mice imnmunizedagainst the MCE proteins received 50 ,ug of protein (MCE)subcutaneously in incomplete Freund adjuvant on day 0 and50 ,ug of protein (MCE) intraperitoneally in saline on day 32.On day 36, two mice immunized against the MCE proteinswere sacrificed by cervical dislocation for use in a fusion.Mice immunized against the region A proteins received 50,ug of protein (region A) subcutaneously in incompleteFreund adjuvant on day 0 and 50 jig of protein (region A)

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intraperitoneally in alum on day 43. On day 157, the micewere inoculated with 37 ,g of protein (region A) subcutane-ously in incomplete Freund adjuvant, and on day 247 themice received 50 ,ug of protein (region A) intraperitoneally insaline. On day 251, two mice were sacrificed for a fusion.

(iii) Fusion and screening. Cell fusion with SP2/0-Ag 14myeloma cells and selection and growth of hybrids wereperformed essentially as described by McKearn (9) andT. Gopalakrishnan (Abstr. 19th Annu. Meet. Am. Soc. CellBiol., p. 448a, abstr. no. SG2622, 1979). Fusion of cells wasperformed in an adherent monolayer on a ConA-coatedtissue culture dish (no. 3060; Costar, Data Packaging, Cam-bridge, Mass.). Cells were mixed at a ratio of 2 to 3 immunespleen cells to 1 myeloma cell, and 7 x 107 to 10 x 107 totalcells were added to each fusion dish. The fusion dishes wereprepared by incubating tissue culture dishes with 1 ml of 0.1M sodium acetate (pH 4.8) containing 15 mg of ConA(Calbiochem-Behring, La Jolla, Calif.) per ml and with 1 mlof 0.1 M sodium acetate (pH 4.8) containing 50 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (Calbiochem-Beh-ring) per ml for 1 h on a rocker platform at room tempera-ture. The dishes were then washed three times with Hanksbalanced salt solution (GIBCO Laboratories, Grand Island,N.Y.) and stored empty at -20°C until use.

Cell culture supernatants were screened for Candida-specific monoclonal antibodies by an enzyme-linked immu-nosorbent assay. Tissue culture plates (no. 3596; Costar)were antigen coated overnight at 4°C with 50 ,ul of a 1-mg/mlsolution of MCE or a 1-,ug/ml solution of region A proteins in0.015 M carbonate buffer, pH 9.6. The plates were blockedwith 200 ,ul of a 3% (wt/vol) solution of bovine serumalbumin in a 0.015 M carbonate buffer (pH 9.6) overnight at4°C. The bovine serum albumin solution was then tappedout, and the plates were stored empty at -80°C until use orrinsed with 0.05 M phosphate-buffered saline (pH 7.4) con-taining 0.05% (wt/vol) Tween 20 (PBST) for immediate use.Monoclonal supernatants (50 ,ul) were added to each welland incubated overnight at 4°C or for 3 h at 37°C. Thesupernatants were tapped out, and the plates were rinsedfour times with PBST. A 50-,u portion of a 1:100 dilution inPBST of peroxidase-labeled, affinity-purified anti-mouseimmunoglobulin G (IgG) plus IgM (heavy plus light chains)(Kirkegaard and Perry Laboratories, Gaithersburg, Md.)was added to each well and incubated for 3 h at 37°C. Thesecond antibody was removed, and the plates were washedas described above. A 100-pI portion of substrate [0.2 mM2,2'-azino-di-(3-ethylbenzthiazoline sulfonate); SigmaChemical Co., St. Louis, Mo.] in 0.1 M citrate buffer (pH 4.0containing 0.2% (vol/vol) hydrogen peroxide was added toeach well and allowed to react for 30 min at room tempera-ture. Antibody-containing wells were visually identified.

Positive hybrids were subcloned twice by limiting dilutionand retested for antibody production by enzyme-linkedimmunosorbent assay. Isotypes were determined byimmunodiffusion with class-specific antisera (Litton Bione-tics, Kensington, Md.).

(iv) Antibody preparation and purification. Ascites fluidwas prepared in pristane (500 pd)-primed BALB/c mice byintraperitoneal injection of 107 hybridoma cells. Ascites fluidwas collected, clarified by centrifugation at 800 x g, andfrozen at -80°C. IgG was prepared from culture superna-tants by precipitation with 50%-saturated (NH4)2SO4, pH7.0, followed by dialysis against 0.04 M phosphate buffer,pH 6.8.

Characterization of monoclonal antibody. (i) Immunodiffu-sion. Monoclonal antibodies were tested for reactivity

against the MCE of region A proteins by double immunodif-fusion. Immunodiffusion tests were performed on plasticdishes (50 by 9 mm) (no. 1006; Falcon Plastics, Oxnard,Calif.) containing 5 ml of 1% (wt/vol) Noble agar (DifcoLaboratories, Detroit, Mich.) in 0.05 M sodium boratebuffer, pH 8.6. A center well and six surrounding wells (3-mm diameter) were cut into the agar so that each surround-ing well was 7 mm from the center of the middle well.Antigen (ca. 35 ,ug of MCE, 20 ,ug of MD-MCE, or 15 ,g ofregion A proteins) was added to the center well, and 10 pl ofmonoclonal antibody in the form of concentrated cell culturesupernatant, ascites fluid, or serum was added to the sur-rounding wells. The dishes were incubated 72 h at roomtemperature in a humid chamber and inspected over indirectlighting for precipitin bands. Faint bands were vis4alizedafter washing, drying, and staining with Coomassie blueG250 as previously described (13).

(ii) Agglutination. Monoclonal antibodies at several dilu-tions were tested for their ability to agglutinate a 2% (vol/vol)suspension of Formalin-fixed C. albicans cells (4). Theagglutination reaction was performed in a tray of flat-bottomed dishes by incubating 3 to 5 drops of the cellsuspension with 500 ,l of monoclonal cell culture superna-tants for 30 min at room temperature with gentle agitationfollowed by incubation for 18 h at 4°C.

Staphylococcus aureus protein A immunoprecipitation. (i)lodination procedure. lodination of the region A proteinsfrom the MD-MCE was performed by using the diphenylglycouril technique (Iodogen; Pierce Chemical Co., Rock-ford, Ill.) (5). The labeled proteins were separated from theunreacted iodide by gel filtration through a G-25 Sephadex(Pharmacia) column (10 by 200 mm). Gelatin phosphatebuffer (0.1 M phosphate buffer [pH 7.4] containing 0.2%[wt/vol] gelatin and 0.02% [wt/vol] NaN3) was used as theequilibration and running buffer. The excluded peak (labeledproteins) was pooled and stored frozen as samples at -80°C.

(ii) Immunoprecipitation. Immunoprecipitation was per-formed essentially as described by Kessler (6). The washingbuffer was RIPA (0.01 M Tris-hydrochloride [pH 7.4], 0.5 MNaCl, 0.001 M EDTA, 1.0% [wt/vol] Triton X-100, 1.0%[wt/vol] sodium deoxycholate, 0.1% [wt/vol] sodium dodecylsulfate [SDS], 0.02% [wt/vol] NaN3). The labeled antigenextract from region A in gelatin phosphate buffer wasprecleared by adding it to the pellet from 8.0 ml of a 10%(wt/vol) suspension of heat-killed, formalinized S. aureusCowan I (Pansorbin; Calbiochem-Behring). The antigen andthe S. aureus Cowan I were incubated on ice for 1 h at 4°Cwith periodic vortexing, and the bacteria were removed bycentrifugation. Labeled antigen (precleared, 5 x 106 tri-chloroacetic acid-precipitable cpm) was then added to 100 pdof ammonium sulfate-concentrated monoclonal superna-tants, 10 ,u of mouse serum or ascites fluid, 20 RI of rabbitserum, or 5 RI of human serum and incubated for 18 h at 4°C.A 5-pl portion of a second antibody (rabbit anti-mouse IgG,IgM, and IgA; Calbiochem-Behring) was added to each tubecontaining mouse antibody and incubated for 18 h at 4°C.Excess S. aureus Cowan I (500 RI) was added and incubatedfor an additional 2 h at 4°C. The bacteria were collected bycentrifugation and washed three times with RIPA buffer. Thepellets were then resuspended in double-concentration sam-ple buffer (7) and heated for 5 min in a boiling-water bath.The bacteria were removed by centrifugation, and the super-natants were applied to SDS-12% polyacrylamide gels. Afterelectrophoresis of the immunoprecipitates, the gels werefixed and stained with Coomassie blue G250 (0.3% [wt/vol]in 10% [vol/vol] CH3COOH, 10% [vol/vol] CH30H, 80%

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[vol/vol] dH20), destained, and dried on filter paper. Thegels were exposed for 24 h to Kodak AR-5 film (EastmanKodak Co., Rochester, N.Y.).

(iii) Immunoblot analysis. Proteins from the MCE or regionA were prepared for SDS-polyacrylamide gel electrophore-sis>(7) and separated on 12% acrylamide gels. When electro-phoresis was completed, the gels were removed and theseparated proteins were electrophoretically transferred tonitrocellulose paper (NCP) (0.45 ,um; BA 85; Schleicher &Schuell, Inc., Keene, N.H.) essentially as described byTowbin et al. (16). The NCP gel was electrophoresed over-night under constant current at 150 mA at 10°C in a Transblotcell (Bio-Rad Laboratories, Richmond, Calif.). After electro-phoresis, the NCP was stained either immunochemically orwith amido black (0.1% [wt/vol] in 45% [vol/vol] CH30H,10% [vol/vol] CH3COOH, 45% [vol/vol] dH2O). Immuno-chemical staining was performed by first blocking the NCPwith 2.5% (wt/vol) casein in Tris-buffered saline (pH 7 to pH8) for 4 h at 45°C. All subsequent manipulations of the NCPwere done at room temperature. The blots were incubatedovernight with serum from a mouse bearing the F19 hybridcell line (1:50 dilution in PBST), serum from a mouseimmunized against region A proteins (1:200 dilution inPBST), or normal mouse serum (1:100 dilution in PBST).The blots were washed four times in PBST and then incubat-ed in a 1:100 dilution in PBST of affinity-purified, horserad-ish peroxidase-labeled anti-mouse IgG plus IgM (heavy pluslight chain) (Kirkegaard and Perry) for 2 h. The blots were

washed as described above and developed with 0.005%(wt/vol) 3,3'-diaminobenzidine tetrahydrochloride (LittonBionetics) and 0.03% (vol/vol) hydrogen peroxide in PBSTfor 30 min.

(iv) Limited proteolytic digestion. Limited proteolytic di-gestion of the 125I-labeled proteins identified by immunopre-cipitation was performed essentially as described by Cleve-land et al. (3). Bands were cut out of the dried-down gel witha razor blade and rehydrated overnight in a solution of 7%(vol/vol) CH3COOH in dH20. The digestion procedure forproteins in gel slices was followed in which 0.15 ,ug (0.10 Uof enzyme activity) of S. aureus V8 protease (Miles Labora-tories, Inc., Elkhart, Ind.) was used. The substrates (125I_labeled proteins) and enzyme were incubated for 45 min atthe interface of the stacking and separating gel. The peptidefragments were separated by electrophoresis on an SDS-15%polyacrylamide gel by the method of Laemmli (7). The gelswere fixed, stained, dried on filter paper, and exposed toKodak AR-5 film with DuPont Cronex Hi-Plus intensifyingscreens as described above.

Reagents. (i) Human sera. Human sera were provided bythe Mycology Serology Laboratory, Harvard School ofPublic Health, Boston, and by N. A. Hyslop, InfectiousDisease Unit, Massachusetts General Hospital, Boston.Serum 618 came from a patient diagnosed as having endocar-ditis due to Candida parapsilosis. Serum C-21 came from aburn patient who had repeated blood cultures positive for C.albicans (five separated by 24 h) and skin grafts on the faceand thighs culture positive for C. albicans (deep surfaceinfection).

(ii) Rabbit serum. Hyperimmune rabbit serum against a

purified protein of molecular weight 48,000 (48K) (44K to52K) from C. albicans was raised by multiple inoculations ofthis protein in incomplete Freund adjuvant.

(iii) Molecular weight standards. Molecular weight stan-dards were obtained from Pharmacia, Uppsala, Sweden. Thestandard preparation contained phosphorylase-b, 94K; bo-vine serum albumin, 67K; ovalbumin, 43K; carbonic anhy-

TABLE 1. Characterization of monoclonal antibodies to C.albicans

Results for hybrid cell linea:Expt A2C7 C2C7 F19

Isotype IgGl (kappa) IgGl (kappa) IgGl (kap4a)Immunodiffusion - - -AgglutinationS. aureus A immuno- 120K-135K, 120K-135K,

precipitation 44K-52K, 44K-52K,35K-38K 35K-38K

Immunoblot analysis NT NT 42K, 43K,50K

a NT, Not tested; -, negative results; and +, presence of aprecipitin band.

drase, 30K; soybean trypsin inhibitor, 20.1K; and a-lactal-bumin, 14.4K.

RESULTS

Isolation and characterization of monoclonal antibodies.Three hybrid cell lines, designated A2C7, C2C7, and F19,were recovered from fusions involving the SP2/0 myelomacell line and spleen cells from BALB/c mice immunized asdescribed above. Hybrid cell lines A2C7 and C2C7 wereproduced from mice immunized against region A proteins,and F19 was produced from mice immunized against MCEproteins. All three hybrid lines secrete monoclonal antibod-ies of the IgGl heavy chain and kappa light chain class (seeTable 1). Monoclonal antibody F19 reacted with antigen(s) inthe cytoplasmic extract to form a precipitin band byimmunodiffusion. Fractions from the diethylaminoethyl col-umn were tested by fused rocket immunoelectrophoresis(12) for their reactivity with monoclonal antibody F19 (datanot shown). A precipitin band was seen with fractionseluting within region A (0.048 to 0.068 M NaCl in 0.05 MTris-hydrochloride, pH 7.8). No precipitin bands were seenagainst the MCE, MD-MCE, or region A proteins withmonoclonal antibodies A2C7 or C2C7.

Agglutination of Formalin-fixed Candida cells was per-formed to determine whether the monoclonal antibodiesrecognized determinants on the cell surface and were able toagglutinate cells. All three monoclonal antibodies were un-able to agglutinate the whole Candida cell. Monoclonalantibodies A2C7 and C2C7 were tested in preliminary immu-nofluorescence studies for their reactivity with C. albicans.No binding of these antibodies to the surface of the cells wasseen (A. Adams, personal communication). The results ofthe immunodiffusion, agglutination, S. aureus protein Aimmunoprecipitation, and immunoblot analysis experimentsare summarized in Table 1.

S. aureus A immunoprecipitation. To identify antigensreactive with those monoclonal antibodies which were posi-tive in the initial enzyme-linked immunosorbent assayscreening of the hybridoma supernatants, S. aureus A im-munoprecipitation with labeled Candida proteins was em-ployed. Two monoclonal antibodies, A2C7 and C2C7, pre-cipitated three proteins of apparent molecular weights 120Kto 135K, 44K to 52K, and 35K to 38K (Fig. 1, lanes 7 and 8).Most of the antibodies did not immunoprecipitate any anti-gens in this analysis, as exemplified by lane 9 which is theimmunoprecipitate obtained from the malignant ascites fluidfrom a mouse inoculated with another hybridoma secreting anon-precipitating monoclonal antibody against C. albicans.

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FIG. 1. S. aureus A immunoprecipitation with A2C7 and C2C7; sera from two patients with invasive candidiasis and from a rabbitimmunized against a 48K protein from C. albicans. M. W. Std., Molecular weight standard. Lanes la and b, 2.5 x 105 and 5 x 105trichloroacetic acid-precipitable cpm, respectively, of the "25I-labeled region A proteins which were used as the antigen in the immunoprecipi-tation. Lanes 2 through 12, antigens which were precipitated by the rabbit, human, and monoclonal antibodies. Lanes 2a and b, 70- and 24-hexposure, respectively, of the antigens recognized by the immune rabbit serum. Lane 3, antigens precipitated by normal rabbit serum. Lane 4,antigens precipitated by serum (no. 618) from a patient with endocarditis due to C. parapsilosis. Lanes 5a and b, 70- and 24-h exposure,respectively, of the antigens recognized by serum (C-21) from a patient with invasive candidiasis; lane 6, serum from a normal, healthyindividual. Lanes 7 and 8, antigens precipitated by the malignant ascites fluid from mice inoculated with hybridomas A2C7 and C2C7,respectively. Lanes 9 and 10, antigens precipitated by the malignant ascites fluid from a mouse inoculated with another hybridoma secreting amonoclonal antibody against C. albicans (IgGl [kappa]) and the culture supernatant from a hybridoma secreting an irrelevant monoclonalantibody, respectively. Lanes 11 and 12, proteins precipitated by S. aureus cells incubated with antigen and second antibody (rabbit anti-mouse), and S. aureus cells incubated with antigen alone, respectively.

It was also seen that there was some small amount ofnonspecific background as evidenced in the negative controlimmunoprecipitates: culture supernatant from an irrelevantmonoclonal antibody (lane 10), proteins precipitated by S.aureus A incubated with antigen and second antibody (rabbitanti-mouse Ig) (lane 11), and S. aureus A incubated withlabeled antigen alone (lane 12).Since the antigenic specificity of F19 could not be deter-

mined by S. aureus A immunoprecipitation, immunoblotanalysis was employed to identify the antigen(s) reactivewith this antibody. Immunoblot analysis with F19 and par-tially purified fractions of cytoplasmic proteins from C.albicans showed that this antibody recognized several pro-teins within region A from the diethylaminoethyl column(Fig. 2). The antigenic determinants were not destroyed by2-mercaptoethanol and boiling for 5 min in SDS samplebuffer. The apparent molecular weights of these proteins are42K, 43K, and 50K.

S. aureus A immunoprecipitation with human and rabbitsera. We have previously shown (lla) that patients withinvasive candidiasis have antibodies in their sera whichrecognize antigens of the same apparent molecular weight asthose recognized by A2C7 and C2C7. To make this compari-son in a more direct way, two human sera from patients withinvasive candidiasis and serum from a rabbit immunizedagainst a 48K (44K to 52K) protein were studied by S.aureus A immunoprecipitation. Antigens precipitated byserum (no. 618) from a patient with endocarditis due to C.parapsilosis are of the same apparent molecular weights asthose recognized by the two monoclonal antibodies (Fig. 1,lane 4). Lanes 5a and b show 70- and 24-h exposures,respectively, of the autoradiographs of immunoprecipitatedantigens recognized by serum (C-21) from a patient with

invasive candidiasis, and lane 6 shows the antigen precipitat-ed by serum from a normal, healthy individual. Lanes 2a andb show that these same antigens are precipitated by amonospecific polyclonal antibody prepared by immunizing arabbit with an apparently homogeneous protein of 48K (44Kto 52K). Lane 3 shows that these antigens are not precipitat-

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FIG. 3. Limited proteolysis of the three antigens (the 44K to 52K protein, the 120K to 135K protein, and the 35K to 38K protein)immunoprecipitated by A2C7 and C2C7, two human sera, and serum from a rabbit immunized against a 48K Candida protein. The threeantigens immunoprecipitated by each serum were cut out of the original gel and treated with S. aureus V8 protease. The fragments generatedby the enzymatic digestion were separated on 15% (wt/vol) acrylamide-SDS gels. The patterns of the major peptide fragments are shown.M. W. Std., Molecular weight standard. The lanes and their contents are: 1, the 44K to 52K antigen precipitated by S. aureus cells alone; 2,the 44K to 52K antigen precipitated by serum C-21 from a patient with invasive candidiasis; 3, the 44K to 52K antigen precipitated by serumfrom the immune rabbit; 4, the 44K to 52K antigen precipitated by serum (no. 618) from a patient with endocarditis due to C. parapsilosis; 5,the 44K to 52K antigen precipitated by C2C7; 6, 44K to 52K antigen precipitated by A2C7; 7, the 120K to 135K antigen precipitated by serumno. 618; 8, the 120K to 135K antigen precipitated by C2C7; 9, the 120K to 135K antigen precipitated by C-21; 10, the 120K to 135K antigenprecipitated by serum from the immune rabbit; 11, the 120K to 135K antigen precipitated by A2C7; 12, the 35K to 38K antigen precipitated byA2C7; 13, the 35K to 38K antigen precipitated by serum from the immune rabbit; 14, the upper band of a doublet that was precipitated by no.618 in the molecular weight range of 35K to 38K; 15, the lower band of a doublet that was precipitated by serum no. 618 in the molecularweight range of 35K to 38K; 16, the 35K to 38K antigen precipitated by C-21; and 17, the 35K to 38K antigen precipitated by C2C7. Lanes 1and 2, 400-h exposure; lanes 3a through 6a, 22.5-h exposure; lanes 3b through 6b, 7-h exposure; lanes 7 and 8, 400-h exposure; lanes 9 through11, 215-h exposure; lanes 12a, 12b, and 13a through 16a, 215-h exposure; and lanes 13b through 16b and 17, 91-h exposure. All exposures weredone in the presence of intensifying screens.

ed in appreciable amounts by normal rabbit serum. It canalso be seen that the 48K (44K to 52K) antigen is apredominant protein in the labeled antigen preparation (lanesla and b) which was also used as an immunogen, asdescribed above.Limited proteolytic digestion analysis. Limited proteolysis

with S. aureus V8 protease was employed to study therelatedness of the three proteins (the 120K to 135K protein,the 44K to 52K protein, and the 35K to 38K protein) thatwere recognized by A2C7 and C2C7, the two human sera,and the immune rabbit serum. Proteins with a similar pri-mary sequence give a series of similar molecular weightpolypeptides when digested with the V8 protease from S.aureus. The three different apparent molecular weight pro-teins precipitated by the monoclonal antibodies, the sera

from the patients, as well as the rabbit antiserum preparedagainst the 48K (44K to 52K) protein showed strong similar-ities in the molecular weights of the proteolytic peptidesgenerated (Fig. 3). Lanes 1 through 6 show different autora-diographic exposures of the peptides generated by limitedproteolysis of the 44K to 52K antigens; lanes 7 through 11show the peptide maps of the 120K to 135K antigens; andlanes 12 through 17 show the peptide maps of the 35K to 38Kantigens immunoprecipitated by the various antisera. It canalso be seen that the 44K to 52K protein precipitated non-specifically by S. aureus A alone (lane 1) is the same as, or isclosely related to, the antigens specifically precipitated bythe various antisera. These proteins share a significantdegree of primary sequence homology. The proteins recog-nized by F19 were not analyzed by limited proteolysis.

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DISCUSSIONMonoclonal antibodies A2C7 and C2C7 recognize a mini-

mum of three proteins with apparent molecular weights of120K to 135K, 44K to 52K, and 35K to 38K (Fig. 1). S.aureus cells, alone or in the presence of a second antibody(rabbit anti-mouse immunoglobulins), also bind a smallamount of the 44K to 52K protein (Fig. 1). At present, thenature of this nonspecific binding is not clear; however, thepresence of the 44K to 52K protein in the immunoprecipi-tates of the monoclonal antibodies and the human sera isinterpreted as a specific reaction. This interpretation isfavored because the amount of this protein immunoprecipi-tated by the monoclonal and human antibodies was 40 to 80times greater than the amount precipitated by the S. aureuscells alone.Data from the limited proteolytic digestion experiment

presented in Fig. 3 demonstrate that A2C7 and C2C7 recog-nize the same three proteins and that these three antigenicproteins share a significant amount of primary structure. Thenature of the common determinant and the relationship ofthe three proteins to each other have not been determined atthis time. The limited proteolysis data are consistent with thepossibility that the shared antigenic determinant may bedefined by the primary structure of the 35K to 38K, 44K to52K, and 120K to 135K proteins; however, the possibility ofa shared carbohydrate determinant cannot be excluded atthis time. One explanation for the existence of three antigen-ically and structurally related proteins with different appar-ent molecular weights is that the three proteins occur as theresult of differential, post-translational modifications of acommon protein. Another possibility is that the three pro-teins may represent polymerized states of a small protein(s)that are not dissociated under the conditions that wereemployed for protein separation in this study. A thirdpossibility is that a precursor-product relationship existsbetween the three proteins. Additional experiments areneeded to determine the precise molecular relationship ofthese proteins to each other.A2C7 and C2C7 recognize serodiagnostically important

proteins. Proteins that are recognized by these antibodiesare also recognized by antibodies in the sera of patientsduring invasive Candida infections. In addition, the proteinsrecognized by the monoclonal antibodies are not antigenical-ly unique to C. albicans, since serum from a patient withendocarditis due to C. parapsilosis contains antibodiesagainst the same three proteins.The 48K protein is highly antigentic, as it is able to elicit

an antibody response in mice, rabbits and humans. The 48K(44K to 52K) protein alone is able to elicit antibodies in arabbit which cross-react with the 120K to 135K and 35K to38K proteins from region A. Contamination of the 48K (44Kto 52K) antigen that was used to immunize the rabbit withthe high- and low-molecular-weight antigens is unlikely,since the 48K (44K to 52K) antigen was cut out of a 12%polyacrylamide preparative gel before immunization (Buck-ley, Vigilante, Strockbine, Zlotnik, and Largen, manuscriptin preparation).F19 does not immunoprecipitate; however, it does recog-

nize proteins from region A by immunoblot analysis (Fig. 2).One of the proteins is a major protein from region A with anapparent molecular weight of 50K (Fig. 2). Comparison ofthis protein by limited proteolysis with the proteins recog-nized by the other monoclonal antibodies was not per-formed. If the 42K, 43K, and 50K proteins are related to the44K to 52K protein recognized by A2C7 and C2C7, the

determinant recognized by F19 is likely to be different fromthat recognized by A2C7 and C2C7. This observation issupported by the fact that F19 fails to recognize the 120K to135K and 35K to 38K proteins recognized by A2C7 andC2C7 and instead recognizes two different proteins withapparent molecular weights of 42K and 43K. At the presenttime the relationship among the 50K, 42K, and 43K proteinsis unclear.One application for monoclonal antibodies with the above

specificities is in the development of a reliable diagnostic testfor disseminated candidiasis. A2C7 and C2C7 recognizeantigens that are released during invasive Candida infec-tions. The 44K to 52K antigen recognized by A2C7 andC2C7 was recently shown to be serodiagnostically important(lla). Patients with invasive Candida infections had signifi-cantly higher antibody levels against this protein than didpatients with noninvasive, superficial candidiasis, patientswith other fungal infections, or normal, healthy individuals(P < 0.001) (lla). These monoclonal antibodies should beuseful in the development of a test(s) for antigen detection tofacilitate the diagnosis of disseminated candidiasis.

ACKNOWLEDGMENTSWe sincerely thank Gerald Pearlman for his photographic assist-

ance and Gregory Harvey for his editorial assistance in the prepara-tion of the manuscript. We thank Dianne Jackson for her advice andhelp during the production of the monoclonal antibodies. We arealso grateful to Lisa Nurmi-McKernan and Susan J. Vigilante forexcellent technical assistance.

This work was supported by Public Health Service grant no. Al14695 and by medical mycology training grant no. Al 07141, bothfrom the National Institutes of Health.

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