canis isolates by monoclonal antibodies.Identification and serotyping of Microsporum

1986, 23(3):609.

J. Clin. Microbiol.

L Polonelli, M Castagnola and G Morace

canis isolates by monoclonal antibodies.

Identification and serotyping of Microsporum

http://jcm.asm.org/content/23/3/609

Updated information and services can be found at:

These include:

CONTENT ALERTS

more»

cite this article),

Receive: RSS Feeds, eTOCs, free email alerts (when new articles

http://journals.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders:

http://journals.asm.org/site/subscriptions/

To subscribe to to another ASM Journal go to:

on December 10, 2013 by PROFESSOR OF RESEARCHhttp://jcm.asm.org/Downloaded from on December 10, 2013 by PROFESSOR OF RESEARCHhttp://jcm.asm.org/Downloaded from

Vol. 23, No. 3 JOURNALOF CLINICALMICROBIOLOGY, Mar. 1986, p.609-615

0095-1137/86/030609-07$02.00/0

Identification and Serotyping of Microsporum canis Isolates by Monoclonal Antibodies

LUCIANO

POLONELLI,1*

MASSINO CASTAGNOLA,2 ANDGIULIA MORACE'

Istituto di

Microbiologial

andIstitutodi Chimica,2 Facolta di Medicina eChirurgia "Agostino Gemelli," Universita Cattolicadel Sacro

Cuore,

00168

Rome, Italy

Received 26 August1985/Accepted6 December1985

Hybridomacellswereproduced byfusingmousemyeloma cells with spleen cells from mice primedwith^an exoantigen of Microsporum canis. Threeclones produced antibodies whichwereexamined by the Westernblot technique for their potential usefulness in theidentification ofM. canisisolates and differentiation of strains within the species. Based on reactions with immunological determinants, all of the M. canis isolates tested presentedeither species-orstrain-specificdomains.Monoclonal antibodies provedtobeusefulreagentsforthe identification ofM. canis isolatesandforthe differentiation of strains within the species. A purified antigen depleted ofcommon antigenic determinants was obtained in affinity chromatography by using monoclonal antibody.

Theapplication of hybridomatechnology hasproven to be useful in the identification of a wide range ofmicroorgan- isms. Monoclonal antibodies may be usedcomparatively in differentimmunologicalassays for phylogenetic studiesand species andstrain identification.

In a previous report, we described the potential for serologically analyzing dermatophytes by precipitating monoclonal antibodies produced against an exoantigen of Microsporum canis(5).

This report deals with the properties of dermatophyte antigenic determinants as determined by the reaction of monoclonalantibodies with polypeptidesseparated by elec- trophoresis in polyacrylamide gels and then transferred electrically to nitrocellulose.

Purification of crude M. canis exoantigen by affinity chromatography with monoclonal antibodies might also be achieved to obtainantigens specific to M. canis.

MATERIALS ANDMETHODS

Cultures. Dermatophyte isolates were obtained from clinical specimens inour institute (Universita Cattolica del Sacro Cuore; UCSC) or were graciously furnished by the Division ofMycotic Diseases, Centers for Disease Control, Atlanta, Ga. (see Tables 1 to 3). All the cultures were maintained inour collection at room temperature in sterile distilled water.

Referenceantigens.M.canisCDC B2094wasthestandard strain used for the production ofthe reference

exoantigen

andformouseimmunization. Itwasextracted in 24 hfroma 7-day-old Sabouraud dextrose agar slant culture

by using

a merthiolate solution(1:5,000) indistilledwater.The solution wasfiltered andconcentrated 50-fold by-lyophilization.The protein andcarbohydrate contents ofthe reference

antigen

wereadjustedtofinal concentrations of2,500and550,ug/ml, respectively. The same procedure was used to

produce

exoantigens (protein content, 2,500

,ug/ml)

from all the cultures used in this study.

Immunizationprotocol. For the

fusion,

BALB/c micewere

*Correspondingauthor.

609

immunized intraperitoneally with 0.1 ml of the reference antigen mixed with an equal amount of incomplete Freund adjuvant. The procedure was repeated once a week for 1 month. Three days before the fusion, the mice were reinoculated intraperitoneally with 0.1 ml of the reference antigen only. Before the fusion, blood samples were col- lected from each mouse and placed in microtiter plates sensitized withthe M. canisreference antigen for enzyme- linked immunosorbentassay of antibody titer.

Production and screening of hybridomas. The procedures for fusion and selection ofthe hybridoma cells producing monoclonal antibodies against M. canis exoantigen were performed asdescribed elsewhere(5).

Western blot analysis. Dermatophyte

lyophilized

exo- antigens (5 ml) were solubilized with 0.5 ml of disruptor bufferconsisting of1 MTris (pH 7),60% (wt/vol) sucrose, 2% (wt/vol) sodium dodecyl sulfate, 5% (wt/vol)B-mercap- toethanol,and0.02% bromphenol blue-saturated solution.A

50-,xg

sample of proteinstandards(150

RI),

includingRNase A (13,700 daltons),chymotripsinogen (25,000 daltons), and ovalbumin(43,000

daltons),

wasaddedperml oftheantigen solution, and it wasboiled for5min. Antigen samples were electrophoresed through 10% polyacrylamide gels (vertical gel system; Bethesda Research Laboratories, Inc.,

Gaithersburg,

Md.).

Electrophoresis

was

performed

at 15 mAand continuedfor18 h toallow the solvent fronttoreach the bottom of the gel. Immunoblotting of the gels was performed by amodificationofthe method ofTowbinetal.

(6). Briefly, the proteins were

electrically

transferred to nitrocellulose sheets

(0.45-jim

pore

size;

Schleicher &

Schuell, Inc., Keene,N.H.)at60 Vfor3 h(Transblot

cell;

Bio-Rad

Laboratories, Richmond, Calif.).

After 90min,the transfer buffer (25 mM Tris base, 192 mM

glycine,

20%

methanol)

was changed. Thenitrocellulose sheetswere cut intostripsandincubated with5%horseserumin

phosphate-

buffered saline(PBS; pH 7.6) for 30 min at room tempera- ture.Thestripswere subsequentlyincubated inashaker for 30minat

37°C

in a1:10 dilution of

hybridoma

culture fluid.

The stripswerethen washed with

PBS,

blockedonce more with 5%horseserumin

PBS,

and incubated for 30 min ina shakerat 37°C with 3 mlofhorseradish

peroxidase-coupled

on December 10, 2013 by PROFESSOR OF RESEARCHhttp://jcm.asm.org/Downloaded from

0

CA

0

00a,

ON ONON ON ON all 0% ON ON as

0%

00

WI

0 0

00

00

00

00

N N N brNN N N N

00 00

oo 00

0

00

tn

00

N-

00

rN

e

0

00

00

N-

00 u

U 0

ffi00

^00tJ< *- > *- v G ° > o) ° -4 61 5:^{61 t sD} ° °

WmC(_>bm .^m>t(tScStSQJoU k1- uU2> .U =DU i

0z 0~0

000 Z00 0t u CQ 2

C -0400 -4C-,^-4 *W '- -C

.c

-. 00 QCO

UV)V)UzUcnV V) V)V)QQQ/

Q.0

~ ~ ~ ~ ~~~-

0

tc -c"Dt,ct

0uuu s00U 2 )V ) ) 2

z u uC a

U

610

00 00

Nq

0'IC 00

inC;

C)9

v .N V)

C1

u 0 Q

Q

0

0 C) 0 D.0

Q r.

ct

"7t

C0

5 E

.t~

C) .0

0

._

C) 0

-

0 CO

0 CZ 4- Q

Lo.

-

CZ

c Cl7

4-

C Ct rD

E-

en

on December 10, 2013 by PROFESSOR OF RESEARCHhttp://jcm.asm.org/Downloaded from

MONOCLONAL ANTIBODIES FOR IDENTIFYING M. CANIS

o

s ID

so WI

O

N-

N-

0%l 00

00

00

0

o~~~~~~~~~~~~o

U

-4

L.o U E

U~~~~s U

T0

^{sqCU V)}

8.:

8.3:

3on U3D °U

rabbit anti-mouse immunoglobulin G antiserum (1:500 in PBScontaining 5% horse serum). After being washed for 30 min with two changes of PBS, the strips were stained with 100 ml of distilled watercontaining 1 ml ofa 1% solution of 4-chloro-1-naphthol and 0.01% hydrogen peroxide. A few strips were differentially stained with Coomassie blue for evaluating protein marker migration in thegel.

Reverse-phase HPLC. All common reagents were ofana- lytical grade. The acetronile was Chromasolv grade pur- chased from Riedel-De Haen(Seelze-Hannover, Hannover, FederalRepublic ofGermany), and trifluoroacetic acid was from Sigma Chemical Co. (St. Louis, Mo.). The high- performance liquid chromatography (HPLC) water was bidistillationfiltered, before use,on aGelman GA6 Metricel membrane filter (0.45-,um pore size). Each exoantigen (5 lyophilized ml) was dissolved in 0.10 ml of solution A for HPLC;0.010 mlwasthen used for eachHPLC analysis. The HPLC conditionswereasfollows.HPLCcolumn, Aquapore RP300 (Brownlee Laboratories,Santa Clara, Calif.); 220 by 4.1 plus 30 by 4.1 mm guard column; solution A, 90%

H20-10% CH3CN plus trifluoroacetic acid (0.1% [wt/vol]);

solution B, CH3CN (100%) plus trifluoroacetic acid (0.1%

[wt/vol]). Gradient applied, percentage B: 0%, 1 min; 0 to 70% in 18 min; 70%, 2 min; reequilibration, 0% at 8 min;

wavelength, 220 nm; adsorbance scale, 1.280. The HPLC apparatus was a two-pump Perkin-Elmer LC1O equipped withan LC85 UV-Vis spectrophotometric recorder and LC

autocontrol,

connected with a LKB two-channel recorder.

Affinity chromatography. All common reagents were ana- lytical grade: Affigel 10 was purchased from Bio-Rad, and ethanolaminewaspurchased from Carlo Erba (Milan, Italy).

Asciticfluid(5 ml) ofM. canis monoclonalantibody UCSC 7 was partially purified by precipitation with ammonium sulfate (50% saturation) at 4°C overnight. The pellet was collected aftercentrifugation (2,200 x g for 15 min at 4°C) and dissolved in 5 ml ofa potassiumphosphate buffer (0.05 mol/liter[ph 7.44; coupling buffer]).Afterdialysis against the buffer(three changes,200 x,4°C),theproteinconcentration was determined by the method of Lowry et al. (3). The

coupling

wasachievedby adding2.5 mlofAffigel10slurry to 5 mlof

antibody

solution(48mgofprotein). Beforeuse, the

Affigel

10wasfilteredonWhatmanno.1cellulose paper and gently washed with 20 ml of cold distilled water. The suspension was stirred at 4°C for 6 h. Then 200 IL of an ethanolamineHCI solution(1mol/liter; pH 8.0)wasaddedto the suspension to ensure complete blocking ofactive ester groupswhich

might

haveremained onthe resin. After1hof

stirring,

the suspension was poured into a small column to obtain a

chromatographic

bed of 1 cm (height) by 2 cm2 (area).Theresinwas

exhaustively

washed with thecoupling buffer, the eluent A278 was checked by means ofan LKB 2238 Uvicord SII connected to an LKB 2210 two-channel recorder. The

uncoupled

antibody was detached from the resin

by washing

the bed firstwithasodiumphosphatebuffer

(0.05 mol/liter; pH 7.11;

buffer A) and then with NaCI (4 mol/liter in buffer A

[buffer B]).

Thisbuffer sequencewasthe same as that

subsequently

used for the

affinity

chromatog- raphy. The resin was then reequilibrated by buffer A. A 120-,ul

sample

of the reference

antigen

(approximately 300 and66 ,ugof'proteinand

carbohydrate

content,respectively)

was

applied

tothe resin bed and allowedtostandfor4 hat

4°C. Washing

with buffer A was

performed

to elute the unbound

antigens

untilthe eluentA278reachedthe baseline.

Then the

antigen against

the monoclonal

antibody

was

separated by

elution oftheresin with bufferB. The purified

antigen

was collected and

dialyzed against

buffer A (three

VOL.23, 1986 611

on December 10, 2013 by PROFESSOR OF RESEARCHhttp://jcm.asm.org/Downloaded from

0000000000 00000000 0% 0%cr0>0% 0% 0% 0% 0

000

NtN

00 0o

000 00 00

N r

00N-

rN

00

o o

0 rN

0

o o 0

00

N- N-

0

o

004

-

S-4-k

4!~~~~~~~

CO~~~~~~~~~~~~~~~0C

z, 0%0 _Z°

0:unc zc v zc tc 0 tO 0 tu t0 0 0

S53D2u :DDDDDDDDWSDDDCD⁰⁰W<R X t ⁰ t2^co,) t.,tD Dn

U U

612

N

Q

D ._C- 0 C) 0

0

0) .0

-c

0.

0) 0

S:

.

.0

E

).

.C (A 0 co6

4-

>1

mL Ca

0)N

Cu

0

0) CO .5

N-

VI)

%n

1-

on December 10, 2013 by PROFESSOR OF RESEARCHhttp://jcm.asm.org/Downloaded from

changes, 200 x, 4°C). The unbound, purified antigen was

collected and lyophilized. The resin was used repeatedly withoutloss of binding activity.

Immunospot assay. A modification of the procedure of Hawkes et al. (2)forthe assay ofantibodies, based on the application of the antigen as aspot to nitrocellulosepaper, wasusedtodetect thereactivity ofimmunoadsorbedmono-

clonalantibodyinaffinitychromatography with either bound

orunbound

antigen.

RESULTS

Threehybridoma cell lines producing monoclonal antibod- ies of the immunoglobulinG classwereselected accordingto their different reactivity with the reference antigen as de- tected by the Western blot technique. This procedure also permitted study of the properties of thedermatophyte pro-

teins with the monoclonal antibodies. Our results were as

follows.

M. canismonoclonal antibody UCSC 3 recognized all of the M. canis isolates tested by reaction with specific anti- genic determinants of 74 and 95 kilodaltons (kDa) (Table 1).

The detection of reactiveantigenic determinants of different molecular weights in the different isolates within the species suggeststheexistence ofnumerousserotypes. Noheterolog-

ousdermatophyte species, showing cross-reactivity, exhib- ited the simultaneous presence of the 74- and 95-kDa anti- genic determinants.

M. canis monoclonal antibody UCSC 7 characterized single M. canis isolated by eithersingle species-specific (98- kDa) or strain-specific antigenic determinants of different molecularweights (Table 2). It wasinterestingto note that the five M. gypseum isolates tested showed a common

domain of 25 kDa.

M.canis monoclonalantibody UCSC 13 (Fig. 1) provedto

^be themostinteresting and specificone.Itreactedonly with

a single 16-kDa antigenic determinant in each M. canis isolate tested. This reaction was also shared by the

go g>

Microsporum distortum isolate tested, which concurs with

the identity of thetwo species as demonstratedpreviously by mating experiments (4). Cross-reactions, although at different molecular weights, were observed only in the Microsporumferrugineum, Microsporum gallinae, and Tri- chophyton soudanense isolates tested. These species are

considered strictly relatedmorphologically (Table 3).

Affinity chromatography was successful inpurifying one

00 Q

_

08

C*lU54coC-) o o

z8uu

mzc

a8u

C~~u~| ~~~~~D u.. ...

1.4 -4tO Ezx t

TABLE 3.

Western

blot analysis of

dermatophyte isolatesa

with M. canismonoclonalantibody UCSC 13

Isolate Molecularsize (kDa)

M.canis

CDC B2094 16

UCSC 1 16

UCSC 2 16

UCSC4 16

UCSC 6 16

UCSC 8 16

UCSC 10 16

UCSC12 16

UCSC 14 16

UCSC 52 16

M.distortum CDC B2174 16

M.ferrugineum CDC83-056097 11

M.gallinaeCDCB3801 23

T.soudanenseCDC 83-048430 12

aThe otherdermatophytes listed in Tables 1 and 2werenegativewith this monoclonalantibody.

613

on December 10, 2013 by PROFESSOR OF RESEARCHhttp://jcm.asm.org/Downloaded from

614 POLONELLI ET AL.

16., 16go _16t

11D

a b c d

FIG. 1. Westernblotanalysis ofdermatophyteisolates withM.canismonoclonalantibody UCSC13(molecularsizeexpressedinkDa).

Lanes: a, M. canisCDC B2094 exoantigen; b, M. canisUCSC 1 exoantigen; c, M. canis UCSC 12 exoantigen; d,M.ferrugineum CDC 83-056097exoantigen; e,Microsporum audouiniiCDC B3800exoantigen.

component of the reference antigen. In a preliminary ap- proach usingM. canis monoclonal antibody UCSC 7 as an immunoadsorbent,itwaspossibleto separatepeak 1(unad- sorbedportion of theantigen) and peak 2 (adsorbed portion of the antigen) (Fig. 2).

E

E

CN

Go

The immunospot assay performed with the reference antigen in toto, peak 1 (unadsorbed antigen), and peak 2 (adsorbed antigen) with M. canis monoclonal antibody UCSC7showedthelossofreactivityofpeak1(unadsorbed antigen) (Fig. 3).

Comparison by reverse-phase HPLC of the reference

aI

I

0.

z

a ^2A

C

50 190

volume (ml)

FIG. 2. Affinity chromatography ofM. c-anis CDC B2094 total

exoantigen againstmonoclonal antibody UCSC7coupledtoAffigel 10resin. Peaka representsthe unadsorbed antigenic fraction, and peakbrepresentsthefraction eluted by sodium chloride(4.0 M)in elution buffer(arrow).

FIG. 3. Evaluation of reaction of in toto (R1), unadsorbed (1A), and adsorbed (2A) exoantigen of M. canis CDC B2094 by im- munospot withM. canis monoclonal antibody UCSC 7. Antigenic fractions 1A and 2A wereobtainedafteraffinity chromatography of theexoantigenwith M.canismonoclonalantibodyUCSC 7. Control (C)was PBSused inplace oftheantigen.

J. CLIN.MICROBIOL.

on December 10, 2013 by PROFESSOR OF RESEARCHhttp://jcm.asm.org/Downloaded from

MONOCLONAL ANTIBODIES FOR IDENTIFYING M. CANIS

E

CM

coc

C4

a .0o 0

.0

T

0.020

1a

@

/

70%

20

time (min)

FIG. 4. Reverse-phase HPLC. t, M. canis CDC B2094 total exoantigen;^a,unadsorbedantigenicfraction; b, the fraction eluted by sodiumchloride (4.0M) in elution buffer after affinity chroma- tography against monoclonalantibody UCSC 7onAffigel 10 resin.

Thedashed line shows the position of the reactiveantigenic deter- minant in the chromatographic pattern. The acetonitrile gradient appliedinthechromatography isalso indicated.

antigen and peak 1 (portioneluted) confirmed the disappear-

anceofonepeak of the original pattern(Fig. 4).

DISCUSSION

Our study clearly showed the enormous potential of monoclonal antibodies for the immunoidentification and typing ofdermatophytes. The effectiveness ofthe monoclo- nalantibodiesmaybecompletely evaluated by usingahighly

sensitive andanalytical procedure, suchastheWesternblot

technique.

It is of

primary

importance, moreover, that the immunization antigen be an exoantigen which can very

easily

and rapidly be produced from the

dermatophyte

isolates tobe identified.

Whenthedermatophyte antigenswereelectrophoretically separatedindenaturing gels and thenimmobilizedon nitro- cellulose strips,wedetectedagreaterdiversity of monoclo- nalantibody reactivitytofungal proteinsthan whenweused thetechniqueofimmunodiffusion ofsolublenondenaturated dermatophyte antigens(5).Analogoustotheresults obtained

by

Braun et al. (1) in the study of viral proteins, the preliminary advantageof thetechnique is in the detection of nonprecipitating antibody and of antibodyto poorlysoluble antigens.

Thefindingthatmultiple-molecular-weightcomponentsof the exoantigen reacted with monoclonal antibodies might suggest either modifications of a common protein or the polymerized states of a protein which was not separated under the denaturingconditions used in this study. Finally, the procedure of affinity chromatography by the im- munoadsorption of nonspecific monoclonal antibodiesmight lead tothepurification ofantigens specific to M. canis.

ACKNOWLEDGMENTS

This studywassupported bygrantsfrom the Ministero Pubblica Istruzione and Consiglio Nazionale delle Ricerche (Progetto Finalizzato:CMI Contrattono. 84.02040.52).

We thankLibero Ajello for hiseditorial assistance in theprepa- rationof thismanuscript.

LITERATURE CITED

1. Braun, D. K., L. Pereira, B. Norrild, and B. Roizman. 1983.

Application of denatured,electrophoretically separated, andim- mobilized lysates of herpes simplex virus-infected cells for de- tectionof monoclonal antibodies and for studies of the properties of viral proteins. J. Virol. 46:103-112.

2. Hawkes, R., E. Niary, and J. Gordon. 1982. A dot-immuno- blotting assay for monoclonal and other antibodies. Anal.

Biochem. 119:142-147.

3. Lowry, 0. H.,N.J.Rosebrough,A. L.Farr,and R.J.Randall.

1951. Protein measurement with the Folin phenol reagent. J.

Biol. Chem. 193:265-275.

4. Matsumoto, T., A. A. Padhye, and L. Ajello. 1983. Successful mating of Microsporum distortum with Nanniziaotae.Trans.Br.

Mycol. Soc. 81:645-650.

5. Polonelli, L., and G. Morace. 1985. Serological analysis of dermatophyte isolates with monoclonal antibodies produced against Microsporumcanis. J. Clin. Microbiol.21:138-139.

6. Towbin,H., T. Staehelin, andJ. Gordon. 1979. Electrophoretic transfer ofproteins from polyacrylamide gels to nitrocellulose sheets and someapplications. Proc. Natl. Acad. Sci. USA 76:

265-275.

VOL.23, 1986 ₆₁₅

on December 10, 2013 by PROFESSOR OF RESEARCHhttp://jcm.asm.org/Downloaded from