JOURNAL OF CLINICAL MICROBIOLOGY, June 1992, p. 1544-1550 Vol. 30, No. 60095-1137/92/061544-07$02.00/0Copyright © 1992, American Society for Microbiology

Monoclonal Antibodies Specific for Clostridium difJicileToxin B and Their Use in ImmunoassaysFRANK MULLER, CLAUDIA STIEGLER, AND ULRICH HADDING*

Institut fiir Medizinische Mikrobiologie und Virologie, Heinrich-Heine-Universitat,Moorenstraf3e 5, D-4000 Dusseldorf, Gennany

Received 9 January 1992/Accepted 12 March 1992

Five mouse monoclonal antibodies (MAbs) against Clostridium difficile toxin B have been raised andcharacterized. Three of them were immunoglobulin M (IgM) antibodies (6B10, 6G3, and 10B9), and the othertwo were of the IgGl isotype (9E5 and 17G2), recognizing specifically two distinct epitopes on the toxin Bmolecule. No MAb was able to neutralize cytotoxic activity significantly. The two IgGl MAbs were purified andapplied to various immunodiagnostic assays. MAbs coupled to latex beads were used for specific removal oftoxin B from cytotoxic samples and for agglutination assay. An indirect sandwich enzyme-linked immunosor-bent assay with MAb 9E5 or 17G2 as the capture antibody was established for identification of toxin B with alower detection limit of 5 ng/ml.

Clostndium difficile toxin A and toxin B are considered tobe etiological agents of antibiotic-associated pseudomembra-nous colitis and some cases of diarrhea in humans. Toxin Aacts primarily as an enterotoxin, damaging the intestinalmucosa and causing hemorrhagic fluid accumulation in rab-bit ileal loops. In addition, it causes hemagglutination andexhibits a slight cytotoxic activity. Toxin B is a highly potentcytotoxin effective against most tissue-cultured mammaliancells and lacks any enterotoxic activity (for a review, seereference 17). Monospecific antisera as well as cross-react-ing monoclonal antibodies (MAbs) raised against purifiedtoxins A and B have led to different interpretations abouttheir structural and immunological relationship, suggestingthat the two toxins are unrelated molecules as well as thatthey share structural and immunological similarities. Fur-thermore, it has been shown that toxin A and toxin B areable to bind several mouse MAbs by a nonimmune reaction(1, 15, 16, 18, 24, 28). Cloning and sequencing of the genescoding for toxins A and B (2, 7, 9, 25) revealed regions ofsignificant homology in the deduced N-terminal proteinsequences (29). This might explain why some of the MAbsdescribed are cross-reacting with both toxins. For a rapiddiagnosis of C. difficile colitis and the presence of C. difficiletoxins in the stool specimens of patients, specific MAbscould represent suitable reagents for establishing immunoas-says of high sensitivity, like the enzyme-linked immunosor-bent assay (ELISA). The most commonly used test to screenC. difficile colitis is still the tissue culture assay, detectingtoxin B because of its high specific cytotoxic activity in fecalspecimens with high sensitivity, provided that a neutralizingantiserum is included in the controls. This test, however, istime-consuming and requires laboratory equipment for tis-sue culture facilities. A commercial latex agglutination test(Culturette rapid latex test) is not specific for the toxins andtherefore also detects nontoxigenic strains of C. difficile (21).The advantages of immunodiagnostic tests such as ELISAspecific for toxin A and/or toxin B have been discussedpreviously (19, 23, 31). In most cases of C. difficile colitis,both toxins are present in the stool specimens of patients andtheir identification by ELISA would offer a substantial

* Corresponding author.

saving of time and costs, standardized results, and simplifiedhandling compared with other methods, in particular thetissue culture assay. Recently, a rapid enzyme immunoassayfor the detection of C. difficile toxin A by use of a MAb anda combined toxin A and toxin B ELISA using specific MAbs(Cytoclone A+B enzyme immunoassay; Cambridge Bio-tech) have been presented (4, 6). In this report we describethe generation of monoclonal immunoglobulin G (IgG) anti-bodies monospecific for toxin B, their characterization, andtheir application to several C. difficile cytotoxin-specificimmunoassays.

MATERIALS AND METHODS

Purification of C. dificile toxins A and B. The purificationof toxins followed the method described previously (5, 28)with minor modifications. Briefly, C. difficile VPI 10463 wasgrown in brain heart infusion medium (Difco, Detroit, Mich.)for 72 h at 37°C. The culture supernatant was clarified bycentrifugation at 10,000 x g for 15 min at 4°C. Toxin A andtoxin B were precipitated successively by addition of solid(NH4)2SO4 to achieve 40 and 70% saturation, respectively.Each precipitate was collected by centrifugation at 10,000 xg for 30 min. The protein pellets were dissolved in 50 mMTris-HCI (pH 7.5), dialyzed against 50 mM Tris-HCl-25 mMNaCl (pH 7.5) overnight at 4°C, and further purified by MonoQ anion-exchange chromatography (fast protein liquid chro-matography system; Pharmacia, Freiburg, Germany). With alinear salt gradient in 50 mM Tris-HCl (pH 7.5), toxin Aeluted at 180 mM NaCl and toxin B eluted at 550 mM NaCl.Fractions were checked by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) and cytotoxic-ity assay. For some purposes toxin fractions were rechro-matographed; toxin B rechromatography in the presence of50 mM CaCl2 removed low-molecular-weight contaminants(22). Protein concentration was determined according to themethod of Bradford (3) with bovine IgG as a standard.

Generation of toxin B-specific MAbs. For immunization ofmice, SDS-PAGE-purified toxin B was prepared. Toxin Bfractions eluted from a Mono Q column were separated in a5% polyacrylamide gel and blotted onto nitrocellulose, andthe proteins were shortly stained with India ink. The 270-kDa toxin B bands (approximately 10 ,ug of protein) were

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C. DIFFICILE TOXIN B-SPECIFIC MONOCLONAL ANTIBODIES 1545

excised and stored at -20°C. Prior to each injection a pieceof nitrocellulose was dissolved in 200 ,u1 of dimethyl sulfox-ide and mixed with an equal volume of Freund's adjuvant.Three-month-old female BALB/c mice were injected fourtimes intraperitoneally with this solution at 3-week intervals.Three days after the last boost, spleen cells were fused withmouse myeloma cell line X63 Ag 8-653 by standard methods(8, 10). Hybridomas were screened for production of toxinB-reactive antibodies by a direct ELISA described belowand were cloned by limited dilution. MAb isotypes weredetermined with a monoclonal isotyping kit (Amersham,Aylesbury, United Kingdom). The toxin A-reactive MAb1337C8 has been described by von Eichel-Streiber et al. (30).Ascitic fluid was induced by injecting MAb-producing hy-bridoma cells intraperitoneally into pristane-primed BALB/cmice (8).

Purification of MAbs. MAbs were purified from hybridomaculture supernatants or ascitic fluid by affinity chromatogra-phy using a protein G-Superose matrix (Pharmacia) accord-ing to the instructions of the manufacturer.SDS-PAGE and immunoblotting. Proteins were fraction-

ated on 7.5% polyacrylamide gels according to the method ofLaemmli (12) and stained with Coomassie brilliant blue or,alternatively, electroblotted to nitrocellulose sheets (27).Western blots (immunoblots) or dot blots of unfractionatednative proteins were blocked with 5% skim milk powder(Oxoid, London, United Kingdom) in phosphate-bufferedsaline (PBS), incubated with an appropriate dilution of MAb,and developed with alkaline phosphatase-labeled anti-mouseimmunoglobulin (Dianova, Hamburg, Germany). As a chro-mogen, 5-bromo-4-chloro-3-indolylphosphate-nitroblue tet-razolium chloride (Boehringer, Mannheim, Germany) wasused.

Cytotoxicity assay. Cytotoxic activity of toxin B wasdetermined by rounding of tissue-cultured Buffalo greenmonkey (BGM) cells basically as described previously (5,28). BGM cells (5 x 103) were seeded into the well of amicrotiter plate and allowed to adhere overnight in Dulbeccomodified Eagle medium (GIBCO, Paisley, United Kingdom).Toxin samples were added in serial fivefold dilutions. Afterincubation for 24 h at 37°C, cell rounding was determined.Confirmatory neutralization tests were done by adding goatantiserum against C. difficile culture filtrate (purchased fromPaesel and Lorei, Frankfurt, Germany) at a dilution of 1:250.One tissue culture dose (TCD) was defined as the reciprocalof the highest dilution of toxin that caused rounding of 100%of the cells.ELISA techniques. (i) Screening ELISA. For screening

hybridoma culture supernatants or ascitic fluids for thepresence of toxin B-reactive antibodies, Mono Q-purifiedtoxin B (1 ,ug in 50 ,u1 per well) was adsorbed to a 96-wellmicrotiter plate (Nunc Immunoplate Maxisorb, Wiesbaden,Germany) overnight at 4°C in PBS (150 mM NaCl in 20 mMphosphate buffer, pH 7.4). For testing the cross-reactivitywith toxin A, wells were coated with Mono Q-purified toxinA (1 ,ug in 50 ,u1 per well) by the same procedure. Wells wereblocked with 1% skim milk powder (Oxoid) in PBS for 30min at room temperature (RT) and washed with PBS.Fifty-microliter aliquots of the culture supernatants takenfrom wells with growing hybridomas were added for 2 h atRT. After washing with PBS, 50 p.1 of a 1:5,000 dilution ofgoat anti-mouse immunoglobulin-peroxidase conjugate (Dia-nova) was added and incubated for a further 2 h at RT. Aftera final washing step the substrate solution (100 ,ul) containing0.1% (wt/vol) o-phenylenediamine (Sigma) and 0.02% (vol/vol) H202 in 100 mM citrate buffer, pH 5.5, was added, and

the color reaction was stopped after 20 min by addition of100 p1 of 2 M HCl solution. The A490 was measured with aNunc Immunoreader NJ 2000. A reaction was defined aspositive if the A490 of the sample was increased by at least0.3 absorbance units compared with that of the controlwithout primary antibody.

(ii) Direct ELISA. For quantitation of toxin B in C. difficileculture supernatants or purified preparations, the wells of amicrotiter plate were coated with serial dilutions of thesample to be tested overnight at 4°C. After blocking with 1%skim milk powder in PBS and washing with PBS, MAbs fromhybridoma culture supernatants or purified from ascitic fluidwere added in appropriate dilutions and incubated for 2 h atRT. Detection of antigen-bound mouse antibodies was per-formed as described above.

(iii) Sandwich ELISA. A sandwich ELISA for quantitationof toxin B was established with MAbs 17G2 and 9E5, resp.,as capture antibodies. The protein G-Superose-purifiedMAbs (10 mg/ml) originating from ascitic fluid were diluted1:1,000 and adsorbed to the wells of a microtiter plate (50 ,ul)overnight at 4°C. After blocking with 1% skim milk powderin PBS and washing with PBS, serial dilutions of toxin Bsamples (C. difficile culture supernatants, stool samples, orpurified toxin B, 50 ,ul) were added to the wells and incu-bated for 2 h at RT. Unbound material was washed away,and the captured toxin was marked with goat antiserumagainst C. difficile VPI 10643 culture filtrate (purchased fromPaesel and Lorei) diluted 1:750 in 1% skim milk powder inPBS (50 ,ul). After washing, toxin-bound goat antibodieswere detected by incubation with 50 p.l of a donkey anti-goatimmunoglobulin-peroxidase conjugate (1:1,000; Dianova) for2 h at RT followed by the color reaction described above.For testing stool specimens, fresh samples were suspendedin 4 volumes of PBS and stored overnight at 4°C. Immedi-ately before the test, the samples were centrifuged for 5 minat 10,000 x g and the supernatant (50 ,ul) was applied to thewells of a prepared microtiter plate either directly or after aserial dilution in PBS.

Coupling MAbs to latex beads. Latex beads (Polybeadpolystyrene microspheres, diameter = 1 ,um; Polysciences,Warrington, Pa.) were coated with protein G-purified MAbs9E5 and 17G2 in equimolar amounts (1 mg/ml), blocked with5% skim milk powder in PBS, washed, and diluted in PBS toa suitable concentration. Macroscopic agglutination wasobserved within 30 min after mixing 10 ,ul of coupled beadswith 10 ,ul of the toxin B-containing sample. For removal oftoxin B from C. difficile culture filtrates or toxin B prepara-tions, 50 ,ul of coupled beads was incubated with 200 p.l ofthe toxin solution for 1 h at RT with occasional shaking. Thebeads were removed by centrifugation for 1 min at 5,000 x g,and the resulting supernatants were subjected to the cyto-toxicity assay as described above.

RESULTS

Generation and characterization of toxin B-specific MAbs.For vaccination of mice we prepared highly purified C.difficile toxin B to avoid a possible cross-reactivity of MAbswith toxin A due to contamination of the immunizing anti-gen. Mono Q-fractionated toxin B was subjected to SDS-PAGE and blotted onto nitrocellulose. The toxin B bandswere excised from the blot and used for immunization. FiveMAbs were obtained from a single successful fusion exper-iment, whereas several other fusions did not yield any toxinB-reactive MAb. The primary screening of hybridomas wasdone with native toxin B by a direct ELISA. Isotyping of the

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1546 MULLER ET AL.

A B A B A B A B A B A B A B

C

* 0 *17G2 9E5 6B10 109B 6G3

dilution factor

2.0

1 .6

1.20

08,

0.8

0.4

0.0

B

10 1

dilution factor

five MAbs identified two of the IgGl(K) subtype, designated9E5 and 17G2, and the other three of the IgM(K) subtype,designated 6B10, 6G3, and 10B9. A further characterizationof the reactivity patterns of these MAbs with native ordenatured toxin A and toxin B was done by screeningELISAs, dot blots, or Western blots. As a control, MAb1337C8, which is monospecific for toxin A (30), was includedin the assays. The results are shown in Fig. 1 and summa-rized in Table 1. All of the MAbs were monospecific fortoxin B, except for the IgM antibody 10B9, which cross-reacted weakly with toxin A in the screening ELISA (Fig.1B). Two of the IgM antibodies, 6G3 and 10B9, recognizedonly the native toxin in ELISA or dot blot, whereas the twoIgGl MAbs 9E5 and 17G2 and the IgM antibody 6B10 alsoreacted specifically with the SDS-denatured toxin B mole-cule on Western blots (Fig. 1A and C).

Analyzing the reactivity patterns of MAbs 9E5, 17G2, and6B10 with proteolytic fragments generated from highly pu-rified toxin B by staphylococcal endoproteinase Glu-Cclearly demonstrated that both IgGl MAbs recognize differ-ent epitopes (Fig. 2). MAb 6B10 bound to fragments of thesame length as did MAb 9E5, suggesting that they detectneighboring or identical antigenic sites. Interestingly, MAb

FIG. 1. Characterization of the reactivity of MAbs with C.difficile toxin A and toxin B. (A and B) Screening ELISA with toxinB and toxin A. Serial fivefold dilutions of hybridoma culturesupernatants were tested for their reactivity with purified toxin B(panel A) and toxin A (panel B) coated onto the wells of a microtiterplate (1 ,ug per well). All determinations were done in duplicate.Solid triangle, MAb 17G2; solid square, MAb 9E5; solid circle, MAb6B10; open triangle, MAb 6G3; open square, MAb 10B9; opencircle, MAb 1337C8. (C) Immunoblot analysis. Purified toxin A (5,ug; lanes A) and toxin B (5 ,ug; lanes B) were run in several parallellanes on a denaturing SDS-polyacrylamide gel and electroblotted toa nitrocellulose membrane or were spotted directly as native protein(5 ,ug per dot) onto the membrane. These Western blots (top) and dotblots (bottom) were developed with the MAb solutions as indicated.CBB, Coomassie brilliant blue-stained lanes of the polyacrylamidegel. Additionally, the precipitation of toxin B (applied in 10-folddilutions in the outer wells) by the MAb 6B10 (center) in anOuchterlony double-diffusion assay is shown.

6B10 was able to precipitate toxin B in Ouchterlony double-diffusion assay (Fig. 1). None of the other MAbs, even incombination, showed this characteristic. This would implythat the epitope recognized by MAb 6B10 is present inseveral copies on the toxin B molecule.

In order to specify the target sites of the MAbs on thetoxin molecule in more functional terms, we checkedwhether one of the MAbs would affect the biological activityof the cytotoxin. For instance, masking of the toxic domain

TABLE 1. Characterization of C. difficile toxin A-and toxin B-reactive MAbs

Reactivity' in:

MAb Ig ELISA Dot blot Western blotSubclassToxin A Toxin B Toxin A Toxin B Toxin A Toxin B

9E5 IgGl(K) - ++ - +++ - +17G2 IgGl(K) - + + - +++ - ++6B10 IgM(K) - +++ - +++ - +++6G3 IgM(K) - +++ - + - -10B9 IgM(K) + + + (+) + + - -1337C8 IgG2b(K) +++ - +++ - + -

a Indicated on a scale from - (no reactivity with the indicated toxin) to+++ (strong reactivity).

A2.5

2.0

CD

1.5

1.0

0.5

0.0 .S.i:3_"I

1 337C8 CBB

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C. DIFFICILE TOXIN B-SPECIFIC MONOCLONAL ANTIBODIES

1 2 3 4 1 2 3 4 1 2 3 4

17G2 9E5 68 0

1 2 3 4

COOMASSIE

FIG. 2. Epitope mapping for MAbs 9E5, 17G2, and 6B10. Mono Q-fractionated toxin B was further purified by a denaturing gel filtrationstep on a Superose 6 column (Pharmacia) in the presence of 0.1% SDS to remove contaminants with lower molecular weights. Aliquots ofthis preparation were incubated with different amounts of staphylococcal endoproteinase Glu-C (Boehringer), subjected to SDS-PAGE,electroblotted to a nitrocellulose membrane, and immunostained with the MAbs indicated. Lanes 1, 10 ,ug of protease; lanes 2, 1 ,ug ofprotease; lanes 3, 0.1 ,ug of protease; lanes 4, no protease added. On the right the Coomassie brilliant blue-stained polyacrylamide gel isshown.

or of the attachment domain involved in binding to the targetcell should inhibit the cytopathic activity of the toxin. So wetested all five MAbs individually as well as in combinationsfor neutralizing activity in cytotoxicity assays. Titrated C.difficile culture filtrates or purified toxin B preparations werepreincubated with serially diluted antibody solutions andthen subjected to the standard cytotoxicity assay. In Fig. 3 isshown one example in which we observed a 10-fold reduc-

106

1 05

10~4

0

1 01

a-tox 9E5 17G2 6B10 6G3 10B9 mixFIG. 3. Neutralization of cytotoxicity. Mono Q-purified toxin B

was preincubated with antitoxin serum or with MAbs as indicatedand tested in the standard cytotoxicity assay. One example of aseries of antibody titrations, in which the antibodies were added atthe highest concentration, is shown. In no case-with the exceptionof the antitoxin serum-could a significant neutralization of morethan a factor of 10 be observed. 0, no antibody added; a-tox, C.difficile antitoxin serum; mix, combination of all five MAbs.

tion of the cytotoxic activity of purified toxin B by MAbs17G2 and 6B10 and the antibody mixture applied at thehighest possible concentration. However, this neutralizingactivity was observed only at the highest toxin dilutions andwas readily abolished when a 5- or 10-fold dilution of theantibody was applied. In comparison, the control antitoxinserum reduced the cytotoxicity by a factor of 1,000. There-fore, we conclude that none of the epitopes recognized bythe five MAbs described here represents a biologically activesite of the cytotoxin.

Coupling of MAbs 9E5 and 17G2 to latex beads. Althoughthe toxin B-specific MAbs obviously do not directly neutral-ize cytotoxicity, we tried to exploit the monospecificity ofthe MAbs to remove specifically toxin B from samples to betested for their biological activities. This was achieved byimmobilizing the IgG MAbs 9E5 and 17G2 in equimolaramounts to polystyrene microspheres. The combination oftwo MAbs was chosen to increase the avidity of an immunecomplex between the sensitized latex beads and the toxinantigen. When toxin B samples (C. difficile culture filtrateand Mono Q-purified fractions) were incubated with theselatex beads to bind the cytotoxin and then centrifuged toremove the beads, a drastic decrease of the tissue culturedose of the samples was observed in the cytotoxicity assay.For Mono Q-purified toxin B the tissue culture dose was

reduced from 312,500 to 2,500, and for culture filtrate it wasreduced from 12,500 to 100 (Fig. 4). This decrease incytotoxic activity of the samples was comparable to thatseen with the positive control, i.e., a neutralizing antitoxinserum. Unsensitized beads served as a negative control.These results suggest that the toxin B-specific MAbs coupledto latex beads could provide a toxin B-specific control inmany assays in which biological functions of the C. difficiletoxins are being investigated.

106 000-

80 000-

49500-

3250-

27500-

18500-

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106

105

C)H--

104

103

10* antiserum beads mAb-beads

FIG. 4. Removal of cytotoxic activity from toxin B fractionswith MAbs 9E5 and 17G2 coupled to latex beads. Mono Q-purifiedtoxin B (solid bars) or C. difficile culture filtrate (hatched bars) waspreincubated without (0) or with (MAb-beads) sensitized latexbeads, with unsensitized latex beads (beads), or with antitoxinserum; centrifuged to remove the beads; and subjected to thestandard cytotoxicity assay.

A further application of the latex-coupled MAbs 9E5 and17G2 with diagnostic relevance would be the detection oftoxin B in samples by a simple agglutination assay. Toevaluate the suitability of the sensitized beads in such asystem, we mixed equal amounts of MAb-coupled beadswith purified toxin B on latex agglutination test plates andexamined the plates macroscopically for agglutination bycomparison with a control with unsensitized beads. Bytitrating the toxin B concentration, we determined the de-tection limit of this method to be 200 ng/ml.

Detection and quantitation of toxin B by ELISA. With thetwo MAbs of the IgGl subtype, 9E5 and 17G2, we developed

A

2.5

2.0

1.5

1.0

0.5

0.0

two ELISA systems for detection and quantitation of toxin Bin various samples. The first approach was based on thedirect screening ELISA used for the detection of toxinB-reactive MAbs. Highly purified toxin B samples werediluted serially, coated onto a microtiter plate, and incubatedwith MAb 9E5 or 17G2 purified from ascitic fluid. BoundMAbs were detected with anti-mouse IgG-peroxidase con-jugate. In this direct ELISA system a minimum of 5 ng ofantigen could be detected with both capture antibodies. Thiscorresponds to a toxin B concentration of about 100 ng/ml inthe sample.

In order to increase the sensitivity of the ELISA, weperformed an indirect sandwich ELISA. This should allowthe specific capture of toxin B molecules from highly com-plex protein mixtures, e.g., fecal specimens or C. difficileculture filtrates, which could interfere in the direct ELISAsystem by competition for the binding sites of the microtiterplate. Therefore, microtiter plates were coated with MAb9E5 or 17G2 as a capture antibody. Titrated toxin samples(highly purified toxin B, C. difficile culture filtrate, or cyto-toxin-positive stool samples) were added, and finally thebound toxin B molecules were detected with a commerciallyavailable C. difficile antitoxin serum in combination with aperoxidase-labeled anti-goat IgG antibody. With both MAbspurified toxin B could be detected at a minimal concentrationof 20 ng/ml (Fig. 5A). This corresponds to a fivefold increasein sensitivity compared with that of the direct ELISAsystem. Analyzing C. difficile culture filtrate samples with upto a 128- or 256-fold dilution of the samples gave clearlypositive results with the sandwich ELISA for MAb 17G2 or9E5, respectively. This sensitivity of the assay is sufficient todetect cytotoxin in stool specimens of patients which havebeen diagnosed as positive in the conventional cytotoxicityassay even at a 125-fold dilution (Fig. 5B). These datasuggest that the toxin B-specific MAbs presented in thisstudy-in particular the IgGl antibodies 9E5 and 17G2-arevery useful tools for detection and/or quantitation of C.difficile cytotoxin.

B

2.0-I A Oe 9E5

00)

1.5

1.0

0.5

0.01 10 102 103 104 105

toxin B concentration (ng/ml) dilution factorFIG. 5. Indirect sandwich ELISA for detection of C. difficile toxin B. (A) Determination of the lower detection limit. Purified toxin B was

titrated in fivefold dilution steps. The MAb indicated was used as the capture antibody. (B) Clinical stool samples diagnosed as cytotoxinnegative (open symbols) or cytotoxin positive (filled symbols) by the standard cytotoxicity assay were titrated in fivefold dilution steps. TheMAb indicated was used as the capture antibody. All determinations were done in duplicate.

0aw)

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DISCUSSION

The most specific and sensitive method available forlaboratory diagnosis of C. difficile colitis is the tissue cultureassay for detection of toxin B in fecal specimens of patients.Other immunodiagnostic tests, like counterimmunoelectro-phoresis, latex agglutination, dot immunobinding assay, andELISA, have been described, but some of them lack speci-ficity for toxigenic strains of C. difficile (1, 11, 13, 14, 19, 20,32). MAbs have been raised against C. difficile enterotoxin(19), and ELISAs specific for toxin A are now developed (6).They offer a substantial saving of time in identifying toxi-genic C. difficile in clinical stool samples compared with thestandard cytotoxicity assay. Each method by itself is re-

stricted to detect the presence of only one of the toxins.Although most toxigenic C. difficile strains analyzed produceboth toxins, their relative concentrations may vary andToxA- ToxB+ or ToxA+ ToxB- strains may occur (26).Therefore, our aim was to generate toxin B-specific MAbssuited for detection of cytotoxin by a rapid and simpleELISA system. Furthermore, the ability to identify mono-

specifically toxin A or toxin B by MAbs would contribute tothe elucidation of the mechanistic role of both toxins inpathogenicity.Of the five cytotoxin-reactive MAbs obtained, only one

(10B9) showed a weak cross-reactivity with native toxin A.The remaining four reacted monospecifically with toxin B.Two of them are IgGl antibodies, recognizing distinctepitopes. Strikingly, all toxin B-reactive MAbs and some ofthe toxin A-reactive MAbs described in previous studiescross-reacted with toxin A or toxin B, respectively (see,e.g., references 1, 15, 16, 24, and 28). This phenomenoncould reflect the ability of both toxins to bind mouse immu-noglobulins by a nonimmune reaction (16); on the otherhand, it could also suggest an immunological relationshipbetween both toxins. von Eichel-Streiber et al. (29) demon-strated a high degree of sequence homology (64%) betweenthe N-terminal regions of both toxins. Aligning the completeamino acid sequences of toxin A and toxin B-deduced fromthe published nucleotide sequences (2, 7)-by the PC/Genecomputer program (A. Bairoch, University of Geneva, Swit-zerland; IntelliGenetics, Inc., version 6.50), we found thatthis high degree of homology is not restricted to the N-ter-minal region but extends over the entire toxin B sequence

(47.2% identity, 13.2% similarity; i.e., 60.4% homology).Therefore, the frequently observed cross-reactivity of MAbswith both toxins (e.g., MAb 10B9 presented here) mightpresumably be due to their close structural similarity, al-though we cannot exclude also unspecific binding (16). Ofthe antibodies described in this study, only MAb 17G2 andMAb 6B10 showed a weak neutralizing activity when theywere preincubated in very high concentrations with purifiedcytotoxin. We therefore conclude that none of the five MAbsis able to interfere directly with a biologically active site ofthe toxin molecule, e.g., a putative binding or toxic domain.However, we cannot rule out the possibility that in our assay

system a putative toxin receptor on the test cells with a very

high affinity to the toxin molecule could displace a MAbdirected against the binding domain of the toxin or that a

putative toxic domain of the native cytotoxin is not accessi-ble for an antibody and becomes activated only after modi-fication (e.g., cleavage) inside the target cell. The ability ofMAb 6B10 to precipitate toxin B in an Ouchterlony double-diffusion assay should in principle lead to neutralization oftoxin B in vitro, analogous to a polyclonal antitoxin serum.

But we did not succeed in purifying this IgM antibody to therequired concentration.

Yet, coupling the MAbs 9E5 and 17G2 to latex beadsallowed the specific capture and physical removal of cyto-toxic activity from toxin solutions, demonstrating that theMAbs in fact are directed against the cytotoxin and could beused as a specific control in experiments testing biologicalfunctions of the toxin B molecule. Moreover, they are wellsuited to screen samples for the presence of toxin B atconcentrations above 200 ng/ml by a rapid agglutinationassay. However, this sensitivity is not sufficient to detectreliably toxin B in fecal specimens, e.g., clinical stoolsamples. The indirect sandwich ELISA described in thisstudy might be a useful tool for this purpose as well as for aprecise quantitation of the toxin B concentration in samplesdown to 5 ng/ml. Although the tissue culture assay detectingabout 50 pg of toxin B per ml is 100-fold more sensitive, theELISA described provides a sensitivity which is sufficient todetect toxin B in fecal specimens of patients suffering fromC. difficile colitis. Moreover, it is much faster, cheaper, andeasier to perform than the cytotoxicity assay, which requirestissue culture facilities and is not standardized. Furtherstudies are in progress in order to evaluate this method forclinical diagnostic use and to increase the sensitivity of theassay, e.g., by combining MAbs as capture antibodies or byuse of antitoxin sera as capture antibodies and the MAbsdescribed here as specific detection antibodies.

ACKNOWLEDGMENTS

We thank E. Lubatschowski and G. Tillmann for skillful technicalassistance and K. Schmitt for helpful discussions.

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Y. Nozawa. 1984. Biochemical characterization and biologicactions of two toxins (D-1 and D-2) of Clostridium difficile. Rev.Infect. Dis. 6:S11-S20.

2. Barroso, L. A., S.-Z. Wang, C. J. Phelps, J. L. Johnson, andT. D. Wilkins. 1990. Nucleotide sequence of Clostridium difficiletoxin B gene. Nucleic Acids Res. 18:4004.

3. Bradford, M. M. 1976. A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal. Biochem. 72:248-254.

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