JOURNAL OF CLINICAL MICROBIOLOGY, June 1992, p. 1544-1550 0095-1137/92/061544-07$02.00/0 Copyright © 1992, American Society for Microbiology

Vol. 30, No. 6

Monoclonal Antibodies Specific for Clostridium difJicile Toxin B and Their Use in Immunoassays FRANK 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 and characterized. Three of them were immunoglobulin M (IgM) antibodies (6B10, 6G3, and 10B9), and the other two were of the IgGl isotype (9E5 and 17G2), recognizing specifically two distinct epitopes on the toxin B molecule. No MAb was able to neutralize cytotoxic activity significantly. The two IgGl MAbs were purified and applied to various immunodiagnostic assays. MAbs coupled to latex beads were used for specific removal of toxin B from cytotoxic samples and for agglutination assay. An indirect sandwich enzyme-linked immunosorbent assay with MAb 9E5 or 17G2 as the capture antibody was established for identification of toxin B with a lower detection limit of 5 ng/ml.

Clostndium difficile toxin A and toxin B are considered to be etiological agents of antibiotic-associated pseudomembranous colitis and some cases of diarrhea in humans. Toxin A acts primarily as an enterotoxin, damaging the intestinal mucosa and causing hemorrhagic fluid accumulation in rabbit ileal loops. In addition, it causes hemagglutination and exhibits a slight cytotoxic activity. Toxin B is a highly potent cytotoxin effective against most tissue-cultured mammalian cells and lacks any enterotoxic activity (for a review, see reference 17). Monospecific antisera as well as cross-reacting monoclonal antibodies (MAbs) raised against purified toxins A and B have led to different interpretations about their structural and immunological relationship, suggesting that the two toxins are unrelated molecules as well as that they share structural and immunological similarities. Furthermore, it has been shown that toxin A and toxin B are able to bind several mouse MAbs by a nonimmune reaction (1, 15, 16, 18, 24, 28). Cloning and sequencing of the genes coding for toxins A and B (2, 7, 9, 25) revealed regions of significant homology in the deduced N-terminal protein sequences (29). This might explain why some of the MAbs described are cross-reacting with both toxins. For a rapid diagnosis of C. difficile colitis and the presence of C. difficile toxins in the stool specimens of patients, specific MAbs could represent suitable reagents for establishing immunoassays of high sensitivity, like the enzyme-linked immunosorbent assay (ELISA). The most commonly used test to screen C. difficile colitis is still the tissue culture assay, detecting toxin B because of its high specific cytotoxic activity in fecal specimens with high sensitivity, provided that a neutralizing antiserum is included in the controls. This test, however, is time-consuming and requires laboratory equipment for tissue culture facilities. A commercial latex agglutination test (Culturette rapid latex test) is not specific for the toxins and therefore also detects nontoxigenic strains of C. difficile (21). The advantages of immunodiagnostic tests such as ELISA specific for toxin A and/or toxin B have been discussed previously (19, 23, 31). In most cases of C. difficile colitis, both toxins are present in the stool specimens of patients and their identification by ELISA would offer a substantial *

Corresponding author.

saving of time and costs, standardized results, and simplified handling compared with other methods, in particular the tissue culture assay. Recently, a rapid enzyme immunoassay for the detection of C. difficile toxin A by use of a MAb and a combined toxin A and toxin B ELISA using specific MAbs (Cytoclone A+B enzyme immunoassay; Cambridge Biotech) have been presented (4, 6). In this report we describe the generation of monoclonal immunoglobulin G (IgG) antibodies monospecific for toxin B, their characterization, and their application to several C. difficile cytotoxin-specific immunoassays. MATERIALS AND METHODS Purification of C. dificile toxins A and B. The purification of toxins followed the method described previously (5, 28) with minor modifications. Briefly, C. difficile VPI 10463 was grown in brain heart infusion medium (Difco, Detroit, Mich.) for 72 h at 37°C. The culture supernatant was clarified by centrifugation at 10,000 x g for 15 min at 4°C. Toxin A and toxin 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 x g for 30 min. The protein pellets were dissolved in 50 mM Tris-HCI (pH 7.5), dialyzed against 50 mM Tris-HCl-25 mM NaCl (pH 7.5) overnight at 4°C, and further purified by Mono Q anion-exchange chromatography (fast protein liquid chromatography system; Pharmacia, Freiburg, Germany). With a linear salt gradient in 50 mM Tris-HCl (pH 7.5), toxin A eluted at 180 mM NaCl and toxin B eluted at 550 mM NaCl. Fractions were checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and cytotoxicity assay. For some purposes toxin fractions were rechromatographed; toxin B rechromatography in the presence of 50 mM CaCl2 removed low-molecular-weight contaminants (22). Protein concentration was determined according to the method of Bradford (3) with bovine IgG as a standard. Generation of toxin B-specific MAbs. For immunization of mice, SDS-PAGE-purified toxin B was prepared. Toxin B fractions eluted from a Mono Q column were separated in a 5% polyacrylamide gel and blotted onto nitrocellulose, and the proteins were shortly stained with India ink. The 270kDa toxin B bands (approximately 10 ,ug of protein) were 1544

VOL. 30, 1992

C. DIFFICILE TOXIN B-SPECIFIC MONOCLONAL ANTIBODIES

excised and stored at -20°C. Prior to each injection a piece of nitrocellulose was dissolved in 200 ,u1 of dimethyl sulfoxide and mixed with an equal volume of Freund's adjuvant. Three-month-old female BALB/c mice were injected four times intraperitoneally with this solution at 3-week intervals. Three days after the last boost, spleen cells were fused with mouse myeloma cell line X63 Ag 8-653 by standard methods (8, 10). Hybridomas were screened for production of toxin B-reactive antibodies by a direct ELISA described below and were cloned by limited dilution. MAb isotypes were determined with a monoclonal isotyping kit (Amersham, Aylesbury, United Kingdom). The toxin A-reactive MAb 1337C8 has been described by von Eichel-Streiber et al. (30). Ascitic fluid was induced by injecting MAb-producing hybridoma cells intraperitoneally into pristane-primed BALB/c mice (8). Purification of MAbs. MAbs were purified from hybridoma culture supernatants or ascitic fluid by affinity chromatography using a protein G-Superose matrix (Pharmacia) according to the instructions of the manufacturer. SDS-PAGE and immunoblotting. Proteins were fractionated on 7.5% polyacrylamide gels according to the method of Laemmli (12) and stained with Coomassie brilliant blue or, alternatively, electroblotted to nitrocellulose sheets (27). Western blots (immunoblots) or dot blots of unfractionated native proteins were blocked with 5% skim milk powder (Oxoid, London, United Kingdom) in phosphate-buffered saline (PBS), incubated with an appropriate dilution of MAb, and developed with alkaline phosphatase-labeled anti-mouse immunoglobulin (Dianova, Hamburg, Germany). As a chromogen, 5-bromo-4-chloro-3-indolylphosphate-nitroblue tetrazolium chloride (Boehringer, Mannheim, Germany) was used. Cytotoxicity assay. Cytotoxic activity of toxin B was determined by rounding of tissue-cultured Buffalo green monkey (BGM) cells basically as described previously (5, 28). BGM cells (5 x 103) were seeded into the well of a microtiter plate and allowed to adhere overnight in Dulbecco modified Eagle medium (GIBCO, Paisley, United Kingdom). Toxin samples were added in serial fivefold dilutions. After incubation for 24 h at 37°C, cell rounding was determined. Confirmatory neutralization tests were done by adding goat antiserum against C. difficile culture filtrate (purchased from Paesel and Lorei, Frankfurt, Germany) at a dilution of 1:250. One tissue culture dose (TCD) was defined as the reciprocal of 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 the presence of toxin B-reactive antibodies, Mono Q-purified toxin B (1 ,ug in 50 ,u1 per well) was adsorbed to a 96-well microtiter plate (Nunc Immunoplate Maxisorb, Wiesbaden, Germany) overnight at 4°C in PBS (150 mM NaCl in 20 mM phosphate buffer, pH 7.4). For testing the cross-reactivity with toxin A, wells were coated with Mono Q-purified toxin A (1 ,ug in 50 ,u1 per well) by the same procedure. Wells were blocked with 1% skim milk powder (Oxoid) in PBS for 30 min at room temperature (RT) and washed with PBS. Fifty-microliter aliquots of the culture supernatants taken from wells with growing hybridomas were added for 2 h at RT. After washing with PBS, 50 p.1 of a 1:5,000 dilution of goat anti-mouse immunoglobulin-peroxidase conjugate (Dianova) was added and incubated for a further 2 h at RT. After a final washing step the substrate solution (100 ,ul) containing 0.1% (wt/vol) o-phenylenediamine (Sigma) and 0.02% (vol/ vol) H202 in 100 mM citrate buffer, pH 5.5, was added, and

1545

the color reaction was stopped after 20 min by addition of 100 p1 of 2 M HCl solution. The A490 was measured with a Nunc Immunoreader NJ 2000. A reaction was defined as positive if the A490 of the sample was increased by at least 0.3 absorbance units compared with that of the control without primary antibody. (ii) Direct ELISA. For quantitation of toxin B in C. difficile culture supernatants or purified preparations, the wells of a microtiter plate were coated with serial dilutions of the sample to be tested overnight at 4°C. After blocking with 1% skim milk powder in PBS and washing with PBS, MAbs from hybridoma culture supernatants or purified from ascitic fluid were added in appropriate dilutions and incubated for 2 h at RT. Detection of antigen-bound mouse antibodies was performed as described above. (iii) Sandwich ELISA. A sandwich ELISA for quantitation of toxin B was established with MAbs 17G2 and 9E5, resp., as capture antibodies. The protein G-Superose-purified MAbs (10 mg/ml) originating from ascitic fluid were diluted 1:1,000 and adsorbed to the wells of a microtiter plate (50 ,ul) overnight at 4°C. After blocking with 1% skim milk powder in PBS and washing with PBS, serial dilutions of toxin B samples (C. difficile culture supernatants, stool samples, or purified toxin B, 50 ,ul) were added to the wells and incubated for 2 h at RT. Unbound material was washed away, and the captured toxin was marked with goat antiserum against C. difficile VPI 10643 culture filtrate (purchased from Paesel and Lorei) diluted 1:750 in 1% skim milk powder in PBS (50 ,ul). After washing, toxin-bound goat antibodies were detected by incubation with 50 p.l of a donkey anti-goat immunoglobulin-peroxidase conjugate (1:1,000; Dianova) for 2 h at RT followed by the color reaction described above. For testing stool specimens, fresh samples were suspended in 4 volumes of PBS and stored overnight at 4°C. Immediately before the test, the samples were centrifuged for 5 min at 10,000 x g and the supernatant (50 ,ul) was applied to the wells of a prepared microtiter plate either directly or after a serial dilution in PBS. Coupling MAbs to latex beads. Latex beads (Polybead polystyrene microspheres, diameter = 1 ,um; Polysciences, Warrington, Pa.) were coated with protein G-purified MAbs 9E5 and 17G2 in equimolar amounts (1 mg/ml), blocked with 5% skim milk powder in PBS, washed, and diluted in PBS to a suitable concentration. Macroscopic agglutination was observed within 30 min after mixing 10 ,ul of coupled beads with 10 ,ul of the toxin B-containing sample. For removal of toxin B from C. difficile culture filtrates or toxin B preparations, 50 ,ul of coupled beads was incubated with 200 p.l of the toxin solution for 1 h at RT with occasional shaking. The beads were removed by centrifugation for 1 min at 5,000 x g, and the resulting supernatants were subjected to the cytotoxicity 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 MAbs with toxin A due to contamination of the immunizing antigen. Mono Q-fractionated toxin B was subjected to SDSPAGE and blotted onto nitrocellulose. The toxin B bands were excised from the blot and used for immunization. Five MAbs were obtained from a single successful fusion experiment, whereas several other fusions did not yield any toxin B-reactive MAb. The primary screening of hybridomas was done with native toxin B by a direct ELISA. Isotyping of the

1546

MULLER ET AL.

J. CLIN. MICROBIOL.

A

A

2.5

B

A B

* 17G2

0

A B

A B

A B

A B

A

B

C

2.0 1.5 CD

1.0

0.5

0.0

6B10

9E5

3_"I

.S.i:

* 109B

6G3

1 337C8

CBB

dilution factor B

FIG. 1. Characterization of the reactivity of MAbs with C.

difficile toxin A and toxin B. (A and B) Screening ELISA with toxin

2.0

B and toxin A. Serial fivefold dilutions of hybridoma culture supernatants were tested for their reactivity with purified toxin B (panel A) and toxin A (panel B) coated onto the wells of a microtiter plate (1 ,ug per well). All determinations were done in duplicate. Solid triangle, MAb 17G2; solid square, MAb 9E5; solid circle, MAb 6B10; open triangle, MAb 6G3; open square, MAb 10B9; open circle, MAb 1337C8. (C) Immunoblot analysis. Purified toxin A (5 ,ug; lanes A) and toxin B (5 ,ug; lanes B) were run in several parallel lanes on a denaturing SDS-polyacrylamide gel and electroblotted to a nitrocellulose membrane or were spotted directly as native protein (5 ,ug per dot) onto the membrane. These Western blots (top) and dot blots (bottom) were developed with the MAb solutions as indicated. CBB, Coomassie brilliant blue-stained lanes of the polyacrylamide gel. Additionally, the precipitation of toxin B (applied in 10-fold dilutions in the outer wells) by the MAb 6B10 (center) in an Ouchterlony double-diffusion assay is shown.

1 .6

1.2 0

08,

0.8 0.4 0.0

10

1

dilution factor five MAbs identified two of the IgGl(K) subtype, designated 9E5 and 17G2, and the other three of the IgM(K) subtype, designated 6B10, 6G3, and 10B9. A further characterization of the reactivity patterns of these MAbs with native or denatured toxin A and toxin B was done by screening ELISAs, dot blots, or Western blots. As a control, MAb 1337C8, which is monospecific for toxin A (30), was included in the assays. The results are shown in Fig. 1 and summarized in Table 1. All of the MAbs were monospecific for toxin B, except for the IgM antibody 10B9, which crossreacted weakly with toxin A in the screening ELISA (Fig. 1B). Two of the IgM antibodies, 6G3 and 10B9, recognized only the native toxin in ELISA or dot blot, whereas the two IgGl MAbs 9E5 and 17G2 and the IgM antibody 6B10 also reacted specifically with the SDS-denatured toxin B molecule on Western blots (Fig. 1A and C). Analyzing the reactivity patterns of MAbs 9E5, 17G2, and 6B10 with proteolytic fragments generated from highly purified toxin B by staphylococcal endoproteinase Glu-C clearly demonstrated that both IgGl MAbs recognize different epitopes (Fig. 2). MAb 6B10 bound to fragments of the same length as did MAb 9E5, suggesting that they detect neighboring or identical antigenic sites. Interestingly, MAb

6B10 was able to precipitate toxin B in Ouchterlony doublediffusion assay (Fig. 1). None of the other MAbs, even in combination, showed this characteristic. This would imply that the epitope recognized by MAb 6B10 is present in several copies on the toxin B molecule. In order to specify the target sites of the MAbs on the toxin molecule in more functional terms, we checked whether one of the MAbs would affect the biological activity of the cytotoxin. For instance, masking of the toxic domain TABLE 1. Characterization of C. difficile toxin Aand toxin B-reactive MAbs Reactivity' in: MAb

Ig Subclass

Dot blot

ELISA

Western blot

Toxin A Toxin B Toxin A Toxin B Toxin A Toxin B

9E5 17G2

IgGl(K)

IgGl(K)

-

6B10

IgM(K) IgM(K) 10B9 IgM(K) + 1337C8 IgG2b(K) +++

6G3

++ ++ +++ +++ ++ -

-

-

(+) +++

+++ +++ +++ + ++ -

-

+ ++ +++

-

-

+

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

(strong reactivity).

C. DIFFICILE TOXIN B-SPECIFIC MONOCLONAL ANTIBODIES

VOL. 30, 1992

1

2

3

4

1 2

3

4

1

2

68

0

3

4

1

2

3

1547

4

106 00080 000-

49500-

32 50-

27500-

18500-

17G2

9E5

COOMASSIE

FIG. 2. Epitope mapping for MAbs 9E5, 17G2, and 6B10. Mono Q-fractionated toxin B was further purified by a denaturing gel filtration a Superose 6 column (Pharmacia) in the presence of 0.1% SDS to remove contaminants with lower molecular weights. Aliquots of this 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 of protease; lanes 3, 0.1 ,ug of protease; lanes 4, no protease added. On the right the Coomassie brilliant blue-stained polyacrylamide gel is shown. step on

or of the attachment domain involved in binding to the target cell should inhibit the cytopathic activity of the toxin. So we tested all five MAbs individually as well as in combinations for neutralizing activity in cytotoxicity assays. Titrated C. difficile culture filtrates or purified toxin B preparations were preincubated with serially diluted antibody solutions and then subjected to the standard cytotoxicity assay. In Fig. 3 is shown 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 mix FIG. 3. Neutralization of cytotoxicity. Mono Q-purified toxin B was preincubated with antitoxin serum or with MAbs as indicated and tested in the standard cytotoxicity assay. One example of a series of antibody titrations, in which the antibodies were added at the highest concentration, is shown. In no case-with the exception of the antitoxin serum-could a significant neutralization of more than 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 MAbs 17G2 and 6B10 and the antibody mixture applied at the highest possible concentration. However, this neutralizing activity was observed only at the highest toxin dilutions and was readily abolished when a 5- or 10-fold dilution of the antibody was applied. In comparison, the control antitoxin serum reduced the cytotoxicity by a factor of 1,000. Therefore, we conclude that none of the epitopes recognized by the five MAbs described here represents a biologically active site of the cytotoxin. Coupling of MAbs 9E5 and 17G2 to latex beads. Although the toxin B-specific MAbs obviously do not directly neutralize cytotoxicity, we tried to exploit the monospecificity of the MAbs to remove specifically toxin B from samples to be tested for their biological activities. This was achieved by immobilizing the IgG MAbs 9E5 and 17G2 in equimolar amounts to polystyrene microspheres. The combination of two MAbs was chosen to increase the avidity of an immune complex between the sensitized latex beads and the toxin antigen. When toxin B samples (C. difficile culture filtrate and Mono Q-purified fractions) were incubated with these latex beads to bind the cytotoxin and then centrifuged to remove the beads, a drastic decrease of the tissue culture dose 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 was reduced from 12,500 to 100 (Fig. 4). This decrease in cytotoxic activity of the samples was comparable to that seen with the positive control, i.e., a neutralizing antitoxin serum. Unsensitized beads served as a negative control. These results suggest that the toxin B-specific MAbs coupled to latex beads could provide a toxin B-specific control in many assays in which biological functions of the C. difficile toxins are being investigated.

1548

MULLER ET AL.

J. CLIN. MICROBIOL.

106 105 104

C) H--

103

10 * antiserum beads mAb-beads FIG. 4. Removal of cytotoxic activity from toxin B fractions with MAbs 9E5 and 17G2 coupled to latex beads. Mono Q-purified toxin B (solid bars) or C. difficile culture filtrate (hatched bars) was preincubated without (0) or with (MAb-beads) sensitized latex beads, with unsensitized latex beads (beads), or with antitoxin serum; centrifuged to remove the beads; and subjected to the standard cytotoxicity assay.

A further application of the latex-coupled MAbs 9E5 and 17G2 with diagnostic relevance would be the detection of toxin B in samples by a simple agglutination assay. To evaluate the suitability of the sensitized beads in such a system, we mixed equal amounts of MAb-coupled beads with purified toxin B on latex agglutination test plates and examined the plates macroscopically for agglutination by comparison with a control with unsensitized beads. By titrating the toxin B concentration, we determined the detection limit of this method to be 200 ng/ml. Detection and quantitation of toxin B by ELISA. With the two MAbs of the IgGl subtype, 9E5 and 17G2, we developed

two ELISA systems for detection and quantitation of toxin B in various samples. The first approach was based on the direct screening ELISA used for the detection of toxin B-reactive MAbs. Highly purified toxin B samples were diluted serially, coated onto a microtiter plate, and incubated with MAb 9E5 or 17G2 purified from ascitic fluid. Bound MAbs were detected with anti-mouse IgG-peroxidase conjugate. In this direct ELISA system a minimum of 5 ng of antigen could be detected with both capture antibodies. This corresponds to a toxin B concentration of about 100 ng/ml in the sample. In order to increase the sensitivity of the ELISA, we performed an indirect sandwich ELISA. This should allow the specific capture of toxin B molecules from highly complex protein mixtures, e.g., fecal specimens or C. difficile culture filtrates, which could interfere in the direct ELISA system by competition for the binding sites of the microtiter plate. Therefore, microtiter plates were coated with MAb 9E5 or 17G2 as a capture antibody. Titrated toxin samples (highly purified toxin B, C. difficile culture filtrate, or cytotoxin-positive stool samples) were added, and finally the bound toxin B molecules were detected with a commercially available C. difficile antitoxin serum in combination with a peroxidase-labeled anti-goat IgG antibody. With both MAbs purified toxin B could be detected at a minimal concentration of 20 ng/ml (Fig. 5A). This corresponds to a fivefold increase in sensitivity compared with that of the direct ELISA system. Analyzing C. difficile culture filtrate samples with up to a 128- or 256-fold dilution of the samples gave clearly positive results with the sandwich ELISA for MAb 17G2 or 9E5, respectively. This sensitivity of the assay is sufficient to detect cytotoxin in stool specimens of patients which have been diagnosed as positive in the conventional cytotoxicity assay even at a 125-fold dilution (Fig. 5B). These data suggest that the toxin B-specific MAbs presented in this study-in particular the IgGl antibodies 9E5 and 17G2-are very useful tools for detection and/or quantitation of C.

difficile cytotoxin.

A

B

2.5

2.0-I

2.0 0

aw)

1.5

0

0)

A

Oe 9E5

1.5

1.0

1.0

0.5

0.5

0.0

0.0 1

10

102

103

104

toxin B concentration (ng/ml)

105

dilution factor

FIG. 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 cytotoxin negative (open symbols) or cytotoxin positive (filled symbols) by the standard cytotoxicity assay were titrated in fivefold dilution steps. The MAb indicated was used as the capture antibody. All determinations were done in duplicate.

C. DIFFICILE TOXIN B-SPECIFIC MONOCLONAL ANTIBODIES

VOL. 30, 1992

DISCUSSION

The most specific and sensitive method available for laboratory diagnosis of C. difficile colitis is the tissue culture assay for detection of toxin B in fecal specimens of patients. Other immunodiagnostic tests, like counterimmunoelectrophoresis, latex agglutination, dot immunobinding assay, and ELISA, have been described, but some of them lack specificity 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 toxigenic C. difficile in clinical stool samples compared with the standard cytotoxicity assay. Each method by itself is restricted to detect the presence of only one of the toxins. Although most toxigenic C. difficile strains analyzed produce both toxins, their relative concentrations may vary and ToxA- ToxB+ or ToxA+ ToxB- strains may occur (26). Therefore, our aim was to generate toxin B-specific MAbs suited for detection of cytotoxin by a rapid and simple ELISA system. Furthermore, the ability to identify monospecifically toxin A or toxin B by MAbs would contribute to the elucidation of the mechanistic role of both toxins in pathogenicity. 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 distinct epitopes. Strikingly, all toxin B-reactive MAbs and some of the toxin A-reactive MAbs described in previous studies cross-reacted with toxin A or toxin B, respectively (see, e.g., references 1, 15, 16, 24, and 28). This phenomenon could reflect the ability of both toxins to bind mouse immunoglobulins by a nonimmune reaction (16); on the other hand, it could also suggest an immunological relationship between both toxins. von Eichel-Streiber et al. (29) demonstrated a high degree of sequence homology (64%) between the N-terminal regions of both toxins. Aligning the complete amino acid sequences of toxin A and toxin B-deduced from the published nucleotide sequences (2, 7)-by the PC/Gene computer program (A. Bairoch, University of Geneva, Switzerland; IntelliGenetics, Inc., version 6.50), we found that this high degree of homology is not restricted to the N-terminal 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 MAbs with both toxins (e.g., MAb 10B9 presented here) might presumably be due to their close structural similarity, although we cannot exclude also unspecific binding (16). Of the antibodies described in this study, only MAb 17G2 and MAb 6B10 showed a weak neutralizing activity when they were preincubated in very high concentrations with purified cytotoxin. We therefore conclude that none of the five MAbs is able to interfere directly with a biologically active site of the 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 MAb directed against the binding domain of the toxin or that a putative toxic domain of the native cytotoxin is not accessible for an antibody and becomes activated only after modification (e.g., cleavage) inside the target cell. The ability of MAb 6B10 to precipitate toxin B in an Ouchterlony doublediffusion assay should in principle lead to neutralization of toxin B in vitro, analogous to a polyclonal antitoxin serum.

1549

But we did not succeed in purifying this IgM antibody to the required concentration. Yet, coupling the MAbs 9E5 and 17G2 to latex beads allowed the specific capture and physical removal of cyto-

toxic activity from toxin solutions, demonstrating that the MAbs in fact are directed against the cytotoxin and could be used as a specific control in experiments testing biological functions of the toxin B molecule. Moreover, they are well suited to screen samples for the presence of toxin B at concentrations above 200 ng/ml by a rapid agglutination assay. However, this sensitivity is not sufficient to detect reliably toxin B in fecal specimens, e.g., clinical stool samples. The indirect sandwich ELISA described in this study might be a useful tool for this purpose as well as for a precise quantitation of the toxin B concentration in samples down to 5 ng/ml. Although the tissue culture assay detecting about 50 pg of toxin B per ml is 100-fold more sensitive, the ELISA described provides a sensitivity which is sufficient to detect toxin B in fecal specimens of patients suffering from C. difficile colitis. Moreover, it is much faster, cheaper, and easier to perform than the cytotoxicity assay, which requires tissue culture facilities and is not standardized. Further studies are in progress in order to evaluate this method for clinical diagnostic use and to increase the sensitivity of the assay, e.g., by combining MAbs as capture antibodies or by use of antitoxin sera as capture antibodies and the MAbs described here as specific detection antibodies. ACKNOWLEDGMENTS We thank E. Lubatschowski and G. Tillmann for skillful technical assistance and K. Schmitt for helpful discussions. REFERENCES 1. Banno, Y., T. Kobayashi, H. Kono, K. Watanabe, K. Ueno, and Y. Nozawa. 1984. Biochemical characterization and biologic 2. 3. 4.

5. 6.

7.

8. 9.

10.

actions of two toxins (D-1 and D-2) of Clostridium difficile. Rev. Infect. Dis. 6:S11-S20. Barroso, L. A., S.-Z. Wang, C. J. Phelps, J. L. Johnson, and T. D. Wilkins. 1990. Nucleotide sequence of Clostridium difficile toxin B gene. Nucleic Acids Res. 18:4004. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. Coughlin, R., C. Fong, S. Brecher, D. Marciani, and M. Pickett. 1991. Comparison of Clostridium difficile cytotoxin titer with titers of toxin A and toxin B by enzyme immunoassay (EIA), abstr. C99. Abstr. 91st Gen. Meet. Am. Soc. Microbiol. 1991. American Society for Microbiology, Washington, D.C. Daubener, W., E. Leiser, C. von Eichel-Streiber, and U. Hadding. 1988. Clostridium difficile toxins A and B inhibit human immune response in vitro. Infect. Immun. 56:1107-1112. DiPersio, J. R., F. J. Varga, D. L. Conwell, J. A. Kraft, K. J. Kozak, and D. H. Willis. 1991. Development of a rapid enzyme immunoassay for Clostridium difficile toxin A and its use in the diagnosis of C. difficile-associated disease. J. Clin. Microbiol. 29:2724-2730. Dove, C. H., S.-Z. Wang, S.-B. Price, C. J. Phelps, D. M. Lyerly, T. D. Wilkins, and J. L. Johnson. 1990. Molecular characterization of the Clostridium difficile toxin A gene. Infect. Immun. 58:480-488. Harlow, E., and D. Lane. 1988. Antibodies: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Johnson, J. L., C. Phelps, L. Barroso, M. D. Roberts, D. M. Lyerly, and T. D. Wilkins. 1990. Cloning and expression of the toxin B gene of Clostridium difficile. Curr. Microbiol. 20:397401. Kearney, J. F., A. Radbruch, B. Liesegang, and K. Rajewski. 1979. A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-se-

1550

MULLER ET AL.

creted hybrid cell lines. J. Immunol. 123:1548-1550. 11. Krishnan, C. 1986. Detection of Clostndium difficile toxins by enzyme immunoassay. J. Hyg. Camb. 96:5-12. 12. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature (London) 227:680-685. 13. Laughon, B. E., R. P. Viscidi, S. L. Gdovin, R. H. Yolken, and J. G. Bartlett. 1984. Enzyme immunoassays for detection of Clostridium difficile toxins A and B in fecal specimens. J. Infect. Dis. 149:781-788. 14. Levine, H. G., M. Kennedy, and J. T. La Mont. 1982. Counterimmunoelectrophoresis vs. cytotoxicity assay for the detection of Clostridium difficile toxin. J. Infect. Dis. 145:398. 15. Libby, J. M., and T. D. Wilkins. 1982. Production of antitoxins to two toxins of Clostridium difficile and immunological comparison of the toxins by cross-neutralization studies. Infect. Immun. 35:374-376. 16. Lyerly, D. M., P. E. Carrig, and T. D. Wilkins. 1989. Nonspecific binding of mouse monoclonal antibodies to Clostridium difficile toxins A and B. Curr. Microbiol. 19:303-306. 17. Lyerly, D. M., H. C. Krivan, and T. D. Wilkins. 1988. Clostridium difficile: its disease and toxins. Clin. Microbiol. Rev. 1:1-18. 18. Lyerly, D. M., C. J. Phelps, J. Toth, and T. D. Wilkins. 1986. Characterization of toxins A and B of Clostridium difficile with monoclonal antibodies. Infect. Immun. 54:70-76. 19. Lyerly, D. M., C. J. Phelps, and T. D. Wilkins. 1985. Monoclonal and specific polyclonal antibodies for immunoassay of Clostridium difficile toxin A. J. Clin. Microbiol. 21:12-14. 20. Lyerly, D. M., N. M. Sullivan, and T. D. Wilkins. 1983. Enzyme-linked immunosorbent assay for Clostridium difficile toxin A. J. Clin. Microbiol. 17:72-78. 21. Lyerly, D. M., and T. D. Wilkins. 1986. Commercial latex test for Clostridium difficile toxin A does not detect toxin A. J. Clin. Microbiol. 23:622-623. 22. Meador, J., III, and R. K. Tweten. 1988. Purification and characterization of toxin B from Clostridium difficile. Infect. Immun. 56:1708-1714. 23. Nguyen, V. K., B. Rihn, C. Heckel, F. Bisseret, R. Girardet, and H. Monteil. 1990. Enzyme immunoassay (ELISA) for detection

J. CLIN. MICROBIOL.

24.

25.

26. 27.

28.

29.

30.

31.

32.

of Clostridium difficile toxin B in specimens of faeces. Med. Microbiol. 31:251-257. Rothman, S. W., M. K. Gentry, J. E. Brown, D. A. Foret, M. J. Stone, and M. P. Strickler. 1988. Immunochemical and structural similarities in toxin A and toxin B of Clostridium difficile shown by binding to monoclonal antibodies. Toxicon 26:583597. Sauerborn, M., and C. von Eichel-Streiber. 1990. Nucleotide sequence of Clostridium difficile toxin A. Nucleic Acids Res. 18:1629-1630. Torres, J. F. 1991. Purification and characterisation of toxin B from a strain of Clostridium difficile that does not produce toxin A. J. Med. Microbiol. 35:40-44. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354. von Eichel-Streiber, C., U. Harperath, D. Bosse, and U. Hadding. 1987. Purification of two high molecular weight toxins of Clostridium difficile which are antigenetically related. Microb. Pathog. 2:307-318. von Eichel-Streiber, C., R. Laufenberg-Feldmann, S. Sartingen, J. Schulze, and M. Sauerborn. 1990. Cloning of Clostridium difficile toxin B gene and demonstration of high N-terminal homology between toxin A and B. Med. Microbiol. Immunol. 179:271-279. von Eichel-Streiber, C., D. Suckau, M. Wachter, and U. Hadding. 1989. Cloning and characterization of overlapping DNA fragments of the toxin A gene of Clostridium difficile. J. Gen. Microbiol. 135:55-64. Walker, R. C., P. J. Ruane, J. E. Rosenblatt, D. M. Lyerly, C. A. Gleaves, T. F. Smith, P. F. Pierce, Jr., and T. D. Wilkins. 1986. Comparison of culture, cytotoxicity assays and enzyme linked immunosorbent assay for toxin A and toxin B in the diagnosis of Clostridium difficile-related enteric disease. Diagn. Microbiol. 5:61-69. Woods, G. L., and P. C. Iwen. 1990. Comparison of dot immunobinding assay, latex agglutination, and cytotoxin assay for laboratory diagnosis of Clostridium difficile-associated diarrhea. J. Clin. Microbiol. 28:855-857.