NOTES
Purification of an enterotoxin produced by BaciNus cereus by immunoaffinity chromatography using a monoclonal antibody KUNIHIROSHINAGAWA,TEIMITAKECHI,AND NAONORIMATSUSAKA Department of VeterinaryMedicine, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020, Japan
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SHUNJISUGII Department of Serology and Immunologv, School of Medical Technology, Kitasato University, Kitasato, Sagamihara, Kanagawa 228, Japan Received May 6, 1991 Revision received September 19, 1991 Accepted October 15, 1991 SHINAGAWA, K., TAKECHI,T., MATSUSAKA,N., and SUGII,S. 1992. Purification of an enterotoxin produced by Bacillus cereus by imrnunoaffinity chromatography using a monoclonal antibody. Can. J. Microbiol. 38: 153-156. A murine monoclonal antibody (MAb) with high reactivity to an enterotoxin produced by Bacillus cereus was used to prepare an immunoadsorbent for purification of the enterotoxin. By immunoaffinity chromatography using the immunoadsorbent, approximately 25% of crude enterotoxin applied was recovered in the eluate. The purified enterotoxin was found to be electrophoretically and antigenically homogeneous. It also showed vascular permeability activity and mouse lethality, and caused fluid accumulation in mouse ligated intestinal loops, whereas it did not show any hemolytic and lecithinase activities. Thus, immunoaffinity chromatography proved useful in the purification of enterotoxin produced by B. cereus in terms of recovery, purity, and relative ease of performing the purification. Key words: purification, immunoaffinity chromatography, enterotoxin, monoclonal antibody, Bacillus cereus. SHINAGAWA, K., TAKECHI,T., MATSUSAKA, N., et SUGII,S. 1992. Purification of an enterotoxin produced by Bacillus cereus by immunoaffinity chromatography using a monoclonal antibody. Can. J. Microbiol. 38 : 153-156. Un anticorps monoclonal murin (MAb), qui possede une reactivite elevee envers une enterotoxine produite par Bacillus cereus, a ete utilisee afin de preparer un immuno-adsorbant pour la purification de l'entkrotoxine. A l'aide de cet immunoadsorbant, environ 25% de la quantite initiale de l'enterotoxine a ete recuperee dans l'eluat par chromatographie d'immuno-affinite. L'enterotoxine purifiee fut trouvee homogene a l'electrophorese et au point de vue antigenique. Elle a aussi montre une activite de permeabilite vasculaire, une capacite de provoquer la mort de souris et de causer l'accumulation de liquide dans des anses intestinales ligaturees de souris, tandis qu'au contraire, elle n'a pas montre d'activite d'hemolyse et de lecithinase. Ainsi, la chromatographie d'immuno-affinite s'est revklee utile pour la purification de l'enterotoxine produite par B. cereus en termes de recuperation, de purete et de facilitk relative d'execution. Mots clks : purification, chromatographie d'immuno-affinite, enterotoxine, anticorps monoclonal, Bacillus cereus. [Traduit par la redaction]
Bacillus cereus is known to cause either diarrheal-type or vomiting-type food poisoning (Gilbert 1979; Gilbert et al. 1981; Turnbull 1981). The diarrheal-type food poisoning by B. cereus is attributed to the production of a diarrheagenic enterotoxin (ET) (Gilbert 1979; Spira and Goepfert 1975; Thompson et al. 1984; Turnbull et al. 1979). Although attempts have been made to isolate ET (Beecher and Macmillan 1990, 1991;Bitaev and Ezepchuk 1987; Ezepchuk et al. 1979; Kramer 1984; Spira and Goepfert 1975; Thompson et al. 1984; Turnbull et al. 1979), it is not yet concluded whether ET is composed of a single or more than one component. Most recently, isolation of almost homogeneous ET has been demonstrated by chromatography on DEAE cellulose, Sephadex G- 100, and Sephadex G-75 (Shinagawa et al. 1991d). The ET obtained by these methods was found to be composed of a single protein entity with a molecular weight of 45 000, although it contained a trace contaminant, which was a component(s) of brain heart infusion broth (Shinagawa et al. 1991d). A single protein entity is in contrast to the previous reports (Beecher and Macmillan ' ~ u t h o rto whom all correspondence should be addressed. Printed in Canada / Imprime au Canada
1990, 1991; Bitaev and Ezepchuk 1987; Thompson et al. 1984) that a bi- or multi-component ET was isolated from culture supernatant of B. cereus. The single protein entity possessed vascular permeability activity and mouse lethality, and caused fluid accumulation in mouse and rabbit ligated intestinal loops, whereas it did not show any hemolytic and lecithinase activities (Shinagawa et al. 1991d). These findings were confirmed with the further purified ET (Shinagawa et al. 1991~).To obtain homogeneous ET in a high yield, immunoaffinity chromatography is more useful than conventional methods for purification. It has proved to be useful in the purification of the staphylococcal enterotoxins, particularly the ones produced in small amounts (Shinagawa et al. 1991a, 1991b). Therefore, to obtain high-purity ET and to elucidate whether only one component is required for the biological activities attributed to ET, immunoaffinity chromatography using murine monoclonal antibodies (MAbs) against B. cereus ET prepared in this laboratory (Shinagawa et al. 1991e) was investigated. The results obtained demonstrated that a single protein component ET in the biologically active form could be easily isolated by immunoaffinity chromatography in a much higher yield than with conventional met hods.
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CAN. J . MICROBIOL. VOL. 38, 1992
TABLE1 . Adsorption and recovery of enterotoxin from MAbs coupled to Affi-Gel 10 MAb
Adsorption (%)
TABLE3. Purification of enterotoxin by immunoaffinity column chromatography using MAb B-10 Volume Protein ET Total ET Recovery (mL) (pg/mL) (pg/mL) (pg) (yo)
Recovery (%)*
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Crude ET* Purified ET NOTE:The immunoadsorbent, MAb (400 pg), was coupled to AffiGel 10 (0.5 mL), and culture supernatant containing 200 pg ET was mixed at 4°C for 10 min. ET was eluted from the immunoadsorbent with 6 M guanidine-HC1, pH 7.2. *Recovery was estimated from ET bound to the immunoadsorbent.
TABLE2. Recovery of enterotoxin from MAb B-10 coupled to Affi-Gel 10, using different eluants Eluant* 6 3 3 3 3 3
M M M M M M
guanidine-HC1 guanidine-HC1 urea KSCN NaCl - 0.01 M Tris-HC1 NaCl - 0.2 M acetic acid
Recovery of ET (yo)+ 16-34 14-18 13-16 12-16 3-5 5-9
NOTE:The immunoadsorbent, MAb (400 pg), was coupled to AffiGel 10 (0.5 mL), and culture supernatant containing 200 pg ET was mixed at 4°C for 10 min. Different elution buffers were used for elution of the bound ET. *All buffers were at pH 7.2, except the 3 M NaCl - 0.2 M acetic acid buffer, which was at pH 2.5. + ~ e c o v e was r ~ estimated from ET bound to the immunoadsorbent.
Bacillus cereus strain FM-1 was used to produce ET for its purification for reference purposes (Shinagawa et al. 1991c, 1991d). The ET was produced by culturing in brain heart infusion medium containing 1% glucose. The purified ET was used in the preparation of specific rabbit antiserum (Shinagawa et al. 1991d). Male BALB/c mice (6 weeks old) were immunized by intraperitoneal injection of 20 pg of ET purified by conventional methods (Shinagawa et al. 1991e). Cell hybridization was performed by the methods described previously (de St. Groth and Sheidegger 1980; Galfre and Milstein 1981; Goding 1980). A detailed procedure for preparation of MAbs is described elsewhere (Shinagawa et al. 1991e). For preparation of an immunoadsorbent, 9 mg of affinitypurified MAbs was dialyzed extensively against 0.2 M NaHC03, pH 8.0, containing 0.3 M NaCl. Two millilitres of swollen Affi-Gel 10 (Bio-Rad Laboratories, Richmond, Calif., U.S.A.) was washed and suspended in the same buffer. The dialyzed MAb was mixed with 2 mL of washed Affi-Gel 10 by end over end rotation at 4OC for 4 h. The mixture was washed and suspended in 0.1 M ethanolamineHCl, pH 8.0. The suspension was mixed gently at room temperature in 0.01 M phosphate-buffered saline, pH 7.8 (PBS). For purification of ET by immunoaffinity chromatography, culture supernatant containing crude ET was dialyzed against PBS at 4OC. After dialysis, the culture supernatant was applied to the immunoadsorbent column (9 x 28 mm). After washing the column with the same buffer, the adsorbed ET was eluted with different eluants. The pH of the eluate was neutralized by addition of 0.1 M NaOH or dialyzed against PBS.
10 9.9
1750 12.4
20 5.1
200 50.3
25.2
*Culture supernatant concentrated by 70% saturated ammonium sulfate was used as crude ET.
Protein concentration was determined by the methods of Lowry et al. (195 I), using bovine serum albumin as the standard. The concentration of MAb was estimated by absorbance values at 280 nm. ET was detected by a microslide double gel diffusion system, using rabbit antiserum specific for ET. An immunodiffusion unit (IDU) was defined as the reciprocal of the highest dilution giving a precipitin line@). Purity of the ET was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli 1970). The reactivity of crude and purified ETs with antibodies was determined by Western blotting (Shinagawa et al. 1991e; Towbin et al. 1979). Biological activities of ET were assayed by the methods described previously (Shinagawa et al. 1991c, 1991d). For determination of hemolytic activity, 0.25 mL of 1% sheep or rabbit erythrocytes was incubated at 37OC for 1 h with 0.25 mL of purified ET. After incubation, the mixture was centrifuged to determine absorbance of the supernatant at 600 nm. We obtained three different MAbs (B-10, D-8, and H-1) against B. cereus ET as reported previously (Shinagawa et al. 1991e). These MAbs were found to specifically react with a single protein entity with a molecular weight of 45 000 (Shinagawa et al. 1991e; Fig. 2). The binding abilities of these three different MAbs to the ET were studied to determine which MAb was most suitable to prepare an immunoadsorbent. As presented in Table 1, two MAbs (B-10 and D-8) demonstrated high binding capacity. The adsorptive capacity of ,these two MAbs when used as immunoadsorbents was essentially equal (Table l), but elution of ET from the immunoadsorbent using MAb B-10 was better than when D-8 was used. However, very poor binding and yield were obtained with H-1 as immunoadsorbent (Table I). To effectively elute the adsorbed ET from the immunoadsorbent prepared using B-10, different eluants were used (Table 2). With 6 M guanidine-HC1 as an eluant, 16-34% of the ET bound by the immunoadsorbent was recovered, whereas a slightly smaller amount of the ET was eluted with 3 M guanidineHCl, 3 M urea, or 3 M KSCN (Table 2). On the other hand, only 3-9% of the ET applied was recovered with 0.01 M Tris-HC1 buffer, pH 7.2, containing 3 M NaCl or 0.2 M acetic acid, pH 2.5, containing 3 M NaCl. From these findings, 6 M guanidine-HC1, pH 7.2, was chosen for elution of the ET from the immunoadsorbent. To purify ET from culture supernatant by affinity chromatography, 10 mL of the culture supernatant containing 200 pg of ET was applied to the immunoadsorbent prepared using MAb B-10. Although about 30% of the ET applied was detected in the unbound fractions, 50.3 pg of ET was eluted from the immunoadsorbent column (Fig. 1; Table 3). The yield was found to be 25.2% (Table 3). Purified ET was found to be electrophoretically and antigenically homogeneous (Fig. 2). In Western blotting with crude ET a single
NOTES
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FIG. 1. Elution pattern of enterotoxin in immunoaffinity chromatography. Washing buffer, 0.05 M NaC1,0.01 M phosphate-buffered saline, p H 7.2; elution buffer, 6 M guanidine-HC1, pH 7.2; column size, 9 x 28 mm; flow rate, 10 mL/h.
band was also observed (Fig. 2B). These results suggest the ET is composed of a single protein component, as reported previously (Shinagawa et al. 1991c, 1991d). It also showed vascular permeability activity to rabbit and mouse lethal toxicity, and caused fluid accumulation in mouse ligated intestinal loops. The minimum amount of purified ET required to give these activities was 40-60 ng for vascular permeability activity, 10-1 5 pg for mouse lethality, and 50 pg for the mouse intestinal loop test, respectively. These biological activities were slightly weaker than those of ET obtained by conventional methods (Shinagawa et al. 1991c), suggesting that exposure of ET to 6 M guanidine-HC1 as an eluant may result in some loss of the biological activities. No lecithinase activity was detected with purified ET at the highest concentration tested (100 pg/mL). With sheep and rabbit erythrocytes, no hemolytic activity was induced by purified ET at up to 112 pg/mL. Similar findings were also obtained with ET purified by conventional methods. Thus, these findings strongly suggest that the single protein entity obtained in the present and previous (Shinagawa et al. 1991c) studies is associated with biological activities attributed to ET. The ET produced by B. cereus has been shown previously to be composed of at least two protein components (Bitaev and Ezepchuk 1987; Thompson et al. 1984). Although each of the components was biologically inactive, the mixture showed vascular permeability and hemolytic activities, and caused fluid accumulation in rabbit ligated intestinal loops. These previous findings differ from the present results in that the single protein component ET possessed vascular permeability activity and caused fluid accumulation in mouse ligated intestinal loops, whereas it did not show any
FIG. 2. SDS-PAGE and Western blot of crude and purified ETs. (A) ETs purified by conventional methods (Shinagawa et a[. 1991c) (lanes 1 and 3) and by immunoaffinity chromatography (lanes 2 and 4) were stained in Coomassie Brilliant Blue (lanes 1 and 2) and in silver reagent (lanes 3 and 4). (B) Western blots of crude (lanes 1 and 4) and purified (lanes 2, 3, 5, and 6) ETs with polyclonal antibody to ET (Shinagawa et a[. 1991d) (lanes 1, 2, and 3) and with monoclonal antibody t o ET (Shinagawa et al. 1991e) (lanes 4, 5, and 6). ETs purified by conventional methods (Shinagawa et al. 1991c) and by immunoaffinity chromatography were used for lanes 2 and 5 and for lanes 3 and 6, respectively. Crude E T was fivefold concentrated culture supernatant.
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CAN. J. MICROBIOL.
hemolytic activity to sheep and rabbit erythrocytes. Beecher and Macmillan (1990, 1991) have recently obtained the twoor three-component hemolysin to possess vascular permeability activity and strong hemolytic activity to sheep erythrocytes. Although they strongly suggest that the hemolysin may be ET produced by B. cereus, since the vascular permeability activity of the hemolysin is supposed to correlate with enterotoxic activity, Thompson et al. (1984) reported that use of .the vascular permeability test alone to analyze the enterotoxic activity can cause misleading results, although vascular permeability activity frequently correlated with enterotoxic activity. Thus, we cannot conclude at present whether the three-component hemolysin is the substance responsible for biological activities attributed to ET, since neither the rabbit nor mouse intestinal loop test for assaying the enterotoxic activity of the hemolysin has been carried out (Beecher and Macmillan 1991). From these findings, the single protein component ET obtained in the previous (Shinagawa et al. 1991c) and present studies may be distinct biologically and structurally from one of the bi- or multi-component ETs (or hemolysin) reported previously (Beechers and Macmillan 1990, 1991; Bitaev and Ezepchuk 1987; Thompson et al. 1984). At present, therefore, there is inadequate information on whether ET produced by all B. cereus strains is composed of one or more than one component since we do not know how many different ETs are produced by B. cereus and whether ET produced by different B. cereus is biologically and antigenically the same. To address these questions, attempts to isolate ET produced by different B. cereus strains isolated from diarrheal-type food poisoning will be made in future using the same procedure for isolation of ET in the same laboratory. From the present findings, the recovery of ET by immunoadsorbent chromatography was 25%, which is much higher than the recovery by conventional methods (Shinagawa et al. 1991~). Thus, immunoaffinity chromatography is the method of choice for purification of ET in terms of purity, percent recovery, and ease of purification. Acknowledgments The authors thank Dr. M. S. Bergdoll, University of Wisconsin, for valuable comments in preparing the manuscript. This study was supported in part by a grantin-aid from the Ministry of Education, Science, and Culture of Japan. Beecher, D. J., and Macmillan, J.D. 1990. A novel bicomponent hemolysin from Bacillus cereus. Infect. Immun. 58: 2220-2227. Beecher, D. J., and Macmillan, J .D. 1991. Characterization of the component of hemolysin BL from Bacillus cereus. Infect. Immun. 59: 1778-1784. Bitaev, A.R., and Ezepchuk, Y .V. 1987. The molecular nature of the pathogenic effect induced by B. cereus. Mol. Genet. Mikrobiol. Virusol. 7: 8-23. de St. Groth, S.F., and Scheidegger, D. 1980. Production of monoclonal antibodies: strategy and tactics. J. Immunol. Methods, 35: 1-21.
VOL. 38,
1992
Ezepchuk, Y.V., Bondarenko, V.M., Yakovlera, E.A., and Koryagina, I.P. 1979. The Bacillus cereus toxins: isolation of permeability factor. Zentralbl. Bakteriol. Parasitenkd. Infecktionskr. Hyg. Abt. 1 Orig. Reihe A , 244: 275-284. Galfre, G., and Milstein, C. 1981. Preparation of monoclonal antibodies: strategies and procedures. Methods. Enzymol. 73: 3-46. Gilbert, R. J . 1979. Bacillus cereus gastroenteritis. In Food-borne infections and intoxications. 2nd ed. Edited b y H . Riemann and F.L. Bryan. Academic Press Inc., New York. pp. 495-518. Gilbert, R. J., Turnbull, P.C.B., Parry, J.M., and Kramer, J.M. 1981. Bacillus cereus and other Bacillus species: their part in food poisoning and other clinical infections. In The aerobic endosporeforming bacteria. Edited b y R.C.W. Berkley and M. Goodfellow. Academic Press Inc., London. pp. 297-3 14. Goding, J. W. 1980. Antibody production by hybridomas. J. Immunol. Methods, 39: 285-308. Kramer, J .M. 1984. Bacillus cereus enterotoxins: production, isolation, detection, and properties. In Bacterial protein toxins. Edited b y J.E. Alouf, J.H. Freer, F.J. Fehrenbach, and J. Jeljaszewicz. Academic Press, London. pp. 385-386. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227: 680-685. Lowry, O.H., Rosebrough, N. J., Farr, A.L., and Randall, R. J . 195 1. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. Shinagawa, K., Mitsumori, M., Matsusaka, N., and Sugii, S. 1991a. Purification of staphylococcal enterotoxins A and E by immunoaffinity chromatography using a murine monoclonal antibody with dual specificity for both of these toxins. J. Immunol. Methods, 139: 49-53. Shinagawa, K., Omoe, K., Matsusaka, N., and Sugii, S. 1991b. Immunological studies on staphylococcal enterotoxin D: production of murine monoclonal antibodies and immunopurification. Can. J. Microbiol. 37: 586-589. Shinagawa, K., Sugiyama, J., Terada, T., et al. 1991c. Improved methods for purification of an enterotoxin produced by Bacillus cereus. FEMS Microbiol. Lett. 80: 1-6. Shinagawa, K., Ueno, S., Konuma, H., et al. 1991d. Purification and characterization of the vascular permeability factor produced by Bacillus cereus. J. Vet. Med. Sci. (formerly named Jpn. J . vet. Sci.), 53: 281-286. Shinagawa, K., Yokoi, R., Matsusaka, N., and Sugii, S. 1991e. Development of murine monoclonal antibodies against an enterotoxin produced by Bacillus cereus. J. Vet. Med. Sci. (formerly named Jpn. J . Vet. Sci.), 53: 419-422. Spira, W.M., and Goepfert, J.M. 1975. Biological characteristics of an enterotoxin produced by Bacillus cereus. Can. J. Microbiol. 21: 1236-1246. Thompson, N.E., Ketterhagen, M.J., Bergdoll, M.S., and Schantz, E. J . 1984. Isolation and some properties of an enterotoxin produced by Bacillus cereus. Infect. Immun. 43: 887-894. Towbin, H., Staehlin, T., and Gordon, J . 1979. Electrophoretic transfer of proteins from polyacrylamide gels t o nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76: 4350-4354. Turnbull, P.C.B. 198 1. Bacillus cereus toxins. Pharmacol. Ther. 13: 453-505. Turnbull, P.C.B., Kramer, J.M., Jgrgensen, K., et al. 1979. Properties and production characteristics of vomiting, diarrheal, and necrotizing toxins of Bacillus cereus. Am. J. Clin. Nutr. 32: 2 19-228.
Purification of an enterotoxin produced by BaciNus cereus by immunoaffinity chromatography using a monoclonal antibody KUNIHIROSHINAGAWA,TEIMITAKECHI,AND NAONORIMATSUSAKA Department of VeterinaryMedicine, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020, Japan
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SHUNJISUGII Department of Serology and Immunologv, School of Medical Technology, Kitasato University, Kitasato, Sagamihara, Kanagawa 228, Japan Received May 6, 1991 Revision received September 19, 1991 Accepted October 15, 1991 SHINAGAWA, K., TAKECHI,T., MATSUSAKA,N., and SUGII,S. 1992. Purification of an enterotoxin produced by Bacillus cereus by imrnunoaffinity chromatography using a monoclonal antibody. Can. J. Microbiol. 38: 153-156. A murine monoclonal antibody (MAb) with high reactivity to an enterotoxin produced by Bacillus cereus was used to prepare an immunoadsorbent for purification of the enterotoxin. By immunoaffinity chromatography using the immunoadsorbent, approximately 25% of crude enterotoxin applied was recovered in the eluate. The purified enterotoxin was found to be electrophoretically and antigenically homogeneous. It also showed vascular permeability activity and mouse lethality, and caused fluid accumulation in mouse ligated intestinal loops, whereas it did not show any hemolytic and lecithinase activities. Thus, immunoaffinity chromatography proved useful in the purification of enterotoxin produced by B. cereus in terms of recovery, purity, and relative ease of performing the purification. Key words: purification, immunoaffinity chromatography, enterotoxin, monoclonal antibody, Bacillus cereus. SHINAGAWA, K., TAKECHI,T., MATSUSAKA, N., et SUGII,S. 1992. Purification of an enterotoxin produced by Bacillus cereus by immunoaffinity chromatography using a monoclonal antibody. Can. J. Microbiol. 38 : 153-156. Un anticorps monoclonal murin (MAb), qui possede une reactivite elevee envers une enterotoxine produite par Bacillus cereus, a ete utilisee afin de preparer un immuno-adsorbant pour la purification de l'entkrotoxine. A l'aide de cet immunoadsorbant, environ 25% de la quantite initiale de l'enterotoxine a ete recuperee dans l'eluat par chromatographie d'immuno-affinite. L'enterotoxine purifiee fut trouvee homogene a l'electrophorese et au point de vue antigenique. Elle a aussi montre une activite de permeabilite vasculaire, une capacite de provoquer la mort de souris et de causer l'accumulation de liquide dans des anses intestinales ligaturees de souris, tandis qu'au contraire, elle n'a pas montre d'activite d'hemolyse et de lecithinase. Ainsi, la chromatographie d'immuno-affinite s'est revklee utile pour la purification de l'enterotoxine produite par B. cereus en termes de recuperation, de purete et de facilitk relative d'execution. Mots clks : purification, chromatographie d'immuno-affinite, enterotoxine, anticorps monoclonal, Bacillus cereus. [Traduit par la redaction]
Bacillus cereus is known to cause either diarrheal-type or vomiting-type food poisoning (Gilbert 1979; Gilbert et al. 1981; Turnbull 1981). The diarrheal-type food poisoning by B. cereus is attributed to the production of a diarrheagenic enterotoxin (ET) (Gilbert 1979; Spira and Goepfert 1975; Thompson et al. 1984; Turnbull et al. 1979). Although attempts have been made to isolate ET (Beecher and Macmillan 1990, 1991;Bitaev and Ezepchuk 1987; Ezepchuk et al. 1979; Kramer 1984; Spira and Goepfert 1975; Thompson et al. 1984; Turnbull et al. 1979), it is not yet concluded whether ET is composed of a single or more than one component. Most recently, isolation of almost homogeneous ET has been demonstrated by chromatography on DEAE cellulose, Sephadex G- 100, and Sephadex G-75 (Shinagawa et al. 1991d). The ET obtained by these methods was found to be composed of a single protein entity with a molecular weight of 45 000, although it contained a trace contaminant, which was a component(s) of brain heart infusion broth (Shinagawa et al. 1991d). A single protein entity is in contrast to the previous reports (Beecher and Macmillan ' ~ u t h o rto whom all correspondence should be addressed. Printed in Canada / Imprime au Canada
1990, 1991; Bitaev and Ezepchuk 1987; Thompson et al. 1984) that a bi- or multi-component ET was isolated from culture supernatant of B. cereus. The single protein entity possessed vascular permeability activity and mouse lethality, and caused fluid accumulation in mouse and rabbit ligated intestinal loops, whereas it did not show any hemolytic and lecithinase activities (Shinagawa et al. 1991d). These findings were confirmed with the further purified ET (Shinagawa et al. 1991~).To obtain homogeneous ET in a high yield, immunoaffinity chromatography is more useful than conventional methods for purification. It has proved to be useful in the purification of the staphylococcal enterotoxins, particularly the ones produced in small amounts (Shinagawa et al. 1991a, 1991b). Therefore, to obtain high-purity ET and to elucidate whether only one component is required for the biological activities attributed to ET, immunoaffinity chromatography using murine monoclonal antibodies (MAbs) against B. cereus ET prepared in this laboratory (Shinagawa et al. 1991e) was investigated. The results obtained demonstrated that a single protein component ET in the biologically active form could be easily isolated by immunoaffinity chromatography in a much higher yield than with conventional met hods.
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CAN. J . MICROBIOL. VOL. 38, 1992
TABLE1 . Adsorption and recovery of enterotoxin from MAbs coupled to Affi-Gel 10 MAb
Adsorption (%)
TABLE3. Purification of enterotoxin by immunoaffinity column chromatography using MAb B-10 Volume Protein ET Total ET Recovery (mL) (pg/mL) (pg/mL) (pg) (yo)
Recovery (%)*
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Crude ET* Purified ET NOTE:The immunoadsorbent, MAb (400 pg), was coupled to AffiGel 10 (0.5 mL), and culture supernatant containing 200 pg ET was mixed at 4°C for 10 min. ET was eluted from the immunoadsorbent with 6 M guanidine-HC1, pH 7.2. *Recovery was estimated from ET bound to the immunoadsorbent.
TABLE2. Recovery of enterotoxin from MAb B-10 coupled to Affi-Gel 10, using different eluants Eluant* 6 3 3 3 3 3
M M M M M M
guanidine-HC1 guanidine-HC1 urea KSCN NaCl - 0.01 M Tris-HC1 NaCl - 0.2 M acetic acid
Recovery of ET (yo)+ 16-34 14-18 13-16 12-16 3-5 5-9
NOTE:The immunoadsorbent, MAb (400 pg), was coupled to AffiGel 10 (0.5 mL), and culture supernatant containing 200 pg ET was mixed at 4°C for 10 min. Different elution buffers were used for elution of the bound ET. *All buffers were at pH 7.2, except the 3 M NaCl - 0.2 M acetic acid buffer, which was at pH 2.5. + ~ e c o v e was r ~ estimated from ET bound to the immunoadsorbent.
Bacillus cereus strain FM-1 was used to produce ET for its purification for reference purposes (Shinagawa et al. 1991c, 1991d). The ET was produced by culturing in brain heart infusion medium containing 1% glucose. The purified ET was used in the preparation of specific rabbit antiserum (Shinagawa et al. 1991d). Male BALB/c mice (6 weeks old) were immunized by intraperitoneal injection of 20 pg of ET purified by conventional methods (Shinagawa et al. 1991e). Cell hybridization was performed by the methods described previously (de St. Groth and Sheidegger 1980; Galfre and Milstein 1981; Goding 1980). A detailed procedure for preparation of MAbs is described elsewhere (Shinagawa et al. 1991e). For preparation of an immunoadsorbent, 9 mg of affinitypurified MAbs was dialyzed extensively against 0.2 M NaHC03, pH 8.0, containing 0.3 M NaCl. Two millilitres of swollen Affi-Gel 10 (Bio-Rad Laboratories, Richmond, Calif., U.S.A.) was washed and suspended in the same buffer. The dialyzed MAb was mixed with 2 mL of washed Affi-Gel 10 by end over end rotation at 4OC for 4 h. The mixture was washed and suspended in 0.1 M ethanolamineHCl, pH 8.0. The suspension was mixed gently at room temperature in 0.01 M phosphate-buffered saline, pH 7.8 (PBS). For purification of ET by immunoaffinity chromatography, culture supernatant containing crude ET was dialyzed against PBS at 4OC. After dialysis, the culture supernatant was applied to the immunoadsorbent column (9 x 28 mm). After washing the column with the same buffer, the adsorbed ET was eluted with different eluants. The pH of the eluate was neutralized by addition of 0.1 M NaOH or dialyzed against PBS.
10 9.9
1750 12.4
20 5.1
200 50.3
25.2
*Culture supernatant concentrated by 70% saturated ammonium sulfate was used as crude ET.
Protein concentration was determined by the methods of Lowry et al. (195 I), using bovine serum albumin as the standard. The concentration of MAb was estimated by absorbance values at 280 nm. ET was detected by a microslide double gel diffusion system, using rabbit antiserum specific for ET. An immunodiffusion unit (IDU) was defined as the reciprocal of the highest dilution giving a precipitin line@). Purity of the ET was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli 1970). The reactivity of crude and purified ETs with antibodies was determined by Western blotting (Shinagawa et al. 1991e; Towbin et al. 1979). Biological activities of ET were assayed by the methods described previously (Shinagawa et al. 1991c, 1991d). For determination of hemolytic activity, 0.25 mL of 1% sheep or rabbit erythrocytes was incubated at 37OC for 1 h with 0.25 mL of purified ET. After incubation, the mixture was centrifuged to determine absorbance of the supernatant at 600 nm. We obtained three different MAbs (B-10, D-8, and H-1) against B. cereus ET as reported previously (Shinagawa et al. 1991e). These MAbs were found to specifically react with a single protein entity with a molecular weight of 45 000 (Shinagawa et al. 1991e; Fig. 2). The binding abilities of these three different MAbs to the ET were studied to determine which MAb was most suitable to prepare an immunoadsorbent. As presented in Table 1, two MAbs (B-10 and D-8) demonstrated high binding capacity. The adsorptive capacity of ,these two MAbs when used as immunoadsorbents was essentially equal (Table l), but elution of ET from the immunoadsorbent using MAb B-10 was better than when D-8 was used. However, very poor binding and yield were obtained with H-1 as immunoadsorbent (Table I). To effectively elute the adsorbed ET from the immunoadsorbent prepared using B-10, different eluants were used (Table 2). With 6 M guanidine-HC1 as an eluant, 16-34% of the ET bound by the immunoadsorbent was recovered, whereas a slightly smaller amount of the ET was eluted with 3 M guanidineHCl, 3 M urea, or 3 M KSCN (Table 2). On the other hand, only 3-9% of the ET applied was recovered with 0.01 M Tris-HC1 buffer, pH 7.2, containing 3 M NaCl or 0.2 M acetic acid, pH 2.5, containing 3 M NaCl. From these findings, 6 M guanidine-HC1, pH 7.2, was chosen for elution of the ET from the immunoadsorbent. To purify ET from culture supernatant by affinity chromatography, 10 mL of the culture supernatant containing 200 pg of ET was applied to the immunoadsorbent prepared using MAb B-10. Although about 30% of the ET applied was detected in the unbound fractions, 50.3 pg of ET was eluted from the immunoadsorbent column (Fig. 1; Table 3). The yield was found to be 25.2% (Table 3). Purified ET was found to be electrophoretically and antigenically homogeneous (Fig. 2). In Western blotting with crude ET a single
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FIG. 1. Elution pattern of enterotoxin in immunoaffinity chromatography. Washing buffer, 0.05 M NaC1,0.01 M phosphate-buffered saline, p H 7.2; elution buffer, 6 M guanidine-HC1, pH 7.2; column size, 9 x 28 mm; flow rate, 10 mL/h.
band was also observed (Fig. 2B). These results suggest the ET is composed of a single protein component, as reported previously (Shinagawa et al. 1991c, 1991d). It also showed vascular permeability activity to rabbit and mouse lethal toxicity, and caused fluid accumulation in mouse ligated intestinal loops. The minimum amount of purified ET required to give these activities was 40-60 ng for vascular permeability activity, 10-1 5 pg for mouse lethality, and 50 pg for the mouse intestinal loop test, respectively. These biological activities were slightly weaker than those of ET obtained by conventional methods (Shinagawa et al. 1991c), suggesting that exposure of ET to 6 M guanidine-HC1 as an eluant may result in some loss of the biological activities. No lecithinase activity was detected with purified ET at the highest concentration tested (100 pg/mL). With sheep and rabbit erythrocytes, no hemolytic activity was induced by purified ET at up to 112 pg/mL. Similar findings were also obtained with ET purified by conventional methods. Thus, these findings strongly suggest that the single protein entity obtained in the present and previous (Shinagawa et al. 1991c) studies is associated with biological activities attributed to ET. The ET produced by B. cereus has been shown previously to be composed of at least two protein components (Bitaev and Ezepchuk 1987; Thompson et al. 1984). Although each of the components was biologically inactive, the mixture showed vascular permeability and hemolytic activities, and caused fluid accumulation in rabbit ligated intestinal loops. These previous findings differ from the present results in that the single protein component ET possessed vascular permeability activity and caused fluid accumulation in mouse ligated intestinal loops, whereas it did not show any
FIG. 2. SDS-PAGE and Western blot of crude and purified ETs. (A) ETs purified by conventional methods (Shinagawa et a[. 1991c) (lanes 1 and 3) and by immunoaffinity chromatography (lanes 2 and 4) were stained in Coomassie Brilliant Blue (lanes 1 and 2) and in silver reagent (lanes 3 and 4). (B) Western blots of crude (lanes 1 and 4) and purified (lanes 2, 3, 5, and 6) ETs with polyclonal antibody to ET (Shinagawa et a[. 1991d) (lanes 1, 2, and 3) and with monoclonal antibody t o ET (Shinagawa et al. 1991e) (lanes 4, 5, and 6). ETs purified by conventional methods (Shinagawa et al. 1991c) and by immunoaffinity chromatography were used for lanes 2 and 5 and for lanes 3 and 6, respectively. Crude E T was fivefold concentrated culture supernatant.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by SAVANNAHRIVNATLABBF on 11/21/14 For personal use only.
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CAN. J. MICROBIOL.
hemolytic activity to sheep and rabbit erythrocytes. Beecher and Macmillan (1990, 1991) have recently obtained the twoor three-component hemolysin to possess vascular permeability activity and strong hemolytic activity to sheep erythrocytes. Although they strongly suggest that the hemolysin may be ET produced by B. cereus, since the vascular permeability activity of the hemolysin is supposed to correlate with enterotoxic activity, Thompson et al. (1984) reported that use of .the vascular permeability test alone to analyze the enterotoxic activity can cause misleading results, although vascular permeability activity frequently correlated with enterotoxic activity. Thus, we cannot conclude at present whether the three-component hemolysin is the substance responsible for biological activities attributed to ET, since neither the rabbit nor mouse intestinal loop test for assaying the enterotoxic activity of the hemolysin has been carried out (Beecher and Macmillan 1991). From these findings, the single protein component ET obtained in the previous (Shinagawa et al. 1991c) and present studies may be distinct biologically and structurally from one of the bi- or multi-component ETs (or hemolysin) reported previously (Beechers and Macmillan 1990, 1991; Bitaev and Ezepchuk 1987; Thompson et al. 1984). At present, therefore, there is inadequate information on whether ET produced by all B. cereus strains is composed of one or more than one component since we do not know how many different ETs are produced by B. cereus and whether ET produced by different B. cereus is biologically and antigenically the same. To address these questions, attempts to isolate ET produced by different B. cereus strains isolated from diarrheal-type food poisoning will be made in future using the same procedure for isolation of ET in the same laboratory. From the present findings, the recovery of ET by immunoadsorbent chromatography was 25%, which is much higher than the recovery by conventional methods (Shinagawa et al. 1991~). Thus, immunoaffinity chromatography is the method of choice for purification of ET in terms of purity, percent recovery, and ease of purification. Acknowledgments The authors thank Dr. M. S. Bergdoll, University of Wisconsin, for valuable comments in preparing the manuscript. This study was supported in part by a grantin-aid from the Ministry of Education, Science, and Culture of Japan. Beecher, D. J., and Macmillan, J.D. 1990. A novel bicomponent hemolysin from Bacillus cereus. Infect. Immun. 58: 2220-2227. Beecher, D. J., and Macmillan, J .D. 1991. Characterization of the component of hemolysin BL from Bacillus cereus. Infect. Immun. 59: 1778-1784. Bitaev, A.R., and Ezepchuk, Y .V. 1987. The molecular nature of the pathogenic effect induced by B. cereus. Mol. Genet. Mikrobiol. Virusol. 7: 8-23. de St. Groth, S.F., and Scheidegger, D. 1980. Production of monoclonal antibodies: strategy and tactics. J. Immunol. Methods, 35: 1-21.
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