Immunological studies on staphylococcal enterotoxin D: production of murine monoclonal antibodies and immunopurification KUNIHIRO SHINAGAWA,' KATSUHIKO OMOE,AND NAONORI MATSUSAKA Department of Veterinary Medicine, Faculty of Agriculture, lwate University, Ueda, Morioka, lwate 020, Japan AND
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SHUNJISUGII Department of Serology and Immunology, School of Medical Technology, Kitasato University, Kitasato, Sagamihara, Kanaga wa 228, Japan Received August 30, 1990 Accepted January 17, 199 1
SHINAGAWA, K., OMOE,K., MATSUSAKA, N., and SUGII,S. 1991. Immunological studies on staphylococcal enterotoxin D: production of murine monoclonal antibodies and immunopurification. Can. J. Microbiol. 37: 586-589. Eight murine monoclonal antibodies (MAbs) against staphylococcal enterotoxin D (SED) were obtained by fusion of myeloma cells with mouse spleen cells immunized with SED only or a combination of SED and either enterotoxin A (SEA) or enterotoxin E (SEE). When only SED was used as an immunogen, six MAbs were specific for SED only, whereas one MAb was reactive with both SED and SEE when both SEs were used as immunogens. One MAb reacted with SEA, SED, and SEE when both SEA and SED were used as immunogens. A MAb with the highest reactivity to SED was used to prepare an immunosorbent for purification of SED by immunoaffinity chromatography. Approximately 70% of the partially purified SED was recovered in the eluate. The purified SED was electrophoretically and antigenically pure. Immunoaffinity chromatography proved useful in the purification of SED in terms of ease of purification, percent enterotoxin, and enterotoxin purity. Key words: enterotoxin D, monoclonal antibodies, Staphylococcus aureus.
SHINAGAWA, K., OMOE,K., MATSUSAKA, N., et SUGII,S. 1991. Immunological studies on staphylococcal enterotoxin D: production of murine monoclonal antibodies and immunopurification. Can. J. Microbiol. 37 : 586-589. Huit anticorps monoclonaux murins (MAbs) diriges contre l'enterotoxine D du staphylocoque (SED) ont ete obtenus par fusion de cellules myelomateuses avec des cellules de rate de souris immunisees avec SED seule ou avec un melange SED - enterotoxine A (SEA) ou SED - enterotoxine E (SEE). Suite a une immunisation faite avec SED seule, six MAbs etaient specifiques contre SED seule et un MAb reagissait contre SED et SEE lorsque ces deux enterotoxines etaient utilisees comme immunogknes. Un MAb reagissait avec SED, SED et SEE et il provenait de la fusion de cellules immunisees avec SEA et SED. Le MAb presentant la plus forte reactivite contre SED a servi d'immunosorbant pour la purification de la SED,parchromatographie d'immunoaffinite. Environ 70% de la SED partiellement purifiee se retrouvait dans l'eluat. La SED purifiee etait tout a fait pure a l'electrophorkse et comme antigkne. Ce type de chromatographie s'est revele rentable pour la purification de la SED en tenant compte de la facilite de la purification, le pourcentage d'enterotoxine et la purete de l'enterotoxine. Mots clks: enterotoxine D, anticorps monoclonaux, Staphylococcus aureus. [Traduit par la redaction]
Introduction Staphylococcal enterotoxins (SEs) are known as exotoxins with molecular masses of 27-30 kDa that cause emesis and diarrhea in humans and primates (Bergdoll 1977, 1979). SEs are serologically classified into A (SEA), B (SEB), C (SEC,, SEC,, and SEC,), D (SED), and E (SEE) (Bergdoll 1977, 1979). SEs have been purified by various methods such as combinations of ion-exchange chromatography and gel filtration (Chu et al. 1966; Borja and Bergdoll 1967; Chang et al. 197 1; Borja et al. 1972; Schantz et al. 1972; Shinagawa et al. 1975, 1977; Chang and Bergdoll 1979; Meyer et al. 1984). These methods are time consuming and give low recoveries of SEs. SED has not been easily isolated by these methods because it is produced in much smaller amounts than the other SEs (Bergdoll 1977, 1979). On the other hand, one-step purification of cell surface antigen and bacterial toxin has been carried out by immunoaffinity chromatography with use of monoclonal antibodies (MAbs) (Schneider et al. 1982; Potomski et al. 1987). This study was undertaken to
' ~ u t h o to r whom all correspondence should be addressed. Printed in Canada 1 Imprime au Canada
prepare MAbs to SED and to utilize them in the purification of SED by immunoaffinity chromatography.
Materials and methods Purification of SEs and preparation of rabbit anti-SED Staphylococcus aureus strains FR 1-722 (SEA), 1 151-7NG (SED), and FRl-326 (SEE) were cultured in 4% NZ-arnine type NAK (Sheffield Chemical, Norwich, NY) medium for production of the SEs (Kato et al. 1966) for their purification (Shinagawa et al. 1974, 1975). Purified SED was used for the preparation of specific antisera in rabbits (Shinagawa et al. 1974). Preparation of MAbs to SED Male BALBIc mice (6-8 weeks old) were immunized by intraperitoneal injection of purified SED (30 kg) with Freund's complete adjuvant (Difco Laboratories, Detroit, MI). Two weeks after the initial injection, 40 kg was injected intraperitoneally without adjuvant. After 8-9 weeks, 60 kg of SED, 50 kg of SEA, or 50 k g of SEE was given intravenously. Spleen cells were removed 3 days later. Cell hybridization was performed by the methods described previously (Galfre et al. 1977; Galfre and Milstein 1981). Hybridomas producing anti-SED were intraperitoneally injected into BALBIc mice to obtain ascitic fluids (Galfre et al. 1977; Galfre and Milstein 1981). MAbs in ascitic fluids
587
SHINAGAWA ET AL.: II
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were purified with Affi-Gel protein A (Bio-Rad Laboratories Richmond, CA). Enzyme-linked immunosorbent assay Specificities of the MAbs to the SEs were determined in triplicate in ELIS-A using polystyrene microtiter plates (Dynatech ~aboratdriesInc., VA). Each well of the plates was coated with 100 pL of purified SEs (10 pg/mL in 0.1 M carbonate buffer, pH 9.5) and incubated at 37°C for 3 h. After coating, the wells were blocked with 1% bovine serum albumin in 0.02 M phosphate-buffered saline (PBS), pH 7.2, at 4°C overnight. After washing the plates 5 times with PBS containing 0.05% Tween-20 (PBST), 100 pL of affinity-purified MAb was added to each well and incubated at 3 ? ~for 2 h. After washing, 100 pL of horseradish peroxidase conjugated goat anti-mouse I ~ (diluted G 1 :1000) was added to each well and incubated at 37°C for 1 h. After washing, 200 pL of the substrate solution containing 0.02% H202and 3 mg/mL o-phenylenediamine(NakaraiChemicals Ltd., Kyoto, Japan) was added to each well; the plates were held in the dark at room temperature for 30 min for color development. Two hundred microlitres of the reaction mixture from each well was,transferred to test tubes, to which 3 mL of H2S04 was added to stop the reaction. The reaction was determined spectrophotometrically with Shimadzu doublebeam spectrophotometer UV- 150 (Shimadzu Co. Ltd., Japan) at 492 nrn
11111.
For determination of binding abilities of MAbs to SED, 100 pL of the purified MAbs at 100 ng/mL was incubated at 37°C for 30 min with 100 pL of SED at 0-10 pg/mL. After incubation, 100 pL of the mixture was added to each well coated with 100 pL of SED at 10 pg/mL to determine the remaining antibody activity in the mixture. Preparation of immunosorbent and immunoaflnity chromatography Two milligrams of affinity-purified MAbs to SED was extensively dialyzed against 0.2 M NaHCO,, pH 8.0, containing 0.3 M NaCl. A total of 0.4 mL of swollen Affi-Gel 10 (Bio-Rad Laboratories, Richmond, CA) was washed in the same buffer. Two milligrams of dialyzed MAbs was mixed at 4°C for 4 h with 0.4 mL of washed Affi-Gel 10 by end-over-end rotation. Then the gel was washed and suspended in 0.1 M ethanolamine-HC1, pH 8.0. After 1 h at room temperature, the suspension was extensively washed in PBS and used for preliminary experiments for immunoaffinity chromatography. To prepare the immunosorbent for large scale isolation of SED, 4.2 mg of MAb was coupled to 2 mL of swollen Affi-Gel 10 as described above. SED was partially purified from culture supernatants by chromatography on SPSephadex C-25 as described previously (Shinagawa et al. 1975, 1977). Partially purified SED was used as the crude material containing SED (crude SED) for immunoaffinity chromatography. After extensive dialysis against PBS, crude SED was applied to the immunosorbent column. The adsorbed material was eluted with 0.2 M acetic acid containing 0.15 M NaCl (pH 2.5). The eluate was immediately neutralized by addition of 1 M Na,CO, (pH 12.2). Other methods Protein contents were determined by the methods of Lowry et al. ( 1951 ) using bovine serum albumin as standard. The concentration of murine MAb was determined by absorbance at 280 nm, calibrating OD,,,,, = 14. SED was quantitatively detected by a double-gel immunodiffusion system (microslide) using rabbit anti-SED serum following the methods described previously (Shinagawa et al. 1974). Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were carried out following the methods described previously (Laemmli 1970; Towbin et al. 1979).
Results Reactivities of murine MAbs against SED Six murine MAbs against SED were obtained by fusion of myeloma cells with mouse spleen cells immunized with SED, whereas two MAbs (DE-2 1 1 and AD-4 1) were obtained by fusion of the same myelomacells with mouse spleen cells immu-
TABLE1. Subclasses and reactivities of murine MAbs against SED MAb
Immunogen
Reactivity of MAb
SED
KD-I (IgGI) KD-2 (IgGI) KD-3 (IgGI) KD-4 (IgG 1 ) KD-5 (IgG,) KD-6 (IgG,)
SED SED SED SED SED SED
SED and SEE
DE-2 1 1 (IgG - I)
SED, SEEt
SED and SEA
AD-4 1 (nd*)
SEA, SED,t SEEt
*Not determined.
tThese SEs were weakly
reactive with the M ~ b s .
nized with either SEE or SEA as a final immunogen. s i x of them (KD-1 to KD-6) were specific for only SED in ELISA, whereas DE-211 reacted with both SED and SEE. AD-41 was reactive with SEA, SED, and SEE (Table 1). These MAbs were not reactive with either SEB or SEC at the highest concentrations used. Similar results were also obtained by Western blotting (data not shown). Table 1 also presents the subclasses of MAbs to SED. T o study reactivities of six MAbs specific for only SED, 100 ng/mL of each purified MAb was incubated at 37°C for 3 0 min with SED at up to 10 bg/mL. After incubation, the remaining antibody activity in the mixture was determined in ELISA. No remaining antibody reactivity was found after any of the MAbs at 100 ng/mL were incubated with SED at 10 bg/mL. KD- 1, KD-3, KD-4, and KD-5 were more reactive with SED than KD-2 and KD-6 (Fig. 1). Of six MAbs to SED tested, KD- 1 was most effectively inactivated by SED (Fig. 1). The amount of SED to be required for 50% inhibition was 10 ng/mL for KD- 1 and 220 ng/mL for KD-6 (Fig. I), respectively.
Immunoaflnity purification of SED T o study which MAbs to SED were most suitable for preparation of the immunosorbent, 2 mg of different purified MAbs (such as KD- 1, KD-3, KD-5, and KD-6) was coupled to 0.4 mL of swollen Affi-Gel 10. Approximately 700 b g of purified MAbs was found to couple to the gel. Crude material containing 100 b g SED was subjected to each of the immunosorbents prepared as above. Of the crude SED, 76.8,34.5, 12.5, and 5.5% were found to bind to the immunosorbents coupled to KD- 1, KD-3, KD-5, and KD-6, respectively (Table 2). By use of 0.2 M acetic acid containing 0.15 M NaCl as an eluant, about 70% of bound SED was eluted from the immunosorbents with KD- 1, KD-3, and KD5. In contrast much less bound SED was eluted from the immunosorbent with KD-6 (Table 2). These findings suggest that KD- 1 is most suitable to prepare the immunosorbent. Thus, KD1 was used to prepare the immunosorbent for large-scale isolation of SED. Twelve millilitres of crude material containing 480 b g of SED was subjected to the column. Although a small amount of SED was detected in the unbound fractions, approximately 365 b g of SED (76%) was eluted using 0.2 M acetic acid as an eluant (Table 3). Purified SED was subjected to SDS-PAGE. As shown in Fig. 2, affinity-purified SED showed a single protein band in SDS-PAGE, whereas crude SED showed multiple protein bands. In immunogel double diffusion, affinity-purified SED formed a
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CAN. J. MICROBIOL. VOL. 37, 1991
SED (ng/mL) FIG. 1. Inhibitory activities of affinity-purified MAbs to SED. MAbs used were KD- 1 (@), KD-2 (U),KD-3 (A), KD-4 (O), KD-5 (El), and KD-6
(4. TABLE2. Adsorption and recovery of SED on immunoadsorbents coupled to different MAbs
MAb coupled to immunosorbent
Adsorption (%) of applied SED to immunosorbent
Recovery (%) of bound SED in eluate
KD- 1 KD-3 KD-5 KD-6
TABLE3. Recovery of SED by irnrnunoaffinity chromatography using MAb KD-1
Crude SED* Purified SED
Volume (mL)
Total protein (Fg)
Total SED (Fg)
Recovery
12 4
2,864 368
480 368
100 76.7
(%I
*Crude SED was partially purified SED prepared by ion-exchange chromatography on SP-Sephadex C-25 as described in Materials and methods.
single precipitin line against rabbit anti-SED serum (Fig. 3). The precipitin line of affinity-purified SED was identical with that of reference pure SED prepared by the conventional methods (Fig. 3).
Discussion Although MAbs have been prepared against the SEs (Edwin et al. 1984, 1986a, 1986b; Meyer et al. 1984; Thompson et al. 1984, 1986; Lapeyre et al. 1987), few MAbs against SED have been reported. In the present study, eight MAbs to SED were isolated. When only SED was used as the immunogen, the MAbs obtained were specific for SED; it was not determined whether the combining sites of these MAbs were the same. MAbs KD- 1,
FIG.2. SDS-PAGE of SED. Lane P, purified SED (1.6 ~ g )lane ; C, crude SED (1.2 ~ g ) .
KD-3, KD-4, and KD-5 were more reactive than MAbs KD-2 and KD-6 (Fig. I), with MAb KD-1 being about 20 times more reactive than MAb KD-6. The fact that MAbs reactive with SEA, SED, and SEE when SEA was used as the final immunogen and MAbs reactive with SED and SEE when SEE was used as the final immunogen were isolated indicates that these SEs contain
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SHINAGAWA ET AL.: I1
FIG.3. Immunogel double diffusion with SED. Center well contains undiluted polyclonal anti-SED. Peripheral wells: purified SED at 80 pg/mL (P), purified SED as reference at 60 pg/mL (R), and crude SED at 60 pg/mL (C).
common epitopes. The cross-reacting MAbs should prove useful in identification of the epitopes and the biological active site(s) of the SEs. Although the recovery of SED from the immunoaffinity chromatography using a MAb to SED was 76.7% of partially purified SED applied to the column, the necessity for chromatofocusing the culture supernatant fluid on SP-Sephadex C-25 to remove protein A reduced the overall recovery. This is probably a reflection on the very small amounts of SED produced by the staphylococci. Even so, this combination of purification steps resulted in a much higher recovery of purified SED than has been possible with other methods (Bergdoll 1977, 1979; Chang and Bergdoll 1979; Lei et al. 1988).
Acknowledgements The authors express gratitude to Dr. Merlin S. Bergdoll, University of Wisconsin, for his critical comments and advice to the manuscript. This study was supported in part by a Grant-inAid for Scientific Research from the Ministry of Education, Science, and Culture of Japan. BERGDOLL, M. S. 1977. Immunological aspects of staphylococcal enterotoxins. In Immunological aspects of foods. Edited by N. Catsmpoolas. AVI Publishing Co. Inc., Westport, CT. pp. 199-220. 1979. Staphylococcal intoxications. In Food-borne infections and intoxications. 2nd ed. Edited by H. Riemann and F.L. Bryan. Academic Press, New York. pp. 443494. BORJA,C. R., and BERGDOLL, M. S. 1967. Purification and partial characterization of enterotoxin C produced by Staphylococcus aureus strain 137. Biochemistry, 6: 1467-1473. I.-Y., and BERGDOLL, M. S. 1972. BORJA,C. R., FANNING, E., HUANG, Purification and some properties of staphylococcal enterotoxin E. J. Biol. Chem. 247: 2456-2463. CHANG,H. C., and BERGDOLL, M. S. 1979. Purification and some physicochemical properties of staphylococcal enterotoxin D. Biochemistry, 18: 1937- 1942. CHANG, P.-C., DICKIE, N., and THATCHER, F. S. 197 1. Hydroxylapatite column chromatography of enterotoxin B. Can.J. Microbiol. 17: 296-297. CHU,S. F., THADHANI, K., SCHANTZ, E. J., and BERGDOLL, M. S. 1966. Purification and characterization of staphylococcal enterotoxin A. Biochemistry, 5: 328 1-3289. EDWIN,C., TATINI,S. R., STROBEL,R. S., and MAHESWARAN, S. K. 1984. Production of monoclonal antibodies to staphylococcal enterotoxin A. Appl. Environ. Microbiol. 48: 1 171-1 175.
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EDWIN, C., TATINI, S. R., and MAHESWARAN, S. K. 1 9 8 6 ~Nature . and reactivity of staphylococcal enterotoxin A monoclonal antibodies. Appl. Environ. Microbiol. 52: 1247-1 252. 19866, Specificity and cross-reactivity of staphylococcal enterotoxin A monoclonal antibodies with enterotoxins B, C,, D, and E. Appl. Environ. Microbiol. 52: 1253-1 257. GALFRE,G., and MILSTEIN,C. 1981. Preparation of monoclonal antibodies: strategies and procedures. Methods Enzymol. 73: 3 4 6 . GALFRE,G., HOWE,S. C., MILSTEIN,C., BUTCHER, G. W., and HOWARD,J. C. 1977. Antibodies to major histocompatibility antigens produced by hybrid cell lines. Nature (London), 266: 550-552. KATO, E., KHAN,M., KUJOVICH, L., and BERGDOLL, M. S. 1966. Purification of enterotoxin A. Appl. Microbiol. 14: 966-972. LAEMMLI,U. K. 1970. Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature (London), 227: 680-685. LAPEYRE, C., KAVERI, S. V., JANIN,F., and STROSBERG, A. D. 1987. Production and characterization of monoclonal antibodies to staphylococcal enterotoxin: use in immunodetection and immunopurification. Mol. Immunol. 24: 1243- 1254. M. S. 1988. Chromatofocusing LEI,Z., REISER,R. F., and BERGDOLL, in the purification of staphylococcal enterotoxin D. J. Clin. Microbiol. 26: 1236-1 237. LOWRY,0. H., ROSEBROUGH, N. J., FARR,A. L., and RANDOLL, R. J. 195 1. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. MEYER,R. F., MILLER,L., BENNETT, R. W., and MACMILLAN, J. D. 1984. Development of a monoclonal antibody capable of interacting with five serotypes of Staphylococcus aureus enterotoxins. Appl. Environ. Microbiol. 47: 283-287. POTOMSKI, J., BURKE,V., WATSON,I., and GRACY,M. 1987. Purification of cytotoxic enterotoxin of Aeromonas sobria by use of monoclonal antibodies. J. Clin. Microbiol. 23: 17 1- 177. SCHANTZ, E. J., ROESSLER, W. G., WOODBURN, M. J., LYNCH,J. M., JACOBY,H. M., SILVERMAN, S. J., GORMAN J. G., and SPERO,L. 1972. Purification and some chemical and physical properties of staphylococcal enterotoxin A. Biochemistry, 11: 360-366. SCHNEIDER, C., NEWMAN, R. A,, SUTHERLAND, R.D., ASSER,U., and GREAVES, M. F. 1982. A one-step purification of membrane protein using a high efficiency immunomatrix. J. Biol. Chem. 257: 10 76610 769. SHINAGAWA, K., ISHIBASHI, M., YAMAMOTO, H., KUNITA, N., HISA, K., and SAKAGUCHI, G. 1974. A consideration to immune doses of staphylococcal enterotoxin B to rabbits. Jpn. J. Med. Sci. Biol. 27: 309-3 14. SHINAGAWA, K., KUNITA,N., and SAKAGUCHI, G. 1975. Simplified methods for purification of staphylococcal enterotoxins A and C and preparation of anti-enterotoxin sera. Jpn. J. Bacteriol. 30: 683-692. (In Japanese with English summary.) 1977. Purification of staphylococcal enterotoxin E and antigenic similarity between enterotoxin A and E. Jpn. J. Bacteriol. 32: 829-835. (In Japanese with English summary.) THOMPSON, N. E., KETTERHAGEN, M. J., and BERGDOLL, M. S. 1984. Monoclonal antibodies to enterotoxins B and C: cross-reaction and localization of epitopes of tryptic fragments. Infect. Immun. 45: 28 1-285. THOMPSON, N. E., BERGDOLL, M. S., MEYER,R. F., BENNETT, R. W., MILLER,L., and MACMILLAN, J. D. 1986. Monoclonal antibodies to the enterotoxins and the toxic shock syndrome toxin produced by Staphylococcus aureus. In Monoclonal antibodies against bacteria. Edited by A. T. J. Macario and C. E. de Macario. Academic Press, London. pp. 23-59. TOWBIN,H., STAEHLIN, T., and GORDON,J. 1979. Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76: 43504354.
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SHUNJISUGII Department of Serology and Immunology, School of Medical Technology, Kitasato University, Kitasato, Sagamihara, Kanaga wa 228, Japan Received August 30, 1990 Accepted January 17, 199 1
SHINAGAWA, K., OMOE,K., MATSUSAKA, N., and SUGII,S. 1991. Immunological studies on staphylococcal enterotoxin D: production of murine monoclonal antibodies and immunopurification. Can. J. Microbiol. 37: 586-589. Eight murine monoclonal antibodies (MAbs) against staphylococcal enterotoxin D (SED) were obtained by fusion of myeloma cells with mouse spleen cells immunized with SED only or a combination of SED and either enterotoxin A (SEA) or enterotoxin E (SEE). When only SED was used as an immunogen, six MAbs were specific for SED only, whereas one MAb was reactive with both SED and SEE when both SEs were used as immunogens. One MAb reacted with SEA, SED, and SEE when both SEA and SED were used as immunogens. A MAb with the highest reactivity to SED was used to prepare an immunosorbent for purification of SED by immunoaffinity chromatography. Approximately 70% of the partially purified SED was recovered in the eluate. The purified SED was electrophoretically and antigenically pure. Immunoaffinity chromatography proved useful in the purification of SED in terms of ease of purification, percent enterotoxin, and enterotoxin purity. Key words: enterotoxin D, monoclonal antibodies, Staphylococcus aureus.
SHINAGAWA, K., OMOE,K., MATSUSAKA, N., et SUGII,S. 1991. Immunological studies on staphylococcal enterotoxin D: production of murine monoclonal antibodies and immunopurification. Can. J. Microbiol. 37 : 586-589. Huit anticorps monoclonaux murins (MAbs) diriges contre l'enterotoxine D du staphylocoque (SED) ont ete obtenus par fusion de cellules myelomateuses avec des cellules de rate de souris immunisees avec SED seule ou avec un melange SED - enterotoxine A (SEA) ou SED - enterotoxine E (SEE). Suite a une immunisation faite avec SED seule, six MAbs etaient specifiques contre SED seule et un MAb reagissait contre SED et SEE lorsque ces deux enterotoxines etaient utilisees comme immunogknes. Un MAb reagissait avec SED, SED et SEE et il provenait de la fusion de cellules immunisees avec SEA et SED. Le MAb presentant la plus forte reactivite contre SED a servi d'immunosorbant pour la purification de la SED,parchromatographie d'immunoaffinite. Environ 70% de la SED partiellement purifiee se retrouvait dans l'eluat. La SED purifiee etait tout a fait pure a l'electrophorkse et comme antigkne. Ce type de chromatographie s'est revele rentable pour la purification de la SED en tenant compte de la facilite de la purification, le pourcentage d'enterotoxine et la purete de l'enterotoxine. Mots clks: enterotoxine D, anticorps monoclonaux, Staphylococcus aureus. [Traduit par la redaction]
Introduction Staphylococcal enterotoxins (SEs) are known as exotoxins with molecular masses of 27-30 kDa that cause emesis and diarrhea in humans and primates (Bergdoll 1977, 1979). SEs are serologically classified into A (SEA), B (SEB), C (SEC,, SEC,, and SEC,), D (SED), and E (SEE) (Bergdoll 1977, 1979). SEs have been purified by various methods such as combinations of ion-exchange chromatography and gel filtration (Chu et al. 1966; Borja and Bergdoll 1967; Chang et al. 197 1; Borja et al. 1972; Schantz et al. 1972; Shinagawa et al. 1975, 1977; Chang and Bergdoll 1979; Meyer et al. 1984). These methods are time consuming and give low recoveries of SEs. SED has not been easily isolated by these methods because it is produced in much smaller amounts than the other SEs (Bergdoll 1977, 1979). On the other hand, one-step purification of cell surface antigen and bacterial toxin has been carried out by immunoaffinity chromatography with use of monoclonal antibodies (MAbs) (Schneider et al. 1982; Potomski et al. 1987). This study was undertaken to
' ~ u t h o to r whom all correspondence should be addressed. Printed in Canada 1 Imprime au Canada
prepare MAbs to SED and to utilize them in the purification of SED by immunoaffinity chromatography.
Materials and methods Purification of SEs and preparation of rabbit anti-SED Staphylococcus aureus strains FR 1-722 (SEA), 1 151-7NG (SED), and FRl-326 (SEE) were cultured in 4% NZ-arnine type NAK (Sheffield Chemical, Norwich, NY) medium for production of the SEs (Kato et al. 1966) for their purification (Shinagawa et al. 1974, 1975). Purified SED was used for the preparation of specific antisera in rabbits (Shinagawa et al. 1974). Preparation of MAbs to SED Male BALBIc mice (6-8 weeks old) were immunized by intraperitoneal injection of purified SED (30 kg) with Freund's complete adjuvant (Difco Laboratories, Detroit, MI). Two weeks after the initial injection, 40 kg was injected intraperitoneally without adjuvant. After 8-9 weeks, 60 kg of SED, 50 kg of SEA, or 50 k g of SEE was given intravenously. Spleen cells were removed 3 days later. Cell hybridization was performed by the methods described previously (Galfre et al. 1977; Galfre and Milstein 1981). Hybridomas producing anti-SED were intraperitoneally injected into BALBIc mice to obtain ascitic fluids (Galfre et al. 1977; Galfre and Milstein 1981). MAbs in ascitic fluids
587
SHINAGAWA ET AL.: II
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were purified with Affi-Gel protein A (Bio-Rad Laboratories Richmond, CA). Enzyme-linked immunosorbent assay Specificities of the MAbs to the SEs were determined in triplicate in ELIS-A using polystyrene microtiter plates (Dynatech ~aboratdriesInc., VA). Each well of the plates was coated with 100 pL of purified SEs (10 pg/mL in 0.1 M carbonate buffer, pH 9.5) and incubated at 37°C for 3 h. After coating, the wells were blocked with 1% bovine serum albumin in 0.02 M phosphate-buffered saline (PBS), pH 7.2, at 4°C overnight. After washing the plates 5 times with PBS containing 0.05% Tween-20 (PBST), 100 pL of affinity-purified MAb was added to each well and incubated at 3 ? ~for 2 h. After washing, 100 pL of horseradish peroxidase conjugated goat anti-mouse I ~ (diluted G 1 :1000) was added to each well and incubated at 37°C for 1 h. After washing, 200 pL of the substrate solution containing 0.02% H202and 3 mg/mL o-phenylenediamine(NakaraiChemicals Ltd., Kyoto, Japan) was added to each well; the plates were held in the dark at room temperature for 30 min for color development. Two hundred microlitres of the reaction mixture from each well was,transferred to test tubes, to which 3 mL of H2S04 was added to stop the reaction. The reaction was determined spectrophotometrically with Shimadzu doublebeam spectrophotometer UV- 150 (Shimadzu Co. Ltd., Japan) at 492 nrn
11111.
For determination of binding abilities of MAbs to SED, 100 pL of the purified MAbs at 100 ng/mL was incubated at 37°C for 30 min with 100 pL of SED at 0-10 pg/mL. After incubation, 100 pL of the mixture was added to each well coated with 100 pL of SED at 10 pg/mL to determine the remaining antibody activity in the mixture. Preparation of immunosorbent and immunoaflnity chromatography Two milligrams of affinity-purified MAbs to SED was extensively dialyzed against 0.2 M NaHCO,, pH 8.0, containing 0.3 M NaCl. A total of 0.4 mL of swollen Affi-Gel 10 (Bio-Rad Laboratories, Richmond, CA) was washed in the same buffer. Two milligrams of dialyzed MAbs was mixed at 4°C for 4 h with 0.4 mL of washed Affi-Gel 10 by end-over-end rotation. Then the gel was washed and suspended in 0.1 M ethanolamine-HC1, pH 8.0. After 1 h at room temperature, the suspension was extensively washed in PBS and used for preliminary experiments for immunoaffinity chromatography. To prepare the immunosorbent for large scale isolation of SED, 4.2 mg of MAb was coupled to 2 mL of swollen Affi-Gel 10 as described above. SED was partially purified from culture supernatants by chromatography on SPSephadex C-25 as described previously (Shinagawa et al. 1975, 1977). Partially purified SED was used as the crude material containing SED (crude SED) for immunoaffinity chromatography. After extensive dialysis against PBS, crude SED was applied to the immunosorbent column. The adsorbed material was eluted with 0.2 M acetic acid containing 0.15 M NaCl (pH 2.5). The eluate was immediately neutralized by addition of 1 M Na,CO, (pH 12.2). Other methods Protein contents were determined by the methods of Lowry et al. ( 1951 ) using bovine serum albumin as standard. The concentration of murine MAb was determined by absorbance at 280 nm, calibrating OD,,,,, = 14. SED was quantitatively detected by a double-gel immunodiffusion system (microslide) using rabbit anti-SED serum following the methods described previously (Shinagawa et al. 1974). Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were carried out following the methods described previously (Laemmli 1970; Towbin et al. 1979).
Results Reactivities of murine MAbs against SED Six murine MAbs against SED were obtained by fusion of myeloma cells with mouse spleen cells immunized with SED, whereas two MAbs (DE-2 1 1 and AD-4 1) were obtained by fusion of the same myelomacells with mouse spleen cells immu-
TABLE1. Subclasses and reactivities of murine MAbs against SED MAb
Immunogen
Reactivity of MAb
SED
KD-I (IgGI) KD-2 (IgGI) KD-3 (IgGI) KD-4 (IgG 1 ) KD-5 (IgG,) KD-6 (IgG,)
SED SED SED SED SED SED
SED and SEE
DE-2 1 1 (IgG - I)
SED, SEEt
SED and SEA
AD-4 1 (nd*)
SEA, SED,t SEEt
*Not determined.
tThese SEs were weakly
reactive with the M ~ b s .
nized with either SEE or SEA as a final immunogen. s i x of them (KD-1 to KD-6) were specific for only SED in ELISA, whereas DE-211 reacted with both SED and SEE. AD-41 was reactive with SEA, SED, and SEE (Table 1). These MAbs were not reactive with either SEB or SEC at the highest concentrations used. Similar results were also obtained by Western blotting (data not shown). Table 1 also presents the subclasses of MAbs to SED. T o study reactivities of six MAbs specific for only SED, 100 ng/mL of each purified MAb was incubated at 37°C for 3 0 min with SED at up to 10 bg/mL. After incubation, the remaining antibody activity in the mixture was determined in ELISA. No remaining antibody reactivity was found after any of the MAbs at 100 ng/mL were incubated with SED at 10 bg/mL. KD- 1, KD-3, KD-4, and KD-5 were more reactive with SED than KD-2 and KD-6 (Fig. 1). Of six MAbs to SED tested, KD- 1 was most effectively inactivated by SED (Fig. 1). The amount of SED to be required for 50% inhibition was 10 ng/mL for KD- 1 and 220 ng/mL for KD-6 (Fig. I), respectively.
Immunoaflnity purification of SED T o study which MAbs to SED were most suitable for preparation of the immunosorbent, 2 mg of different purified MAbs (such as KD- 1, KD-3, KD-5, and KD-6) was coupled to 0.4 mL of swollen Affi-Gel 10. Approximately 700 b g of purified MAbs was found to couple to the gel. Crude material containing 100 b g SED was subjected to each of the immunosorbents prepared as above. Of the crude SED, 76.8,34.5, 12.5, and 5.5% were found to bind to the immunosorbents coupled to KD- 1, KD-3, KD-5, and KD-6, respectively (Table 2). By use of 0.2 M acetic acid containing 0.15 M NaCl as an eluant, about 70% of bound SED was eluted from the immunosorbents with KD- 1, KD-3, and KD5. In contrast much less bound SED was eluted from the immunosorbent with KD-6 (Table 2). These findings suggest that KD- 1 is most suitable to prepare the immunosorbent. Thus, KD1 was used to prepare the immunosorbent for large-scale isolation of SED. Twelve millilitres of crude material containing 480 b g of SED was subjected to the column. Although a small amount of SED was detected in the unbound fractions, approximately 365 b g of SED (76%) was eluted using 0.2 M acetic acid as an eluant (Table 3). Purified SED was subjected to SDS-PAGE. As shown in Fig. 2, affinity-purified SED showed a single protein band in SDS-PAGE, whereas crude SED showed multiple protein bands. In immunogel double diffusion, affinity-purified SED formed a
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CAN. J. MICROBIOL. VOL. 37, 1991
SED (ng/mL) FIG. 1. Inhibitory activities of affinity-purified MAbs to SED. MAbs used were KD- 1 (@), KD-2 (U),KD-3 (A), KD-4 (O), KD-5 (El), and KD-6
(4. TABLE2. Adsorption and recovery of SED on immunoadsorbents coupled to different MAbs
MAb coupled to immunosorbent
Adsorption (%) of applied SED to immunosorbent
Recovery (%) of bound SED in eluate
KD- 1 KD-3 KD-5 KD-6
TABLE3. Recovery of SED by irnrnunoaffinity chromatography using MAb KD-1
Crude SED* Purified SED
Volume (mL)
Total protein (Fg)
Total SED (Fg)
Recovery
12 4
2,864 368
480 368
100 76.7
(%I
*Crude SED was partially purified SED prepared by ion-exchange chromatography on SP-Sephadex C-25 as described in Materials and methods.
single precipitin line against rabbit anti-SED serum (Fig. 3). The precipitin line of affinity-purified SED was identical with that of reference pure SED prepared by the conventional methods (Fig. 3).
Discussion Although MAbs have been prepared against the SEs (Edwin et al. 1984, 1986a, 1986b; Meyer et al. 1984; Thompson et al. 1984, 1986; Lapeyre et al. 1987), few MAbs against SED have been reported. In the present study, eight MAbs to SED were isolated. When only SED was used as the immunogen, the MAbs obtained were specific for SED; it was not determined whether the combining sites of these MAbs were the same. MAbs KD- 1,
FIG.2. SDS-PAGE of SED. Lane P, purified SED (1.6 ~ g )lane ; C, crude SED (1.2 ~ g ) .
KD-3, KD-4, and KD-5 were more reactive than MAbs KD-2 and KD-6 (Fig. I), with MAb KD-1 being about 20 times more reactive than MAb KD-6. The fact that MAbs reactive with SEA, SED, and SEE when SEA was used as the final immunogen and MAbs reactive with SED and SEE when SEE was used as the final immunogen were isolated indicates that these SEs contain
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SHINAGAWA ET AL.: I1
FIG.3. Immunogel double diffusion with SED. Center well contains undiluted polyclonal anti-SED. Peripheral wells: purified SED at 80 pg/mL (P), purified SED as reference at 60 pg/mL (R), and crude SED at 60 pg/mL (C).
common epitopes. The cross-reacting MAbs should prove useful in identification of the epitopes and the biological active site(s) of the SEs. Although the recovery of SED from the immunoaffinity chromatography using a MAb to SED was 76.7% of partially purified SED applied to the column, the necessity for chromatofocusing the culture supernatant fluid on SP-Sephadex C-25 to remove protein A reduced the overall recovery. This is probably a reflection on the very small amounts of SED produced by the staphylococci. Even so, this combination of purification steps resulted in a much higher recovery of purified SED than has been possible with other methods (Bergdoll 1977, 1979; Chang and Bergdoll 1979; Lei et al. 1988).
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