Letters in Applied Microbiology 1992, 15, 23-25
A highly sensitive enzyme-linked immunosorbent assay for Clostridium perfringens enterotoxin T. U E M U R A S ,. Y O S H I T A K ED. , H u & T. K A J I K A W ADepartment of Veterinary Medicine, College of Agriculture, University of Osaka Prefecture, Mozu Ume-machi 4-cho, Sakai-shi, Osaka 591, Japan DSTIOI: received 9 March 1992 and accepted 14 March 1992 U E M U R AT, . , Y O S H I T A K E ,S., H u , D. & K A J I K A W AT. , 1992. A highly sensitive enzyme-linked immunosorbent assay for Clostridium perfringens enterotoxin. Letters in Applied Microbiology 15, 23-25.
A highly sensitive enzyme-linked immunosorbent assay (ELISA)for quantitation of Clostridium perfringens enterotoxin (CPE) by a sandwich method with polystyrene beads was elaborated. The ELISA was very sensitive with a detection limit of 1 pg/ml of CPE. Clostridium perfringens culture fluid did not interfere with the assay. This ELISA may be useful for the mass screening for CI. perfringens producing small amounts of CPE.
An enterotoxin (CPE) produced by Clostridium perfringens is responsible for a common form of bacterial food poisoning (Walker 1975). CPE is a protein and preparation of specific antiserum against CPE has accelerated the development of different immunological methods for CPE determination. Of the methods available enzymelinked immunosorbent assay (ELISA) is widely employed. The most sensitive ELISA for CPE was reported by Olsvik et al. (1982), detecting 100 pg/ml of CPE. Kato et al. (1976) developed a highly sensitive ELlSA which could detect 0.3 fM of human IgG. This report describes the application of this ELISA for CPE determination and discusses some problems in the detection of CPE in etiological studies of Cl. perfringens food poisoning. Materials and Methods ENTEROTOXIN A N D
ANTI-ENTEROTOXIN
Clostridium perfringens enterotoxin (CPE) was purified from the extract of sporulating cells of Cl. perfringens type A NCTC 8239 by the methods previously reported (Uemura et al. 1985). Anti-CPE IgG was isolated from the serum of a rabbit immunized with purified CPE
by gel filtration on a column of Sephacryl S-300 Superfine (Pharmacia, Uppsala, Sweden) equilibrated with 50 mM sodium phospate buffer, pH 7.0. Anti-CPE antibody was purified from the rabbit immune serum by affinity chromatography on a CNBr-activated Sepharose 4B column coupled with purified CPE as previously reported (Uemura & Skjelkvile 1976). Anti-CPE F a b fragment was prepared from the anti-CPE antibody by pepsin digestion and reduction by mercaptoethanol as described by Kato et al. (1976). F A B ' - 8 - D - G A L A C T O S I DCAOSNEJ U G A T E Thiol groups in the hinge of anti-CPE F a b were conjugated to maleimide groups which were introduced into the 8-D-galactosidase (Boehringer Mannheim Yamanouchi, Tokyo) with N,N'-o-phenylenedimaleimideas described by Kato et al. (1976). CULTURES
A non-enterotoxigenic stock strain of Cl. perfringens was incubated overnight at 37°C in Gifu Anaerobic Medium (GAM; Nissui, Tokyo). Escherichia coli K 12 was cultured
T . Uemura et al. overnight in Brain Heart Infusion Broth (BHI; Nissui) at 37°C.
400 x
ELISA PROCEDURE
Polystyrene beads (diam. 3.2 mm; Paesel Lorei(PSL) GmbH, Frankfurt, Germany) were coated with anti-CPE IgG (10 pg/ml solution in 0.1 M Tris-HC1 buffer, pH 9.0) by incubating the beads-antibody mixture in a glass vessel overnight at 4°C. The beads were washed twice by the addition and aspiration of Buffer A (0.01 M phosphate buffer, pH 7) containing 0.1 M NaCl, 1 mM MgCl,, and both BSA and NaN, at 0.1% (w/v) and stored at 4°C until use. One polystyrene bead coated with anti-CPE IgG was put into a well in a microtitre plate (U-form; Corning Lab., Corning, NY, USA) with 0.1 ml of a test solution. The test solution (0-100 ng/ml) was prepared by diluting CPE with PBS, GAM, Duncan and Strong medium (DS medium; Duncan & Strong, 1968), GAM culture, or BHI culture containing E. coli. The plate was sealed and incubated for 2 h at 37°C and kept at 4°C overnight. After incubation, the bead was washed five times by the addition and aspiration of Buffer A and incubated with 0.1 ml of Fab’$?-D-galactosidase conjugate diluted 104-fold with Buffer A for 2 h at 37°C and kept at 4°C overnight; the bead was washed as described above and finally transferred from the well to a small test-tube (12 mm by 150 mm). Buffer A (0.2 ml) containing 0.1 mM 4methylumbelliferyl-/3-D-galactoside as a substrate was added to the bead in the tube and the reaction was developed for 30 min at 30°C. The reaction was stopped by adding 2 ml of 0.1 M glycine-NaOH buffer, pH 10.3, and the fluorescence intensity of the 4-methylumberliferone released was read against a 100% standard of 1 pg/ml quinine in 50 mM H , S 0 4 . The wavelengths for excitation and emission were 360 nm and 450 nm, respectively. Results and Discussion
The standard curve obtained by this modified ELISA is shown in Fig. 1. The procedure proved to be at least 100 times more sensitive than any other immunological method reported for CPE determination. In mass screening for CPE-producing Cl. perfringens, it is possible to determine CPE directly in sporulating cultures
5
rI
8
200
c 0) +
c
I
o
1
1
lo-’ loo 10’ lo2 10’
1
1
lo4 lo5
Enterotoxtn (pg/ml)
Fig. 1. Standard curve for ELISA determination of purified CIostridiurn perfringens enterotoxin in PBS.
with stock reagents. To assess whether there was interference by media or culture fluid or a decrease in the sensitivity with the stock reagents, tests were done with either CPE diluted with GAM culture containing Cl. perfringens or with 3-month-old stock reagents of the polystyrene beads coated with anti-CPE IgG (kept at 4°C) and the Fab’$-D-galactosidase conjugate (kept at -20°C). There was no interference from the culture fluid and only a slight decrease in sensitivity with stock reagents (Fig. 2). Finally, the influence of E. coli culture on the ELISA was tested by the use of /3-Dgalactosidase from E. coli as a label enzyme; this confirmed that E. coli did not influence the result (Fig. 2). These results show that the modified ELISA is useful in the direct determination of CPE in Cl. perfringens cultures. Some strains of Cl. perfringens produce CPE during sporulation. Many media have been developed to enhance sporulation of Cl. perfringens, e.g. DS medium, but CPE production by C1. perfringens in sporulation media is known to vary from time to time even if the same strain and the same type of medium are used. To detect the ability of Cl. perfringens, isolated from food and the natural environment, to produce CPE is of great importance from the food hygiene viewpoint. But, obtaining CPE production in uitro constantly is quite difficult. Yasukawa e t al. (1975) divided Cl. perfringens into two types, CPE-positive and CPE-negative
Sensitive ELISA for enterotoxin 400
-
which normally produces a small amount of CPE as pointed out by Olsvik et al. (1982) will change to produce a large amount of CPE after heating. Mass screening for Cl. perfringens which produce a small amount of CPE may be the first step in such a study, and this modified ELISA may serve as a sensitive method.
H Buffer A
25
GAM
200
L
ul
a3 c
c
References
o
10-I
loo
lo1
lo2
lo3 to4
lo5
Enterotoxin (pg/ml)
Fig. 2. Standard curve for ELISA determination of purified Clostridium perfringens enterotoxin. Enterotoxin was diluted with PBS (B), Cl. perfringens vegetative-cell-culture in Gifu Anaerobic Medium (A) or Escherichia coli BHI culture (a).The ELISA was set up with 3-month-old stock reagents, polystyrene beads coated with anti-CPE IgG and Fab+?-D-gaIactosidase conjugate.
strains by an immuno-diffusion test at the lowest detection level of 0.5 pg CPE. Olsvik e t al. (1982) showed, however, that most Cl. perfringens strains produced a very small amount (less than 0.1 ng/ml). Conventional assay methods necessitate laborious concentration procedures for the determination of CPE at such a low level. Some isolates from Cl. perfringens food poisoning outbreaks produce a large amount of CPE (100 ng/ml or more). Heat-shock is known to stimulate Cl. perfringens to produce CPE (Uemura, 1978). It is of great interest to clarify whether Cl. perfringens
DUNCAN,C.L. & STRONG,D.H. 1968 Improved medium for sporulation of Clostridium perfringens. Applied Microbiology 16,82-89. KATO,K., FUKUI,H., HAMAGUCHI, Y. & ISHIKAWA, E. 1976 Enzyme-linked immunoassay : Conjugation of the F a b fragment of rabbit IgG with j3-D-galactosidase from E . coli and its use for immunoassay. Journal of Immunology 116, 1554-1560. OLSVIK, O., GRANUM, P.E. & BERDAL,P. 1982 Detection of Clostridium perfringens type A enterotoxin by ELISA. Acta Pathologica, Microbiologica et Immunologica Scandinavica Sect. B 90,445-447. UEMURA, T. 1978 Incidence of enterotoxigenic Clostridium perfringens in healthy humans in relation to the enhancement of enterotoxin production by heat treatment. Journal of Applied Bacteriology 44, 41 1419.
UEMURA, T. & SKJELKVALE, R. 1976 An enterotoxin produced by Clostridium perfringens type D. Purification by affinity chromatography. Acta Pathologica et Microbiologica Scandinavica, Sect. B 84, 414-420.
UEMURA, T., AOI, Y., HORIGUCHI, Y., WADANO,A., N. & SAKAGUCHI, G. 1985 Purification of GOSHIMA, Clostridium perfringens enterotoxin by high performance liquid chromatography. FEMS Microbiology Letters 29, 293-297. WALKER, H.W. 1975 Food-borne illness from Clostridium perfringens. Critical Review of Food Science and Nutrition 7 , 71-104. YASUKAWA, A,, OKADA,Y., KITASE,T. & MIYAMOTO, S. 1975 Distribution of enterotoxin-producing strains of Clostridium perfringens type A in human beings, foods and soils. Journal of Food Hygienic Society of Japan 16,313-317.
A highly sensitive enzyme-linked immunosorbent assay for Clostridium perfringens enterotoxin T. U E M U R A S ,. Y O S H I T A K ED. , H u & T. K A J I K A W ADepartment of Veterinary Medicine, College of Agriculture, University of Osaka Prefecture, Mozu Ume-machi 4-cho, Sakai-shi, Osaka 591, Japan DSTIOI: received 9 March 1992 and accepted 14 March 1992 U E M U R AT, . , Y O S H I T A K E ,S., H u , D. & K A J I K A W AT. , 1992. A highly sensitive enzyme-linked immunosorbent assay for Clostridium perfringens enterotoxin. Letters in Applied Microbiology 15, 23-25.
A highly sensitive enzyme-linked immunosorbent assay (ELISA)for quantitation of Clostridium perfringens enterotoxin (CPE) by a sandwich method with polystyrene beads was elaborated. The ELISA was very sensitive with a detection limit of 1 pg/ml of CPE. Clostridium perfringens culture fluid did not interfere with the assay. This ELISA may be useful for the mass screening for CI. perfringens producing small amounts of CPE.
An enterotoxin (CPE) produced by Clostridium perfringens is responsible for a common form of bacterial food poisoning (Walker 1975). CPE is a protein and preparation of specific antiserum against CPE has accelerated the development of different immunological methods for CPE determination. Of the methods available enzymelinked immunosorbent assay (ELISA) is widely employed. The most sensitive ELISA for CPE was reported by Olsvik et al. (1982), detecting 100 pg/ml of CPE. Kato et al. (1976) developed a highly sensitive ELlSA which could detect 0.3 fM of human IgG. This report describes the application of this ELISA for CPE determination and discusses some problems in the detection of CPE in etiological studies of Cl. perfringens food poisoning. Materials and Methods ENTEROTOXIN A N D
ANTI-ENTEROTOXIN
Clostridium perfringens enterotoxin (CPE) was purified from the extract of sporulating cells of Cl. perfringens type A NCTC 8239 by the methods previously reported (Uemura et al. 1985). Anti-CPE IgG was isolated from the serum of a rabbit immunized with purified CPE
by gel filtration on a column of Sephacryl S-300 Superfine (Pharmacia, Uppsala, Sweden) equilibrated with 50 mM sodium phospate buffer, pH 7.0. Anti-CPE antibody was purified from the rabbit immune serum by affinity chromatography on a CNBr-activated Sepharose 4B column coupled with purified CPE as previously reported (Uemura & Skjelkvile 1976). Anti-CPE F a b fragment was prepared from the anti-CPE antibody by pepsin digestion and reduction by mercaptoethanol as described by Kato et al. (1976). F A B ' - 8 - D - G A L A C T O S I DCAOSNEJ U G A T E Thiol groups in the hinge of anti-CPE F a b were conjugated to maleimide groups which were introduced into the 8-D-galactosidase (Boehringer Mannheim Yamanouchi, Tokyo) with N,N'-o-phenylenedimaleimideas described by Kato et al. (1976). CULTURES
A non-enterotoxigenic stock strain of Cl. perfringens was incubated overnight at 37°C in Gifu Anaerobic Medium (GAM; Nissui, Tokyo). Escherichia coli K 12 was cultured
T . Uemura et al. overnight in Brain Heart Infusion Broth (BHI; Nissui) at 37°C.
400 x
ELISA PROCEDURE
Polystyrene beads (diam. 3.2 mm; Paesel Lorei(PSL) GmbH, Frankfurt, Germany) were coated with anti-CPE IgG (10 pg/ml solution in 0.1 M Tris-HC1 buffer, pH 9.0) by incubating the beads-antibody mixture in a glass vessel overnight at 4°C. The beads were washed twice by the addition and aspiration of Buffer A (0.01 M phosphate buffer, pH 7) containing 0.1 M NaCl, 1 mM MgCl,, and both BSA and NaN, at 0.1% (w/v) and stored at 4°C until use. One polystyrene bead coated with anti-CPE IgG was put into a well in a microtitre plate (U-form; Corning Lab., Corning, NY, USA) with 0.1 ml of a test solution. The test solution (0-100 ng/ml) was prepared by diluting CPE with PBS, GAM, Duncan and Strong medium (DS medium; Duncan & Strong, 1968), GAM culture, or BHI culture containing E. coli. The plate was sealed and incubated for 2 h at 37°C and kept at 4°C overnight. After incubation, the bead was washed five times by the addition and aspiration of Buffer A and incubated with 0.1 ml of Fab’$?-D-galactosidase conjugate diluted 104-fold with Buffer A for 2 h at 37°C and kept at 4°C overnight; the bead was washed as described above and finally transferred from the well to a small test-tube (12 mm by 150 mm). Buffer A (0.2 ml) containing 0.1 mM 4methylumbelliferyl-/3-D-galactoside as a substrate was added to the bead in the tube and the reaction was developed for 30 min at 30°C. The reaction was stopped by adding 2 ml of 0.1 M glycine-NaOH buffer, pH 10.3, and the fluorescence intensity of the 4-methylumberliferone released was read against a 100% standard of 1 pg/ml quinine in 50 mM H , S 0 4 . The wavelengths for excitation and emission were 360 nm and 450 nm, respectively. Results and Discussion
The standard curve obtained by this modified ELISA is shown in Fig. 1. The procedure proved to be at least 100 times more sensitive than any other immunological method reported for CPE determination. In mass screening for CPE-producing Cl. perfringens, it is possible to determine CPE directly in sporulating cultures
5
rI
8
200
c 0) +
c
I
o
1
1
lo-’ loo 10’ lo2 10’
1
1
lo4 lo5
Enterotoxtn (pg/ml)
Fig. 1. Standard curve for ELISA determination of purified CIostridiurn perfringens enterotoxin in PBS.
with stock reagents. To assess whether there was interference by media or culture fluid or a decrease in the sensitivity with the stock reagents, tests were done with either CPE diluted with GAM culture containing Cl. perfringens or with 3-month-old stock reagents of the polystyrene beads coated with anti-CPE IgG (kept at 4°C) and the Fab’$-D-galactosidase conjugate (kept at -20°C). There was no interference from the culture fluid and only a slight decrease in sensitivity with stock reagents (Fig. 2). Finally, the influence of E. coli culture on the ELISA was tested by the use of /3-Dgalactosidase from E. coli as a label enzyme; this confirmed that E. coli did not influence the result (Fig. 2). These results show that the modified ELISA is useful in the direct determination of CPE in Cl. perfringens cultures. Some strains of Cl. perfringens produce CPE during sporulation. Many media have been developed to enhance sporulation of Cl. perfringens, e.g. DS medium, but CPE production by C1. perfringens in sporulation media is known to vary from time to time even if the same strain and the same type of medium are used. To detect the ability of Cl. perfringens, isolated from food and the natural environment, to produce CPE is of great importance from the food hygiene viewpoint. But, obtaining CPE production in uitro constantly is quite difficult. Yasukawa e t al. (1975) divided Cl. perfringens into two types, CPE-positive and CPE-negative
Sensitive ELISA for enterotoxin 400
-
which normally produces a small amount of CPE as pointed out by Olsvik et al. (1982) will change to produce a large amount of CPE after heating. Mass screening for Cl. perfringens which produce a small amount of CPE may be the first step in such a study, and this modified ELISA may serve as a sensitive method.
H Buffer A
25
GAM
200
L
ul
a3 c
c
References
o
10-I
loo
lo1
lo2
lo3 to4
lo5
Enterotoxin (pg/ml)
Fig. 2. Standard curve for ELISA determination of purified Clostridium perfringens enterotoxin. Enterotoxin was diluted with PBS (B), Cl. perfringens vegetative-cell-culture in Gifu Anaerobic Medium (A) or Escherichia coli BHI culture (a).The ELISA was set up with 3-month-old stock reagents, polystyrene beads coated with anti-CPE IgG and Fab+?-D-gaIactosidase conjugate.
strains by an immuno-diffusion test at the lowest detection level of 0.5 pg CPE. Olsvik e t al. (1982) showed, however, that most Cl. perfringens strains produced a very small amount (less than 0.1 ng/ml). Conventional assay methods necessitate laborious concentration procedures for the determination of CPE at such a low level. Some isolates from Cl. perfringens food poisoning outbreaks produce a large amount of CPE (100 ng/ml or more). Heat-shock is known to stimulate Cl. perfringens to produce CPE (Uemura, 1978). It is of great interest to clarify whether Cl. perfringens
DUNCAN,C.L. & STRONG,D.H. 1968 Improved medium for sporulation of Clostridium perfringens. Applied Microbiology 16,82-89. KATO,K., FUKUI,H., HAMAGUCHI, Y. & ISHIKAWA, E. 1976 Enzyme-linked immunoassay : Conjugation of the F a b fragment of rabbit IgG with j3-D-galactosidase from E . coli and its use for immunoassay. Journal of Immunology 116, 1554-1560. OLSVIK, O., GRANUM, P.E. & BERDAL,P. 1982 Detection of Clostridium perfringens type A enterotoxin by ELISA. Acta Pathologica, Microbiologica et Immunologica Scandinavica Sect. B 90,445-447. UEMURA, T. 1978 Incidence of enterotoxigenic Clostridium perfringens in healthy humans in relation to the enhancement of enterotoxin production by heat treatment. Journal of Applied Bacteriology 44, 41 1419.
UEMURA, T. & SKJELKVALE, R. 1976 An enterotoxin produced by Clostridium perfringens type D. Purification by affinity chromatography. Acta Pathologica et Microbiologica Scandinavica, Sect. B 84, 414-420.
UEMURA, T., AOI, Y., HORIGUCHI, Y., WADANO,A., N. & SAKAGUCHI, G. 1985 Purification of GOSHIMA, Clostridium perfringens enterotoxin by high performance liquid chromatography. FEMS Microbiology Letters 29, 293-297. WALKER, H.W. 1975 Food-borne illness from Clostridium perfringens. Critical Review of Food Science and Nutrition 7 , 71-104. YASUKAWA, A,, OKADA,Y., KITASE,T. & MIYAMOTO, S. 1975 Distribution of enterotoxin-producing strains of Clostridium perfringens type A in human beings, foods and soils. Journal of Food Hygienic Society of Japan 16,313-317.
