APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1977, Copyright X) 1977 American Society for Microbiology
p.
125-128
Vol. 34, No. 2
Printed in U.S.A.
Rapid Detection and Quantitation of Clostridium perfringens Enterotoxin by Counterimmunoelectrophoresis HARI S. NAIK
AND
CHARLES L. DUNCAN*
Department of Food Microbiology and Toxicology, Food Research Institute, University of Wisconsin, Madison, Wisconsin 53706 Received for publication 25 March 1977
Conditions for detection and quantitation of Clostridium perfringens enterotoxin by counterimmunoelectrophoresis are described. As little as 0.2 ,ug of enterotoxin per ml could be detected. The test was found to be rapid, sensitive, specific, and easy for the detection and quantitation of enterotoxin.
Clostridium perfringens enterotoxin can be detected on the basis of its biological activity. The toxin induces fluid accumulation in ligated ileal loops of rabbits and lambs (4, 9), is lethal to mice (8, 12), and produces erythema in the skin of guinea pigs and rabbits (7, 12). For the purpose of quantitation, the assay based on erythemal activity is more sensitive than the loop technique or the measurement of lethality in mice (8, 12). All of these methods suffer from variations in the sensitivity of the animal being used, the requirement for sufficient controls to eliminate the activity of other perfringens toxins or nonspecific responses, and the cost of the animals involved. We have reported that by using an electroimmunodiffusion technique, as little as 1 ,tg of enterotoxin per ml could be quantitated (2), whereas the detection limit by reversed passive hemagglutination (RPHA) was reported to be as little as 0.5 ng/ml (13). Counterimmunoelectrophoresis (CIEP) is a specific, rapid, sensitive, easy, and accurate means of inducing a precipitin reaction between a particular antigen and its antibody. CIEP has been used in the past for the routine identification of various kinds of antigens (1, 5, 6, 10, 11). Here, we describe the use of this technique for rapid detection of C. perfringens enterotoxin. MATERIALS AND METHODS For CIEP, a projector slide cover glass (Eastman Kodak Co., Rochester, N.Y.), 8.3 by 10.2 cm, was used for support of the agar gel. A very thin coating of 2.0% Ionagar no. 2 (Oxoid Ltd., London) in 0.025 ionic-strength barbital-acetate buffer (pH 8.6) was applied to the glass slide with a brush and allowed to harden for 15 min. The slide was then layered with 15 ml of 0.6% agarose (Fisher Scientific Co., Pittsburgh, Pa.) in 0.025 ionic-strength barbital-acetate buffer (pH 8.6). The slides were allowed to harden 125
for at least 20 min and were stored in a humidity chamber until used. Barbital-acetate buffer (pH 8.6) was prepared in distilled demineralized water. The composition of stock buffer was 3.09% sodium barbital, 0.552% barbital, and 1.23% sodium acetate. The buffer solution was heated at 55°C for 4 h to dissolve the buffer salts and was then diluted with 2 volumes of water to 0.1 ionic strength. For 0.025 ionic-strength buffer, the stock was diluted with 3 volumes of water. Parallel rows of wells 3 mm in diameter were cut 5 mm apart in the agar. A slide ofthis size accommodated as many as three double rows, with nine paired wells in each row. Thus 27 samples could be analyzed for the presence of enterotoxin in one slide. Ten microliters of enterotoxin antigen to be tested (C. perfringens-purified enterotoxin, enterotoxin in crude cell extract, culture supernatant, or food extract) was diluted using doubling dilutions, in demineralized distilled water (pH 6.7), 0.025 ionicstrength barbital-acetate buffer, or 0.85% saline and placed in the rows of wells near the cathode. Antienterotoxin (12), produced in rabbits against purified enterotoxin and diluted 1:2 with 0.85% saline, was placed in the wells near the anode in similar volume. The electrode vessel contained barbital-acetate buffer of the same ionic strength, 0.025, as was used for the gel preparation. Electrophoresis was carried out in an electrophoresis chamber (Gelman Instrument Co., Ann Arbor, Mich.) at room temperature or at 4°C with constant current of 12 mA/slide for 30 min. Whatman no. 1 filter paper was used as an electrode wick. After electrophoresis, the slides were removed to a humidity chamber and allowed to develop for 5 to 10 min. This process made the precipitin line very sharp, clear, and easily observable. Even a very faint precipitin line not observable with the naked eye could be intensified by overnight incubation in a humidity chamber followed by staining with 2% tannic acid for 5 min. For a permanent record, slides could be fixed and processed as described in the Millipore counterimmunoelectrophoresis system manual (Millipore Corp., Acton, Mass.). A linear relationship existed between the concentration of enterotoxin and the CIEP titer of toxin as
iV
E.
IF
I
FIG. 1. (a) CIEP plate showing nonspecific food (cooked chicken) proteins partly masking the precipitin
lines obtained using C. perfringens enterotoxin and specific anti-enterotoxin serum. The top wells ofeach pair of rows contained decreasing quantities of enterotoxin from left to right. The bottom wells contained a constant amount of anti-enterotoxin serum. Cathode is the top and anode is the bottom of the figure. The plate was stained with tannic acid as described in the text. (b) CIEP plate containing samples identical to that in (a). In this plate, nonspecific food proteins have been removed by deproteinization overnight in phosphate-buffered saline containing sodium azide (see text) before staining with tannic acid. 126
VOL. 34, 1977
C. PERFRINGENS ENTEROTOXIN DETECTION BY CIEP
determined by the highest dilution of toxin exhibiting a visible precipitin band. This allowed quantitation of enterotoxin in unknown samples based on the titer obtained with a standard enterotoxin preparation of known concentration.
RESULTS AND DISCUSSION In CIEP, on application of current, negatively charged antigen migrates towards the anode, and the antibodies having no significant charge are moved cathodically by electroendosmosis. In our studies, a sharp, clear precipitin line appearing in the area between the paired wells as a result of reaction between enterotoxin and its antibody was readily visible at the end of 30 min. It was observed that for quantitation, a twofold dilution of enterotoxin in demineralized distilled water (pH 6.7) was better than dilution in 0.85% saline or 0.025 ionicstrength barbital-acetate buffer. No nonspecific reaction was observed with various controls including 0.85% NaCl, 0.025 ionic-strength barbital-acetate buffer, distilled water, D-S sporulation medium (3), and various food extracts such as chicken gravy, beef gravy, moist cooked ground beef, chicken, turkey, mutton, and pork. An example of enterotoxin detection in the presence of cooked chicken is shown in Fig. la, Nonspecific food proteins could be easily washed away (Fig. lb) in a deproteinizing solution prepared by dissolving 1.88 g of sodium phosphate dibasic heptahydrate, 8.0 g of sodium chloride, 0.08 g of sodium phosphate monobasic, and 0.1 g of sodium azide in demineralized distilled water to make a final volume of 1 liter. No difference was obtained when electrophoresis was at 4°C or at room temperature. Different antisera used for the detection of enterotoxin had CIEP titers of 1:4, 1:8, 1:16, 1:32, and 1:64. Titration of antisera was done using a constant toxin concentration of 1.05 gg/ml. It was observed that even antiserum with a 1:4 titer was as effective for enterotoxin detection as antiserum having a high CIEP titer of 1:64. Using CIEP, the detection limit for C. perfringens type A enterotoxin was 0.2 ,ug/ml, or 0.002 ,ug on a weight basis. This was more sensitive than various other techniques we have used, such as erythemal activity (3.8 ,tg/ ml), electroimmunodiffusion (1.0 gg/ml), and single-gel diffusion (3.0 gg/ml) (2). It was possible to readily detect enterotoxin in the supernatant fluid of various sporulating cultures grown in D-S sporulation medium (3) for 24 h at 37°C. Using CIEP, in addition to rapid screening of cultures or mutants for their enterotoxin-producing ability, cultures could be grouped as low and high enterotoxin producers. CIEP has also been useful in identifying toxin-
127
containing fractions obtained from various chromatographic columns used during purification of the enterotoxin. In addition, we have used CIEP for detection and quantitation of enterotoxin in foods and for titration of antisera produced in rabbits against purified enterotoxin. CIEP is quicker, easier, and more specific than RPHA. It requires only 30 min, whereas RPHA requires at least 2 h at room temperature. In our laboratory, even though as little as 0.001 ,ug of enterotoxin per ml could be detected using RPHA, variable results were often obtained with the RPHA test because of different batches of sheep erythrocytes as well as nonspecific hemagglutination (unpublished data). Compared with electroimmunodiffusion, CIEP quantitation based on determination of the end point is easy, as one observes only for a precipitin line in between paired wells, whereas electroimmunodiffusion quantitation is based on cone height. Since the cone heights are affected by the menstruum in which the toxin is contained, quantitation may not be as accurate wtih electroimmunodiffusion as with CIEP. ACKNOWLEDGMENTS This research was supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison; Public Health Service research grant AI-11865-06 from the National Institute of Allergy and Infectious Diseases; Public Health Service research grant FD-00203-07 from the Food and Drug Administration; and by contributions to the Food Research Institute by member industries. C.L.D. is the recipient of Public Health Service Research Career Development award AI-70721-03 from the National Institute of Allergy and Infectious Diseases. H.S.N. is the recipient of a Postdoctoral Research Fellowship from the Government of India in the field of veterinary public health. LITERATURE CITED 1. Cho, H. J., and E. V. Langford. 1974. Rapid detection of bovine mycoplasma antigens by counterimmunolectrophoresis. Appl. Microbiol, 28:897-899. 2. Duncan, C. L., and E. B. Somers. 1972. Quantitation of Clostridium perfringens type A enterotoxin by elec-
troimmunodiffusion. Appl. Microbiol. 24:801-804. 3. Duncan, C. L., and D. H. Strong. 1968. Improved medium for sporulation of Clostridium perfringens. Appl. Microbiol. 16:82-89. 4. Duncan, C. L., and D. H. Strong. 1969. Ileal loop fluid accumulation and production of diarrhea in rabbits by cell-free products of Clostridium perfringens. J. Bacteriol. 100:86-94. 5. Edwards, E. A. 1971. Immunologic investigations of meningococcal disease. I. Group-specific Neisseria meningitidis antigens present in the serum of patients with fulminant meningococcemia. J. Immunol. 106:314-317. 6. Gocke, D. J., and C. Howe. 1970. Rapid detection of Australia antigen by counterimmunoelectrophoresis. J. Immunol. 104:1031-1032. 7. Hauschild, A. H. W. 1970. Erythemal activity of the cellular enteropathogenic factor of Clostridium perfringens type A. Can. J. Microbiol. 16:651-654. 8. Hauschild, A. H. W., and R. Hilsheimer. 1971. Purifica-
128
NAIK AND DUNCAN tion and characteristics ofthe enterotoxin ofClostrid-
ium perfiingens type A. Can. J. Microbiol. 17:14251433. 9. Hauschild, A. H. W., L. Niilo, and W. J. Dorward. 1971. The role of enterotoxin in Clostridium perfringens type A enteritis. Can. J. Microbiol. 17:987-991. 10. Hill, H. R., M. E. Riter, S. K. Menge, D. R. Johnson, and J. M. Matsen. 1975. Rapid identification of group B streptococci by counterimmunoelectrophoresis. J. Clin. Microbiol. 1:188-191.
APPL. ENVIRON. MICROBIOL. 11. Meher-Homji, K. M. 1975. Counterimmunoelectrophoresis in rapid diagnosis of pox virus infections. Med. J. Aust. 1:790. 12. Stark, R. L., and C. L. Duncan. 1971. Biological characteristics of Clostridium perfringens type A enterotoxin. Infect. Immun. 4:89-96. 13. Uemura, T., G. Sakaguchi, and H. P. Riemann. 1973. In vitro production ofClostridium perfringens enterotoxin and its detection by reversed passive hemagglutination. Appl. Microbiol. 26:381-385.
p.
125-128
Vol. 34, No. 2
Printed in U.S.A.
Rapid Detection and Quantitation of Clostridium perfringens Enterotoxin by Counterimmunoelectrophoresis HARI S. NAIK
AND
CHARLES L. DUNCAN*
Department of Food Microbiology and Toxicology, Food Research Institute, University of Wisconsin, Madison, Wisconsin 53706 Received for publication 25 March 1977
Conditions for detection and quantitation of Clostridium perfringens enterotoxin by counterimmunoelectrophoresis are described. As little as 0.2 ,ug of enterotoxin per ml could be detected. The test was found to be rapid, sensitive, specific, and easy for the detection and quantitation of enterotoxin.
Clostridium perfringens enterotoxin can be detected on the basis of its biological activity. The toxin induces fluid accumulation in ligated ileal loops of rabbits and lambs (4, 9), is lethal to mice (8, 12), and produces erythema in the skin of guinea pigs and rabbits (7, 12). For the purpose of quantitation, the assay based on erythemal activity is more sensitive than the loop technique or the measurement of lethality in mice (8, 12). All of these methods suffer from variations in the sensitivity of the animal being used, the requirement for sufficient controls to eliminate the activity of other perfringens toxins or nonspecific responses, and the cost of the animals involved. We have reported that by using an electroimmunodiffusion technique, as little as 1 ,tg of enterotoxin per ml could be quantitated (2), whereas the detection limit by reversed passive hemagglutination (RPHA) was reported to be as little as 0.5 ng/ml (13). Counterimmunoelectrophoresis (CIEP) is a specific, rapid, sensitive, easy, and accurate means of inducing a precipitin reaction between a particular antigen and its antibody. CIEP has been used in the past for the routine identification of various kinds of antigens (1, 5, 6, 10, 11). Here, we describe the use of this technique for rapid detection of C. perfringens enterotoxin. MATERIALS AND METHODS For CIEP, a projector slide cover glass (Eastman Kodak Co., Rochester, N.Y.), 8.3 by 10.2 cm, was used for support of the agar gel. A very thin coating of 2.0% Ionagar no. 2 (Oxoid Ltd., London) in 0.025 ionic-strength barbital-acetate buffer (pH 8.6) was applied to the glass slide with a brush and allowed to harden for 15 min. The slide was then layered with 15 ml of 0.6% agarose (Fisher Scientific Co., Pittsburgh, Pa.) in 0.025 ionic-strength barbital-acetate buffer (pH 8.6). The slides were allowed to harden 125
for at least 20 min and were stored in a humidity chamber until used. Barbital-acetate buffer (pH 8.6) was prepared in distilled demineralized water. The composition of stock buffer was 3.09% sodium barbital, 0.552% barbital, and 1.23% sodium acetate. The buffer solution was heated at 55°C for 4 h to dissolve the buffer salts and was then diluted with 2 volumes of water to 0.1 ionic strength. For 0.025 ionic-strength buffer, the stock was diluted with 3 volumes of water. Parallel rows of wells 3 mm in diameter were cut 5 mm apart in the agar. A slide ofthis size accommodated as many as three double rows, with nine paired wells in each row. Thus 27 samples could be analyzed for the presence of enterotoxin in one slide. Ten microliters of enterotoxin antigen to be tested (C. perfringens-purified enterotoxin, enterotoxin in crude cell extract, culture supernatant, or food extract) was diluted using doubling dilutions, in demineralized distilled water (pH 6.7), 0.025 ionicstrength barbital-acetate buffer, or 0.85% saline and placed in the rows of wells near the cathode. Antienterotoxin (12), produced in rabbits against purified enterotoxin and diluted 1:2 with 0.85% saline, was placed in the wells near the anode in similar volume. The electrode vessel contained barbital-acetate buffer of the same ionic strength, 0.025, as was used for the gel preparation. Electrophoresis was carried out in an electrophoresis chamber (Gelman Instrument Co., Ann Arbor, Mich.) at room temperature or at 4°C with constant current of 12 mA/slide for 30 min. Whatman no. 1 filter paper was used as an electrode wick. After electrophoresis, the slides were removed to a humidity chamber and allowed to develop for 5 to 10 min. This process made the precipitin line very sharp, clear, and easily observable. Even a very faint precipitin line not observable with the naked eye could be intensified by overnight incubation in a humidity chamber followed by staining with 2% tannic acid for 5 min. For a permanent record, slides could be fixed and processed as described in the Millipore counterimmunoelectrophoresis system manual (Millipore Corp., Acton, Mass.). A linear relationship existed between the concentration of enterotoxin and the CIEP titer of toxin as
iV
E.
IF
I
FIG. 1. (a) CIEP plate showing nonspecific food (cooked chicken) proteins partly masking the precipitin
lines obtained using C. perfringens enterotoxin and specific anti-enterotoxin serum. The top wells ofeach pair of rows contained decreasing quantities of enterotoxin from left to right. The bottom wells contained a constant amount of anti-enterotoxin serum. Cathode is the top and anode is the bottom of the figure. The plate was stained with tannic acid as described in the text. (b) CIEP plate containing samples identical to that in (a). In this plate, nonspecific food proteins have been removed by deproteinization overnight in phosphate-buffered saline containing sodium azide (see text) before staining with tannic acid. 126
VOL. 34, 1977
C. PERFRINGENS ENTEROTOXIN DETECTION BY CIEP
determined by the highest dilution of toxin exhibiting a visible precipitin band. This allowed quantitation of enterotoxin in unknown samples based on the titer obtained with a standard enterotoxin preparation of known concentration.
RESULTS AND DISCUSSION In CIEP, on application of current, negatively charged antigen migrates towards the anode, and the antibodies having no significant charge are moved cathodically by electroendosmosis. In our studies, a sharp, clear precipitin line appearing in the area between the paired wells as a result of reaction between enterotoxin and its antibody was readily visible at the end of 30 min. It was observed that for quantitation, a twofold dilution of enterotoxin in demineralized distilled water (pH 6.7) was better than dilution in 0.85% saline or 0.025 ionicstrength barbital-acetate buffer. No nonspecific reaction was observed with various controls including 0.85% NaCl, 0.025 ionic-strength barbital-acetate buffer, distilled water, D-S sporulation medium (3), and various food extracts such as chicken gravy, beef gravy, moist cooked ground beef, chicken, turkey, mutton, and pork. An example of enterotoxin detection in the presence of cooked chicken is shown in Fig. la, Nonspecific food proteins could be easily washed away (Fig. lb) in a deproteinizing solution prepared by dissolving 1.88 g of sodium phosphate dibasic heptahydrate, 8.0 g of sodium chloride, 0.08 g of sodium phosphate monobasic, and 0.1 g of sodium azide in demineralized distilled water to make a final volume of 1 liter. No difference was obtained when electrophoresis was at 4°C or at room temperature. Different antisera used for the detection of enterotoxin had CIEP titers of 1:4, 1:8, 1:16, 1:32, and 1:64. Titration of antisera was done using a constant toxin concentration of 1.05 gg/ml. It was observed that even antiserum with a 1:4 titer was as effective for enterotoxin detection as antiserum having a high CIEP titer of 1:64. Using CIEP, the detection limit for C. perfringens type A enterotoxin was 0.2 ,ug/ml, or 0.002 ,ug on a weight basis. This was more sensitive than various other techniques we have used, such as erythemal activity (3.8 ,tg/ ml), electroimmunodiffusion (1.0 gg/ml), and single-gel diffusion (3.0 gg/ml) (2). It was possible to readily detect enterotoxin in the supernatant fluid of various sporulating cultures grown in D-S sporulation medium (3) for 24 h at 37°C. Using CIEP, in addition to rapid screening of cultures or mutants for their enterotoxin-producing ability, cultures could be grouped as low and high enterotoxin producers. CIEP has also been useful in identifying toxin-
127
containing fractions obtained from various chromatographic columns used during purification of the enterotoxin. In addition, we have used CIEP for detection and quantitation of enterotoxin in foods and for titration of antisera produced in rabbits against purified enterotoxin. CIEP is quicker, easier, and more specific than RPHA. It requires only 30 min, whereas RPHA requires at least 2 h at room temperature. In our laboratory, even though as little as 0.001 ,ug of enterotoxin per ml could be detected using RPHA, variable results were often obtained with the RPHA test because of different batches of sheep erythrocytes as well as nonspecific hemagglutination (unpublished data). Compared with electroimmunodiffusion, CIEP quantitation based on determination of the end point is easy, as one observes only for a precipitin line in between paired wells, whereas electroimmunodiffusion quantitation is based on cone height. Since the cone heights are affected by the menstruum in which the toxin is contained, quantitation may not be as accurate wtih electroimmunodiffusion as with CIEP. ACKNOWLEDGMENTS This research was supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison; Public Health Service research grant AI-11865-06 from the National Institute of Allergy and Infectious Diseases; Public Health Service research grant FD-00203-07 from the Food and Drug Administration; and by contributions to the Food Research Institute by member industries. C.L.D. is the recipient of Public Health Service Research Career Development award AI-70721-03 from the National Institute of Allergy and Infectious Diseases. H.S.N. is the recipient of a Postdoctoral Research Fellowship from the Government of India in the field of veterinary public health. LITERATURE CITED 1. Cho, H. J., and E. V. Langford. 1974. Rapid detection of bovine mycoplasma antigens by counterimmunolectrophoresis. Appl. Microbiol, 28:897-899. 2. Duncan, C. L., and E. B. Somers. 1972. Quantitation of Clostridium perfringens type A enterotoxin by elec-
troimmunodiffusion. Appl. Microbiol. 24:801-804. 3. Duncan, C. L., and D. H. Strong. 1968. Improved medium for sporulation of Clostridium perfringens. Appl. Microbiol. 16:82-89. 4. Duncan, C. L., and D. H. Strong. 1969. Ileal loop fluid accumulation and production of diarrhea in rabbits by cell-free products of Clostridium perfringens. J. Bacteriol. 100:86-94. 5. Edwards, E. A. 1971. Immunologic investigations of meningococcal disease. I. Group-specific Neisseria meningitidis antigens present in the serum of patients with fulminant meningococcemia. J. Immunol. 106:314-317. 6. Gocke, D. J., and C. Howe. 1970. Rapid detection of Australia antigen by counterimmunoelectrophoresis. J. Immunol. 104:1031-1032. 7. Hauschild, A. H. W. 1970. Erythemal activity of the cellular enteropathogenic factor of Clostridium perfringens type A. Can. J. Microbiol. 16:651-654. 8. Hauschild, A. H. W., and R. Hilsheimer. 1971. Purifica-
128
NAIK AND DUNCAN tion and characteristics ofthe enterotoxin ofClostrid-
ium perfiingens type A. Can. J. Microbiol. 17:14251433. 9. Hauschild, A. H. W., L. Niilo, and W. J. Dorward. 1971. The role of enterotoxin in Clostridium perfringens type A enteritis. Can. J. Microbiol. 17:987-991. 10. Hill, H. R., M. E. Riter, S. K. Menge, D. R. Johnson, and J. M. Matsen. 1975. Rapid identification of group B streptococci by counterimmunoelectrophoresis. J. Clin. Microbiol. 1:188-191.
APPL. ENVIRON. MICROBIOL. 11. Meher-Homji, K. M. 1975. Counterimmunoelectrophoresis in rapid diagnosis of pox virus infections. Med. J. Aust. 1:790. 12. Stark, R. L., and C. L. Duncan. 1971. Biological characteristics of Clostridium perfringens type A enterotoxin. Infect. Immun. 4:89-96. 13. Uemura, T., G. Sakaguchi, and H. P. Riemann. 1973. In vitro production ofClostridium perfringens enterotoxin and its detection by reversed passive hemagglutination. Appl. Microbiol. 26:381-385.
