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Detection of Staphylococcal Enterotoxin☆
Detection of Staphylococcal
Enterotoxin~:;
N. Dickie Division of Microbiology Food and Dru~ D:rectorate Ottawa, Canada
Abstract Current methodology for the detection of staphylococcal enterotoxins in food is evaluated. The potential advantages of the solid-phase radioimmunoassy technique for the quantitative estimation of enterotoxins are discussed.
Resume Dne evalUfltion est faite de la methodologie usuelle de la detection des enterotoxines de staphylocoques dans les aliments. La discussion est orientee sur les avantages potentiels de la technique de la radioimmunologie sur phase solide pour I'estimation quantitative des enterotoxines.
'.L'he enterotoxins have been identified as specific proteins produced by the staphylococci, under certain conditions, in foods and in culture media. The ingestion of these substances by humans results in what is called staphylococcal food poisoning, characterized by vomiting and diarrhea, which occur usually within 6 hours after eating the contaminated food. Purification studies employing the monkey as a bioassay system revealed the existence of several enterotoxins which provoke emesis at the level of a few micrograms (10- 6 g) per kg body wt and which could be differentiated on the basis of their reaction with specific antibodies. Accordingly, a nomenclature was established for the enterotoxins, designating them enterotoxin A, B, C, etc. based on the criteria of immunologic specificity and the induction of an illness in monkeys analagous to the symptomology of the foodborne illness occurring in man. Inasmuch as this type of food-borne illness is of world-wide occurrence, the need for a practical and quantitative method for estimation of enterotoxin in food has posed a major problem in this field. Staphylococcal enterotoxin in food is most reliably detected in the laboratory by the emetic reaction provoked in monkeys fed the test material. However, the use of monkeys is an unwieldy and costly procedure in which relatively large numbers of animals are required to obtain quantitative data relating emetic dose to weight of toxin administered. Moreover, animals other than the monkey are relatively insensitive to enterotoxin unless they are injected intraperitoneally or intravenously, and animals such as rodents, which have no vomiting mechanism, cannot be used. Cats and kittens have been used with some success, but frequently specimens require inactivation of substances that provoke symptoms similar to those caused by enterotoxin when administered by parental routes. Another disadvantage with the cat assay is that cats are relatively insensitive to enterotoxin C.
Immunoassay Because the enterotoxins are antigenic, serological • Presented at the C.I.F.T. Annual Conference, July 12, 1969. Ottawa. Canada. J. lnst. Can. Technol. Aliment. Vol. 3, No 4, 1970
procedures based on the antigen-antibody reaction have been developed and are the only practical means presently available for the detection of staphylococcal enterotoxins in foods. To detect traces of enterotoxin in food, the toxin is first separated from food constituents and then concentrated enough to permit its serological identification. Methods such as single and double diffusion tube or microslide gel diffusion techniques are used qualitatively for the detection of the enterotoxins, although a single diffusion tube method has been adopted for their quantitative determination (Bergdoll, 1967). In this method, the rate of movement of the enterotoxin-antienterotoxin precipitin band formed in the agar column is measured, and enterotoxin concentration is calculated from the measured rates. The standard procedure, employing this technique, requires a period of several days, however, for completion.
Radioimmunoassay Immunochemical methods employing radio-active iodine-labeled antigens are potentially capable of providing greater specificity and considerably greater sensitivity in the measurement of antigen bound to antibody, compared with the standard precipitin methods. An immunoassay of this type is based on a reaction between radioactive iodine-labeled antigen and specific antibody, which results in formation of a labeled antigen-antibody complex. The addition of unlabeled antigen to this system reduces the amount of labeled antigen-antibody complexes by competitive inhibition (Fig. 1). Immunoassay procedures of this type have found application to the measurement of several peptide hormones in plasma (Yalow and Berson, 1964), and recently the successful radioimmunoassay of enterotoxin B has been reported. In this study (Gruber and Wright, 1967), the attachment of 1.5 atoms of iodine per molecule of enterotoxin B did not affect the immunologic specificity of the antigen as judged by the precipitin reaction. Iodine is preferentially SUbstituted on tyrosyl residues of proteins. Since amino acid analysis of the purified enterotoxins has revealed ~igh molar ratios of tyrosine, these proteins are theoretIcally amenable to iodination for use in the radioimmunoassay. Radioactivity contained in the antigen-antibody complexes can be measured by collecting the complexes on membrane filters, or by selective precipitation of the complex with ammonium sulfate.
Solid-phase Radioimmunoassay A new method of solid-phase radioimmunoassay (Catt and Tregear, 1967), based on the ability of antibody-coated polymers specifically to bind radioactive tracer antigen, is probably most suitable for meeting the requirements of a simple, rapid and
143
antibody +
r l25 -antigen ~
1
125
-antigen-antibody
+ antigen (from food. unlabeled)
H antigen - antibody Figure 1.
Competitive inhibition of 112 5 antigen reaction with antibody by unlabeled antigen.
inexpensive method for the quantitative determination of enterotoxins. It has been found that certain unsllb~tituted polymers such as polypropylene can adsorb antibody and the antibody can then bind an adequate f}lHll1tity of radioactive tracer antigen for use in the assay. ''''''hereas adsorption of antibody to glass is negligilJle, antibody from sera of relatively high titer ean be adsorlJed to polymeric surfaces, such as polypropylene tubes, by forces that are unaffected by conditions far more rigorous than those occurring in the assay. The procedure has been applied successfully to the detection of human placental lactogen and human growth hormone at the level of a few nanograms (10- 9 g)jml of plasma. All phases of the assay, including incubation and counting of bound radioactivity, are performed in antibody-coated plastic tubes. This provides for simplicity and for uniformity of results. In addition to providing the potential for a highly specific and sensitive measure of the antigen-antibo?~- reaction, the radioimmunoassay obviates problems tll'lsmg from secondary effects of the antigen·antibody reaction, such as the precipitin reaction in immunodiffusion experiments in which difficulties may be encountered in staining of bands for visualization. Moreover, by providing a sensitive measure of antiO'enbinding capacity, the usefulness of the method is indicated in the selection and evaluation of antisera in a variety of biological phenomena. Efforts in our laboratory to apply the solid-phase radioimmunoassay method for the detection of staphylococcal enterotoxins began with purification of enterotoxin B to obtain pure antigen for labeling studies. A purified preparation of the toxin, when subjected to the technique of isoelectric focusing, yielded three major components which could be distinguished ac-
144
cording to differences in their isoelectric points. The three components were found to produce a single, identical line of antigen-antibody in immunodiffusion experiments employing enterotoxin B antiserum, and provoked emesis in monkeys following intravenous administration. The existence of multiple electrophoretic forms of enterotoxin B has been recognized earlier in other laboratories (Schantz et al., 1965) and it has been suggested that these entities may differ from each other in their tertiary structure or conformation. Moreover, differences in toxicity of the electrophoretically distinct proteins have been demonstrated in piglets. The foregoing discussion serves to illustrate another instance where the potential usefulness of radioimmunoassay may be realized, viz., an evaluation of antigen-binding capacity of homologous antiserum for conformational isomers of a single protein. The list of potential applications of the method can be extended. Recently, the enterotoxin B molecule has been subjected to selective cleavage to determine the complete amino acid sequence of the toxin by analysis of the recovered fragments (Bergdoll et al., 1969). Radioimmunoassay provides a convenient method whereby isolated fragments of the molecule containing the amino acid sequences responsible for antigenicity may conceivably be identified. In general, the advantages of specificity, sensitivity and simplicity inherent in solid-phase radioimmunoassay appear to meet the requirements for a practical method for the quantitative determination of enterotoxins in foods.
References Bergdoll, M. S. 1967. The staphylococcal enterotoxins, p. 1. In R.I. Mateles and G. N. Wogan. (eds.), Biochemistry of Some Foodborne Microbial Toxins. The M.LT. Press, Cambridge. BergdOll, M. S., Huang, 1.-Y., Chu, F.S. and Borja, C. 1969. The chemistry of bacterial toxins: the staphylococcal enterotoxins. Paper presented to the Symposium on the Chemical Control of the Human Environment, Johannesburg. Catt, K. and Tregear, G. W. 1967. Solid-phase radioimmunoassay in antibody-coated tubes. Science, 158 :1570. Gruber, J. and Wright, G. G. 1967. Iodine-131 labeling of purified microbial antigens by micro-diffusion. Proc. Soc. Exp. BioI. Med., 126:282. SChantz, E. J., Roessler, W. G., Wagman, J., Spero, L., Dunnery, D.A. and BergdOll, M. S. 1965. Purification of staphylococcal enterotoxin B. Biochemistry, 4:1011. Yalow, R. S. and Berson, S. A. 1964. Immunoassay of plasma insulin. In D. Glick, (ed.), Methods of Biochemical Analysis. Interscience publishers, New York, 12:69. Received April 8, 1970
Can. Inst. Food Technol. J. Vol. 3, No.4, 1970
Enterotoxin~:;
N. Dickie Division of Microbiology Food and Dru~ D:rectorate Ottawa, Canada
Abstract Current methodology for the detection of staphylococcal enterotoxins in food is evaluated. The potential advantages of the solid-phase radioimmunoassy technique for the quantitative estimation of enterotoxins are discussed.
Resume Dne evalUfltion est faite de la methodologie usuelle de la detection des enterotoxines de staphylocoques dans les aliments. La discussion est orientee sur les avantages potentiels de la technique de la radioimmunologie sur phase solide pour I'estimation quantitative des enterotoxines.
'.L'he enterotoxins have been identified as specific proteins produced by the staphylococci, under certain conditions, in foods and in culture media. The ingestion of these substances by humans results in what is called staphylococcal food poisoning, characterized by vomiting and diarrhea, which occur usually within 6 hours after eating the contaminated food. Purification studies employing the monkey as a bioassay system revealed the existence of several enterotoxins which provoke emesis at the level of a few micrograms (10- 6 g) per kg body wt and which could be differentiated on the basis of their reaction with specific antibodies. Accordingly, a nomenclature was established for the enterotoxins, designating them enterotoxin A, B, C, etc. based on the criteria of immunologic specificity and the induction of an illness in monkeys analagous to the symptomology of the foodborne illness occurring in man. Inasmuch as this type of food-borne illness is of world-wide occurrence, the need for a practical and quantitative method for estimation of enterotoxin in food has posed a major problem in this field. Staphylococcal enterotoxin in food is most reliably detected in the laboratory by the emetic reaction provoked in monkeys fed the test material. However, the use of monkeys is an unwieldy and costly procedure in which relatively large numbers of animals are required to obtain quantitative data relating emetic dose to weight of toxin administered. Moreover, animals other than the monkey are relatively insensitive to enterotoxin unless they are injected intraperitoneally or intravenously, and animals such as rodents, which have no vomiting mechanism, cannot be used. Cats and kittens have been used with some success, but frequently specimens require inactivation of substances that provoke symptoms similar to those caused by enterotoxin when administered by parental routes. Another disadvantage with the cat assay is that cats are relatively insensitive to enterotoxin C.
Immunoassay Because the enterotoxins are antigenic, serological • Presented at the C.I.F.T. Annual Conference, July 12, 1969. Ottawa. Canada. J. lnst. Can. Technol. Aliment. Vol. 3, No 4, 1970
procedures based on the antigen-antibody reaction have been developed and are the only practical means presently available for the detection of staphylococcal enterotoxins in foods. To detect traces of enterotoxin in food, the toxin is first separated from food constituents and then concentrated enough to permit its serological identification. Methods such as single and double diffusion tube or microslide gel diffusion techniques are used qualitatively for the detection of the enterotoxins, although a single diffusion tube method has been adopted for their quantitative determination (Bergdoll, 1967). In this method, the rate of movement of the enterotoxin-antienterotoxin precipitin band formed in the agar column is measured, and enterotoxin concentration is calculated from the measured rates. The standard procedure, employing this technique, requires a period of several days, however, for completion.
Radioimmunoassay Immunochemical methods employing radio-active iodine-labeled antigens are potentially capable of providing greater specificity and considerably greater sensitivity in the measurement of antigen bound to antibody, compared with the standard precipitin methods. An immunoassay of this type is based on a reaction between radioactive iodine-labeled antigen and specific antibody, which results in formation of a labeled antigen-antibody complex. The addition of unlabeled antigen to this system reduces the amount of labeled antigen-antibody complexes by competitive inhibition (Fig. 1). Immunoassay procedures of this type have found application to the measurement of several peptide hormones in plasma (Yalow and Berson, 1964), and recently the successful radioimmunoassay of enterotoxin B has been reported. In this study (Gruber and Wright, 1967), the attachment of 1.5 atoms of iodine per molecule of enterotoxin B did not affect the immunologic specificity of the antigen as judged by the precipitin reaction. Iodine is preferentially SUbstituted on tyrosyl residues of proteins. Since amino acid analysis of the purified enterotoxins has revealed ~igh molar ratios of tyrosine, these proteins are theoretIcally amenable to iodination for use in the radioimmunoassay. Radioactivity contained in the antigen-antibody complexes can be measured by collecting the complexes on membrane filters, or by selective precipitation of the complex with ammonium sulfate.
Solid-phase Radioimmunoassay A new method of solid-phase radioimmunoassay (Catt and Tregear, 1967), based on the ability of antibody-coated polymers specifically to bind radioactive tracer antigen, is probably most suitable for meeting the requirements of a simple, rapid and
143
antibody +
r l25 -antigen ~
1
125
-antigen-antibody
+ antigen (from food. unlabeled)
H antigen - antibody Figure 1.
Competitive inhibition of 112 5 antigen reaction with antibody by unlabeled antigen.
inexpensive method for the quantitative determination of enterotoxins. It has been found that certain unsllb~tituted polymers such as polypropylene can adsorb antibody and the antibody can then bind an adequate f}lHll1tity of radioactive tracer antigen for use in the assay. ''''''hereas adsorption of antibody to glass is negligilJle, antibody from sera of relatively high titer ean be adsorlJed to polymeric surfaces, such as polypropylene tubes, by forces that are unaffected by conditions far more rigorous than those occurring in the assay. The procedure has been applied successfully to the detection of human placental lactogen and human growth hormone at the level of a few nanograms (10- 9 g)jml of plasma. All phases of the assay, including incubation and counting of bound radioactivity, are performed in antibody-coated plastic tubes. This provides for simplicity and for uniformity of results. In addition to providing the potential for a highly specific and sensitive measure of the antigen-antibo?~- reaction, the radioimmunoassay obviates problems tll'lsmg from secondary effects of the antigen·antibody reaction, such as the precipitin reaction in immunodiffusion experiments in which difficulties may be encountered in staining of bands for visualization. Moreover, by providing a sensitive measure of antiO'enbinding capacity, the usefulness of the method is indicated in the selection and evaluation of antisera in a variety of biological phenomena. Efforts in our laboratory to apply the solid-phase radioimmunoassay method for the detection of staphylococcal enterotoxins began with purification of enterotoxin B to obtain pure antigen for labeling studies. A purified preparation of the toxin, when subjected to the technique of isoelectric focusing, yielded three major components which could be distinguished ac-
144
cording to differences in their isoelectric points. The three components were found to produce a single, identical line of antigen-antibody in immunodiffusion experiments employing enterotoxin B antiserum, and provoked emesis in monkeys following intravenous administration. The existence of multiple electrophoretic forms of enterotoxin B has been recognized earlier in other laboratories (Schantz et al., 1965) and it has been suggested that these entities may differ from each other in their tertiary structure or conformation. Moreover, differences in toxicity of the electrophoretically distinct proteins have been demonstrated in piglets. The foregoing discussion serves to illustrate another instance where the potential usefulness of radioimmunoassay may be realized, viz., an evaluation of antigen-binding capacity of homologous antiserum for conformational isomers of a single protein. The list of potential applications of the method can be extended. Recently, the enterotoxin B molecule has been subjected to selective cleavage to determine the complete amino acid sequence of the toxin by analysis of the recovered fragments (Bergdoll et al., 1969). Radioimmunoassay provides a convenient method whereby isolated fragments of the molecule containing the amino acid sequences responsible for antigenicity may conceivably be identified. In general, the advantages of specificity, sensitivity and simplicity inherent in solid-phase radioimmunoassay appear to meet the requirements for a practical method for the quantitative determination of enterotoxins in foods.
References Bergdoll, M. S. 1967. The staphylococcal enterotoxins, p. 1. In R.I. Mateles and G. N. Wogan. (eds.), Biochemistry of Some Foodborne Microbial Toxins. The M.LT. Press, Cambridge. BergdOll, M. S., Huang, 1.-Y., Chu, F.S. and Borja, C. 1969. The chemistry of bacterial toxins: the staphylococcal enterotoxins. Paper presented to the Symposium on the Chemical Control of the Human Environment, Johannesburg. Catt, K. and Tregear, G. W. 1967. Solid-phase radioimmunoassay in antibody-coated tubes. Science, 158 :1570. Gruber, J. and Wright, G. G. 1967. Iodine-131 labeling of purified microbial antigens by micro-diffusion. Proc. Soc. Exp. BioI. Med., 126:282. SChantz, E. J., Roessler, W. G., Wagman, J., Spero, L., Dunnery, D.A. and BergdOll, M. S. 1965. Purification of staphylococcal enterotoxin B. Biochemistry, 4:1011. Yalow, R. S. and Berson, S. A. 1964. Immunoassay of plasma insulin. In D. Glick, (ed.), Methods of Biochemical Analysis. Interscience publishers, New York, 12:69. Received April 8, 1970
Can. Inst. Food Technol. J. Vol. 3, No.4, 1970