APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1976, p. 274-279 Copyright © 1976 American Society for Microbiology

Vol. 31, No. 2 Printed in U.S.A.

Recovery of Staphylococcal Enterotoxin from Foods by Affinity Chromatography CONSTANTIN GENIGEORGIS* AND JASON K. KUO Department of Epidemiology and Preventive Medicine, School of Veterinary Medicine, University of California, Davis, California 95616

Received for publication 9 October 1975

Extraction, concentration, and serological detection of staphylococcal enterotoxins from foods are laborious and time consuming. By exposing food extracts to an insoluble matrix tagged with specific anti-enterotoxin B, we have been able to recover the toxin from foods in a sensitive and rapid way. After mixing the reagents for 2 h at room temperature, immunoglobulin G antibodies were attached to CNBr-activated Sepharose 4B at pH 8.5 (0.1 M carbonate buffer with 0.5 M NaCl). Sepharose-antibody complex (1 ml) specifically recovered 0.1 to 30 ,g of enterotoxin B from 400 ml of food extract (100 g of food) after mixing for 2 h at 4 C. The Sepharose-antibody-toxin complex was washed with 0.02 M phosphate-buffered saline at pH 7.2, and the toxin was dissociated by 2 to 4 ml of 0.2 M HCl-glycine plus 0.5 M NaCl buffer at pH 2.8. The recovered enterotoxin was free of interfering food components and could be detected serologically. Work to couple antibodies A, B, C, D, and E to Sepharose to recover all five toxins in one step is under study.

Staphylococcal food poisoning remains one of the two most common types of food poisoning in this country (5, 12). During the last 10 years, outstanding advances have been made in the elucidation of the environmental conditions under which staphylococci grow and produce their enterotoxins and in the development of diagnostic procedures for the detection of these toxins in foods. Regulatory agencies, the food industry, and academic researchers are always in need of simple, sensitive, reliable, rapid, and inexpensive diagnostic procedures. Such procedures allow regulatory agencies to screen large numbers of food samples, enable the food industry to establish mass screening programs, and permit researchers to plan additional experiments to learn more about the physiology of enterotoxin production. Detection of enterotoxins present in pure form in solution by the use of a number of different serological methods based on antigenantibody reactions is highly successful (2). In the presence of increasing amounts of organic compounds present in the culture media or food or produced by staphylococci, the serological methods become less reliable and require a certain degree of enterotoxin purification. It is this purification that remains the bottleneck of the whole detection effort. In practice, the procedure for the detection of enterotoxins by serological methods involves 274

four steps: extraction of the enterotoxins from foods, purification, concentration to detectable levels, and detection. The current recommended procedures for the detection of staphyloccal enterotoxins in foods are laborious and time consuming. The most extensively tested method for extraction, purification, and concentration of enterotoxins is the method of Casman and Bennett (6). Depending on the type of food, a number of modifications of the Casman and Bennett method (14, 24, 32) are available that, with the detection step, might take from 1 to 7 days. Most of the methods for the recovery of entertoxins from foods are based on the use of ion-exchange chromatography. Recently, solidphase radioimmunoassay methods for the detection of enterotoxins have been reported (9, 18). The methods are fast, simple, and sensitive, but they require special handling of reagents and equipment. One of the most outstanding advances in biochemical separation methods is the development of affinity chromatography (10). The new method permits the isolation of substances according to their biological function and, thus, differs radically from conventional chromatographic techniques in which separation depends on physical and chemical differences between substances. Because of the unique specificity of antigen-antibody interactions, affinity chromatography allows the separation of either

VOL. 31, 1976

RECOVERY OF STAPHYLOCOCCAL ENTEROTOXIN

antigen or antibody in highly purified form (11, 21, 28, 31). In this paper we report the development of a sensitive and rapid method for the recovery of staphylococcal enterotoxin B from culture media and from foods using affinity chromatography. MATERIALS AND METHODS Enterotoxin reagents. Pure enterotoxin B was supplied by M. S. Bergdoll and E. H. Schantz, both of the Food Research Institute, University of Wisconsin, Madison. Specific rabbit antisera and enterotoxin C were prepared in the laboratory (13). Pure immunoglobulin G (IgG) was prepared by heating 5 ml of antiserum for 30 min at 56 C, centrifuging, and then filtering the supernatant through a Sephadex (Pharmacia, Uppsala, Sweden) column (2.5 by 85 cm). The Sephadex had been previously equilibrated with 0.1 M tris(hydroxymethyl) aminomethane (Tris) buffer, pH 8.1, with 0.2 M NaCl. All fractions of 5 ml of the protein peak at an optical density at 280 nm (OD2,.) with antibody activity as determined by the microslide gel diffusion method (6) against enterotoxin B were pooled. The IgG of the pooled fractions was precipitated with 50% saturated (NH4)2SO4 overnight, and the precipitates were redissolved in 5 ml of Tris buffer and refiltered through the Sephadex column. The main fractions of the antibody peak were pooled and stored in the freezer until needed. Microbial species. Staphylococcus aureus strain 137 (ATCC 19095) was furnished by M. S. Bergdoll; S. aureus S-6, 196-E, 243 (ATCC 14458), 472, 587, 790, and Wood-46 were orginally obtained from the late E. P. Casman and R. Bennett of the Food and Drug Administration, Washington, D.C.; S. epidermidis strains and Micrococcus lysodeikticus (ATCC ISU-1) came from our collection. Enterotoxin assay. The microslide (6), the singlegel diffusion (29), and the reversed passive hemagglutination (RPHA) (25) methods were used for qualitative and quantitative assay of enterotoxin B. Sensitization of formalinized sheep erythrocytes with the antibody and the RPHA test were performed as described by Uemura et al. (27). Coupling of antibody to Sepharose. The coupling of enterotoxin B IgG to the Sepharose 4B (Pharmacia) matrix was done by the method of Cuatrecasas and Anfinsen (10). Ten milliliters Sepharose 4B activated with CNBr was washed with 15 volumes of ice-cold coupling buffer (0.1 M carbonate buffer, pH 8.5, with 0.5 M NaCl) in a Buchner funnel. Three milliliters of antibody solution was then added to the funnel and mixed with the moist Sepharose, and the suspension was transferred to a 25-ml Erlenmeyer flask. The flask was then placed on a reciprocal water bath shaker (140 rpm) and incubated for 2 h at room temperature. At the end of the incubation, the coupled Sepharose was washed with carbonate buffer and with glycine buffer (0.2 M HCl-glycine, pH 2.8, with 0.5 NaCl) alternatively until the OD20o of the washings was not measurable. The washed Sepharose was finally

275

equilibrated in buffered saline (0.02 M phosphate buffer, pH 7.2, with 0.15 M NaCl and 1:10,000 merthiolate) and stored in the refrigerator until needed. Protein determination. Protein measurements were done by the method of Lowry et al. (20).

RESULTS The recovery of staphylococcal enterotoxin B by affinity chromatography was investigated in a number of experiments, each of which evaluated the various steps of the procedure. Coupling of antibody. Early studies indicated that more than 75% of the antibody was coupled to Sepharose in the presence of carbonate buffer, pH 8.5, and less than 56% in the presence of phosphate buffer (0.2 M, pH 6.5, with 0.5 M NaCl). The carbonate buffer was used for the rest of the study. The efficiency of the coupling procedure was increased to over 95% antibody attachment when the initial antibody was previously dialyzed against the coupling buffer. This approach was used routinely for the rest of the study. Amounts as high as 7.5 mg of antibody could be coupled to 1 ml of Sepharose, but for reasons of economy of reagents smaller amounts were coupled. Recovery of enterotoxin from buffers. Amounts of 0.1 ,ug of pure enterotoxin B were added to 2 ml to 100 ml of buffered saline (0.02 M phosphate, pH 7.2, with 0.15 M NaCl). The toxin solution was filtered through columns (0.6 by 3.5 cm) containing 1 ml of Sepharose-antibody at rates of 1 to 2 ml/min. Using the singlegel diffusion test (29), it was determined that 1 ml of Sepharose containing 500 jig of antibody could bind up to 30 ,ug of pure toxin. Elution of enterotoxin from Sepharose-antibody complex. The use of glycine buffer (pH 2.8), urea (4 M), propionic acid (1 M), guanidine hydrochloride (5 M), and potassium thiocyanate (2 to 4 M) was evaluated for their efficiency in dissociating the antigen-antibody complex to permit recovery of enterotoxin. Urea, guanidine hydrochloride, and potassium thiocyanate failed to show any antigen-antibody dissociation. Propionic acid was also unsatisfactory because of the release of large amounts of protein from the column, possibly due to dissociation of antibody-antigen complex from the Sepharose. Two to four milliliters of glycine buffer per 1 ml of Sepharose was found to be the most efficient buffer in dissociating more than 95% of the enterotoxin attached to Sepharose-antibody complex. Recovery of enterotoxin B from staphylococcal culture supernatants. Supernatants of toxigenic and nontoxigenic staphylococcal cul-

276

APPL. ENVIRON. MICROBIOL.

GENIGEORGIS AND KUO

tures and micrococcal cultures grown in brain heart infusion (Difco) or PHP-NAK broth (11) for 24 to 36 h were lyophilized or kept frozen until use as sources of enterotoxins or controls. In a typical experiment, fresh or reconstituted lyophilized culture supernatant adjusted to pH 7.2 was filtered through a column (0.6 by 3.5 cm) containing 1 ml of Sepharose-anti-enterotoxin complex. Next the column was washed with at least 20 ml of buffered saline, and the enterotoxin was eluted with 9 ml of glycine buffer. The OD280 and the amount of enterotoxin in the eluant in one such experiment are shown in Fig. 1. Since the 120 gg of enterotoxin B present in the culture supernatant was much higher than the maximum amount that can be taken up by the 1 ml of Sepharose, most of the toxin passed through the column without reacting with the antibody. Obviously all of the toxin could be recovered if the conjugated Sepharose was more than 4 ml. Recovery of enterotoxin B from foods. Effort was made to recover enterotoxin B added to Italian style dry salami of various ages, ched-

dar cheese, and fresh milk by affinity chroma-

tography.

Solid food (100 g) was homogenized in a blender with 400 ml of distilled water and 75 ,ug of enterotoxin B (1 ml of culture supernatant). The pH of the homogenate or the milk was brought to 4.6 with 1.0 N HCl before it was centrifuged at 20,000 x g for 30 min at 4 C. The supernatant was not treated with chloroform as described by others (24, 32) because the latter had some effect on the ability of the Sepharoseantibody complex to bind enterotoxin. The pH of the supernatant was adjusted to 7.2, centrifuged if there was any turbidity, and then mixed with 3 ml of wet Sepharose-antibody conjugate in a beaker for 2 h at 4 C (batch method). The suspension was then placed in a column (2 cm in diameter) with a sintered-glass filter, drained, and then washed with 50 ml of buffered saline. The enterotoxin was eluted from the column with 12 ml of glycine buffer. Each fraction of 3 ml was adjusted immediately to pH 7.2 with 1 N NaOH and then was tested for the presence of enterotoxin by gel diffusion and RPHA. More than 75% of enterotoxin added to foods was recovered by the afflnity chromatography method as determined by the D {ADING gel diffusion tube test. Nearly 95% of the toxin 1.8F recovered was present in the first 9 ml of GEIL DIFFUSION TEST eluent. Limit of enterotoxin recovery. Amounts of 1.6 enterotoxin (pure or in culture supernatant) ranging from 0.1 to 200 ug were added to 500 ml 1.4 of buffer or 100 g of food. By using the batch absorption method, we were able to recover HCI-GLYCIE BUFFE R_ II 1.2 _ more than 75% of the pure enterotoxin added to (pH 2.8) food, even at the 0.1 ,ug level, and more than 95% when the toxin was added to the buffer. 1.0 I, Reverse passive hemagglutination was used to determine the pure toxin at levels below 0.3 ,ug/ Ls 0.8 ml. This method could detect a minimum of 0.001 ug of enterotoxin B/ml when the pure enterotoxin was dissolved in phosphateor buffered saline. Food extracts without added 04 \ toxin, but treated with conjugated Sepharose and formalinized erythrocytes, served as negaII II tive controls when we determnined the mini02 mum amounts of enterotoxin recovered by the J" 2 4 Sepharose-antibody complex. 6 8 10 12 When fresh or reconstituted culture supernatants of enterotoxigenic strains other than type FRACTIONS B, non-enterotoxigenic S. aureus, S. epidermiFIG. 1. Recovery of enterotoxin B from 4 ml of dis, micrococcal species, and salami, cheese, brain heart infusion broth culture supernatant by 1 ml of conjugated Sepharose. Column size, 0.6 by 3.5 and milk extracts were exposed to conjugated Sepharose, no cross-reacting substances to type cm; flow rate, 6 drops/min. Column washed with 20 ml of buffered saline and toxin dissociated with 9 ml B enterotoxin were eluted with glycine buffer, of glycine buffer and collected in 3-ml fractions. as determined by the microslide test (Table 1). Naturally occurring hemagglutinins in foods Quantitation of toxin based on tube single-gel diffusion test. were removed completely from the food extracts

r',,

.1

I

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RECOVERY OF STAPHYLOCOCCAL ENTEROTOXIN

277

TABLE 1. Reaction of food extracts and culture supernatants of various cocci purified by affinity chromatography and tested for the presence of enterotoxin B by the microslide and RPHA methods Preparation Preparation

Type of enterotoxin pro-

~~duced

RPHA recin reaction

RPHA end point well no.

+ + -

10 8

-

+ + + + + + + +

18 16 10 4 8 9 10 5

Microslide action

re-

Pure enterotoxins B,1 ug/ml Cl, 5 ug/ml C,, 500 ug/ml

C, C,

S. aureus strains S-6 243 137 196-E 472 790 587 Wood-46

A,B B C A D E Toxin type unknown Nontoxigenic

Other species S. epidermidis (BP5, 15, 23, 47) M. lysodeikticus

Nontoxigenic

-

-

0

Nontoxigenic

-

-

0

-

-

0

-

-

0 0 0

B

Food-media Brain heat infusion Nontoxigenic broth Milk Nontoxigenic Cheese Nontoxigenic Various dry salamis Nontoxigenic a For purification, see references 4 and 11. '9 For purification, see text and reference 8.

by the affinity chromatography approach, as determined by RPHA. Yet, affinity chromatography did not eliminate completely hemagglutinins present in culture supernatants of all of the S. aureus strains and pure enterotoxin C tested by RPHA. Apparently, these substances reacted with the antibodies to pure enterotoxin B and were recovered when the conjugated Sepharose was treated with the glycine buffer. Absorption of the elutates three times with formalinized sheep erythrocytes did not remove the hemagglutinating principle. Further studies indicated that the hemagglutinins were non-dialyzable and not inactivated by trypsin, but their activity was decreased by 50% after heating at 100 C for 35 min (Table 2). Filtration through Sephadex G-10 to G-100 indicated chromatographic behavior very similar to enterotoxins A, B, and C. DISCUSSION In the present work, affinity chromatography has been shown to be a powerful tool for the recovery of at least 0.1 ,tg of enterotoxin B from 100 g of food (0.001 ,ugg). It has been suggested that as little as 1 ,ug of enterotoxin can make a person sick (2). Assuming also that a person

+

+ + -

-

-

-

-

0

TABLE 2. Heat resistance (at 100 C) of a hemagglutinin" produced by S. aureus strains and reacting with antibodies to pure enterotoxin B after purification by affinity chromatography Min

RPHA end point well no.

0 20 35 45

8 6 4 4

"Prepared from S. aureus strain 137 grown in brain heart infusion broth for 24 h.

might eat 100 g of a toxic food, then a food with 0.01 gg of enterotoxin per g can cause food poisoning. Gilbert and Wieneke (14) reported that they detected 0.01 to 0.075 ,ug of enterotoxin per g in foods involved in food poisoning in England. It is obvious from the above that a satisfactory method for the recovery of enterotoxins from food should be able to detect at least 0.01 ,ug/g of food. The current Food and Drug Administration ion-exchange chromatographic method has been claimed to recover 0.0006 ,g/g of foods, as determined by the microslide gel diffusion (1). Our approach recovered at least

278

GENIGEORGIS AND KUO

0.001 Ag/g of food. In two experiments we failed recover 0.0001 ,ug/g using the RPHA technique. The great advantage of affinity chromatography lies in the fact that it is very rapid and allows, in one step, purification and concentration of enterotoxins to be detected by one of the available serological methods. If the molecular weight of IgG is considered to

to be 1.5 x 10; and enterotoxin B about 3 x 104,

and assuming that one molecule of IgG can bind at the most two molecules of enterotoxin, then 500 ,.g of pure and specific antibody in 1 ml of Sepharose should bind about 200 ,tg of enterotoxin instead of the maximum 30 ,g observed. The antibody inefficiency in recovering enterotoxin is understandable since CNBr couples with free amino groups and the IgG molecules are attached in a random fashion. Antigenbinding (Fab) sites are not all available for binding. The presence of other antibodies unrelated to enterotoxin might also contribute to the inefficiency observed. We are currently trying to increase the efficiency of coupled antibody to bind enterotoxin by using a C6 to C,,, chain molecule as an arm between the Sepharose and antibody molecules (10). Hemagglutinins exhibiting some of the characteristics observed in this study and interfering with the detection of enterotoxin B have been reported previously as being present in staphylococcal cultures (19). There are three possible explanations for the observed '"nonspecific" hemagglutinations. (i) The IgG used in the study has not only specific enterotoxin B antibodies but also minor antibodies to other products produced by various S. aureus strains. These products may be chemically related to enterotoxin B, or they may be different. However, they behave similarly to enterotoxin chromatographically or electrophoretically. (ii) The enterotoxins contain varying amounts of different, but structurally related or similar, antigenic determinants. (iii) Both of the above factors contribute to the problem. The heterogeneity of staphylococcal enterotoxins B and C has been demonstrated by resolving the pure toxins by hydroxyl apatite chromatography or isoelectric focusing to toxic and nontoxic components immunologically identical for each toxin (7, 22). A number of workers (3, 15-17) have demonstrated antigenic and chemical similarities among staphylococcal enterotoxins. In the present study, testing of pure enterotoxin C1 purified in 1971 (13) according to Borja and Bergdoll (4) by RPHA suggested that enterotoxin Cl has some similarity to enterotoxin B. Yet, when the

APPL. ENVIRON. MICROBIOL.

Cl toxin was chromatographed in a hydroxyl apatite column, as described by Chang et al. (7) for enterotoxin B, the major toxic peak obtained was free of any hemagglutinating property (Table 1). As far as we know, the immunochemical similarities among the various components obtained by isoelectric focusing or by hydroxyl apatite chromatography of pure enterotoxins have not been evaluated, though components of each serological type have been considered immunologically identical. The presence of beta-hemolysin as a persistent impurity in preparations of staphylococcal enterotoxin and nuclease has been demonstrated (8). Such impurity induces production of minor antihemolytic antibodies during the immunization of animals for the production of specific antibodies to enterotoxin. Thus, hemolysin in the enterotoxin or antihemolytic antibodies in the anti-enterotoxin serum may cause problems during the application of the highly sensitive hemagglutination method. The accuracy of this method depends wholly on the monovalency of the antiserum and the purity of antigen used for its production (8). Although beta-hemolysin is heat labile, some activity may survive 10 to 30 min of boiling (8, 26, 30). Beta-hemolysin is also sensitive to trypsin (30). In our studies, trypsin treatment of the interfering hemagglutinin(s) did not reduce its activity. We have also been unable to observe any hemolytic activity at a level of 1 mg of the pure enterotoxin B per ml that was used for the production of the antiserum in this study. If there is, indeed, activity, it should be below the level detectable by the sheep blood agar method (8). The possible role of protein A to the nonspecific hemagglutinations observed is minimized by the fact that this staphylococcal product is sensitive to trypsin (23). The present study has demonstrated the applicability of affinity chromatography for the recovery of enterotoxin B from culture media and foods in a rapid and simple way. Minute amounts of staphylococcal products possibly related to enterotoxin B, but still enough to cause cross-reactions in the RPHA test, demonstrated the need for highly purified enterotoxins to be used for the production of specific antibodies. Work is now in progress for the production of such specific antibodies. Work is also in progress for the attachment of type B and other types of enterotoxin antibodies to the same preparation of Sepharose. This would allow the recovery of all known enterotoxins from foods in one simple step. For the time being, the use of gel diffusion methods offers the best ap-

VOL. 31, 1976

RECOVERY OF STAPHYLOCOCCAL ENTEROTOXIN

proach for the specific detection of enterotoxin B recovered from foods by affinity chromatography. ACKNOWLEDGMENTS This investigation was supported by Pan American Health Organization grant RD-AMRo-3189. We wish to thank M. S. Bergdoll and E. J. Schantz for supplying pure staphylococcal enterotoxin B. LITERATURE CITED 1. Bennett, R. W., S. A. Keoseyan, S. R. Tatini, H. Thota, and W. S. Collins. 1973. Staphylococcal enterotoxin: a

2. 3.

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14.

comparative study of serological detection methods. Can. Inst. Food Sci. Technol. J. 6:131-134. Bergdoll, M. S. 1970. Enterotoxins, p. 265-326. In T. C. Monti, S. Kadis, and S. J. Ajl (ed.), Microbial toxins, vol. 3. Academic Press Inc., New York. Bergdoll, M. S., R. N. Robbins, K. Weiss, E. R. Broja, I. Y. Huang, and F. S. Chu. 1973. The staphylococcal enterotoxins: similarities, p. 390-396. In J. Jeljaszewicz (ed.), Staphylococci and staphylococcal infections. Polish Medical Publishers, Warsaw. Borja, C. R., and M. S. Bergdoll. 1967. Purification and partial characterization of enterotoxin C produced by Staphylococcus aureus strain 137. Biochemistry 6:1467-1473. Bryan, L. F. 1970. The epidemiology of staphylococcal food poisoning, p. 1-14. In New York State Agricultural Experiment Station Research Circular, no. 23, Geneva, N.Y. Casman, E. P., and W. Bennett. 1965. Detection of staphylococcal enterotoxin in food. Appl. Microbiol. 13:181-189. Chang, P. C., N. Dickie, and F. S. Thatcher. 1971. Hydroxyl apatite column chromatography of enterotoxin B. Can. J. Microbiol. 17:296-297. Chesbro, W., and V. Kucic. 1971. Beta hemolysin: persistent impurity in preparations of staphylococcal nuclease and enterotoxin. Appl. Microbiol. 22:233241. Collins, W. S., A. D. Johnson, J. F. Metzger, and R. W. Bennett. 1973. Rapid solid-phase radioimmunoassay for staphylococcal enterotoxin A. Appl. Microbiol. 25:774-777. Cuatrecasas, P., and E. B. Anfinsen. 1970. Affinity chromatography. Methods Enzymol. 22:345-377. Dandiliker, W. B., and V. A. de Saussure. 1968. Antibody purification and neutral pH utilizing immunospecific adsorbents. Biochemistry 5:357-365. Genigeorgis, C. (ed.). 1974. Recent developments on staphylococcal food poisoning, p. 34-93. Procedures of the 16th Annual Food Hygiene Symposium. Academy of Health Science, U.S. Army Veterinary Division, San Antonio, Tex. Genigeorgis, C., M. S. Foda, A. Mantis, and W. W. Sadler. 1971. Effect of sodium chloride and pH on enterotoxin C production. Appl. Microbiol. 21:862866. Gilbert, R. J., and A. A. Wieneke. 1973. Staphylococcal food poisoning with special reference to the detection a

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of enterotoxin in food, p. 273-283. In B. Hobbs and J. H. B. Christian (ed.), Microbiological safety of food. Academic Press Inc., New York. Gruber, J., and G. G. Wright. 1969. Ammonium sulfate coprecipitation antibody determination with purified staphylococcal enterotoxins. J. Bacteriol. 99:18-24. Huang, I. Y., J. Schantz, and M. S. Bergdoll. 1975. The amino acid sequence of the staphylococcal enterotoxins. Jpn. J. Med. Sci. Biol. 28:73-75. Johnson, H. M., J. A. Bukovic, and P. E. Kauffman. 1972. Antigenic cross-reactivity of staphylococcal enterotoxins. Infect. Immun. 5:645-647. Johnson, H. M., J. A. Bukovic, and P. E. Kauffman. 1973. Stap;aylococcal A and B: solid-phase radioimmunoassay. Appl. Microbiol. 26:309-313. Johnson, H. M., H. F. Hall, and M. Simon. 1967. Enterotoxin B: serological assay in cultures by passive hemagglutination. Appl. Microbiol. 15:815-818. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. Mannik, M., and D. E. Stage. 1971. Antibody-agarose immunoadsorbents: complete removal of classes of immunoglobulins from serum. J. Immunol. 196:1670-

1672. 22. Metzger, J. F., A. D. Johnson, and L. Spero. 1975. Intrinsic and chemically produced microheterogeneity of Staphylococcus aureus enterotoxin type C. Infect. Immun. 12:93-97. 23. Oeding, P., and A. Grov. 1972. Cellular antigens, p. 333-356. In J. 0. Cohen (ed.), The staphylococci. Wiley-Interscience, New York. 24. Reiser, R., D. Conaway, and M. S. Bergdoll. 1974. Detection of staphylococcal enterotoxin in foods. Appl. Microbiol. 27:83-85. 25. Silverman, S. J., A. R. Knott, and M. Howard. 1968. Rapid, sensitive assay for staphylococcal enterotoxin and a comparison of serological methods. Appl. Microbiol. 16:1019-1023. 26. Thatcher, F. S., and B. H. Matheson. 1955. Studies with staphylococcal toxins. II. The specificity of enterotoxin. Can. J. Microbiol. 1:382-400. 27. Uemura, T., G. Sakaguchi, and H. P. Riemann. 1974. In vitro production of Clostridium perfringens enterotoxin and its detection by reversed passive hemagglutination. Appl. Microbiol. 26:381-385. 28. Weintraub, B. 1970. Concentration and purification of human chorionic somatomammotropin (HCS) by affinity chromatography: application to radioimmunoassay. Biochem. Biophys. Res. Commun. 39:83-89. 29. Weirether, F. J., E. E. Lewis, A. J. Rosenwald, and R. E. Lincoln. 1966. Rapid quantitative serological assay of staphylococcal enterotoxin B. Appl. Microbiol. 14:284-291. 30. Wiseman, G. M. 1965. Some characteristics of the betahemolysin of Staphylococcus aureus. J. Pathol. Bacteriol. 89:187-207. 31. Wofsy, L., and B. Burr. 1969. The use of affinity chromatography for the specific purification of antibodies and antigens. J. Immunol. 103:380-382. 32. Zehren, V. L., and V. F. Zehren. 1968. Examination of large quantities of cheese for staphylococcal enterotoxin A. J. Dairy Sci. 51:635-644.

Recovery of staphylococcal enterotoxin from foods by affinity chromatography.

Extraction, concentration, and serological detection of staphylococcal enterotoxins from foods are laborious and time consuming. By exposing food extr...
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