Production of a monovalent staphylococcal enterotoxic antiserum type A and its use for typing staphylococcal enterotoxins

A monovalent specific staphylococcal antiserum, type A, was obtained by means of the isolated and purified preparation of type A staphylococcal enterotoxin. This antiserum was proved to be identical to antiserum of the same type, manufactured by Serva Feinbiochemica GmbH & Co. (West Germany). The titer of the newly obtained antiserum in the precipitation test was 1 : 16, and its use allowed one to detect enterotoxin of the above-mentioned type at a concentration of 0.004 mg/ml. The study of 320 staphylococcal strains with the use of this antiserum revealed that 25 strains (7.8%) produced type A enterotoxin.     

Evaluation of ELISA and GM1-ELISA for detection of Salmonella enterotoxin.

The ELISA and GM1-ELISA, by using antiserum to purified Salmonella enterotoxin (SE), were standardized and carried out to screen salmonellae isolated from foods of animal origin for enterotoxigenicity. Of the 101 strains of Salmonella belonging to 15 different serogroups tested, 76 (75.24%) strains from 13 serogroups were found enterotoxigenic. ELISA correlated well with rabbit ligated ileal loop (RLIL) test for the detection of enterotoxin producing salmonellae with 24 test strains. ELISA also yielded positive reaction with 7 of 13 RLIL negative strains. GM1-ELISA could not be carried out as none of the 101 cell free culture supernatants (CFCS) were able to bind with GM1-ganglioside. ELISA and GM1-ELISA were also standardized with antiserum to cholera toxin for the detection of salmonellae producing cholera related enterotoxin. None of the 101 strains was found to produce cholera related enterotoxin. ELISA could detect as low as 15 ng/100 microliters of purified SE and 10 ng/100 microliters of cholera toxin when tested with their homologous antisera.     

Simplified Method for Purification of Clostridium perfringens Type A Enterotoxin

Purification of Clostridium perfringens type A enterotoxin from sporulated cells was simplified. The method consisted of precipitation of the enterotoxin from the extract of sonically treated cells at 40% saturation of ammonium sulfate at pH 7, differential solubilization in 0.02 M phosphate buffer, pH 6.7, and repeated gel filtration on Sephadex G-200. The purified enterotoxin was at least 98% pure in ultracentrifugation, polyacrylamide gel electrophoresis, and agar gel double diffusion. Recovery was over 74% from the sporulated cell extract. The toxin had biological activities of at least 4,700 mouse intravenous minimal lethal doses/mg of N, 3,900 capillary permeability-increasing U/mg of N in the guinea pig skin, and 210 rabbit intestinal loop distension U/mg of N. The toxin, containing no hexose, lipid, or nucleic acid, appeared to be identical in sedimentation constant, isoelectric point, and ultraviolet absorption spectrum to the toxin purified previously by different procedures.     

Thermal Inactivation of Staphylococcal Enterotoxin B in Veronal Buffer

The times and temperatures required to inactivate staphylococcal enterotoxin B were studied by use of the double-gel-diffusion technique to assay enterotoxin. Enterotoxin B (99 +% pure) was suspended in 0.04 M Veronal buffer, dispensed into borosilicate vials, and the vials were sealed and heated in an oil bath. An amount of 30 mug/ml of this toxin was reduced to less than 0.7 mug/ml in 103.0, 87.1, 70.5, 57.2, 39.1, 27.6, 16.4, and 12.0 min, respectively, at temperatures of 96, 99, 101.7, 104.4, 110, 115.6, 121, and 126.7 C. The end point for enterotoxin inactivation by gel diffusion was identical to that by intravenous injection of cats. Limited studies with crude enterotoxin B showed that the crude preparation was slightly more thermostable. The respective D values of crude and purified enterotoxin B were 64.5 and 52.3, 40.5 and 34.4, 29.7 and 23.5, 18.8 and 16.6, and 11.4 and 9.9 min at temperatures of 99, 104.4, 110, 115.6, and 121 C. The z value for purified enterotoxin B was 32.4 C. The experimental activation energy was 20,700 cal/g mole, standard enthalpy of activation at 120 C was 19,900 cal/g mole, standard entropy of activation at 120 C was -21.4 cal/g mole K, and the standard free energy of activation at 120 C was 28,200 cal/g mole.     

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 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 mug 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.     

Double-antibody solid-phase enzyme immunoassay for the detection of staphylococcal enterotoxin A.

A simple double-antibody enzyme immunoassay that uses a microtechnique was developed for detecting staphylococcal enterotoxin A in food products. Sample preparation can be completed in less than 15 min. Assay sensitivity ranges from 0.4 ng (20-h test time) to 3.2 ng (1- to 3-h test time) of toxin per ml of prepared sample. Separation and detection of enterotoxin from spiked food products ranged between 72 and 98% of the amount added.     

Rapid quantitative serological assay of staphylococcal enterotoxin B

A simple, rapid method, based on the Oudin single diffusion technique, is described for the quantitative assay of staphylococcal enterotoxin B. The method yields reproducible results without close control of such assay variables as temperature, antiserum dilution, and assay time, provided that the ionic strength is maintained above 0.2 n sodium chloride equivalent.     

Double-antibody solid-phase enzyme immunoassay for the detection of staphylococcal enterotoxin A.

A simple double-antibody enzyme immunoassay that uses a microtechnique was developed for detecting staphylococcal enterotoxin A in food products. Sample preparation can be completed in less than 15 min. Assay sensitivity ranges from 0.4 ng (20-h test time) to 3.2 ng (1- to 3-h test time) of toxin per ml of prepared sample. Separation and detection of enterotoxin from spiked food products ranged between 72 and 98% of the amount added.     

Quantitative Detection of Type A Staphylococcal Enterotoxin by Laurell Electroimmunodiffusion

The detection of staphylococcal enterotoxin A by the quantitative technique of electroimmunodiffusion is described. High dilutions of type-specific rabbit antiserum were used in 1% agarose gels, 1 mm thick, and prepared in 0.05-mug barbital buffer, pH 8.6. Volumes of 10 muliters containing 1.5 to 10 ng of toxin were electrophoresed out of 4-mm diameter wells at 5 mA/cm width of gel. The precipitin cones formed were made visible by first immersing the agarose gels in 0.2 M NaCl and then overlaying the surface with the purified globulin fraction of sheep serum against rabbit globulin, followed by soaking of the gels in 1% aqueous cadmium acetate and staining with 0.1% thiazine red in 1% glacial acetic acid. Fully extended cones, 4 to 23 mm in length depending on toxin concentration and antiserum dilution, were developed in 2 to 5 h of electrophoresis, and visualization was achieved within 2 to 3 h. Because the method is qualitative, quantitative, simple, rapid, and sensitive, it offers a practical tool for the detection of small amounts of bacterial toxins in contaminated foods. The method should also qualify as a sensitive detection device in biochemical procedures which attempt to trace, detect, and identify biological substances in nanogram quantities, provided these substances are antigenic and capable of forming a precipitate with their specific antibodies.     

Nature and reactivity of staphylococcal enterotoxin A monoclonal antibodies

Monoclonal antibodies from four clones (C5, C3, B2II, and B2I) directed against staphylococcal enterotoxin A were tested by the indirect enzyme-linked immunosorbent assay and double-gel immunodiffusion (micro-Ouchterlony) assay for the nature of heavy and light chain types. The reactivities of monoclonal antibodies were also tested by indirect enzyme-linked immunosorbent assay with various levels of purified staphylococcal enterotoxin A and various levels (dilutions) of monoclonal antibodies and saturation analysis-competitive indirect enzyme-linked immunosorbent assay. The heavy-chain isotype of monoclonal antibodies was found to be an unspecified subclass of immunoglobulin G1, and the light chain was the kappa type. Monoclonal antibodies from all of the clones exhibited high reactivity and nearly the same affinity to staphylococcal enterotoxin A in saturation analysis-competitive enzyme-linked immunosorbent assay. Purified immunoglobulin G from B2I yielded very high absorbance (1.2) at 405 nm with 1 ng of staphylococcal enterotoxin A as the coating antigen in the enzyme-linked immunosorbent assay. Monoclonal antibodies from B2I also neutralized the biological activity of staphylococcal enterotoxin A when tested by the kitten bioassay.     

Suitability of the enzyme treatment and ammonium sulfate precipitation method for detection of staphylococcal enterotoxin from different foods

Summary An enzyme treatment and ammonium sulfate precipitation procedure was adapted to the detection of staphylococcal enterotoxin from high-protein foods. The enterotoxin is extracted from food with distilled water, after which soluble proteins are acid-precipitated (pH 4.5) and the supernatant washed with chloroform (pH 7.5). The extract is then treated with trypsin andPseudomonas peptidase for 2 h at +37°C. Residual unhydrolyzed material is precipitated with 60% ammonium sulfate for 15 min at +4°C. The precipitate is redissolved in phosphate buffer and concentrated by dialysis against polyethyleneglycol. The concentrate is washed with chloroform and lyophilized. The dry material is dissolved in 0.2 ml distilled water and enterotoxin detected by the micro-slide method with 24 h incubation at +37°C. Using this method, it has been possible within three days to detect 0.2–1.0 g staphylococcal enterotoxin A added to minced meat, dry sausage, smoked fish, cheese and milk.     

The use of polyethylene glycol in radioimmunoassay of staphylococcal enterotoxins

The double-antibody radioimmunoassay of enterotoxins A, B, and C was modified by the addition of aqueous polyethylene glycol which precipitated double-antibody bound staphylococcal enterotoxins with little or no precipitation of free enterotoxin. The precipitate formed appeared as a thin coating at the bottom of the test tube and was not removed by aspiration. The procedure obviates the need for normal rabbit serum and for an additional wash of the precipitate.     

Identification of functionally active fragments of staphylococcal enterotoxin B.

It has been found that staphylococcal enterotoxin B contains a proteolysis-sensitive sequence in the cysteine loop formed by two half-cystines located in the middle of the toxin polypeptide chain. Fragments of the enterotoxin formed as a result of its digestion in this region have been isolated, their N-terminal sequences have been determined and sites of proteolysis have been identified. It has been demonstrated that the N-terminal fragment of staphylococcal enterotoxin B is capable of activating T cell proliferation in the culture of human mononuclear cells practically to the same degree as the intact enterotoxin. The toxin's C-terminal fragment possesses an ability to activate calmodulin-dependent enzymes and is probably the toxicogenic part of the enterotoxin.     

Capillary Tube Immunological Assay for Staphylococcal Enterotoxins

A simple assay is reported in which 1 mug of staphylococcal enterotoxins A, B, and D per ml was detected in less than 1 hr. Interfacial reaction of antisera and enterotoxin solutions in a 1-ml internal diameter capillary tube allowed rapid detection of sera type.     

Enterotoxin B: Serological Assay in Cultures by Passive Hemagglutination

Bis-diazotized benzidine hemagglutination with Formalinized sheep erythrocytes was adapted to the rapid and specific detection of enterotoxin B in staphylococcal culture fluids. There was complete agreement between hemagglutination inhibition (HI) and gel diffusion in detecting 8 enterotoxin B-positive cultures from a total of 68 staphylococcal cultures tested. The sensitivity of HI equals or exceeds that of gel diffusion. Also, results can be obtained in several hours, even with extremely low concentrations of enterotoxin, whereas it may require 24 hr to 1 week to obtain comparable results with gel diffusion. Problems associated with the presence of potent hemagglutinins for sheep erythrocytes in several staphylococcal culture fluids are discussed.     

Purification and some physicochemical properties of staphylococcal enterotoxin D.

A method was developed for the isolation of staphylococcal enterotoxin D in highly purified form from cultures of Staphylococcus aureus strain 1151m. The method involves removal of the toxin from the culture supernatant fluid with the ion-exchange resin CG-50 followed by chromatography on carboxymethylcellulose (twice) and by gel filtration on Sephadex G-75 (twice). The purified toxin is homogeneous by polyacrylamide gel and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and double gel diffusion tests. It is a simple, colorless, antigenic protein with an isoelectric point of 7.4 as determined by isoelectric focusing. Its molecular weight was determined to be 27 300 +/- 700 by molecular sieve chromatography on Sephadex G-100 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its serological activity is stable over a wide range of pH values (1.2--10.7). The enterotoxin consists of 236 amino acid residues and contains no free sulfhydryl groups. End-group analysis showed serine to be the NH2-terminal amino acid and lysine to be the COOH-terminal amino acid.     

Synthesis of a precursor to the B subunit of heat-labile enterotoxin in Escherichia coli.

Escherichia coli K-12 minicells were employed to investigate the biosynthesis of plasmid-encoded, heat-labile enterotoxin of E. coli. Two polypeptide species related to the B subunit of the toxin were expressed in the minicells. One of these polypeptides (molecular weight, 11,500) was immunoprecipitated by antiserum to cholera toxin. Because the B subunits of heat-labile enterotoxin and cholera toxin have common antigenic sites, we concluded that this species was the mature B subunit. The larger polypeptide (molecular weight, 13,000) is likely to be a precursor of the B subunit because it could be chased into the mature form. This conversion was inhibited by compounds which dissipate proton motive force, suggesting that processing requires energy.     

Nucleotide sequence of the type A staphylococcal enterotoxin gene.

We determined the nucleotide sequence of the gene encoding staphylococcal enterotoxin A (entA). The gene, composed of 771 base pairs, encodes an enterotoxin A precursor of 257 amino acid residues. A 24-residue N-terminal hydrophobic leader sequence is apparently processed, yielding the mature form of staphylococcal enterotoxin A (Mr, 27,100). Mature enterotoxin A has 82, 72, 74, and 34 amino acid residues in common with staphylococcal enterotoxins B and C1, type A streptococcal exotoxin, and toxic shock syndrome toxin 1, respectively. This level of homology was determined to be significant based on the results of computer analysis and biological considerations. DNA sequence homology between the entA gene and genes encoding other types of staphylococcal enterotoxins was examined by DNA-DNA hybridization analysis with probes derived from the entA gene. A 624-base-pair DNA probe that represented an internal fragment of the entA gene hybridized well to DNA isolated from EntE+ strains and some EntA+ strains. In contrast, a 17-base oligonucleotide probe that encoded a peptide conserved among staphylococcal enterotoxins A, B, and C1 hybridized well to DNA isolated from EntA+, EntB+, EntC1+, and EntD+ strains. These hybridization results indicate that considerable sequence divergence has occurred within this family of exotoxins.     

On the cross-reactivity of staphylococcal enterotoxins A, B, and C.

Strong cross-reactions were demonstrated for staphylococcal enterotoxins B (SEB) and C1 (SEC1) by antigen-binding capacity and by competitive binding ability. Both SEB and SEC1 combined completely with the heterologous antibody although requiring four times as much antiserum as the homologous enterotoxin and both displaced about one-third of the other enterotoxin from a heterologous antigen-antibody system. It is proposed that one of the three major antigenic determinants of these enterotoxins possesses a significant similarity but probably not an identity of structure. SEB and SEC1 did not combine with antiserum to enterotoxin A nor inhibit the reaction of SEA with anti-SEA. SEA had no intrinsic binding capacity for anti-SEB or anti SEC1 nor did it inhibit the binding of either enterotoxin to its own antibody. Affinity chromatography was employed to demonstrate that a small apparent binding of SEA to anti-SEB was due to antibody to SEA in the anti-SEB serum and that an almost complete displacement of SEC1 binding to anti-SEC1 was caused by contaminating SEC (about 0.01%) in preparations of enterotoxin A.     

Heat Inactivation of Enterotoxin A from Staphylococcus aureus in Veronal Buffer

Serological tests were used to determine the slope of the thermal inactivation curve of crude enterotoxin A in Veronal buffer (pH 7.2), and the resulting z value was 27.8 C. (50 F). Serological assays also showed that the heat inactivation at each time-temperature depended on the original concentration of enterotoxin A. The usefulness of the Oudin tube serological test for determining end points of inactivation of naturally produced enterotoxin A (not concentrated) is discussed. We concluded that this test cannot be used to determine end points of heat inactivation for enterotoxin A in the minute quantities naturally produced in foods.