Modified method for production and purification of Staphylococcus aureus enterotoxin B.

A medium containing 4% bio-trypcase and 1% yeast extract was used for the production of Staphylococcus aureus enterotoxin B. The yield obtained was estimated at 200 micrograms of enterotoxin per ml of S. aureus S-6 culture supernatant. The purification method involves chromatography on Biorex 70 resin, isoelectric focusing, and gel filtration on Sephadex G-100. The purified enterotoxin (isoionic point, pH 8.55) was shown to be homogenous protein with a molecular weight of 29,000 when tested by gel electrophoresis.     

Staphylococcal enterotoxin type D: isolation, purification, identification

The three step scheme of isolation and purification of staphylococcal type D enterotoxin was elaborated to obtain the homogeneous protein. The scheme includes protein concentration by (NH4)2SO4 saturation and subsequent fractionation on DEAE-cellulose and Sephacryl S-200. The yield of homogeneous protein is 25.5%. The molecular mass of the 29,600 D protein was identified by electrophoresis in polyacrylamide gel in the presence of SDS. The enterotoxic dose for staphylococcal type D enterotoxin is 5 mkg per kg of body mass as identified in experiments with cats. Immunological identity of the obtained protein was established to the commercial preparation of staphylococcal enterotoxin D produced by "Serva".     

Comparison of different purification procedure for extraction of staphylococcal enterotoxin A from foods.

Different procedures commonly used for extraction, purification, and concentration of staphylococcal enterotoxins from foods were investigated with 131I- and 125I-labeled staphylococcal enterotoxin A. Loss of labeled enterotoxin A was compared with loss of total nitrogen. The results showed that in most of the common procedures, such as gel filtration, ion exchange, and heat treatment, the percentage of loss of labeled enterotoxin A was greater than the loss of total nitrogen. Chloroform extraction and acid precipitation with hydrochloric acid had nearly the same effect on the purification of both labeled enterotoxin A and total nitrogen. Ammonium sulfate precipitation proved to be practical and was successfully used for purification of enterotoxin A from sausage extract. Simultaneous use of trypsin and Pseudomonas peptidase for treatment of food extracts considerably reduced food proteins capable of interfering with serological detection of enterotoxins but did not essentailly influence the loss of enterotoxin A.     

Scaled-up production and purification of Clostridium perfringens type A enterotoxin

Methods for small-scale production of Clostridium perfringens type A enterotoxin were unsuitable for large-scale culture of this organism. Rapid, efficient harvesting of 40 1 batch culture of Cl. perfringens was achieved by tangential flow micro-filtration with the Millipore Pellicon cassette system. Enterotoxin-containing extracts were prepared by passing concentrated suspensions of the harvested cells through a French pressure cell. The overall yield of purified enterotoxin was 38.8%. The toxin gave a single band on native polyacrylamide gels but formed high molecular weight aggregates in the presence of sodium dodecyl sulphate. These aggregates frequently occurred during storage of non-sterile enterotoxin preparations but could be separated from the monomer toxin by gel filtration on Sephadex G-100. Purified monomer enterotoxin had biological activities of 119.3 micrograms/kg mouse lethal dose when injected intraperitoneally and 3333 capillary permeability increasing units/mg protein in guinea pig skin. Thirty micrograms of the enterotoxin caused fluid accumulation in ligated rabbit ileal loops. Aggregated enterotoxin had no demonstrable biological or immunological activity.     

An enhanced protocol for expression and purification of heat-stable enterotoxin of enterotoxigenic Escherichia coli

We present an improved protocol for expression and purification of heat-stable enterotoxin (STa) of enterotoxigenic Escherichia coli (ETEC). In this protocol, controlled growth conditions at different pHs (7.4, 8.0, and 8.6) were adopted using a bioreactor. In addition, specific adsorbent resins, methacrylate, were used for STa purification. The bioreactor provided optimal ETEC growth at pH 7.4 with high STa production. Furthermore, methacrylate bounded specifically to STa and dramatically enhanced the purification process of STa. The STa-specific activity was high (8.9 × 10(6) units/mg protein), and the minimal effective dose of STa required for production of gut weight to remaining body weight ratio ≥ 0.083 was recorded as less than 0.2 ng in 2-3 days old suckling mice. The protocol presented, produces highly purified STa as documented by matrix-assisted laser desorption ionization-time of flight mass spectroscopy/. Also, as compared with the traditional methods, this procedure is trouble-free and practical for scale-up production and purification of STa peptides.     

Our experiences with isolation of heat-labile enterotoxin from Escherichia coli

From the Escherichia coli strain isolated from a patient suffering from diarrhoea a homogenate and concentrated culture filtrate were prepared. From these materials the heat-labile enterotoxin was isolated after its elution with 0.2 M D-galactose from Sepharose 6B column. The obtained enterotoxin was positive in the rabbit ileal loop test up to a concentration of 1 microgram protein/ml. In the immunodiffusion test it reacted in a concentration up to 5 micrograms protein/ml with anticholeragen and in a concentration up to 30 micrograms protein/ml with its specific antiserum. This antiserum was prepared by intramuscular immunization of rabbits by enterotoxin with complete Freund's adjuvant.     

Purification and properties of an enterotoxin from a coatless spore mutant of Clostridium perfringens type A

A method is described for isolating an enterotoxin from a coatless spore mutant (8-6) of Clostridium perfringens type A. The characteristics of this enterotoxin only slightly resembled those of previously isolated enterotoxins of C. perfringens. The type A (8-6) enterotoxin was found to be composed of two subunits of Mr 18 000 with isoelectric points of 3.8 and 4.3. The LD50 for mice was 39 micrograms/kg with 0.10 micrograms corresponding to one erythemal unit. The type A (8-6) enterotoxin was inactivated by heating for 10 min at 60 degrees C. The amino acid composition data of type A (8-6) and delta toxins was similar, but type A (8-6) and type A enterotoxins showed less similarity. This lack of similarity between type A and type A (8-6) enterotoxins was confirmed by the failure of anti-sera to type A enterotoxin to neutralize the type A (8-6) enterotoxin, in both the mouse and erythemal tests.     

Degree of purification of a chemical epidemic typhus vaccine of the heterogeneous ballast proteins

The degree of purification of chemical typhus vaccine from the ballast proteins of the substrate has been studied by chemical and immunochemical methods. The analysis of 11 production batches of the vaccine and a number of model preparations has revealed that chemical typhus vaccine is completely free from nonprotein nitrogen-containing components and, to a considerable extent, purified from ballast protein, its concentration being 0.033-0.242 mg per vaccination dose, and also possesses a high specific activity (198-1454 antigenic units per mg of protein. Ballast proteins include at least 5 egg antigens. The concentration of one of them varies between 0 and 5 micrograms per dose. A number of recommendations is made; their realization will make it possible to increase the degree of purification of chemical typhus vaccine.     

Production and characterisation of Campylobacter jejuni enterotoxin in a synthethic medium and its assay in rat ileal loops

A synthetic medium for production of Campylobacter jejuni enterotoxin was developed for the purposes of its purification by modifying syncase medium, replacing sucrose with glucose, and supplementing with 0.025% sodium pyruvate, 0.25% sodium metabisulphite, 0.001% ferric chloride and 0.1% L-cysteine, adjusted to pH 6.7. Culture filtrates of a human diarrhoeal and a chicken isolate, grown in this medium caused fluid accumulation ranging between 0.50-0.70 ml/cm of rat ileal loop. The kinetics of toxin production indicated a peak at 36 h and decline by 72 h, coinciding with the period of release of protease by the organism. At least 0.4 rat ileal loop units of enterotoxic activity was recovered per ml of culture filtrates and one unit of this toxin contained only 14 micrograms of protein. The toxin is heat-labile, pH dependent, nonhaemolytic, resistant to trypsin, sensitive to papain and pronase and may show subunit molecular weight analogy with CT subunits.     

Free-solution isoelectric focusing for the purification of Staphylococcus aureus enterotoxin C1

A free-solution isoelectric focusing protocol was developed for the preparative purification of Staphylococcus aureus enterotoxin C1 (SEC1). A toxin consisting of a single isoelectric species, pI 8.8, was purified. Thirty-nine milligrams of SEC1 was recovered from 3 liters of culture supernatant. This significantly improved purification scheme utilized ammonium sulfate precipitation and the Bio-Rad Rotofor isoelectric cell to complete isolation in 2 days, thereby avoiding the protein degradation prevalent when published procedures are used. The purification protocol developed here for SEC1 is used to illustrate the utility of Rotofor fractionation in the general purification of bacterial exotoxins.     

Fluid accumulation in mouse ligated intestine inoculated with Clostridium perfringens enterotoxin.

Clostridium perfringens enterotoxin, when inoculated into the ligated intestinal loop of mice, caused marked distension due to fluid accumulation. The increase in weight of the intestinal loop was proportional to the log dose of enterotoxin within a range from 1 to 16 micrograms. The fluid accumulation was arrested by washing the loop with saline or by injection of the specific anti-enterotoxin serum into the loop 5 or even 30 min after inoculation of the enterotoxin. A significant increase in weight of the loop was found as early as 10 min after inoculation of the toxin. These results may suggest that entergotoxin is neither bound firmly to the mucosal membrane nor permeates into the cells of the intestinal wall. The mouse intestinal loop test is economical, simple to perform, and applicable for quantitative determination of the enteropathogenic activity of C. perfringens enterotoxin.     

Fluid accumulation in mouse ligated intestine inoculated with Clostridium perfringens enterotoxin.

Clostridium perfringens enterotoxin, when inoculated into the ligated intestinal loop of mice, caused marked distension due to fluid accumulation. The increase in weight of the intestinal loop was proportional to the log dose of enterotoxin within a range from 1 to 16 micrograms. The fluid accumulation was arrested by washing the loop with saline or by injection of the specific anti-enterotoxin serum into the loop 5 or even 30 min after inoculation of the enterotoxin. A significant increase in weight of the loop was found as early as 10 min after inoculation of the toxin. These results may suggest that entergotoxin is neither bound firmly to the mucosal membrane nor permeates into the cells of the intestinal wall. The mouse intestinal loop test is economical, simple to perform, and applicable for quantitative determination of the enteropathogenic activity of C. perfringens enterotoxin.     

Purification of an enterotoxin produced by Bacillus cereus by immunoaffinity chromatography using a monoclonal antibody.

A murine monoclonal antibody (MAb) with high reactivity to an enterotoxin produced by Bacillus cereus was used to prepare an immunoadsorbent for purification of the enterotoxin. By immunoaffinity chromatography using the immunoadsorbent, approximately 25% of crude enterotoxin applied was recovered in the eluate. The purified enterotoxin was found to be electrophoretically and antigenically homogeneous. It also showed vascular permeability activity and mouse lethality, and caused fluid accumulation in mouse ligated intestinal loops, whereas it did not show any hemolytic and lecithinase activities. Thus, immunoaffinity chromatography proved useful in the purification of enterotoxin produced by B. cereus in terms of recovery, purity, and relative ease of performing the purification.     

Novel method for purification of staphylococcal enterotoxin A

A novel single-step procedure for the purification of staphylococcal enterotoxin A (SEA), namely, dye ligand affinity chromatography with the triazine dye Red A, was developed. SEA purified by this method produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The yield from 5 liters of culture supernatant was 0.113 g, corresponding to an overall yield of 55%. In some instances, purification of SEA from culture supernatants by dye ligand affinity chromatography produced two enterotoxin peaks that could be eluted from the column with 300 and 500 mM phosphate buffer (pH 6.8). Enterotoxin from these peaks produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but multiple bands were observed on isoelectric focusing gels. This method of purification represents a significant improvement in time, yields, and purity of enterotoxin over previously published purification methods.     

Improvements in the microtitre GM1 ganglioside enzyme-linked immunosorbent assay for Escherichia coli heat-labile enterotoxin

A variant of the microtitre GM1-ELISA for Escherichia coli heat-labile enterotoxin was studied. The test was improved by both reducing the assay time from 2 1/2 d to 8 h and by determining the most appropriate GM1 coating concentration. Coating the plates with greater than or equal to 3 micrograms of GM1/ml yielded a maximal sensitivity and ensured a linear relationship between the enterotoxin concentration and the extinction observed when using the final assay-procedure. Thus an optimal accuracy was obtained. This ELISA was 4- to 8-times more sensitive than the Vero cell monolayer assay. The sensitivity of this ELISA and of the chinese hamster ovary cell monolayer assay were identical.     

Novel method for purification of staphylococcal enterotoxin A.

A novel single-step procedure for the purification of staphylococcal enterotoxin A (SEA), namely, dye ligand affinity chromatography with the triazine dye Red A, was developed. SEA purified by this method produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The yield from 5 liters of culture supernatant was 0.113 g, corresponding to an overall yield of 55%. In some instances, purification of SEA from culture supernatants by dye ligand affinity chromatography produced two enterotoxin peaks that could be eluted from the column with 300 and 500 mM phosphate buffer (pH 6.8). Enterotoxin from these peaks produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but multiple bands were observed on isoelectric focusing gels. This method of purification represents a significant improvement in time, yields, and purity of enterotoxin over previously published purification methods.     

Identification of a new enterotoxin as enterotoxin C

Bergdoll, Merlin S. (University of Chicago, Chicago, Ill.), Concordia R. Borja, and Remedios M. Avena. Identification of a new enterotoxin as enterotoxin C. J. Bacteriol. 90:1481-1485. 1965.-Identification of a new enterotoxin was accomplished by purification of the enterotoxins produced by staphylococcal strains 137 and 361 and by the preparation of specific antitoxin to the enterotoxin. Toxicity of the preparations was determined in rhesus monkeys, and specificity of the enterotoxin-antitoxin reaction was determined in gel-diffusion plates. The enterotoxin has been designated enterotoxin C, and staphylococcal strain 137 (ATCC 19095) was selected as the prototype strain.     

Radioimmunofixation of human ferritin following serum isoelectric focusing

Radioimmunofixation of human ferritin following isoelectric focusing of serum was developed to study the microheterogeneity of this protein in native serum without previous purification or concentration. This method requires only 2-10 microliter of serum and can be used with levels of ferritin as low as 10 micrograms/l. In this way, the extensive microheterogeneity of this protein was revealed, since in some cases it produced as many as 35 bands with isoelectric points in a pH range of 4.95-5.9. Very different isoelectric focusing patterns (spectrotypes) of ferritin were observed during the investigation of pathological sera. The high sensitivity of this technique makes it useful for the investigation of serum ferritin in diseases involving modifications of the metabolism of this protein.     

Improved method for purification of enterotoxin from Clostridium perfringens type A.

The purification procedure of Clostridium perfringens type A enterotoxin has been improved. The cell sonic extract was precipitated twice with ammonium sulfate, first 40% saturated to concentrate the enterotoxin and then 15% saturated. The two precipitations were followed by gel filtration on Sephadex G-100. The enterotoxin appeared to be homogeneous on 7% polyacrylamide gel electrophoresis after this three-step purification procedure, with a recovery of 56% and a 12.3-fold purification. The solubility properties at different pH values, temperatures, and ammonium sulfate concentrations are also given as basis for the purification procedure.     

Improved method for purification of enterotoxin from Clostridium perfringens type A

The purification procedure of Clostridium perfringens type A enterotoxin has been improved. The cell sonic extract was precipitated twice with ammonium sulfate, first 40% saturated to concentrate the enterotoxin and then 15% saturated. The two precipitations were followed by gel filtration on Sephadex G-100. The enterotoxin appeared to be homogeneous on 7% polyacrylamide gel electrophoresis after this three-step purification procedure, with a recovery of 56% and a 12.3-fold purification. The solubility properties at different pH values, temperatures, and ammonium sulfate concentrations are also given as basis for the purification procedure.