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Staphylococcus aureus enterotoxins C1 (SEC1) and C2 (SEC2) produced from 50-liter quantities of crude culture supernatants were purified chromatographically in a neutral or acid milieu. Microheterogenity of SEC1 was markedly increased by treatment of the purified toxin with alkali, and new more acidic charged species appeared. SEC2 was more heterogenous than any of the other S. aureus enterotoxins and was affected only slightly by treatment with alkali. Prolonged incubation of the organism during production of the SEC2 produced changes in charged species that may be related to a bacterial deamidase, since similar changes were not seen with alkaline treatment of the purified toxin. Although SEC1 and SEC2 showed complete identity immunologically, they are separate, distinct toxins, and alkali treatment of SEC1 did not produce SEC2.

Summary This paper describes a rapid sensitive solid phase radioimmunoassay procedure for staphylococcal enterotoxin B, which can be applied to A and C. The procedure is 100-fold more sensitive thandescribed immunodiffusion and precipitin methods and is reliable at a level of 0.01 µg/ml. It is usable for detection ad measurement of enterotoxin in foodstuffs, biologic fluids and toxin preparations, both purified and crude.

A rapid solid-phase radioimmunoassay for staphylococcal enterotoxin A is described. The assay procedure requires 3 to 4 h for completion by using a competitive inhibition system in which the antibody is attached to bromacetyl cellulose particles. It is accurate to a level of 0.01 μg of enterotoxin A/ml in a variety of media such as ham, milk products, crab meat, custard, etc. No significant interference was found with any media or food product tested.

Methods for the production and isolation of exfoliative toxin are described. Fermentation conditions were established under which large quantities of the crude material can be produced. Column chromatography methods, including carboxymethyl cellulose and hydroxyapatite, were utilized to purify the toxic protein. The pure toxin had a molecular weight of 26,000 as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The pure toxin is a simple protein composed of 17 amino acids. Tests for carbohydrate and for alpha- and beta-hemolysin were negative. The mean effective dose of the purified toxin was 0.5 mug per newborn mouse.

Release of Staphylococcus aureus enterotoxin B (SEB) into the culture medium was initiated during the mid-log phase of growth. A medium consisting of 4% N-Z Amine A (Sheffield), 0.2% dextrose, and 1% yeast extract supported maximum production of SEB. Although pH of the medium during cultivation did not significantly affect the growth curve of the organism, the time required for detectable excretion was affected, as was the final yield. Optimal conditions for SEB production were achieved with pH control at 7.0; alkaline control (pH 8.0) produced only minimal amounts of toxin, whereas acid control (pH 6.0) resulted in 50% reduction in yield. Slightly less SEB was produced when there was no extrinsic pH control, and cultures were buffered only by media constituents and by-products of growth. With pH control at 7.0, deletion of 0.2% dextrose from the medium resulted in 40% reduction in the 8-h yield. There was also a delay in production during early stages of fermentation.

Electrofocusing fractionation of purified Staphylococcus enterotoxin B was performed utilizing pH 7–9, 7–10, and 9.0–9.5 ampholine—sucrose gradients. On the pH 7–10 focusing experiments, four physically distinct, but antigenically similar entities were isolated. The major component (isoionic point pH 9.4 at 4°C) was refocused and found to be stable.