Detection of staphylococcal enterotoxin A by Laurell electroimmunodiffusion Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/g445cg71m

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  • The serological detection of staphylococcal enterotoxin A by the quantitative technique of Laurell electroimmunodiffusion is described as well as the application of the technique to the detection of small quantities of enterotoxin A in foods. High dilutions of type-specific rabbit antiserum were used in 1% agarose gels, 1 mm thick, and prepared in 0.05 1.μ barbital buffer pH 8.6. Volumes of 10 μ 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, then overlaying the surface with the purified globulin fraction of sheep serum against rabbit globulin, followed by soaking the gels in 1% aqueous cadmium acetate, and staining with 0. 1% Thiazine Red R 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 hr of electrophoresis and visualization was achieved within 2 to 3 hr. The usefulness of the technique for the detection of small quantities of enterotoxin A in foods was investigated. Dairy as well as nondairy food items were artificially contaminated with purified staphylococcal enterotoxin A in amounts varying from 0. 25 to 1. 0μg per 100 g sample. The toxin was extracted with 0. 05 μ. barbital buffer pH 8. 6 and the extract partially purified and concentrated to 1. 0 ml by membrane ultrafiltration. The presence or absence of toxin in the concentrate was determined by Laurell electroimmunodiffusion. The combined use of membrane ultrafiltration and electroimmunodiffusion permitted the detection of 0. 75 μg staphylococcal enterotoxin A per 100 g of food within 8 to 10 hr.
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