High performance liquid chromatography of the staphylococcal enterotoxins Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/79408113x

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  • Many methods in use today for the isolation, purification, and identification of the staphylococcal enterotoxins involve lengthy steps with significant loss of enterotoxin, are subject to interference from contaminating sample components, and lack sufficient specificity. Culture extracts of enterotoxin producing Staphylococcus aureus strains (enterotoxins A-E), crude enterotoxin preparations (enterotoxins A-E), by high performance anion exchange, and and purified enterotoxin B (SEB) were liquid chromatography (HPLC). cation exchange supports were Steric investigated exclusion, employed using ammonium acetate and potassium phosphate mobile phase buffers. Chromatographically resolved sample components were analyzed by sodium dodecyl sulfate-microslab polyacrylamide gel electrophoresis and the Ouchterlony microslide gel double diffusion test. High performance steric exclusion chromatography with the MicroPak TSK 3000 SW support was capable of partial separation of enterotoxin from sample components. The potential of high performance steric exclusion chromatography to serve as a rapid prefractionation step in the purification of enterotoxins is discussed. High performance anion exchange chromatography with the SynchroPak AX-300 support was ineffective in resolving enterotoxins from sample components. High performance cation exchange chromatography with the IEX 530 CM support was determined to be an effective technique for resolution of multiple forms of SEB from crude and purified enterotoxin preparations. A rapid gradient elution method employing the IEX 530 CM support with an ammonium acetate buffer as the mobile phase is presented for the isolation, purification, and presumptive identification of the staphylococcal enterotoxins. The use of high performance steric exclusion chromatography as a rapid prefractionation step coupled to high performance cation exchange chromatography is discussed. The potential of HPLC methods to improve existing serological assays for enterotoxin is also discussed.
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