Purpose: The scope of the study was an in-depth purity and identity characterisation of commercially available Staphylococcal enterotoxin A (SEA) and the accurate determination of the protein concentration of a solution prepared from the lyophilised toxin, with the aim to produce a suitable calibration solution for quantitative SEA measurements.
Methods: The set of methods applied to assess the purity and identity of the toxin comprised of SDS-PAGE (initial mass determination), Q-TOF (accurate mass determination), multi-enzyme LC-MS/MS (sequencing of the protein for identity), and ELISA (cross-reactivity check and indicative concentration). The prepared SEA stock solution was value assigned for its protein concentration by amino acid analysis (AAA), using an in-house isotope dilution LC-MS/MS method.
Results: SDS-PAGE indicated one major band, confirming the >95% purity statement issued by the material provider. Sequencing confirmed the published SEA sequence, and ELISA for Staphylococcal enterotoxins A-D confirmed the absence of those toxins in the preparation. However, a strong cross-reaction was obtained with a SEE-specific ELISA (sequence similarity of SEA and SEE). AAA provided a reliable means to establish the protein concentration of the SEA solution. Moreover, AAA measurements spread over a period of 3 years confirmed excellent sample stability for a storage at -20⁰C.
Significance: The comprehensively characterised and thoroughly value-assigned SEA solution provides the basis for accurate quantitative SEA analyses in complex food matrices; and thus, will contribute to establish and maintain reliable measurement results for effective consumer protection.