A biosensor method for a competitive immunoassay detection of staphylococcal enterotoxin B (SEB) in milk

A sensitive and more rapid biosensor method for the detection of staphylococcal enterotoxins (SE) is needed by the food industry. Staphylococcal enterotoxin B (SEB) is highly heat resistant and is a potential bioterrorism agent. Our research objective was to develop a competitive immunoassay using a surface plasmon resonance (SPR) biosensor for the detection of SEB below 1 ng/mL [part per billion (ppb)] in fresh fluid milk. The assay consisted of SEB immobilization on the sensor surface. An anti-SEB was allowed to bind with the SEB in samples off line prior to the biosensor analysis. The excess and unbound anti-SEB was then captured by SEB sensor The assay conditions were optimized to detect SEB in HEPES buffer and in whole milk. An analysis of milk samples spiked with 0.312-50 ppb. SEB consisted of heating the samples at 95C followed by rapid cooling and centrifugation at 2961 x g to separate the skim fraction. Aliquots of the skim fraction containing SEB were allowed to bind with anti-SEB for 30 or 60 min. The SEB and anti-SEB complex were separated front the free anti-SEB by centrifugation, and the supernatants were injected over the sensor SEB was detectable in buffer at 0.78-50 ppb and in spiked whole and skim milk from 0.312-25 ppb. The biosensor analysis including the sensor regeneration was 15 min per sample in a fully automated system. The competitive assay format resulted in higher detection sensitivity and greater sample throughput than the SPR biosensor sandwich assay. The competitive assay will be utilized for the detection of SEB in various foods and will be optimized for the detection of other staphylococcal toxins in foods.