A BIOSENSOR METHOD FOR A COMPETITIVE IMMUNOASSAY DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN B (SEE) IN MILK

摘要

A sensitive and more rapid biosensor method for the detection of staphylococcal enterotoxins (SE) is needed by the food industry. Staphylococcal enterotoxin B (SEE) is highly heat resistant and is a potential bioterrorism agent. Our research objectivewas to develop a competitive immunoassay using a surface plasman resonance (SPR) biosensor for the detection of SEE below 1 ng/mL [pan per billion (ppb)J in fresh fluid milk. The assay consisted of SEE immobilization on the sensor surface. An anti-SEB was allowed to bind with the SEE in samples off line prior to the biosensor analysis. The excess and unbound anti-SEB was then captured by SEE sensor. The assay conditions were optimized to detect SEE in HEPES buffer and in whole milk. An analysis of milksamples spiked with 0.312-50ppb. SEE 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 SEE were allowed to bind with anti-SEB for 30 or 60 min. The SEE and anti-SEB complex were separated from the free anti-SEB by centrifugation, and the supernatants were injected over the sensor. SEE 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 competitiveassay will be utilized for the detection of SEE in various foods and will be optimized for the detection of other staphylococ-cal toxins in foods.