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Title: DEVELOPMENT OF COMPETITIVE IMMUNOASSAY FOR DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN B(SEB) IN MILK

Author
item Medina, Marjorie

Submitted to: International Food Technology Meeting Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: 2/12/2003
Publication Date: 7/12/2004
Citation: Medina, M.B. 2004. Development of competitive immunoassay for detection of staphylococcal enterotoxin b(seb) in milk. International Food Technology Meeting Abstracts. Paper number 491-4.

Interpretive Summary:

Technical Abstract: A sensitive and more rapid biosensor method for detection of staphylococcal enterotoxins is needed by the food industry. Staphylococcus aureus enterotoxin B (SEB) is highly heat resistant and is a potential bioterrorism agent. Our research objective is to develop a competitive immunoassay using a surface plasmon resonance (SPR) biosensor for the detection of SEB below 1 ng/mL (ppb) in fresh fluid milk. The assay consisted of SEB immobilization on the sensor surface. Anti-SEB was allowed to bind with 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. Analysis of milk samples spiked with 0.312 ' 50 ppb SEB consisted of heating the samples at 95 degrees C followed by rapid cooling and centrifugation at 2961g 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 was separated from 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 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 detection of SEB in various foods and will be optimized for detection of other staphylococcal toxins in foods.