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United States Department of Agriculture

Agricultural Research Service


item Medina, Marjorie

Submitted to: Journal of Rapid Methods and Automation in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/7/2005
Publication Date: 5/20/2005
Citation: Medina, M.B. 2005. Development of a competitive immunoassay for detection of staphylococcal enterotoxin b ((seb) in milk. Journal of Rapid Methods and Automation in Microbiology. 13. pp. 37-55.

Interpretive Summary: Foodborne pathogenic bacteria, Staphylococcus aureus, produce toxins that can cause serious gastroenteritis and many other discomforts even with trace quantities. Common heat processing of foods and normal cooking temperatures can kill the bacteria but the produced toxins, staphylococcal enterotoxin B (SEB), are not destroyed. Therefore, ingestion of contaminated foods can still cause illness. The heat resistance of the SEB makes it a potential bioterrorism agent. Thus, there is a need of good methods to detect trace levels of SEB in foods. In this work, we developed a rapid and semi-automated method to detect SEB in milk using a biosensor. The method successfully detected the presence of 0.3 to 20 parts per billion of SEB in fresh whole milk. This technique has advantages over previously reported methods because the sample preparation is simple and the analysis is rapid (15 min per sample). This information will be useful for food industry and regulatory agencies to enhance the safety and the security of our foods.

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.

Last Modified: 06/26/2017
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