Author
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Medina, Marjorie |
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Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 2/20/2006 Publication Date: 6/8/2006 Citation: Medina, M.B. 2006. Development of a fluorescent latex microparticle immunoassay for detection of staphylococcal enterotoxin b (seb). Journal of Agricultural and Food Chemistry.54(14):4937-4942. Interpretive Summary: Staphylococcal enterotoxins are food safety and food security concerns. These toxins 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. The heat resistance of the SEB makes it a potential biothreat agent. Rapid, sensitive and inexpensive (low cost) methods with high throughput are needed for food safety and biosecurity monitoring purposes. In this work, we are developing such a method for monitoring purposes where the test reagents can be easily prepared by the laboratory and can be used for testing a large number of samples. The method utilizes a fluorescent chemical where the presence of SEB toxin can be measured by the amount of fluorescence (glow) in the samples. We also simplified the sample preparation for detection of SEB toxin at trace and toxic levels below 1 ng/mL or 1 part per billion in drinking water and milk. This technique has advantages over previously reported methods because milk and water are directly tested and 25 – 50 samples can be tested in less than 3 hrs. In addition, the cost of reagents per test is < US $1. This approach will be useful for the food industry and regulatory agencies to enhance the safety and the security of our foods. Technical Abstract: Staphylococcus aureus enterotoxin B (SEB) is a highly heat resistant enteric toxin with a potential as a bio-threat agent. A sensitive method for detection of staphylococcal enterotoxins is needed for food safety and food defense monitoring. Our research objectives were to develop a competitive fluorescent immunoassay with detection of SEB below toxic levels of 1 ng/mL or 1 part per billion (ppb) and to minimize sample preparation. Anti-SEB was immobilized onto carboxylated polystyrene micro-particles and SEB was labeled with fluorescein isothiocyanate (FITC). The concentrations of these reagents were optimized for detection of SEB at <1 ng/mL and other assay conditions (sample volumes and incubation periods) were optimized. Drinking water and milk samples were spiked with 0.125 – 10 ng/mL SEB and were equilibrated overnight prior to analysis. The water and milk samples were directly analyzed but heating the milk samples for 10 min at 90C improved the assay performance. SEB in samples bound with the anti-SEB linked to the latex followed by the competitive binding of SEB-FITC tracer. The excess, unbound tracer was separated by centrifugation and the fluorescent density of the supernatant was measured. SEB was detected below the toxic levels, 0.125 ng/mL in drinking water and 0.5 ng/mL in whole milk. This fluorescent latex micro-particle immunoassay will be utilized for detection of SEB in various foods matrices. |
