Title: DEVELOPING A FLUORESCENT LATEX MICRO-PARTICLE IMMUNOASSAY USING ALEXA FLUOR 568 FOR DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN A (SEA) Author
Submitted to: Journal of Rapid Methods and Automation in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 30, 2007
Publication Date: March 1, 2007
Citation: Medina, M.B. 2007. Developing a fluorescent latex micro-particle immunoassay using alexa fluor 568 for detection of staphylococcal enterotoxin a (sea). Journal of Rapid Methods and Automation in Microbiology. 15:33-48. Interpretive Summary: Trace amounts of toxins produced by the foodborne pathogenic bacteria, Staphylococcus aureus cause major gastroenteritis. Common heat processing of foods and normal cooking temperatures can kill the bacteria but the produced toxins, are not destroyed. Therefore, eating the toxin-contaminated foods can still cause an illness. Staphylococcal enterotoxin A (SEA) is the most recovered toxin in staphylococcal food poisoning outbreaks in the United States and also globally. Rapid and inexpensive (low cost) methods that can detect trace amounts of toxins are needed for food safety and bio-defense monitoring purposes. In this research, we developed a method where the test reagents can be easily prepared in the laboratory and can be used for testing a large number of samples. The method utilizes a red fluorescent chemical where the presence of SEA toxin can be measured by the amount of fluorescence (glow) in the samples. We are reporting for the first time, the use of SEA labeled with red fluorescent dye as a tracer (indicator). The sample preparation is simple followed by detection of SEA below 1 part per billion in buffer and hotdogs. Analysis of 20 samples can be completed in less than 2.5 hrs. In addition, the cost of reagents per test is < US $2. This fluorescent-labeled SEA can be mixed with the green fluorescent-labeled SEB to simultaneously detect SEA and SEB in a single sample. 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 produces heat stable toxins that cause major foodborne gastroenteritis. Staphylococcal enterotoxin A (SEA) is the most recovered in staphylococcal food poisoning outbreaks. A sensitive method for detection of SEA is needed for food safety and food defense monitoring. Our research objectives were to develop a fluorescent immunoassay with detection of SEA below toxic levels of 1 ng/mL and to minimize sample preparation. An affinity purified anti-SEA was covalently linked to the polystyrene micro-particles. The SEA toxin was labeled with Alexa Fluor 568 (SEA-F). The concentrations of these reagents were optimized for detection of SEA in buffer and spiked hotdogs. SEA in samples was captured by the anti-SEA linked to the latex followed by the binding of SEA-F tracer. The latex-anti-SEA complex, bound with SEA and SEA-F, was separated by centrifugation, and the fluorescent density of the supernatant was measured. SEA was detected at 0.25 to 10 ng/mL in buffer and in spiked hot dogs (ng/g). This fluorescent latex particle immunoassay can be utilized for detection of SEA in a multi-toxin detection system.