DEVELOPING A FLUORESCENT LATEX MICROPARTICLE IMMUNOASSAY USING ALEXA FLUOR 568 FOR DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN A¹

Abstract

Staphylococcus aureus produces heat-stable toxins that cause major foodborne gastroenteritis. Staphylococcal enterotoxin A (SEA) is the most recovered toxin in staphylococcal food poisoning outbreaks. A sensitive method for the detection of SEA is needed for food safety and food defense monitoring. The objectives of this study were to develop a fluorescent immunoassay for the detection of SEA below the toxic level of 1 ng/mL and to minimize sample preparation. An affinity-purified anti-SEA was covalently linked to polystyrene microparticles. The SEA toxin was labeled with Alexa Fluor 568 (A568). The concentrations of these reagents were optimized for the 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 the SEA-A568 tracer. The latex-anti-SEA complex, bound with SEA and SEA-A568, was separated by centrifugation, and the fluorescent density of the supernatant was measured. SEA was detected at 0.25-10 ng/mL in buffer and in spiked hotdogs (ng/g). This fluorescent latex particle immunoassay can be utilized for the detection of SEA in a multitoxin detection system. PRACTICAL APPLICATIONSRapid and inexpensive methods are needed to detect trace levels of enterotoxins produced by Staphylococcus aureus organisms. Methods with 1 Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. 2 Corresponding 33 detection at or below 1 ng/g or 1 ng/mL are desired in testing food systems for food safety and food defense monitoring. Fluorescent immunoassays have the potential of providing high throughput analysis and increased sensitivity compared with enzyme immunoassays. The fluorescent latex microparticle immunoassay (FLMIA) using Alexa Fluor 568 as SEA antigen label detected SEA at 0.25-10 ng/mL in buffer solutions and 0.5 ng/g in spiked hot dogs. The FLMIA can be used for trace analysis in foods. This research shows the use of a fluorescent-labeled antigen as opposed to the use of labeled antibody (IgG). This research has shown the feasibility of using a long-wavelength fluorescent dye (Alexa Fluor 568) in immunodiagnostic assays. The use of the red fluorescent label for SEA will allow a multitoxin assay with green fluorescentlabeled SEB (staphylococcal enterotoxin B) in a single sample. 34 M.B. MEDINA 36 M.B. MEDINA