Abstract
Objectives: Staphylococcus aureus is a Gram-positive microorganism. S. aureus can grow in various foods and cause food poisoning by secreting enterotoxins. The most common enterotoxins involved in food poisoning are staphylococcal enterotoxin A and staphylococcal enterotoxin B, but Staphylococcal Enterotoxin A (SEA) is predominant. The main types of food contaminated with SEs are meat and meat products, poultry and eggs, milk and dairy products. The aim of this study was to develop a rapid and sensitive fluorescence immunoassay for detection of staphylococcal enterotoxin A in milk. Methods: Monoclonal and polyclonal antibodies for SEA were produced and characterized. Competitive fluorescence immunoassay based on Magnetic Nanoparticles (MNPs) was performed and optimized. MNPs were used as a solid carrier of the antibodies. The first step of the assay was immunoreaction between the immobilized antibody onto MNPs and SEA in milk sample. Then the fluorescein-SEA conjugate was added to the sample. Thus, competitive immunoreaction between MNP-mAb/MNP-pAb with SEA and SEA-FITC was performed. These immuno-complexes were separated by a magnetic separator and the obtained supernatants were analyzed. The fluorescent signal from the excess of conjugated SEA was proportional to the SEA contained in the milk. The assay duration was only 30 min. Results: The fluorescence immunoassays performed with polyclonal antibody had linear ranges from 5 pg/mL to 100 ng/mL SEA in a buffer, and from 50 pg/mL to 50 ng/mL SEA in spiked milk samples. While the same assays performed with monoclonal antibody had linear ranges from 1 pg/mL to 20 ng/mL SEA in buffer, and from 10 pg/mL to 10 ng/mL SEA in spiked milk samples. The detection limits of the developed immunoassays performed in milk were: 48 pg/mL with polyclonal antibody and 9 pg/mL with monoclonal antibody. Conclusion: A rapid and sensitive fluorescence immunoassay based on magnetic nanoparticles with a polyclonal and monoclonal antibody for determination of staphylococcal enterotoxin A in milk was developed.
Highlights
IntroductionIt is usually found in nasal passages and skin of healthy individuals
A rapid and sensitive fluorescence immunoassay based on magnetic nanoparticles with a polyclonal and monoclonal antibody for determination of staphylococcal enterotoxin A in milk was developed
Staphylococcal enterotoxins (SEA, SEB, SEC, SED, SEE), Freund’s adjuvant, Tween 20, Tween 80, bovine serum albumin, polyethylene glycol 4000, ammonium sulfate, dimethylsulphoxide, fetal calf serum, 8-azaguanine, Lglutamine, HiTrap Protein G HP column, anti-sheep immunoglobulin G (IgG) whole molecule – peroxydase antibody, anti-mouse IgG whole molecule – peroxidase antibody, 3,3',5,5'-tetramethylbenzidine, 3% H2O2, H2SO4, dimethylformamide, fluorescein 5(6)isothiocyanate, Sephadex G25 Medium, 2-amino-2(hydroxymethyl)-1,3-propanediol (Tris), glycine, goat antimouse IgG antibody conjugated with alkaline phosphatase, (3aminopropyl)triethoxysilane (APTES), glutaraldehyde, HiPrepTM 26/10 Desalting, and 1-stepTM NBT/BCIP substrate solution were delivered by Sigma-Aldrich, Germany
Summary
It is usually found in nasal passages and skin of healthy individuals. S. aureus can adapt to grow in various foods and cause food poisoning by secreting enterotoxins [1, 2]. Staphylococcal Enterotoxins (SEs) are proteins with 220 240 amino acids and have similar molecular weights (25–30 kDa). The most common SEs involved in food poisoning are SEA and SEB, and SEA is predominant [2, 3]. The main types of food contaminated with SEs are meat and meat products, poultry and eggs, milk and dairy products [2]
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