Summary
A competitive immunoassay for staphylococcal enterotoxin A (SEA) detection in milk was developed, using immobilised antibody onto magnetic nanoparticles (MNPs). MNPs were prepared and then modified to introduce amino groups on them. The morphology and size of the obtained both unmodified and modified MNPs were characterized using TEM analyses. Monoclonal anti‐SEA antibody was immobilised onto the modified MNPs (MNP‐Ab). Staphylococcal enterotoxin A was conjugated with fluorescent dye ATTO620NHS. The characteristics of fluorescence conjugate were examined. The amount of MNP‐Ab and concentration of the fluorescent conjugate used for competitive immunoassay were optimized: 0.25 mg and 53 μg mL−1, respectively. The detection limit of developed immunoassay was determined – 0.23 ng mL−1 SEA in spiked milk samples. The immunoassay takes only 30 min, the magnetic separation is fast (<10 s) and the volume of the sample for analysis is very small (200 μL).
Ochratoxins are possible human carcinogens. The aim of this study is to develop a rapid and sensitive competitive immunofluorescent analysis for determination of ochratoxin A (OTA) on the base of immobilized polyclonal antibody against ochratoxin and immobilized F(ab′) 2 fragment on magnetic nanoparticles (MNPs). F(ab′) 2 fragment of anti-OTA antibody was obtained by pepsin hydrolysis of polyclonal antibody against OTA. The competitive fluorescent conjugate OTA-OVA-FITC (OTA coupled to ovalbumin (OVA) and then conjugated to fluorescein isothiocyanate (FITC)) was prepared and purified by size-exclusion chromatography. Competitive immunoassay was performed by using obtained immobilized antibody or F(ab′) 2 fragment on magnetic nanoparticles and the conjugate OTA-OVA-FITC. The analytical characteristics of the analysis with immobilized polyclonal antibody and F(ab′) 2 fragment were compared. The linear measuring range of OTA in milk, obtained with immobilized whole antibody, was from 0.1 to 2.5 ng/mL OTA and with immobilized F(ab′) 2 fragment from 0.1 to 7.5 ng/mL OTA. The detection limit of immunoassay with immobilized whole antibody was 0.1 ng/mL OTA and with immobilized F(ab′) 2 fragment was 0.08 ng/mL OTA. Milk samples were spiked with OTA at different levels. The recovery rates when using immobilized F(ab′) 2 fragment were between 99.4 and 118.0%, and the relative standard deviations (RSDs) were 6.5-7.7%. The results indicate that this fluorescence immunoassay with MNPs was accurate and has good reproducibility.
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