Magnetic Nanoparticle-Based Fluorescence Immunoassay for Determination of Ochratoxin A in Milk

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.