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

Abstract

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.

Occurrence of Ochratoxin A in Different Types of Cheese Offered for Sale in Italy

“…Since in these animals the bioavailability of OTA is low to begin with, the chance of OTA transferring to milk is minimal. Thus, detection of the toxin in milk generally occurs only in cases of high concentrations of OTA in animal feed [ 60 , 61 ].…”

Magnetic nanoparticle‐based immunosensors and aptasensors for mycotoxin detection in foodstuffs: An update

“…Benefiting from their inherent ferromagnetic properties, large specific surface area, physicochemical stability, biocompatibility, and cost‐effective synthesis, MNPs have emerged as shining stars in biosensing applications (Becheva et al., 2020; Gao, Torrente‐Rodríguez et al., 2022; He et al., 2023; Zeng et al., 2022). Among the various types of MNPs that have been synthesized and applied for different purposes, iron oxides (Fe 3 O 4 magnetite, α‐Fe 2 O 3 hematite, and γ‐Fe 2 O 3 maghemite) are the most widely used ones.…”

“…To overcome those limitations of natural Abs, recombinant Ab fragments have been investigated in immunosensing applications, including single‐domain variable fragments (scFv), antigen‐binding fragments (Fab), and single‐domain Abs (nanobodies, or Nbs). In some instances, F(abʹ) 2 ‐based immunoassay was observed with a slightly lower limit of detection (LOD) compared to the one based on whole Ab (Becheva et al., 2020), probably due to the decreased steric hindrance. Among the Ab fragments, Nbs with the smallest antigen‐binding domain have demonstrated superior properties than natural Abs for the fabrication of immunosensors.…”

A Chemiluminescence Enzyme Immunoassay Based on Biotinylated Nanobody and Streptavidin Amplification for Diazinon Sensitive Quantification

“…However, polyclonal and monoclonal antibodies (pAbs and mAbs), the widely used two types of antibodies, are raised by a traditional strategy of preparing immunogens and coating antigens, which are born with defects. The instability of pAb production results in an obvious difference between batches, cannot be replicated on a large scale, and is prone to the production of non-specific antibodies [4]. mAbs are generated by hybridoma cells with high specificity and yields that are increasingly being used in immunoassays, Biosensors 2023, 13, 577 2 of 12 but they demand complex preparation procedures and a strict preservation condition [5].…”