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

  • PDF / 747,111 Bytes
  • 11 Pages / 595.276 x 790.866 pts Page_size
  • 77 Downloads / 216 Views

DOWNLOAD

REPORT


Magnetic Nanoparticle-Based Fluorescence Immunoassay for Determination of Ochratoxin A in Milk Zlatina Rumenova Becheva 1 & Milka Koycheva Atanasova 1 & Yavor Lukanov Ivanov 1 & Tzonka Ivanova Godjevargova 1 Received: 19 June 2020 / Accepted: 25 August 2020 # Springer Science+Business Media, LLC, part of Springer Nature 2020

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. Keywords Ochratoxin . Magnetic nanoparticles . Antibody fragmentation . OTA-OVA-FITC

Introduction Ochratoxins are highly toxic secondary metabolites produced by several Aspergillus and Penicillium species that can be found in various agricultural commodities such as cereals, coffee beans (either roasted or instant), cocoa, milk, beer, wine, grape juice, spices, and dried fruits (Malir et al. 2016). Ochratoxins include ochratoxin A (OTA), ochratoxin B (OTB), ochratoxin C (OTC), and ochratoxin α (OTα), of which OTA is considered the most toxic. OTA is classified by the International Agency of Research on Cancer as class 2B, possible human carcinogens. The enforceable maximum levels (MLs) for OTA in some food are stipulated in the Ochratoxin A detection in milk * Zlatina Rumenova Becheva [email protected] 1

Department of Biotechnology, Faculty of Technical Science, “Prof. Dr Assen Zlatarov” University, Burgas, Bulgaria

Commission Regulation of EC (EC 2006). The MLs for OTA in foodstuffs such as cereals, dried vine fruit, coffee, and some spices for several years are in range 3–10 μg/kg (Eskola et al. 2019). The highest level in this EU legislation is set at 10 ppb (equal to 10 μ