• PDF / 348,892 Bytes
• 10 Pages / 595.276 x 790.866 pts Page_size
• 111 Downloads / 242 Views

DOWNLOAD

REPORT

---

Fluorescence Polarization Immunoassay for Rapid, Accurate and Sensitive Determination of Ochratoxin A in Wheat Vincenzo Lippolis & Michelangelo Pascale & Stefania Valenzano & Anna Chiara Raffaella Porricelli & Michele Suman & Angelo Visconti

Received: 27 February 2013 / Accepted: 15 April 2013 # Springer Science+Business Media New York 2013

Abstract A sensitive and accurate fluorescence polarization (FP) immunoassay has been developed for the determination of ochratoxin A (OTA) in naturally contaminated wheat samples. A fluorescein-labeled OTA tracer was synthesized, and its binding response with three monoclonal antibodies was tested. The most sensitive competitive FP immunoassay showed an IC50 value of 0.48 ng/mL with a negligible cross-reactivity for ochratoxin B (1.7 %) and no cross-reactivity with other mycotoxins commonly occurring in wheat. The wheat sample was extracted with acetonitrile/water (60:40, v/v) and purified by a rapid solid-phase extraction procedure using an aminopropyl column prior to the FP immunoassay. The overall time of analysis was less than 20 min. The average recovery from spiked wheat samples (3 to 10 μg/kg) was 87 %, with relative standard deviations generally lower than 6 %. Limits of detection and quantification were 0.8 and 2.0 μg/kg, respectively. The trueness of the method was assessed by using two reference materials for OTA showing good accuracy and precision. A good correlation (r=0.995) was observed between OTA contamination of 19 naturally contaminated wheat samples analyzed by both FP immunoassay and high-performance liquid chromatography/immunoaffinity clean-up used as reference method. These results show that the developed FP method is suitable for high-throughput screening, as well as for reliable V. Lippolis : M. Pascale (*) : S. Valenzano : A. C. R. Porricelli : A. Visconti Institute of Sciences of Food Production (ISPA), National Research Council of Italy (CNR), via G. Amendola 122/O, 70126 Bari, Italy e-mail: [email protected] M. Suman Barilla Food Research Labs, via Mantova 166, 43122 Parma, Italy

quantitative determination of OTA in wheat at level far below the EU regulatory limits. Keywords Ochratoxin A . Fluorescence polarization immunoassay . Solid phase extraction . Wheat

Introduction Ochratoxin A (OTA) is a mycotoxin produced by several Penicillium and Aspergillus species growing in different agricultural commodities in the field or during storage (Bayman and Baker 2006; Miller and Trenholm 1994). Several surveys have shown the presence of OTA in a wide range of foods and beverages, such as cereals, beer, wine, cocoa, coffee, dried fruit, spices, and meat products (Walker 2002; Jørgensen 2005; Duarte et al. 2010). According to the EU SCOOP project, Scientific Cooperation Task 3.2.7, cereals are the most important dietary source of OTA contributing to more than 50 % of the total intake (SCOOP 2002). OTA is a strong nephrotoxic agent, and it has been shown to be teratogenic, mutagenic, hepatotoxic, and immunosuppressive to animal species (Barl