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Fluorescence Polarization Immunoassay for Rapid, Accurate and Sensitive Determination of Ochratoxin A in Wheat

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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 quantitative determination of OTA in wheat at level far below the EU regulatory limits.

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Acknowledgments

This work has been supported by the Italian Ministry of Education, University and Research, MIUR (P.O.N. “Ricerca & Competitività” 2007–2013), project no. 02_00186_341751212792-S.I.Mi.S.A. “New Strategies for Improvement of Food Safety: Prevention, Control, Correction”. The authors would like to thank Giuseppe Panzarini (ISPA-CNR) for his technical help in the HPLC analysis.

Conflict of Interest

Vincenzo Lippolis declares that he has no conflict of interest. Michelangelo Pascale declares that he has no conflict of interest. Stefania Valenzano declares that she has no conflict of interest. Anna Chiara Raffaella Porricelli declares that she has no conflict of interest. Michele Suman declares that she has no conflict of interest. Angelo Visconti declares that he has no conflict of interest. This article does not contain any studies with human or animal subjects.

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Authors and Affiliations

  1. Institute of Sciences of Food Production (ISPA), National Research Council of Italy (CNR), via G. Amendola 122/O, 70126, Bari, Italy

    Vincenzo Lippolis, Michelangelo Pascale, Stefania Valenzano, Anna Chiara Raffaella Porricelli & Angelo Visconti

  2. Barilla Food Research Labs, via Mantova 166, 43122, Parma, Italy

    Michele Suman

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  1. Vincenzo Lippolis
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  2. Michelangelo Pascale
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  3. Stefania Valenzano
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  4. Anna Chiara Raffaella Porricelli
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  5. Michele Suman
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  6. Angelo Visconti
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Correspondence to Michelangelo Pascale.

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Lippolis, V., Pascale, M., Valenzano, S. et al. Fluorescence Polarization Immunoassay for Rapid, Accurate and Sensitive Determination of Ochratoxin A in Wheat. Food Anal. Methods 7, 298–307 (2014). https://doi.org/10.1007/s12161-013-9627-3

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  • DOI: https://doi.org/10.1007/s12161-013-9627-3

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