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Sensitive Flow-Through Immunoassay for Rapid Multiplex Determination of Cereal-Borne Mycotoxins in Feed and Feed Ingredients Natalia V. Beloglazova, Kinga Graniczkowska, Emmanuel Njumbe Ediage, Olga Averkieva, and Sarah De Saeger J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03172 • Publication Date (Web): 25 Dec 2016 Downloaded from http://pubs.acs.org on December 26, 2016

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

Sensitive Flow-Through Immunoassay for Rapid Multiplex

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Determination of Cereal-Borne Mycotoxins in Feed and Feed Ingredients

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Natalia V. Beloglazova †*, Kinga Graniczkowska †, Emmanuel Njumbe Ediage †, Olga Averkieva ‡

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, Sarah De Saeger. †

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Ottergemsesteenweg 460, 9000 Ghent, Belgium

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Ghent University, Faculty of Pharmaceutical Sciences, Laboratory of Food Analysis,

Nutriad, Hoogveld 93, 9200 Dendermonde, Belgium

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*

Corresponding author (Tel.: +32 9 2648127; Fax: +32 9 2648199; E-mail address:

[email protected])

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Abstract

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An easy-to-operate membrane-based flow-through test for multiplex screening of four

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mycotoxins (zearalenone, deoxynivalenol, aflatoxin B1, and ochratoxin A) in a variety of cereal

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based feed ingredients and compound feed, such as wheat, barley, soybean, wheat bran, rice, rice

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bran, maize, rapeseed meal, sunflower meal and various types of complete feed (ducklings feed,

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swine feed, broiler feed, piglet feed) was developed and validated. First, the antibodies were

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evaluated by enzyme-linked immunosorbent assay, then employed in the membrane rapid test.

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The cut-off levels for zearalenone, deoxynivalenol, aflatoxin B1 and ochratoxin A were 50, 200,

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1 and 10 µg/kg, respectively, based on the European regulations and consumers’ requirements.

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As sample pretreatment, consecutive steps of extraction, dilution, solid-phase extraction by

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addition of C18 sorbent and final filtration of supernatant were followed. Both the sample

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preparation and the analysis procedure were simple, cost-effective and easy to perform on-site in

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a non-laboratory environment. The impact of sample processing on the result of experiment was

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investigated supported by experimental design. The validation procedure was performed based

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on the Commission Regulation 2006/401/EC. The amount of false-positive and false-negative

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outcomes were below 5%, going along with the Commission Decision 2002/657/EC. Liquid

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chromatography–tandem mass spectrometry was performed as a confirmatory technique.

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Keywords: multi-assay, membrane test, flow-through, rapid test, immunoassay, multiplex

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screening, mycotoxins.

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Journal of Agricultural and Food Chemistry

Introduction

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The globalized food supply system including storage and transportation of all ingredients

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can easily result in the spread of food related risks. It has already reported that about a quarter of

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all crops worldwide are affected by mycotoxins.1 This has a large impact on food and feed

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production and livestock farming, holding an increased risk for human health. Mycotoxins are

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comparatively small secondary metabolites, formed by fungi, e.g. Aspergillus, Penicillium,

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Fusarium etc. growing on agricultural crops alike in field and during storage.2 Although

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prevention in the field is the major part in the mycotoxin risk management, contamination of

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various commodities with mycotoxins is unavoidable under certain environmental conditions. So

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far more than 400 chemically diverse compounds have been identified in this group and

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depending on the type, mycotoxins can trigger diverse biochemical, functional and

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morphological syndromes in human and animal which can lead to mortality.3 Among the most

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widespread and theoretically toxic mycotoxins classified till date ochratoxin A (OTA, Figure

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1.1), aflatoxin B1 (AFB1, Figure 1.2), zearalenone (ZEN, Figure 1.3) and deoxynivalenol (DON,

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Figure 1.4) receive a great attention.

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Numerous mycotoxigenic fungi can established the same niche and generate their toxic

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metabolites under similar conditions resulting in co-contamination of mycotoxins in food and

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feed. Apart from that, mixtures of several raw materials in compound feed can raise the risk of

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feed pollution with numerous toxins.4 The simultaneous presence of several mycotoxins and the

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strong need for their control contributed to the development of various multi-mycotoxin

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detection approaches. The most common technique for mycotoxin determination is liquid-

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chromatography tandem mass spectrometry (LC-MS/MS), which allows simultaneous detection

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of dozens of mycotoxins, sometimes including their modified forms, in one run.5-7 Despite its

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high sensitivity and accuracy this technique is time-consuming, requiring use of advanced

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equipment, expensive internal standards and a high volume of organic solvents.

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When a large sample number has to be monitored for multiple toxins, sample throughput

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is an important criterion. In this regard, a screening method can be used. Among all screening

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methods two main groups can be distinguished: spectroscopic and receptor-based techniques.

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Spectroscopic methods, which are much cheaper and easier to perform than LC, are not

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appropriate for direct determination of mycotoxins in complex matrices due to their limited

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sensitivity and specificity.8 One of the most widely used receptor-based techniques for rapid

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mycotoxin monitoring is enzyme-linked immunosorbent assay (ELISA). Despite its high matrix

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dependence, ELISA is simple, specific and sensitive approach, it also provides high sample

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throughput, but to perform ELISA an instrument is required, also time needed to obtain results is

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much longer in comparison with the following mentioned rapid tests. Nowadays, multiplex

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screening tests for mycotoxins are in high demand. Lateral flow immunoassay (LFA) has been

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extensively used for quick detection of single or multiple analytes,9,10 however this technique

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has some limitation related to

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influence of immunoreagents specific to each compound on another. It is affected by the liquid

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running from the front and passing all lines. Another popular on-site format applied for rapid

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screening is a flow-through membrane-based assay (MBA).11 The flow-through approach allows

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to separate different test zones, and therefore to minimize this cross-influence. Furthermore, to

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reduce the abovementioned saturation problem, additional absorption layers can be always

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applied.

saturation of the membrane and a quite pronounced cross-

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However in the field of mycotoxin analysis, a very limited number of multiplex MBA

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have been reported.12-15 All previously designed flow-through tests, including our prior research

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commercially important cereal-based products, especially feed and feed ingredients. In this work

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for the first time a multi-analyte flow-through immunoassay for fast screening of four

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mycotoxins, deoxynevalenol, zearalenone, ochratoxin A and aflatoxin B1 (Figure 1) in different

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feed matrices was designed at low cut-off levels. Different parameters were investigated for

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optimization of the assay and its validation for various industrially important feed matrices.

, were either characterized by quite high cut-off values or were not validated for a variety of

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Materials and methods

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Reagents and materials

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Deoxynevalenol and ochratoxin A

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Israel). Aflatoxin B1, zearalenone, casein sodium salt from bovine milk, bovine serum albumin

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(BSA), phosphate buffered saline (PBS) sachets, carbonate bicarbonate buffered saline tablets

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(CBS), Tween 20 (Tween; polyoxyethylenesorbitan monolaurate), skim milk powder, sealing

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film for 96-well multiwell plates were purchased from Sigma-Aldrich (Bornem, Belgium). The

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substrate chromogenic solution Colorburst Blue TMB/Peroxide was supplied by Thermo Fisher

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Scientific (Leuven, Belgium). Methanol, HPLC-grade was purchased from VWR International

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(Zaventem, Belgium). Polyclonal rabbit anti-mouse immunoglobulins (IgG) (2.1 g/L) were

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obtained from Dako Denmark A/S (Glostrup, Denmark). The monoclonal antibodies: anti-

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zearalenone (MAbZEN#1, 1 mg/mL, characterized with 36% cross-reaction with α-zearalenol),

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anti-ochratoxin A (MAbOTA#1, 1 mg/mL, characterized with 32% cross-reaction with

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ochratoxin B), anti-aflatoxin B1 (MAbAFB1#1, 1.3 mg/mL, described with 79% cross-reaction

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towards aflatoxin M1, 33% towards aflatoxin M2, 76% towards aflatoxin B2 (AFB2), 55%

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towards aflatoxin G1 (AFG1), 6% towards aflatoxin G2 (AFG2) and none at all with AFB2a and

standards were purchased from Fermentek (Jerusalem,

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AFG2a) were provided by Soft Flow Inc. (Pécs, Hungary). A monoclonal anti-deoxynivalenol

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antibody (clone 4, MAbDON#1, 1 mg/mL, characterized with 429% cross-reactivity for 15-

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acetyl–deoxynivalenol and