Sensitive Flow-through Immunoassay for Rapid Multiplex

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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

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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Journal of Agricultural and Food Chemistry

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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