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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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†
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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
