Fluorescence Polarization Immunoassay Based on a New Monoclonal

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A Fluorescence Polarization Immunoassay Based on a New Monoclonal Antibody for the Detection of the Zearalenone Class of Mycotoxins in Maize Xiya Zhang, Sergei Alexandrovich Eremin, Kai Wen, Xuezhi Yu, Chenglong Li, Yuebin Ke, Haiyang Jiang, Jianzhong Shen, and Zhanhui Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05614 • Publication Date (Web): 24 Feb 2017 Downloaded from http://pubs.acs.org on February 26, 2017

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Journal of Agricultural and Food Chemistry

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A Fluorescence Polarization Immunoassay Based on

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a New Monoclonal Antibody for the Detection of the

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Zearalenone Class of Mycotoxins in Maize

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Xiya Zhang†, Sergei A. Eremin‡, Kai Wen†, Xuezhi Yu†, Chenglong Li†, Yuebin Ke§, Haiyang Jiang†,

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Jianzhong Shen†, Zhanhui Wang*,†

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†

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Medicine, China Agricultural University, Beijing Key Laboratory of Detection Technology for

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Animal-Derived Food Safety, Beijing Laboratory for Food Quality and Safety, 100193 Beijing,

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People’s Republic of China

Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Veterinary

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‡

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University, Moscow 119991, Russia

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§

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Shenzhen, People’s Republic of China

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*Corresponding author (Tel.: +86-106-273-4565; Fax: +86-106-273-1032; E-mail:

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[email protected].)

Department of Chemical Enzymology, Faculty of Chemistry, M.V. Lomonosov Moscow State

Department of Genetic Toxicology, Shenzhen Center for Disease Control and Prevention, 518020

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ACS Paragon Plus Environment


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ABSTRACT

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To develop a sensitive fluorescence polarization immunoassay (FPIA) for screening

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the zearalenone class of mycotoxins in maize, two new monoclonal antibodies

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(MAbs) with uniform affinity to zearalenone class and four fluorescein-labeled

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tracers were prepared. After careful selection of appropriate tracer-antibody pairs in

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term of sensitivity and specificity, a FPIA that could simultaneously detect

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zearalenone class with similar sensitivity was developed. Under optimum conditions,

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the half maximal inhibitory concentrations (IC50) of the FPIA in buffer were 1.89,

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1.97, 2.43, 1.99, 2.27 and 2.44 µg/L for zearalenone, α-zearalenol, β-zearalenol,

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α-zearalanol, β-zearalanol and zearalanone, respectively. The limit of detection of

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FPIA for the zearalenone class was around 12 µg/kg in maize and the recoveries

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ranged from 84.6-113.8% with coefficients of variation below 15.3% in spiked

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samples. Finally, the FPIA was applied for screening naturally contaminated maize

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samples and the results indicated a good correlation with that of HPLC-MS/MS.

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KEYWORDS: zearalenone, monoclonal antibody, fluorescence polarization

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immunoassay, maize, detection

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INTRODUCTION

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Humans and animals are continuously exposed to a variety of mycotoxins that

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naturally occur in various food products such as maize.1 Zearalenone, 1, is an

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estrogenic mycotoxin that is an important secondary metabolite produced by several

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species of the genus Fusarium, such as Fusarium graminearum, F. culmorum, F.

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cerealis, F. equiseti, F. crookwellense and F. semitectum.2 It is one of the most

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prevalent mycotoxins worldwide in maize, barley, wheat and other cereal grains.2-5 It

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has been classified as a group III carcinogen by the International Agency for

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Research on Cancer because it has been associated with early puberty, hyperplastic

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and neoplastic endometria, and human cervical cancer, and it exhibits a genotoxic

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potential in vitro and in vivo through induction of micronuclei, chromosome

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aberrations, DNA fragmentation and cell cycle arrest.6-9 Due to the high toxicity of 1

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in both animals and humans, the European Union (EU) has set a maximum residue

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levels (MRLs) of 100 µg/kg for cereals and 350 µg/kg for unprocessed maize, while

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the legal limits of 60 µg/kg has been set for cereal and cereal-based products in

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China.3, 10 Actually, two other structurally similar mycotoxins α- and β-zearalenol, 2

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and 3, usually co-occur with 1 in cereals infected with Fusarium (see Figure 1).3,11-13

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In addition, it has been reported that 1 could be metabolized to 2 and 3, α- and

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β-zearalanol, 4 and 5, in some animal species.12,14 These mycotoxins and metabolites,

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i.e., 2, 3, 4, 5 and zearalanone, 6 (see Figure 1), have also been reported to be

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harmful because of their estrogenic activity.12,15,16 Thus, monitoring the presence of

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the zearalenone class of resorcylic acid lactones in maize is necessary to reduce the 3



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risk of zearalenones to humans and livestock. Considering the speed, cost and

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efficiency, a detection method that can simultaneously detect the zearalenone class,

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instead of each individual analog, would be preferable.

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Antibody-based methods, i.e., immunoassays, are relatively inexpensive, rapid

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and sensitive screening methods that are used to detect chemical contaminants in

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large-scale monitoring programs.17 There have been some reports of the

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immunoassays that can be used for the detection of the zearalenone class, mainly

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enzyme-linked immunosorbent assays (ELISA)3,18,19 and lateral-flow immunoassays

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(LFAs).20,21 ELISA, although very sensitive, requires multiple incubations, washing

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steps, and extensive pipetting and, thus, is time-consuming.22 LFAs are usually used

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for qualitative analysis and low throughput. Compared with ELISA and LFA, the

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fluorescence polarization immunoassay (FPIA) takes only a few minutes before

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measuring, needs no separations and washing steps,23 and also can give the

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quantitative results. These advantages make this method be suitable for monitoring

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large-scale samples. For example, commercial FPIA kits have been developed for

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aflatoxins, deoxynivalenol and fumonisins in cereals. Recently, some FPIA methods

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for the detection of 1 in corn have been reported.22,24-26 However, these methods

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were less accuracy for detection those zearalenone structural analogs due to the

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diverse cross-reactivity (CR) values of 35.3%≤CR%≤522.2%25 or 20%≤CR%≤

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195%22 derived from large variations in affinities of antibodies to each zearalenone

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analog. Thus, establishing a FPIA with high sensitivity and accuracy for

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simultaneous detection of the zearalenone class is necessary. 4


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Antibodies and tracers are two main factors that could directly improve the

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sensitivity and accuracy of an FPIA for the zearalenone class detection. All reported

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antibodies against zearalenones do not meet the requirement of detection of the

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zearalenone class with highly accuracy because of diverse CR% values. For example,

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a monoclonal antibody (MAb) named 2D3 was prepared with an IC50 value of 0.02

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µg/L for 1, but this exhibited a low CR with 2 (4.4 %).27 Thus, an antibody that could

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be employed to develop a multi-analyte FPIA should provide not only high but also

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uniform affinity to all the targets of interest. In addition, the fluorescein thiocarbamyl

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hexamethylenediamine-labeled zearalenone (1-HMDF) as a tracer was designed to

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establish a FPIA for screening 1 in corn with the limit of detection (LOD) value of

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137 µg/kg.24 The sensitivity of the FPIA was improved 2-fold (LOD of 77 µg/kg)

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when 4'-(aminomethyl) fluorescein-labelled zearalenone (1-AMF) was selected as a

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tracer using the same antibody in a follow-up study.25 In a word, the sensitivity of

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FPIA is significantly influenced by the structure of the tracer. Therefore, we have to

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search a better tracer.

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The aim of the present study was to produce new MAbs with uniform affinity

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towards the zearalenone structural analogs and tracers to develop a FPIA for the

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simultaneous detection of the zearalenone class with highly sensitivity and accuracy.

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The feasibility of the developed method was confirmed by HPLC-MS/MS and the

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results showed that the FPIA could be used as a screening method for the

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simultaneous detection of the zearalenone class in maize.

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MATERIALS AND METHODS 5



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Reagents and materials

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Zearalenone, 1, α-zearalanol, 2, β-zearalanol, 3, α-zearalenol, 4, β-zearalenol, 5,

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zearalanone, 6, aflatoxin B1, ochratoxin A, fumonisins B1, deoxynivalenol, keyhole

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limpet

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3,3',5,5'-tetramethylbenzidine (TMB), Freund's complete/incomplete adjuvant,

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polyethylene glycol 1500 (PEG 1500) and hypoxanthine aminopterin thymidine

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(HAT) were purchased from Sigma-Aldrich (St. Louis. MO). Fetal bovine serum was

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provided by Gibco BRL (Paisley, Scotland).

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(DMEM) were obtained from Shanghai Lifei Biotechnology Co., Ltd (Shanghai,

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China). Ethylenediamine fluoresceinthiocarbamyl (EDF) was previously synthesized

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by our group.28 4'-(aminomethyl) fluorescein (AMF) was obtained from Molecular

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Probes (Eugene, OR). Horseradish peroxidase (HRP)-conjugated goat anti-mouse

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IgG (H+L) was purchased from Jackson ImmunoResearch Laboratories, Inc. (West

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Grove,

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N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide

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pyridine, dimethyformamide and N-hydroxysuccinimide (NHS) were obtained from

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Aladdin Chemistry Co., Ltd (Shanghai, China). 0.05 M borate buffer (pH 8.0) was

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selected as a working buffer for FPIA experiment. The fluorescein-labeled tracers of

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1-EDF/AMF were previously synthesized (Figure 1B).24

hemocyanin

PA).

(KLH),

bovine

serum

albumin

(BSA),

Dulbecco's modified Eagle's medium

O-Carboxymethyl

oxime hydrochloride

(CMO), (EDC.HCl),

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Non-binding surface, non-sterile and 96-well black polystyrene microplates

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were purchased from Corning Life Sciences (New York). Thin layer chromatography

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(TLC) aluminum sheets (M5554 silica gel 60 F254) were obtained from Merck 6


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(Darmstadt, Germany).

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Five female BALB/c mice which supplied by Vital River Laboratory Animal

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Technology Co. Ltd. (Beijing, China) were raised in specific pathogen free

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conditions. And those mice were disposed in compliance with Animal Care Center of

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China Agricultural University (2012-SYXK-0037) and Chinese laws and guidelines

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(GKFCZ2001545).

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Apparatus

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Optical density (OD450) signals, fluorescence intensity (FI) and fluorescence

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polarization (FP) values were measured by a Spectramax M5 microplate reader

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(Sunnyvale, CA). Deionized water for the FPIA detection system was provided by a

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Milli-Q synthesis system (Bedford, MA).

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Production of MAbs against zearalenone class

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The hapten of 1-CMO (see Figure 2) was synthesized according to the 133

previously study.29 Briefly, 16 µmol of 1 were dissolved in 1 mL of pyridine and

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reacted with 80 µmol of CMO at 90 °C for 8 h. Those reaction solutions were dried 135

by a vacuum pump, and then redissolved in 5 mL of distilled water (adjusted to pH 8 136

with 1 M sodium bicarbonate buffer). Unchanged 1 was extracted from the sodium 137

bicarbonate buffer by 10 mL of ethyl acetate. Subsequently, the aqueous phase was 138

adjusted to pH 3 by 0.1 M HCl, and the hapten 1-CMO was extracted from the water 139

phase with 5 mL of ethyl acetate for three times. The extraction was dried to obtain 140

the hapten 1-CMO. The hapten 1-CMO was coupled to carrier protein (KLH and 7



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BSA) using the active ester method as previously described.30

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The female BALB/c mouse was injected subcutaneously with 100 µg of 1-KLH 143

(100 µL) emulsified with 100 µL adjuvant for three times.31 The mouse that showed

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competitive inhibition of antibodies binding to 1-BSA by the free zearalenone 145

structural analogs was sacrificed for fusion. The culture supernatants of the 146

hybridoma were screened with an indirect competitive ELISA (icELISA) as 147

previously described.32 The positive hybridomas were used to produce the ascites

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fluids. 149

Preparation of the fluorescein-labeled tracers

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6 was selected for coupling to EDF and AMF (see Figure 2). Since 6 has no

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reactive groups to enable coupling reactions, the first step was converting it into

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6-CMO, prepared in the same way as for 1-CMO. Then, 6-CMO was conjugated to

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amino-fluorescein derivatives (EDF/AMF) with a 6-carbon bridge using the active

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ester method.24,25 Briefly, 6-CMO (2 µmol), EDC (6 µmol) and NHS (6 µmol) were

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dissolved in 0.2 mL of DMF and reacted at room temperature for 12 h. The

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precipitates were removed by centrifugation. EDF or AMF (3 µmol) was added into

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the clear solutions and reacted for another 12 h at RT. And TLC with a

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trichloromethane/methanol (4:1, v/v) as a mobile phase was used to purify the

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fluorescein-labeled tracers of 6-EDF/AMF as described previously.23 The major

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yellow band (Rf around 0.5) was scraped off and soaked into 100 µL of methanol for

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5 min, and centrifuged at 3,000 g for 5 min. The clear solutions were the tracers of

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6-EDF/AMF, and further diluted by 0.05 M borate buffer (pH 8.0) to obtain the

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working concentration or stored at -20 °C in the dark until used.

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Protocol of the FPIA

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The procedure of FPIA was as follows: 70 µL/well of standards, 70 µL/well of

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fluorescein-labeled tracers, and 70 µL/well of the working solution of optimally

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diluted MAb were sequentially added into a microplate. Following a shaking step in

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the Spectramax M5 microplate reader, FP values were measured at λex 485 nm and

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λem 530 nm with an emission cutoff of 515 nm. The standard curve was calculated as

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the reference.33

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To determine the specificity of the developed FPIA, CR% values with other

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mycotoxins including 1, 2, 3, 4, 5, 6, aflatoxin B1, ochratoxin A, fumonisins B1 and

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deoxynivalenol were calculated as the following equation:31

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CR = (IC50 of zearalenone/IC50 of competitor) × 100% Preparation of the maize samples

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Zearalenone-free and naturally contaminant maize flour samples were kindly

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supplied by Prof. De Saeger (Ghent University). 5 g of the maize flour were weighed

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into 50 mL polypropylene centrifuge tubes and 15 mL of methanol/borate buffer (7/3,

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v/v) was added to each sample for extraction. Vortex the mixture for 5 min and

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centrifuge at 3,000 g for 5 min. The obtained supernatant was diluted by 0.05 M

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borate buffer (pH 8.0) for analysis without any further processing. A total of 20

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blank samples were measured by the development of FPIA as a control. The limit of 9



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detection (LOD) was calculated using the following formula: _

LOD = (X + 3SD)

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_

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The X was the average detection concentration of the 20 blank samples. The

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working range corresponds to the concentration of standard varying from IC20-IC80

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of the calibration curves. Naturally contaminated maize samples confirmed by

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HPLC-MS/MS 3, 34 were prepared as the same procedure and submitted to the FPIA.

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RESULTS AND DISCUSSION

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MAbs production

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Zearalenone structural analogs are small molecules (MW

Fluorescence Polarization Immunoassay Based on a New Monoclonal

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