Fluorescence Polarization Immunoassay Based on ... - ACS Publications

Subscriber access provided by University of Newcastle, Australia

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

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.

Page 1 of 33

Journal of Agricultural and Food Chemistry

1

A Fluorescence Polarization Immunoassay Based on

2

a New Monoclonal Antibody for the Detection of the

3

Zearalenone Class of Mycotoxins in Maize

4

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

5

Jianzhong Shen†, Zhanhui Wang*,†

6

†

7

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

8

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

9

People’s Republic of China

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

10

‡

11

University, Moscow 119991, Russia

12

§

13

Shenzhen, People’s Republic of China

14

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

15

[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

1

ACS Paragon Plus Environment


16

ABSTRACT

17

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

18

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

19

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

20

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

21

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

22

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

23

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

24

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

25

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

26

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

27

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

28

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

29

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

30

KEYWORDS: zearalenone, monoclonal antibody, fluorescence polarization

31

immunoassay, maize, detection

2


Page 2 of 33

Page 3 of 33


32

INTRODUCTION

33

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

34

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

35

estrogenic mycotoxin that is an important secondary metabolite produced by several

36

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

37

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

38

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

39

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

40

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

41

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

42

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

43

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

44

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

45

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

46

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

47

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

48

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

49

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

50

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

51

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

52

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

53

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



54

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

55

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

56

instead of each individual analog, would be preferable.

57

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

58

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

59

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

60

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

61

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

62

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

63

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

64

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

65

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

66

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

67

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

68

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

69

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

70

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

71

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

72

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

73

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

74

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

75

simultaneous detection of the zearalenone class is necessary. 4


Page 4 of 33

Page 5 of 33


76

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

77

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

78

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

79

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

80

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

81

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

82

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

83

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

84

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

85

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

86

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

87

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

88

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

89

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

90

search a better tracer.

91

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

92

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

93

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

94

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

95

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

96

simultaneous detection of the zearalenone class in maize.

97

MATERIALS AND METHODS 5



98

Page 6 of 33

Reagents and materials

99

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

100

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

101

limpet

102

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

103

polyethylene glycol 1500 (PEG 1500) and hypoxanthine aminopterin thymidine

104

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

105

provided by Gibco BRL (Paisley, Scotland).

106

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

107

China). Ethylenediamine fluoresceinthiocarbamyl (EDF) was previously synthesized

108

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

109

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

110

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

111

Grove,

112

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

113

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

114

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

115

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

116

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),

117

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

118

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

119

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


Page 7 of 33


120

(Darmstadt, Germany).

121

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

122

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

123

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

124

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

125

(GKFCZ2001545).

126

Apparatus

127

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

128

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

129

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

130

Milli-Q synthesis system (Bedford, MA).

131

Production of MAbs against zearalenone class

132

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

134

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



141

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

142

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

144

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

148

fluids. 149

Preparation of the fluorescein-labeled tracers

150

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

151

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

152

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

153

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

154

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

155

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

156

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

157

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

158

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

159

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

160

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

161

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

8


Page 8 of 33

Page 9 of 33


162

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

163

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

164

Protocol of the FPIA

165

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

166

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

167

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

168

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

169

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

170

the reference.33

171

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

172

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

173

deoxynivalenol were calculated as the following equation:31

174

175

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

176

Zearalenone-free and naturally contaminant maize flour samples were kindly

177

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

178

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

179

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

180

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

181

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

182

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



183

Page 10 of 33

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

LOD = (X + 3SD)

184

_

185

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

186

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

187

of the calibration curves. Naturally contaminated maize samples confirmed by

188

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

189

RESULTS AND DISCUSSION

190

MAbs production

191

Zearalenone structural analogs are small molecules (MW

Fluorescence Polarization Immunoassay Based on ... - ACS Publications

Recommend Documents