Development of an Enzyme-Linked Immunosorbent Assay for the

Nov 5, 2016 - Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology & Business University (BTBU), Beijing 100048,...
2 downloads 3 Views 873KB Size
Subscriber access provided by UNIV OF WATERLOO

Article

Development of an Enzyme-linked Immunosorbent Assay for the Detection of Tyramine as an Index of Freshness in Meat and Seafood Wei Sheng, Congcong Sun, Guozhen Fang, Xuening Wu, Gaoshuang Hu, Yan Zhang, and Shuo Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04422 • Publication Date (Web): 05 Nov 2016 Downloaded from http://pubs.acs.org on November 7, 2016

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 30

Journal of Agricultural and Food Chemistry

1

Development of an Enzyme-linked Immunosorbent Assay for the Detection of

2

Tyramine as an Index of Freshness in Meat and Seafood

3 4

Wei Sheng,† Congcong Sun,† Guozhen Fang,† Xuening Wu,† Gaoshuang Hu,† Yan

5

Zhang,† and Shuo Wang*,†, ‡

6



7

Tianjin University of Science and Technology, Tianjin 300457, China.

8



9

Technology & Business University (BTBU), Beijing 100048, China

Key Laboratory of Food Nutrition and Safety, Ministry of Education of China,

Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing

10 11 12 13

*Corresponding author. Tel: +86 22 6091 2483; Fax: +86 22 6091 2489 Email: [email protected]

14 15 16 17 18 19 20 21 22 1

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

23

ABSTRACT

24

A competitive indirect enzyme-linked immunosorbent assay (ciELISA) using

25

polyclonal antibody was developed to detect tyramine in meat and seafood. This

26

ciELISA had a 50% inhibition concentration (IC50) of 0.20 mg/L and a limit of

27

detection (LOD) of 0.02 mg/L, and showed no cross-reactivity with tyrosine or other

28

biogenic amines. The average recoveries of tyramine from spiked samples for this

29

ciELISA ranged from 85.6 to 102.6%, and the results exhibited good correlation with

30

the high-performance liquid chromatography (HPLC) results. The LOD of this assay

31

for tyramine in meat and seafood samples was 1.20 mg/kg. The ciELISA was

32

successfully applied to detect tyramine in positive fish samples, and the results were

33

validated by HPLC to be reliable. The developed ciELISA allows for rapid, specific

34

and accurate detection of tyramine in meat and seafood samples, and it could be a

35

potential useful tool for the evaluation of the freshness of protein-rich foods.

36

KEYWORDS:

tyramine, ELISA, detection, meat, seafood, freshness

37 38 39 40 41 42 43

2

ACS Paragon Plus Environment

Page 2 of 30

Page 3 of 30

Journal of Agricultural and Food Chemistry

44

INTRODUCTION

45

Tyramine is a biogenic amine (Figure 1), and it is widely present in protein-rich foods,

46

such as meats, meat products, aquatic products, and fermented products. The

47

formation of tyramine is mainly through microbic decarboxylation of amino acids.

48

Tyrosine is the main precursor of tyramine. A small amount of biogenic amines does

49

not cause toxic effects on the human due to the detoxification of the amine oxidases in

50

the human intestine for the amines. Nevertheless, high concentrations of biogenic

51

amines will produce a serious health risk for the human.1 Tyramine causes an increase

52

in blood pressure indirectly by inducing the release of noradrenaline from the

53

sympathetic nervous system. Tyramine also may cause lachrymation and salivation,

54

and increase respiration and blood glucose concentrations.2 Taking in large amounts

55

of tyramine can induce severe headaches and may cause brain hemorrhaging or

56

cardiac failure.3 The European food safety authority (EFSA) has indicated that the

57

exposure to 600 mg/person/meal of tyramine in food show no adverse health effects

58

on individuals who did not take monoamino oxidase inhibitor (MAOI) drugs.

59

However, the safe limits were only 50 mg and 6 mg for the individuals taking third

60

generation and classical MAOI drugs, respectively. 4

61

It is necessary to detect the tyramine content in food not only because of its

62

toxicological effects but also due to its role as an index of freshness for protein-rich

63

foods. The formation and accumulation of some biogenic amines under the action of

64

decarboxylase-positive microorganisms present naturally in food or introduced by

65

contamination during storage and processing seriously affects the freshness and 3

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 4 of 30

66

quality of food. The consumption of foods with high amounts of these biogenic

67

amines can pose health risks for consumers.1 Some studies showed that tyramine

68

could be used as an important freshness index for red meat. The change in its content

69

could objectively reflect the spoilage process.5,6 Several

70

analytical

methods

such

as

HPLC7-11

coupled

with

mass

71

spectrometry,12-14 gas chromatography mass spectrometry,15 ion chromatography,16-18

72

thin

73

sensors,25 and biosensors26-28 have been established to detect tyramine in foods.

layer

chromatography,19-21

capillary

electrophoresis,22-24

electrochemical

74

However, the application of the methods mentioned above has been limited for

75

the routine monitoring of a large number of samples due to the requirement of

76

complex and time-consuming sample pretreatment procedures, professional operators

77

and expensive instruments. Compared with the above instrumental methods,

78

immunoassays have several advantages including high specificity, easy accessibility,

79

reduced analysis time and lower costs. Some immunoassays including the ELISA and

80

immunochromatographic assay have been reported for determining histamine in

81

food.29,30 However, studies relating to the preparation of specific antibody toward

82

tyramine and the development of immunoassay to detect tyramine in foods have not

83

been reported. In this study, our goal was developing a tyramine-specific ciELISA,

84

which would be applied to monitor the spoilage of protein-rich foods by detecting the

85

tyramine content.

86

MATERIALS AND METHODS

87

Materials and Instruments. 4

ACS Paragon Plus Environment

Page 5 of 30

Journal of Agricultural and Food Chemistry

88

Tyramine,

phenylethylamine,

histamine,

tryptamine,

5-hydroxy

tryptamine,

89

cadaverine, putrescine, spermine, spermidine, ovalbumin (OVA), bovine serum

90

albumin (BSA), formaldehyde, glutaraldehyde (50%, v/v), Freund’s adjuvants, fish

91

skin glue, and 3,3',5,5'-tetramethylbenzidine (TMB) were purchased from

92

Sigma-Aldrich (St. Louis, MO). HPLC-grade acetonitrile and dimethyl sulfoxide were

93

purchased from Merck (Darmstadt, Germany). Goat anti-rabbit IgG (H+L)

94

horseradish peroxidase conjugate (HRP-labeled secondary antibody) was obtained

95

from Promega (Madison, WI). Polyvinylpyrrolidone and tween-20 were obtained

96

from Sangon Biotech Co., Ltd. (Shanghai, China). Protein A-Sepharose 4B was

97

purchased from Amersham (Chalfont St Giles, UK). Polystyrene microplates were

98

purchased from Nunc (Roskilde, Denmark)

99

Ultrapure water was prepared by Milli-Q system (Millipore, Billerica, MA).

100

Microplates were washed in a Bio-Rad microplate washer (Hercules, CA).

101

Absorbance measurement was achieved on a Multiskan Spectrum plate reader

102

(Labsystems Diagnostics Oy, Vantaa, Finland). HPLC system (Shimadzu, Tokyo,

103

Japan) was composed of two LC-10ATvp pumps, an SPD-10Avp UV detector and a

104

CTO-10ASvp column oven.

105

Preparation of Protein Conjugates.

106

As shown in Figure 2, the tyramine was coupled to carrier protein using formaldehyde

107

and glutaraldehyde as linkers to prepare the immunogen and coating antigen.

108 109

According

to

the

formaldehyde

coupling

method,

the

immunogen

tyramine-formaldehyde-BSA and coating antigen tyramine-formaldehyde-OVA were 5

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 6 of 30

110

prepared as follows: 20 mg of BSA or OVA was dissolved in the coupling buffer (PBS,

111

pH 7.4, 10 mmol/L, 4 mL). 4.2 mg of tyramine for immunogen or 3.7 mg of tyramine

112

for coating antigen was dissolved in 200 µL of dimethyl sulfoxide, and then, the

113

resulting tyramine solution was allowed to add drop by drop to the foregoing protein

114

solution under magnetic stirring. Then, 5 µL of 37% formaldehyde solution was added

115

to the foregoing mixture, which was then held in a water bath at 30 °C for 8 h with

116

stirring. Finally, the protein conjugates were dialyzed in PBS (pH 7.4, 10 mmol/L) for

117

3 d. According

118

to

the

glutaraldehyde

coupling

method,

the

immunogen

119

tyramine-glutaraldehyde-BSA and coating antigen tyramine-glutaraldehyde-OVA

120

were prepared as follows: 20 mg of BSA or OVA was dissolved in the coupling buffer

121

(PBS, pH 7.4, 10 mmol/L, 4 mL). 4.2 mg of tyramine for immunogen or 3.7 mg of

122

tyramine for coating antigen was dissolved in 200 µL of dimethyl sulfoxide, and then,

123

the resulting tyramine solution was allowed to add drop by drop to the foregoing

124

protein solution under magnetic stirring. Then, 15 µL of 25% glutaraldehyde solution

125

was added to the foregoing mixture, which was then held at 4 °C overnight with

126

stirring. Finally, the protein conjugates were dialyzed in PBS (pH 7.4, 10 mmol/L) for

127

3 d.

128

Antibody Production.

129

The animal experiments were performed in compliance with the Regulations for the

130

Administration of Affairs Concerning Experimental Animals issued by the Ministry of

131

Science and Technology of the People’s Republic of China and Tianjin Municipal 6

ACS Paragon Plus Environment

Page 7 of 30

Journal of Agricultural and Food Chemistry

132

Science and Technology Commission. Four New Zealand white rabbits were equally

133

divided into two groups for immunogen tyramine-formaldehyde-BSA and

134

tyramine-glutaraldehyde-BSA, respectively. Each immunogen at 1 mg/mL was used

135

to immunize two rabbits subcutaneously six times at two-week intervals. Freund’s

136

complete and incomplete adjuvant were used to emulsify with an equal volume of the

137

immunogen for the initial injection and the subsequent booster immunizations,

138

respectively. After the rabbits were bled, the collected whole blood was allowed to

139

coagulate and centrifuge for the separation of the antisera at 4 °C. The antisera

140

purification was performed by protein A-Sepharose 4B affinity column.

141

Competitive Indirect ELISA (ciELISA).

142

The coating antigens (100 µL/well) diluted in 50 mmol/L sodium carbonate coating

143

buffer (pH 9.6) were added into microwell plate and incubated overnight at 4 °C. The

144

microwells were then washed thrice with the washing buffer (PBST, 10 mmol/L PBS

145

with 0.05% Tween-20), and the blocking solution (200 µL/well) was added to block

146

the unbound active sites at 37 °C for 1 h. After the microwells were washed, 50 µL of

147

tyramine standards or diluted sample solutions and 50 µL of anti-tyramine antibodies

148

in PBS were added into each well, and the mixtures were allowed to incubate at 25 °C

149

for 1 h. The microwells were washed again, and the HRP-labeled secondary antibody

150

diluted in PBS (100 µL/well) was then added followed by incubation for 0.5 h at

151

25 °C. After washing, 100 µL per well of TMB substrate solution was added and the

152

enzymatic reaction was kept for 15 min. And then, the reaction was stopped by the

153

addition of 50 µL per well of 1.25 mol/L H2SO4. Finally, the absorbance was 7

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 8 of 30

154

measured in a dual-wavelength mode plate reader with the test wavelength set at 450

155

nm and the reference wavelength set at 650 nm.

156

Determination of Cross-reactivity.

157

Cross-reactivities

with

158

phenylethylamine,

histamine,

159

putrescine, spermine and spermidine were determined by the developed ciELISA to

160

evaluate the antibody specificity. The cross-reactivity (%) was calculated as:

161

Cross-reactivity (%) = IC50 (tyramine) / IC50 (other compound) ×100

162

HPLC Analysis.

163

HPLC analysis was applied to validate the results obtained from the ciELISA at 254

164

nm for UV detection. The mobile phase was formed from ultrapure water (solvent A)

165

and acetonitrile (solvent B). Separation was performed on an INERTSIL ODS-3 C18

166

column (5 µm, 25 cm×4.6 mm) at a flow rate of 1.0 mL/min with the column

167

temperature set at 35 °C. An optimal gradient elution program was 0-5 min: 65-70%

168

B; 5-20 min: 70-100% B; 20-24 min: 100% B; 24-25 min: 100-65% B; 25-30 min: 65%

169

B, stop. The injection volume was 20 µL.

170

Sample Treatment and Recovery.

171

Pork, beef, squid and codfish samples were purchased from local supermarkets.

172

Aliquots of each sample (1.0 g) were transferred into 15 mL plastic centrifuge tubes,

173

and 4 mL of 3% (w/v) trichloroacetic acid (TCA) in water was added. The mixtures

174

were horizontally vibrated for 1 h and the supernatants were separated by the

175

centrifugation at 3,214 × g for 15 min at 4 °C. Hexane (4 mL) was added to the

tyrosine

and

tryptamine,

other

biogenic

5-hydroxy

8

ACS Paragon Plus Environment

amines,

tryptamine,

including cadaverine,

Page 9 of 30

Journal of Agricultural and Food Chemistry

176

supernatants, and the resultant mixtures were thoroughly vortexed for 5 min to

177

remove the fat. The upper organic phases were discarded, and the pH values of the

178

lower solutions were adjusted to 7 by adding 1 mol/L NaOH. The final solutions were

179

diluted with sample dilution buffer for ciELISA analysis.

180

For the recovery study, all samples with known background contents of tyramine

181

were fortified with tyramine to give final concentrations of 12, 24, and 60 mg/kg and

182

analyzed simultaneously by ciELISA and HPLC to evaluate the accuracy of the

183

developed ciELISA.

184

Analysis and Validation of Tyramine in Incurred Samples.

185

Several positive incurred fish samples including yellow croaker, besugo, Japanese

186

besugo, and brown-striped mackerel scad were analyzed using this ciELISA to survey

187

the tyramine concentration. HPLC analysis was applied simultaneously to determine

188

the tyramine contents in these fish samples to validate the reliability of the developed

189

ciELISA.

190

RESULTS AND DISSCUSSION

191

Antibody Characterization.

192

The

193

tyramine-formaldehyde-OVA were prepared using formaldehyde as the crosslinking

194

agent. The immunogen tyramine-glutaraldehyde-BSA and the coating antigen

195

tyramine-glutaraldehyde-OVA were prepared using glutaraldehyde as the crosslinking

196

agent.

197

tyramine-formaldehyde-BSA showed high inhibition by tyramine. That is, all of the

immunogen

In

Table

tyramine-formaldehyde-BSA

1,

the

A1

and

A2

and

from

the

rabbits

9

ACS Paragon Plus Environment

coating

antigen

immunized

with

Journal of Agricultural and Food Chemistry

198

reactive tyramine groups, such as the amino and phenolic hydroxyl groups on the

199

immunogen tyramine-formaldehyde-BSA prepared via coupling the ortho-position

200

hydrogen atoms of the phenolic hydroxyl group on tyramine with the protein using

201

formaldehyde as the crosslinking agent, were retained and exposed, which contributed

202

to obtaining highly specific antibodies against tyramine. Moreover, when coated with

203

heterogeneous coating antigen tyramine-glutaraldehyde-OVA, the A1 from rabbit 1

204

immunized with tyramine-formaldehyde-BSA showed the highest inhibition. Finally,

205

the A1 and coating antigen tyramine-glutaraldehyde-OVA were selected for further

206

experiment.

207

Competitive Indirect ELISA Development.

208

For ciELISA, the concentration of the coating antigen, the dilution of the HRP-labeled

209

secondary antibody, the blocking solution, the pH of the assay buffer, and the

210

incubation temperature may influence the assay performance. To develop a sensitive

211

and reliable ciELISA, the effects of the above factors on the maximal absorbance

212

(Amax) reflecting the maximum binding of the antibody with the antigen and IC50 value

213

reflecting the sensitivity of assay were examined.

214

Coating with less coating antigen per well and using a low concentration of

215

HRP-labeled secondary antibody made the assay more sensitive, so a concentration of

216

0.1 µg/well of coating antigen and a dilution of 1:15000 for the HRP-labeled

217

secondary antibody were finally used. Two kinds of blocking solutions were tested,

218

and the assay is more sensitive when using skim milk powder/PBS. Therefore, 0.5%

219

skim milk powder/PBS (w/v) was finally chosen as the blocking solution. The 10

ACS Paragon Plus Environment

Page 10 of 30

Page 11 of 30

Journal of Agricultural and Food Chemistry

220

resulting IC50 was lower (0.24 mg/L) at pH 7.4. Therefore, a pH of 7.4 for the assay

221

buffer was selected for further study. In this assay, an incubation temperature of 25 °C

222

was used to achieve the highest sensitivity.

223

The standard inhibition curve for the ciELISA to detect tyramine is shown in

224

Figure 3. This ciELISA had an IC50 value of 0.20±0.015 mg/L and a LOD (calculated

225

as IC15) of 0.02±0.004 mg/L.

226

Determination of Cross-reactivity.

227

The evaluation of antibody specificity was performed by measuring the

228

cross-reactivities with tyrosine and eight other biogenic amines. No cross-reactivity

229

(