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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

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Journal of Agricultural and Food Chemistry

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

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Tyramine as an Index of Freshness in Meat and Seafood

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

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Zhang,† and Shuo Wang*,†, ‡

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†

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

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‡

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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

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

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ACS Paragon Plus Environment


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ABSTRACT

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

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

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

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

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

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

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

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

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

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

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

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

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KEYWORDS:

tyramine, ELISA, detection, meat, seafood, freshness

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INTRODUCTION

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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generation and classical MAOI drugs, respectively. 4

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

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

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

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decarboxylase-positive microorganisms present naturally in food or introduced by

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contamination during storage and processing seriously affects the freshness and 3



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

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

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

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could objectively reflect the spoilage process.5,6 Several

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analytical

methods

such

as

HPLC7-11

coupled

with

mass

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

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thin

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

layer

chromatography,19-21

capillary

electrophoresis,22-24

electrochemical

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

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

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complex and time-consuming sample pretreatment procedures, professional operators

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and expensive instruments. Compared with the above instrumental methods,

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immunoassays have several advantages including high specificity, easy accessibility,

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

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immunochromatographic assay have been reported for determining histamine in

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

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

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

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

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tyramine content.

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MATERIALS AND METHODS

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Materials and Instruments. 4


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Tyramine,

phenylethylamine,

histamine,

tryptamine,

5-hydroxy

tryptamine,

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

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

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

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

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

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

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

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

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

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purchased from Nunc (Roskilde, Denmark)

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

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

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Absorbance measurement was achieved on a Multiskan Spectrum plate reader

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

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

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CTO-10ASvp column oven.

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Preparation of Protein Conjugates.

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

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

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According

to

the

formaldehyde

coupling

method,

the

immunogen

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



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

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

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

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

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

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

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

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3 d. According

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to

the

glutaraldehyde

coupling

method,

the

immunogen

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

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

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

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

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

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

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

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

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3 d.

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Antibody Production.

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

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Administration of Affairs Concerning Experimental Animals issued by the Ministry of

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


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

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

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

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

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

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immunogen for the initial injection and the subsequent booster immunizations,

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

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

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

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Competitive Indirect ELISA (ciELISA).

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

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

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

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

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

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

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

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

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

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

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

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



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

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nm and the reference wavelength set at 650 nm.

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Determination of Cross-reactivity.

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Cross-reactivities

with

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phenylethylamine,

histamine,

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

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

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

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HPLC Analysis.

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

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

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

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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%

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

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B, stop. The injection volume was 20 µL.

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Sample Treatment and Recovery.

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

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

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

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

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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


amines,

tryptamine,

including cadaverine,

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

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

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

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diluted with sample dilution buffer for ciELISA analysis.

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

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

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

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developed ciELISA.

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Analysis and Validation of Tyramine in Incurred Samples.

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

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

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the tyramine concentration. HPLC analysis was applied simultaneously to determine

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

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ciELISA.

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RESULTS AND DISSCUSSION

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Antibody Characterization.

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The

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

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

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

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agent.

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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


coating

antigen

immunized

with


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

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

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

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

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

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

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

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

206

experiment.

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Competitive Indirect ELISA Development.

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

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

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

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

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

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reflecting the sensitivity of assay were examined.

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

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

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

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

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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


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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

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was used to achieve the highest sensitivity.

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

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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.

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Determination of Cross-reactivity.

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The evaluation of antibody specificity was performed by measuring the

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

229

(

Development of an Enzyme-Linked Immunosorbent Assay for the

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