Development and Application of a Gel-Based Immunoassay for the

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Development and Application of a Gel-based Immunoassay for Rapid Screening of Salbutamol and Ractopamine Residues in Pork Chenglong Li, Jingya Li, Wenxiao Jiang, Suxia Zhang, Jianzhong Shen, Kai Wen, and Zhanhui Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04203 • Publication Date (Web): 23 Nov 2015 Downloaded from http://pubs.acs.org on November 23, 2015

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Journal of Agricultural and Food Chemistry

Development and Application of a Gel-based Immunoassay for Rapid Screening of Salbutamol and Ractopamine Residues in Pork Chenglong Li †, , Jingya Li †, , Wenxiao Jiang †, ‡, Suxia Zhang †, Jianzhong Shen †, #, Kai Wen †, #, *, Zhanhui Wang † †

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

of Veterinary Medicine, China Agricultural University, Beijing Key Laboratory of Detection Technology for Animal-Derived Food Safety, Beijing Laboratory for Food Quality and Safety, 100193 Beijing, People’s Republic of China ‡

The Engineering Lab of Synthetic Biology, Key Lab of Biomedical Engineering,

School of Medicine, Health Science Center, Shenzhen University, 518060 Shenzhen, People’s Republic of China #

National Reference Laboratory for Veterinary Drug Residues, 100193 Beijing,

People’s Republic of China



*

These authors contributed equally to this paper Author to whom correspondence should be addressed

Tel: +86-10-6273 1032 Fax: +86-10-6273 1032 E-mail: [email protected]

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Abstract

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Salbutamol (SAL) and ractopamine (RAC) have been illegally used to promote

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protein synthesis and to increase the feed conversion rate in livestock. However, the

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residues of SAL and RAC could cause potential hazards for human health. The

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Ministry of Agriculture of China banned the use of SAL and RAC as growth

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promoters. In this paper, we enclosed detail information on developing a rapid and

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sensitive gel-based immunoassay for on-site screening SAL and RAC residues in pork.

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The detection time was shortened to 20 min. The limits of detection were 0.5 μg/kg

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for SAL and RAC by visual detection, while, the quantitative gel-based immunoassay

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enabled the detection of SAL (0.051 μg/kg) and RAC (0.020 μg/kg) in spiked pork

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samples. The gel-based immunoassay showed promise as a multiplexed immunoassay

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for on-site surveilling of SAL and RAC residues in pork.

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Keywords: salbutamol; ractopamine; gel-based multiplexed immunoassay; pork

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1. Introduction

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β-Agonists, a class of phenylethanolamine compounds, are widely used for the

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treatment of respiratory diseases. Among them, salbutamol (SAL) and ractopamine

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(RAC) are two typical representatives, and they are originally used as tocolytics,

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bronchodilators, and heart tonics in human and veterinary medicine.

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years, β-agonists have been illegally used to promote protein synthesis and to increase

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the feed conversion rate in animal husbandry.

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chemicals could pose potential hazard on human health, such as food poisoning,

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cardiovascular and central nervous diseases. 6-8 Therefore, the Ministry of Agriculture

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of China and some other countries banned the use of β-agonist as growth promoters in

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animal husbandary. 9

3-5

1, 2

In recent

However, the residues of these

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Because these compounds could enhance the growth rates and feed efficiency,

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β-agonists were still illegally used in food-animal producing. Thus, it is urgent to

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develop rapid and sensitive analytical methods for screening of trace residues for

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surveillance. 10 During the last two decades, some analytical methods were developed

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for the analysis of β-agonists residues in various food matrices. Of all, GC-MS,

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LC-MS and LC-MS/MS are widely used for the determination of SAL and RAC

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residues.

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instruments,

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procedures. Hence, they are not suitable for on-site screening purpose.

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Immunoassays are widely used as screening method in food safety, environmental

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monitoring and clinic diagnosing. Recently, enzyme linked immunosorbent assay

11-16

However, these chromatographic techniques require expensive professional

technicists

and

time-consuming

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sample

treatment 17

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(ELISA), lateral-flow tests, surface plasmon resonance sensors and other analytical

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methods were developed for rapid screening of veterinary drug residues.

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However, lateral-flow tests have low sensitivity; ELISA is time-consuming, and SPR

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needs special equipment. 23, 24 Thus, a rapid and sensitive analytical method is needed

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for screening purpose.

3, 9, 18-22

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In this study, a rapid and sensitive non-instrumental method was developed for

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simultaneous detection of SAL and RAC residues. The principle of gel-based

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immunoassay was similar to traditional ELISAs.

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the specific combination of antigen and antibody reacted. After the addition of

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chromogenic substrate, a blue color development was visible. The cut-off value was

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defined as the concentration of target analyte at which there was no color

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development. Furthermore, the colour intensity of the color development was

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evaluated to make a quantitative detection. To our knowledge, it is the first time that a

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qualitative and quantitative gel-based immunoassay was developed for simultaneously

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screening two kinds of β-agonists residues.

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2. Materials and methods

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Reagents and materials

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Gel was used as a carrier where

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SAL and RAC standards were purchased from China Institutes for Food and

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Drug Control (Beijing, China). Anti-SAL antibody, anti-RAC antibody, SAL-HRP and

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RAC-HRP conjugates were obtained from Beijing WDWK Biotechnology Co., Ltd

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(Beijing, China). CNBr-activited sepharose 4B was purchased from GE Healthcare 4

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(New Jersey, USA). Goat anti-mouse IgG were purchased from Jackson

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Immuno-Research Inc. (Pennsylvania, USA). Bond Elut SPE cartridges (1 mL) and

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polyethylene frits were purchased from Agilent Technologies (California, USA).

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Solutions and buffers

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Phosphate-buffered saline (PBS, 0.01 M, pH 7.4), chromogenic substrate (0.1%

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TMB, H2O2 in 0.05 M citrate buffer, pH 4.5), coupling buffer (NaHCO3 buffer

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containing 0.5 M NaCl, pH 8.3), blocking buffer (coupling buffer containing 0.2 M of

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glycine, pH 8.0) and acetate buffer (pH 4.0, containing 0.5 M NaCl) were prepared in

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this study.

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Preparation of the gels

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Gel coupled with anti-mouse IgG (AM-CG) was carried out according to the

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instruction of the manufacturer and previous researches. 23 The anti-SAL antibody and

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anti-RAC antibody were immobilized onto the gel to prepare the anti-SAL gel and

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anti-RAC gel. Preparation of blocked gel (BG, gel coupled with glycine) was similar

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with the AM-CG that described above, by using glycine to block the active site of the

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coupled gels. The prepared gel was suspended in PBS containing 0.03% Proclin 300,

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and stored at 4 °C before use.

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Gel-based immunoassay

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The first frit was placed on the bottom at the 1 mL Bond Elut Reservoir, and 200

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μL of anti-RAC gel was added to the column. The second frit was used to cover the

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RAC test-layer, and the third frit was added to the column to produce an air gap layer

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(0.3 cm). Then, 200 μL of anti-SAL gel was added to the column above the third frit, 5

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and the fourth frit was used to cover the SAL test-layer. The prepared columns were

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stored at 4 °C until use.

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Sample treatment and trace residues analysis

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The pork samples were purchased from outlet of Beijing, and all samples were

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verified as free of SAL and RAC using LC-MS/MS. 8 Then, 1 g of pork sample was

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extracted using 3 mL of trichloroacetic acid. After centrifugation, the pH of the

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extracting solution was adjusted to pH 7.0 using 1 M NaOH. Then, about 3 mL of the

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extracting solutions were added to the test column from the inlet. The sample extracts

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should pass through the column at the speed of 1 drop per second. Then, 200 μL of

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diluted RAC-HRP and SAL-HRP conjugates solution was added to the test layer, and

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incubated for 6 min. Then, 6 mL of PBST-NaCl solution was added in order to

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remove the unbound conjugates. After adding 300 μL of the chromogenic substrate,

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the visual color was observed after incubation for 5 min.

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Quantitative interpretation and assay validation

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The qualitative gel-based immunoassay could only provide a rough result with

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naked eyes, which resulted in a low sensitivity. In order to improve its sensitivity, the

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images of the gel-based immunoassays were record and were saved in JPG format,

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and the colour intensity of test layers were evaluated by “Adobe Photoshop CS5”, and

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values of saturation (parameter S, HSB mode) were obtained for quantitative analysis.

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26, 27

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quantitative analysis.

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The value of parameter S was obtained and calculated for curve fitting and

The gel-based immunoassay was validated by a well-established LC-MS/MS 6

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method. 8 Blank pork samples spiked with SAL and RAC standards were analyzed by

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both the quantitative gel-based immunoassay and the well-established LC-MS/MS

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method which detection limits for SAL and RAC were 0.04 μg/kg and 0.10 μg/kg,

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respectively. Correlation of the two methods was compared, and further validation

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was carried out according to Commission Decision 2002/657/EC.

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3 Results and discussion

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Principle of the gel-based immunoassay

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The principle of the gel-based immunoassay was similar with traditional ELISAs.

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While, the gel-based immunoassay used sepharose 4B gels as carrier for immobilizing

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antibody. There were two test layers and one air gap layer in the developed

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immunoassay. For test layer, CNBr-activated sepharose 4B bead was utilized as

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carrier media to immobilize anti-SAL and anti-RAC antibodies. Then target analytes

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in samples would compete with HRP-analyte conjugates to combine with antibody in

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the test layer. After the addition of chromogenic substrate and the color development,

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the results can be observed by naked eyes. The negative results would present a

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visible blue color on the respective test layers, while the positive results presented no

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or negligible color. The cut-off value was defined as the lowest concentration of

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anlayte which results in no color development. Compared with traditional

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immunoassays, the gel-based immunoassays have a larger loading volume, which

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could greatly improve the sensitivity.

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Optimization of gel-based immunoassay 7

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Compared with traditional immunoassays, the non-specific adsorption effects

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could greatly affect the sensitivity of the gel-based immunoassay. In this study, the

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concentration of the antibodies and HRP-analyte conjugates were optimized first.

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Dilutions of SAL-HRP ranged from 1:8000 to 1:40000 in 0.01 M PBS (pH 7.4), while,

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dilutions of anti-SAL antibody ranged from 1:4000 to 1:20000 were used for

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optimization. In consideration of color development and non-specific adsorption

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effect, SAL-HRP dilution at 1:8000 and anti-SAL antibody dilution at 1:10000 were

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chosen. The optimization of RAC test-layer was similar to the SAL test-layer, and the

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RAC-HRP dilution at 1:20000 and anti-RAC antibody dilution at 1:10000 were

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chosen as optimal. If the concentrations of antibody and HRP tracer are higher than

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the selective level, non-specific adsorption’s interference can influence on the visual

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color development results, and lower concentrations of antibody and HRP tracer may

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lead to weak color development.

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Washing buffer is also important for eliminating nonspecific adsorption.

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Different volumes (2, 4, 6 and 8 mL) and different ionic strength (NaCl concentration

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at 0, 0.05, 0.1, 0.5, 1.0 M in washing buffer) were investigated. Increasing both of the

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volume and ionic strength under a certain level could greatly minimize the

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non-specific adsorption effect. The results revealed that 6 mL of the washing buffer

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(NaCl concentration was 0.5 M) could effectively decrease the nonspecific adsorption

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to insignificant levels.

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In order to simultaneously screen two analytes, the reaction time (incubation

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time of HRP conjugates and detection time of chromogenic substrate) should be 8

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optimized for both test layers. As shown in Figure 1A, the parameter S (saturation, %)

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value could increase when prolong the incubation time with HRP conjugates. The

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non-specific adsorption effect appeared when the incubation time was over 6 min.

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Thus, 6 min was chosen as optimal for incubation time of HRP conjugates. The

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optimization procedure of detection time was similar to the optimization procedure of

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incubation time. As shown in Figure 1B, 5 min was chosen as optimal detection time

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in consideration of the non-specific adsorption effect and the color development

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intensity.

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Assay specificity

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β-Agonists are a class of phenylethanolamine compounds with synthetic

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chemical structure. The specificity of the gel-based immunoassay was evaluated by

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determining the cross-reactivity using other β-agonists. In this study, clenbuterol

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(CLE), cimaterol, terbutaline, zipaterol, clorprenaline, brobuterol and bambuterol

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were tested. There was no elimination of the color intensity in the test layers for the

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other β-agonists at a high concentration (100 ng/mL), which indicated that the

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gel-based immunoassay is high specific for detecting SAL and RAC residues (Figure

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2).

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Sample preparation and trace residues analysis

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The complex food matrix effect could greatly influent the immunoassays. Due to

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the high sample loading capacity, sample matrix effect could be reduced greatly using

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a comparatively simple preparation procedure. The results of the gel-based

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immunoassay in standard solution were shown in Figure 3A. The color of the test 9

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layers fades when increasing analyte concentration in standard solution. When the

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concentration was higher than the cut-off level (defined as qualitative LODs), it could

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occupy enough binding sites of antibodies, and lead to the color disappeared. From

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the results of Figure 3A, the qualitative LODs were 0.5 μg/kg for SAL and RAC,

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respectively. Spiked pork samples were prepared and tested by the developed

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gel-based immunoassays. As shown in Figure 3B, the cut-off values were 0.5 μg/kg

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for screening of SAL and RAC residues in pork, and these results indicated that the

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matrix effects showed negligible effects on the assay sensitivity.

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Probabilities of positive and negative results were tested to prevent false negative

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results due to matrix interferences. In this study, 50 blank samples and 50 spiked

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samples (spiked with SAL and RAC at 0.5 μg/kg) were tested by the developed

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gel-based immunoassays. As summarized in Table 1, the false positive and false

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negative rates were 8% and 4% for SAL residue analysis, while the results were 2%

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and 4% for RAC residue analysis, which indicated the permission of this method to be

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used in the detection of these two analytes.

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Quantitative analysis and assay validation

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More intuitive results could be obtained by quantifying the colour intensity of the

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test layers in Figure 3A. According to previous research, the colour intensity was

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obtained from the images, and the results were tested for three times and the average

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value and standard deviation was calculated. B/B0 ratio was selected to express the

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competitive inhibition curve where B0 and B were the parameter S values obtained

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from binding at zero and certain concentrations of standards. A four parameter logistic 10

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equation was used to fit the immunoassay data. The calibration curve and analytical

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performance were shown in Figure 4. Comparing with observed by naked eye, it has

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higher resolution to decide the cut-off value. The calculated LODs of SAL and RAC

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were 0.051 μg/kg and 0.020 μg/kg, respectively. The color quantization plays an

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important role for improving the sensitivity, which were about 10 to 20 times better

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than qualitative gel-based immunoassays.

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To evaluate the accuracy and precision of the quantitative gel-based

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immunoassay, an LC-MS/MS method was used as a reference method for comparison.

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As shown in Table 2, both methods had satisfactory recoveries and the coefficient of

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variations (CVs). The average recoveries of SAL and RAC of the gel-based

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immunoassay were in the range of 78-109%, and CV was in the range of 8.5-16.7%.

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As shown in Figure 5, the linear regression of the two methods was

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y=0.7231x+0.1196 with the R2 of 0.9439 for SAL residue analysis, and

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y=0.8566x+0.0311 with the R2 of 0.9538 for RAC residue analysis. These results

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indicated that the correlation of the two methods was satisfying, and the described

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gel-based assay was proved to be rapid, sensitive and precise for screening of SAL

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and RAC residues in pork.

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Comparison with other multiplexed immunoassays

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Several immunoassays have been developed for multi-residue detection of

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-agonists. Zuo et al. established a hapten microarray for simultaneous determination

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of SAL, RAC and CLE in pig urine, which obtained the detection limits for SAL and

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RAC

were

0.01

g/L

and

0.5

g/L

respectively.

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A

disposable

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electrochemiluminescent immunosensors array for measurement of SAL and RAC in

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swine urine was recently reported. 29 The detection limits were 17 pg/mL for SAL and

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8.5 pg/mL for RAC. Han et al. also developed a time-resolved chemiluminescence

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immunoassay for multiplexed detection of RAC and CLE in swine urine.

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flow immunoassays based on different reporters, such as colloidal gold, Ru(phen)32+

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doped silica nanoparticle, and fluorescent beads have been widely used for screening

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of β-agonists.

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chemiluminescent determination of SAL and RAC in swine urines.

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gel-based immunoassay combined the preconcentration and immunochemical

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measurement was developed for SAL and RAC detection on different sepharose gel

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test layers. The detection time was shortened within 20 min, which was comparable

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with lateral flow immunoassays, while the sensitivity was lower in the developed

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gel-based immunoassays.

31-33

30

Lateral

Gao et al. also described an immunochromatographic test strip for 34

In this assay, a

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In conclusion we described a gel-based immunoassay for multiplexed detection

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of SAL and RAC residues in pork samples, which capable of sifting a minimum of 0.5

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g/kg of both SAL and RAC by naked eye, 0.051 g/kg of SAL and 0.020 g/kg of

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RAC by digital camera within 20 min. These qualitative and quantitative results

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revealed the suitability of this assay as a tool for fast, sensitive, cost-effective and

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field screening of chemical residues in animal origin foods.

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Funding Sources

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This work was supported by grants from the Special Fund for Agro-scientific 12

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Research in the Public Interest (201203088-02A).

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immunochemically-based

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test

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monitoring

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hapten microarray. Talanta 2010, 82, 61-66.

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near-simultaneous assay of multiple “lean meat agent” residues in swine urine using a

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disposable electrochemiluminescent immunosensors array. Biosens. Bioelectron. 2013,

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of ractopamine and salbutamol. Anal. Chim. Acta 2014, 839, 91-96.

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Figure Captions

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Figure 1. Optimization of the incubation time (A) and the detection time (B) of

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gel-based immunoassays.

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Figure 2. Color saturation of different β-agonists compounds (SAL; RAC;

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clenbuterol, CLE; cimaterol, CIM; terbutaline, TER; zipaterol, ZIP; clorprenaline,

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CLO; brobuterol, BRO; bambuterol, BAM; 100 ng/mL)

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Figure 3. The gel-based immunoassay for screening of SAL and RAC in standard

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solution (A) and spiked pork samples (B).

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Figure 4. The calibration curve of the gel-based immunoassay for screening of SAL

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and RAC residues in pork. a: The LOD refers to the analyte concentration

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corresponding to 20% specific binding. b: The IC50 refers to the analyte concentration

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corresponding to 50% specific binding. c: The linear range represent the analyte

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concentrations obtained between 20% and 80% specific bindings.

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Figure 5. The correlation analysis of pork samples by the developed gel-based

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immunoassay and LC-MS/MS method.

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Table 1 Analytical performance of the gel-based immunoassay Parameter False positive rate, % (Nfalse positive/N-×100) False negative rate, % (Nfalse negative/N+×100) Specificity, % (Nnegative/N-×100) Specificity, % (Npositive/N+×100)

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SAL 8 4 92 96

RAC 2 4 98 96

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Table 2 SAL and RAC determination in pork sample by gel-based immunoassay and LC-MS/MS (n = 3) Analyte SAL

RAC

Spiked (μg/kg) 0.3 0.5 1.0 0.3 0.5 1.0

Gel-based Immunoassay Recovery (%) CV (%) 86 8.5 78 12.4 91 15.3 82 14.8 109 9.3 84 16.7

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LC-MS/MS Recovery (%) CV (%) 107 8.9 93 7.1 89 6.8 87 8.2 105 8.3 95 10.2

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Figure 1. Optimization of the incubation time (A) and the detection time (B) of gel-based immunoassays. 145x58mm (216 x 216 DPI)

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Figure 2. Color saturation of different β-agonists compounds (SAL; RAC; clenbuterol, CLE; cimaterol, CIM; terbutaline, TER; zipaterol, ZIP; clorprenaline, CLO; brobuterol, BRO; bambuterol, BAM; 100 ng/mL) 81x58mm (219 x 219 DPI)

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Figure 3. The gel-based immunoassay for screening of SAL and RAC in standard solution (A) and spiked pork samples (B). 145x51mm (227 x 227 DPI)

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Figure 4. The calibration curve of the gel-based immunoassay for screening of SAL and RAC residues in pork. a: The LOD refers to the analyte concentration corresponding to 20% specific binding. b: The IC50 refers to the analyte concentration corresponding to 50% specific binding. c: The linear range represent the analyte concentrations obtained between 20% and 80% specific bindings. 145x49mm (300 x 300 DPI)

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Figure 5. The correlation analysis of pork samples by the developed gel-based immunoassay and LC-MS/MS method. 145x57mm (217 x 217 DPI)

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Graphic for table of contents 96x46mm (300 x 300 DPI)

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