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

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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 †, #, *, 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

3

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

4

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

5

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

6

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

7

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

8

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

9

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

10

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

11

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

12

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

13

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

2



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1. Introduction

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

16

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

17

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

18

bronchodilators, and heart tonics in human and veterinary medicine.

19

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

20

the feed conversion rate in animal husbandry.

21

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

22

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

23

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

24

animal husbandary. 9

3-5

1, 2

In recent

However, the residues of these

25

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

26

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

27

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

28

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

29

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

30

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

31

residues.

32

instruments,

33

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

34

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

35

monitoring and clinic diagnosing. Recently, enzyme linked immunosorbent assay

11-16

However, these chromatographic techniques require expensive professional

technicists

and

time-consuming

3


sample

treatment 17

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

37

methods were developed for rapid screening of veterinary drug residues.

38

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

39

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

40

for screening purpose.

3, 9, 18-22

41

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

42

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

43

immunoassay was similar to traditional ELISAs.

44

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

45

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

46

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

47

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

48

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

49

qualitative and quantitative gel-based immunoassay was developed for simultaneously

50

screening two kinds of β-agonists residues.

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2. Materials and methods

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Reagents and materials

25

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

56

(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

59

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

65

this study.

66

Preparation of the gels

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

68

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

71

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

72

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

73

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

78

(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.

81

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

94

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

95

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.

98

26, 27

99

quantitative analysis.

100

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.

106

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.

109

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

110

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

112

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

114

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

115

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

116

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

120

could greatly improve the sensitivity.

121

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

124

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

127

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

128

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

132

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

133

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

134

lead to weak color development.

135

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

141

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



144

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

145

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.

147

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

150

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

151

intensity.

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Assay specificity

153

β-Agonists are a class of phenylethanolamine compounds with synthetic

154

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

155

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

158

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

159

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

160

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

163

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

164

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

168

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

185

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

187

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

192

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

195

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

197

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

200

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

202

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

203

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.

11


28

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

225

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



231

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

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References

233

1. Price, A. H.; Clissold, S. P., Salbutamol in the 1980s. Drugs 1989, 38, 77-122.

234

2. Ho, J. K.; Huo, T. I.; Lin, L. C.; Tsai, T. H., Pharmacokinetics of ractopamine and

235

its organ distribution in rats. J. Agric. Food Chem. 2014, 62, 9273-9278.

236

3. Shelver, W. L.; Smith, D. J.; Berry, E. S., Production and characterization of a

237

monoclonal antibody against the β-adrenergic agonist ractopamine. J. Agric. Food

238

Chem. 2000, 48, 4020-4026.

239

4. Garssen, G. J.; Geesink, G. H.; Hovingbolink, A. H.; Verplanke, J. C., Effects of

240

dietary clenbuterol and salbutamol on meat quality in veal calves. Meat Sci. 1995, 40,

241

337-350.

242

5. Brambilla, G.; Cenci, T.; Franconi, F.; Galarini, R.; Macri, A.; Rondoni, F.;

243

Strozzi, M.; Loizzo, A., Clinical and pharmacological profile in a clenbuterol

244

epidemic poisoning of contaminated beef meat in Italy. Toxicol. Lett. 2000, 114,

245

47-53.

246

6. Malucelli, A.; Ellendorff, F.; Meyer, H. H. D., Tissue distribution and residues of

247

clenbuterol, salbutamol, and terbutaline in tissues of treated broiler-chickens. J. Anim.

248

Sci. 1994, 72, 1555-1560.

249

7. Martínez-Navarro, J. F., Food poisoning related to consumption of illicit

250

β-agonist in liver. Lancet 1990, 336, 1311.

251

8. Shao, B.; Jia, X.; Zhang, J.; Meng, J.; Wu, Y.; Duan, H.; Tu, X., Multi-residual

252

analysis of 16 β-agonists in pig liver, kidney and muscle by ultra performance liquid

253

chromatography tandem mass spectrometry. Food Chem. 2009, 114, 1115-1121. 14



254

9. Jiang, W.; Beier, R. C.; Wang, Z.; Wu, Y.; Shen, J., Simultaneous screening

255

analysis of 3-methyl-quinoxaline-2-carboxylic acid and quinoxaline-2-carboxylic acid

256

residues in edible animal tissues by a competitive indirect immunoassay. J. Agric.

257

Food Chem. 2013, 61, 10018-10025.

258

10. Jiang, W.; Luo, P.; Wang, X.; Chen, X.; Zhao, Y.; Shi, W.; Wu, X.; Wu, Y.; Shen,

259

J., Development of an enzyme-linked immunosorbent assay for the detection of

260

nitrofurantoin metabolite, 1-amino-hydantoin, in animal tissues. Food Control 2012,

261

23, 20-25.

262

11. Bocca, B.; Fiori, M.; Cartoni, C.; Brambilla, G., Simultaneous determination of

263

zilpaterol and other beta agonists in calf eye by gas chromatography/tandem mass

264

spectrometry. J. AOAC Int. 2003, 86, 8-14.

265

12. Sniegocki, T.; Posyniak, A.; Zmudzki, J., Improved gas chromatography-mass

266

spectrometry method for the determination of clenbuterol and salbutamol in animal

267

urine. Bull. Vet. Inst. Pulawy 2005, 49, 443-447.

268

13. Kootstra, P. R.; Kuijpers, C.; Wubs, K. L.; van Doorn, D.; Sterk, S. S.; van Ginkel,

269

L. A.; Stephany, R. W., The analysis of beta-agonists in bovine muscle using

270

molecular imprinted polymers with ion trap LCMS screening. Anal. Chim. Acta 2005,

271

529, 75-81.

272

14. Wu, Y.; Miao, H.; Fan, S.; Zhao, Y., Determination of 23 β2-agonists and 5

273

β-blockers in animal muscle by high performance liquid chromatography-linear ion

274

trap mass spectrometry. Sci. China Chem. 2010, 53, 832-840.

275

15. Fan, S.; Miao, H.; Zhao, Y.; Chen, H.; Wu, Y., Simultaneous detection of residues 15


Page 16 of 34

Page 17 of 34


276

of 25 β2-agonists and 23 β-blockers in animal foods by high-performance liquid

277

chromatography coupled with linear ion trap mass spectrometry. J. Agric. Food Chem.

278

2012, 60, 1898-1905.

279

16. Wang, L.; Zeng, Z.; Su, Y.; Zhang, G.; Zhong, X.; Liang, Z.; He, L., Matrix

280

effects in analysis of β-agonists with LC-MS/MS: Influence of analyte concentration,

281

sample source, and SPE type. J. Agric. Food Chem. 2012, 60, 6359-6363.

282

17. Jiang, W.; Wang, Z.; Beier, R. C.; Jiang, H.; Wu, Y.; Shen, J., Simultaneous

283

determination of 13 fluoroquinolone and 22 sulfonamide residues in milk by a

284

dual-colorimetric enzyme-linked immunosorbent assay. Anal. Chem. 2013, 85,

285

1995-1999.

286

18. Li, X.; Zhang, G.; Deng, R.; Yang, Y.; Liu, Q.; Xiao, Z.; Yang, J.; Xing, G.; Zhao,

287

D.; Cai, S., Development of rapid immunoassays for the detection of ractopamine in

288

swine urine. Food Addit. Contam., Part A 2010, 27, 1096-1103.

289

19. Sheu, S. Y.; Lei, Y. C.; Tai, Y. T.; Chang, T. H.; Kuo, T. F., Screening of

290

salbutamol residues in swine meat and animal feed by an enzyme immunoassay in

291

Taiwan. Anal. Chim. Acta 2009, 654, 148-153.

292

20. Lei, Y. C.; Tsai, Y. F.; Tai, Y. T.; Lin, C. Y.; Hsieh, K. H.; Chang, T. H.; Sheu, S. Y.;

293

Kuo, T. F., Development and fast screening of salbutamol residues in swine serum by

294

an enzyme-linked immunosorbent assay in Taiwan. J. Agric. Food Chem. 2008, 56,

295

5494-5499.

296

21. He, P.; Shen, L.; Liu, R.; Luo, Z.; Li, Z., Direct detection of β-agonists by use of

297

gold nanoparticle-based colorimetric assays. Anal. Chem. 2011, 83, 6988-6995. 16



Page 18 of 34

298

22. Lu, X.; Zheng, H.; Li, X.; Yuan, X.; Li, H.; Deng, L.; Zhang, H.; Wang, W.; Yang,

299

G.; Meng, M.; Xi, R.; Aboul-Enein, H. Y., Detection of ractopamine residues in pork

300

by surface plasmon resonance-based biosensor inhibition immunoassay. Food Chem.

301

2012, 130, 1061-1065.

302

23. Jiang, W.; Beloglazova, N. V.; Wang, Z.; Jiang, H.; Wen, K.; De Saeger, S.; Luo,

303

P.; Wu, Y.; Shen, J., Development of a multiplex flow-through immunoaffinity

304

chromatography test for the on-site screening of 14 sulfonamide and 13 quinolone

305

residues in milk. Biosens. Bioelectron. 2015, 66, 124-128.

306

24. Li, M.; Yang, H.; Li, S.; Zhao, K.; Li, J.; Jiang, D.; Sun, L.; Deng, A.,

307

Ultrasensitive and quantitative detection of a new β-agonist phenylethanolamine A by

308

a novel immunochromatographic assay based on surface-enhanced raman scattering

309

(SERS). J. Agric. Food Chem. 2014, 62, 10896-10902.

310

25. Beloglazova, N. V.; Goryacheva, I. Y.; Mikhirev, D. A.; De Saeger, S.; Niessner,

311

R.;

312

benzo[a]pyrene in aqueous samples. Anal. Sci. 2008, 24, 1613-1617.

313

26. Basova, E. Y.; Goryacheva, I. Y.; Rusanova, T. Y.; Burmistrova, N. A.; Dietrich,

314

R.; Martlbauer, E.; Detavernier, C.; Van Peteghem, C.; De Saeger, S., An

315

immunochemical test for rapid screening of zearalenone and T-2 toxin. Anal. Bioanal.

316

Chem. 2010, 397, 55-62.

317

27. Beloglazova, N. V.; Goryacheva, I. Y.; Rusanova, T. Y.; Yurasov, N. A.; Galve, R.;

318

Marco, M. P.; De Saeger, S., Gel-based immunotest for simultaneous detection of

319

2,4,6-trichlorophenol and ochratoxin A in red wine. Anal. Chim. Acta 2010, 672, 3-8.

Knopp,

D.,

New

immunochemically-based

field

17


test

for

monitoring

Page 19 of 34


320

28. Zuo, P.; Zhang, Y.; Liu, J.; Ye, B., Determination of β-adrenergic agonists by

321

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

322

29. Li, Z.; Wang, Y.; Kong, W.; Li, C.; Wang, Z.; Fu, Z., Highly sensitive

323

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

324

disposable electrochemiluminescent immunosensors array. Biosens. Bioelectron. 2013,

325

39, 311-314.

326

30. Han, J.; Gao, H.; Wang, W.; Wang, Z.; Fu, Z., Time-resolved chemiluminescence

327

strategy for multiplexed immunoassay of clenbuterol and ractopamine. Biosens.

328

Bioelectron. 2013, 48, 39-42.

329

31. Zhang, M.; Wang, M.; Chen, Z.; Fang, J.; Fang, M.; Liu, J.; Yu, X., Development

330

of a colloidal gold-based lateral-flow immunoassay for the rapid simultaneous

331

detection of clenbuterol and ractopamine in swine urine. Anal. Bioanal. Chem. 2009,

332

395, 2591-2599.

333

32. Xu, W.; Chen, X.; Huang, X.; Yang, W.; Liu, C.; Lai, W.; Xu, H.; Xiong, Y.,

334

Ru(phen)32+ doped silica nanoparticle based immunochromatographic strip for rapid

335

quantitative detection of β-agonist residues in swine urine. Talanta 2013, 114,

336

160-166.

337

33. Wang, P.; Wang, Z.; Su, X., A sensitive and quantitative fluorescent

338

multi-component immuno-chromatographic sensor for β-agonist residues. Biosens.

339

Bioelectron. 2015, 64, 511-516.

340

34. Gao, H.; Han, J.; Yang, S.; Wang, Z.; Wang, L.; Fu, Z., Highly sensitive

341

multianalyte immunochromatographic test strip for rapid chemiluminescent detection 18



342

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

19


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Figure Captions

344

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

345

gel-based immunoassays.

346

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

347

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

348

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

349

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

350

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

351

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

352

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

353

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

354

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

355

concentrations obtained between 20% and 80% specific bindings.

356

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

357

immunoassay and LC-MS/MS method.

20



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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)

21


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

22


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


Figure 1

23


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Figure 2

24



Figure 3

25


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Figure 4

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Figure 5

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Graphic for table of contents

28



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)



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)



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)


Development and Application of a Gel-Based Immunoassay for the

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