Journal o f Food Protection, Vol. 77, No. 11, 2014, Pages 1998-2003 doi: 10.4315/0362-028X.JFP-14-086 Copyright © , International Association for Food Protection

Research Note

Sample Preincubation Strategy for Sensitive and Quantitative Detection of Clenbuterol in Swine Urine Using a Fluorescent Microsphere-Based Immunochromatographic Assay SHENG L. DENG,12 SHAN SHAN,1 CHAO L. XU,1 DAO F. L IU ,1 YONG H. XIO N G ,1 HUA W E I,1 AND WEI H. L A I1* 1State Key Laboratory o f Food Science and Technology, Nanchang University, Nanchang 330047, People’s Republic o f China; and institute o f Microbiology, Jiangxi Academy o f Sciences, Nanchang 330096, People’s Republic o f China MS 14-086: Received 21 February 2014/Accepted 18 June 2014

ABSTRACT We describe an ultrasensitive and quantitative immunochromatographic assay to determine the amount of clenbuterol (CLB) in swine urine. In this study, fluorescein isothiocyanate polystyrene fluorescent microspheres were used as probes. A sample preincubation strategy was introduced to this immunochromatographic assay. Results showed that the strategy evidently improved the sensitivity and accuracy of lateral flow assay. The method was completed in 20 min, and a half-maximal inhibitory concentration of 0.13 pg liter-1 was obtained. The limit of detection of the proposed method to determine CLB in swine urine was 0.01 pg liter ', which was lower than the limit of detection of immunochromatographic assays without preincubation. Intraand interday recoveries of spiked swine urine ranged from 85.0 to 107.5%. The relative standard deviation values of the preincubated test strip ranged from 2.7 to 12.5%. Analysis of the CLB in swine urine samples showed that the result obtained from the lateral flow assay is consistent with that obtained from a commercial enzyme-linked immunosorbent assay kit. Our results suggest that the developed fluorescent microsphere-based immunochromatographic assay may be useful as a rapid screening method to detect CLB quantitatively.

C lenbuterol (4-am ino-a-[t-butylam inom ethyl]-3,5-dichlorobenzyl alcohol hydrochloride; CLB) is a sym patho­ m im etic am ine used as a decongestant and bronchodilator. CLB has been frequently used in anim al agriculture because this substance induces w eight gain with a high proportion of m uscle relative to fat (5 , 11). H ow ever, the effects o f CLB residues in food products for hum an consum ption from treated anim als have raised public health concerns. Intoxication and other side effects have been associated w ith m eat or liver products containing CLB residues ( 12). F or this reason, the use o f CLB as a grow th prom oter has been banned in the People’s Republic o f C hina and the E uropean U nion. A lthough the E uropean U nion has established a m axim um residue limit o f 0.1 pg k g -1 for CLB in bovine m uscle (3 ), incidents o f CLB poisoning still occur in som e areas ( 13). In China, m ore than 1,000 individuals suffered from illness in G uangdong Province in 2001 after they consum ed contam inated sw ine livers and hearts (8). In another case, 300 individuals were adversely affected in Shanghai on 15 Septem ber 2006 (4 ). Several m ethods, including liquid chrom atographytandem m ass spectrom etry (2), gas chrom atography-m ass spectrom etry (8 , 13), and enzym e-linked im m unosorbent assay (ELISA ) ( 7, 19), have been developed to determ ine

CLB w ith various matrices. Som e o f these m ethods feature high sensitivity and reproducibility but require tim e, welltrained personnel, and sophisticated instrum ents. Therefore, a sim ple, rapid, sensitive, and precise on-site decision­ m aking m ethod to detect CLB should be developed. Im m unochrom atographic assay is a one-step m ethod w ith several advantages, such as sim ple operation, rapid results, relatively low costs, and m odest instrum entation. In the past decades, gold nanoparticles w ere com m only used as probes in CLB test strip kits. H ow ever, this m ethod is neither sensitive nor accurate in quantifying CLB ( 7- 9 , 1618). Thus far, several probes, such as Ru(phen) 3 + doped silica nanoparticle ( 15), Eu (III)-B H H C T -coated silica nanoparticles ( 14), and quantum dots ( 10), have been developed for CLB test strips. This study aimed to develop a m ore sensitive procedure to determ ine CLB in sw ine urine. Fluorescein isothiocya­ nate polystyrene fluorescent m icrospheres (FM S) w ere used to facilitate the covalent attachm ent o f m onoclonal antibod­ ies against CLB (anti-CLB mAb). A sam ple preincubation strategy was introduced to the im m unochrom atographic assay to im prove sensitivity and offset the inherent heterogeneity o f the test strips. M A T E R IA L S A N D M E T H O D S

* Author for correspondence. Tel: + 8 6 13879178802; Fax: + 8 6 791 8833 3708; E-mail: [email protected]

M aterials. CLB, bovine serum albumin (BSA), and goat anti­ mouse antibody were purchased from Sigma (St. Louis, MO).

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FM S-mAb





Secondary antibody







FIGURE 1. Principle o f CLB detection by the FMS-based test strip. (A) CLB detection by the FMS-based test strip using a preincubation format and a portable fluorescence reader connected to an online computer. (B) Schematic o f the FMS-based test strip to detect swine urine samples (negative and positive). (C) Fluorescence intensities o fT and C lines, as measured using a portable reader. Ractopamine, salbutamol, terbutaline, epinephrine, norepinephrine, fenoterol, phenylbutazone, and ambroxol were obtained from Dr. Ehrenstorfer, GmbH (Augsburg, Germany). Fluorescein isothio­ cyanate FMS (diameter, 175 nm; excitation wavelength, 470 nm; emission wavelength, 525 nm; COOH, 443 peq g~' ) were obtained from Merck (Darmstadt, Germany). Anti-CLB mAb was provided by Wuxi Zodoboer Biotech. Co., Ltd. (Wuxi, China). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride

(EDC) was obtained from Shanghai Medpep Co., Ltd. (Shanghai, China). Nitrocellulose membrane, absorbent pad, sample pad, and conjugate pad were purchased from Millipore (Bedford, MA). Polystyrene 96-well microtiter plates were purchased from Coming Inc. (Coming, NY). Solvents and other chemicals were of analytical reagent grade. Apparatus. An XYZ-3050 Platform was purchased from BioDot (Irvine, CA). Fluorescence reader was procured from Shanghai Huguo Science Instrument Co., Ltd. (Shanghai, China) (excitation wavelength, 470 nm; emission wavelength, 520 nm). Preparation of FMS-mAb. A 25-pl aliquot of 10 mg ml-1 FMS was dissolved in 5 ml of 0.02 M phosphate buffer (pH 6.0); next, 2.7 pg of anti-CLB mAb was added to the suspension. Approximately 43 pi (0.1 mg m f 1) of EDC was also added to the mixture and then stirred. The dispersion was allowed to react for 2 h at room temperature, and the reaction was blocked with 500 pi of 10% BSA for 30 min. The mixture was centrifuged at 12,000 rpm (14,790 x g) for 10 min at 4°C and then washed twice with phosphate buffer (0.02 M; pH 6.0). The pellet was resuspended in phosphate buffer (0.02 M; pH, 7.4) with 1% BSA, 0.05% Tween 20, 3% sucrose, and 0.05% sodium azide. Part of the FMS-mAb was dotted in the wells of 96-well ELISA plates and dried at 30°C for 2 h; the rest was applied to a glass fiber membrane and dried at 37°C for 6 h.

FIGURE 2. Standard curve o f the test strip with and without sample preincubation to detect CLB in swine urine (n = 5 ) .

Preparation of test strips. The sample pad was treated with 50 mM borate buffer (pH 7.4) containing 1% BSA, 0.5% Tween 20, and 0.05% sodium azide. The sample pad was then dried at



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TABLE 1. Accuracy ancl precision o f the test strip in detecting CLB-supplemented swine urine samplesa Intraday Supplemented concn of CLB (pg l i t e r 1)

CLB detected (pg liter ')

0.1 0.2 0.5 5.0

0.089 0.196 0.472 5.263

Interday Recovery ± RSD (%) 89.0 98.0 94.4 105.3

± + + +

5.8 3.7 4.8 2.7

CLB detected (pg liter ') 0.085 0.179 0.457 5.374

Recovery ± RSD (%) 85.0 89.5 91.4 107.5

+ + + ±

12.5 10.4 7.8 6.6

n = 5. Intraday, five replicate measurements of each sample on the same day; interday, five different days of the interassay experiment. 60°C for 2 h. CLB-BSA (0.5 mg ml ’) and goat anti-mouse antibody (1.0 mg m f 1) were dispensed onto the nitrocellulose membrane, using a XYZ-3050 dispensing workstation at the test (T) and control (C) lines, respectively; the sample pad was then dried at 30°C for 12 h for prolonged storage. An untreated absorption pad was used. For the traditional strip, the glass fiber membrane was sprayed with FMS-mAb. For the new strip, the glass fiber was used without treatment. Nitrocellulose membrane, absorption pad, glass fiber membrane, and pretreated sample pad were then assembled as strips. Test strip testing and measurement. For the traditional strip, each 100-pl swine urine sample was added to the sample well of a test strip. After 20 min, the test strip was placed in a fluorescence reader. For the new strip, each 100-pl swine urine sample was added to ELISA microplates containing FMS-mAb and was incubated for 5 min at room temperature. The mixture was pipetted into a sample well of a test strip. After 15 min, the treated test strip was placed in the fluorescence reader. The signals of T and C lines were immediately determined. A calibration standard curve was obtained by plotting the ratio between the fluorescent intensities of T line to the fluorescent intensities of C line (T/C) against the logarithm of CLB concentrations. Scanning software (Shanghai Huguo Science Instrument Co., Ltd.) was used to convert the ratio between line intensities to a concentration value based on the standard curve. Sensitivity and specificity of the test strip. Fifty negative swine urine samples (detected by liquid chromatography-tandem mass spectrometry) were randomly collected from Jiangxi, China. All of the samples were mixed to yield a negative control group. Supplemented swine urine samples containing CLB (0, 0.01, 0.03, 0.09, 0.27, 0.81, 2.43, and 7.29 pg l i t e r 1) were prepared. Each concentration was then analyzed in five replicates. For quantitative detection, the limit of detection (LOD) is defined as the concentration of CLB in the sample solution that causes a 10% decrease in the T/C ratio compared with that produced by the negative control (14). The specificity of the test strip was evaluated using other adrenergic agonists, such as ractopamine, salbutamol, terbutaline, epinephrine, norepinephrine, fenoterol, phenylbutazone, and ambroxol. Experiments at each concentration were performed in five replicates. Cross-reactivity was calculated as the ratio of the IC50 value of CLB to that of other adrenergic agonists: cross-reactivity = I C 50 ( C L B t/I C s o (adrenergic agonists) ^ 1 0 0 % (15). Stability of the test strip. The stability of the test strip was determined using the same batch of test strips at varying concentrations of CLB after these strips were stored for 0, 2, 4, 6, 8, 10, and 12 months at 4 and 25 °C, respectively. The signals of T and C lines were also determined. The T/C ratio against the logarithm of the CLB concentrations was analyzed.

Assay validation. Fifty supplemented swine urine samples containing various concentrations of CLB were used to compare the level of detection of an ELISA kit (R-Biopharm AG, Darmstadt, Germany) used for a lateral flow assay with preincubation.

RESULTS AND DISCUSSION Principle of the method. Immunochromatographic assay directed to small molecules usually relies on a competitive pattern, in which signal intensity is inversely proportional to analyte concentration in a particular sample. Quantitative results rely on the signal of the T line or the ratio of the T line intensity to the C line intensity (6, 14). The assay performed in this study is shown schematically in Figure 1. In our study, an additional preincubation step is introduced before the sample is added to the strip. CLBs then blind to anti-CLB mAb in the microplate when small amounts of CLB are present in the sample. After 5 min, the incubated sample is pipetted into a sample well of the cassette. The mixture moves across the membrane via capillary action. As the T line is reached, some binding sites of FMS-mAb are rapidly occupied by CLB-BSA. The remaining complex is then allowed to migrate to the C line. The goat anti-mouse antibody captures the remaining FMSmAb. Therefore, a high CLB concentration in the sample corresponds to a low T/C ratio obtained in the test strip. Comparison between traditional and new strips in terms of sensitivity and relative standard deviation (RSD). Under optimal conditions, the calibration standard curves were established by plotting (B/B0) against the logarithm of the CLB concentration. Figure 2 shows that the IC50 values of traditional and new strips were 0.46 and 0.13 pg liter 1 (P < 0.05), respectively. An LOD of 0.043 pg liter-1 was observed in the traditional strip. By contrast, an LOD of 0.011 pg liter-1 was obtained in the new strip. Sensitivity was also improved by approximately 3.5-fold when the sample was preincubated. The low LOD reached the levels required by the European regulations related to CLB determination. When a preincubation step is performed, the LOD obtained is lower than that published in previous papers using the CLB test strip (9, 14, 1 6-18). Thus, the proposed method is highly sensitive and is suitable to primarily screen CLB at low levels. Although traditional quantitative lateral flow assay often shows high imprecision when complex matrix samples are detected (1), the precision of the test strips was improved when a preincubation step was performed. The RSD values

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TABLE 2. Comparison o f test strip and commercial ELISA kit results for the detection o f CLB in swine urine ELISA kit (n = 2)

Proposed method in = 3)

Sample no.

Supplemented level (pg liter- ')

CLB detected (pg liter-1)

Recovery (%)

RSD (%)

CLB detected (pg liter-1)

Recovery (%)

RSD (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50

0.2 0.2 0.2 0.2 0.2 0.5 0.5 0.5 0.5 0.5 1.0 1.0 1.0 1.0 1.0 1.5 1.5 1.5 1.5 1.5 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0

0.23 0.21 0.22 0.23 0.18 0.63 0.59 0.60 0.47 0.55 0.82 1.19 0.81 1.09 1.16 1.32 1.25 1.77 1.45 1.43 1.85 1.76 2.28 2.09 2.18 1.91 1.76 1.82 2.34 2.30 2.36 2.14 2.18 2.81 2.83 2.62 2.74 2.36 2.48 3.06 2.79 2.58 3.22 2.63 3.14 2.81 2.79 3.26 2.89 3.38

113.0 104.0 108.0 112.5 87.5 125.4 118.0 120.0 93.4 109.2 82.0 119.0 81.3 109.0 116.0 88.0 83.3 118.0 96.7 95.3 92.5 88.0 114.0 104.5 109.0 95.5 88.0 91.0 117.0 115.0 94.4 85.6 87.2 112.4 113.2 104.8 109.6 94.4 99.2 102.0 93.0 86.0 107.3 87.7 104.7 93.7 93.0 108.7 96.3 112.7

4.6 7.8 11.5 3.2 9.1 2.9 3.3 11.7 3.2 4.5 2.4 4.7 7.1 9.8 3.9 4.4 4.6 5.6 10.4 3.7 5.3 4.8 3.5 6.9 5.1 3.6 2.7 7.9 6.4 4.3 6.7 12.2 4.7 3.8 8.6 5.6 6.4 5.5 10.5 7.3 6.9 5.4 6.2 9.2 10.7 8.3 5.1 6.8 3.4 5.2

0.22 0.18 0.22 0.21 0.19 0.52 0.59 0.46 0.42 0.49 0.86 1.06 0.95 0.86 1.09 1.28 1.29 1.65 1.58 1.56 1.89 1.85 2.16 1.93 2.05 1.81 1.72 1.89 2.13 1.92 2.41 2.30 2.38 2.69 2.76 2.58 2.41 2.28 2.67 3.28 3.05 2.81 3.09 2.95 3.08 2.76 2.86 3.08 3.07 3.09

110.5 92.0 110.0 102.5 97.0 104.6 117.6 92.0 84.6 98.6 86.0 106.0 95.0 86.0 109.0 85.3 86.0 110.0 105.3 104.0 94.5 92.5 108.0 96.5 102.5 90.5 86.0 94.5 106.5 96.0 96.4 92.0 95.2 107.6 110.4 103.2 96.4 91.2 106.8 109.3 101.7 93.7 103.0 98.3 102.7 92.0 95.3 102.7 102.3 103.0

8.3 7.9 12.2 5.4 9.6 4.5 5.8 13.8 9.3 7.7 6.3 8.2 4.6 13.5 7.8 4.7 3.9 10.6 12.6 5.7 6.3 7.6 5.1 8.4 6.7 3.5 3.6 5.5 7.3 9.2 8.5 10.6 2.4 5.0 14.6 10.4 7.8 4.6 7.2 9.2 8.1 5.9 7.2 6.8 14.7 5.9 4.6 7.2 9.3 4.1

of the preincubated test strip ranged from 2.7 to 5.8% and were lower than those that were not preincubated (5.9 to 10.2%; P < 0.05). A possible explanation for this result is that preincubation may induce complete binding between antibody and antigen in liquid environments and release

antigen-antibody complexes uniformly on sample pad and the nitrocellulose membrane. Cross-reactivity of the test strip. Adrenergic agonists, including ractopamine, salbutamol, terbutaline, epinephrine,



norepinephrine, fenoterol, phenylbutazone, and ambroxol, were selected to verify the cross-reactivity of the test strip. Anti-CLB mAb, as a label antibody of the test strip, presented negligible cross-reactivity with similarly struc­ tured adrenergic agonists; this result indicated that the test exhibited high selectivity for CLB. Stability of the test strip. The prepared strips were stored at 4 and 25°C for 12 months. The stability of the strips was determined using negative (0 pg liter-1) and positive (0.1 pig liter-1) samples. The results showed that the measured values did not significantly change (P > 0.05). Therefore, test strips could be stored at 4 or 25°C for 12 months. Analysis of CLB-supplemented swine urine. The prepared FMS-based test strip and a fluorescence reader were used to evaluate the applicability of the strips in actual samples. CLB standard solutions were added to the blank swine urine (50 negative mixed swine urine), and a calibration curve of the T/C ratio was established. The accuracy and precision of the test strip used to detect CLBsupplemented swine urine samples are shown in Table 1. Intraday recoveries ranged from 89.0 to 105.3%, and interday recoveries ranged from 85.0 to 107.5%. Intra- and interday RSD values 0.05) indicated that our results are adequately reproducible; thus, these strips can be applied to detect CLB in swine urine. To further confirm the reliability of the proposed quantitative test strips, we evaluated 50 negative urine samples supplemented with CLB standards at different concentrations (0.2 to 3.0 ptg liter-1 ) by using the test strip (LOD was 0.01 pig liter-1 in swine urine) and a commercial ELISA kit (LOD was 0.2 pg liter-1 in swine urine). The average recoveries of CLB in spiked swine urine ranged from 81.3 to 125.4% and from 84.6 to 117.6% when the prepared test strip and the ELISA kit were used, respectively. The results obtained using the two methods were not significantly different (R 2 = 0.9759, P > 0.05), indicating that the results produced by the test strip are highly similar to those obtained using the ELISA kit (Table 2). The new strip was more rapid and sensitive than the commercial ELISA kit. The detection time periods of the new strip and the commercial ELISA kit were approxi­ mately 20 and 90 min, respectively. The LODs of the new strip and the commercial ELISA kit were 0.01 and 0.2 pg liter- 1 in swine urine, respectively. In conclusion, a simple and sensitive quantitative immunochromatographic assay was developed on the basis of fluorescein isothiocyanate polystyrene FMS to determine CLB. Using a preincubation step increased the sensitivity, accuracy, and reproducibility of the test strip. An LOD value of 0.01 pg liter-1 CLB, which is lower than that published in the literature for previous test strips of CLB, was obtained using our proposed method. The entire analytical procedure could be completed in 20 min. High recoveries with acceptable RSD were also achieved. The performance of the proposed test strip was compared with that of a commercial ELISA kit. The results indicated that

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the FMS-based strip assay could provide an alternative method to determine CLB in swine urine rapidly, sensitively, and precisely. ACKNOWLEDGMENTS This work was supported by the National Key Technology R&D Program (2012BAK17B02), the National Natural Science Foundation of China (30960301), the Science and Technology Program of Jiangxi Province of China (20121BBF60050), Jiangxi Agriculture Research System (JXARS-03), and Jiangxi Education Bureau Technology Project (2013KJLD13009).












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Sample preincubation strategy for sensitive and quantitative detection of clenbuterol in swine urine using a fluorescent microsphere-based immunochromatographic assay.

We describe an ultrasensitive and quantitative immunochromatographic assay to determine the amount of clenbuterol (CLB) in swine urine. In this study,...
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