Development of an Ultrasensitive Immunochromatographic Assay (ICA) Strip for the Rapid Detection of Phenylethanolamine A in Urine and Pork Samples Li Junhua, Li Chunsheng, Wu Meng, Zhang Yan, Ma Xiaofei, Cheng Hua, and Yan Jinghui

In this study a one-step immunochromatographic assay based on competitive format was developed for the rapid detection of phenylethanolamine A (PEAA) residues in urine and pork samples. A monoclonal antibody against PEAA was produced from BALB/c mice immunized with the PEAA-BSA conjugate. The results of this qualitative test strip were to be interpreted visually. The visual detection limit (VDL) and threshold level of the optimized immunochromatographic assay for PEAA were 0.1 ng/mL and 0.5 ng/mL, respectively. Cross-reactions with other β-agonists were not significant inhibitions to the performance of the test strip assay. The results from the test strip were in a good agreement with those obtained using a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay. The immunochromatographic assay developed here was a useful on-site screening tool that is rapid to use, low in cost, and extremely convenient for the detection of PEAA in urine samples and pork samples.

Abstract:

Keywords: immunoassay, monoclonal antibody, nanoparticles, phenylethanolamine A, pork

Phenylethanolamine A (PEAA) is one of the β-agonists, which has been used not only to treat diseases, but also to promote growth in animals. However, concerning the harm to function of human body, a new fast and convenient method for monitoring PEAA should be established. We describe the development of a new test assay for rapid detection of PEAA in urine and pork samples.

Practical Application:

Introduction β-agonists are originally used in the treatment of asthma, bronchitis, emphysema, and premature birth (Price and Clissold 1986; Shishani and others 2003). There are also advantages to feeding animals with β-agonists, such as increasing the amount of lean meat, conserving feed, improving the efficiency of feed conversion, and shortening the time to market (Jones and others 1985; Chen and others 2005). However, eating pork that contains β-agonists can cause a series of symptoms in humans, such as nausea, dizziness, weakness, and tremor. Phenylethanolamine A [PEAA, C19H24N2O4, MW 344.17 g/mol], also known as clenrapmine (CLRA), is a new type of β-agonist following salbutamol (SAL), ractopamine (LAC), and clenbuterol (CL). Bulletin no. 1519 issued by the Ministry of Agriculture of China proclaimed that “PEAA is prohibited from being used in feeds and animal drinking water.” PEAA as a new type of β-agonist use in animals in the food industry has raised health concerns. Although many analytical methods like high-performance liquid chromatography (HPLC) (Li and others 2003; Zhang and others 2003; Mazhar and others 2009; Yan and others 2012), liquid chromatography−mass spectrometry (LC−MS) (Blanca and

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MS 20141696 Submitted 10/13/2014, Accepted 1/5/2015. Authors Junhua, Chunsheng, Meng, Hua, and Jinghui are with Biology Inst. of Hebei Academy of Sciences, Shijiazhuang, Hebei, 050051, PR China. Authors Yan and Xiaofei are with Hebei Food Inspection and Research Inst., Shijiazhuang, Hebei, 050091, PR China. Direct inquiries to author Chunsheng (E-mail: [email protected]).

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others 2005; Dickson and others 2005; Melwanki and others 2005; Nielen and others 2008; Garcia and others 2011), gas chromatography-mass spectrometry (GC−MS) (Garc´ıa and others 2004), and enzyme-linked immunosorbent assay (ELISA) (Sheu and others 2009; Fang and others 2011) have been used to monitor β-agonists in biological samples, there are very few analytical methods that have been developed for the detection of PEAA. In 2010, a standard procedure for the detection of PEAA in biological samples using high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) detection method is issued by the Ministry of Agriculture of China (2010). An LCMS/MS method is developed to detect PEAA in swine muscle (Sun and Yan 2011). A UHPLC-ESI-MS/MS with QuEChERS method is developed to detect PEAA in animal hair, tissues, and feed (Zhang and others 2012). A method of direct competitive enzyme-linked immunosorbent assay (ELISA) is reported to detect PEAA residues in urine samples (Bai and others 2012). In 2014, a method of immunochromatographic assay based on SERS is developed to detect PEAA in urine (Li and others 2014). These biochemical techniques for monitoring the abuse of PEAA are highly sensitive and reliable. However, the time-consuming need for extensive sample processing, use of specialized instrumentation, and skilled analysts, put certain limitations on the use of these detection methods. As such, the development of rapid, cheap, convenient, out-of-laboratory techniques for monitoring PEAA is extremely important. In this article, the polyclonal antibodies, which were produced with the synthesized immunogen PEAA-BSA, were highly sensitive to PEAA. Our work shows the advantages of good specificity, R  C 2015 Institute of Food Technologists

doi: 10.1111/1750-3841.12814 Further reproduction without permission is prohibited

high sensitivity, convenient for the detection of PEAA in samples of PEAA·HCl (380.87 MW) was dissolved in 600 μL methanol. on-site, and could be performed within 5 min without compli- 1.2 mL of 1 mol/L HCl was then added to the solution. The pH value of the solution was less than 1. Then 10 mg of zinc powder cated handling procedures. was added and the mixture was stirred at room temperature for 6 h. After completion of the reaction, the excessive zinc power Materials and Methods was removed by filtration. (2) Diazotization reaction: a total of Reagents and materials 1.2 mL of ice cold 0.1 mol/L NaNO2 was added into solution Phenylethanolamine A (PEAA), cimaterol (CIM), salbuta- dropwise (0. 6 mL was added initially and subsequently 0.1 mL per mol (SAL), ractopamine (LAC), clenbuterol (CL), formoterol, 30 min was added), the color of starch iodide test paper remained norepinephrine, sulfamethazine (SM2), chloramphenicol (CAP), navy blue throughout this period. The mixture was stirred for Proclin 300, bovine serum albumin (BSA), ovalbumin (OVA), about 3 h at 0 to 4 °C. The excessive NaNO2 was removed using sodium azide, Hydrogen tetra-chloroaurate hydrate (HAuCl4), ammonium sulfamate. (3) Conjugation reaction : 35 mg of BSA Tri-sodium citrate and 3,3 ,5,5 -tetramethylbenzidine (TMB) was dissolved in 0.7 mL of 0.05 mol/L PBS, then the mixture was were purchased from Sigma (St. Louis, Mo., U.S.A). Tween-20 added to the prepared solutions dropwise. The same process was and sucrose were purchased from Sinopharm Chemical Reagent repeated for the OVA reaction. The pH values of the 2 mixtures (Shanghai, China). All other reagents used in this study were of were readjusted to 8.0 with 1 mol/L NaOH. The mixtures were analytical grade and obtained from standard sources. BALB/c mice stirred overnight at 0 to 4 °C. Following the reaction, the 2 reaged 6 to 8 weeks-old were purchased from the Laboratory Animal action mixtures were dialyzed against 0.01 mol/L PBS (pH 7.4). Center of Hebei Province (Shijiazhuang, China). The myeloma UV-Vis spectral measurements were employed to determine the cell line of SP2/0 origin was produced in our laboratory. extent of conjugation. Vivid 170 Nitrocellulose membranes, grade 8964 conjugate pads, and type 133 absorbent pads were from Pall (Saint GermainPreparation and identification of monoclonal antibody en-Laye, France). Rabbit anti-mouse immunoglobulin G (IgG) was produced in our laboratory. All glassware used was siliconized (MAb) Five female BALB/c mice (each 6 to 8 wk of age) were imwith Sigmacote obtained from Xi-Cheng Technology Co., Ltd munized with PEAA-BSA. The immunization was multiple-site (Beijing, China). subcutaneously injected to the back of the mice. In the initial imPhenylethanolamine A stock solution was prepared by dissolving 10.0 mg of PEAA in 10 mL of methanol and then kept at 4 °C munization, 0.15 mg of PEAA-BSA in 0.5 mL of NaCl solution prior to further dilution. Phosphate-buffered saline (PBS; sodium (0.9%) was emulsified with an equal volume of Freund’s complete phosphate 10 mmol/L, NaCl 137 mmol/L, KCl 2.7 mmol/L; adjuvant. In subsequent injections, the same dosage of PEAA-BSA was emulsified with an equal volume of Freund’s incomplete adjupH 7.4). vant. The booster injection 3 d before cell fusion was carried out with a 2-fold dosage of PEAA-BSA without emulsification with Apparatus the adjuvant. Milli-Q Ultra Pure System (Millipore, Bedford, Mass., U.S.A.), Hybridomas were produced by fusioning between SP2/0 an HM 3030 XYZ Dispensing Platform, ZQ 4200 Guillotine myeloma and spleen cells as previously described by Yang and othCutter, and YK 725 assembly roller used for preparation of test ers (2010). High-affinity and stable monoclonal cell strains against strips were purchased from Jinbiao Technology Co., Ltd (ShangPEAA were screened out by repeatedly confirming detections. hai, China). The high-speed freezing centrifuge (CF16 RX) was Cultured cells which achieved a certain density were injected into from Hitachi (Tokyo, Japan). The IC assay was validated with a pristane-pretreated BALB/c mice to produce ascitic fluids. The asSHIMADZU 20A series HPLC (SHIMADZU, Japan) coupled to citic fluids were purified by the octylic acid-ammonium method. an AB SCIEX QTRAP5500 mass spectrometer equipped with an electrospray ionization source (AB SCIEX, U.S.A). Colloidal gold particles was validated with H-7650 transmission electron mi- Preparation of gold-MAb probes The purified anti-PEAA MAb were dialyzed against 10 mM croscope (Hitachi, Tokyo, Japan); UV-Visible spectrophotometer (UV-2600) was from SHIMADZU, Japan. Nanodrop 2000 was phosphate buffer (pH 7.4). A total of 0.1 mg of MAb was added drop-wise to 10 mL of colloidal gold solution (pH 8.0) and stirred from Thermo. for 1 h at room temperature. The solution was blocked by 0.1% (w/v) BSA for 1 h, then centrifuged at 8000 rpm for 30 min to Preparation of colloidal gold particles The colloidal gold particles with an average diameter of 25 remove the uncoordinated protein from the solution. The final nm were prepared using Frens’ method with slight modification gold-MAb probes were concentrated and resuspended in 5 mL of (Frens and Kolloid 1972). When 96.25 mL of water was heated 10 mM phosphate buffer (pH7.4) containing 0.5% (w/v) BSA, to boiling, 1.75 mL of 2% sodium citrate solution and 2 mL of 5% sucrose, and 0.1% sodium azide, and stored at 4 °C. 1% HAuCl4 solution were added simultaneously. The mixture was stirred continuously until the color turned bright wine red, Preparation of test strips then the mixture was boiled for an additional 5 min. The product Gold-MAb probe solution coated and dried on the bottom of was cooled and stored at 4 °C. The average size of the colloidal microwells. Rabbit antimouse IgG and PEAA-OVA were coated, gold particles was measured by transmission electron microscopy respectively, on nitrocellulose (NC) membrane (30 × 2.5 cm) as (TEM) and UV-Vis spectrometry. test (T) line and control (C) line. The distance between the test line and control line was about 5 mm. As is shown in Figure 1, Conjugation of PEAA to BSA and OVA Gold-MAb probe solution coated on the bottom of microwThe conjugates of protein to PEAA were conducted as de- ells instead of conjugate pad. The sample pad, NC membrane, scribed by Bai and others (2012). (1) Reduction reaction: 10 mg and absorbent pad were laminated onto a plastic backing plate. Vol. 80, Nr. 4, 2015 r Journal of Food Science T895

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ICA for phenylethanolamine A residues . . .

ICA for phenylethanolamine A residues . . . The plate was cut into 4 mm wide strips, and the dried wells were Table 1–MS/MS parameters for detection of PEAA and PEAAD3. encapsulated in aluminum foil with desiccant for further use.

Sample preparation The pork sample 5.0 g were mixed with 10 mL of methanol, ultrasonically extracted for 20 min, and the mixture was then filtrated using filter paper .The filtrate was evaporated to dryness by nitrogen, and the residue was redissolved in PBS. The urine sample was not required special treatment, and could be used directly, if turbid, the impurities could be removed by 4000 rpm centrifugation 10 min or filtration, the supernatant or filtrate was used for detection.

Compound

Precursor ion MRM Collision Cone (m/z) transition(m/z) energy (eV) voltage (V)

PEAA

345

PEAA-D3

348

345>327 345>150 348>153

25 25 25

20 20

Chromatographic conditions A SHIMADZU 20A series HPLC instrument was used for PEAA analysis. The chromatographic separation was carried out on a Kinetex C18 (2.1 mm × 100 mm, 2.6 μm). The column oven was set at 40 °C. The mobile phase consisted of solvent A (0.1% formic acid in water) and solvent B (acetonitrile). The gradient elution program was as follows: Mobile phase gradient used (A:B; v/v) was 90:10 at 0 min, 80:20 at 5.0 min, from 5 to 10 min the gradient was returned to the initial conditions. The flow rate was set at 300 μL/min, and the sample injection volume was 2.0 μL.

Immunochromatographic assay procedure A series of concentrations of the PEAA standard (0, 0.1, 0.2, 0.5, 1, 5, 10, 50 ng/mL) or sample solution were assayed by test strip. 90 μL of standard solution or sample solution were added to each well and mixed with the gold-Ab probe with gentle shaking. The samples were incubated for 3 min at room temperature. The test strip was then inserted into the mixture and allowed to develop color for a further 2 min. The test results were then determined Mass spectrometry conditions visually. The above HPLC system was coupled to an AB SCIEX QTRAP5500 mass spectrometer. The empirically determined opEvaluation of the immunochromatographic assay (ICA) A series of PEAA concentrations were spiked into PBS solution, timal conditions for sample analysis were as follows: Ion source: yielding final concentrations of 0, 0.1, 0.2, 0.5, 1, 5, 10, 50 ng/mL, electrospray ionization (ESI); detection method was performed respectively. Ten replicates were assayed for each of the standard using the multiple reaction monitoring (MRM) ion mode; ion concentrations. The standard solutions were assayed by ICA to find source temperature, 500 °C; ionspray voltage, 5500 V; nebulizer the VDL and threshold level that gave a complete disappearance gas, 0.7 MPa; curtain gas, 0.4 MPa; collision gas, 0.4 MPa. Optimized acquisition parameters of PEAA was presented in Table 1, of color in the test line (Sun and others 2006). To evaluate the cross-reactivity of the anti-PEAA MAb with and the standard map of PEAA was shown in Figure 2. competing compounds such as clenbuterol, ractopamine, salbutamol, tulobuterol, formoterol, norepinephrine, sulfamethazine, Results and Discussion and chloramphenicol in the assay, these competing compounds Antigen synthesis appraisal were prepared standard solutions to final concentrations of as 0, 1, A UV-vis absorbance method was employed to determine 5, 10, 50, 102 , 103 , 104 ng/mL. Cross-reactivity was investigated whether the linking had been a success. As shown in Figure 3, as previously described (Wang and others 2007). Cross-reactivity the maximum absorption peak of BSA and PEAA were all at 280 (CR%) = concentration of standard PEAA inhibiting of antibody nm, the superposition peak of the coupling was at 280 nm, still binding/concentration of other structural analogue inhibiting of PEAA – BSA had a characteristic absorption peak (– N = N –) at antibody binding×100%. 360 nm. It showed that coupling success. Validation PEAA-contaminated natural urine samples and pork samples, which were supplied by the Hebei Food Inspection and Research Inst, were analyzed using the developed test strips. The same samples were also analyzed by using HPLC-MS/MS for comparison of performance.

Production and characterization of antibodies Indirect ELISA was developed for assessing the preliminarily characterized of the anti-PEAA MAb (Lu and others 2006; Liu and others 2007). MAb 1C11E4 exhibited high competition between free PEAA and PEAA-OVA as determined by indirect ELISA, and the titer was determined to be 3.0 × 106 . MAb 1C11E4 was used for subsequent immunoassay development,such as the evaluation of sensitivity and specificity.

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Characterization of the nanogold particles and conjugates As shown in Figure 4, a peak at 522 nm in the curve “a” was the result of the surface plasmon resonance of nanogold particles. After the addition of antibody, the antibody was adsorbed directly on the nanogold particles by simple electrostatic interaction (Gandhi and others 2009). The peak shifted farther to 525 nm due to the combination of antibody and nanogold particles as shown in curve “b.” Figure 4(a) presets the form of electron microscopy of the particles, the nanogold particles in monodispersed state with an average diameter of the colloidal gold particles 25 nm (±5 nm), Figure 1–Schematic description for the principle of competitive assay in a the prepared gold colloid had good reproducibility and stability. test strip format.

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ICA for phenylethanolamine A residues . . .

Figure 2–The standard map of PEAA was obtained by HPLC-MS/MS.

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Figure 3–UV−vis spectrum.

ICA for phenylethanolamine A residues . . . Figure 4(b) presets the form of electron microscopy of conjugates Optimization of ICA after negative staining. The image displays that the colloidal gold Optimization of the immobilization concentration of the particles were surrounded by antibody. PEAA-OVA conjugate (0.2, 0.3, 0.4 mg/mL) and optimization of dilution ratio of the gold-MAb probe (1:5, 1:10, 1:15). As shown in Figure 6, the optimal combinations were as follows: the Test principle and result assessment of ICA concentration of PEAA-OVA was 0.3 mg/mL and the dilution The ICA was based on the competitive theory. Due to capratio of gold-Ab probe was 1:15. This combination provided the illary action, the gold-Ab probe, which was solubilized, moves desired cut-off level, low consumption of reagents, and sufficient upward as the sample solution flows chromatographically across intensity of the test line and clear background. the membrane. The competition between PEAA molecules in the samples and the immobilized PEAA-OVA conjugate for binding to monoclonal anti-PEAA antibodies conjugated with colloidal Evaluation of the immunochromatographic assay gold particles, the gold-MAb probe which were not captured by Sensitivity. The visual detection limit (VDL) of an imPEAA-OVA would move continuously to the control line. The munochromatographic assay could be defined as the minimum result of the test with a clear background and comparison of the analyte concentration producing color on the test line that was intensity of the color among the test and the control could be significantly weaker than that of negative control strip (Zhou and easily distinguished with the naked eye. The result was shown in others 2009). As shown in Figure 7, The VDL of PEAA was 0.1 Figure 5. The appearance of a red line on the test line for negative ng/mL. The test line was invisible when the concentrations of sample, so as a weaker or disappeared red line on the test line for a PEAA ࣙ 0.5 ng/mL, so the threshold level was 0.5 ng/mL. positive sample. While the control line didn’t show a red line, the Specificity. The results were shown in Table 2. These results gold conjugate was invalid. indicated that no significant inhibition was found and demonstrated cross-reactivity of lower than 0.01%. Application and validation. Since immunochromatographic assays are primarily aimed at qualitative assays, a comparison with HPLC-MS/MS was carried out to confirm the accuracy. 15 urine samples and 6 pork samples were analyzed using both the developed test strip and HPLC-MS/MS. The results (Table 2) showed an agreement between the qualitative conclusions based on HPLC-MS/MS analysis and the immunochromatographic assay. The developed immunochromatographic assay was shown to be applicable to the semiquantitative estimation of the PEAA content in samples.

Figure 4–UV/Vis spectra of (a) nanogold, (b) antibody-nanogold conjugate. Electron microscopic images of (A) nanogold and (B) antibodynanogold conjugate.

Figure 6–Optimization of the antigen-antibody combination conditions.

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Figure 5–Assessment illustrations of the test strip results. C: control line, T: test line. 1: negative; 2 and 3: positive; 4 and 5: invalid.

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Figure 7–Sensitivity of the test strip in analyzing using different concentrations of PEAA solutions of various concentrations.

ICA for phenylethanolamine A residues . . .

Phenylethanolamine A Clenbuterol Ractopamine Salbutamol Tulobuterol Formoterol Norepinephrine Sulfamethazine Chloramphenicol

0

1

5

10

50

102

-

+ -

+ -

+ -

+ -

+ -

103

104

+ -

+ -

Note: “+”, Positive:PEAA final concentration in test solution was higher than 0.5 ng/mL; “-”, Negative:PEAA final concentration in test solution was less than 0.5 ng/mL.

Table 3–Detection results of UPLC-MS/MS and ICA for PEAA in urine and pork samples. Samples

UPLC-MS/MS(mg/L±SD)

ICA (n = 5)

ND ND 3.9 ± 0.11 1.16 ± 0.02 0.81 ± 0.05 1.9 ± 0.03 1.29 ± 0.12 ND 0.89 ± 0.07 0.28 ± 0.02 ND 0.69 ± 0.02 0.92 ± 0.12 3.42 ± 0.09 ND

–,–,–,–,– –,–,–,–,– +,+,+,+,+ +,+,+,+,+ +,+,+,+,+ +,+,+,+,+ +,+,+,+,+ –,–,–,–,– +,+,+,+,+ ±,±,±,±,± –,–,–,–,– +,+,+,+,+ +,+,+,+,+ +,+,+,+,+ –,–,–,–,–

1.83 ± 0.05 1.03 ± 0.06 ND 2.14 ± 0.03 0.92 ± 0.02 0.72 ± 0.04

+,+,+,+,+ +,+,+,+,+ –,–,–,–,– +,+,+,+,+ +,+,+,+,+ +,+,+,+,+

Urine samples 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Pork samples 1 2 3 4 5 6

Note: ND, not detectable. “+” and “-” indicate positive and negative, respectively; “±” indicates a weak positive.

Conclusion An immunochromatogrphic assay for PEAA detection was developed based on the high specificity, high sensitivity MAb1C11E4. The immunochromatogrphic assay was optimized and evaluated. The VDL and threshold level of the optimized test strips for PEAA were 0.1 and 0.5 ng/mL, respectively. For validation, both the test strip and a standard HPLC-MS/MS detection method were used to analyze the same urine samples and pork samples, and the results showed good agreement. Although the assay provides only a preliminary semiquantitative result, it was extremely rapid, low cost, and convenient for the detection of PEAA in samples on-site, and could be performed within 5 min without complicated handling procedures.

Acknowledgments The authors are grateful for financial support from the Ministry of Science and Technology of Hebei Province (project nr. 13222902D) and the Budget Dept. of Hebei Province (project nr. 13341).

Author Contributions Li Chunsheng designed the study; Wu Meng and Cheng Hua interpreted the results; Li Junhua executed the study and drafted

the manuscript; Ma Xiaofei and Zhang Yan validated the results; and Yan Jinghui modified the manuscript.

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Table 2–Cross-reactivity of test strip.

Development of an ultrasensitive immunochromatographic assay (ICA) strip for the rapid detection of phenylethanolamine A in urine and pork samples.

In this study a one-step immunochromatographic assay based on competitive format was developed for the rapid detection of phenylethanolamine A (PEAA) ...
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