Research Article Received: 9 August 2013

Revised: 9 November 2013

Accepted article published: 6 January 2014

Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jsfa.6557

Development of an enzyme-linked immunosorbent assay for detection of clopidol residues in chicken tissues Jin-qing Jiang, Hai-tang Zhang, Hui-hui Zhang, Zi-liang Wang,∗ Xue-feng Yang and Guo-ying Fan Abstract BACKGROUND: Clopidol is mainly used for the prevention and treatment of coccidiosis, which poses a serious potential hazard to public health, in veterinary medicine. The aim of this study was to prepare monoclonal antibodies (mAbs) against clopidol (CLOP) and develop an immunoassay for detecting CLOP residues in chicken tissues. After derivation, CLOP hapten was conjugated to carrier proteins to synthesize the artificial antigen, and immunized Balb/C mice were employed to screen mAbs. RESULTS: A sensitive hybridoma named C1G3 was screened out and two indirect competitive enzyme-linked immunosorbent assay (icELISA) standard curves were established. For the traditional two-step assay the linear range was from 0.06 to 98 ng mL−1 , with half-maximal inhibitory concentration (IC50 ) and limit of detection (LOD) values of 2.76 ng mL−1 and 0.03 ng mL−1 respectively, while the rapid one-step icELISA had a working range from 0.08 to 102 ng mL−1 , with IC50 and LOD values of 3.52 ng mL−1 and 0.03 ng mL−1 respectively. It was also indicated that a 10-fold dilution in chicken muscles gave an inhibition curve almost the same as that obtained in phosphate-buffered saline. When applied to spiking tests in chicken samples, the correlation coefficient (R2 ) between concentrations added and measured was 0.9534. CONCLUSION: The results of this study suggest that the immunoassay described is a promising alternative for screening CLOP residues in biological matrices and is suitable for routine diagnostics. c 2014 Society of Chemical Industry  Keywords: clopidol; artificial antigen; monoclonal antibody; immunoassay; chicken tissues

INTRODUCTION Clopidol (3,5-dichloro-2,6-dimethyl-4-pyridinol, CLOP) is mainly used for the prevention and treatment of coccidiosis in veterinary medicine. Because of a very high curative effect, it has been widely used in the chicken-raising industry all over the world. However, long-term use or non-prescribed medication may cause CLOP residues in animals and their tissues, which pose a serious potential hazard to public health. Research results show that CLOP displays certain teratogenic and embryo-toxic effects and also a weak mutagenic effect in mice. When foods containing high clopidol residues are consumed by humans, expansion of coronary heart and blood flow is induced, which is very dangerous for people with coronary artery disease.1 To protect human health, various countries and international organizations have laid out stringent regulations on its residue limit in foodstuffs of animal origin, and China’s government legislation has established a maximum permissible level of 50 µg kg−1 for CLOP in chicken tissues.2 This has resulted in the development of a broad range of methods to detect CLOP residues in biological matrices. Conventional analytical methods include high-performance liquid chromatography (HPLC),3 liquid chromatography/tandem mass spectrometry (LC/MS/MS)4,5 and gas chromatography/tandem mass spectrometry (GC/MS/MS).6,7 These methods include multistep procedures J Sci Food Agric (2014)

such as hydrolysis, extraction, derivative formation and purification. While these techniques are accurate and can serve as quantitative and confirmatory methods, they are time-consuming and require skilled personnel as well as expensive equipment. Competition within the food and feed industry is forcing manufacturers to reduce costs, so rapid, less expensive and easier-to-use immunoassay methods have become increasingly important. However, commercial kits using immunological methods are expensive and can be problematic to import in many developing countries. Morever, there are few published immunoassays for CLOP detection with respect to animal tissues, except that the artificial antigen of CLOP has been synthesized by Chinese researchers.8 For this reason, we aimed in the present study to generate highly sensitive antibodies for the precise analysis of CLOP residues in chicken samples using hybridoma technology. Monoclonal antibodies are preferred over polyclonal antibodies because their affinity and specificity do not vary from bleed to bleed. Following the minimization of sample pretreatment, we also discuss matrix effects and establish different standard curves. ∗

Correspondence to: Zi-liang Wang, Henan Institute of Science and Technology, Xinxiang 453003, China. E-mail: [email protected] Henan Institute of Science and Technology, Xinxiang 453003, China

www.soci.org

c 2014 Society of Chemical Industry 

www.soci.org

MATERIALS AND METHODS

OH

Chemicals and reagents CLOP was obtained from Sigma (St Louis, MO, USA). GaMIgG-HRP (whole molecule specific) was purchased from Sino-American Biotechnology Company (Shanghai, China). N,N Dicyclohexylcarbodiimide (DCC), Freund’s complete adjuvant (FCA) and Freund’s incomplete adjuvant (FIA) were obtained from Pierce (Rockford, IL, USA). Bovine serum albumin (BSA, molecular weight (MW) 67 000 Da) and ovalbumin (OVA, MW 45 000 Da) were supplied by Sigma. Dialysis bags (8000–14 000 Da) were from Beijing Solarbio Science & Technology Co., Ltd (Beijing, China). N-Hydroxysuccinimide (NHS), hypoxanthine/thymidine/ aminopterin (HAT) and hypoxanthine/thymidine (HT) were obtained from Sigma-Aldrich (St Louis, MO, USA). RPMI-1640 with L-glutamine was obtained from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS) was from Hangzhou Sijiqing Biological Engineering Materials Co., Ltd (Hangzhou, China). Cell culture plates (24 and 96 wells) and culture flasks were obtained from Costar Inc. (Bethesda, MD, USA). All other solvents and reagents were of analytical grade or higher unless stated otherwise. Instruments A Multiskan MK3 spectrophotometric microtiter reader (Thermo Company, Boston, MA, USA) with a 450 nm filter was used for absorbance measurements. Ultraviolet (UV)–visible spectra were obtained with a DU800 UV–visible spectrophotometer (Beckman-Coulter, Fullerton, CA, USA). Infrared (IR) spectra were acquired using a TENSOR 27 IR spectrometer (Bruker, Bremen, Germany). Transferpettes (eLINE, Proline) were obtained from Biohit (Helsinki, Finland). A Legend Micro 17 microcentrifuge and GS15R high-speed refrigerated centrifuge were supplied by Thermo Company. A Galaxy S + CO2 incubator (RS-Biotech, Ayrshire, UK) was used for cell cultivation. An SW-CJ-2FD superclean bench was purchased from Suzhou Purification Equipment Co., Ltd (Suzhou, China). A TS100-F inverted microscope (Nikon Company, Tokyo, Japan) was used for cell observation. Derivation of clopidol hapten Under alkali and acidification, a nucleophilic substitution reaction was employed to introduce an ester bond (&bond;COO group) and then to synthesize the clopidol hapten containing a carboxyl group. To a solution of 15 mL of 0.5 mol L−1 NaOH, 5 mmol of CLOP was added and completely dissolved, then the water was removed through vacuum distillation. After being washed with water, and dried at room temperature, the primary product was dissolved in 20 mL of dimethylformamide (DMF), then 0.56 mL of ethyl bromoacetate was added dropwise. The chemical reaction was continued for 3 h at 60 ◦ C. After cooling to room temperature, 150 mL of distilled water was added and the reaction mixture was placed in a 4 ◦ C refrigerator to cool overnight. After vacuum filtration, water washing and filtration, the CLOP derivative containing an ester bond was obtained. Then 20 mL of distilled water was added and the product was heated to 85 ◦ C. Next 1 mol L−1 NaOH solution was added dropwise so that all the solid was dissolved. After cooling to room temperature, 1 mol L−1 HCl was added dropwise in order to achieve a pH value of 4. The reaction mixture was placed in a 4 ◦ C refrigerator to cool overnight. After being filtered, recrystallized and dried, the clopidol hapten containing a carboxyl group, named the carboxymethyl ether derivative (CLOP-2C), was acquired. The synthetic pathways are illustrated in Fig. 1.

wileyonlinelibrary.com/jsfa

J-Q Jiang et al.

Cl CH3

ONa Cl

N

vacuum distillation

CH3

Cl

Cl

NaOH CH3

N

CH3

BrCH2COOCH2CH3 (60 °C)

OCH2COOCH2CH3

OCH2COOH Cl

Cl

NaOH

Cl

Cl

HCl

CH3

N

CH3

CH3

N

CH3

Figure 1. Synthesis scheme of clopidol hapten-carboxymethyl ether derivative (CLOP-2C).

IR identification Under heating from an IR lamp, 2 mg of CLOP-2C and approximately 200 mg of dry KBr were evenly mixed and ground in an agate mortar. By placing part of the mixture in a tablet mold at a pressure of 8 t for 3–5 min, a transparent KBr pellet sample piece of thickness 1 mm was made, whose IR spectrum was acquired with an IR spectrometer. Synthesis of immunogen and coating antigen The synthetic procedures for the complete antigen used for immunization and the coating antigen used for detection are presented below. The NHS method was employed to synthesize the artificial immunogen of CLOP-2C-BSA.9 Briefly, 75 mg of the CLOP carboxymethyl ether derivative was suspended in 3 mL of DMF and then 18 mg of NHS and 35 mg of DCC were added. During the following 12 h of incubation in a dark chamber, the mixture was stirred with an HY-4 reciprocal shaker at 37 ◦ C. After centrifugation, the supernatant was added dropwise to 20 mg of BSA dissolved in 2 mL of 0.01 mol L−1 phosphate-buffered saline (PBS, pH 7.4) at room temperature. The resulting mixture was stirred by rotor in a dark chamber overnight. After centrifugation at 3000 × g for 10 min, the obtained supernatant was dialyzed against PBS for 7 days. When the micromolecule absorption peak of the dialyzed solution had disappeared, as checked by the UV–visible spectrum, the artificial immunogen was stored in an ampoule at −20 ◦ C. The UV absorbance spectrum was employed to determine whether the linking had been successful. To synthesize the CLOP coating antigen conjugate (CLOP-2COVA), the mixed anhydride technique of Shelver et al.10 was employed, with slight modification. Briefly, 75 mg of CLOP hapten was dissolved in 3 mL of DMF and then 90 µL of triethylamine was added. During the following 30 min of incubation in an ice bath, 45 µL of isobutyl chloroformate was added and stirred for another 1 h. Then the resulting mixture was added dropwise to 20 mg of OVA dissolved in 3 mL of 0.05 mol L−1 carbonate-buffered saline (CBS, pH 9.6) and incubated under shaking at 4 ◦ C in a cryostat for 12 h. The reaction mixture was dialyzed under stirring against CBS for 7 days with frequent changes of CBS buffer to remove the uncoupled free hapten. The solution was lyophilized and the coating antigen of CLOP-2C-OVA conjugate obtained was stored at −20 ◦ C.

c 2014 Society of Chemical Industry 

J Sci Food Agric (2014)

ELISA for detection of clopidol residues in chicken tissues Immunization Eight-week-old Balb/C female mice were vaccinated intraperitoneally with CLOP-2C-BSA as immunogen following the low-dose multipoint method.11 The first injection was given using an emulsion containing FCA and immunogen (1:1 v/v; a dose of 150 µg CLOP-2C-BSA conjugate per mouse). Four similar booster vaccines were given at 3 week intervals, but the immunogen was emulsified in FIA. After the second booster immunization, mouse blood samples were obtained from the coccygeal vein section and checked for their titers and ability to compete with CLOP. The mouse whose antiserum showed the highest titer and gave the best dose–response curve toward CLOP was selected for the fusion experiment. Four days prior to cell fusion, the mouse was injected with 200 µg of immunogen in 0.2 mL of PBS, divided equally into intravenous and intraperitoneal injections. Preparation of CLOP monoclonal antibodies Portions of the cell fusion procedure and cloning conditions were detailed previously12 and are only briefly described here. The spleen from the mouse with the strongest competition toward CLOP and highest titer was aseptically harvested and fused with NS0 cells at a 10:1 ratio using PEG 1500 as fusing agent. Then hybridoma cells were distributed into 96-well culture plates in which mouse peritoneal macrophages had been prepared the day before and grown with selective HAT medium. At 12–15 days after cell fusion, culture supernatants were screened for the presence of antibodies that recognized the analyte. Then these positive clones were transferred into 24-well plates. Seven days later, hybridomas from wells showing a positive response in the enzyme-linked immunosorbent assay (ELISA) procedure described below were cloned three times by the limiting dilution method and expanded to guarantee their monoclonal origin. Colonies of interest were frozen in culture medium containing 100 g L−1 dimethyl sulfoxide and cryopreserved in liquid nitrogen, followed by defrosting three times to select the stable antibody-producing clones. A mature female Balb/C mouse was injected intraperitoneally with 0.8 mL of paraffin 10 days before receiving an intraperitoneal injection of the interesting hybridoma cells, and ascitic fluid was collected 2 weeks later. The antibodies were purified using the saturated ammonium sulfate method and stored at −20 ◦ C in 50 µL aliquots. ELISA procedures Indirect ELISA protocol The protocol used for the indirect ELISA was similar to that described in a previous study.13 Briefly, coating antigen of CLOP2C-OVA was appropriately diluted in CBS and 100 µL aliquots were added to each well of flat-bottom polystyrene ELISA plates. After 2 h of incubation at 37 ◦ C, the plates were washed with washing buffer (PBS containing 0.05% Tween-20) three times and unbound active sites were blocked with 250 µL per well of blocking buffer, followed by overnight incubation at 4 ◦ C. After the blocking solution had been removed, appropriate dilution of the antiserum or monoclonal antibody (50 µL per well) was added across the plate, which was incubated for 15 min at 37 ◦ C and then washed as described above. Then GaMIgG-HRP (1:1000, 50 µL per well) was added, followed by incubation for 25 min at 37 ◦ C. After three washings, 60 µL per well of enzyme substrate solution was added, followed by 15 min of incubation at room temperature. The enzymatic reaction was inhibited with 2 mol L−1 H2 SO4 (100 µL per well) and the absorbance was read spectrophotometrically in single-wavelength mode at 450 nm. Pre-immune withdrawal serum (the serum before J Sci Food Agric (2014)

www.soci.org immunisation) and PBST were used as a negative control and a blank control respectively, which were included in all assays. The antibody titer was defined as the reciprocal of the dilution that resulted in an absorbance value that was twice the blank value. Indirect competitive ELISA (icELISA) Bidimensional checkerboard titration was used to obtain the optimized concentrations of coating antigen and antibody, and the protocol used for icELISA was also carried out according to the general assay conditions previously described. After being coated and blocked, 50 µL of monoclonal antibody and 50 µL of varying concentrations of standard analyte or target samples were added to each well. During the traditional two-step icELISA procedure, GaMIgG-HRP (1:1000, 50 µL per well) was added 15 min later, and the subsequent steps were similar to those of the indirect ELISA. In this study we also tried the one-step icELISA procedure14 and analyzed the competitive inhibition curves against CLOP. In this case, 50 µL of antibody, 50 µL of standard analyte and 50 µL of GaMIgG-HRP were added simultaneously. The plates were then incubated, washed and measured with a microplate reader at 450 nm as described above. The calibration curves were fitted based on the average of three separate assays in triplicate, and sensitivity was evaluated according to the half-maximal inhibitory concentration (IC50 ) values, where IC50 represents the concentration of CLOP that produces 50% inhibition of antibody binding to the hapten conjugate. The limit of detection (LOD) was defined as the lowest concentration of CLOP that exhibits a signal of 15% inhibition.15 The working range for icELISA was calculated as the concentration of the analyte providing a 20–80% inhibition rate (IC20 –IC80 values) of the maximum signal. Pretreatment of chicken tissues Fresh meat samples of chicken with known backgrounds and certified as free of CLOP were purchased in retail outlets in Xinxiang, China. Prior to running the immunoassay detection, chicken samples were homogenized and then 5 g portions of finely ground materials were weighed into 10 mL centrifugal screw cap vials. Next 5 mL of 0.01 mol L−1 NaOH/acetonitrile (1:10 v/v) was added and the homogenates were mixed on a vortex mixer for 30 s and vigorously shaken for 10 min. The samples were centrifuged at 3500 × g for 20 min at 25 ◦ C and then 1 mL aliquots of the supernatants were diluted in 0.01 mol L−1 PBS (total 2-, 4-, 6-, 10- and 20-fold dilution). A 50 µL aliquot of each buffer solution was used for ELISA detection. Typical experimental standard curves were obtained by plotting absorbance values against different concentrations of CLOP. B and B0 values from each dilution curve were compared with those generated in PBS to determine the appropriate dilution factor. For spiking tests, a standard stock solution of CLOP was prepared by diluting the initial drug dissolved in methanol to a final concentration of 1 mg mL−1 . The stock solution was then serially diluted with PBS (0.01 mol L−1 ) to give working standard solutions. Recoveries were calculated by interpolation of the mean absorbance values on a standard curve constructed by one-step icELISA in PBS.

RESULTS AND DISCUSSION IR identification of clopidol derivative For the production of high-quality monoclonal antibodies and the development of sensitive immunoassays, it is necessary to modify the molecular structure of target analytes properly, e.g. to

c 2014 Society of Chemical Industry 

wileyonlinelibrary.com/jsfa

www.soci.org

Transmittance (%)

(B)

Transmittance (%)

(A)

J-Q Jiang et al.

Wavenumber (cm−1)

Wavenumber (cm−1)

Figure 2. IR spectral identification of (A) clopidol and (B) clopidol hapten derivative.

synthesize a hapten derivative that bears a functional group to be coupled covalently to the carrier protein. Clopidol is not soluble in organic solvents, because it is an amphiphatic compound and only has good solubility under alkaline conditions. Therefore in this study a nucleophilic substitution reaction was employed to introduce an ester bond (&bond;CH2 COOCH2 CH3 ) and then to synthesize the clopidol derivative containing a carboxyl group under alkali and acidification. Figure 2 shows the IR spectra of (A) clopidol and (B) clopidol hapten derivative. In Fig. 2A the IR absorption bands at 3249.2133 and 1561.3671 cm−1 are extremely sharp, representing the characteristic absorption peaks of &bond;OH and the pyridine ring in clopidol respectively, while in Fig. 2B the absorption peak at 3249.2133 cm−1 has disappeared owing to the loss of &bond;OH in the pyridine ring. As compared with clopidol, a strong sharp absorption peak at 1738.4146 cm−1 and a blunt absorption peak at 3442.9772 cm−1 appear in Fig. 2B, attributable to &bond;C&dbond;O and &bond;OH in the carboxy group of the clopidol derivative respectively. The absorption peak at 1561.3671 cm−1 ascribed to the pyridine ring is almost the same in Figs 2A and 2B, proving that the characteristic functional group of clopidol remains in the hapten derivative. The results indicated that the clopidol hapten derivative was synthesized successfully. UV spectrogram of clopidol immunogen For artificial antigen synthesis, a good option is to place a linking group opposite the characteristic group and maximally reserve the parent molecular property. The ideal spacer arm should be of three- to six-chain atom length, and longer or shorter than this length is of no advantage. If the spacer arm is too short, the hapten will be submerged in the ‘sea’ of the carrier protein molecule. Conversely, if the spacer arm is too long, it will be folded.16 Following this approach, clopidol immunogen was synthesized by introducing a three-atom spacer length at the pyridine ring of the hapten derivative, which preserves the specific moieties of clopidol and achieves an optimal hapten density and thus the highest antigenicity of conjugate. In this study, UV absorbance was used to check the extent of coupling and to determine whether the linking had been successful, because of its simplicity and low sample consumption. The spectra from 200 to 500 nm were recorded for BSA (0.5 mg

wileyonlinelibrary.com/jsfa

Figure 3. UV scanning spectra of BSA, CLOP-2C and BSA-CLOP-2C.

mL−1 ), BSA-CLOP-2C (1 mg mL−1 ) and CLOP-2C (0.2 mg mL−1 ) and the UV–visible results are presented in Fig. 3. The absorbance for BSA-CLOP-2C gave a significantly shifted peak at 278 nm compared with the 276 nm peak for CLOP-2C, while the maximum absorbance of BSA was at 280 nm in this work, which indicated that CLOP was successfully conjugated with BSA. On the basis of UV absorbance, an average hapten/protein conjugation ratio was confirmed as 14.3:1. The results are satisfactory considering that the highest immunogenicity would be produced with a conjugation ratio between 8 and 25.17 Production and characteristics of monoclonal antibodies (mAbs) Two cell fusions were performed with lymphocytes obtained from the spleen of selected mice, and mAb-producing hybridomas were screened by simultaneous non-competitive and competitive ELISA procedures as described above. Although cell fusion efficiency was normal (the fusion rate was about 76%) and the number of positive wells was not unusually low (the positive well rate was 22%), only a low number of them significantly recognized CLOP in the culture supernatants. Finally, six hybridomas were cloned and expanded and the mAbs were purified and characterized. Using a mouse monoclonal antibody isotyping kit, all six antibodies were of the IgG1 isotype with κ light chain. The protein concentrations of all mAbs were between 5.2 and 6.8 mg mL−1 . Based on the results of checkerboard titration, the antibody titers and IC50 values were

c 2014 Society of Chemical Industry 

J Sci Food Agric (2014)

ELISA for detection of clopidol residues in chicken tissues

www.soci.org

100

2.0 Two-step assay (IC50 = 2.76 ng mL-1)

60 40 20 0 1E-3

0.01

0.1 1 10 100 1000 10000 Concentration of CLOP (ng mL-1)

Figure 4. Optimized standard icELISA inhibition curves for CLOP. Data were obtained by averaging three independent curves, each run in triplicate.

determined (data not shown). From the inhibition curves obtained, the most sensitive hybridoma, named C1G3, was selected for further evaluation and subsequent immunoassay development. Two-step and one-step icELISA standard curves To ascertain the applicability of the C1G3 mAb generated in this study, both two-step and one-step icELISA formats were investigated. It is well known that the working concentrations of coating antigen and antibody are crucial factors for the sensitivity of ELISAs. Therefore checkerboard titrations were performed taking into account the optimal concentrations. The optimal reagent dilutions were determined when Amax was between 1.5 and 2 and the standard curve of inhibition ratio versus CLOP pursued the lowest IC50 values. Based on the checkerboard results, coating antigen concentrations were determined to be 1 and 1.2 µg mL−1 for the two-step and one-step assays respectively. The mAb was allowed at 0.5 µg mL−1 (1:10000 dilution) for the twostep assay and at 0.6 µg mL−1 (1:8000 dilution) for the one-step assay. Other assay conditions were as described above. Under these optimized conditions, representative standard inhibition curves of the two icELISA formats (two-step and onestep) are shown in Fig. 4. The competitive curve obtained with the two-step assay allowed the detection of CLOP from 0.06 to 98 ng mL−1 , with an IC50 value of 2.76 ng mL−1 and a LOD value of 0.03 ng mL−1 . Using the one-step icELISA format, the analyte could be determined in the range 0.08–102 ng mL−1 , with IC50 and LOD values of 3.52 ng mL−1 and 0.03 ng mL−1 respectively. Apparently, the IC50 values achieved in C1G3 mAb were about one order of magnitude lower than those in pAbs collected from immunized mice, indicating higher sensitivity of the developed ELISAs. Although the IC50 and LOD values of the two ELISA formats were comparable, the one-step icELISA assay was quicker, because it saved 40 min by omitting one washing procedure and shortening the incubation time. With this tremendous saving in time, the onestep icELISA method provided higher efficiency and was therefore evaluated further. Determination of matrix effects and assay parameters It is known that immunoassays often have high potential for nonspecific binding, which will reduce the precision and accuracy of the analysis. Chemical substances present in samples or sample extracts, such as small-molecule proteins, lipids, free J Sci Food Agric (2014)

Absorbance

1.6

1.5 1.0 0.5 0.0 -1 1 -3 10 10 10

1.2

3 10

CLOP (ng mL-1)

0.8 1:2 1:4 1:6 1:10 1:20

0.4

0.0 10-3

10-2

10-1

100

101 102 CLOP (ng mL-1)

103

104

Figure 5. CLOP standard curves in diluted chicken tissues. Each point represents the average of three separate assays in triplicate. The inset shows the one-step icELISA standard curve in PBS.

amino acids and certain more complex compounds, might affect antigen–antibody interaction, reduce sensitivity and lower the extent of color development. Two methods are commonly used to reduce matrix effects. The first method generally requires hydrolysis and extraction procedures, i.e. chemical or physical technology, which is time-consuming and laborious; the second method involves dilution of the extracts, which is simple and efficient and was employed in the present research. In this study, chicken tissues were simply extracted with NaOH/acetonitrile and no further clean-up step was employed. A comparison between calibration curves for CLOP prepared in PBS and in sample extracts is displayed in Fig. 5. As the dilution increased from 1:2 to 1:20, the sensitivity gradually improved to that in PBS. The average B0 values for dilutions at 1:2, 1:4, 1:6, 1:10 and 1:20 had absorbances of 1.264, 1.413, 1.672, 1.868 and 1.895 respectively compared with 1.901 in PBS. As Fig. 5 shows, 20-fold dilution had higher goodness-of-fit than other dilution folds. However, a large dilution factor may reduce sensitivity, even causing the CLOP concentration to move out of the quantitative detection range of the developed immunoassay. It can be seen from the data that the two standard curves of 1:10 and 1:20 dilutions are superimposable, which suggests that matrix interference can actually be ignored at a dilution of 1:10.

Concentrations measured (µg kg-1)

One-step assay (IC50 = 3.52 ng mL-1)

Absorbance at 450 nm

B/B0 (%)

80

2.0

70 60 50 40 30 20

y = 1.008x - 1.0928 R2 = 0.9534

10 0

0

20

40

60

Concentrations spiked (µg

80

kg-1)

Figure 6. Spiking tests in chicken muscles between concentrations spiked and measured.

c 2014 Society of Chemical Industry 

wileyonlinelibrary.com/jsfa

www.soci.org Taking into account that 10-fold dilution of chicken tissue is advised during sample preparation, the LOD value of the one-step icELISA may be corrected to 0.3 ng mL−1 (corresponded to 0.3 µg kg−1 ). Spiking tests in chicken samples The spiked recoveries were used to evaluate the potential application of C1G3 mAb to detect CLOP residues in chicken by one-step icELISA. Samples were obtained from CLOP-spiked chicken muscles with three replicates per concentration. Figure 6 displays the correlation between concentrations spiked and determined and the linear regression between the two data groups. From Fig. 6 we can see that the data points are closely distributed on both sides of the standard line, i.e. the data added and measured are very similar and with no significant differences, although there is a slight tendency for the ELISA values to be slightly lower than the practical values. We believe that the extraction process prior to analysis caused some loss of the target compound, resulting in lower recovery. The results demonstrate that this onestep icELISA can be used as a screening method for detecting CLOP residues in chicken tissues and that it provides a noticeable advantage over methods requiring tedious clean-up procedures.

CONCLUSIONS In this study, CLOP artificial antigen was synthesized and highquality mAbs were obtained from immunized Balb/C mice. Under optimal assay conditions, two specific icELISA standard curves were established. The one-step icELISA was more favorable for detecting CLOP residues in chicken tissues and could also be potentially applied to the analysis of CLOP contents in other agroproducts. From a practical point of view, the developed icELISAs provide a rapid, accurate and inexpensive way to detect CLOP residues and can be carried out in any routine laboratory without the need for special facilities.

ACKNOWLEDGEMENTS This project was supported by the National Natural Science Foundation of China (U1204310), the Twelfth Five-Year Plan for National Science and Technology of China (2011BAK10B01), the Henan Innovation Project for University Prominent Research Talents (2010HASTIT026) and the Natural Science Research Program of the Education Department in Henan Province of China (2010A230004).

REFERENCES 1 Huang XY, Liu ZS and Guo YL, Veterinary Pharmacopoeia of the People’s Republic of China. China Agriculture Press, Beijing, pp. 285–286 (2005) (in Chinese).

wileyonlinelibrary.com/jsfa

J-Q Jiang et al.

2 Ministry of Agriculture of the People’s Republic of China, Pollutionfree poultry food and poultry by-products. NY 5034-2005 (2005) (in Chinese). 3 Pang GF, Cao YZ, Fan CL, Zhang JJ, Li XM and MacNeil JD, Determination of clopidol residues in chicken tissues by liquid chromatography: collaborative study. J AOAC Int 86:685–693 (2003). 4 Yang W, Xu J, Yang G, Ding T, Qi J, Liu H, et al, Determination of clopidol residue in poultry products by liquid chromatography-electrospray ion trap tandem mass spectrometry. Se Pu 27:144–148 (2009) (in Chinese). 5 Pang GF, Cao YZ, Fan CL, Zhang JJ, Li XM and Wang C, Determination of clopidol residues in chicken tissues by high-performance liquid chromatography-mass spectrometry. J Chromatogr A 882:85–88 (2000). 6 Liu X, Su Y, Fang B, Ding H, He L, Zeng Z, et al, Determination of clopidol residues in chicken muscle by gas chromatography-mass spectrometry. Se Pu 27:86–90 (2009) (in Chinese). 7 Fang B, Su Y, Ding H, Zhang J and He L, Determination of residual clopidol in chicken muscle by capillary gas chromatography-negative chemical ionization-mass spectrometry. Anal Sci 25:1203–1206 (2009). 8 Xie ZL, Li W and He JG, Synthesis and characterization of artificial antigen of clopidol. Food Sci 30:228–231 (2009) (in Chinese). 9 Sakamoto S, Putalun W, Tsuchihashi R, Morimoto S, Kinjo J and Tanaka H, Development of an enzyme-linked immunosorbent assay (ELISA) using highly-specific monoclonal antibodies against plumbagin. Anal Chim Acta 607:100–105 (2008). 10 Shelver WL, Smith DJ and Berry ES, Production and characterization of a monoclonal antibody against the β-adrenergic agonist ractopamine. J Agric Food Chem 48:4020–4026 (2000). 11 Wu JX, Zhang SE and Zhou XP, Monoclonal antibody-based ELISA and colloidal gold-based immunochromatographic assay for streptomycin residue detection in milk and swine urine. J Zhejiang Univ Sci B 11:52–60 (2010). 12 Liu W, Li W, Yin W, Meng M, Wan Y, Feng C, et al, Preparation of a monoclonal antibody and development of an indirect competitive ELISA for the detection of chlorpromazine residue in chicken and swine liver. J Sci Food Agric 90:1789–1795 (2010). 13 Liu Z, Lu S, Zhao C, Ding K, Cao Z, Zhan J, et al, Preparation of anti-danofloxacin antibody and development of an indirect competitive enzyme-linked immunosorbent assay for detection of danofloxacin residue in chicken liver. J Sci Food Agric 89:1115–1121 (2009). ´ C, Agullo´ C, Abad-Somovilla A and 14 Mercader JV, Su´arez-Pantaleon Abad-Fuentes A, Hapten synthesis and monoclonal antibodybased immunoassay development for the detection of the fungicide kresoxim-methyl. J Agric Food Chem 56:1545–1552 (2008). 15 Jiang JQ, Wang ZL, Zhang HT, Zhang XJ, Liu XY and Wang SH, Monoclonal antibody-based ELISA and colloidal gold immunoassay for detecting 19-nortestosterone residue in animal tissues. J Agric Food Chem 59:9763–9769 (2011). 16 Jiang JQ, Zhang HT, Li GL, Wang ZL, Wang JH and Zhao HZ, Preparation of anti-nortestosterone antibodies and development of an indirect heterologous competitive enzyme-linked immunosorbent assay to detect nortestosterone residues in animal urines. Anal Lett 44:2373–2393 (2011). 17 Singh KV, Kaur J, Varshney GC, Raje M and Suri CR, Synthesis and characterization of hapten–protein conjugates for antibody production against small molecules. Bioconjugate Chem 15:168–173 (2004).

c 2014 Society of Chemical Industry 

J Sci Food Agric (2014)

Development of an enzyme-linked immunosorbent assay for detection of clopidol residues in chicken tissues.

Clopidol is mainly used for the prevention and treatment of coccidiosis, which poses a serious potential hazard to public health, in veterinary medici...
261KB Sizes 0 Downloads 0 Views