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ANIMAL PRODUCTION |
Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, China Agricultural University, Beijing 100094, People’s Republic of China
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Key Words: enzyme-linked immunosorbent assay • high-performance liquid chromatography • ractopamine • swine feed
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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; Maclennan and Edwards, 1989). Ractopamine has been shown to improve BW gain, leanness, and feed efficiency in pigs (Nelson et al., 1987; Yen et al., 1990; Xiao et al., 1999).
Because residues in animal tissues have been linked to some poisoning cases in humans (Kuiper et al., 1998), most of the ß-adrenergic agonists except ractopamine, which was recently licensed as a swine feed additive in the United States, are now prohibited from use as feed additives in food animals. Because of the growth improvement in animals fed ractopamine, there is a possibility that adulteration of feed with ractopamine could result in residues in animal tissues and lead to human poisoning. Therefore, a need exists for a rapid, sensitive, and economical method for detecting ractopamine in animal feeds.
Several assays for determination of ractopamine residues in animal tissues have been reported, including immunoassays (Shelver and Smith, 2000, 2002a), liquid chromatography (Turberg et al., 1995, 1996; Smith and Shelver, 2002), liquid chromatography/mass spectrometry (Antignac et al., 2002; Shishani et al., 2003), gas chromatography/mass spectrometry (Bocca et al., 2003), and other methods (Inoue and Chang, 2003; Shelver and Smith, 2003).
However, few assays are available for detecting ractopamine in animal feeds, except for an HPLC method with coulometric detection (Turberg et al., 1994). No studies are available in which ractopamine in animal feeds was determined by ELISA. The objective of this paper was to develop a rapid, sensitive ELISA method to determine ractopamine in swine feeds using HPLC as the reference assay.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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The extract solvent was prepared by adding 100 mL of water and 2 mL of hydrochloric acid into 900 mL of methanol. The ractopamine standard stock solution (1 mg/mL) was prepared by dissolving 100 mg of ractopamine in 100 mL of methanol and was stored at –20°C before being used for the preparation of standard solutions. The assay buffer was PBS containing 0.01 M phosphate buffer (pH 7.2) with 0.145 mM NaCl, and the washing buffer was composed of PBS with 0.05% Tween20.
Production and Preparation of AntiRactopamine Monoclonal Antibody
Ractopamine immunogens were synthesized according to Shelver and Smith (2000). Briefly, the hapten was synthesized by coupling ractopamine with glutarate anhydride, and then the hapten was coupled to BSA (ractopamine-BSA) as the immunogen and coupled to ovalbumin as a coating antigen using the mixed anhydride method.
The monoclonal antibody (MAb) against ractopamine was produced as described by Shelver et al. (2000) with some modifications. Briefly, five 8-wk-old female BALB/c mice (Inst. of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China) were injected s.c. on the dorsal region with ractopamine-BSA at a dosage of 100 µg/kg of BW in Freund’s complete adjuvant. The mice were boosted with ractopamine-BSA in Freund’s incomplete adjuvant subcutaneously 2 wk later. Through 4 boosters (each at 2-wk intervals), serum was collected and the antibody titer was monitored. The spleen from the mouse with the greatest titer was removed, and splenocytes were fused with SP2/O myeloma cells.
The splenocyte-fused myeloma cells were cultured in 96-well plates. The positive hybridomas were rescreened using the indirect competitive ELISA described later, with ractopamine as the competitor. Hybridomas, which produced specific antibodies against free ractopamine, were subcloned twice with the limiting dilution method. The isotype of the antibodies was detected by using mouse heavy-chain- and light-chain-specific antisera in a commercial ELISA kit (Southern Biotech Co., Beijing, China). The MAb that was obtained was used to develop the indirect competitive ELISA.
Sample Extraction and Purification
Ractopamine was added to swine feed samples or supplements known to be free of ractopamine at levels of 2.5, 5.0, 10.0, 20.0, or 40.0 µg/g in the complex feed and 50.0 to 400.0 µg/g in the supplement, and air-dried for 10 min. Ten grams of the prepared samples and 100 mL of the extract solvent were added to a glass jar and then mixed for 30 min on a variable speed reciprocal shaker (Changzhou Guohua Electrical Equipment Co., Jiangsu, China). The jar was centrifuged for 10 min at 4,000 xg (Mikro 22 R; Hettich Co., Kirchlengern, Germany). Then 1.0 mL of the supernatant was pipetted into a plastic centrifuge tube and evaporated to dryness under a stream of nitrogen in a water bath at 45°C. The residue was dissolved in 4.0 mL of 0.3% acetic acid by stirring for 60 s, before 2.0 mL of dichloromethane was added. The mixture was stirred for 30 s before centrifugation for 10 min at 3,000 xg. The supernatant was transferred into another clean centrifuge tube. Then 2.0 mL of hexane was added, and the mixture was stirred for 30 s and then centrifuged for 5 min at 1,000 xg. The upper organic solvent phase was discarded and the aqueous phase was stored at 4°C until analyses by ELISA and HPLC.
Indirect Competitive ELISA
The checkerboard procedure was used to optimize the coating antigen and the antibody concentrations. For the ELISA, each well of a microtitre plate was coated with 100 µL of the coating antigen at the optimal dilution and incubated overnight at 4°C. The excess binding sites were blocked with 0.5% gelatin (Sigma). After removing the blocking solution, 50 µL of the optimal antibody dilution (1:300,000 for the antibody and 1:500,000 for the coating antigen) and 50 µL of the ractopamine standard solution or sample extract were added to the wells (in triplicate), and then the plate was incubated for 1 h at 37°C. After 3 washes with PBS with 0.05% Tween20, 100 µL of horseradish peroxidase-labeled goat antimouse IgG (Huamei Biological Technical Co., Beijing, China) solution was added to each well of the plate before the plate was incubated for 30 min at 37°C. After incubation, the plate was washed and then 100 µL of the tetramethylbenzidine (TMB, Cxbio Biotechnology Ltd., Shanghai, China) substrate system was added.
After the plate was incubated for another 15 min at 37°C, 50 µL of 2 M H2SO4 was added to stop the reaction, and the plate was read using an ELISA plate reader (Sunrise, Tecan Inc., Durham, NC) at 450 nm.
Liquid Chromatography
A Waters 2695 liquid chromatography unit equipped with a reversed-phase Supelco C18 column (250 x4.6mm, 5 µm) and a Waters 2475 fluorescence detector was used (Waters Co., Milford, CT). The fluorescence detector was set at the excitation wavelength of 226 nm and emission wavelength at 305 nm. The mobile phase was prepared by adding 680 mL of purified water, 20 mL of acetic acid, and 0.87 g/L of 1-pentanesulfonic acid sodium salt to 320 mL of acetonitrile, and then filtered under vacuum through a 0.45-µm filter. The HPLC was performed at a flow rate of 1.0 mL/min and an injection volume of 50 µL.
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RESULTS AND DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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. The MAb produced in our laboratory has good specificity toward ractopamine (Tables 1 and 2), similar to the report of Shelver et al. (2000). In the current study, cross-reactivity of the MAb with ractopamine metabolites was not determined because the aim was to develop a method for detecting ractopamine in swine feeds, in which ractopamine metabolites are unlikely to be present. The MAb produced in our study had an inhibitory concentration 50 (IC50) of 5.3 ng/mL toward ractopamine, which was greater than that reported by Shelver et al. (2000) but probably sensitive enough for detecting ractopamine in swine feeds.
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); therefore this mixture was chosen as the extraction solvent. We also used dichloromethane or hexane to clean up the extract; however, using both dichloromethane and hexane gave better results than either solvent alone in terms of minimizing interference peaks on liquid chromatography or nonspecific binding in the ELISA. Turberg et al. (1994) reported recoveries ranging from 85 to 100% from animal feeds by solvent extraction with 0.2% HCl in methanol followed by liquid-liquid partition and solid phase extraction, which is a complex and time-consuming procedure. Shelver and Smith (2002b) used an IAC column to extract and purify ractopamine from cattle and sheep tissues, with recoveries of 82.1 to 92.5% and 91.7 to 92.3%, respectively.
Ractopamine Assay
ELISA Method.
In swine complex feeds, the recoveries (Table 2) ranged from 73.1 to 86.5% (CV of 3.3 to 9.0%). The recovery from swine supplement (Table 2) was from 105.5 to 111.4% (CV of 5.5 to 7.7%). Because ractopamine is prohibited in food animals in China, no detectable ractopamine should be present in swine feeds. The limit of detection in this study, defined as 10% of the inhibition, was 0.6 ng/g (equal to 0.24 µg/g ractopamine in feeds). Two studies reporting detection of ractopamine residues in cattle and sheep urine by ELISA (Shelver and Smith, 2000, 2002a) reported a limit of detection of 0.3 and 0.76 ng/mL, respectively.
HPLC Method.
The present HPLC method was a modification of the validated procedure for ractopamine determination in swine muscle and liver samples (Center for Veterinary Medicine’s Document Control Unit [HFV-199], FDA, Rockville, MD), The limit of detection, defined as the lowest concentration of ractopamine that can be reliably detected, is 0.48 µg/g at a signal-noise ratio of >3, so the method was sufficiently sensitive for detecting ractopamine in animal feeds. In addition, the recoveries were high (83.6 to 98.2% from complex feeds and 89.1 to 92.9% from supplement; Table 2) for the HPLC method.
Comparison of ELISA and HPLC
As shown in Table 2, the recovery values with the HPLC method were more consistent at different ractopamine concentrations in complex feeds and supplement than those of the ELISA method. In addition, the 2 methods did not show good correlation (P = 0.009 and 0.005, respectively).
In summary, in this study both methods gave satisfactory results in recovery studies from the feed samples containing ractopamine, and the ELISA method was more sensitive and rapid and less expensive than the HPLC method, but the former was less consistent and specific than the latter. Therefore, in practice the ELISA method could be used for ractopamine screening in swine feeds before confirmation and quantification by HPLC.
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Footnotes |
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2 Corresponding author: sjz{at}cau.edu.cn
Received for publication August 22, 2005. Accepted for publication November 28, 2005.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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