In ternationa l Scholars Journa ls African Journal of Food Science Research ISSN 2375-0723 Vol. 3 (8), pp. 190-195, August, 2015. Available online at www.internationalscholarsjournals.org © International Scholars Journals Author(s) retain the copyright of this article. Full Length Research Paper Immunoaffinity column as cleanup tool for an enzyme linked immunosorbent assay of ractopamine detection in various tissues of swine Wentao Xu 1 , Kunlun Huang 1 , Aike Deng 1 , Baiqiang Zhai 1 , Heng Zhao 1,2 , Yingcong Li 1 , Zhihong Liang 1 and Yunbo Luo 1 * 1 College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China. 2 Current address: Cell Biology and Immunology Group, Wageningen University, P.O. Box 338, 6700 AH, The Netherlands. Accepted 09 April, 2015 Ractopamine has been developed to be the main -agonist substance used illegally in meat producing animals. A simple and efficient extraction and purification procedure for ractopamine was developed by means of the immunoaffinity column (IAC) as a cleanup tool. Purified polyclonal antibodies against RCT were produced and coupled covalently to CNBr-activated Sepharose 4B. Both the binding conditions and elution protocols were optimized and the capacity, reusability, precision and accuracy of IAC were determined. The IAC was successfully employed to isolate and purify the RCT from the various tissues of swine. Subsequently, enzyme linked immunosorbent assay (ELISA) procedures were established further on to measure RCT. The antibodies showed negligible cross-reactivity with other -agonists. IAC-ELISA allowed 0.2 ng/mL of RCT to be detected in urine and 0.5 ng/mL to be detected in other various tissues of swine, which makes this method an acceptable screening tool to access RCT. IAC-ELISA for the detection of RCT was validated by LC-MS and the correlations between the results from LC-MS and those from IAC-ELISA were all high (above 0.89). Key words: Purification, ractopamine, swine, immunoaffinity column, enzyme linked immunosorbent assay. INTRODUCTION -agonists have been misused as growth promoting agents in meat producing animals over 20 years. Ractopamine (RCT) has been developed to be the main -agonist substance used illegally for this purpose. Ractopamine is a phenethanolamine leanness- enhancing agent that has been approved by U.S. Food and Drug Administration (FDA, 2000; Muirhead, 2000) and regula-tory agencies in Brazil, Venezuela, Colombia, Guatemala, Dominican Republic, and the Philippines as a feed addi-tive for swine (Shelver and Smith, 2002). Phenethanola-mine -agonists have a history of being used for unautho-rized purposes by livestock producers (Kuiper et al., 1998; Mitchell and Dunnavan, 1998; Shiu and Chong, 2001). The positive influence of ractopamine hydrochlo-ride on these economically important traits should make the product attractive to swine growers, and to producers *Corresponding author. E-mail: lyb@cau.edu.cn. of livestock species for which ractopamine is not approve The presence of drug residues in animal tissues is a potential food safety concern, especially when the com- pound has been used illegally or in a manner not pres- cribed by regulatory official. In an effort to combat the illicit use of -agonists com- pounds, regulatory organizations worldwide test animal tissues for the presence of illicit drugs through both screening and confirmatory assays. For residue analysis, the analyte is separated from the matrix by liquid- liquid or solid-phase extraction (SPE), followed by liquid or gas chromatography or immunological analysis. Sample pre- parations steps are often time consuming and tedious, making an efficient cleanup method very desirable when high throughput is required. High sensitivities of immu- noassays are practically desirable for off-label drug moni- oring because it may be desirable to detect the analyte even after extended withdrawal periods. -agonists immunoassays have been developed for clenbuterol (Yamamoto and Iwata, 1982), fenoterol (Haasnoot et al., Wentao et al. 191 1994) and ractopamine (Elliott et al., 1998; Ba, 1998). MATERIALS AND METHODS Reagents and instruments: CNBr-activated Sepharose 4B was obtained from Pharmacia Biotech. Protein A-Sepharose 4B was purchased from Sigma. Ractopamine was provided by Eli-lilly (USA). Liquid scintillation cocktail (EcoliteTM) was purchased from ICN Biomedicaal, Inc. Animal specimens including Swine live and muscle samples were obtained from swine known to be free of ractopamine and were pooled (Beijing Laboratory Animal Research center, China). Microplate Reader MK3 was from Finland LAB- SYSTEMS. All other chemicals or solvents were obtained from Sigma or Amerscreso and were analytical grade or better. Immunogen and enzyme conjugate preparation: RCT (99.999%, Daming Company Henan, China) was coupled to a carrier, bovine serum albumin (BSA), and to an enzyme, horseradish peroxidase (HRP), using the coupling agent, butane-1,4-diol diglycidyl ether (Yamamoto and Iwata, 1982; Haasnoot et al., 1994). Immunogen: A 30 mg amount of BSA was dissolved in 0.5 mL of de-ionized water and the pH was adjusted to 10.8 using 0.5 mol/L sodium hydroxide. A 6 uL volume (30 umol) of butane-1,4-diol digly- cidyl ether was added and the mixture was incubated for 20 h at room temperature under a nitrogen atmosphere. A 15 mg (50 umol) amount of RCT was dissolved in 0.5 ml of 0.1 mol/L sodium hydroxide, 200 uL dimethylformamide being added to help solu- bility. This solution was degassed using nitrogen then added to the epoxy-activated BSA solution. The reaction mixture was incubated for 20 h at room temperature under a nitrogen atmosphere. Purifi- cation of the RCT-BSA immunogen was achieved using extensive dialysis (10 000 molecular mass off) against 0.15 mol/L -1 sodium hydroxide. The immunogen was diluted with 0.15 mol/L sodium hy- droxide to give a final protein concentration of 1 mg/mL and stored at –20 o C until required. Enzyme conjugate: A 20 mg amount of HRP was dissolved in 0.5 mL of de-ionized water and the pH was adjusted to 10.8 using 0.5 mol/L sodium hydroxide and the solution was de-gassed with nitro- gen. A 50 L volume (5 mol) of a solution of butane-1,4-diol digly- cidyl ether was added and the mixture was incubated for 20 h at room temperature under a nitrogen atmosphere. Unreacted butane- 1,4-diol diglycidyl ether was removed by gel filtration (Sephadex G25-M, equilibrated with 1 mmol/L sodium acetate). A 20 mg amount of RCT was added to the activated HRP solution, 200 uL dimethylformamide being added to increase solubility. This solution was degassed using nitrogen. The enzyme-labelled conjugant was then purified by gel filtration (Sephadex G25-M, equilibrated with 1 mmol/L sodium acetate) and by use of dextran-coated charcoal. The HRP-RCT was stored at –20 o C until required. Production and purification of polyclonal antibodies. The poly- clonal antibodies against RCT-BSA were obtained from New Zea- land White rabbits (Beijing laboratory research center, China). In brief, immunogen was emulsified with an equal volume of Freund’s complete adjuvant to give a final concentration of 0.5 mg/mL. This mixture was given in four intradermic injections. Boosting injections of Freund’s incomplete adjuvant were made at 2-week intervals. Blood samples were collected 1-week after the third booster injections and tested for titer determination. Blood collected from rabbits was first allowed to stand overnight at 4 o C, then centrifuged at 200 × g for 15 min. The obtained antiserum was purified in three steps by firstly using a saturated ammonium sulfate method (Elliott et al., 1998). The 20 mL antiserum was purified by the saturated ammonium sulfate method. At last the precipitates were dissolved in 2 mL of PBS and dialyzed against PBS until no sulfate ion could be detected in the dialysis solution with 0.5 M BaCl2 acidified with HCl. The obtained immunoglobulin (IgG) was purified in the second step by the way of protein A-Sepharose 4B (Zhao et al., 2003). The eluates (0.1 M glycine buffer pH 7.2, 0.5 mL) were collected in tubes containing 50 mL of 1 M Tris (pH 8.0) and mixed gently to bring the pH back to neutral. At last, the antiserum was purified to remove the anti-BSA antibodies by adsorption of anti-BSA antibodies to a column containing BSA cross-linked to an Affiprep 10 matrix (Bio-Rad) and the column could be regenerated by elution with 50 mM glycine (pH 2.3)-0.5 M NaCl-0.02% Triton X-100 (Giraudi et al., 1998). The IgG concentration in the obtained solution was calculated based on the UV absorption difference between 280 and 260 nm (Chen et al., 1993). The formula used for the calculation was Cprotein (mg/mL) = 1.45 × A280 nm - 0.74 × A260 nm. The purity of the purified IgG was checked on SDS-PAGE gel. Enzyme immunoassay procedure: A direct competition ELISA for-mat was utilized to measure ractopamine binding and cross reac-tivity to related compounds. The checker board procedure was used to optimize the enzyme tracer and the anti-RCT antibody concen-trations. After optimization, the ELISAs were processed as follows: Microtiter plates were coated with 200 uL diluted antiserum in bicarbonate buffer (0.05 M, pH 9.6) overnight, 4 o C or 2 h at room temperature. The plate was washed with PBST (10 mM PBS con- taining 0.05% Tween 20, pH 7.4) three times. Serial dilutions (50 uL) of the analyte or sample solution in PBS-organic solvent, toge- ther with 50 uL of the tracer, was added to the wells and incubated for an appropriate time. After another washing step, 100 uL per well of TMB solution (400 uL of 0.6% TMB-DMSO and 100 uL of 1% H2O2 diluted with 25 mL of citrate-acetate buffer, pH 5.5) was added. Followed by incubation at 37 o C for 30 min. Color develop- ment was stopped by adding 50 uL/well of 2 M sulfuric acid. The plates were read at 450 nm with Microplate Reader MK3 (Thermo Labsystems, Vantaa, Finland) and the resulting curves fitted with a four-parameter logistic equation to determine the IC50. The IC50 was defined as the concentration of inhibitor required to inhibit color development by 50% compared to control wells containing no competitors. Antiserum cross-reactivities: Competitors used for the compete- tion studies are fenoterol, isoxsuprine, salmeterol, fomoterol and clenbuterol, which have comparable structure to ractopamine. The sensitivity of the purified rabbit antiserum was determined by con- structing calibration curves and calculating the 50% inhibition of control index (IC50), that is, the mid-point of the calibration curve. Competitive ELISA was performed using direct assay format with competitor concentration of 1 ug/mL. Compounds that produced sigmoidal like curves were fitted to the four parameter logistic equation using SAS; y (%B/B0) = (A - D)/ [1 + (x/B) C ] + D (Raab, 1983). Coupling antibody to CNBr-activated sepharose (Primot et al., 2000): Purified antibodies against RCT were coupled to CNBr- activated Sepharose to generate the immunoaffinity column. The IgG solution was dialyzed against 0.1 M NaHCO3, 0.1 M Na2CO3, 0.2 M NaCl, pH 8.3, and was added to the Sepharose beads active- ted with 1 liter of 1 mM HCl under constant rotation at 4 o C overnight at a concentration of 2 mg/mL (IgG/Sepharose beads). Then the supernatant was removed and the beads were incubated with 1 M ethanolamine pH 9.0 to deactivate the remaining active sites for 2 h under constant rotation at 4 o C. Beads were washed with 0.1 M acetate, pH 4.0, 0.5 M NaCl, then with bead buffer (PBS pH 7.4, 0.02 NaN3) and stored in 20% suspension in bead buffer at 4 o C until further use. Negative rabbit antiserum containing no antibody against RCT was applied to generate the blank column as a negative control (Xu et al., 2005). Determination of the optimal conditions of ractopamine on the immunoaffinity column (Xu et al., 2005): IACs were subjected to multiple loadings and elutions to determine column stability. Col-umns were loaded with 5 ug ractopamine, washed with both 10 mL 10% MeOH and 10 mL PBS (pH 7.4), and eluted with either 10 mL 100% MeOH or 10 mL 0.2 M glycine buffe (pH 2.5). All eluants were measured by ELISA and LC to determine recovery. Between uses, 192 Afr. J. Food Sci. Res. columns were stored at 4 o C in PBS-0.02 NaN3 (pH 7.4). To select the optimal pH condition for elution of RCT. 1 mL solution of a mixture of antibody RCT-Sepharose beads were washed with 5 mL of bead buffer. The matrix was incubated with 2 mL of RCT solution (0.25 mg/mL) and 2 m of OVA (0.25 mg/mL) for 40 min under constant rotation at 4 o C. After 7 times washing, the column was eluted with 0.2 M glycine buffer (2 mL) at various pH conditions (pH 5.5, 5.0, 4.5, 4.0, 3.5, 3.0 and 2.5). The collected fractions were assayed for RCT by ELISA. Column capacity and reusability of IAC: Column capacity for RCT was determined by passing 10 ug RCT in 10 mL PBS through an affinity column at 1 mL/min. To ensure maximum binding, the effluent was loaded to the column once more and washed with 10 mL of 10% MeOH (v/v) and 10 mL PBS (pH 7.4). Bound RCT was eluted with 0.2 M glycine buffer (pH 2.5) at 0.5 mL/min. RCT in the glycine eluate was determined by ELISA and LC. To determine column reusability, 1 mL samples (100 ppb) were loaded onto 2 mL IAC for ractopamine. After the column was washed and eluted as the above, the column regenerated with equilibrating buffer and allowed to stand for various periods, from 10 min to 24 h. The capacity of the regene- rated column was then determined by reloading with standards. This process was repeated ten times. RCT both in the washing eluate and in the glycine eluate was determined by ELISA and LC. Precision and accuracy of IAC. Samples of 100, 1000, 2500, and 5000 ng ractopamine mixture were fortified in 10 mL urine and loaded onto a 1 mL IAC. These represent 10, 100, 250, and 500 ppb fortified ractopamine samples. Columns were washed with 10 mL of 10% MeOH (v/v) and 10 mL 0.1 M sodium phosphate buffer (pH 7.4), and then eluted with 0.2 M glycine buffer (pH 2.5). Ractopamine in the glycine eluant was quantified by ELISA and LC. The procedure was repeated 3 times for each fortification level. Preparation of urine, muscle, kidney, and liver samples, and application of IAC-ELISA: A 10 g test portion of tissue was added to 10 mL PBS (pH 7.4) and the mixture was homogenized for 1 min with a Tissumizer (Tekmar Company, Cincinnati, USA) set a high torque. Samples were then cooled in an ice bath. This was repea- ted until no large particles were visually observed (usually 2 - 4 homogenization cycles) . Supernatants were separated from tissue debris by centrifugation at 12 000 × g for three times and stored at – 20 o C until used. Ractopamine (10 and 50 ng) was dis-solved in PBS (pH 7.4) or various supernatants as mentioned above from various issues of swine. These samples were to be assayed by ELISA and IAC-ELISA in order to determine the matrix effect and to calculate the detection limit of various issues. Validation of ractopa- mine LC/MS (Kootstra et al., 2005). All the samples were evapo- rated to dryness at 55 under a stream of nitrogen. The residue is dissolved in 100 uL 10:90 (v/v) methanol: water. Calibration curve was set up with ten-fold dilution of Ractopamine in 10:90 (v/v) met- hanol:water, from 100 to 0.1 ng/mL. These solutions were transfer- red to a LC vial and 20 uL is injected onto the LC-MS system. Equipment The MS system is a Agilent LCQ Classic-system equipped with an APCI+ interface. The LC system consists of a P4000 Spectra SYSTEM quaternary pump and an AS3000 SpectraSYSTEM auto- sampler (Agileng, Wilmington, DE, USA). Chromatographic separations are carried out on a Phenomenex Luna 5 m C18 250 mm × 4.6 mm column at an oven temperature of 50. The LC gradient uses two solvents: 95% methanol 10 mM ammonium acetate (A) and 5% methanol 10 mM ammonium ace-tate (B) . Flow rate 0.20 mL/min, linear gradient from 6% A- 80% A in 20 min. After 20 min the system is reconditioned for 10 min at 6% A. MS detection and conditions The analytes are detected in MRM (MS2) mode. The mass frag- ments are used for the screening method. The instrument was tuned and calibrated according to manufacturer’s specifications. RESULTS AND DISCUSSION Production and purification of polyclonal antibodies The polyclonal antibody against RCT was firstly purified through a saturated ammonium sulfate and a protein A sepharose 4B affinity column. This two- step purification using 20 mL of antiserum resulted in a 70 mg of pure antibody. The antibody purity was checked by SDS- PAGE, which showed one strong band (the heavy chain) and one weak band (the light chain) under the reducing conditions (data not shown). The purified antibodies were repeatedly flown through the column for over three times and the anti-BSA antibodies were efficiently separated from the anti-RCT antibodies. Both polyclonal antibody and monoclonal antibody were used in immunoaffinity chromatography. The most usable antibody was the poly- clonal antibody (James et al., 2004). Polyclonal antibo- dies, obtained by immunizing a rabbit or goat and purify- ing the immunoglobulin fraction from the resulting serum. One can use quite pure antigen to avoid raising unwant- ed antibodies to minor impurities in the protein prepara- tion. The obtained antiserum can be purified to remove many impurities and other unwanted antibodies by met- hods such as saturated ammonium sulfate, protein A sepharose 4B affinity column or Affiprep 10 matrix. Control column When the blank column was loaded with RCT spiked water using the modified immunoaffinity protocol as the above, RCT was not specifically adsorbed with the blank column containing the control IgG that not generated toward ractopamine. The amount of RCT in the washing and elution fractions was the same as that of columns containing anti- RCT antibodies. The above result demon-strated that the retention of RCT was primarily due to the anti-RCT antibody. The presence of RCT in the blank column eluate indicated that the RCT was only slightly re-tained by the blank column. This might possibly be due to insufficient blocking of active sites or might indicate the wash volume was not adequate. These results show that ractopamine was specifically retained by the ractopamine IAC, but not by the control column. Determination of the optimal conditions of ractopamine on the immunoaffinity column Elution of ractopamine from the IAC with glycine resulted in excellent recovery of 80 - 100% and offered two main advantages over elution with methanol. Firstly, elution with glycine buffer resulted in cleaner chromatograms of parent ractopamine after fortification in samples. Elution with methanol resulted in the co-elution of fluorescent compounds that interfered with chromatographic analysis Wentao et al. 193 Figure 1. pH reducing elution of antibody against RCT-sepharose beads with 0.2 M glycine buffer. The eluting amount of RCT increased with the decrease of eluting pH. Figure 2. IAC stability with multiple elution using 0.2 M glycine, pH 2.5 or 100% MeHO. Results are expressed as percentage of loaded ractopamine in the elution fraction (n = 3). Almost half of the error bars for the glycine elution are too small to be visible. Ractopamine IACs quickly degraded when eluted with > 50% methanol and showed marked performance problems in the second use. However, when 0.2 M glycine, pH 2.5, was used to eluate the analytes, only minimal decreases in column capacity occurred after 10 uses. of ractopamine (Shelver and Smith, 2002). Secondly, IACs were stable after glycine buffer was used, whereas columns eluted sequentially with solvents containing a high percentage of methanol rapidly degraded (discussed below). The major disadvantage of using glycine as the eluting buffer is the increased amount of time required to concentrate it (that is by evaporation) comparing to methanol. Figure 1 showed that the antibody (anti-RCT)-sepha- rose beads was specifically combined with RCT, and the eluting amount of RCT increased with the decrease of eluting pH. If the eluting pH was too low, the antibody (against RCT)-sepharose beads were unstable which affected the combination between antibody and antigen, so the prime pH of elution buffer was selected as pH 2.5. In stability tests, ractopamine IACs quickly degraded when eluted with >50% methanol and showed marked performance problems in the second use. However, when 0.2 M glycine, pH 2.5, was used to eluate the analytes, only minimal decreases in column capacity occurred after 10 uses (Figure 2). Furthermore, ractopamine IACs stored at 4 o C in PBS-0.02% sodium azide were stable over 3 months. Because of the apparent advantages of glycine elution, subsequent studies on the effects of matrix (urine, muscle, kidney, and liver) were performed with glycine as the elution solvent. Spiked ractopamine urine samples were used to deter- mine the precision and recovery at various ractopamine levels. The recoveries of ractopamine from the urine sam- ples for 10, 50, 100, 250, 500 ng/mL were 81.6, 87.3, 90.8, 86.5 and 83.4%, respectively. The standard errors of the measurements were 6.5, 7.8, 10.3, 8.4, and 5.6 ng/mL respectively. The IAC gave excellent recovery and reproducibility. The clean sample from IAC allows the sensitivity to be easily controlled by concentrating the eluant to obtain greater sensitivity. The method was confirmed useful over a very wide range of concentra- tions with only minor alterations in the LC procedure. Matrices such as urine, tissues, and serum have great impact on the results of ELISA when these samples were to be detected directly by ELISA. But after these samples were purified by IAC, the effect can be eliminated. So IAC-ELISA enhances not only the sensitivity but also the accuracy of detection for ractopamine. Antiserum cross-reactivity Under these conditions, cross-reactivity to-wards other - agonists was determined. A number of -agonists, which Table 1. Cross-reactivity profile toward -adrenergic agonists from antiserum raised to ractopamine. Compound Ractopamine Fenoterol Isoxsuprine Salmeterol Fomoterol Clenbuterol IC50 3.3 650 >80.000 >80.000 >80.000 >80.000 % CR 100% 0.5 <0.05 <0.05 <0.05 <0.05 Figure 3. Representative standard curve for RCT by IAC-ELISA. Each point represents the mean of 5 determinants. Vertical bars indicate + SD about the mean. Figure 4A and 4B. The precursor ion and the daughter ions of ractopamine in all samples detected and qualified by MS-MS. The precursor ion of ractopamine is 302.1 and the daughter ions of ractopamine are 284 and 164. were fenoterol, isoxsuprine, salmeterol, fomoterol and clenbuterol, tested for their cross-reactivity because they have comparable structure to ractopamine. Fenoterol showed a cross-activity of 0.5% and other -agonists re- 194 Afr. J. Food Sci. Res. Wentao et al. 195 reacted 0.05 % (Table 1). Thus, this ELISA can only recognize ractopamine of the compounds tested. Detec- tion limit and standard curve: The results obtained from the analysis of residue-free urine and other samples were used to estimate the detection limit. Figure 3 showed a representative standard curve for ractopamine. The detection limit was 0.2 ng/mL of ractopamine in urine, based on the mean response for the control sample ex- tracts plus three times the standard deviation with IC50 of 3.3 + 0.25 ng/mL (Figure 3). In addition, IAC-ELISA allowed 0.5 ng/mL of RCT to be detected in other various tissues of swine. Validation of IAC-ELISA by LC-MS The validation indicated that all the eluates contain the ractopamine (The precursor ion of ractopamine is 302.1 and the daughter ions of ractopamine are 284 and 164) (Figure 4A and 4B) and the correlations between the results from LC-MS and those from IAC-ELISA were all high (above 0.8943). 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