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1790 F F O O N S N www.soci.org W Liu et al. Acepromazine Chloropromazine Azaperone Haloperidol Figure 1. Chlorpromazine and other structurally related sedatives analysed in this study. raphy–mass spectrometry14 – 16 have been used. These methods require extensive sample preparation, sometimes expensive apparatus, highly trained personnel to operate sophisticated instruments and interpret complicated results, and can only determine a limited number of samples at one time. Therefore these methods are not suitable as screening tests. In contrast, because of the rapidity, mobility, convenience, high sensitivity, and low detection limit, enzyme-linked immunosorbent assay (ELISA) methods have been N N N OH used for the detection of various drug residues in real systems. O N 17 – 22 A key factor for an ELISA test is whether a polyclonal antibody or monoclonal antibody (MAb) towards the compound detected was used. Compared with a polyclonal antibody, the application of a monoclonal antibody is advantageous in terms of better purity, satisfactory sensitivity and high specificity. 23 – 26 In this paper, an ELISA test kit based on a monoclonal antibody toward chlorpromazine was developed and applied to detect chlorpromazine spiked in swine liver and chicken samples. This study is the first to prepare a monoclonal antibody of chlorpromazine and develop an immunoassay based on an antibody to detect residues of chlorpromazine in swine liver and chicken. EXPERIMENTAL Chemicals and materials Bovine serum albumin (BSA), ovalbumin (OVA) and goat anti-mouse IgG–horseradish peroxidase (HRP) conjugate were provided by Beijing Wanger Biotechnology Co., Ltd. (Beijing, China). o-Phenylenediamine (OPD) and 3,3 ′ ,5,5 ′ - F Cl N S OH N S Azaperol N Promethazine N N Cl N tetramethylbenzidine (TMB), N,N ′ -dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) were purchased from Xinjingke Biotechnology (Beijing, China). Freund’s complete (cFA) and incomplete adjuvants (iFA) were obtained from Sigma–Aldrich (St Louis, MO, USA). n-Hexane, hydrochloric acid, dimethylsulfoxide (DMSO), sulfuric acid, sodium hydroxide, acetonitrile, hydrogen peroxide (30% H2O2) and other reagents used were provided by Guangmang Chemical Co. (Jinan, China). Chicken samples were purchased from commodity exchange and swine liver samples were from a supermarket in Jinan City, China. Instrumentation and supplies ELISA was performed on polystyrene 96-well microtitre plates (Bio Basic Inc., Ontario, Canada) and spectrophotometrically read with a GF-M3000 microplate reader (Ruicong Shanghai Technology Development Co., Ltd, Shanghai, China). Centrifugation was carried out with a Biofuge Stratos refrigerated centrifuge (Heraeus, Hanau, Germany). Protein dialyses were performed using dialysis bags from Aibo Economic & Trade Co., Ltd (Jinan, China). LC-MS was performed on a LC/MS-2010A instrument from Shimadzu (Kyoto, Japan). Buffers Ultra-pure deionised water was used for the preparation of all buffers and reagents for the immunoassays, unless especially indicated. Phosphate-buffered saline (PBS, pH 7.4) consisted of 138 mmol L −1 NaCl, 1.5 mmol L −1 KH2PO4, 7 mmol L −1 Na2HPO4 and 2.7 mmol L −1 KCl. The wash buffer (PBST) was a PBS buffer www.interscience.wiley.com/jsfa c○ 2010 Society of Chemical Industry J Sci Food Agric 2010; 90: 1789–1795 N
Detection of chlorpromazine in swine liver and chicken by ELISA www.soci.org added 0.05% (v/v) Tween 20. 0.05 M carbonate buffer (15 mmol L −1 Na2CO3 and 35 mmol L −1 NaHCO3, pH9.6)wasusedasacoating buffer. The blocking buffer was a solution of PBS mixed with 1% of OVA and 0.05% (v/v) Tween 20. The substrate buffer was 0.1 mol L −1 sodium acetate/citrate buffer (pH 5.0). Eighty millilitres of acetonitrile added to 20 mL of 0.1 mol L −1 HCl was used as the extractive solution. To prepare the substrate solution, 10 mg of TMB + 5 mL DMSO was defined as substrate solution A and 5 µLof H2O2 [30% (w/w)] + 15 mL citrate buffer was defined as solution B. The stopping solution was 2 mol L −1 H2SO4. Preparation of immunogen and coating antigen Modification of hapten As described in Fig. 2 (Scheme 1), the mixture of acepromazine (compound I) (10 mg, 0.031 mmol) in 2.5 mL of ethanol and hydroxylamine hydrochloride (2.5 mg) in 1.0 mL of distill water was stirring under reflux in water bath for 2.5 h. A solution of NaOH (1.0 mL, 0.05 mol L −1 ) was added dropwise during the procedure. An acetate buffer (1.0 mL, pH 4.0) was added dropwise followed by adding 4 mg of ice until a white precipitate appeared. The white solid was isolated by centrifugation (10 000 × g)after1day. The white solid was dissolved in DMF (2.5 mL) and the mixture was kept reaction at room temperature for 2 h after succinic anhydride was added. The reaction was continuing for 4 h after 100 mL of triethylamine was added to yield chlorpromazine hapten (compound III). Preparation of immunogen The immunogen of chlorpromazine was prepared via a mixed acid anhydride reaction (Fig. 2, Scheme I). In this procedure, 15 µL of isobutyl chloroformate was added into the 1.0 mL of hapten prepared above at 10 ◦ C to obtain solution A. The solution B was prepared by dissolving 36 mg of BSA in 2 mL of sodium carbonate (50 mmol L −1 ). Under stirring, the solution A was added dropwise into solution B with molar ratio of hapten : BSA = 10 : 1 for 4 h at 10 ◦ C to get the immunogen of cationic BSA (cBSA)–chlorpromazine (compound V). The mixture was stirred overnight at 4 ◦ C and dialysed for 3 days against PBS (0.01 mol L −1 ), exchanging the dialysis solution twice each day. The solution obtained was stored at −20 ◦ C for future use. Preparation of coating antigen The mixture of 1.0 mL of hapten (compound III) obtained in procedure Scheme I, 20 mg of DCC, and 12.5 mg of NHS in 0.5 mL of DMF was stirring for 24 h at room temperature to obtain solution C. The solution D was prepared by dissolving 50 mg OVA in 3.5 mL of PBS (0.01 mol L −1 , pH 7.2). The solution C was added dropwise into solution D and the mixture obtained was stirred at room temperature for 3 h to remove small amounts of impurities. The precipitation was removed by centrifugation at 13 000 × g for 30 min and the supernatant was collected to obtain the coating of cationic OVA (cOVA)–chlorpromazine (compound VII), which was stored at −20 ◦ C for future use. Immunisation, cell fusion and purification of MAbs BALB/c mice were initially immunised by an intraperitoneal injection of 150 µg of cBSA–chlorpromazine in an equal volume of cFA. Two weeks later, a booster injection was performed using the same amount of cBSA–chlorpromazine in iFA. More double boosts were continued with 100 mg cBSA–chlorpromazine given in the tail vein at an interval of 2 weeks. After the immune response had been validated, the splenocytes from immunised mice were fused with a logarithmically growing hypoxanthine–aminopterin–thymidine (HAT)-sensitive mouse myeloma cells Sp2/0 (7 : 1) by the polyethylene glycol (PEG) method. The hybridoma cells were screened by indirect ELISA and cloned by the limited dilution method. The hybridoma cells were cultured in the medium (pH 7.4) containing 0.2% NaHCO3 and RPMI1640 added 20% newborn calf serum in 37 ◦ C to obtain MAbs for chlorpromazine. The purification was performed according to the acid–ammonium sulfate method, and the purified MAbs obtained was stored at −20 ◦ C for future use. Antibody titre determination by indirect competitive ELISA The antibody titre was tested by indirect ELISA. The procedure was carried out as described below. The microplates were coated with coating antigen cOVA–chlorpromazine at 1/500, 1/1000 and 1/2000 by overnight incubation at 4 ◦ C. Plates were washed with wash buffer three times and blocked with 250 µLwell −1 of blocking buffer, followed by incubation for 1 h at room temperature. Plates were washed three times again, then the appropriate dilution of antisera was added, and the plates were incubated for 2 h at room temperature. After the plates had been washed three times, goat anti-mouse IgG-HRP (1 : 1000, 100 µL well −1 ) was added, followed by incubation for 2 h at room temperature. Plates were washed three times and TMB substrate solution A and B was added (50 µL well −1 ) in turn. After that, the plates were incubated for another 15 min at room temperature. The colour development was inhibited by adding stopping solution (100 µL well −1 ), and absorbances were measured at 450 nm. Absorbance values were corrected by blank reading. Pre-immune withdrawal serum (the serum before immunisation) was used as a negative control, and the antibody titre was defined as the reciprocal of the dilution that resulted in an absorbance value of twice the blank value. Development of indirect competitive ELISA The checker-board procedure was used to obtain the optimised coating antigen and the primary antibody concentrations. To each well of a 96-well plate, 100 µL of10µg mL −1 of cOVA–chlorpromazine in bicarbonate buffer (0.05 mol L −1 , pH 9.6) was added, and the mixture was incubated overnight at 4 ◦ C. The plate was washed with wash buffer three times between each step, and blocked with 250 µL well −1 of blocking buffer, followed by incubation for 1 h at room temperature. After the blocking solution was removed, 100 µL of primary antibody was added to each well followed by the addition of PBST buffer or competitor in PBST buffer, and the plate was incubated for 2 h. Then, the goat anti-mouse IgG HRP (1 : 1000, 100 µL well −1 ) was added, followed by incubation for 2 h at room temperature. Substrate solutions A and B were added (50 µL well −1 ) in turn, and the plate was incubated for another 15 min at room temperature. The colour development was inhibited by adding stopping solution (2 mol L −1 H2SO4, 100 µL well −1 ), and absorbance was measured at 450 nm. Absorbance was corrected by blank reading. Pre-immune withdrawal serum was used as a negative control. The result was expressed in % inhibition as follows: % inhibition = %B/B0, where B is the absorbance of the well with competitor and B0 is the absorbance of the well without competitor. Standard curve generation The cOVA–chlorpromazine (1/1000) was used as coating antigen, and indirect cELISA was performed as described above. The J Sci Food Agric 2010; 90: 1789–1795 c○ 2010 Society of Chemical Industry www.interscience.wiley.com/jsfa 1791
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