Food Chemistry 166 (2015) 372–379
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
An enzyme-linked chemiluminescent immunoassay developed for detection of Butocarboxim from agricultural products based on monoclonal antibody Qingkui Fang a,1, Limin Wang a,1, Xiude Hua a,1, Yulong Wang a, Suyan Wang a, Qi Cheng a, Jia Cai a, Fengquan Liu a,b,⇑ a b
Key Laboratory of Integrated Management of Crop Diseases and Pests (Ministry of Education), College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, PR China Institute of Plant Protection, Jiangsu Academy of Agricultural Science, Nanjing 210014, PR China
a r t i c l e
i n f o
Article history: Received 6 September 2013 Received in revised form 6 May 2014 Accepted 10 June 2014 Available online 18 June 2014 Keywords: Butocarboxim Oxime moiety Monoclonal antibody Enzyme-linked chemiluminescent immunoassay
a b s t r a c t In this study, four different haptens around the oxime moiety of Butocarboxim were designed and synthesised. Two of the haptens were conjugated with bovine serum albumin (BSA) to serve as the immunogen and all the haptens were conjugated with ovalbumin (OVA) for the coating antigen. The anti-Butocarboxim monoclonal antibody (Mab) was selected based on eight immunogen/coating antigen combinations. The first enzyme-linked chemiluminescent immunoassay (ELCIA) for determining Butocarboxim in agricultural products was developed. Under the optimised conditions, the detection limit for the ELCIA was 20 ngmL1 and the linear range was 27–2700 ngmL1. Analyte recoveries for extracts of spiked agricultural (apple and greengrocery) products and tap water ranged from 97.18% to 107.00%. The developed immunoassay has great potential to be developed as a test kit offering a simple and cost-effective approach (such as lateral flow test strip) for screening purposes and evaluating environmental exposure to Butocarboxim. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Environmental contamination by persistent pesticides is a major health concern because of human consumption of agricultural products containing residues of the chemicals. There is thus an ongoing need to develop rapid and reliable method for determination of pesticide residues in such food samples. Carbamates pesticides, such as Butocarboxim, have been increased progressively used as pest control in recent years, as has the use of organophosphorus (OP) pesticides, as alternatives to organochlorine (OC) pesticides (Bogialli et al., 2004). Owing to its broad spectrum of biological activity, Butocarboxim can be used as an insecticide and miticide (Hassal, 1982). The residues in agricultural products are a serious health concern because of the compound’s high toxicity to human and animals (LD50 values in the rat of 153 mgkg1). The toxic effect of the Butocarboxim relates to the ability to inhibit the enzyme activity of acetylcholinesterase (AChE), and several adverse effects have been reported (Li,
⇑ Corresponding author. 1
E-mail address: [email protected] (F. Liu). These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.foodchem.2014.06.060 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
Hammock, & Seiber, 1991). Thus, it is highly desirable to devise a sensitive and rapid method for determination of Butocarboxim in agricultural products to ensure health and safety and protect the environment. There have been several analytical methods designed and developed to detect the Butocarboxim, such as ultra-performance liquid chromatography (Leandro, Hancock, Fussell, & Keely, 2007) liquid chromatography–mass spectrometry (Jansson, Pihlström, Österdahl, & Markides, 2004), the calibration curves for the two techniques exhibiting linearity over the concentration ranges 5–250 and 10–200 ngmL1, respectively. Such approaches, however, require expensive equipment and involve complex and time-consuming sample treatments followed by pre-concentration steps performed by skilled professionals (Liu, Jin, & Wang, 2012; Wang, Du, Lu, Lin, & Smith, 2011). Furthermore, the large amounts of organic solvents used in sample preparation are not desirable from an environmental disposal standpoint (Wang, Lu, Wang, Du, & Zou, 2011). To address such environmental and analytical challenges, the enzyme-linked chemiluminescent immunoassay has received attention as an attractive and cost-effective tool for measurement of Butocarboxim. The key step in the development of a chemiluminescent immunoassay for small molecules detection is hapten design for the
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production of the monoclonal antibody (Mab) (Li, Zhang, & Zhang, 2009a; Zhang, Sun et al., 2008). However, to the best of our knowledge, antibodies have not been produced because of the low immunogenicity of the small molecule, Butocarboxim. Therefore, we designed and synthesised four haptens of Butocarboxim around the oxime moiety to prepare the anti-Butocarboxim antibody. Two of these haptens were conjugated with BSA to serve as the immunogen while all four haptens were conjugated with ovalbumin (OVA) to serve as the coating antigen. The best antigen/coating antigen group would be selected based on results from the eight combinations. Consequently, we were able to prepare the first anti-Butocarboxim Mab was developed an enzyme-linked chemiluminescent immunoassay. Based on the production of the monoclonal antibody, the goal of this study was to develop a competitive ELCIA (Co-ELCIA) for the detection of Butocarboxim in agricultural products. Such an approach could provide a basis for developing kits, which would offer a simple and cost-effective route (such as lateral flow test strip) for screening purposes and evaluating exposure to Butocarboxim in the environment.
2. Materials and methods 2.1. Reagents and equipment Butocarboxim, Methomyl, Thiofanox, Butoxycarboxim, and Thiodicarb were purchased from Dr. Ehrenstorfer (Augsburg, Germany). Chemical reagents for hapten synthesis were supplied by Jiangsu Pesticide Research Institute (Nanjing, China). Analytical grade solvents were from Sinopharm Group Chemical Reagent Co., Ltd. (Shanghai, China). Dulbecco’s modified eagle’s medium (DMEM), HAT medium (Hypoxantin, Aminopterin, Thymidin), and HT medium (Hypoxantin, Aminopterin) were purchased from Gibco (USA). Tween 20, N-hydroxysuccinimide (NHS), N,N-dicyclohexylcarbodiimide (DCC), bovine serum albumin (BSA), ovalbumin (OVA), complete or incomplete Freund’s adjuvant and luminol were all purchased from Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from Hangzhou ‘Sijiqing’ Company (Hangzhou, China). Horseradish peroxidase (HRP)-labeled goat anti-mouse IgG was purchased from Boster Biological Technology Co., Ltd. (Wuhan, China). The mouse Mab isotyping kit was purchased from Roche Co. (Mannheim, Germany). SP2/0 cells were stored in the plant quarantine and applied immunology laboratory of Nanjing Agricultural University (Nanjing, China), and BALB/c mice were purchased from the Center of Comparative Medicine of Yangzhou University (Yangzhou, China). All animals used in this study, and animal experiments, were approved by the Department of Science and Technology of Jiangsu Province. The license number was SYXK (SU) 2010-0005. White polystyrene 96-well microtiter plates were from Costar (Corning, Tewksbury, MA, USA). Polystyrene 96-well microtiter plates were washed with ELX405TM (BioTek, Winooski, VT, USA). Chemiluminescent intensity was measured with a SpectraMax M5 instrument (Molecular Devices, Sunnyvale, CA, USA). High performance liquid chromatography (HPLC) was performed using an Agilent Technologies 1200 series instrument (CA, USA). Centrifugation was performed with an Allegra x-12 R centrifuge (Beckman Coulter, USA). The protein concentration of the immunogen and coating antigen was determined by a Nanodrop 1000 UV-VIA (Thermo, USA). The buffers and solutions used include carbonate-buffered saline (CBS, 0.05 molL1, 15 mmolL1 Na2CO3, 35 mmolL1 NaHCO3, pH 9.6), a phosphate-buffered saline (PBS, 0.15 molL1, 1.47 mmolL1 KH2PO4, 8.09 mmolL1 Na2HPO412H2O, 136.9 mmolL1 NaCl, 2.68 mmolL1 KCl, pH 7.4), a PBS containing 0.05% (v/v) Tween-20 (PBST), a borate buffer (0.2 M, 40 mmolL1
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Na2B4O7H2O, 40 mmolL1 H2BO3, pH 9.0), and a chemiluminescent substrate solution(1.0 mmolL1 luminol, 0.025 mmolL1 p-iodophenol, 1.7 mmolL1 H2O2 in 0.1 molL1 Tris–HCl buffer, pH 8.6). In this study, B and B0 are the absorbances of the analyte at the standard point and at zero concentration respectively. 2.2. Synthesis of haptens The first and important step in development of an immunoassay for small molecules is hapten design and synthesis. Four different haptens were designed and synthesised based on the oxime moiety of Butocarboxim. The synthetic routes for these haptens are illustrated in Fig. 1. 2.3. O-(4-oxobutanoic acid)-acetoxime (H1) Under a nitrogen atmosphere, 2.15 mL of trithylamine was dropped into a mixture of acetone oxime (1 g) and succinic anhydride (1.88 g), which were dissolved in tetrahydrofuran (20 mL). The mixture was heated under reflux for 24 h. The aqueous phase was acidified with 3 M HCl, extracted with ethyl acetate (10 mL 3 times) and the products (2.6 g) were recovered. The products were dissolved in a mixture of ethyl acetate:petroleum ether (1:1, v/v), filtered and then recrystallised, resulting in a product (1.74 g), yield of 66.9%. 2.4. O-(N-butyric acid carbamoyl)-acetoxime (H2) Solution I: consisted of 2.95 g of triphosgene and 8 mL of pyridine dissolved in CH2Cl2 (20 mL). Solution II: consisted of 3.3 mmol of acetone oxime dissolved in CH2Cl2 (20 mL). Solution II was slowly added into (dropwise) solution I at 15 °C. Then the mixture was raised to room temperature. Five hours later, the products (1, acyl chloride) were filtered and washed three times with cold water, and then spin dried with anhydrous sodium sulfate. The acyl chloride (0.45 g, 3.3 mmol) was dissolved in 4 mL of chilled NaOH solution (4 molL1), forming solution A. c-Aminobutyric acid (0.63 g, 6.1 mmol) was dissolved in 4 mL of NaOH solution (4 molL1), and the solution was cooled at 4 °C to obtain solution B. Solution C was 3 mL of NaOH (4 molL1), which was cooled at 4 °C. Both solutions A and C were divided into five equal portions. All portions of solutions A and C were added to solution B with an interval of at least 5–10 min between each addition. The reaction mixture was stirred in an ice-water bath for 2 h. After acidification to pH 4 with concentrated HCl, the carboxylic acid derivative was extracted with ethyl acetate (50 mL 3 times). The ethyl acetate phase was washed several times with diluted HCl and extracted with 1 M bicarbonate solution (50 mL 2 times). The aqueous phase was acidified again with concentrated HCl, extracted with ethyl acetate, and dried over anhydrous sodium sulfate, and the solvent was evaporated, yielding a white solid, H2, as product (0.4 g, yield: 60%). 2.5. O-(4-oxobutanoic acid)-3-methylthio butanone oxime (H3) 3-methylthio-2-butanone (0.2 g, 1.69 mmol) was dissolved into 2 mL pyridine, and hydroxylamine hydrochloride (0.36 g, 5.13 mmol) was added to the solution. The reaction was stopped after stirring at room temperature for 24 h. The pH of the solution was adjusted with 3 molL1 HCl in an ice-water bath, and the solution was then extracted with ethyl acetate (10 mL 3 times), dried over anhydrous sodium sulfate, filtered and spin dried. A colorless oily mixture (2, 0.2 g, 88.9%) was obtained. The product of 2 (0.2 g, 1.50 mmol) and succinic anhydride (0.15 g, 1.50 mmol) were dissolved in tetrahydrofuran (5 mL),
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ൕ R1, R2, R3 is atom or groups
ൖ
H1
ൗ
1
H2
൘
H3
2
൙
3
5
4
H4
൚
Fig. 1. I: The oxim group; II: Synthetic routes for H1 [O-(4-oxobutanoic acid)-acetoxime]; III: Synthetic routes for H2 [O-(N-butyric acid carbamoyl)-acetoxime]; IV: Synthetic routes for H3 [O-(4-oxobutanoic acid)-3-methylthio butanone oxime]; V: Synthetic routes for H4 [O-(N-ethyl carbamoyl)-3-(2-carboxyethyl) mercapto butanone oxime]; VI: The active ester method.
containing 1 mL of triethylamine. The solution was stirred for 24 h at room temperature. The solvent was spin dried after the reaction had stopped. Then ethyl acetate was added and the solution pH value was adjusted to 3–4 with 3 M HCl. The solution was next extracted with ethyl acetate (10 mL 3 times), dried with
anhydrous sodium sulfate, filtered and then the mixture was concentrated. Separation of the products was done by silica-gel flash chromatography, using a mixture of ethyl acetate:petroleum ether (1:2, v/v), and a yellowish oily mixture, H3 (0.2 g, 57.3%), was obtained.
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2.6. O-(N-ethyl carbamoyl)-3-(2-carboxyethyl) mercapto butanone oxime (H4) A mixture of 3-Cl-2-buranone (1.06 g, 10 mmol) and 3-mercaptopropionate acid methyl ester (1.22 g, 10 mmol) was dissolved in CH2Cl2 (11 mL), and was added with triethylamine (1 mL) was then added. The mixture was stirred in an ice-water bath for 30 min, the bath was then removed and stirring continued overnight at room temperature, during which time insoluble material formed. On completion of the reaction, water (2.5 mL) was added to the mixture and the insolubles material underwent dissolution. The organic layer was extracted with water (10 mL) and the organic phase was then dried with anhydrous sodium sulfate, filtered and the mixture concentrated. Separation of the products was achieved by silica-gel flash chromatography, using a mixture of ethyl acetate:petroleum ether (1:4, v/v) and thus yielded a colorless oily mixture 3 (0.68 g, 35.8%). Product 3 (0.68 g, 3.58 mmol) was dissolved in pyridine (3.5 mL), which was then added to hydroxylamine hydrochloride (0.74 g, 10.7 mmol). After stirring at room temperature for 24 h to complete the reaction, the mixture was placed in an ice-water bath and the solution pH was adjusted with 3 molL1 HCl. The solution was then extracted with ethyl acetate (10 mL 3 times), dried over anhydrous sodium sulfate, filtered and spin dried. A colorless oily mixture 4 (0.57 g, 78%) resulted. Product 4 (0.57 g, 2.78 mmol) and ethyl isocyanate (0.21 g, 3.02 mmol) were dissolved in ether (5 mL). This mixture was added to trithamine (3 mL), which was then stirred at room temperature for 12 h. On completion of the reaction, the solution was extracted with a mixture of water:ether (20 mL:10 mL), and then the aqueous layer was washed with ethyl acetate (20 mL 2 times), while the organic layer was dried with anhydrous sodium sulfate, filtered and the mixture then concentrated. Separation of these products was performed by silica-gel flash chromatography using a mixture of ethyl acetate:petroleum ether (1:5, v/v) and a colorless oily mixture 5 (0.51 g, 67.1%) was obtained. The mixture 5 (0.51 g,1.84 mmol) was dissolved in a solution of Na2CO3 (1.43 g, 36 mL water), which was then added to methanol, and after stirring at 75 °C for 1 h, dissolution resulted. The mixture was allowed to cool to room temperature and the organic phase was spin dried. The aqueous layer was acidified with 3 M HCl and adjusted to pH 3–4. The solution was then extracted with ethyl acetate (20 mL 3 times), dried with anhydrous sodium sulfate, filtered and the mixture concentrated. The separation of products was performed by silica-gel flash chromatography using ethyl acetate:petroleum ether (1:2, v/v), and a yellowish oily mixture H4 (0.28 g, 58.1%) was obtained. 2.7. Preparation of immunogens and coating antigens To generate immunogens, haptens 3 and 4 were covalently attached through their carboxylic acid moieties to the lysine groups of BSA using the active ester method (Zhang et al., 2007; Zhang, Wu et al., 2008; Liang, Liu, Liu, Yu, & Fan, 2008; Ju, Tang, Fan, & Chen, 2008; Lee, Kim, Park, Chung, & Lee, 2005). Briefly, Haptens were activated as a result of treatment with a 50% molar excess of NHS and DCC for 6 h at room temperature. Next, the mixture was centrifuged and the supernatant collected. To this solution, 15 mgmL1 BSA in borate buffer was added and the mixture was stirred at room temperature for 4 h. Using the same method, haptens 1–4 were coupled to OVA to obtain coating antigens. The immunogens and coating antigens were purified by dialysis in phosphate buffer saline (PBS, 0.15 M).
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2.8. Immunisation BALB/C female mice of about 7 weeks old were immunised with the BSA-hapten 3 and BSA-hapten 4 conjugates. Five mice were treated for each kind of immunogen. The first dose consisted of 100 lg of conjugate intraperitoneally injected as an emulsion of PBS and Freund’s complete adjuvant. The subsequent injections, emulsified in Freund’s incomplete adjuvant were given at 3-week intervals. One week after the last injection, the anti-Butocarboxim antiserums were obtained from the tail vein of each mouse. Ten sera were obtained: 3-1, 3-2, 3-3, 3-4, 3-5 for BSA-hapten 3 and 4-1, 4-2, 4-3, 4-4, 4-5 for BSA-hapten 4. The sera were tested for antibody titers and for analyte recognition by indirect competitive ELISA. The process of indirect competitive ELISA was performed as described previously (Qian et al., 2007). 2.9. Production of monoclonal antibody The mouse showing the highest serum reactivity was given a peritoneal cavity injection of 100 lg immunogen in PBS at 1 week intervals. Three to four days after the last injection, the donor mouse was sacrificed. SP2/0 murine myeloma cells were cultured in DMEM supplemented with 20% FBS. Splenocytes of selected mice were harvested aseptically. Cell fusion and hybridoma selection procedures were performed essentially as described previously (Li, Zhang, Zhang, Zhang, & Chen, 2009b; Liu et al., 2009; Wang, Zhang, Chen, Liu, & Li, 2011; Wang et al., 2009; Zeng et al., 2007; Zhang et al., 2007). Briefly, the splenocytes were added to myeloma cells to give a ratio of five splenocytes per myeloma cell and the mixture was centrifuged. One mL of PEG 1500 at 37 °C was dropped on a cell pellet over 1 min and left to stand for 1 min, then 30 mL of DMEM was added over 4 min and the pellet was left standing for a further 10 min. The fused cells were then spun down and resuspended in HAT selection medium before they were distributed in a dose of 100 lL per well in a 96-well culture plates which was previously coated with feeding cells. The HAT selection medium consisted of DMEM, 20% FBS, 10 mmolL1 hypoxanthine, 0.4 mmolL1 aminopterin and 1.6 mmolL1 thymidine. Half of the media in the wells were replaced by fresh HAT media every 4th day. The HAT media were changed to HT media with no aminopterin, when most of the non-fused cells were eliminated. After the hybridoma cells had grown to 30–40% confluence in the well, the culture supernatants were initially screened by indirect ELISA for the ability to bind to H2-OVA, culture supernatant from unfused SP2/0 cells serving as the negative control. Positive hybridoma cells were subcloned by limiting dilution, and stable antibody-producing clones were expanded and cryopreserved in liquid nitrogen. Competitive indirect ELISA using H2-OVA as a coating antigen was then employed to determine if the antibodies from the finally expanded clones could recognise the pesticides. The selected clones were cryopreserved in liquid nitrogen. 2.10. Development of a competitive enzyme-linked chemiluminescent immunoassay (Co-ELCIA) The basic steps in the Co-ELCIA process are as follows. Microtiter plates were coated with optimised concentrations of hapten-OVA in carbonate–bicarbonate buffer (50 mM, pH 9.6) by incubating for overnight at 4 °C. Plates were then blocked by incubating with 1% gelatin in PBS (120 lL per well) for 1.5 h. Aliquots (25 lL per well) of analyte dissolved in working solution and aliquots of Mab (25 lL per well), at a previously determined concentration, were added to the blocked plate, the Mab having been diluted with
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working solution. After incubation for 1 h, 50 lL per well of diluted (1/20,000) goat anti-mouse IgG-HRP was added to the plate. The mixture was incubated for 1 h. Finally, 150 lL of chemiluminescent substrate solution was pipetted into each well. The reading was done after 3 min of incubation at room temperature in the dark, and the emitted photons were measured at 425 nm (endpoint method). All incubations were performed at 37 °C and the plates were washed five times with PBST (0.15 M PBS containing 0.05% Tween 20, pH 7.4) after each incubation, unless specified otherwise. The Microtitre plates are working as below: via the use of radiation, the resulting polystyrene surface is primarily hydrophobic with intermittent carboxyl groups capable of ionic interactions with positively charged groups on biomolecules. The mechanism of immobilisation is passive adsorption through hydrophobic and ionic interactions. This is considered a general purpose surface capable of binding medium (>10 kD) and large biomolecules that possess ionic groups and/or hydrophobic regions (http:// www.corning.com/lifesciences/us_canada/en/technical_resources/ surfaces/assay/high_binding_polystyrene.aspx). 2.11. Optimisation of Co-ELCIA Selection of the coating antigen/antibody combination was performed by Co-ELCIA. Four coating antigens (H1, H2, H3, H4-OVA) that were able to conjugate with the MAbs 1C9 were screened out by Co-ELCIA. Then the optimum combination of coating antigen and antibody was confirmed based on the sensitivity of the Co-ELCIA. The dilution ratios of antibody (1:6000–1:24,000) and coating antigen (1:6000–1:14,000) were confirmed based on the sensitivity of the Co-ELCIA. The working solutions, prepared at a series of pH values (6.0– 8.5) and ionic strengths (0.1–3.2 M), were used to dilute the Butocarboxim standards and were tested using the established ELCIA. The tested working solution that resulted in the best sensitivity for the ELCIA was selected as the optimised working solution. 3. Evaluation of the optimised Co-ELCIA 3.1. Cross-reactivity studies To evaluate the selectivity of the Co-ELCIA, four carbamate oxime esters pesticides (Butoxycarboxim, Thiofanox, Thiodicarb and Methomyl) were evaluated using the established ELCIA. The cross-reactivity (CR) was calculated as follows: CR ¼ ½IC50 ðButocarboximÞ=IC50 ðcompoundÞ 100%. Here, the CR of Butocarboxim was defined as 100%. 3.2. Accuracy Two different agricultural samples (apple and greengrocery) and tap water were chosen to evaluate the performance of the ELCIA. The apples and greengrocery were bought from the local market. All samples were verified by HPLC not to contain Butocarboxim before undertaking spiking and recovery studies. Apples and greengrocery were cut into pieces and homogenised. 5.0 g of each sample were weighed and placed in a 50 mL centrifuge tube, spiked with known concentrations of Butocarboxim standard solution dissolved in methanol. The samples were thoroughly mixed and allowed to stand at room temperature for 1 h. 10 mL of methanol was added to each sample and the samples were shaken thoroughly on the rotary shaker for 10 min, and then centrifuged at 4000g (Allegra x-12R centrifuge, Beckman coulter, USA) for 10 min. The supernatants were then transferred to a 25 mL volumetric flask, following 10 mL of methanol was added
again, thoroughly shaken for 10 min, and then centrifuged at 4000g for 10 min. The supernatant was transferred to the volumetric flask. And set the volume to 25 mL (sample extract) with optimal working buffer (without organic solvent). The sample extracts were diluted with optimal working buffer (without organic solvent) and then mixed with antibody (diluted in PBST) as 1:1 (v/v) for ELCIA analysis. Also the tap water was spiked with known concentrations (2.5, 5.0, and 10 lgmL1) of Butocarboxim in methanol. HPLC analysis of Butocarboxim was conducted at 210 nm (UV), and the mobile phase was acetonitrile:water (28:72) at a flow rate of 1.4 mLmin1. XDB-C18 column (4.6 250 mm). The temperature of the column oven was maintained at room temperature. Retention time for Butocarboxim was 6.8 ± 0.2 min.
4. Results and discussion 4.1. Verification of haptens The haptens H1–H4 were synthesised and structures were clarified by 1H NMR. The results of 1H NMR were described as follows: H1: 1H NMR (300 MH, CDCl3) d: 2.97 (s, 3H), 3.11 (s, 3H), 4.00 (s, 4H) H2: 1H NMR (300 MHz, DMSO) d: 2.22(2H, t, J = 7.2 Hz), 1.67 (m, 2H), 3.07 (m, 2H), 1.98 (s, 3H), 1.97 (s, 3H) H3: 1H NMR (300 MHz, CDCl3) d: 3.60 (m, 1H), 2.75 (s, 4H), 2.02 (s, 3H), 2.00 (s, 3H), 1.38 (d, 3H, J = 7.2 Hz) H4: 1H NMR (300 MHz, CDCl3) d: 3.63 (m, 1H), (m, 2H), 2.66 (m, 4H), 2.06 (s, 3H), 1.38 (d, 3H, J = 7.2 Hz), 1.20 (t, 3H, J = 7.2 Hz) 4.2. Screening of the antiserums There were 10 antiserums (5 from H3-BSA (3-1 to 3-5) and 5 from H4-BSA (4-1 to 4-5) were investigated. The homologous noncompetitive ELISA (with the homologous coating antigen) indicated that antiserums 3-4 and 4-2 had the highest titer. Then, the competitive ELISA was operated to select the best antiserum/ coating antigen combination through the IC50 values. The Table 1 shows that the 4-2/H2-OVA combination gave the best IC50. Therefore, the mouse 4-2 was selected for subsequent hybridoma production, and the H2-OVA was used as the coating antigen. 4.3. Production and characterisation of monoclonal antibody After selection of the antiserum, the mouse produced sera 4-2 was selected for subsequent hybridoma production because of its highest relative reactivity to Butocarboxim. Hybridoma clones were initially screened by indirect Co-ELCIA for their reactivities with H2-OVA, and a total of 20 hybridoma cell lines were obtained. After the 5 screen cycles, the hybridoma cell line 1C9 was found to secrete antibodies of the IgG1, kappa type. The MAb, present in the ascites fluid in mouse, was purified by precipitation with 50%
Table 1 ELISA sensitivity (IC50, IC50 values (lgmL1) of the combination between serum and coating antigen) for different combination of antibodies and coating antigens (n = 3)a. IC50 was higher than 1000 lgmL1. Antiserum
Immunogen
Coating antigen (IC50, lgmL1) H1-OVA
H3-4 H4-2 a
BSA-hapten 3 BSA-hapten 4
N N
a
IC50 was higher than 1000 lgmL1.
H2-OVA
H3-OVA
H4-OVA
10 1
N N
N N
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ammonium sulfate (Reik et al., 1987) and stored at 35 °C for following characterisation studies. 4.4. Optimisation of the Co-ELCIA Experimental parameters, which may affect the analytical performance of the assay, were examined and included the concentrations of coating antigen and antibody, pH, and the ionic strength of the Co-ELCIA system. Fig. 2 shows the results of these experiments. In this study, the highest ratio of CLUmax/IC50 was considered as the main criterion for evaluating the ELCIA assay (Long, Shi, He, Sheng, & Wang, 2009). From the results of Fig. 2, the optimised conditions for the Co-ELCIA were selected as: pH = 8.0, ionic strength was 1.6 M. Both of the dilution ratio of coating antigen and antibody were 1:8000.
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4.5. Measurement of Butocarboxim by Co-ELCIA Under optimised conditions, the analytical performance for the Co-ELCIA for detection of Butocarboxim was examined with different concentrations of standard Butocarboxim in PBST. The results presented in Fig. 3 indicated that the developed Co-ELCIA was suitable for the determination of Butocarboxim. After conversion of Fig. 3a, it was observed that in the range of 27–2700 ngmL1, the graph between B/B0 and logarithm of concentration of butocarboxim (ngmL1) was linear (Fig. 3b), the regression equation was obtained (y = 0.4009x + 1.5076, R2 = 0.9906). The limit detection (LD) was 20 ngmL1 by the extrapolation of B0-2SD extrapolation. The Mean ± SD were described as below: 0.082 ± 0.0030, 0.090 ± 0.0037, 0.13 ± 0.0022, 0.33 ± 0.0051, 0.58 ± 0.013, 0.77 ± 0.011, 0.90 ± 0.015, 0.97 ± 0.0060, 0.99 ± 0.041,
Fig. 2. Effect of pH values, ionic strengths, dilution ratio of coating antigen and dilution ratio of antibody (monoclonal from mouse) on the performance of assay: pH [(a) 6.0 (185,431 ± 5110), 6.5 (201,020 ± 6719), 7.0 (234,909 ± 1061), 7.5 (268,210 ± 15,575), 8.0 (330,885 ± 10,081), 8.5 (250,856 ± 6007)]; ionic strengths [(b) 0.1 (32,522 ± 1890), 0.2 (56,085 ± 2006), 0.4 (59,339 ± 3474), 0.8 (65,318 ± 3306), 1.6 (97,551 ± 5216), 3.2 (44,185 ± 2176) molL1]; dilution ratio of coating antigen [(c), 1:6000 (107,197 ± 4947), 1:8000 (112,373 ± 6417), 1:10,000 (100,346 ± 689), 1:12,000 (90,246 ± 7738), 1:14,000 (78,537 ± 4946)] and dilution ratio of antibody [(d) 1:6000 (112,354 ± 2298), 1:8000 (149,197 ± 5377), 1:12,000 (103,245 ± 5907), 1:16,000 (94,815 ± 1695), 1:20,000 (50,429 ± 6871), 1:24,000 (42,198 ± 975) ]. Results are the means of three repetitions experiments (Mean ± SD, n = 3).
Fig. 3. Indirect competitive enzyme-linked chemiluminescent immunoassay (Co-ELCIA) curve for Butocarboxim. (a) Standard inhibition curve of Butocarboxim; (b) the calibration curve from ‘‘a’’. It was observed from ‘‘a’’ that in the range of 27–2700 ngmL1, the graph between B/B0 and logarithm of concentration of Butocarboxim (ngmL1) was linear, and the regression equation was obtained (y = 0.4009x + 1.5076, R2 = 0.9906). The Mean ± SD of ‘‘a’’ were 0.082 ± 0.0030, 0.090 ± 0.0037, 0.13 ± 0.0022, 0.33 ± 0.0051, 0.58 ± 0.013, 0.77 ± 0.011, 0.90 ± 0.015, 0.97 ± 0.0060, 0.99 ± 0.041, 1.0 ± 0.011, respectively. (n = 3).
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Table 2 Cross-reactivity (CR) of a set of analogs related to Butocarboxim by Co-ELCIA (n = 3). Compound
Chemical structure CH 3
Butocarboxim S
O
O
O
H 3C
16.7
1.95
41.0
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Analytical Methods
An enzyme-linked chemiluminescent immunoassay developed for detection of Butocarboxim from agricultural products based on monoclonal antibody Qingkui Fang a,1, Limin Wang a,1, Xiude Hua a,1, Yulong Wang a, Suyan Wang a, Qi Cheng a, Jia Cai a, Fengquan Liu a,b,⇑ a b
Key Laboratory of Integrated Management of Crop Diseases and Pests (Ministry of Education), College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, PR China Institute of Plant Protection, Jiangsu Academy of Agricultural Science, Nanjing 210014, PR China
a r t i c l e
i n f o
Article history: Received 6 September 2013 Received in revised form 6 May 2014 Accepted 10 June 2014 Available online 18 June 2014 Keywords: Butocarboxim Oxime moiety Monoclonal antibody Enzyme-linked chemiluminescent immunoassay
a b s t r a c t In this study, four different haptens around the oxime moiety of Butocarboxim were designed and synthesised. Two of the haptens were conjugated with bovine serum albumin (BSA) to serve as the immunogen and all the haptens were conjugated with ovalbumin (OVA) for the coating antigen. The anti-Butocarboxim monoclonal antibody (Mab) was selected based on eight immunogen/coating antigen combinations. The first enzyme-linked chemiluminescent immunoassay (ELCIA) for determining Butocarboxim in agricultural products was developed. Under the optimised conditions, the detection limit for the ELCIA was 20 ngmL1 and the linear range was 27–2700 ngmL1. Analyte recoveries for extracts of spiked agricultural (apple and greengrocery) products and tap water ranged from 97.18% to 107.00%. The developed immunoassay has great potential to be developed as a test kit offering a simple and cost-effective approach (such as lateral flow test strip) for screening purposes and evaluating environmental exposure to Butocarboxim. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Environmental contamination by persistent pesticides is a major health concern because of human consumption of agricultural products containing residues of the chemicals. There is thus an ongoing need to develop rapid and reliable method for determination of pesticide residues in such food samples. Carbamates pesticides, such as Butocarboxim, have been increased progressively used as pest control in recent years, as has the use of organophosphorus (OP) pesticides, as alternatives to organochlorine (OC) pesticides (Bogialli et al., 2004). Owing to its broad spectrum of biological activity, Butocarboxim can be used as an insecticide and miticide (Hassal, 1982). The residues in agricultural products are a serious health concern because of the compound’s high toxicity to human and animals (LD50 values in the rat of 153 mgkg1). The toxic effect of the Butocarboxim relates to the ability to inhibit the enzyme activity of acetylcholinesterase (AChE), and several adverse effects have been reported (Li,
⇑ Corresponding author. 1
E-mail address: [email protected] (F. Liu). These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.foodchem.2014.06.060 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
Hammock, & Seiber, 1991). Thus, it is highly desirable to devise a sensitive and rapid method for determination of Butocarboxim in agricultural products to ensure health and safety and protect the environment. There have been several analytical methods designed and developed to detect the Butocarboxim, such as ultra-performance liquid chromatography (Leandro, Hancock, Fussell, & Keely, 2007) liquid chromatography–mass spectrometry (Jansson, Pihlström, Österdahl, & Markides, 2004), the calibration curves for the two techniques exhibiting linearity over the concentration ranges 5–250 and 10–200 ngmL1, respectively. Such approaches, however, require expensive equipment and involve complex and time-consuming sample treatments followed by pre-concentration steps performed by skilled professionals (Liu, Jin, & Wang, 2012; Wang, Du, Lu, Lin, & Smith, 2011). Furthermore, the large amounts of organic solvents used in sample preparation are not desirable from an environmental disposal standpoint (Wang, Lu, Wang, Du, & Zou, 2011). To address such environmental and analytical challenges, the enzyme-linked chemiluminescent immunoassay has received attention as an attractive and cost-effective tool for measurement of Butocarboxim. The key step in the development of a chemiluminescent immunoassay for small molecules detection is hapten design for the
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production of the monoclonal antibody (Mab) (Li, Zhang, & Zhang, 2009a; Zhang, Sun et al., 2008). However, to the best of our knowledge, antibodies have not been produced because of the low immunogenicity of the small molecule, Butocarboxim. Therefore, we designed and synthesised four haptens of Butocarboxim around the oxime moiety to prepare the anti-Butocarboxim antibody. Two of these haptens were conjugated with BSA to serve as the immunogen while all four haptens were conjugated with ovalbumin (OVA) to serve as the coating antigen. The best antigen/coating antigen group would be selected based on results from the eight combinations. Consequently, we were able to prepare the first anti-Butocarboxim Mab was developed an enzyme-linked chemiluminescent immunoassay. Based on the production of the monoclonal antibody, the goal of this study was to develop a competitive ELCIA (Co-ELCIA) for the detection of Butocarboxim in agricultural products. Such an approach could provide a basis for developing kits, which would offer a simple and cost-effective route (such as lateral flow test strip) for screening purposes and evaluating exposure to Butocarboxim in the environment.
2. Materials and methods 2.1. Reagents and equipment Butocarboxim, Methomyl, Thiofanox, Butoxycarboxim, and Thiodicarb were purchased from Dr. Ehrenstorfer (Augsburg, Germany). Chemical reagents for hapten synthesis were supplied by Jiangsu Pesticide Research Institute (Nanjing, China). Analytical grade solvents were from Sinopharm Group Chemical Reagent Co., Ltd. (Shanghai, China). Dulbecco’s modified eagle’s medium (DMEM), HAT medium (Hypoxantin, Aminopterin, Thymidin), and HT medium (Hypoxantin, Aminopterin) were purchased from Gibco (USA). Tween 20, N-hydroxysuccinimide (NHS), N,N-dicyclohexylcarbodiimide (DCC), bovine serum albumin (BSA), ovalbumin (OVA), complete or incomplete Freund’s adjuvant and luminol were all purchased from Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from Hangzhou ‘Sijiqing’ Company (Hangzhou, China). Horseradish peroxidase (HRP)-labeled goat anti-mouse IgG was purchased from Boster Biological Technology Co., Ltd. (Wuhan, China). The mouse Mab isotyping kit was purchased from Roche Co. (Mannheim, Germany). SP2/0 cells were stored in the plant quarantine and applied immunology laboratory of Nanjing Agricultural University (Nanjing, China), and BALB/c mice were purchased from the Center of Comparative Medicine of Yangzhou University (Yangzhou, China). All animals used in this study, and animal experiments, were approved by the Department of Science and Technology of Jiangsu Province. The license number was SYXK (SU) 2010-0005. White polystyrene 96-well microtiter plates were from Costar (Corning, Tewksbury, MA, USA). Polystyrene 96-well microtiter plates were washed with ELX405TM (BioTek, Winooski, VT, USA). Chemiluminescent intensity was measured with a SpectraMax M5 instrument (Molecular Devices, Sunnyvale, CA, USA). High performance liquid chromatography (HPLC) was performed using an Agilent Technologies 1200 series instrument (CA, USA). Centrifugation was performed with an Allegra x-12 R centrifuge (Beckman Coulter, USA). The protein concentration of the immunogen and coating antigen was determined by a Nanodrop 1000 UV-VIA (Thermo, USA). The buffers and solutions used include carbonate-buffered saline (CBS, 0.05 molL1, 15 mmolL1 Na2CO3, 35 mmolL1 NaHCO3, pH 9.6), a phosphate-buffered saline (PBS, 0.15 molL1, 1.47 mmolL1 KH2PO4, 8.09 mmolL1 Na2HPO412H2O, 136.9 mmolL1 NaCl, 2.68 mmolL1 KCl, pH 7.4), a PBS containing 0.05% (v/v) Tween-20 (PBST), a borate buffer (0.2 M, 40 mmolL1
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Na2B4O7H2O, 40 mmolL1 H2BO3, pH 9.0), and a chemiluminescent substrate solution(1.0 mmolL1 luminol, 0.025 mmolL1 p-iodophenol, 1.7 mmolL1 H2O2 in 0.1 molL1 Tris–HCl buffer, pH 8.6). In this study, B and B0 are the absorbances of the analyte at the standard point and at zero concentration respectively. 2.2. Synthesis of haptens The first and important step in development of an immunoassay for small molecules is hapten design and synthesis. Four different haptens were designed and synthesised based on the oxime moiety of Butocarboxim. The synthetic routes for these haptens are illustrated in Fig. 1. 2.3. O-(4-oxobutanoic acid)-acetoxime (H1) Under a nitrogen atmosphere, 2.15 mL of trithylamine was dropped into a mixture of acetone oxime (1 g) and succinic anhydride (1.88 g), which were dissolved in tetrahydrofuran (20 mL). The mixture was heated under reflux for 24 h. The aqueous phase was acidified with 3 M HCl, extracted with ethyl acetate (10 mL 3 times) and the products (2.6 g) were recovered. The products were dissolved in a mixture of ethyl acetate:petroleum ether (1:1, v/v), filtered and then recrystallised, resulting in a product (1.74 g), yield of 66.9%. 2.4. O-(N-butyric acid carbamoyl)-acetoxime (H2) Solution I: consisted of 2.95 g of triphosgene and 8 mL of pyridine dissolved in CH2Cl2 (20 mL). Solution II: consisted of 3.3 mmol of acetone oxime dissolved in CH2Cl2 (20 mL). Solution II was slowly added into (dropwise) solution I at 15 °C. Then the mixture was raised to room temperature. Five hours later, the products (1, acyl chloride) were filtered and washed three times with cold water, and then spin dried with anhydrous sodium sulfate. The acyl chloride (0.45 g, 3.3 mmol) was dissolved in 4 mL of chilled NaOH solution (4 molL1), forming solution A. c-Aminobutyric acid (0.63 g, 6.1 mmol) was dissolved in 4 mL of NaOH solution (4 molL1), and the solution was cooled at 4 °C to obtain solution B. Solution C was 3 mL of NaOH (4 molL1), which was cooled at 4 °C. Both solutions A and C were divided into five equal portions. All portions of solutions A and C were added to solution B with an interval of at least 5–10 min between each addition. The reaction mixture was stirred in an ice-water bath for 2 h. After acidification to pH 4 with concentrated HCl, the carboxylic acid derivative was extracted with ethyl acetate (50 mL 3 times). The ethyl acetate phase was washed several times with diluted HCl and extracted with 1 M bicarbonate solution (50 mL 2 times). The aqueous phase was acidified again with concentrated HCl, extracted with ethyl acetate, and dried over anhydrous sodium sulfate, and the solvent was evaporated, yielding a white solid, H2, as product (0.4 g, yield: 60%). 2.5. O-(4-oxobutanoic acid)-3-methylthio butanone oxime (H3) 3-methylthio-2-butanone (0.2 g, 1.69 mmol) was dissolved into 2 mL pyridine, and hydroxylamine hydrochloride (0.36 g, 5.13 mmol) was added to the solution. The reaction was stopped after stirring at room temperature for 24 h. The pH of the solution was adjusted with 3 molL1 HCl in an ice-water bath, and the solution was then extracted with ethyl acetate (10 mL 3 times), dried over anhydrous sodium sulfate, filtered and spin dried. A colorless oily mixture (2, 0.2 g, 88.9%) was obtained. The product of 2 (0.2 g, 1.50 mmol) and succinic anhydride (0.15 g, 1.50 mmol) were dissolved in tetrahydrofuran (5 mL),
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ൕ R1, R2, R3 is atom or groups
ൖ
H1
ൗ
1
H2
൘
H3
2
൙
3
5
4
H4
൚
Fig. 1. I: The oxim group; II: Synthetic routes for H1 [O-(4-oxobutanoic acid)-acetoxime]; III: Synthetic routes for H2 [O-(N-butyric acid carbamoyl)-acetoxime]; IV: Synthetic routes for H3 [O-(4-oxobutanoic acid)-3-methylthio butanone oxime]; V: Synthetic routes for H4 [O-(N-ethyl carbamoyl)-3-(2-carboxyethyl) mercapto butanone oxime]; VI: The active ester method.
containing 1 mL of triethylamine. The solution was stirred for 24 h at room temperature. The solvent was spin dried after the reaction had stopped. Then ethyl acetate was added and the solution pH value was adjusted to 3–4 with 3 M HCl. The solution was next extracted with ethyl acetate (10 mL 3 times), dried with
anhydrous sodium sulfate, filtered and then the mixture was concentrated. Separation of the products was done by silica-gel flash chromatography, using a mixture of ethyl acetate:petroleum ether (1:2, v/v), and a yellowish oily mixture, H3 (0.2 g, 57.3%), was obtained.
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2.6. O-(N-ethyl carbamoyl)-3-(2-carboxyethyl) mercapto butanone oxime (H4) A mixture of 3-Cl-2-buranone (1.06 g, 10 mmol) and 3-mercaptopropionate acid methyl ester (1.22 g, 10 mmol) was dissolved in CH2Cl2 (11 mL), and was added with triethylamine (1 mL) was then added. The mixture was stirred in an ice-water bath for 30 min, the bath was then removed and stirring continued overnight at room temperature, during which time insoluble material formed. On completion of the reaction, water (2.5 mL) was added to the mixture and the insolubles material underwent dissolution. The organic layer was extracted with water (10 mL) and the organic phase was then dried with anhydrous sodium sulfate, filtered and the mixture concentrated. Separation of the products was achieved by silica-gel flash chromatography, using a mixture of ethyl acetate:petroleum ether (1:4, v/v) and thus yielded a colorless oily mixture 3 (0.68 g, 35.8%). Product 3 (0.68 g, 3.58 mmol) was dissolved in pyridine (3.5 mL), which was then added to hydroxylamine hydrochloride (0.74 g, 10.7 mmol). After stirring at room temperature for 24 h to complete the reaction, the mixture was placed in an ice-water bath and the solution pH was adjusted with 3 molL1 HCl. The solution was then extracted with ethyl acetate (10 mL 3 times), dried over anhydrous sodium sulfate, filtered and spin dried. A colorless oily mixture 4 (0.57 g, 78%) resulted. Product 4 (0.57 g, 2.78 mmol) and ethyl isocyanate (0.21 g, 3.02 mmol) were dissolved in ether (5 mL). This mixture was added to trithamine (3 mL), which was then stirred at room temperature for 12 h. On completion of the reaction, the solution was extracted with a mixture of water:ether (20 mL:10 mL), and then the aqueous layer was washed with ethyl acetate (20 mL 2 times), while the organic layer was dried with anhydrous sodium sulfate, filtered and the mixture then concentrated. Separation of these products was performed by silica-gel flash chromatography using a mixture of ethyl acetate:petroleum ether (1:5, v/v) and a colorless oily mixture 5 (0.51 g, 67.1%) was obtained. The mixture 5 (0.51 g,1.84 mmol) was dissolved in a solution of Na2CO3 (1.43 g, 36 mL water), which was then added to methanol, and after stirring at 75 °C for 1 h, dissolution resulted. The mixture was allowed to cool to room temperature and the organic phase was spin dried. The aqueous layer was acidified with 3 M HCl and adjusted to pH 3–4. The solution was then extracted with ethyl acetate (20 mL 3 times), dried with anhydrous sodium sulfate, filtered and the mixture concentrated. The separation of products was performed by silica-gel flash chromatography using ethyl acetate:petroleum ether (1:2, v/v), and a yellowish oily mixture H4 (0.28 g, 58.1%) was obtained. 2.7. Preparation of immunogens and coating antigens To generate immunogens, haptens 3 and 4 were covalently attached through their carboxylic acid moieties to the lysine groups of BSA using the active ester method (Zhang et al., 2007; Zhang, Wu et al., 2008; Liang, Liu, Liu, Yu, & Fan, 2008; Ju, Tang, Fan, & Chen, 2008; Lee, Kim, Park, Chung, & Lee, 2005). Briefly, Haptens were activated as a result of treatment with a 50% molar excess of NHS and DCC for 6 h at room temperature. Next, the mixture was centrifuged and the supernatant collected. To this solution, 15 mgmL1 BSA in borate buffer was added and the mixture was stirred at room temperature for 4 h. Using the same method, haptens 1–4 were coupled to OVA to obtain coating antigens. The immunogens and coating antigens were purified by dialysis in phosphate buffer saline (PBS, 0.15 M).
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2.8. Immunisation BALB/C female mice of about 7 weeks old were immunised with the BSA-hapten 3 and BSA-hapten 4 conjugates. Five mice were treated for each kind of immunogen. The first dose consisted of 100 lg of conjugate intraperitoneally injected as an emulsion of PBS and Freund’s complete adjuvant. The subsequent injections, emulsified in Freund’s incomplete adjuvant were given at 3-week intervals. One week after the last injection, the anti-Butocarboxim antiserums were obtained from the tail vein of each mouse. Ten sera were obtained: 3-1, 3-2, 3-3, 3-4, 3-5 for BSA-hapten 3 and 4-1, 4-2, 4-3, 4-4, 4-5 for BSA-hapten 4. The sera were tested for antibody titers and for analyte recognition by indirect competitive ELISA. The process of indirect competitive ELISA was performed as described previously (Qian et al., 2007). 2.9. Production of monoclonal antibody The mouse showing the highest serum reactivity was given a peritoneal cavity injection of 100 lg immunogen in PBS at 1 week intervals. Three to four days after the last injection, the donor mouse was sacrificed. SP2/0 murine myeloma cells were cultured in DMEM supplemented with 20% FBS. Splenocytes of selected mice were harvested aseptically. Cell fusion and hybridoma selection procedures were performed essentially as described previously (Li, Zhang, Zhang, Zhang, & Chen, 2009b; Liu et al., 2009; Wang, Zhang, Chen, Liu, & Li, 2011; Wang et al., 2009; Zeng et al., 2007; Zhang et al., 2007). Briefly, the splenocytes were added to myeloma cells to give a ratio of five splenocytes per myeloma cell and the mixture was centrifuged. One mL of PEG 1500 at 37 °C was dropped on a cell pellet over 1 min and left to stand for 1 min, then 30 mL of DMEM was added over 4 min and the pellet was left standing for a further 10 min. The fused cells were then spun down and resuspended in HAT selection medium before they were distributed in a dose of 100 lL per well in a 96-well culture plates which was previously coated with feeding cells. The HAT selection medium consisted of DMEM, 20% FBS, 10 mmolL1 hypoxanthine, 0.4 mmolL1 aminopterin and 1.6 mmolL1 thymidine. Half of the media in the wells were replaced by fresh HAT media every 4th day. The HAT media were changed to HT media with no aminopterin, when most of the non-fused cells were eliminated. After the hybridoma cells had grown to 30–40% confluence in the well, the culture supernatants were initially screened by indirect ELISA for the ability to bind to H2-OVA, culture supernatant from unfused SP2/0 cells serving as the negative control. Positive hybridoma cells were subcloned by limiting dilution, and stable antibody-producing clones were expanded and cryopreserved in liquid nitrogen. Competitive indirect ELISA using H2-OVA as a coating antigen was then employed to determine if the antibodies from the finally expanded clones could recognise the pesticides. The selected clones were cryopreserved in liquid nitrogen. 2.10. Development of a competitive enzyme-linked chemiluminescent immunoassay (Co-ELCIA) The basic steps in the Co-ELCIA process are as follows. Microtiter plates were coated with optimised concentrations of hapten-OVA in carbonate–bicarbonate buffer (50 mM, pH 9.6) by incubating for overnight at 4 °C. Plates were then blocked by incubating with 1% gelatin in PBS (120 lL per well) for 1.5 h. Aliquots (25 lL per well) of analyte dissolved in working solution and aliquots of Mab (25 lL per well), at a previously determined concentration, were added to the blocked plate, the Mab having been diluted with
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working solution. After incubation for 1 h, 50 lL per well of diluted (1/20,000) goat anti-mouse IgG-HRP was added to the plate. The mixture was incubated for 1 h. Finally, 150 lL of chemiluminescent substrate solution was pipetted into each well. The reading was done after 3 min of incubation at room temperature in the dark, and the emitted photons were measured at 425 nm (endpoint method). All incubations were performed at 37 °C and the plates were washed five times with PBST (0.15 M PBS containing 0.05% Tween 20, pH 7.4) after each incubation, unless specified otherwise. The Microtitre plates are working as below: via the use of radiation, the resulting polystyrene surface is primarily hydrophobic with intermittent carboxyl groups capable of ionic interactions with positively charged groups on biomolecules. The mechanism of immobilisation is passive adsorption through hydrophobic and ionic interactions. This is considered a general purpose surface capable of binding medium (>10 kD) and large biomolecules that possess ionic groups and/or hydrophobic regions (http:// www.corning.com/lifesciences/us_canada/en/technical_resources/ surfaces/assay/high_binding_polystyrene.aspx). 2.11. Optimisation of Co-ELCIA Selection of the coating antigen/antibody combination was performed by Co-ELCIA. Four coating antigens (H1, H2, H3, H4-OVA) that were able to conjugate with the MAbs 1C9 were screened out by Co-ELCIA. Then the optimum combination of coating antigen and antibody was confirmed based on the sensitivity of the Co-ELCIA. The dilution ratios of antibody (1:6000–1:24,000) and coating antigen (1:6000–1:14,000) were confirmed based on the sensitivity of the Co-ELCIA. The working solutions, prepared at a series of pH values (6.0– 8.5) and ionic strengths (0.1–3.2 M), were used to dilute the Butocarboxim standards and were tested using the established ELCIA. The tested working solution that resulted in the best sensitivity for the ELCIA was selected as the optimised working solution. 3. Evaluation of the optimised Co-ELCIA 3.1. Cross-reactivity studies To evaluate the selectivity of the Co-ELCIA, four carbamate oxime esters pesticides (Butoxycarboxim, Thiofanox, Thiodicarb and Methomyl) were evaluated using the established ELCIA. The cross-reactivity (CR) was calculated as follows: CR ¼ ½IC50 ðButocarboximÞ=IC50 ðcompoundÞ 100%. Here, the CR of Butocarboxim was defined as 100%. 3.2. Accuracy Two different agricultural samples (apple and greengrocery) and tap water were chosen to evaluate the performance of the ELCIA. The apples and greengrocery were bought from the local market. All samples were verified by HPLC not to contain Butocarboxim before undertaking spiking and recovery studies. Apples and greengrocery were cut into pieces and homogenised. 5.0 g of each sample were weighed and placed in a 50 mL centrifuge tube, spiked with known concentrations of Butocarboxim standard solution dissolved in methanol. The samples were thoroughly mixed and allowed to stand at room temperature for 1 h. 10 mL of methanol was added to each sample and the samples were shaken thoroughly on the rotary shaker for 10 min, and then centrifuged at 4000g (Allegra x-12R centrifuge, Beckman coulter, USA) for 10 min. The supernatants were then transferred to a 25 mL volumetric flask, following 10 mL of methanol was added
again, thoroughly shaken for 10 min, and then centrifuged at 4000g for 10 min. The supernatant was transferred to the volumetric flask. And set the volume to 25 mL (sample extract) with optimal working buffer (without organic solvent). The sample extracts were diluted with optimal working buffer (without organic solvent) and then mixed with antibody (diluted in PBST) as 1:1 (v/v) for ELCIA analysis. Also the tap water was spiked with known concentrations (2.5, 5.0, and 10 lgmL1) of Butocarboxim in methanol. HPLC analysis of Butocarboxim was conducted at 210 nm (UV), and the mobile phase was acetonitrile:water (28:72) at a flow rate of 1.4 mLmin1. XDB-C18 column (4.6 250 mm). The temperature of the column oven was maintained at room temperature. Retention time for Butocarboxim was 6.8 ± 0.2 min.
4. Results and discussion 4.1. Verification of haptens The haptens H1–H4 were synthesised and structures were clarified by 1H NMR. The results of 1H NMR were described as follows: H1: 1H NMR (300 MH, CDCl3) d: 2.97 (s, 3H), 3.11 (s, 3H), 4.00 (s, 4H) H2: 1H NMR (300 MHz, DMSO) d: 2.22(2H, t, J = 7.2 Hz), 1.67 (m, 2H), 3.07 (m, 2H), 1.98 (s, 3H), 1.97 (s, 3H) H3: 1H NMR (300 MHz, CDCl3) d: 3.60 (m, 1H), 2.75 (s, 4H), 2.02 (s, 3H), 2.00 (s, 3H), 1.38 (d, 3H, J = 7.2 Hz) H4: 1H NMR (300 MHz, CDCl3) d: 3.63 (m, 1H), (m, 2H), 2.66 (m, 4H), 2.06 (s, 3H), 1.38 (d, 3H, J = 7.2 Hz), 1.20 (t, 3H, J = 7.2 Hz) 4.2. Screening of the antiserums There were 10 antiserums (5 from H3-BSA (3-1 to 3-5) and 5 from H4-BSA (4-1 to 4-5) were investigated. The homologous noncompetitive ELISA (with the homologous coating antigen) indicated that antiserums 3-4 and 4-2 had the highest titer. Then, the competitive ELISA was operated to select the best antiserum/ coating antigen combination through the IC50 values. The Table 1 shows that the 4-2/H2-OVA combination gave the best IC50. Therefore, the mouse 4-2 was selected for subsequent hybridoma production, and the H2-OVA was used as the coating antigen. 4.3. Production and characterisation of monoclonal antibody After selection of the antiserum, the mouse produced sera 4-2 was selected for subsequent hybridoma production because of its highest relative reactivity to Butocarboxim. Hybridoma clones were initially screened by indirect Co-ELCIA for their reactivities with H2-OVA, and a total of 20 hybridoma cell lines were obtained. After the 5 screen cycles, the hybridoma cell line 1C9 was found to secrete antibodies of the IgG1, kappa type. The MAb, present in the ascites fluid in mouse, was purified by precipitation with 50%
Table 1 ELISA sensitivity (IC50, IC50 values (lgmL1) of the combination between serum and coating antigen) for different combination of antibodies and coating antigens (n = 3)a. IC50 was higher than 1000 lgmL1. Antiserum
Immunogen
Coating antigen (IC50, lgmL1) H1-OVA
H3-4 H4-2 a
BSA-hapten 3 BSA-hapten 4
N N
a
IC50 was higher than 1000 lgmL1.
H2-OVA
H3-OVA
H4-OVA
10 1
N N
N N
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ammonium sulfate (Reik et al., 1987) and stored at 35 °C for following characterisation studies. 4.4. Optimisation of the Co-ELCIA Experimental parameters, which may affect the analytical performance of the assay, were examined and included the concentrations of coating antigen and antibody, pH, and the ionic strength of the Co-ELCIA system. Fig. 2 shows the results of these experiments. In this study, the highest ratio of CLUmax/IC50 was considered as the main criterion for evaluating the ELCIA assay (Long, Shi, He, Sheng, & Wang, 2009). From the results of Fig. 2, the optimised conditions for the Co-ELCIA were selected as: pH = 8.0, ionic strength was 1.6 M. Both of the dilution ratio of coating antigen and antibody were 1:8000.
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4.5. Measurement of Butocarboxim by Co-ELCIA Under optimised conditions, the analytical performance for the Co-ELCIA for detection of Butocarboxim was examined with different concentrations of standard Butocarboxim in PBST. The results presented in Fig. 3 indicated that the developed Co-ELCIA was suitable for the determination of Butocarboxim. After conversion of Fig. 3a, it was observed that in the range of 27–2700 ngmL1, the graph between B/B0 and logarithm of concentration of butocarboxim (ngmL1) was linear (Fig. 3b), the regression equation was obtained (y = 0.4009x + 1.5076, R2 = 0.9906). The limit detection (LD) was 20 ngmL1 by the extrapolation of B0-2SD extrapolation. The Mean ± SD were described as below: 0.082 ± 0.0030, 0.090 ± 0.0037, 0.13 ± 0.0022, 0.33 ± 0.0051, 0.58 ± 0.013, 0.77 ± 0.011, 0.90 ± 0.015, 0.97 ± 0.0060, 0.99 ± 0.041,
Fig. 2. Effect of pH values, ionic strengths, dilution ratio of coating antigen and dilution ratio of antibody (monoclonal from mouse) on the performance of assay: pH [(a) 6.0 (185,431 ± 5110), 6.5 (201,020 ± 6719), 7.0 (234,909 ± 1061), 7.5 (268,210 ± 15,575), 8.0 (330,885 ± 10,081), 8.5 (250,856 ± 6007)]; ionic strengths [(b) 0.1 (32,522 ± 1890), 0.2 (56,085 ± 2006), 0.4 (59,339 ± 3474), 0.8 (65,318 ± 3306), 1.6 (97,551 ± 5216), 3.2 (44,185 ± 2176) molL1]; dilution ratio of coating antigen [(c), 1:6000 (107,197 ± 4947), 1:8000 (112,373 ± 6417), 1:10,000 (100,346 ± 689), 1:12,000 (90,246 ± 7738), 1:14,000 (78,537 ± 4946)] and dilution ratio of antibody [(d) 1:6000 (112,354 ± 2298), 1:8000 (149,197 ± 5377), 1:12,000 (103,245 ± 5907), 1:16,000 (94,815 ± 1695), 1:20,000 (50,429 ± 6871), 1:24,000 (42,198 ± 975) ]. Results are the means of three repetitions experiments (Mean ± SD, n = 3).
Fig. 3. Indirect competitive enzyme-linked chemiluminescent immunoassay (Co-ELCIA) curve for Butocarboxim. (a) Standard inhibition curve of Butocarboxim; (b) the calibration curve from ‘‘a’’. It was observed from ‘‘a’’ that in the range of 27–2700 ngmL1, the graph between B/B0 and logarithm of concentration of Butocarboxim (ngmL1) was linear, and the regression equation was obtained (y = 0.4009x + 1.5076, R2 = 0.9906). The Mean ± SD of ‘‘a’’ were 0.082 ± 0.0030, 0.090 ± 0.0037, 0.13 ± 0.0022, 0.33 ± 0.0051, 0.58 ± 0.013, 0.77 ± 0.011, 0.90 ± 0.015, 0.97 ± 0.0060, 0.99 ± 0.041, 1.0 ± 0.011, respectively. (n = 3).
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Table 2 Cross-reactivity (CR) of a set of analogs related to Butocarboxim by Co-ELCIA (n = 3). Compound
Chemical structure CH 3
Butocarboxim S
O
O
O
H 3C
16.7
1.95
41.0
