Toxicon 106 (2015) 89e96
Contents lists available at ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Identification of a high-affinity monoclonal antibody against ochratoxin A and its application in enzyme-linked immunosorbent assay Xian Zhang a, Mengjiao Sun a, Yue Kang a, Hui Xie a, Xin Wang a, Houhui Song b, Xiaoliang Li a, Weihuan Fang a, * a
Zhejiang University Institute of Preventive Veterinary Medicine, and Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, 388 Yuhangtang Road, Hangzhou, Zhejiang, 310058, China College of Animal Science and Technology, Zhejiang A&F University, 88 Huanbei Road, Lin'an, Zhejiang, 311300, China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 August 2015 Received in revised form 16 September 2015 Accepted 21 September 2015 Available online 26 September 2015
Ochratoxin A (OTA) is one of the most commonly occurring mycotoxins produced by some species of Aspergillus and can contaminate cereal and cereal products. A high-affinity anti-OTA monoclonal antibody (mAb) was generated from a hybridoma cell line 2D8 using splenocytes from a BALB/c mouse immunized with synthesized OTA-bovine serum albumin conjugate. The mAb 2D8 is specific with high affinity (3.75 109 L/M). An indirect competitive ELISA (ic-ELISA) was then developed using this mAb for quantitative determination of OTA in corn and feed samples. Using the optimized conditions, there was good linearity between OTA concentration and competitive inhibition (y ¼ 0.6076x þ 0.2441, R2 ¼ 0.9923) with the working range from 2.4 to 23.6 mg/kg, IC50 at 7.6 mg/kg and lower limit of detection at 1.4 mg/kg. The recovery rates in spiked samples were 91.2e110.3%. Of the 56 corn and feed samples, this ic-ELISA and a commercial kit both found the same 13 samples positive for OTA with good linear correlation between the two methods in OTA quantification (R2 ¼ 0.9706). We conclude that this ic-ELISA can be used for rapid and quantitative screening of corn and feed samples for the presence of OTA. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Ochratoxin A Artificial antigen Monoclonal antibody ic-ELISA Quantification
1. Introduction Mycotoxins are toxic secondary metabolites of fungal species and often contaminate agricultural commodities during storage or processing (Shephard, 2008; Sorrenti et al., 2013). Ochratoxin A (OTA) is produced by several fungi of the Aspergillus or Penicillium species growing in different agricultural commodities and has been shown to be nephrotoxic, teratogenic, carcinogenic and immunotoxic to several animal species (Malir et al., 2013; Mateo et al., 2011). The International Agency for Research on Cancer (IARC) has classified OTA as a possible human carcinogen (group 2B) (Humans et al., 2004; Mateo et al., 2011). Maximum limit of OTA in cereals has been set at 5 mg/kg in the European Union (EU) (Yang et al., 2012). Therefore, accurate and rapid detection of OTA is important to prevent contaminated food from entering into the food chain.
* Corresponding author. E-mail address: [email protected] (W. Fang). http://dx.doi.org/10.1016/j.toxicon.2015.09.028 0041-0101/© 2015 Elsevier Ltd. All rights reserved.
A number of methods have been developed for OTA detection in cereal and cereal products, including mass spectrometry (MS), high-performance liquid chromatography (HPLC) (Huang et al., 2014; Marino-Repizo et al., 2015; Zecevic et al., 2004). While these methods can be reliable, they have several drawbacks, such as expensive instrumentation, time-consuming procedures, extensive clean-up and purification, and trained personnel required in operation (Shim et al., 2009; Wang et al., 2013). Immunoassays, especially the enzyme-linked immunosorbent assay (ELISA), are robust methods for rapid and high throughput screening of different samples. In recent years, diagnostic kits based on monoclonal antibodies are widely used in detection of foodborne pathogens or drug residues because of their adaptability, simplicity, selectivity, and low cost (Le et al., 2012; Lupo et al., 2010). The main purposes of the present study were to obtain a monoclonal antibody (mAb) against OTA with high specificity and affinity and to develop a sensitive indirect competitive ELISA (icELISA) for detection of OTA in corn and feed samples.
90
X. Zhang et al. / Toxicon 106 (2015) 89e96
2. Materials and methods 2.1. Materials Ochratoxin A (OTA), Ochratoxin B (OTB), Zearalenone (ZEN), Fumonisin B1 (FB1), Aflatoxin B1 (AFB1), Patulin (PAT), Citrinin (CIT), Deoxynivalenol (DON), N,N0 -dicyclohexylcarbodiimide (DCC), N-hydroxy-succinimide (NHS), bovine serum albumin (BSA), ovalbumin (OVA), polyethylene glycol 1450 (PEG 1450), dimethyl sulfoxide (DMSO), Freund's complete adjuvant, Freund's incomplete adjuvant and polyethylene glycol solution (PEG 1450) were purchased from the Sigma Chemical Co. (St. Louis, MO, USA). Hypoxanthine-aminopterin-thymidine (HAT) medium, hypoxanthine-thymidine (HT) medium and RPMI 1640 were obtained from Invitrogen (Carlsbad, CA, USA). Horseradish peroxidase (HRP) conjugated goat anti-mouse IgG was obtain from Sungene Biotech Co. (Tianjin, China). HRP conjugated goat anti-mouse (IgG1, IgG2a, IgG2b, IgG3, IgM, IgA) were purchased from UcallM Biotechnology Co. (Jiangsu, China). Female BALB/c mice (5 weeks old) were obtained from Zhejiang Chinese Medical University (Hangzhou, China). Polyclonal antibodies against OTA (pAb-OTA) were obtained from Huaan Megnech (Beijing, China). Ochratoxin A ELISA kit (R1311) was from R-Biopharm (Darmstadt, Germany). Myeloma cells SP2/0 were stored in our laboratory. The ELISA substrate 3, 3, 5, 5-tetramethylbenzidine (TMB) was purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). The OTA-free cereal samples (corn, wheat, and soybean) were purchased from a local Wal-Mart Store. Fifty-six corn and feed samples were from Dr F.Q. Wang, Institute of Feed Science, Zhejiang University where local feed companies send their samples as part of the quality assurance program. 2.2. Conjugation of OTA with bovine serum albumin and ovalbumin The complete antigens OTA-BSA and OTA-OVA were prepared by two-step approach according to the previously described methods (Kawamura et al., 1989) with slight modifications. Ochratoxin A (2.0 mg) was dissolved in 500 mL anhydrous tetrahydrofuran (THF) and then mixed with 2.0 mg NHS and 3.0 mg DCC (Fig. 1). The mixture was then allowed to react for 12 h by shaking gently at room temperature (RT). The reaction mixture was centrifuged at 10,000g for 15 min. The supernatant sample was dried by vacuum concentrator (CentriVap, Labconoco, MO, USA) and the residue was dissolved in 0.5 mL dimethyl sulfoxide (DMSO). BSA (15 mg) was dissolved in 2.0 mL of 0.13 M phosphate buffer (pH
7.4). Activated OTA was added drop-wise to the BSA solution. Reaction was allowed to proceed for 4 h by vigorous shaking at RT. The solution was then centrifuged and the supernatant containing OTABSA was dialyzed against 0.01 M/L phosphate buffered saline (PBS, pH 7.4) at 4 C for 72 h to remove the residual free OTA and DMSO. The same method was employed to prepare OTA-OVA conjugate. Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) (Tseng et al., 2010) was used to determine the molecular weight of the conjugates OTA-BSA and OTA-OVA. ELISA was also used to confirm the conjugates as follows: OTA-OVA or OTA-BSA conjugate solution (1.0 mg/mL) dissolved in carbonate buffer solution (CBS, pH 9.6) was immobilized onto a microtiter plate (100 mL/well) Maxisorp polystyrene 96-well plates (Nunc, Roskilde, Denmark) for overnight coating at 4 C, followed by blocking with 200 mL of 5% (m/v) skimmed milk and 0.05% Tween-20 in 10 mM PBS (pH 7.4). The coated antigens were probed with 100 mL of pAb-OTA (0.5 mg/mL), followed by addition of 100 mL of goat anti-rabbit IgG-HRP (0.05 mg/mL) to each well. Color development was achieved by addition of 100 mL TMB and incubated for 10 min at 37 C. Stop solution (2 M H2SO4, 50 mL) was added and absorbance was monitored at 450 nm (SpectroMax M2, Molecular Device, CA, USA). All incubations proceeded at 37 C for 1 h. Between incubations, the ELISA plates were washed three times with washing buffer PBST (PBS containing 0.05% Tween 20, v/ v; pH 7.4). The antibody solutions were prepared in 0.5% (m/v) skimmed milk PBST. 2.3. Generation of monoclonal antibodies against OTA Female BALB/c mice (5 weeks old) were given three intradermal injections of OTA-BSA at multiple sites for mAb production by the standard procedure (Sheng et al., 2012). The OTA-BSA conjugate (200 mg) was dissolved in 200 mL of sterile 0.01 M PBS, emulsified with an equal volume of complete Freund's adjuvant and injected into the female BALB/c mice on the dorsal side at multiple sites. All booster injections (100 mg per 100 mL) used Freund's incomplete adjuvant for emulsification. Seven days after the third immunization, the titer of antiserum was tested by indirect ELISA, as described above, with the OTA-OVA conjugate as the coating antigen. The use of mice was approved by Zhejiang University Committee on Laboratory Animal Welfare and Ethics (Approval No. 20140023). One of the mice with the highest antibody titer was euthanized and the splenocytes were fused with SP2/0 myeloma cells use PEG 1450. The fused cells were propagated in HAT medium (Lu et al.,
Fig. 1. Schematic illustration of conjugation of ochratoxin A with bovine serum albumin (BSA) and ovalbumin (OVA).
X. Zhang et al. / Toxicon 106 (2015) 89e96
2011). About two weeks after cell fusion, culture supernatants were screened for mAb specific to OTA using the OTA-OVA conjugate as the coating antigen. The culture supernatants which showed higher OD were chosen for clonal selection by the limiting dilution method prior to ascites production according to a previous study (Skinner et al., 2013). The cells from the positive clone secreting specific mAb were cultured and injected into the abdomen of BALB/c mice to produce ascites. The anti-OTA mAb was obtained from ascites and purified by caprylic/ammonium sulfate precipitation (CA-AS) method (Liu et al., 1999). Purification was verified by SDS-PAGE. Protein concentration was determined using the BCA method. The purified mAb was aliquoted and stored at 20 C for later use. 2.4. Characterization of the monoclonal antibodies against OTA Conjugate OTA-OVA was used as coating antigen (100 mL per
91
well, 1 mg/mL) The plate was incubated at 37 C for 1 h. After blocking and washing, the mAb was added (100 mL/well, 1:5000 dilution) and incubated at 37 C for 1 h. Different HRP conjugated goat anti-mouse antibodies to IgG1, IgG2a, IgG2b, IgG3, IgM and IgA (1:5000 dilutions) were pipetted into plates for further incubation at 37 C for 45 min. The remaining ELISA procedure was the same as described above. Chromosomes of hybridomas cell were analyzed according to previous study with some modifications (Ling et al., 2014a). The hybridomas cells were cultivated for 2 days and then treated with 0.2 mg/mL colchicine for 7 h in 5% CO2 at 37 C. The cells were harvested by centrifugation at 1000g for 15 min and resuspended in 15 mL of 0.075 mol/L potassium chloride hypotonic solution for 20 min at 37 C. The cells were harvested by centrifugation at 1000g for 15 min, resuspended with 5 mL of fixing solution (methanol:acetic acid, v/v 3:1) and kept overnight at 4 C. The hybridoma cells were resuspended with 0.5 mL fixing solution. The
Fig. 2. Analysis of the conjugates ochratoxin A-bovine serum albumin (OTA-BSA) (A) and ochratoxin A-ovalbumin (OTA-OVA) (B) by MALDI-TOF-MS and by indirect ELISA using rabbit polyclonal antibodies against ochratoxin A (C). The top panels in A and B were BSA or OVA control and the bottom panels were OTA-BSA or OTA-OVA conjugates.
92
X. Zhang et al. / Toxicon 106 (2015) 89e96
Fig. 3. Antibody titers of serum samples from four mice immunized with Ochratoxinbovine serum albumin conjugate as measured by indirect ELISA using ochratoxinovalbumin conjugate as the coating antigen. Control was the mouse immunized with the adjuvant only.
numbers of chromosomes of the hybridomas cells were counted on the glass-slide smears after staining with Giemsa. Cross-reactivity between the anti-OTA mAb and other mycotoxins was determined by ic-ELISA according to the previous study (Jin et al., 2014). Free OTB, AFB1, FB1, ZEN, PAT, CIT and DON were used as potential binding competitors. Cross-reactivity was calculated as percent inhibition using the formula: IC50 of OTA/IC50 of other mycotoxins 100%. To determine the affinity of the purified anti-OTA mAb, ic-ELISA was performed as the previous study (Ling et al., 2014b). OTA-OVA were coated at different concentrations (100 mL per well, 1, 0.5, 0.25 and 0.125 mg/mL). The plate was incubated at 37 C for 1 h. Serial 2fold dilutions of the anti-OTA mAb (1000e8,192,000 dilutions from the 4 mg/mL stock solution) were used (100 mL/well). Each concentration was in triplicate wells. HRP conjugated goat anti-mouse IgG was used at 1:20,000 dilution (0.05 mg/mL). All other procedures were the same as described above. Affinity constant (Kaff) was calculated using the reported method described previously (Loomans et al., 1995). 2.5. Optimization of ic-ELISA for quantitation of OTA The concentrations of the coating antigen and antibody, temperature and time of incubation were optimized by checkerboard design according to the previous report (Jiang et al., 2012). The ELISA plates were coated with OTA-OVA (100 mL/well) at 4 C for overnight. After blocking and washing, 75 mL of serial dilutions of OTA standard solutions and 75 mL of diluted anti-OTA mAb at optimal concentration were mixed. The mixtures were pipetted into the wells (100 mL/well), each OTA concentration in triplicate wells. The plates were incubated for 1 h at 37 C. The remaining procedures were the same as described above. 2.6. Calibration curve and matrix interference analysis The OTA-OVA conjugate at optimum concentration was used to coat the plate wells. Different concentrations of OTA (serial 2-fold dilution from 7.8 to 0.015 ng/mL and zero control) were mixed with equal volume of anti-OTA mAb and pipetted into plate wells (100 mL/well). HRP conjugated goat anti-mouse IgG was used at 1:5000 dilution (0.2 mg/mL). The remaining procedures were the same as described above. The calibration curve was prepared with GraphPad Prism 5: x-axis represents the log concentration of OTA (ng/mL) and y-axis (B/B0), the division of OD450 nm value of
Fig. 4. Characterization of monoclonal antibodies against ochratoxin A by antibody subtypes (A), titers of ascites (B) and chromosome numbers of the 2D8 hybridoma cells (C).
standard solutions by OD450 nm of zero OTA control. The linear range of detection was defined as the concentration of OTA leading to 20%e80% inhibition (Kido et al., 2008). The lower detection limit (LOD) was calculated with the method of 3SD from standard zero. Methanol was often used for mycotoxin extraction from samples. Methanol in the extraction buffer and the matrixes of sample extracts, including proteins and vitamins are known to affect reaction between antigen and antibody (Melnikova et al., 2000; Rehan and Younus, 2006). These interferences can be minimized n et al., 2006). by proper sample preparation procedures (Alarco Dilution is a commonly used method (Ono et al., 2000; Quan et al., 2006). In this study, the matrix effect was minimized by diluting the extracts with PBS in different ratios. To study possible matrix interferences in different cereal samples (corn, wheat and soybean), serial calibration curves were prepared using different diluted extracts (three levels: 1:1, 1:3 and 1:7) with PBS as control.
X. Zhang et al. / Toxicon 106 (2015) 89e96
93
Fig. 6. Tri-parametric curve fitting of log concentration of ochratoxin A vs inhibition index (B/B0) by ic-ELISA. The insert shows the calibration curve for quantification of ochratoxin A.
2.7. Sample preparation, recovery analysis and OTA determination in corn and feed samples Different cereal samples (corn, wheat and soybean) were purchased from local markets. All samples were tested by LCeMS/MS before spiking and recovery tests to ensure that they were OTAfree. The cereal samples were ground and dried by overnight incubation in a 60 C incubator. The standard OTA solution was spiked at levels of 2.5, 5, 10 and 20 mg/kg by dropwise addition, mixed thoroughly and allowed to stand at RT overnight. Five gram of samples was weighed into a 50-mL plastic centrifuge tube, and the samples were extracted with 12.5 mL of methanol/water (7/3, v/v) at RT. The samples were vigorously mixed by vortexing for 5 min. After centrifugation at 4000g for 10 min, the supernatant samples were used for ic-ELISA. To evaluate the precision of this ic-ELISA, each sample was tested three times in one day and repeated three times on different days. Ground corn and feed samples were tested simultaneously by the ic-ELISA established in this study and a commercial kit (with indicated detection limit at 1.25 mg/kg). These samples were extracted with methanol/water (7/3, v/v) and diluted 1:7 with PBS, each sample was tested in triplicate wells. Correlation between the two assays was investigated using linear regression (Microsoft Excel software, 2010 version). 3. Results 3.1. Preparation and identification of the OTA conjugates Fig. 5. Purification of 2D8 monoclonal antibody against ochratoxin A (A), and its specificity (B) and affinity (C). A: M is protein marker; 1 ascites and 2 purified monoclonal antibody. B: Cross-reactivity of the monoclonal antibody to other mycotoxins as determined by ic-ELISA. C: Affinity as determined by indirect ELISA.
Table 1 Cross-reactivity of the 2D8 anti-ochratoxin A monoclonal antibody to other mycotoxins. Analyte
IC50 (ng/ml)
Cross-reactivity (%)
Ochratoxin A Ochratoxin B Aflatoxin B1 Fumonisin B1 Zearalenone Deoxynivalenol Citrinin Patulin
0.38 7.1 >1 >1 >1 >1 >1 >1
100 5.4
Contents lists available at ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Identification of a high-affinity monoclonal antibody against ochratoxin A and its application in enzyme-linked immunosorbent assay Xian Zhang a, Mengjiao Sun a, Yue Kang a, Hui Xie a, Xin Wang a, Houhui Song b, Xiaoliang Li a, Weihuan Fang a, * a
Zhejiang University Institute of Preventive Veterinary Medicine, and Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, 388 Yuhangtang Road, Hangzhou, Zhejiang, 310058, China College of Animal Science and Technology, Zhejiang A&F University, 88 Huanbei Road, Lin'an, Zhejiang, 311300, China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 August 2015 Received in revised form 16 September 2015 Accepted 21 September 2015 Available online 26 September 2015
Ochratoxin A (OTA) is one of the most commonly occurring mycotoxins produced by some species of Aspergillus and can contaminate cereal and cereal products. A high-affinity anti-OTA monoclonal antibody (mAb) was generated from a hybridoma cell line 2D8 using splenocytes from a BALB/c mouse immunized with synthesized OTA-bovine serum albumin conjugate. The mAb 2D8 is specific with high affinity (3.75 109 L/M). An indirect competitive ELISA (ic-ELISA) was then developed using this mAb for quantitative determination of OTA in corn and feed samples. Using the optimized conditions, there was good linearity between OTA concentration and competitive inhibition (y ¼ 0.6076x þ 0.2441, R2 ¼ 0.9923) with the working range from 2.4 to 23.6 mg/kg, IC50 at 7.6 mg/kg and lower limit of detection at 1.4 mg/kg. The recovery rates in spiked samples were 91.2e110.3%. Of the 56 corn and feed samples, this ic-ELISA and a commercial kit both found the same 13 samples positive for OTA with good linear correlation between the two methods in OTA quantification (R2 ¼ 0.9706). We conclude that this ic-ELISA can be used for rapid and quantitative screening of corn and feed samples for the presence of OTA. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Ochratoxin A Artificial antigen Monoclonal antibody ic-ELISA Quantification
1. Introduction Mycotoxins are toxic secondary metabolites of fungal species and often contaminate agricultural commodities during storage or processing (Shephard, 2008; Sorrenti et al., 2013). Ochratoxin A (OTA) is produced by several fungi of the Aspergillus or Penicillium species growing in different agricultural commodities and has been shown to be nephrotoxic, teratogenic, carcinogenic and immunotoxic to several animal species (Malir et al., 2013; Mateo et al., 2011). The International Agency for Research on Cancer (IARC) has classified OTA as a possible human carcinogen (group 2B) (Humans et al., 2004; Mateo et al., 2011). Maximum limit of OTA in cereals has been set at 5 mg/kg in the European Union (EU) (Yang et al., 2012). Therefore, accurate and rapid detection of OTA is important to prevent contaminated food from entering into the food chain.
* Corresponding author. E-mail address: [email protected] (W. Fang). http://dx.doi.org/10.1016/j.toxicon.2015.09.028 0041-0101/© 2015 Elsevier Ltd. All rights reserved.
A number of methods have been developed for OTA detection in cereal and cereal products, including mass spectrometry (MS), high-performance liquid chromatography (HPLC) (Huang et al., 2014; Marino-Repizo et al., 2015; Zecevic et al., 2004). While these methods can be reliable, they have several drawbacks, such as expensive instrumentation, time-consuming procedures, extensive clean-up and purification, and trained personnel required in operation (Shim et al., 2009; Wang et al., 2013). Immunoassays, especially the enzyme-linked immunosorbent assay (ELISA), are robust methods for rapid and high throughput screening of different samples. In recent years, diagnostic kits based on monoclonal antibodies are widely used in detection of foodborne pathogens or drug residues because of their adaptability, simplicity, selectivity, and low cost (Le et al., 2012; Lupo et al., 2010). The main purposes of the present study were to obtain a monoclonal antibody (mAb) against OTA with high specificity and affinity and to develop a sensitive indirect competitive ELISA (icELISA) for detection of OTA in corn and feed samples.
90
X. Zhang et al. / Toxicon 106 (2015) 89e96
2. Materials and methods 2.1. Materials Ochratoxin A (OTA), Ochratoxin B (OTB), Zearalenone (ZEN), Fumonisin B1 (FB1), Aflatoxin B1 (AFB1), Patulin (PAT), Citrinin (CIT), Deoxynivalenol (DON), N,N0 -dicyclohexylcarbodiimide (DCC), N-hydroxy-succinimide (NHS), bovine serum albumin (BSA), ovalbumin (OVA), polyethylene glycol 1450 (PEG 1450), dimethyl sulfoxide (DMSO), Freund's complete adjuvant, Freund's incomplete adjuvant and polyethylene glycol solution (PEG 1450) were purchased from the Sigma Chemical Co. (St. Louis, MO, USA). Hypoxanthine-aminopterin-thymidine (HAT) medium, hypoxanthine-thymidine (HT) medium and RPMI 1640 were obtained from Invitrogen (Carlsbad, CA, USA). Horseradish peroxidase (HRP) conjugated goat anti-mouse IgG was obtain from Sungene Biotech Co. (Tianjin, China). HRP conjugated goat anti-mouse (IgG1, IgG2a, IgG2b, IgG3, IgM, IgA) were purchased from UcallM Biotechnology Co. (Jiangsu, China). Female BALB/c mice (5 weeks old) were obtained from Zhejiang Chinese Medical University (Hangzhou, China). Polyclonal antibodies against OTA (pAb-OTA) were obtained from Huaan Megnech (Beijing, China). Ochratoxin A ELISA kit (R1311) was from R-Biopharm (Darmstadt, Germany). Myeloma cells SP2/0 were stored in our laboratory. The ELISA substrate 3, 3, 5, 5-tetramethylbenzidine (TMB) was purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). The OTA-free cereal samples (corn, wheat, and soybean) were purchased from a local Wal-Mart Store. Fifty-six corn and feed samples were from Dr F.Q. Wang, Institute of Feed Science, Zhejiang University where local feed companies send their samples as part of the quality assurance program. 2.2. Conjugation of OTA with bovine serum albumin and ovalbumin The complete antigens OTA-BSA and OTA-OVA were prepared by two-step approach according to the previously described methods (Kawamura et al., 1989) with slight modifications. Ochratoxin A (2.0 mg) was dissolved in 500 mL anhydrous tetrahydrofuran (THF) and then mixed with 2.0 mg NHS and 3.0 mg DCC (Fig. 1). The mixture was then allowed to react for 12 h by shaking gently at room temperature (RT). The reaction mixture was centrifuged at 10,000g for 15 min. The supernatant sample was dried by vacuum concentrator (CentriVap, Labconoco, MO, USA) and the residue was dissolved in 0.5 mL dimethyl sulfoxide (DMSO). BSA (15 mg) was dissolved in 2.0 mL of 0.13 M phosphate buffer (pH
7.4). Activated OTA was added drop-wise to the BSA solution. Reaction was allowed to proceed for 4 h by vigorous shaking at RT. The solution was then centrifuged and the supernatant containing OTABSA was dialyzed against 0.01 M/L phosphate buffered saline (PBS, pH 7.4) at 4 C for 72 h to remove the residual free OTA and DMSO. The same method was employed to prepare OTA-OVA conjugate. Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) (Tseng et al., 2010) was used to determine the molecular weight of the conjugates OTA-BSA and OTA-OVA. ELISA was also used to confirm the conjugates as follows: OTA-OVA or OTA-BSA conjugate solution (1.0 mg/mL) dissolved in carbonate buffer solution (CBS, pH 9.6) was immobilized onto a microtiter plate (100 mL/well) Maxisorp polystyrene 96-well plates (Nunc, Roskilde, Denmark) for overnight coating at 4 C, followed by blocking with 200 mL of 5% (m/v) skimmed milk and 0.05% Tween-20 in 10 mM PBS (pH 7.4). The coated antigens were probed with 100 mL of pAb-OTA (0.5 mg/mL), followed by addition of 100 mL of goat anti-rabbit IgG-HRP (0.05 mg/mL) to each well. Color development was achieved by addition of 100 mL TMB and incubated for 10 min at 37 C. Stop solution (2 M H2SO4, 50 mL) was added and absorbance was monitored at 450 nm (SpectroMax M2, Molecular Device, CA, USA). All incubations proceeded at 37 C for 1 h. Between incubations, the ELISA plates were washed three times with washing buffer PBST (PBS containing 0.05% Tween 20, v/ v; pH 7.4). The antibody solutions were prepared in 0.5% (m/v) skimmed milk PBST. 2.3. Generation of monoclonal antibodies against OTA Female BALB/c mice (5 weeks old) were given three intradermal injections of OTA-BSA at multiple sites for mAb production by the standard procedure (Sheng et al., 2012). The OTA-BSA conjugate (200 mg) was dissolved in 200 mL of sterile 0.01 M PBS, emulsified with an equal volume of complete Freund's adjuvant and injected into the female BALB/c mice on the dorsal side at multiple sites. All booster injections (100 mg per 100 mL) used Freund's incomplete adjuvant for emulsification. Seven days after the third immunization, the titer of antiserum was tested by indirect ELISA, as described above, with the OTA-OVA conjugate as the coating antigen. The use of mice was approved by Zhejiang University Committee on Laboratory Animal Welfare and Ethics (Approval No. 20140023). One of the mice with the highest antibody titer was euthanized and the splenocytes were fused with SP2/0 myeloma cells use PEG 1450. The fused cells were propagated in HAT medium (Lu et al.,
Fig. 1. Schematic illustration of conjugation of ochratoxin A with bovine serum albumin (BSA) and ovalbumin (OVA).
X. Zhang et al. / Toxicon 106 (2015) 89e96
2011). About two weeks after cell fusion, culture supernatants were screened for mAb specific to OTA using the OTA-OVA conjugate as the coating antigen. The culture supernatants which showed higher OD were chosen for clonal selection by the limiting dilution method prior to ascites production according to a previous study (Skinner et al., 2013). The cells from the positive clone secreting specific mAb were cultured and injected into the abdomen of BALB/c mice to produce ascites. The anti-OTA mAb was obtained from ascites and purified by caprylic/ammonium sulfate precipitation (CA-AS) method (Liu et al., 1999). Purification was verified by SDS-PAGE. Protein concentration was determined using the BCA method. The purified mAb was aliquoted and stored at 20 C for later use. 2.4. Characterization of the monoclonal antibodies against OTA Conjugate OTA-OVA was used as coating antigen (100 mL per
91
well, 1 mg/mL) The plate was incubated at 37 C for 1 h. After blocking and washing, the mAb was added (100 mL/well, 1:5000 dilution) and incubated at 37 C for 1 h. Different HRP conjugated goat anti-mouse antibodies to IgG1, IgG2a, IgG2b, IgG3, IgM and IgA (1:5000 dilutions) were pipetted into plates for further incubation at 37 C for 45 min. The remaining ELISA procedure was the same as described above. Chromosomes of hybridomas cell were analyzed according to previous study with some modifications (Ling et al., 2014a). The hybridomas cells were cultivated for 2 days and then treated with 0.2 mg/mL colchicine for 7 h in 5% CO2 at 37 C. The cells were harvested by centrifugation at 1000g for 15 min and resuspended in 15 mL of 0.075 mol/L potassium chloride hypotonic solution for 20 min at 37 C. The cells were harvested by centrifugation at 1000g for 15 min, resuspended with 5 mL of fixing solution (methanol:acetic acid, v/v 3:1) and kept overnight at 4 C. The hybridoma cells were resuspended with 0.5 mL fixing solution. The
Fig. 2. Analysis of the conjugates ochratoxin A-bovine serum albumin (OTA-BSA) (A) and ochratoxin A-ovalbumin (OTA-OVA) (B) by MALDI-TOF-MS and by indirect ELISA using rabbit polyclonal antibodies against ochratoxin A (C). The top panels in A and B were BSA or OVA control and the bottom panels were OTA-BSA or OTA-OVA conjugates.
92
X. Zhang et al. / Toxicon 106 (2015) 89e96
Fig. 3. Antibody titers of serum samples from four mice immunized with Ochratoxinbovine serum albumin conjugate as measured by indirect ELISA using ochratoxinovalbumin conjugate as the coating antigen. Control was the mouse immunized with the adjuvant only.
numbers of chromosomes of the hybridomas cells were counted on the glass-slide smears after staining with Giemsa. Cross-reactivity between the anti-OTA mAb and other mycotoxins was determined by ic-ELISA according to the previous study (Jin et al., 2014). Free OTB, AFB1, FB1, ZEN, PAT, CIT and DON were used as potential binding competitors. Cross-reactivity was calculated as percent inhibition using the formula: IC50 of OTA/IC50 of other mycotoxins 100%. To determine the affinity of the purified anti-OTA mAb, ic-ELISA was performed as the previous study (Ling et al., 2014b). OTA-OVA were coated at different concentrations (100 mL per well, 1, 0.5, 0.25 and 0.125 mg/mL). The plate was incubated at 37 C for 1 h. Serial 2fold dilutions of the anti-OTA mAb (1000e8,192,000 dilutions from the 4 mg/mL stock solution) were used (100 mL/well). Each concentration was in triplicate wells. HRP conjugated goat anti-mouse IgG was used at 1:20,000 dilution (0.05 mg/mL). All other procedures were the same as described above. Affinity constant (Kaff) was calculated using the reported method described previously (Loomans et al., 1995). 2.5. Optimization of ic-ELISA for quantitation of OTA The concentrations of the coating antigen and antibody, temperature and time of incubation were optimized by checkerboard design according to the previous report (Jiang et al., 2012). The ELISA plates were coated with OTA-OVA (100 mL/well) at 4 C for overnight. After blocking and washing, 75 mL of serial dilutions of OTA standard solutions and 75 mL of diluted anti-OTA mAb at optimal concentration were mixed. The mixtures were pipetted into the wells (100 mL/well), each OTA concentration in triplicate wells. The plates were incubated for 1 h at 37 C. The remaining procedures were the same as described above. 2.6. Calibration curve and matrix interference analysis The OTA-OVA conjugate at optimum concentration was used to coat the plate wells. Different concentrations of OTA (serial 2-fold dilution from 7.8 to 0.015 ng/mL and zero control) were mixed with equal volume of anti-OTA mAb and pipetted into plate wells (100 mL/well). HRP conjugated goat anti-mouse IgG was used at 1:5000 dilution (0.2 mg/mL). The remaining procedures were the same as described above. The calibration curve was prepared with GraphPad Prism 5: x-axis represents the log concentration of OTA (ng/mL) and y-axis (B/B0), the division of OD450 nm value of
Fig. 4. Characterization of monoclonal antibodies against ochratoxin A by antibody subtypes (A), titers of ascites (B) and chromosome numbers of the 2D8 hybridoma cells (C).
standard solutions by OD450 nm of zero OTA control. The linear range of detection was defined as the concentration of OTA leading to 20%e80% inhibition (Kido et al., 2008). The lower detection limit (LOD) was calculated with the method of 3SD from standard zero. Methanol was often used for mycotoxin extraction from samples. Methanol in the extraction buffer and the matrixes of sample extracts, including proteins and vitamins are known to affect reaction between antigen and antibody (Melnikova et al., 2000; Rehan and Younus, 2006). These interferences can be minimized n et al., 2006). by proper sample preparation procedures (Alarco Dilution is a commonly used method (Ono et al., 2000; Quan et al., 2006). In this study, the matrix effect was minimized by diluting the extracts with PBS in different ratios. To study possible matrix interferences in different cereal samples (corn, wheat and soybean), serial calibration curves were prepared using different diluted extracts (three levels: 1:1, 1:3 and 1:7) with PBS as control.
X. Zhang et al. / Toxicon 106 (2015) 89e96
93
Fig. 6. Tri-parametric curve fitting of log concentration of ochratoxin A vs inhibition index (B/B0) by ic-ELISA. The insert shows the calibration curve for quantification of ochratoxin A.
2.7. Sample preparation, recovery analysis and OTA determination in corn and feed samples Different cereal samples (corn, wheat and soybean) were purchased from local markets. All samples were tested by LCeMS/MS before spiking and recovery tests to ensure that they were OTAfree. The cereal samples were ground and dried by overnight incubation in a 60 C incubator. The standard OTA solution was spiked at levels of 2.5, 5, 10 and 20 mg/kg by dropwise addition, mixed thoroughly and allowed to stand at RT overnight. Five gram of samples was weighed into a 50-mL plastic centrifuge tube, and the samples were extracted with 12.5 mL of methanol/water (7/3, v/v) at RT. The samples were vigorously mixed by vortexing for 5 min. After centrifugation at 4000g for 10 min, the supernatant samples were used for ic-ELISA. To evaluate the precision of this ic-ELISA, each sample was tested three times in one day and repeated three times on different days. Ground corn and feed samples were tested simultaneously by the ic-ELISA established in this study and a commercial kit (with indicated detection limit at 1.25 mg/kg). These samples were extracted with methanol/water (7/3, v/v) and diluted 1:7 with PBS, each sample was tested in triplicate wells. Correlation between the two assays was investigated using linear regression (Microsoft Excel software, 2010 version). 3. Results 3.1. Preparation and identification of the OTA conjugates Fig. 5. Purification of 2D8 monoclonal antibody against ochratoxin A (A), and its specificity (B) and affinity (C). A: M is protein marker; 1 ascites and 2 purified monoclonal antibody. B: Cross-reactivity of the monoclonal antibody to other mycotoxins as determined by ic-ELISA. C: Affinity as determined by indirect ELISA.
Table 1 Cross-reactivity of the 2D8 anti-ochratoxin A monoclonal antibody to other mycotoxins. Analyte
IC50 (ng/ml)
Cross-reactivity (%)
Ochratoxin A Ochratoxin B Aflatoxin B1 Fumonisin B1 Zearalenone Deoxynivalenol Citrinin Patulin
0.38 7.1 >1 >1 >1 >1 >1 >1
100 5.4
