3.1. Chemicals and Instruments
Aflatoxin G1 (solid), aflatoxin G2, B1, B2, and M1 standard solutions, Freund′s complete adjuvant and Freund’s incomplete adjuvant, bovine serum albumin (BSA), ovalbumin (OVA), goat anti-mouse immunoglobulin G horseradish peroxidase (IgG/HRP), 3,3,5,5-tetra-methylbenzidine (TMB), hypoxanthine aminopterin thymidine (HAT), hypoxanthine thymidine (HT), polyethylene glycol 1450 (PEG 1450, 50%), and mouse monoclonal antibody ISO2-1 kit were purchased from Sigma-Aldrich (St. Louis, MO, USA). Culture media RPMI-1640 with l-glutamine and HEPES (free acid, 238.3 g·L−1) was obtained from Thermo Hyclone (Logan, UT, USA). Penicillin (+10,000 Units per milliliter) and streptomucin (+10,000 μg·mL−1) was from Gibco (Grand Island, NY, USA). Hybridoma Fusion and Cloning Supplement (HFCS) was obtained from Invitrogen (Waltham, MA, USA). All chemicals and organic solvents were analytical reagent grade. Female Balb/c mice of 8–10 weeks old were purchased from Wuhan Institute of Biologic Products (Wuhan, China). SP2/0 myeloma cells were purchased from China Center for Type Culture Collection (CCTCC, Wuhan, China). Cell culture plates (6, 24, and 96 wells) were from Shanghai Sunub Bio-Tech Development, Inc. (Shanghai, China) Cell culture flasks were from Iwaki Co. (Iwaki, Japan). 96-well polystyrene microplates were from Costar (Cambridge, MA, USA). A microtiter plate reader of Spectra Max M2e with a computer-controlled system was from PerkinElmer (Waltham, MA, USA). HPLC series (Agilent 1100, Agilent Technologies, Santa Clara, CA, USA) consisted of a fluorescence detector, a C18-column (5 µm particle size, 150 mm × 4.6 mm I.D., Agilent Technologies, Santa Clara, CA, USA), and a post-column derivation system.
3.2. Solutions, Buffers, and Growth Media
The following solutions and media were used: (1) stock solutions of aflatoxin B1, B2, G1, G2, and M1 at a concentration of 1 μg·mL−1 were prepared in methanol and diluted to standard solution with PBS; (2) coating buffer was 0.1 M carbonate buffer, pH 9.6; (3) phosphate buffer saline (PBS), pH 7.4; (3) washing buffer was 0.05% Tween20 (v/v) in PBS (PBST); (4) blocking buffer was 1% OVA (w/v) in PBST; (5) substrate solution was freshly prepared with 10 mL pH 5.0 phosphate-citrate buffer, 1 mL 2 mg·mL−1 TMB (in ethanol) and 25 μL 3% H2O2; (6) stop solution was 2 mol·L−1 H2SO4; (7) complete medium was 78 mL RPMI-1640 medium, 20 mL fetal bovine (inactivated at 56 °C for 30 min), 1 mL antibiotics (+10,000 Units per milliliter penicillin, +10,000 μg per milliliter streptomucin) and 1 mL HEPES; (8) freezing solution was 10% (v/v) dimethyl sulphoxide (DMSO) in complete medium.
3.3. Preparation of Artificial Antigen
was conjugated with BSA and OVA to yield immunogens and coating antigens, respectively, according to the method of reductive alkylation [20
]. Briefly, 2.5 mg of aflatoxin G1
was dissolved in 2.1 mL acetone, then 30 μL of 10% H2
was added into the solution. After the mixture was stirred with a magnetic bar at 60 °C for 5 h, 1.5 mL of Milli-Q water was added. The acetone in the mixture was removed with a vacuum rotary evaporator, and the product was extracted with chloroform (5 mL × 4) and washed with 4.5 mL of Milli-Q water. After the removal of the chloroform, the resulting powder was deep yellow. The powder (0.6 mg) was added into a PBS buffer containing 10 mg BSA (or OVA) and reacted at 37 °C for 30 min. After that, 70 μL of 1 mg/mL NaBH4
in water was added. The reaction was continued for another 30 min at 4 °C and then stopped by adding 35 μL of 0.1 N HCl. The conjugates were dialyzed against two liters of PBS for three days (two changes of buffer each day). The AFG1
-BSA conjugate was stored at −20 °C and the AFG1
-OVA conjugate was mixed with an equal volume of glycerol and stored at −20 °C until use.
3.5. Cell Fusion and Cloning
SP2/0 myeloma cells were injected subcutaneously into a free Balb/c mouse to produce SP2/0 solid tumor. The SP2/0 myeloma cells were collected aseptically from the solid tumor and homogenized in a glass homogenizer. The cell supernatant was separated by density gradient centrifugation with lymphocyte separation medium, and then stored at 4 °C in RPMI 1640 medium for later use. Cell fusion was carried out according to the conventional method [21
]. The mouse of interest was sacrificed and spleen cells were aseptically harvested. Spleen cells (108
) were fused with SP2/0 myeloma cells (107
) at a ratio of 5–10:1 in the presence of PEG1450. The fused cells were suspended in 20 mL of complete medium and then mixed with 80 mL semi-solid complete medium containing 1% HAT, methylcelluose, hybridoma fusion and cloning supplement (HFCS), and non-essential amino acids. The fused cells were evenly distributed into six-well plates and incubated in 5% CO2
at 37 °C. After 12 days of cultivation, many tips of monoclonal hybridomas that could be seen by naked eyes in the semi-solid medium were single picked out to 96-well culture plates containing 1% HT complete medium. Culture supernatants from each well were screened when the hybridomas reached half-confluence (15–17 days) [22
]. The hybridomas whose culture supernatants gave an absorption value of over 2.0 by indirect noncompetitive ELISA were transferred to 24-well microculture plates. Supernatants from 24-well plates were tested again both in indirect noncompetitive and competitive ELISA. Only those hybridomas that retained high absorption values and had good cross-reactivity with aflatoxins G1
were selected for further screening. Hybridomas of interest were cultivated for three months for detecting the stability then propagated, and stored in freezing solution according to the freezing procedure: 30 min at 4 °C, overnight in gas of liquid nitrogen and cryopreserved in liquid nitrogen.
3.6. ELISA Screening
The indirect noncompetitive ELISA procedure [15
] was carried out with minor modifications. AFG1
-OVA of 2 μg·mL−1
was diluted with coating buffer, coated onto microtiter plates (100 μL per well), and incubated overnight at 4 °C. The coated plates were washed three times with PBST and blocked with blocking buffer for 2 h at 37 °C (200 μL per well). After an additional wash, 100 μL of supernatant was added to each well and incubated for 2 h at 37 °C. Following the same washing step, 100 μL per well of IgG-HRP solution (1:20,000 in PBST) was added and incubated for 1 h at 37 °C. After the sixth wash, color development was carried out by adding freshly prepared substrate solution (100 μL per well), gentle shaking, and incubation for 15 min at 37 °C [23
]. Stop solution (50 μL) was added to each well to stop the enzymatic reaction. The absorbance was measured at 450 nm with a microplate reader. The competitive indirect ELISA procedure was carried out to screen clones that had specificity for G-group aflatoxins. This method was similar to that of indirect ELISA, except that mixing the same volume of aflatoxin standard solutions and of supernatant (50 μL: 50 μL) was done before adding the mixture to each well [24
3.7. Determination of Isotype, Sensitivity, and Specificity
Ascetic fluids were prepared according to the method described previously [25
] with minor modifications. The fluids were purified by octanoic acid-octanoic acidoctanoic acidoctanoic acidammonium sulfate precipitation and dialyzed with deionized water for three days. The isotype of each monoclonal antibody was determined by a commercially available isotyping kit from Sigma (St. Louis, MO, USA).
The competitive indirect ELISA format described above was carried out to evaluate the sensitivity of monoclonal antibodies. Standard solutions of aflatoxin B1, B2, G1, G2, M1, with eight concentrations ranging from 0.125 to 640 ng·mL−1 were prepared. The absorbance at each aflatoxin concentration and the absorbance without aflatoxin were referred as B and B0, respectively. Sigmoidal curves were plotted as inhibitions (B/B0) versus logarithm of analyte concentration. The cross-reactivities (CRs) of antibody for different analogies were measured with the IC50 values (analyte concentration resulting in half-maximum inhibition) and calculated as follow: CR (%) = (IC50 AFG1/IC50 analyte) × 100.
The indirect noncompetitive ELISA was carried to determine the titer. The ELISA titer of ascetic fluid was defined as the highest ascetic dilution which gave an absorbance greater than the pre-immuned control serum in the first dilution.
3.8. Optimization of CI-ELISA
Initial experiments were performed to assess the optimal working concentrations of coating antigen and antibody. Different concentrations of coating antigen (0.5, 1, 2, and 4 μg·mL−1
) and antibody dilutions (1:16,000, 1:32,000, 1:64,000, 1:128,000) were used in combination and optimized by comparing the resulting dose-response curves. According to an optimized experimental design [26
], the optimal combination was selected when optical density was about 1.0.
With the optimized working concentrations of coating antigen and antibody, a series of parameters including blocking buffer, organic solvent concentration, and reaction buffer were evaluated to improve the sensitive of CI-ELISA [27
]. The optimum conditions were determined by IC50
, which is the most significant criterion for the immunoassay. The effect of organic solvent (10%, 20%, and 40% of methanol), blocking buffer (1% OVA, 1% BSA, 1% gelatin and without blocking agent) and reaction buffer (PBS and PBST) on sensitivity of ELISA was studied sequentially. Based on the optimal conditions, CI-ELISA using different aflatoxin G1
standards (0.125–640 ng·mL−1
) was repeated five times to establish quantitive standard curve for aflatoxin G1
and evaluate the precision and sensitivity.
3.9. Analysis of Peanut Samples Spiked with and Naturally Contaminated by Aflatoxin G1
The spike and recovery study was carried out by spiking uncontaminated peanuts samples with different amounts of G-group aflatoxins (G1
]. Three working concentrations (5, 50, and 200 ng·mL−1
) were prepared. Each peanut sample (cut into pieces) 1.0 g in 20-mL glass tube was added with 15, 150, or 600 ng the analytes (spiking levels were 15, 150, and 600 ng·g−1
respectively). And then the spiked samples were extracted with an 80% methanol (v
) solution that contained 4% (w
) NaCl and treated by ultrasound for 5 min. The mixture was allowed to stand for 30 min to separate the supernatant. Each clear supernatant was diluted in five volumes of PBS and analyzed by both the developed CI-ELISA and a HPLC equipped with a fluorescence detector (wavelength of excitation 360 nm and emission 420 nm), which is the Chinese detection standard method [29