ABSTRACT
Simultaneous contamination of ochratoxin A (OTA) and aflatoxin B1 (AFB1) in cereals are an ongoing global concern. For this reason, a sensitive method for the simultaneous detection of OTA and AFB1 in soybean, corn, and rice is required. Herein, we prepared a bispecific monoclonal antibody (BsMAb) that specifically recognized OTA and AFB1, and subsequently developed a quantum dot nanobead (QB) lateral flow immunoassay (QB-LFIA) based on BsMAb. The half-inhibitory concentrations (IC50) for OTA and AFB1 detection were 0.73 and 0.01 µg/kg in soybean, 1.14 and 0.03 µg/kg in corn, and 1.69 and 0.07 µg/kg in rice, respectively. The average recoveries of OTA and AFB1 were 80.20–130.05% with coefficients of variation below 11%. The performance of the proposed method was compared with that of traditional LFIA, and higher sensitivity was observed. The BsMAb-based QB-LFIA was sensitive and specific for the simultaneous detection of OTA and AFB1 in cereals.
1. Introduction
Cereals such as rice, wheat, soybean, and corn are the major dietary sources around the world (Kim et al., Citation2022). However, more than 25% of agricultural products are contaminated with mycotoxins, primarily due to insufficient drying or improper storage conditions (Jin et al., Citation2021; Yang et al., Citation2018). Mycotoxins are toxic secondary metabolites produced by fungi (Kong et al., Citation2017; Liu et al., Citation2021). So far, there are more than 30 fungal strains producing toxins, with ochratoxin A (OTA) and aflatoxin B1 (AFB1) being the main contaminants in cereals, such as soybean, peanuts, wheat, rice, and corn (Liu, Hua, et al., Citation2020; Zhang et al., Citation2016). OTA and AFB1 are considered to be widespread and highly toxic mycotoxins, which have associated with child growth impairment and cause renal diseases and liver cancer (Ademola et al., Citation2021; Wu et al., Citation2014). Given that mycotoxins can travel up the food chain and become carcinogenic to humans (Liu et al., Citation2018; Tian et al., Citation2022), strict national standards are set to limit the levels of mycotoxins in cereals. OTA is listed as a potential carcinogen (Group 2B) by the International Agency for Research on Cancer (IARC) (Cheng et al., Citation2020; Liu, Huang, et al., Citation2020; Lu et al., Citation2022). The maximum residue levels (MRLs) of OTA are set at 3.0–5.0 µg/kg by the European Union (EU) and 5.0 µg/kg in all cereals by the Ministry of Agriculture in China (Bu et al., Citation2019; Xiong et al., Citation2021). As regards AFB1, it is categorized as a Group 1 carcinogen by the IARC. The MRLs of AFB1 in cereals are 2.0 µg/kg in EU and 5.0–20 µg/kg in China (Zhu et al., Citation2020). Simultaneous contamination of OTA and AFB1 in cereals is an ongoing global concern and has a toxic additive effect (Lee & Ryu, Citation2017; Wei et al., Citation2021). Accordingly, establishing a sensitive and accurate multiple detection to simultaneously monitor OTA and AFB1 in cereals is urgent.
Currently, numerous detection methods of OTA and AFB1 individually have been developed, including instrumental methods and immunoassay methods (Mejri-Omrani et al., Citation2016; Wang et al., Citation2019). Methods for simultaneously monitoring OTA and AFB1 have focused on instrumental methods, including liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Manizan et al., Citation2018; Shuib & Saad, Citation2022) and high-performance liquid chromatography (HPLC) (Keskin & Eyupoglu, Citation2023). However, these methods require expensive apparatus, tedious operations, trained operators, and long analysis time. Fortunately, lateral flow immunoassay (LFIA) is simple, rapid, low-cost, and suitable for on-site testing (Wu et al., Citation2021). Nevertheless, the LFIA based on antibodies reported for the detection of mycotoxins in cereals could recognize only one analyte because of antibody specificity. Moreover, the traditional dual LFIA based on two antibodies has the interference between different antibodies and complex steps in labelling the probes. Notably, preparing an antibody that simultaneously detects two analytes could solve the above problems.
Bispecific monoclonal antibody (BsMAb), whose Fab fragments are distinct, can specifically recognize different antigen molecules (Li et al., Citation2022). Few reports on using BsMAb in food safety have been made. Zhang (Zhang et al., Citation2022) prepared BsMAb by the hybrid-hybridoma technique and developed an immunochromatographic assay for simultaneously detecting amantadine and AFB1 in feed samples. Wang (Wang et al., Citation2016) developed multianalyte enzyme-linked immunosorbent assay (ELISA) to detect furaltadone metabolite, leucomalachite green, and malachite green based on BsMAb. Ouyang et al. (Citation2015) prepared BsMAb to determine imidacloprid and methyl parathion simultaneously in pesticides. However, BsMAb with high sensitivity for the simultaneous detection of OTA and AFB1 in cereals has not been reported. In the current work, a sensitive and specific BsMAb that simultaneously bound OTA and AFB1 was prepared by hybrid-hybridoma technology.
Moreover, novel labelling materials have the potential to improve the detection efficiency (Huang et al., Citation2020; Shan et al., Citation2015). Compared with the traditional labelling materials, quantum dot nanobead (QB) are formed by embedding quantum dots in nanoscale microspheres, which have the advantages of large molar extinction coefficient, narrow fluorescence emission, wide ultraviolet excitation, and high quantum yield (Hou et al., Citation2020; Lai et al., Citation2020). Therefore, we have prepared BsMAb that simultaneously detected OTA and AFB1 by the hybrid-hybridoma technique. Using QB as the labelling material, a BsMAb-based LFIA was established for the simultaneous detection of OTA and AFB1 in soybean, corn, and rice.
2. Materials and methods
2.1. Materials and reagents
OTA, AFB1, aflatoxin B2 (AFB2), aflatoxin M1 (AFM1), ochratoxin B (OTB), ochratoxin C (OTC), zearalenone (ZEN), fumonisin B1 (FB1), T-2 toxin (T-2), deoxynivalenol (DON), 8-azaguanine (8-AG), and 5-bromodeoyuridine (5-BrdU) were obtained from J&K Scientific Ltd. (Shanghai, China). Culture media RPMI 1640, fetal bovine serum (FBS), HAT medium supplements, HT medium supplements, and polyethylene glycol (PEG) were purchased from Gibco (NY, USA). Bovine serum albumin (BSA), 3,3,5,5-tetramethylbenzidine (TMB), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), sodium dodecyl sulphonate (SDS), and goat anti-mouse IgG-HRP were purchased from Sigma-Aldrich (St. Louis, MO, USA). Carboxylate-modified CdSe/ZnS QDs were purchased from Ocean Nano Tech, LLC (San Diego, CA, USA). The poly (maleic anhydride-alt-1-octadecene) (PMAO), CHCl3, and polymethyl methacrylate (PMMA) were purchased from Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). OTA-BSA and AFB1-BSA were prepared in our laboratory. Sample pad, absorbent pad, polyvinylchloride (PVC) backing pad, and nitrocellulose (NC) membrane were purchased from Shanghai Kinbio Tech. Co., Ltd. (Wuxi, China). A BioDot XYZ platform was purchased from BioDot (Irvine, CA, USA). A fluorescence intensity strip reader was purchased from Hangzhou Hemai Technology Co., Ltd. (Hangzhou, China). Soybean, corn, and rice were purchased from Tian Hong supermarket (Nanchang, China).
2.2. Cultivation of HGPRT and TK deficient hybridoma cells
As shown in , to obtain enzyme-deficient hybridoma cells, the chemical mutation was performed as follows: OTA hybridoma cells were cultured until logarithmic growth stage in an RPMI 1640 culture medium containing 10% FBS. After they were mutagenized with different concentration gradients (20, 40, 60, 80, and 100 µg/mL) of 8-AG for 15 days, the 8-AG resistant cells were cultured in HAT medium and more than 80% of cells death in HAT medium was taken as an indication of HGPRT deficiency. The eligible HGPRT deficient hybridoma cells were screened and named as OTA-HGPRT-. Meanwhile, AFB1 hybridoma cells were cultured with different concentration gradients (20, 40, 60, 80, and 100 µg/mL) of 5-BrdU for 15 days. Through the same process, TK deficient hybridoma cells named AFB1-TK– were obtained.
Figure 1. Process of BsMAb generation by hybrid-hybridoma technology. (a) OTA hybridoma cells were mutagenized with different concentration gradients of 8-AG. (b) AFB1 hybridoma cells were mutagenized with different concentration gradients of 5-BrdU. (c) Tetrasomal hybridoma cells were obtained by fusing the OTA-HGPRT– cell and AFB1-TK– cell.
2.3. Preparation of BsMAb
As shown in , OTA-HGPRT– cells were fused with AFB1-TK– cells at a ratio of 1:1, and tetramal hybridoma cells were obtained according to hybrid-hybridoma technology (Guo et al., Citation2018). The fused cells were grown on 96-well plates by using HAT medium for the first three days. Six days after cell fusion, the cells were cultured with HT solution. On the seventh day, the cell supernatants from each well were detected by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) to screen hybridoma cells with high affinity and inhibition to OTA and AFB1 simultaneously. Subsequently, the tetramal hybridoma cells capable of producing the highest titre and inhibition of BsMAb were selected after four rounds of subcloning. Ultimately, the designated cells were injected into the intraperitoneal cavity of female BALB/C mice (8–10 weeks of age) to produce ascites, and the ascitic fluid solution was purified with the caprylic acid-ammonium sulphate for subsequent experiments.
2.4. ic-ELISA method
ic-ELISA was performed as follows (Wen et al., Citation2020). 100 µL/well of OTA-BSA and AFB1-BSA were separately encapsulated in 96-well microplate plates for 2 h. After the first washing step with washes buffer, all wells were blocked with 200 µL/well blocking buffer and incubated for 2 h. Following the second washing step, 50 µL/well of OTA or AFB1 standard solution and 50 µL/well of gradient dilution BsMAb were added to react for 30 min. After washing, 100 µL/well of HRP-labeled goat anti-mouse IgG (3 µg/mL) was added to each well for 30 min. The excess solution was then washed, and 100 µL/well TMB substrate (2 µg/mL) was added. Following incubation for 15 min, the reaction was terminated using 50 µL/well H2SO4. At last, the results were obtained at 450 nm under a microplate spectrophotometer. All incubations were performed at 37°C.
2.5. Characterization of BsMAb
The calibration curves of BsMAb were evaluated by ic-ELISA. Meanwhile, the subtype of BsMAb was identified based on an isotyping ELISA kit for mouse monoclonal antibody, and the molecular weight of BsMAb was estimated by SDS-PAGE.
2.6 BsMAb-based QB-LFIA
2.6.1. Preparation of QB
The QB were synthesized by microemulsion technique as follows (Duan et al., Citation2015). 20 mg of CdSe/ZnS QDs was dissolved in 2 mL of CHCl3 containing PMMA (60 mg/mL) and PMAO (40 mg/mL). Then, 5 mL of SDS (3 mg/mL) was added into the organic phase to form an emulsion by sonicating in an ultrasonic cell disintegrator. Finally, the mixture was centrifuged at 4°C at 13,000 r/min for 15 min after rotary evaporation and washed three times with ultrapure water.
2.6.2. Preparation and characterization of QB-BsMAb probe
BsMAb (structure shown in Scheme 1(B)) was directly conjugated to QB with some modifications (Shen et al., Citation2015). In a typical procedure, 2 µL of QB (15 mg/mL), 30 µL of EDC (1 mg/mL), and 11 µg of BsMAb (0.2 mg/mL) were added to 0.5 mL of PB (0.01 M, pH 4.5). After stirring for 1 h, 100 µL of 10% BSA (w/v) was used to block the QB for another 1 h. Subsequently, the reaction product was centrifuged at 4°C at 13,500 r/min for 15 min and resuspended with 100 µL of PBS (0.01 M, pH 7.4). The size and morphology of QB was determined by a transmission electron microscopy (TEM, JEOL JEM-2100, Tokyo, Japan). Dynamic light scattering (DLS) analysis of QB and QB-BsMAb were conducted using a particle size analyzer (Malvern Instruments Ltd., Worcestershire, UK).
Scheme 1. Schematic illustration of the BsMAb-based QB-LFIA for the simultaneous detection of OTA and AFB1. (A) Composition of test strip. (B) Structure of BsMAb. (C) Principle of the BsMAb-based QB-LFIA. (D) Interpretation of different test results.
2.6.3. Assembly of the BsMAb-based QB-LFIA
The strip was made of three sections (Scheme 1(A)) as follows: sample pad, NC membrane, and absorbent pad. OTA-BSA and AFB1-BSA and goat anti-mouse IgG antibody were applied to the NC membranes as the T1 and T2 and C lines, respectively. After drying at 37°C for 4 h, the sample pad, NC membrane, and absorbent pad that were connected with one another from top to bottom were placed in a plastic box. The above strips were divided into 4.0 mm width and stored in a drying box for subsequent usage.
2.6.4. Detection strategy of the BsMAb-based QB-LFIA
As illustrated in Scheme 1(C,D), BsMAb-based QB-LFIA for the simultaneous detection of OTA and AFB1 was based on competitive immunoassay, whereby the OTA and AFB1 in the sample solution competed with the detection antigens on the NC and bound to the QB-BsMAb conjugates. After premixing 2 µL of QB-BsMAb probe with 100 µL of sample solution in an ELISA well for 5 min and then adding to the sample well of the strip, the mixture slowly migrated forward under capillary force. After a 15 min reaction, the fluorescence intensity of the QB-BsMAb probe on the T1, T2, and C lines could be scanned with a QB portable reader for quantitative analysis.
2.7. Optimization of key parameters
To obtain the optimum sensitivity and appropriate fluorescence intensity, we designed a single-factor analysis to optimize the key parameters of the BsMAb-based QB-LFIA, such as the labelling pH (4.0, 4.5, 5, 5.5, and 6.0), amount of BsMAb (9.0, 10.0, 11.0, 12.0, and 13.0 µg), amount of EDC (10, 20, 30, 40, and 50 µg), volume of QB-BsMAb probe (1.0, 1.5, 2.0, 2.5, and 3.0 µL), concentration of OTA-BSA (0.5, 0.6, 0.7, 0.8, and 0.9 mg/mL), and concentration of AFB1-BSA (0.6, 0.7, 0.8, 0.9, and 1.0 mg/mL).
2.8. Immunological kinetic analysis of the BsMAb-based QB-LFIA
The detection time of the quantitative assay was determined by the immunological kinetic analysis of the BsMAb-based QB-LFIA. The specific process was as follows. First, 100 µL of negative PBS (without OTA and AFB1) and positive PBS (with 5 ng/mL of OTA and 0.05 ng/mL of AFB1) were incubated with 2 µL of QB-BsMAb probe for 5 min. Then, they were dropped into the well of the test strip. After the reaction was performed for 1 min, the fluorescence intensity of the T1 and T2 line were recorded every 1 min and tracked for 50 min. Finally, the immunodynamic curves were drawn to select the optimum detection time.
2.9. Establishment of standard curves in PBS
OTA standard solution (0, 0.5, 1, 2.5, 5, 10, 25, 50, 100, and 200 ng/mL) and AFB1 standard solution (0, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2.5, and 5 ng/mL) were prepared in PBS (0.01 M, pH 7.4). Using the logarithm of the concentration of OTA and AFB1 as abscissa and T-line fluorescence intensity as the ordinate. IC50 was defined as the concentration with 50% inhibition of the signal. Each experiment was repeated in triplicate.
2.10. Specificity assessment
OTA, AFB1, AFB2, AFM1, OTC, OTB, ZEN, DON, FB1, and T-2 were used to determine the specificity of the developed assay. Each experiment was repeated in triplicate.
2.11. Stability experiment
To verify the stability of the BsMAb-based QB-LFIA, the accelerated aging experiment was applied. The assembled test strips were placed in a drying cabinet at 60°C for 7 days, and then the negative and positive samples were detected every day. Each experiment was repeated in triplicate.
2.12. Detection of OTA and AFB1 with the BsMAb-based QB-LFIA in actual sample
2.12.1. Sample pretreatment
Three different cereal samples (soybean, corn, and rice) were certified as OTA-free and AFB1-free to evaluate the proposed BsMAb-based QB-LFIA. Samples were extracted according to a reported procedure with some modifications (Pei et al., Citation2018). In a typical procedure, 5.0 g of pulverized rice, soybean, and corn samples were spiked with working standard solution and then extracted by vibrating with 25 mL of deionized water containing 80% methanol for 2 min. After centrifuging at 4000 rpm at room temperature for 2 min, the supernatant was diluted 16-fold with PBS (0.01 M, pH 7.4) for subsequent analysis.
2.12.2. Establishment of standard curves in cereal samples
The above real samples were fortified with OTA concentrations ranging within 0.5–200 µg/kg and AFB1 concentrations ranging within 0.01–5 µg/kg for the proposed BsMAb-based QB-LFIA. Standard curves were established using the log values of the concentration of OTA and AFB1 as abscissa and T-line fluorescence intensity as the ordinate. Each experiment was repeated in triplicate.
2.12.3. Recovery experiment
OTA at 1, 2.5, and 5 µg/kg and AFB1 at 0.025, 0.05, and 0.1 µg/kg were added to the above three matrices, respectively. Subsequently, the recovery rates and coefficients of variation (CVs) of OTA and AFB1 were calculated using the BsMAb-based QB-LFIA. Each experiment was repeated in triplicate.
2.13. Comparing BsMAb-based QB-LFIA with OTA-based QB-LFIA and AFB1-based QB-LFIA
To determine whether the proposed method had the advantage of detection compared with traditional QB-LFIA, OTA-based QB-LFIA and AFB1-based QB-LFIA were simultaneously prepared. Under the optimum conditions for each, the standard curves in PBS were established. Meanwhile, three spiked samples were determined by OTA-based QB-LFIA and AFB1-based QB-LFIA.
3. Results and discussion
3.1. Evaluation of BsMAb
TK deficient hybridoma cells were fused with HGPRT deficient hybridoma cells to obtain tetramal hybridoma cells. After the primary screening of HAT medium and four rounds of subcloning, the tetramal hybridoma cell (2F7) with high affinity and inhibition toward OTA and AFB1 simultaneously was screened out. The calibration curves were evaluated by ic-ELISA. The standard curve for OTA detection was y = −0.7298 lg (x) + 0.4470, the IC50 value was 0.58 ng/mL, and the linear range was 0.25–2.5 ng/mL ((A)). The standard curve for AFB1 detection was y = −0.9941 lg (x) + 0.0383, the IC50 value was 0.22 ng/mL, and the linear range was 0.05–0.5 ng/mL ((B)). As shown in (C), the mouse immunoglobulin isotyping ELISA kit indicated that the BsMAb was IgG1subclass. (D) shows that two distinct bands of light chain (approximately 25 kDa) and heavy chain (approximately 55 kDa) from BsMAb. This result showed that BsMAb was a monoclonal antibody.
Figure 2. Characterization of BsMAb. (A) Standard curve for BsMAb detection of OTA. (B) Standard curve for BsMAb detection of AFB1. (C) Subtype of BsMAb. (D) SDS-PAGE of BsMAb.
3.2. Characterization of QB and QB-BsMAb probe
As shown in the TEM image in (A,B), the QB had good dispersion and regular spherical shape with an average diameter of 164 nm. Results of DLS analysis in (C) revealed that the hydrodynamic size of the QB-BsMAb probe was 255 nm, which was 91 nm higher than that of the QB. As shown in (D), the zeta potential of the QB was −29 mV, whereas that of the QB-BsMAb probe increased it to −20 mV. These results proved that QB-BsMAb probe was successfully prepared.
Figure 3. Characterization of QB and QB-BsMAb probe. (A) TEM image of individual QB at high magnification. (B) High-resolution TEM image of the QB. (C) Hydrodynamic diameter variations of QB and QB-BsMAb probe. (D) Zeta potential values of QB and QB-BsMAb probe.
3.3. Optimization of the detection conditions of the BsMAb-based QB-LFIA
The coupling efficiency and activity of the antibodies affected the conjugation between the BsMAb and QB (Lou et al., Citation2019). The labelling pH was first optimized. As shown in (A), the inhibition ratios of OTA and AFB1 increased gradually with increased pH. However, considering that the signal strength of OTA and AFB1 at pH 5.0 was less than that at pH 4.5, we selected pH 4.5 as the optimum labelling pH. To explore the influence of the amount of BsMAb, BsMAb with different amounts were prepared. As shown in (B), the signal strength of the OTA reached maximum when 10 µg of BsMAb was applied, but the inhibition rates of OTA and AFB1 were lower than the amount of BsMAb at 11 µg. Hence, the optimum amount of BsMAb was 11 µg. However, the surface of QB was covered with carboxyl groups, and activation was required before covalently coupling the amino group of BsMAb. Accordingly, the influence of the amount of EDC was explored in (C). The signal strength of OTA and AFB1 increased gradually with increased additive amount of EDC consumption. However, the inhibition rates of OTA and AFB1 reached the maximum when the amount of EDC was 30 µg. Thus, the optimum amount of EDC was 30 µg. Excessive volumes of QB-BsMAb probe are known to result usually in decreased inhibition rate. Thus, as shown in (D), the signal strength of OTA and AFB1 increased with increased volume of QB-BsMAb probe. Although the highest signal strength of OTA and AFB1 was obtained with 3.0 µL of QB-BsMAb probe, the inhibition rates were low. When the volume of QB-BsMAb probe was 2.0 µL, the inhibition rates were the highest while the signal strength was high. Therefore, 2.0 µL of QB-BsMAb probe was selected for the BsMAb-based QB-LFIA.
Figure 4. Optimization of the key parameters of the BsMAb-based QB-LFIA. (A) Labelling pH (4.0, 4.5, 5, 5.5, and 6.0). (B) Amount of BsMAb (9.0, 10.0, 11.0, 12.0, and 13.0 µg). (C) Amount of EDC (10, 20, 30, 40, and 50 µg). (D) Volume of QB-BsMAb probe (1.0, 1.5, 2.0, 2.5, and 3.0 µL). (E) Concentration of OTA-BSA (0.5, 0.6, 0.7, 0.8, and 0.9 mg/mL). (F) Concentration of AFB1-BSA (0.6, 0.7, 0.8, 0.9, and 1.0 mg/mL). (G) Immunological kinetic curve of OTA. (H) Immunological kinetic curve of AFB1.
Furthermore, the effects of different concentrations of OTA-BSA and AFB1-BSA on the T line were studied. As can be seen from (E), the signal strength reached its maximum when the OTA-BSA concentration was 0.8 mg/mL, but the inhibition rate was lower than that at 0.7 mg/mL. In (F), the signal strength of AFB1 increased with increased concentration of AFB1-BSA. The highest inhibition rate of AFB1 was obtained and the signal strength was high when the AFB1-BSA concentration was 0.8 mg/mL. Therefore, the optimum concentration of OTA-BSA and AFB1-BSA were 0.7 and 0.8 mg/mL, respectively.
3.4. Immunological kinetic analysis of the BsMAb-based QB-LFIA
Immunological kinetic curves were drawn by plotting FT and immune response time within 50 min. PBS buffer served as a negative control, and PBS diluted OTA standard solution (5 ng/mL) and AFB1 (0.05 ng/mL) standard solution served as a positive control. As the trends in (G,H), the fluorescence intensity of OTA reached a stable state at 19 min and that of AFB1 attained equilibrium after 24 min. Therefore, optimum detection time was 24 min.
3.5. Detection of OTA and AFB1 with the BsMAb-based QB-LFIA in PBS and cereal samples
Under the optimum experimental conditions, the sensitivity for simultaneous detection of OTA and AFB1 in PBS, soybean, corn, and rice samples were assessed. The linear regression equation of the standard curve for OTA detection in PBS was y = −1935.4596 lg (x) – 2065.1067 (R2 = 0.9897), the IC50 value was 1.83 ng/mL, and the linear range was 0.5–10 ng/mL ((A)). For the quantitative detection of OTA in soybean, corn, and rice samples ((B–D)), the linear regression equations were y = −1198.0649 lg (x) + 1207.0191 (R2 = 0.9881), y = −1568.7037 lg (x) + 2144.3751 (R2 = 0.9823), and y = −1436.5792 lg (x) + 2208.9969 (R2 = 0.9937), respectively. The IC50 values were 0.73, 1.14, and 1.69 µg/kg, and the corresponding linear ranges were 0.1–5, 0.5–10, and 1–10 µg/kg. Similarly, we established the standard curves for AFB1 detection by using the BsMAb-based QB-LFIA. As shown in (E), the linear regression equation for AFB1 detection in PBS was y = −870.7719 lg (x) – 551.1567 (R2 = 0.9947), the IC50 value was 0.03 ng/mL, and the linear range was 0.01–0.1 ng/mL. For the quantitative detection of AFB1 in soybean, corn, and rice samples ((F–H)), the linear regression equations were y = −505.9941 lg (x) – 148.8251 (R2 = 0.9904), y = −748.8796 lg (x) – 527.4102 (R2 = 0.9997), and y = −750.7227 lg (x) – 265.9100 (R2 = 0.9873). The IC50 values were 0.01, 0.03, and 0.07 µg/kg, and the corresponding linear ranges were 0.0025–0.25, 0.01–0.1, and 0.0025–0.25 µg/kg. Due to the different content of water, protein, and fat, resulting in the large variation in the detection performance of three cereals sample. Notably, lists the IC50 values and linear range of the BsMAb-based QB-LFIA for the simultaneous detection of OTA and AFB1 in PBS, soybean, corn, and rice samples. Results indicated that the BsMAb-based QB-LFIA met the national testing requirements for OTA and AFB1 in soybean, corn, and rice samples.
Figure 5. Standard curves of the BsMAb-based QB-LFIA for the simultaneous detection of OTA and AFB1 in PBS and cereal samples. (A) PBS spiked with OTA. (B) Soybean spiked with OTA. (C) Corn spiked with OTA. (D) Rice spiked with OTA. (E) PBS spiked with AFB1. (F) Soybean spiked with AFB1. (G) Corn spiked with AFB1. (H) Rice spiked with AFB1.
Table 1. Calibration curves of the BsMAb-based QB-LFIA for the analysis of OTA and AFB1.
3.6. Specificity of the BsMAb-based QB-LFIA
Ten kinds of mycotoxins (OTA, AFB1, AFB2, OTC, AFM1, OTB, ZEN, DON, FB1, and T-2) were selected for the specificity experiment by using the proposed BsMAb-based QB-LFIA. In (A), the BsMAb-based QB-LFIA for the simultaneous detection of OTA and AFB1 exhibited negligible cross-reactivity with the other four mycotoxins except OTB, OTC, AFM1 and AFB2. However, a certain extent of CR with OTB, OTC, AFM1 and AFB2 detection was consistent with previous finding (He et al., Citation2021). All these results verified that the BsMAb-based QB-LFIA had good specificity for OTA and AFB1.
Figure 6. Specificity and stability of the BsMAb-based QB-LFIA. (A) Specificity of the BsMAb-based QB-LFIA. Stability results of (B) OTA and (C) AFB1.
3.7. Stability of the BsMAb-based QB-LFIA
As shown in (B,C), no significant change occurred in fluorescence intensity after 7 days of storage in a drying cabinet at 60°C. According to the Arrhenius equation (Wang et al., Citation2022), the shelf life of the test strip at room temperature (25°C) was 6 months. These results indicated that the BsMAb-based QB-LFIA was stable.
3.8. Recovery experiment of the BsMAb-based QB-LFIA
The recoveries and CVs were calculated to obtain the precision and accuracy of the BsMAb-based QB-LFIA in soybean, corn, and rice samples. shows the average recoveries and CVs of OTA in spiked soybean, corn, and rice samples. The average recoveries were 102.26–111.44% with CVs 1.09–5.16% in spiked soybean samples, 98.82–129.35% with CVs 1.32–10.41% in spiked corn samples, and 80.20–106.78% with CVs 2.41–10.31% in spiked rice samples for OTA detection. Furthermore, the average recoveries were 86.81–104.99% with CVs 2.50–5.63% in spiked soybean samples, 82.02–130.05% with CVs 1.86–5.59% in spiked corn samples, and 80.50–114.09% with CVs 1.62–5.97% in spiked rice samples for AFB1 detection. These data indicated that the BsMAb-based QB-LFIA could precisely and reliably monitor OTA and AFB1 contamination in soybean, corn, and rice samples.
Table 2. Results of recovery experiment (n = 3).
3.9. Comparing BsMAb-based QB-LFIA with OTA-based QB-LFIA and AFB1-based QB-LFIA
Under the optimum experimental conditions, the OTA-based QB-LFIA and AFB1-based QB-LFIA were established to detect OTA and AFB1, respectively. The standard curves of OTA-based QB-LFIA in PBS, soybean, corn, and rice samples were drawn. The IC50 values for OTA detection in PBS, soybean, corn, and rice samples were 4.35, 3.21, 2.73, and 1.47 µg/kg, respectively ((A–D)). The IC50 values for AFB1 detection in PBS, soybean, corn, and rice samples were 0.08, 0.05, 0.07, and 0.09 µg/kg ((E–H)). The sensitivity of three different QB-LFIA for OTA and AFB1 detection in PBS, soybean, corn, and rice samples was compared to evaluate whether the BsMAb had the advantage of detection in LFIA. Fmax (maximum fluorescence intensity value) and IC50 value are the key parameters to evaluate the performance of LFIA (Zhang et al., Citation2013). Higher Fmax/IC50 value indicated better performance in detection. As shown in , regardless of OTA or AFB1 detection, the calculated Fmax/IC50 values of the BsMAb-based QB-LFIA were higher than that of the traditional test strip in PBS and three samples. This finding indicated that the proposed method had the higher sensitivity and advantages of detection compared with traditional QB-LFIA.
Figure 7. Standard curves of the OTA-based QB-LFIA and AFB1-based QB-LFIA in PBS and cereal samples. (A) PBS spiked with OTA. (B) Soybean spiked with OTA. (C) Corn spiked with OTA. (D) Rice spiked with OTA. (E) PBS spiked with AFB1. (F) Soybean spiked with AFB1. (G) Corn spiked with AFB1. (H) Rice spiked with AFB1.
Table 3. Contrast of sensitivity in the proposed method for OTA and AFB1 detection compared with traditional OTA-based QB-LFIA and AFB1-based QB-LFIA.
The sensitivity of the proposed method was at least 2-fold than that of the traditional LFIA for OTA and AFB1 detection in PBS, and the degree of increase in sensitivity varied in different samples due to the matrix effect. Guo et al. (Citation2009) prepared a BsMAb and compared it with a monoclonal antibody based on colloidal gold in water samples, whose visual result was that the monoclonal antibody had higher sensitivity for two pesticides. They thought it may be attributed to the structure of BsMAb IgG, which has only one binding site for two analytes compared with the monoclonal antibody whose molecule has two binding sites for one analyte. However, this result is the opposite of what we found. The reasons for the higher sensitivity of BsMAb obtained was speculated as follows. First, the gene of hybridoma cells changed during the mutagenesis of 8-AG and 5-BrdU, and different mutation effects were generated with different mutagenic agents. Second, the affinity of BsMAb for OTA and AFB1 improved, resulting in higher binding efficiency of antigen and antibody. Third, the BsMAb had higher targeting than traditional single specific bivalent monoclonal antibody. Lastly, the mode of double T lines reduced steric hindrance and facilitated the binding of the probe and antigen on the T line. Accordingly, we established a new detection method and provided a positive example for the application of BsMAb through the hybrid-hybridoma technique in food safety.
4. Conclusions
We obtained tetramal hybridoma cells producing BsMAb that had high affinity and inhibition to OTA and AFB1 simultaneously. The developed BsMAb presented good specificity. Using the prepared BsMAb, a convenient multiplexed LFIA based on QB was established to simultaneously detect OTA and AFB1 in soybean, corn, and rice. The IC50 values for OTA and AFB1 detection were 0.73 and 0.01 µg/kg in soybean, 1.14 and 0.03 µg/kg in corn, and 1.69 and 0.06 µg/kg in rice, which met the national testing requirements for OTA and AFB1 in cereals. Additionally, the recovery rate varied from 80.20% to 130.05% with CVs lower than 11%. The comparison between the proposed BsMAb-based QB-LFIA and traditional LFIA illustrated that our method had higher sensitivity for OTA and AFB1 detection. To the best of our knowledge, a QB-LFIA based on BsMAb with high sensitivity in the simultaneous detection of OTA and AFB1 in cereals has not yet been reported. This BsMAb-based QB-LFIA has a promising application in monitoring OTA and AFB1 contamination in soybean, corn, and rice, and provides new ideas for the simultaneous detection.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Additional information
Funding
References
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