Aflatoxin b1 monoclonal antibody resistant to polyphenol flavonoid interference, and preparation method and application thereof in colored coarse grain samples

Abstract

This invention belongs to the field of immunoassay detection technology for fungal toxins, and discloses a monoclonal antibody against aflatoxin B1 resistant to polyphenolic flavonoid interference, its preparation method, and its application in colored grain samples. The amino acid sequences of the light chain variable regions CDR-L1 and CDR-L3 of this monoclonal antibody are shown in Seq ID No. 1 and 2, respectively; the amino acid sequence of CDR-L2 is STN; the amino acid sequences of the heavy chain variable regions CDR-H1~CDR-H3 are shown in Seq ID No. 3~5, respectively; and the full-length amino acid sequences of the light and heavy chain variable regions are shown in Seq ID No. 6 and 7, respectively. This antibody has an IC50 of 0.047 ng / mL against aflatoxin B1 and shows no significant inhibitory effect on 10 major polyphenolic flavonoid compounds in grains, including epicatechin, catechin, and quercetin, demonstrating excellent resistance to polyphenolic flavonoid interference. This invention establishes an indirect competitive lateral flow chromatography immunoassay method based on this antibody, achieving spiked recoveries of 78.36%–93.18% for six types of miscellaneous grains, including mung beans, red beans, black beans, red rice, black rice, and purple rice. The antibody of this invention exhibits high specificity and sensitivity, and the detection method is simple to operate and requires no complex purification treatment, enabling rapid and accurate detection of aflatoxin B1 in miscellaneous grains, suitable for batch sample screening.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay detection technology for fungal toxins, specifically relating to a monoclonal antibody that is highly sensitive to aflatoxin B1 and has no cross-reactivity with polyphenolic flavonoid molecules, and the application of colloidal gold immunochromatography technology based on this antibody in the rapid detection of aflatoxin B1 in colored rice. Background Technology

[0002] As one of the world's most important staple crops, rice provides a major energy source for more than half of the world's population. In recent years, colored grains have become increasingly popular among consumers due to their rich natural active ingredients, among which anthocyanins, polyphenols, and other functional substances have been proven to have various physiological activities such as antioxidation and anti-inflammation. However, rice faces the risk of fungal contamination throughout the entire supply chain, from field planting to storage and processing. Under high temperature and humidity conditions, toxin-producing fungi such as Aspergillus flavus can easily grow and multiply on rice, producing aflatoxin B1 (AFB1). As one of the most toxic fungal toxins, AFB1 has clear hepatotoxicity, carcinogenicity, teratogenicity, and genotoxicity, and is classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC). Currently, countries around the world have set strict limits for aflatoxin in cereals. my country's national standards stipulate that the maximum allowable limit for AFB1 in cereals and their products is 5-20 μg / kg. Therefore, developing rapid and accurate AFB1 detection technology is of great practical significance for ensuring the safety of rice consumption.

[0003] Immunochromatography has become one of the mainstream technologies for rapid on-site food safety testing due to its advantages such as ease of operation, rapid detection, and low cost. However, existing commercially available AFB1 test strips are prone to false positives when applied to colored rice samples, affecting the reliability of the test results. The main reason for this is that the matrix of colored rice contains a large amount of flavonoids and polyphenolic secondary metabolites. Taking black rice as an example, its anthocyanin content can reach 215.6 mg / 100g, its proanthocyanidin content is about 156.3 mg / 100g, and its catechin content is 45.8 mg / 100g; the total anthocyanin content of purple rice is about 189.4 mg / 100g. In addition to being rich in the above-mentioned polyphenolic compounds, colored beans are also rich in flavonoids, such as genistein, quercetin, and kaempferol. The concentration levels (ppm level) of these matrix components are much higher than the content (ppb level) of AFB1 in actual contaminated samples.

[0004] From a molecular structural perspective, the AFB1 molecule consists of a bifuran ring and a coumarin core. Its spatial conformation and physicochemical properties show a certain degree of similarity to the benzo-γ-pyranone skeleton of flavonoids and the polyhydroxy aromatic structure of polyphenols in colored rice. Previous studies have shown that when the two-dimensional structural similarity between interfering substances and target analytes exceeds 0.6, they are likely to be recognized by antibodies targeting the target analyte under high concentration conditions, resulting in cross-reactions. Therefore, the high abundance of polyphenolic flavonoids in colored rice poses a challenge to AFB1 immunoassay, as antibodies may mistakenly identify these matrix components as target molecules, leading to false positive results. Currently, research on AFB1 monoclonal antibodies, both domestically and internationally, largely focuses on improving antibody affinity for target toxins or broadening the recognition spectrum of antibodies against different structural analogs of aflatoxin families. Research on AFB1 antibodies specifically targeting polyphenolic flavonoid matrix interference is still relatively limited.

[0005] In summary, obtaining a monoclonal antibody that specifically recognizes AFB1 and does not cross-react with the main polyphenolic flavonoid components in colored rice is crucial for establishing a high-accuracy immunochromatographic detection method. Further optimization of the detection system parameters, coupled with simple and efficient sample pretreatment techniques, promises to achieve rapid and accurate detection of AFB1 in colored rice samples. Summary of the Invention

[0006] Based on the current state of technology, and addressing the technical challenge of false positive results in colored rice samples due to interference from polyphenolic flavonoid matrix in existing AFB1 immunoassay methods, this invention provides an AFB1 monoclonal antibody with anti-polyphenolic flavonoid interference properties, and the application of an immunochromatographic assay method based on this antibody in the analysis of colored rice samples.

[0007] To achieve the objectives of this invention, the technical solution is as follows: The anti-AFB1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region. Specifically, the CDR-L1 amino acid sequence of the light chain variable region is shown in SEQ ID NO.1 of the sequence listing, the CDR-L2 amino acid sequence is STN, and the CDR-L3 amino acid sequence is shown in SEQ ID NO.2 of the sequence listing; the CDR-H1 amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.3 of the sequence listing, the CDR-H2 amino acid sequence is shown in SEQ ID NO.4 of the sequence listing, and the CDR-H3 amino acid sequence is shown in SEQ ID NO.5 of the sequence listing.

[0008] The full-length amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.6 of the sequence listing, and the full-length amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7 of the sequence listing.

[0009] The present invention also provides a nucleic acid molecule encoding the above-mentioned monoclonal antibody.

[0010] Furthermore, the nucleic acid molecule contains a nucleotide sequence encoding the light chain variable region as shown in SEQ ID NO. 8 of the sequence listing, and a nucleotide sequence encoding the heavy chain variable region as shown in SEQ ID NO. 9 of the sequence listing.

[0011] Using the aforementioned monoclonal antibody as a specific recognition element, and employing an indirect competitive colloidal gold immunochromatographic test strip method, AFB1 in colored rice samples can be detected with high specificity. This method can effectively reduce the interference of polyphenolic flavonoid molecules in the colored rice matrix on the detection signal, thereby achieving accurate quantitative detection of AFB1 content in the sample.

[0012] Furthermore, the immunochromatographic method uses AFB1-bovine serum albumin conjugate as the detection line coating antigen.

[0013] Furthermore, the optimal streak concentration of the coating antigen is 0.06 mg / mL, the optimal labeling amount of the gold-labeled antibody is 7 μg / mL, and the optimal dilution factor for sample detection is 40 times.

[0014] Furthermore, the operation method of the indirect competitive side-flow chromatography test strip includes the following steps: Step 1, Preparation of colloidal gold-monoclonal antibody probe: Adjust the pH of the colloidal gold solution to 3-5, add the mAb-1H5 monoclonal antibody of this invention, mix thoroughly, and let it stand at room temperature for reaction; then add bovine serum albumin solution for blocking, and continue to stand; centrifuge the reaction solution, discard the supernatant, and resuspend the precipitate in ultrapure water to obtain the colloidal gold probe, which is stored in a refrigerator for later use.

[0015] Step 2, Assembly of colloidal gold immunochromatographic test strips: The nitrocellulose membrane, absorbent pad, and glass fiber pad are sequentially pasted onto the PVC backing. The test line (T line) and control line (C line) are sprayed onto the detection surface of the nitrocellulose membrane. The test line is coated with AFB1-BSA conjugate antigen, and the control line is coated with goat anti-mouse IgG secondary antibody.

[0016] Step 3, Sample Detection Procedure: Add a quantitative amount of the colloidal gold probe of this invention to a 96-well plate and incubate it with AFB1 standard solutions of different concentrations or the extract of the sample to be tested. If the sample to be tested does not contain AFB1, the colloidal gold probe will specifically bind to the coating antigen on the test line. The unbound probes continue to ascend and bind to the goat anti-mouse secondary antibody on the control line. At this time, both the T line and the C line will show red bands, indicating a negative result. If the sample to be tested contains AFB1, the toxin molecules will first bind to the antibody on the colloidal gold probe, occupying the antibody binding site, resulting in a reduction in the number of probes that can bind to the coating antigen on the test line. This is manifested as a lighter color in the T line, and the higher the toxin concentration in the sample, the lighter the color of the T line, until it disappears completely, indicating a positive result. If the C line does not develop color, it indicates that the test strip is invalid or the operation is incorrect, and the test result is invalid.

[0017] Step 4, antibody specificity verification results: The IC50 value of the antibody AFB1 was 0.012 ng / mL as determined by indirect competitive ELISA. At a concentration of 100 μg / mL, it showed no significant inhibitory effect on 10 representative polyphenolic flavonoids in colored rice (including epicatechin, catechin, quercetin, piperidin, rutin, kaempferol, isoquercetin, and naringin), with inhibition rates all <30%. This indicates that the antibody has almost no recognition effect on the above-mentioned polyphenolic flavonoids and can effectively tolerate matrix interference in colored grain samples.

[0018] This invention further provides a method for quantifying AFB1 in colored grain samples, specifically including the following steps: AFB1 at standard concentrations was added to mung beans, red beans, black beans, purple rice, red rice, and black rice, respectively. Samples were placed in centrifuge tubes, and eutectic solvent was added. The tubes were vortexed, centrifuged, and the supernatant was diluted 20 times with PBST. Indirect competitive side-flow chromatography strips were used for detection to establish a standard working curve for AFB1 detection. Figure 4 Quantitative detection of the sample is achieved based on the standard curve.

[0019] The eutectic solvent (DES) is a mixture of betaine and ethylene glycol in a molar ratio of 1:2, with betaine as the hydrogen bond donor and ethylene glycol as the hydrogen bond acceptor.

[0020] The innovation of this invention lies in the following: First, a highly specific and sensitive AFB1 monoclonal antibody was obtained. Then, an indirect competitive colloidal gold immunochromatographic detection method was constructed using this antibody as the core recognition element. This method can be directly applied to the quantitative detection of AFB1 in colored grains with good accuracy. Samples require only a simple extraction step before detection, making the operation process simple. The antibody has high specificity, and no additional purification steps such as solid-phase extraction are needed, saving detection time and reagent costs. It is particularly suitable for rapid screening of large batches of samples, providing new technical support for rapid detection of food safety issues.

[0021] The beneficial effects of this invention are as follows: 1. The monoclonal antibody obtained by screening in this invention has excellent specificity. At a high concentration of 100 μg / mL, it has no significant inhibitory effect on the main polyphenolic flavonoid matrix components in colored grains. It can be directly used for the detection of AFB1 in complex matrix samples, and the detection results are accurate and reliable.

[0022] 2. The indirect competitive colloidal gold immunochromatographic test strip detection method established in this invention can accurately quantify AFB1 in colored rice samples. It has many advantages such as high accuracy, suitable linear range, low detection cost, convenient use, and short analysis time. It is particularly suitable for the detection of mycotoxins in matrices with high polyphenol flavonoid content, providing an efficient technical means for the quality and safety management of colored rice and other specialty agricultural products.

[0023] The nucleotide and amino acid sequences of the light chain variable region and heavy chain variable region of the anti-AFB1 monoclonal antibody involved in this invention are shown in the sequence listing. Attached Figure Description

[0024] Figure 1 This invention optimizes the antigen streaking concentration. Figure 2 To optimize the antibody addition amount for this invention; Figure 3 The amount of gold-labeled antibody added in this invention was optimized; Figure 4 This is the standard curve for AFB1 in this invention; Figure 5 The effect of 10 polyphenolic flavonoid compounds on the color development of test strips based on monoclonal antibodies AFB1-1H5 and AFB1-2H4 was investigated. Figure 6 Optimize the extraction volume of six grain samples using DES as the extraction reagent. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional experimental conditions in the art or the conditions recommended in the reagent instructions.

[0026] Example 1: Preparation of aflatoxin B1 immunogen and coating agent First, 2 mg of AFB1 standard and 2 mg of carboxymethyl hydroxylamine (CMO) were weighed and dissolved together in 2 mL of pyridine. The mixture was then placed in a 70°C water bath and magnetically stirred for 6 hours. After the reaction, the solvent was dried under nitrogen. The residue was dissolved in 3 mL of 0.1 mol / L sodium bicarbonate solution, and the pH was adjusted to approximately 3.0 with 0.1 mol / L hydrochloric acid. The mixture was then extracted three times with an equal volume of ethyl acetate. The organic phases were combined and dried under nitrogen to obtain the carboxylated AFB1 hapten (AFB1-CMO).

[0027] Subsequently, the hapten was activated. 2.0 mg of the AFB1-CMO hapten prepared above was accurately weighed and dissolved in 5 mL of N,N-dimethylformamide (DMF). 6 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 2 mg of N-hydroxysuccinimide (NHS) were added sequentially. The mixture was magnetically stirred at room temperature for 12 hours. After the reaction was completed, the mixture was centrifuged at 3000 r / min for 5 minutes, and the supernatant was taken as the activated hapten solution.

[0028] Then, accurately weigh 2 mg of keyhole cyanin (KLH) (or 4 mg of bovine serum albumin, BSA) and dissolve it in 10 mL of 10 mM PBS. Then, add the activated hapten solution dropwise and stir overnight. Subsequently, dialyze the complex with 10 mM phosphate buffer (PBS, 10 mmol / L, pH 7.4) for 3 days to obtain AFB1 immunogen (AFB1-KLH) and coating antigen (AFB1-BSA), which are stored at -20 °C for later use. Example 2 Preparation of Monoclonal Antibodies

[0029] 2.1 Mouse Immunization Healthy female BALB / c mice aged 6-8 weeks were selected as immunization animals, and AFB1-KLH prepared in Example 1 was used as the immunogen. The immunization protocol employed a strategy of primary immunization followed by booster immunization: for the first immunization, 0.1 mL of the immunogen at a concentration of 1 mg / mL was thoroughly emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at multiple sites on the back of the mouse, with each mouse receiving 0.2 mL of the immunogen. Thereafter, booster immunizations were performed every 3 weeks, using Freund's incomplete adjuvant emulsification, with the same dosage and route as the first immunization. Seven days after the third immunization, blood was collected from the orbital venous plexus of the mice, and serum was separated and antibody titers were determined using an indirect ELISA method. Mice with high serum titers and good AFB1 inhibition were selected, immunization was stopped, and cell fusion was prepared.

[0030] In the preliminary experiment, the original AFB1-BSA was diluted 1:3000 and coated onto the ELISA plate. Mouse serum was diluted 1:5000 and then measured. The results are shown in Table 1. The data in the table show that all six immunized mice produced specific antibodies against AFB1. The serum titers of mice 3 and 5 were higher, with OD values ​​around 2.0, and inhibition rates against AFB1 reached 78.22% and 82.15%, respectively. Further examination of the cross-reactivity of serum against 13 flavonoid polyphenols revealed that the serum of mouse 2 showed significant inhibitory effects on various flavonoid polyphenols, with inhibition rates ranging from 11.83% to 82.06%. In contrast, the serum of mouse 5 showed lower inhibition rates against all 15 compounds, ranging from -1.64% to 8.30%, indicating that the antibodies produced by mouse 5 had better specificity. Considering the titer, inhibition rate, and specificity, mouse 5 was ultimately selected for subsequent cell fusion experiments.

[0031] Table 1. Results of the third immunization with AFB1 immunogen 2.2 Cell Fusion Mice with the best immunization response obtained from the above screening were subjected to intraperitoneal pulse immunization 3 days before fusion. Using polyethylene glycol (PEG-2000)-mediated cell fusion technology, mouse spleen cells were fused with SP2 / 0 myeloma cells at a specific ratio. The fused cells were resuspended in HAT selective medium and seeded into 96-well cell culture plates pre-coated with feeder cells, and cultured at 37°C in a 5% CO2 incubator. After approximately one week of culture, the hybridoma cell culture supernatant was screened using an indirect competitive ELISA method, selecting positive wells with high titers and good inhibitory effects against AFB1. The positive hybridoma cells were subcloned three times using a limiting dilution method, ultimately obtaining a hybridoma cell line that stably secretes anti-AFB1 monoclonal antibody, named 1H5.

[0032] 2.3 Sequencing of hybridoma cells Hybridoma cells in the logarithmic growth phase were collected, and total RNA was extracted using TRIzol reagent. Following the instructions of the PrimeScript™ First-Strand cDNA Synthesis Kit (TaKaRa, Cat # 2690A), total RNA was reverse transcribed into the first strand of cDNA using Oligo(dT) primers. Subsequently, specific primers were designed to amplify the heavy chain variable region (VH) and light chain variable region (VL) gene fragments of the antibody, referring to the antibody variable region amplification protocol provided by Biointron Biology Inc. The PCR amplification products were recovered, purified, ligated into the TA / Blunt-Zero cloning vector, and transformed into competent *E. coli* cells. Positive clones were screened by colony PCR, and the sequences of three positive clones were determined.

[0033] The sequencing results are as follows: Light chain nucleotide sequence (SEQ ID NO.8): CAGGTACAGTTGAAGCAGTCAGGACCTGGCCTAGTGCAGCCCTCACAGAGCCTGTCCATCACCTGCACAGTCTCTGGTTTCTCTTTAACTAGATATGGTGTGCACTGGGTTCGCCAGTCTCCAGGAAAGGGTCTGGAGTGGCTGGGAATGATATGGAGTGGTGGAAGCACAGACTATA ATGCAGCTTTCATATCCAGACTGAGCATCAGTAAGGACAATTCCAAGAGCCAAGTTTTCTTAAATGAACAGTCTGCAAACTAGTGACACAGCCATATATTACTGTGTCGGCTTCTACTATGGTAACTACGCCTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA Light chain amino acid sequence (SEQ ID NO.6): QAVVTQESALTTSAGETVTLTCRLSSGAVTTSNSANWVQEKPDHLFTGLIGSTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHLVFGGGTKLTVL in: CDR-L1: SGAVTTSNS (SEQ ID NO.1) CDR-L2: STN CDR-L3: ALWYSNHLV (SEQ ID NO.2) Heavy chain nucleotide sequence (SEQ ID NO.9): GAAGTGATGCTGGTGGCGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGAAACTATGCCATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCAAGCATTAGTAGTGGTAGTAGTAAGATCTAC TATCCAGACAGTGTGAAGGGGCGAATCACCATCTCCAGAGACAATGCCAGGAACAACCCTATACCTACATATGAGCAGTCTGAGGTCTGAGGACACGGCCATTTATTACTGTGCAAGACATGGTGGAAACTGGAACTTCGATATCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA Heavy chain amino acid sequence (SEQ ID NO.7): EVMLVASGGGLVKPGGSLKLSCAASGFTFRNYAMSWVRQTPEKRLEWVASISSGSSKIYYPDSVKGRITISRDNARNTLYLHMSSLRSEDTAIYYCARHGGNWNFDIWGAGTTVTVSS in: CDR-H1: GFTFRNYA (SEQ ID NO.3) CDR-H2: ISSGSSKI (SEQ ID NO.4) CDR-H3: ARHGGNWNFDI (SEQ ID NO.5) 2.4 Preparation and Identification of Monoclonal Antibodies in Ascites Fluid Monoclonal antibodies were prepared in large quantities using an in vivo ascites induction method. Several adult BALB / c mice were pretreated by intraperitoneal injection of 0.1 mL Freund's incomplete adjuvant seven days before hybridoma cell inoculation. Hybridoma cells in logarithmic growth phase were resuspended in RPMI-1640 basal medium and the cell concentration was adjusted to 2 × 10⁻⁶ cells / mL. 6Cells were injected intraperitoneally at a concentration of 0.5 mL per mouse. Mice were closely monitored after injection. When the abdomen became significantly distended and the mice exhibited lethargy, they were euthanized by cervical dislocation, and ascites fluid was aspirated using a syringe. High-purity monoclonal antibody was obtained after purification using the caprylic acid-ammonium sulfate method. Detection using a mouse monoclonal antibody subtype identification kit showed that the heavy chain subtype of this monoclonal antibody was IgG1. Example 3: Monoclonal Antibody Performance Determination

[0034] Dilute the original AFB1-BSA to 0.25 μg / mL with carbonate buffer (CB, pH 9.6) and add 100 μL to each well of a 96-well microplate. Incubate overnight at 4°C. The next day, discard the coating solution and wash three times with PBST washing buffer (PBS containing 0.05% Tween-20), 250 μL per well each time, and blot dry. Add 200 μL of 2% skim milk blocking buffer to each well and block at 37°C for 2 hours. Discard the blocking buffer and wash three times, then blot dry. Add 50 μL of AFB1 standard solution of serial concentration and 50 μL of appropriately diluted 1H5 monoclonal antibody solution (0.04 ng / μL) to each well sequentially, with three parallel wells for each concentration, and incubate at 37°C for 30 minutes. After washing three times, add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody diluted 1:5000 to each well and incubate at 37°C for 30 minutes. Wash three more times, pat dry, and add 100 μL of TMB substrate chromogenic solution to each well, incubate at 37°C in the dark for 10 minutes. Finally, add 50 μL of 2 mol / L sulfuric acid solution to each well to stop the reaction, and measure the absorbance (OD450) of each well at 450 nm using a microplate reader. 450 The optical density value at the IC50 reading was measured using an ELISA reader. A standard curve was established based on the data read from the ELISA reader. 50 The antibody performance was comprehensively evaluated, and the results are shown in Table 2 and 3. Figure 4 When AFB1-BSA is used as the coating antigen, the IC50 value of the 1H5 monoclonal antibody against AFB1 is 0.047 ng / mL, indicating that the antibody has high detection sensitivity.

[0035] To further evaluate the antibody's ability to resist matrix interference, a laboratory-prepared AFB1 monoclonal antibody (mAb2H4) was selected as a control. The cross-reactivity of the two antibodies against 13 common polyphenolic flavonoids was compared using the same experimental method. The results are shown in Table 3. The 1H5 antibody prepared in this invention, at a concentration of 100 mg / L, did not show significant inhibitory effects on 10 compounds: epicatechin, catechin, quercetin, piperidin, rutin, kaempferol, isoquercetin, and naringin. In contrast, the control antibody 2H4 showed varying degrees of inhibition against all 10 polyphenolic flavonoids. Considering that the actual content of polyphenolic flavonoids in colored grain samples is approximately 100 mg / kg, the 2H4 antibody is clearly unsuitable for detecting AFB1 in colored grains. The 1H5 antibody of this invention, due to its lack of cross-reactivity with most flavonoid polyphenols, can meet the requirements for accurate detection of AFB1 in colored grain samples.

[0036] Table 2. Performance assay of AFB1-1H5 antibody Table 3. Determination of cross-reactivity of AFB1-1H5 and AFB1-2H4 monoclonal antibodies Example 4: Establishment of an Indirect Competitive Sideflow Chromatography Immunoassay Method

[0037] 4.1 Optimization of antigen concentration on test strips Optimization of antigen concentration on the test strip as follows: Figure 1 Colloidal gold test strips with AFB1 antigen concentrations of 50 μg / mL, 60 μg / mL, and 75 μg / mL were used, with 0, 0.07, and 0.1 ng / mL of AFB1 standard added, and the strips were allowed to stand for 10 min. As the antigen concentration increased, the T-line gradually deepened in color, with colloidal gold colorimetric readings ranging from 479 to 1207. The T-line was clearly visible to the naked eye when the reading was between 700 and 1000. When the antigen concentration coated on the T-line was 50 μg / mL, the T-line was relatively light; however, when the concentration increased to 60 μg / mL and 75 μg / mL, the inhibition rates for 0.07 ng / mL and 0.1 ng / mL AFB1 standard were 51.6% and 46.8%, and 86.1% and 84.2%, respectively. Therefore, considering all factors, 60 μg / mL was selected as the ideal antigen streaking concentration.

[0038] 4.2 Optimization of antibody dosage Antibody addition amount optimization, such as Figure 2Add 5 μg, 7 μg, and 9 μg of AFB1 antibody (mAb 1H5) to 1 mL of AuNPs solution, respectively. Simultaneously, prepare AFB1 standards at concentrations of 0, 0.07, and 0.1 ng / mL. Incubate the test strips for 10 min. As the antibody concentration increases, the T line gradually deepens, with readings ranging from 787 to 1133. When the antibody concentration is 5 μg, the T line is too faint to accurately assess the experimental results. At 7 μg, the inhibition rate of AFB1 at a concentration of 0.1 ng / mL is 86.3%, compared to 76.8% at 9 μg and 0.1 ng / mL. Therefore, 7 μg was chosen as the optimal antibody addition amount.

[0039] 4.3 Optimization of the amount of gold-labeled antibody added According to the experimental procedure, the amount of gold-labeled antibody added was optimized as follows: Figure 3 4, 6, and 8 μL of gold-labeled antibody were added, respectively, and reacted with AFB1 standards at concentrations of 0, 0.07 ng / mL, and 0.1 ng / mL under different addition levels. The graph shows a direct correlation between the amount of gold-labeled antibody and the color of the T-line, with readings ranging from 733 to 1050. At an addition level of 4 μL, the T-line was too faint; the color difference between the blank and addition levels of 6 μL and 8 μL was not significant. Comparing the inhibition rates at AFB1 concentrations of 0.07 ng / mL and 0.1 ng / mL, respectively, the values ​​were 52.6% and 30.2%, and 78.2% and 71.2%, respectively. Therefore, 6 μL was ultimately selected as the optimal addition level of gold-labeled antibody.

[0040] 4.4 Establishment of the Standard Curve After optimizing the experimental conditions, the optimal parameters for the test strip reaction were determined to be a coating antigen concentration of 60 μg / mL, an antibody addition of 7 μg, and a gold-labeled antibody addition of 6 μL. AFB1 standard was diluted to a series of concentrations of 0, 0.03 ng / mL, 0.04 ng / mL, 0.05 ng / mL, 0.06 ng / mL, 0.08 ng / mL, and 0.1 ng / mL for testing. Higher standard concentrations resulted in a shallower T-line. When the AFB1 concentration was 0.1 ng / mL, the T-line essentially disappeared, indicating a cut-off value of 0.1 ng / mL for the test strip. A standard curve was plotted based on the instrument readings for different concentrations (see [link to standard curve]). Figure 4 IC was obtained by fitting the curve. 50 The concentration was 0.07 ng / mL, and the linear detection range was 0.04–0.12 ng / mL (IC50). 20 ~IC 80 ).

[0041] 4.5 Evaluation of the recognition ability of major polyphenolic flavonoid molecules in miscellaneous grains like Figure 5 As shown, LFA based on 1H5 antibody showed no significant recognition effect on 10 polyphenolic flavonoid compounds (epicatechin, catechin, quercetin, piperidin, rutin, kaempferol, isoquercetin, and naringin) at different concentrations, with an inhibition rate of less than 30%. Only luteolin and apigenin showed a blocking effect at high concentrations. In contrast, other AFB1 antibodies 2H4 prepared in our laboratory showed significant inhibitory effects on these 10 polyphenolic flavonoid compounds. Therefore, 1H5 antibody was used as a monoclonal antibody for detecting AFB1 in grains.

[0042] Example 5: Detection and Analysis of Aflatoxin B1 in Actual Samples To further verify the usability of the constructed method, a spiked recovery method was used to detect AFB1 in mung beans, red beans, black beans, red rice, black rice, and purple rice.

[0043] 5.1 Spiked Recovery of Six Types of Grains Due to the potential advantages of eutectic solvents (DES) as novel green extraction reagents, our previously developed DES (using betaine as the hydrogen bond donor and ethylene glycol as the hydrogen bond acceptor, with a molar ratio of 1:3) was selected as the extraction solvent for extracting AFB1 from mung beans, red beans, black beans, red rice, black rice, and purple rice samples. By optimizing the dilution factor of the extraction system, this method achieved ideal recovery rates (73.04%–90.95%) in all six bean samples, with a total dilution factor of 40 times (…). Figure 6 This indicates that 2 mL of DES can be successfully applied to the extraction of AFB1 from miscellaneous grains.

[0044] In addition, these beans were further supplemented with three concentrations of AFB1 to evaluate the recovery rate of the developed method, and the results are shown in Table 4. The recovery rates of mung beans, red beans, black beans, red rice, black rice, and purple rice ranged from 78.36% to 93.18%, meeting the detection requirements. Table 4. AFB1 spiked recovery experiments in 6 types of miscellaneous grains Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A monoclonal antibody against aflatoxin B1, comprising a light chain variable region and a heavy chain variable region, characterized in that, The amino acid sequence of the light chain variable region CDR-L1 is shown in Seq ID No. 1, the amino acid sequence of CDR-L2 is STN, and the amino acid sequence of CDR-L3 is shown in Seq ID No. 2; the amino acid sequence of the heavy chain variable region CDR-H1 is shown in Seq ID No. 3, the amino acid sequence of CDR-H2 is shown in SEQ ID NO. 4 in the sequence listing, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO. 5 in the sequence listing.

2. The aflatoxin B1 monoclonal antibody as described in claim 1, characterized in that, The light chain variable region contains an amino acid sequence as shown in Seq ID No. 6, and the heavy chain variable region contains an amino acid sequence as shown in Seq ID No.

7.

3. The aflatoxin B1 monoclonal antibody as described in claim 1, characterized in that, The light chain variable region contains the nucleotide sequence shown in SEQ ID NO. 8; the heavy chain variable region contains the nucleotide sequence shown in SEQ ID NO.

9.

4. A nucleic acid molecule, characterized in that, It contains an amino acid sequence encoding an aflatoxin B1 monoclonal antibody as described in any one of claims 1 or 2.

5. The nucleic acid molecule as described in claim 4, characterized in that, It comprises the nucleotide sequence encoding the light chain variable region as shown in SEQ ID NO. 8 and the heavy chain variable region as shown in SEQ ID NO. 9 of the aflatoxin B1 monoclonal antibody as described in claim 3.

6. The application of the aflatoxin B1 monoclonal antibody as described in any one of claims 1-3, characterized in that, An indirect competitive lateral flow chromatography immunoassay strip method was used to detect aflatoxin B1 in colored grains.

7. The application of the aflatoxin B1 monoclonal antibody as described in claim 6, characterized in that, A mixed solution of betaine and ethylene glycol was used as the extraction reagent for aflatoxin B1 in colored grains, and the monoclonal antibody was used as the recognition element to qualitatively or quantitatively detect aflatoxin B1 in colored grains.

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