A zearalenone monoclonal antibody against flavonoid interference and a preparation method thereof and application thereof in legume sample detection

Abstract

The application belongs to the field of mycotoxin immunoassay detection, and discloses a zearalenone monoclonal antibody with anti-flavonoid interference and a preparation method thereof and application thereof in detection of legume samples. The amino acid sequences of the light chain variable region LCDR1 and LCDR3 of the monoclonal antibody are respectively shown as Seq ID No. 1~2, the amino acid sequence of CDR-L2 is WTS, the amino acid sequences of the heavy chain variable region HCDR1~HCDR3 are respectively shown as Seq ID No. 3~5, and the full-length sequences of the light chain and the heavy chain variable region are respectively shown as Seq ID No. 6, 7. The antibody has an IC50 of 0.052 ng / mL for zearalenone, has no obvious inhibitory effect on 13 main flavonoid compounds in legumes such as daidzein and soybean isoflavone, and has excellent anti-flavonoid interference performance. Rapid and accurate detection of zearalenone in legumes can be realized, and the application is 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 zearalenone and has no cross-reaction with flavonoid molecules, and the application of colloidal gold side-flow immunochromatography based on this antibody to the detection of zearalenone in legumes. Background Technology

[0002] Legumes, as an important source of plant-based foods, occupy a crucial position in our daily diet. During storage, legumes are often susceptible to infection by Fusarium fungi, especially under high humidity conditions, leading to the accumulation of mycotoxins such as zearalenone (ZEN). The concentration of ZEN can range from 10 ng / mL to 807 ng / mL, exhibiting potential reproductive toxicity, hepatotoxicity, genotoxicity, immunotoxicity, teratogenicity, and carcinogenicity. It has been classified as a Group 3 carcinogen by the International Agency for Research on Cancer (IARC) of the World Health Organization. Many countries and organizations have established strict limits for ZEN; for example, China sets the legal limit at 60 μg / kg for its use in grains and their products. Therefore, establishing accurate and rapid ZEN detection methods is of great significance for ensuring the safety of legume foods.

[0003] Lateral flow chromatography (LFA), based on antigen-antibody specific reactions, is currently an important method for rapid detection of ZEN in food safety. However, existing ZEN monoclonal antibodies are prone to producing false positive results when applied to legume samples. For example, the LFA method constructed using ZEN mAb 3D4 has poor accuracy in detecting ZEN in legume samples. The reason for this may be that legume matrices are rich in flavonoids. For instance, the main components in soybeans include genistein (44.8 mg / 100g), daidzein (33.45 mg / 100g), and kaempferol (5.3 mg / 100g); peas are mainly composed of daidzein, genistein, and glycyrrhizin. Cowpeas contain 24.7 mg / 100g of quercetin, while green soybeans contain only 0.3 mg / 100g of quercetin. Furthermore, the red and black soybean varieties not only contain the aforementioned flavonoids but are also rich in anthocyanins (such as cyanidin), with contents ranging from 1.1 mg / 100g to 1411.46 mg / 100g. The content of these flavonoids (at the ppm level) is significantly higher than that in ZEN soybeans. Structurally, the ZEN molecule contains a phenolic ring and a macrocyclic lactone ring, and its spatial conformation and chemical properties are significantly similar to the phenolic hydroxyl group and benzo-γ-pyranone structure characteristic of flavonoids in soybeans. Studies have shown that compounds with highly similar structures to the target analyte (two-dimensional similarity > 0.7), especially under high concentration conditions, may be recognized by monoclonal antibodies targeting the analyte. Therefore, this structural similarity poses a potential challenge to the detection of zearalenone monoclonal antibodies in soybean samples—the antibody may simultaneously recognize and bind to these flavonoids, leading to unexpected cross-reactions. Current research on the preparation of ZEN monoclonal antibodies mainly focuses on improving the affinity for zearalenone and enhancing the broad-spectrum reactivity to zearalenone and its main derivatives, while there is a lack of research on developing ZEN monoclonal antibodies against interference from flavonoid compounds.

[0004] Therefore, obtaining monoclonal antibodies that do not recognize flavonoids in legumes but can highly recognize ZEN is the key to constructing a highly accurate LFA method for detecting ZEN in legumes, so as to better achieve rapid and accurate detection of ZEN in legume samples. Summary of the Invention

[0005] Based on the current state of technology, in order to solve the problem that the direct detection of zearalenone in legume samples in the existing technology is often subject to interference from flavonoid compounds and thus produces false positives, the present invention aims to provide a zearalenone monoclonal antibody with high detection sensitivity and strong resistance to flavonoid interference; another objective is to provide its preparation method and its application in the detection of legume samples.

[0006] To achieve the objectives of this invention, the technical solution is as follows: The first aspect of the present invention provides a monoclonal antibody against zearalenone, wherein the light chain variable region contains the amino acid sequence shown in SEQ ID NO. 6, wherein the amino acid sequences of CDR-L1 and CDR-L3 are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively, and the amino acid sequence of CDR-L2 is WTS; Its heavy chain variable region contains the amino acid sequence shown in SEQ ID NO. 7, wherein the amino acids of CDR-H1, CDR-H2, and CDR-H3 are shown in SEQ IN NO. 3, SEQ IN NO. 4, and SEQ IN NO. 5, respectively.

[0007] The present invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding the light chain variable region of the above-mentioned anti-zearalenone monoclonal antibody as shown in SEQ ID NO.8 and / or the heavy chain variable region of the above-mentioned anti-zearalenone monoclonal antibody as shown in SEQ ID NO.9.

[0008] The second aspect of this invention provides a method for indirect competitive lateral flow chromatography immunoassay strips for detecting zearalenone in legume samples using the above-mentioned monoclonal antibody with high specificity: using the above-mentioned monoclonal antibody as the recognition element can reduce the interference of flavonoid molecules in legumes on detection, and using zearalenone-bovine serum albumin as the coating antigen to achieve accurate detection of zearalenone content in legumes.

[0009] The specific method is as follows: Step 1, Preparation of the probe colloidal gold-monoclonal antibody: Adjust the pH of the colloidal gold solution to 3-5, then add mAb-4A4 and mix thoroughly. After standing, add bovine serum albumin solution to the above solution and let it stand again. Finally, centrifuge the mixture and discard the supernatant. Dissolve the precipitate in ultrapure water to obtain the probe.

[0010] Step 2, Assembly of colloidal gold test strips: Cover the liner surface with nitrocellulose membrane, absorbent pad, and glass fiber membrane in sequence. Set the detection line (T line) and control line (C line) in the transverse direction on the non-covered surface of the nitrocellulose membrane. Use zearalenone-BSA as the T line and goat anti-mouse immunoglobulin as the C line.

[0011] Step 3, detection using the colloidal gold test strip: Add different volumes of probe to a 96-well plate and incubate with different concentrations of zearalenone standard solution. When the sample solution does not contain zearalenone, the probe specifically binds to the antigen on the T line, and the remaining probe then binds to the goat anti-mouse secondary antibody on the C line. Simultaneous color development of both the C and T lines indicates a negative result. When the sample solution contains zearalenone, the probe first reacts with the zearalenone, and the remaining probe then binds to the antigen on the T line, resulting in color development. The more zearalenone present, the lighter the T line becomes, until the T line completely disappears, indicating a positive result. If the C line does not develop color, it indicates incorrect operation or that the test strip is invalid. This method achieves qualitative detection of zearalenone in legume samples.

[0012] The antibody has an IC50 of 0.052 ng / mL against zearalenone and cannot recognize 13 major flavonoid compounds (100 μg / mL) in legumes, including daidzein, genistein, quercetin, kaempferol, silymarin, rutin, baicalin, puerarin, apigenin, hyperoside, and morin, with an inhibition rate of <20%. This indicates that the established method has no significant recognition effect on flavonoid compounds, and the antibody is resistant to interference from flavonoid compounds in legume samples. Figure 5 ).

[0013] Preferably, the optimal concentration of the antigen for streaking is 0.3 mg / mL, the optimal amount of antibody added is 11 μg / mL, and the optimal dilution factor of the sample is 40 times.

[0014] This invention further provides a method for the quantitative detection of zearalenone in legume samples, specifically including: Standard concentrations of zearalenone were added to cowpeas, red beans, broad beans, purple kidney beans, mung beans, soybeans, and black beans, respectively. Samples were placed in centrifuge tubes, and eutectic solvents were added. After vortexing and centrifugation, the supernatant was diluted with PBST and directly detected using the aforementioned indirect competitive side-flow chromatography immunoassay method. A standard curve was established. Figure 4 ), and quantitative detection of the sample to be tested is achieved based on the standard curve.

[0015] The eutectic solvent is selected from betaine and ethylene glycol in a molar ratio of 1:3. Betaine is the hydrogen bond donor, and ethylene glycol is the hydrogen bond acceptor.

[0016] The innovations of this invention are as follows: First, it provides a monoclonal antibody with high specificity and sensitivity for zearalenone, and constructs an indirect competitive lateral flow chromatography immunoassay strip method using its recognition element. This method is applied to the detection of zearalenone in legumes, demonstrating high accuracy. The sample can be detected after simple extraction, making the operation simple and highly specific. No further purification treatment is required before use in the lateral flow chromatography immunoassay strip, saving operation time and costs, and possessing the potential for rapid batch screening. This provides support for establishing a rapid, sensitive, and accurate detection method.

[0017] The beneficial effects of this invention are as follows: 1. The monoclonal antibody provided by this invention has high specificity, enabling excellent qualitative and quantitative detection of ZEN, which is convenient, rapid, and accurate. It has no significant inhibitory effect on high flavonoid molecules (100 μg / mL concentration) in legumes.

[0018] 2. The indirect competitive lateral flow chromatography immunoassay strip method is used to quantitatively detect zearalenone in legume samples. It has advantages such as high accuracy, wide linear range, low cost, convenient use and short operation time. It can be used to detect zearalenone in samples with high flavonoid content, providing a new method for the control of mycotoxins in samples with high flavonoid content.

[0019] The nucleotide and amino acid sequences of the light chain variable region and heavy chain variable region of the monoclonal antibody against zearalenone involved in this invention are shown in the sequence listing. Attached Figure Description

[0020] 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 of the zearalenone-4A4 monoclonal antibody against zearalenone in this invention; Figure 5 The effect of 13 flavonoid compounds on the color development of test strips based on ZEN 4A4 monoclonal antibodies; Figure 6 Standard curves for flavonoid compounds that cross-react with ZEN 3D4 antibody (for comparison); Figure 7 The test strips of this invention exhibit tolerance results at different methanol concentrations; Figure 8 The test strips of this invention exhibit tolerance results at different acetonitrile concentrations; Figure 9 Optimize the extraction volume for 8 types of legumes. Detailed Implementation

[0021] The present invention is further illustrated below by way of examples, but these examples should not be construed as limiting the scope of protection of the present invention. Experimental methods in the following examples, unless otherwise specified, were performed according to conventional methods and conditions or as per the product instructions. Unless otherwise specified, all percentages are mass percentages. Example 1: Preparation of Zearalenone Immunogen and Coating Genome

[0022] First, 5 mg of zearalenone and 5 mg of carboxymethyl hydroxylamine (CMO) were dissolved in 5 mL of pyridine solution and reacted with magnetic stirring at 70 °C for 6 h. The resulting reaction solution was dried under nitrogen, dissolved in 3 mL of 0.1 M NaHCO3 solution, and the pH was adjusted to 3.0 with 0.1 M HCl. The solution was then extracted three times with 5 mL of ethyl acetate and dried under nitrogen to obtain the hapten ZEN-CMO.

[0023] Subsequently, 2.0 mg of the above hapten was accurately weighed and dissolved in 5 mL of N,N-dimethylformamide (DMF). 6 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 2 mg of N-hydroxythiosuccinimide (NHS) were added. The mixture was stirred at room temperature for 12 h and centrifuged at 3000 rpm for 5 min to obtain the activated ZEN hapten.

[0024] Then, accurately weigh 5 mg of keyhole blue protein (KLH) (or 10 mg of bovine serum albumin, BSA) and dissolve it in 10 mL of 10 mM PBS. Then, add the activated solution dropwise to the mixture and react at 4°C. Subsequently, dialyze the complex with 10 mM phosphate buffer (PBS, 10 mmol / L, pH 7.4) for 3 days to obtain the purified complete antigen, which is stored at -20 °C for later use. Example 2 Preparation of monoclonal antibodies

[0025] 2.1 Mouse Immunization BALB / c mice (8 weeks old) were immunized subcutaneously with the zearalenone-KLH immunogen described in Example 1. The immunization program consisted of a single primary immunization and several booster immunizations. For the first immunization, 0.1 mL of zearalenone-KLH immunogen (1 mg / mL) was emulsified in 0.1 mL of Freund's complete adjuvant, and 0.2 mL of the zearalenone-KLH immunogen was injected into the mice. Over three weeks, Freund's incomplete adjuvant was administered using two identical doses of the booster. Seven days after the third immunization, blood was collected from the corner of the eye, and serum was separated and the serum antibody titer was detected by indirect ELISA. Immunization was discontinued for mice that passed the initial immunization in preparation for fusion. The original zearalenone-BSA was diluted 3000 times, and mouse serum was diluted 5000 times for assay. The results are shown in Table 1. All mice showed good immunity and good recognition of zearalenone. Mice No. 2 and No. 3 showed a colorimetric index of around 2.0, with inhibition rates of 71.68% and 78.34%, respectively. However, mice No. 2 showed significant inhibitory effects on 13 flavonoid compounds, including daidzein, genistein, genistein, quercetin, kaempferol, silymarin, rutin, baicalin, puerarin, apigenin, hyperoside, and morin, with inhibition rates ranging from 1.34% to 80.45%. Mice No. 3 showed inhibition rates of -0.96% to 7.5% on the 13 flavonoid compounds. Therefore, mice No. 3 were selected for fusion. Table 1. Results of the third immunization with zearalenone immunogen

[0026] 2.2 Cell Fusion Mice exhibiting high titers and high inhibition rates were selected for fusion. Spleen cells from immunized mice were fused with SP2 / 0 mouse myeloma cells using PEG-2000. The fused cells were resuspended in HAT medium and transferred to 96-well plates containing feeder cells. One week later, the hybridoma cell supernatant was analyzed by icELISA to screen for high-titer hybridoma cells. Cells secreting anti-zearalenone antibodies were subcloned three times using a limiting dilution method, ultimately obtaining a hybridoma cell line that stably secretes anti-zearalenone antibodies, named ZEN-4A4.

[0027] 2.3 Sequencing of hybridoma cells Following the technical manual of the PrimeScript™ First-Strand cDNA Synthesis Kit (TaKaRa, Cat # 2690A), total RNA was reverse transcribed into cDNA using reverse transcription primers. Then, antibody fragments of VH and VL were amplified according to the operating procedure of Biointron Biology Inc., and cloned into the TA / Blunt-Zero cloning vector. Clones were screened by PCR, and the sequences of three positive clones were determined.

[0028] The sequencing results are as follows: Its light chain nucleotide sequence (SEQ ID NO.8): GACATTTGTGATGACACAGTCTCCATCCTCCCTGACTGTGACAGCAGGAGAAGGTCACTATGAACTGCAAGTCCGGTCAGAGCCTATTACACAGTGGAAATCAAAAGAACTACTTGACCTGGTTCCAGCAGAAACCAGGGCAGCCTCCTAAACTGTTGTTCTACTGGA CATCCACTCGGGAATCTGGGGTCCCTGATCGCTTCACCGGCAGTGGGTCTGGAACAGATTTCACTCTCACCATCAGTAGTGTGCAGCCTGAAGACCTGGCAGTTTTATTACTGTCAGAACAATTATCTTTATCCGTACACGTTCGGAGGGGGAACCAAACTGGAGATAAAG Its light chain amino acid sequence (SEQ ID NO.6): DIVMTQSPSSLTVTAGEKVTMNCKSGQSLLHSGNQKNYLTWFQQKPGQPPKLLFYWTSTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAVYYCQNNYLYPYTFGGGTKLEIK in: CDR-L1: QSLLHSGNQKNY (SEQ ID NO.1) CDR-L2: WTS CDR-L3: QNNYLYPYT(SEQ ID NO.2) Its heavy chain nucleotide sequence (SEQ ID NO.9): CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTATTGGGGCTGTTACAACTAGTAACTACGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTGGTCTAATAGGTGGTACCAACA ACCGAGCTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCGACCACGGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA Its heavy chain amino acid sequence (SEQ ID NO.7): QVQLQQPGAELVKPGTSVKLSCKTFGYTFTSYWMHWVKQRPGQGLEWIGEISPSNGRTHYIEKFRNKATLSVDKSSSTAYMELRRSLTSEDSAVYFCTRSGGYDVGYGLDYWGQGTSVTVSS in: CDR-H1: GYTFTSYW (SEQ ID NO.3) CDR-H2: ISPSNGRT (SEQ ID NO.4) CDR-H3: TRSGGYDVGYGLDY (SEQ ID NO.5) 2.4 Preparation and Identification of Monoclonal Antibodies in Ascites Fluid Seven days prior to inoculation, seven Balb / c mice were pretreated by intraperitoneal injection of 0.1 mL Freund's incomplete adjuvant. Hybridoma cells (zearalenone-4A4) were suspended in RPMI-1640 basal medium and the cell number was adjusted to 2 × 10⁻⁶ cells / mL. 6 The concentration of the monoclonal antibody was 0.5 mL / mL, and each mouse was intraperitoneally inoculated. Ascites fluid was collected when the mice's abdomens became significantly distended, their condition deteriorated, and they became immobile and near death. The monoclonal antibody was confirmed to be IgG type I using ELISA. Example 3: Monoclonal Antibody Performance Determination

[0029] First, the coated protozearene-BSA was diluted to 0.3 μg / mL with carbonate buffer (CB, pH 9.6), and 100 μL was added to each well of a 96-well plate. After incubation at 4 °C for 10 h, the plate was washed three times with 250 μL of PBST solution and then dried. Next, each well was blocked with 200 μL of 2% skim milk powder at 37 °C for 2 h. After rinsing, 50 μL of different concentrations of zearalenone standard solution and 50 μL of zearalenone-4A4 monoclonal antibody (0.05 ng / L) were added to the wells, and the plate was incubated at 37 °C for 30 min. After washing, 100 μL of HRP-goat anti-mouse IgG (1:5000) diluted in PBS was added to each well, and the plate was incubated again at 37 °C for 30 min. The plate was washed three times, 100 μL of substrate solution was added, and the plate was incubated at 37 °C for 10 min. Finally, 50 μL of 2M H2SO4 was added to each well to terminate the reaction. The reaction was stopped at 450 nm (OD). 450 The optical density value at the IC50 reading was measured using an ELISA reader. A standard curve was calculated 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 The results show that, using zearalenone-BSA as the coating agent, the IC50 of zearalenone-4A4 monoclonal antibody against zearalenone is [missing information]. 50 The value was 0.052 ng / mL. Simultaneously, the cross-reactivity rates of the antibody prepared in this invention and the previously reported monoclonal antibody ZEN-3D4 against 13 common flavonoids were compared. The results showed that the ZEN-4A4 antibody prepared in this invention had no significant inhibitory effect on 100 mg / L of daidzein, genistein, quercetin, kaempferol, silymarin, rutin, baicalin, puerarin, apigenin, hyperoside, and morin, while ZEN-3D4 showed no significant inhibitory effect on 100 mg / L of daidzein, genistein, genistein, quercetin, kaempferol, silymarin, rutin, baicalin, puerarin, apigenin, hyperoside, and morin. ZEN-3D4 showed significant inhibitory effects on aglycones, genistein, quercetin, and kaempferol. It also showed some inhibitory effects on 100 mg / L of silymarin, silymarin, rutin, baicalin, puerarin, apigenin, hyperoside, and morin. Considering that the flavonoid content in legume samples is approximately 100 mg / kg, ZEN-3D4 is not suitable for detecting ZEN in legumes. However, ZEN-4A4, which does not show significant inhibition on most flavonoids, is suitable for the accurate detection of ZEN in legumes. Table 2. Performance determination of zearalenone-4A4 antibody

[0030]

[0031] Table 3. Determination of cross-reactivity between ZEN-4A4 and ZEN-3D4 monoclonal antibodies

[0032] Example 4: Establishment of an Indirect Competitive Sideflow Chromatography Immunoassay Method 4.1 Optimization of antigen concentration on test strips Optimization of antigen concentration on the test strip streak line, such as Figure 1 Colloidal gold test strips with zearalenone antigen concentrations of 0.15 mg / mL, 0.3 mg / mL, and 0.45 mg / mL were used, with 0, 0.5, and 4 ng / mL of zearalenone 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 563 to 1216. The T-line was clearly visible to the naked eye when the reading was between 700 and 1000. When the T-line antigen concentration was 0.15 mg / mL, the T-line was lighter; however, when the concentration increased to 0.30 mg / mL and 0.45 mg / mL, the inhibition rates for 0.5 ng / mL and 4 ng / mL zearalenone standard were 49.8% and 70.5%, and 38.1% and 66.3%, respectively. Therefore, considering all factors, 0.30 mg / mL was selected as the ideal antigen streaking concentration.

[0033] 4.2 Optimization of antibody dosage Antibody addition amount optimization, such as Figure 2 9 μg, 11 μg, and 13 μg of zearalenone antibody (mAb 4A4) were added to 1 mL of AuNPs solution, respectively. Simultaneously, zearalenone standards at concentrations of 0, 0.5, and 4 ng / mL were prepared, and the solutions were allowed to react with the test strips for 10 min. As the antibody concentration increased, the T-line color gradually deepened, with readings ranging from 696 to 1068. At a antibody concentration of 9 μg, the T-line color was too faint to accurately assess the experimental results. At an antibody concentration of 11 μg, the inhibition rate of zearalenone at a concentration of 8 ng / mL was 75.5%, compared to 63.2% at 13 μg and 8 ng / mL, indicating a higher inhibition rate and better efficacy. Therefore, 11 μg was selected as the optimal antibody addition amount.

[0034] 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 3Gold-labeled antibody was added at concentrations of 6, 8, and 10 μL, respectively, and reacted with standards at zearalenone concentrations of 0, 0.5 ng / mL, and 4 ng / mL. The results show a direct correlation between the amount of gold-labeled antibody and the color of the T-line, with readings ranging from 697 to 1127. At a concentration of 6 μL, the T-line was too faint; at concentrations of 8 μL and 10 μL, the color difference in the blank was minimal. Comparing the inhibition rates at zearalenone concentrations of 1 ng / mL and 8 ng / mL, the values ​​were 49.8% and 79.5%, and 35.4% and 67.6%, respectively. Therefore, 8 μL was ultimately selected as the optimal amount of gold-labeled antibody.

[0035] 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 0.30 mg / mL, an antibody addition of 11 μg, and a gold-labeled antibody addition of 8 μL. Zearalenone standards were diluted to a series of concentrations of 0, 0.25 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, and 4 ng / mL for testing. Higher standard concentrations resulted in a shallower T-line. When the zearalenone concentration was 4 ng / mL, the T-line essentially disappeared, indicating a cut-off value of 4 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 The regression equation for the curve is y=136.84+762.76 / ((1+X / 0.46)^1.31), the correlation coefficient R2 is 0.994, the half-inhibition concentration IC50 is 0.46 ng / mL, and the linear detection range is 0.16~1.33 ng / mL (IC20~IC80).

[0036] 4.5 Assessment of the recognition ability of major flavonoid molecules in legumes like Figure 5 As shown, the LFA based on the 4A4 antibody showed no significant recognition effect on 13 high-concentration flavonoid compounds (10 μg / mL and 100 μg / mL), with an inhibition rate of less than 20%. These compounds included daidzein, genistein, genistein, quercetin, kaempferol, silymarin, rutin, baicalin, puerarin, apigenin, hyperoside, and morin. The T and C lines of the test strip were not interfered with by the flavonoid compounds, and the color development of the T line was basically the same as when the flavonoid concentration was 0 μg / mL. The recognition effect of the ZEN 3D4 antibody on these flavonoid compounds was also compared. Figure 6The study found that ZEN 3D4 exhibited significant recognition activity for five flavonoids: daidzein, genistein, quercetin, and kaempferol, with IC50 values ​​of 30.52 μg / mL, 30.61 μg / mL, 25.01 μg / mL, 19.91 μg / mL, and 20.68 μg / mL, respectively. Therefore, using the 4A4 antibody as an antibody for detecting zearalenone in legumes demonstrates excellent anti-interference activity.

[0037] Example 5: Detection and analysis of zearalenone in actual samples To further verify the applicability of the constructed method, a spiked recovery method was used to detect zearalenone in red beans.

[0038] 5.1 Organic Reagent Tolerance Test Different concentrations of methanol and acetonitrile have a significant impact on the experimental performance of antibodies. Excessive concentrations can affect antibody activity, leading to intolerance. The appropriate extraction solution for matrix spike recovery should be selected based on the tolerance observed.

[0039] Prepare methanol solutions with concentrations of 2.5%, 5%, and 7.5%, and plot standard curves based on the readings. Figure 7 It can be seen that the inhibition rate decreased when the methanol concentration was 7.5%, and the IC50 increased from 0.49 to 0.83, indicating that intolerance and inactivation occurred (Table 4), that is, the methanol tolerance of the antibody was 5%.

[0040] Prepare acetonitrile solutions with concentrations of 0% and 2.5%, and plot a standard curve based on the readings. Figure 8 It can be seen that when the acetonitrile concentration of the extract is 2.5%, the competition becomes significantly worse (Table 4), that is, the monoclonal antibody has a tolerance of 0% to acetonitrile.

[0041] Table 4. Tolerance of mAb 4A4 to methanol and acetonitrile

[0042] In summary, this study indicates that the antibody has poor tolerance to methanol and acetonitrile, and is not suitable for extracting ZEN from actual samples using conventional organic reagents.

[0043] 5.2 Spiked recovery of 8 types of legume samples Because mAb 4A4 has weak tolerance to methanol and acetonitrile, and because eutectic solvents (DES) have potential advantages 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 zearalenone from cowpea, red bean, broad bean, purple kidney bean, mung bean, soybean, and black bean samples. This method achieved ideal recoveries (81.86%–102.80%) in all eight bean samples, with a total dilution factor of 40-fold (…). Figure 9 This indicates that 2 mL of DES can be successfully applied to the extraction of ZEN from legumes.

[0044] In addition, three concentrations of ZEN were added to these beans to evaluate the recovery rate of the developed method, and the results are shown in Table 5. The limits of detection for cowpeas, red beans, broad beans, purple kidney beans, mung beans, soybeans, and black beans were between 0.39 μg / kg and 0.88 μg / kg, and the recoveries (83.94%–106.73%) met the detection requirements.

[0045] Table 5. Spiking recovery experiments of zearalenone in 8 legume samples 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 zearalenone monoclonal antibody, 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 WTS, 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, and the amino acid sequence of CDR-H3 is shown in Seq ID No.

5.

2. The zearalenone 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 zearalenone monoclonal antibody as described in claim 1, characterized in that, The light chain variable region comprises a nucleotide sequence as shown in SEQ ID NO. 8; the heavy chain variable region comprises a nucleotide sequence as shown in SEQ ID NO.

9.

4. A nucleic acid molecule, characterized in that, It contains an amino acid sequence encoding a 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 of the anti-zearalenone monoclonal antibody as shown in SEQ ID NO. 8 and the heavy chain variable region as shown in SEQ ID NO.

9.

6. The application of the zearalenone monoclonal antibody according to any one of claims 1-3, characterized in that, An indirect competitive side-flow chromatography immunoassay strip method was used to detect zearalenone in legume samples.

7. The application of the zearalenone 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 zearalenone in soybeans, and the monoclonal antibody was used as the recognition element to qualitatively or quantitatively detect zearalenone in soybeans.

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