Monoclonal antibody for detecting Bt Cry1Fa toxin and application thereof

Abstract

The invention provides a Bt Cry1Fa toxin detection monoclonal antibody and application, and belongs to the technical field of pesticide immunological detection. Amino acid sequences of a heavy chain variable region and a light chain variable region of the monoclonal antibody Cry1Fa-mAb are respectively shown as SEQ ID NO.3 and SEQ ID NO.4. The monoclonal antibody Cry1Fa-mAb can recognize Cry1Fa toxin with high sensitivity, can be used for high-sensitivity detection of Bt Cry1Fa toxin in food and environmental samples, such as DAS-ELISA detection, colloidal gold immunochromatography test paper detection (AuNPs-LFIA) or aggregation-induced emission immunochromatography test paper detection (AIEFMs-LFIA), and has wide application value.

Description

Technical Field

[0001] This invention belongs to the field of immunology technology, specifically a monoclonal antibody and detection method for detecting Bacillus thuringiensis Cry1Fa toxin. Background Technology

[0002] Bacillus thuringiensis (Bt) is a Gram-positive, facultative anaerobic bacillus that synthesizes and accumulates highly specific insecticidal protein-based parasporal crystal toxins (Crytoxins) during its growth cycle. These toxins primarily act on the midgut cells of target pests, recognizing and binding to specific receptor proteins on the midgut epithelial cell membrane surface, such as cadherin-like proteins, ATP-binding cassette transporters (ABC transporters), aminopeptidase N (APN), and alkaline phosphatase (ALP). This triggers a series of signal transduction events, ultimately leading to midgut cell membrane perforation, osmotic imbalance, and cell rupture, resulting in pest death. As of February 2024, the International Bt Toxin Insect-Resistant Protein Database (http: / / www.lifesci.sussex.ac.uk / home / Neil_Crickmore / Bt / ) has published and certified BtCry toxins covering 78 gene families (Cry1 to Cry78), with a total of 818 genotypes identified. Among these Cry toxins, some highly toxic and widely used subtypes have been developed into biopesticides or implanted into the genomes of transgenic crops to achieve green control of specific pests, such as Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C, Cry1Fa, Cry1Ie, Cry1J, Cry2Aa, Cry2Ab, Cry2Ae, Cry3Aa, and Cry3Ab.

[0003] In agricultural production, BtCry toxin-based biotechnology has been widely applied to the breeding and promotion of insect-resistant crops. According to the latest data released by the International Service for the Acquisition of Agri-biotech Applications (ISAAA) (ISAAA Brief No. 55, 2020), the average annual planting area of ​​commercially grown genetically modified Bt insect-resistant crops worldwide has exceeded 100 million hectares, with an average annual increase in the value of related agricultural products approaching US$19 billion. The application of the BtCry toxin gene has not only effectively reduced the use of traditional chemical pesticides and lowered the risk of farmland environmental pollution, but also improved agricultural production efficiency, providing a strong guarantee for global food security.

[0004] Cry1Fa toxin exhibits significant toxicity against the Asian corn borer and shows promise for use alone or in combination with other toxins. Currently, enzyme-linked immunosorbent assay (ELISA) is the reported detection method for Cry1Fa toxin, but it suffers from low sensitivity and is cumbersome. Therefore, developing a simple, rapid, and effective detection technology for tracking and screening Bt Cry1Fa toxin residues has become a pressing technical challenge in this field. Summary of the Invention

[0005] To address the aforementioned issues, this application provides a method for designing monoclonal antibodies for the detection of Bt Cry1Fa toxin, obtaining relevant antibody materials based on this method, and establishing a specific detection method for Bt Cry1Fa toxin, thereby improving detection sensitivity and simplifying the detection process.

[0006] Specifically, the present invention is implemented as follows:

[0007] First, this application provides a monoclonal antibody that can specifically recognize Bt Cry1Fa toxin, which the applicant has named Cry1Fa-mAb. The antibody is composed of a heavy chain and a light chain, and the amino acid sequences of the variable regions of the heavy chain and the light chain are shown in SEQ ID NO.3 and SEQ IN NO.4, respectively.

[0008] Secondly, this application provides the encoding gene of the monoclonal antibody Cry1Fa-mAb that can specifically recognize Bt Cry1Fa toxin, and the encoding nucleotide sequences of its heavy chain variable region and light chain variable region are shown in SEQ ID NO.1 and SEQ INNO.2, respectively.

[0009] Third, this application provides a recombinant expression vector and recombinant engineered bacteria containing the genes encoded as shown in SEQ ID NO.1 and SEQ IN NO.2.

[0010] Fourth, this application also provides the application of the aforementioned monoclonal antibody Cry1Fa-mAb in the detection of Bt Cry1Fa toxin. Specifically, in the embodiments of this application, the monoclonal antibody Cry1Fa-mAb is used as the detection antibody and applied to the detection of Bt Cry1Fa toxin. For example, in the embodiments of this application, Cry1Fa-mAb is used as the detection antibody, and the Cry1Fa-pAb polyclonal antibody is used as the capture antibody, applied to sandwich ELISA (DAS-ELISA), colloidal gold immunochromatographic strip (AuNPs-LFIA), or aggregation-induced fluorescence immunochromatographic strip (AIEFMs-LFIA) for Bt Cry1Fa toxin.

[0011] Fifth, this application provides an ELISA kit for the detection of Bt Cry1Fa toxin, which contains the aforementioned monoclonal antibody. Furthermore, the kit also includes conventional ELISA kit components, such as PBST buffer, secondary antibody (anti-mouse IgG-HRP), and chromogenic solution.

[0012] Compared with the prior art, this application has the following beneficial effects:

[0013] 1. The monoclonal antibody Cry1Fa-mAb created in this application can specifically recognize Bt Cry1Fa toxin, and it is the first reported monoclonal antibody that can specifically recognize Cry1Fa toxin. It also does not have cross-recognition with existing Cry1, Cry2, Cry3 and Vip3Aa classes, and has high specificity and sensitivity.

[0014] 2. The amino acid sequence and encoding gene of the monoclonal antibody Cry1Fa-mAb protein of this application have been amplified by PCR using mouse universal single-chain antibody primers and sequenced. Therefore, this antibody material can not only be prepared by generating ascites fluid from traditionally immunized mice to produce the target antibody, but also be prepared by soluble expression through various expression systems (such as prokaryotic expression), which is beneficial for practical production applications. Attached Figure Description

[0015] Figure 1 The results show the binding activity of Cry1Fa toxin in the serum of mice with different numbers after the last round of immunization with Cry1Fa toxin in Example 1.

[0016] Figure 2 SDS-PAGE analysis of the purification effect of the target monoclonal antibody and the results of antibody subtype identification.

[0017] Figure 3 The results show the affinity of Cry1Fa-mAb and Cry1Fa-pAb for Cry1Fa toxin.

[0018] Figure 4 The results are from the DAS-ELISA detection of Cry1Fa toxin.

[0019] Figure 5 The results are for the Cry1Fa toxin colloidal gold assay (AuNPs-LFIA).

[0020] Figure 6 The results are from the Cry1Fa toxin aggregation-induced fluorescence assay (AIEFMs-LFIA).

[0021] Figure 7 The results show the cross-identification detection results of six toxins using DAS-ELISA, AuNPs-LFIA, and AIEFMs-LFIA. Detailed Implementation

[0022] The reagent formulations involved in the examples are as follows:

[0023] (1) PBS solution

[0024] Weigh out 8g of NaCl, 0.2g of KCl, 2.9g of Na2HPO4·12H2O, and 0.2g of KH2PO4, add them separately to distilled water, dissolve them completely, and then bring the volume to 1L.

[0025] (2) PBST solution

[0026] Add 0.05% Tween-20 by volume to the PBS solution.

[0027] (3) Tetramethylbenzidine (TMB) solution:

[0028] Weigh 10 mg of tetramethylbenzidine and dissolve it in 1 ml of dimethyl sulfoxide. Protect from light and store at 4 °C for later use.

[0029] (4) Substrate colorimetric solution:

[0030] 10ml formulation components: 9.875mL CPBS, 100μL TMB solution, 25μL H2O2 (volume ratio 20%).

[0031] Sources of materials involved in the embodiments:

[0032] Bt Cry1Fa toxin was purchased from Meiyan (Beijing) Agricultural Technology Co., Ltd.

[0033] The Balb / c model mice used in the experiment were provided by the Center for Comparative Medicine, Yangzhou University.

[0034] The New Zealand White Rabbit was purchased from Nanjing Zhongding Biotechnology Co., Ltd.

[0035] Goat anti-rabbit / mouse IgG-HRPs were purchased from Beijing Qingke Biotechnology Co., Ltd.

[0036] TMB substrate chromogenic solution was purchased from Beijing Pulilai Gene Technology Co., Ltd.

[0037] Freund's complete adjuvant, Freund's incomplete adjuvant, hypoxanthine thymidine (HT), hypoxanthine aminopterin thymidine (HAT), and polyethylene glycol (PEG) were all purchased from Sigma-Aldrich.

[0038] DMEM high-glucose culture medium was purchased from Nanjing Youqing Biotechnology Co., Ltd.

[0039] Fetal bovine serum was purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0040] HAT medium: purchased from Sigma-Aldrich, USA, model H0262-10VL;

[0041] HiTrap Protein A / G HP was purchased from GE.

[0042] The Mouse monoclonal antibody isotype ELISA Kit was purchased from Beijing Baicaotai Biotechnology Co., Ltd.

[0043] Cell culture plates (9, 24 and 96 wells), cell culture flasks and ELISA plates were purchased from Corning.

[0044] All other reagents used were of analytical grade.

[0045] The nucleotide and amino acid sequences involved in the examples are as follows:

[0046] SEQ IN NO.1 (351bp):

[0047] CAGGTGCAACTGCAGGAGTCTGGACCTGGCCTGGTGAAACCTTCTCAGTCTCCGTCCCTCACCTGCACTGTCACTGGCTTCTCAATCACCAGTGATTATGCCTGGAGCTGGATCCGGCAGTTTCCAGGAAGCAAACTGGAGTGGATGGGCTACATAACCTTCAGTGGTAGTACTGACTACAACCCATCTCTCAAAAGTCGAATCTCTATCACTCGTGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATACTTTTGTGCAAGAGGGAGCTGGGACGCGCGGGCTTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

[0048] SEQ IN NO.2(321bp):

[0049] GACATTGAGCTCACCCAGTCTCCAGCAATCCTGTCTGCATCTCCAGGGGAGAAGGTCACAATGACTTGCAGGGCCAGCTCAAGTGTAAGTTTCATGCACTGGTACCAGCAGAAGGCAGGATCCTCCCCCAAAACCTGGATTTATGCCACATCCAACCTGGCTTCTGGAGTCCCTGACCGTTTCAGTGGCAGTGGGTCTGGGACCTCTTATTCTCTCACAATCAAAAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTAAGCCATTCACGTTCGGCTCGGGCACCAAGCTGGAAATCAAACGG

[0050] SEQ IN NO.3(117aa):

[0051] QVQLQESGPGLVKPSQSPSLTCTVTGFSITSDYAWSWIRQFPGSKLEWMGYITFSGSTDYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYFCARGSWDARAYWGQGTTVTVSS

[0052] SEQ IN NO.4(107aa):

[0053] DIELTQSPAILSASPGEKVTMTCRASSSVSFMHWYQQKAGSSPKTWIYATSNLASGVPDRFSGSGSGTSYSLTIKRVEAEDAATYYCQQWSSKPFTFGSGTKLEIKR

[0054] Example 1: Preparation of mouse fusion cells with high binding activity to Cry1Fa toxin

[0055] 1. Immunization and efficacy testing process:

[0056] First round of immunization: 100 μg of Cry1Fa toxin was dissolved in 100 μL of PBS solution, and 100 μL of complete Freund's adjuvant was added and thoroughly mixed and emulsified. This mixture was then injected intraperitoneally into healthy female Balb / c mice aged 6 weeks. Second round of immunization: Two weeks later, 120 μg of Cry1Fa toxin was dissolved in 100 μL of PBS solution, and 100 μL of incomplete Freund's adjuvant was added and thoroughly mixed and emulsified. This mixture was then injected intraperitoneally into the test mice. Third round of immunization: Two weeks later, 120 μg of Cry1Fa toxin was dissolved in 100 μL of PBS solution, and 100 μL of incomplete Freund's adjuvant was added and thoroughly mixed and emulsified. This mixture was then injected intraperitoneally into the test mice. Fourth round of immunization: Two weeks after that, 120 μg of Cry1Fa toxin was dissolved in 100 μL of PBS solution... 100 μL of incomplete Freund's adjuvant was added to the PBS solution and thoroughly mixed and emulsified, and then injected into the peritoneal cavity of the test mice. One week later, the fifth round of immunization was performed: 160 μg of Cry1Fa toxin was dissolved in 100 μL of PBS solution and then directly injected into the peritoneal cavity of the test mice. One week later, spleen B cells of the test mice were collected to prepare monoclonal antibody fusion cell lines.

[0057] Before each round of immunization, a small amount of blood was collected by tail clipping to extract serum (blood from immunized mice was obtained by tail clipping, left to stand at room temperature for 6 hours, and the extracted serum was collected, which is the serum of the test mice) for indirect noncompetitor ELISA (INC-ELIS, see the literature "Xu et al., "Microcystin-LR nanobody screening from an alpaca phage display nanobody library and its expression and application, 2018, Ecotoxicology and Environmental Safety") to determine and analyze the corresponding immunization effect.

[0058] Specific steps: Add 100 μL of Cry1Fa toxin solution (2 μg / mL) to each well of a 96-well plate and incubate at 37°C for 2 hours. Remove the plate, wash with 300 μL / well of PBST buffer, then add 300 μL / well of MPBS solution and incubate at 37°C for 2 hours. Remove the plate, wash with 300 μL / well of PBST buffer, then add 100 μL / well of mouse serum solution diluted 10-fold (V:V) with PBS buffer and incubate at 37°C for 2 hours. Remove the plate, wash with 300 μL / well of PBST buffer, then add 100 μL / well of secondary antibody (anti-mouse IgG-HRP diluted with PBS buffer at a volume ratio of 1:5000) and incubate at 37°C for 2 hours. After washing the plate with 300 μL / well of PBST buffer, add 100 μL / well of TMB chromogenic buffer. Incubate at 37°C in the dark for 15 minutes, then measure the OD of the relevant reaction wells. 450 Values. The ICN-ELISA binding activity of Cry1Fa toxin in serum samples from different numbered groups of test mice (NO.1-NO.5) collected after the final round of immunization is shown in the figure. Figure 1 As shown, this indicates that after five rounds of immunization, antibodies that efficiently bind to the antigen Cry1Fa toxin were produced in the mouse serum.

[0059] 2. Cell fusion process

[0060] This will determine if the immune response has reached a usable level (positive OD). 450 Value > 1.0, titer reaches 10 4Mice subjected to cervical dislocation were then immersed in 75% alcohol for 10 minutes, drained, and their spleens were removed under sterile conditions and ground in incomplete culture medium (HAT medium). After filtering, the homogenate was collected and aliquoted into 20 mL tubes. Splenic cells were counted under a microscope. Splenic cells and SP2 / 0 myeloma cells were mixed in equal volumes at a cell ratio of 5:1 and centrifuged at 1000 rpm for 10 minutes at 37°C. The supernatant was discarded, and the precipitated mixed cells were resuspended in 1 mL of PEG at 40°C and allowed to stand for 1 minute. Then, 15 mL of HAT medium was added, and the mixture was incubated at 37°C for 10 minutes before centrifugation at 800 rpm for 8 minutes. The precipitated cells were resuspended in 5 mL of HAT medium, and then the volume was adjusted to 50 mL. Finally, the mixture was aliquoted into 96-well plates at 100 μL per well and incubated at 37°C. Five days later, fresh HAT medium was added to the cells at 100 μL / well. Two days later, half of the culture medium was aspirated from the wells, and fresh HAT medium was added again at the same volume. The cells were then cultured until they occupied about 1 / 10 of the bottom of the well. The cell culture medium was then used for INC-ELISA to determine the binding activity of the Cry1Fa toxin. The detection method was the same as above.

[0061] Example 2: Purification effect and antibody subtype identification results of the target monoclonal antibody analyzed by SDS-PAGE

[0062] The fusion cell wells of the Cry1Fa toxin subtype identified in Example 1 as having a high binding value to Cry1Fa toxin were serially diluted and cultured until a subclonal fusion cell line that could stably secrete the monoclonal antibody appeared.

[0063] The cell count of the target subclonal fusion cell line was quantified to 10⁻⁶ cells using DMEM high-glucose medium. 6 Then, 0.5 mL / mouse was injected into the peritoneal cavity of female mice. After 5 days of feeding, ascites fluid was extracted from the peritoneal cavity of the test mice. Finally, the target monoclonal antibody protein was separated and purified from the extracted ascites fluid by ammonium sulfate precipitation method (see the literature "Preparation and Detection Application of Bt(Cry1Fa) Toxin Polyclonal Antibody", Xu Chongxin et al., Agricultural Product Quality and Safety, 2016, No. 4) and HiTrap Protein G HP column purification method (steps refer to the product operation manual). The purified monoclonal antibody protein was dissolved in PBS.

[0064] This embodiment ultimately yielded a high-affinity monoclonal antibody, which the applicant named Cry1Fa-mAb. After RNA extraction and reverse conversion to cDNA, the antibody was expanded and sequenced using universal mouse monoclonal primers. The nucleotide and amino acid sequences of the heavy chain variable region are shown in SEQ IN NO.1 and SEQ ID NO.3, respectively, and the nucleotide and amino acid sequences of the light chain variable region are shown in SEQ IN NO.2 and SEQ ID NO.4, respectively.

[0065] The purification effect and antibody subtype identification results of Cry1Fa-mAb are as follows: Figure 2 As shown. Figure 2 In the diagram, A represents the target monoclonal antibody analyzed by SDS-PAGE, B represents the antibody purification effect and antibody subtype identification results, M represents the protein marker, lane 1 represents ascites fluid, lanes 2-3 represent purified exudate, and lanes 4-5 represent purified monoclonal antibodies.

[0066] Example 3: Determination of the affinity of Cry1Fa-mAb and Cry1Fa-pAb for Cry1Fa toxin

[0067] The assay method used in this embodiment refers to the method disclosed in the literature "Shen et al., Establishment of novel receptor-antibody sandwich assays to broadly detect Bacillus thuringiensis Cry1 and Cry2 toxins, 2023, International Journal of Biological Macromolecules". The specific steps of analyzing the affinity between Cry1Fa-mAb and Cry1Fa toxin using an Octet Red 96 molecular interaction analyzer (Pall ForteBio Corp., Menlo Park, CA, USA) are as follows:

[0068] First, the APS biosensor was immersed in PBS for 10 min. Then, Cry1Fa-mAb or Cry1Fa-pAb was loaded onto the APS biosensor (PallForteBio Corp., Menlo Park, CA, USA) for 600 s. The affinity of Cry1Fa-mAb and Cry1Fa-pAb for the immobilized Cry1Fa toxin was determined by binding over 600 s and dissociation over 300 s. The assay buffer was supplemented with PBS containing 0.1% BSA (protective protein) and 0.02% Tween-20 (to remove non-specific adsorption). The instrument calculated the binding constant (k). a) and dissociation constant (k d The data was then analyzed using Octet System Data Analysis software to determine the affinity constant (K) between the two components. D ).

[0069] Test results as follows Figure 3 As shown, A and B are the affinity test results of Cry1Fa-mAb and Cry1Fa-pAb for Cry1Fa toxin, respectively.

[0070] Example 4: A DAS-ELISA standard curve for the detection of Cry1Fa toxin was established using Cry1Fa-mAb monoclonal antibody as the detection antibody and Cry1Fa-pAb polyclonal antibody as the capture antibody.

[0071] The assay method used in this embodiment is based on the method disclosed in the reference "Shen et al., Establishment of novel receptor-antibody sandwich assays to broadly detect Bacillus thuringiensis Cry1 and Cry2 toxins, 2023, International Journal of Biological Macromolecules". Using the Cry1Fa-mAb monoclonal antibody and the Cry1Fa polyclonal antibody Cry1Fa-pAb prepared in Example 2 (preparation method referenced in "Jin et al., Rational design and application of broad-spectrum antibodies for Bt Cry toxins determination, 2024, Analytical biochemistry"), a dual-antibody sandwich ELISA (DAS-ELISA) for the detection of Cry1Fa toxin is established. The specific steps are as follows:

[0072] Add 100 μL of 1.25 μg / mL Cry1Fa-pAbs to each well of a 96-well microplate and incubate overnight at 4°C. Remove the coated plate, wash with 300 μL / well of PBST buffer, then add 300 μL / well of MPBS solution and incubate at 37°C for 2 hours. Remove the coated plate, wash with 300 μL / well of PBST buffer, then perform a 2-fold serial dilution of Cry1Fa toxin (0-2 μg / mL), adding 100 μL / well. Use PBS as a negative control and incubate at 37°C for 1 hour. Remove the coated plate, wash with 300 μL / well of PBST buffer, then add 100 μL / well of 2.5 μg / mL Cry1Fa-mAb and incubate at 37°C for 1 hour. After washing the plate with 300 μL / well of PBST buffer, add 100 μL / well of anti-mouse IgG-HRP secondary antibody (diluted 1:5000 with PBS buffer) and incubate at 37°C for 1 hour. Finally, after washing, add TMB chromogenic buffer and incubate at 37°C in the dark for 15 minutes. Then, measure the OD of the relevant reaction wells. 450 A DAS-ELISA method for Cry1Fa toxin was established. Four-parameter fitting was performed using GraphPad Prism8 software, and the SC value of Cry1Fa toxin was calculated using ELISA-Calc. 10 and SC 90 All values ​​are the average of three repeated measurements. The test results are as follows: Figure 4 As shown in the figure. A and B represent the DAS-ELISA detection standard curve and linear equation, respectively. Detection sensitivity and effective linear range are shown in Table 1 below.

[0073] Table 1 establishes the sensitivity and linear range of different detection methods for Cry1Fa toxin detection.

[0074]

[0075] Example 5 establishes colloidal gold immunochromatographic assay strips (AuNPs-LFIA) and aggregation-induced fluorescence immunochromatographic assay strips (AIEFMs-LFIA) for the detection of Cry1Fa toxin, using Cry1Fa-mAb monoclonal antibody as the detection antibody and Cry1Fa-pAb polyclonal antibody as the capture antibody.

[0076] Detection Procedure: Optimized control and test line antibodies (0.5 mg / mL HRP-labeled goat anti-mouse IgG and 1 mg / mL Cry1Fa-pAbs) were sprayed onto an NC membrane as the control and test lines, respectively, and vacuum-dried at 37°C for 3 hours. The test strips were then compressed and cut into 3 mm wide strips using an automatic cutter. The completed test strips were sealed and stored under dry conditions. The optimal volume (1 μL) was determined based on signal-to-noise ratio and reproducibility. For the detection procedure, 1 μL of each probe (for the optimization method of test strip detection conditions and the synthesis method of colloidal gold nanoparticles, refer to Zeng et al., Ultrastable Luminescent Organic-Inorganic Perovskite Quantum Dots via Surface Engineering: Coordination of Methylammonium Bromide and Covalent Silica Encapsulation, ACS Appl Mater Interfaces, 2018) was mixed with 100 μL of PBS containing continuous concentrations of Cry1Fa toxin (0-2000 ng / mL) and incubated for 5 minutes. The mixture is then applied to the sample pad, allowing it to pass through the test paper under capillary drive. After the reaction is complete, the test line and control line signals are recorded using GR-S1 and FIC-S1 brand test paper readers (China), and gray intensity and fluorescence values ​​are collected for quantitative analysis.

[0077] Standard curves and linear regression models were generated using GraphPad Prism 8 software. The limits of detection (LOD) and linear range were determined using the same statistical methods as DAS-ELISA. Triple replicates were performed for each concentration point to ensure robustness and reproducibility of the analysis.

[0078] Test results as follows Figure 5 , Figure 6 As shown. Figure 5 In the table, A and B are the linear equation and standard curve of AuNPs-LFIA, respectively, and C is the test strip detection result. The sensitivity and effective linear range are shown in Table 1. Figure 6 In the table, A and B are the linear equation and standard curve of AIEFMs-LFIA, respectively, and C is the test strip detection result. The sensitivity and effective linear range are shown in Table 1.

[0079] Example 5 Cross-reactivity of the Cry1Fa detection method

[0080] At the optimal working concentration screened in the above embodiments, different Cryotoxins (Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry2Aa, and Cry2Ab) were used as detection targets, and the cross-reactivity of different detection methods was investigated at a maximum concentration of 25 μg / mL. Each sample was analyzed at least three times. The detection results are as follows: Figure 7 As shown, AC represent the detection results of DAS-ELISA, AuNPs-LFIA, and AIEFMs-LFIA, respectively. These results indicate that the three detection systems provided in the above embodiments did not show significant cross-reactivity with any of the other six common Cryotoxins.

[0081] Example 6: Addition and recovery test of Cry1Fa toxin

[0082] 1. Addition and recycling of corn leaves

[0083] First, cut the blank corn leaves to 1mm. 2 The samples were fragmented into small pieces and then freeze-dried at -75°C for 24 hours. The freeze-dried samples were collected, and different concentrations of Cry1Fa toxin were added to the leaves. 5 mg of the sample was weighed into a 1.5 mL centrifuge tube, and 1 mL of extraction buffer was added to dissolve it. The centrifuge tube was placed on a centrifuge, shaken at 40 rpm for 40 min, and then centrifuged at 10,000 rpm for 20 min. The supernatant was collected and analyzed by DSA-ELISA and AuNPs-LFIA methods. The results are shown in Table 2.

[0084] Table 2. Cry1Fa toxin addition and recovery test

[0085]

[0086] The recovery experiments on maize leaf samples shown in Table 2 demonstrate that the methods for detecting Cry1Fa toxin established based on DSA-ELISA, AuNPs-LFIA, and AIEFMs-LFIA achieve recovery rates of 87% to 97.79% and coefficients of variation of 3.12% to 6.65% for Cry1Fa toxin in maize leaves. These results indicate that the established methods are reliable and suitable for the detection of relevant samples in practice.

Claims

1. A monoclonal antibody for detecting Bt Cry1Fa toxin, characterized in that, The amino acid sequences of the heavy and light chains of the antibody are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

2. The coding sequence of the monoclonal antibody for detecting Bt Cry1Fa toxin as described in claim 1, characterized in that, The nucleotide sequences of the heavy and light chains of the antibody are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

3. Recombinant expression vectors, transgenic cell lines, or recombinant engineered bacteria containing the genes encoded as shown in SEQ ID NO.1 and SEQ IN NO.

2.

4. The application of the Bt Cry1Fa toxin detection monoclonal antibody as described in claim 1 in the detection of Cry1Fa toxin.

5. The application as described in claim 4, characterized in that, The application refers to using the monoclonal antibody as a detection antibody in the detection of Bt Cry1Fa toxin.

6. The application according to claim 5, characterized in that, The detection methods include DAS-ELISA detection of Bt Cry1Fa toxin, colloidal gold immunochromatographic assay, or aggregation-induced fluorescence immunochromatographic assay.

7. An ELISA kit for detecting Bt Cry1Fa toxin, characterized in that, The kit contains monoclonal antibodies with heavy and light chain amino acid sequences as shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

Images