A monoclonal antibody for broad-spectrum detection of Bt Cry toxin and its application

Abstract

The present invention provides a monoclonal antibody for broad-spectrum detection of Bt Cry toxins and its application, belonging to the technical field of pesticide immunological detection. The cloned antibody JJF-GP-mAb provided in the present application can simultaneously and highly sensitively identify Bt Cry toxin subtypes such as Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C, Cry1F, Cry2Ab and Cry3Aa, and can be used for broad-spectrum and highly sensitive detection of Bt Cry toxins in food and environmental samples, and has broad application value.

Description

Technical Field

[0001] The present invention relates to the technical field of pesticide immunological detection, in particular to an antibody for broad-spectrum detection of Bt Cry toxins, and a preparation method and application thereof. Background Art

[0002] Bt Cry toxins are parasporal crystal proteins derived from Bacillus thuringiensis (Bt) that are highly lethal to common agricultural and forestry pests, including Lepidoptera, Coleoptera, Diptera, and Hymenoptera. They primarily inhibit the activity of specialized proteins or enzymes in the target insect's midgut, such as cadherins, ATP-binding cassette transporters, aminopeptidases, and alkaline phosphatases. This triggers a series of signaling pathways, perforating midgut cells and ultimately killing the insects. According to the International Bt Toxin Insecticide Protein Database (http: / / www.lifesci.sussex.ac.uk / home / Neil_Crickmore / Bt / ), as of February 2024, Bt Cry toxins have been discovered and certified, encompassing 78 gene families (Cry1 to Cry78) and a total of 818 subtypes. Among them, at least Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C, Cry1F, Cry1Ie, Cry1J, Cry2Aa, Cry2Ab, Cry2Ae, Cry3Aa, and Cry3Ab have been used in the form of biopesticide formulations or genetically modified insect-resistant crops for green pest control of target pests. According to the latest statistics from the International Service for the Acquisition of Agri-biotech Applications (ISAAA) (Global status of commercialized biotech / GM crops in 2019: biotech crops drive socio-economic development and sustainable environment in the new frontier, ISAAA Brief No. 55. ISAAA Ithaca (NY), (2020-11-30)

[0003] https: / / www.isaaa.org / resources / publications / briefs / 55 / default.asp.), currently, the global planting area of ​​Bt Cry toxin gene insect-resistant crops exceeds 100 million hectares per year, and the annual increase in the value of related agricultural products is close to US$19 billion.

[0004] However, with the long-term and large-scale promotion and application of Bt Cry toxin formulations and their transgenic insect-resistant crops, the frequency of abnormal resistance evolution induced by them in target pests (such as the diamondback moth, fall armyworm, tobacco hornworm, cotton bollworm, and rice leaf roller) has increased, and the serious threat to the safety of non-target organisms (such as silkworm, Coccinella bispinata, and Caenorhabditis elegans) has become increasingly prominent ("Mechanisms of Resistance to Insecticidal Proteins from Bacillus thuringiensis", Jurat-Fuentes et al., Annual Review of Entomology, 2021, No. 66; "A Historical Overview of Analysis Systems for Bacillus thuringiensis (Bt) Cry Proteins", J. Gu et al., Microchemical Journal, 2021, No. 165). In addition, transgenic Bt Cry toxin insect-resistant crops may pose safety risks, such as the development of drug tolerance in target organisms, gene cross-species escape, imbalance in the structure of microbial biodiversity, and damage to the immune systems of non-target organisms. The papers "Diversity of Rhizosphere Microorganisms and Bacterial Physiological Groups of Transgenic Bt Corn" (Wang Min et al., Journal of Ecology, Issue 3, 2010) and "Effects of Transgenic Bt Corn on the Diversity of Soil Bacteria" (Liu Ling et al., Journal of Ecology and Rural Environment, Issue 3, 2011) analyzed the number and diversity of bacteria in the soil of indoor and outdoor Bt corn cultivation. The results showed that there were significant differences in the number and diversity of soil bacteria between the Bt corn cultivation group and the blank control group. The paper "Cry1Ac protoxin from Bacillus thuringiensis sp. kurstaki HD73 binds to surface proteins in the mouse small intestine" (Vázquez-Padrón et al., Biochem Biophys Res Commun, January 2000) found in animal experiments that when mice ingested 10 mg / kg and 100 mg / kg of Bt endotoxin and exotoxin, the mice showed significant inhibition of T cell ANAE positivity, spleen index, and macrophage phagocytic function. This inhibitory effect became increasingly pronounced with increasing doses. The study also found that when the accumulation coefficient of Bt toxin protein in the animal body exceeded 6.24, it could cause damage to the liver, kidneys, and gastrointestinal tract. Cell swelling and vacuolar degeneration were observed in the liver and kidneys, and lesions in the glomerular vascular epithelial cells were also observed.Long-term, high-dose exposure to Bt toxins can also lead to significant decreases in total white blood cell counts and hemoglobin levels in animals, demonstrating their immunosuppressive toxicity. Therefore, developing a simple, convenient, and effective detection technology for broad-spectrum tracking of multiple Bt Cry toxin residues is of great scientific significance and holds broad market potential.

[0005] Currently, the most widely used Bt Cry toxin detection methods are immunological detection methods based on traditional polyclonal antibodies, monoclonal antibodies, or new genetically engineered antibodies. Conventionally, a single Bt Cry toxin subtype is used as an immunization or coating agent. However, whether using animal immunization or antibody library screening strategies, it is difficult to obtain highly active, broad-spectrum antibodies that recognize multiple Bt Cry toxin subtypes. Studies have found that the amino acid sequences of most common Bt Cry toxin subtypes are highly conserved in evolution, and the corresponding proteins are also highly similar in three-dimensional structure ("Bacillus thuringiensis: a century of research, development and commercial applications", Sanahuja et al., Plant Biotechnol J, 2011, No. 9; "Structural, functional, and evolutionary analysis of Cry toxins of Bacillus thuringiensis: an in silico study", Das et al., Egyptian Journal of Biological Pest Control, 2021, No. 31). In their previous studies on the common structure and function analysis of Bt Cry toxins, the applicant team compared the amino acid sequence information of seven Bt Cry toxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1E, and Cry1F) and obtained three common structural short peptides that mimicked part of the structure of Bt Cry toxins ("Production and Characterization of Monoclonal Antibody Broadly Recognizing Cry1 Toxins by Use of Designed Polypeptide as Hapten", Dong et al., Analytical Chemistry, Vol. 88, No. 14, 2016). By coupling these short peptides to haptens of large molecular weight proteins for immunization, monoclonal and polyclonal antibodies that could recognize these seven toxin subtypes were successfully prepared. The minimum detection sensitivity of the established DAS-ELISAs for these toxins was close to 15 ng / mL. However, the molecular weight of these common structural short peptides was too small to be cloned, expressed, and purified by conventional methods, nor could they be used directly as immunogens for immunizing animals. This greatly limited the application of this method in Bt Practical application in the preparation of broad-spectrum antibodies against Cry toxins.In addition, the applicant team also designed an alternating antigen coating method to successfully obtain genetically engineered antibodies with broad recognition of Cry1Ab, Cry1Ab, Cry1B, Cry1B, and Cry1F toxin subtypes from a phage-displayed antibody library. The indirect competitive enzyme-linked immunosorbent assay method established for these toxins has a minimum detection sensitivity of 29-74 ng / mL. However, the antibodies prepared by this method have a narrow spectrum of recognition and relatively low detection sensitivity. Therefore, developing methods for preparing broad-spectrum antibodies for broad-spectrum detection of Bt Cry toxins and obtaining antibodies with broad binding activity has become a technical challenge that urgently needs to be solved in this field. Summary of the Invention

[0006] To address the above issues, the present application provides a method for designing monoclonal antibodies for broad-spectrum detection of Bt Cry toxins, obtains relevant antibody materials based on this method, and establishes a method for broad-spectrum detection of Bt Cry toxins. Specifically, the present invention is implemented as follows:

[0007] First, the present application provides a monoclonal antibody that can highly sensitively identify 8 Bt Cry toxin subtypes, which the applicant named JJF-GP-mAb; the antibody consists of a heavy chain and a light chain, and the amino acid sequences of the heavy chain and light chain are shown in SEQ ID NO.3 and SEQ IN NO.4, respectively.

[0008] Secondly, the present application provides the coding gene of the monoclonal antibody JJF-GP-mAb that can highly sensitively identify 8 Bt Cry toxin subtypes, and the coding nucleotide sequences of its heavy chain and light chain are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0009] Third, the present application provides a hybridoma cell line 1A10-C5-B11 that can secrete the monoclonal antibody JJF-GP-mAb, and its deposit number is CCTCCNO: C202403.

[0010] Fourth, the present application provides a recombinant expression vector, a transgenic cell line or a recombinant engineered bacterium containing the coding gene shown in SEQ ID NO.1 or SEQ ID NO.2.

[0011] Fifth, this application also provides the use of the monoclonal antibody JJF-GP-mAb described above in the detection of Bt Cry toxins; the Bt Cry toxins include at least one of Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C, Cry1F, Cry2Ab, and Cry3Aa. Specifically, the monoclonal antibody JJF-GP-mAb is used as a detection antibody in a competitive ELISA (ic-ELISA) for Bt Cry toxins, or as a capture antibody in a sandwich ELISA (DAS-ELISA) for Bt Cry toxins.

[0012] In the examples of the present application, the amino acid sequence information of six Bt Cry toxins, Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C and Cry1F, is compared from a public Bt toxin information database. Three-dimensional structural models of the corresponding toxins are simultaneously established, and their similarities are compared. Then, an alternating screening immunization strategy is designed based on the evolutionary relationships of the amino acid sequences and the similarities of the three-dimensional structures of the corresponding toxins. The monoclonal antibody (JJF-GP-mAb) prepared by this design method can simultaneously and highly sensitively identify eight Bt Cry toxin subtypes, including Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C, Cry1F, Cry2Ab and Cry3Aa.

[0013] Compared with the prior art, this application has the following beneficial effects:

[0014] 1. The monoclonal antibody JJF-GP-mAb created in this application can simultaneously recognize 8 and 10 common commercially used Bt Cry toxin subtypes. It is the first reported broad-spectrum antibody that can simultaneously span Cry1, Cry2, and Cry3 toxins, greatly improving the broad-spectrum recognition range of Bt Cry toxins compared with existing reports.

[0015] 2. The amino acid sequence of the monoclonal antibody JJF-GP-mAb protein of the present application and its encoding gene have been determined. Therefore, this antibody material can not only be used to prepare the target antibody by traditionally immunizing mice to produce ascites, but can also be prepared by soluble expression through various expression systems, which is beneficial for actual production and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The three-dimensional structural models of the six Bt Cry toxins obtained in Example 1;

[0017] Figure 2 The three-dimensional structural model of Bt Cry toxin in Example 1 is an overlapping complex and the associated RMSD values;

[0018] Figure 3 The results of ICN-ELISA binding activity assays of the test mouse sera collected after the last round of immunization in Example 2 against the six immunogen Bt Cry toxin subtypes are shown;

[0019] Figure 4 The purification effect of the target monoclonal antibody and the antibody subtype identification results analyzed by SDS-PAGE in Example 2;

[0020] Figure 5 The results show that JJF-GP-mAb can recognize the broad spectrum of Bt Cry toxin subtypes.

[0021] Figure 6 The results of ICN-ELISA binding activity assays of the rabbit serum collected after the last round of immunization in Example 3 against the six immunogen Bt Cry toxin subtypes are shown;

[0022] Figure 7 The purification effect of the target polyclonal antibody analyzed by SDS-PAGE in Example 3;

[0023] Figure 8 The results show that the JJF-GP-pAbs polyclonal antibody has a broad spectrum of recognition capabilities against Bt Cry toxin subtypes.

[0024] Figure 9 An IC-ELISA standard curve for Bt Cry toxin was established based on the JJF-GP-mAb monoclonal antibody;

[0025] Figure 10 Standard curve for DAS-ELISAs for broad-spectrum detection of Bt Cry toxins using JJF-GP-mAb as capture antibody and JJF-GP-pAbs as tracer antibody. DETAILED DESCRIPTION

[0026] The reagent formula involved in the embodiment:

[0027] (1) PBS solution

[0028] Weigh 8 g of NaCl, 0.2 g of KCl, 2.9 g of Na2HPO4·12H2O, and 0.2 g of KH2PO4, add them respectively into distilled water, dissolve them fully, and adjust the volume to 1 L.

[0029] (2) PBST solution

[0030] Tween-20 was added to the PBS solution at a volume ratio of 0.05%.

[0031] (3) Tetramethylbenzidine (TMB) solution:

[0032] Weigh 10 mg of tetramethylbenzidine and dissolve it in 1 ml of dimethyl sulfoxide. Protect from light and store at 4°C until use.

[0033] (4) Substrate color development solution:

[0034] 10ml formula ingredients: 9.875mL CPBS, 100μL TMB solution, 25μL 20% H2O2 by volume.

[0035] Sources of materials involved in the examples:

[0036] Bt Cry toxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C, Cry1F, Cry1Ie, Cry2Aa, Cry2Ab, Cry3Aa, Cry3Ab, Bt Vip3) were purchased from Meiyan (Beijing) Agricultural Science and Technology Co., Ltd.;

[0037] The Balb / c model mice were provided by the Center for Comparative Medicine of Yangzhou University.

[0038] New Zealand white rabbits were purchased from Nanjing Zhongding Biological Co., Ltd.

[0039] Goat anti-rabbit / mouse IgG-HRPs was purchased from Beijing Qingke Biotechnology Co., Ltd.

[0040] TMB substrate colorimetric solution was purchased from Beijing Pulilai Gene Technology Co., Ltd.

[0041] Freund's complete adjuvant, Freund's incomplete adjuvant, hypoxanthine thymidine (HT), hypoxanthine aminopterin thymidine (HAT), and polyethylene glycol (PEG) were purchased from Sigma.

[0042] DMEM high-glucose culture medium was purchased from Nanjing Youqing Biotechnology Co., Ltd.;

[0043] Fetal bovine serum (Fetal Bovine Serum) was purchased from Nanjing Novozymes Biotechnology Co., Ltd.

[0044] HAT medium: purchased from Sigma-Aldrich, USA, model number H0262-10VL;

[0045] HiTrap Protein A / G HP was purchased from GE;

[0046] Mouse monoclonal antibody isotype ELISA Kit was purchased from Beijing Biotech Biotechnology Co., Ltd.;

[0047] Cell culture plates (9-, 24-, and 96-well), cell culture flasks, and ELISA plates were purchased from Corning;

[0048] The rest of the reagents used were of analytical grade.

[0049] Example 1 Comparative Analysis of Bt Cry Toxin Structure Simulators

[0050] From Genbank( http: / / www.ncbi.nlm.nih.gov / ) Download the gene sequences of Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C and Cry1F toxins and translate them into corresponding amino acid sequences. Then, the three-dimensional structures of these toxins were modeled with the help of the SWISS-MODEL online free website (http: / / swissmodel.expasy.org / ). The relevant toxin models were evaluated based on online bioinformatics websites such as Ramachandran plot, ERRAT and Verify3D (Dong et al., "Production and Characterization of Monoclonal Antibody Broadly Recognizing Cry1 Toxins by Use of Designed Polypeptide as Hapten", 2016, Analytical Chemistry), and the rationality and similarity of the relevant toxin models were predicted and compared. The three-dimensional structure models of the six Bt Cry toxins are as follows: Figure 1 The rationality evaluation results are shown in Table 1 below.

[0051] Table 1 Model structure evaluation

[0052]

[0053]

[0054] Figure 2 The overlapping complex of the three-dimensional structure model of Bt Cry toxin and the related RMSD values ​​are shown, where (a) is the result of the overlapping complex of the three-dimensional structure model of Bt Cry toxin, and (b) is the related RMSD value result. Figure 1 、 Figure 2It can be seen that the predicted three-dimensional structural models of Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C and Cry1F toxins selected in this example are highly credible and reasonable. Through overlap composite and root mean square deviation (RMSD) analysis, it is further confirmed that the similarity relationship order of these Bt Cry toxin subtypes is Cry1Ab>Cry1Ah>Cry1Ac>Cry1C>Cry1F>Cry1B, which is consistent with the evolutionary conservation relationship order of their amino acid sequences (see the document "Bacillus thuringiensis: a century of research, development and commercial applications", Sanahuja et al., Plant Biotechnol J, Issue 9, 2011).

[0055] Example 2 Preparation and purification of broad-spectrum monoclonal antibodies to Bt Cry toxin

[0056] Based on the evolutionary relationships of the amino acid sequences of the six Bt Cry toxin subtypes obtained in Example 1 and the similarities in the three-dimensional structures of the corresponding toxins, the following immunization strategy was designed:

[0057] 1. Immunization and effect determination process:

[0058] 1.1) Mix Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C, and Cry1F in a mass ratio of 1:1:1:3:2:2 to obtain toxin mixture I, which is set aside;

[0059] Cry1Ah and Cry1B were mixed uniformly in a mass ratio of 1:1 to obtain toxin mixture II, which was set aside;

[0060] Cry1C and Cry1F were mixed uniformly in a mass ratio of 1:1 to obtain toxin mixture III for later use;

[0061] Cry1Ab and Cry1C were mixed evenly in a mass ratio of 1:1 to obtain toxin mixture IV, which was set aside;

[0062] Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C and Cry1F were uniformly mixed in a mass ratio of 1:1:1:1:1:1 to obtain a toxin mixture V, which was set aside;

[0063] 1.2) First round of immunization: 100 μg of toxin mixture I was dissolved in 100 μL PBS solution, 100 μL complete Freund's adjuvant was added and thoroughly mixed and emulsified, and then injected into the peritoneal cavity of 6-week-old healthy female Balb / c mice by intraperitoneal injection; Two weeks later, the second round of immunization was performed: 120 μg of toxin mixture II was dissolved in 100 μL PBS solution, 100 μL incomplete Freund's adjuvant was added and thoroughly mixed and emulsified, and then injected into the peritoneal cavity of the test mice; Two weeks later, the third round of immunization was performed: 120 μg of toxin mixture III was dissolved in 100 μL To the PBS solution, 100 μL of incomplete Freund's adjuvant was added, mixed and emulsified thoroughly, and then injected into the peritoneal cavity of the test mice; two weeks later, the fourth round of immunization was performed: 120 μg of toxin mixture IV was taken, dissolved in 100 μL of PBS solution, 100 μL of incomplete Freund's adjuvant was added, mixed and emulsified thoroughly, and then injected into the peritoneal cavity of the test mice; one week later, the fifth round of immunization was performed: 160 μg of toxin mixture V was taken, 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 for the preparation of monoclonal antibody fusion cell lines.

[0064] Before each round of immunization, a small amount of blood was collected by tail clipping to precipitate serum (immunized mouse blood was obtained by tail clipping, allowed to stand at room temperature for 6 hours, and the precipitated serum was collected, which was the test mouse serum) for indirect noncompetitor enzyme-linked immunosorbent assay (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 measure and analyze the corresponding immune effect. Specific steps: A toxin mixture V solution (Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C and Cry1F were mixed in equal amounts and dissolved in PBS buffer) with a concentration of 2 μg / mL was added to a 96-well plate at 100 μL / well and allowed to stand for 2 hours at 37°C. The coated plate was removed and washed with 300 μL / well of PBST buffer. Add MPBS solution at a rate of 300 μL / well and incubate at 37°C for 2 hours. Remove the coated plate, wash the plate with 300 μL / well PBST buffer, then add a 10-fold dilution (V:V) of the test mouse serum solution with PBS buffer at a rate of 100 μL / well, and incubate at 37°C for 2 hours. Remove the coated plate, wash the plate with 300 μL / well PBST buffer, then add secondary antibody (anti-mouse IgG-HRP diluted with PBS buffer at a volume ratio of 1:5000) at a rate of 100 μL / well, and incubate at 37°C for 2 hours. Wash the plate again with 300 μL / well PBST buffer, then add TMB color development solution at a rate of 100 μL / well, incubate at 37°C in the dark for 15 minutes, and then measure the OD of the relevant reaction wells. 450 The results of ICN-ELISA binding activity test of the test mouse serum collected after the last round of immunization against the six immunogen Bt Cry toxin subtypes are as follows: Figure 3 shown.

[0065] 2. Cell fusion process

[0066] It will be determined that the immune effect has reached a usable level (positive OD 450 Value>1.0, the titer reaches 10 4The mice were dislocated by cervical dislocation and then soaked in 75% alcohol for 10 minutes. They were then removed and drained. In a sterile environment, the spleen was removed and ground in an incomplete culture medium (HAT culture medium). After filtering, the grinding liquid was collected and aliquoted at 20 mL / tube, and the spleen cells were counted under a microscope. Splenocytes and SP2 / 0 myeloma cells were mixed at an equal volume ratio of 5:1 according to the cell number, and then centrifuged at 1000 rpm in a 37°C centrifuge for 10 minutes. The supernatant was discarded, and the precipitated mixed cells were resuspended with 1 mL of PEG at 40°C and allowed to stand for 1 minute. Then 15 mL of HAT culture medium was added, and the cells were allowed to stand in a 37°C incubator for 10 minutes and then centrifuged at 800 rpm for 8 minutes. The precipitated cells were first resuspended with 5 mL of HAT culture medium, and then HAT culture medium was added to make the volume 50 mL. Finally, 100 μL / well was aliquoted into a 96-well plate and placed in a 37°C incubator for culture. After five days, fresh HAT medium was added to the cells at a volume of 100 μL / well. Two days later, half of the culture medium in the wells was aspirated and an equal volume of fresh HAT medium was added. The cells were then cultured until the cells occupied approximately 1 / 10 of the bottom of the wells. The cell culture medium was then used to analyze the binding activity of the cells to the six Bt Cry toxins described above using the same INC-ELISA method as in step 1.2 above.

[0067] 3. Purification and preparation of target monoclonal antibodies and analysis of their broad-spectrum recognition capabilities

[0068] The fusion cell wells that have been identified as having broad-spectrum and high activity (antibodies that can recognize more than three substances) to recognize the above six immunogen BtCry toxin subtypes are cultured in gradient dilution until a subclone fusion cell line that can stably secrete the monoclonal antibody appears.

[0069] The cell number of the target subclone fusion cell line was quantified to 10 using DMEM high glucose medium. 6 The target monoclonal antibody protein was isolated and purified from the ascites using ammonium sulfate precipitation (see "Preparation and Detection of Polyclonal Antibodies to Bt (Cry1F) Toxin," Xu Chongxin et al., Agricultural Product Quality and Safety, Issue 4, 2016) and HiTrap Protein G HP column purification (refer to the product manual for procedures).

[0070] This example finally obtained a monoclonal antibody with broad spectrum and high activity, which the applicant named JJF-GP-mAb. After sequencing, the nucleotide sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ IN NO.1, and the nucleotide sequence of the light chain variable region is shown in SEQ IN NO.2; the amino acid sequence of the heavy chain variable region is shown in SEQ IN NO.3, and the amino acid sequence of the light chain variable region is shown in SEQ IN NO.4. The purification effect and antibody subtype identification results are shown in Figure 4 shown. Figure 4 In the figure, (a) is the target monoclonal antibody analyzed by SDS-PAGE, and (b) is the antibody purification effect and antibody subtype identification results; M: Protein marker; Lanes 1-4: purified target monoclonal antibody protein after treatment with β-thiol denaturing buffer.

[0071] The hybridoma cell line secreting the monoclonal antibody JJF-GP-mAb (designated 1A10-C5-B11 by the applicant) was deposited with the China Center for Type Culture Collection (CCTCC) on January 9, 2024, at Wuhan University, Wuhan 430072, China, under the accession number CCTCC NO: C202403. Antibody isotype determination was performed according to the Mouse Monoclonal Antibody Isotype ELISA Kit manual. The broad-spectrum recognition of the prepared monoclonal antibody against BtCry toxins was also determined using an INC-ELISA. A 2 μg / mL mixed toxin solution (equal amounts of Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C, and Cry1F dissolved in PBS) was added to a 96-well plate at a rate of 100 μL / well. The plates were incubated at 37°C for 2 hours. Remove the coated plate, wash with 300 μL / well PBST buffer, add 300 μL / well MPBS solution, and incubate at 37°C for 2 hours. Remove the coated plate, wash with 300 μL / well PBST buffer, add 100 μL / well 2.5 μg / mL JJF-GP-mAb protein solution (JJF-GP-mAb protein dissolved in PBS buffer), and incubate at 37°C for 2 hours. Remove the coated plate, wash with 300 μL / well PBST buffer, and add 100 μL / well secondary antibody (anti-mouse IgG-HRP diluted 1:5000 in PBS buffer by volume) and incubate at 37°C for 2 hours. After washing the plate with 300 μL / well PBST buffer, add TMB colorimetric solution at 100 μL / well and develop the color for 15 minutes at 37°C in the dark. Then measure the OD of the relevant reaction wells. 450 The result is as follows Figure 5shown.

[0072] above Figure 3-5 This shows that the immunization strategy designed in this example is successful. The titer of the immunized mouse serum for the six immunogen BtCry toxin subtypes reached 107, which is a high binding activity level. In addition, the pure JJF-GP-mAb monoclonal antibody prepared by screening the spleen cell fusion cell line of the immunized mice can simultaneously have high recognition and binding activity for eight Bt Cry toxin subtypes, including Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C, Cry1F, Cry2Ab and Cry3Aa. This shows that the monoclonal antibodies prepared by the designed immunization strategy have the characteristics of broad-spectrum and high binding activity to Bt Cry toxins.

[0073] Example 3 Preparation and purification of broad-spectrum polyclonal antibodies to Bt Cry toxin

[0074] The initial rabbit immunization strategy for the preparation of the broad-spectrum monoclonal antibody to Bt Cry toxin in this example was the same as the mouse immunization strategy for the polyclonal antibody in Example 2. The only difference was that the immunization dose of the toxin mixture was doubled in each round, and subcutaneous injection was used, as follows:

[0075] 1. Immunization and effect determination process

[0076] 1.1) Mix Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C, and Cry1F toxins in a mass ratio of 1:1:1:3:2:2 to obtain toxin mixture I, which is set aside;

[0077] 1.2) Dissolve 200 μg of toxin mixture I in 100 μL of PBS solution, add 100 μL of complete Freund's adjuvant and mix thoroughly to emulsify to obtain the immunogen;

[0078] 1.3) First round of immunization: The immunogen was injected into the subcutaneous tissue of healthy female New Zealand white rabbits weighing approximately 2 kg using multiple subcutaneous injections. Two weeks later, a second round of immunization was performed: 240 μg of toxin mixture II (obtained by mixing Cry1Ah and Cry1B at a mass ratio of 1:1) was dissolved in 100 μL of PBS solution, and 100 μL of incomplete Freund's adjuvant was added for thorough mixing and emulsification, and the same was injected subcutaneously into the test rabbit. Two weeks later, a third round of immunization was performed: 240 μg of toxin mixture III (obtained by mixing Cry1C and Cry1F at a mass ratio of 1:1) was dissolved in 100 μL of PBS solution, and 100 μL of incomplete Freund's adjuvant was added for thorough mixing and emulsification, and the same was injected subcutaneously into the test rabbit. Two weeks later, a fourth round of immunization was performed: 240 μg of toxin mixture IV (obtained by mixing Cry1Ab and Cry1C at a mass ratio of 1:1) was dissolved in 100 μL of PBS solution, and 100 μL of incomplete Freund's adjuvant was added to fully mix and emulsify, and the mixture was also injected subcutaneously into the test rabbits; one week later, the fifth round of immunization was carried out: 320 μg of toxin mixture V (obtained by mixing Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C and Cry1F at a mass ratio of 1:1:1:1:1:1) was dissolved in 100 μL PBS solution, and then directly injected into the subcutaneous part of the test rabbits at multiple points; after another week, serum was collected: blood was drawn from the immune rabbits by cardiac blood sampling, and the blood was allowed to stand at room temperature for 6 hours. The precipitate on the upper layer of the solution was the precipitated serum (immune serum solution).

[0079] Before each round of immunization, a small amount of blood was collected from the ear vein to separate the serum for analysis of the corresponding immune effect by indirect non-competitive enzyme-linked immunosorbent assay. The detection method was the same as in Example 2, except that the secondary antibody was replaced with anti-rabbit IgG-HRP in equal amounts from anti-mouse IgG-HRP. The results of ICN-ELISA binding activity of the test rabbit serum collected after the last round of immunization to the six immunogens Bt Cry toxin subtypes are shown as follows: Figure 6 shown.

[0080] 2. Purification and preparation of target polyclonal antibodies and analysis of their broad-spectrum recognition ability

[0081] Referring to the saturated ammonium sulfate precipitation method previously disclosed by the applicant team (see the document "Preparation and Detection Application of Bt (Cry1F) Toxin Polyclonal Antibodies", Xu Chong-chang et al., Quality and Safety of Agricultural Products, Issue 4, 2016) and the HiTrap ProteinA HP (for steps, refer to the product operation manual) column purification method, polyclonal antibody proteins were separated and purified from the test rabbit serum collected after the last round of immunization. The polyclonal antibody protein obtained in this example was named JJF-GP-pAbs. The purification effect of the target polyclonal antibody JJF-GP-pAbs was shown in the following figure using SDS-PAGE analysis (Xu Chong-chang et al., "Detection of Cry 1C Toxin in Rice by Indirect Competitive Time-Resolved Fluorescence Immunoassay", Journal of Nanjing Agricultural University, 2014). Figure 7 shown. Figure 7 Middle, M: Protein marker; Lanes 1-2: Purified target polyclonal antibody protein after treatment with β-thiol denaturation buffer.

[0082] The broad-spectrum recognition of the prepared polyclonal antibody JJF-GP-pAbs against Bt Cry toxins was also determined using the INC-ELISA method: A 2 μg / mL mixed toxin solution (equal amounts of Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C, and Cry1F dissolved in PBS) was added to a 96-well plate at 100 μL / well and incubated at 37°C for 2 hours. The coated plate was removed, washed with 300 μL / well of PBST buffer, and then blocked with 300 μL / well of MPBS at 37°C for 2 hours. The coated plate was removed, washed with 300 μL / well of PBST buffer, and then 100 μL / well of a 2.5 μg / mL JJF-GP-pAbs protein solution (dissolved in PBS) was added at 37°C for 2 hours. Remove the coated plate, wash the plate with 300 μL / well PBST buffer, add secondary antibody (anti-rabbit IgG-HRP diluted with PBS buffer at a volume ratio of 1:5000) at 100 μL / well, and incubate at 37°C for 2 hours. Wash the plate again with 300 μL / well PBST buffer, add TMB colorimetric solution at 100 μL / well, develop the color at 37°C in the dark for 15 minutes, and measure the OD450 value of the relevant reaction wells. The results are as follows: Figure 8 shown.

[0083] above Figure 6-8The test results demonstrated that the immunization strategy designed in this example was successful. The titers of the immunized rabbit serum against the six immunogen Bt Cry toxin subtypes all reached 108, indicating a high binding activity level. Furthermore, the pure JJF-GP-pAbs polyclonal antibodies isolated and prepared therefrom simultaneously exhibited high recognition and binding activity against ten Bt Cry toxin subtypes, including Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C, Cry1F, Cry2Aa, Cry2Ab, and Cry3Aa. This indicates that the polyclonal antibodies prepared using this designed immunization strategy exhibit broad-spectrum, high-binding activity against Bt Cry toxins.

[0084] Example 4 Establishment of IC-ELISA for broad-spectrum detection of Bt Cry toxins

[0085] The detection method in this example is based on the literature reference: Xu et al., "Selection and application of broad-specificity human domain antibody for simultaneous detection of Bt Crytoxins", 2016, Analytical Biochemistry. Utilizing the broad-spectrum recognition of Bt Cry toxins by the monoclonal antibody JF-GP-mAb prepared in Example 2, an IC-ELISA method for broad-spectrum detection of eight Bt Cry toxin subtypes, including Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C, Cry1F, Cry2Ab, and Cry3Aa, was established. The specific steps are as follows:

[0086] Six Cry1 toxins were diluted with CBS to concentrations of 5, 2.5, 1.25, 0.625, 0.3125, and 0.15625 μg / mL. 100 μL per well was added to a 96-well microtiter plate and incubated overnight at 4°C. The coated plate was removed and washed with 300 μL / well of PBST buffer. MPBS solution was added at a rate of 300 μL / well and blocked at 37°C for 2 hours. The coated plate was removed and washed with 300 μL / well of PBST buffer. Antibodies were diluted 2-fold to concentrations of 5, 2.5, 1.25, 0.625, 0.3125, and 0.15625 μg / mL at a rate of 100 μL / well. PBS was used as a negative control and incubated at 37°C for 1 hour. The coated plate was removed and washed with 300 μL / well of PBST buffer. Anti-mouse IgG-HRP secondary antibody (diluted 1:5000 in PBS buffer) was added at 100 μL / well and incubated at 37°C for 1 hour. The plate was then washed again with 300 μL / well of PBST buffer. TMB colorimetric solution was added at 100 μL / well and incubated at 37°C in the dark for 15 minutes. The OD450 values ​​of the relevant wells were measured to determine the optimal antigen and antibody concentrations. Using these determined antigen and antibody concentrations, IC-ELISA assays were developed for six Cry toxins. Ten two-fold dilutions of Cry toxins were prepared in PBS starting at 400 μg / mL. Six standard curves were generated at the optimal antibody concentration. Four-parameter fitting was performed using GraphPad Prism software, and IC20 and IC50 values ​​for the six Cry toxins were calculated using ELISACalc. All values ​​are the average of three replicates.

[0087] Figure 9 Based on the JF-GP-mAb prepared in Example 2, an IC-ELISA standard curve for Bt Cry toxin was established. The inhibition ratios were the mean ± SDs of three measurements.

[0088] Table 2 shows the activity parameter values ​​for the IC-ELISA established with JJF-GP-mAb for broad-spectrum detection of BtCry toxins, calculated according to the corresponding standard curve equation.

[0089] Table 2

[0090]

[0091] Example 5 Addition recovery experiment

[0092] To evaluate the practicality and applicability of the IC-ELISA method established in Example 4, tap water, corn, rice, soybean, and soil samples were selected for spiking and recovery testing of the eight Bt Cry toxin subtype standards. Tap water samples were obtained from the laboratory of the Jiangsu Academy of Agricultural Sciences and filtered through a 0.45 μm filter membrane before spiking. Corn, rice, soybean, and soil (obtained from the experimental fields of the Jiangsu Academy of Agricultural Sciences) were oven-dried at 45°C, ground into a powder, and filtered through a 100-mesh sieve before spiking.

[0093] According to the linear detection range of the DAS-ELISAs method for different Bt Cry toxin subtypes established in Example 4 (Table 2), the final concentrations of Cry1Ab toxin added to tap water were 3, 8.5, and 20 μg / mL, the final concentrations of Cry1Ac toxin added were 1.2, 4.5, and 20 μg / mL, the final concentrations of Cry1Ah toxin added were 2.5, 12, and 55 μg / mL, the final concentrations of Cry1B toxin added were 5, 20, and 50 μg / mL, the final concentrations of Cry1C toxin added were 1.5, 4.5, and 16.5 μg / mL, the final concentrations of Cry1F toxin added were 1.5, 12, and 77 μg / mL, the final concentrations of Cry2Ab toxin added were 3, 18, and 75 μg / mL, and the final concentrations of Cry3Aa toxin added were 2, 6, and 20 μg / mL. The final concentrations of Cry1Ab toxin added to corn, rice, soybean and soil were 30, 85 and 200 μg / g, the final concentrations of Cry1Ac toxin added were 12, 45 and 200 μg / g, the final concentrations of Cry1Ah toxin added were 25, 120 and 550 μg / g, the final concentrations of Cry1B toxin added were 50, 200 and 500 μg / g), the final concentrations of Cry1C toxin added were 15, 45 and 165 μg / g), the final concentrations of Cry1F toxin added were 15, 120 and 770 μg / g, the final concentrations of Cry2Ab toxin added were 30, 180 and 750 μg / g and the final concentrations of Cry3Aa toxin added were 20, 60 and 200 μg / g. The specific operation is that the corresponding concentration of Bt Cry toxin to be tested is added to the tap water sample and then directly measured according to the DAS-ELISA method established in Example 4; and the corresponding concentration of Bt Cry toxin to be tested is added to 1g powder samples such as corn, rice, soybeans and soil, and vortexed at room temperature for 30 minutes to promote sufficient mixing of the toxin and the sample. Then, the sample is placed at 4°C and left to stand overnight to allow the toxin to fully react with the sample. The next day, an appropriate amount of shaking agent is added to the 10 ml extract containing the sample and shaken at room temperature for 1 hour. Subsequently, it is immediately centrifuged at 8000g for 10 minutes. After centrifugation, the precipitate is discarded and the supernatant is retained as the extract required for the experiment. The determination was carried out according to the DAS-ELISA method established in Example 4, and the results are shown in Table 3.

[0094] Table 3 Recovery and coefficient of variation of IC-ELISA using JJF-GP-mAb for Bt Cry toxin-spiked samples

[0095]

[0096]

[0097]

[0098]

[0099] The above results showed that the IC-ELISA established with JJF-GP-mAb was effective for detecting different concentrations of Cry 1Ab, Cry1Ac, Cry1Ah, Cry 1B, Cry 1C, Cry 1F, Cry 2Ab and Cry The recovery rate of 3Aa toxin in tap water samples was 87.66%-101.70% with a coefficient of variation of 0.89–5.39%, the recovery rate in corn samples was 82.69%-93.62% with a coefficient of variation of 0.51%-9.28%, the recovery rate in rice samples was 76.97%-98.54% with a coefficient of variation of 0.45%-10.39%, the recovery rate in soybean samples was 76.65%-93.75% with a coefficient of variation of 0.87%-8.56%, and the recovery rate in soil samples was 74.47%-86.59% with a coefficient of variation of 1.16%-10.47%. The recoveries of all added Bt Cry toxin concentrations in the co-test samples were 74.47%-101.70% and the coefficient of variation was 0.45%-10.47%, indicating that the IC-ELISA established with JJF-GP-mAb in this application has good accuracy and stability for the detection of eight Bt Cry toxins, including Cry 1Ab, Cry 1Ac, Cry 1Ah, Cry 1B, Cry 1C, Cry 1F, Cry 2Ab and Cry 3Aa. Example 6 Establishment of a DAS-ELISA method for the broad-spectrum detection of Bt Cry toxins

[0100] The detection method of this example refers to the method disclosed in the literature "Dong et al., Production and Characterization of Monoclonal Antibody Broadly Recognizing Cry1 Toxins by Use of Designed Polypeptide as Hapten, 2016, Analytical Chemistry". In combination with the characteristics of JJF-GP-mAb and JJF-GP-pAbs prepared in Examples 2 and 3 for broad-spectrum recognition of Bt Cry toxins, a DAS-ELISA method for broad-spectrum detection of eight Bt Cry toxin subtypes, including Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C, Cry1F, Cry2Ab and Cry3Aa, was finally established using the prepared JJF-GP-mAb as the capture antibody and JJF-GP-pAbs as the tracer antibody. The specific steps are as follows:

[0101] A 2.5 μg / mL JJF-GP-mAb solution (antibody protein dissolved in PBS buffer) was added to a 96-well plate at 100 μL / well and incubated at 37°C for 2 hours. The coated plate was removed and washed with 300 μL / well PBST buffer. MPBS solution was then added at 300 μL / well and blocked at 37°C for 2 hours. The coated plate was removed and washed with 300 μL / well of PBST buffer. Then, 100 μL / well of a gradient of Bt Cry toxins (test subtypes: Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C, Cry1F, Cry2Ab, and Cry3Aa) diluted in PBS buffer was added (10, 20, 50, 80, 100, 200, 500, 800, 1000, 1500, 2000, 2500, 5000, 8000, and 10000 ng / mL) and incubated at 37°C for 2 hours. The coated plate was removed and washed with 300 μL / well of PBST buffer. Then, 100 μL / well of a 5.0 μg / mL JJF-GP-pAbs solution diluted in PBS buffer was added and incubated at 37°C for 2 hours. Remove the coated plate and wash with 300 μL / well of PBST buffer. Then, add 100 μL / well of anti-rabbit IgG-HRP secondary antibody (diluted 1:5000 in PBS buffer) and incubate at 37°C for 2 hours. Wash the plate again with 300 μL / well of PBST buffer and add 100 μL / well of TMB colorimetric solution. After developing at 37°C in the dark for 15 minutes, measure the OD450 value of the relevant reaction wells to establish a DAS-ELISA standard curve for the corresponding Bt Cry toxin subtype.The 10% saturation concentration value (10% of saturating concentrations (SC10), also known as the limit of detection (LOD)), 50% of saturating concentration value (50% of saturating concentration (SC50), and 10%-90% of saturating concentrations (SC10-SC90), also known as the linear detection range (Linear detection range)) were all determined based on the established DAS-ELISAs standard curve according to the method reported in the literature "Establishment of novel receptor-antibody sandwich assays to broadly detect Bacillus thuringiensis Cry1 and Cry2 toxins" (Sheng et al., International Journal of Biological Macromolecules, Issue 254, 2024).

[0102] Figure 10 Standard curve for DAS-ELISAs for broad-spectrum detection of Bt Cry toxins using JJF-GP-mAb as capture antibody and JJF-GP-pAbs as tracer antibody.

[0103] Table 4 shows the key evaluation indicators of DAS-ELISAs established with JJF-GP-mAb as capture antibody and JJF-GP-pAbs as tracking antibody for broad-spectrum detection of Bt Cry toxins.

[0104] Table 4

[0105]

[0106] Figure 10The results in Table 4 show that the DAS-ELISAs established with JJF-GP-mAb and JJF-GP-pAbs prepared based on the immune strategy designed in the present invention are used for the broad-spectrum detection of Bt Cry toxins. Compared with the research examples introduced in the research background, they have the dual advantages of a wider detection spectrum (covering 8 commonly commercialized subtypes of the three major types of Cry1, Cry2 and Cry3) and higher detection sensitivity for toxins (LOD values ​​for Cry1Ab, Cry1Ac, Cry1Ah, Cry1F and Cry2Ab are all <10 ng / mL; LOD values ​​for Cry1B, Cry1C and Cry3Aa also reached 17.55, 34.28 and 50.35 ng / mL, respectively).

[0107] Example 7 Addition recovery experiment

[0108] In order to evaluate the practicality and applicability of the DAS-ELISAs method established in Example 6, tap water, corn, rice, soybean and soil samples were selected as the objects of the recovery test of the above-mentioned 8 Bt Cry toxin subtype standards. The tap water sample was taken from the laboratory of Jiangsu Academy of Agricultural Sciences and filtered through a 0.45μm filter membrane for sample addition, while the corn, rice, soybean and soil (taken from the experimental field of Jiangsu Academy of Agricultural Sciences) were air-dried in a 45℃ oven, ground into powder, and then filtered through a 100-mesh sieve before being used for sample addition. The linear detection range of Cry toxin subtypes (Table 1) was selected as follows: Cry1Ab toxin was added at a final concentration of 10, 30, and 150 μg / mL or μg / kg), Cry1Ac toxin was added at a final concentration of 5, 20, and 110 μg / mL or μg / kg), Cry1Ah toxin was added at a final concentration of 10, 60, and 1000 μg / mL or μg / kg, and Cry1B toxin was added at a final concentration of 10, 30, and 500 μg / mL or μg / kg), Cry1C toxin added at a final concentration of 40, 300, and 4000 μg / mL or μg / kg), Cry1F toxin added at a final concentration of 10, 30, and 100 μg / mL or μg / kg, Cry2Ab toxin added at a final concentration of 10, 300, and 3000 μg / mL or μg / kg), and Cry3Aa toxin added at a final concentration of 20, 300, and 2500 μg / mL or μg / kg. Specifically, the corresponding concentration of the Bt Cry toxin to be tested was added to the tap water sample and then directly measured according to the DAS-ELISA method established in Example 4. When the corresponding concentration of the Bt Cry toxin to be tested was added to powder samples such as corn, rice, soybeans, and soil, an equal volume of protein extract (PBS buffer containing 0.1% Tween 20 and 0.1% BSA) was added. The mixture was shaken and mixed at room temperature for 1 hour, and then centrifuged at 8000g in a 37°C centrifuge for 30 minutes. The supernatant was aspirated, diluted 10-fold with PBS buffer, and then measured according to the DAS-ELISA method established in Example 4. The results are shown in Table 5.

[0109] Table 5 Recovery and coefficient of variation of DAS-ELISAs using JJF-GP-mAb as capture antibody and JJF-GP-pAbs as tracer antibody for BtCry toxin-spiked samples

[0110] Table 5

[0111]

[0112]

[0113]

[0114] The test results in Table 5 show that the DAS-ELISAs established with JJF-GP-mAb as the capture antibody and JJF-GP-pAbs as the tracking antibody (other polyclonal antibodies can also be used in the specific implementation) were effective for different concentrations of Cry 1Ab, Cry 1Ac, Cry 1Ah, Cry 1B, Cry 1C, Cry 1F, Cry 2Ab and Cry The recovery rate of 3Aa toxin in tap water samples was 90.05%-98.28% with a coefficient of variation of 0.77-8.20%; the recovery rate in corn samples was 83.42%-93.94% with a coefficient of variation of 1.90%-8.61%; the recovery rate in rice samples was 75.21%-96.46% with a coefficient of variation of 1.19%-9.76%; the recovery rate in soybean samples was 77.62%-93.90% with a coefficient of variation of 1.48%-8.88%; and the recovery rate in soil samples was 74.12%-93.77% with a coefficient of variation of 0.90%-7.95%. The recoveries of all added BtCry toxin concentrations in the co-test samples were 74.12%-98.28%, with coefficients of variation of 0.77%-9.76%. This indicates that the DAS-ELISAs established in this application using JJF-GP-mAb as the capture antibody have good accuracy and stability for the detection of eight Bt Cry toxins, including Cry 1Ab, Cry 1Ac, Cry 1Ah, Cry1B, Cry 1C, Cry 1F, Cry 2Ab and Cry 3Aa, and are more conducive to the broad-spectrum tracking screening of multiple toxin residues in Bt Cry preparations or their genetically modified insect-resistant crops.

Claims

1. A monoclonal antibody for broad-spectrum detection of Bt Cry toxins, characterized in that: The antibody consists of a heavy chain and a light chain, and the amino acid sequences of the heavy chain and the light chain are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

2. The gene encoding the monoclonal antibody for broad-spectrum detection of Bt Cry toxins according to claim 1, characterized in that: The coding nucleotide sequences of the heavy chain and light chain are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.

3. A hybridoma cell line secreting a monoclonal antibody, whose deposit number is CCTCC NO: C202403.

4. A recombinant expression vector, transgenic cell line or recombinant engineered bacteria containing the coding genes shown in SEQ ID NO. 1 and SEQ ID NO.

2.

5. Use of the monoclonal antibody according to claim 1 in the non-diagnostic detection of Bt Cry toxin, wherein the Bt Cry toxin is selected from at least one of Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C, Cry1F, Cry2Ab and Cry3Aa.

6. The use according to claim 5, characterized in that The application refers to using the monoclonal antibody as a detection antibody or a capture antibody in ELISA detection of Bt Cry toxin.

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