Insect-resistant protein cry1da hybridoma cell strain, antibody produced by the same and use thereof

Abstract

The application discloses an insect-resistant protein Cry 1Da hybridoma cell strain, an antibody produced by the hybridoma cell strain and application of the hybridoma cell strain. The hybridoma cell strain is 7B1, which has been preserved in the China General Microbiological Culture Collection Center on May 29, 2026, and the preservation number is CGMCC No. 46851. The monoclonal antibody produced by the cell strain has a titer of 1:12280000, a subtype of IgG2b, and can be used as a capture antibody or a detection antibody. The monoclonal antibody is paired with another anti-Cry 1Da monoclonal antibody to construct a double antibody sandwich ELISA detection system, and is used for detection of the insect-resistant protein Cry 1Da.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to an insect-resistant protein Cry 1Da hybridoma cell line and the monoclonal antibody produced therefrom and its applications. Background Technology

[0002] Bacillus thuringiensis (Bt) Bacillus thuringiensis Bt (Bacillus subtilis) is a widely distributed group of Gram-positive bacteria that produces insecticidal crystal proteins during its growth. Bt crystal proteins mainly include Cry protein and Cyt protein. Cry protein exhibits insecticidal activity against various pests, including Lepidoptera, Diptera, and Coleoptera, and has been widely used in the development of biopesticides and transgenic insect-resistant crops.

[0003] With the advancement of research and industrialization of genetically modified insect-resistant crops, Cry-type insect-resistant proteins have been introduced into various crops such as corn, cotton, rice, and potatoes to enhance their resistance to target pests. Cry 1Da protein is one type of Cry-type insect-resistant protein. Accurate detection of Cry 1Da protein in crop materials and their derivatives is necessary during the research, development, seed production, preservation, and subsequent monitoring of genetically modified insect-resistant crops.

[0004] Currently, common methods for detecting specific insect-resistant proteins in genetically modified crops include immunological detection methods such as Western blotting and enzyme-linked immunosorbent assay (ELISA). Monoclonal antibodies, due to their high specificity, good batch stability, and suitability for establishing standardized detection systems, are an important basic material for constructing methods for detecting insect-resistant proteins. Therefore, obtaining hybridoma cell lines that stably secrete monoclonal antibodies against Cry1Da protein and providing the monoclonal antibodies produced by them is of great significance for the qualitative and / or quantitative detection of Cry1Da protein. Summary of the Invention

[0005] The purpose of this invention is to provide a hybridoma cell line containing the insect-resistant protein Cry 1Da, the monoclonal antibody produced therefrom, and its applications. The hybridoma cell line stably secretes monoclonal antibodies against the Cry 1Da protein, and these monoclonal antibodies can be used as capture or detection antibodies in a double-antibody sandwich ELISA detection system.

[0006] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a hybridoma cell line 7B1 containing the insect-resistant protein Cry 1Da. The hybridoma cell line 7B1 was deposited on May 29, 2026, at the China General Microbiological Culture Collection Center (CGMCC), classified and named as a monoclonal antibody hybridoma cell line. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, and the deposit number is CGMCC No. 46851.

[0007] In a second aspect, the present invention provides a monoclonal antibody produced from the Cry 1Da hybridoma cell line 7B1, which is an insect-resistant protein described in the first aspect.

[0008] Furthermore, the titer of the monoclonal antibody obtained by indirect ELISA detection is 1:12280000.

[0009] Furthermore, the subtype of the monoclonal antibody is IgG2b.

[0010] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2.

[0011] Thirdly, the present invention provides the monoclonal antibody described in the second aspect as a capture antibody, and the anti-Cry 1Da monoclonal antibody produced by hybridoma cell line 7F1 as a detection antibody, for use in the qualitative and / or quantitative detection of the insect protein Cry 1Da using a double-antibody sandwich ELISA.

[0012] Furthermore, the hybridoma cell line 7F1 was deposited on May 29, 2026, at the China General Microbiological Culture Collection Center (CGMCC), classified and named as a monoclonal antibody hybridoma cell line. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession number CGMCC No. 46852.

[0013] Compared with the prior art, the present invention has the following beneficial effects: This application utilizes a recombinant protein composed of amino acid residues 84-440 of the recombinant insect-resistant protein Cry 1Da, purified from prokaryotic expression, as an immunogenic antigen. A hybridoma cell line, 7B1, capable of stably secreting anti-Cry 1Da monoclonal antibodies was obtained using hybridoma technology. The monoclonal antibody, as detected by indirect ELISA, has a titer of 1:12,280,000, an antibody isotype of IgG2b, and clearly defined amino acid sequences in both the heavy and light chain variable regions. This monoclonal antibody can be used as a capture or detection antibody, and can be paired with another anti-Cry 1Da monoclonal antibody to construct a double-antibody sandwich ELISA detection system, providing antibody material for the detection of the insect-resistant protein Cry 1Da. Attached Figure Description

[0014] Figure 1 The image shows the SDS-PAGE electrophoresis results of the purified monoclonal antibody produced from hybridoma cell line 7B1.

[0015] Figure 2The figure shows the titer results of monoclonal antibodies produced by hybridoma cell line 7B1.

[0016] Figure 3 The image shows the results of Western blot analysis of the specific recognition of prokaryotic Cry 1Da protein by the monoclonal antibody generated from hybridoma cell line 7B1.

[0017] Figure 4 The image shows the results of a sandwich ELISA assay for detecting Cry 1Da protein in transgenic maize, using a monoclonal antibody produced by hybridoma cell line 7B1 as the capture antibody and another anti-Cry 1Da monoclonal antibody 7F1 as the detection antibody; "1 / 80" indicates that the sample was diluted 80 times.

[0018] Figure 5 The image shows the results of a double-antibody sandwich ELISA assay using another anti-Cry 1Da monoclonal antibody 7F1 as the capture antibody and a monoclonal antibody produced by hybridoma cell line 7B1 as the detection antibody, to detect Cry 1Da, Cry 1Ah, and Cry 1Ab+Cry 1Fa samples. Detailed Implementation

[0019] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments. All equivalent substitutions, conventional modifications, or improvements made based on the present invention should fall within the scope of protection of the present invention.

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art; unless otherwise specified, the reagents, materials and instruments used are commercially available.

[0021] Example 1: Obtaining the hybridoma cell line 7B1 and preparing its monoclonal antibody. 1.1 Preparation of Immunogens Amplification of genomic DNA from transgenic maize Cry 1Da Gene fragments were used to construct the expression vector pET28a- Cry 1Da (84-440aa) was transformed into Escherichia coli BL21 competent cells, activated by seed, expanded culture, and induced to express by IPTG overnight at 16℃. The His-Cry 1Da (84-440aa) recombinant protein, which is composed of amino acid residues 84-440 of Cry 1Da protein, was purified by Ni affinity chromatography and gel filtration.

[0022] 1.2 Immunized Animals Eight 8-week-old specific pathogen-free (SPF) female BALB / c mice (purchased from Hubei Provincial Laboratory Animal Research Center, license No.: SCXK (E) 2015-0018) were immunized with His-Cry 1Da (84-440aa) recombinant protein as antigen. The antigen was mixed with an equal volume of complete Freund's adjuvant (for primary immunization) or incomplete Freund's adjuvant (for booster immunization) and emulsified. After sufficiently mixing until a water-in-oil state was achieved, multi-point subcutaneous immunization was performed, followed by 2-3 booster immunizations with an interval of 2 weeks between each immunization. Titer detection was performed thereafter, and when the titer was >1:10000, intraperitoneal booster immunization was performed within 1 week, and the immunization dose of antigen was directly dissolved in 250 μL of PBS solution. The specific immunization procedure and immunization dose are shown in Table 1.

[0023] Table 1 Immunization procedure and immunization dose

[0024] Immunization example: In the first immunization, 50 μg of antigen is dissolved in PBS, then mixed with adjuvant at a volume ratio of 1:1.

[0025] 1.3 Cell fusion Three days after the final booster immunization, positive control blood is collected, the spleen is taken and prepared into a single cell suspension; after treatment, SP2 / 0 cells in logarithmic phase are mixed with spleen cells at a certain ratio (1:5-1:10), treated with 50% PEG1450 for 1 min, diluted with basal medium DMEM to terminate the reaction. After low-speed centrifugation, the cells are gently suspended and mixed with HAT medium containing 20% fetal bovine serum, according to 2×10 7 cells per plate are seeded into a pre-prepared feeder cell plate, and cultured at 37°C in an environment containing 5% CO2.

[0026] 1.4 Cell line establishment 1) Detection of fusion plates After the medium change of the fusion plate, when the cells grow to a medium size with more than about 10,000 cells, detection is started. After the ELISA quality control is qualified (that is, negative control OD 450 < 0.2 and positive control OD 450 > 1.0), positive wells (generally OD 450 ≥ 0.5) are selected for subcloning.

[0027] 2) Subcloning method and detection Wells with high positive values (OD 450 > 2.0) detected in the fusion plate are picked out for limiting dilution. Subcloning is performed by counting the number of monoclonal wells at 60% per plate. Monoclonal wells with higher positive values are picked out for limiting dilution each time. ELISA detection can be performed 5-7 days after each subcloning, until monoclonal cell lines that can stably secrete positive antibodies are finally screened out for expanded culture.

[0028] 3) Cell line establishment: Stable cell lines secreting positive antibodies, selected during the subcloning stage, were expanded into 24-well plates. After expansion, the supernatant was collected for antigen detection. Stability was verified using serially diluted ELISA and Western blotting. The monoclonal antibody secreted by the Cry 1Da hybridoma cell line 7B1 specifically detected Cry 1Da protein in transgenic maize samples. Cells were collected and expanded into 10 cm culture dishes. The supernatant was collected again, and the antibody titer was measured. Cells with high OD were selected. 450 Cell lines with a growth rate of >2.0 were cultured in cell flasks and cryopreserved, resulting in hybridoma cell line 7B1.

[0029] Hybridoma cell line 7B1 was deposited on May 29, 2026, at the China General Microbiological Culture Collection Center (CGMCC), classified as a monoclonal antibody hybridoma cell line. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession number CGMCC No. 46851.

[0030] Hybridoma cell line 7F1 was deposited on May 29, 2026, at the China General Microbiological Culture Collection Center (CGMCC), classified as a monoclonal antibody hybridoma cell line. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession number CGMCC No. 46852.

[0031] 4) Cell cryopreservation identification For cryopreserved cell lines, one cell line from the same batch was revived and identified using the following criteria: ① The number of revived live cells should be ≥1 million cells / vial; ② The number of viable cells among the live cells should be ≥500,000 / cell line; ③ The revived cells should not contain any microorganisms other than the cell line (e.g., bacteria, fungi, mycoplasma, etc.); ④ After the revived cells have grown to a certain number, select the well-grown cells for monoclonal counting and plate-laying, and test the antibody secretion ability of the monoclonal cells to see if they are all positive or have antibody secretion; ⑤ The cell culture supernatant should also be subjected to ELISA (OD). 450 >2.0), while performing Western blotting to determine whether positive antibodies are secreted.

[0032] pass Figure 3 It is known that the monoclonal antibody secreted by the Cry 1Da hybridoma cell line 7B1 can specifically detect the Cry 1Da protein expressed in prokaryotes.

[0033] 1.5 Preparation of ascites Mice were first injected intraperitoneally with phytane or liquid paraffin. One week later, hybridoma cell line 7B1 was inoculated into the peritoneum of mice. After cell line establishment, the cells were expanded in 10% fetal bovine serum medium. When the cell density reached 1×10⁻⁶ cells / mL, the cells were cultured. 6 -2×10 6 When the concentration of the sample was 100 μg / mL, the sample was centrifuged at 800 rpm, the precipitate was collected, and after resuspending in PBS, it was injected intraperitoneally into mice (liquid paraffin). After 7-10 days, the ascites fluid was collected for purification.

[0034] 1.6 Antibody Purification After pretreatment, the collected ascites fluid was purified using a Protein A-agarose affinity chromatography column. The specific steps are as follows: 1) Buffer: The starting buffer is pH 7.0, 20 mM phosphate buffer; the elution buffer is pH 2.7, 0.1 mM glycine hydrochloride.

[0035] 2) Prepare collection tubes: Take 1.5 mL centrifuge tubes and add 70 μL of pH9.0 1M Tris-HCl to each centrifuge tube.

[0036] 3) Sample preparation: The sample obtained by precipitation with 50% SAS was dialyzed overnight in the starting buffer and filtered through a 0.22μm microporous membrane.

[0037] 4) Purification process: Equilibrate the Protein A-agarose affinity chromatography column (HiTrap Protein A 1 mL, Pharmacia Biotech) with sufficient starting buffer (8-10 mL). Load 15-25 mL of the sample to be purified (containing 10.2-21.1 mg of protein per mL) onto the column at a flow rate of 0.5 mL / min. Then wash sequentially with 7-8 mL of starting buffer, 6-7 mL of elution buffer, and 5 mL of starting buffer at the same flow rate, collecting 1 mL of eluent from each tube.

[0038] 5) Purity and Activity Assay: The purity of the purified monoclonal antibody (McAb) was determined by SDS-PAGE. See details. Figure 1 The hybridoma cell line 7B1 monoclonal antibody was purified to remove almost all impurities and showed two specific main bands (55 kDa and 30 kDa).

[0039] 1.7 Determination of Monoclonal Antibody Titer Using recombinant His-Cry 1Da protein as the antigen, the titer of the purified monoclonal antibody was detected by indirect ELISA. The specific procedures are as follows: (1) Antigen coating: Dilute the antigen to 1 μg / ml with coating solution, add 100 μL / well to a polystyrene 96-well reaction plate, and incubate overnight at 4°C.

[0040] (2) Washing: The coating liquid is discarded the next day, and the well is washed once with 200 μL of washing solution.

[0041] (3) Sealing: Add 200 μL / well sealing solution and place at 37℃ for 2 h.

[0042] (4) Washing: Discard the coating liquid and wash 3 times with 200 μL / well of washing liquid.

[0043] (5) Add the primary antibody 7B1 to be tested: Dilute the 7B1 monoclonal antibody stock solution serially according to the ratio to make the detection concentration from 1 μg / mL to 0.498 ng / mL, add 100 μL / well to the reaction plate, and incubate at 37℃ for 1 h. Calculate the antibody dilution factor based on the 7B1 monoclonal antibody stock solution concentration of 12.0 mg / mL.

[0044] (6) Washing: Discard the sample to be tested, and wash 3 times with 200 μL of washing solution per well.

[0045] (7) Add enzyme-labeled secondary antibody: Add HRP-labeled goat anti-mouse IgG (1:10000, diluted with enzyme), 100 μL / well, and incubate at 37℃ for 40 min.

[0046] (8) Washing: Discard the enzyme-labeled secondary antibody and wash 5 times with 200 μL / well washing buffer.

[0047] (9) Color development: Add 90 μL of freshly prepared substrate solution per well and place in the dark at 37°C for 15-20 min.

[0048] (10) Termination of reaction and colorimetric analysis: After adding 50 μL / well of stop solution, measure the absorbance of each well at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0049] Using the OD of the hole to be tested 450 Values ​​and blank control well OD 450 A value ratio of not less than 2.1 is considered positive, and the highest antibody dilution factor that can be used to determine a positive result is taken as the antibody titer.

[0050] See results Figure 2 The titer of the purified 7B1 monoclonal antibody was determined by ELISA, based on a stock concentration of 12.0 mg / mL.

[0051] Table 2. Concentrations of monoclonal antibodies produced by hybridoma cell line 7B1 7B1 12.0 mg / mL Antibody subtype identification revealed that the monoclonal antibody produced by hybridoma cell line 7B1 is subtype IgG2b. The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2; the amino acid sequence of the heavy chain variable region of the 7F1 monoclonal antibody is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4.

[0052] The amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 1 is as follows: EVQLVESGGGLVKPGGSLKLSCAASGFTFSTYAMSWVRQTPEKRLEWVATITSGGSHTHYPDSVKGRFTISRDNAKNTLDLKMSSLRSEDTAMYYCVRGVRGLYAMDYWGQGTSVTVSS.

[0053] The amino acid sequence of the variable region of the light chain shown in SEQ ID NO: 2 is as follows: DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQRPGQSPKPLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLTVYSCQQHYSTPYTFGGGTKLEIR.

[0054] The amino acid sequence of the 7F1 heavy chain variable region shown in SEQ ID NO: 3 is as follows: EVQLVESGGDLVKPGGSLKVSCAASGFTFSSYAMSWVRQTPEKRLEWVATITSGGTYTHYPDSVKGRFTISRDNAKDTLYLQMSSLRSEDTAMYFCVRGVRGVYAIDYWGQGTSVTVSS.

[0055] The amino acid sequence of the 7F1 light chain variable region shown in SEQ ID NO: 4 is as follows: DIVMTQSHKLMSTSVGDRVSITCKASKDVSNAVAWYQQKPGQSPKPLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAIYYCQQHYNTPYTFGGGTKMEIK.

[0056] 1.8 Double Antibody Sandwich ELISA Detection In this embodiment, another anti-Cry 1Da monoclonal antibody, 7F1, was paired with the 7B1 monoclonal antibody to construct a double-antibody sandwich ELISA detection system. The 7F1 monoclonal antibody was produced from the Cry 1Da hybridoma cell line 7F1. The hybridoma cell line 7F1 was deposited on May 29, 2026, at the China General Microbiological Culture Collection Center (CGMCC), classified as a monoclonal antibody hybridoma cell line, with the deposit address at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, and accession number CGMCC No. 46852.

[0057] In this embodiment, the 7F1 monoclonal antibody is used as a paired antibody to verify the application of the 7B1 monoclonal antibody in a double-antibody sandwich ELISA detection system.

[0058] The main detection conditions for the double-antibody sandwich ELISA are as follows: the capture antibody coating concentration is 2 μg / mL, the working concentration of the detection antibody is 2 μg / mL, the detection antibody is biotin-labeled, and the detection is performed using a biotin-avidin enzyme-linked colorimetric system; the amount of standard and sample added is 100 μL / well, the incubation conditions are 37℃ for 1 hour, the colorimetric conditions are 37℃ in the dark for 15 minutes, and the absorbance value is read at 450 nm.

[0059] (1) Endogenous proteins were extracted from maize leaves transgenic with the Cry 1Da gene. A sandwich ELISA was performed using monoclonal antibody 7B1 as the capture antibody and another monoclonal antibody 7F1 (Cry 1Da) as the detection antibody. The results are shown in [Figure number missing]. Figure 4 .

[0060] The results showed that the double-antibody sandwich ELISA system constructed from 7B1 and 7F1 monoclonal antibodies could detect Cry 1Da protein in transgenic maize samples.

[0061] The method for extracting protein from genetically modified corn is as follows: The tissue was quick-frozen in liquid nitrogen, ground, and 1 mL (1-2 mL depending on the sample amount, generally 0.5 g) of protein extraction solution was added. The mixture was stirred at 4℃ for 30 minutes, centrifuged at 12000 rpm and 4℃ for 15 minutes, and the supernatant was collected. The formula of the protein extraction solution is shown in Table 3.

[0062] Table 3 Protein Extract Formulation

[0063] (2) Specific detection of Bt Cry insect-resistant protein commonly used in transgenic crops Besides Cry 1Da, other commonly used Cry insect-resistant proteins in transgenic crops include Cry 1Fa and Cry 1Ah Bt proteins. To detect the specificity of a double-antibody sandwich ELISA system constructed from 7B1 and 7F1 monoclonal antibodies, another anti-Cry 1Da monoclonal antibody, 7F1, was used as the capture antibody, and the monoclonal antibody produced by the hybridoma cell line 7B1 of this invention was used as the detection antibody for sandwich ELISA detection of transgenic crops. Cry 1Ah (HGK60) and Cry 1Ab+Cry 1Fa (BFL4-2) insect-resistant maize material, results are shown in […]. Figure 5 .

[0064] The results showed that the detection signals of Cry 1Ah and Cry 1Ab+Cry 1Fa samples were lower than those of Cry 1Da samples, indicating that the double-antibody sandwich ELISA system constructed from 7B1 and 7F1 monoclonal antibodies has good detection specificity for Cry 1Da protein. These results also indicate that the 7B1 monoclonal antibody can be used both as a capture antibody and a detection antibody in the double-antibody sandwich ELISA system for the detection of the insect-resistant protein Cry 1Da.

[0065] (3) Sensitivity detection of the double-antibody sandwich ELISA system The sensitivity of the constructed double-antibody sandwich ELISA detection system was tested using 7B1 monoclonal antibody as the capture antibody and 7F1 monoclonal antibody as the detection antibody. In the sensitivity test, the coating concentration of 7B1 monoclonal antibody was 2 μg / mL, the working dilution of 7F1 monoclonal antibody was 1:2000, and the working dilution of avidin-HRP was 1:4000.

[0066] Cry 1Da protein standards were prepared at concentrations of 0, 1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 ng / mL, with two parallel wells set up for each concentration. The results are shown in Table 4.

[0067] Table 4. Results of double-antibody sandwich ELISA detection of Cry 1Da protein standards.

[0068] Based on the detection results of Cry 1Da protein standards, using OD 450 A standard curve equation was obtained by fitting a value of x and a Cry 1Da protein concentration of y to the curve. y=(0.054734327+9.684830778x) / (1+0.033651005x−0.123750117x 2 ).

[0069] Simultaneously, 16 repeated tests were performed using blank samples that did not contain Cry 1Da protein, and the resulting OD values ​​were... 450 Substituting the values ​​into the standard curve above, the corresponding Cry 1Da protein concentration was calculated. Statistically, the average converted concentration of the blank sample was approximately 0.344 ng / mL, and the standard deviation was approximately 0.038 ng / mL. Using the average converted concentration of the blank sample plus twice the standard deviation as the limit of detection in this embodiment, the limit of detection for Cry 1Da protein calculated from the raw data was 0.421 ng / mL.

[0070] The results showed that the double-antibody sandwich ELISA detection system constructed from 7B1 monoclonal antibody and 7F1 monoclonal antibody had good sensitivity for detecting Cry 1Da protein.

Claims

1. A hybridoma cell line 7B1 that secretes a monoclonal antibody against the insect-resistant protein Cry 1Da, characterized in that, The hybridoma cell line 7B1 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 29, 2026, with accession number CGMCC No. 46851.

2. The monoclonal antibody produced by the hybridoma cell line 7B1 according to claim 1.

3. The monoclonal antibody according to claim 2, characterized in that, The titer of the monoclonal antibody, as determined by indirect ELISA, was 1:12280000.

4. The monoclonal antibody according to claim 2, characterized in that, The subtype of the monoclonal antibody is IgG2b.

5. The monoclonal antibody according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

2.

6. The monoclonal antibody according to any one of claims 2-5, as a capture antibody, and the anti-Cry1Da monoclonal antibody produced by hybridoma cell line 7F1 with accession number CGMCC No. 46852, as a detection antibody, are used in the qualitative and / or quantitative detection of the insect protein Cry1Da using a double-antibody sandwich ELISA.

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