Enzyme linked immunosorbent assay kit for quantitatively detecting GAT protein and detection method of enzyme linked immunosorbent assay kit

Abstract

The invention discloses an enzyme linked immunosorbent assay kit for quantitatively detecting herbicide-resistant protein GAT in transgenic soybeans and a detection method of the enzyme linked immunosorbent assay kit. The enzyme linked immunosorbent assay kit comprises a capture antibody and a detection antibody, the capture antibody is secreted by a hybridoma cell strain 1C2 with the preservation number of CGMCC No.46332, and the detection antibody is secreted by a hybridoma cell strain 1C2 with the preservation number of CGMCC No.46332. The detection antibody is obtained by secreting a hybridoma cell strain 1D5 with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.46333. The enzyme linked immunosorbent assay kit and the detection method thereof have the characteristics of simplicity and convenience in operation, capability of simultaneously and quickly detecting a large number of samples, high sensitivity and high specificity.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and in particular to an enzyme-linked immunosorbent assay kit for quantitatively detecting a herbicide-resistant protein GAT in transgenic soybeans and a detection method thereof. Background Art

[0002] The glyphosate acetyltransferase expressed by the gat gene (glyphosate-degrading gene) in Bacillus bacteria confers herbicide tolerance to transgenic crops. The cultivation of herbicide-tolerant crops has made field spraying of herbicides an effective weed control method, making herbicide tolerance one of the most advantageous traits in agricultural production. Currently, herbicide-tolerant crops account for over 80% of the global genetically modified crop population, and the proportion of genetically modified crops continues to increase.

[0003] The development and advancement of transgenic technology has driven the advancement of biology. While genetically modified foods can meet consumer expectations for yield, insect resistance, and herbicide tolerance, they also pose potential threats to human health. These include the potential for certain genes to become toxic after introduction into the host; the potential for allergens and drug resistance in genetically modified foods; and altered nutritional value. During the research, development, and commercialization of genetically modified foods, comprehensive safety assessments are crucial to enable consumers to quickly distinguish genetically modified foods from natural foods. Establishing appropriate methods for identifying and detecting genetically modified components in genetically modified foods can facilitate the safety management of agricultural genetically modified organisms, ensuring the safety of humans, animals, and microorganisms, while also protecting the ecological environment and promoting further research in agricultural genetically modified biotechnology. Therefore, the development of a GAT enzyme-linked immunosorbent assay (ELISA) kit is crucial for the rapid and quantitative analysis of herbicide-tolerant GAT proteins in genetically modified crops and their derivatives. Summary of the Invention

[0004] The present invention aims to provide an enzyme-linked immunosorbent assay kit and a detection method thereof for quantitatively detecting the herbicide-resistant protein GAT in transgenic soybeans, which has the characteristics of simple operation, ability to rapidly detect a large number of samples simultaneously, high sensitivity and high specificity.

[0005] To achieve the above objectives, the present invention provides an enzyme-linked immunosorbent assay kit for quantitatively detecting the herbicide-tolerant protein GAT in transgenic soybeans. The enzyme-linked immunosorbent assay kit comprises a capture antibody and a detection antibody. The capture antibody is secreted by the hybridoma cell line 1C2 with a deposit number of CGMCC No. 46332, and the detection antibody is secreted by the hybridoma cell line 1D5 with a deposit number of CGMCC No. 46333.

[0006] The hybridoma cell lines 1C2 and 1D5 were obtained by immunizing BALB / c mice with prokaryotic expression of purified herbicide-resistant protein GAT, fusing the immunized mouse spleen cells with commercial mouse hybridoma cells SP2 / 0, and screening them with HAT culture medium.

[0007] Furthermore, the amino acid sequence of the heavy chain variable region of the capture antibody is shown in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2 in the sequence listing; the amino acid sequence of the heavy chain variable region of the detection antibody is shown in SEQ ID NO: 3 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4 in the sequence listing. The specific sequences are: Furthermore, the detection antibody is a biotin-labeled detection antibody.

[0008] Furthermore, the enzyme-linked immunosorbent assay kit also includes a microporous reaction plate, a sample processing solution, a biotin-labeled detection antibody, a herbicide-resistant protein GAT standard, a horseradish peroxidase-labeled avidin, a biotin-labeled detection antibody working solution, a horseradish peroxidase-labeled avidin working solution, a washing solution, a substrate solution, and a stop solution.

[0009] The present invention also provides a method for quantitatively detecting the herbicide-tolerant protein GAT in transgenic soybeans using the above-mentioned enzyme-linked immunosorbent assay kit, comprising the following steps: 1) Detection: Set up wells for herbicide-resistant protein GAT standard, wells for test samples, and blank control wells. Add capture antibody to each well for incubation, then discard the liquid. After drying, add herbicide-resistant protein GAT standard, test sample or sample treatment solution to each well for incubation, then discard the liquid. After drying, add biotin-labeled detection antibody working solution to each well for incubation, discard the liquid in the wells, wash and dry, add horseradish peroxidase-labeled avidin working solution to each well, incubate, discard the liquid in the wells, wash and dry, add substrate solution to each well, protect from light and color development, add stop solution to each well, mix well, and measure the optical density (OD) value of each well using a microplate reader after the reaction is terminated; 2) Data Processing: Subtract the OD value of the blank control well S0 from the OD value of each standard well to obtain the corrected OD value of each standard well. Use the corrected OD value of each standard well to plot a working curve against the concentration of the herbicide-resistant protein GAT standard added to each standard well. Subtract the OD value of the blank control well S0 from the OD value of each sample well to obtain the corrected OD value of each sample well to be tested, and substitute it into the working curve to calculate the concentration of the herbicide-resistant protein GAT in each sample well to be tested.

[0010] Furthermore, before the detection in step 1), each solution in the enzyme-linked immunosorbent assay kit is placed at 18-25° C. and equilibrated for at least 30 minutes.

[0011] Furthermore, the stop solution is 1M sulfuric acid solution.

[0012] Furthermore, the optical density (OD) value of each well was measured using a microplate reader at a wavelength of 450 nm.

[0013] Furthermore, the incubation temperature in each well was 37°C.

[0014] Furthermore, the biotin-labeled detection antibody working solution is prepared by diluting the biotin-labeled detection antibody solution with the biotin-labeled antibody diluent at a ratio of 1:100, such as adding 10 μL of biotin-labeled antibody to 990 μL of biotin-labeled antibody diluent, gently mixing, and preparing within 10 minutes before use.

[0015] Furthermore, the horseradish peroxidase-labeled avidin working solution is obtained by diluting the horseradish peroxidase-labeled avidin with the horseradish peroxidase-labeled avidin diluent at a ratio of 1:100, such as adding 10 μL of horseradish peroxidase-labeled avidin to 990 μL of the horseradish peroxidase-labeled avidin diluent, gently mixing, and preparing the solution within 10 minutes before use.

[0016] Further, in step 1), add 80-120 μL of standard or sample to be tested or sample treatment solution to each well, shake gently to mix, cover with plate sticker, and incubate at 37°C for 1.5-2.5 hours; discard the liquid, spin dry, and do not wash; add 80-120 μL of biotin-labeled antibody working solution to each well, cover with a new plate sticker, and incubate at 37°C for 0.5-1.5 hours; discard the liquid in the well, spin dry, wash the plate 2-4 times, soaking for 1-3 minutes each time, 150-250 μL / well, spin dry; add 80-120 μL of horseradish peroxidase-labeled avidin working solution to each well, cover with a new plate sticker, and incubate at 37°C for 0.5-1.5 hours; discard the liquid in the well, spin dry, wash the plate 4-6 times, soaking for 1-3 minutes each time, 150-250 μL / well, spin dry; add 80-100 μL of substrate solution to each well, and incubate at 37 Incubate in the dark for 15-30 minutes; add 50 μL of stop solution to each well to terminate the reaction.

[0017] Further, in step 1), 100 μL of standard or test sample or sample treatment solution was added to each well, the mixture was gently shaken, the plate was covered with a plate sticker, and incubated at 37°C for 2 hours; the liquid was discarded, the plate was dried without washing; 100 μL of biotin-labeled antibody working solution was added to each well, the plate was covered with a new plate sticker, and the plate was incubated at 37°C for 1 hour; the liquid in the well was discarded, the plate was dried, and the plate was washed 3 times, soaking for 2 minutes each time, 200 μL / well, and dried; 100 μL of horseradish peroxidase-labeled avidin working solution was added to each well, the plate was covered with a new plate sticker, and the plate was incubated at 37°C for 1 hour; the liquid in the well was discarded, the plate was dried, the plate was washed 5 times, soaking for 2 minutes each time, 200 μL / well, and dried; 90 μL of substrate solution was added to each well in sequence.

[0018] Furthermore, the detection method is an ELISA double antibody sandwich method.

[0019] Compared with the existing technology, the present invention has the following beneficial effects: the kit of the present invention is a detection kit based on the ELISA double antibody sandwich method, which is low in cost, the main reagents are provided in the form of working solution, the operation is simple, and a large number of samples can be quickly detected simultaneously; it has the characteristics of high sensitivity and high specificity; it is also the first kit in China and even in the world that can quantitatively and specifically detect the herbicide-resistant protein GAT in genetically modified soybeans.

[0020] Preservation Information The hybridoma cell lines 1C2 and 1D5 provided herein were deposited with the General Microbiology Center of the China Culture Collection Administration on March 14, 2025, and are classified as GAT monoclonal antibody hybridoma cell lines. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101. The deposit numbers are CGMCC No. 46332 and CGMCC No. 46333, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a standard curve established according to the enzyme-linked immunosorbent assay method of the present invention.

[0022] Figure 2 This is a graph showing the sensitivity test results of the monoclonal antibody produced by the hybridoma cell line 1C2 of the present invention, wherein: lane 1 is 5 ng of GAT protein and 10 μg / ml of 1C2 antibody; lane 2 is 10 ng of GAT protein and 10 μg / ml of 1C2 antibody; lane 3 is 20 ng of GAT protein and 10 μg / ml of 1C2 antibody; lane 4 is 40 ng of GAT protein and 10 μg / ml of 1C2 antibody; lane 5 is 60 ng of GAT protein and 10 μg / ml of 1C2 antibody; and lane 6 is 80 ng of GAT protein and 10 μg / ml of 1C2 antibody.

[0023] Figure 3The figures are the specificity detection results of the monoclonal antibodies produced by the hybridoma cell line 1C2 according to the present invention, wherein: the left figure is a staining diagram, lane 1 is GAT protein, lane 2 is Pat / pat protein, lane 3 is Pat / bar protein, and lane 4 is G2 EPSPS protein; the right figure is a Western result diagram, lane 1 is GAT protein 100 ng, 1C2 antibody 10 μg / ml, lane 2 is Pat / pat protein 100 ng, 1C2 antibody 10 μg / ml, lane 3 is Pat / bar protein 100 ng, 1C2 antibody 10 μg / ml, lane 4 is G2 EPSPS protein 100 ng, 1C2 antibody 10 μg / ml.

[0024] Figure 4 This is a graph showing the sensitivity test results of the monoclonal antibody produced by the hybridoma cell line 1D5 according to the present invention, wherein: lane 1 is 5 ng of GAT protein and 10 μg / ml of 1D5 antibody; lane 2 is 10 ng of GAT protein and 10 μg / ml of 1D5 antibody; lane 3 is 20 ng of GAT protein and 10 μg / ml of 1D5 antibody; lane 4 is 40 ng of GAT protein and 10 μg / ml of 1D5 antibody; lane 5 is 60 ng of GAT protein and 10 μg / ml of 1D5 antibody; and lane 6 is 80 ng of GAT protein and 10 μg / ml of 1D5 antibody.

[0025] Figure 5 The figures are the specificity detection results of the monoclonal antibodies produced by the hybridoma cell line 1D5 according to the present invention, wherein: the left figure is a staining diagram, lane 1 is GAT protein, lane 2 is Pat / pat protein, lane 3 is Pat / bar protein, and lane 4 is G2 EPSPS protein; the right figure is a Western result diagram, lane 1 is GAT protein 100 ng, 1D5 antibody 10 μg / ml, lane 2 is Pat / pat protein 100 ng, 1D5 antibody 10 μg / ml, lane 3 is Pat / bar protein 100 ng, 1D5 antibody 10 μg / ml, lane 4 is G2 EPSPS protein 100 ng, 1D5 antibody 10 μg / ml. DETAILED DESCRIPTION

[0026] The specific embodiments of the present invention are described in detail below with reference to the accompanying drawings, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0028] Unless otherwise specified, the materials and reagents used in the following examples can be purchased from commercial sources.

[0029] Example 1: Quantitative detection of herbicide-tolerant protein GAT in transgenic soybeans using an enzyme-linked immunosorbent assay kit The invention discloses an enzyme-linked immunosorbent assay kit for quantitatively detecting the herbicide-resistant protein GAT in genetically modified soybeans. The enzyme-linked immunosorbent assay kit comprises a microporous reaction plate coated with a capture antibody, a sample processing solution, a biotin-labeled detection antibody, a herbicide-resistant protein GAT standard, avidin labeled with horseradish peroxidase, a biotin-labeled detection antibody diluent, a diluent of the avidin labeled with horseradish peroxidase, a concentrated washing solution, a substrate solution, a stop solution, and a plate sticker.

[0030] Among them, the formula of the sample treatment solution is: Biotinylated detection antibody: 100 μg / mL biotinylated GAT1D5 mouse monoclonal antibody solution (PBS, 50 v / v% glycerol); Standard: 100ng of His-GAT recombinant protein (lyophilized powder). The His-GAT recombinant protein is obtained by amplifying and sequencing the GAT coding gene, and then connecting it to the pET28a plasmid after correct identification. gat The recombinant protein GAT expression strain was transformed into Escherichia coli BL21 (DE3) competent cells, and the recombinant protein GAT expression strain was revived and cultured, induced with IPTG at 16°C overnight, and the protein was purified. Horseradish peroxidase-labeled avidin: 1:40 horseradish peroxidase-labeled avidin stock solution (Jinkairui Company), working solution diluted 1:100; Biotinylated Antibody Dilution Buffer / Horseradish Peroxidase-Conjugated Avidin Dilution Buffer Formula: Concentrated washing solution: 10× PBS containing 1.0 wt% Tween-20; Substrate solution: 0.5 mL 2 mg / mL TMB in anhydrous ethanol, 10 mL substrate buffer, 32 μL 30% H2O2, mix and prepare immediately. Stop solution: 1M H2SO4.

[0031] Characteristics of the kit: 1) Sensitivity: 55.04 ng / mL 2) Precision: Intra-assay CV% < 8%, Inter-assay CV% < 10% 3) Specificity: This kit specifically detects GAT and has no cross-reaction with other related proteins.

[0032] The capture antibody is secreted by the hybridoma cell line 1C2 with a deposit number of CGMCC No. 46332. The detection range of 1C2 as a capture antibody is: 78.125-625 ng / mL.

[0033] The detection antibody is secreted by the hybridoma cell line 1D5 with a preservation number of CGMCC No.46333.

[0034] The hybridoma cell lines 1C2 and 1D5 were obtained by immunizing BALB / c mice with prokaryotic expression of the herbicide-resistant protein GAT, fusing the immunized mouse spleen cells with commercial mouse hybridoma cells SP2 / 0, and screening them with HAT culture medium.

[0035] The specific detection method includes the following steps: 1) Place each solution in the ELISA kit at 18-25°C and equilibrate for at least 30 minutes; 2) Set up wells for herbicide-resistant protein GAT standard, wells for test samples, and blank control wells, add capture antibody to each well for incubation, then discard the liquid, spin dry, add herbicide-resistant protein GAT standard, test sample or sample treatment solution (see Table 1) to each well for incubation, then discard the liquid, spin dry, add biotin-labeled detection antibody working solution to each well for incubation, discard the liquid in the wells, wash and spin dry, add horseradish peroxidase-labeled avidin working solution to each well, incubate, discard the liquid in the wells, wash and spin dry, add substrate solution to each well, protect from light and color development, add stop solution 1M sulfuric acid solution to each well, mix well, and after the reaction is terminated, measure the optical density (OD) value of each well at a wavelength of 450 nm using a microplate reader; Table 1 Sample treatment solution formula 3) Data Processing: Subtract the OD value of the blank control well S0 from the OD value of each standard well to obtain the corrected OD value of each standard well. Use the corrected OD value of each standard well to plot a working curve against the concentration of the herbicide-resistant protein GAT standard added to each standard well. Subtract the OD value of the blank control well S0 from the OD value of each sample well to be tested to obtain the corrected OD value of each sample well to be tested, and substitute it into the working curve to calculate the concentration of the herbicide-resistant protein GAT in each sample well to be tested.

[0036] in: The biotin-labeled detection antibody working solution is prepared by diluting the biotin-labeled detection antibody solution with the biotin-labeled antibody diluent at a ratio of 1:100. For example, add 10 μL of biotin-labeled antibody to 990 μL of biotin-labeled antibody diluent, mix gently, and prepare within 10 minutes before use.

[0037] Concentrated washing solution: Salt may precipitate when stored at low temperatures. Warm the solution in a water bath to aid dissolution during dilution. Working solution: Dilute the concentrated washing solution with deionized water at a ratio of 1:25. For example, use a graduated cylinder to measure 240 mL of deionized water, pour it into a beaker or other clean container, then measure 10 mL of concentrated washing solution and evenly add. Stir to mix thoroughly. Prepare the solution immediately before use.

[0038] The horseradish peroxidase-labeled avidin working solution is prepared by diluting the horseradish peroxidase-labeled avidin with the horseradish peroxidase-labeled avidin diluent at a ratio of 1:100, such as adding 10 μL of horseradish peroxidase-labeled avidin to 990 μL of horseradish peroxidase-labeled avidin diluent, gently mixing, and preparing within 10 minutes before use.

[0039] In step 2), add 100 μL of standard or test sample or sample treatment solution to each well, shake gently to mix, cover with a plate sticker, and incubate at 37°C for 2 hours; discard the liquid, spin dry, and do not wash; add 100 μL of biotin-labeled antibody working solution to each well, cover with a new plate sticker, and incubate at 37°C for 1 hour; discard the liquid in the well, spin dry, wash the plate three times, soaking for 2 minutes each time, 200 μL / well, and spin dry; add 100 μL of horseradish peroxidase-labeled avidin working solution to each well, cover with a new plate sticker, and incubate at 37°C for 1 hour; discard the liquid in the well, spin dry, wash the plate five times, soaking for 2 minutes each time, 200 μL / well, and spin dry; add 90 μL of substrate solution to each well in sequence.

[0040] The standard was diluted from 25 ng to a series of concentrations. The standard was added to each well and incubated according to the detection method of the present invention. The liquid was then discarded, and a biotin-labeled detection antibody working solution was added and incubated. The liquid in the wells was discarded, and after drying, a horseradish peroxidase-labeled avidin working solution was added to each well and incubated. The liquid in the wells was discarded, and after drying, a substrate solution was added to each well. After color development in the dark, a stop solution was added to each well. Within 5 minutes after the reaction was terminated, the optical density (OD) value of each well was measured in sequence at a wavelength of 450 nm using a microplate reader to prepare a standard curve. For details, see [see References] Figure 1 , R of the standard curve 2 >0.99, linear detection range 78.125-625 ng / mL, and the formula for calculating sample concentration is: x=(y+0.2419) / 0.0043.

[0041] Transgenic soybean leaves were ground with liquid nitrogen, thoroughly mixed with a sample treatment solution, and allowed to stand on ice for 30 minutes. The supernatant was collected and appropriately diluted. The sample was added to each well for incubation according to the detection method of the present invention, and the liquid was discarded. A biotin-labeled detection antibody working solution was added and incubated. The liquid in the wells was discarded, and after drying, a horseradish peroxidase-labeled avidin working solution was added to each well and incubated. The liquid in the wells was discarded, and after drying, a substrate solution was added to each well. After color development in the dark, a stop solution was added to each well. Within 5 minutes after the reaction was terminated, the optical density (OD) value of each well was measured in sequence at a wavelength of 450 nm using a microplate reader. The GAT content in the transgenic soybean leaves was calculated as x = 6.23±0.03 μg / mL.

[0042] Key points: 1. To ensure the accuracy of the test results, double-hole determination is set for both standards and samples, and a standard curve is required for each test.

[0043] 2. If the content of the substance to be tested in the specimen is too high, it is necessary to dilute it with sample diluent first so that the sample meets the detection range of the kit, and then multiply it by the corresponding dilution factor in the final calculation.

[0044] 3. Sample Addition: When adding samples, use clean, disposable pipette tips to avoid cross-contamination. Add samples gently to avoid foaming. Add samples to the bottom of the plate wells, not along the sides. The optimal time for each sample addition is 10 minutes. For large numbers of samples, use a dispenser.

[0045] 4. Incubation: To prevent sample evaporation or contamination, the ELISA plate must be covered with a plate sticker during incubation. The plate should not be allowed to dry out during the experiment. During incubation, the incubator temperature should be monitored to ensure it remains constant at 37°C and adjusted promptly. During incubation, the incubator should not be opened too many times to avoid disrupting temperature balance.

[0046] 5. Washing: The washing process is very important. Insufficient washing can easily cause false positives.

[0047] (1) Manual plate washing method: Aspirate (do not touch the well walls or bottom) or shake off the liquid in the ELISA plate; place several layers of absorbent paper on the laboratory bench and tap the ELISA plate facing downward several times; inject 200 μL / well of the recommended wash buffer into the wells and soak for 2 minutes. Repeat this process several times as described in the operating procedures.

[0048] (2) Automatic plate washing: If there is an automatic plate washing machine, it should be used in the formal experiment process only after you are proficient in its use.

[0049] 6. Color Development: To ensure the accuracy of experimental results, add the stop solution as soon as possible after the substrate reaction time expires. After adding the substrate solution, observe the color development at regular intervals (e.g., every 10 minutes) to control the reaction time. When a clear blue gradient is visible in the first 3-4 wells of the standard sample and no color is evident in the last 3-4 wells, add the stop solution to terminate the reaction. The blue color will immediately turn yellow. The stop solution should be added in the same order as the substrate solution.

[0050] 7. The substrate solution should be light blue or colorless. If the color becomes too dark, it must be discarded. The substrate solution is easily contaminated and should be stored away from light.

[0051] Example 2: Verification of sensitivity and specificity of antibodies used in enzyme-linked immunosorbent assay kits GAT protein gradient was loaded (the loading amount of lanes 1-6 of 5 ng / μl GAT protein was 1, 2, 4, 8, 12, and 16 μl, respectively), and SDS-PAGE gel was run. The membrane was transferred using purified monoclonal antibody (1C2, 10 μg / ml) as the primary antibody and goat anti-mouse IgG (H+L)-HRP (1:10000) as the secondary antibody. The western blotting results were detected by chemiluminescence, and the ultrasensitive color solution was exposed for 30 s. Figure 2 It can be seen that the sensitivity of the purified 1C2 monoclonal antibody WB is 5 ng.

[0052] In addition to GAT, other commonly used herbicide-resistant proteins in transgenic crops include PAT / pat, PAT / bar, CP4 EPSPS, G2EPSPS, and G10 EPSPS. Monoclonal antibody 1C2 (10 μg / ml) was used for Western detection of GAT, PAT / pat, PAT / bar, and G2EPSPS (all concentrations were 10 ng / μl, and the sample volume was 10 μl) (SDS-PAGE gel was run, and the membrane was transferred using purified monoclonal antibody (1C2) as the primary antibody, goat anti-mouse IgG (H+L)-HRP (1:10000) as the secondary antibody, and the western blotting results were detected by chemiluminescence, with the ultrasensitive color development solution exposed for 2 seconds). The detection results are shown in the table. Figure 3 The results showed that the monoclonal antibody produced by the purified 1C2 (ascites purified by Protein A-agarose affinity chromatography column) could specifically recognize GAT, but could not effectively recognize other herbicide-resistant proteins.

[0053] GAT protein gradient was loaded (the loading amount of lanes 1-6 of 5 ng / μl GAT protein was 1, 2, 4, 8, 12, and 16 μl, respectively), and SDS-PAGE gel was run. The membrane was transferred using purified monoclonal antibody (1D5, 10 μg / ml) as the primary antibody and goat anti-mouse IgG (H+L)-HRP (1:10000) as the secondary antibody. The western blotting results were detected by chemiluminescence, and the ultrasensitive color solution was exposed for 30 s. Figure 4 It can be seen that the sensitivity of the purified 1D5 monoclonal antibody WB is 5 ng.

[0054] Western blotting was performed using monoclonal antibody 1D5 (10 μg / ml) to detect GAT, PAT / pat, PAT / bar, and G2 EPSPS (all concentrations were 10 ng / μl, and the sample volume was 10 μl) (SDS-PAGE gel was run, and the membrane was transferred using purified monoclonal antibody (1D5) as the primary antibody, goat anti-mouse IgG (H+L)-HRP (1:10,000) as the secondary antibody, and the western blotting results were detected by chemiluminescence, with ultrasensitive color development solution exposure for 2 seconds). The detection results are shown in the table. Figure 5 The results showed that the monoclonal antibody produced by the purified 1D5 (ascites purified by Protein A-agarose affinity chromatography column) could specifically recognize GAT, but could not effectively recognize other herbicide-resistant proteins.

Claims

1. An enzyme-linked immunosorbent assay kit for quantitatively detecting the herbicide-resistant protein GAT in transgenic soybeans, characterized in that: The enzyme-linked immunosorbent assay kit comprises a capture antibody and a detection antibody. The capture antibody is secreted by the hybridoma cell line 1C2 with a deposit number of CGMCC No. 46332, and the detection antibody is secreted by the hybridoma cell line 1D5 with a deposit number of CGMCC No. 46333.

2. The enzyme-linked immunosorbent assay kit according to claim 1, wherein The amino acid sequence of the heavy chain variable region of the capture antibody is shown in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2 in the sequence listing; the amino acid sequence of the heavy chain variable region of the detection antibody is shown in SEQ ID NO: 3 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4 in the sequence listing.

3. The enzyme-linked immunosorbent assay kit according to claim 1 or 2, wherein The detection antibody is a biotin-labeled detection antibody.

4. The enzyme-linked immunosorbent assay kit according to claim 3, wherein The enzyme-linked immunosorbent assay kit further comprises a microporous reaction plate, a sample processing solution, a biotin-labeled detection antibody, a herbicide-resistant protein GAT standard, a horseradish peroxidase-labeled avidin, a biotin-labeled detection antibody working solution, a horseradish peroxidase-labeled avidin working solution, a washing solution, a substrate solution, and a stop solution.

5. A method for quantitatively detecting the herbicide-tolerant protein GAT in transgenic soybeans using the enzyme-linked immunosorbent assay kit according to claim 4, characterized in that: The following steps are involved: 1) Detection: Set up wells for herbicide-resistant protein GAT standard, wells for test samples, and blank control wells. Add capture antibody to each well for incubation, then discard the liquid. After drying, add herbicide-resistant protein GAT standard, test sample or sample treatment solution to each well for incubation, then discard the liquid. After drying, add biotin-labeled detection antibody working solution to each well for incubation, discard the liquid in the wells, wash and dry, add horseradish peroxidase-labeled avidin working solution to each well, incubate, discard the liquid in the wells, wash and dry, add substrate solution to each well, protect from light and color development, add stop solution to each well, mix well, and measure the optical density (OD) value of each well using a microplate reader after the reaction is terminated; 2) Data processing: Subtract the OD value of the blank control well S0 from the OD value of each standard well to obtain the corrected OD value of each standard well. Use the corrected OD value of each standard well to plot a working curve against the concentration of the herbicide-tolerant protein GAT standard added to each standard well. The corrected OD value of each sample well to be tested, obtained by subtracting the OD value of the blank control well S0 from the OD value of each sample well to be tested, was substituted into the working curve to calculate the concentration of the herbicide-resistant protein GAT in each sample well to be tested.

6. The method according to claim 5, characterized in that Before the detection in step 1), each solution in the enzyme-linked immunosorbent assay kit is placed at 18-25° C. and equilibrated for at least 30 minutes.

7. The method according to claim 5, characterized in that The stop solution is 1M sulfuric acid solution.

8. The method according to claim 5, characterized in that The optical density (OD) value of each well was measured at a wavelength of 450 nm using a microplate reader.

9. The method according to claim 5, characterized in that The temperature of each incubation in each well was 37°C.

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