A hybridoma cell line 2D2 and its monoclonal antibody and its application

Abstract

The present invention discloses a hybridoma cell line 2D2 and a monoclonal antibody and application thereof. The present invention also discloses the application of the anti-light stinking smut monoclonal antibody in the detection of light stinking smut winter spores. The present invention also discloses an HRP-coupled light stinking smut monoclonal antibody. The present invention also discloses a detection kit. The monoclonal antibody and the coupled antibody and the kit involved in the present invention have the characteristics of specifically recognizing light stinking smut, and have broad application prospects for detecting light stinking smut wheat in the fields of wheat import and export trade and market circulation.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a hybridoma cell line 2D2 and a monoclonal antibody and application thereof. Background Art

[0002] Wheat light bunt is a global plant fungal disease with a history of more than a hundred years. The disease is caused by Ustilago sutchusii. Wheat infected with the pathogen will emit a distinct fishy odor, which not only leads to a decrease in wheat yield, affects its quality and grade, and causes economic losses, but may also pose a threat to animal and human health through the food chain. The secondary basidiospores produced after the germination of the winter spores of Ustilago sutchusii are the key way to infect wheat, and the extremely high tolerance and vitality of the winter spores are the main reasons for the difficulty in preventing and controlling wheat light bunt.

[0003] In wheat infected with light stinky smut, the formation of galls (infected wheat grains are called galls) is a significant pathological feature of the disease. During grain storage and transportation, the galls in wheat are prone to rupture, releasing the winter spores of light stinky smut. The diameter of the winter spores of light stinky smut is about 14 μm, which is beyond the scope of human eye recognition. At present, conventional methods for detecting wheat light stinky smut, such as microscopy, have problems such as long detection cycle, inability to accurately quantify, and difficulty in judging the degree of infection of samples. This method is not only time-consuming, but also prone to missed detection when dealing with actual samples containing trace or trace amounts of winter spores. Although molecular biological methods, such as PCR, have been applied to the detection of this fungus, its detection process is complicated, the detection cycle is long, and it is difficult to quantify, and it is also prone to false positive results.

[0004] Enzyme-linked immunosorbent assay (ELISA) has the advantages of fast speed and good specificity, and is widely used in pathogen detection. The specificity and sensitivity of this technology depend on the specific recognition and capture performance between the antibody and antigen used. The key to ELISA is the preparation of the corresponding monoclonal antibody. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a hybridoma cell line 2D2.

[0006] The technical problem that the present invention also aims to solve is to provide the application of the hybridoma cell line in the preparation of anti-Tilletia leucoderma monoclonal antibodies.

[0007] The technical problem that the present invention needs to solve is to provide an anti-light Tilletia monoclonal antibody.

[0008] The technical problem that the present invention needs to solve is to provide the application of anti-Tinella glabra monoclonal antibody in the detection of Tinella glabra teliospores.

[0009] The technical problem that the present invention needs to solve is to provide an HRP-coupled Tilletia sutchuenensis monoclonal antibody.

[0010] The final technical problem to be solved by the present invention is to provide a detection kit capable of detecting the winter spores of Tilletia sutchuenensis with high sensitivity and high specificity.

[0011] Technical solution: The present invention provides a hybridoma cell line 2D2, which was deposited in the China Center for Type Culture Collection on October 22, 2024, and its classification name is hybridoma cell line 2D2 Monoclonal antibody hybridoma cell line against Tilletia foetida 2D2, the deposit number is CCTCC NO: C2024296, and the deposit address is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, Postal Code: 430072.

[0012] The present invention also includes the use of the hybridoma cell line in preparing anti-Tilletia leucoderma monoclonal antibodies.

[0013] The present invention also includes an anti-Tilletia sutchuenensis monoclonal antibody, which is secreted by the hybridoma cell line.

[0014] Among them, the anti-light smut fungus monoclonal antibody includes a heavy chain variable region and a light chain variable region, the heavy chain variable region includes CDRH1 with an amino acid sequence as shown in SEQ ID NO.1, CDRH2 with an amino acid sequence as shown in SEQ ID NO.2, and CDRH3 with an amino acid sequence as shown in SEQ ID NO.3, and the light chain variable region includes CDRL1 with an amino acid sequence as shown in SEQ ID NO.4, CDRL2 with an amino acid sequence of KVS, and CDRL3 with an amino acid sequence as shown in SEQ ID NO.5.

[0015] Among them, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.7.

[0016] The nucleotide sequence of the heavy chain is shown in SEQ ID NO.8, and the nucleotide sequence of the light chain is shown in SEQ ID NO.9.

[0017] The amino acid sequence of the heavy chain is shown in SEQ ID NO.10, and the amino acid sequence of the light chain is shown in SEQ ID NO.11.

[0018] The present invention also includes the use of the anti-U. luminifera monoclonal antibody in the detection of U. luminifera teliospores.

[0019] The present invention also includes an HRP-coupled anti-smut fungus monoclonal antibody, which contains the anti-smut fungus monoclonal antibody.

[0020] The present invention also includes a detection kit, which includes the anti-U. tiliaceus monoclonal antibody or the HRP-coupled U. tiliaceus monoclonal antibody.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the present invention obtains a high titer hybridoma cell line for the first time, and the monoclonal antibody secreted by the hybridoma cell line has the ability to detect the winter spores of light stinking smut with high sensitivity and high specificity. The conjugated antibody and detection kit based on the monoclonal antibody of light stinking smut of wheat in the present invention have been verified to have the specificity of recognizing and binding the winter spores of light stinking smut, and can be used as a monoclonal antibody for the detection of light stinking smut of wheat, which can lay a foundation for the inspection and quarantine of wheat import and export trade and domestic market circulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the detection system of Ustilago farnesii based on immunomagnetic separation and chemiluminescence;

[0023] Figure 2 It is a linear graph of the detection system of Tillandsia spores based on immunomagnetic separation and chemiluminescence. DETAILED DESCRIPTION

[0024] Example 1 Screening of Hybridoma Cell Lines of Tilletia glabra

[0025] 1. Immunization of mice with winter spores of Smut fungus

[0026] 1) Weigh 0.1 mg of winter spores of wheat spores (wheat spores of wheat spores were provided by Jiangsu Grain and Oil Quality Monitoring Center) and mix with 675 μL of saline, then 675 μL of Freund's complete adjuvant. After the spore solution is completely emulsified, 6-8 week old Blbc mice are immunized for the first time. The second to fourth immunizations are performed by mixing winter spore saline solution with Freund's incomplete adjuvant and emulsifying them. Each immunization is performed 14 days apart. Blood is collected on the 7th day after the four immunizations, and 10 μL of blood is collected from the tail vein. ELISA verifies whether the antiserum has a positive reaction.

[0027] 2) ELISA verification: 100 μL of the winter spore suspension of S. tiliaceus was added to a 96-well plate in each well, incubated in a 4°C refrigerator overnight, and then washed three times with PBST (PBS buffer solution containing 0.05% Tween-20). Then 200 μL of 5% bovine serum albumin solution (PBS buffer solution was used as the solvent) was added to each well, and the plates were blocked at 37°C for 1 hour. After being removed, the plates were washed three times with PBST to remove excess bovine serum albumin solution.

[0028] 3) Add 100 μL of the gradient diluted antiserum to the plate, mix well and incubate at 37°C for 1 hour, then add HRP-goat anti-mouse secondary antibody diluted 5000 times with PBS, incubate at 37°C for 0.5 hour, then wash the plate twice with PBST, add 100 μL of chemiluminescent substrate solution (luminol, A:B=1:1), mix well, and place the plate in an ELISA instrument to detect the chemiluminescence intensity. At the same time, use the serum of normal mice as a negative control, and use the same method to detect the chemiluminescence intensity of normal mouse serum.

[0029] 2. Screening of hybridoma cell lines of Ustilago sutchuenensis and preparation of monoclonal antibodies

[0030] 1) According to the results of step 1, select mice with high ELISA response intensity and perform the fifth immunization with teliospore suspension on the 7th day after blood collection;

[0031] 2) Cell fusion: On the 5th day after the fifth immunization, the spleen of the mice was obtained, ground and fused with SP2 / 0 cells, and plated.

[0032] 3) Subclone screening: On the 14th to 16th day after cell fusion, observe the cells under a microscope and take the supernatant for ELISA detection; for cells detected with specific antibody positive wells, timely transfer and conduct the first round of subclone screening; 6-7 days later, observe the cells under a microscope and take the supernatant for ELISA detection; for cells detected with specific antibody positive wells, timely transfer and conduct the second round of subclone screening; 5-6 days later, observe the cells under a microscope and take the supernatant for ELISA detection; for cells detected with specific antibody positive wells, timely transfer and conduct the third round of subclone screening;

[0033] 4) After three rounds of subclone screening, 4 hybridoma cell lines that can stably secrete antibodies against S. leucoderma were screened by ELISA. These positive cell lines were expanded and subcultured, and the antibody titer of the cell supernatant was tested in each generation to verify whether the cells could continue to stably secrete antibodies during the subculture process. The results of subculture showed that these 4 hybridoma cells could normally secrete antibodies in 6 consecutive generations of culture, showing good antibody secretion stability. One of the positive cell lines showed a significant response intensity in the test. The response value ratio of the enzyme-labeled wells without spores was greater than 2.0 compared with the normal mouse serum negative control group using the enzyme-labeled wells without spores as the blank control. This cell line has been selected for preservation (preservation number: CCTCC No. C2024296). Subsequently, we further expanded the culture of this cell line. When the cells grew to 90% of the culture dish, a small amount of cells were taken out and inoculated into a T25 flask for continued culture. At the same time, the remaining cells were collected and frozen for subsequent research.

[0034] 5) Preparation of ascites: One week in advance, inject mineral oil into the mouse peritoneum. Culture the cells in the T25 flask until they are 90% confluent, collect the cells, and inject them into the mouse peritoneum. Observe the mouse peritoneum for 8-20 days, and collect ascites when it is swollen to the point of impeding movement.

[0035] 6) Ascites purification: After centrifugation and filtration, the ascites was diluted with an equal volume of PBS, purified by ProteinAG, and the antibodies were collected.

[0036] 7) Validation of monoclonal antibodies, spores of wheat dwarf stinking powder powder, Aspergillus flavus spores, Aspergillus niger spores, and Paecilomyces varroa spores (spores of wheat dwarf stinking powder powder were purchased from the Royal Culture Collection of the Netherlands, and other spores were all well-known and commonly used conventional spores, provided by the Microbiological Testing Center of Nanjing Product Quality Supervision and Inspection Institute) were selected as cross-validation materials, and the antibody was packaged and validated according to the method of step 1. The validation results are shown in Table 1 below. According to the chemiluminescent signal, when the coating antigen is the winter spore of wheat light stinking powder powder, the chemiluminescent signal is higher than 0.71, while when the spores of other strains are used as the coating antigen, the chemiluminescent signal is significantly reduced, which is lower than 0.6521. It is shown that the monoclonal antibody of wheat light stinking powder powder prepared by the present invention has significant specific adsorption to the winter spore of wheat light stinking powder powder, and can be used to identify wheat light stinking powder powder.

[0037] Table 1 Monoclonal antibody validation results

[0038]

[0039] In the present invention, the enzyme-labeled wells without spores were used as blank controls, and the hybridoma cell line 2D2 of wheat light smut fungus with a response value ratio greater than 2.0 to the normal mouse serum negative control group was screened out. The cell line was officially deposited in the China Center for Type Culture Collection on October 22, 2024, and its classification was named: Monoclonal antibody hybridoma cell line 2D2 against light smut fungus Tilletia foetida 2D2, deposit number: CCTCC NO: C2024296, deposited in China Center for Type Culture Collection; the depository address is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University; Postal Code: 430072;

[0040] Example 2 Sequencing of Monoclonal Antibodies to Tilletia tritici

[0041] In Example 1, a monoclonal antibody against Tilletia sutchuenensis was successfully prepared. To further analyze its molecular structure, Beijing Sino Biological Technology Co., Ltd. was commissioned to perform sequencing analysis on the monoclonal antibody, thereby obtaining the detailed amino acid sequence and nucleotide sequence of the monoclonal antibody.

[0042] The monoclonal antibody against wheat smut comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDRH1 as shown in SEQ ID NO.1, CDRH2 as shown in SEQ ID NO.2, and CDRH3 as shown in SEQ ID NO.3, and the light chain variable region comprises CDRL1 as shown in SEQ ID NO.4, CDRL2 with an amino acid sequence of KVS, and CDRL3 as shown in SEQ ID NO.5;

[0043] SEQ ID NO.1 GFNFSDYY

[0044] SEQ ID NO.2 ISDGGSYT

[0045] SEQ ID NO.3 ARPINWAWFAY

[0046] SEQ ID NO.4 QSLVHSNGNTY

[0047] SEQ ID NO.5 SQSTHVPYT

[0048] The heavy chain variable region and light chain variable region of the monoclonal antibody are 118 and 112 amino acids long, respectively. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.7.

[0049] Heavy chain variable region sequence

[0050] SEQ ID NO.6

[0051] EVQLQQSGGGLVKPGGSLKLSCAASGFNFSDYYIYWVRQTPEKRLEWVATISDGGSYTYYPDSVKGRFTISRDNAKNNLYLQMSSLKSEDTAMYYCARPINWAWFAYWGQGTLVTVSA

[0052] Light chain variable region sequence

[0053] SEQ ID NO.7

[0054] DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTKLEIK

[0055] Heavy chain full length nucleotide sequence:

[0056] SEQ ID NO.8

[0057]

[0058] Full-length nucleotide sequence of the light chain:

[0059] SEQ ID NO.9

[0060] ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAAGGAGTGCTGA

[0061] Amino acid sequence of the full-length heavy chain:

[0062] SEQ ID NO.10

[0063] MGWSLILLFLVAVATRVLSEVQLQQSGGGLVKPGGSLKLSCAASGFNFSDYYIYWVRQTPEKRLEWVATISDGGSYTYYPDSVKGRFTISRDNAKNNLYLQMSSLKSEDTAMYYC ARPINWAWFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKV DKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAP IEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0064] Light chain full length amino acid sequence

[0065] SEQ ID NO.11

[0066] MKLPVRLLLVLMFWIPASSSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVP YTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRKEC

[0067] Example 3 Application of monoclonal antibody 2D2 - Detection of winter spores of Tilbury fungus by immunomagnetic separation combined with chemiluminescence technology

[0068] 1. Biotin-labeled polyclonal antibody

[0069] Preparation of polyclonal antibodies against wheat smut fungus: (1) Emulsification: Take 10 5The winter spore solution of Ustilago farfara with a concentration of 100 / mL was mixed with an equal volume of Freund's complete adjuvant and emulsified. (2) Animal immunization: 13-week-old male New Zealand white rabbits were selected as immunized animals. The immunization volume for each rabbit was 1 mL, and the immunization method was subcutaneous injection at the back of the neck. Booster immunizations were performed at the second and sixth weeks after immunization, with the same immunization dose. (3) Separation and purification: Blood was collected from the carotid artery 2 weeks after the last immunization. After standing at 37°C for 1 hour, the serum was collected by centrifugation at 3500 rpm, and then the serum was affinity purified to obtain polyclonal antibodies to Ustilago farfara.

[0070] Biotin-labeled polyclonal antibody: Use DMSO to prepare a biotin solution with a concentration of 2 mg / mL, mix 300 μL (1.1 mg / mL) of the triticum tartaria polyclonal antibody with 10 μL of the biotin solution (2 mg / mL), shake the reaction at room temperature for 30 minutes, then transfer to a 10K dialysis membrane for dialyzation, and dialyze in PBS buffer (0.01 M, pH 7.5) for 24 hours, changing the buffer every 6 hours. After the dialysis, collect the biotin-labeled polyclonal antibody in a centrifuge tube and store it at -20°C for later use.

[0071] 2. HRP-conjugated monoclonal antibody 2D2

[0072] The HRP coupling kit (Shanghai Sangon Biotech Co., Ltd., Catalog No. D601047-0001) was used to couple the Ustilago sutchuenensis monoclonal antibody 2D2 prepared in Example 1. The coupling principle is to activate horseradish peroxidase (HRP) with periodate, and the activated horseradish peroxidase cross-links with the primary amine on the monoclonal antibody through Schiff base, and then reduce the Schiff base to produce a stable coupling product.

[0073] The specific operation steps of HRP conjugation to the monoclonal antibody 2D2 of light stinking powder fungus are as follows: add 200 μL of HRP activation buffer to 500 μL of HRP solution, react at room temperature for 30 minutes on a suspension instrument; then add 200 μL of HRP conjugation reagent and let it stand for 30 minutes; add 1 mL of 0.5 mg / mL monoclonal antibody 2D2 of light stinking powder fungus, mix gently with a pipette, transfer the liquid to a dialysis bag and place it in 2L of conjugated dialysis solution for dialysis, and place the container containing the dialysis solution on a magnetic stirrer for dialysis at room temperature for 2 hours. After dialysis, transfer the liquid in the dialysis bag to a 5 mL centrifuge tube, add 100 μL of reducing agent, let it stand at room temperature for 2 hours, and mix once every half an hour. At this point, HRP conjugation to the monoclonal antibody 2D2 of light stinking powder fungus is completed.

[0074] 3. Establishment of a system and standard curve for immunomagnetic separation combined with chemiluminescence detection of winter spores of Tilletia sutchuenensis

[0075] 0.1 mL of 10 mg / mL streptavidin magnetic beads (Nanjing Dongna Biotechnology Co., Ltd., catalog number: MB1003) was transferred and washed three times with PBST (0.01 mol / L PBS, pH 7.6, containing 0.05% Tween-20) buffer solution, and magnetic separation was performed, and the supernatant was discarded; then, 10 μL of the biotin-labeled polyclonal antibody prepared in step 1 was diluted 500 times with 0.5% BSA-PBST (0.01 mol / L PBS, pH 7.6, containing 0.5% BSA and 0.05% Tween-20) buffer. The concentration of the polyclonal antibody after dilution was 2 μg / mL. 100 μL of the diluted polyclonal antibody was added to the washed magnetic beads, incubated at 37°C with shaking for 30 min, then washed with PBST and magnetically separated, and the washing step was repeated three times to obtain nanoimmunomagnetic beads. 100 μL of nanoimmunomagnetic beads were taken and mixed with 200 μL of 10, 50, 100, 250, 500, 1000, 25000, 50000, 100000, 250000, 500000 spores / mL of U. sutsugamushi winter spore solution (the winter spore solution was diluted with 0.5% BSA-PBST), and incubated at 37°C for 30 min with shaking; then washed with PBST three times and magnetically separated. Then, 200 μL of HRP-conjugated U. sutsugamushi monoclonal antibody 2D2 diluted 500 times with 0.5% BSA-PBST was added to the streptavidin magnetic beads (the concentration of the diluted U. sutsugamushi monoclonal antibody 2D2 was 1 μg / mL), incubated at 37°C with shaking for 30 min, and washed again with PBST three times. Finally, add 100 μL of luminol chemiluminescent reagent (Huzhou Yingchuang Biotechnology Co., Ltd., liquid A, product number: CL-A-001, liquid B, product number: CL-B-001, liquid A: liquid B = 1:1), mix well and immediately detect the chemiluminescent signal. Thus, the system of immunomagnetic separation combined with chemiluminescence detection of winter spores of Ticklish powder fungus was established. Subsequently, a standard curve was drawn with the logarithm of spore concentration as the horizontal axis and the chemiluminescence response intensity as the vertical axis, see Figure 2 .

[0076] According to the test results, the linear range of the method based on magnetic immunochemiluminescence detection of spores of Smut fungus is 5.0×10-1.0×10 5 spores / mL, linear correlation R 2 =0.97.

[0077] 4. Sensitivity test

[0078] The detection method of this step is basically the same as that of step 3. The standard curve in step 3 is used to test the spore concentrations at low concentrations of 10, 20, 50, and 100 spores / mL, respectively. The final detection result is that the minimum detection limit is 50 spores / mL.

[0079] Comparative Example:

[0080] The target spores were detected by conventional ELISA method, and the steps were as follows: (1) Plate coating: 0.1 mol / L Na2CO3 solution was used to prepare the winter spore solution of Ustilago farnesii (concentrations were 100, 1000, and 2000 spores / mL), 100 μL was taken to a 96-well plate, incubated at room temperature for 2 h, and stored in a refrigerator at 4°C overnight; (2) Washing: The 96-well plate was washed 4 times with 0.05% PBST, 200 μL each time; (3) Blocking: 200 μL of 5% BSA-PBST was transferred and incubated at 37°C for 2 h; (4) Washing: Wash 4 times with PBST, 200 μL each time; (5) Polyclonal antibody coupling: 100 μL of 0.5% Add the polyclonal antibody of Ustilago farinae (1.1 mg / mL) diluted with BSA-PBS to a 96-well plate and incubate at 37°C for 2 h; (6) Washing: Wash 4 times with PBST, 200 μL each time; (7) Bind the secondary antibody: dilute HRP-labeled mouse anti-rabbit IgG (Shanghai Sangon Biotechnology Co., Ltd., Catalog No. D110065-0001) 5000 times with 0.5% BSA-PBS solution, transfer 100 μL, and incubate at 37°C for 2 h; (8) Washing: Wash the 96-well plate 5 times with PBS solution, 200 μL each time; (9) Detection: Add 100 μL of chemiluminescent substrate solution (Huzhou Yingchuang Biotechnology Co., Ltd., Catalog No. A solution: CL-A-001, Catalog No. B solution: CL-B-001, A solution: B solution = 1:1), mix well, and perform chemiluminescence detection using an enzyme reader. Experimental verification showed that this method can detect the winter spores of S. tiliaceus in about 24 hours, and the detection limit is 1000 spores / mL.

[0081] The immunomagnetic separation combined with chemiluminescence detection system for the winter spores of the light stinking powder powder established in the present invention can control the detection time within 2 hours, and the detection limit reaches 50 spores / mL. Therefore, the immunomagnetic separation combined with chemiluminescence detection method for the winter spores of the light stinking powder powder provided by the present invention significantly improves the efficiency and sensitivity of the detection.

Claims

1. A hybridoma cell line 2D2, characterized in that: The hybridoma cell line 2D2 was deposited in the China Center for Type Culture Collection on October 22, 2024, and its classification was named as hybridoma cell line 2D2 Monoclonal antibody hybridoma cell line against Tilletia foetida 2D2, the deposit number is CCTCC NO: C2024296.

2. Use of the hybridoma cell line according to claim 1 in the preparation of anti-Tilletia leucoderma monoclonal antibodies.

3. An anti-Tilletia smut monoclonal antibody, characterized in that: The anti-Tilletia leucoderma monoclonal antibody is secreted by the hybridoma cell line according to claim 1.

4. The anti-Tilletia leucoderma monoclonal antibody according to claim 3, characterized in that: The anti-light smut fungus monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDRH1 with an amino acid sequence as shown in SEQ ID NO.1, CDRH2 with an amino acid sequence as shown in SEQ ID NO.2, and CDRH3 with an amino acid sequence as shown in SEQ ID NO.3, and the light chain variable region comprises CDRL1 with an amino acid sequence as shown in SEQ ID NO.4, CDRL2 with an amino acid sequence of KVS, and CDRL3 with an amino acid sequence as shown in SEQ ID NO.

5.

5. The anti-Tilletia leucoderma monoclonal antibody according to claim 4, characterized in that The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

7.

6. The anti-Tilletia leucoderma monoclonal antibody according to claim 4, characterized in that: The nucleotide sequence of the heavy chain is shown in SEQ ID NO.8, and the nucleotide sequence of the light chain is shown in SEQ ID NO.

9.

7. The anti-Tilletia sutchuenensis monoclonal antibody according to claim 4, characterized in that: The amino acid sequence of the heavy chain is shown in SEQ ID NO.10, and the amino acid sequence of the light chain is shown in SEQ ID NO.

11.

8. Use of the anti-Tinous smut fungus monoclonal antibody according to any one of claims 3 to 7 in the detection of Tinous smut fungus teliospores.

9. An HRP-conjugated monoclonal antibody against Tilletia glabra, characterized in that: It contains the anti-Tilletia smut monoclonal antibody according to any one of claims 3 to 7.

10. A detection kit, characterized in that: The method comprises the anti-U. tina sutchuenensis monoclonal antibody according to any one of claims 3 to 7 or the HRP-coupled U. tina sutchuenensis monoclonal antibody according to claim 9.

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