A hybridoma cell line 1D6, its monoclonal antibody and applications

Abstract

The present invention discloses a hybridoma cell line 1D6, its monoclonal antibody and applications. The present invention also discloses the application of the monoclonal antibody against Tilletia foetida in the detection of Tilletia foetida teliospores. The present invention also discloses a single-stranded DNA with a single-base sequence for modifying the monoclonal antibody against Tilletia foetida. The present invention also discloses a detection kit. The monoclonal antibody, its labeling agent and the kit exhibit a high specific recognition ability for Tilletia foetida. The method of detecting single bases by mass spectrometry has a signal amplification effect, and the detection operation is simple, so that the method has been significantly improved in terms of sensitivity and detection efficiency. With the progress and continuous popularization and application of mass spectrometry technology, this technology has broad application potential and prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to a hybridoma cell line 1D6, its monoclonal antibody and applications. Background Art

[0002] Tilletia foetida is a devastating plant fungal disease caused by Tilletia foetida. After Tilletia foetida infects wheat, it will cause the wheat ears to turn black and the grains to shrink, seriously affecting the yield and quality of wheat. According to statistics, the yield of wheat infected with Tilletia foetida can be reduced by 20%-30%, and can reach more than 50% in severe cases. The infected grains will release trimethylamine during the processing, producing a strong fishy smell, resulting in the inedibility of the flour and seriously reducing its economic value. In addition, the pathogen spores have extremely strong environmental tolerance and transmission ability, can survive in the soil for many years, and are prone to cause repeated outbreaks and large-scale spread of the disease.

[0003] At present, the detection methods of Tilletia foetida mainly include morphological identification, molecular biology detection and immunological detection, etc. Morphological identification relies on microscopic observation of the morphological characteristics of spores, but this method is time-consuming and the accuracy is affected by the operator's experience. Molecular biology methods (such as PCR, qPCR) have high sensitivity and specificity, but there are problems such as complex operation, dependence on professionals and long time consumption. Traditional immunological detection methods also have some disadvantages. For example, for some target molecules in low-concentration or complex samples, their detection sensitivity may not be sufficient; due to the existence of cross-reactions, traditional immunoassays may produce false positive or false negative results, affecting the specificity of detection. The common problems in existing Tilletia foetida detection methods are difficult extraction of the target and insufficient detection sensitivity.

[0004] Using a single-stranded DNA with a single-base sequence as a mass tag for mass spectrometry immunoassay, after the nucleic acid sequence is labeled on the monoclonal antibody, the DNA nucleic acid strand is enzymatically hydrolyzed into single bases by exonuclease and alkaline phosphatase, and the concentration of the enzymatically hydrolyzed bases is detected by liquid chromatography-tandem mass spectrometry method, so as to achieve the quantification of the target. Tilletia foetida poses a serious threat to global wheat production. Developing rapid and accurate detection methods is crucial for disease prevention and control. The single-base sequence mass tag mass spectrometry detection method provides an innovative technical means for the diagnosis and prevention and control of Tilletia foetida with its advantages such as high sensitivity. In the future, with the further development and popularization of mass spectrometry technology, this method is expected to play a greater role in the field of agricultural disease detection and provide strong support for ensuring food security and sustainable agricultural development.

[0005] In the technical exploration of the binding between single-base sequence DNA single-strands and monoclonal antibodies, we face several key challenges: Firstly, achieving efficient directional coupling; secondly, ensuring the stability of the binding; thirdly, enhancing the signal amplification ability; in addition, there are also issues regarding the feasibility of large-scale production and adaptability in different complex scenarios. To overcome these problems, it is necessary to integrate multidisciplinary technologies such as materials science, molecular biology, and engineering, and conduct comprehensive innovation and breakthroughs. Summary of the Invention

[0006] Object of the Invention: The technical problem to be solved by the present invention is to provide a hybridoma cell line 1D6.

[0007] Another technical problem to be solved by the present invention is to provide the application of the hybridoma cell line in the preparation of a monoclonal antibody against Tilletia foetida.

[0008] Another technical problem to be solved by the present invention is to provide a monoclonal antibody against Tilletia foetida.

[0009] Another technical problem to be solved by the present invention is to provide the application of the monoclonal antibody against Tilletia foetida in the detection of Tilletia foetida teliospores.

[0010] Another technical problem to be solved by the present invention is to provide a single-base sequence DNA single-strand labeled monoclonal antibody against Tilletia foetida.

[0011] Another technical problem to be solved by the present invention is to provide a detection kit.

[0012] The last technical problem to be solved by the present invention is to provide a detection method capable of highly sensitive and highly specific detection of Tilletia foetida teliospores.

[0013] Technical Solution: The present invention provides a hybridoma cell line 1D6. The hybridoma cell line 1D6 was deposited with the China Center for Type Culture Collection (CCTCC) on October 22, 2024. Its taxonomic name is the hybridoma cell line 1D6 Monoclonal antibody hybridoma cell line against Tilletia foetida Tilletia foetida 1D6, and the deposit number is CCTCC NO: C2024295. The address of the deposit unit is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, within Wuhan University, Postcode: 430072.

[0014] The present invention also includes the application of the hybridoma cell line 1D6 in the preparation of a monoclonal antibody against Tilletia foetida.

[0015] The present invention also includes a monoclonal antibody against Tilletia foetida, and the monoclonal antibody against Tilletia foetida is secreted by the hybridoma cell line 1D6.

[0016] Among them, the monoclonal antibody against Tilletia foetida includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes CDRH1 with the amino acid sequence shown in SEQ ID NO.1, CDRH2 with the amino acid sequence shown in SEQ ID NO.2, and CDRH3 with the amino acid sequence shown in SEQ ID NO.3. The light chain variable region includes CDRL1 with the amino acid sequence shown in SEQ ID NO.4, CDRL2 with the amino acid sequence SAS, and CDRL3 with the amino acid sequence shown in SEQ ID NO.5.

[0017] 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.

[0018] Among them, 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.

[0019] Among them, 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.

[0020] The present invention also includes the application of the monoclonal antibody against Tilletia foetida in the detection of Tilletia foetida teliospores.

[0021] The present invention also includes a monoclonal antibody for modifying Tilletia foetida with a single-base sequence DNA single strand, which contains the monoclonal antibody against Tilletia foetida.

[0022] The present invention also includes a detection kit, which includes the monoclonal antibody against Tilletia foetida or the monoclonal antibody for labeling Tilletia foetida with a single-base sequence DNA single strand.

[0023] The present invention also includes a detection method for detecting Tilletia foetida teliospores, which specifically includes the following steps:

[0024] 1) Add solutions containing Tilletia foetida teliospores at different concentrations to nano-immunomagnetic beads and incubate to obtain complex system solutions of immunomagnetic beads-teliospores at different concentrations.

[0025] 2) Modify the monoclonal antibody against Tilletia foetida with a single-base sequence DNA single strand to obtain a DNA strand-modified antibody.

[0026] 3) Incubate the DNA strand-modified antibody with the complex systems of immunomagnetic beads-teliospore at different concentrations in step 1) respectively to obtain an immunomagnetic beads-teliospore-DNA strand-modified antibody solution;

[0027] 4) Add Exonuclease I and alkaline phosphatase solution to the immunomagnetic beads-teliospore-DNA strand-modified antibody solution for a mixed reaction. After the reaction, collect the supernatant solution and detect the single-base sequence to obtain a standard curve of the concentration of Tilletia foetida teliospores and the detection signal of adenine single-base sequence;

[0028] 5) Add the test solution containing Tilletia foetida teliospores to the nano-immunomagnetic beads for incubation to obtain a test solution. Add the DNA strand-modified antibody, Exonuclease I and alkaline phosphatase solution to the test solution for a mixed reaction, detect the signal of adenine single-base sequence, and substitute it into the linear equation of the standard curve for calculation to obtain the corresponding concentration of Tilletia foetida teliospores.

[0029] Beneficial effects: Compared with the prior art, the present invention has the following advantages: The application of the monoclonal antibody against Tilletia foetida in the detection of Tilletia foetida teliospores provides a powerful tool for the early diagnosis and prevention and control of wheat bunt. The monoclonal antibody of the present invention can specifically recognize Tilletia foetida teliospores, greatly improving the accuracy and efficiency of detection, helping to take timely prevention and control measures and reducing agricultural production losses; The single-base sequence DNA single-strand labeling technology of the present invention combined with this monoclonal antibody for the detection of Tilletia foetida teliospores further enhances the sensitivity of the detection signal, and even extremely low concentrations of pathogens can be effectively detected, thereby reducing the missed detection rate; The detection method of the present invention reduces the harmful chemical substances that may be used in traditional detection methods, is safer and more environmentally friendly, and is beneficial to the health of laboratory staff and environmental protection. Therefore, the present invention not only provides an efficient solution for the detection of wheat bunt, but also provides important support for the quality safety of agricultural products, the prevention and control of plant diseases and related scientific research. Brief Description of the Drawings

[0030] Figure 1 It is a schematic diagram of immunomagnetic separation combined with single-base sequence mass tag mass spectrometry for detecting Tilletia foetida teliospores;

[0031] Figure 2 It is a chromatogram of liquid chromatography-tandem mass spectrometry for detecting single-base sequence mass tags and a chromatogram of extracted quantitative and qualitative ion pairs;

[0032] Figure 3 It is a linear diagram of immunomagnetic separation combined with single-base sequence mass tag mass spectrometry for detecting Tilletia foetida teliospores. Detailed implementation mode

[0033] Example 1 Screening of hybridoma cell lines of Tilletia foetida

[0034] 1. Immunize mice with Tilletia foetida teliospores as antigens

[0035] 1) Weigh 0.1 mg of Tilletia foetida teliospores (provided by Jiangsu Grain and Oil Quality Monitoring Center), mix with 675 μL of normal saline, and then mix with 675 μL of Freund's complete adjuvant. After the spore solution is completely emulsified, immunize 6-8-week-old Blbc mice for the first time. The second to fourth immunizations are carried out after mixing and emulsifying the teliospore normal saline solution with Freund's incomplete adjuvant. The interval between each immunization is 14 days. Seven days after the four immunizations, collect blood from the tail vein, collect 10 μL of blood, and verify whether the antiserum has a positive reaction by ELISA.

[0036] 2) ELISA verification: Add 100 μL of the Tilletia foetida teliospore suspension to each well of a 96-well plate, incubate overnight in a 4 °C refrigerator, take it out, wash the plate 3 times with PBST solution (PBS buffer solution (0.01 mol / L PBS, pH 7.6, the same below) containing 0.05% Tween-20, the same below), then add 200 μL of 5% bovine serum albumin solution (using PBS buffer as the solvent) to each well, and block at 37 °C for 1 h. After taking it out, wash it 3 times with PBST solution to remove the excess bovine serum albumin solution.

[0037] 3) Add 100 μL of the serially diluted antiserum to the wells, mix well, incubate at 37 °C for 1 h, then add HRP-goat anti-mouse secondary antibody diluted 5000 times with PBS (Shanghai Sangon Biotech Co., Ltd., product number C65021-0100), incubate at 37 °C for 0.5 h, then wash the plate 2 times with PBST solution, add 100 μL of chemiluminescent substrate solution (luminol, A:B = 1:1, Innoreagent Company, Huzhou, Zhejiang. The luminol reagent formula is divided into two parts: one part is the alkaline solution of luminol, and the other part is the hydrogen peroxide solution. Storing them separately can extend the shelf life of the reagent because hydrogen peroxide decomposes in an alkaline environment and luminol is more stable in an acidic condition), mix well, and place the microplate in a microplate reader to detect the chemiluminescence intensity. At the same time, use the serum of normal mice as a negative control and detect the chemiluminescence intensity of the serum of normal mice in the same way.

[0038] 2. Screening of hybridoma cell lines of Tilletia foetida and preparation of monoclonal antibodies

[0039] 1) According to the measurement results in step 1, select mice with high ELISA response intensity (chemiluminescence intensity greater than 1.2), and perform the fifth immunization with teliospores suspension on the 7th day after blood collection.

[0040] 2) Cell fusion: On the 5th day after the fifth immunization, take the spleens of the mice, grind them and perform cell fusion with SP2 / 0, and then plate them.

[0041] 3) Subcloning screening: On the 14th - 16th day after cell fusion, observe the cells under the microscope and take the supernatant for ELISA detection; for the cells in the wells with positive specific antibody detected, transfer them in time and perform the first round of subcloning screening; after 6 - 7 days, observe the cells under the microscope and take the supernatant for ELISA detection; for the cells in the wells with positive specific antibody detected, transfer them in time and perform the second round of subcloning screening; after 5 - 6 days, observe the cells under the microscope and take the supernatant for ELISA detection; for the cells in the wells with positive specific antibody detected, transfer them in time and perform the third round of subcloning screening.

[0042] 4) After three rounds of subcloning screening, 4 hybridoma cell lines that can stably secrete antibodies against Tilletia foetida were screened out by ELISA detection. These positive cell lines were expanded and passaged, and the antibody titers of the cell supernatants were detected in each generation to verify whether the cells could continuously and stably secrete antibodies during the passage process. The results of subculture showed that these 4 hybridoma cells could secrete antibodies normally during 6 consecutive generations of culture, showing good antibody secretion stability. One of the positive cell lines showed a significant response intensity in the detection. Using the enzyme - labeled wells without spores as the blank control, the ratio of its response value to that of the normal mouse serum negative control group was greater than 2.2. This cell line has been selected for preservation (Preservation No.: CCTCC No. C2024295). Subsequently, we further expanded the culture of this cell line. When the cells grew to 90% confluence in the culture dish, a small amount of cells were taken and inoculated into a T25 flask for continuous culture, while the remaining cells were collected and cryopreserved for subsequent research use.

[0043] 5) Ascites preparation: Inject mineral oil into the abdominal cavity of mice 1 week in advance. When the cells in the T25 flask grow to 90% confluence, collect the cells and inject them into the abdominal cavity of mice. Observe the abdominal cavity of mice within 8 - 20 days. When the abdomen bulges to the point where movement is restricted, collect the ascites.

[0044] 6) Ascites purification: After centrifuging and filtering the ascites, dilute it with an equal volume of PBS, and purify the antibody using the Smart - antibody Protein G antibody purification kit (purchased from Engibody Company, USA, product number: P4144). Collect the antibody after purification.

[0045] 7) Verification of monoclonal antibody. Select Tilletia controversa Kühn spores, Aspergillus flavus spores, Aspergillus niger spores, and Paecilomyces variotii spores (Tilletia controversa Kühn spores were purchased from the Centraalbureau voor Schimmelcultures, and the other spores are well-known and commonly used conventional spores provided by the Microbial Testing Center of Nanjing Institute of Product Quality Supervision and Inspection) as materials for cross-verification. Verify the antibody by coating plates according to the method in step 1, and the verification results are shown in Table 1 below. According to the chemiluminescence signal, when the coating antigen is Tilletia foetida teliospores, the chemiluminescence signal is all higher than 0.8120, while when other strain spores are used as the coating antigen, the chemiluminescence signal decreases significantly, lower than 0.6430. It shows that the monoclonal antibody against Tilletia foetida prepared by the present invention has significant specific adsorption to Tilletia foetida teliospores and can be used to identify Tilletia foetida.

[0046] Table 1 Verification results of monoclonal antibody

[0047]

[0048] In the present invention, the enzyme-labeled wells without spore coating are used as blank controls, and a Tilletia foetida hybridoma cell line 1D6 with a response value ratio greater than 2.2 to the normal mouse serum negative control group is screened out. This cell line was officially deposited at the China Center for Type Culture Collection (CCTCC) on October 22, 2024. Its taxonomic name is: Monoclonal antibody hybridoma cell 1D6 against Tilletia foetida Tilletia foetida 1D6, deposit number: CCTCC NO: C2024295, deposited at the China Center for Type Culture Collection (CCTCC); the address of the depositary institution is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, within Wuhan University; postal code: 430072.

[0049] Example 2 Sequencing of monoclonal antibody against Tilletia foetida

[0050] In Example 1, a monoclonal antibody against Tilletia foetida was successfully prepared. To further analyze its molecular structure, Beijing Sino Biological Inc. was entrusted to perform sequencing analysis on this monoclonal antibody, and thus the detailed amino acid sequence and nucleotide sequence of this monoclonal antibody were obtained.

[0051] The monoclonal antibody against Tilletia foetida includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes CDRH1 with the amino acid sequence shown in SEQ ID NO.1, CDRH2 with the amino acid sequence shown in SEQ ID NO.2, and CDRH3 with the amino acid sequence shown in SEQ ID NO.3. The light chain variable region includes CDRL1 with the amino acid sequence shown in SEQ ID NO.4, CDRL2 with the amino acid sequence SAS, and CDRL3 with the amino acid sequence shown in SEQ ID NO.5;

[0052] SEQ ID NO.1 GYTFSSYW

[0053] SEQ ID NO.2 ILAGSGST

[0054] SEQ ID NO.3 ARSGYGDQ

[0055] SEQ ID NO.4 QNVGSN

[0056] SEQ ID NO.5 QQYNSYPLT

[0057] The lengths of the heavy chain variable region and the light chain variable region of this monoclonal antibody are 115 and 107 amino acids respectively. The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO.6, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO.7;

[0058] Heavy chain variable region sequence

[0059] SEQ ID NO.6

[0060] EVKLVESGAELMKPGASVKISCKATGYTFSSYWLEWVKQRPGHGPEWIGEILAGSGSTKYNEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYCARSGYGDQWGQGTTLTVSS

[0061] Light chain variable region sequence

[0062] SEQ ID NO.7

[0063] DIVMTQSQKFMSTSVGDRVRVTCKASQNVGSNIVWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVESEDLAEYFCQQYNSYPLTFGTGTQLELK

[0064] Full-length nucleotide sequence of the heavy chain:

[0065] SEQ ID NO.8

[0066]

[0067] Full-length nucleotide sequence of the light chain:

[0068] SEQ ID NO.9

[0069] ATGGAGACACAGTCCCAGGTCTTTGTATACATGTTGCTGTGGTTGTCTGGTGTTGATGGAGACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACTTCAGTAGGAGACAGGGTCAGAGTCACCTGCAAGGCCAGTCAGAATGTGGGTAGTAATATAGTCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCGCTGATTTACTCGGCATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGTAATGTGGAGTCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATATAACAGCTATCCGCTCACGTTCGGTACTGGGACCCAGCTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGCTGA

[0070] Amino acid sequence of the full-length heavy chain:

[0071] SEQ ID NO.10

[0072] MGWSLILLFLVAVATRVLSEVKLVESGAELMKPGASVKISCKATGYTFSSYWLEWVKQRPGHGPEWIGEILAGSGSTKYNEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYCARSGYGDQWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0073] Full-length amino acid sequence of the light chain

[0074] SEQ ID NO.11

[0075] METQSQVFVYMLLWLSGVDGDIVMTQSQKFMSTSVGDRVRVTCKASQNVGSNIVWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVESEDLAEYFCQQYNSYPLTFGTGTQLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0076] Example 3 Application of monoclonal antibody 1D6 - Immunomagnetic separation combined with single-base sequence quality tag mass spectrometry for detecting Tilletia foetida teliospores

[0077] 1. Biotin-labeled polyclonal antibody

[0078] Preparation of polyclonal antibody against Tilletia foetida: (1) Emulsification: Take 10 5The teliospores solution of Tilletia foetida at a concentration of [[[ID=]]] cells / mL was mixed with an equal volume of Freund's complete adjuvant and emulsified. (2) Animal immunization: Male New Zealand white rabbits at 13 weeks of age 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 2 weeks and 6 weeks after the initial immunization, with the same immunization dose. (3) Isolation and purification: Blood was collected from the carotid artery 2 weeks after the last immunization. After standing at 37 °C for 1 h, the serum was collected by centrifugation at 3500 rpm. Then, the serum was affinity purified to obtain the polyclonal antibody against Tilletia foetida. A biotin solution with a concentration of 2 mg / mL was prepared using dimethyl sulfoxide (CAS: 67-68-5, Sigma, catalog number: D9170) (prepared using biotin 3-sulfo-N-hydroxysuccinimide ester sodium salt, CAS: 119616-38-5, Sigma, catalog number: B5161). 300 μL (1.1 mg / mL) of the polyclonal antibody against Tilletia foetida was mixed with 10 μL of the biotin solution (2 mg / mL). After reacting with shaking at room temperature for 30 min, the mixture was transferred to a 10K dialysis membrane for dialysis. It was dialyzed in PBS buffer (0.01 M, pH 7.5) for 24 h, and the buffer was changed every 6 h. After dialysis, the biotin-labeled polyclonal antibody was collected in a centrifuge tube and stored at -20 °C for later use.

[0079] 2. Preparation of immunomagnetic beads

[0080] Transfer 0.1 mL of streptavidin magnetic beads at 10 mg / mL (Nanjing Dongna Biotechnology Co., Ltd., catalog number: MB1003), wash them 3 times with PBST (0.01 mol / L PBS, pH 7.6, containing 0.05% Tween-20) buffer solution, and perform magnetic separation to discard the supernatant. Then, take 10 μL of the biotin-labeled polyclonal antibody prepared in step 1, dilute it 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 solution. The diluted polyclonal antibody concentration is 2 μg / mL. Take 100 μL of the diluted polyclonal antibody and add it to the washed magnetic beads. Incubate with shaking at 37 °C for 30 min, then wash with PBST and perform magnetic separation. Repeat the washing step 3 times to obtain nano-immunomagnetic beads.

[0081] DNA single-strand modification of antibody

[0082] A single-stranded DNA consisting of a single-base sequence of adenine (A) was customized (ordered from Sangon Biotech (Shanghai) Co., Ltd.). The sequence length of the adenine single-base sequence DNA strand was 30 bp, and the concentration was 100 μM. Using chemical synthesis, during the solid-phase synthesis of the adenine single-base sequence DNA strand, a phosphoramidite monomer with an aldehyde group was introduced as the last nucleotide. After synthesis, the 5'-end of the adenine single-base sequence DNA strand would carry an aldehyde group modification. Pipette 100 μL of the adenine single-base sequence DNA single strand (concentration 100 μM) and 10 μL of the monoclonal antibody 1D6 of Tilletia foetida (concentration 1 mg / mL), react in 0.01 M carbonate buffer solution for 12 h (the pH of the carbonate buffer solution is 8.5), and then perform dialysis to remove the free adenine single-base sequence DNA single strand that has not bound to the antibody; use an ultrafiltration tube to concentrate the DNA strand-modified antibody solution to obtain a DNA strand-modified antibody with a concentration of 2 mg / mL. Storage method: Add an equal volume of glycerol and store at -20°C.

[0083] The principle of the binding of the amino group in the monoclonal antibody 1D6 to the aldehyde group-modified adenine single-base sequence DNA single strand is based on the Schiff base reaction, and an imine bond is formed through the specific condensation of the amino group and the aldehyde group. This method has the characteristics of high efficiency, specificity, and flexibility, and is widely used in fields such as immunoassay, targeted therapy, and molecular diagnosis. The reaction equation is: R-NH 2 +R’-CHO→R-N=CH-R’+H 2 O

[0084] 3. Detection of the adenine single-base sequence by liquid chromatography-tandem mass spectrometry (LC-MS / MS)

[0085] The method for detecting the adenine single-base sequence by LC-MS / MS (the instrument was purchased from Agilent Technologies, model: 6470 LC / TQ) is described in detail below.

[0086] Chromatographic conditions:

[0087] Mobile phase: aqueous phase (A), 0.1% formic acid in water; organic phase (B), acetonitrile.

[0088] Gradient elution program: 0 - 2 min, 30 - 60% B; 3 - 6 min, 60 - 80% B.

[0089] Post-run: 2 min;

[0090] Injection volume: 2 μL.

[0091] Flow rate: 0.2 ml / min.

[0092] Column temperature: 30°C.

[0093] Chromatographic column: C18 reversed-phase column, model: ZORBAX Eclipse Plus (provided by Agilent Technologies).

[0094] Mass spectrometry conditions:

[0095] Ion source: Electrospray ionization (ESI), negative ion mode.

[0096] Scan mode: Multiple reaction monitoring (MRM).

[0097] Nebulizer pressure: 40 psi.

[0098] Gas temperature: 300 °C, gas flow rate: 10 L / min.

[0099] Sheath gas temperature: 300 °C, sheath gas flow rate: 11 L / min.

[0100] Capillary voltage: 3000 v.

[0101] Fragmentation voltage: 0 v.

[0102] Ion pair settings: Quantitative ion pair: 134.0 → 92.0, qualitative ion pair: 134.0 → 79.0.

[0103] Through the above optimized liquid chromatography and mass spectrometry conditions, highly sensitive and highly specific detection of adenine single-base sequence can be achieved. Figure 2 Shown are the MRM detection chromatogram of adenine single-base sequence and the chromatograms of the quantitative and qualitative ion pairs extracted from the MRM chromatogram of adenine single-base sequence, with the concentration of adenine single-base sequence being 0.1 μM in the figure.

[0104] 4. Establishment of an immunomagnetic separation combined with single-base sequence mass tag mass spectrometry detection system and drawing of a standard curve

[0105] (1) Accurately weigh 50 μg of nano-immunomagnetic beads. After washing twice with PBS buffer (0.01 mol / L, pH 7.6), add 100 μL of 1% skim milk powder solution (the skim milk powder solution is dissolved with PBS) and perform blocking treatment for 30 min.

[0106] (2) Use magnetic separation technology to remove the blocking solution. Add 100 μL of a solution containing Tilletia foetida teliospores (solutions with concentrations of 10, 50, 100, 500, 1000, 5000, 10000, 25000 cells / mL are prepared with PBS) to the nano-immunomagnetic beads. After mixing, place them in a constant temperature shaker at 37 °C and incubate at a rotation speed of 120 rpm for 45 min to form an immunomagnetic bead-teliospore complex.

[0107] After incubation, the supernatant was removed using magnetic separation technology and washed once with PBS.

[0108] (4)Mix 10 μL of the prepared DNA strand-modified antibody with 90 μL of the complex system solution of immunomagnetic beads-teliospore, react at room temperature for 30 min, perform magnetic separation to remove the DNA strand-modified antibody that did not bind to the immunomagnetic beads-teliospore complex, and then add DNA exonuclease Exonuclease I and alkaline phosphatase solution to the immunomagnetic beads-teliospore-DNA strand-modified antibody system. React for 1 h at 37 °C in the buffer solution supporting the exonuclease, extract twice, and collect the aqueous phase into the injection vial. Use LC-MS / MS to detect the adenine single-base sequence in teliospore solutions with different concentrations. The specific detection method is the same as step 3 above, so as to obtain different chromatograms of the adenine single-base sequence. With the teliospore concentration as the abscissa and the mass spectrometry signal value as the ordinate, draw a standard curve, as shown in Figure 3 . Based on immunomagnetic separation combined with single-base sequence mass tag mass spectrometry for detecting Tilletia foetida teliospore, the linear range is 10 - 2500 / mL, and the linear correlation R 2 = 0.9873, and the lowest detection limit is 10 / mL. When using this method to detect Tilletia foetida teliospore samples, the detection time can be controlled within 2 hours.

[0109] (5)Verification of accuracy and stability

[0110] Verify the established method through a spiking experiment. Specifically as follows:

[0111] Prepare spiked Tilletia foetida teliospore solutions with blank sample matrices, and the spiked concentrations are 50, 500, and 5000 / mL respectively. Set up 3 parallels for each spiked concentration. According to the immunomagnetic separation combined with single-base sequence mass tag mass spectrometry detection system established in step 4, detect the Tilletia foetida teliospore solutions with known spiked concentrations respectively, and calculate the recovery rate and stability after detection. The specific spiked recovery results are shown in Table 2.

[0112] Table 2 Spiked verification results of the detection method

[0113]

[0114] It can be seen from the results that the spiked recovery rate of the detection method ≥ 78.25%, and RSD ≤ 7.15%. Therefore, this detection method has good accuracy and stability.

Claims

1. A hybridoma cell line 1D6, characterized in that: The hybridoma cell line 1D6 was deposited in the China Center for Type Culture Collection on October 22, 2024, and its classification was named as monoclonal antibody hybridoma cell line against light smut fungus 1D6 Tilletia foetida 1D6, the deposit number is CCTCC NO:C2024295.

2. Use of the hybridoma cell line 1D6 according to claim 1 in the preparation of monoclonal antibodies against Tilletia sutchuenensis.

3. A monoclonal antibody against Tilletia leucoderma, characterized in that: The monoclonal antibody against Tilletia sutchuenensis is secreted by the hybridoma cell line 1D6 according to claim 1.

4. A monoclonal antibody against Tilletia leucoderma, characterized in that: The monoclonal antibody against light smut fungus 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 SAS, and CDRL3 with an amino acid sequence as shown in SEQ ID NO.

5.

5. The monoclonal antibody against Tilletia leucoderma 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 monoclonal antibody against Tilletia leucoderma 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 monoclonal antibody against Tilletia leucoderma 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 monoclonal antibody against Tilletia glabra according to any one of claims 3 to 7 in the detection of Tilletia glabra teliospores.

9. A single-base sequence DNA single-stranded modified Tilletia monoclonal antibody, characterized in that: The invention comprises the monoclonal antibody against Smut fungus according to any one of claims 3 to 7.

10. A detection kit, characterized in that: The invention comprises the anti-Ustilago farfara monoclonal antibody according to any one of claims 3 to 7 or the single-base sequence DNA single-stranded modified Ustilago farfara monoclonal antibody according to claim 9.

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