Hybridoma cell strain 1D6 as well as monoclonal antibody and application thereof

Abstract

The invention discloses a hybridoma cell strain 1D6 as well as a monoclonal antibody and application thereof. The invention also discloses an application of the monoclonal antibody for resisting the Tilletia foetida in the detection of teliospore of the Tilletia foetida. The invention also discloses a single base sequence DNA single chain modified Tilletia foetida monoclonal antibody. The invention also discloses a detection kit. The monoclonal antibody as well as the marker and the kit thereof show high specific recognition capability on Tilletia foetida. The method for detecting the single base by mass spectrometry has a signal amplification effect and is simple and convenient in detection operation, so that the sensitivity and the detection efficiency of the method are remarkably improved. With the progress and continuous popularization and application of the mass spectrum technology, the technology has wide application potential and prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a hybridoma cell line 1D6 and a monoclonal antibody thereof and application thereof. Background Art

[0002] Wheat light stinky smut is a devastating plant fungal disease caused by Ustilago farfara. When wheat light stinky smut infects wheat, it causes 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 light stinky smut can be reduced by 20%-30%, and in severe cases it can reach more than 50%. The infected grains will release trimethylamine during processing, producing a strong fishy smell, making the flour inedible and seriously reducing its economic value. In addition, the pathogen spores have extremely strong environmental tolerance and transmission capabilities, and can survive in the soil for many years, which can easily cause repeated outbreaks and large-scale spread of the disease.

[0003] At present, the detection methods of light stinking smut mainly include morphological identification, molecular biology detection and immunological detection. Morphological identification relies on the observation of the morphological characteristics of spores under a microscope, but this method is time-consuming and its 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, such as: for certain target molecules in low concentrations or complex samples, the detection sensitivity may not be enough; due to the existence of cross-reactions, traditional immunoassays may have false positive or false negative results, affecting the specificity of the detection. The common problems in the existing light stinking smut detection methods are the difficulty in extracting the target and the insufficient detection sensitivity.

[0004] Using a single-base sequence DNA single strand as a mass label for mass spectrometry immunoassay, after the nucleic acid sequence is labeled on a monoclonal antibody, the DNA nucleic acid chain is enzymatically hydrolyzed into a single base by exonuclease and alkaline phosphatase, and the concentration of the base after enzymatic hydrolysis is detected by liquid chromatography-tandem mass spectrometry, thereby achieving quantification of the target. Wheat light stinking smut poses a serious threat to global wheat production, and the development of rapid and accurate detection methods is crucial for disease prevention and control. The single-base sequence mass label mass spectrometry detection method provides an innovative technical means for the diagnosis and prevention and control of light stinking smut 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 combining single-base sequence DNA strands with monoclonal antibodies, we face several key challenges: first, achieving efficient directional coupling, second, ensuring the stability of the binding, and third, improving the signal amplification capability. In addition, there are also issues of feasibility of large-scale production and adaptability in different complex scenarios. To overcome these challenges, it is necessary to integrate multidisciplinary technologies such as materials science, molecular biology, and engineering to carry out comprehensive innovation and breakthroughs. Summary of the invention

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

[0007] The technical problem that the present invention needs to solve is to provide the use of the hybridoma cell strain in preparing monoclonal antibodies against Tilletia sutchuenensis.

[0008] The technical problem that the present invention needs to solve is to provide a monoclonal antibody against Tilletia smut.

[0009] The technical problem that the present invention needs to solve is to provide the application of the monoclonal antibody against Tilletia glabra in the detection of Tilletia glabra teliospores.

[0010] The technical problem that the present invention needs to solve is to provide a single-base sequence DNA single-stranded labeling Tilletia monoclonal antibody.

[0011] The technical problem that the present invention needs to solve is to provide a detection kit.

[0012] The final technical problem to be solved by the present invention is to provide a detection method capable of detecting the winter spores of Tilletia sutchuenensis with high sensitivity and high specificity.

[0013] Technical solution: The present invention provides a hybridoma cell line 1D6, which was deposited in the China Center for Type Culture Collection (CCTCC) on October 22, 2024, and its classification name is hybridoma cell line 1D6 Monoclonal antibody hybridoma cell line against Tilletia foetida 1D6, the deposit number is CCTCC NO: C2024295, and the deposit address is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, Postal Code: 430072.

[0014] The present invention also includes the use of the hybridoma cell line 1D6 in the preparation of monoclonal antibodies against Tilletia smutata.

[0015] The present invention also includes a monoclonal antibody against T. tina, which is secreted by the hybridoma cell line 1D6.

[0016] Among them, the monoclonal antibody against light smut fungus 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 SEQID NO.4, CDRL2 with an amino acid sequence of SAS, and CDRL3 with an amino acid sequence as 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] 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] 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 use of the monoclonal antibody against S. tiliaceus in the detection of S. tiliaceus teliospores.

[0021] The present invention also includes a monoclonal antibody against Ustilago sutchuenensis modified with a single-base sequence DNA single strand, which contains the monoclonal antibody against Ustilago sutchuenensis.

[0022] The present invention also includes a detection kit, which includes the monoclonal antibody against Ustilago sutchuenensis or the monoclonal antibody against Ustilago sutchuenensis labeled with a single-base sequence DNA strand.

[0023] The present invention also includes a method for detecting the teliospores of Tilletia smut, which specifically comprises the following steps: 1) Add different concentrations of solutions containing winter spores of wheat smut fungus to the nano-immunomagnetic beads and incubate them to obtain different concentrations of immunomagnetic beads-winter spore complex system solutions. 2) Modify the monoclonal antibody of S. tiliaceus with a single-base sequence DNA strand to obtain a DNA strand-modified antibody; 3) incubating the DNA chain modified antibody with the immunomagnetic beads-teliospore complex system of different concentrations in step 1) to obtain an immunomagnetic beads-teliospore-DNA chain modified antibody solution; 4) Add DNA exonuclease Exonuclease I and alkaline phosphatase solution to the immunomagnetic beads-tiolar spore-DNA chain modified antibody solution for mixed reaction. After the reaction is completed, collect the supernatant solution and detect the single base sequence to obtain a standard curve of the concentration of wheat spores and the adenine single base sequence detection signal; 5) A test solution containing winter spores of Ustilago serrata is added to nano-immunomagnetic beads for incubation to obtain a test solution, a DNA chain modification antibody, a DNA exonuclease Exonuclease I and an alkaline phosphatase solution are added to the test solution for mixed reaction, a signal of adenine single base sequence is detected, and the signal is substituted into the linear equation of the standard curve for calculation to obtain the corresponding concentration of winter spores of Ustilago serrata.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following advantages: the application of the monoclonal antibody against light stinking smut of the present invention in the detection of light stinking smut winter spores provides a powerful tool for the early diagnosis and prevention and control of wheat light stinking smut. The monoclonal antibody of the present invention can specifically identify the light stinking smut winter spores, greatly improving the accuracy and efficiency of detection, and helping to take prevention and control measures in time to reduce agricultural production losses; the single-base sequence DNA single-stranded labeling technology of the present invention is combined with the monoclonal antibody to detect the light stinking smut winter spores, further enhancing 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 chemicals that may be used in traditional detection methods, is safer and more environmentally friendly, and is beneficial to the health and environmental protection of laboratory workers. Therefore, the present invention not only provides an efficient solution for the detection of wheat light stinking smut, but also provides important support for agricultural product quality and safety, plant disease prevention and control, and related scientific research. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of immunomagnetic separation combined with single base sequence mass tag mass spectrometry for detection of wheat teliospores of Tilletia serrata; Figure 2 Chromatograms of single-base sequence mass tags detected by liquid chromatography-tandem mass spectrometry and chromatograms of extracted quantitative and qualitative ion pairs; Figure 3 Linearity diagram for immunomagnetic separation combined with single base sequence mass tag mass spectrometry detection of Tilletia oleracea teliospores. DETAILED DESCRIPTION

[0026] Example 1 Screening of Hybridoma Cell Lines of Tilletia glabra 1. Immunization of mice with winter spores of Smut fungus 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, and the plate was incubated in a 4°C refrigerator overnight. The plate was washed three 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), and then 200 μL of 5% bovine serum albumin solution (PBS buffer as solvent) was added to each well. The plate was blocked at 37°C for 1 h, and then washed three times with PBST solution to remove excess bovine serum albumin solution.

[0028] 3) Add 100 μL of the gradient diluted antiserum to the well plate, mix well and incubate at 37°C for 1 hour, then add HRP-goat anti-mouse secondary antibody (Shanghai Sangon Biotechnology Co., Ltd., Cat. No. C65021-0100) diluted 5000 times with PBS, incubate at 37°C for 0.5 hour, then wash the plate twice with PBST solution, add 100 μL of chemiluminescent substrate solution (luminol, A:B=1:1, Zhejiang Huzhou innoreagent Company, 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. Separate storage can extend the shelf life of the reagent, because hydrogen peroxide will decompose in an alkaline environment, while luminol is more stable under acidic conditions), mix well, and place the ELISA plate in an ELISA instrument to detect the chemiluminescence intensity. At the same time, the serum of normal mice was used as a negative control, and the chemiluminescence intensity of normal mouse serum was detected by the same method.

[0029] 2. Screening of hybridoma cell lines of Ustilago sutchuenensis and preparation of monoclonal antibodies 1) According to the results of step 1, select mice with high ELISA response intensity (chemiluminescence intensity greater than 1.2) and perform the fifth immunization with teliospore suspension on the 7th day after blood collection; 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.

[0030] 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; 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 2.2 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. C2024295). 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.

[0031] 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.

[0032] 6) Purification of ascites: After centrifugation and filtration, the ascites was diluted with an equal volume of PBS and purified using the Smart-antibody Protein G antibody purification kit (purchased from Engibody, USA, catalog number: P4144). After purification, the antibodies were collected.

[0033] 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 0.8120, while when the spores of other strains are used as the coating antigen, the chemiluminescent signal is significantly reduced, which is lower than 0.6430. 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.

[0034] Table 1 Monoclonal antibody validation results

[0035] In the present invention, the enzyme-labeled wells without spores were used as blank controls, and the hybridoma cell line 1D6 of wheat smut fungus with a response value ratio greater than 2.2 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 (CCTCC) on October 22, 2024, and its classification was named: Monoclonal antibody hybridoma cell line 1D6 against smut fungus Tilletia foetida 1D6, the deposit number is: CCTCC NO: C2024295, deposited in China Center for Type Culture Collection (CCTCC); the address of the deposit unit is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province; Postal Code: 430072.

[0036] Example 2 Sequencing of Monoclonal Antibodies to Tilletia tritici 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.

[0037] The monoclonal antibody of wheat 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; SEQ ID NO.1 GYTFSSYW SEQ ID NO.2 ILAGSGST SEQ ID NO.3 ARSGYGDQ SEQ ID NO.4 QNVGSN SEQ ID NO.5 QQYNSYPLT The heavy chain variable region and light chain variable region of the monoclonal antibody are 115 and 107 amino acids in length, 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; Heavy chain variable region sequence SEQ ID NO.6 EVKLVESGAELMKPGASVKISCKATGYTFSSYWLEWVKQRPGHGPEWIGEILAGSGSTKYNEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYCARSGYGDQWGQGTTLTVSS Light chain variable region sequence SEQ ID NO.7 DIVMTQSQKFMSTSVGDRVRVTCKASQNVGSNIVWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVESEDLAEYFCQQYNSYPLTFGTGTQLELK Heavy chain full length nucleotide sequence: SEQ ID NO.8 Full-length nucleotide sequence of the light chain: SEQ ID NO.9 ATGGAGACACAGTCCCAGGTCTTTGTATACATGTTGCTGTGGTTGTCTGGTGTTGATGGAGACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACTTCAGTAGGAGACAGGGTCAGAGTCACCTGCAAGGCCAGTCAGAATGTGGGTAGTAATATAGTCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCGCTGATTTACTCGGCATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGTAATGTGGAGTCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATATAACAGCTATCCGCTCACGTTCGGTACTGGGACCCAGCTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGCTGA Amino acid sequence of the full-length heavy chain: SEQ ID NO.10 MGWSLILLFLVAVATRVLSEVKLVESGAELMKPGASVKISCKATGYTFSSYWLEWVKQRPGHGPEWIGEILAGSGSTKYNEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYY CARSGYGDQWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDK KIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPI EKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK Light chain full length amino acid sequence SEQ ID NO.11 METQSQVFVYMLLWLSGVDGDIVMTQSQKFMSTSVGDRVRVTCKASQNVGSNIVWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVESEDLAEYFCQQYNSYPLT FGTGTQLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC Example 3 Application of monoclonal antibody 1D6 - Detection of winter spores of Tilletia sphaeroides by immunomagnetic separation combined with single base sequence mass tag mass spectrometry 1. Biotin-labeled polyclonal antibody 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. A 2 mg / mL biotin solution was prepared with dimethyl sulfoxide (CAS: 67-68-5, Sigma, catalog number: D9170) (biotin 3-sulfo-n-hydroxysuccinimide ester sodium salt was used for preparation, CAS: 119616-38-5, Sigma, catalog number: B5161), 300 μL (1.1 mg / mL) of the polyclonal antibody against wheat smut fungus was mixed with 10 μL of the biotin solution (2 mg / mL), and the mixture was shaken for 30 min at room temperature and then transferred to a 10K dialysis membrane for dialysis. The mixture was dialyzed in PBS buffer (0.01 M, pH 7.5) for 24 h, during which the buffer was changed every 6 h. After the dialysis, the biotin-labeled polyclonal antibody was collected in a centrifuge tube and stored at -20°C for later use.

[0038] 2. Preparation of Immunomagnetic Beads 0.1 mL of 10 mg / mL streptavidin magnetic beads (Nanjing Dongna Biotechnology Co., Ltd., catalog number: MB1003) was transferred, 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 taken, and 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 with shaking at 37°C for 30 min, then washed with PBST and magnetically separated, and the washing step was repeated three times to obtain nanoimmunomagnetic beads.

[0039] DNA single-stranded modified antibody A DNA single strand consisting of a single base sequence of adenine (A) was customized (ordered by Sangon Biotech (Shanghai) Co., Ltd.). The sequence length of the adenine single base sequence DNA chain was 30 bp and the concentration was 100 μM. Using the chemical synthesis method, during the solid phase synthesis of the adenine single base sequence DNA chain, 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 chain will be modified with an aldehyde group. Take 100 μL of adenine single-base sequence DNA single strand (concentration of 100 μM) and 10 μL (concentration of 1 mg / mL) monoclonal antibody 1D6 of Ustilago glabra, react in 0.01M carbonate buffer solution for 12 h (pH of carbonate buffer solution is 8.5), then dialyze to remove free adenine single-base sequence DNA single strand that is not bound to the antibody; use ultrafiltration tube to concentrate the DNA chain modified antibody solution to obtain DNA chain modified antibody, the concentration of which is 2 mg / mL. Storage method: add an equal volume of glycerol and store at -20℃.

[0040] The principle of the amino group in the monoclonal antibody 1D6 binding to the single-stranded DNA of the adenine base sequence modified with an aldehyde group is based on the Schiff base reaction, which generates an imine bond through the specific condensation of the amino group and the aldehyde group. This method is highly efficient, specific and flexible, and is widely used in immunoassays, targeted therapy and molecular diagnosis. The reaction equation is: R-NH2+R'-CHO→RN=CH-R'+H2O 3. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) detection of adenine single base sequence The following is a detailed description of the method for detecting adenine single base sequence by LC-MS / MS (the instrument was purchased from Agilent, equipment model: 6470 LC / TQ).

[0041] Chromatographic conditions: Mobile phase: aqueous phase (A), 0.1% formic acid in water; organic phase (B), acetonitrile.

[0042] Gradient elution program: 0-2 min, 30-60% B; 3-6 min, 60-80% B.

[0043] Post-run: 2min; Injection volume: 2 μL.

[0044] Flow rate: 0.2ml / min.

[0045] Column temperature: 30℃.

[0046] Chromatographic column: C18 reverse phase column, model: ZORBAX Eclipse Plus (provided by Agilent).

[0047] Mass spectrometry conditions: Ion source: electrospray ionization (ESI), negative ion mode.

[0048] Scan mode: Multiple reaction monitoring (MRM).

[0049] Atomizer pressure: 40psi.

[0050] Gas temperature: 300°C, gas flow rate: 10L / min.

[0051] Sheath gas temperature: 300°C, sheath gas flow rate: 11 L / min.

[0052] Capillary voltage: 3000v.

[0053] Fragmentation voltage: 0v.

[0054] Ion pair settings: quantitative ion pair: 134.0→92.0, qualitative ion pair: 134.0→79.0.

[0055] Through the above-mentioned optimized liquid chromatography and mass spectrometry conditions, highly sensitive and highly specific detection of adenine single base sequences can be achieved. Figure 2 Shown are the MRM detection chromatogram of the adenine single base sequence and the chromatograms of the quantitative and qualitative ion pairs extracted from the MRM chromatogram of the adenine single base sequence, where the concentration of the adenine single base sequence is 0.1 μM.

[0056] 4. Establishment of immunomagnetic separation combined with single-base sequence mass label mass spectrometry detection system and drawing of standard curve (1) Accurately weigh 50 μg of nanoimmunomagnetic beads, wash twice with PBS buffer (0.01 mol / L, pH 7.6), add 100 μL of 1% skim milk powder solution (skim milk powder solution is dissolved with PBS), and block for 30 min.

[0057] (2) The blocking solution was removed by magnetic separation technology, and 100 μL of a solution containing winter spores of Ustilago farinae was added to the nano-immunomagnetic beads (the solution was prepared with PBS to a concentration of 10, 50, 100, 500, 1000, 5000, 10000, and 25000 / mL). After mixing, the solution was placed in a 37°C constant temperature shaker and incubated at 120 rpm for 45 min to form an immunomagnetic bead-winter spore complex.

[0058] (3) After incubation, remove the supernatant using magnetic separation technology and wash once with PBS.

[0059] (4) Mix 10 μL of the prepared DNA chain modification antibody with 90 μL of the immunomagnetic beads-winter spore complex system solution, react at room temperature for 30 minutes, and perform magnetic separation to remove the DNA chain modification antibody that is not bound to the immunomagnetic beads-winter spore complex. Then add DNA exonuclease Exonuclease I and alkaline phosphatase solution to the immunomagnetic beads-winter spore-DNA chain modification antibody system, react for 1 hour at 37°C in the buffer solution of the exonuclease, extract twice, and collect the aqueous phase in an injection bottle. Use liquid LC-MS / MS to detect the adenine single base sequence in winter spore solutions of different concentrations. The specific detection method is the same as step 3 above, thereby obtaining different chromatograms of the adenine single base sequence. Use the winter spore concentration as the horizontal axis and the mass spectrometry signal value as the vertical axis to draw a standard curve, see Figure 3 The linear range of immunomagnetic separation combined with single base sequence mass tag mass spectrometry for the detection of winter spores of Tilletia serrata was 10-2500 / mL, and the linear correlation R 2 =0.9873, and the minimum detection limit is 10 / mL. This method can be used to detect the winter spores of S. tiliaceus within 2 hours.

[0060] (5) Verification of accuracy and stability The established method was verified by spiked experiments. The details are as follows: The blank sample matrix was used to prepare the spiked wheat spore solution, with the spike concentrations of 50, 500, and 5000 / mL, respectively, and 3 parallels were set up for each spike concentration. According to the immunomagnetic separation combined with single base sequence mass label mass spectrometry detection system established in step 4, the wheat spore solution with known spike concentrations was tested respectively, and the recovery rate and stability were calculated after the test. The specific spike recovery results are shown in Table 2.

[0061] Table 2 Results of spike verification of the detection method

[0062] From the results, we can see that the spiked recovery rate of the detection method is ≥78.25%, and RSD is ≤7.15%, so 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 Tilletia leucoderma 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. The monoclonal antibody against Tilletia leucoderma according to claim 3, 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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