A hybridoma cell line 2B1 and its monoclonal antibody and its application

Abstract

The present invention discloses a hybridoma cell line 2B1 and its monoclonal antibody and application. The present invention also discloses the application of the monoclonal antibody against light tartaria in the detection of tartaria winter spores. The present invention also discloses a monoclonal antibody against light tartaria coupled with nanogold. The present invention also discloses a detection kit. The monoclonal antibody and its conjugate and the kit exhibit highly specific recognition ability for light tartaria, can realize rapid, accurate and sensitive detection of fungal spores, and have good anti-interference performance, and have broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a hybridoma cell line 2B1 and a monoclonal antibody thereof and application thereof. Background Art

[0002] Light bunt is a fungal disease that seriously threatens wheat production and is mainly caused by Tilletia sutchuenensis. Therefore, the research on the detection technology of light bunt wheat is of great significance to ensure food security and people's health.

[0003] In recent years, with the development of molecular biology, bioinformatics and other technologies, new technical means have been provided for the detection of light bunt wheat. However, traditional methods such as morphological identification are time-consuming, PCR requires complex pre-treatment, and immunological methods such as ELISA may not be sensitive enough. Therefore, it is particularly urgent to develop a more efficient and specific detection method.

[0004] Since its introduction in the 1970s, monoclonal antibody preparation technology has been widely used in biology, medicine, agriculture and other fields. Monoclonal antibodies are highly specific and can bind to specific antigens, so they have significant advantages in pathogen detection. However, there are a series of technical bottlenecks and biological challenges in the construction of hybridoma cell lines and the preparation of monoclonal antibodies, which directly affect the development efficiency and application value of antibodies. The main difficulties include: (1) low cell fusion efficiency, involving the compatibility of different cell lines, or poor effect of fusion agents; (2) positive clone screening involves a huge workload, or the sensitivity of the screening method is insufficient; (3) hybridoma cells will lose the ability to secrete antibodies during long-term passage, which involves cell genetic instability; (4) monoclonal antibody specificity issues, such as cross-reaction; (5) various problems will arise during the production process, such as cell apoptosis and product degradation during large-scale culture, and challenges in the purification step, such as removing host cell proteins and endotoxins. 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 2B1.

[0006] 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.

[0007] The technical problem that the present invention needs to solve is to provide a monoclonal antibody against Tilletia smut.

[0008] 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.

[0009] The technical problem that the present invention needs to solve is to provide a nano-gold-coupled monoclonal antibody against Tilletia smut.

[0010] The technical problem that the present invention also aims to solve is to provide a detection method capable of detecting the winter spores of Tilletia sutchuenensis with high sensitivity and high specificity.

[0011] The final technical problem to be solved by the present invention is to provide a detection kit. The immunomagnetic separation combined with inductively coupled plasma mass spectrometry (ICP-MS) detection kit of the present invention forms a "three-in-one design" of antibody precise capture of target spores-metal tag signal amplification-ICP-MS ultra-sensitive quantification, which overcomes the problems of insufficient overall sensitivity, complex operation and inaccurate quantification of wheat light stinking smut detection.

[0012] Technical solution: In order to solve the above technical problems, the present invention provides a hybridoma cell line 2B1, which 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 2B1 Monoclonal antibody hybridoma cell lineagainst Tilletia foetida 2B1, the deposit number is CCTCC NO: C2024294, and the deposit address is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, Postal Code: 430072.

[0013] The present invention also includes the use of the hybridoma cell strain in preparing monoclonal antibodies against Tilletia smut.

[0014] The present invention also includes a monoclonal antibody against Tilletia sutchuenensis, which is secreted by the hybridoma cell line.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] The present invention also includes the use of the monoclonal antibody against S. tiliaceus in the detection of S. tiliaceus teliospores.

[0020] The present invention also includes a nano-gold-coupled monoclonal antibody against S. tina, which contains the monoclonal antibody against S. tina.

[0021] The present invention also includes a detection kit, which includes the monoclonal antibody against S. tina, or the monoclonal antibody against S. tina, coupled with nanogold.

[0022] The present invention also includes a method for detecting the teliospores of Tilletia smut, comprising the following steps:

[0023] 1) The polyclonal antibody of Tilletia tritici was mixed with the biotin solution to obtain the biotin-labeled polyclonal antibody;

[0024] 2) Incubate streptavidin magnetic beads with biotin-labeled polyclonal antibodies to obtain nanoimmunomagnetic beads;

[0025] 3) The nanogold solution was mixed with the monoclonal antibody 2B1 of wheat smut fungus to obtain the nanogold probe;

[0026] 4) adding solutions containing different concentrations of winter spores of wheat smut fungus to the nano-immunomagnetic beads to form a complex of immunomagnetic beads and winter spores;

[0027] 5) The nanogold probe is added to a solution containing an immunomagnetic bead-tio spore complex of different concentrations. After incubation, the unbound nanogold probe is removed, and then a citric acid solution is added to the immunomagnetic bead-tio spore-nanogold probe complex system to desorb the nanogold in the immunomagnetic bead-tio spore-nanogold complex system, and the magnetic beads are separated by magnetic adsorption to obtain nanogold solutions containing different concentrations. The gold element in the above solution is determined by ICP-MS to obtain a standard curve between the mass spectrometry signal of the gold element and the different concentrations of T. tio spores of T. septoria.

[0028] 6) The Tiliospore solution of the Tiliospore to be tested is mixed with the nano-immunomagnetic beads and the nano-gold probe, and after adding the citric acid solution for reaction, the gold element is determined by ICP-MS to obtain the gold element mass spectrometry signal, and the concentration of the Tiliospore solution to be tested is obtained by comparing with the standard curve.

[0029] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0030] 1. 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 to the winter spores of light stinking smut, and can be used as a monoclonal antibody for detecting light stinking smut of wheat.

[0031] 2. The kit and detection method for detecting winter spores of wheat smut fungus by immunomagnetic separation combined with ICP-MS provided by the present invention have innovative technology integration, breaking through the traditional detection mode, combining the biospecific capture of immunomagnetic separation with the trace metal detection capability of ICP-MS, realizing full-chain innovation from "biological recognition" to "physical signal conversion", and greatly improving the detection sensitivity; the signal method mechanism is innovative, breaking through the sensitivity limit, and achieving an ultra-high sensitivity detection limit through the signal amplification effect of nanogold bound to the winter spores. At the same time, ICP-MS detection adopts internal standard correction, which has the effect of eliminating matrix interference and improving the signal-to-noise ratio; the detection process is innovative to achieve rapid and accurate quantification: the target spores are quickly enriched and separated by immunomagnetic separation, without the need for spore culture or DNA extraction, and the detection cycle is shortened from several days to 1.5 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of detection of teliospores of Ustilago farnesii based on immunomagnetic separation-ICP-MS;

[0033] Figure 2 This is a transmission electron microscopy image of a monoclonal antibody labeled with nanogold and bound to the winter spores of Smut fungus;

[0034] Figure 3 It is a linear graph of the detection of Tiliospores of Tillandella glabra based on immunomagnetic separation-ICP-MS;

[0035] Figure 4 To highlight the effects of different immunomagnetic capture times on the detection signal;

[0036] Figure 5 The effect of the time for gold nanoparticle labeling of target spores on the detection signal emphasis. DETAILED DESCRIPTION

[0037] Example 1 Screening of Hybridoma Cell Lines of Tilletia glabra

[0038] 1. Immunization of mice with winter spores of Smut fungus

[0039] 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.

[0040] 2) ELISA verification: 100 μL of the winter spore suspension of S. tiliaceus was added to a 96-well plate in each well and incubated in a 4°C refrigerator overnight. The plate was washed three times with PBST (PBS buffer solution with a concentration of 0.01 mol / L and pH 7.6 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 blocked at 37°C for 1 h. The plate was washed three times with PBST to remove excess bovine serum albumin solution.

[0041] 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 buffer (concentration 0.01 mol / L, pH 7.6), 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) (Zhejiang Huzhou innoreagent Company, the luminol reagent formula is divided into two parts: one is the alkaline solution of luminol, and the other 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.

[0042] 2. Screening of hybridoma cell lines of Ustilago sutchuenensis and preparation of monoclonal antibodies

[0043] 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;

[0044] 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.

[0045] 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;

[0046] 4) After three rounds of subclone screening, 4 hybridoma cell lines that can stably secrete antibodies against S. leucoderma were screened out by ELISA test. 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 subculture results showed that the 4 hybridoma cells could secrete antibodies normally 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 enzyme-labeled wells with no spores were used as blank controls, and its response value ratio with the normal mouse serum negative control group was greater than 2.2. The cell line was officially deposited in the China Center for Type Culture Collection on October 22, 2024, and its classification name was: Monoclonal antibody hybridoma cell line 2B1 against S. leucoderma Tilletia foetida 2B1, the deposit number is: CCTCC NO: C2024294, the deposit address is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University; Postal Code: 430072. 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 T25 bottles for further culture. At the same time, the remaining cells were collected and frozen for subsequent research.

[0047] 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.

[0048] 6) Purification of ascites: After centrifugation and filtration, the ascites was diluted with an equal volume of PBS buffer and purified using a Smart-antibody Protein G antibody purification kit (purchased from Engibody, USA, catalog number: P4144). After purification, the antibodies were collected.

[0049] 7) Validation of monoclonal antibodies, spores of wheat dwarf stinking powder, Aspergillus flavus spores, Aspergillus niger spores, and Paecilomyces varroa spores (spores of wheat dwarf stinking 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, the chemiluminescent signal is higher than 0.79, while when the spores of other strains are used as the coating antigen, the chemiluminescent signal is significantly reduced, lower than 0.6340. It is shown that the monoclonal antibody of wheat light stinking powder prepared by the present invention has significant specific adsorption to the winter spore of wheat light stinking powder, and can be used to identify wheat light stinking powder.

[0050] Table 1 Monoclonal antibody validation results

[0051]

[0052] In the present invention, the enzyme-labeled wells without spores were used as blank controls, and the hybridoma cell line 2B1 of wheat light 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 on October 22, 2024, and its classification was named: Monoclonal antibody hybridoma cell line 2B1 against light smut fungus Tilletia foetida 2B1, the deposit number is: CCTCC NO: C2024294, deposited in China Center for Type Culture Collection; the depository address is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province; Postal Code: 430072.

[0053] Example 2 Sequencing of monoclonal antibodies against Tilletia serrata

[0054] 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.

[0055] The monoclonal antibody of 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 SAS, and CDRL3 as shown in SEQ ID NO.5;

[0056] SEQ ID NO.1: GFTFSDYY

[0057] SEQ ID NO.2: ISDGGTYT

[0058] SEQ ID NO.3: AKDRGFGPDY

[0059] SEQ ID NO.4: QDVSTA

[0060] SEQ ID NO.5: QQHYSTPYT

[0061] The heavy chain variable region and light chain variable region of the monoclonal antibody are 117 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.

[0062] Heavy chain variable region sequence

[0063] SEQ ID NO.6

[0064] EVKLEESGGGLVKPGGSLKLSCAASGFTFSDYYMYWVRQTPEKRLEWVATISDGGTYTFYPDSVKGRFTISRDNAKNNLYLQMSSLKSEDTAMYYCAKDRGFGPDYWGQGTTLTVSS

[0065] Light chain variable region sequence

[0066] SEQ ID NO.7

[0067] DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWHQQKPGQSPKLLIHSASYRYTGVPDRFTGSGSGTDFSFTISSVQAEDLAVYYCQQHYSTPYTFGGGTKLEIK

[0068] Heavy chain full length nucleotide sequence:

[0069] SEQ ID NO.8

[0070]

[0071] Full-length nucleotide sequence of the light chain:

[0072] SEQ ID NO.9

[0073] ATGGAGTCACAGTCTCAGGTCTTTGTATTCGTGTTTCTCTGGTTGTCTGGTGTTGACGGAGACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGAGTACTGCTGTAGCCTGGCATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTCACTCGGCATCCTACCGGTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCTCTTTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGCTGA

[0074] Amino acid sequence of the full-length heavy chain:

[0075] SEQ ID NO.10

[0076] MGWSLILLFLVAVATRVLSEVKLEESGGGLVKPGGSLKLSCAASGFTFSDYYMYWVRQTPEKRLEWVATISDGGTYTFYPDSVKGRFTISRDNAKNNLYLQMSSLKSEDTAMYYC AKDRGFGPDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVD KKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPI EKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0077] Light chain full length amino acid sequence

[0078] SEQ ID NO.11

[0079] MESQSQVFVFVFLWLSGVDGDIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWHQQKPGQSPKLLIHSASYRYTGVPDRFTGSGSGTDFSFTISSVQAEDLAVYYCQQHYSTPYT FGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0080] Example 3 Application of monoclonal antibody 2B1 - Detection of winter spores of Tilletia sphaeroides by immunomagnetic separation combined with inductively coupled plasma mass spectrometry

[0081] 1. Biotin-labeled polyclonal antibody

[0082] 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.

[0083] Biotin-labeled polyclonal antibody: Prepare a 2 mg / mL biotin solution with dimethyl sulfoxide (CAS: 67-68-5, Sigma, catalog number: D9170) (biotin 3-sulfo-n-hydroxysuccinimide ester sodium salt is used for preparation, CAS: 119616-38-5, Sigma, catalog number: B5161), mix 300 μL (1.1 mg / mL) of the polyclonal antibody against wheat smut fungus with 10 μL of the biotin solution (2 mg / mL), shake the reaction at room temperature for 30 min, then transfer to a 10K dialysis membrane for dialysis, and dialyze in PBS buffer (0.01 mol / L, pH 7.5) for 24 h, changing the buffer every 6 h. After dialysis, collect the biotin-labeled polyclonal antibody in a centrifuge tube and store it at -20°C for later use.

[0084] Immunomagnetic bead preparation

[0085] 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.

[0086] Preparation of gold nanoparticles and conjugation of gold nanoparticles with monoclonal antibodies

[0087] Slowly add 3mL of 2% (mg / L) gold chloride (HAuCl4) solution to 180mL of deionized water, and heat the mixture to boiling while stirring continuously, and maintain for 15min. Add 10mL of 1% (g / L) sodium citrate solution dropwise, continue heating for 15min, until the color of the solution changes to wine red, and stop heating. Continue stirring, let the solution cool naturally to room temperature, and the nanogold (AuNPs) solution is ready. Use 0.1mol / L potassium carbonate (K2CO3) solution to adjust the pH value of the nanogold solution to 8.0. Pipette 0.5mL of the nanogold solution and 0.5mL of 1mg / mL wheat light smut powder fungus monoclonal antibody 2B1, respectively, mix the two evenly, place in a 4℃ refrigerator, and let stand overnight to promote the binding of the antibody to the nanogold. The next day, add 5% BSA to the mixture, react at room temperature for 2h, and perform blocking treatment. The mixed solution was centrifuged at 10,000 rpm, 5 min, 4°C to remove unbound free monoclonal antibodies. The precipitate-labeled nanogold probe was resuspended in 0.01 mol / L PBS. At this point, the nanogold probe was prepared.

[0088] 4. Establishment of immunomagnetic separation combined with inductively coupled plasma mass spectrometry detection system and drawing of standard curve

[0089] 50 μg of nano-immunomagnetic beads were accurately weighed, washed twice with PBS (0.01 mol / L, pH 7.6, the same below), and then 100 μL of 1% skim milk powder solution (skim milk powder solution was dissolved with PBS) was added for blocking for 30 min. The blocking solution was removed by magnetic separation technology, and 100 μL of solution containing winter spores of wheat light smut fungus (PBS was used to prepare the solution at concentrations of 200, 1000, 2500, 5000, 10000, 7500, 10000, 25000, and 50000 / mL) was added to the nano-immunomagnetic beads, mixed well, and placed in a 37°C constant temperature shaker, incubated at 120 rpm for 45 min to form a complex of immunomagnetic beads-winter spores. After incubation, the supernatant was removed by magnetic separation technology and washed once with PBS. 10 μL of nanogold probe was mixed with 100 μL of 1% skim milk powder solution and blocked for 1 hour to reduce the nonspecific adsorption of nanogold by magnetic beads and reduce the background signal. The blocked nanogold probe was added to the above-mentioned solutions containing immunomagnetic beads-telopores of different concentrations and incubated on a constant temperature shaker at 37°C, 120 rpm, and reacted for 1 hour. After the incubation, the unbound nanogold probe was removed by magnetic separation and washed three times with PBST (0.01 mol / L PBS containing 0.1% Tween-20), 200 μL each time, and then washed once with 200 μL PBS. 100 μL of 1 mol / L citric acid solution was added to the above-mentioned solutions containing the immunomagnetic beads-tiolar spores-nanogold composite system of different concentrations, and the nanogold in the immunomagnetic beads-tiolar spores-nanogold composite system was desorbed. The desorption was carried out at room temperature for 10 min, and the magnetic beads were separated by magnetic adsorption to obtain solutions containing nanogold. The gold element was determined in the above-mentioned solutions using ICP-MS.

[0090] Since the content of nano-gold labeled on different target spores is different, after the nano-gold is desorbed by acid hydrolysis, the content of gold element is determined by ICP-MS, and different mass spectrometry signals of gold element can be measured. According to the test results, the concentration of winter spores of wheat spores is used as the horizontal axis and the detection signal value is used as the vertical axis to draw a standard curve. The linear range of the detection of winter spores of wheat spores based on immunomagnetic separation combined with inductively coupled plasma mass spectrometry is 200-50000 / mL, and the linear correlation R 2 =0.9909, linear graph see Figure 3 After verification, the minimum detection limit of this method is 200 / mL, and the entire detection time can be controlled within 1.5h.

[0091] At the same time, the accuracy and stability of the detection method were verified using the manual spike mode, as follows: a spiked wheat smut fungus spore solution was configured with a blank sample matrix, and the spiked concentrations were 200, 1000, 10000, and 50000 / mL, respectively, and 3 parallels were set up for each spiked concentration. According to the immunomagnetic separation combined with inductively coupled plasma mass spectrometry detection system established in Example 3, the wheat smut fungus spore solutions with known spiked concentrations were detected respectively, and the recovery rate and stability were calculated after detection. The specific spiked recovery results are shown in Table 2.

[0092] Table 2 Results of spike verification of the detection method

[0093]

[0094] From the results, we can see that the spiked recovery rate of the detection method is ≥72.32% and RSD≤11.26%, so this detection method has good accuracy and stability.

[0095] Example 4 Condition Optimization

[0096] 1. Optimization of immunomagnetic capture time

[0097] The winter spores of T. tritici were diluted to a concentration of 5000 / mL with PBS buffer, 50 μg of the immune nanomagnetic beads prepared in Example 3 (blocked with 100 μL 1% skim milk powder for 30 min before use) were taken, and reacted at 37°C and 120 rpm for 15, 30, 45, 60, 75, and 90 min, respectively, and then magnetically separated to collect the immune magnetic beads enriched with the target spores, and then washed with PBS for 3 times to obtain the spore-magnetic bead complex, which was characterized by electron microscopy. Figure 2 . 200 μL of 1% skimmed milk powder was used to block 10 μL of the nanogold probe prepared in Example 3 for 1 h, and then added to the spore-magnetic bead complexes with different incubation times, mixed and incubated at 37 °C, 120 rpm for 1 h. After labeling, excess nanogold was removed by magnetic separation, washed twice with 200 μL of PBST and once with PBS, and a desorption step was performed after magnetic separation. 100 μL of 1 mol / L citric acid solution was added to the complex, vortexed and mixed, and desorbed at room temperature for 10 min. After magnetic separation, 100 μL of the desorbed solution was taken for ICP-MS detection of the gold content. In this way, the influence of different capture times on the detection signal can be obtained. For specific analysis, see Figure 4 From the experimental results, it can be seen that as the capture time increases, the detection signal gradually decreases. 15 min is the best time to capture the target spores, and 15 min was selected for subsequent experiments.

[0098] 2. Optimization of gold nanoparticle labeling time

[0099] Take 0.5 mL of the nanogold solution prepared in Example 3 and adjust the pH to 8-9 with 0.1 mol / L K2CO3 solution, add 15 μL of the monoclonal antibody 2B1 (1 mg / mL) of wheat smut fungus, react at 4°C overnight, then add 100 μL of 5% BSA solution to block for 1 h, then centrifuge, centrifugation conditions: 12000 rpm, 10 min, remove unbound antibodies after centrifugation, and re-dissolve with 0.01 mol / L PBS. The capture process of the target spores is the same as above. Block 10 μL of nanogold probe with 200 μL 1% skimmed milk powder for 1 h, and then add the winter spore-magnetic bead complex of wheat smut fungus prepared according to the method of Example 2 (spore concentration is 1000 / mL) for labeling, respectively, labeling for 15, 30, 45, 60, 75, and 90 min, after labeling, wash off the excess nanogold with PBST, and the desorption and testing steps are the same as above. See the specific optimization experimental results for details. Figure 5 From the optimization experiment results, it can be seen that with the extension of labeling time, the detection signal first increases and then decreases, so the optimal labeling time is 45 min.

Claims

1. A hybridoma cell line 2B1, characterized in that: The hybridoma cell line 2B1 was deposited in the China Center for Type Culture Collection on October 22, 2024, and its classification was named Monoclonal antibody hybridoma cell line against Tilletia foetida 2B1, the deposit number is CCTCC NO: C2024294.

2. Use of the hybridoma cell line 2B1 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 2B1 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 nano-gold-coupled monoclonal antibody against Tilletia glabra, 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 monoclonal antibody against Ustilago sutchuenensis according to any one of claims 3 to 7 or the monoclonal antibody against Ustilago sutchuenensis coupled with nanogold according to claim 9.

Images