A hybridoma cell line 1B5, its monoclonal antibody and applications

Abstract

The present invention discloses a hybridoma cell line 1B5, 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 SERS nanolabel for labeling the monoclonal antibody against Tilletia foetida. The present invention also discloses a detection kit. The monoclonal antibody, its label and the kit exhibit a high specific recognition ability for Tilletia foetida. Detecting the nanolabel signal using a Raman spectrometer has the advantages of simple detection operation, which significantly improves the sensitivity and detection efficiency of the method. Quantitative analysis can also be performed according to the intensity of the Raman spectral signal.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and particularly relates to a hybridoma cell line 1B5, its monoclonal antibody and applications. Background Art

[0002] Tilletia foetida is a serious disease caused by the fungus (Tilletia foetida), mainly infecting wheat and other gramineous crops. This disease not only causes significant yield losses, but also seriously affects the grain quality. Infected grains release trimethylamine during processing, producing a strong fishy smell, making the flour inedible and severely reducing its economic value. The pathogen spores have extremely strong environmental tolerance and transmission ability, and can spread through various channels such as wind, rain, seeds and farm tools, increasing the difficulty of disease prevention and control. The outbreak of Tilletia foetida poses a serious threat to global wheat production. Therefore, developing a rapid and accurate detection method is crucial for disease prevention and control.

[0003] In recent years, thanks to the rapid development of fields such as molecular biology and bioinformatics, the detection technology for wheat infected with Tilletia foetida has been significantly innovated. Nevertheless, traditional morphological identification methods are time-consuming, while PCR technology relies on cumbersome pretreatment steps. At the same time, immunological detection methods such as ELISA may be insufficient in terms of sensitivity. Although existing methods meet the detection requirements to a certain extent, there are still problems such as insufficient sensitivity, complex operation and high cost. Therefore, developing a rapid, sensitive and economical detection method has become the focus of current research.

[0004] Since the advent of monoclonal antibody preparation technology in the 1970s, this technology has been widely applied in multiple disciplinary fields such as biology, medicine, and agriculture. Monoclonal antibodies, with their high specificity, can precisely recognize and bind to specific antigens, thus occupying a prominent advantageous position in pathogen detection. However, in the construction of hybridoma cell lines and the preparation process of monoclonal antibodies, technical bottlenecks and biological challenges emerge in an endless stream, and these problems directly affect the efficiency of antibody development and its application value. The main difficulties currently faced include: (1) Low efficiency of cell fusion, which involves the compatibility between different cell lines and the insufficient efficacy of fusogens; (2) High labor intensity in the positive clone screening process, and the sensitivity of screening methods needs to be improved; (3) Hybridoma cells may lose their antibody-secreting ability during long-term subculture, reflecting the problem of cell genetic stability; (4) Specificity issues of monoclonal antibodies, especially the trouble of cross-reactivity; (5) Various obstacles encountered in the production process, such as cell apoptosis and product degradation during large-scale culture, and challenges faced in the purification step, such as removing impurities like host cell proteins and endotoxins. The existence of these problems not only poses higher technical requirements for the research and development of monoclonal antibodies but also prompts scientific researchers to continuously explore and innovate to optimize and improve the existing preparation processes.

[0005] The surface-enhanced Raman scattering-based vertical flow immunoassay (SERS-VFIA) method, which is a novel immunoassay method based on SERS technology, has the advantages of high sensitivity, rapid detection, and multiplex analysis. In the application of the SERS-VFIA detection method, successfully labeling monoclonal antibodies to SERS nanolabels constitutes the core link of the technical process. However, this process has encountered a number of technical challenges and problems. Specifically, they include: (1) The binding efficiency problem between antibodies and SERS nanolabels: This binding process may be limited by the structural characteristics of antibodies, the surface properties of nanolabels, and the diverse influence of environmental factors. The combined action of these factors may lead to the instability of binding efficiency; (2) Maintenance of antibody activity: During the labeling stage, the biological activity of antibodies may be damaged due to chemical modification or physical effects, thereby affecting their binding efficacy to specific antigens; (3) Homogeneity and stability of nanolabels: The homogeneity of SERS nanolabels is the key to ensuring the reliability of detection signals. The differences between batches of nanolabels and their long-term stability are technical problems that must be overcome in the production process; (4) Controllability of the labeling process: Precisely regulating the binding ratio and orientation between antibodies and nanolabels is a process with extremely high technical requirements, which requires a high degree of precision in experimental design and excellent operation skills.

[0006] These challenges and difficulties require scientific researchers to conduct in-depth research and innovation when developing SERS-VFIA-based detection methods to achieve efficient, stable, and reliable detection technologies. Summary of the Invention

[0007] Object of the Invention: The technical problem to be solved by the present invention is to provide a hybridoma cell line 1B5.

[0008] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned hybridoma cell line in the preparation of a monoclonal antibody against Tilletia foetida.

[0009] Another technical problem to be solved by the present invention is to provide a monoclonal antibody against Tilletia foetida.

[0010] 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.

[0011] Another technical problem to be solved by the present invention is to provide a monoclonal antibody against Tilletia foetida labeled with SERS nanolabels.

[0012] Another technical problem to be solved by the present invention is to provide a detection method capable of highly sensitively, quickly, and conveniently detecting Tilletia foetida teliospores.

[0013] The last technical problem to be solved by the present invention is to provide a detection kit.

[0014] Technical Solution: To solve the above technical problems, the present invention provides a hybridoma cell line 1B5. The hybridoma cell line 1B5 was deposited with the China Center for Type Culture Collection on October 22, 2024. Its taxonomic name is Monoclonal antibody hybridoma cell line against Tilletia foetida 1B5, and the deposit number is CCTCC NO: C2024293. The address of the depositary institution is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, within Wuhan University, Postcode: 430072. Tilletia foetida The content of the present invention also includes the application of the above-mentioned hybridoma cell line 1B5 in the preparation of a monoclonal antibody against Tilletia foetida.

[0015] The content of the present invention also includes a monoclonal antibody against Tilletia foetida, which is secreted by the above-mentioned hybridoma cell line 1B5.

[0016]

[0017] ​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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] The content of the present invention also includes the application of the monoclonal antibody against Tilletia foetida in the detection of Tilletia foetida teliospores.

[0022] The content of the present invention also includes a SERS nanolabel-labeled monoclonal antibody against Tilletia foetida, which contains the monoclonal antibody against Tilletia foetida.

[0023] The content of the present invention also includes a detection kit, which includes the monoclonal antibody against Tilletia foetida or the SERS nanolabel-labeled monoclonal antibody against Tilletia foetida.

[0024] The content of the present invention also includes the application of the monoclonal antibody against Tilletia foetida in the detection of Tilletia foetida teliospores, providing strong technical support for the rapid detection of wheat bunt.

[0025] The content of the present invention also includes a method for detecting Tilletia foetida teliospores, comprising the following steps:

[0026] 1) Fix the polyclonal antibody against Tilletia foetida of wheat on the NC membrane;

[0027] 2) Incubate the SERS nanolabel solution with the monoclonal antibody against Tilletia foetida of wheat to obtain a biofunctionalized SERS nanolabel;

[0028] 3) Mix the bio-functionalized SERS nanolabels with sample solutions of Tilletia foetida teliospores at different concentrations to obtain mixtures at different concentrations. Drop the mixtures at different concentrations onto the NC membrane in step 1). After multiple washings, use a microscopic Raman instrument to detect the Raman signal. Use the logarithm of the Tilletia foetida teliospores concentration as the abscissa and the Raman response intensity as the ordinate to make a standard curve.

[0029] 4) Mix the test solution containing Tilletia foetida teliospores with the bio-functionalized SERS nanolabels and then drop the mixture onto the NC membrane in step 1). After multiple washings, use a microscopic Raman instrument to detect the Raman signal. Substitute the corresponding intensity of this Raman signal into the linear equation of the standard curve for calculation to obtain the concentration of the test Tilletia foetida teliospores.

[0030] Advantages: Compared with the prior art, the present invention has the following advantages: The monoclonal antibody prepared by the present invention has high specificity and can accurately recognize the specific antigen of Tilletia foetida. The signal molecule of the monoclonal antibody of the present invention combined with the SERS nanolabel has a unique Raman fingerprint spectrum, which can effectively avoid background interference and further improve the specificity of detection; Through the surface plasmon resonance effect of metal nanoparticles, the SERS technology can enhance the Raman signal by millions of times, thereby realizing the trace detection of target molecules. For the detection of low-concentration pathogens such as Tilletia foetida, the SERS nanolabel can significantly improve the detection sensitivity and reduce the missed detection rate; The present invention combines a vertical flow immunochromatography device to achieve rapid flow of the sample through capillary action. The detection time is within 10 - 15 minutes, which is suitable for on-site rapid screening. The sample can be directly loaded without complex extraction or purification steps, simplifying the operation process; Since SERS detection equipment is usually small in size and easy to carry, it is suitable for on-site detection in the field or outside the laboratory. The monoclonal antibody of the present invention combined with the vertical flow immunochromatography device is simple to operate and can be completed without professional training; The present invention combines monoclonal antibodies and can achieve quantitative analysis of Tilletia foetida spores or antigens by measuring the intensity of Raman signals, providing more accurate detection results. At the same time, due to the wide dynamic range of SERS technology, it can detect target molecules at low and high concentrations simultaneously. In summary, the monoclonal antibody, its detection kit and detection method of the present invention provide strong technical support for the detection of wheat bunt and have broad application prospects. Description of the Drawings

[0031] Figure 1 It is a schematic diagram of the preparation and detection of SERS-VFIA. A, The preparation process of the core-shell structure SERS nanolabel; B, The schematic diagram of SERS-VFIA for detecting Tilletia foetida teliospores.

[0032] Figure 2It is the standard curve for detecting Tilletia foetida teliospores by SERS-VFIA;

[0033] Figure 3 It is the Raman spectrum control chart after detecting each spore solution by SERS-VFIA. Specific implementation manners

[0034] Example 1 Screening of hybridoma cell lines of Tilletia foetida

[0035] 1. Immunize mice with Tilletia foetida teliospores as antigens

[0036] 1) Weigh 0.1 mg of Tilletia foetida teliospores (Tilletia foetida teliospores are provided by Jiangsu Grain and Oil Quality Monitoring Center), mix it with 675 μL of normal saline, and then mix it 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.

[0037] 2) ELISA verification: Add 100 μL of the Tilletia foetida teliospore suspension to each well of a 96-well plate, incubate it overnight in a 4°C refrigerator, take it out, wash the plate 3 times with PBST solution (PBS buffer solution with a concentration of 0.01 mol / L and pH 7.6 containing 0.05% Tween-20, the same below), then add 200 μL of 5% bovine serum albumin solution (using PBS buffer solution (0.01 mol / L, pH 7.6, the same below) as the solvent) to each well, and block it at 37°C for 1 h. Take it out and wash it 3 times with PBST solution to remove the excess bovine serum albumin solution.

[0038] 3) Add 100 μL of the serially diluted antiserum to the well plate. After mixing, incubate at 37 °C for 1 h. Then add the HRP-goat anti-mouse secondary antibody (Shanghai Sangon Biotech Co., Ltd., product number C65021-0100) diluted 5000-fold with PBS (concentration 0.01 mol / L, pH 7.6), and incubate at 37 °C for 0.5 h. Then wash the plate twice with PBST solution, add 100 μL of the chemiluminescent substrate solution (luminol, A:B = 1:1, Innoreagent Co., Ltd., 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 while luminol is more stable under acidic conditions). After mixing, 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 using the same method.

[0039] 2. Screening of Tilletia foetida hybridoma cell lines and preparation of monoclonal antibodies

[0040] 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 a teliospores suspension on the 7th day after blood collection.

[0041] 2) Cell fusion. On the 5th day after the fifth immunization, take the spleen of the mouse, grind it and perform cell fusion with SP2 / 0, and plate it.

[0042] 3) Subcloning screening. On the 14th - 16th day after cell fusion, observe the cells under a microscope and take the supernatant for ELISA detection; for the cells in the wells with positive specific antibody detected, transfer and inoculate them in time for the first round of subcloning screening; after 6 - 7 days, observe the cells under a microscope and take the supernatant for ELISA detection; for the cells in the wells with positive specific antibody detected, transfer and inoculate them in time for the second round of subcloning screening; after 5 - 6 days, observe the cells under a microscope and take the supernatant for ELISA detection; for the cells in the wells with positive specific antibody detected, transfer and inoculate them in time for the third round of subcloning screening.

[0043] 4) After three rounds of subcloning and screening, 4 hybridoma cell lines that could stably secrete antibodies against Tilletia foetida were screened out by ELISA. 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 passage. The results of passage culture showed that these 4 hybridoma cells could normally secrete antibodies during 6 consecutive generations of culture, demonstrating good antibody secretion stability. One of the positive cell lines showed a significant response intensity in the detection. Taking 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. C2024293). 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 out and inoculated into a T25 flask for continuous culture, while the remaining cells were collected and frozen for subsequent research use.

[0044] 5) Ascites preparation: Mineral oil was intraperitoneally injected into mice 1 week in advance. The cells in the T25 flask were cultured until 90% confluent, then the cells were collected and injected into the peritoneal cavity of mice. The peritoneal cavity of mice was observed within 8 - 20 days. When it bulged to the point where movement was hindered, the ascites was collected.

[0045] 6) Ascites purification: After centrifugation and filtration of the ascites, it was diluted with an equal volume of PBS, and the antibody was purified using a Smart-antibody Protein G antibody purification kit (purchased from Engibody Company, USA, Catalog No.: P4144). The antibody was collected after purification.

[0046] 7) Verification of monoclonal antibody: Ustilago tritici spores, Aspergillus flavus spores, Aspergillus niger spores, and Paecilomyces variotii spores (Ustilago tritici spores were purchased from the Royal Netherlands Culture Collection of Microorganisms, and the other spores are well-known and commonly used conventional spores provided by the Microbial Detection Center of Nanjing Institute of Product Quality Supervision and Inspection) were selected as materials for cross-verification. The antibody was verified by coating the plate 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 was Tilletia foetida teliospores, the chemiluminescence signal was all higher than 0.8041, while when other strain spores were used as the coating antigen, the chemiluminescence signal decreased significantly, lower than 0.5230. 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.

[0047] Table 1 Verification results of monoclonal antibody

[0048]

[0049] In the present invention, an enzyme-linked immunosorbent assay (ELISA) well without spore-containing plates was used as a blank control, and a hybridoma cell line 1B5 of Tilletia foetida was screened out with a response value ratio greater than 2.2 compared to the normal mouse serum negative control group. This cell line was officially deposited at the China Center for Type Culture Collection on October 22, 2024, and its taxonomic name is: Monoclonal antibody hybridoma cell line 1B5 against Tilletia foetida Tilletia foetida 1B5, deposit number: CCTCC NO:C2024293, deposited at the China Center for Type Culture Collection; the address of the deposit unit is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, within Wuhan University; postal code: 430072.

[0050] Example 2 Sequencing of the monoclonal antibody against Tilletia foetida

[0051] In Example 1, a monoclonal antibody against Tilletia foetida was successfully prepared. To further analyze its molecular structure, Beijing Sino Biological Inc. was commissioned to perform sequencing analysis on this monoclonal antibody, thereby obtaining the detailed amino acid sequence and nucleotide sequence of this monoclonal antibody.

[0052] 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 shown in SEQ ID NO.1, CDRH2 shown in SEQ ID NO.2, and CDRH3 shown in SEQ ID NO.3, and the light chain variable region includes CDRL1 shown in SEQ ID NO.4, CDRL2 with the amino acid sequence SAS, and CDRL3 shown in SEQ ID NO.5;

[0053] SEQ ID NO.1 GYSITTIYY

[0054] SEQ ID NO.2 ITYDGGN

[0055] SEQ ID NO.3 ARVDGYYVFAT

[0056] SEQ ID NO.4 QNVGTN

[0057] SEQ ID NO.5 QQYNTYPLT

[0058] The lengths of the heavy chain variable region and the light chain variable region of this monoclonal antibody are 118 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;

[0059] Heavy chain variable region sequence

[0060] SEQ ID NO.6

[0061] EVMLVESGPGLARPSQSLSLTCSVTGYSITTIYYWTWVRQFPGNKLEWMGYITYDGGNYYNPSLNNRISITRDTSQNQFFLKLNSVTTEDTATYYCARVDGYYVFATWGQGTLVTVSA

[0062] Light chain variable region sequence

[0063] SEQ ID NO.7

[0064] DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNIVWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYNTYPLTFGPGTKLELK

[0065] Full-length nucleotide sequence of heavy chain:

[0066] SEQ ID NO.8

[0067]

[0068] Full-length nucleotide sequence of the light chain:

[0069] SEQ ID NO.9

[0070] ATGGAGTCACAGTCTCAGGTCTTTGTATACATGTTGCTGTGGTTGTCTGGTGTTGATGGAGACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGGGTACTAATATAGTCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTACTCGGCATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGACTATTTCTGTCAGCAATATAACACCTATCCGCTCACGTTCGGTCCTGGGACCAAGCTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAATGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGCTGA

[0071] Amino acid sequence of the full-length heavy chain:

[0072] SEQ ID NO.10

[0073] MGWSLILLFLVAVATRVLSEVMLVESGPGLARPSQSLSLTCSVTGYSITTIYYWTWVRQFPGNKLEWMGYITYDGGNYYNPSLNNRISITRDTSQNQFFLKLNSVTTEDTATYYCARVDGYYVFATWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0074] Full-length amino acid sequence of the light chain

[0075] SEQ ID NO.11

[0076] MESQSQVFVYMLLWLSGVDGDIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNIVWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYNTYPLTFGPGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYE-HNSYTCEATHKTSTSPIVKSFNRNEC

[0077] Example 3 Application of monoclonal antibody 1B5 - Detection of Tilletia foetida teliospores by vertical flow immunoassay based on surface-enhanced Raman nanoprobes

[0078] 1. Immobilize polyclonal antibody

[0079] 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 in wheat.

[0080] Immobilization of polyclonal antibody: The nitrocellulose membrane (NC) was ultrasonically treated in a 95% ethanol solution for 30 s, then laid flat on a glass slide and washed multiple times with PBS buffer (0.01 mol / L, pH 7.6). The polyclonal antibody against Tilletia foetida in wheat (1.0 μg / mL) was immobilized on the NC membrane to form a sensing membrane for the detection area. The NC membrane was placed on a shaker and incubated statically at 37 °C for 1 h. Then, the non-specific adsorption sites on the NC membrane were blocked with a 2% BSA solution, and then washed 3 times with PBS buffer. The prepared NC membrane was stored at 4 °C for later use.

[0081] Preparation of gold nanoparticles by citrate thermal reduction method

[0082] In a magnetically stirred oil bath, 1 mL of 2 mg / L HAuCl4 solution was added to 199 mL of ultrapure water. While continuously stirring, the solution was heated to 120 °C and boiled for 3 - 5 minutes. Subsequently, 1.5 mL of 1 g / L sodium citrate aqueous solution was quickly added to the boiling solution under vigorous stirring until the color of the solution turned red - orange. After boiling for about 10 minutes, the solution was cooled to room temperature and then stored at 4 °C to obtain the gold nanoparticle solution.

[0083] Preparation of SERS nanotags

[0084] First, the gold nanoparticles prepared by the above citrate thermal reduction method were used as the seeds for preparing SERS nanotags. Then, Raman dye NBA (1.0 μM, 500 μL) was added dropwise to 5 mL of the gold nanoparticle solution and continuously stirred for 1 h. Then, the gold particles conjugated with Raman tags were centrifuged at 8000 rpm for 15 min to remove the unbound Raman tags. The precipitate obtained by centrifugation was resuspended in 5 mL of ultrapure water to obtain an Au@ NBA colloidal solution. Then, 500 μL of 0.1 M ascorbic acid was added dropwise under magnetic stirring to the Au@ NBAIn the colloidal solution. Immediately, 500 μL of 1 mM AgNO3 solution was added dropwise with a micropipette under stirring and stirred continuously for 40 min. Since ascorbic acid reduced AgNO3, the color of the colloidal solution gradually changed from purple-pink to orange. The appearance of this phenomenon indicates that the silver shell layer gradually grew on the periphery of the colloidal gold. Then the obtained solution was centrifuged at 10000 rpm for 10 min to remove the supernatant, and then the obtained Au NBA @Ag precipitate was resuspended in an equal volume of ultrapure water to obtain the SERS nanolabel solution for standby.

[0085] 4. Preparation of Biologically Functionalized SERS Nanolabels

[0086] First, the pH of the SERS nanolabel solution was adjusted to 8.5 using 0.1 M K2CO3 and HCl. Pipette 2 mL of the SERS nanolabel solution (Au NBA @Ag solution, 1 μg / mL) and 5 μL of the monoclonal antibody 1B5 of Tilletia foetida (10 μg / mL), mix well and incubate at 37 °C for 1 h; then add 2% BSA for overnight blocking. After the blocking, the biologically functionalized nanolabel solution was centrifuged to remove the unbound antibody and reduce non-specific adsorption. Then, the biologically functionalized core-shell structured nanolabels were resuspended in an equal volume of PBS solution and stored in a refrigerator at 4 °C for standby.

[0087] 5. Establishment of SERS VFA Detection System and Plotting of Standard Curve

[0088] First, 30 μL of the sample solution containing the teliospores of Tilletia foetida (solutions with concentrations of 5, 10, 50, 100, 500, 1000, 5000, 10000, 20000, 50000 cells / mL prepared with PBS) were mixed with the prepared biologically functionalized SERS nanolabels at room temperature for 2 min. Then, 10 μL of the mixture was slowly dropped onto the detection area of the antibody-modified NC membrane and kept for 3 min. Then, the detection area was washed three times with PBST solution, 20 μL of PBST solution was used for each washing, and the time interval was 40 s. Finally, the detection area on the membrane was detected using a micro-Raman spectrometer. The Raman shift of the NBA SERS nanolabel is 593 cm -1 . Using the logarithm of the concentration of Tilletia foetida teliospores as the abscissa and the Raman response intensity as the ordinate, a standard curve was plotted, as shown in Figure 2 . Thus, the linear range for detecting Tilletia foetida teliospores by SERS VFA is 10 - 10000 cells / mL, the linear correlation R 2 = 0.9671, and the lowest detection limit is 10 cells / mL.

[0089] Example 4 Verification of the Specificity of SERS VFA for Detecting Tilletia foetida Teliospores

[0090] Use PBS buffer solution (0.01 mol / L, pH 7.6) to prepare solutions of Tilletia foetida teliospores, Tilletia controversa teliospores, Aspergillus flavus spores, Aspergillus niger spores, and Paecilomyces variotii spores respectively (Tilletia controversa spores were purchased from the Dutch Royal Culture Collection, and the other spores are well-known and commonly used conventional spores, provided by the Microbial Detection Center of Nanjing Institute of Product Quality Supervision and Inspection). The concentration of each spore solution is 1000 / mL. Then, according to the SERS VFA detection steps in Example 3, each solution was detected. The Raman spectra of each spore solution are shown in Figure 3 . According to the detection results, it can be seen that when detecting Tilletia foetida teliospores, the Raman spectral signal at 593 cm -1 is strong, and there is almost no Raman spectral signal at 593 cm -1 for other spores. Therefore, the specificity of this detection method is verified.

[0091] Example 5 Verification of the Accuracy and Stability of the SERS VFA Detection Method

[0092] The detection method was verified by a spike addition experiment. The specific steps are as follows: Use a blank sample matrix to prepare spike addition solutions of Tilletia foetida teliospores with spike addition concentrations of 10, 100, 1000, and 10000 / mL respectively, and set 3 parallels for each spike addition concentration. According to the SERS VFA detection system established in Example 3, the sample solutions with known spike addition concentrations were detected respectively, and the recovery rate and stability were calculated after detection. The specific spike addition recovery results are shown in Table 2.

[0093] Table 2 Spike Addition Verification Results of the Detection Method

[0094]

[0095] From the results, it can be seen that the spike addition recovery rate of the detection method ≥ 70.26%, and RSD ≤ 12.56%. Therefore, this detection method has good accuracy and stability.

Claims

1. A hybridoma cell line 1B5, characterized in that: The hybridoma cell line 1B5 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 1B5, the deposit number is CCTCC NO:C2024293.

2. Use of the hybridoma cell line 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 1B5 according to claim 1.

4. An anti-Tilletia smut monoclonal antibody, 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. Use of the monoclonal antibody against T. tiliaceus according to any one of claims 3 to 5 in the detection of T. tiliaceus teliospores.

7. A SERS nano-tag labeled monoclonal antibody against Tilletia smut, characterized in that: The invention comprises the monoclonal antibody against Tilletia smut described in any one of claims 3 to 5.

8. A detection kit, characterized in that: The method comprises the monoclonal antibody against light smut fungus according to any one of claims 3 to 5 or the monoclonal antibody for light smut fungus labeled with a SERS nano-tag according to claim 7.

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