Hybridoma cell strain 1B5 as well as monoclonal antibody and application thereof

Abstract

The invention discloses a hybridoma cell strain 1B5 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 monoclonal antibody of the Tilletia foetida labeled by the SERS nano-label. 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 Raman spectrometer is adopted to detect the nano-label signal, so that the detection operation is simple and convenient, the sensitivity and the detection efficiency of the method are remarkably improved, and quantitative analysis can be performed according to the strength of the Raman spectrum signal.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a hybridoma cell line 1B5 and a monoclonal antibody thereof and application thereof. Background Art

[0002] Wheat light bunt is a serious disease caused by a fungus (Ustilago farfara), which mainly infects wheat crops. The disease not only causes significant yield losses, but also seriously affects the quality of grains. Infected grains release trimethylamine during processing, producing a strong fishy smell, rendering the flour inedible and seriously reducing its economic value. The pathogen spores have extremely strong environmental tolerance and transmission capabilities, and can spread through wind, rain, seeds, and agricultural tools, increasing the difficulty of disease prevention and control. The outbreak of light bunt poses a serious threat to global wheat production, so the development of rapid and accurate detection methods 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 of wheat with light stinky smut has been significantly innovated. Despite this, traditional morphological identification methods are time-consuming, and PCR technology relies on cumbersome pre-treatment steps. At the same time, immunological detection methods such as ELISA may still be insufficient in terms of sensitivity. Although the existing methods have met the detection needs to a certain extent, there are still problems such as insufficient sensitivity, complex operation, and high cost. Therefore, the development of 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 used in many disciplines such as biology, medicine, and agriculture. Monoclonal antibodies have a significant advantage in pathogen detection due to their high specificity and ability to accurately identify and bind to specific antigens. However, in the construction of hybridoma cell lines and the preparation of monoclonal antibodies, technical bottlenecks and biological challenges emerge one after another, which directly affect the efficiency of antibody development and its application value. The main difficulties currently faced include: (1) low cell fusion efficiency, which involves the compatibility between different cell lines and the insufficient efficacy of fusion agents; (2) the labor intensity of the positive clone screening process is high, and the sensitivity of the screening method needs to be improved; (3) hybridoma cells may lose their antibody secretion ability during long-term passage, reflecting the problem of cell genetic stability; (4) the specificity of monoclonal antibodies, especially the problem of cross-reaction; (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 such as host cell proteins and endotoxins. The existence of these problems not only puts forward higher technical requirements for the research and development of monoclonal antibodies, but also prompts scientific researchers to continuously explore and innovate in order to optimize and improve the existing preparation process.

[0005] Surface-Enhanced Raman Scattering-based Vertical Flow Immunoassay (SERS-VFIA) is a new immunoassay method based on SERS technology, which has the advantages of high sensitivity, rapid detection and multiplex analysis. In the application of SERS-VFIA detection method, the successful labeling of monoclonal antibodies to SERS nanotags constitutes the core link of the technical process. However, this process encounters many technical challenges and difficulties. Specifically, they include: (1) The binding efficiency of antibodies and SERS nanotags: The binding process may be limited by the structural characteristics of antibodies, the surface properties of nanotags, and the diverse influence of environmental factors, which together may lead to instability of binding efficiency; (2) Maintenance of antibody activity: During the labeling stage, the biological activity of antibodies may be damaged by chemical modification or physical effects, thereby affecting their binding efficiency with specific antigens; (3) Uniformity and stability of nanotags: The uniformity of SERS nanotags is the key to ensuring the reliability of detection signals. The batch-to-batch differences in nanotags and their long-term stability are technical challenges that must be overcome in the production process; (4) Controllability of the labeling process: Precisely regulating the binding ratio and positioning of antibodies and nanotags is a technically demanding process that requires highly precise experimental design and sophisticated operating skills.

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

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

[0008] 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.

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

[0010] 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.

[0011] The technical problem that the present invention needs to solve is to provide a monoclonal antibody for marking Tilletia smut with a SERS nano-tag.

[0012] The technical problem that the present invention also aims to solve is to provide a detection method that can detect the winter spores of Tine Ustilago officinalis with high sensitivity, rapidity and convenience.

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

[0014] Technical solution: In order to solve the above technical problems, the present invention provides a hybridoma cell line 1B5, 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 1B5 Monoclonal antibody hybridoma cell lineagainst Tilletia foetida 1B5, the deposit number is CCTCC NO: C2024293, and the deposit address is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, Postal Code: 430072.

[0015] The present invention also includes the use of the hybridoma cell line 1B5 in the preparation of monoclonal antibodies against Tilletia sutchuenensis.

[0016] The present invention also includes a monoclonal antibody against T. tina, which is secreted by the hybridoma cell line 1B5.

[0017] 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.

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

[0022] The present invention also includes a SERS nano-tag labeled monoclonal antibody against S. tina, which contains the monoclonal antibody against S. tina.

[0023] The present invention also includes a detection kit, which includes the anti-U. sutchuenensis monoclonal antibody or the SERS nano-tag labeled U. sutchuenensis monoclonal antibody.

[0024] The present invention also includes the application of monoclonal antibodies against light stinking smut in the detection of teliospores of light stinking smut, which provides strong technical support for the rapid detection of wheat light stinking smut.

[0025] The present invention also includes a method for detecting the teliospores of Smut fungus, comprising the following steps: 1) Immobilize the polyclonal antibody of Tilletia tritici on the NC membrane; 2) The SERS nanotag solution was incubated with the monoclonal antibody of wheat smut fungus to obtain the biofunctionalized SERS nanotag; 3) The biofunctionalized SERS nanotags were mixed with sample solutions of wheat spores of different concentrations to obtain mixed solutions of different concentrations, and the mixed solutions of different concentrations were dripped onto the NC membrane of step 1). After multiple washings, the Raman signal was detected by a micro-Raman instrument, and a standard curve was made with the logarithm of the wheat spores concentration as the abscissa and the Raman response intensity as the ordinate; 4) The test solution containing the winter spores of wheat stinking smut fungus is mixed with the biofunctionalized SERS nanotag and then dripped onto the NC membrane in step 1). After multiple washings, the Raman signal is detected by a micro-Raman instrument. The corresponding intensity of the Raman signal is substituted into the linear equation of the standard curve for calculation to obtain the concentration of the winter spores of wheat stinking smut fungus to be tested.

[0026] Beneficial effects: 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 identify the specific antigen of the light smut fungus. The monoclonal antibody of the present invention combined with the signal molecule of the SERS nanotag has a unique Raman fingerprint spectrum, which can effectively avoid background interference and further improve the specificity of detection; the SERS technology can enhance the Raman signal by millions of times through the plasma resonance effect of metal nanoparticles, thereby realizing trace detection of target molecules. For the detection of low-concentration pathogens such as light stinking smut, SERS nanotags can significantly improve the detection sensitivity and reduce the missed detection rate; the present invention combines a vertical flow immunochromatographic device to achieve rapid flow of samples through capillary action, and the detection time is within 10-15min, which is suitable for rapid on-site screening. The sample can be directly added without complicated extraction or purification steps, simplifying the operation process; since the SERS detection equipment is usually small in size and easy to carry, it is suitable for on-site detection outside the field or laboratory. The monoclonal antibody of the present invention is combined with a vertical flow immunochromatographic device for simple operation, and the detection can be completed without professional training; the present invention combines monoclonal antibodies and measures the intensity of Raman signals to achieve quantitative analysis of light stinking smut spores or antigens, providing more accurate detection results. At the same time, due to the wide dynamic range of SERS technology, low and high concentrations of target molecules can be detected simultaneously. In summary, the monoclonal antibody and its detection kit and detection method of the present invention provide strong technical support for the detection of wheat light stinking smut, and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of SERS-VFIA preparation and detection. A, Preparation process of SERS nanotag based on core-shell structure; B, Schematic diagram of SERS-VFIA detection of winter spores of wheat smut fungus; Figure 2 This is the standard curve for detecting the teliospores of wheat smut fungus by SERS-VFIA; Figure 3 This is a comparison chart of the Raman spectra of each spore solution after SERS-VFIA detection. DETAILED DESCRIPTION

[0028] 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.

[0029] 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 with a concentration of 0.01 mol / L and pH 7.6 containing 0.05% Tween-20, the same below), and then 200 μL of 5% bovine serum albumin solution (PBS buffer (0.01 mol / L, pH 7.6, the same below) was added to each well, and the plate was blocked at 37°C for 1 h. The plate was washed three times with PBST solution to remove excess bovine serum albumin solution.

[0030] 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 (concentration 0.01 mol / L, pH 7.6), 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.

[0031] 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.

[0032] 3) Subclone screening: On the 14th to 16th day after cell fusion, observe the cells under a microscope and take the supernatant for ELISA detection; for cells detected with specific antibody positive wells, timely transfer and conduct the first round of subclone screening; 6-7 days later, observe the cells under a microscope and take the supernatant for ELISA detection; for cells detected with specific antibody positive wells, timely transfer and conduct the second round of subclone screening; 5-6 days later, observe the cells under a microscope and take the supernatant for ELISA detection; for cells detected with specific antibody positive wells, timely transfer and conduct the third round of subclone screening; 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. C2024293). 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.

[0033] 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.

[0034] 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.

[0035] 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 0.8041, while when the spores of other strains are used as the coating antigen, the chemiluminescent signal is significantly reduced, which is lower than 0.5230. 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.

[0036] Table 1 Monoclonal antibody validation results

[0037] In the present invention, the enzyme-labeled wells without spores were used as blank controls, and the hybridoma cell line 1B5 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 against light smut fungus 1B5 Tilletia foetida 1B5, the deposit number is: CCTCC NO: C2024293, 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.

[0038] 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.

[0039] The monoclonal antibody against wheat smut comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDRH1 as shown in SEQ ID NO.1, CDRH2 as shown in SEQ ID NO.2, and CDRH3 as shown in SEQ ID NO.3, and the light chain variable region comprises CDRL1 as shown in SEQ ID NO.4, CDRL2 with an amino acid sequence of SAS, and CDRL3 as shown in SEQ ID NO.5; SEQ ID NO.1 GYSITTIYY SEQ ID NO.2 ITYDGGN SEQ ID NO.3 ARVDGYYVFAT SEQ ID NO.4 QNVGTN SEQ ID NO.5 QQYNTYPLT The heavy chain variable region and light chain variable region of the monoclonal antibody are 118 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 EVMLVESGPGLARPSQSLSLTCSVTGYSITTIYYWTWVRQFPGNKLEWMGYITYDGGNYYNPSLNNRISITRDTSQNQFFLKLNSVTTEDTATYYCARVDGYYVFATWGQGTLVTVSA Light chain variable region sequence SEQ ID NO.7 DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNIVWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYNTYPLTFGPGTKLELK Heavy chain full length nucleotide sequence: SEQ ID NO.8 Full-length nucleotide sequence of the light chain: SEQ ID NO.9 ATGGAGTCACAGTCTCAGGTCTTTGTATACATGTTGCTGTGGTTGTCTGGTGTTGATGGAGACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGGGTACTAATATAGTCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTACTCGGCATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGACTATTTCTGTCAGCAATATAACACCTATCCGCTCACGTTCGGTCCTGGGACCAAGCTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAATGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGCTGA Amino acid sequence of the full-length heavy chain: SEQ ID NO.10 MGWSLILLFLVAVATRVLSEVMLVESGPGLARPSQSLSLTCSVTGYSITTIYYWTWVRQFPGNKLEWMGYITYDGGNYYNPSLNNRISITRDTSQNQFFLKLNSVTTEDTATYYC ARVDGYYVFATWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKV DKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAP IEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK Light chain full length amino acid sequence SEQ ID NO.11 MESQSQVFVYMLLWLSGVDGDIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNIVWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYNTYPLT FGPGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYE-HNSYTCEATHKTSTSPIVKSFNRNEC Example 3 Application of monoclonal antibody 1B5 - Detection of winter spores of Tilbury fungus based on vertical flow immunochromatography with surface enhanced Raman nanotags 1. Fixed polyclonal antibody Preparation of polyclonal antibodies against wheat smut fungus: (1) Emulsification: Take 10 5The winter spore solution of Ustilago fargesii 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 3500rpm, and then the serum was affinity purified to obtain polyclonal antibodies to Ustilago fargesii.

[0040] Fixation of polyclonal antibodies: The nitrocellulose membrane (NC) was placed in a 95% ethanol solution for ultrasonic treatment for 30 seconds, then spread on a glass slide and washed several times with PBS buffer (0.01 mol / L, pH 7.6). The polyclonal antibody against wheat smut fungus (1.0 μg / mL) was fixed on the NC membrane to form a sensing membrane with a detection area. The NC membrane was placed in a shaker and incubated at 37°C for 1 hour. The nonspecific adsorption sites on the NC membrane were then blocked with a 2% BSA solution, and then washed three times with PBS buffer. The prepared NC membrane was stored at 4°C for later use.

[0041] Preparation of Gold Nanoparticles by Citrate Thermal Reduction 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 changed to red-orange. After boiling for about 10 minutes, the solution was cooled to room temperature and then stored at 4°C to obtain a nanogold solution.

[0042] Preparation of SERS nanotags First, the gold nanoparticles prepared by the citrate thermal reduction method were used as seeds for preparing SERS nanotags. Then, the Raman dye NBA (1.0 μM, 500 μL) was added dropwise to 5 mL of the nanogold solution and stirred for 1 hour. The gold particles bound to the Raman tag were then centrifuged at 8000 rpm for 15 minutes to remove the unbound Raman tags. The precipitate obtained by centrifugation was resuspended in 5 mL of ultrapure water to obtain Au@ NBA Then, 500 μL of 0.1 M ascorbic acid was added dropwise to the Au@ NBAcolloidal solution. Then, 500 μL of 1 mM AgNO3 solution was added dropwise with a micropipette under stirring, and stirring was continued for 40 min. As ascorbic acid reduced AgNO3, the color of the colloidal solution gradually changed from purple pink to orange. This phenomenon indicated that the silver shell gradually grew on the periphery of the colloidal gold. The resulting solution was then centrifuged at a speed of 10,000 rpm for 10 min to remove the supernatant, and then the resulting Au NBA @The Ag precipitate was resuspended in an equal volume of ultrapure water to obtain a SERS nanotag solution for later use.

[0043] 4. Preparation of biofunctionalized SERS nanotags First, adjust the pH of the SERS nanotag solution to 8.5 using 0.1M K2CO3 and HCl. NBA 2 mL of @Ag solution (1 μg / mL) and 5 μL of monoclonal antibody 1B5 (10 μg / mL) were mixed thoroughly and incubated at 37 °C for 1 h; then 2% BSA was added for overnight blocking. After blocking, the biofunctionalized nanotag solution was centrifuged to remove unbound antibodies and reduce nonspecific adsorption. Afterwards, the biofunctionalized core-shell nanotag was resuspended in an equal volume of PBS solution and stored in a 4 °C refrigerator for later use.

[0044] 5. Establishment of SERS VFA detection system and drawing of standard curve First, 30 μL of sample solution containing winter spores of wheat spores (PBS was used to prepare solutions with concentrations of 5, 10, 50, 100, 500, 1000, 5000, 10000, 20000, and 50000 / mL) was mixed with the prepared biofunctionalized SERS nanotags for reaction at room temperature for 2 minutes. Then, 10 μL of the mixture was slowly dropped on the detection area of ​​the antibody-modified NC membrane and kept for 3 minutes. Then, the detection area was washed three times with PBST solution, using 20 μL PBST solution each time, with a time interval of 40 seconds. Finally, a micro-Raman instrument was used to detect the detection area on the membrane. The Raman shift of the NBA SERS nanotag is 593 cm -1 The standard curve was drawn with the logarithmic value of the concentration of wheat spores of Tilletia sutsuga as the abscissa and the Raman response intensity as the ordinate. Figure 2 It can be concluded that the linear range of SERS VFA detection of wheat spores is 10-10000 / mL, and the linear correlation R 2 =0.9671, and the minimum detection limit is 10 / mL.

[0045] Example 4 Verification of the specificity of SERS VFA in detecting winter spores of Tilletia sutchuenensis PBS buffer solution (0.01 mol / L, pH 7.6) was used to prepare winter spores of T. tritici, winter spores of T. dwarfi, Aspergillus flavus spores, Aspergillus niger spores and Paecilomyces variotii spore solutions (T. tritici spores were purchased from the Royal Culture Collection of the Netherlands, and other spores were well-known and commonly used conventional spores provided by the Microbiological Testing Center of Nanjing Product Quality Supervision and Inspection Institute). The concentration of each spore solution was 1000 spores / mL, and then each solution was tested according to the SERS VFA detection steps in Example 3. The Raman spectra of each spore solution are shown in Figure 2. Figure 3 According to the test results, the detection of winter spores of Smut fungus is at 593 cm -1 The Raman spectrum signal at 593 cm -1 There is almost no Raman spectral signal at the site, thus verifying the specificity of this detection method.

[0046] Example 5 Verification of the accuracy and stability of the SERS VFA detection method The detection method was verified by a spike experiment. Specifically, a spike solution of wheat light spores of ...

[0047] Table 2 Results of spike verification of the detection method

[0048] From the results, we can see that the spiked recovery rate of the detection method is ≥70.26%, and RSD is ≤12.56%, so 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 light smut fungus 1B5 Tilletia foetida 1B5, the deposit number is CCTCC NO:C2024293.

2. Use of the hybridoma cell line 1B5 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. 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 SERS nano-tag labeled monoclonal antibody against Tilletia sutchuenensis, 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 method comprises the monoclonal antibody against light smut fungus according to any one of claims 3 to 7 or the monoclonal antibody for light smut fungus labeled with a SERS nano-tag according to claim 9.

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