A monoclonal antibody against dihydromyricetin and its application

Abstract

This invention discloses a monoclonal antibody against dihydromyricetin and its applications, relating to the field of dihydromyricetin detection technology. The invention screened a hybridoma cell line that secretes an antibody specifically binding to dihydromyricetin. Immunoassay experiments demonstrated that the monoclonal antibody against dihydromyricetin provided by this invention has high specificity, high detection sensitivity, and a wide linear detection range for dihydromyricetin. This invention provides a detection tool and method for rapid quality assessment of vine tea, promoting the healthy development of the vine tea industry.

Description

Technical Field

[0001] This invention relates to the field of dihydromyricetin detection technology, and more specifically, to a monoclonal antibody against dihydromyricetin and its application. Background Technology

[0002] Dihydromyricetin (DMY) is a polyphenolic hydroxy dihydroflavonol, belonging to the flavonoid class of compounds. The chemical formula of dihydromyricetin is C0. 15 H 12 O8 has a molecular weight of 320.25. Its structure is shown below:

[0003] .

[0004] Vine tea, also known as moldy tea, berry tea, or Maoyan green frost tea, is rich in flavonoids. Among them, dihydromyricetin is the main active monomer in the total flavonoids of vine tea, accounting for over 30% in the dried young stems and leaves.

[0005] Currently, most vine tea on the market is processed from wild vine leaves. However, wild vine tea can no longer meet the growing market demand, necessitating artificial cultivation to satisfy this need. Traditional vine tea garden construction involves fully cultivating and preparing the land on soil below 20 degrees Celsius, followed by trenching and planting, with the application of farmyard manure at the time of planting. However, due to the extensive use of various feeds in modern livestock and poultry farming, their manure contains large amounts of heavy metals. This accumulation of heavy metals after fertilization inevitably affects the quality of the tea garden, and will certainly impact the content of dihydromyricetin.

[0006] Wild berry tea is a traditional herbal beverage of the Tujia ethnic group in the Wuling Mountains region, western Hunan, western Hubei, and eastern Guizhou. However, high-quality wild-harvested and organically grown teas are difficult to assess. Therefore, the industry urgently needs rapid testing and evaluation tools and methods to promote the healthy development of the wild berry tea industry and to ensure premium prices for high-quality products. Some tea farmers have introduced wild berry tea cultivation into farmland, which involves the use of agricultural inputs such as fertilizers, thus reducing the content of functional components in the tea. Therefore, developing rapid testing tools is of great significance to the industry's development.

[0007] Currently, the determination of dihydromyricetin content mainly includes spectroscopic methods, chromatographic methods, capillary electrophoresis, electrochemical sensor methods, and hydrogen nuclear magnetic resonance quantification. Traditional methods in the laboratory require expensive equipment and techniques, and involve lengthy experiments to obtain results. Traditional detection methods suffer from complex operations, high costs, and significant reagent consumption. Rapid detection technologies can simplify and accelerate the testing process, reducing costs. Currently, there are no reports on dihydromyricetin antibody preparation and immunoassay methods.

[0008] In view of this, the present invention is proposed. Summary of the Invention

[0009] The purpose of this invention is to provide a monoclonal antibody against dihydromyricetin and its application to solve the above-mentioned technical problems.

[0010] This invention is implemented as follows:

[0011] In a first aspect, the present invention provides a dihydromyricetin hapten having the structure shown in Formula I:

[0012] .

[0013] Secondly, the present invention provides a dihydromyricetin hapten having the structure shown in Formula II:

[0014] .

[0015] Thirdly, the present invention provides a dihydromyricetin antigen, which has the structure shown in Formula III:

[0016] Among them, the carrier protein is the transport protein;

[0017] Fourthly, the present invention provides a hybridoma cell, which is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.46124.

[0018] Fifthly, the present invention provides a monoclonal antibody against dihydromyricetin or its antigen-binding fragment, comprising: a heavy chain complementarity-determining region and a light chain complementarity-determining region, wherein the heavy chain complementarity-determining region comprises: CDR-H1, CDR-H2 and CDR-H3, the amino acid sequences of which are shown in SEQ ID NO: 1-3 in sequence, and the light chain complementarity-determining region comprises: CDR-L1, CDR-L2 and CDR-L3, wherein the amino acid sequences of CDR-L1 and CDR-L3 are shown in SEQ ID NO: 4-5 in sequence, and the amino acid sequence of CDR-L2 is WSA.

[0019] In a sixth aspect, the present invention provides the use of a monoclonal antibody against dihydromyricetin or its antigen-binding fragment, or the dihydromyricetin antigen, in any of the following:

[0020] (1) Detection of dihydromyricetin;

[0021] (2) Quality assessment of vine tea;

[0022] (3) Preparation of dihydromyricetin detection products;

[0023] The testing products include reagents, kits, test strips, antibody chips, antibody probes, or testing instruments.

[0024] In a seventh aspect, the present invention provides a dihydromyricetin detection product, wherein the detection product is a reagent, kit, test strip, antibody chip, antibody probe or detector; the detection product includes the above-mentioned anti-dihydromyricetin monoclonal antibody or its antigen-binding fragment, or the above-mentioned antibody secreted by hybridoma cells.

[0025] Eighthly, the present invention provides a method for detecting dihydromyricetin:

[0026] (1) Add diluent and anti-dihydromyricetin monoclonal antibody or its antigen-binding fragment to the control well of the microplate coated with dihydromyricetin antigen, and incubate; add dihydromyricetin standard or test sample and anti-dihydromyricetin monoclonal antibody or its antigen-binding fragment to the inhibition well of the microplate coated with dihydromyricetin antigen, and incubate; by adding enzyme-labeled secondary antibody to the control well and inhibition well respectively and incubating, the absorbance of the control well and inhibition well is obtained by colorimetric reaction; by plotting the standard curve of absorbance versus dihydromyricetin concentration, the concentration of dihydromyricetin in the test sample is obtained according to the standard curve;

[0027] (2) Apply the dihydromyricetin standard or the sample to be tested onto the sample pad of the test strip, and plot the standard curve of the ratio of the color values ​​of the T line and the C line on the test strip to the concentration of dihydromyricetin. Obtain the concentration of dihydromyricetin in the sample based on the standard curve.

[0028] The test strip includes a sample pad, an antibody conjugation pad, a fiber membrane, and absorbent paper. The antibody conjugation pad is coated with a labeled monoclonal antibody against dihydromyricetin or its antigen-binding fragment. The fiber membrane has T lines and C lines. The T lines are coated with dihydromyricetin antigen, and the C lines are coated with a secondary antibody against anti-dihydromyricetin monoclonal antibody or its antigen-binding fragment.

[0029] In a ninth aspect, the present invention provides an antibody conjugate formed by conjugating the above-mentioned monoclonal antibody against dihydromyricetin or its antigen-binding fragment with a label, wherein the label is selected from at least one of fluorescent dyes, enzymes that catalyze substrate color development, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

[0030] In a tenth aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned monoclonal antibody against dihydromyricetin or its antigen-binding fragment.

[0031] The present invention has the following beneficial effects:

[0032] This invention provides a hapten and a complete antigen of dihydromyricetin. By immunizing mice with the complete antigen of dihydromyricetin (i.e., the antigen itself), hybridoma fusion is performed, and a hybridoma cell line is selected that secretes an antibody specifically binding to dihydromyricetin. Immunoassay experiments demonstrate that the monoclonal antibody against dihydromyricetin provided by this invention has high specificity, high sensitivity, and a wide linear detection range for dihydromyricetin. Therefore, the monoclonal antibody against dihydromyricetin provided by this invention can be used to develop dihydromyricetin detection kits, test strips, and other products. This invention provides a detection tool and method for rapid quality assessment of vine tea, promoting the healthy development of the vine tea industry. Attached Figure Description

[0033] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0034] Figure 1 Chemical reaction route for preparing haptens by esterification of succinic anhydride with the hydroxyl group in dihydromyricetin;

[0035] Figure 2 A standard curve for ELISA detection of dihydromyricetin;

[0036] Figure 3 The dihydromyricetin hapten shown in Formula I 1 H-NMR spectrum;

[0037] Figure 4 The mass spectrum of the dihydromyricetin hapten shown in Formula I is shown below.

[0038] Figure 5 The molecular weights of complete antigens and BSA and OVA standards were detected using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

[0039] Figure 6 The standard curve constructed for the detection of dihydromyricetin in vine tea using test strips. Detailed Implementation

[0040] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0041] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a person skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by DM. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and MP. Calos, 1987); *Current Protocols in Molecular Biology* (edited by FM. Ausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JE Coligan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0042] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0043] Definition of noun

[0044] The term "antigen-binding fragment" broadly refers to all proteins / protein fragments containing a CDR region, particularly antibodies or antibody functional fragments. "Antigen-binding fragment" includes antigen-binding fragments of the aforementioned antibodies, including Fab, F(ab')2, Fd, Fv, scFv, bispecific antibodies, multispecific antibodies, and the smallest antibody recognition unit, as well as single-chain derivatives of these antibodies and fragments. Antibody types can include IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD, etc. Furthermore, the term "antibody" includes both naturally occurring and non-naturally occurring antibodies, including, for example, chimeric, bifunctional, and humanized antibodies, as well as related synthetic isoforms. The term "antibody" is used interchangeably with "immunoglobulin."

[0045] The term “antibody” as used in this article is used in the broadest sense and can include full-length monoclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antibody fragments, provided they exhibit the desired biological activity, such as specific binding to dihydromyricetin antigen or fragments thereof.

[0046] In this invention, the terms "complementarity-determining region" or "CDR" refer to highly variable regions of the heavy and light chains of an immunoglobulin, specifically regions containing one or more, or even all, of the major amino acid residues that contribute to the binding affinity of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In specific embodiments of this invention, CDRs refer to highly variable regions of the heavy and light chains of the antibody.

[0047] In this invention, the heavy chain complementarity-determining region (CDR) is represented by HCDR, which includes HCDR1, HCDR2, and HCDR3; the light chain complementarity-determining region (LCDR) is represented by LCDR, which includes LCDR1, LCDR2, and LCDR3. Commonly used CDR labeling methods in the art include the Kabat numbering scheme, the IMGT numbering scheme, the Chothia and Lesk numbering scheme, and the new standardized numbering system introduced by Lefranc et al. in 1997 for all protein sequences of the immunoglobulin superfamily. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences led to the creation of the Kabat database, and the Kabat numbering scheme is generally considered the widely adopted standard for numbering antibody residues. This invention uses the Kabat annotation standard to label CDR regions, but CDR regions labeled by other methods are also within the scope of this invention.

[0048] Typically, the variable region (VH) of the antibody heavy chain is obtained by linking the following CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4. HCDR1 is synonymous with CDR-H1.

[0049] The variable region (VL) of the antibody light chain can be obtained by linking the following numbered CDRs with FRs in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0050] As used herein, the term "nucleic acid molecule" refers to a sequence of nucleoside or nucleotide monomers composed of natural bases, sugars, and interglycosylation (backbone) bonds. The term also includes modified or substituted sequences containing non-naturally occurring monomers or portions thereof. The nucleic acid molecules of this invention can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences and may contain natural bases, including adenine, guanine, cytosine, thymine, and uracil. Modified bases may also be present. Examples of these modified bases include nitrogenous and denitrogenated adenine, guanine, cytosine, thymine, and uracil; and xanthine and hypoxanthine.

[0051] In a first aspect, the present invention provides a dihydromyricetin hapten having the structure shown in Formula I:

[0052] .

[0053] The synthesis route of the dihydromyricetin hapten follows the method of Murakami et al. The ease of esterification of the six hydroxyl groups in the dihydromyricetin molecule is as follows: 3-OH > 4′-OH > 5′-OH > 5-OH > 3′-OH > 7-OH. Therefore, the hapten is prepared by esterification of the hydroxyl groups in dihydromyricetin with succinic anhydride. The reaction route is as follows... Figure 1 As shown.

[0054] Secondly, the present invention provides a dihydromyricetin hapten having the structure shown in Formula II:

[0055] .

[0056] It is prepared by activating Formula I. The hapten of Formula I is reacted with N-hydroxysuccinimide (NHS) and DCC to prepare the dihydromyricetin hapten of Formula II, which can be directly used for subsequent conjugation to carrier proteins.

[0057] Thirdly, the present invention provides a dihydromyricetin antigen, which has the structure shown in Formula III:

[0058] Among them, the carrier protein is the transport protein;

[0059] In a preferred embodiment of the present invention, the carrier protein includes, but is not limited to, BSA, OVA, keyhole limpet hemocyanin (KLH), diphtheria toxoid, or tetanus toxoid.

[0060] Fourthly, this invention provides a hybridoma cell line, deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC NO.46124. The deposit date was October 23, 2024. The submitted biological material (strain) is DMY-2H3, scientifically described as a mouse hybridoma cell line. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The identification result is viable.

[0061] It can secrete monoclonal antibodies against dihydromyricetin.

[0062] Fifthly, the present invention provides a monoclonal antibody against dihydromyricetin or its antigen-binding fragment, comprising: a heavy chain complementarity-determining region and a light chain complementarity-determining region, wherein the heavy chain complementarity-determining region comprises: CDR-H1, CDR-H2 and CDR-H3, the amino acid sequences of which are shown in SEQ ID NO: 1-3 in sequence, and the light chain complementarity-determining region comprises: CDR-L1, CDR-L2 and CDR-L3, wherein the amino acid sequences of CDR-L1 and CDR-L3 are shown in SEQ ID NO: 4-5 in sequence, and the amino acid sequence of CDR-L2 is WSA.

[0063] Immunoassay experiments demonstrated that the monoclonal antibody against dihydromyricetin provided by this invention exhibits high specificity, high sensitivity, and a wide linear detection range for dihydromyricetin. Therefore, the monoclonal antibody against dihydromyricetin provided by this invention can be used to develop dihydromyricetin detection kits, test strips, and other products. This invention provides a rapid detection tool and method for evaluating the quality of vine tea, promoting the healthy development of the vine tea industry.

[0064] In a preferred embodiment of the present invention, the antibody or its antigen-binding fragment further includes a heavy chain framework region and a light chain framework region; the heavy chain framework region includes HFR1, HFR2, HFR3 and HFR4 having at least 80% homology with the amino acid sequences shown in SEQ ID NO: 6-9; for example, the heavy chain framework region includes HFR1, HFR2, HFR3 and HFR4 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology with the amino acid sequences shown in SEQ ID NO: 6-9.

[0065] The light chain framework region includes LFR1, LFR2, LFR3, and LFR4, which have at least 80% homology with the amino acid sequences shown in SEQ ID NO:10-13. For example, the light chain framework region includes LFR1, LFR2, LFR3, and LFR4, which have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology with the amino acid sequences shown in SEQ ID NO:10-13.

[0066] In one embodiment, the heavy chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO:14, and the light chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO:15.

[0067] In a preferred embodiment of the present invention, the antibody or its antigen-binding fragment further includes a constant region, which includes a heavy chain constant region and / or a light chain constant region. The heavy chain constant region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0068] The antigen-binding fragment is selected from any one of the antibody's F(ab')2, Fab', Fab, Fv, and scFv.

[0069] The antigen-binding fragments of the aforementioned antibodies typically possess the same binding specificity as the antibodies from which they originate. Those skilled in the art will readily understand, based on the description herein, that the functional fragments of the aforementioned antibodies can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds.

[0070] The antigen-binding fragments of the aforementioned antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesizing, for example, automated peptide synthesizers sold by Applied BioSystems.

[0071] In a sixth aspect, the present invention provides the use of a monoclonal antibody against dihydromyricetin or its antigen-binding fragment, or the dihydromyricetin antigen, in any of the following:

[0072] (1) Detection of dihydromyricetin;

[0073] (2) Quality assessment of vine tea;

[0074] (3) Preparation of dihydromyricetin detection products;

[0075] The testing products include reagents, kits, test strips, antibody chips, antibody probes, or testing instruments.

[0076] To improve reagent stability and extend shelf life, those skilled in the art can add functional components such as stabilizers and protectants as needed. Protein stabilizers are selected from sucrose, trehalose, BSA, glycerol, mannitol, Triton X-100, and Tween-20. Protectants are selected from cryoprotectants, such as polyols and sugars. Polyols may be selected from sorbitol, mannitol, or mixtures thereof. The reagent may be in the form of, but is not limited to, solid, liquid, or semi-solid.

[0077] Antibody chips refer to chips formed by immobilizing the aforementioned anti-dihydromyricetin antibody or its antigen-binding fragments on a carrier.

[0078] In a preferred embodiment of the present invention, the kit includes a solid phase, on which an antibody or its antigen-binding fragment is coated; for example, by chemical coupling, the antibody or its antigen-binding fragment is linked to the solid phase.

[0079] In a preferred embodiment of the present invention, the solid phase is selected from microspheres, plates, and membranes;

[0080] In a preferred embodiment of the present invention, the solid phase is selected from magnetic microspheres, plastic microspheres, plastic microparticles, latex microspheres, microporous plates, glass, capillaries, nylon, and nitrocellulose membranes.

[0081] Based on the specific binding properties of anti-dihydromyricetin antibodies or their antigen-binding fragments to dihydromyricetin, these antibodies can be used for quality control of dihydromyricetin in vine tea, enabling efficient and precise monitoring of the dihydromyricetin content in vine tea and thus monitoring the quality of the active ingredients in vine tea.

[0082] In a seventh aspect, the present invention provides a dihydromyricetin detection product, wherein the detection product is a reagent, kit, test strip, antibody chip, antibody probe or detector; the detection product includes the above-mentioned anti-dihydromyricetin monoclonal antibody or its antigen-binding fragment, or the above-mentioned antibody secreted by hybridoma cells.

[0083] In a preferred embodiment of the present invention, the detection product also includes the aforementioned dihydromyricetin antigen.

[0084] Immunoassay is an analytical method that uses the specific binding reaction between antigens and antibodies to detect various substances. When using rapid immunoassay technology based on test strips (colloidal gold) to detect small molecules, the basic principle is direct competition with enzyme-linked immunosorbent assay (ELISA).

[0085] The preferred reagent kit is a competitive ELISA kit.

[0086] A chip, also known as a suspension array or liquid array, consists of a carrier and nucleic acid molecules (such as primers and / or probes) and / or antibodies bound to the surface of the carrier.

[0087] The aforementioned carrier can be made of various materials and in various forms, such as preferably a container with a flat bottom. A more typical preferred example is multi-well plates, microplates, microfluidic-based devices (e.g., microfluidic chips), petri dish-like containers, etc., which are widely used in biochemical assays, and are not limited thereto.

[0088] The microfluidic chip is selected from T-type chip, flow focusing chip or coaxial flow chip PDMS chip or metal droplet generator or PMMA microfluidic chip.

[0089] Furthermore, the kit may also include at least one of the following: buffer solution, detection reagent, diluent, washing solution, dihydromyricetin antigen, and dihydromyricetin standard, and is not limited thereto.

[0090] Eighthly, the present invention provides a method for detecting dihydromyricetin, comprising any one of the following methods:

[0091] (1) Add diluent and the above-mentioned anti-dihydromyricetin monoclonal antibody or its antigen-binding fragment to the control well of the microplate coated with the above-mentioned dihydromyricetin antigen, and incubate; add dihydromyricetin standard or test sample and the above-mentioned anti-dihydromyricetin monoclonal antibody or its antigen-binding fragment to the inhibition well of the microplate coated with the above-mentioned dihydromyricetin antigen, and incubate; by adding enzyme-labeled secondary antibody to the control well and the inhibition well respectively and incubating, the absorbance of the control well and the inhibition well is detected by colorimetric reaction; by plotting the standard curve of absorbance versus dihydromyricetin concentration, the concentration of dihydromyricetin in the test sample is obtained according to the standard curve;

[0092] (2) Apply the dihydromyricetin standard or the sample to be tested onto the sample pad of the test strip, and plot the standard curve of the ratio of the color values ​​of the T line and the C line on the test strip to the concentration of dihydromyricetin. Obtain the concentration of dihydromyricetin in the sample based on the standard curve.

[0093] When plotting the standard curve of absorbance versus dihydromyricetin concentration, this includes, but is not limited to, plotting: the standard curve of the absorbance ratio of the inhibition well / control well versus the concentration of the dihydromyricetin standard, or plotting the standard curve of the absorbance ratio of (control well - inhibition well) / control well versus the concentration of the dihydromyricetin standard.

[0094] The coating concentration of dihydromyricetin antigen on the microplate was 12.5 × 10⁻⁶. -6 -1×10 -3 mg / mL; the dilution of the monoclonal antibody against dihydromyricetin or its antigen-binding fragment was 1:2000-8000, correspondingly, the added concentration of the monoclonal antibody against dihydromyricetin or its antigen-binding fragment was 12.5 × 10 mg / mL. -6 -5×10 -4 mg / mL. At the above coating concentration and dilution, the dihydromyricetin antigen showed the best inhibitory effect on dihydromyricetin.

[0095] The coating concentration of dihydromyricetin antigen on the microplate was 12.5 × 10⁻⁶. -6 mg / mL, 20×10 -6 mg / mL, 3×10 -5 mg / mL, 5×10 -5 mg / mL, 1×10-4 mg / mL, 5×10 -4 mg / mL or 1×10 -3 mg / mL, etc.

[0096] The concentration of the monoclonal antibody against dihydromyricetin or its antigen-binding fragment added was 12.5 × 10⁻⁶. -6 mg / mL, 3×10 -5 mg / mL, 5×10 -5 mg / mL, 1×10 -4 mg / mL, 2×10 -4 mg / mL or 5×10 -4 mg / mL, etc.

[0097] The test strip includes a sample pad, an antibody-binding pad, a fiber membrane, and absorbent paper. The antibody-binding pad is coated with the aforementioned anti-dihydromyricetin monoclonal antibody or its antigen-binding fragment, which is labeled. The fiber membrane has T lines and C lines. The T lines are coated with dihydromyricetin antigen, and the C lines are coated with a secondary antibody containing an anti-dihydromyricetin monoclonal antibody or its antigen-binding fragment.

[0098] For example, the marker is a colloid.

[0099] The colloid is selected from colloidal gold, colloidal silver, or colloidal selenium.

[0100] In a ninth aspect, the present invention provides an antibody conjugate formed by conjugating the above-mentioned monoclonal antibody against dihydromyricetin or its antigen-binding fragment with a label, wherein the label is selected from at least one of fluorescent dyes, enzymes that catalyze substrate color development, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

[0101] The aforementioned markers refer to substances possessing properties that can be directly observed with the naked eye or detected by instruments, such as luminescence, color development, and radioactivity. These properties enable qualitative or quantitative detection of the corresponding target analytes. In practical applications, those skilled in the art can select appropriate markers based on detection conditions or actual needs. Regardless of the marker used, it falls within the scope of protection of this invention.

[0102] Fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5...). .5, Cy3, etc. or similar substances), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or similar substances) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP), etc.).

[0103] In optional embodiments, the enzymes that catalyze substrate color development include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxygenase.

[0104] In optional embodiments, radioactive isotopes include, but are not limited to, those mentioned above. 212 Bi、 131 I, 111 In、 90 Y、 186 Re、 211 At、 125 I, 188 Re、 153 Sm、 213 Bi、 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn, 97 Ru、 62 Cu、 64 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb, 166 Ho、 105 Rh、 177 Lu、 172 Lu and18 F.

[0105] In optional embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0106] In optional embodiments, nanoparticle-based markers include, but are not limited to, nanoparticles and colloids; nanoparticles include, but are not limited to, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0107] The colloid is selected from colloidal gold, colloidal silver, or colloidal selenium.

[0108] In a tenth aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned monoclonal antibody against dihydromyricetin or its antigen-binding fragment.

[0109] In one embodiment, the coding sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO:16, and the coding sequence of the light chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO:17.

[0110] In another aspect, the present invention also provides a carrier containing the above-mentioned nucleic acid molecules.

[0111] The term "vector" is used herein in its most common sense and includes any intermediate medium for nucleic acids that enables the nucleic acids to be introduced, for example, into prokaryotic and / or eukaryotic cells and, where appropriate, integrated into the genome. Vectors of this type preferably replicate and / or are expressed in cells. The term "vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art. The term "plasmid," as used herein, generally refers to a construct of extrachromosomal genetic material, typically a circular double-stranded DNA that can replicate independently of chromosomal DNA. Any plasmid and vector can be used as long as it can replicate and remain stable within the host.

[0112] In one alternative embodiment, the vector is an expression vector, and an important feature of an expression vector is that it typically contains an origin of replication, a promoter, a marker gene, and translation control elements.

[0113] In a twelfth aspect, the present invention also provides a recombinant cell containing the aforementioned carrier.

[0114] The term "recombinant cell" refers to any cell that can be transformed or transfected with exogenous nucleic acids. According to the invention, the term "recombinant cell" includes prokaryotic (e.g., *Escherichia coli*) or eukaryotic cells (e.g., mammalian cells, particularly human cells, yeast cells, and insect cells). Mammalian cells, such as those derived from humans, mice, hamsters, pigs, goats, or primates, are particularly preferred. Cells can originate from multiple tissue types and comprise primary cells and cell lines. Nucleic acids may be present in the host cell in single-copy or two or more copies, and in one embodiment, are expressed in the recombinant cell.

[0115] In one alternative implementation, the recombinant cells are eukaryotic cells.

[0116] In one alternative implementation, the recombinant cells are mammalian cells.

[0117] In one alternative implementation, the recombinant cells are HEK293.

[0118] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0119] Example 1

[0120] This embodiment describes the preparation of dihydromyricetin hapten.

[0121] (1) In this embodiment, a dihydromyricetin hapten with the structure shown in Formula I was prepared:

[0122] .

[0123] The synthesis route of the dihydromyricetin hapten was based on the method of Murakami et al. The ease of esterification of the six hydroxyl groups in the dihydromyricetin molecule was as follows: 3-OH > 4′-OH > 5′-OH > 5-OH > 3′-OH > 7-OH. Therefore, the hapten was prepared by esterification of the hydroxyl groups in dihydromyricetin with succinic anhydride. The molar amounts of succinic anhydride and dihydromyricetin were 6.3 mmol and 9.2 mmol, respectively. Under the catalytic conditions of 4-dimethylaminopyridine (DMAP), pyridine, acting as a base catalyst, nucleophilic catalyst, and solvent, is the key reagent for the efficient selective esterification of dihydromyricetin. Co-occurrence with DMAP can improve the yield. The reaction conditions were: vigorous stirring at room temperature for 16 hours, followed by the addition of methanol (120 mL) and water (1 mL) to the reaction mixture at room temperature and stirring for 1 hour. The reaction mixture was further purified and concentrated to obtain the desired product, a light yellow powder (450 mg), with a purity of approximately 80-85%. The dihydromyricetin hapten with the structure shown in Formula I was prepared by referring to the reaction route. Figure 1 As shown.

[0124] The chemical structure of the product was identified, and the dihydromyricetin hapten represented by Formula I was... 1 H-NMR spectrum reference Figure 3 As shown, the mass spectrum is referenced. Figure 4 As shown.

[0125] (2) In this embodiment, the dihydromyricetin hapten shown in Formula I was activated to prepare a dihydromyricetin hapten with the structure shown in Formula II:

[0126] .

[0127] The specific steps are as follows:

[0128] Weigh out 0.036 mmol of the dihydromyricetin hapten (Formula I), 0.047 mmol of N-hydroxysuccinimide (NHS), and 0.040 mmol of DCC, and dissolve them in 0.5 mL of anhydrous N,N-dimethylformamide (DMF). After stirring at room temperature (25°C) for 6 hours, centrifuge the reaction solution at 8000 rpm for 5 minutes and collect the supernatant for later use. The reaction product containing the dihydromyricetin hapten with the structure shown in Formula II can be directly used for subsequent conjugation of carrier proteins.

[0129] Example 2

[0130] This embodiment describes the preparation of dihydromyricetin complete antigen (dihydromyricetin hapten conjugated with BSA with the structure shown in Formula II).

[0131] The supernatant of the dihydromyricetin-activated hapten was slowly added dropwise to the carrier protein solution (the carrier protein solution was prepared by dissolving 20 mg BSA in phosphate-buffered saline (PBS) at pH 6.4, with a concentration of 10 mg / mL). The molar ratio of hapten to carrier protein was 30:1. After stirring overnight at 4°C, the resulting reaction solution was dialyzed six times with PBS solution at pH 6.4 and a concentration of 0.01 mol / L. The completely dialyzed reaction product solution was diluted to a 4 mg / mL solution and stored at -20°C for later use.

[0132] The complete antigen of the product was named dihydromyricetin complete antigen C1. The chemical structure of the product was identified, and the molecular weights of the complete antigen and BSA standard were determined using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The results were referenced... Figure 5 As shown in Figure III, the structural formula of the dihydromyricetin complete antigen C1 is shown below:

[0133] .

[0134] Example 3

[0135] This embodiment describes the preparation of dihydromyricetin complete antigen (dihydromyricetin hapten coupled with OVA with the structure shown in Formula II).

[0136] The supernatant of the dihydromyricetin-activated hapten was slowly added dropwise to the carrier protein OVA solution (which was prepared by dissolving 20 mg of OVA in phosphate-buffered saline (PBS) at pH 6.4, with a concentration of 10 mg / mL). The molar ratio of hapten to carrier protein was 30:1. After stirring overnight at 4°C, the resulting reaction solution was dialyzed six times with PBS solution at pH 6.4 and a concentration of 0.01 mol / L. The completely dialyzed reaction product solution was diluted to a 4 mg / mL solution and stored at -20°C for later use.

[0137] The complete antigen of the product was named dihydromyricetin complete antigen C2. The chemical structure of the product was identified, and the molecular weights of the complete antigen and OVA standard were determined using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The results are referenced from [reference needed]. Figure 5 As shown. The structural formula of the dihydromyricetin complete antigen C2 is shown in Formula IV below:

[0138]

[0139] Example 4

[0140] Antibodies were prepared using the dihydromyricetin complete antigen C1.

[0141] (1) Balb / C mice aged 6-8 weeks were used as experimental animals.

[0142] (2) Basic immunization: The diluted dihydromyricetin complete antigen C1 antigen solution (concentration of 1 mg / mL) was filtered through a sterile filter and an equal volume of Freund's complete adjuvant was added. The mixture was then thoroughly emulsified with a magnetic stirrer until it did not diffuse when dropped into water. Balb / c mice were injected intraperitoneally and subcutaneously at multiple sites on the back with the emulsified complete antigen at a dose of 0.1 mg emulsified antigen per mouse.

[0143] (3) Booster Immunization: Two weeks after the primary immunization, diluted complete antigen C1 solution (concentration 1 mg / mL) was emulsified with an equal volume of Freund's incomplete adjuvant using a magnetic stirrer and then injected subcutaneously into mice at multiple sites on the back. The immunization dose was 0.1 mg of emulsified antigen per mouse. Booster immunizations were performed every two weeks, starting from the third booster immunization. Blood was collected from the orbital rim on the 7th day after each immunization, and the antibody titer and inhibitory effect were detected by indirect competitive ELISA. The coating antigen used in this method was complete antigen C2. Using C2 as the coating antigen helps to avoid screening for antibodies with immunogenicity against BSA protein, thereby increasing the success rate of screening for antibodies that specifically bind to dihydromyricetin.

[0144] (4) Sprint immunization: Select mice with high titer and good inhibition and perform sprint immunization on the 10th day after the 5th immunization. 1 mg / mL 100uL is injected intraperitoneally without adjuvant.

[0145] (5) Cell fusion

[0146] On day 3 post-immunization, hybridoma fusion was performed by fusing mouse spleen cells with mouse myeloma cells sp2 / 0 using PEG 1450. The cells were then selectively cultured in 2% HAT medium. Indirect competitive ELISA was used to screen wells with high titers and good inhibition. Subcloning was performed through limiting dilutions to obtain the DMY-2H3 hybridoma cell line containing a monoclonal antibody against dihydromyricetin. The antibody secreted by this cell line exhibited good specificity for dihydromyricetin, with a detection sensitivity reaching 2.1 μg / L. This cell line is deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC NO. 46124.

[0147] (6) Cell cryopreservation and thawing

[0148] The obtained hybridoma cell line DMY-2H3 was expanded and cultured, then resuspended in DMEM and centrifuged. Cells were then frozen in cell cryopreservation medium at a concentration of 1×10⁻⁶. 9 Cryopreservation was performed at a rate of 1 / mL using liquid nitrogen for long-term storage. Upon thawing, the cryopreservation tubes were removed from the liquid nitrogen tank and immediately placed in a 37°C water bath for thawing. They were then transferred to preheated 10 mL DMEM via pipette, centrifuged to remove the cryopreservation solution, and finally transferred to a culture plate for incubation.

[0149] (7) Preparation and purification of monoclonal antibodies.

[0150] Eight 10-12 week old female BALB / c mice were intraperitoneally injected with sterile paraffin oil, 0.3 mL per mouse. One week later, each mouse was intraperitoneally injected with the monoclonal cell line DMY-2H3, with approximately 10 cells injected per mouse. 6Once the mice's abdomens swelled, ascites fluid was collected and purified using the saturated ammonium sulfate method before being stored at -20°C. This yielded purified monoclonal antibodies secreted by the hybridoma cell line DMY-2H3.

[0151] Example 5

[0152] The monoclonal antibody obtained in Example 4 was tested for antibody efficacy.

[0153] The buffer solutions used in the following experiments are as follows:

[0154] Coating buffer (pH 9.6 0.05 M carbonate buffer): Na2CO3 1.5 g; NaHCO3 2.94 g, add pure water to a final volume of 1000 mL;

[0155] Phosphate-buffered saline (PBS) (0.01M pH 7.4): 0.2 g KH2PO4; 8 g NaCl; 2.92 g NaH2PO4·12H2O, add pure water to 1000 mL;

[0156] Washing buffer (PBST): Add 1 mL of Tween-20 to 1000 mL of prepared PBS solution;

[0157] Sample dilution buffer (PBSTG): Add 1 mL Tween-20 and 1 g gelatin (melted by microwave heating) to the prepared PBS, and bring the volume to 1 L;

[0158] Colorimetric solutions: TMB stock solution (375 mg TMB solid + 30 mL DMSO (prepared according to the ratio, stored at room temperature and protected from light)), BUFFER (0.1 g potassium sorbate + 46.04 g potassium dihydrogen citrate hydrate + 1 L pure water). The colorimetric solution is prepared fresh each time: 200 μL TMB stock solution + 11 mL BUFFER + 3.34 μL 30% hydrogen peroxide solution.

[0159] Termination solution (2M H2SO4): 445.6 mL of distilled water, add 54.4 mL of concentrated sulfuric acid (98%) dropwise while stirring.

[0160] The following is a checkerboard experiment involving antigen and antibody:

[0161] 1) Wrapped in blankets:

[0162] 1 mg / mL of dihydromyricetin complete antigen C1 was serially diluted with coating buffer at 1:1000, 1:2000, 1:4000, and 1:8000 to obtain coated antigen solutions of different concentrations of dihydromyricetin complete antigen C1. 100 μL of the dihydromyricetin complete antigen C1 coated antigen solution prepared in Example 2 was added to each well of a 96-well microplate and incubated overnight at 4°C. The plate was then washed three times with PBST.

[0163] 2) Competition:

[0164] Dihydromyricetin solid standard was purchased from Chengdu Desite Biotechnology Co., Ltd. Because dihydromyricetin is easily oxidized, the standard was prepared fresh for each use. It was prepared as a 1 mg / mL liquid standard using methanol. The 1 mg / mL dihydromyricetin standard was diluted to 10 ng / mL using PBSTG. 50 μL of sample diluent was added to each well in the zero well, and 50 μL of the diluted 10 ng / mL dihydromyricetin standard solution was added to each well in the inhibition well.

[0165] Dihydromyricetin antibody (1 mg / mL) was serially diluted with PBSTG at 1:1000, 1:2000, 1:4000, 1:8000, and 1:16000 to obtain dihydromyricetin antiserum dilution (50 μL / well). The solution was placed in a humidified chamber at 37°C for 30 min and washed 3 times.

[0166] 3) Add enzyme-labeled secondary antibody: Dilute goat anti-mouse enzyme-labeled secondary antibody (IgG-HRP, Jackson) 10,000 times with PBSTG, add 100 μL to each well, place in a humidified chamber at 37°C for 30 min, and wash the plate 3 times.

[0167] 4) Color development: The color development solution should be prepared fresh for use. Mix the prepared TMB solution with hydrogen peroxide in the specified ratio, add 100 μL to each well, and develop the color at room temperature in the dark for 10 min.

[0168] 5) Termination: Add 50 μL of 2 M H2SO4 to each well and measure the OD value of each well at 450 nm using a microplate reader.

[0169] The formula for calculating the inhibition rate is: Inhibition rate = (B0 - B) / B0 × 100%), where B0 is the OD value of the control well and B is the OD value of the inhibition well.

[0170] The results are shown in the table below:

[0171]

[0172] Note: I represents the inhibition well in the ELISA plate, and C represents the control well in the ELISA plate.

[0173] The results in Table 1 show that the best inhibitory effect on dihydromyricetin was achieved when the coating antigen dilution was 1:4000 and the antibody dilution was 1:2000, with an inhibition rate of 81.8%. This indicates that the antibody produced by the hybridoma cells DMY-2H3 can detect dihydromyricetin.

[0174] Example 6

[0175] This embodiment provides a standard curve for detecting dihydromyricetin and tests the sensitivity of the detection method.

[0176] The dihydromyricetin standard solution was diluted with sample diluent to the following concentrations: 200 ng / mL, 100 ng / mL, 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 6.25 ng / mL, 3.125 ng / mL, 1.56 ng / mL, 0.78 ng / mL, 0.39 ng / mL, and 0.195 ng / mL. A standard curve experiment was performed following the checkerboard test procedure, with three replicates for each standard concentration. The coating antigen was diluted 1:4000, and the antibody was diluted 1:2000 for detection.

[0177]

[0178] Plotting a standard curve: Using dihydromyricetin standard solutions of different concentrations (ng / mL) as the X-axis and the ratio of absorbance values ​​(B / B0, where B is the average absorbance value of the dihydromyricetin standard solution and B0 is the average absorbance value of the control wells) as the Y-axis, plot the standard curve as follows: Figure 2 As shown.

[0179] The results show that its sensitivity (IC) 50 The concentration was 2.1 ng / mL, and the sensitivity (IC50) was 2.1 ng / mL. 50 ( ) is the concentration value of dihydromyricetin standard with an inhibition rate of 50%.

[0180] The linear detection range was 0.46 ng / mL - 9.54 ng / mL. This indicates that the antibody prepared in Example 4 above has excellent detection performance and high sensitivity.

[0181] Example 7

[0182] This embodiment performs specific detection on the antibody prepared in Example 4.

[0183] Solid standards of myricetin, myricetin, naringenin, and quercetin purchased from Chengdu Desite Biotechnology Co., Ltd. were prepared into 1 mg / mL liquid standards using methanol solvent. The solutions were prepared fresh for each use. The above four analog liquid standards were diluted with the sample diluent of Example 5 to the following concentrations: 1000 ng / mL, 500 ng / mL, 250 ng / mL, 125 ng / mL, 62.5 ng / mL, 31.25 ng / mL, 15.6 ng / mL, and 7.8 ng / mL, respectively.

[0184] A standard curve was established according to the method in Example 6, and the inhibition concentration IC50 was determined. 50 (The standard concentration value at which the inhibition rate reaches 50%).

[0185] Cross-reactivity rate (%) = (dihydromyricetin IC50) 50 ) / (analog IC 50 ) × 100%.

[0186]

[0187] The results showed that the antibody produced by hybridoma cells DMY-2H3 had good specificity for dihydromyricetin.

[0188] Example 8

[0189] This embodiment provides a method for detecting dihydromyricetin in vine tea, as detailed below:

[0190] 1. Development of colloidal gold immunochromatographic test strips

[0191] 1) Preparation of gold-labeled antibody pads: Take 1 mL of colloidal gold solution (40 nm) into a 2 mL centrifuge tube (rinse with pure water), add 3 μL of 0.2 M K2CO3 solution, then add 6 μg of antibody, mix by inverting, and let stand for 20 min. Then add 50 μL of 3% BSA for blocking, mix by inverting, and let stand for 20 min. Then centrifuge the mixture at 10000 r / min for 10 min (4℃) using a high-speed refrigerated centrifuge. Discard the supernatant, add 0.5 mL of reconstitution solution (0.01 M PBS containing 3% sucrose) to the precipitate for reconstitution, spread the solution onto a 1 cm × 15 cm sample pad, and dry in a 37℃ oven (1.5 h).

[0192] 2) Scribing: The nitrocellulose membrane attached to the substrate was scribed using a gold-spraying scribing instrument. The concentration of T-line antigen (dihydromyricetin complete antigen C1) was 1.5 mg / mL, and the concentration of C-line secondary antibody was 1.0 mg / mL.

[0193] 3) Assembly: Assemble the sample pad, colloidal gold pad, nitrocellulose membrane and absorbent paper in that order, and then cut them into 4mm wide test strips using a chopper for subsequent testing.

[0194] 2. Establishment of the standard curve

[0195] Dihydromyricetin standards at concentrations of 5, 10, 20, 50, 100, 200, 500, 1000, and 2000 ng / mL were prepared using pure water. A standard curve was established based on the test strip results. Figure 6 An equation was established for the concentration of dihydromyricetin and the T / C value of the test strip: y = A2 + (A1-A2) / (1 + (x / x0)^p), A1=1.00818 ± 0.05365, A2=0.04562 ± 0.0434, x0=34.63145 ± 9.03569, p=0.85805 ± 0.1557. The concentration of dihydromyricetin in the sample can be calculated based on the T / C value.

[0196] 3. Actual sample testing

[0197] Based on the established standard curve, the dihydromyricetin content of dried and fresh vine tea from different origins was detected using colloidal gold immunochromatographic assay strips (Lateral Flow Immunoassay, LFIA). Simultaneously, high-performance liquid chromatography (HPLC) was used to detect the same samples as a control. The tea processing method for LFIA detection is as follows:

[0198] 1) Dried tea leaves: 1 g + 50 ml (80 - 100 ℃ hot water) soak for 3 minutes, take one drop (about 30 μL) and add it to a centrifuge tube containing 4 ml of diluent, and dilute according to the dilution factor for testing.

[0199] 2) Fresh tea leaves: Grind 1 g of fresh tea leaves with a grinding rod, add 50 ml of 80-100 ℃ hot water and soak for 3 minutes, take one drop (about 30 μL) and add it to a centrifuge tube containing 4 ml of diluent, and dilute according to the dilution factor for testing.

[0200]

[0201] The results of LFIA and HPLC detections were basically consistent, indicating that the test strips prepared in this invention are reliable in detecting the concentration of dihydromyricetin in vine tea.

[0202] The sequence information involved in this invention is as follows:

[0203]

[0204] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A dihydromyricetin hapten, characterized in that, It has the structure shown in Equation II: 。 2. A dihydromyricetin antigen, characterized in that, The dihydromyricetin antigen has the structure shown in Formula III: The carrier protein is the transport protein.

3. The dihydromyricetin antigen according to claim 2, characterized in that, The carrier protein is BSA, OVA, keyhole hemocyanin, diphtheria toxoid, or tetanus toxoid.

4. A hybridoma cell, characterized in that, It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.46124.

5. A monoclonal antibody against dihydromyricetin or its antigen-binding fragment, characterized in that, It includes: The heavy chain complementarity-determining region (CDR) includes CDR-H1, CDR-H2, and CDR-H3, whose amino acid sequences are shown in SEQ ID NO: 1-3. The light chain complementarity-determining region includes CDR-L1, CDR-L2, and CDR-L3, wherein the amino acid sequences of CDR-L1 and CDR-L3 are shown in SEQ ID NO: 4-5, and the amino acid sequence of CDR-L2 is WSA.

6. The monoclonal antibody against dihydromyricetin or its antigen-binding fragment according to claim 5, characterized in that, The antibody or its antigen-binding fragment further includes a heavy chain framework region and a light chain framework region; the heavy chain framework region includes HFR1, HFR2, HFR3 and HFR4, which have at least 80% homology with the amino acid sequences shown in SEQ ID NO:6-9; the light chain framework region includes LFR1, LFR2, LFR3 and LFR4, which have at least 80% homology with the amino acid sequences shown in SEQ ID NO:10-13.

7. The monoclonal antibody against dihydromyricetin or its antigen-binding fragment according to claim 5, characterized in that, The antibody or its antigen-binding fragment further includes a constant region, which includes a heavy chain constant region and / or a light chain constant region, wherein the heavy chain constant region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; and the light chain constant region is selected from the κ-type or λ-type light chain constant region. The antigen-binding fragment is selected from any one of the antibody's F(ab')2, Fab', Fab, Fv, and scFv.

8. The use of the monoclonal antibody against dihydromyricetin as described in any one of claims 5-7 or its antigen-binding fragment, or the dihydromyricetin antigen as described in any one of claims 2-3, in any of the following: (1) Detection of dihydromyricetin; (2) Quality assessment of vine tea; (3) Preparation of dihydromyricetin detection products; The detection products include reagents, kits, test strips, antibody chips, antibody probes, or detection instruments.

9. A dihydromyricetin detection product, characterized in that, The detection product is a reagent, kit, test strip, antibody chip, antibody probe or detector; the detection product includes the monoclonal antibody against dihydromyricetin as described in any one of claims 5-7 or its antigen-binding fragment, or the antibody secreted by hybridoma cells as described in claim 4.

10. The dihydromyricetin detection product according to claim 9, characterized in that, The testing product also includes the dihydromyricetin antigen as described in claim 3.

11. A method for detecting dihydromyricetin, characterized in that, It includes any of the following methods: (1) Add diluent and monoclonal antibody against dihydromyricetin or its antigen-binding fragment as described in any one of claims 5-7 to the control wells of a microplate coated with dihydromyricetin antigen as described in any one of claims 2-3, and incubate; add dihydromyricetin standard or sample to be tested and monoclonal antibody against dihydromyricetin or its antigen-binding fragment as described in any one of claims 5-7 to the inhibition wells of a microplate coated with dihydromyricetin antigen as described in any one of claims 2-3, and incubate; by adding enzyme-labeled secondary antibody to the control wells and inhibition wells respectively and incubating, the absorbance of the control wells and inhibition wells is detected by colorimetric reaction; by plotting a standard curve of absorbance versus dihydromyricetin concentration, the concentration of dihydromyricetin in the sample is obtained according to the standard curve; (2) Apply the dihydromyricetin standard or the sample to be tested onto the sample pad of the test strip, and plot the standard curve of the ratio of the color values ​​of the T line and the C line on the test strip to the concentration of dihydromyricetin. Obtain the concentration of dihydromyricetin in the sample based on the standard curve. The test strip comprises a sample pad, an antibody conjugation pad, a fiber membrane, and absorbent paper. The antibody conjugation pad is coated with a monoclonal antibody against dihydromyricetin as described in any one of claims 5-7, or an antigen-binding fragment thereof, bearing a marker. The fiber membrane has T lines and C lines. The T lines are coated with dihydromyricetin antigen as described in any one of claims 2-3, and the C lines are coated with a secondary antibody against the monoclonal antibody against dihydromyricetin, or an antigen-binding fragment thereof.

12. The method for detecting dihydromyricetin according to claim 11, characterized in that, The coating concentration of dihydromyricetin antigen on the microplate was 12.5 × 10⁻⁶. -6 -1×10 -3 mg / mL; the concentration of the monoclonal antibody against dihydromyricetin or its antigen-binding fragment added was 12.5 × 10 mg / mL. -6 -5×10 -4 mg / mL.

13. The method for detecting dihydromyricetin according to claim 11, characterized in that, The marker is a colloid.

14. The method for detecting dihydromyricetin according to claim 13, characterized in that, The colloid is selected from colloidal gold, colloidal silver, or colloidal selenium.

15. An antibody conjugate, characterized in that, It is formed by conjugation of a monoclonal antibody against dihydromyricetin as described in any one of claims 5-7 or its antigen-binding fragment and a marker, wherein the marker is selected from at least one of fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent reagents and nanoparticle markers.

16. A nucleic acid molecule, characterized in that, It encodes the monoclonal antibody against dihydromyricetin or its antigen-binding fragment as described in any one of claims 5-7.

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