Monoclonal antibody for resisting dihydromyricetin and application thereof

Abstract

The invention discloses an anti-dihydromyricetin monoclonal antibody and application thereof, and relates to the technical field of dihydromyricetin detection. According to the invention, a hybridoma cell strain is screened out, and the hybridoma cell strain can secrete an antibody specifically combined with dihydromyricetin. Immunodetection experiments prove that the monoclonal antibody for resisting dihydromyricetin provided by the invention has the technical advantages of high specificity to dihydromyricetin, high detection sensitivity to dihydromyricetin and wide linear detection range. The invention provides a detection tool and means for rapid detection and evaluation of ampelopsis grossedentata quality, and promotes the benign development of the ampelopsis grossedentata industry.

Description

Technical Field

[0001] The present invention relates to the technical field of dihydromyricetin detection, and in particular, to a monoclonal antibody against dihydromyricetin and its application. Background Art

[0002] Dihydromyricetin (dihydroflavonol, Dihydromyricetin, DMY) is a polyphenolic hydroxyl dihydroflavonol and belongs to the flavonoid compound. The chemical formula of dihydromyricetin is C 15 H 12 O8, and the molecular weight is 320.25. Its structure is shown as follows:

[0003]

[0004] Tengcha, also known as mold tea, berry tea or Maoyan qingshuang tea. Tengcha is rich in flavonoid compounds. Among them, dihydromyricetin is the main monomer active ingredient in the total flavonoids of Tengcha, and its content in the dry young stems and leaves exceeds 30%.

[0005] At present, the Tengcha on the market is mainly processed by picking wild Tengcha leaves, and the wild Tengcha can no longer meet the growing market demand. It is urgent to cultivate Tengcha artificially to meet the market demand. The traditional construction of Tengcha tea gardens is to carry out full reclamation and land preparation on land below 20 degrees, and then open ditches for planting. When planting, farmyard manure is applied. Since a large amount of various feeds are now used in the artificial breeding of livestock and poultry, there are a large amount of heavy metals in their feces, and heavy metal accumulation occurs after fertilization. This accumulation will inevitably affect the quality of the tea garden and will also inevitably affect the content of dihydromyricetin.

[0006] It is not easy to judge the good quality of wild-picked and organically grown berry tea. Therefore, there is an urgent need for rapid detection and evaluation tools and means in the industry to promote the healthy development of the berry tea industry and promote high-quality and high-price products. Some tea farmers introduce berry tea to farmland for planting, and there will be agricultural inputs such as chemical fertilizers in these processes, which will reduce the content of functional components in berry tea. Therefore, the development of rapid detection tools is of great significance for the industrial development.

[0007] At present, the determination of dihydromyricetin content mainly includes spectroscopy, chromatography, capillary electrophoresis, electrochemical sensor method, hydrogen nuclear magnetic resonance quantitative method, etc. When using traditional methods for inspection in the laboratory, expensive instrument equipment and technical means are required, and it takes a long time of experimentation to draw a conclusion. The traditional detection methods have problems such as complex detection operations, high costs, and large reagent consumption. Through rapid detection technology, the inspection process can be simplified and speeded up, and the detection cost can be reduced. There is no report on the preparation of dihydromyricetin antibodies and immunoassay methods yet.

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

[0009] The object of the present invention is to provide a monoclonal antibody against dihydromyricetin and its application to solve the above technical problems.

[0010] The present invention is implemented as follows:

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

[0012]

[0013] In the second aspect, the present invention provides a dihydromyricetin hapten, which has the structure shown in Formula II below:

[0014]

[0015] In the third aspect, the present invention provides a dihydromyricetin antigen, which has the structure shown in Formula III below:

[0016] wherein Carrier protein is a carrier protein;

[0017] In the fourth aspect, the present invention provides a hybridoma cell, which is deposited in the China General Microbiological Culture Collection Center with the deposit number of CGMCC NO. 46124.

[0018] In the fifth aspect, the present invention provides a monoclonal antibody against dihydromyricetin or its antigen-binding fragment, which includes: heavy chain complementarity-determining regions and light chain complementarity-determining regions. The heavy chain complementarity-determining regions include: CDR-H1, CDR-H2, and CDR-H3, and their amino acid sequences are shown as SEQ ID NO: 1-3 in sequence. The light chain complementarity-determining regions include: CDR-L1, CDR-L2, and CDR-L3. Among them, the amino acid sequences of CDR-L1 and CDR-L3 are shown as SEQ ID NO: 4-5 in sequence, and the amino acid sequence of CDR-L2 is WSA.

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

[0020] (1) Detecting dihydromyricetin;

[0021] (2) Assessing the quality of Ampelopsis grossedentata;

[0022] and (3) Preparing a dihydromyricetin detection product;

[0023] The detection product is a reagent, a kit, a test strip, an antibody chip, an antibody probe, or a detector.

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

[0025] In an eighth aspect, the present invention provides a method for detecting dihydromyricetin:

[0026] (1) Add a diluent and a monoclonal antibody against dihydromyricetin or its antigen-binding fragment to the control wells of a microplate coated with dihydromyricetin antigen, and incubate; add a dihydromyricetin standard or a test sample and a monoclonal antibody against dihydromyricetin or its antigen-binding fragment to the inhibition wells of the microplate coated with dihydromyricetin antigen, and incubate; add an enzyme-labeled secondary antibody to the control wells and the inhibition wells respectively for incubation, and after a color reaction, detect the absorbance of the control wells and the inhibition wells. By plotting a standard curve of absorbance versus dihydromyricetin concentration, the concentration of dihydromyricetin in the measured sample is obtained according to the standard curve;

[0027] (2) Load a dihydromyricetin standard or a test sample onto the sample pad of a test strip respectively, plot a standard curve of the ratio of the chromaticity values of the T line and the C line on the test strip versus dihydromyricetin concentration, and obtain the concentration of dihydromyricetin in the measured sample according to the standard curve;

[0028] The test strip includes a sample pad, an antibody-binding pad, a fiber membrane and a blotting paper. The antibody-binding pad is coated with a monoclonal antibody against dihydromyricetin or its antigen-binding fragment with a label. The fiber membrane has a T line and a C line. The T line is coated with dihydromyricetin antigen, and the C line is coated with a secondary antibody against the monoclonal antibody against dihydromyricetin or its antigen-binding fragment.

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

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

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

[0032] The present invention provides a hapten and a complete antigen of dihydromyricetin. By immunizing mice with the complete antigen of dihydromyricetin (i.e., the antigen), hybridoma fusion is carried out, and a hybridoma cell strain is screened out, which can secrete an antibody specifically binding to dihydromyricetin. Through immunodetection experiments, it is proved that the monoclonal antibody against dihydromyricetin provided by the present invention has high specificity for dihydromyricetin, high detection sensitivity for dihydromyricetin, and a wide linear detection range. Therefore, the monoclonal antibody against dihydromyricetin provided by the present invention can be used to develop products such as detection kits and test strips for dihydromyricetin. The proposal of the present invention provides detection tools and means for the rapid detection and evaluation of the quality of Ampelopsis grossedentata, and promotes the healthy development of the Ampelopsis grossedentata industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

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

[0035] Figure 2 Standard curve graph for ELISA detection of dihydromyricetin;

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

[0037] Figure 4 Mass spectrum of the hapten of dihydromyricetin shown in Formula I;

[0038] Figure 5 Molecular weights of the complete antigen and BSA and OVA standards detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS);

[0039] Figure 6 Standard curve constructed for the test strip detection of dihydromyricetin in Ampelopsis grossedentata. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0040] Reference will now be made in detail to embodiments of the present invention, one or more examples of which are described below. Each example is provided by way of explanation and not limitation of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used in another embodiment to yield a still further embodiment.

[0041] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the capabilities of those of ordinary skill in the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Animal Cell Culture (R.I. Freshney ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al. eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al. eds., 1994); and Current Protocols in Immunology (J.E. Coligan et al. eds., 1991), each of which is hereby incorporated by reference in its entirety.

[0042] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not specified by the manufacturer can all be obtained as conventional products through commercial purchase.

[0043] Definition of Terms

[0044] The term "antigen-binding fragment" generally refers to all proteins / protein fragments containing CDR regions, especially antibodies or antibody functional fragments. "Antigen-binding fragments" include antigen compound-binding fragments of the above-mentioned antibodies, including Fab, F(ab’)2, Fd, Fv, scFv, bispecific antibodies, multispecific antibodies and the minimum recognition units of antibodies, as well as single-chain derivatives of these antibodies and fragments. The types of antibodies can be selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD, etc. In addition, the term "antibody" includes naturally occurring antibodies and non-naturally occurring antibodies, including, for example, chimeric, bifunctional and humanized antibodies, as well as related synthetic isoforms. The term "antibody" can be used interchangeably with "immunoglobulin".

[0045] The term "antibody" 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, as long as they exhibit the required biological activities, such as specifically binding to the dihydromyricetin antigen or its fragments.

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

[0047] In the present invention, the heavy chain complementarity determining regions are denoted as HCDR, which include HCDR1, HCDR2, and HCDR3; the light chain complementarity determining regions are denoted as LCDR, which include 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 the immunoglobulin variable region. Over the past few decades, the accumulation of sequences has led to the creation of the KABATMAN database, and the Kabat numbering scheme is generally considered the widely adopted standard for numbering antibody residues. The present invention uses the Kabat annotation standard to label the CDR regions, but the CDR regions labeled by other methods also fall within the scope of protection of the present invention.

[0048] Generally, the variable region VH of the heavy chain of an antibody can be obtained by connecting the CDRs and FRs numbered as follows in the following combined arrangement: HFR1 - HCDR1 - HFR2 - HCDR2 - HFR3 - HCDR3 - HFR4. HCDR1 is synonymous with CDR - H1.

[0049] The variable region VL of the light chain of an antibody can be obtained by connecting the CDRs and FRs numbered as follows in the following combined arrangement: 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 inter - sugar (backbone) linkages. The term also includes modified or substituted sequences containing non - naturally occurring monomers or portions thereof. The nucleic acid molecules of the present invention can be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA), and can contain natural bases, including adenine, guanine, cytosine, thymine, and uracil. Modified bases can also be included. Examples of these modified bases include nitrogen - containing and de - nitrogenated adenine, guanine, cytosine, thymine, and uracil; as well as xanthine and hypoxanthine.

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

[0052]

[0053] The synthesis route of the dihydromyricetin hapten refers to the method of Murakami et al. The ease of esterification reaction of the 6 hydroxyl groups in the dihydromyricetin molecule is: 3-OH > 4′-OH > 5′-OH > 5-OH > 3′-OH > 7-OH. Therefore, the hapten is prepared by the esterification reaction of succinic anhydride with the hydroxyl groups in dihydromyricetin. The reaction route refers to Figure 1 as shown.

[0054] In the second aspect, the present invention provides a dihydromyricetin hapten having the structure shown in Formula II below:

[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 the subsequent conjugation with carrier protein.

[0057] In the third aspect, the present invention provides a dihydromyricetin antigen having the structure shown in Formula III below:

[0058] wherein Carrier protein is a carrier protein;

[0059] In a preferred embodiment of the application 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] In the fourth aspect, the present invention provides a mouse hybridoma cell line, which is deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC NO. 46124. The deposit time is: October 23, 2024. The biological material (strain) submitted is DMY-2H3, and the scientific description is a mouse hybridoma cell line. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the identification result is viable.

[0061] It can secrete monoclonal antibodies against dihydromyricetin.

[0062] In a fifth aspect, the present invention provides a monoclonal antibody against dihydromyricetin or an antigen-binding fragment thereof, which comprises: heavy chain complementarity-determining regions and light chain complementarity-determining regions. The heavy chain complementarity-determining regions include: CDR-H1, CDR-H2 and CDR-H3, and their amino acid sequences are shown as SEQ ID NO: 1-3 in sequence. The light chain complementarity-determining regions include: CDR-L1, CDR-L2 and CDR-L3. Among them, the amino acid sequences of CDR-L1 and CDR-L3 are shown as SEQ ID NO: 4-5 in sequence, and the amino acid sequence of CDR-L2 is WSA.

[0063] Immunodetection experiments prove that the monoclonal antibody against dihydromyricetin provided by the present invention has high specificity for dihydromyricetin, high detection sensitivity for dihydromyricetin, and a wide linear detection range. Therefore, the monoclonal antibody against dihydromyricetin provided by the present invention can be used to develop products such as detection kits and test strips for dihydromyricetin. The proposal of the present invention provides detection tools and means for the rapid detection and evaluation of the quality of Ampelopsis grossedentata, and promotes the healthy development of the Ampelopsis grossedentata industry.

[0064] In a preferred embodiment of the application of the present invention, the antibody or its antigen-binding fragment further comprises 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 as SEQ ID NO: 6-9 in sequence; for example, the heavy chain framework region includes HFR1, HFR2, HFR3 and HFR4 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 as SEQ ID NO: 6-9 in sequence.

[0065] The light chain framework region includes LFR1, LFR2, LFR3 and LFR4 which have at least 80% homology with the amino acid sequences shown as SEQ ID NO: 10-13 in sequence. 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 as SEQ ID NO: 10-13 in sequence.

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

[0067] In a preferred embodiment of the application of the present invention, the antibody or its antigen-binding fragment further comprises a constant region, and the constant region comprises 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 F(ab’)2, Fab’, Fab, Fv and scFv of the antibody.

[0069] The antigen-binding fragment of the above antibody generally has the same binding specificity as the antibody from which it is derived. Those skilled in the art can easily understand from the content recorded in the present invention that the functional fragment of the above antibody can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemically reducing and cleaving disulfide bonds.

[0070] The antigen-binding fragment of the above antibody can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesis using, for example, an automatic peptide synthesizer, such as those 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 a dihydromyricetin antigen in any one of the following:

[0072] (1) Detecting dihydromyricetin;

[0073] (2) Evaluating the quality of Ampelopsis grossedentata;

[0074] And (3) preparing a dihydromyricetin detection product;

[0075] The detection product is a reagent, a kit, a test strip, an antibody chip, an antibody probe or a detector.

[0076] In order to improve the stability of the reagent and extend its shelf life, those skilled in the art can add functional components such as stabilizers and protectants to the reagent as needed. The protein stabilizer is selected from: sucrose, trehalose, BSA, glycerol, mannitol, TritonX-100 and Tween-20. The protectant is selected from cryoprotectants, such as polyols and sugars. The polyols are selected from sorbitol, mannitol or a mixture thereof. The form of the reagent includes, but is not limited to, solid, liquid and semi-solid.

[0077] The antibody chip refers to a chip formed by immobilizing the above antibody against dihydromyricetin or its antigen-binding fragment on a carrier.

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

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

[0080] In a preferred embodiment of the application of the present invention, the solid phase is selected from magnetic microspheres, plastic microspheres, plastic particles, latex microspheres, microtiter plates, glass, capillary tubes, nylon and nitrocellulose membranes.

[0081] Based on the property that the antibody against dihydromyricetin or its antigen-binding fragment specifically binds to dihydromyricetin, this antibody can be used for the quality control of dihydromyricetin in Ampelopsis grossedentata, and can efficiently and accurately determine the content of dihydromyricetin in Ampelopsis grossedentata, and monitor the quality of the active ingredients in Ampelopsis grossedentata.

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

[0083] In a preferred embodiment of the application of the present invention, the detection product further comprises the above-mentioned dihydromyricetin antigen.

[0084] Immunoassay is an analytical method for detecting various substances by using the specific binding reaction of antigen and antibody. When the immunochromatographic assay (colloidal gold) based rapid detection technology is used to detect small molecules, its basic principle is the direct competitive enzyme-linked immunosorbent assay method.

[0085] The kit is preferably a competitive ELISA kit.

[0086] The chip can also be called a suspension array or a liquid array. It includes 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 of various materials and forms. For example, it can be preferably selected from containers with a flat bottom. A more typical preferred example is the multi-well plates widely used in biochemical detection, microplates, microfluidics-based devices (such as microfluidic chips), petri dish-like containers, etc., and is not limited thereto.

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

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

[0090] In an eighth aspect, the present invention provides a method for detecting dihydromyricetin, which includes any one of the following methods:

[0091] (1) Add a diluent and the above-mentioned monoclonal antibody against dihydromyricetin or its antigen-binding fragment to the control wells of a microplate coated with the above-mentioned dihydromyricetin antigen, and incubate; add a dihydromyricetin standard or a sample to be tested and the above-mentioned monoclonal antibody against dihydromyricetin or its antigen-binding fragment to the inhibition wells of a microplate coated with the above-mentioned dihydromyricetin antigen, and incubate; add an enzyme-labeled secondary antibody to the control wells and the inhibition wells respectively for incubation, and after a color reaction, detect the absorbance of the control wells and the inhibition wells, and by plotting a standard curve of absorbance versus dihydromyricetin concentration, obtain the concentration of dihydromyricetin in the sample to be tested according to the standard curve;

[0092] (2) Load a dihydromyricetin standard or a sample to be tested onto the sample pad of a test strip respectively, plot a standard curve of the ratio of the chromaticity values of the T line and the C line on the test strip versus the dihydromyricetin concentration, and obtain the concentration of dihydromyricetin in the sample to be tested according to the standard curve;

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

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

[0095] The coating concentration of the dihydromyricetin antigen on the microplate is 12.5×10 -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 addition concentration of the monoclonal antibody against dihydromyricetin or its antigen-binding fragment is 12.5×10 -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 an absorbent paper. The antibody-binding pad is coated with the above-mentioned monoclonal antibody against dihydromyricetin or its antigen-binding fragment with a label. The fiber membrane has a T line and a C line. The T line is coated with dihydromyricetin antigen, and the C line is coated with a secondary antibody against the monoclonal antibody against dihydromyricetin or its antigen-binding fragment.

[0098] For example, the label 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, and 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 above-mentioned label refers to a class of substances with characteristics such as luminescence, color development, radioactivity, etc. that can be directly observed by the naked eye or detected or detected by an instrument. Through these characteristics, qualitative or quantitative detection of the corresponding target can be achieved. In the actual use process, those skilled in the art can select a suitable label according to the detection conditions or actual needs. No matter which label is used, it falls within the protection scope of the present invention.

[0102] Fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (such as, including but not limited to, fluorescein isothiocyanate (FITC), hydroxy fluorescein (FAM), tetrachloro fluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (such as, including but not limited to, rhodamine B isothiocyanate (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (such as, including but not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, etc. or their analogs), Alexa series dyes and their derivatives (such as, including but not limited to, Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 647, 680, 700, 750, etc. or their analogs), and protein dyes and their derivatives (such as, including but not limited to, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (PerCP), etc.).

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

[0104] In alternative embodiments, the radioisotopes include, but are not limited to 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 an alternative embodiment, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacean luciferin and its derivatives, ruthenium bipyridine and its derivatives, acridinium ester and its derivatives, dioxetane and its derivatives, rhodamine alkaloid and its derivatives, and peroxyoxalate and its derivatives.

[0106] In an alternative embodiment, the nanoparticle-based labels include, but are not limited to, nanoparticles, colloids; the 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 monoclonal antibody against dihydromyricetin or its antigen-binding fragment described above.

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

[0110] In an eleventh aspect, the present invention further provides a vector containing the nucleic acid molecule described above.

[0111] The term "vector" is used herein in its most general sense and includes any intermediate agent for nucleic acids that can enable the nucleic acid to be introduced into prokaryotic and / or eukaryotic cells, for example, and integrated into the genome where appropriate. This type of vector preferably replicates and / or expresses in cells. The term "vector" refers to bacterial plasmids, phages, 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, usually a circular double-stranded DNA, which can replicate independently of chromosomal DNA. Any plasmid and vector can be used as long as it can replicate and be stable in the host.

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

[0113] In a twelfth aspect, the present invention further provides a recombinant cell containing the above vector.

[0114] The term "recombinant cell" refers to any cell that can be transformed or transfected with exogenous nucleic acid. The term "recombinant cell" according to the present invention includes prokaryotes (e.g., Escherichia coli) or eukaryotic cells (e.g., mammalian cells, especially human cells, yeast cells, and insect cells). Mammalian cells are particularly preferred, such as cells from humans, mice, hamsters, pigs, goats, or primates. The cells can be derived from multiple tissue types and include primary cells and cell lines. The nucleic acid can be present in the host cell in a single copy or in two or more copies, and in one embodiment, is expressed in the recombinant cell.

[0115] In an alternative embodiment, the recombinant cell is a eukaryotic cell.

[0116] In an alternative embodiment, the recombinant cell is a mammalian cell.

[0117] In an alternative embodiment, the recombinant cell is HEK293.

[0118] The features and properties of the present invention will be further described in detail below in conjunction with the examples.

[0119] Example 1

[0120] In this example, the preparation of dihydromyricetin hapten was carried out.

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

[0122]

[0123] The synthesis route of the dihydromyricetin hapten refers to the method of Murakami et al. The ease of esterification reaction of the 6 hydroxyl groups of the dihydromyricetin molecule is: 3-OH > 4′-OH > 5′-OH > 5-OH > 3′-OH > 7-OH. Therefore, the hapten was prepared by the esterification reaction of succinic anhydride with the hydroxyl groups in dihydromyricetin. The reaction molar amounts of succinic anhydride and dihydromyricetin were 6.3 mmol and 9.2 mmol respectively. Under the condition of 4-dimethylaminopyridine (DMAP) catalyst, pyridine, as a base catalyst, nucleophilic catalyst, and solvent, is a key reagent for efficiently realizing the selective esterification of dihydromyricetin. Combining with DMAP can improve the yield. The reaction conditions were vigorous stirring at room temperature for 16 hours, and then methanol (120 mL) and water (1 mL) were added to the reaction mixture at room temperature and stirred 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 about 80 - 85%. The dihydromyricetin hapten having the structure shown in Formula I was prepared, and the reaction route is referred to Figure 1 as shown.

[0124] The chemical structure of the product was identified, and the 1H-NMR spectrum of the dihydromyricetin hapten shown in Formula I was referred to 1 as shown, and the mass spectrum was referred to Figure 3 as shown. Figure 4 as shown.

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

[0126]

[0127] The specific steps are as follows:

[0128] Weigh the prepared dihydromyricetin hapten shown in Formula I (0.036 mmol), N-hydroxysuccinimide (NHS, 0.047 mmol) and DCC (0.040 mmol), dissolve them with 0.5 mL of anhydrous N,N-dimethylformamide (DMF), stir and react at room temperature of 25 °C for 6 hours, then centrifuge the reaction solution at 8000 rpm for 5 minutes, take the supernatant for standby, and obtain a reactant containing the dihydromyricetin hapten with the structure shown in Formula II. This reaction product can be directly used for the subsequent conjugation with carrier protein.

[0129] Example 2

[0130] In this example, a complete antigen of dihydromyricetin (dihydromyricetin hapten with the structure shown in Formula II conjugated with BSA) was prepared.

[0131] Slowly drip the supernatant of the activated dihydromyricetin hapten into the carrier protein solution (this carrier protein solution is obtained by dissolving 20 mg of BSA in phosphate buffer (PBS) with a pH value of 6.4 and mixing evenly, and the concentration is 10 mg / mL). The feeding molar ratio of the hapten to the carrier protein is 30:1. After stirring overnight at 4 °C, dialyze the obtained reaction solution 6 times with PBS solution with a pH value of 6.4 and a concentration of 0.01 mol / L, dilute the completely dialyzed reaction product solution to a solution with a concentration of 4 mg / mL, and store it at -20 °C for standby.

[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 detected by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were referred to Figure 5 as shown. The structural formula of dihydromyricetin complete antigen C1 is as shown in the following Formula III:

[0133]

[0134] Example 3

[0135] In this example, the preparation of the dihydromyricetin complete antigen (dihydromyricetin hapten conjugated with OVA having the structure shown in Formula II) was carried out.

[0136] The supernatant of the activated dihydromyricetin hapten was slowly added dropwise to the carrier protein OVA solution (this solution was obtained by dissolving 20 mg of OVA in phosphate buffered saline (PBS) with a pH of 6.4 and mixing well, and the concentration was 10 mg / mL). The feeding molar ratio of the hapten to the carrier protein was 30:1. After stirring overnight at 4 °C, the obtained reaction solution was dialyzed 6 times with a PBS solution having a pH of 6.4 and a concentration of 0.01 mol / L. The dialyzed complete reaction product solution was diluted to a solution with a concentration of 4 mg / mL and stored at -20 °C for later use.

[0137] The product complete antigen was named dihydromyricetin complete antigen C2. The chemical structure of the product was identified. The molecular weights of the complete antigen and the OVA standard were detected using a matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS). The results are shown in Figure 5 The structural formula of dihydromyricetin complete antigen C2 is as shown in Formula IV below:

[0138]

[0139] Example 4

[0140] An antibody was prepared using dihydromyricetin complete antigen C1.

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

[0142] (2) Primary immunization: The diluted dihydromyricetin complete antigen C1 antigen solution (concentration 1 mg / mL) was filtered through a sterile filter and then equal volume of Freund's complete adjuvant was added. It was fully emulsified with a magnetic stirrer until it did not disperse when dropped into water. The emulsified complete antigen was used to inject Balb / c mice intraperitoneally and subcutaneously at multiple points on the back. The injection dose was 0.1 mg of emulsified antigen per mouse.

[0143] (3) Booster immunization: Two weeks after the primary immunization, the diluted complete antigen C1 solution (concentration 1 mg / mL) and equal volume of Freund's incomplete adjuvant were fully emulsified with a magnetic stirrer and then used to immunize mice by subcutaneous multiple point injection on the back. The immunization dose was 0.1 mg of emulsified antigen per mouse. The booster immunization was carried out once every 2 weeks. Starting from the third booster immunization, blood was collected from the orbital cavity on the 7th day after each immunization, and the antibody titer and inhibitory effect were detected by the indirect competitive ELISA method. The coating antigen used in the method was complete antigen C2. Using C2 as the coating antigen helped to avoid screening antibodies with immunological activity against BSA protein, thereby increasing the success rate of screening antibodies that specifically bind dihydromyricetin.

[0144] (4) Boost immunization: Select mice with high titer and good inhibition. Conduct boost immunization on the 10th day after the 5th immunization by intraperitoneal injection of 100 μL of 1 mg / mL without adjuvant.

[0145] (5) Cell fusion

[0146] On the 3rd day after boost immunization, perform hybridoma fusion. Fuse spleen cells of mice with mouse myeloma cells sp2 / 0 using PEG 1450, and conduct selective culture using 2% HAT medium. Screen cell wells with high titer and good inhibition by indirect competitive ELISA method, and perform subcloning by limited dilution. Finally, obtain the monoclonal antibody hybridoma cell line DMY-2H3 of dihydromyricetin. The antibody secreted by this cell line has good specificity for dihydromyricetin, and the detection sensitivity can reach 2.1 μg / L. This cell line is preserved in the China General Microbiological Culture Collection Center with the preservation number CGMCC NO.46124.

[0147] (6) Cryopreservation and resuscitation of cells

[0148] After expanding the obtained hybridoma cell line DMY-2H3, resuspend it by pipetting with DMEM and centrifuge. Freeze it in liquid nitrogen at a density of 1×10 9 cells / mL for long-term preservation. For resuscitation, take out the cryopreservation tube from the liquid nitrogen tank and immediately place it in a 37°C water bath for thawing. Then transfer it to pre-warmed 10 mL DMEM using a pipette, centrifuge to remove the cryopreservation solution, and then transfer it to a culture plate for culture.

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

[0150] Take 8 female BALB / C mice aged 10 - 12 weeks, and inject 0.3 mL of sterile paraffin oil intraperitoneally per mouse. One week later, inject each mouse intraperitoneally with the monoclonal cell line DMY-2H3, with approximately 10 6 cells per mouse. When the abdomen of the mice becomes swollen, start collecting mouse ascites, and purify the ascites by the saturated ammonium sulfate method and store it at -20°C. Thus, obtain the purified monoclonal antibody secreted by the hybridoma cell line DMY-2H3.

[0151] Example 5

[0152] Detect the antibody effect of the monoclonal antibody obtained in Example 4.

[0153] The various buffers used in the following experiments are as follows:

[0154] Coating buffer (0.05M carbonate buffer, pH 9.6): 1.5 g of Na2CO3; 2.94 g of NaHCO3, make up to 1000 mL with pure water;

[0155] Phosphate buffer PBS (0.01M, pH 7.4): 0.2 g of KH2PO4; 8 g of NaCl; 2.92 g of NaH2PO4·12H2O, make up to 1000 mL with pure water;

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

[0157] Sample diluent (PBSTG): Add 1 mL of Tween-20 and 1 g of gelatin (melted by microwave heating) to the prepared PBS, make up to 1 L;

[0158] Chromogenic solution: TMB stock solution (375 mg of TMB solid + 30 mL of DMSO (prepared according to the ratio, stored at room temperature in the dark)), BUFFER (0.1 g of potassium sorbate + 46.04 g of potassium dihydrogen citrate hydrate + 1 L of pure water), the chromogenic solution is prepared immediately before use: 200 μL of TMB stock solution + 11 mL of BUFFER + 3.34 μL of 30% hydrogen peroxide solution;

[0159] Stop solution (2M H2SO4): 445.6 mL of distilled water, add 54.4 mL of concentrated sulfuric acid (98%) drop by drop and stir.

[0160] The following is the checkerboard experiment of antigen and antibody:

[0161] 1) Coating:

[0162] Dihydromyricetin complete antigen C1 at 1 mg / mL was serially diluted with coating buffer at 1:1000, 1:2000, 1:4000, and 1:8000 to obtain coating antigen solutions of dihydromyricetin complete antigen C1 at different concentrations. Add 100 μL of the dihydromyricetin complete antigen C1 coating antigen solution prepared in Example 2 to each well of a 96-well ELISA plate, incubate overnight at 4°C, and wash 3 times with PBST;

[0163] 2) Competition:

[0164] The solid standard of dihydromyricetin was purchased from Chengdu Dest Biotechnology Co., Ltd. Since dihydromyricetin is easily oxidized, the standard was prepared immediately before use. It was prepared into a liquid standard of 1 mg / mL with methanol solvent, and the 1 mg / mL dihydromyricetin standard was diluted to 10 ng / mL with PBSTG. Add 50 μL of sample diluent to each zero well, and add 50 μL of the diluted 10 ng / mL dihydromyricetin standard solution to each inhibition well.

[0165] The dihydromyricetin antibody (1 mg / mL) was serially diluted 1:1000, 1:2000, 1:4000, 1:8000, 1:16000 with PBSTG to obtain antiserum dilutions of dihydromyricetin (50 μL / well), incubated in a humid chamber at 37 °C for 30 min, and the plates were washed 3 times.

[0166] 3) Add enzyme-labeled secondary antibody: Dilute the goat anti-mouse enzyme-labeled secondary antibody (IgG-HRP, Jackson) 10000-fold with PBSTG, add 100 μL to each well, incubate in a humid chamber at 37 °C for 30 min, and wash the plates 3 times.

[0167] 4) Color development: The color developer was prepared freshly. After mixing the prepared TMB solution and hydrogen peroxide in proportion, 100 μL was added to each well and the color was developed 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 an enzyme-linked immunosorbent assay reader.

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

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

[0171]

[0172]

[0173] Note: I represents the inhibition wells in the enzyme-linked immunosorbent assay plate, and C represents the control wells in the enzyme-linked immunosorbent assay plate.

[0174] Table 1 results showed that when the coating antigen dilution was 1:4000 and the antibody dilution was 1:2000, the inhibitory effect on dihydromyricetin was the best, with an inhibition rate of 81.8%, indicating that the antibody produced by the above hybridoma cell DMY-2H3 could detect dihydromyricetin.

[0175] Example 6

[0176] This example provided a standard curve for detecting dihydromyricetin and tested the sensitivity of the detection method.

[0177] Dihydromyricetin reference substance solution was diluted with sample diluent to the following different 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, 0.195 ng / mL. Referring to the checkerboard experiment procedure, a standard curve experiment was carried out. Three parallels were made for each standard concentration. The dilution factor of the coated antigen was selected as 1:4000, and the antibody dilution was 1:2000 for detection.

[0178]

[0179] Drawing the standard curve: Using the dihydromyricetin reference substance solution with 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 reference substance solution and B0 is the average absorbance value of the control well) as the Y-axis, draw the standard curve graph as Figure 2 shown.

[0180] The results show that its sensitivity (IC 50 ) is 2.1 ng / mL, and the sensitivity (IC 50 ) is the concentration value of the dihydromyricetin reference substance when the inhibition rate reaches 50%.

[0181] The linear detection range is 0.46 ng / mL - 9.54 ng / mL. It shows that the antibody prepared in Example 4 above has a good detection effect and high sensitivity.

[0182] Example 7

[0183] In this example, the antibody prepared in Example 4 was specifically detected.

[0184] Solid reference substances of myricetin, myricitrin, naringenin, and quercetin purchased from Chengdu Deste Biotechnology Co., Ltd. were prepared into liquid standards of 1 mg / mL with methanol solvent, prepared as needed, and the above 4 kinds of analog liquid standards were respectively diluted into the following concentrations with the sample diluent in Example 5: 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.

[0185] According to the method in Example 6, a standard curve was established, and the inhibitory concentration IC 50 (the concentration value of the standard sample when the inhibition rate reaches 50%) was measured.

[0186] Cross-reactivity rate (%) = (dihydromyricetin IC 50 ) / (analog IC 50)×100%.

[0187]

[0188]

[0189] The results showed that the antibody produced by the hybridoma cell DMY-2H3 had good specificity for dihydromyricetin.

[0190] Example 8

[0191] This example provides a method for detecting dihydromyricetin in Ampelopsis grossedentata, which is as follows:

[0192] 1. Development of colloidal gold immunochromatographic test strip

[0193] 1) Preparation of gold-labeled antibody pad: Take 1 mL of colloidal gold solution (40 nm) in a 2 mL centrifuge tube (rinsed with pure water), add 3 μL of 0.2 M K2CO3 solution to it, then add 6 μg of antibody, mix well by inverting up and down and let it stand for 20 min, then add 50 μL of 3% BSA for blocking, mix well by inverting up and down and let it stand for 20 min. Then use a high-speed refrigerated centrifuge to centrifuge the mixture at 10000 r / min for 10 min (4 °C). 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 on a 1 cm × 15 cm sample pad, and dry it in an oven at 37 °C (1.5 h).

[0194] 2) Membrane scribing: Use a gold-spraying membrane scribing instrument to scribe the nitrocellulose membrane attached to the bottom plate. The concentration of the T-line antigen (dihydromyricetin complete antigen C1) is 1.5 mg / mL, and the concentration of the C-line secondary antibody is 1.0 mg / mL.

[0195] 3) Assembly: Assemble in the order of sample pad, colloidal gold pad, nitrocellulose membrane and absorbent paper in turn, and then use a cutting machine to cut it into test strips 4 mm wide for subsequent testing.

[0196] 2. Establishment of standard curve

[0197] Prepare dihydromyricetin standard products with concentrations of 5, 10, 20, 50, 100, 200, 500, 1000, 2000 ng / mL with pure water, and establish a standard curve according to the test results of the test strip for reference Figure 6, an equation for the concentration of dihydromyricetin and the T / C value of the test strip was established: y = A2 + (A1 - A2) / (1 + (x / x0)^p), where 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 test sample can be calculated based on the T / C value.

[0198] 3. Detection of actual samples

[0199] According to the standard curve established above, the content of dihydromyricetin in dried and fresh Ampelopsis grossedentata tea from different origins was detected using a colloidal gold immunochromatographic test strip (Lateral Flow Immunoassay, LFIA), and the same samples were detected by high performance liquid chromatography as a control. The treatment method of tea leaves during LFIA detection is as follows:

[0200] 1) Dried tea leaves: 1 g + 50 ml (soaked in hot water at 80 - 100 °C for 3 minutes, take a drop of about 30 μL and add it to a centrifuge tube containing 4 ml of dilution solution, and dilute for detection according to the dilution factor.

[0201] 2) Fresh tea leaves: Grind 1 g of fresh tea leaves with a grinding rod, add 50 ml (soaked in hot water at 80 - 100 °C for 3 minutes, take a drop of about 30 μL and add it to a centrifuge tube containing 4 ml of dilution solution, and dilute for detection according to the dilution factor.

[0202]

[0203] The detection results of LFIA and HPLC are basically the same, indicating that the test strip prepared by the present invention is reliable in detecting the concentration of dihydromyricetin in Ampelopsis grossedentata tea.

[0204] The sequence information involved in the present invention is as follows:

[0205]

[0206]

[0207] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A dihydromyricetin hapten, characterized in that, It has the structure shown in Formula I as follows:

2. A dihydromyricetin hapten, characterized in that, It has the structure shown in Formula II as follows:

3. A dihydromyricetin antigen, characterized in that, The dihydromyricetin antigen has the structure shown in Formula III as follows: wherein Carrier protein is a carrier protein; Preferably, the carrier protein is BSA, OVA, keyhole limpet hemocyanin, diphtheria toxoid or tetanus toxoid.

4. A hybridoma cell, characterized in that, It is preserved in the China General Microbiological Culture Collection Center with the preservation number of CGMCC NO. 46124.

5. A monoclonal antibody against dihydromyricetin or an antigen-binding fragment thereof, characterized in that, It includes: Heavy chain complementarity determining regions and light chain complementarity determining regions. The heavy chain complementarity determining regions include CDR-H1, CDR-H2 and CDR-H3, and their amino acid sequences are shown as SEQ ID NO: 1-3 in sequence. The light chain complementarity determining regions include CDR-L1, CDR-L2 and CDR-L3. Among them, the amino acid sequences of CDR-L1 and CDR-L3 are shown as SEQ ID NO: 4-5 in sequence, and the amino acid sequence of CDR-L2 is WSA; Preferably, 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 as SEQ ID NO: 6-9 in sequence; the light chain framework region includes LFR1, LFR2, LFR3 and LFR4 which have at least 80% homology with the amino acid sequences shown as SEQ ID NO: 10-13 in sequence; Preferably, the antibody or its antigen-binding fragment further includes a constant region. The constant region 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 regions; The antigen-binding fragment is selected from any one of F(ab’)2, Fab’, Fab, Fv and scFv of the antibody.

6. Use of the monoclonal antibody against dihydromyricetin or its antigen-binding fragment according to claim 5, or the dihydromyricetin antigen according to claim 3 in any one of the following: (1) Detecting dihydromyricetin; (2) Evaluating the quality of Ampelopsis grossedentata; and (3) Preparing a dihydromyricetin detection product; The detection product is a reagent, a kit, a test strip, an antibody chip, an antibody probe or a detector.

7. A dihydromyricetin detection product, characterized in that The detection product is a reagent, a kit, a test strip, an antibody chip, an antibody probe or a detector; the detection product includes the monoclonal antibody against dihydromyricetin or its antigen-binding fragment according to claim 5, or the antibody secreted by the hybridoma cell according to claim 4; Preferably, the detection product further includes the dihydromyricetin antigen according to claim 3.

8. A method for detecting dihydromyricetin, characterized in that, It includes any one of the following methods: (1) Add dilution solution and the monoclonal antibody against dihydromyricetin or its antigen-binding fragment as claimed in claim 5 to the control wells of the microplate coated with the dihydromyricetin antigen as claimed in claim 3, and incubate; add dihydromyricetin standard or the sample to be tested and the monoclonal antibody against dihydromyricetin or its antigen-binding fragment as claimed in claim 5 to the inhibition wells of the microplate coated with the dihydromyricetin antigen as claimed in claim 3, and incubate; add enzyme-labeled secondary antibody to the control wells and the inhibition wells respectively for incubation, and after color development reaction, detect the absorbance of the control wells and the inhibition wells, and obtain the concentration of dihydromyricetin in the sample to be tested by plotting the standard curve of absorbance against dihydromyricetin concentration and according to the standard curve; (2) Load the dihydromyricetin standard or the sample to be tested onto the sample pad of the test strip respectively, plot the standard curve of the ratio of the chromaticity values of the T line and the C line on the test strip against the dihydromyricetin concentration, and obtain the concentration of dihydromyricetin in the sample to be tested according to the standard curve; The test strip includes a sample pad, an antibody-binding pad, a fiber membrane and a blotting paper. The antibody-binding pad is coated with the monoclonal antibody against dihydromyricetin or its antigen-binding fragment as claimed in claim 5 with a label. The fiber membrane has a T line and a C line. The T line is coated with the dihydromyricetin antigen as claimed in claim 3, and the C line is coated with a secondary antibody against the monoclonal antibody against dihydromyricetin or its antigen-binding fragment; Preferably, the coating concentration of dihydromyricetin antigen on the microplate is 12.5×10 -6 -1×10 -3 mg / mL; the addition concentration of the monoclonal antibody against dihydromyricetin or its antigen-binding fragment is 12.5×10 -6 -5×10 -4 mg / mL; Preferably, the label is a colloid; Preferably, the colloid is selected from colloidal gold, colloidal silver or colloidal selenium.

9. An antibody conjugate, characterized in that, It is formed by conjugating the monoclonal antibody against dihydromyricetin or its antigen-binding fragment as claimed in claim 5 with a label, and the label is selected from at least one of fluorescent dyes, enzymes that catalyze substrate color development, radioisotopes, chemiluminescent reagents and nanoparticle-based labels.

10. A nucleic acid molecule, characterized in that, It encodes the monoclonal antibody against dihydromyricetin or its antigen-binding fragment as claimed in claim 5.

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