Anti-atropine antibodies and uses thereof

Abstract

The present application belongs to the field of biotechnology, and specifically relates to an anti-atropine antibody or antigen-binding fragment thereof, an isolated nucleic acid molecule, a vector, a cell, a preparation, a conjugate, an immunochromatography test strip, a detection kit, a method for the anti-atropine antibody or antigen-binding fragment thereof, and use of the anti-atropine antibody or antigen-binding fragment thereof in detecting the presence or level of atropine alkaloids in a sample.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to anti-atropine antibodies and their uses. Background Technology

[0002] Atropine (ATR) is an anticholinergic drug, an M-receptor blocker. These drugs exhibit pharmacological activities such as inhibiting glandular secretion, relieving smooth muscle spasms, stimulating the respiratory center, and relieving vagal nerve inhibition of the heart. For years, research on atropine has primarily focused on the medical field, with few reports on the dangers of exceeding therapeutic doses or monitoring of illegal use. Extensive research data indicates that excessive atropine intake can induce adverse reactions such as blurred vision, pupil dilation, dry mouth, skin redness and swelling, vomiting, and tachycardia; in severe cases, it can lead to death. Driven by profit, some unscrupulous vendors exploit atropine's ability to inhibit glandular secretion by artificially inducing thirst and excessive water consumption in livestock to illegally gain weight. After slaughter and processing, incompletely metabolized atropine residues remain in the meat products. Long-term or excessive consumption of meat products containing atropine residues by consumers can easily lead to acute poisoning and chronic cumulative poisoning, seriously endangering human health and food safety. Therefore, it is particularly important to test the content of atropine alkaloids in finished meat products.

[0003] Currently, commonly used methods for detecting atropine residues in animal-derived finished meat products include spectrophotometry, high-performance liquid chromatography (HPLC), and coupled-instrument methods. Spectrophotometry does not rely on sophisticated equipment and offers advantages such as ease of operation and rapid detection; however, its detection limit is relatively high, making it unsuitable for trace analysis of low concentrations of atropine residues in food. HPLC and coupled-instrument methods, on the other hand, require expensive equipment, are time-consuming, and demand high levels of expertise from both the laboratory environment and the operators. In recent years, immunological methods have become an important means of initial screening for large batches of samples before confirmatory testing for veterinary drug residues due to their advantages of ease of operation, rapid detection, high sensitivity, and low operator barriers. Summary of the Invention

[0004] In order to solve one of the aforementioned technical problems in the prior art, the present invention provides an anti-atropine antibody and its uses.

[0005] A first aspect of the present invention provides an anti-atropine antibody or an antigen-binding fragment thereof, the anti-atropine antibody or the antigen-binding fragment thereof comprising: a1) having CDR-H1, CDR-H2, and CDR-H3 included in the heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 2; and / or, having CDR-L1, CDR-L2, and CDR-L3 included in the light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 4; or a2) having one or more amino acid substitutions, deletions, or additions compared to CDR-H1, CDR-H2, and CDR-H3 as shown in a1); and / or having one or more amino acid substitutions, deletions, or additions compared to CDR-L1, CDR-L2, and CDR-L3 as shown in a1); The CDR is defined according to the Kabat, Chothia, IMGT, Contact, or AbM numbering system.

[0006] In some embodiments, the anti-atropine antibody or its antigen-binding fragment comprises: b1) A VH comprising the following three CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 5, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 6, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 7; and / or, a VL comprising the following three CDRs: CDR-L1 having the amino acid sequence shown in SEQ ID NO: 8, CDR-L2 having the amino acid sequence shown in SEQ ID NO: 9, and CDR-L3 having the amino acid sequence shown in SEQ ID NO: 10; or b2) VH comprising the following three CDRs: having one or more amino acid substitutions, deletions, or additions compared to CDR-H1, CDR-H2, and CDR-H3 as shown in b1); and / or VL comprising the following three CDRs: having one or more amino acid substitutions, deletions, or additions compared to CDR-L1, CDR-L2, and CDR-L3 as shown in b1); The CDR is defined according to the Kabat numbering system.

[0007] In some embodiments, the heavy chain variable region of the anti-atropine antibody or its antigen-binding fragment further includes a framework region of the heavy chain variable region.

[0008] In some embodiments, the framework region of the heavy chain variable region includes the framework region of the heavy chain variable region derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, or geese, or a mutant thereof. In some embodiments, the framework region of the heavy chain variable region includes the framework region of the heavy chain variable region derived from immunoglobulins of mice, rats, lemurs, macaques, chimpanzees, or humans, or a mutant thereof. In some embodiments, the framework region of the heavy chain variable region includes the framework region of the heavy chain variable region derived from mouse immunoglobulins, or a mutant thereof.

[0009] In some embodiments, the light chain variable region of the anti-atropine antibody or its antigen-binding fragment further includes a framework region of the light chain variable region.

[0010] In some embodiments, the framework region of the light chain variable region includes the framework region of the light chain variable region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, or geese. In some embodiments, the framework region of the light chain variable region includes the framework region of the light chain variable region or a mutant thereof derived from immunoglobulins of mice, rats, lemurs, macaques, chimpanzees, or humans. In some embodiments, the framework region of the light chain variable region includes the framework region of the light chain variable region or a mutant thereof derived from mouse immunoglobulins.

[0011] In some embodiments, the anti-atropine antibody or its antigen-binding fragment comprises: The heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and / or the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0012] In some embodiments, the anti-atropine antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region.

[0013] In some embodiments, the heavy chain constant region includes at least a portion of a heavy chain constant region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, or geese. In some embodiments, the heavy chain constant region includes at least a portion of a heavy chain constant region or a mutant thereof derived from immunoglobulins of mice, rats, lemurs, macaques, chimpanzees, or humans. In some embodiments, the heavy chain constant region includes a heavy chain constant region or a mutant thereof derived from mouse immunoglobulins.

[0014] In some embodiments, the light chain constant region includes at least a portion of a light chain constant region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, or geese. In some embodiments, the light chain constant region includes at least a portion of a light chain constant region or a mutant thereof derived from immunoglobulins of mice, rats, lemurs, macaques, chimpanzees, or humans. In some embodiments, the light chain constant region includes a light chain constant region or a mutant thereof derived from mouse immunoglobulins.

[0015] In some embodiments, the heavy chain constant region includes the heavy chain constant region or a mutant thereof derived from IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 or IgM immunoglobulin.

[0016] In some embodiments, the light chain constant region includes light chain constant regions derived from κ and λ immunoglobulins.

[0017] In some embodiments, the heavy chain constant region includes the heavy chain constant region derived from IgG1 immunoglobulin or a mutant thereof.

[0018] In some embodiments, the light chain constant region includes a light chain constant region derived from κ-type immunoglobulin.

[0019] In some embodiments, the anti-atropine antibody or its antigen-binding fragment is a murine antibody, a chimeric antibody, or a humanized antibody.

[0020] In some embodiments, the antigen-binding fragment of the anti-atropine antibody is a Fab fragment, a Fab' fragment, a Fab'-SH fragment, an F(ab')2 fragment, an Fv fragment, a single-chain Fv (scFv), a dsFv, or an Fd fragment.

[0021] A second aspect of the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the anti-atropine antibody or an antigen-binding fragment thereof described in the first aspect.

[0022] Those skilled in the art will understand that nucleotides in nucleic acid molecules can be substituted based on codon degeneracy. In some embodiments, the nucleotide sequence of the nucleic acid molecule is codon-optimized.

[0023] In some embodiments, the nucleic acid molecule encoding the heavy chain variable region of the anti-atropine antibody or its antigen-binding fragment according to the first aspect of the invention comprises: SEQ ID NO: 1, or a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0024] In some embodiments, the nucleic acid molecule encoding the light chain variable region of the anti-atropine antibody or its antigen-binding fragment according to the first aspect of the invention comprises: SEQ ID NO: 3, or a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0025] A third aspect of the present invention provides a carrier comprising the nucleic acid molecule described in the second aspect.

[0026] In some embodiments, the vector may be an expression vector. In some embodiments, the expression vector may include eukaryotic expression vectors and / or prokaryotic expression vectors. In some embodiments, the eukaryotic expression vector includes, but is not limited to, yeast expression vectors, mammalian expression vectors, and insect expression vectors. For example, the expression vector may include, but is not limited to, plasmids, retroviral vectors, lentiviral vectors, bacteriophage vectors, adenovirus vectors, adeno-associated vectors, or herpes simplex vectors.

[0027] In some embodiments, the carrier may be selected from nanoparticles, liposomes, exogenous bodies, microbubbles, or gene guns.

[0028] A fourth aspect of the present invention provides a cell comprising the anti-atropine antibody or its antigen-binding fragment as described in the first aspect, the nucleic acid molecule as described in the second aspect, or the vector as described in the third aspect.

[0029] In some embodiments, the cells do not involve reproductive material.

[0030] In some embodiments, the cell can be a host cell conventionally used in the art, as long as the expression vector stably expresses the carried nucleic acid molecule as the LMP1 antibody or its antigen-binding fragment described above. In some embodiments, the host cell can be a prokaryotic cell and / or a eukaryotic cell. The prokaryotic cell may include, for example, *Escherichia coli*, and the eukaryotic cell may include, for example, CHO cells, HEK293 cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, HeLa cells, Vero cells, Expi293 cells, hybridoma cells, yeast cells, and insect cells.

[0031] The fifth aspect of the present invention provides a method for preparing the anti-atropine antibody or its antigen-binding fragment as described in the first aspect, the method comprising: culturing the cells described in the fourth aspect, and collecting the anti-atropine antibody or its antigen-binding fragment from the cultured cell culture.

[0032] A sixth aspect of the present invention provides a conjugate comprising the anti-atropine antibody or an antigen-binding fragment thereof described in the first aspect; and a conjugation portion.

[0033] In some implementations, the coupling portion is a detectable marker.

[0034] In some embodiments, the detectable marker can be any substance detectable by means of fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, chemical, etc. Such markers are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridine esters, magnetic beads, calorimetric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) microbeads, and biotin for binding avidin (e.g., streptavidin) modified with the above markers. In some embodiments, such markers are suitable for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable marker is selected from radioactive isotopes, fluorescent substances, luminescent substances, colored substances, or enzymes. In some embodiments, the detectable markers described above can be linked to the anti-atropine antibody or its antigen-binding fragment of the present invention via linkers of different lengths to reduce potential steric hindrance.

[0035] In some embodiments, the detectable marker may include, but is not limited to, enzymes (e.g., horseradish peroxidase), radionuclides, fluorescent dyes, luminescent substances (e.g., chemiluminescent substances), colored substances, biotin, etc.

[0036] A seventh aspect of the present invention provides an immunochromatographic test strip, the test strip comprising a base plate, a conjugate pad, a sample pad, a nitrocellulose membrane, and an absorbent pad, wherein the sample pad, the conjugate pad, the nitrocellulose membrane, and the absorbent pad are sequentially connected and fixed to the base plate in a horizontal direction, the conjugate pad is loaded with a fluorescent microsphere-antibody conjugate, and the antibody is the anti-atropine antibody or its antigen-binding fragment described in the first aspect; the nitrocellulose membrane is provided with a detection line and a control line, and the detection line is coated with complete atropine antigen.

[0037] In some embodiments, the atropine complete antigen is obtained by conjugation of atropine and ovalbumin.

[0038] In some embodiments, the control line is coated with goat anti-mouse IgG antibody.

[0039] The eighth aspect of the present invention provides a detection kit comprising: the anti-atropine antibody or its antigen-binding fragment as described in the first aspect, the nucleic acid molecule as described in the second aspect, the vector as described in the third aspect, the cell as described in the fourth aspect, the conjugate as described in the sixth aspect, or the immunochromatographic test strip as described in the seventh aspect.

[0040] In some embodiments, the test kit is used to detect the presence or level of atropine alkaloids in a sample.

[0041] In some embodiments, the atropine alkaloids include atropine, scopolamine, or anisodamine.

[0042] The ninth aspect of the present invention provides the use of the anti-atropine antibody or its antigen-binding fragment described in the first aspect, the nucleic acid molecule described in the second aspect, the vector described in the third aspect, the cell described in the fourth aspect, the conjugate described in the sixth aspect, the immunochromatographic test strip described in the seventh aspect, or the detection kit described in the eighth aspect in at least one of (1)-(2): (1) To detect the presence or level of atropine alkaloids in the sample, this test is not for diagnostic purposes; (2) Prepare a product, which is used to detect the presence or level of atropine alkaloids in a sample.

[0043] In some embodiments, the atropine alkaloids include atropine, scopolamine, or anisodamine.

[0044] The tenth aspect of this invention provides a method for detecting the presence or level of atropine alkaloids in a sample, the method comprising the steps of: contacting the sample with the anti-atropine antibody or its antigen-binding fragment as described in the first aspect, allowing the anti-atropine antibody or its antigen-binding fragment to form a complex with the atropine alkaloids, and detecting the formation of the complex. This detection is for non-diagnostic purposes.

[0045] In some embodiments, the atropine alkaloids include atropine, scopolamine, or anisodamine. Attached Figure Description

[0046] Figure 1 : Schematic diagram of the synthesis process of ATR complete antigen by succinic anhydride method.

[0047] Figure 2 The results of MS identification of ATR hapten.

[0048] Figure 3 : Schematic diagram of the one-step synthesis process of ATR complete antigen by CDI.

[0049] Figure 4UV spectrum of ATR immunogen prepared by CDI method (ATR immunogen was dissolved in two different solutions: left image is physiological saline, right image is PBS).

[0050] Figure 5 UV spectrum of ATR immunogen prepared by succinic anhydride method (ATR immunogen was dissolved in two different solutions: left image is physiological saline, right image is PBS).

[0051] Figure 6 UV spectra of CDI-coated and succinic anhydride-coated antigens (left: CDI-coated antigen; right: succinic anhydride-coated antigen).

[0052] Figure 7 ATR hybridoma cell screening results.

[0053] Figure 8 Results of 5D8 antibody affinity constant determination.

[0054] Figure 9 Electrophoresis image of 5D8 antibody.

[0055] Figure 10 The results are as follows: sensitivity test results of the test strips, where 1 to 9 represent the test results of atropine at 0.0 μg / kg, 0.1 μg / kg, 0.2 μg / kg, 0.3 μg / kg, 0.5 μg / kg, 1 μg / kg, 2 μg / kg, 3 μg / kg, and 5 μg / kg, respectively.

[0056] Figure 11 The results of the test strip sensitivity test are shown below. 1 to 9 represent the test results of scopolamine at concentrations of 0.0 μg / kg, 0.1 μg / kg, 0.2 μg / kg, 0.3 μg / kg, 0.5 μg / kg, 1 μg / kg, 2 μg / kg, 3 μg / kg, and 5 μg / kg, respectively.

[0057] Figure 12 The results of the sensitivity test of the test strip are shown in the figure. 1 to 9 represent the detection results of 0.0 μg / kg, 0.1 μg / kg, 0.2 μg / kg, 0.3 μg / kg, 0.5 μg / kg, 1 μg / kg, 2 μg / kg, 3 μg / kg and 5 μg / kg, respectively.

[0058] Figure 13 The test strip specificity test results include: 1. Atropine standard; 2. Clenbuterol; 3. Sulfamethazine; 4. Zopiclone; 5. Tetracycline. Detailed Implementation

[0059] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention. Such structures and techniques have also been described in many publications.

[0060] definition Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0061] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0062] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0063] As used herein, the term "antibody" refers to a globulin produced by plasma cells, which are produced by the proliferation and differentiation of B lymphocytes in response to antigen stimulation. Antibodies specifically bind to the corresponding antigens and mediate immune effects. They are mainly found in serum and body fluids and are important immune molecules mediating humoral immunity. Antibodies can encompass various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific, trispecific, or tetraspecific antibodies), single-chain molecules, and antigen-binding fragments. The chemical basis of antibodies is immunoglobulin (Ig). It should be understood that the antibodies described herein can be used to prepare chimeric antigen receptors (CARs), for example, by integrating the antigen-binding domain of the antibody into a CAR construct, or to prepare immune cells (such as T cells or NK cells) expressing the CAR, as well as for therapeutic methods or pharmaceutical compositions for related diseases.

[0064] As used herein, the term "monoclonal antibody" refers to antibodies derived from a substantially homogeneous group of antibodies, meaning that, apart from possible trace amounts of variant antibodies (e.g., containing naturally occurring mutations or generated during the production of the monoclonal antibody formulation, typically present in small quantities), the individual antibodies within the group are identical and / or bind to the same epitopes. Unlike polyclonal antibody formulations, which typically comprise different antibodies targeting different antigenic determinants (epitaxes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen.

[0065] As used herein, the term "multispecific antibody" is used in its broadest sense to encompass antibodies exhibiting multi-epitope specificity. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit exhibits multi-epitope specificity; antibodies having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; antibodies having two or more single variable regions, each single variable region binding to a different target or a different epitope of the same target; full-length antibodies, antibody fragments, bispecific antibodies, and trispecific antibodies, antibody fragments covalently or non-covalently linked, etc.

[0066] The terms “full-length antibody” and “intact antibody” as used herein are used interchangeably to refer to antibodies that are structurally similar to natural antibodies. “Natural antibody” refers to a naturally occurring immunoglobulin molecule. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH) (also called a variable heavy chain domain or heavy chain variable domain) and three constant domains (CH1, CH2, and CH3) (also called heavy chain constant regions, CH). From the N-terminus to the C-terminus, each light chain has a variable region (VL) (also called a variable light chain domain or light chain variable domain) and a light chain constant domain (CL) (also called light chain constant region). The heavy chain of an antibody can be one of five types: α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), and can be further subdivided into subtypes such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chain of an antibody, based on the amino acid sequence of its constant domain, can be one of two types: the κ (kappa) light chain and the λ (lambda) light chain.

[0067] Within the light and heavy chains, variable and constant regions are linked by a "J" region containing approximately 12 or more amino acid residues, and the heavy chain also contains a "D" region containing approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0068] The term "Fd fragment" as used herein refers to an antibody fragment consisting of VH and CH1 domains. The term "dAb fragment" as used herein refers to an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544 546 (1989)). The term "Fab fragment" as used herein refers to an antibody fragment consisting of VL, VH, CL, and CH1 domains. The term "F(ab')2 fragment" as used herein refers to an antibody fragment containing two Fab fragments linked by disulfide bridges on the hinge region. The term "Fab' fragment" as used herein refers to the fragment obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light and heavy chain Fd fragment (composed of VH and CH1 domains). The term "Fab'-SH" as used herein refers to a Fab fragment containing free thiol groups.

[0069] As used in this article, the term "Fv fragment" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. The Fv fragment is generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as the Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.

[0070] As used herein, the term "scFv" refers to a single polypeptide chain containing VL and VH domains linked by a linker. In some cases, a disulfide bond may also exist between the VH and VL domains of the scFv.

[0071] As used herein, the term "variable region" or "variable domain" refers to the domain of the antibody heavy or light chain involved in the binding of the antigen-binding molecule to the antigen. The variable regions (VH and VL) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved framework regions (FR1-4) and three hypervariable regions (HVR1-3), arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL domain is sufficient to confer antigen-binding specificity. The three HVRs within the VH and VL together constitute the antigen-binding site of Ig, which can bind complementary to the corresponding antigenic epitope; therefore, the HVRs are also called complementarity-determining regions (CDRs), denoted as CDR1, CDR2, and CDR3, respectively. The VH or VL chain of the antibody may further contain all or part of the constant regions of the heavy or light chain.

[0072] The CDR of the antibody or antigen-binding fragment of the present invention can be determined according to various numbering systems known in the art. In some embodiments, the CDR contained in the antibody or antigen-binding fragment of the present invention is preferably determined by the IMGT, Kabat, Contact, Chothia, or AbM numbering system.

[0073] Antibody "classes" refer to the types of constant structural domains or constant regions possessed by the antibody's heavy chain. Based on differences in heavy chain structure and antigenicity, they can be classified into five classes: μ chain, γ chain, α chain, δ chain, and ε chain. Immunoglobulins composed of different heavy and light chains are respectively called IgA, IgD, IgE, IgG, and IgM. Even within the same class of Ig, the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain differ, thus further subdividing the same class of Ig into different subclasses. For example, human IgG can be divided into IgG1–IgG4; IgA can be divided into IgA1 and IgA2. Based on differences in light chain structure and antigenicity, immunoglobulin (Ig) light chains are divided into κ (kappa) chains and λ (lambda) chains, thus classifying Ig into two types: κ type and λ type.

[0074] "Humanized antibodies" comprise amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, humanized antibodies comprise at least one, typically two, variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to the HVRs of the non-human antibody, and all or substantially all FRs correspond to the FRs of the human antibody. Humanized antibodies may optionally comprise at least a portion of the antibody constant region derived from a human antibody. Antibodies in a "humanized form," such as non-human antibodies, refer to antibodies that have undergone humanization.

[0075] As used herein, the terms “polynucleotide,” “nucleic acid,” or “nucleotide sequence” refer to isolated nucleic acid molecules or constructs, such as messenger RNA (mRNA), virus-derived RNA, or plasmid DNA (pDNA). Polynucleotides may contain conventional phosphodiester bonds or unconventional bonds (such as amide bonds, as found in peptide nucleic acids (PNAs)). The term “nucleic acid molecule” refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide.

[0076] An "antibody fragment" or "antigen-binding fragment" contains a portion of a complete antibody that retains the antibody's antigen-binding activity. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv; bisomatic antibodies, trisomatic antibodies, tetrasomatic antibodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments and single-domain antibodies (single-domain antibodies).

[0077] As used herein, the terms "antigen-binding domain" or "antigen-binding site" refer to the portion of an antigen-binding molecule that specifically binds to an antigenic determinant. More specifically, the term "antigen-binding domain" refers to a portion of an antibody containing a region that specifically binds to and is complementary to a portion or all of the antigen. In cases where the antigen molecule is large, the antigen-binding molecule may bind only a specific portion of the antigen, called an epitope. The antigen-binding domain may be provided by, for example, one or more variable domains (also called variable regions). Preferably, the antigen-binding domain comprises a variable region (VL) of the antibody light chain and a variable region (VH) of the antibody heavy chain. In one aspect, the antigen-binding domain is capable of binding its antigen and blocking or partially blocking the function of said antigen.

[0078] The term "hapten," used in this article, is also known as an incomplete antigen. Its key characteristic is that it cannot induce an immune response when it exists alone, meaning it lacks immunogenicity. However, when a hapten is cross-linked or bound (usually in the form of a covalent bond) to a large protein molecule or a non-antigenic carrier such as polylysine, it acquires immunogenicity and thus induces an immune response. Therefore, a hapten itself only possesses immunoreactivity (antigenicity) and lacks immunogenicity. A "complete antigen," on the other hand, refers to a substance that simultaneously possesses both immunogenicity and immunoreactivity—two crucial characteristics.

[0079] The specific “binding strength” or “affinity” of an antibody or its antigen-binding fragment to an antigen refers to the strength of the non-covalent interaction between a single binding site and its binding ligand (e.g., antigen), and can be distinguished from unwanted or non-specific binding. The ability of an antigen-binding molecule to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) techniques and conventional binding assays.

[0080] As used herein, the term "isolated" nucleic acid molecule or polynucleotide refers to a nucleic acid molecule, DNA, or RNA, that has been separated from its natural environment. In this invention, the recombinant polynucleotide encoding an antibody or its antigen-binding fragment contained in the vector is also isolated. Other examples of isolated polynucleotides include recombinant polynucleotides in heterologous host cells or polynucleotides purified in solution. Isolated polynucleotides include polynucleotide molecules typically found in cells containing the polynucleotide molecule, but which are located extrachromosomally or at chromosomal locations different from their natural chromosomal locations. Isolated RNA molecules include in vivo or in vitro RNA transcripts of this invention, in positive and negative strand forms, and in double strand forms. The isolated polynucleotides or nucleic acids of this invention further include synthetically generated molecules of this type. Additionally, the polynucleotide or nucleic acid may be or may include regulatory elements such as promoters, ribosome binding sites, or transcription terminators.

[0081] As used herein, the terms "vector" or "expression vector" and "expression construct" are used interchangeably to refer to a DNA molecule to which a specific gene, operatively linked, is introduced into a target cell and directed for expression. The vector includes a vector as a self-replicating nucleic acid structure and a vector incorporated into the genome of the host cell into which it has been introduced. The expression vector of the present invention comprises an expression cassette. The expression vector can be transcribed into a large amount of stable mRNA. Once the expression vector is in the target cell, a ribonucleic acid molecule or protein encoded by the gene is generated by cellular transcription and / or translation mechanisms. In one embodiment, the expression vector of the present invention comprises an expression cassette containing a polynucleotide sequence encoding an antibody of the present invention or an antigen-binding fragment thereof. The term "expression cassette" of the present invention refers to a recombinant or synthetically generated polynucleotide having a series of nucleic acid elements that allow a specific nucleic acid to be transcribed in a target cell. The recombinant expression cassette can be introduced into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Typically, in addition to other sequences, the recombinant expression cassette portion of the expression vector includes the nucleic acid sequence to be transcribed and a promoter. In some embodiments, the expression cassette of the present invention comprises a polynucleotide sequence encoding an antibody of the present invention or an antigen-binding fragment thereof.

[0082] As used herein, the terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” including primary transformed cells and their derived progeny. The nucleic acids of the progeny may not be completely identical to those of the parent cells and may contain mutations. Host cells are any type of cell that can be used to generate the antibodies or antigen-binding fragments of the present invention. Host cells include cultured cells, such as cultured mammalian cells, such as CHO cells, HEK293 cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells, yeast cells, insect cells, and plant cells, as well as cells contained within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0083] The "sequence identity percentage" or "identity percentage" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location where the same nucleotide or amino acid is present in both the target and reference sequences. Vacancies are not nucleotides or amino acids and are not counted in the target sequence. Similarly, vacancies in the reference sequence are not counted because nucleotides or amino acids from the target sequence are counted, but those from the reference sequence are not.

[0084] The percentage of sequence identity can be calculated as follows: determine the number of positions in both sequences where the same amino acid residue or nucleic acid base appears (the number of matching positions), divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of the percentage of sequence identity between two sequences can be accomplished using software that is readily available online and downloadable. Suitable software programs are available from various sources for protein and nucleotide sequence alignment. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST program suite available from the NCBI BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparing two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa.

[0085] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0086] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the invention. The reagents and / or kits used in the following embodiments are commercially available or can be synthesized by known methods.

[0087] It should be noted that, unless specific conditions are specified in the examples, experimental conditions should be performed according to standard conditions, manufacturer recommendations, or publicly reported experimental conditions. Reagents or instruments whose manufacturers are not specified are all commercially available, standard products. For reagents whose manufacturers are specified, similar products from other manufacturers are substitutes.

[0088] Unless otherwise specified, the experimental materials and instruments used in the embodiments of this invention are as follows: Experimental materials Atropine (ATR), scopolamine, anisodamine, succinic anhydride (butanediamine), ovalbumin (OVA), keyhole hemocyanin (KLH), N,N-dimethoxyformamide (DMF), pyridine, tetrahydrofuran (THF), dimethylformamide (DMF), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide ester (NHS) were all purchased from Shanghai Sangon Biotech Co., Ltd. Fluorescent microspheres (FCEU002) were purchased from Bangs Chemical Company; 2-morpholine ethanesulfonamide, glycine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich; atropine standard, Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and HRP-labeled secondary antibody (goat anti-mouse) were purchased from Shanghai Sangon Biotech Co., Ltd. Fetal bovine serum, RPMI-1640 cell culture medium, HAT supplement, HT additive, trypsin, and penicillin antibodies were purchased from Gibco (Shanghai), China. All water used in this experiment was distilled water. Myeloma cells SP2 / 0 were stored in the applicant's laboratory. All other reagents used to prepare solutions were purchased from Shanghai Guoyao Chemical Reagent Co., Ltd. Nitrocellulose membranes (Millipore Hiflow-140), white substrates, and glass fibers were purchased from Shanghai Jiening Biotechnology Co., Ltd.

[0089] Experimental instruments The following instruments were used: a 6540 UHD Accurate-Mass Q-TOF LC / MS system (Agilent Technologies); a GENESYS UV spectrophotometer (Thermo Fisher Scientific); an MS-H280-Pro magnetic stirrer (Dalong Xingchuang Experimental Instruments Co., Ltd.); a three-position spray system (XYZ3050TM) and a paper cutter system (CM4000TM) (BIO DOT, USA); an ultrasonic cell disruptor (Ningbo Xinzhi Biotechnology Co., Ltd.); an electrically heated constant temperature drying oven (DHG-9030A) (Shanghai Zhongyou Instrument Equipment Co., Ltd.); a benchtop refrigerated ultra-high speed centrifuge (BECKMAN COULTER); an ultrapure water system (Milipore, USA); a JN615 fluorescence detector (Shanghai Jiening Biotechnology Co., Ltd.); and a 365 nm flashlight (UltraFire BG-42-A).

[0090] The sequence information involved in the embodiments of the present invention is as follows: Table 1: Sequence Information

[0091] Example 1: Preparation and Validation of ATR Hapten and Complete Antigen 1. Preparation of ATR haptens and complete antigens ATR is a small molecule compound and belongs to the category of haptens. It only has immunoreactivity but no immunogenicity. Therefore, it cannot be used as an immunogen to stimulate the body to produce corresponding antibodies. It needs to be coupled with a corresponding protein to become a complete antigen. It is chosen to be coupled with KLH, which has strong immunogenicity and large molecular weight, as the immunogen, while OVA, which is easy to adsorb on the ELISA plate and has weak immunogenicity, is chosen as the coating antigen.

[0092] This embodiment uses two methods to prepare the ATR hapten and complete antigen, as detailed below: (1) Succinic anhydride method First, the process of modifying the ATR hapten using the succinic anhydride method is as follows: Figure 1 As shown, using pyridine as a solvent, 30 mg of ATR and 11.4 mg of succinic anhydride were weighed and dissolved in a round-bottom flask. A reflux reflux apparatus was installed, and the mixture was stirred magnetically in an oil bath at 70°C for 24 hours. Then, 1.03 mg of succinic anhydride was added to the reaction solution, and after stirring for 6 hours, most of the pyridine was removed by nitrogen purging in a fume hood, followed by standing to allow it to evaporate and form brown crystals. Finally, mass spectrometry (MS) was used to determine whether the hapten had been successfully modified. Figure 2 The results of MS identification of ATR hapten, the molecular formula of hapten C 21 H 25 The actual measured molecular weight of O4N3 was 390.2, which is close to the theoretical value, indicating that the ATR hapten modification was successful.

[0093] Haptens prepared using the succinic anhydride method are used to conjugate proteins. Protein conjugation is performed using the activated ester method. Immunogens are conjugated to KLH, and coating antigens are conjugated to OVA. The reaction formula is as follows: Figure 1 As shown. Specific steps: Weigh 7.3 mg of the hapten, 8.6 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and 5.2 mg of N-hydroxysuccinimide ester (NHS), and dissolve them in 1 mL of N,N-dimethylformamide (DMF). Stir with a magnetic stirrer for 16 h to obtain solution A (i.e., the hapten solution). Dissolve 20 mg of KLH in 10 mL of PBS and physiological saline, respectively. Dissolve 20 mg of OVA in 10 mL of PBS. Pre-cool the solutions at -20°C and then stir with a magnetic stirrer to obtain solutions B (i.e., KLH-PBS solution, KLH-physiological saline solution, and OVA-PBS solution). Add hapten solution A dropwise to solution B. Stir at 4°C for 24 h, then transfer to a dialysis bag and dialyze in PBS at 4°C for 3 days, changing the dialysate three times daily. After preparing the artificial antigen, aliquot and store at -20°C. Labeled as ATR-OVA, ATR-KLH1 (physiological saline), and ATR-KLH2 (PBS).

[0094] (2) N-N'-Carboxydiimidazole method (CDI method) The process of modifying ATR haptens using the CDI method is as follows: Figure 3 As shown, since CDI is easily decomposed in water, anhydrous tetrahydrofuran (THF) was chosen as the solvent. 1.2 mg of ATR and 13 mg of CDI were weighed and dissolved in 500 µL of anhydrous tetrahydrofuran. After reacting at room temperature in the dark for 4 hours, the solution was dried at low temperature to remove THF. The product was then dissolved in 100 µL of dimethylformamide (DMF), designated as solution A (i.e., hapten solution), for subsequent use with conjugated proteins.

[0095] The immunogen-coupled protein chosen is KLH, which has extremely high immunogenicity; the coating antigen-coupled protein chosen is OVA. The reaction formula is as follows: Figure 3 As shown. Specific steps: Dissolve 20 mg of KLH in 10 mL of PBS and physiological saline, respectively. Dissolve 20 mg of OVA in 10 mL of PBS. Pre-cool the solutions to -20°C and then stir on a magnetic stirrer to obtain solutions B (i.e., KLH-PBS solution, KLH-physiological saline solution, and OVA-PBS solution). Add hapten solution A dropwise to solution B. Stir at 4°C for 24 hours, then transfer to a dialysis bag and dialyze in PBS at 4°C for 3 days, changing the dialysate three times daily. After preparing the artificial antigen, aliquot and store at -20°C. Label as ATR-OVA-CDI, ATR-KLH3-CDI (physiological saline), and ATR-KLH4-CDI (PBS).

[0096] 2. Identification of complete antigens The complete antigen was identified using a UV spectrophotometer, scanning in the wavelength range of 0-400 nm to determine whether the complete antigen was successfully synthesized. Based on the different positions of the maximum UV absorption peaks of different substances, if the maximum absorption peak of the complete antigen shifts relative to that of the ATR hapten and carrier protein, it indicates that the complete antigen conjugation was successful.

[0097] The identification results of the immunogen (i.e., the antigen obtained by conjugation of ATR and KLH) are as follows: Figure 4 and Figure 5 As shown, the maximum absorption peak of the immunogen shifted compared to the maximum absorption peaks of the hapten and the carrier protein, indicating that the immunogen was successfully conjugated.

[0098] The identification results of the coating antigen (i.e., the antigen obtained by conjugating ATR with OVA) are as follows: Figure 6 As shown, its maximum absorption peak shifted compared to ATR and OVA, verifying the successful preparation of the coating material.

[0099] Example 2: Preparation of Monoclonal Antibodies 1. Mouse immunization Due to variations in immunization efficacy among different mice, this experiment selected 12 healthy BALB / c mice and divided them into 4 groups. Each group was injected with one of four prepared immunogens (ATR-KLH1, ATR-KLH2, ATR-KLH3-CDI, and ATR-KLH4-CDI), with 3 mice in each group performing parallel experiments. All mice were numbered and immunized according to the immunization schedule. Starting with the third immunization, tail blood was collected and serum was tested one week after each immunization. Mice with good titers received booster immunizations, and mice with high titers and good inhibition rates were selected for cell fusion. The entire immunization process is shown in Table 1. In the fourth immunization, immunogen #3 (prepared using the CDI method with PBS solution, ATR-KLH4-CDI) showed the best effect. Mice using immunogen #3 were flushed with PBS and their serum was tested again. The serum test results are shown in Tables 2, 3, and 4. The mouse with the best titer was selected for cell fusion based on the calculated inhibition rate and OD value. Ultimately, the second mouse (ATR-2 mouse) was selected for cell fusion.

[0100] Table 1: Mouse Immunization Procedure

[0101] Table 2: Serum results from ATR-1 mice (using ATR-KLH4-CDI as the immunogen)

[0102] Table 3: Serum results from ATR-2 mice (using ATR-KLH4-CDI as the immunogen)

[0103] Table 4: Serum results from ATR-3 mice (using ATR-KLH4-CDI as the immunogen)

[0104] The specific steps for immunization are as follows: ATR complete antigen (immunogen) prepared using the succinic acid dry method and CDI method was adjusted to a concentration of 1 mg / mL with physiological saline, then mixed with an equal volume of Freund's complete adjuvant (FCA), placed in an ice box, and emulsified using an emulsifier until the solvent reached a milky white, non-flowing state. Mice were immunized via multiple subcutaneous injections on the back. The initial immunization dose was 100 µg, followed by booster immunizations every three weeks at a dose of 50 µg using Freund's complete adjuvant (FIA), emulsified using an emulsifier before immunization. Starting from the third immunization, blood was collected from the tail of mice one week after immunization for serum testing. After blood collection, the serum was analyzed using an indirect competitive ELISA (ic-ELISA) method to screen mice with high titers and high inhibition rates for subsequent cell fusion.

[0105] The specific steps for serum testing are as follows: (1) Coating: The prepared coating original ATR-OVA-CDI was diluted to 1 mg / mL with 0.01 M CBS buffer solution, and then diluted to 1K, 3K, 9K and 27K. 100 μL of coating solution was added to each well and incubated at 37 °C for 2 h.

[0106] (2) Washing: After incubation, remove the liquid from the wells, add 250 μL of washing solution to each well, gently shake for 1 minute and then remove the liquid. Repeat this process four times. Then, spin dry using a spinner.

[0107] (3) Sealing: Add 200 μL of sealing solution (5% skim milk powder solution) to each well and incubate at 37 °C for 2 h. After incubation, first remove the liquid from the well, then wash four times as described above, and store at 4 °C for later use.

[0108] (4) Add primary antibody: Dilute mouse serum to 1K, 3K, 9K, and 27K. Add 50 μL of 0.01M PBS to the top four rows of the ELISA plate as the blank control group, and add 50 μL of standard to the bottom four rows as the experimental group. Then, add 50 μL of serum of different concentrations to the corresponding wells, incubate at 37℃ for 30 min, first discard the liquid in the wells, and then wash four times as described above.

[0109] (5) Add secondary antibody: It needs to be prepared and used immediately. Dilute HRP-labeled goat anti-mouse secondary antibody 2000 times, add 100 μL to each well, incubate at 37 ℃ for 30 min, first remove the liquid in the well, and then wash four times as described above.

[0110] (6) Color development reaction: It needs to be prepared and used immediately. Add 50 μL of substrate color development solution A and color development solution B (Solepro TMB two-component color development solution) to each well, mix gently, and incubate at 37 ℃ in the dark for 30 min.

[0111] (7) Termination of reaction: Add 50 μL of 2M H2SO4 to each well and immediately place it in a microplate reader to measure OD. 450 .

[0112] The formula for calculating the inhibition rate is as follows: 2. Cell fusion Splenocytes and SP2 / 0 cells from ATR-2 mice were mixed evenly at a ratio of 5:1 and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded after centrifugation. Double-distilled water at 37 °C and alcohol swabs were prepared, and the fusion procedure was performed in a 37 °C water bath. The double-distilled water at 37 °C was poured into a beaker. The centrifuged SP2 / 0 cells were then inverted onto the alcohol swabs to absorb the culture medium.

[0113] (1) At the first minute, take 1 mL of PEG1500, place the cells in the centrifuge tube in a water bath at 37 °C, start timing, add a drop of culture medium and gently stir with the pipette tip, then slowly add more culture medium, and remove the pipette tip after 60 seconds.

[0114] (2) At the second minute, the centrifuge tubes were placed in a water bath at 37 ℃ for 1 minute.

[0115] (3) At 3 min and 4 min, slowly add 1 mL of preheated RPMI-1640 medium while gently stirring with a centrifuge tube.

[0116] (4) At 5 min and 6 min, slowly add 2 mL of preheated RPMI-1640 medium, stirring gently with a centrifuge tube while adding.

[0117] (5) At the 7th minute, take 1 mL of PEG1500, place the cells in the centrifuge tube in a water bath at 37°C, start timing, add a drop of culture medium and gently stir evenly with the pipette tip, then slowly add more culture medium, and remove the pipette tip after 60 seconds.

[0118] (6) After fusion, place the cells in an incubator and let stand for 10 minutes, then centrifuge at 1000 rpm for 10 minutes, discarding the supernatant. After dispersing the cells, slowly add HAT medium and mix thoroughly by pipetting. Slowly add 100 μL to each well of a 96-well plate and incubate in a cell culture incubator.

[0119] 3. Hybridoma cell screening After successful hybridoma cell fusion, screening and subcloning are required. Specifically, after culturing the hybridoma fusion cells for a period of time, they are cultured in HAT medium with a partial medium change. After one week, the medium is completely replaced with fresh HAT medium. After 10 days, HT medium replaces the HAT medium. Wells producing ATR antibodies are then screened using indirect ELISA. ATR-OVA is used as the coating antigen, and cell lines from positive wells are selected. The inhibition rate is measured using ic-ELISA (see section "1. Mouse Immunization" for the method). After three subcloning processes, three ATR hybridoma cell lines are obtained. The results of ic-ELISA testing on the three cell lines are as follows... Figure 7 As shown, 5D8 exhibits the best overall antibody affinity.

[0120] 4. Preparation and purification of ascites fluid (1) Preparation of ascites Monoclonal antibodies were prepared using an in vivo induction method. One week before injecting hybridoma cells into mice, each mouse was injected intraperitoneally with 0.5 mL of sterile paraffin to enhance the mouse's immune response. The selected frozen 5D8 hybridoma cells were then thawed and injected into mice intraperitoneally after reaching a suitable growth stage. The mice's abdomens were observed for one week post-injection; once a significant bulge was observed, ascites fluid was collected for subsequent purification.

[0121] (2) Purification of ascites Ascites fluid was purified using the octanoic acid-ammonium sulfate method. The specific steps are as follows: After centrifugation of ascites fluid, the supernatant was collected and diluted 2-fold with 0.06 mol / L, pH 5.0 acetate buffer. The pH was then adjusted to 4.5 with 1 mol / mL HCl solution. Caprylic acid was added dropwise with stirring at a ratio of 33 μL / mL serum to remove impurities. After thorough mixing, the solution was incubated at 4°C for at least 2 hours. The supernatant was collected and filtered after centrifugation at 9000 rpm for 30 minutes at 4°C. One-tenth the volume of the supernatant was added to 10×PBS solution, and the pH was adjusted to 7.4. Ammonium sulfate was added at a ratio of 0.277 g / mL, and the solution was stirred thoroughly and incubated at 4°C for at least 4 hours. The supernatant was discarded after centrifugation under the same conditions. The precipitate was collected and purified using an antibody purification column.

[0122] 5. Determination of antibody affinity constant and antibody subtype The original ATR-OVA-CDI coating was diluted to 1 μg / mL, 0.3 μg / mL, and 0.1 μg / mL, respectively, and coated onto plates according to the ic-ELISA method. The antibody concentration was tripled starting from 100 μg / mL, for a total of 8 concentration gradients (see "1. Mouse Immunization" for the method). Finally, the OD value of each well was measured, the affinity curve was plotted using Origin, and the affinity constant was calculated according to the following formula.

[0123] In the formula, n represents the concentration multiple of different coating agents, and n is 3. and Representing different original coating concentrations The corresponding antibody concentration.

[0124] Antibody affinity represents the strength of the ability of an antigen and antibody to bind. Generally, when the antibody affinity is greater than 1 / 3, the affinity is higher than 1 / 3. The measured values ​​indicate good antibody binding ability. In the experiment, the coating antigen was selected at concentrations of 1 μg / mL, 0.3 μg / mL, and 0.1 μg / mL, and the antibody was tripled starting at 100 μg / mL. OD was measured using an indirect competitive ELISA method. 450 Values, fitted to curves using Origin, such as Figure 8 As shown, substituting into the formula and calculating yields... , , Calculate the average value to obtain This indicates that 5D8 is a high-affinity monoclonal antibody. The hybridoma cells corresponding to the high-affinity antibody were sequenced. The sequencing results are shown in Table 1.

[0125] The isotype of ATR antibody was determined using a mouse monoclonal antibody Ig subclass identification kit (Bio-Lon, BF16001). By changing the type of secondary antibody, the monoclonal antibody type was determined by color and absorbance values. The isotype corresponding to the well with the darkest color and highest OD value on the ELISA plate is the antibody type. Based on color and absorbance values, the isotype corresponding to the 5D8 antibody was determined to be IgG1, with its light chain being Kappa.

[0126] Antibody molecular weight was detected by SDS-PAGE. In the SDS-PAGE experiment, β-mercaptoethanol in the loading buffer acts as a reducing agent to break the disulfide bonds of the antibody molecules. Combined with high-temperature denaturation in a metal bath, this ensures complete separation of the heavy and light chains of the antibody. The electrophoresis results are then analyzed. Figure 9 It can be seen that the ATR antibody was successfully purified, with the heavy chain molecular weight being approximately 50 kDa and the light chain molecular weight being approximately 25 kDa.

[0127] The above results indicate that mice immunized with the four prepared immunogens were successfully screened for high titers using ic-ELISA, demonstrating that the immunogen prepared using the CDI method and dissolved in PBS had the best immunizing effect. Hybridoma cells were successfully fused from the spleen cells and myeloma cells of these high-titer mice. After cell screening and subcloning, the required monoclonal antibodies were selected and cultured. Ascites fluid was generated through in vivo culture in mice, and the ascites fluid was purified to obtain the monoclonal antibodies. Antibody affinity assays showed that the 5D8 antibody exhibited the best overall affinity.

[0128] Example 3: Preparation of fluorescent microsphere test strips and detection of sensitivity and specificity 1. Preparation of fluorescent microsphere test strips (1) In this embodiment, the fluorescent microsphere test strip was prepared using the 5D8 antibody prepared in Example 2. The specific steps are as follows: (2) Sample pad treatment: Immerse the sample pad in a solution containing 10% sucrose, 1% Casein and 0.5% Tween20 for 30 min and then dry it in a constant temperature oven at 37 ℃ for later use.

[0129] (3) Treatment of conjugate pads: Immerse the conjugate pads in a solution containing 0.3% surfactant S9 (Tetronic 1307), 10% trehalose and 0.25% PVP40 for 30 min and then dry them in a constant temperature oven at 37 ℃ for later use.

[0130] (4) Sample processing solution: 20mM MES, 2% PVP40, 0.05% SDS, 0.5% Tween20, 5% methanol, adjust pH to 6.0.

[0131] (5) Microsphere dilution solution: 20mM MES, 10% sucrose, 10% trehalose, 0.5% BSA, 0.2% Tween 20, 0.2% PEG 20000, adjust pH to 6.0.

[0132] (6) Assembly of fluorescent microsphere test strips: 1) Activation of microspheres: Centrifuge 420 µL of microspheres at 14000 rpm for 20 min, then resuspend the microspheres in 1 mL of 20 mM MES pH 6.0 buffer and sonicate to mix (20% sonication for 3 s, pause for 5 s, 2 min). Then add 42 µL of 4 mg / mL NHS and 2 mg / mL EDC respectively to activate for 30 min, centrifuge at 14000 rpm for 20 min, and resuspend in 420 µL of 20 mM borax.

[0133] 2) Microsphere conjugation: Add 400 µL of borax and 100 µL of activated fluorescent microspheres to a new centrifuge tube, then add 200 µg of anti-ATR monoclonal antibody and conjugate for 2 h.

[0134] 3) Blocking: Block with 100 µL of 10% glycine for 15 min, then block with 100 µL of 10% BSA for 15 min, and centrifuge at 14000 rpm for 30 min.

[0135] 4) Spray microspheres: Resuspend (3) microspheres in 1 mL of microsphere diluent and sonicate. Then dilute the microspheres 20 times with microsphere diluent. Spray 810 µL of microspheres onto a 30 cm × 8 mm conjugate pad at a speed of 9 µL / cm. Place the microspheres in a 37 ℃ forced-air drying oven overnight to dry for later use.

[0136] 5) ATR-OVA-CDI was administered at a concentration of 1.0 mg / mL and 0.5 mg / mL of goat anti-mouse IgG (Boster Biological, BA1038) using XYZ3050. TMThe three-point spray system was sprayed onto the nitrocellulose membrane (NC) at a speed of 1 µL / cm as the test line (T) and the quality control line (C), and then placed in a 37 ℃ forced-air drying oven overnight to dry for later use.

[0137] 6) Attach the NC membrane, conjugate pad, sample pad, and absorbent paper to the PVC fluorescent substrate, and then use a BIO-Dot cutter to cut the assembled strip into 3mm wide test strips. Seal and store them in an aluminum foil bag containing desiccant.

[0138] Result Interpretation: Use a pipette to add 75 μL of the diluted sample to the sample well. After 15 min, observe the results with a flashlight with an excitation light source of 365 nm. If both the T line and the C line show red bands, the result is negative. If the C line shows a red band but the T line shows no band, the result is positive. If the C line shows no band, the test strip is invalid regardless of whether the T line shows a band.

[0139] 2. Testing the sensitivity of test strips ATR was dissolved in methanol, and the dissolved ATR was diluted with 0.01M PBS at concentrations of 0.0 μg / kg, 0.1 μg / kg, 0.2 μg / kg, 0.3 μg / kg, 0.5 μg / kg, 1 μg / kg, 2 μg / kg, 3 μg / kg, and 5 μg / kg. The prepared test strips were used to detect the ATR standards diluted at these gradients. Different concentrations of ATR were detected using a 365 nm UV excitation source. When the dilution concentration was 3 μg / kg, the detection line on the test strip disappeared, indicating that the sensitivity of the test strip was 3 μg / kg. Figure 10 ).

[0140] Scopolamine was dissolved in methanol. The dissolved scopolamine was then diluted with 0.01M PBS at concentrations of 0.0 μg / kg, 0.1 μg / kg, 0.2 μg / kg, 0.3 μg / kg, 0.5 μg / kg, 1 μg / kg, 2 μg / kg, 3 μg / kg, and 5 μg / kg. The prepared test strips were used to detect the scopolamine standards at these gradient concentrations. Different concentrations of scopolamine were detected using a 365 nm UV excitation source. When the dilution concentration was 3 μg / kg, the detection line on the test strip disappeared, indicating that the sensitivity of the test strip was 3 μg / kg. Figure 11 ).

[0141] Anisodamine was dissolved in methanol. The dissolved anisodamine was then diluted with 0.01M PBS at concentrations of 0.0 μg / kg, 0.1 μg / kg, 0.2 μg / kg, 0.3 μg / kg, 0.5 μg / kg, 1 μg / kg, 2 μg / kg, 3 μg / kg, and 5 μg / kg. The prepared test strips were used to detect the anisodamine standard at these gradient concentrations. Different concentrations of anisodamine were detected using a 365 nm UV excitation source. When the dilution concentration was 3 μg / kg, the detection line on the test strip disappeared, indicating that the sensitivity of the test strip was 3 μg / kg. Figure 12 ).

[0142] 3. Detection of test strip specificity The prepared test strips were used to detect ATR standard (5 μg / kg), clenbuterol (5 μg / kg), sulfadiazine (5 μg / kg), zopiclone (5 μg / kg), and tetracycline (5 μg / kg). The results showed that only the C line of the ATR standard showed color, while both the T and C lines of the other samples showed color, indicating that the test strip has good specificity. Figure 13 ).

[0143] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. An anti-atropine antibody or its antigen-binding fragment, characterized in that, The anti-atropine antibody or its antigen-binding fragment includes: a1) having CDR-H1, CDR-H2, and CDR-H3 included in the heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 2; and / or, having CDR-L1, CDR-L2, and CDR-L3 included in the light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 4; or a2) having one or more amino acid substitutions, deletions, or additions compared to CDR-H1, CDR-H2, and CDR-H3 as shown in a1); and / or having one or more amino acid substitutions, deletions, or additions compared to CDR-L1, CDR-L2, and CDR-L3 as shown in a1); The CDR is defined according to the Kabat, Chothia, IMGT, Contact, or AbM numbering system.

2. The anti-atropine antibody or its antigen-binding fragment according to claim 1, characterized in that, The anti-atropine antibody or its antigen-binding fragment includes: b1) A VH comprising the following three CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 5, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 6, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 7; and / or, a VL comprising the following three CDRs: CDR-L1 having the amino acid sequence shown in SEQ ID NO: 8, CDR-L2 having the amino acid sequence shown in SEQ ID NO: 9, and CDR-L3 having the amino acid sequence shown in SEQ ID NO: 10; or b2) VH comprising the following three CDRs: having one or more amino acid substitutions, deletions, or additions compared to CDR-H1, CDR-H2, and CDR-H3 as shown in b1); and / or VL comprising the following three CDRs: having one or more amino acid substitutions, deletions, or additions compared to CDR-L1, CDR-L2, and CDR-L3 as shown in b1); The CDR is defined according to the Kabat numbering system.

3. The anti-atropine antibody or its antigen-binding fragment according to any one of claims 1 to 2, characterized in that, The heavy chain variable region of the anti-atropine antibody or its antigen-binding fragment also includes the framework region of the heavy chain variable region; Preferably, the framework region of the heavy chain variable region includes the framework region of the heavy chain variable region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, or geese; further includes the framework region of the heavy chain variable region or a mutant thereof derived from immunoglobulins of mice, rats, lemurs, macaques, chimpanzees, or humans; and even further includes the framework region of the heavy chain variable region or a mutant thereof derived from immunoglobulins of mice. Preferably, the light chain variable region of the anti-atropine antibody or its antigen-binding fragment further includes the framework region of the light chain variable region; Preferably, the framework region of the light chain variable region includes the framework region of the light chain variable region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, or geese; further includes the framework region of the light chain variable region or a mutant thereof derived from immunoglobulins of mice, rats, lemurs, macaques, chimpanzees, or humans; and even further includes the framework region of the light chain variable region or a mutant thereof derived from immunoglobulins of mice. Preferably, the anti-atropine antibody or its antigen-binding fragment comprises: The heavy chain variable region (VH) includes the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 80% sequence identity with it; and / or the light chain variable region (VL) includes the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence having at least 80% sequence identity with it. Preferably, the anti-atropine antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region; Preferably, the heavy chain constant region includes at least a portion of the heavy chain constant region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese; further includes at least a portion of the heavy chain constant region or a mutant thereof derived from immunoglobulins of mice, rats, lemurs, macaques, chimpanzees, or humans; and even more particularly includes the heavy chain constant region or a mutant thereof derived from immunoglobulins of mice. Preferably, the light chain constant region includes at least a portion of the light chain constant region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese; further includes at least a portion of the light chain constant region or a mutant thereof derived from immunoglobulins of mice, rats, lemurs, macaques, chimpanzees, or humans; and even more particularly includes the light chain constant region or a mutant thereof derived from immunoglobulins of mice. Preferably, the heavy chain constant region includes the heavy chain constant region derived from IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 or IgM immunoglobulin or a mutant thereof; Preferably, the light chain constant region includes light chain constant regions derived from κ and λ immunoglobulins; Preferably, the heavy chain constant region includes the heavy chain constant region derived from IgG1 immunoglobulin or a mutant thereof; Preferably, the light chain constant region includes a light chain constant region derived from type κ immunoglobulin; Preferably, the anti-atropine antibody or its antigen-binding fragment is a murine antibody, a chimeric antibody, or a humanized antibody; Preferably, the antigen-binding fragment of the anti-atropine antibody is a Fab fragment, Fab' fragment, Fab'-SH fragment, F(ab')2 fragment, Fv fragment, single-chain Fv (scFv), dsFv or Fd fragment.

4. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the anti-atropine antibody or an antigen-binding fragment thereof as claimed in any one of claims 1 to 3.

5. A vector comprising the nucleic acid molecule of claim 4.

6. A cell comprising the anti-atropine antibody or its antigen-binding fragment as described in any one of claims 1 to 3, the nucleic acid molecule as described in claim 4, or the vector as described in claim 5.

7. A method for preparing the anti-atropine antibody or its antigen-binding fragment according to any one of claims 1 to 3, the method comprising: The cell culture of claim 6 is used to collect the anti-atropine antibody or its antigen-binding fragment from the cultured cell culture.

8. A conjugate comprising the anti-atropine antibody or its antigen-binding fragment as described in any one of claims 1 to 3; and a conjugation portion; Preferably, the coupling portion is a detectable marker.

9. An immunochromatographic test strip, characterized in that, The test strip includes a base plate, a conjugate pad, a sample pad, a nitrocellulose membrane, and an absorbent pad. The sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad are sequentially connected and fixed to the base plate in a horizontal direction. The conjugate pad is loaded with a fluorescent microsphere-antibody conjugate. The antibody is the anti-atropine antibody or its antigen-binding fragment as described in any one of claims 1 to 3. The nitrocellulose membrane is provided with a detection line and a control line. The detection line is coated with complete atropine antigen.

10. A test kit, comprising: The anti-atropine antibody or its antigen-binding fragment as described in any one of claims 1 to 3, the nucleic acid molecule as described in claim 4, the vector as described in claim 5, the cell as described in claim 6, the conjugate as described in claim 8, or the immunochromatographic test strip as described in claim 9.

11. Use of the anti-atropine antibody or its antigen-binding fragment according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the vector according to claim 5, the cell according to claim 6, the conjugate according to claim 8, the immunochromatographic test strip according to claim 9, or the test kit according to claim 10 in at least one of (1)-(2): (1) Detect the presence or level of atropine alkaloids in the sample; (2) Prepare a product, said product being used to detect the presence or level of atropine alkaloids in a sample; Preferably, the atropine alkaloids include atropine, scopolamine, or anisodamine.

12. A method for detecting the presence or level of atropine alkaloids in a sample, the method comprising the following steps: The sample is brought into contact with the anti-atropine antibody or its antigen-binding fragment according to any one of claims 1 to 3, so as to allow the anti-atropine antibody or its antigen-binding fragment to form a complex with atropine alkaloids, and the formation of the complex is detected; Preferably, the atropine alkaloids include atropine, scopolamine, or anisodamine.

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