Detection method for detecting botulinum toxin A HA-70 and product

Abstract

The invention discloses a detection method for detecting botulinum toxin A HA-70 and a product. The detection product disclosed by the invention comprises an antibody capable of being combined with the botulinum toxin A HA-70. The antibody has good affinity and stability, can be widely applied to detection of botulinum toxin type A HA-70, can be combined with a capillary immunoelectrophoresis method to establish a standard map for detection of botulinum toxin component antibodies, and provides a quality control means for commercialization and self-research of antibodies, so that the antibody has good application prospects. According to the method, more possibilities are provided for establishment of related botulinum toxin detection methods, and detection requirements of more scenes can be met.

Description

Technical Field

[0001] This invention belongs to the field of botulinum toxin detection, and in particular relates to a detection method and product for detecting botulinum toxin type A HA-70. Background Technology

[0002] Botulinum toxin (BoNT) is produced by Clostridium botulinum (Clostridium botulinum). Clostridium botulinum Botulinum toxin type A is a protein-based neurotoxin produced by botulinum toxin, which has eight serotypes: A, H, and V. The botulinum toxin complex protein is composed of neurotoxins, non-toxic non-hemagglutinin, and hemagglutinin components. Most of the toxin protein components possess certain immunogenicity and can produce corresponding antibodies.

[0003] Currently, the following problems exist in the detection of botulinum toxin and corresponding antibodies: First, in the research and development and production of botulinum toxin-related products, antibodies targeting toxin complex proteins or neurotoxin proteins are inevitably used. Whether commercially available or self-developed, there is a lack of standards and effective quality control methods, making it difficult to verify the true specificity of antibodies and their binding activity against component proteins. Second, there is a lack of commercially available antibodies targeting the complete set of botulinum toxin components. When developing self-developed antibodies, there is a lack of commercially available specific component proteins as antigens, and there are currently no effective purification methods for specific component proteins of natural toxins. Third, the lack of antibodies and specific component proteins also hinders the establishment of some botulinum toxin-related detection methods, such as: using immunological methods to monitor the integrity and purity of toxin complex proteins, identifying different component botulinum toxin products, and verifying the expression of botulinum toxin components.

[0004] Therefore, there is an urgent need for products and methods for detecting botulinum toxin component proteins. Summary of the Invention

[0005] To address at least some of the technical problems in the prior art, the present invention provides a method and product for detecting botulinum toxin type A hemagglutinin-70 (HA-70). Specifically, the present invention includes the following:

[0006] A first aspect of the present invention provides a method for detecting botulinum toxin type A HA-70, comprising the following steps: (a) Provide the sample to be tested; (b) The test sample is contacted with an antibody or an antigen-binding fragment thereof for a time and under conditions sufficient to form an antibody or an antigen-binding fragment thereof, the antibody or the antigen-binding fragment thereof comprising the heavy chain CDR1-3 as shown in SEQ ID NO. 1-3 and / or the light chain CDR1-3 as shown in SEQ ID NO. 4-6; (c) detecting the binding of the antibody or antigen-binding fragment thereof to the test sample, or detecting the presence of the antibody or antigen-binding fragment thereof / antigen complex, to determine the presence or amount of HA-70 in the sample.

[0007] In a second aspect of the present application, there is provided a detection product for detecting botulinum toxin type A HA-70, wherein the detection product comprises an antibody or antigen-binding fragment thereof, which antibody or antigen-binding fragment thereof comprises heavy chain CDR1-3 having the amino acid sequences of SEQ ID NO. 1-3 and light chain CDR1-3 having the amino acid sequences of SEQ ID NO. 4-6.

[0008] In certain embodiments, the detection product for detecting botulinum toxin type A HA-70 according to the present application comprises a kit, a test strip or a protein chip.

[0009] In a third aspect of the present application, there is provided use of a reagent in the manufacture of a product for detecting botulinum toxin type A HA-70, wherein the reagent is an antibody or antigen-binding fragment thereof, which antibody or antigen-binding fragment thereof comprises heavy chain CDR1-3 having the amino acid sequences of SEQ ID NO. 1-3 and light chain CDR1-3 having the amino acid sequences of SEQ ID NO. 4-6.

[0010] In a fourth aspect of the present application, there is provided an antibody or antigen-binding fragment thereof, which antibody or antigen-binding fragment thereof is capable of specifically binding to botulinum toxin type A HA-70, which antibody or antigen-binding fragment thereof comprises heavy chain CDR1-3 having the amino acid sequences of SEQ ID NO. 1-3 and light chain CDR1-3 having the amino acid sequences of SEQ ID NO. 4-6.

[0011] In certain embodiments, the antibody or antigen-binding fragment thereof according to the present application has any one of the amino acid sequences of (I) to (III): (I) a heavy chain amino acid sequence as set forth in SEQ ID NO. 7 and a light chain amino acid sequence as set forth in SEQ ID NO. 8; (II) an amino acid sequence having at least 90% homology to the amino acid sequence as set forth in (I) and having the same function; (III) an amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids to the amino acid sequence as set forth in (I) or (II) and having the same function.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof according to the present invention includes a monoclonal antibody, a chimeric antibody, a humanized antibody or a murine antibody; and the antigen-binding fragment includes Fab, Fab', F(ab')2 or scFv.

[0013] In a fifth aspect, the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof as described in the fourth aspect of the invention.

[0014] A sixth aspect of the invention provides a carrier molecule, wherein the carrier molecule comprises a nucleic acid molecule according to a fifth aspect of the invention.

[0015] A seventh aspect of the invention provides a host cell, wherein the host cell comprises a nucleic acid molecule according to a fifth aspect of the invention or a carrier molecule according to a sixth aspect of the invention.

[0016] An eighth aspect of the present invention provides a method for preparing an antibody or an antigen-binding fragment thereof according to a third aspect of the present invention, wherein the antibody or the antigen-binding fragment thereof is prepared by artificial synthesis or genetic engineering.

[0017] This invention purifies the natural botulinum toxin type A complex component protein HA-70, solving the problem of difficult purification of natural botulinum toxin complex component proteins and providing an effective tool for component protein purification. Furthermore, it uses hybridoma technology to prepare antibodies against botulinum toxin type A HA-70, preserving the antibody's affinity maturation in vivo and the original pairing of the antibody variable region and antigen constant region gene combination, thus enriching the botulinum toxin complex component protein antibody library. The antibodies of this invention can specifically recognize botulinum toxin type A HA-70 and exhibit good affinity and stability, making them widely applicable to the detection of botulinum toxin type A HA-70. They can be combined with capillary immunoelectrophoresis to establish standard patterns for botulinum toxin component antibodies, providing quality control for commercial and self-developed antibodies. Therefore, this invention provides more possibilities for the establishment of botulinum toxin-related detection methods and can meet the detection needs of more scenarios, such as screening for Clostridium botulinum contamination in food and environmental samples, and assisting in the laboratory diagnosis of suspected botulism cases. Attached Figure Description

[0018] Figure 1 The SDS-PAGE results of the HA-70 antigen are shown.

[0019] Figure 2 The results of subclass identification of the anti-HA-70 antibody of the present invention are shown.

[0020] Figure 3Capillary immunoelectrophoresis detection results of the anti-HA-70 antibody of the present application and natural type A botulinum toxin complex are shown.

[0021] Figure 4 Affinity detection results of the anti-HA-70 antibody of the present application are shown. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present application will now be described in detail, which should be considered to be merely illustrative in nature and are not to be considered as limiting the scope of the present application, and are understood to be within the scope of the present application, with some aspects, features and embodiments of the present application being described in more detail.

[0023] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of a parameter, unless otherwise stated, the inclusion of either a lower or an upper limit of a range of values is expressly stated. Each smaller range between any stated value or inferred value within the stated range and any other stated or inferred value in the stated range is also expressly stated. The inclusion of either a lower or an upper limit of a range of values also includes the exclusion of that either lower or upper limit. It is to be understood that such terminology as "about" and "substantially" as used herein is intended to advance more fully description of the present application and

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0025] I. Methods In one aspect of the present application, a detection method for detecting HA-70 of type A botulinum toxin is provided, which comprises the following steps: (a) providing a sample to be tested; (b) contacting the sample to be tested with an antibody or an antigen binding fragment thereof comprising heavy chain CDR1-3 of an amino acid sequence as shown in SEQ ID NO. 1-3 and / or light chain CDR1-3 of an amino acid sequence as shown in SEQ ID NO. 4-6, under time and conditions sufficient to form an antibody or antigen binding fragment thereof / antigen complex; (c) detecting the binding of the antibody or antigen binding fragment thereof to the sample to be tested, or detecting the presence of the antibody or antigen binding fragment thereof / antigen complex, to determine the presence of HA-70 in the sample or to determine the amount of HA-70 in the sample.

[0026] In the present application, the sample is not particularly limited, and examples thereof include, but are not limited to, a food sample (for example, but not limited to, a vegetable, a meat product, a dairy product, honey, a fermented food, etc.), an environmental sample (for example, but not limited to, soil, a water sample, etc.), a pharmaceutical sample, a biological sample, etc.

[0027] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present application can carry any detectable label. Examples of labels that can be used include, but are not limited to, radioactive substances, fluorescent groups, chemical labels, biological agents (such as, but not limited to, biotin / streptavidin detection), or enzyme substrate labels. In additional embodiments, detection antibodies that can carry detectable labels can be used in the present application, the use of detection antibodies can in some cases enhance the signal of the antibodies of the present application.

[0028] In the present application, the "time and conditions sufficient to form an antibody or antigen-binding fragment thereof / antigen complex" are not particularly limited, as long as the HA-70 in the sample binds and forms a stable antibody or antigen-binding fragment thereof / antigen complex or conjugate after being contacted with the antibody or antigen-binding fragment thereof of the present application.

[0029] It is well known to those skilled in the art how to detect the binding of HA-70 to the antibody or antigen-binding fragment thereof in the sample, or to detect the presence of the antibody or antigen-binding fragment thereof / antigen complex, thereby determining whether HA-70 is present in the sample or determining the amount of HA-70. Such detection techniques include, but are not limited to, immunofluorescence, radioimmunoassay, enzyme-linked immunoassay, agglutination test, immunoadsorption, immunoelectrophoresis, immunoelectron microscopy, etc.

[0030] II. Detection products In one aspect of the present application, a detection product for detecting (or quantifying) botulinum toxin type A HA-70 is provided, which comprises the antibody or antigen-binding fragment thereof described in the present application, and optionally an instruction on how to perform the detection method of the present application on botulinum toxin type A HA-70.

[0031] Examples of the detection product of the present application include, but are not limited to, kits, test papers, protein chips, etc.

[0032] Herein, the term "kit" refers to a set of standardized reagents and consumables that are systematically combined and provided to accomplish a specific biological detection or experimental operation. Exemplarily, the kit is expected to comprise core reaction reagents, reaction system supports, signal generation and detection systems, solid phase carriers, auxiliary components, etc., wherein examples of the core reaction reagents include, but are not limited to, the antibody or antigen-binding fragment thereof of the present application, antigens, controls, etc.; examples of the reaction system supports include, but are not limited to, buffers, protein stabilizers, blocking agents, enzymes, etc.; examples of the signal generation and detection systems include, but are not limited to, substrates, color developing agents, or labels for chemiluminescence, fluorescence, or colorimetric detection; examples of the solid phase carriers include, but are not limited to, enzyme-labeled plates, test paper strips, chips, etc.; examples of the auxiliary components include, but are not limited to, instructions, packaging containers, etc.

[0033] In certain embodiments, the kit can further comprise at least one of a washing solution, a substrate solution, a dilution solution, and a standard solution. The components of the washing solution are not particularly limited, and examples thereof include, but are not limited to, a buffer (such as, but not limited to, PBS, Tris-HCl, etc.), a surfactant (such as, but not limited to, Tween-20), a preservative (such as, but not limited to, isothiazolinone). The substrate solution can use known substrates, examples of which include, but are not limited to, a chromogenic substrate (such as, but not limited to, TMB, OPD, etc.), a fluorescent substrate (such as, but not limited to, AMC, 4-MU, etc.), a luminescent substrate (such as, but not limited to, luminol, ECL, etc.), etc. The components of the dilution solution are not particularly limited, and examples thereof include, but are not limited to, a buffer, a surfactant, etc. The components of the standard solution are not particularly limited, and examples thereof include, but are not limited to, a BSA solution, a trehalose solution, an animal serum, etc.

[0034] In the present application, examples of the kit include, but are not limited to, an ELISA detection kit, a plate-based chemiluminescence detection kit, a fully automatic chemiluminescence detection kit, a radioimmunoassay kit, a fluorescent immunoassay kit, a chemiluminescence immunoassay kit, an immunoblotting kit, a flow cytometry detection kit, a colloidal gold method detection kit, a bioluminescence immunoassay kit, an immunochromatography detection kit, a latex-based kit, a kit based on direct competition or indirect competition, etc.

[0035] The present application further provides use of a reagent in the preparation of a product for detecting (or quantifying) botulinum toxin type A HA-70, wherein the reagent comprises the antibody or antigen-binding fragment thereof of the present application.

[0036] III. Antibody or antigen-binding fragment thereof In one aspect of the present application, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof is capable of specifically binding to botulinum toxin type A HA-70, and the antibody or antigen-binding fragment thereof comprises heavy chain CDR1-3 having the amino acid sequence shown in SEQ ID NO. 1-3 and light chain CDR1-3 having the amino acid sequence shown in SEQ ID NO. 4-6.

[0037] The term "antibody" herein refers to an immunoglobulin molecule that specifically binds to an antigen. A typical antibody monomer has two identical heavy chains and two identical light chains connected by disulfide bonds, forming a "Y" shaped structure. An antibody usually comprises a variable region and a constant region in each of the heavy and light chains. The variable region is located at the N-terminus of the heavy and light chains, and its amino acid sequence is highly variable, which collectively constitutes the antigen-binding fragment of the antibody. The heavy chain variable region and the light chain variable region cooperate closely through non-covalent interactions and disulfide bonds to form complementarity determining regions, which directly determine the specificity and affinity of the antibody in recognizing antigens. The constant region is located at the C-terminus of the chains, and its sequence is relatively conserved within the same genus. This region mainly mediates the effector functions of the antibody, such as binding to Fc receptors on the surface of immune cells, activating the complement system, etc., thereby realizing the regulation and transmission of immune responses.

[0038] The "light chain variable region (VL)" or "heavy chain variable region (VH)" of the present application is composed of "framework" regions interspersed with three "complementarity determining regions (CDRs)". The framework regions serve to adjust the CDRs for specific binding to epitopes of antigens. From the amino terminus to the carboxyl terminus, both VL and VH domains comprise the following framework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The complementarity determining regions include three prominent loop-like structures (CDR1, CDR2, CDR3), which have extremely high variability in amino acid sequence, directly contact and determine the specificity and affinity of the antibody in binding to antigens. FR1-FR4 constitute the β-sheet barrel scaffold of the variable region, and their main function is to provide the correct spatial folding, positioning and orientation for the CDR loops, ensuring that the CDRs form a stable, available antigen-binding pocket. Although relatively conserved, certain FR residues also participate in antigen contact or affect antibody stability. Therefore, the six CDRs of VHand VLcollectively constitute the antigen-binding site, while the eight framework regions provide precise structural support for these key sites.

[0039] In the present application, an "epitope" includes any determinant capable of specific binding to an antibody. An epitope is the region of an antigen that is bound by an antibody specifically targeted to that antigen, and when the antigen is a protein, the epitope comprises specific amino acids that directly contact the antibody. Epitopes are usually located on proteins, but in some cases can also be located on other types of molecules, such as nucleic acids. Epitope determinants can include chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl groups or sulfonyl groups, and can have specific three-dimensional structural features and / or specific charge features. In general, a specific antibody against a particular target antigen will preferentially recognize the epitope on the target antigen in a complex mixture of proteins and / or macromolecules.

[0040] In certain embodiments, the antibody or antigen-binding fragment thereof has any one of the amino acid sequences shown in (I)-(III): (I) the heavy chain amino acid sequence shown in SEQ ID NO. 7 and the light chain amino acid sequence shown in SEQ ID NO. 8; (II) an amino acid sequence having at least 90% homology to the amino acid sequence shown in (I) and having the same function; (III) an amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (I) or (II) and having the same function.

[0041] Herein, the terms "homology" and "identity" are used interchangeably. Homologous sequences include amino acid sequences that are at least 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequences of the present application. To determine sequence identity, sequence alignments can be performed, which can be done in various ways known to those skilled in the art, e.g., using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software, etc. Those skilled in the art will be able to determine appropriate parameters for alignment, including any algorithms needed to achieve optimal alignment over the full length of the sequences being compared.

[0042] Herein, variant antibody sequences obtained by modifying, substituting, deleting or adding one or more amino acids are also within the scope of the present application. The term "modifying" refers to covalent chemical modifications of the side chains of amino acid residues in the sequence (e.g., glycosylation, pegylation, acetylation, etc.). The term "substituting" refers to replacing one or more residues in the original sequence with one or more different amino acid residues. The term "deleting" refers to removing one or more amino acid residues from the original sequence. The term "inserting" or "adding" refers to introducing one or more additional amino acid residues into the original sequence. To maximize the preservation of the core binding function of the antibody, the above sequence changes are preferably made outside of the variable regions (VH / VL), e.g., at non-critical positions in the framework regions or in the constant regions. In particular, the variants should avoid disruptive changes to critical residues in the complementarity determining regions, unless the change is intended to be a purposeful affinity maturation. All variants falling within the scope of the present application must retain the desired biological function of the original antibody or antigen binding fragment thereof, in particular its ability to specifically bind the target antigen. The binding activity of these variants can be determined by standard methods in the art (e.g., ELISA, surface plasmon resonance). In certain embodiments, the variants can have equivalent, or improved functional properties, e.g., enhanced affinity, stability or reduced immunogenicity.

[0043] In the present application, conservative amino acid substitutions are preferred, and these conservative antibody variants are preferably generated by amino acid substitutions according to Table 1: Table 1 In the present application, the antibody includes a monoclonal antibody, a chimeric antibody, a humanized antibody, or a murine antibody.

[0044] The term "monoclonal antibody" (sometimes referred to as "monoclonal" or "mAb"), as used herein, refers to a highly uniform population of immunoglobulin molecules that are produced by a clonal population of B cells (or engineered cell lines) that are genetically identical, having identical structure and chemical properties, and are specific for a single antigenic determinant. Monoclonal antibodies differ from conventional polyclonal antibody preparations, which typically include different antibodies directed against different determinants. Each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the benefits of monoclonal antibodies are that they are produced by hybridoma or recombinant engineered cell culture, and are not admixed with other immunoglobulins. This property contrasts with polyclonal antibody preparations, which generally include antibodies directed against different antigenic determinants. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of identical antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0045] The term "chimeric antibody" is used herein to refer generally to an antibody in which a portion of each heavy or light chain amino acid sequence is homologous to the corresponding amino acid sequence in an antibody from a particular species, or belonging to a particular class, while the remainder of the chain is homologous to the corresponding sequence in another species. For example, a "mouse / human chimeric antibody" has variable regions derived from the VHand VLregions of an antibody from a non-human species (e.g., mouse, rat), which are responsible for providing the high specific recognition and binding ability to the antigen, and constant regions that are of human origin, which determine the effector function and pharmacokinetic properties of the antibody in humans. Chimeric antibodies retain the high affinity and specificity of the parent non-human antibody, as the sequence and structure of the variable regions are unchanged. At the same time, the humanized constant regions significantly reduce the immunogenicity induced in humans, and endow them with more suitable effector functions in humans. The variable regions have the advantage of being easy to prepare, and their specificity is not affected by the source of the constant regions with which they are combined.

[0046] As used herein, the term "humanized antibody" generally refers to a chimeric antibody that contains minimal sequence from a non-human immunoglobulin, thereby reducing the immunogenicity of a foreign antibody when introduced into a human, while retaining the full antigen binding affinity and specificity of the antibody. Humanized antibodies are a more advanced form of chimerism. It not only replaces the constant region with human sequences, but also makes key modifications to the variable region. The core technology is "CDR grafting", that is, the CDRs in the mouse antibody that directly contact the antigen and determine specificity are grafted onto a pre-selected, highly compatible human antibody framework region. In this process, structural biology analysis and computer simulation are often needed to fine-tune key framework region residues to ensure that the humanized antibody after grafting fully inherits and even improves the binding performance of the parent antibody. Humanized antibodies have significantly lower immunogenicity than murine antibodies and chimeric antibodies, have longer half-lives and better safety in humans, while retaining the high specificity and high affinity required for targeting antigens.

[0047] As used herein, the term "murine antibody" generally refers to an antibody whose variable region framework and CDR regions are derived from mouse germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, it is also derived from mouse germline immunoglobulin sequences. In the present invention, a murine antibody can comprise amino acid residues not encoded by mouse germline immunoglobulin sequences, for example, it can include mutations introduced by in vitro random mutation or point mutation or by in vivo somatic mutation.

[0048] As used herein, the term "antigen binding fragment" generally refers to a functional fragment composed of partial domains of an antibody that can retain the antigen binding ability of the specificity of the intact antibody. They do not have the constant region of the intact antibody, so they usually do not mediate effector functions such as antibody-dependent cellular cytotoxicity or complement-dependent cellular cytotoxicity, but their smaller molecular size can bring better tissue penetration. It can be understood that the antigen binding function of an antibody can be achieved by a full-length fragment of an antibody, or by a heavy chain comprising Fab, Fab', F(ab')2 or scFv, or a light chain comprising Fab, Fab', F(ab')2 or scFv. It can be understood that any antibody derivative obtained by genetic engineering or enzymatic method, which contains the necessary variable region mentioned above and can specifically bind to the target antigen, belongs to the category of "antigen binding fragment" described in the present invention.

[0049] In the present application, the terms "bind," "specifically bind," "directed against," and "target" are used interchangeably and generally refer to a non-covalent interaction occurring between an immunoglobulin molecule and an antigen specific for the immunoglobulin. The strength or affinity of an immunological binding interaction can be expressed in terms of a dissociation constant (Kd), where a smaller Kdrepresents a higher affinity. "Affinity" refers to the strength of the total non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen), reflecting the tightness of their association. Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity reflecting the 1 : 1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can be expressed in terms of the dissociation equilibrium constant of the binding. The affinity of an antibody for an antigen can be quantitatively determined by a variety of biophysical methods well established in the art, such as, but not limited to, surface plasmon resonance technology, isothermal titration calorimetry, bio-layer interferometry, etc. As long as the method is capable of accurately measuring the kinetic or thermodynamic parameters of the binding reaction and calculating the Kdvalue, it is suitable for assessing the binding affinity described in the present application.

[0050] IV. Nucleic Acid Molecules In one aspect of the present application, there is provided a nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof described in the present application.

[0051] The term "nucleic acid" as used herein is intended to include polymeric forms of nucleotides of any length, containing deoxyribonucleotides, ribonucleotides and / or their analogs, including DNA, RNA and hybrids thereof, which further include DNA or RNA analogs such as those containing modified backbones (e.g., peptide nucleic acids (PNAs)) or modified bases. Thus, nucleic acids of the present application include DNA, cDNA, mRNA, recombinant nucleic acids, etc.

[0052] Once the coding sequence of an antibody or antigen-binding fragment thereof described in the present application is obtained, the antibody or antigen-binding fragment thereof can be obtained in large quantities using recombinant techniques. An exemplary method is to clone its encoding gene into a vector, which is then introduced into cells, and then isolated from the proliferated host cells by conventional methods.

[0053] V. Vector Molecules In one aspect of the present application, there is provided a vector molecule comprising a nucleic acid molecule described in the present application.

[0054] The vector of this invention refers to an artificial construct capable of delivering and preferably expressing one or more target genes or sequences in host cells. The vector of this invention is not limited and can be an expression vector, viral vector, etc. In some embodiments, the vector contains a target gene encoding an antibody of this invention or an antigen-binding fragment thereof, a promoter, a terminator, or optionally a marker gene. The vector can be a known vector or a self-constructed vector. Known vectors include phage vectors, plasmid vectors, lentiviral vectors, adenovirus vectors, AAV viral vectors, etc.

[0055] VI. Host Cells In one aspect, the present invention provides a host cell comprising the nucleic acid molecule or the carrier molecule described herein.

[0056] The host cell of this invention refers to any cell type suitable for transformation, transfection, transduction, etc., using a nucleic acid construct or expression vector containing the nucleic acid molecules of this invention. Host cells may include bacterial, fungal, plant, or animal cells, wherein examples of bacterial host cells include, but are not limited to, *Escherichia coli* (E. coli). Escherichia coli ),salmonella( Salmonella Bacillus subtilis ( Bacillus subtilis ), pneumococcus ( Pneumococcus Streptococcus ( Streptococcus Haemophilus influenzae ( ) Haemophilus influenzae Examples of fungal host cells include, but are not limited to, Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae Pichia pastoris () Pichia pastoris Examples of plant host cells include, but are not limited to, tobacco Benzodiaceae cells, Arabidopsis thaliana cells, and rice suspension cells; examples of animal host cells include, but are not limited to, CHO (Chinese hamster ovary cell line) and NSO cells.

[0057] VII. Preparation Method One aspect of the present invention provides a method for preparing the antibody or its antigen-binding fragment described herein. The preparation method is not particularly limited, and includes preparation by artificial synthesis or genetic engineering.

[0058] In some embodiments, the antibodies or antigen-binding fragments of the present invention are obtained through artificial synthesis. Methods for artificially synthesizing antibodies or antigen-binding fragments of the present invention are known in the art, for example, by direct amino acid synthesis.

[0059] In certain embodiments, the antibody or antigen-binding fragment thereof of the present application is obtained by genetic engineering expression. Genetic engineering expression systems include prokaryotic cell expression systems, eukaryotic cell expression systems, and cell-free expression systems. Examples of the prokaryotic cell expression system include E. coli expression systems. The eukaryotic cell expression system includes yeast expression systems, insect cell expression systems, and mammalian cell expression systems.

[0060] In one specific embodiment, the antibody preparation method of the present application comprises: (1) constructing an antibody heavy chain recombinant vector expressing the amino acid sequence shown in SEQ ID NO. 7 and an antibody light chain recombinant vector expressing the amino acid sequence shown in SEQ ID NO. 8; (2) transforming the vectors into host cells and culturing under conditions suitable for antibody expression; (3) collecting the antibody and purifying.

[0061] In another preferred embodiment, the antibody of the present application is prepared by immunization with an antigen, wherein the antigen has the amino acid sequence shown in SEQ ID No. 9 (see Table 3).

[0062] Examples The following shows the preparation and analysis of the antibody against botulinum toxin type A HA-70.

[0063] 1. Preparation of antibody (1) Take the natural botulinum toxin complex of Clostridium botulinum Hall strain, purify and culture, and perform electrophoresis on the complex. The electrophoresis result is shown in Figure 1 , and the band corresponding to the subunit HA-70 is recovered and extracted for preservation.

[0064] (2) Prepare the anti-HA-70 monoclonal antibody by hybridoma technology. Add an equal amount of Freund's adjuvant to HA-70 for emulsification, immunize 6-8 week old Balb / c mice, and perform subcutaneous injection in the groin of the mice at 0w, 3w, 5w, 7w, and 9w, respectively, with a dose of 40 μg per mouse and 140 μl per mouse; after the fifth immunization, select the mice with higher titer and perform impact immunization after two weeks, with a dose of 100 μg per mouse.

[0065] (3) Incubate the SP2 / 0 myeloma cells in a CO2 incubator (CO2 content is 5.0%) at 37°C, change the medium regularly, and subculture in time.

[0066] (4) Under sterile conditions, repeatedly flush the abdominal cavity of normal Balb / c mice with complete 1640 medium to prepare feeder cells.

[0067] (5) Spleen cells of immunized mice were isolated under sterile conditions and mixed with SP2 / 0 myeloma cells at a ratio of 5:1. Cell fusion was performed using the PEG method. The fused cell suspension was then dropped into a 96-well plate pre-coated with feeder cells.

[0068] (6) After culturing for a certain period of time, positive clones were screened by microscopic observation combined with ELISA detection. The positive wells were cloned in three stages by limiting dilution to obtain hybridoma cell lines that stably secrete specific antibodies.

[0069] (7) Antibodies were prepared by in vivo induction. Balb / c mice were sensitized with liquid paraffin (0.5 mL / mouse), and 1×10⁻⁶ antibodies were collected after 1 week. 6 Hybridoma cells were injected intraperitoneally, and ascites was collected 10 days later and purified using a Protein-G affinity chromatography column.

[0070] 2. Antibody Analysis The purified monoclonal antibodies were subclassed using a subclass identification kit, and the results are as follows: Figure 2 As shown, the antibody of the present invention is an IgG2a subclass.

[0071] The immunoreactivity of the monoclonal antibody and the natural botulinum toxin type A complex was identified by capillary immunoelectrophoresis, and the results are as follows: Figure 3 As shown, the monoclonal antibody of the present invention can specifically bind to botulinum toxin type A HA-70.

[0072] The affinity of monoclonal antibodies was analyzed using biomembrane interferometry, and the results are as follows: Figure 4 As shown in Table 4, the monoclonal antibody of the present invention has high affinity.

[0073] The antibody was molecularly sequenced, and the amino acid sequence obtained is shown in Table 2.

[0074] Table 2 Antibody amino acid sequences Table 3. Antigen Amino Acid Sequence Table 4 Antibody Affinity Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting botulinum toxin type A HA-70, characterized in that, Includes the following steps: (a) Provide the sample to be tested; (b) The test sample is contacted with an antibody or an antigen-binding fragment thereof for a time and under conditions sufficient to form an antibody or an antigen-binding fragment thereof, the antibody or the antigen-binding fragment thereof comprising the heavy chain CDR1-3 as shown in SEQ ID NO. 1-3 and / or the light chain CDR1-3 as shown in SEQ ID NO. 4-6; (c) Detect the binding of the antibody or its antigen-binding fragment to the sample to be tested, or detect the presence of the antibody or its antigen-binding fragment / antigen complex, to determine the presence of HA-70 in the sample or to determine the amount of HA-70 in the sample.

2. A detection product for detecting botulinum toxin type A HA-70, characterized in that, The detection product contains an antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising heavy chain CDR1-3 as shown in SEQ ID NO. 1-3 and / or light chain CDR1-3 as shown in SEQ ID NO. 4-6.

3. The application of the reagent in the preparation of products for detecting botulinum toxin type A HA-70, characterized in that, The reagent antibody or its antigen-binding fragment comprises heavy chain CDR1-3 as shown in SEQ ID NO. 1-3 and / or light chain CDR1-3 as shown in SEQ ID NO. 4-6.

4. An antibody or its antigen-binding fragment, characterized in that, The antibody or its antigen-binding fragment can specifically bind to botulinum toxin type A HA-70, and the antibody or its antigen-binding fragment includes the heavy chain CDR1-3 as shown in SEQ ID NO. 1-3 and / or the light chain CDR1-3 as shown in SEQ ID NO. 4-6.

5. The antibody or its antigen-binding fragment according to claim 4, characterized in that, The antibody or its antigen-binding fragment has any one of the amino acid sequences shown in (I)-(III): (I) The heavy chain amino acid sequence shown in SEQ ID NO.7 and / or the light chain amino acid sequence shown in SEQ ID NO.8; (II) and (I) show amino acid sequences that have at least 90% homology and the same function; (III) is an amino acid sequence with the same function obtained by modifying, substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (I) or (II).

6. The antibody or its antigen-binding fragment according to claim 4, characterized in that, The antibody includes monoclonal antibody, chimeric antibody, humanized antibody or murine antibody; the antigen-binding fragment includes Fab, Fab', F(ab')2 or scFv.

7. A nucleic acid molecule, characterized in that, The nucleic acid molecule comprises a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof according to any one of claims 4-6.

8. A carrier molecule, characterized in that, The carrier molecule comprises the nucleic acid molecule according to claim 7.

9. A host cell, characterized in that, The host cell contains the nucleic acid molecule according to claim 7 or the carrier molecule according to claim 8.

10. The method for preparing the antibody or its antigen-binding fragment according to any one of claims 4-6, characterized in that, The antibody or its antigen-binding fragment is prepared by artificial synthesis or genetic engineering.

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