A detection product and method for detecting botulinum toxin type A component proteins.

Abstract

This invention discloses a detection product and method for detecting botulinum toxin type A component proteins. The detection product comprises an antibody capable of specifically recognizing botulinum toxin type A component proteins. The antibody exhibits good affinity and stability, enabling its wide application in the detection of botulinum toxin type A component proteins. It can be combined with capillary immunoelectrophoresis to establish standard spectra for the detection of botulinum toxin component antibodies, providing quality control for both commercial and self-developed antibodies. Therefore, this invention offers more possibilities for establishing botulinum toxin-related detection methods and can meet the detection needs of more scenarios.

Description

Technical Field

[0001] This invention belongs to the field of botulinum toxin detection, and in particular relates to a detection product and method for detecting botulinum toxin type A component proteins. 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 detection products and methods that can detect 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 detection product and method for detecting botulinum toxin type A component proteins. Specifically, the present invention includes the following:

[0006] In a first aspect, the present invention provides a detection product for detecting botulinum toxin type A component proteins, wherein the detection product is used to detect hemagglutinin-70, the detection product comprising an antibody or an antigen-binding fragment thereof, the antibody or the antigen-binding fragment thereof comprising a heavy chain CDR1-3 as shown in SEQ ID NO. 1-3 and / or a light chain CDR1-3 as shown in SEQ ID NO. 4-6.

[0007] In some embodiments, the detection product according to the present invention includes a reagent kit, test strip, or protein chip.

[0008] A second aspect of the present invention provides a method for detecting botulinum toxin type A hemagglutinin-70, comprising the following steps:

[0009] (a) Provide the sample to be tested;

[0010] (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;

[0011] (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 hemagglutinin-70 in the sample or to determine the amount of hemagglutinin-70 in the sample.

[0012] In a third aspect, the present invention provides an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof is capable of targeting botulinum toxin type A hemagglutinin-70, the antibody or the antigen-binding fragment thereof comprising a heavy chain CDR1-3 as shown in SEQ ID NO. 1-3 and / or a light chain CDR1-3 as shown in SEQ ID NO. 4-6.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof according to the present invention has any one of the amino acid sequences shown in (I)-(III):

[0014] (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;

[0015] (II) and (I) show amino acid sequences that have at least 90% homology and the same function;

[0016] (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).

[0017] 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.

[0018] A fourth aspect of the invention provides a nucleic acid molecule, wherein the nucleic acid molecule comprises a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof according to a third aspect of the invention.

[0019] A fifth aspect of the invention provides a carrier molecule, wherein the carrier molecule comprises a nucleic acid molecule according to a fourth aspect of the invention.

[0020] A sixth aspect of the invention provides a host cell, wherein the host cell comprises a nucleic acid molecule according to a fourth aspect of the invention or a carrier molecule according to a fifth aspect of the invention.

[0021] A seventh 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.

[0022] An eighth aspect of the invention provides the use of a reagent in the preparation of a product for detecting botulinum toxin type A hemagglutinin-70, wherein the reagent comprises an antibody or an antigen-binding fragment thereof according to a third aspect of the invention.

[0023] This invention purifies the natural botulinum toxin type A complex component protein hemagglutinin-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 hemagglutinin-70, preserving the antibody's affinity maturation in vivo and the original pairing of the antibody's 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 hemagglutinin-70 and have good affinity and stability, enabling widespread application in the detection of botulinum toxin type A hemagglutinin-70. They can be combined with capillary immunoelectrophoresis to establish standard patterns for botulinum toxin component antibodies, providing quality control methods 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

[0024] Figure 1 The SDS-PAGE results of hemagglutinin-70 antigen are shown.

[0025] Figure 2 The results of subclass identification of the anti-hemagglutinin-70 antibody of the present invention are shown.

[0026] Figure 3The capillary immunoelectrophoresis results of the anti-hemagglutinin-70 antibody and the natural botulinum toxin type A complex of the present invention are shown. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, 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 invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention.

[0030] I. Product Testing

[0031] In one aspect, the present invention provides a detection product for detecting (or quantifying) botulinum toxin type A hemagglutinin-70, the detection product comprising an antibody or an antigen-binding fragment thereof, and optionally instructions on how to perform the detection method of the present invention on botulinum toxin type A hemagglutinin-70, said antibody or antigen-binding fragment 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.

[0032] Examples of the detection products of this invention include, but are not limited to, reagent kits, test strips, protein chips, etc.

[0033] In this document, the term "reagent kit" refers to a set of standardized reagents and consumables systematically combined and provided to perform a specific biological assay or experimental procedure. Exemplarily, a reagent kit may include core reaction reagents, reaction system support, signal generation and detection system, solid-phase carrier, auxiliary components, etc. Examples of core reaction reagents include, but are not limited to, the antibodies or antigen-binding fragments thereof, antigens, and controls of this invention; examples of reaction system support include, but are not limited to, buffer solutions, protein stabilizers, blocking agents, and enzymes; examples of signal generation and detection systems include, but are not limited to, substrates, chromogenic agents, or labels used for chemiluminescence, fluorescence, or colorimetric detection; examples of solid-phase carriers include, but are not limited to, ELISA plates, test strips, and chips; examples of auxiliary components include, but are not limited to, instructions for use, and packaging containers.

[0034] In some embodiments, the kit may further include at least one of a washing solution, a substrate solution, a diluent, and a calibration solution. The washing solution is not particularly limited in composition, and examples include, but are not limited to, buffers (e.g., but not limited to PBS, Tris-HCl, etc.), surfactants (e.g., but not limited to Tween-20), and preservatives (e.g., but not limited to isothiazolinone). The substrate solution may use known substrates, including, but not limited to, chromogenic substrates (e.g., but not limited to TMB, OPD, etc.), fluorescent substrates (e.g., but not limited to AMC, 4-MU, etc.), and luminescent substrates (e.g., but not limited to luminol, ECL, etc.). The diluent is not particularly limited in composition, and examples include, but are not limited to, buffers and surfactants. The calibration solution is not particularly limited in composition, and examples include, but are not limited to, BSA solution, trehalose solution, and animal serum.

[0035] In this invention, examples of reagent kits include, but are not limited to, ELISA detection kits, plate-based chemiluminescence detection kits, fully automated chemiluminescence detection kits, radioimmunoassay kits, fluorescence immunoassay kits, chemiluminescence immunoassay kits, immunoblotting kits, flow cytometry detection kits, colloidal gold detection kits, bioluminescent immunoassay kits, immunochromatographic detection kits, latex-based kits, and kits based on direct or indirect competitive methods.

[0036] II. Methods

[0037] One aspect of the present invention provides a method for detecting botulinum toxin type A hemagglutinin-70, comprising the following steps:

[0038] (a) Provide the sample to be tested;

[0039] (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;

[0040] (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 hemagglutinin-70 in the sample or to determine the amount of hemagglutinin-70 in the sample.

[0041] In this invention, the samples are not particularly limited, and examples include, but are not limited to, food samples (e.g., but not limited to, vegetables, meat products, dairy products, honey, fermented foods, etc.), environmental samples (e.g., but not limited to, soil, water samples, etc.), drug samples, biological samples, etc.

[0042] In some embodiments, the antibodies or antigen-binding fragments of the present invention may carry any detectable label. Examples of labels that may be used include, but are not limited to, radioactive substances, fluorescent groups, chemical labels, biological agents (e.g., but not limited to biotin / streptavidin detection), or enzyme substrate labels. In other embodiments, detection antibodies carrying detectable labels may be used in the present invention, and the use of detection antibodies may, in some cases, enhance the signal of the antibodies of the present invention.

[0043] In this invention, the "time and conditions sufficient to form an antibody or its antigen-binding fragment / antigen complex" are not particularly limited, as long as the hemagglutinin-70 in the sample binds to and forms a stable antibody or its antigen-binding fragment / antigen complex or conjugate after contact with the antibody or its antigen-binding fragment of this invention.

[0044] Those skilled in the art are well aware of how to detect the binding of hemagglutinin-70 to an antibody or its antigen-binding fragment in a sample, or the presence of an antibody or its antigen-binding fragment / antigen complex, thereby determining the presence or amount of hemagglutinin-70 in the sample. Such detection techniques include, but are not limited to, immunofluorescence, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), agglutination assay, immunoadsorption, immunoelectrophoresis, and immunoelectron microscopy.

[0045] III. Antibody or its antigen-binding fragment

[0046] In one aspect of the present invention, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof is capable of targeting botulinum toxin type A hemagglutinin-70, 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.

[0047] In this article, the term "antibody" refers to an immunoglobulin molecule with the ability to specifically bind antigens. A typical antibody monomer consists of two identical heavy chains and two identical light chains linked by disulfide bonds, forming a "Y"-shaped structure. Antibodies typically contain variable and constant regions in each heavy and light chain. The variable region is located at the N-terminus of both the heavy and light chains, and its amino acid sequence is highly variable, collectively constituting the antigen-binding fragment of the antibody. The variable regions of the heavy and light chains cooperate closely through non-covalent interactions and disulfide bonds to form a complement-determining region, which directly determines the specificity and affinity of the antibody for antigen recognition. The constant region is located at the C-terminus of the chain, and its sequence is relatively conserved within the same species. This region mainly mediates the effector functions of the antibody, such as binding to Fc receptors on the surface of immune cells and activating the complement system, thereby regulating and transducing the immune response.

[0048] The "light chain variable region (VL)" or "heavy chain variable region (VH)" of this invention consists of "framework" regions interspersed among three "complementarity-determining regions (CDRs)". The framework regions are used to adjust the CDRs for specific binding of antigenic epitopes. From the amino terminus to the carboxyl terminus, both the VL and VH domains contain the following framework (FR) regions and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The complementarity-determining regions include three prominent ring structures (CDR1, CDR2, CDR3) with extremely high amino acid sequence variability, directly contacting and determining the specificity and affinity of the antibody for binding antigens. FR1-FR4 constitute the β-sheet barrel scaffold of the variable region; their main function is to provide the correct spatial folding, positioning, and orientation for the CDR rings, ensuring that the CDRs form stable, usable antigen-binding pockets. Although relatively conserved, some FR residues also participate in antigen contact or affect antibody stability. Therefore, the six CDRs of VH and VL together constitute the antigen binding site, while the eight framework regions provide precise structural support for these key sites.

[0049] In this invention, an "epitaph" includes any determinant cluster capable of specifically binding to an antibody. An epitope is a region of an antigen that binds to an antibody that specifically targets that antigen. When the antigen is a protein, the epitope contains a specific amino acid that is in direct contact with the antibody. Epitopes are typically located on proteins, but in some cases they can also be located on other types of molecules, such as nucleic acids. Epitope determinants can include chemically active surface groups of a molecule, 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. Typically, specific antibodies against a particular target antigen preferentially recognize epitopes on the target antigen in complex mixtures of proteins and / or macromolecules.

[0050] In some embodiments, the antibody or its antigen-binding fragment has any one of the amino acid sequences shown in (I)-(III):

[0051] (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;

[0052] (II) and (I) show amino acid sequences that have at least 90% homology and the same function;

[0053] (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).

[0054] In this document, 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 this invention. To determine sequence identity, sequence alignment can be performed, which can be done in various ways known to those skilled in the art, such as using BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms required to achieve optimal alignment across the full-length sequences being compared.

[0055] In this invention, variant antibody sequences obtained by modification, substitution, deletion, or addition of one or more amino acids are also within the scope of protection of this invention. The term "modification" refers to covalent chemical modification of the side chains of amino acid residues in the sequence (e.g., glycosylation, polyethylene glycolation, acetylation, etc.). The term "substitution" refers to replacing one or more residues in the original sequence with one or more different amino acid residues. The term "deletion" refers to removing one or more amino acid residues from the original sequence. The terms "insertion" or "addition" refer to introducing one or more additional amino acid residues into the original sequence. To maximize the preservation of the antibody's core binding function, the above sequence changes preferably occur outside the variable region (VH / VL), such as at non-critical sites or constant regions of the frame region. In particular, the variants should avoid disruptive alterations to critical residues in the complementarity-determining region unless the alteration is intended for targeted affinity maturation. All variants falling within the scope of this invention must retain the desired biological function of the original antibody or its antigen-binding fragment, particularly its ability to specifically bind to the target antigen. The binding activity of these variants can be determined by standard methods in the art (e.g., but not limited to ELISA, surface plasmon resonance, etc.). In some implementations, the variant may have equivalent or improved functional properties, such as enhanced affinity, stability, or reduced immunogenicity.

[0056] In this invention, conserved amino acid substitutions are preferred, and these conserved antibody variants are preferably generated by amino acid substitutions according to Table 1:

[0057] Table 1

[0058]

[0059] In this invention, antibodies include monoclonal antibodies, chimeric antibodies, humanized antibodies, or murine antibodies.

[0060] As used in this article, "monoclonal antibody" (sometimes also called "mAb") refers to a class of highly homogeneous immunoglobulin molecules produced from B cell clones (or engineered cell lines) with identical genetic backgrounds. These molecules share the same structure and chemical properties and are specific to a single antigenic determinant. Unlike conventional polyclonal antibody preparations (which typically contain different antibodies targeting different determinants), each monoclonal antibody targets a single determinant on an antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are obtained through hybridoma or recombinant engineered cell culture, free from contamination by other immunoglobulins. This characteristic contrasts with polyclonal antibody products, which generally include antibodies targeting different antigenic determinants. The modifier "monoclonal" indicates the antibody's characteristic of being obtained from a homogeneous group of antibodies, but this should not be interpreted as requiring any special methods to produce the antibody.

[0061] In this article, the term "chimeric antibody" generally refers to an antibody in which a portion of the amino acid sequence of each heavy or light chain is homologous to, or belongs to, a corresponding amino acid sequence from an antibody of a specific species, while the remaining segments of that chain are homologous to a corresponding sequence from another species. For example, a "mouse / human chimeric antibody" has a variable region derived from the VH and VL regions of an antibody from a non-human species (such as mice or rats), responsible for providing high specificity for antigen recognition and binding, and a human-derived constant region that determines the antibody's effector function and pharmacokinetic properties in the human body. Chimeric antibodies retain the high affinity and specificity of the parent non-human antibody because the sequence and structure of the variable region remain unchanged. Simultaneously, its humanized constant region significantly reduces immunogenicity in humans and endows it with more suitable human effector functions. The variable region has the advantage of being easy to prepare, and its specificity is unaffected by the source of the constant region it is combined with.

[0062] In this article, the term "humanized antibody" generally refers to a chimeric antibody containing fewer sequences derived from non-human immunoglobulins, thereby reducing the immunogenicity when xenobiotic antibodies are introduced into humans, while maintaining the antibody's complete antigen-binding affinity and specificity. Humanized antibodies are a more advanced form of chimerism. They not only replace constant regions with human sequences but also critically modify variable regions. The core technology is "CDR transplantation," which involves embedding the specific CDRs from mouse antibodies that directly contact the antigen into the pre-selected, highly compatible human antibody framework region. This process often requires structural biology analysis and computer simulations to fine-tune the residues in key framework regions to ensure that the transplanted humanized antibody fully inherits or even enhances the binding performance of the parent antibody. Humanized antibodies exhibit significantly lower immunogenicity than mouse antibodies and chimeric antibodies, have a longer half-life and better safety in humans, while retaining the high specificity and affinity required for targeting antigens.

[0063] In this document, the term "mouse antibody" generally refers to an antibody whose variable region framework and CDR region are derived from mouse germline immunoglobulin sequences. Additionally, if the antibody contains a constant region, it is also derived from mouse germline immunoglobulin sequences. In this invention, mouse antibodies may contain amino acid residues not encoded by mouse germline immunoglobulin sequences, for example, mutations introduced through in vitro random or point mutations or through in vivo somatic mutations.

[0064] In this document, the term "antigen-binding fragment" generally refers to a functional fragment composed of partial antibody domains that retains the complete antibody-specific antigen-binding ability. These fragments lack the constant regions of a complete antibody and therefore typically do not mediate effector functions such as antibody-dependent cytotoxicity or complement-dependent cytotoxicity. However, their smaller molecular size may result in better tissue penetration. It is understood that the antigen-binding function of an antibody can be achieved through the full-length fragment of the antibody, or through a heavy chain including Fab, Fab', F(ab')2, or scFv, or a light chain including Fab, Fab', F(ab')2, or scFv. It is understood that any antibody derivative obtained through genetic engineering or enzymatic digestion methods that contains the aforementioned necessary variable regions and can specifically bind to the target antigen falls within the scope of the "antigen-binding fragment" described in this invention.

[0065] In this invention, the terms “binding,” “specific binding,” “targeting,” and “specifically binding” are used interchangeably and generally refer to non-covalent interactions occurring between an immunoglobulin molecule and an antigen specific to said immunoglobulin. The strength or affinity of an immunobinding interaction can be expressed as a dissociation constant (Kd), where a smaller Kd represents a higher affinity. “Affinity” refers to the strength of the sum of all 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 strength of the binding. Unless otherwise stated, when used herein, “binding affinity” refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y is generally expressed as a binding dissociation equilibrium constant. The affinity of an antibody for an antigen can be quantitatively determined using a variety of biophysical methods recognized in the art, such as, but not limited to, surface plasmon resonance, isothermal titration calorimetry, and biofilm interferometry. As long as the method can accurately measure the kinetic or thermodynamic parameters of the binding reaction and calculate the Kd value, it is applicable to evaluating the binding affinity described in this invention.

[0066] IV. Nucleic acid molecules

[0067] In one aspect, a nucleic acid molecule is provided that comprises a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof described in this invention.

[0068] As used in this invention, the term "nucleic acid" is intended to include polymeric forms of nucleotides of any length containing deoxyribonucleotides, ribonucleotides, and / or their analogues, including DNA, RNA, and DNA / RNA hybrids, and also including DNA or RNA analogues, such as those containing a modified backbone (e.g., peptide nucleic acid (PNA) or phosphate thioester) or modified bases. Therefore, the nucleic acids of this invention include DNA, cDNA, mRNA, recombinant nucleic acids, etc.

[0069] Once the coding sequence of the antibody or its antigen-binding fragment described in this invention is obtained, recombinant technology can be used to obtain the antibody or its antigen-binding fragment in large quantities. An exemplary method is to clone its coding gene into a vector, transform it into cells, and then isolate it from the proliferated host cells using conventional methods.

[0070] V. Carrier molecules

[0071] In one aspect, the present invention provides a carrier molecule comprising the nucleic acid molecule described herein.

[0072] The term "vector" in this invention refers to an artificial construct that can introduce a foreign gene or nucleic acid sequence into a host cell and guide its replication and / or expression. The vector of this invention is not limited and can be a cloning vector, expression vector, etc. In some embodiments, the vector contains a target gene encoding the antibody of this invention or its antigen-binding fragment, 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.

[0073] VI. Host Cells

[0074] In one aspect, the present invention provides a host cell comprising the nucleic acid molecule or the carrier molecule described herein.

[0075] 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.

[0076] VII. Preparation Method

[0077] 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.

[0078] 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.

[0079] In some embodiments, the antibodies or antigen-binding fragments of the present invention are obtained through genetic engineering expression. Genetic engineering expression systems include prokaryotic cell expression systems, eukaryotic cell expression systems, and cell-free expression systems. Examples of prokaryotic cell expression systems include Escherichia coli expression systems. Eukaryotic cell expression systems include enzyme expression systems, insect cell expression systems, and mammalian cell expression systems.

[0080] In one specific embodiment, the antibody preparation method of the present invention includes:

[0081] (1) Construct 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;

[0082] (2) The vector is transformed into host cells and cultured under conditions suitable for antibody expression;

[0083] (3) Collect antibodies and purify them.

[0084] In another preferred embodiment, the antibody of the present invention is prepared by immunization with an antigen having the amino acid sequence shown in SEQ ID No. 9 (see Table 3).

[0085] The present invention further provides the use of reagents in the preparation of products for detecting (or quantifying) botulinum toxin type A hemagglutinin-70, wherein the reagents comprise the antibody or antigen-binding fragment thereof described in the present invention.

[0086] Example

[0087] The following shows the preparation and analysis of antibodies against botulinum toxin type A hemagglutinin-70.

[0088] 1. Antibody preparation

[0089] (1) Natural botulinum toxin complex was obtained by culturing and purifying the Hall strain of Clostridium botulinum type A. The complex was subjected to electrophoresis, and the electrophoresis results are as follows: Figure 1 As shown, the band corresponding to the subunit hemagglutinin-70 was recovered, extracted, and preserved.

[0090] (2) Prepare anti-hemagglutinin-70 monoclonal antibody using hybridoma technology. Emulsify hemagglutinin-70 with an equal amount of Freund's adjuvant and immunize 6-8 week old Balb / c mice. The mice were injected subcutaneously into the groin at 0, 3, 5, 7 and 9 weeks of age, with doses of 40 μg / mouse and 140 μl / mouse, respectively. After the fifth immunization, mice with higher titers were selected for a shock immunization two weeks later, with 100 μg / mouse.

[0091] (3) SP2 / 0 myeloma cells were cultured in a CO2 incubator at 37°C (CO2 content of 5.0%), with the medium changed regularly and passaged in a timely manner.

[0092] (4) Under sterile conditions, the peritoneal cavity of normal Balb / c mice was repeatedly flushed with complete 1640 culture medium to prepare feeder cells.

[0093] (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.

[0094] (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.

[0095] (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.

[0096] 2. Antibody Analysis

[0097] 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 IgG1 subclass.

[0098] 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 hemagglutinin-70.

[0099] The antibody was molecularly sequenced, and the amino acid sequence obtained is shown in Table 2.

[0100] Table 2 Antibody amino acid sequences

[0101]

[0102] Table 3. Antigen Amino Acid Sequence

[0103]

[0104] 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 detection product for detecting botulinum toxin type A component proteins, characterized in that, The detection product is used to detect hemagglutinin-70. The detection product contains an antibody or its antigen-binding fragment, the antibody or its antigen-binding fragment comprising a heavy chain CDR1-3 with amino acid sequences as shown in SEQ ID NO.1-3 and a light chain CDR1-3 with amino acid sequences as shown in SEQ ID NO.4-6.

2. A method for detecting botulinum toxin type A hemagglutinin-70, characterized in that, The method described is a non-disease diagnostic method and 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 a heavy chain CDR1-3 as shown in SEQ ID NO. 1-3 and a 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 hemagglutinin-70 in the sample or to determine the amount of hemagglutinin-70 in the sample.

3. An antibody or its antigen-binding fragment, characterized in that, The antibody or its antigen-binding fragment can target botulinum toxin type A hemagglutinin-70, and the antibody or its antigen-binding fragment includes the heavy chain CDR1-3 with amino acid sequences as shown in SEQ ID NO.1-3 and the light chain CDR1-3 with amino acid sequences as shown in SEQ ID NO.4-6.

4. The antibody or its antigen-binding fragment according to claim 3, characterized in that, The antibody or its antigen-binding fragment has a heavy chain with an amino acid sequence as shown in SEQ ID NO.7 and a light chain with an amino acid sequence as shown in SEQ ID NO.

8.

5. The antibody or its antigen-binding fragment according to claim 3, characterized in that, The antibody is selected from monoclonal antibodies; the antigen-binding fragment is selected from Fab, Fab', F(ab')2 or scFv.

6. 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 3-5.

7. A carrier molecule, characterized in that, The carrier molecule comprises the nucleic acid molecule according to claim 6.

8. A host cell, characterized in that, The host cell contains the nucleic acid molecule according to claim 6 or the carrier molecule according to claim 7.

9. The method for preparing the antibody or its antigen-binding fragment according to any one of claims 3-5, characterized in that, The antibody or its antigen-binding fragment is prepared by artificial synthesis or genetic engineering.

10. The application of the reagent in the preparation of products for detecting botulinum toxin type A hemagglutinin-70, characterized in that, The reagent comprises an antibody or an antigen-binding fragment thereof according to any one of claims 3-5.

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