Novel Anti-botulinum toxin antibody

Abstract

The present invention relates to a novel monoclonal antibody specifically binding to botulinum toxin and a method for detecting botulinum toxin using same. The novel antibody of the present invention allows botulinum toxin in an analysis sample to be detected or quantified with significantly superior binding force and specificity compared to commercially available antibodies, thereby being able to be effectively used to efficiently evaluate the quality of products containing botulinum toxin preparations.

Description

Novel anti-botulinum toxin antibody

[0001] The present invention relates to a novel antibody that specifically binds to botulinum toxin and a hybridoma cell line that produces the same.

[0002] Botulinum toxin is a neurotoxin produced by the bacterium Clostridium botulinum and is a powerful toxin that causes botulism in the human body. Botulinum toxin induces nerve paralysis by blocking the release of acetylcholine, a neurotransmitter secreted from the axon terminals of the neuromuscular junction. Botulinum toxin is classified into seven serotypes (A, B, C, D, E, F, and G), and among them, types A, B, E, F, and G are known to act on the human nervous system, excluding types C and D. Since it was reported that botulinum toxin is also effective in treating patients with blepharospasm and strabismus, the use of botulinum toxin for therapeutic and cosmetic purposes in clinical immunology has been rapidly increasing, and currently, various manufacturers are marketing products containing botulinum toxin preparations as active ingredients.

[0003] For products containing such botulinum toxin preparations, quality evaluation methods capable of verifying their safety and efficacy are essential. To this end, the development of means to detect botulinum toxin with high specificity and selectivity is critical. Traditionally, animal testing (i.e., in vivo confirmation methods for botulinum toxin) has long been used for the quality evaluation of products containing botulinum toxin preparations. However, these animal testing methods present inconveniences in execution because the testing methods and reagents used vary by manufacturer, and the quantity of samples that can be analyzed at once is very limited. Furthermore, due to the nature of animal testing, experimental results are heavily influenced by the researchers, testing institutions, and facilities. Additionally, a large number of experimental animals are sacrificed, and psychological stress is inflicted on researchers, resulting in unnecessary healthcare costs and serious additional issues such as social backlash. Crucially, these animal tests not only have low detection accuracy but also suffer from the fatal disadvantage that the long testing time makes it impossible to obtain test results quickly. In order to resolve the problems of such animal testing, there is a growing need to develop standardized in vitro identification methods. The 'Standards and Methods for Biological Products (No. 2022-58)' and the European Pharmacopoeia (EP 11.2) present enzyme immunological methods and immunochemical analysis methods, respectively, as in vitro identification methods for botulinum toxin preparations.

[0004] Immunoassays are biological analytical methods that perform qualitative and quantitative analysis of analytes through antigen-antibody reactions. Due to their high specificity and sensitivity, they are widely used in important areas of pharmaceutical analysis, such as disease diagnosis, therapeutic drug monitoring, clinical toxicology, and bioequivalence studies. Enzyme-linked immunoassay (ELISA) is currently the most widely utilized method among various immunoassays and is classified into three types based on the antibody utilization method: Direct ELISA, Indirect ELISA, and Sandwich ELISA. Sandwich ELISA is a method in which an antigen-specific 'capture antibody' is conjugated to an immunoplate, and the primary antibody is detected using an 'enzyme-conjugated secondary antibody conjugate.' This method is applicable only if there are at least two types of antibodies that bind to different sites on the antigen. This detection method selects the same antigen twice through capture and detection, making it the method with the highest specificity for the antigen. Antibodies are classified into polyclonal antibodies and monoclonal antibodies based on their epitopes; polyclonal antibodies are antibodies that possess multiple epitopes for an antigen, whereas monoclonal antibodies possess a specific epitope for the antigen. Therefore, monoclonal antibodies have higher concentration, purity, and specificity compared to polyclonal antibodies, and in terms of production, they offer the advantage of being able to be produced as a continuous and renewable resource when manufacturing desired hybridoma cells.

[0005] Accordingly, the inventors of the present invention sought to develop a method for efficiently evaluating botulinum toxin preparations by detecting botulinum toxin with high specificity and accuracy, and completed the present invention by developing a novel anti-botulinum toxin monoclonal antibody that can be used in sandwich enzyme immunoassay (Sandwich ELISA) by binding to botulinum toxin with high specificity and accuracy to detect the toxin with high specificity and accuracy.

[0006] Throughout this specification, numerous papers and patent documents are referenced and cited. The disclosures of the cited papers and patent documents are incorporated by reference into this specification in their entirety to more clearly explain the state of the art to which the present invention pertains and the content of the present invention.

[0007] [Prior Art Literature]

[0008] (Patent Document) U.S. Patent Publication No. 2019-0256584

[0009] The inventors have made diligent research efforts to develop a method capable of rapidly and reliably evaluating the quality of products containing botulinum toxin preparations by detecting the presence of botulinum toxin in a sample under analysis with high accuracy and specificity, or by quantifying it. As a result, the present invention was completed by newly selecting a monoclonal antibody with particularly high binding affinity and specificity among monoclonal antibodies that specifically bind to botulinum toxin type A, and by confirming that the presence of botulinum toxin in a sample under analysis can be verified and quantified with high accuracy using this antibody.

[0010] Therefore, the objective of the present invention is to provide a monoclonal antibody that specifically binds to botulinum toxin.

[0011] Another objective of the present invention is to provide a hybridoma cell line that produces the monoclonal antibody.

[0012] Another objective of the present invention is to provide a composition for detecting botulinum toxin comprising the monoclonal antibody.

[0013] Another objective of the present invention is to provide a method for detecting botulinum toxin in a target sample.

[0014] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims, and drawings.

[0015] According to one aspect of the present invention, the present invention provides a monoclonal antibody that specifically binds to botulinum toxin, comprising:

[0016] A heavy chain variable region comprising one or more sequences selected from the group consisting of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 17, SEQ ID NO 18, and SEQ ID NO 19; and a light chain variable region comprising one or more sequences selected from the group consisting of SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 20, SEQ ID NO 21, and SEQ ID NO 22.

[0017] The inventors have made diligent research efforts to develop a method capable of rapidly and reliably evaluating the quality of products containing botulinum toxin preparations by detecting or quantifying the presence of botulinum toxin in samples under analysis with high accuracy and specificity. As a result, the present invention was completed by newly selecting a monoclonal antibody with particularly high binding affinity and specificity among monoclonal antibodies that specifically bind to botulinum toxin type A, and by confirming that the presence of botulinum toxin in samples under analysis can be verified and quantified with high accuracy using this antibody.

[0018] In this specification, the term “botulinum toxin” may be used interchangeably with “Clostridium botulinum toxin” and refers to a neurotoxic protein produced by the bacterium Clostridium botulinum and closely related species. Botulinum toxin causes muscle paralysis (muscle relaxation) by preventing the release of acetylcholine (Ach), a type of neurotransmitter secreted from the axon terminals of the neuromuscular junction, which is the synapse between a nerve cell and a muscle fiber. Although this toxicity is extremely fatal to the human body, it can be used for the prevention or treatment of various diseases by being diluted. Diluted solutions of botulinum toxin can be used for the prevention or treatment of various diseases and pathological conditions, including movement disorders such as dystonia and non-dystonia, ophthalmic diseases, pain-related diseases such as migraines, urological diseases, and wrinkle improvement for cosmetic purposes.

[0019] In this specification, the term “antibody” means an antibody against botulinum toxin, particularly serotype A thereof, which specifically recognizes and binds to a specific epitope of the toxin, and includes not only the complete antibody form but also antigen-binding fragments (antibody fragments) of the antibody molecule.

[0020] A complete antibody has a structure consisting of two full-length light chains and two full-length heavy chains, with each light chain connected to the heavy chain by a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and has subclasses gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The light chain constant region has kappa (κ) and lambda (λ) types.

[0021] In this specification, the term “antigen-binding fragment of an antibody” refers to a fragment within the entire antibody molecule that possesses antigen-antibody binding function, and includes Fab, F(ab'), F(ab')2, and Fv, etc. Among the antibody fragments, Fab comprises a variable region of the light chain and heavy chain, a constant region of the light chain, and a first constant region of the heavy chain (C H1 It has a structure containing ) and possesses one antigen-binding site. Fab' is heavy chain C H1 It differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the domain. F(ab')2 antibodies are produced when the cysteine ​​residues in the hinge region of Fab' form disulfide bonds. A recombinant technology for generating Fv fragments from minimal antibody fragments having only a heavy chain variable region and a light chain variable region is disclosed in PCT international published patent applications WO 88 / 10649, WO 88 / 106630, WO 88 / 07085, WO 88 / 07086, and WO 88 / 09344. Two-chain Fvs have a heavy variable region and a light variable region connected by non-covalent bonds, while single-chain Fvs generally have a heavy variable region and a single variable region connected by covalent bonds via a peptide linker or directly connected at the C-terminus, allowing them to form a dimer-like structure similar to two-chain Fvs. These antibody fragments can be obtained using proteolytic enzymes (for example, restriction cleavage of a whole antibody with papain yields Fab, and cleavage with pepsin yields the F(ab')2 fragment), or they can be produced using recombinant DNA technology.

[0022] According to a specific embodiment of the present invention, the antigen-binding fragment of the present invention is a Fab fragment, an F(ab') fragment, an F(ab')2 fragment, or an Fv fragment.

[0023] The antibodies of the present invention include, but are not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, short-chain Fvs(scFV), short-chain antibodies, Fab fragments, F(ab') fragments, disulfide-bound Fvs(sdFV) and anti-idiotype (anti-Id) antibodies, and epitope-bound fragments of said antibodies.

[0024] In this specification, the term “heavy chain” refers to a variable region domain V comprising an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen. H and 3 invariant domains C H1 , C H2 and C H3 It refers to both the full-length heavy chain containing and its fragments.

[0025] In this specification, the term “light chain” refers to a variable region domain V comprising an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen. L and invariant domain C L It refers to the entire length light chain and all fragments thereof that include it.

[0026] In this specification, the term “variable region” refers to a region located at the amino acid terminus of the light or heavy chain of an antibody that has various arrangements different for each antibody and determines the specificity of binding with an antigen; the variable region includes the complementarity determining region (CDR). Accordingly, the “heavy chain variable region” refers to a variable region located in the heavy chain of the antibody, and the “light chain variable region” refers to a variable region located in the light chain of the antibody.

[0027] In this specification, the term “CDR (complementarity determining region)” refers to the amino acid sequence of a hypervariable region of an immunoglobulin heavy chain and light chain (Kabat et al. Sequences of Proteins of Immunological Interest, 4th Ed. US Department of Health and Human Services, National Institutes of Health (1987)). The heavy chain and light chain of an antibody each contain three CDRs (heavy chain: HCDR1, HCDR2, and HCDR3; light chain: LCDR1, LCDR2, and LCDR3), and these CDRs provide major contact residues for the antibody to bind to an antigen or epitope. The monoclonal antibodies of the present invention may have the heavy chain CDRs (HCDRs) of SEQ ID NOs 1 to 3; SEQ ID NOs 9 to 11; and SEQ ID NOs 17 to 19, and SEQ ID NOs 4 to 6; SEQ ID NOs 12 to 14; It may have light chain CDRs (LCDRs) of sequence numbers 20 to 22.

[0028] In this specification, the term “specifically binding” has the same meaning as “specifically recognizing” and may be used interchangeably; it refers to a state or phenomenon in which an antigen and an antibody (or a fragment thereof) interact specifically through an immunological reaction. In the present invention, “specifically binding to botulinum toxin” means that an antibody that specifically and selectively binds to the antigen, botulinum toxin, binds to it.

[0029] In this specification, the term “monoclonal antibody” refers to a protein molecule that specifically binds to a single antigenic site (a single epitope). For the purposes of the present invention, the monoclonal antibody of the present invention is a protein molecule that recognizes botulinum toxin (especially serotype A) and specifically binds to botulinum toxin (especially serotype A). Monoclonal antibodies are also referred to as monoclonal antibodies, and monoclonal antibodies that bind to a single antigen can be produced by producing them in a single type of plasma cell.

[0030] According to a specific embodiment of the present invention, the monoclonal antibody comprises a heavy chain variable region including an HCDR1 region selected from the group consisting of sequences 1, 9, and 17; an HCDR2 region selected from the group consisting of sequences 2, 10, and 18; and an HCDR3 region selected from the group consisting of sequences 3, 11, and 19.

[0031] According to a specific embodiment of the present invention, the monoclonal antibody comprises a light chain variable region including an LCDR1 region selected from the group consisting of sequences 4, 12, and 20; an LCDR2 region selected from the group consisting of sequences 5, 13, and 21; and an LCDR3 region selected from the group consisting of sequences 6, 14, and 22.

[0032] According to a specific embodiment of the present invention, the heavy chain variable region comprises one or more sequences selected from the group consisting of sequence number 1, sequence number 2, and sequence number 3, and the light chain variable region comprises one or more sequences selected from the group consisting of sequence number 4, sequence number 5, and sequence number 6.

[0033] According to a specific embodiment of the present invention, the heavy chain variable region comprises one or more sequences selected from the group consisting of SEQ ID NO. 9, SEQ ID NO. 10, and SEQ ID NO. 11, and the light chain variable region comprises one or more sequences selected from the group consisting of SEQ ID NO. 12, SEQ ID NO. 13, and SEQ ID NO. 14.

[0034] According to a specific embodiment of the present invention, the heavy chain variable region comprises one or more sequences selected from the group consisting of SEQ ID NO. 17, SEQ ID NO. 18, and SEQ ID NO. 19, and the light chain variable region comprises one or more sequences selected from the group consisting of SEQ ID NO. 20, SEQ ID NO. 21, and SEQ ID NO. 22.

[0035] According to a specific embodiment of the present invention, the heavy chain variable region comprises the sequence of SEQ ID NO. 7, and the light chain variable region comprises the sequence of SEQ ID NO. 8.

[0036] According to a specific embodiment of the present invention, the heavy chain variable region comprises the sequence of SEQ ID NO. 15, and the light chain variable region comprises the sequence of SEQ ID NO. 16.

[0037] According to a specific embodiment of the present invention, the heavy chain variable region comprises the sequence of SEQ ID NO. 23, and the light chain variable region comprises the sequence of SEQ ID NO. 24.

[0038] The monoclonal antibody of the present invention comprises three clones, 9H10, 5C2, and 1E10, listed in Table 5, and the CDR sequences of the heavy chain and light chain of each clone are as shown in Table 5.

[0039] According to a specific embodiment of the present invention, the botulinum toxin is botulinum toxin type A.

[0040] In this specification, the term “Botulinum toxin type A” refers to botulinum toxin protein of serotype A.

[0041] In this specification, the term “serotype A” refers to type A among the forms classified according to the serological characteristics of botulinum toxin protein. Botulinum toxin protein is classified into a total of eight types, from A to H, based on physiological characteristics such as serological characteristics, proteolytic ability, and glycolytic ability, and recently, subtypes are also classified. Botulinum toxin “serotype A” is the most lethal to humans among known natural substances, and in addition to serotype A, seven immunologically distinct serotypes B, C, D, E, F, G, and H have been identified. Different serotypes can be identified by binding to antibodies specific to each serotype, and the severity of paralysis caused by each serotype and the animal species most affected differ from one another.

[0042] The molecular weight of the botulinum toxin protein molecule is approximately 150 kDa in the seven known botulinum toxin chronotypes A, B, C, D, E, F, and G. Botulinum toxin is released by Clostridium bacteria as a complex containing a 150 kDa botulinum toxin protein molecule along with associated non-toxic proteins. Therefore, botulinum toxin type A complex can be produced by Clostridium bacteria in 900 kDa, 500 kDa, and 300 kDa forms. Botulinum toxin types B and C appear to be produced only as 700 kDa or 500 kDa complexes. Specifically, the botulinum toxin type C may be C1. Botulinum toxin type D is produced as 300 kDa and 500 kDa complexes. Finally, botulinum toxin types E and F are produced only as approximately 300 kDa complexes. These complexes (i.e., those with a molecular weight greater than about 150 kDa) are believed to include non-toxic erythroggingin proteins and non-toxic and non-toxic non-erythroggingin proteins. These two non-toxic proteins (containing an associated neurotoxin complex with the botulinum toxin molecule) will serve to provide stability against denaturation of the botulinum toxin molecule and protection against digestive acids when the toxin is ingested. Additionally, the larger the botulinum toxin complex (molecular weight greater than about 150 kDa), the more slowly the botulinum toxin will diffuse from the site where the complex is injected intramuscularly. Therefore, in the present invention, the botulinum toxin may include both a form that does not contain complexing proteins and a complex form that contains complexing proteins. The molecular weight of the botulinum toxin protein derived from Clostridium botulinum of types A, B, C, D, E, F, or G that does not contain naturally forming complexing proteins is approximately 150 kDa.However, when toxin proteins are produced by Clostridium botulinum bacteria, botulinum toxin proteins are produced by forming various complexes with various hemagglutinin and non-hemagglutinin proteins that assist and protect the action of botulinum toxin proteins. Botulinum toxin serotype A containing naturally formed complex proteins is a complex form with a molecular weight of approximately 900 kDa, 500 kDa, or 300 kDa; serotypes B and C are complex forms with a molecular weight of approximately 500 kDa; serotype D is a complex form with a molecular weight of approximately 300 kDa or 500 kDa; and serotypes E and F are complex forms with a molecular weight of approximately 300 kDa.

[0043] According to one aspect of the present invention, the present invention provides a hybridoma cell line that produces the monoclonal antibody of the present invention.

[0044] In this specification, the term “hybridoma cell” refers to a cell created by artificially fusing two or more cells. Hybridoma cells are produced by fusing two or more homologous or heterologous cells together using a substance that induces cell fusion, such as polyethylene glycol, or a specific type of virus, so that a single cell can possess the different functions of the different cells.

[0045] Monoclonal antibodies can generally be produced using hybridoma cells. By fusing spleen cells obtained from animals previously exposed to a target antigen with immortalized myeloma cells, monoclonal antibodies that divide indefinitely and specifically bind to the target antigen can be produced. Specifically, in the present invention, monoclonal antibodies that specifically bind to botulinum toxin type A can be produced using hybridoma cells produced by fusing the spleen of a mouse exposed to botulinum toxin type A (antigen) with myeloma cells. According to a specific embodiment of the present invention, the hybridoma cell line used to produce monoclonal antibodies that specifically bind to botulinum toxin type A is a cell line with accession number KCTC15640BP, KCTC15641BP, or KCTC15642BP.

[0046] According to one aspect of the present invention, the present invention provides a composition for detecting botulinum toxin comprising the monoclonal antibody of the present invention.

[0047] In this specification, the term “detection” refers to the act of confirming the presence of botulinum toxin in a target sample, or quantifying the amount of botulinum toxin if it is present.

[0048] In this specification, the term “detection composition” refers to an integrated mixture used to confirm the presence of botulinum toxin in a target sample, or to quantify the amount of botulinum toxin if it is present.

[0049] According to a specific embodiment of the present invention, the botulinum toxin is botulinum toxin type A.

[0050] In this invention, botulinum toxin or its serotypes have already been described above, so their description is omitted to prevent excessive duplication.

[0051] According to another aspect of the present invention, the present invention provides a kit for detecting botulinum toxin comprising the monoclonal antibody of the present invention.

[0052] In this specification, the term “detection kit” refers to a device used to confirm the presence of botulinum toxin in a target sample or to quantify the amount of botulinum toxin if it is present. Specifically, the detection kit of the present invention may be a device that confirms the presence of botulinum toxin in a target sample or quantifies it by including a monoclonal antibody that specifically binds to the botulinum toxin of the present invention.

[0053] The detection kit of the present invention may include not only the monoclonal antibody of the present invention but also tools or reagents commonly used in the field for immunological analysis. Such tools or reagents include, but are not limited to, suitable carriers, labeling substances capable of generating a detectable signal, solvents, cleaning agents, buffers, stabilizers, etc. If the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction stopping agent. Suitable carriers may include soluble carriers, for example, physiologically acceptable buffers known in the field, e.g., PBS; insoluble carriers, for example, polymers such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluoropolymer, cross-linked dextran, polysaccharide, and latex plated with metal, as well as other paper, glass, metal, agarose, and combinations thereof, but are not limited thereto, and any type of suitable carrier may be used. In addition, the detection kit of the present invention may additionally include a label capable of confirming the antigen-antibody reaction between the monoclonal antibody and the botulinum toxin of the present invention.

[0054] According to a specific embodiment of the present invention, the detection kit further comprises a label and a chromogenic substrate.

[0055] In the present invention, the term “label” refers to a means capable of confirming the antigen-antibody reaction between the monoclonal antibody and the botulinum toxin of the present invention. Specifically, such means include, but are not limited to, visual inspection, detection equipment, or detection reagents.

[0056] The above detection reagent comprises a labeling reagent, an auxiliary specific binding member, a component of a signal generation system, or a combination thereof, which enables external identification of the presence of a target substance to be tested (specifically botulinum toxin) through visual inspection or other instruments. Examples of labels include catalysts, enzymes (e.g., phosphatases, peroxidases, etc.; more specifically, basic phosphatases used in combination with enzyme substrates and pepper peroxidase, etc.), enzyme substrates (e.g., nitroblue tetrazolium, 3,5',5,5'-tetranitrobenzidine, 3,3',5,5'-tetramethylbenzidine, 4-methoxy-1-naphthol, 4-chloro-1-naphthol, 5-bromo-4-chloro-3-indoleylphosphate, dioxetane as a chemiluminescent enzyme substrate, and derivatives and analogs thereof), fluorescent compounds (e.g., fluorescein, phycobiliprotein, rhodamine, etc. and their derivatives and analogs), chemiluminescent compounds, metal sols, dye sols, and particulate latex. Examples include latex, color indicators, color matter contained in liposomes, carbon sols, and non-metallic sols such as selenium. As examples of labeling means, HRP (Horseradish peroxidase) is used for enzymes, FITC (Fluorescein isothiocyanate) for fluorescent substances, Luminol, Isoluminol, and Lucigenin for luminescent substances, and 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, 186Re, etc. are used as labeling means, but are not limited thereto. Labeling by the above labeling means can be confirmed using the enzyme substrate or a tool for measuring fluorescence, luminescence, or radiation.

[0057] The label of the present invention can be conjugated to the monoclonal antibody of the present invention.

[0058] According to a specific embodiment of the present invention, the label is selected from the group consisting of Q dot (Quantum dot), HRP (horseradish peroxidase), alkaline phosphatase, glucose oxidase, luciferase, β-D-galactosidase, malate dehydrogenase (MDH), acetylcholinesterase, colloidal gold, fluorescent material, radioactive material, and dye.

[0059] According to a specific embodiment of the present invention, the chromogenic substrate is selected from the group consisting of DAB (diaminobenzidine), AEC (3-amino-9-ethylcarbazole), BCIP / NBT (5-bromo-4-chloro-3-indolyl phosphate / nitroblue tetrazolium), BCIP / INT (5-bromo-4-chloro-3-indolyl phosphate / iodonitrotetrazolium), NF (New fuchsin), FRT (Fast Red TR Salt), TMB (3,3',5,5'-tetramethyl bezidine), ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) and OPD (o-phenylenediamine).

[0060] In this specification, “chromogenic substrate” refers to a substrate (substance) required to induce color development by reacting with the label to produce a colored reaction product. Such a chromogenic substrate that induces color development is generally selected and used according to the type of label. A chromogenic substrate such as TMB is degraded by HRP used as a label for the secondary antibody conjugate to produce a chromogenic precipitate, and the presence or absence of a protein antigen is detected by visually confirming the degree of precipitation of the chromogenic precipitate.

[0061] According to a specific embodiment of the present invention, the botulinum toxin is botulinum toxin type A.

[0062] In this invention, botulinum toxin or its serotypes have already been described above, so their description is omitted to prevent excessive duplication.

[0063] According to another aspect of the present invention, the present invention provides a method for detecting botulinum toxin in a desired sample, comprising the following steps:

[0064] Step of preparing the target sample;

[0065] The step of treating the above-mentioned target sample with a monoclonal antibody of any one of claims 1 to 8; and

[0066] A step of measuring the degree of antigen-antibody reaction between the monoclonal antibody of any one of claims 1 to 8 above and the botulinum toxin in the target sample.

[0067] In this specification, the term “target sample” refers to a sample for which it is to determine whether it contains botulinum toxin (specifically botulinum toxin type A) or, if so, the amount thereof. Specifically, it may be various products (pharmaceuticals) containing botulinum toxin preparations, but is not limited thereto.

[0068] In the present invention, the term “antigen-antibody reaction” refers to a reaction process or reaction result in which an antigen and an antibody specifically bind; specifically, it refers to the specific binding reaction between the monoclonal antibody and the botulinum toxin of the present invention, but is not limited thereto.

[0069] According to a specific embodiment of the present invention, the degree of the antigen-antibody reaction can be measured through a method selected from the group consisting of enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, sandwich ELISA, western blotting, immunodot blotting assay, immunofluorescence assay (IFA), immunochoemiluminescence assay, immunohistochemistry, immunochromatography, and lateral flow immunoassay (LFA), but is not limited thereto, and any type of measurement means may be used.

[0070] According to a specific embodiment of the present invention, the botulinum toxin is botulinum toxin type A.

[0071] In this invention, botulinum toxin or its serotypes have already been described above, so their description is omitted to prevent excessive duplication.

[0072] The features and advantages of the present invention are summarized as follows:

[0073] (a) The present invention provides a novel monoclonal antibody that specifically binds to botulinum toxin and a method for detecting botulinum toxin using the same.

[0074] (b) The novel antibody of the present invention can detect or quantify botulinum toxin in a sample to be analyzed with significantly superior binding strength and specificity compared to commercially available antibodies, and thus can be usefully utilized to efficiently evaluate the quality of products containing botulinum toxin preparations.

[0075] Figure 1 is a figure showing the SDS-PAGE results to verify that structural mutations occurred in the botulinum toxin protein type A toxoid protein.

[0076] Figure 2 shows the results of examining the growth curves of each cell to analyze the characteristics of the hybridoma cell line that produces monoclonal antibodies.

[0077] Figure 3 is a figure showing the results of confirming the isotype of the purified antibody.

[0078] Figure 4 is a figure showing the results of confirming the molecular weight and purity of the purified antibody.

[0079] Figure 5 is a figure showing the results of confirming the binding sites of antibodies against botulinum toxin type A (900 kDa and 150 kDa) through Western blot.

[0080] Figure 6 illustrates sensograms of antibodies against botulinum toxin type A using ForteBio Octet Red96 equipment. Figure 6a shows the sensogram for the 1E10 monoclonal antibody, Figure 6b shows the 5C2 monoclonal antibody, and Figure 6c shows the 9H10 monoclonal antibody, respectively.

[0081] Figure 7 illustrates the sensograms of commercial antibodies against botulinum toxin type A using ForteBio Octet Red96 equipment. Figure 7a illustrates the sensogram for the commercial antibody MBS310475, and Figure 7b illustrates the sensogram for the commercial antibody ab40786, respectively.

[0082] Figure 8 is a figure showing the results of confirming the binding sites of antibodies against botulinum toxin type A (900 kDa and 150 kDa) by comparing them with commercial antibodies through Western blot.

[0083] Figure 9 is a figure illustrating the results of the verification of the specificity of the identification test method for botulinum toxin type A.

[0084] Figure 10 is a figure showing the results of confirming changes in absorbance by treating botulinum toxin type A (900 kDa) at different concentrations using the botulinum toxin type A identification test method.

[0085] The results of the kinetic analysis of the monoclonal antibodies prepared in the present invention show that 9H10, 5C2, and 1E10 (Figs. 6a to 6c) exhibit binding at a concentration of 0.17 μg / mL of botulinum toxin type A (900 kDa), whereas two commercially available antibodies serving as controls (Figs. 7a and 7b) do not bind even at a concentration of 55.6 μg / mL.

[0086] The present invention will be described in more detail below through examples. These examples are intended solely to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these examples according to the gist of the invention.

[0087] Examples

[0088] [Example 1] Preparation of a Hybridoma Cell Line for the Production of Monoclonal Antibodies

[0089] 1) Antigen production

[0090] Botulinum toxin type A protein (900 kDa BoNT / A) was treated with 0.16% (w / v) formaldehyde, incubated at 37°C for 28 days, and toxoids were produced by dialyzing with 1X PBS. SDS-PAGE analysis confirmed that not only neurotoxins (Hc, Lc) but also various bands corresponding to toxin complexes were not observed (Fig. 1), and mouse in vivo tests confirmed that all mice survived without weight loss (Table 1). Therefore, it was confirmed that structural mutations occurred in the botulinum toxin type A protein and that the toxin activity was eliminated.

[0091] Before N (g) After (g) After-Before (g) Test Group 1 2 1.34 25.69 4.35 2 20.77 26.26 5.49 3 18.33 25.44 7.11

[0092] 2) Immunization of mice

[0093] Botulinum toxin type A toxoid was injected into mice (BALB / c) mixed with an adjuvant, and blood was collected from the mice to check for antibody production using ELISA. As a result, it was confirmed that the antibody titer (1:5,000) increased appropriately after two rounds of immunization.

[0094] 3) Cell Fusion and Hybridoma Cell Production

[0095] Spleens were removed from immunized mice, B lymphocytes were isolated, and then fused with cultured myeloma cells (sp2 / 0). The fused cells were cultured in a medium supplemented with hypoxanthine, aminopterine, and thymidine (HAT medium) to selectively select and culture hybridoma cells in which only myeloma and B lymphocytes were fused.

[0096] 4) Selection of hybridoma cells producing monoclonal antibodies specific to botulinum toxin type A

[0097] The fused cells were cultured in a total of 10 96-well plates, and 144 positive hybridoma cells producing antibodies that react with botulinum toxin type A were selected via ELISA. These cells underwent repeated cloning processes to separate positive and negative cells using the serial dilution method and single-cell selection, resulting in the establishment of three final cell lines highly reactive to botulinum toxin type A. The reactivity of the supernatant of the final established cell lines to botulinum toxin type A was confirmed via indirect ELISA, and the results are shown in Table 2.

[0098] CloneName9H105C21E10BoNT / A (100 ng / well)2.3302.3021.984

[0099] [Example 2] Characterization of Hybridoma Cell Lines

[0100] To analyze the characteristics of the cells, cell proliferation and cell cycle were measured for three types of hybridoma cell lines.

[0101] 1) Cell proliferation rate

[0102] After thawing each cell, place 2 x 10⁶ in a T75 flask 5 The proliferation rate was measured after seeding under cells / mL conditions and undergoing two stabilization passages. The cell proliferation rate was measured within 4 to 10 passages, and the proliferation rates of the three cell lines were up to 15-fold, with all exceeding 5-fold. In addition, the doubling time for each cell line was calculated using the following formula, and the results showed a doubling time of 14.6–15.8 hours (Table 3).

[0103] Clone Name Growth (Living Cell) Division Time (h) Division Time Formula Cell Growth Rate Passage 9H 10135 - 1014.6 t: Time N(t) : Number of cells after time t N0: Initial number of cells 5C2156 - 1015.6 1E1094 - 1015.8

[0104] 2) Cell cycle

[0105] The cell cycle was measured three times at 12-hour intervals from 0 to 72 hours using an automated cell counter, and the cell cycles of the lag phase (0-24h), exponential phase (24-48h), stationary phase (48-60h), and death phase (60-72h) were confirmed in two cell lines, excluding the 5C2 cell line which did not reach the death phase by 72 hours (Fig. 2).

[0106] [Example 3] Production and Purification of Monoclonal Antibodies

[0107] Hybridoma cells were stabilized through at least three subcultures, including thawing. For subculture, each cell was placed in a T175 flask at a rate of 2 x 10⁶ 5 Seeding was performed at cells / mL, and on the second day, 25 mL of growth media was added for culture. Afterward, the growth media was replaced with serum-free media, and the cells were cultured for 4 days. The supernatant of the cell culture was collected, and IgG was purified using a Protein G column (HiTrap Protein G HP column).

[0108] FPLC purification was performed as follows. Binding buffer (0.02M Phosphate buffer, pH 7.0) was equilibrated by applying 10 CV (Column volume) at a flow rate of 5 mL / min, and then the filtered culture supernatant was loaded into the column at a flow rate of 5 mL / min. Subsequently, the column was washed with binding buffer at a flow rate of 5 mL / min at 15 CV, and IgG was obtained by applying elution buffer (0.1M Glycine-HCl, pH 2.7) at a flow rate of 1 mL / min at 3.5 CV. The yield of the purified monoclonal antibodies is shown in Table 4.

[0109] Clonal Name Yield (mg / L) Total Amount (mg) 9H 10 19.5 13.6 55 C 2 7.4 54.1 1E 10 5.6 82.8 4

[0110] [Example 4] Characterization of Monoclonal Antibodies

[0111] 1) Amino acid sequence analysis

[0112] The sequences of the heavy chain variable region (VH) and light chain variable region (VL), heavy chain CDR (CDRH) and light chain CDR (CDRL) of three monoclonal antibodies produced from hybridoma cell lines are shown in Table 5.

[0113] 클론서열번호도메인아미노산 서열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

[0114] 2) Check Isotype

[0115] To characterize the above monoclonal antibody, isotype analysis via ELISA confirmed that it possesses an IgG2 heavy chain and a kappa light chain (Table 6, Fig. 3).

[0116] CloneName 5C29H101E10 Type IgG2b / kappa IgG2a / kappa IgG10.1230.1240.118 IgG2a0.0552.2172.495 IgG2b2.5670.1330.122 IgG30.0520.0520.052 IgA0.0550.2780.053 IgM0.0490.0510.051 Kappa0.4410.3630.427 Lambda0.0570.0570.062

[0117] 3) Confirmation of molecular weight and purity of monoclonal antibodies

[0118] SDS-PAGE was performed to confirm the molecular weight and purity of the above monoclonal antibody. Specifically, samples under reducing and non-reducing conditions were prepared using LDS Sample buffer (4X) (Thermo Fisher Scientific, 84788) and NuPAGE sample reducing agent (10X) (Invitrogen, NP0004). Dual color standards (Bio-rad, 1610374) (2.5 μL / well) and the sample (2 μg / well) were loaded onto a 4-20% Tris-glycine SDS-PAGE gel (Bio-rad, 456-1096) and electrophoresis was performed (120V, 1 hour). The gel after electrophoresis was stained with Coomassie Brilliant Blue R-250 stain (Bio-rad, 161-0436) for 10 minutes, destained with Coomassie Brilliant Blue R-250 destaining solution (Bio-rad, 161-0438), and then the bands were checked using the ChemiDoc XRS+ System (Bio-rad).

[0119] As a result, as shown in Figure 4, the IgG form corresponding to 150 kDa was confirmed under non-reducing conditions, and under reducing conditions, it was confirmed to consist of a heavy chain of 50 kDa and a light chain of 25 kDa. In addition, it was confirmed that it exhibited 100% purity through the band % under non-reducing conditions.

[0120] 4) Confirmation of antibody binding sites via Western blotting

[0121] Western blotting was performed on 900 kDa and 150 kDa botulinum toxin type A to confirm the antibody binding sites of the above monoclonal antibodies.

[0122] Specifically, samples were prepared using 5X SDS sample buffer (KOMA Biotech, KTG020-5) and NuPAGE sample reducing agent (10X) (Invitrogen, NP0004). 4 μL / well of Precision Plus Protein Dual Color Standard (Bio-rad, 161-0374), 1 μg / well of 900 kDa Botulinum Toxin Type A, and 0.5 μg / well of 150 kDa Botulinum Toxin Type A were loaded onto a 4-20% Tris-glycine SDS-PAGE gel (Bio-rad, 456-1096) and subjected to electrophoresis (100V 15 min, 130V 45 min), after which the proteins were transferred to an Immobilon-P PVDF membrane (Millipore, IPVH00010) (65V, 3 h). The PVDF membrane was reacted in a blocking buffer (5% BSA) at room temperature for 1 hour, followed by reaction with a diluted monoclonal antibody solution (10 μg / mL in blocking buffer) at room temperature for 2 hours. After washing three times for 10 minutes with 1X TBST (0.1% tween 20 in TBS), the PVDF membrane was reacted with a diluted anti-mouse IgG-HRP conjugate solution (1:1000 dilution, Cell signaling, 7076s) at room temperature for 1 hour. After washing four times with 1X TBST for 10 minutes, the membrane was detected with a Clarity western ECL substrate (Bio-rad, 170-5061) solution and quantified using a ChemiDoc XRS+ System (Bio-rad).

[0123] As a result, it was confirmed that all three antibodies had binding affinity to non-toxin proteins (NTNH, HA70A, HA33, HA17), including the neurotoxin (HC, LC) sites of the 900 kDa complex, and the binding affinity to Hc and Lc was re-verified through Western blot against a 150 kDa pure toxin.

[0124] In addition, to determine the ratio of Hc and Lc binding in the 900 kDa toxin complex, the binding ratio of Hc and Lc among the total bands under 900 kDa reducing conditions was calculated, and it was confirmed that the binding ratio was 2.9 - 19.5% (Fig. 5).

[0125] 5) Confirmation of antigen-antibody reactivity via indirect ELISA

[0126] In order to confirm the antigen-antibody reactivity of the above monoclonal antibody, an indirect ELISA was performed on 900 kDa and 150 kDa botulinum toxin type A.

[0127] Specifically, 100 ng of botulinum toxin (BoNT / A, 900 kDa & 150 kDa) diluted in coating buffer (0.1 M carbonate buffer, pH 9.6) was coated onto an immunoplate (SPL, 32296) at 4°C for at least 16 hours. After washing four times with washing buffer (1XPBST: 0.1 % Tween 20 in 1X PBS), the plate was incubated with 200 μL of blocking buffer (5 % BSA in PBS) at 25°C for 2 hours. Subsequently, after washing four times with washing buffer, 100 μL of monoclonal antibody dilution (5 μL / mL) was added to the plate and incubated at 25°C for 2 hours. The immunoplate was washed four times with wash buffer, 100 μL of anti-mouse IgG-HRP conjugate dilution (1:1000 dilution, Cell signaling, 7076s) was added, and the reaction was carried out at 25°C for 1 hour. This was washed four times with wash buffer, 100 μL of ABTS (Roche, 11684302001) was added, and the reaction was carried out at 25°C for 15 minutes. The reaction was stopped by adding 50 μL of 2M sulfuric acid, and the absorbance was measured at 405 nm using a Bio-Tek Synergy2 instrument.

[0128] Test results confirmed that the three monoclonal antibodies specifically bind to BoNT / A of 900 kDa and 150 kDa, with an S / N ratio (the ratio of absorbance of the test solution to the blank) of more than 5 times. In particular, the S / N ratio of 900 kDa showed high reactivity with 33-42 (Table 7).

[0129] BoNT / A 900 kDa Toxin 150 kDa Toxin Clone Name Absorbance (405 nm) S / N Ratio Absorbance (405 nm) S / N Ratio BoNT / A Blank BoNT / A Blank 9H 10 2.89 30.069 42 0.86 0.087 105C 2 2.63 40.081 33 0.766 0.12 161E 10 2.75 80.065 42 0.79 80.082 10

[0130] 6) Comparison with commercially available antibodies through kinetic analysis of monoclonal antibodies

[0131] The kinetics analysis of monoclonal antibodies was performed using BLI analysis with a ForteBio Octet Red96 instrument. An anti-mouse IgG FC Capture (AMC) Dip and Read™ Biosensors (ForteBio, 18-5088) was used, and all samples were diluted in 10X kinetics buffer (Sartorius, 18-1105). As controls, commercially available mouse monoclonal antibodies against botulinum toxin type A, ab40786 (Abcam) and MBS310475 (Mybiosource), were used.

[0132] Specifically, the AMC tip was equilibrated in 10X kinetic buffer for 60 seconds, after which 3 μg / mL (20 nM) of antibody was loaded for 600 seconds. Subsequently, binding was performed for 1200 seconds with Botulinum Toxin Type A (900 kDa) serially diluted twofold, starting from 5 to 50 μg / mL (5.56–55.56 nM), followed by dissociation in 10X kinetic buffer for 7200 seconds. The sensitogram results are shown in Figures 6 and 7. For 9H10, 5C2, and 1E10 (Figures 6a to 6c), binding was shown at a concentration of 0.17 μg / mL of Botulinum Toxin Type A (900 kDa), whereas for the two commercially available control antibodies (Figures 7a and 7b), no binding was shown even at a concentration of 55.6 μg / mL.

[0133] Data analysis was performed using ForteBio Data Analysis Software v9.0, and the measured binding (kon), dissociation (kdis) rate constants, and equilibrium dissociation constant (KD) are plotted in Table 8. As a result, all three antibodies at 1 pM (10 -12 Very high affinity of ) or lower was confirmed (Table 8).

[0134] Clone Name K D (M)K on (1 / Ms)K dis (1 / s)R^29H10<1.0 x 10 -12 6.93 x 10 5 < 1.0 x 10 -7 0.99555C2<1.0 x 10 -12 9.14 x 10 5 < 1.0 x 10 -7 0.95161E10<1.0 x 10 -12 8.93 x 10 5 < 1.0 x 10 -7 0.9616

[0135] [Example 5] Comparative analysis with a commercially available antibody specific to botulinum toxin type A via indirect ELISA

[0136] In order to compare the specificity for botulinum toxin type A, an indirect ELISA was performed on the three monoclonal antibodies (9H10, 5C2, 1E10) and one commercial antibody (Abcam, ab40786) as a comparison test group.

[0137] Specifically, 100 ng of botulinum toxin (BoNT / A, 900 kDa & 150 kDa) diluted in coating buffer (0.1 M carbonate buffer, pH 9.6) was coated onto an Immunoplate (SPL, 32296) at 4°C for at least 16 hours. After washing four times with washing buffer (1XPBST: 0.05 % Tween 20 in 1X PBS), the plate was incubated with 200 μL of blocking buffer (3 % Skim milk in PBS) at 25°C for 1 hour. Subsequently, after washing four times with washing buffer, 100 μL of monoclonal antibody dilution (5 μL / mL) was added to the Immunoplate and incubated at 25°C for 2 hours. The immunoplate was washed four times with wash buffer, 100 μL of anti-mouse IgG-HRP conjugate dilution (1:1000 dilution, Cell signaling, 7076s) was added, and the reaction was carried out at 25°C for 1 hour. This was washed four times with wash buffer, 100 μL of ABTS (Roche, 11684302001) was added, and the reaction was carried out at 25°C for 15 minutes. The reaction was stopped by adding 50 μL of 2M sulfuric acid, and the absorbance was measured at 405 nm using a Bio-Tek Synergy2 instrument.

[0138] As a result of the test, the three monoclonal antibodies mentioned above all had higher S / N ratios—the ratio of absorbance of the test solution to the 900 kDa and 150 kDa blanks—compared to the commercial antibody in the comparative test group, and it was confirmed that they specifically bound to 900 kDa BoNT / A. On the other hand, for the commercial antibody in the comparative test group, the S / N ratio was 2 or less, confirming that the binding affinity to 900 kDa and 150 kDa BoNT / A was low (Table 9).

[0139] BoNT / A 900 kDa Toxin 150 kDa Toxin Antibody Absorbance (405 nm) S / N Ratio Absorbance (405 nm) S / N Ratio BoNT / A Blank BoNT / A Blank 9H 10 2.18 90.079 28 0.30 10.069 45C 2 2.12 40.066 32 0.15 90.065 21E 10 2.21 30.071 31 0.18 80.067 3Ab 40 78 60.13 00.065 20.068 0.0661

[0140] [Example 6] Comparative analysis with commercially available antibodies specific to botulinum toxin type A via Western blotting

[0141] Western blotting was performed on the three monoclonal antibodies (9H10, 5C2, 1E10) and one commercial antibody (Abcam, ab40786) as a comparison group to compare binding sites for botulinum toxin type A.

[0142] Specifically, samples were prepared using 5X SDS sample buffer (KOMA Biotech, KTG020-5) and NuPAGE sample reducing agent (10X) (Invitrogen, NP0004). Precision Plus Protein Dual Color Standard (Bio-rad, 456-1096) 4 μL / well, 900 kDa Botulinum Toxin Type A 1 μg / well, and 150 kDa Botulinum Toxin Type A 0.5 μg / well were loaded onto a 4-20% Tris-glycine SDS-PAGE gel (Bio-rad, 456-1096) and subjected to electrophoresis (100V 15 min, 130V 45 min), after which the proteins were transferred to an Immobilon-P PVDF membrane (Millipore, IPVH00010) (65V, 3 h). The PVDF membrane was reacted in a blocking buffer (5% BSA) at room temperature for 1 hour, followed by reaction with a diluted monoclonal antibody solution (10 μg / mL in blocking buffer) at room temperature for 2 hours. After washing three times for 10 minutes under conditions of 1X TBST (0.1% tween 20 in TBS), the PVDF membrane was reacted with a diluted anti-mouse IgG-HRP conjugate solution (1:1000 dilution, Cell signaling, 7076s) at room temperature for 1 hour. After washing four times with 1X TBST for 10 minutes, the membrane was detected with a Clarity western ECL substrate (Bio-rad, 170-5061) solution and quantified using a ChemiDoc XRS+ System (Bio-rad).

[0143] As a result, it was confirmed that all three antibodies had binding affinity to non-toxin proteins (NTNH, HA70A, HA33, HA17), including the neurotoxin (HC, LC) sites of the 900 kDa complex, and the binding affinity to Hc and Lc was re-verified through Western blot on the 150 kDa pure toxin.

[0144] In addition, to determine the binding ratio of Hc and Lc in the 900 kDa toxin complex, the binding ratio of Hc and Lc among the total bands under 900 kDa reducing conditions was calculated, and it was confirmed to show a binding ratio of 5.0–12.2%. On the other hand, it was confirmed that one commercial antibody in the comparative test group did not bind to Hc and Lc of 900 kDa botulinum toxin type A, but bound only to the non-toxin protein HA33 (Fig. 8).

[0145] [Example 7] Analytical method for confirming botulinum toxin type A based on Sandwich ELISA

[0146] Among the three types of monoclonal antibodies mentioned above, the most superior monoclonal antibody (9H10) was selected as the final candidate antibody by comparing the hybridoma cell line growth rate, antibody yield, and binding activity with the antigen, and a sandwich ELISA-based botulinum toxin type A confirmation test method was developed.

[0147] Botulinum antitoxin type A (NIBSC, 14 / 174) was used as the capture antibody, a monoclonal antibody (9H10) was used as the first detection antibody, and an antibody specific to mouse IgG conjugated with HRP (Horseradish peroxidase) was used as the second detection antibody to induce a color reaction with the substrate.

[0148] The assay method was optimized under the following conditions. Botulinum antitoxin type A (NIBSC, 14 / 174, 0.001 IU / μL) diluted in coating buffer (0.1M carbonate buffer, pH 9.6) was coated onto Immunoplates (SPL, 32296) at a concentration of 100 μL / well at 4°C for at least 16 hours. After washing four times with washing buffer (1XPBST: 0.1% Tween 20 in 1X PBS), the plates were incubated with 200 μL of blocking buffer (5% BSA in PBS) at 25°C for 2 hours. After washing four times with washing buffer, botulinum toxin protein (50 units / well or 1 ng / well) was added to the plates and incubated at 25°C for 2 hours. After washing four times with wash buffer, 100 μL of monoclonal antibody dilution (1 μg / mL) was added to the plate and incubated at 25°C for 2 hours. Subsequently, the plate was washed four times again with wash buffer, 100 μL of anti-mouse IgG-HRP conjugate dilution (1:500 dilution, Cell Signaling, 7076s) was added, and incubated at 25°C for 1 hour. This was washed four times again with wash buffer, and 100 μL of TMB (Thermo Fisher, 34028) was added and incubated at 25°C for 15 minutes. The reaction was stopped by adding 50 μL of 2M sulfuric acid, and the absorbance was measured at 450 nm using a Bio-Tek Synergy2 instrument. Subsequently, in the examples, analysis was performed under the above optimized conditions.

[0149] [Example 8] Method validation for the identification of botulinum toxin type A

[0150] The validity of the above analytical method was verified for specificity and precision based on the guidelines for the validation of analytical methods for pharmaceuticals, etc. For the verification of specificity, Metabiologics' standard toxin was used, and for the verification of precision, Botulax 100 units was used as a standard.

[0151] 1) Specificity verification

[0152] Specificity refers to the ability to selectively evaluate an analyte within elements expected to be present. Therefore, we aimed to verify the specificity for botulinum toxin type A by analyzing all serotypes of botulinum toxin (BoNT / A ~ G) three times. An absorbance of less than 0.15 was determined to be negative, and an S / N ratio, which is the ratio of the absorbance of the test solution to the blank, was determined to be positive if it was 5 times or more.

[0153] As a result of the analysis, the S / N ratio of botulinum toxin type A was positive with a ratio of 5 or more, and the absorbances of types B to G, excluding botulinum toxin type A, were all negative with an absorbance of less than 0.15 (Table 10, Fig. 9). Therefore, it was confirmed that the monoclonal antibody is specific to botulinum toxin type A.

[0154] SPL Item 9H105C21E10 BoNT / AAbs.2.5912.7152.824 S / N Ratio (샘플 / 블랭크) 262728BoNT / BAbs.0.0980.1010.102BoNT / C0.1000.0960.100BoNT / D0.1040.1060.100BoNT / E0.0970.0990.102BoNT / F0.0980.1020.104BoNT / G0.0970.0980.100Blank0.0950.0980.103

[0155] 2) Precision Verification

[0156] The precision of the botulinum toxin type A confirmation test was evaluated using intra-day, inter-day, and inter-rater relative standard deviation (%RSD).

[0157] a. Test Intensive Precision

[0158] To evaluate the precision within the test period, the same tester performed a total of 6 repeated tests under the above-mentioned optimized conditions. As a result of the analysis, the absorbance of the standard solution and the blank, and the %RSD of the S / N Ratio, were within 15%, satisfying the acceptance criteria (Table 11).

[0159] Item Test 1 Test 2 Test 3 Test 4 Test 5 6%RSDSTD Abs.0.8920.8900.8940.8870.8210.8224.1 Blank Abs.0.0980.1010.1040.1070.1060.1023.2 S / N Ratio (샘플 / 블랭크) 9998885.9

[0160] b. Daily precision of the test

[0161] To evaluate the precision of the test day, the same tester performed a total of 12 repeated tests (6 times / day) on different dates under the above-mentioned optimized conditions. As a result of the analysis, the absorbance of the standard solution and blank, and the %RSD of the S / N Ratio, were within 15%, satisfying the acceptance criteria (Table 12).

[0162] Item Test 1 Test 2 Test 3 Test 4 Test 5 6% RSD STD Abs. 1 day 0.89 20.89 0.89 40.88 70.82 10.82 29. 12 days 0.78 10.74 90.72 50.67 40.77 80.767 Blank Abs. 1 day 0.09 80.10 10.10 40.10 70.10 60.10 28.82 days 0.08 60.09 0.09 0.08 70.08 80.085 S / N Ratio (샘플 / 블랭크) 1 day 9998886.02 day 988899

[0163] c. Inter-rater precision

[0164] To evaluate inter-rater precision, different testers each performed the test six times under the above-mentioned optimized conditions. As a result of the analysis, the absorbance and %RSD of the S / N Ratio of the standard solution and blank were within 15%, satisfying the acceptance criteria (Table 13).

[0165] Item Test 1 Test 2 Test 3 Test 4 Test 5 Test 6%RSDSTD Abs.Ope. A0.8920.8900.8940.8870.8210.8223.1Ope. B0.8350.8610.8510.8450.8610.842Blank Abs.Ope. A0.0980.1010.1040.1070.1060.1022.8Ope. B0.1000.0990.0980.1010.1010.102S / N Ratio (샘플 / 블랭크) Ope. A9998884.2Ope. B899898

[0166] [Example 9] Sandwich ELISA Analysis for Verification of Botulinum Toxin Type A

[0167] 1) Reactivity according to botulinum toxin type A concentration

[0168] By applying the above analytical method, the change in absorbance according to the concentration of botulinum toxin type A (900 kDa) was confirmed using a sandwich ELISA. Botulinum toxin type A was treated at 10, 20, and 40 ng / mL, and it was confirmed that the absorbance increased proportionally as the concentration of botulinum toxin type A increased (Table 14, Fig. 10).

[0169] Item Blank BoNT / A 10 ng / mL 20 ng / mL 40 ng / mL Absorbance 0.10 0.78 11.40 62.419 S / N Ratio N / A 8 14 24

[0170] 2) Analysis of botulinum toxin preparations

[0171] The above analytical method was applied to analyze the toxin protein serotypes of 22 lots of 5 commercially available botulinum toxin preparations. As a result, the S / N ratio of all products was greater than 5, confirming them to be botulinum toxin type A. Therefore, it was confirmed that this analytical method is applicable to the analysis of commercially available botulinum toxin preparations (Table 15).

[0172] Product List | Lot. No. | Absorbance | S / N Ratio Botulex | Note 100 | EX | HGA | 10 | 50.69 | 8 | HGA | 220 | 30.66 | 38 | HGA | 221 | 290.72 | 69 | HGA | 221 | 320.67 | 68 | HGA | 221 | 510.70 | 89 | 50 | EX | HGC | 210 | 10.67 | 38 | HGC | 220 | 270.58 | 87 | HGC | 221 | 60.75 | 29 | HGC | 221 | 70.73 | 39 | HGC | 221 | 80.69 | 48 Botox | Note 100 | C | 730 | 3C | 2.11 | 325 | C | 737 | 5C | 32.16 | 326 | 50C | 7710 | C | 22.33 | 728 Wondertox | Note 100 | UHG | 00 | 32.34 | 228 | HG | 00 | 82.41 | 429 Nabota | Note 100 UX211012.29028X220182.43129X221432.5493150 UX201092.26327X211412.52830X211352.46930LizTox Ju100 UVG20142.49030

[0173] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0174] The novel antibody of the present invention can detect or quantify botulinum toxin in a sample to be analyzed with significantly superior binding strength and specificity compared to commercially available antibodies, thereby being useful for efficiently evaluating the quality of products containing botulinum toxin preparations.

Claims

1. A monoclonal antibody that specifically binds to botulinum toxin, comprising the following: A heavy chain variable region comprising one or more sequences selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 17, SEQ ID NO. 18, and SEQ ID NO. 19; and A light chain variable region comprising one or more sequences selected from the group consisting of sequence no. 4, sequence no. 5, sequence no. 6, sequence no. 12, sequence no. 13, sequence no. 14, sequence no. 20, sequence no. 21 and sequence no.

22.

2. In Paragraph 1, The above heavy chain variable region comprises one or more sequences selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3, and A monoclonal antibody characterized in that the light chain variable region comprises one or more sequences selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO.

6.

3. In Paragraph 1, The above heavy chain variable region comprises one or more sequences selected from the group consisting of SEQ ID NO. 9, SEQ ID NO. 10, and SEQ ID NO. 11, and A monoclonal antibody characterized in that the light chain variable region comprises one or more sequences selected from the group consisting of SEQ ID NO. 12, SEQ ID NO. 13, and SEQ ID NO.

14.

4. In Paragraph 1, The above heavy chain variable region comprises one or more sequences selected from the group consisting of SEQ ID NO. 17, SEQ ID NO. 18, and SEQ ID NO. 19, and A monoclonal antibody characterized in that the light chain variable region comprises one or more sequences selected from the group consisting of SEQ ID NO. 20, SEQ ID NO. 21, and SEQ ID NO.

22.

5. In Paragraph 1, The above heavy chain variable region includes the sequence of SEQ ID NO. 7, and A monoclonal antibody characterized in that the above light chain variable region comprises the sequence of SEQ ID NO.

8.

6. In Paragraph 1, The above heavy chain variable region includes the sequence of SEQ ID NO. 15, and A monoclonal antibody characterized in that the above light chain variable region comprises the sequence of SEQ ID NO.

16.

7. In Paragraph 1, The above heavy chain variable region includes the sequence of SEQ ID NO. 23, and A monoclonal antibody characterized in that the above light chain variable region comprises the sequence of SEQ ID NO.

24.

8. A monoclonal antibody according to claim 1, characterized in that the botulinum toxin is botulinum toxin type A.

9. A hybridoma cell line that produces a monoclonal antibody according to any one of claims 1 to 8.

10. The hybridoma cell line according to claim 9, characterized in that the above hybridoma cell line has accession number KCTC15640BP, KCTC15641BP, or KCTC15642BP.

11. A composition for detecting botulinum toxin comprising a monoclonal antibody according to any one of claims 1 to 8.

12. A composition according to claim 11, characterized in that the botulinum toxin is botulinum toxin type A.

13. A kit for detecting botulinum toxin comprising a monoclonal antibody according to any one of claims 1 to 8.

14. A detection kit according to claim 13, characterized by additionally including a label and a chromogenic substrate.

15. A kit according to claim 14, wherein the label is selected from the group consisting of Q dot (Quantum dot), HRP (horseradish peroxidase), alkaline phosphatase, glucose oxidase, luciferase, β-D-galactosidase, malate dehydrogenase (MDH), acetylcholinesterase, colloidal gold, fluorescent material, radioactive material, and dye.

16. A kit according to claim 14, wherein the chromogenic substrate is selected from the group consisting of DAB (diaminobenzidine), AEC (3-amino-9-ethylcarbazole), BCIP / NBT (5-bromo-4-chloro-3-indolyl phosphate / nitroblue tetrazolium), BCIP / INT (5-bromo-4-chloro-3-indolyl phosphate / iodonitrotetrazolium), NF (New fuchsin), FRT (Fast Red TR Salt), TMB (3,3',5,5'-tetramethyl bezidine), ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) and OPD (o-phenylenediamine).

17. A kit according to claim 13, characterized in that the botulinum toxin is botulinum toxin type A.

18. A method for detecting botulinum toxin in a target sample, comprising the following steps: Step of preparing the target sample; The step of treating the above-mentioned target sample with a monoclonal antibody of any one of claims 1 to 8; and A step of measuring the degree of antigen-antibody reaction between the monoclonal antibody of any one of claims 1 to 8 above and the botulinum toxin in the target sample.

19. A method according to claim 18, characterized in that the intended sample is a botulinum toxin product.

20. The method according to claim 18, wherein the antigen-antibody reaction is measured by a method selected from the group consisting of enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, sandwich ELISA, western blotting, immunodot blotting assay, immunofluorescence assay (IFA), immunochoemiluminescence assay, immunohistochemistry, immunochromatography, and lateral flow immunoassay (LFA).

21. A method according to claim 18, characterized in that the botulinum toxin is botulinum toxin type A.

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