A detection kit and detection method for botulinum neurotoxin type B

Abstract

The present invention discloses a detection kit and a detection method for type B botulinum neurotoxin. The present invention can complete qualitative detection of various suspected cases of botulinum neurotoxin poisoning in a short time, provide evidence support for assisting doctors in diagnosis and timely treatment decisions, and is of great significance in social health and safety. At the same time, the detection method of the present invention can significantly improve the specificity of toxin proteins, avoid the time problems caused by animal experiments such as serotype identification, and can be applied to the research and development and production process of botulinum neurotoxin products. In addition, the present invention can perform quality control on the stability and shelf life of botulinum neurotoxin standards.

Description

Technical Field

[0001] The invention belongs to the field of biological detection, and in particular relates to a detection kit and a detection method for type B botulinum neurotoxin. Background Art

[0002] Cases of botulinum neurotoxin poisoning have occurred in many regions. Globally, food and infant botulinum neurotoxin poisoning incidents are also frequently reported. Among the reported cases, type A and type B botulinum neurotoxin poisoning are the most common. With the widespread use of botulinum neurotoxin in pharmaceutical and cosmetic applications, cases of botulinum neurotoxin poisoning due to improper use have gradually occurred. Therefore, it is of great significance to develop detection methods and detection products that can perform qualitative detection of various suspected botulinum neurotoxin poisoning cases in a short time, thereby providing evidence support for assisting doctors in diagnosis and making timely treatment decisions.

[0003] In addition, a series of tests are also required for botulinum neurotoxin to ensure quality control during the development and production of botulinum neurotoxin products, such as the use of total protein assay to detect the content of related toxin proteins, and the use of mouse in vivo neutralization method for serum type identification (which may take several days), etc. However, these detection methods for botulinum neurotoxin still have the disadvantages of complex operation, long time consumption, and low sensitivity.

[0004] Therefore, there is still a need to develop a rapid and effective botulinum neurotoxin detection antibody or its antigen-binding fragment, detection kit and detection method. Summary of the invention

[0005] In order to solve at least some of the technical problems in the above-mentioned prior art, the present invention uses a natural botulinum neurotoxin complex as an antigen to prepare a natural antibody against type B botulinum neurotoxin, and the antibody or its antigen-binding fragment has good sensitivity, specificity, affinity and stability. Specifically, the present invention includes the following contents.

[0006] In a first aspect of the present invention, a detection kit for botulinum neurotoxin type B is provided, the detection kit comprising an antibody or an antigen-binding fragment thereof capable of binding to botulinum neurotoxin type B, the antibody or the antigen-binding fragment thereof comprising heavy chain CDR1-3 having amino acid sequences as shown in GFAFSSYD, ISSGGSST and ARQAFYTYDGTAMDY, respectively, and / or light chain CDR1-3 having amino acid sequences as shown in QSIVHNNGDTY, KVS and FQGSHIPWT, respectively.

[0007] A second aspect of the present invention provides a method for detecting botulinum neurotoxin type B, the method for detecting botulinum neurotoxin type B comprising the following steps:

[0008] (1) contacting the sample to be tested with an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof is capable of binding to botulinum neurotoxin type B, and the antibody or the antigen-binding fragment thereof comprises heavy chain CDR1-3 having amino acid sequences as shown in GFAFSSYD (SEQ ID NO.3), ISSGGSST (SEQ ID NO.4), and ARQAFYTYDGTAMDY (SEQ ID NO.5), and / or light chain CDR1-3 having amino acid sequences as shown in QSIVHNNGDTY (SEQ ID NO.6), KVS, and FQGSHIPWT (SEQ ID NO.7);

[0009] (2) detecting the binding of the botulinum neurotoxin type B to the antibody or its antigen-binding fragment to determine the presence of the botulinum neurotoxin type B in the test sample or to determine the amount of the botulinum neurotoxin type B in the test sample.

[0010] In a third aspect of the present invention, an antibody or an antigen-binding fragment thereof is provided, which is capable of targeting botulinum neurotoxin type B, and comprises heavy chain CDR1-3 having amino acid sequences as shown in GFAFSSYD, ISSGGSST and ARQAFYTYDGTAMDY, and / or light chain CDR1-3 having amino acid sequences as shown in QSIVHNNGDTY, KVS and FQGSHIPWT, respectively.

[0011] In certain 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):

[0012] (I) Contains EVKLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVATISSGGSSTFYPDSVKGRFTISRDSARNTLYLQMISLRSEDTALYFCARQAFYTYDGTAMDY WGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC GCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISK TKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ ID NO.1) and / or the light chain amino acid sequence of DVLMTQTPLSLPVSLGDQASIYCRSSQSIVHNNGDTYFEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO.2);

[0013] (II) an amino acid sequence that has at least 90% homology to the amino acid sequence shown in (I) and has the same function;

[0014] (III) an amino acid sequence obtained by modifying, replacing, deleting or adding one or more amino acids to the amino acid sequence shown in (I) or (II) and having the same function.

[0015] In certain embodiments, according to the antibody or antigen-binding fragment thereof described in the present invention, the antibody includes a monoclonal antibody, a chimeric antibody, a murine antibody or a humanized antibody; the antigen-binding fragment includes Fab (a fragment comprising the entire variable region (VH and VL) and the constant region CH1), Fab' (a fragment removing the crystallizable region), F(ab)2 (a fragment connected by a disulfide bond), F(ab')2 (a fragment connected by a disulfide bond removing the crystallizable region) or scFv (single-chain variable fragment).

[0016] In a fourth aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to the present invention.

[0017] The fifth aspect of the present invention provides a vector molecule, wherein the vector molecule comprises the nucleic acid molecule described in the present invention.

[0018] The sixth aspect of the present invention provides a host cell, wherein the host cell comprises the nucleic acid molecule of the present invention or the vector molecule of the present invention.

[0019] In a seventh aspect, the present invention provides a method for preparing the antibody or antigen-binding fragment thereof according to the present invention, wherein the antibody or antigen-binding fragment thereof is prepared by artificial synthesis or genetic engineering.

[0020] The antibody or antigen-binding fragment thereof of the present invention can complete qualitative detection of various suspected cases of botulinum neurotoxin poisoning in a short time (such as using the ELISA method to complete the detection within 8 hours). At the same time, the antibody or antigen-binding fragment thereof and the detection method of the present invention can significantly improve the specificity for toxin protein, avoid the time problems caused by animal experiments such as serotype identification, etc., and can be applied to the research and development and production process of botulinum neurotoxin products. In addition, the antibody or antigen-binding fragment thereof and the detection method of the present invention can perform quality control on the stability and shelf life of botulinum neurotoxin standards in many detection processes of scientific research and production work. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The subclass identification results of the monoclonal antibody A1 of the present invention are shown.

[0022] Figure 2 The results of SDS-PAGE analysis of the monoclonal antibody A1 of the present invention are shown.

[0023] Figure 3 The binding activity of the monoclonal antibody A1 of the present invention is shown.

[0024] Figure 4The results of antigen site identification of the monoclonal antibody A1 of the present invention are shown, wherein A is the SDS-PAGE electrophoresis analysis result, 1 is the analysis result under non-reducing conditions, 2 is the analysis result under reducing conditions, and B is the Western Blot (WB) analysis result.

[0025] Figure 5 The specificity of the monoclonal antibody A1 of the present invention is shown.

[0026] Figure 6 The affinity analysis results of the monoclonal antibody A1 of the present invention are shown, and the curves from top to bottom in the figure represent 160 nM, 80 nM, 40 nM, 20 nM, 5 nM, 2.5 nM and 1.3 nM, respectively. DETAILED DESCRIPTION

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

[0028] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0030] Method for detecting botulinum neurotoxin type B

[0031] In one aspect of the present invention, a method for detecting botulinum neurotoxin type B is provided, wherein the method for detecting botulinum neurotoxin type B comprises the following steps:

[0032] (1) contacting a sample to be tested with an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof is capable of binding to botulinum neurotoxin type B, and the antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1-3 having amino acid sequences as shown in GFAFSSYD, ISSGGSST, and ARQAFYTYDGTAMDY, respectively, and / or a light chain CDR1-3 having amino acid sequences as shown in QSIVHNNGDTY, KVS, and FQGSHIPWT, respectively;

[0033] (2) detecting the binding of the botulinum neurotoxin type B to the antibody or its antigen-binding fragment to determine the presence of the botulinum neurotoxin type B in the test sample or to determine the amount of the botulinum neurotoxin type B in the test sample.

[0034] The antibodies or antigen-binding fragments thereof of the present invention may carry any detectable label, and examples of labels that can be used include, but are not limited to, radioactive substances, fluorescent groups, chemical labels, biological agents (such as but not limited to biotin / streptavidin) or enzyme substrate labels.

[0035] Those skilled in the art are familiar with how to detect the binding of botulinum neurotoxin type B and the antibody or its antigen-binding fragment in the test sample, or detect the presence of the immune complex after binding, so as to determine whether botulinum neurotoxin type B is present in the test sample or determine the amount of botulinum neurotoxin type B. Such detection techniques include, but are not limited to, immunofluorescence, radioimmunoassay, enzyme-linked immunosorbent assay, agglutination test, immunoadsorption, immunoelectrophoresis, immunoelectron microscopy, etc.

[0036] Antibodies or antigen-binding fragments thereof

[0037] In one aspect of the present invention, an antibody or an antigen-binding fragment thereof is provided, which is capable of targeting botulinum neurotoxin type B, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1-3 having amino acid sequences as shown in GFAFSSYD, ISSGGSST and ARQAFYTYDGTAMDY, respectively, and / or a light chain CDR1-3 having amino acid sequences as shown in QSIVHNNGDTY, KVS and FQGSHIPWT, respectively.

[0038] As used herein, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a specific antigen. Antibodies typically contain a variable region and a constant region in each heavy chain and light chain. The variable regions of the antibody heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors. Therefore, most antibodies have a heavy chain variable region (VH) and a light chain variable region (VL), which together form the antibody portion that binds to the antigen.

[0039] The "light chain variable region (VL)" or "heavy chain variable region (VH)" of the present invention consists of a "framework" region intercalated with three "complementarity determining regions (CDRs)". The framework region is used to adjust the CDRs for specific binding to antigenic epitopes. The CDRs contain the amino acid residues in the antibody that are primarily responsible for antigen binding. 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.

[0040] In the present invention, the antibody or antigen-binding fragment thereof has any one of the amino acid sequences shown in (I)-(III):

[0041] (I) comprising the heavy chain amino acid sequence shown in SEQ ID NO.1 and / or the light chain amino acid sequence shown in SEQ ID NO.2;

[0042] (II) an amino acid sequence that has at least 90% homology to the amino acid sequence shown in (I) and has the same function;

[0043] (III) an amino acid sequence obtained by modifying, replacing, deleting or adding one or more amino acids to the amino acid sequence shown in (I) or (II) and having the same function.

[0044] As used herein, the terms "homology" and "identity" are used interchangeably. Homologous sequences include amino acid sequences that are at least 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to the sequences of the present invention. In order to determine sequence identity, sequence alignment can be performed, which can be performed in a variety of ways known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software, etc. Those skilled in the art can determine appropriate parameters for alignment, including any algorithm required for achieving optimal alignment in the full-length sequence being compared.

[0045] Herein, variant antibody sequences obtained by modification, substitution, deletion or addition of one or more amino acids also fall within the scope of protection of the present invention. The term "modification" refers to any chemical modification of an amino acid sequence. The term "substitution" refers to the replacement of one or more amino acids by different amino acids. "Deletion" refers to the reduction of one or more amino acids in an amino acid sequence. "Insertion" or "addition" refers to an increase in one or more amino acids compared to a naturally occurring molecule resulting from a change in an amino acid sequence. It should be noted that, in the above-mentioned variant antibodies provided by the present invention, the modification, substitution, deletion or addition preferably occurs in a region outside the variable region, for example, in the framework region or constant region of the antibody, and the modified, substituted, deleted or added antibody or antigen-binding fragment thereof still retains the desired functional properties of the antibody or antigen-binding fragment thereof of the present invention, or has improved properties of binding to the antigen.

[0046] In the present invention, the antibody may include a monoclonal antibody, a chimeric antibody, a murine antibody or a humanized antibody.

[0047] The term "monoclonal antibody" as used herein, sometimes also referred to as "monoclonal antibody" or mAb, refers to an immunoglobulin obtained from a pure cell line, having the same structure and chemical properties, and being specific to a single antigenic determinant. Monoclonal antibodies are different from conventional polyclonal antibody preparations (usually having different antibodies for different determinants), in that each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the benefit of monoclonal antibodies is that they are obtained by hybridoma or recombinant engineering cell culture and are not mixed with other immunoglobulins. This characteristic is in contrast to polyclonal antibody preparations, which generally include antibodies for different antigenic determinants. The modifier "monoclonal" indicates the characteristic of the antibody, which is obtained from a homogeneous antibody population, but this should not be interpreted as requiring any special method to produce the antibody.

[0048] Herein, the term "chimeric antibody" generally refers to an antibody in which a portion of each heavy or light chain amino acid sequence is homologous to the corresponding amino acid sequence in an antibody from a particular species, or belongs to a particular class, while the remaining segments of the chain are homologous to the corresponding sequence in another species. For example, the variable regions of the light and heavy chains are both from the variable regions of an antibody from one animal species, while the constant portions are homologous to the antibody sequences from another species. For example, to obtain a chimeric antibody, a non-human B cell or hybridoma cell can be used to produce the variable region, while the constant region combined therewith is from a human. The variable region has the advantage of being easy to prepare, and its specificity is not affected by the source of the constant region combined therewith.

[0049] Herein, the term "humanized antibody" generally refers to a chimeric antibody that contains fewer sequences from non-human immunoglobulins, thereby reducing the immunogenicity of xenogeneic antibodies when introduced into humans, while maintaining the antibody's full antigen binding affinity and specificity.

[0050] Herein, the term "antigen-binding fragment" generally refers to one or more fragments of an antibody that specifically bind to an antigen. It is understood that the antigen-binding function of an antibody can be achieved by a full-length fragment of the antibody, or by a heavy chain comprising a fragment of Fab, Fab', F(ab)2, F(ab')2, or scFv, or a light chain comprising a fragment of Fab, Fab', F(ab)2, F(ab')2, or scFv.

[0051] In the present invention, the terms "binding", "specific binding", "directed against" and "targeting" are used interchangeably and generally refer to the non-covalent interaction that occurs between an immunoglobulin molecule and an antigen that is specific to the immunoglobulin. The strength or affinity of an immunological binding 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). Unless otherwise indicated, when used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody or its antigen-binding fragment and an antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of a binding dissociation equilibrium constant. Affinity can be measured by conventional methods well known in the art, including those known in the prior art and described herein.

[0052] Nucleic acid molecules

[0053] In one aspect of the present invention, a nucleic acid molecule is provided, which comprises a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of the present invention.

[0054] The term "nucleic acid" as used herein is intended to include polymeric forms of nucleotides of any length, containing deoxyribonucleotides, ribonucleotides and / or their analogs, including DNA, RNA and DNA / RNA hybrids, including DNA or RNA analogs, such as those containing modified backbones (e.g., peptide nucleic acids (PNA) or thiophosphates) or modified bases. Therefore, nucleic acids of the present invention include DNA, cDNA, mRNA, recombinant nucleic acids, etc.

[0055] Once the coding sequence of the antibody or antigen-binding fragment thereof of the present invention is obtained, the antibody or antigen-binding fragment thereof can be obtained in large quantities by recombinant technology. An exemplary method is to clone the coding gene into a vector, transfer it into cells, and then separate it from the propagated host cells by conventional methods.

[0056] Carrier molecules

[0057] One aspect of the present invention provides a vector molecule comprising the nucleic acid molecule of the present invention.

[0058] The vector of the present invention refers to an artificial construct that can deliver and preferably express one or more target genes or sequences in a host cell. The vector of the present invention is not limited and can be an expression vector, a viral vector, etc. In certain embodiments, the vector comprises a target gene, a promoter, a terminator encoding an antibody or an antigen-binding fragment thereof of the present invention, or optionally further comprises a marker gene. The vector can use a known vector or a self-constructed vector. Known vectors include plasmid vectors, lentiviral vectors, adenoviral vectors, AAV viral vectors, etc.

[0059] Host cells

[0060] In one aspect of the present invention, a host cell is provided, which comprises the nucleic acid molecule or the vector molecule of the present invention.

[0061] The host cell of the present invention refers to any cell type suitable for transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a nucleic acid molecule of the present invention. A host cell includes any progeny of a parent cell that differs from the parent cell due to mutations that occur during replication.

[0062] Preparation method

[0063] In one aspect of the present invention, a method for preparing the antibody or antigen-binding fragment thereof of the present invention is provided. The preparation method is not particularly limited and includes preparation by artificial synthesis or genetic engineering.

[0064] In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention can be obtained by artificial synthesis. Methods for artificially synthesizing antibodies or antigen-binding fragments thereof are known in the art, for example, the antibody or antigen-binding fragment thereof of the present invention can be obtained by direct amino acid synthesis.

[0065] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention can be obtained by genetic engineering expression. Genetic engineering expression systems include prokaryotic cell expression systems, eukaryotic cell expression systems, and cell-free expression systems. Examples of prokaryotic cell expression systems include Escherichia coli expression systems. Eukaryotic cell expression systems include zymocyte expression systems, insect cell expression systems, and mammalian cell expression systems.

[0066] Without being bound by any theory, the antibodies of the present invention can be prepared by the following steps:

[0067] (1) constructing a recombinant vector expressing the antibody heavy chain of the amino acid sequence shown in SEQ ID NO.1 and a recombinant vector expressing the antibody light chain of the amino acid sequence shown in SEQ ID NO.2;

[0068] (2) transforming the vector into host cells and culturing the cells under conditions suitable for antibody expression;

[0069] (3) Collect and purify antibodies.

[0070] Detection Kits

[0071] The present invention further provides a detection kit for detecting (or quantifying) botulinum neurotoxin type B, wherein the detection kit comprises the antibody or antigen-binding fragment thereof described in the present invention, and instructions for implementing the detection method of the present invention on botulinum neurotoxin type B.

[0072] As used herein, the term "kit" refers to a combination of reagents and other materials. The kit is expected to include reagents such as buffers, protein stabilizing agents, signal generating systems (e.g., fluorescent signal generating systems), antibodies or antigen binding fragments thereof, control proteins, and test containers (e.g., microtiter plates, etc.). The term "kit" is not limited to a specific combination of reagents and / or other materials, for example, the kit may also include instructions for using the reagents. The kit may be packaged in any suitable manner, typically having components in a single container or (if necessary) in multiple containers and instructions for performing detection instructions. The kit may be prepared by a variety of methods known in the art.

[0073] In certain embodiments, the kit may further include at least one of a cleaning solution, a substrate solution, a diluent, and a calibration solution. The composition of the cleaning solution is not particularly limited, and examples thereof include, but are not limited to, a buffer, a surfactant, and a preservative. The substrate solution may use a known substrate, and examples thereof include, but are not limited to, a chromogenic substrate, a fluorescent substrate, a luminescent substrate, and the like. The composition of the diluent is not particularly limited, and examples thereof include, but are not limited to, a buffer, a surfactant, and the like. The composition of the calibration solution is not particularly limited, and examples thereof include, but are not limited to, a BSA solution, a trehalose solution, an animal serum, and the like.

[0074] Example

[0075] This example shows the preparation process of anti-botulinum neurotoxin type B monoclonal antibody.

[0076] 1. Experimental equipment

[0077] The biological safety cabinet is from Sujing Group Antai Air Technology Co., Ltd., the pulsating vacuum sterilizer is from Shandong Xinhua Medical Instrument Co., Ltd., the convenient liquid suction system is from WIGGENS, the biomolecular interaction system and CM5 chip are both from Cytiva, the gel electrophoresis imaging analysis system is from Bio-Rad, the digital inverted microscope is from Nikon, and the -20℃ low-temperature preservation box is from Haier.

[0078] 2. Experimental Methods

[0079] 1. Preparation

[0080] (1) Natural botulinum neurotoxin type B was prepared and purified from Clostridium botulinum type B strain Okra, detoxified with formaldehyde to a toxoid, and stored in 0.01 M phosphate buffer (PB) (pH 7.0) at a concentration of 1 mg / ml.

[0081] (2) Monoclonal antibodies against botulinum neurotoxin type B were prepared using hybridoma technology. An equal amount of Freund's adjuvant was added to the toxoid for emulsification and used to immunize 6-8 week old Balb / c mice (albino house mouse substrain used to produce mouse myeloma cells). The mice were subcutaneously injected in the groin at 0 w, 3 w, 5 w, 7 w, and 9 w, with doses of 100, 85, 70, 55, and 40 μg / mouse, 120 μl / mouse, respectively. Two weeks after the fifth immunization, the mice were given a shock immunization of 100 μg / mouse.

[0082] (3) SP2 / 0 cells (mouse myeloma cells) were cultured in a 37°C CO2 incubator (CO2 content was 5.0%), the medium was changed regularly, and the cells were subcultured in a timely manner.

[0083] (4) Under sterile conditions, the peritoneal cavity of normal Balb / c mice was repeatedly flushed with complete 1640 medium to prepare feeder cells.

[0084] (5) Under sterile conditions, spleen cells from immunized mice were isolated and mixed with SP2 / 0 myeloma cells at a ratio of 5:1. Cell fusion was performed using the PEG method, and the fused cell suspension was dripped into a 96-well plate pre-coated with feeder cells.

[0085] (6) After a certain period of culture, positive clones are screened by microscopic observation combined with ELISA detection, and positive wells are cloned at the third level using the limiting dilution method to obtain hybridoma cell lines that stably secrete specific antibodies.

[0086] (7) Monoclonal antibodies were prepared by in vivo induction. Balb / c mice were sensitized with liquid paraffin, 0.5 mL / mouse. 1×10 6The hybridoma cells were injected intraperitoneally, and the ascites was collected 10 days later and purified using a Protein-G affinity chromatography column to obtain monoclonal antibody A1.

[0087] 2. Identification of Antibodies

[0088] The prepared monoclonal antibody A1 was identified as follows:

[0089] a. The purified monoclonal antibody was identified by using a subclass identification kit. The results showed that the monoclonal antibody was IgG1 subclass κ type ( Figure 1 ).

[0090] b. SDS-PAGE analysis was used to identify the monoclonal antibody bands, and the results showed a clear band around 150 kDa with a purity of 96% ( Figure 2 ).

[0091] c. The binding activity of the monoclonal antibody was tested by ELISA. The results showed that the titer of the monoclonal antibody was 1:105000. The lowest concentration of the monoclonal antibody when a positive result was detected by ELISA was 6.7 ng / ml ( Figure 3 ).

[0092] d. The specificity of the monoclonal antibody was identified by ELISA. The results showed that the monoclonal antibody exhibited significant binding activity and high specificity to botulinum neurotoxin type B ( Figure 5 ), Figure 5 BONT / A is botulinum neurotoxin type A, BONT / B is botulinum neurotoxin type B, BONT / C is botulinum neurotoxin type C, BONT / D is botulinum neurotoxin type D, BONT / E is botulinum neurotoxin type E, and BONT / F is botulinum neurotoxin type F.

[0093] e. The antigen site bound by the monoclonal antibody was identified by WB. The botulinum neurotoxin type B was subjected to SDS-PAGE electrophoresis and then subjected to WB analysis. The monoclonal antibody was used to bind to the light chain and heavy chain of the reduced botulinum neurotoxin type B. The results showed that the monoclonal antibody bound to the non-reduced toxin around 150 kDa and the light chain of the reduced botulinum neurotoxin type B ( Figure 4 ).

[0094] f. The affinity of the monoclonal antibody was analyzed using surface plasmon resonance technology. The results showed that the Kd of the monoclonal antibody was 3.585×10 -10 M( Figure 6 ).

[0095] The antigen used in the present invention is a 150 kDa botulinum neurotoxin, which is the key component of the botulinum neurotoxin complex that exerts toxicity. Currently, most botulinum neurotoxin products on the market are based on toxin complexes. Compared with toxin complex antigens, the monoclonal antibodies against botulinum neurotoxin in the present invention have more significant sensitivity and specificity, and have a wider range of uses in detection.

[0096] The present invention is directed to botulinum neurotoxin type B in its natural conformation, and can ensure good affinity and stability during detection. The present invention prepares natural antibodies against botulinum neurotoxin type B through hybridoma technology, retains the affinity maturation of the antibody in vivo, and retains the native pairing of the antibody variable region and antigen constant region gene combination. Therefore, the present invention makes up for the lack of natural antibodies against botulinum neurotoxin type B in the domestic market, and can be used as a powerful tool both in the diagnosis of botulinum neurotoxin poisoning types and in the development and production of botulinum neurotoxin products. In addition, the present invention can provide a sequence reference for recombinant anti-botulinum neurotoxin type B antibodies.

[0097] As a powerful detection tool, the antibodies and detection methods provided by the present invention can first complete qualitative detection of various suspected cases of botulinum neurotoxin poisoning in a short time, assist doctors in diagnosis, and provide evidence support for timely treatment decisions. At the same time, in the process of research and development and production of botulinum neurotoxin products, the specificity of the detection method for toxin protein is significantly improved, and the problem of too long time in animal experiments such as serotype identification is avoided. In addition, in the many detection processes of scientific research and production work, it plays a role in quality control of the stability and shelf life of type B botulinum neurotoxin standards.

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A detection kit for botulinum neurotoxin type B, characterized in that: The detection kit comprises an antibody or an antigen-binding fragment thereof that binds to botulinum neurotoxin type B, wherein the antibody or the antigen-binding fragment thereof comprises heavy chain CDR1-3 whose amino acid sequences are respectively shown as GFAFSSYD, ISSGGSST and ARQAFYTYDGTAMDY, and light chain CDR1-3 whose amino acid sequences are respectively shown as QSIVHNNGDTY, KVS and FQGSHIPWT.

2. Use of the reagent in the preparation of a kit for detecting botulinum neurotoxin type B, characterized in that: The reagent comprises an antibody or an antigen-binding fragment thereof that binds to botulinum neurotoxin type B, and the detection comprises the step of contacting the sample to be tested with the antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment comprises heavy chain CDR1-3 whose amino acid sequences are respectively shown as GFAFSSYD, ISSGGSST and ARQAFYTYDGTAMDY, and light chain CDR1-3 whose amino acid sequences are respectively shown as QSIVHNNGDTY, KVS and FQGSHIPWT.

3. Use of the reagent according to claim 2 in preparing a kit for detecting botulinum neurotoxin type B, characterized in that: The detection comprises the following steps: (1) contacting the sample to be tested with the antibody or its antigen-binding fragment; (2) detecting the binding of the botulinum neurotoxin type B to the antibody or its antigen-binding fragment to determine the presence of the botulinum neurotoxin type B in the test sample or to determine the amount of the botulinum neurotoxin type B in the test sample.

4. An antibody or an antigen-binding fragment thereof, characterized in that The antibody or its antigen-binding fragment is capable of targeting botulinum neurotoxin type B, and the antibody or its antigen-binding fragment comprises heavy chain CDR1-3 whose amino acid sequences are respectively shown as GFAFSSYD, ISSGGSST and ARQAFYTYDGTAMDY, and light chain CDR1-3 whose amino acid sequences are respectively shown as QSIVHNNGDTY, KVS and FQGSHIPWT.

5. The antibody or antigen-binding fragment thereof according to claim 4, characterized in that: The antibody or antigen-binding fragment thereof has a heavy chain amino acid sequence shown in SEQ ID NO.1 and a light chain amino acid sequence shown in SEQ ID NO.

2.

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

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

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

9. A host cell, characterized in that The host cell comprises the nucleic acid molecule of claim 7 or the vector molecule of claim 8.

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

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