A method and kit for detecting botulinum toxin activity or synaptosome-related proteins

Abstract

The present invention discloses a method and a kit for detecting botulinum toxin activity or synaptosome-associated proteins. The method comprises the step of contacting a sample to be tested with an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof targets the synaptosome-associated protein or its cleavage fragment. The present invention obtains a humanized single-chain antibody against synaptosome-associated proteins from an antibody library, thereby ensuring that the antibodies are fully humanized, constructs a eukaryotic expression vector with the aid of genetic engineering technology, and expresses the antibodies in eukaryotic cells. In addition, the present invention converts the obtained fully humanized single-chain antibody into a specific fully humanized complete antibody, which is then separated and purified, and its biological properties such as specificity and affinity are evaluated, thereby establishing a method for detecting botulinum toxin activity and a method for detecting synaptosome-associated proteins. In addition, the affinity of the anti-synaptosome-associated protein antibody of the present invention is higher than that of the commercially available rabbit synaptosome-associated protein polyclonal antibody.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method and a kit for detecting botulinum toxin activity or synaptosome-related proteins. Background Art

[0002] Synaptosome-associated protein of 25 kDa (SNAP25) is a 25 kDa (206 amino acids) helical protein first identified by Oyler et al. It consists of two isoforms, SNAP25a and SNAP25b, which appear at different stages of neural development. Structurally, SNAP25 is a helical protein. Two α-helical SNAP25s and two other helical vesicle-associated membrane proteins (VAMP) and syntaxin (STX) together form a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Functionally, SNAP25 is involved in the release of presynaptic neurotransmitters. Moreover, residues 180-197 play a key role in the regulation of calcium dynamics. In addition, the C-terminal zipper of SNAP25 has also been found to trigger the opening of the fusion pore and conformational changes of the SNARE complex.

[0003] Botulinum toxin (BoNTs) is a neurotoxin polypeptide, which is the most toxic natural protein toxin known to humans, with a median lethal dose of 0.1-1.0 ng / kg for humans. Botulinum toxins include BoNT / A, BoNT / C, and BoNT / E. After entering the cell, BoNTs block the release of neurotransmitters at the neuromuscular junction by cleaving SNARE proteins, ultimately leading to flaccid nerve paralysis. Therefore, BoNTs are widely used to treat nervous system-related diseases and the beauty industry, such as chronic migraine, trigeminal neuralgia, hemifacial spasm, dystonia, overactive bladder, frown lines, etc.

[0004] Currently, commercial SNAP25 antibodies are often used to detect the biological activity of botulinum toxin. However, most SNAP25 antibodies are polyclonal or monoclonal antibodies of animal origin (including mouse and rabbit origin). Animal-derived monoclonal antibodies are produced through hybridoma technology, which requires animal immunization, complex hybridoma fusion and screening processes. The entire cycle takes a long time and has poor reproducible performance.

[0005] Therefore, there is still a need to screen fully human antibodies with short cycle times, excellent repeatability and high affinity for detecting botulinum toxin activity. Summary of the invention

[0006] In order to solve at least some of the problems in the above-mentioned prior art, the present invention obtains anti-hSNAP25 humanized single-chain antibody from antibody library screening, constructs eukaryotic expression vector by genetic engineering technology, expresses it in eukaryotic cells, and converts it into specific fully humanized complete hSNAP25-IgG1 antibody, which is then separated and purified, and its biological properties such as specificity and affinity are evaluated, and a method for detecting botulinum toxin activity or synaptosome-related proteins is established. Specifically, the present invention includes the following contents.

[0007] In a first aspect, the present invention provides a method for detecting botulinum toxin activity or synaptosome-associated protein, the method comprising the step of contacting a sample to be tested with an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof targets the synaptosome-associated protein or a cleavage fragment thereof.

[0008] In certain embodiments, according to the method described herein, the antibody or antigen-binding fragment thereof comprises heavy chain antigen complementary determining regions CDR H1-H3 and / or light chain antigen complementary determining regions CDR L1-L3, wherein the heavy chain antigen complementary determining regions CDR H1-H3 respectively have the sequences shown in SEQ ID NO.1-3, and the light chain antigen complementary determining regions CDR L1-L3 respectively have the sequences shown in SEQ ID NO.4-6.

[0009] In certain embodiments, according to the methods described herein, the botulinum toxin comprises BoNT / A, BoNT / C, and / or BoNT / E.

[0010] In a second aspect of the present invention, an antibody or an antigen-binding fragment thereof is provided, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain antigen complementary determining region CDR H1-H3 and / or a light chain antigen complementary determining region CDR L1-L3, wherein the heavy chain antigen complementary determining region CDR H1-H3 respectively has the sequence shown in SEQ ID NO.1-3, and the light chain antigen complementary determining region CDR L1-L3 respectively has the sequence shown in SEQ ID NO.4-6.

[0011] In certain embodiments, the antibody or antigen-binding fragment thereof according to the present invention has any one of the following amino acid sequences:

[0012] (I) the heavy chain amino acid sequence shown in SEQ ID NO.7 and / or the light chain amino acid sequence shown in SEQ ID NO.8;

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

[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] The third aspect of 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.

[0016] The fourth aspect of the present invention provides a vector molecule, wherein the vector molecule comprises the nucleic acid molecule described in the present invention.

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

[0018] In a sixth 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.

[0019] In a seventh aspect, the present invention provides a kit for detecting botulinum toxin activity or synaptosome-associated proteins, the kit comprising the antibody or antigen-binding fragment thereof according to the present invention.

[0020] The anti-synaptosome-associated protein antibody of the present invention has higher affinity than commercially available rabbit synaptosome-associated protein polyclonal antibodies, and has the advantages of short screening cycle, excellent repeatable production performance, and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) image of the recombinant human SNAP25 protein and biotin conjugate (B-hSNAP25) under reducing conditions.

[0022] Figure 2 The SDS-PAGE verification results of SNAP25H02 under reducing (lane 1) and non-reducing (lane 2) conditions are shown.

[0023] Figure 3 The purity of the SNAP25H02 antibody was determined using HPLC.

[0024] Figure 4The affinity test results of SNAP25H02 antibody detected by Biacore. The gray curves from top to bottom in the figure represent 50 nM, 25 nM, 12.5 nM, 6.25 nM and 3.125 nM, respectively, and the black curve is the corresponding fitting curve.

[0025] Figure 5 Comparison of BoNT / A activity detected by SNAP25H02 and commercially available S9684 antibody. DETAILED DESCRIPTION

[0026] 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.

[0027] 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.

[0028] 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.

[0029] In the present invention, "synaptosome-associated protein" and "SNAP25" are used interchangeably and refer to the full-length protein having 206 amino acids.

[0030] method

[0031] Different serotypes of botulinum toxin have unique specificity for their respective target proteins and the peptide bonds cleaved, wherein BoNT / A and BoNT / E cleave SNAP25 at the carboxyl terminus, releasing a 9 or 26 amino acid fragment, respectively, and BoNT / C also cleaves SNAP25 near the carboxyl terminus, releasing an 8 amino acid fragment. Therefore, a method for detecting the activity of BoNT / A, BoNT / C and BoNT / E can be established using SNAP25 antibodies or antigen-binding fragments thereof. In one aspect of the present invention, a method for detecting botulinum toxin activity (or botulinum toxin endopeptidase activity) or synaptosome-associated proteins is provided, comprising the step of contacting a sample to be tested with an antibody or an antigen-binding fragment thereof, wherein the antibody or an antigen-binding fragment thereof is capable of targeting synaptosome-associated proteins or cleaved fragments thereof.

[0032] In a preferred embodiment of detecting the activity of botulinum toxin, the method comprises obtaining a sample to be tested; allowing the sample to be tested to bind to the antibody (also called capture antibody) or its antigen-binding fragment to form a complex or conjugate; allowing the complex or conjugate to further bind to the detection antibody; and determining the amount of synaptosome-associated protein cleaved by the botulinum toxin, thereby determining the activity of the botulinum toxin. Wherein, the capture antibody or its antigen-binding fragment comprises heavy chain antigen complementary determining region CDR H1-H3 and / or light chain antigen complementary determining region CDR L1-L3, the heavy chain antigen complementary determining region CDR H1-H3 has the sequence shown in SEQ ID NO.1-3 respectively, and the light chain antigen complementary determining region CDR L1-L3 has the sequence shown in SEQ ID NO.4-6 respectively, and the capture antibody or its antigen-binding fragment can specifically bind to the synaptosome-associated protein or its cleavage fragment. The detection antibody can specifically bind to the cleavage fragment of the synaptosome-associated protein obtained by cleavage of the botulinum toxin and the detection antibody contains a detectable group, and the detection antibody is an alkaline phosphatase-binding antibody, a horseradish peroxidase-binding antibody, or an antibody bound to a fluorescent dye. In a preferred embodiment, the capture antibody is human SNAP25H02 antibody, and the detection antibody is SNAP25 197 -HRP mouse monoclonal antibody.

[0033] In a preferred embodiment of detecting synaptosome-associated proteins, the method comprises obtaining a sample to be tested; contacting the sample to be tested with the antibody or its antigen-binding fragment; determining the binding of the antibody or its antigen-binding fragment to the synaptosome-associated protein or quantifying the formed antigen-antibody complex to determine the presence of the synaptosome-associated protein in the sample to be tested or the amount of the synaptosome-associated protein in the sample to be tested. Wherein, the antibody or its antigen-binding fragment comprises heavy chain antigen complementary determining regions CDR H1-H3 and / or light chain antigen complementary determining regions CDR L1-L3, the heavy chain antigen complementary determining regions CDR H1-H3 respectively have the sequences shown in SEQ ID NO.1-3, and the light chain antigen complementary determining regions CDRL1-L3 respectively have the sequences shown in SEQ ID NO.4-6.

[0034] In the present invention, the botulinum toxin includes BoNT / A, BoNT / C and / or BoNT / E. In a preferred embodiment, the botulinum toxin is BoNT / A.

[0035] The present invention further provides the use of a reagent in preparing a kit for detecting botulinum toxin activity or synaptosome-related proteins. The reagent comprises the above-mentioned antibody or antigen-binding fragment thereof of the present invention.

[0036] In one embodiment of the present invention, the method for detecting synaptosome-associated proteins in a sample is not used for prediction or diagnosis of a disease, but is used to determine whether synaptosome-associated proteins exist in a sample, or to perform quantitative detection of synaptosome-associated proteins, and the results can be used to assist standard diagnostic techniques or provide treating physicians with intermediate information related to neuropathological diseases in subjects who have been diagnosed or suspected of having the disease. Considering that it is currently known that synaptosome-associated proteins can be used as potential biomarkers and therapeutic targets for diseases, therefore, in another embodiment of the present invention, the detection of synaptosome-associated proteins in a sample can be used for prediction or diagnosis of a disease, such as for prediction, progression, or even prognosis of a neuropathological disease. Based on this, the method of the present invention is also referred to as "the use of a reagent in the preparation of a diagnostic product (e.g., a diagnostic kit) for prediction, progression, or prognosis of a neuropathological disease", and wherein the reagent comprises an antibody or an antigen-binding fragment thereof, and the antibody or an antigen-binding fragment thereof comprises a heavy chain CDR H1-H3 having an amino acid sequence as shown in SEQ ID NO.1-3 and / or a light chain CDR L1-L3 having an amino acid sequence as shown in SEQ ID NO.4-6. In the present invention, neuropathological diseases include but are not limited to Creutzfeldt-Jakob disease, Alzheimer's disease, neuropathic pain, schizophrenia, epilepsy, ataxia and speech disorders.

[0037] In the present invention, the sample to be tested may include at least one of the following: skin, muscle (including but not limited to skeletal muscle and smooth muscle), bladder, glandular tissue (including but not limited to prostate, lacrimal gland, endocrine gland and exocrine gland), blood vessels, spinal cord and brain (including nerve fibers in any or all of these tissues).

[0038] Antibodies or antigen-binding fragments thereof

[0039] In one aspect of the present invention, an antibody or an antigen-binding fragment thereof is provided, which comprises heavy chain antigen complementary determining regions CDRH1-H3 and / or light chain antigen complementary determining regions CDR L1-L3, wherein the heavy chain antigen complementary determining regions CDR H1-H3 respectively have the sequences shown in SEQ ID NO.1-3, and the light chain antigen complementary determining regions CDR L1-L3 respectively have the sequences shown in SEQ ID NO.4-6.

[0040] In a preferred embodiment, the antibody or antigen-binding fragment thereof has any one of the following amino acid sequences:

[0041] (I) the heavy chain amino acid sequence shown in SEQ ID NO.7, and / or the light chain amino acid sequence shown in SEQ ID NO.8;

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

[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] In the present invention, antibody refers to the immunoglobulin produced by B cells proliferating and differentiating into plasma cells after antigen stimulation, which can specifically bind to the corresponding antigen and exert immune function. The basic structure of an antibody is a Y-shaped tetrapeptide chain, which is composed of two identical heavy chains (H chains) and two identical light chains (L chains). The heavy chain and the light chain are named according to their molecular weight, and their relative molecular masses are approximately 50-75 kDa and 25 kDa, respectively. Structurally, the heavy chains and the heavy chains, and the heavy chains and the light chains are connected by disulfide bonds to form a symmetrical molecule with light and heavy chain pairs. The amino acid sequence of the carboxyl terminal (C-terminal) of the H chain and the L chain is relatively stable and changes very little, which is called the constant region (C region). The C region is related to the antibody effector function, and the C region of the same type of antibody is the same in composition. The C region of the antibody H chain is divided into CH1, CH2 and CH3, and the C region of the L chain is called CL. The amino-terminal (N-terminal) of the H chain and the L chain has a large variation in the sequence of about 110 amino acids, which is called the variable region (V region). The V region is related to antigen recognition. The position of the V region where the antibody binds to the antigen, and its amino acid composition and arrangement determine the antigen binding specificity of the antibody. The variable regions of the H chain and L chain of the antibody are called VH and VL, respectively. The variable region of a heavy chain and the variable region of a light chain interact to form the antigen binding site (Fv). The V region of the H chain and the L chain each have 3 regions with highly variable amino acid composition and arrangement order, which are called CDR H1, CDR H2, CDR H3, CDR L1, CDR L2 and CDR L3, respectively.

[0045] In the present invention, the single-chain antibody is a genetically engineered small molecule antibody formed by connecting the heavy chain variable region VH and the light chain variable region VL via an amino acid peptide segment, and has a molecular weight of about 25-27 kDa.

[0046] In the present invention, the antigen-binding fragment includes Fab (referring to a fragment containing 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 removing the crystallizable region connected by a disulfide bond) or scFv (single-chain variable fragment).

[0047] In the present invention, the terms "identity" and "homology" 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 the appropriate parameters for comparison, including any algorithm required for achieving optimal comparison in the full-length sequence compared.

[0048] In the antibodies with modified amino acid sequences provided by the present invention, preferably, the modification occurs in regions other than the variable region, such as in the framework region or constant region of the antibody, and the modified antibody retains the desired functional properties of the antibody of the present invention or its antigen-binding fragment, or has improved antigen-binding properties. The antibodies of the present invention may include glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage or non-natural amino acid modifications, etc.

[0049] In the present invention, 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. "Addition" refers to an alteration in an amino acid sequence that results in an increase of one or more amino acids compared to a naturally occurring molecule.

[0050] Unless otherwise indicated, the antibodies or antigen-binding fragments thereof described herein are isolated antibodies or antigen-binding fragments thereof. The term "isolated" as used herein refers to an antibody or antigen-binding fragment thereof that has been extracted from its natural environment. Peptides and proteins that have been "isolated" thus include peptides and proteins purified by standard purification methods. The term also includes peptides and proteins prepared by recombinant expression in host cells and chemically synthesized peptides and proteins.

[0051] Nucleic acid molecules

[0052] In one aspect of the present invention, a nucleic acid molecule is provided, which encodes the antibody or antigen-binding fragment thereof according to the present invention.

[0053] 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.

[0054] Once the coding sequence of the antibody or antigen-binding fragment thereof of the present invention is isolated, 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.

[0055] Carrier molecules

[0056] One aspect of the present invention provides a vector molecule comprising the nucleic acid molecule of the present invention.

[0057] 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 of the present invention or a precursor thereof, or optionally further comprises a marker gene. The vector can use a known vector or a self-constructed vector. Known vectors include phage vectors, plasmid vectors, lentiviral vectors, adenoviral vectors, AAV viral vectors, etc.

[0058] Host cells

[0059] In one aspect of the present invention, a host cell is provided, which comprises the nucleic acid molecule or the vector molecule described in the present invention.

[0060] 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. Host cells include any progeny of a parent cell that differs from the parent cell due to mutations that occur during replication. In a preferred embodiment, the host cell is a 293F cell.

[0061] Preparation method

[0062] 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.

[0063] In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention is 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 is obtained by direct amino acid synthesis.

[0064] In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention is obtained by genetic engineering expression. Genetic engineering expression systems include prokaryotic cell expression systems, eukaryotic cell expression systems, and cell-free expression systems. Examples of 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.

[0065] In a specific embodiment, the method for preparing an antibody of the present invention comprises:

[0066] (1) constructing a recombinant vector expressing the antibody heavy chain of the amino acid sequence shown in SEQ ID NO.7 and / or a recombinant vector expressing the antibody light chain of the amino acid sequence shown in SEQ ID NO.8;

[0067] (2) transforming the vector into host cells and culturing the cells under conditions suitable for antibody expression;

[0068] (3) Collect and purify antibodies.

[0069] Reagent test kit

[0070] In one aspect of the present invention, a kit for detecting botulinum toxin activity or synaptosome-associated proteins is provided, which comprises the antibody or antigen-binding fragment thereof according to the present invention.

[0071] In a preferred embodiment, the kit includes a capture antibody and a detection antibody. The capture antibody includes heavy chain antigen complementary determining regions CDR H1-H3 and / or light chain antigen complementary determining regions CDR L1-L3, wherein the heavy chain antigen complementary determining regions CDR H1-H3 have the sequences shown in SEQ ID NO.1-3, respectively, and the light chain antigen complementary determining regions CDR L1-L3 have the sequences shown in SEQ ID NO.4-6, respectively. The detection antibody can be any antibody known in the art that can specifically bind to cleaved SNAP25, such as an antibody from mouse, rabbit or humanized. Different detection antibodies can be selected according to the different types of botulinum toxin to be tested. As an example, when the botulinum toxin to be tested is BoNT / A, any suitable anti-SNAP25 antibody that can specifically bind to 197 amino acids can be selected, such as SNAP25 197 Mouse monoclonal antibody, those skilled in the art may also simultaneously or use other antibodies such as SNAP25 198 SNAP25180 In a preferred embodiment, the capture antibody is a human SNAP25H02 antibody, and the detection antibody is a SNAP25 197 -HRP mouse monoclonal antibody.

[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, 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 further comprises at least one of a cleaning solution, a substrate solution, a diluent and a calibration solution. Wherein, 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 can 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] Herein, "capture antibody" and "coating antibody" are used interchangeably, and "detection antibody" and "labeled antibody" are used interchangeably. The detection antibody of the present invention may be combined with a detectable marker, and the term "detectable marker" herein means a compound that produces a detectable signal. The detectable marker can be an MRI contrast agent, a scintigraphy contrast agent, an X-ray imaging contrast agent, an ultrasound contrast agent, an optical imaging contrast agent. Examples of detectable markers include, but are not limited to, radioactive elements, fluorophores such as fluorescein, Alexa or cyanine; chemiluminescent compounds such as luminol; bioluminescent compounds such as alkaline phosphatase, horseradish peroxidase, luciferase, etc.; and contrast agents such as nanoparticles or gadolinium. The choice of a suitable detectable marker depends on the detection system used and is within the capabilities of those skilled in the art. For example, when the detection system is MRI, the detectable marker is preferably iron oxide nanoparticles or gadolinium; when the detection system is fluorescence imaging, the detectable marker is preferably fluorescein, Alexa or cyanine; when the detection system is chemiluminescence imaging, the detectable marker is preferably luminol; when the detection system is bioluminescence imaging, the detectable marker is preferably luciferase or alkaline phosphatase; when the detection system is nuclear imaging, the detectable marker is preferably a radioactive element.

[0075] In the present invention, the kit may be provided in the form of a bottle, a cassette, a strip, etc., without particular limitation.

[0076] Example 1

[0077] 1. Method for preparing conjugate B-hSNAP25 by coupling recombinant human SNAP25 protein (hSNAP25) expressed in Escherichia coli with biotin

[0078] 1) Dissolve biotin in DMSO to 10 mg / mL and add to the protein at a mass ratio of 10:1.

[0079] 2) Seal with sealing film and fix on a shaker at 37°C for 2 hours. Wash the G25 column with PBS for 30 minutes. After PBS flows down, use a pipette to transfer the well-reacted protein to the G25 column on a shaker at 37°C. Add 1 ml of PBS for elution. Do not transfer the first drop and transfer the remaining bound protein to a 2 ml tube until the protein is completely eluted.

[0080] 2. Screening of phage libraries

[0081] Typical biopanning requires 3-5 rounds of screening to achieve acceptable enrichment. In this example, 4 rounds of screening were performed, and pre-absorption was performed before each round of panning to reduce non-specific binders.

[0082] The experimental steps are briefly described as follows:

[0083] 1) Incubate the ELISA plate coated with streptavidin-coupled magnetic beads M-280 for 2 h;

[0084] 2) Coat the ELISA plate with B-hSNAP25 and incubate overnight;

[0085] 3) Block the ELISA plate with blocking buffer and incubate overnight;

[0086] 4) Add the phage library to each well and incubate at 37°C for 2 h;

[0087] 5) Wash 5 times with an ELISA plate washer to remove unbound phages;

[0088] 6) Add elution buffer and incubate at 37°C for 10 min. Pipette up and down vigorously 10 times to elute the positive phages from the target antigen. Then, transfer the filtrate to a microcentrifuge tube containing neutralization buffer.

[0089] 7) The partially eluted phages were -2 and 10 -10Serial dilutions were performed between cfu, and the infection logarithmic phase E.coil TG1 (OD600≈0.5), infected E.coil TG1 was spread on 2×YT-A (2×YT medium supplemented with ampicillin) agar plates, grown overnight at 37°C, and the number of recovered phages was calculated;

[0090] 8) Infect partially eluted phage E.coil After TG1, 2 YT-AG medium (2×YT medium supplemented with ampicillin and glucose) was added, grown overnight at 37°C, stirred at 5000 rpm for 15 min at 4°C, and the pellet was resuspended in 2× yeast extract tryptone medium (2×YT medium) containing glycerol and stored at −80°C;

[0091] 9) Inoculate part of the recovered glycerol stock solution into 2×YT-AG medium and grow to the logarithmic phase (OD 600≈0.5). 11 pfu of M13K07 helper phage, incubated in a 37°C water bath for 30 min, centrifuged at 5000 rpm for 15 min, resuspended the pellet in 2×YT-AK medium (2×YT medium supplemented with ampicillin and kanamycin), and grown overnight at 30°C with shaking (225 rpm);

[0092] 10) Centrifuge the culture at 4000 g for 20 min in a 50 mL centrifuge tube to precipitate the bacteria and collect the supernatant (containing the phage library). To precipitate the phage, add 1 / 5 volume of 5× PEG / NaCl to the supernatant and place on ice for at least 1 h (PEG will promote the precipitation of the phage complex from the solution). Remove the supernatant thoroughly by centrifugation at 12000 g for 15 min at 4°C. Resuspend the pellet in 1 mL of sterile PBS. To eliminate bacterial contamination of the phage in the suspension, centrifuge in a microcentrifuge (2 min, maximum g). Transfer the supernatant to a new tube for the next round of panning.

[0093] 11) Repeat steps 1)-10) 4 times.

[0094] Table 1 Monitoring of phage antibody library screening process

[0095]

[0096] Enrichment factor = number of input phages / number of eluted phages.

[0097] 3. Monoclonal phage ELISA experiment

[0098] 1) Coat a 96-well plate with streptavidin-conjugated B-hSNAP25 (SA+B-hSNAP25) and use PBS as a negative control at 4°C overnight;

[0099] 2) Wash the plate, add blocking buffer, incubate at 37°C for 1 h, and then wash the plate;

[0100] 3) Culture a single colony picked from the plate overnight at 37°C, inoculate 2-5 μL / well from the overnight plate, grow to exponential phase (OD 600 ≈ 0.5), prepare a mixture by adding M13KO7 helper phage, the ratio of phage to bacteria should be about 20:1, incubate and shake for several hours;

[0101] 4) Combine the recovered monoclonal phage supernatant (25 μL / well) with 75 μL of blocking buffer and add to the antigen-coated 96-well plate and incubate at 37°C for 1 h;

[0102] 5) After washing the plate, add 100 μL / well of 1:5000 diluted anti-M13-HRP and incubate at 37°C for 1 h;

[0103] 6) After washing the plate, add 100 μL / well TMB colorimetric solution and incubate at room temperature in the dark for 5-30 min;

[0104] 7) Add 50 μL / well 1 M H2SO4 to terminate the reaction and read the absorbance of each well at 450 nm in a microplate reader.

[0105] An antibody that can bind to the antigen was screened, namely Clone12-scFv, and sequenced. The sequencing results are as follows:

[0106] After sequencing, the amino acid sequence of the Clone12-scFv antibody is shown in SEQ ID No.9, wherein the amino acid sequence of the heavy chain CDRH1-H3 is shown in SEQ ID No.1-3, and the amino acid sequence of the light chain CDR L1-L3 is shown in SEQ ID No.4-6.

[0107] Table 2 Antibody amino acid sequences

[0108] antibody or its antigen-binding fragment amino acid sequence CDR H1 SYGMH(SEQ ID No.1) CDR H2 AISESGGGTHYADFVKG(SEQ ID No.2) CDR H3 DKYSGRYQGSYGLDV(SEQ ID No.3) CDR L1 RSSQSLLHRNGYNYLD(SEQ ID No.4) CDR L2 LGSNRAS(SEQ ID No.5) CDR L3 MQGKTWPIT(SEQ ID No.6) heavy chain EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSAISESGGGTHYADFVKGRFTISRDNSNSTLYLQMNSLRAEDTAVYYCAKDKYSGRYQGSYGLDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID No.7) light chain DVVMTQSPLSLPVTPGEPASISCRSSQSLLHRNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPARFSGSGSGTDFTLKISRVEAEDVGVYYCMQGKTWPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID No.8) Clone12-scFv EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSAISESGGGTHYADFVKGRFTISRDNSNSTLYLQMNSLRAEDTAVYYCAKDKYSGRYQGSYGLDVWGQGTMVTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVTPGEPASISCRSSQSLLHRNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPARFSGSGSGTDFTLKISRVEAEDVGVYYCMQGKTWPITFGQGTRLEIKR(SEQ ID No.9)

[0109] 4. ELISA experiment of soluble phage

[0110] The identified positive clones were subjected to soluble phage ELISA.

[0111] 1) Place it in E.coi l IPTG was used to induce expression in TG1 at 30°C and 37°C;

[0112] 2) After expression is complete, the bacteria are collected by centrifugation for periplasmic extraction;

[0113] 3) The periplasmic extract was diluted 1:10 and added to the pre-coated SA+B-hSNAP25 ELISA plate, and PBS was used as a control;

[0114] 4) Mouse anti-E-tag monoclonal antibody at a dilution of 1:2000 and HRP goat anti-mouse polyclonal antibody at a dilution of 1:5000 were used as the secondary antibody and the tertiary antibody, respectively, to detect the soluble expression of Clone12-scFv. As shown in Table 3, Clone12-scFv was soluble and positively bound to B-hSNAP 25.

[0115] Table 3 Soluble phage ELISA

[0116]

[0117] 5. Expression of fully human SNAP25-IgG1 (SNAP25H02) antibody

[0118] Based on the above research, this example screened out a specific anti-hSNAP25 humanized single-chain antibody, namely Clone12-scFv, from a large-capacity phage antibody library. Since the scFv single-chain antibody has no Fc segment, its affinity is not as good as that of a complete IgG1. Therefore, this example converts the anti-Clone12-scFv single-chain antibody into a complete IgG1 antibody, named SNAP25H02. The amino acid sequence of the SNAP25H02 heavy chain is shown in SEQ ID No.7, and the amino acid sequence of the SNAP25H02 light chain is shown in SEQ ID No.8.

[0119] Genetic engineering technology was used to amplify the heavy chain constant region of human antibodies and the variable region of heavy chains screened from the phage library, the light chain constant region of human antibodies and the variable region of light chains screened from the phage library to the eukaryotic cell expression plasmid pcDNA3.4 to construct the light chain expression plasmid and the heavy chain expression plasmid, respectively. The process is as follows:

[0120] 1) Plasmid preparation: Transform SNAP25H02 antibody light chain plasmid and SNAP25H02 antibody heavy chain plasmid into DH5α competent bacteria respectively, and then inoculate them into LB agar culture plates containing Amp antibiotics for resistance screening and culture. Pick positive clones and inoculate them into 150 ml LB medium containing 150 μl 100X Amp antibiotics, shake well, incubate overnight, and extract the plasmid according to the instructions of the plasmid extraction kit;

[0121] 2) Transfection of 293F cells: 293F cells were cultured in suspension at 37°C, 5% CO2 and 120 rpm, maintaining a cell density of 2×10 5 Cells / ml were cultured every 4 days, and the cell confluence was checked to be between 70-80%. The 293F cell density was adjusted to about 1.1×10 6 cells / mL, dilute the heavy chain and light chain plasmids, and polyethyleneimine (PEI) in Opi-MEM, and then mix them together (light chain: heavy chain = 1:1; PEI: plasmid = 2.7:1). Add the mixture to the flask containing 293F cells at one time, shake gently and move crosswise to ensure sufficient mixing. If the cells are in suspension, transfection is more efficient. Place the flask in a CO2 incubator at 37°C and 5% CO2 for 6 days;

[0122] 3) SNAP25H02 antibody purification: 293F cell suspension was centrifuged at 4°C and 8000 rpm for 30 minutes, and the cell supernatant was filtered through a 0.22 μm filter membrane. The antibody was purified with protein A, and the filtrate was transferred to a dialysis bag and stirred overnight at 4°C. The dialysate was removed and the final antibody concentration was detected with Nanodrop. It was filtered through a 0.22 μm filter membrane and 0.1 mg of protein sample was taken for concentration determination, SDS-PAGE and HPLC. In the SDS-PAGE analysis, the gel was stained with Coomassie Brilliant Blue for 30 minutes. Under reducing conditions, the light and heavy chains migrated at approximately 50 kDa and 25 kDa, respectively. Under non-reducing conditions, the protein migrated at 130-180 kDa ( Figure 2 ), HPLC results showed that the purity of SNAP25H02 antibody was 97.1% ( Figure 3 ).

[0123] 6. Determination of SNAP25H02 Antibody Affinity

[0124] 1) Anti-human IgG (Fc) immobilization: Anti-human IgG (Fc) was diluted to 25 μg / mL in the immobilization buffer and immobilized on the surface of the carboxymethyl dextran (CM 5) sensor chip (BIAcore), 10 μL / min, 420 s. The amount of anti-human IgG (Fc) antibody coupled to the CM 5 sensor chip was approximately 7000-14000 RU;

[0125] 2) SNAP25H02 antibody dilution: dilute SNAP25H02 antibody to 2.5 μg / mL with running buffer and inject it into the sample channel at 10 μL / min to achieve a capture level of approximately 100-130 RU;

[0126] 3) Sample dilution: Different concentrations of SNAP25 protein (3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM) were diluted in running buffer for affinity and kinetics measurements. The 1:1 binding model was used to measure binding affinity and / or kinetics. The results were as follows: Figure 4 shown.

[0127] The specific binding affinity results are shown in Table 4.

[0128] Table 4 SNAP25H02 antibody affinity

[0129]

[0130] Example 2

[0131] The following exemplifies the application of human SNAP25H02 antibody in the detection of botulinum toxin endopeptidase activity.

[0132] 1. Primary Antibody Incubation

[0133] Human SNAP25H02 antibody and commercially available SNAP25 rabbit polyclonal antibody S9684 were diluted 1:1000 with 1× PBS buffer, 100 μL / well, coated with shaking at 4°C overnight, washed three times with a plate washer (PBST), patted dry, blocked with 2% BSA solution at room temperature for 1.5 h, discarded, and patted dry.

[0134] 2. Add the sample to be tested

[0135] The BoNT / A to be tested was diluted with diluent (50 mmol / L HEPES, 20 μmol / L ZnCl2, 10 mmol / L DTT, 1mg / ml BSA, 0.5% Tween20; pH 7.0) from 100 U / mL in a 2-fold gradient for 10 times; 50 μL / well was added to a 96-well transparent plate, and activated in a constant temperature shaker at 37°C, 400 rpm for 30 min; h-SNAP25 protein was diluted to 0.25 μg / ml with the prepared diluent, and 50 μL / well was added to the activated toxin well. Place in a constant temperature shaker at 37°C, shake at 400 rpm, and incubate for 10 min. Then take it out from the constant temperature shaker, add 90 μL / well to the blocked ELISA enzyme labeling plate, shake at 4°C overnight, wash 3 times with a plate washer, and pat dry.

[0136] 3. Secondary Antibody Incubation

[0137] SNAP25 197-HRP mouse monoclonal antibody (50 μg / ml) was diluted 1:10000 with 2% BSA solution, 100 μL / well, shaken at room temperature for 1.5 h, washed three times with a plate washer, and patted dry.

[0138] 4. ELISA color development

[0139] The color developing solution was mixed in a 1:1 ratio (Note: the color developing solution is easily volatile when exposed to light, so it should be prepared and used immediately, and wrapped in tin foil), 100 μL / well of the color developing solution was added, and the optical density value was detected at D450 on an ELISA reader.

[0140] The results are as follows Figure 5 As shown, SNAP25H02 detected the EC of BoNT / A 50 The EC of S9684 for BoNT / A detection was 5.95 U / ml. 50 It is 25.19 U / ml.

[0141] It can be seen that the present invention screened the humanized anti-hSNAP25 single-chain antibody from the human natural single-chain antibody library, ensuring the full humanization of the antibody, constructed a eukaryotic expression vector with the help of genetic engineering technology, and expressed it in the eukaryotic cell 293F. In addition, the present invention converts the obtained anti-hSNAP25 humanized single-chain antibody into a specific fully humanized complete hSNAP25-IgG1 antibody, then separates and purifies it, and evaluates its biological properties such as specificity and affinity, thereby establishing a botulinum toxin activity detection method and a synaptosome-related protein detection method.

[0142] 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 kit for detecting botulinum toxin activity or synaptosome-related proteins, characterized in that: The kit comprises an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises heavy chain antigen complementary determining regions CDR H1-H3 whose sequences are respectively shown in SEQ ID NO.1-3 and light chain antigen complementary determining regions CDR L1-L3 whose sequences are respectively shown in SEQ ID NO.4-6.

2. The kit according to claim 1, characterized in that The kit includes additional antibodies for binding to fragments of synaptosomal associated proteins that have been cleaved or cleaved by botulinum toxin.

3. Use of the reagent in the preparation of a product for detecting botulinum toxin activity or synaptosome-related proteins, characterized in that: The reagent includes an antibody or an antigen-binding fragment thereof, and the detection includes the step of contacting the sample to be tested with the antibody or the antigen-binding fragment thereof; The antibody or antigen-binding fragment thereof comprises heavy chain antigen complementary determining regions CDR H1-H3 whose sequences are respectively shown in SEQ ID NO. 1-3 and light chain antigen complementary determining regions CDR L1-L3 whose sequences are respectively shown in SEQ ID NO. 4-6.

4. Use of the reagent according to claim 3 in the preparation of a product for detecting botulinum toxin activity or synaptosome-related proteins, characterized in that: The botulinum toxin includes BoNT / A, BoNT / C and / or BoNT / E.

5. An antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof comprises heavy chain antigen complementary determining regions CDR H1-H3 whose sequences are respectively shown in SEQ ID NO. 1-3 and light chain antigen complementary determining regions CDR L1-L3 whose sequences are respectively shown in SEQ ID NO. 4-6.

6. The antibody or antigen-binding fragment thereof according to claim 5, characterized in that: The antibody or antigen-binding fragment thereof has a heavy chain amino acid sequence shown in SEQ ID NO.7 and a light chain amino acid sequence shown in SEQ ID NO.

8.

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 claim 5 or 6.

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

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

10. The method for preparing an antibody or an antigen-binding fragment thereof according to claim 5 or 6, characterized in that: The antibody or antigen-binding fragment thereof is prepared by artificial synthesis or genetic engineering.

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