Cell-based method for determining botulinum toxin activity

Abstract

The present invention relates to cells and antibodies for determining botulinum toxin activity, and methods of using them to measure activity. Currently, in the field of measurement of botulinum toxin potency, there is a need to develop a cell-based potency assay (CBPA) to replace the mouse LD50 bioassay (mLD50). The cells and antibodies of the present invention for botulinum toxin activity assay are cells and antibodies that replace mLD50 with CBPA, and the cell line has a significantly shorter division time and a significantly higher sensitivity to botulinum toxin than the conventional SiMa cells used to determine botulinum toxin activity, and thus is very suitable for detecting toxins and determining cell-based botulinum toxin activity. The CBPA assay using the cells and antibodies of the present invention is expected to become a highly reliable and repeatable cell-based potency assay method for botulinum toxin.

Description

[0001] This application is a divisional application of the application filed on August 19, 2019, with application number CN 2019800908385 and invention title "A Cell-Based Method for Determining the Activity of Botulinum Toxin". [Technical Field]

[0002] This invention relates to cells and antibodies used to determine the activity of botulinum toxin, and methods for using them to determine the activity. [Background Technology]

[0003] Currently, mouse LD 50 Bioassay (mLD) 50 mLD (molecularly derived liquid) is a widely accepted method for detecting residual botulinum toxin (BoNT / A) in food, clinical, or environmental samples. Specifically, the pharmaceutical industry uses mLD. 50 As a standard analytical method, mLD50 is used to measure botulinum toxin titers for cosmetic or clinical applications. However, due to the high influence of testing institutions / facilities and researchers on botulinum toxin titer determination using mLD50, it is difficult to accurately and reproducibly quantify the biological efficacy of botulinum toxin. Therefore, in order to minimize the number of animals used in experiments and the suffering involved, and to standardize the method for measuring botulinum toxin titers, the ZEBET conference was held to encourage the development of alternative mLD50 measurement methods (Altern LabAnim. 2010 Aug; 38(4):315-30). In various countries, many research institutions and industries have conducted various studies to develop cell-based titer assays (CBPA) or cell-based bioassays (CBB) that are sufficient to replace mLD50 in terms of specificity, sensitivity and reproducibility. In order to successfully establish CBPA or CBB, they have been trying to obtain (1) the SNAP25 titer. 197 Specific monoclonal or polyclonal antibodies, and (2) antibodies against low levels of botulinum toxin. It exhibits a highly sensitive neuronal cell line. As early as 2004, Dr. Chapman and his colleagues invented a fluorescent reporter gene assay method that fuses botulinum toxin with two fluorescent proteins (Proc Natl Acad Sci USA. 2004 Oct 12; 101(41):14701-6), which uses BoCell… TM The analytical method (Biosentinel Inc.) was named and commercialized. Despite BoCell... TMThe assay is significant as the first to detect the neutral endopeptidase activity of botulinum toxin in animal cells cultured in 98-well dishes. However, it suffers from a drawback: its sensitivity is 2-3 times lower than that of assays using mice (Appl Environ Microbiol vol. 78, 21(2012): 7687-97). As mentioned above, there has been a global effort to develop CBPA to replace mLD50, thus potentially ending the use of animal-based analytical methods. Although challenges remain in developing effective CBPA, it will help expand the applications of various botulinum toxin products, improve quality control, provide consumers with greater confidence, and enhance the competitiveness of botulinum toxin products.

[0004] Therefore, this invention aims to overcome the limitations of conventional CPBA and develop a more effective CBPA. Using the CPBA of this invention, a comparative analysis of 13 different neuronal cell lines was performed, and a novel clone, N2-42F, was developed, which is the most optimized among the wide range of clones constituting neurons. This clone has a very short division time, less than 24 hours, and compared to SiMa, it is expected to be very useful as a cell for CBPA because it can attach to and be stably cultured in culture dishes coated with poly-d-lysine (PDL).

[0005] This invention also relates to an antibody for CPBA, used as an alternative to mouse LD50 bioassay (mLD50). The antibody of this invention is a monoclonal antibody with significantly high binding affinity and specificity to SNAP25. The antibody of this invention overcomes the limitations of conventional CPBA, making it possible to develop more effective CBPAs, and therefore holds promise for positive applications in the pharmaceutical and cosmetic fields.

[0006] Furthermore, this invention relates to an optimal CBPA assay using N2-42F cells and a monoclonal antibody with significantly high binding affinity and specificity to SNAP25, capable of measuring botulinum toxin titers of 0.5 U or less. The CBPA assay using the cells and antibodies of this invention promises to become a cell-based assay for botulinum toxin titers with high reliability and reproducibility.

[0007]

public

[0008] [Technical Issues]

[0009] This invention relates to cells and antibodies used to determine the activity of botulinum toxin, and methods for using them to determine the activity.

[0010] However, the technical objectives to be achieved by the present invention are not limited to the above-mentioned technical objectives. Those skilled in the art can clearly understand other objectives not mentioned above through the following description.

[0011] [Technical Solution]

[0012] In the following description, various embodiments described herein will be described with reference to the accompanying drawings. Numerous specific details, such as particular configurations, compositions, and processes, are set forth in the description to provide a thorough understanding of the invention. However, certain embodiments may be practiced without one or more of these specific details, or in combination with other known methods and configurations. In other instances, known methods and preparation techniques have not been described in detail to avoid unnecessarily obscuring the invention. The reference to “one embodiment” or “one embodiment” throughout the invention means that a particular property, structure, combination, or feature described in association with an example is included in at least one embodiment of the invention. Therefore, the phrase “in one embodiment” or “one embodiment” appearing in various places throughout the specification does not necessarily refer to the same embodiment of the invention. Furthermore, particular properties, structures, combinations, or features may be combined in any suitable manner in one or more embodiments.

[0013] Unless otherwise specified in the specification, all scientific and technical terms used in the specification are the same as those commonly understood by those skilled in the art.

[0014] Allergan, the manufacturer of SNAP25, has successfully constructed a monoclonal antibody specific to SNAP25197 and identified the human neuroblastoma cell line (SiMa) as an ideal host cell line highly sensitive to botulinum toxin (PLoS One. 2012; 7(11):e49516). Using these antibodies and cells, Allergan developed a novel CBPA assay and received FDA approval in 2010 as the first CBPA capable of replacing mLD50 (US8455213B2 and US2010 / 0204559A1). MERZ, another botulinum toxin manufacturer in Germany, developed the CBA-ELISA (WO 2014 / 207109A1) in 2014 and received FDA approval in 2015. The CBA-ELISA uses a fixation method (in situ) of differentiated neuronal cells, followed by permeabilization through the cell membrane, to perform an immunological detection of endogenous SNAP25 (synaptosome neurotransmitter 25). Allergan's CBPA and MERZ's CBA-ELISA exhibited excellent sensitivity comparable to mouse bioassays at subpicomolar concentrations (i.e., <1.0 U / well) at EC50. Both Allergan and MERZ's techniques utilize a commonly used commercial rabbit polyclonal antibody (Sigma S9684) to detect SNAP25 under optimal conditions. In terms of cells, Allergan's CBPA specifically uses the differentiated human neuroblastoma cell line SiMa, while MERZ's CBA-ELISA uses differentiated human induced pluripotent stem cells (iPS) as standardized and optimized host cells. SiMa proliferates very slowly, typically requiring over 70 hours for the cell number to double (population doubling time, PDT). iPS production is also very time-consuming and storage is more difficult. Therefore, there is a need to develop CBPA neuronal cell lines that are highly sensitive to botulinum toxin, have short PDTs, and are easily stored. Furthermore, a research group led by Dr. David Beebe of the University of Wisconsin raised a question regarding the applicability of SiMa as a CBPA cell line, as SiMa does not exhibit motor neuron-like characteristics (J Biomol Screen. 2016 Jan; 21(1):65-73). They developed an alternative CBPA using the motor neuron-like cell line NG108-15, even in EC 50 Sensitivity was also observed at concentrations of 7.9 pM or lower. However, the CBPA developed used Western blots to determine SNAP25. 197 and SNAP25 FL The endogenous levels of these substances are difficult to utilize in high-throughput analyses.

[0015] In one embodiment of the invention, "botulinum toxin" is a neurotoxic protein produced by Clostridium botulinum. The genus Clostridium comprises over 127 species, classified according to their morphology and function. Botulinum toxin, a potent polypeptide neurotoxin produced by the anaerobic, Gram-positive bacterium Clostridium botulinum, causes a neuroparalytic disease in humans and animals known as botulism. Spores of Clostridium botulinum can be found in soil and can be cultured in unsterilized and unsealed household canned food containers, which is the cause of many cases of botulism. Symptoms of botulism typically appear 18 to 36 hours after consuming food contaminated with Clostridium botulinum cultures or spores. Botulinum toxin appears to be able to pass through the intestinal wall without diminishing its toxicity and has a high affinity for cholinergic motor neurons. Symptoms of botulism can progress from difficulty walking, swallowing, and speaking to respiratory muscle paralysis and even death. Botulinum toxin type A is the most lethal naturally occurring biological agent known to humans. The LD50 of commercially available botulinum toxin type A (a purified neurotoxin complex) is approximately 50 picograms (one unit). On a molar (M) basis, botulinum toxin type A is approximately 1.8 billion times more lethal than diphtheria, approximately 600 million times more lethal than sodium cyanide, approximately 30 million times more lethal than cobra toxin, and approximately 12 million times more lethal than cholera. One unit (U) of botulinum toxin can be defined as the LD50 administered intraperitoneally to female Swiss Webster mice weighing 18 to 20 grams each. Seven different botulinum neurotoxins are immunologically described as neurotoxin serotypes A, B, C1, D, E, F, and G, each distinguished by neutralizing type-specific antibodies. The differences in serum botulinum toxin types depend on the animal species they are used in and the severity and duration of the paralysis they induce. For example, based on measurements of paralysis rates in rats, botulinum toxin type A is 500 times more potent than botulinum toxin type B. Furthermore, botulinum toxin type B at a dose of 480 U / kg has been determined to be non-toxic to primates, approximately 12 times the LD50 of botulinum toxin type A in primates. Botulinum toxin is thought to bind with a significant high affinity to cholinergic motor neurons, entering neurons and blocking the release of acetylcholine. Additional uptake can occur through low-affinity receptors, as well as phagocytosis and pinocytosis. Regardless of serotype, the molecular mechanism of toxin poisoning appears to be similar and involves at least three steps. In the first step of this process, the toxin binds to the presynaptic membrane of the target neuron through a specific interaction between the toxin's heavy chain (H chain or HC) and cell surface receptors.The receptors for each type of botulinum toxin and tetanus toxin are believed to be different. The carboxyl terminus of HC appears to be important for targeting botulinum toxin to the cell surface.

[0016] The second step involves botulinum toxin crossing the plasma membrane of the target cell. Botulinum toxin is first engulfed by the cell via receptor-mediated endocytosis, forming an endosome containing the toxin. The toxin then escapes from the endosome into the cell's cytoplasm. This step is thought to be mediated by the amino-terminal segment HN of the heavy chain, which triggers a conformational change in the toxin in response to a pH of approximately 5.5. Endosomals are known to possess proton pumps that lower the pH within the endosome. The conformational change exposes hydrophobic residues in the toxin, allowing it to embed itself into the endosome membrane. Subsequently, the botulinum toxin (or at least the light chain of the botulinum toxin) is translocated across the endosome membrane into the cytoplasm. The final step in the botulinum toxin activity mechanism is believed to involve the reduction of the disulfide bonds connecting the heavy and light chains. The full toxic activity of botulinum toxin and tetanus toxin is contained in the light chain of the whole toxin; the light chain is a zinc (Zn++) neutral endopeptidase that selectively cleaves proteins essential for the recognition and docking of neurotransmitter-containing vesicles with the cytoplasmic surface of the plasma membrane, as well as for vesicle-to-membrane fusion. Tetanus neurotoxins, botulinum toxins of types B, D, F, and G, cause degradation of synaptosome proteins (also known as vesicle-associated membrane proteins (VAMPs)) (synaptosome membrane proteins). The result of any of these degradation processes is the removal of most of the VAMPs present on the cytoplasmic surface of the synaptic vesicles. Serotypes A and E cleave SNAP-25. Serotype C1 was initially thought to cleave the syntaxin, but it was found to cleave both the synaptic fusion protein and SNAP-25. Each toxin specifically cleaves different bonds, except for those cleaved by botulinum toxin type B (and tetanus toxin). Each of these cleavages prevents vesicle-membrane docking, thus preventing exocytosis of vesicle contents.

[0017] Botulinum toxin has been used in clinical settings to treat neuromuscular disorders characterized by skeletal muscle hyperactivity (i.e., dyskinesia). In 1989, a botulinum toxin type A complex was approved by the U.S. Food and Drug Administration (FDA) for the treatment of blepharospasm, strabismus, and hemifacial spasm. Subsequently, botulinum toxin type A was also approved by the FDA for the treatment of cervical dystonia and glabellar lines, while botulinum toxin type B was also approved for the treatment of cervical dystonia. Compared with botulinum toxin type A, non-type A botulinum toxin serotypes have apparently lower potency and / or shorter duration of activity. Clinical effects of peripheral intramuscular botulinum toxin type A typically appear within one week after injection. The typical duration of symptom relief after a single intramuscular injection of botulinum toxin type A is approximately 3 months, but the duration of therapeutic activity has been reported to be significantly longer.

[0018] Although all botulinum toxin serotypes significantly inhibit the release of the neurotransmitter acetylcholine at the neuromuscular junction, they do so by affecting different neurosecretory proteins and cleaving these proteins at different sites. For example, both botulinum toxin types A and E cleave the 25 kDa synaptosome-associated protein (SNAP-25), but they target different amino acid sequences within this protein. Botulinum toxin types B, D, F, and G act on vesicle-associated membrane proteins (VAMPs, also known as synaptobrevins), with each serotype cleaving different sites within the protein. Finally, botulinum toxin type C1 appears to cleave both synaptic fusion proteins and SNAP-25 simultaneously. These differences in mechanisms of action may affect the relative potency and / or duration of action of various botulinum toxin serotypes. In particular, the substrates for botulinum toxins can be found in a wide variety of cell types.

[0019] For all seven known botulinum toxin serotypes, the molecular weight of botulinum toxin is approximately 150 kDa. Botulinum toxin is released as a complex by *Clostridial bacterium*, containing a 150 kDa botulinum toxin protein molecule and associated non-toxin proteins. Therefore, type A botulinum toxin complexes can be produced by *Clostridial* in sizes of 900 kDa, 500 kDa, or 300 kDa. Types B and C1 botulinum toxins are apparently produced only as 700 kDa or 500 kDa complexes. Types B and C1 botulinum toxins are produced as 300 kDa or 500 kDa complexes. Finally, types E and F botulinum toxins are produced only as complexes of approximately 300 kDa. Complexes (i.e., with a molecular weight greater than approximately 150 kDa) are thought to contain non-toxin hemagglutinin proteins, non-toxin, and non-toxin non-hemagglutinin proteins. These two non-toxin proteins (which, together with the botulinum toxin molecule, form the associated neurotoxin complex) can provide stability, preventing denaturation of the botulinum toxin molecule and protecting it from digestive acid during ingestion. Furthermore, larger botulinum toxin complexes (molecular weight greater than approximately 150 kDa) may result in slower diffusion of botulinum toxin from the intramuscular injection site. Therefore, in this invention, botulinum toxin can include both a form without the complex protein and a complex form containing the complex protein. Botulinum toxin proteins derived from Clostridium botulinum types A, B, C, D, E, F, or G, without the natural complex protein, have a molecular weight of approximately 150 kDa. However, when the toxin protein is produced by Clostridium botulinum, various complexes formed by the botulinum toxin protein with various hemagglutinin and non-hemagglutinin proteins can support and protect the function of the botulinum toxin protein. Botulinum toxin serotype A contains naturally occurring complex proteins in 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. Serotypes E and F are complex forms with a molecular weight of approximately 300 kDa.

[0020] Furthermore, in vitro studies have shown that botulinum toxin inhibits potassium cation-induced release of acetylcholine and norepinephrine from primary cell cultures of brainstem tissue. Additionally, botulinum toxin has been reported to inhibit the induced release of glycine and glutamate in primary cultures of spinal cord neurons, and in brain synaptosome formulations, it inhibits the release of neurotransmitters acetylcholine, dopamine, norepinephrine, CGRP, substance P, and glutamate. Therefore, when used at sufficient concentrations, the stimulus-induced release of most neurotransmitters can be blocked by botulinum toxin.

[0021] Type A botulinum toxin can be obtained by establishing and culturing a culture of Clostridium botulinum in a fermenter, followed by harvesting and purifying the fermentation mixture according to known procedures. All botulinum toxin serotypes are initially synthesized as inactive single-chain proteins and must be cleaved or incised by proteases to acquire neuroactive properties. Bacterial strains that produce botulinum toxin serotypes A and G possess endogenous proteases; therefore, serotypes A and G can be recovered from bacterial cultures in their predominantly active form. In contrast, botulinum toxin serotypes C1, D, and E are synthesized by non-proteolytic strains and are therefore typically inactivated upon recovery from cultures. Serotypes B and F can be produced by both proteolytic and non-proteolytic strains and can therefore be recovered in either active or inactive forms. However, even proteolytic strains that produce, for example, type B botulinum toxin serotypes can only cleave a portion of the produced toxin. The exact ratio of cleaved to uncleaved molecules depends on the culture time and temperature. Therefore, it is well known that a certain proportion of any formulation, such as botulinum toxin type B, may be inactive, possibly due to its significantly lower potency compared to botulinum toxin type A. The presence of inactive botulinum toxin molecules in clinical formulations increases the total protein load of the formulation, which is associated with increased antigenicity but does not affect its clinical efficacy. Furthermore, botulinum toxin type B is known to have a shorter duration of activity upon intramuscular injection and is also less active than botulinum toxin type A at the same dose levels.

[0022] High-quality crystals of botulinum toxin type A can be obtained from Hall A botulinum strain, characterized by ≥3×10⁻⁶ crystals. 7 U / mg, A260 / A278 less than or equal to 0.60, and exhibiting a distinct banding pattern on gel electrophoresis. The known Schantz method can be used to obtain crystalline botulinum toxin type A. Typically, botulinum toxin type A complexes can be isolated and purified from anaerobic fermentation by culturing *Botulinum toxin type A* in a suitable culture medium. Known methods can also be used to isolate pure botulinum toxin from non-toxin proteins, such as purified botulinum toxin type A with a molecular weight of approximately 150 kDa and a specific potency of 1-2 x 10⁻⁶. 8 LD50 U / mg or higher; purified botulinum toxin type B, molecular weight approximately 156 kDa, specific potency 1-2 × 10⁻⁶. 8 LD50 U / mg or higher, and purified botulinum toxin type F with a molecular weight of approximately 155 kDa and a specific potency of 1-2 × 10⁻⁶. 7 LD50 U / mg or higher.

[0023] Botulinum toxin and / or botulinum toxin complexes are commercially available from manufacturers of compounds known in the art, and pure botulinum toxin can also be used to prepare pharmaceutical compositions.

[0024] Like enzymes, the biological activity of botulinum toxins (which are intracellular peptidases) depends at least in part on their three-dimensional conformation. Therefore, type A botulinum toxin is detoxified by heating, surface stretching with different chemicals, and surface drying. Furthermore, it is known that diluting botulinum toxin complexes obtained through known cultivation, fermentation, and purification to very low toxin concentrations for use in pharmaceutical formulations results in rapid detoxification unless a suitable stabilizer is present. Diluting the toxin from milligrams to solutions containing nanograms per milliliter presents significant challenges because specific toxicity is rapidly lost at such large dilutions. Since botulinum toxins can be used months or years after formulation of toxin-containing pharmaceutical compositions, suitable stabilizers should be used to stabilize the toxin. Therefore, as disclosed in this invention, there is a need to develop optimal stabilizer techniques to control the sustained-release of botulinum toxin in vivo.

[0025] According to reports, botulinum toxin type A has been used in the following clinical settings:

[0026] The typical duration of an intramuscular injection of botulinum toxin is about 3 to 4 months. However, in some cases, subtype A botulinum toxin, when used to treat glandular disorders such as hyperhidrosis, can be effective for up to 12 months, and in some cases up to 27 months.

[0027] In addition to its pharmacological effects in peripheral sites, botulinum toxin can also exhibit inhibitory effects in the central nervous system and can ascend to the spinal region via retrograde transport. Therefore, botulinum toxin injected at peripheral sites (e.g., intramuscularly) can be retrogradely transported to the spinal cord.

[0028] Botulinum toxin has also been proposed for, or has been used for, the treatment of skin, bone, and tendon wounds; intrathecal pain; various autonomic nervous system disorders, including sweat gland disorders; tension headaches; migraines; postoperative pain and visceral pain; hair growth and prevention of hair loss; psoriasis and dermatitis; muscle injuries; various cancers; smooth muscle disorders; nerve entrapment syndromes; acne; neurogenic inflammation; optic nerve disorders; pancreatic diseases; prostate diseases, including benign prostatic hyperplasia, prostate cancer, and urinary incontinence; fibromyalgia; and piriformis syndrome. Modified clostridium neurotoxins or fragments thereof, preferably botulinum toxin, chemically conjugated or recombined with a specific target moiety, are also known to be administered as agents to the spinal cord for the treatment of pain. Furthermore, targeted botulinum toxin (i.e., having a non-naturally bound moiety) is known for its use in the treatment of various diseases. Additionally, botulinum toxin has been injected into the pectoral muscles to control pectoral muscle spasms. Controlled release of toxin implants is known, for example, in the case of transdermal administration of botulinum toxin. Botulinum toxin is well known to be used to: weaken the chewing or masseter muscles in the mouth, promote healing of self-inflicted wounds and resulting ulcers, heal benign cystic lesions or tumors; treat anal fissures; and treat certain types of atopic dermatitis. Furthermore, botulinum toxin may have an effect in reducing inflammatory pain induced in a rat formalin model. Additionally, botulinum toxin nerve blocks have been reported to lead to a reduction in epidermal thickness. Finally, botulinum toxin is known to be applied to the feet to treat excessive sweating, toe cramps, idiopathic toe gait, and foot dystonia.

[0029] Tetanus toxin, and its derivatives (i.e., those with non-natural targeting components), fragments, hybrids, and chimeras can also have therapeutic uses. Tetanus toxin shares many similarities with botulinum toxin. Therefore, both tetanus toxin and botulinum toxin are polypeptides made from closely related Clostridium tetani and Clostridium botulinum. Furthermore, both tetanus toxin and botulinum toxin are double-chain proteins composed of a light chain (molecular weight: approximately 50 kDa) covalently linked to a heavy chain (molecular weight: approximately 100 kDa) via a single disulfide bond. Thus, the molecular weight of tetanus toxin and each of the seven non-complex botulinum toxins is approximately 150 kDa. Additionally, for both tetanus toxin and botulinum toxin, the light chain contains domains exhibiting intracellular biological (protease) activity, while the heavy chain contains receptor-binding (immunogenicity) and cell membrane potential domains. Furthermore, both tetanus toxin and botulinum toxin exhibit high specific affinity for ganglioside receptors on the surface of presynaptic cholinergic neurons. Receptor-mediated endocytosis of tetanus toxin in peripheral cholinergic neurons leads to retrograde axonal transport, blocks the release of inhibitory neurotransmitters at the central synapse, and causes spastic paralysis. Conversely, receptor-mediated endocytosis of botulinum toxin in peripheral cholinergic neurons is believed to cause almost no retrograde transport, inhibition of acetylcholine exocytosis from poisoned peripheral motor neurons, or flaccid paralysis. Finally, tetanus toxin and botulinum toxin are similar to each other in biosynthesis and molecular structure. Therefore, the overall sequence identity between tetanus toxin and botulinum toxin type A is 34%, with sequence identity of up to 62% in some functional domains.

[0030] In one embodiment of the invention, "acetylcholine" is an ester of choline and acetic acid, and it is the first known neurotransmitter. It is distributed throughout neurons and has the chemical formula C7H. 16 NO2 has a molecular weight of 146.21 kDa.

[0031] Typically, each type of neuron in the mammalian nervous system releases only one type of small-molecule neurotransmitter, although there is evidence that the same neuron can release multiple neuromodulators. The neurotransmitter acetylcholine is secreted by neurons in many regions of the brain, particularly by the large pyramidal cells of the motor cortex, several different neurons in the basal ganglia, motor neurons innervating skeletal muscles, preganglionic neurons of the autonomic nervous system (sympathetic and parasympathetic), the first sac-like fibers of muscle spindle fibers, postganglionic neurons of the parasympathetic nervous system, and some postganglionic neurons of the sympathetic nervous system. Essentially, only the postganglionic sympathetic fibers leading to sweat glands, arrector pili muscles, and a few blood vessels are cholinergic, as most postganglionic neurons of the sympathetic nervous system secrete the neurotransmitter norepinephrine. In most cases, acetylcholine has an excitatory effect. However, acetylcholine is known to have an inhibitory effect on some peripheral parasympathetic nerve endings (e.g., the vagus nerve inhibits heart rate). Efferent signals from the autonomic nervous system are transmitted to the body via either the sympathetic or parasympathetic nervous system. Preganglionic neurons of the sympathetic nervous system extend from the cell bodies of preganglionic sympathetic neurons located in the middle and lateral horns of the spinal cord. Preganglionic sympathetic fibers extending from the cell bodies synapse with postganglionic neurons located in the paravertebral sympathetic ganglia or prevertebral ganglia. Since both sympathetic and parasympathetic preganglionic neurons are cholinergic, applying acetylcholine to the ganglia will simultaneously stimulate both sympathetic and parasympathetic postganglionic neurons. Acetylcholine activates two types of receptors, muscarinic and nicotinic receptors. Muscarinic receptors are present in all effector cells stimulated by postganglionic neurons of the parasympathetic nervous system, as well as in effector cells stimulated by postganglionic cholinergic neurons of the sympathetic nervous system. Nicotinic receptors are present in the adrenal medulla and in autonomic ganglia, specifically on the cell surface of postganglionic neurons at the synapses between preganglionic and postganglionic neurons of the sympathetic and parasympathetic nervous systems. Nicotinic receptors are also found in many nonautonomic nerve endings, such as in the skeletal muscle fiber membrane at the neuromuscular junction. When small, transparent intracellular vesicles fuse with the presynaptic neuron cell membrane, acetylcholine is released from the cholinergic neuron. Various non-neuronal secretory cells, such as adrenal medulla (and the PC12 cell line) and pancreatic islet cells, release catecholamines and parathyroid hormone, respectively, from large, densely packed nuclear vesicles. The PC12 cell line, a clone of rat pheochromocytoma cells, is widely used as a tissue culture model for studying sympathetic adrenal gland development. When denervated cells are permeabilized (e.g., via electroporation) or directly injected with the toxin, botulinum toxin inhibits the release of both types of compounds from both cell types in vitro. Botulinum toxin is also known to prevent the release of the neurotransmitter glutamate from cortical synaptic cell cultures. Neuromuscular junctions are formed in skeletal muscle through the proximity of axons to muscle cells.Signals transmitted through the nervous system generate action potentials in terminal axons, activating ion channels and causing the release of the neurotransmitter acetylcholine from synaptic vesicles within neurons, such as at the motor endplates of the neuromuscular junction. Acetylcholine crosses the extracellular space and binds to acetylcholine receptor proteins on the surface of the muscle endplate. Once sufficient binding occurs, the muscle cell's action potential triggers specific changes in membrane ion channels, leading to muscle cell contraction. Acetylcholine is then released from the muscle cell and metabolized by cholinesterases in the extracellular space. The metabolites are recycled back to the terminal axons for further processing into acetylcholine.

[0032] In one embodiment of the invention, "toxin activity" refers to the intrinsic potency of botulinum toxin, specifically the 50% mortality rate in mice weighing 18-20g when measured by mouse LD50 bioassay (mLD50) (a standard assay). Botulinum toxin, particularly type A botulinum toxin, is the most lethal natural biological agent known to humans, with a lethality 1.8 billion times that of diphtheria toxin, 600 million times that of sodium cyanide, 30 million times that of cobra venom, and 12 million times that of cholera toxin. Therefore, a difference of approximately 20% in botulinum toxin potency will result in a significant difference in efficacy, for example, 360 million times that of diphtheria toxin or 2.4 million times that of cholera toxin.

[0033] Botulinum toxin preparations for medical or cosmetic purposes are typically marketed as lyophilized or liquid formulations. The problem is that, because botulinum toxin itself is a protein, its activity becomes highly unstable due to temperature, pH, light, physical shock, or gases (air, nitrogen, oxygen, etc.). When the potency of botulinum toxin decreases as described above, it becomes almost impossible to achieve its intended effect. Therefore, it is essential to accurately predict the potency of botulinum toxin during the preparation or application process.

[0034] In one embodiment of the present invention, "antibody" is a term known in the art and refers to a specific protein molecule targeting an antigenic site. For the purposes of this invention, an antibody refers to an antibody that specifically binds to the SNAP25 protein. This antibody can be produced according to conventional methods. The antibodies of the present invention comprise partial peptides that can be produced from proteins, and the partial peptides of the present invention contain at least 7 amino acids, preferably at least 9 amino acids, more preferably at least 12 amino acids. The form of the antibodies of the present invention is not particularly limited, and the antibodies of the present invention include polyclonal antibodies, monoclonal antibodies, or portions thereof with antigen-binding ability, and the antibodies of the present invention include all immunoglobulin antibodies. In addition, the antibodies of the present invention also include special antibodies such as humanized antibodies. The antibodies of the present invention include not only complete antibodies having light and heavy chains, but also functional fragments of antibody molecules. The expression "functional fragment of antibody molecule" means a fragment with at least antigen-binding ability, and examples of functional fragments include Fab, F(ab'), F(ab')2, Fv, etc.

[0035] In one embodiment of the invention, the term "kit" refers to a set of compositions and accessories required for a specific purpose. For the purposes of this invention, the kit comprises components similar to SNAP25. FL Or SNAP25 197 The activity of botulinum toxin is measured using a specifically binding antibody, a composition containing the antibody, or a cell culture dish coated with the antibody.

[0036] In one embodiment of the present invention, a cell line is provided, the clone of which is selected from the parental neuronal cell line Neu ro-2a.

[0037] The inventors selected Neuro-2a cells, which are sensitive to botulinum toxin, from 13 different neuronal cell lines. Their sensitivity is similar to that of the SiMa cell line. Finally, through a clonal selection process, N2-42F (accession number: KCTC 13712BP) was selected from Neuro-2a cells. All of them showed high sensitivity to botulinum toxin, thus completing the present invention.

[0038] The cell lines in this invention can be used to determine the activity of botulinum toxin or to detect botulinum toxin.

[0039] Even with continued passage, the cell lines of this invention retain their sensitivity to botulinum toxin, making them ideal for use in cell-based assay platforms.

[0040] The cell line used to determine the activity of botulinum toxin according to the present invention refers to a homogeneous single cell isolated from a parental neuronal cell line corresponding to a population comprising various cell types, and refers to cells with common genetic characteristics, such as high or low gene expression levels of specific genes.

[0041] Cell lines used to determine botulinum toxin activity can be isolated from parental neuronal cell lines through methods such as clonal selection, or generated by regulating gene expression levels. Regulation of gene expression levels can be achieved using conventional gene expression regulation methods, such as transformation and promoter manipulation.

[0042] The parental neuronal cell line of this invention can include any immortalized cell line derived from nerves, preferably Neuro-2a cells, more preferably Neuro-2a cells (accession number: KCTC AC28106), but is not limited thereto. Neuro-2a cells are mouse neuronal cells, generally used for determining LD50. The doubling time of conventional SiMa cells used to determine botulinum toxin activity is 34-100 hours, while the doubling time of Neuro-2a cells is only 24 hours, indicating that Neuro-2a cells are not only very suitable for the cellular assay of botulinum toxin activity, but also very suitable for the detection of botulinum toxin. Furthermore, when observed under a microscope, Neuro-2a cells represent a population containing various cell types, thus making it very suitable for selecting individual cells that are more sensitive to botulinum toxin.

[0043] The cell line of the present invention is cloned and selected from the parental neuronal cell line Neuro-2a, and may be N2-42F (accession number: K CTC 13712BP), but is not limited thereto.

[0044] The cell line of this invention for determining botulinum toxin activity can be used to detect botulinum toxin or determine its activity. This cell line is sensitive to botulinum toxin, therefore it can detect the presence of botulinum toxin in target samples and measure the toxicity of botulinum toxin based on its concentration.

[0045] The botulinum toxin of this invention is a neurotoxic protein produced by Clostridium botulinum, which can be classified into seven serotypes: A, B, C (C1, C2), D, E, F, and G. Botulinum toxin affects different neurosecretory proteins according to serotype and cleaves these proteins at different sites. Specifically, botulinum toxin serotypes A and E can cleave SNAP25 (synaptosome neurotransmitter 25), while botulinum toxin serotypes B, D, F, and G can cleave VAMP (vesicle-associated membrane protein), and botulinum toxin serotype C1 can cleave synaptic fusion proteins and SNAP25, thereby inducing neurotoxicity. Preferably, the botulinum toxin can be botulinum toxin serotype A or botulinum toxin serotype B, more preferably botulinum toxin serotype A, but is not limited thereto.

[0046] Since the botulinum toxin serotypes A and B of the present invention are purified and widely used in the treatment of dystonia, cosmetic applications, etc., when the cell lines used in the present invention to determine the activity of botulinum toxin are used to measure the efficacy of botulinum toxin, the advantage is that the concentration at which side effects may occur can be determined, thereby solving the problems that may arise when botulinum toxin is used in the above-mentioned applications.

[0047] In another embodiment of the invention, a cell-based method for determining botulinum toxin activity is provided.

[0048] The method of the present invention includes the following steps: culturing a cell line according to the present invention; treating the cultured cell line with botulinum toxin; and measuring the sensitivity of the botulinum toxin-treated cell line to botulinum toxin.

[0049] The cell-based method for determining botulinum toxin activity of the present invention can be achieved by treating a cell line used to determine botulinum toxin activity with botulinum toxin and measuring the sensitivity of the cell line to botulinum toxin. Therefore, to avoid excessive complexity based on the repeated description in the specification, details regarding the cell line used to determine botulinum toxin activity, botulinum toxin, neurosecretory proteins cleaved by botulinum toxin, parental neuronal cell lines, etc., have been omitted.

[0050] In the step of culturing cell lines according to the present invention, the cell lines can be cultured in culture plates coated with poly-D-lysine. Compared with plates commonly used for culturing cell lines or plates coated with gelatin or collagen, when using plates coated with poly-D-lysine, the cell lines according to the present invention can be evenly distributed, firmly attached, and maintain a healthy cell state.

[0051] In this invention, the step of measuring the sensitivity of cell lines to botulinum toxin may include measuring the cleavage of endogenous neurosecretory proteins induced by botulinum toxin. Specifically, in the case of botulinum toxin serotypes A and E, SNAP25 cleavage may be measured; in the case of botulinum toxin serotypes B, D, F, and G, VAMP cleavage may be measured; and for botulinum toxin serotype C1, synaptic fusion protein and / or SNAP25 cleavage may be measured.

[0052] The determination of cleavage in this invention can be achieved by using methods such as detecting proteins that have specificity for cleavage peptides of endogenous neurosecretory proteins.

[0053] The antibodies of the present invention refer to protein molecules that can recognize all or cleaved peptides of neurosecretory proteins as antigens and can specifically bind to neurosecretory proteins, including polyclonal antibodies, monoclonal antibodies and recombinant antibodies.

[0054] The method for detecting the protein of the present invention can be any conventional method for detecting proteins, including but not limited to Western blotting, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), radioimmunodiffusion, Ouchterlony double diffusion immunoassay, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, fluorescence activated cell sorting instrument (FACS), protein microarray, etc.

[0055] In another embodiment of the invention, a cell-based method for detecting botulinum toxin is provided.

[0056] The method of the present invention includes the following steps: culturing a cell line according to the present invention; treating the cultured cell line with a sample of interest; and measuring the sensitivity of the sample-treated cell line to botulinum toxin.

[0057] The cell-based method for detecting botulinum toxin according to the present invention can be implemented by treating cell lines with a sample of interest instead of botulinum toxin, in order to determine the activity of botulinum toxin, and by measuring the sensitivity of the cell line to botulinum toxin. Therefore, to avoid excessive complexity based on a repetitive description, details regarding cell lines for determining botulinum toxin activity, botulinum toxin, neurosecretory proteins cleaved by botulinum toxin, parental neuronal cell lines, coated plates, protein detection methods, antibodies, etc., are omitted.

[0058] The sample used for the purposes of this invention is a sample intended to contain botulinum toxin, examples of which may include biological samples, including cell culture supernatant, blood, saliva, sputum, cerebrospinal fluid, secretions, lymph, dialysis fluid, body fluids, urine, etc., as well as chemical samples containing compounds.

[0059] In yet another embodiment of the invention, an antibody is provided that specifically binds to SNAP25, wherein SNAP25 is SNAP25. Fl Or SNAP25 197The antibody has the heavy chain CDR1 region shown in SEQ ID NO: 11 to 13, 28 to 33, 55 to 56; the heavy chain CDR2 region shown in any of the following groups: SEQ ID NO: 14 to 16, 34 to 39, 57 to 58; the heavy chain CDR3 region shown in any of the following groups: SEQ ID NO: 17 to 19, 40 to 46 and 59 to 60; the light chain CDR1 region shown in any of the following groups: SEQ ID NO: 20 to 22, 47 to 49 and 61 to 62; the light chain CDR2 region shown in any of the following groups: SEQ ID NO: 23 to 24, 50 to 51 and 63 to 64; and the light chain CDR3 region shown in any of the following groups: SEQ ID NO: 25 to 27, 52 to 54 and 65 to 66.

[0060] More specifically, the antibody preferably specifically binds to SNAP25. FL The antibody comprises, but is not limited to, the heavy chain CDR1 region shown in SEQ ID NO: 11; the heavy chain CDR2 region shown in SEQ ID NO: 14; the heavy chain CDR3 region shown in SEQ ID NO: 17; the light chain CDR1 region shown in SEQ ID NO: 20; the light chain CDR2 region shown in SEQ ID NO: 23; and the light chain CDR3 region shown in SEQ ID NO: 25. More specifically, the antibody may be the antibody shown in SEQ ID NO: 83 and 84, but is not limited thereto.

[0061] Furthermore, the preferred antibody is one that specifically binds to SNAP25. FL The antibody comprises: the heavy chain CDR1 region shown in SEQ ID NO: 12; the heavy chain CDR2 region shown in SEQ ID NO: 15; the heavy chain CDR3 region shown in SEQ ID NO: 18; the light chain CDR1 region shown in SEQ ID NO: 21; the light chain CDR2 region shown in SEQ ID NO: 24; and the light chain CDR3 region shown in SEQ ID NO: 26. More specifically, the antibody may be, but is not limited to, the antibodies shown in SEQ ID NO: 87 and 88.

[0062] Additionally, the antibody preferably specifically binds to SNAP25. FLThe antibody comprises: the heavy chain CDR1 region shown in SEQ ID NO: 13; the heavy chain CDR2 region shown in SEQ ID NO: 16; the heavy chain CDR3 region shown in SEQ ID NO: 19; the light chain CDR1 region shown in SEQ ID NO: 22; the light chain CDR2 region shown in SEQ ID NO: 24; and the light chain CDR3 region shown in SEQ ID NO: 27. More specifically, the antibody may be, but is not limited to, the antibodies shown in SEQ ID NO: 89 and 90.

[0063] Furthermore, the preferred antibody is one that specifically binds to SNAP25. 197 The antibody, wherein: the heavy chain CDR1 region shown in SEQ ID NO: 28; the heavy chain CDR2 region shown in SEQ ID NO: 34; the heavy chain CDR3 region shown in SEQ ID NO: 40; the light chain CDR1 region shown in SEQ ID NO: 47; the light chain CDR2 region shown in SEQ ID NO: 50; and the light chain CDR3 region shown in SEQ ID NO: 52. More specifically, the antibody may be, but is not limited to, the antibodies shown in SEQ ID NO: 71 and 72.

[0064] Furthermore, the preferred antibody is one that specifically binds to SNAP25. 197 The antibody comprises: the heavy chain CDR1 region shown in SEQ ID NO: 29; the heavy chain CDR2 region shown in SEQ ID NO: 35; the heavy chain CDR3 region shown in SEQ ID NO: 41; the light chain CDR1 region shown in SEQ ID NO: 48; the light chain CDR2 region shown in SEQ ID NO: 50; and the light chain CDR3 region shown in SEQ ID NO: 52. More specifically, the antibody may be, but is not limited to, the antibodies shown in SEQ ID NO: 73 and 74.

[0065] Furthermore, the preferred antibody is one that specifically binds to SNAP25. 197 The antibody comprises: the heavy chain CDR1 region shown in SEQ ID NO: 29; the heavy chain CDR2 region shown in SEQ ID NO: 36; the heavy chain CDR3 region shown in SEQ ID NO: 42; the light chain CDR1 region shown in SEQ ID NO: 47; the light chain CDR2 region shown in SEQ ID NO: 50; and the light chain CDR3 region shown in SEQ ID NO: 52. More specifically, the antibody may be, but is not limited to, the antibodies shown in SEQ ID NO: 75 and 76.

[0066] Furthermore, the preferred antibody is one that specifically binds to SNAP25. 197 The antibody, wherein: the heavy chain CDR1 region shown in SEQ ID NO: 33; the heavy chain CDR2 region shown in SEQ ID NO: 35; the heavy chain CDR3 region shown in SEQ ID NO: 43; the light chain CDR1 region shown in SEQ ID NO: 48; the light chain CDR2 region shown in SEQ ID NO: 50; and the light chain CDR3 region shown in SEQ ID NO: 52. More specifically, the antibody may be the antibody shown in SEQ ID NO: 77 and 78, but is not limited thereto.

[0067] Furthermore, the preferred antibody is one that specifically binds to SNAP25. 197 The antibody, wherein: the heavy chain CDR1 region shown in SEQ ID NO: 30; the heavy chain CDR2 region shown in SEQ ID NO: 37; the heavy chain CDR3 region shown in SEQ ID NO: 44; the light chain CDR1 region shown in SEQ ID NO: 48; the light chain CDR2 region shown in SEQ ID NO: 50; and the light chain CDR3 region shown in SEQ ID NO: 52. More specifically, the antibody may be the antibody shown in SEQ ID NO: 79 and 80, but is not limited thereto.

[0068] Furthermore, the antibody is preferably one that specifically binds to SNAP25. 197 The antibody comprises: the heavy chain CDR1 region shown in SEQ ID NO: 31; the heavy chain CDR2 region shown in SEQ ID NO: 38; the heavy chain CDR3 region shown in SEQ ID NO: 45; the light chain CDR1 region shown in SEQ ID NO: 47; the light chain CDR2 region shown in SEQ ID NO: 50; and the light chain CDR3 region shown in SEQ ID NO: 53. More specifically, the antibody may be, but is not limited to, the antibodies shown in SEQ ID NO: 81 and 82.

[0069] Furthermore, the preferred antibody is one that specifically binds to SNAP25. 197 The antibody, wherein: the heavy chain CDR1 region shown in SEQ ID NO: 32; the heavy chain CDR2 region shown in SEQ ID NO: 39; the heavy chain CDR3 region shown in SEQ ID NO: 46; the light chain CDR1 region shown in SEQ ID NO: 49; the light chain CDR2 region shown in SEQ ID NO: 51; and the light chain CDR3 region shown in SEQ ID NO: 54. More specifically, the antibody may be, but is not limited to, the antibodies shown in SEQ ID NO: 85 and 86.

[0070] In addition, the preferred antibody is one that is compatible with SNAP25. FL and SNAP25 197 The antibody specifically binds to the following regions: the heavy chain CDR1 region shown in SEQ ID NO: 55; the heavy chain CDR2 region shown in SEQ ID NO: 57; the heavy chain CDR3 region shown in SEQ ID NO: 59; the light chain CDR1 region shown in SEQ ID NO: 61; the light chain CDR2 region shown in SEQ ID NO: 63; and the light chain CDR3 region shown in SEQ ID NO: 65. More specifically, the antibody may be, but is not limited to, the antibodies shown in SEQ ID NO: 67 and 68.

[0071] In addition, the preferred antibody is one that is compatible with SNAP25. FL and SNAP25 197 The antibody specifically binds to the following regions: the heavy chain CDR1 region shown in SEQ ID NO: 56; the heavy chain CDR2 region shown in SEQ ID NO: 58; the heavy chain CDR3 region shown in SEQ ID NO: 60; the light chain CDR1 region shown in SEQ ID NO: 62; the light chain CDR2 region shown in SEQ ID NO: 64; and the light chain CDR3 region shown in SEQ ID NO: 66. More specifically, the antibody may be, but is not limited to, the antibodies shown in SEQ ID NO: 69 and 70.

[0072] In yet another embodiment of the invention, an antibody composition comprising an antibody, or a culture dish coated with an antibody, or a kit comprising an antibody composition or a culture dish is provided.

[0073] In another embodiment of the invention, hybridoma cells capable of producing antibodies are provided, wherein the hybridoma cells are a fusion of mouse spleen cells and myeloma cells injected with any one or more peptides selected from the group consisting of SEQ ID NO: 1 to 10.

[0074] In another embodiment of the invention, a method for determining the activity of botulinum toxin is provided, comprising the steps of: (a) treating neuronal cells with botulinum toxin; and (b) measuring SNAP25 in the neuronal cells using one or more antibodies selected from those shown in SEQ ID NO: 67 to 90. FL Or SNAP25 197 Among them, botulinum toxin is type A botulinum toxin.

[0075] In another embodiment of the present invention, a method for detecting botulinum toxin is provided, comprising the steps of: (a) processing a target sample into nerve cells; and (b) measuring SNAP25 in the neuronal cells using one or more antibodies selected from those shown in SEQ ID NO:67 to 8, SEQ ID NO:85, or SEQ ID NO:86. 197 ; and (c) determine when SNAP25 is measured 197 At that time, botulinum toxin was present in the sample, and the botulinum toxin was type A botulinum toxin.

[0076] In another embodiment of the invention, a cell-based method is provided for analyzing neurotoxin titers, comprising the steps of: (a) culturing a Neuro-2a-derived neuronal cell line; (b) treating the neuronal cell line with a neurotoxin; and (c) using a neurotoxin that specifically binds to SNAP25. FL and SNAP25 197 (d) antibody treatment of neuronal cell lines or samples obtained from neuronal cell lines; and (d) using antibodies that specifically bind to SNAP25. 197 But without combining SNAP25 FL The neuronal cell line (c) of the antibody treatment step (c), wherein the Neuro-2a-derived cell line is the N2-42F cell line (accession number: KCTC 137 12BP), the neurotoxin is botulinum toxin, and the neurotoxin in step (b) is diluted with a medium containing GT1b (ganglioside GT1b trisodium salt) and used to treat the cell line.

[0077] The antibody used in step (c) of the cell-based method for analyzing neurotoxin potency includes: the heavy chain CDR1 region composed of SEQ ID NO: 55 or 56; the heavy chain CDR2 region composed of SEQ ID NO: 57 or 58; the heavy chain CDR3 region composed of SEQ ID NO: 59 or 60; the light chain CDR1 region composed of SEQ ID NO: 61 or 62; the light chain CDR2 region composed of SEQ ID NO: 63 or 64; and the light chain CDR3 region composed of SEQ ID NO: 65 or 66. More specifically, the antibody used in step (c) of the cell-based method for analyzing neurotoxin potency includes: the heavy chain CDR1 region composed of SEQ ID NO: 55; the heavy chain CDR2 region composed of SEQ ID NO: 57; the heavy chain CDR3 region composed of SEQ ID NO: 59; the light chain CDR1 region composed of SEQ ID NO: 61; the light chain CDR2 region composed of SEQ ID NO: 63; and the light chain CDR3 region composed of SEQ ID NO: 65. Additionally, the antibody used in step (c) of the cell-based method for analyzing neurotoxin potency includes: the heavy chain CDR1 region composed of SEQ ID NO: 56; the heavy chain CDR2 region composed of SEQ ID NO: 58; the heavy chain CDR3 region composed of SEQ ID NO: 60; the light chain CDR1 region composed of SEQ ID NO: 62; the light chain CDR2 region composed of SEQ ID NO: 64; and the light chain CDR3 region composed of SEQ ID NO: 66.

[0078] In addition, the antibodies used in step (d) of the cell-based method for analyzing neurotoxin potency include: heavy chain CDR1 regions selected from any of the following groups: SEQ ID NO: 28 to 33; heavy chain CDR2 regions selected from any of the following groups: SEQ ID NO: 34 to 39; heavy chain CDR3 regions selected from any of the following groups: SEQ ID NO: 40 to 46; light chain CDR1 regions selected from any of the following groups: SEQ ID NO: 47 to 49; light chain CDR2 regions selected from any of the following groups: SEQ ID NO: 50 to 51; and light chain CDR3 regions selected from any of the following groups: SEQ ID NO: 52 to 54. More specifically, the antibody used in step (d) of the cell-based method for analyzing neurotoxin potency includes: the heavy chain CDR1 region composed of SEQ ID NO: 28; the heavy chain CDR2 region composed of SEQ ID NO: 34; the heavy chain CDR3 region composed of SEQ ID NO: 40; the light chain CDR1 region composed of SEQ ID NO: 47; the light chain CDR2 region composed of SEQ ID NO: 50; and the light chain CDR3 region composed of SEQ ID NO: 52. Additionally, the antibody used in step (d) of the cell-based method for analyzing neurotoxin potency includes: the heavy chain CDR1 region composed of SEQ ID NO: 29; the heavy chain CDR2 region composed of SEQ ID NO: 35; the heavy chain CDR3 region composed of SEQ ID NO: 41; the light chain CDR1 region composed of SEQ ID NO: 48; the light chain CDR2 region composed of SEQ ID NO: 50; and the light chain CDR3 region composed of SEQ ID NO: 52. Additionally, the antibody used in step (d) of the cell-based method for analyzing neurotoxin potency includes: the heavy chain CDR1 region composed of SEQ ID NO: 29; the heavy chain CDR2 region composed of SEQ ID NO: 36; the heavy chain CDR3 region composed of SEQ ID NO: 42; the light chain CDR1 region composed of SEQ ID NO: 47; the light chain CDR2 region composed of SEQ ID NO: 50; and the light chain CDR3 region composed of SEQ ID NO: 52.Furthermore, the antibody used in step (d) of the cell-based method for analyzing neurotoxin potency includes: the heavy chain CDR1 region composed of SEQ ID NO: 33; the heavy chain CDR2 region composed of SEQ ID NO: 35; the heavy chain CDR3 region composed of SEQ ID NO: 43; the light chain CDR1 region composed of SEQ ID NO: 48; the light chain CDR2 region composed of SEQ ID NO: 50; and the light chain CDR3 region composed of SEQ ID NO: 52. Additionally, the antibody used in step (d) of the cell-based method for analyzing neurotoxin potency includes: the heavy chain CDR1 region composed of SEQ ID NO: 30; the heavy chain CDR2 region composed of SEQ ID NO: 37; the heavy chain CDR3 region composed of SEQ ID NO: 44; the light chain CDR1 region composed of SEQ ID NO: 48; the light chain CDR2 region composed of SEQ ID NO: 50; and the light chain CDR3 region composed of SEQ ID NO: 52. Additionally, the antibodies used in step (d) of the cell-based method for analyzing neurotoxin potency include: the heavy chain CDR1 region composed of SEQ ID NO: 31; the heavy chain CDR2 region composed of SEQ ID NO: 38; the heavy chain CDR3 region composed of SEQ ID NO: 45; the light chain CDR1 region composed of SEQ ID NO: 47; the light chain CDR2 region composed of SEQ ID NO: 50; and the light chain CDR3 region composed of SEQ ID NO: 53. Furthermore, the antibodies used in step (d) of the cell-based method for analyzing neurotoxin potency include: the heavy chain CDR1 region composed of SEQ ID NO: 32; the heavy chain CDR2 region composed of SEQ ID NO: 39; the heavy chain CDR3 region composed of SEQ ID NO: 46; the light chain CDR1 region composed of SEQ ID NO: 49; the light chain CDR2 region composed of SEQ ID NO: 51; and the light chain CDR3 region composed of SEQ ID NO: 54.

[0079] In another embodiment of the invention, a cell culture medium for treating neuronal cells with neurotoxins is provided, the cell culture medium comprising GT1b (ganglioside GT1b trisodium salt). The concentration of GT1b in the cell culture medium can be from 25 to 75 μg / ml, and the cell culture medium further contains creatine and arginine. Furthermore, the concentration of creatine in the cell culture medium can be from 0.1 to 10 mM, and the concentration of arginine in the cell culture medium can be from 0.5 to 50 mM. Additionally, the cell culture medium can be RPMI 1640 (Roswell Park Memorial Institute 1640) medium.

[0080] Each step of the invention will be described in detail below.

[0081] [Beneficial Effects]

[0082] In recent years, the demand for botulinum toxin for medical and cosmetic purposes has increased rapidly; however, there is still no stable and reproducible cell-based titer assay for measuring botulinum toxin titers. Because botulinum toxin is a highly potent neurotoxin protein, there is a particular need to develop highly specific and sensitive cells and antibodies for accurate cell-based measurements of botulinum toxin potency.

[0083] This invention relates to antibodies for determining botulinum toxin activity and antibody compositions comprising such antibodies. Compared to conventional SiMa cells used to determine or detect botulinum toxin activity, the novel cell lines of this invention exhibit significantly shorter doubling times and significantly higher sensitivity to botulinum toxin compared to parental cell lines, indicating that they are well-suited for cell-based determination or detection of botulinum toxin activity. Furthermore, the cell lines according to the invention can be stably cultured in culture dishes coated with poly-d-lysine (PDL), thus making them highly effective for cell-based determination or detection of botulinum toxin activity.

[0084] The anti-botulinum toxin antibody according to the present invention is effective against ①SNAP25 FL ,②SNAP25 197 , or ③SNAP25 FL and SNAP25 197 These are monoclonal antibodies with binding specificity, exhibiting remarkably superior specificity and sensitivity. Therefore, they are expected to be widely used in the pharmaceutical and cosmetic fields.

[0085] Furthermore, this invention relates to an optimal CBPA assay using N2-42F cells and a monoclonal antibody exhibiting significantly high binding affinity and specificity to SNAP25, and this CBPA assay can measure botulinum toxin titers of 0.5 U or less. The CBPA assay using the cells and antibodies of this invention promises to become a cell-based titer assay for botulinum toxin with high reliability and reproducibility. [Brief Description of the Attached Image]

[0086] Figure 1 This image shows the results of a Western blot analysis measuring sensitivity to botulinum toxin A (BoNT / A) in neuronal cells according to one embodiment of the present invention. Lane M represents a protein size marker; lane 1 represents the expression level of SNAP25 protein in total cell lysates without BoNT / A toxin; and lane 2 represents the expression level of SNAP25 protein in total cell lysates with BoNT / A toxin. Furthermore, N2a represents Neuro-2a cells, and K-BM1 represents KP-N-RT-BM-1 cells.

[0087] Figure 2 The results of Western blot analysis examining the extent of SNAP25 cleavage during a three-step clone selection process, according to one embodiment of the invention, are shown. In the second clone selection process (second clone selection), clone 42, which showed significant SNAP25 cleavage caused by BoNT / A, was selected from only six clones, including clone 42. In the third clone selection process (third clone selection), clone 24 (42F) consistently showed SNAP25 cleavage caused by BoNT / A in multiple identical experiments.

[0088] Figure 3 This is a graph showing the results of measuring the division time (doubling time) between N2-42F cells and their parent Neruo-2a cells according to one embodiment of the present invention.

[0089] Figure 4 The images show 20x images of Neruo-2a (parental cell line), SiMa cells, and clone N2-42F imaged using a Leica DM18, with cell morphology confirmed when 60% confluence was reached, according to one embodiment of the invention.

[0090] Figure 5 Images of N2-42F cells cultured in plates coated with type IV collagen, gelatin, and poly-D-lysine, according to one embodiment of the invention, are shown.

[0091] Figures 6a and 6b show the results of Western blot analysis according to an embodiment of the present invention, used to detect the degree of SNAP25 lysis when SiMa cells and N2-42F were treated with different concentrations of BoNT / A, and the degree of SANP25 or Vamp2 lysis when N2-42F and Neuro-2a were treated with different types of botulinum toxin.

[0092] Figure 7A and 7B The following is a Western blot analysis result showing the stability of N2-42F strains obtained through the clonal selection process of the present invention, according to an embodiment of the present invention.

[0093] Figure 8 This is a schematic diagram showing the location of the SNAP25 antigenic peptide used to produce monoclonal or polyclonal antibodies using synthetic peptides according to one embodiment of the present invention.

[0094] Figure 9 This is a schematic diagram showing a method for forming hybridoma cells for producing monoclonal antibodies, and a method for screening clones according to an embodiment of the present invention.

[0095] Figures 10A to 10C The results of initial screening of hybridoma cells using ELISA to generate monoclonal antibodies according to one embodiment of the present invention are shown.

[0096] Figures 11A to 11C The results of re-screening cells for single-cell clone generation from initial hybridoma cell screening results are shown according to one embodiment of the invention.

[0097] Figures 12A to 12C The results of a second reselection for generating single-cell clones are shown according to one embodiment of the invention.

[0098] Figures 13A to 13C The results of a third reselection for generating single-cell clones are shown according to one embodiment of the invention.

[0099] Figure 14A and 14B A pattern of IgG for generating polyclonal antibodies isolated from rabbit serum protein is shown according to one embodiment of the present invention.

[0100] Figure 15A and 15B The results of kinetic analysis of the monoclonal antibody prepared according to one embodiment of the present invention are shown.

[0101] Figures 16a and 16b show the results of kinetic analysis of a monoclonal antibody produced in this invention and bound to / dissociated with stepwise diluted recombinant GST-SNAP25, according to one embodiment of the invention.

[0102] Figure 17 The results of Western blot analysis of the antigen-binding specificity of the monoclonal antibody produced according to an embodiment of the present invention are shown.

[0103] Figures 18a and 18b show the results of Western blot analysis performed according to one embodiment of the present invention to confirm the antigen-binding specificity of the monoclonal antibody generated in the present invention and conjugated with HRP.

[0104] Figures 19A to 19C The results of SDS-PAGE electrophoresis of a monoclonal antibody generated and conjugated with biotin according to one embodiment of the present invention are shown.

[0105] Figure 20A and 20B The following is an illustration of the SDS-PAGE electrophoresis results of a polyclonal antibody prepared according to the invention and crosslinked with AP.

[0106] Figures 21A to 21C The results of optimizing the toxin time in a method for determining the activity of botulinum toxin according to one embodiment of the present invention are shown.

[0107] Figure 22 The results of optimizing the toxin culture medium in a method for determining the activity of botulinum toxin according to one embodiment of the present invention are shown.

[0108] Figure 23A and 23B The results of optimizing a sensitizer in a method for determining the activity of botulinum toxin, according to one embodiment of the present invention, are shown.

[0109] Figures 24A to 24C The results of optimizing GT1b in a method for determining the activity of botulinum toxin according to one embodiment of the present invention are shown.

[0110] Figure 25A and 25B The results of optimizing N2 / B27 in a method for determining the activity of botulinum toxin according to one embodiment of the present invention are shown.

[0111] Figure 26A and 26B The invention illustrates the results of optimizing the capture antibody treatment in a method for determining the activity of botulinum toxin, according to one embodiment of the invention.

[0112] Figure 27A and 27B An embodiment of the invention is shown, which optimizes the detection of antibody treatment results in a method for determining the activity of botulinum toxin.

[0113] Figure 28 The results show the optimization of the method for detecting HRP conjugate activity in a method for determining the activity of botulinum toxin according to one embodiment of the present invention.

[0114] Figure 29 This is a schematic diagram illustrating a sandwich ELISA method for determining the activity of botulinum toxin according to an embodiment of the present invention.

[0115] Figures 30A to 30C The results show the accuracy and linearity of a sandwich enzyme-linked immunosorbent assay (ELISA) method for determining the activity of botulinum toxin according to one embodiment of the present invention.

[0116] Figures 31A to 31C The results of measuring the biopotency of botulinum toxin activity by sandwich ELISA according to one embodiment of the present invention are shown.

[0117] [Best Implementation of the Invention]

[0118] This invention aims to overcome the limitations of conventional CPBA and develop a more efficient CBPA. Using the CPBA of this invention, a comparative analysis of 13 different neuronal cell lines was performed, and a novel clone, N2-42F, was developed, optimized among the wide range of clones constituting neurons. This clone has a division time of less than 24 hours compared to SiMa and can adhere to and be stably cultured in poly-d-lysine (PDL)-coated culture dishes. The antibody in this invention is a monoclonal antibody with significantly high binding affinity and specificity for SNAP25. Furthermore, this invention relates to a cell-based optimal CBPA assay using N2-42F cells and a monoclonal antibody with significantly high binding affinity and specificity for SNAP25, which can measure botulinum toxin titers of 0.5 U or less.

[0119]

Embodiments of the Invention

[0120] The present invention will be further described in detail below by way of examples. It will be apparent to those skilled in the art that these examples are merely for illustrating the invention in more detail and are not intended to limit the scope of the invention.

Example

[0121] Preparation Example 1: Preparation of Cell Preparation Materials

[0122] The materials used for cell production in this invention are described below.

[0123] 0.25% trypsin EDTA (Gibco) TM 25200056), GlutaMAX TM (Gibco TM 35050061), MEM (Gibco) TM 11095080), MEM non-essential amino acids (1xNEAA; Gibco) TM 11140050), Sodium pyruvate (Gibco) TM 11360070), TrypLE TM Expression enzyme (1x) (Gibco) TM 12604021), Antibiotic antifungal solution (AA; 100x; Sigma A5955), Boric acid (Sigma B6768), Collagen from human placenta (Sigma C533), Dithiothreitol (DTT; Sigma D0632), DMSO (Sigma D2650), Gelatin solution (Sigma G1393), Poly-D-lysine hydrobromide (Sigma P6407), Polysorbate (Sigma P7949), Sodium tetraborate (Sigma 221732), DPBS (WelgeneLB001-02), Fetal bovine serum (FBS; YI Frontier US-FBS-500), Glycerol (Affymatrix USB 16374), PCR mycoplasma detector (Takara Bio 6601), RIPA buffer (10x; abcam) ab156034), 6-well plate (Falcon 353046), 12-well plate (Corning CLS3513), 24-well plate (Falcon 353047), T75 flask (Falcon BD353136), and TaKaRa EX Taq TM (Takara Bio RR001).

[0124] Preparation Example 2. Botulinum toxin A (botulinum toxin serotype A; BoNT / A) stock solution, diluent and Preparation of BoNT / A toxin culture medium

[0125] Botulinum toxin A ( Botulinum toxin serotype A; BoNT / A, hereinafter referred to as BoNT / A Provided by Hugel (ExbII1501).

[0126] Preparation Example 2-1. Preparation of BoNT / A stock solution and toxin culture medium

[0127] BoNT / A was diluted with a toxin dilution buffer (containing 50 mM sodium phosphate, pH 7.0, 1 mM dithiothreitol (DTT), 0.05% polysorbate, 20% glycerol, and 0.2 mg / ml acetylated BSA) to prepare a stock solution (10 nM). The BoNT / A stock solution was stored aliquots at -80°C until used in the following examples. A stock solution was prepared by resuspending lyophilized BoNT / A (200 U) in 1 ml of toxic medium or physiological saline and allowing the suspension to stand at room temperature for 10 minutes. Stock solution A was prepared by mixing 150 μl of the stock solution with 450 μl of toxic medium at room temperature. Stock solution B was prepared by mixing 20 μl of stock solution A with 180 μl of toxic medium at room temperature. Stock solution C was prepared by mixing 20 μl of stock solution B with 180 μl of toxic medium at room temperature.

[0128] Preparation Example 2-2. Preparation of Standard Reference Samples for Deriving Standard Curves

[0129] Standard stock solution A (50 pM, 113.6 U / ml) was prepared by resuspending lyophilized BoNT / A (100 U) in 880 μl of toxin medium or physiological saline and allowing the suspension to stand at room temperature for 10 minutes. Standard stock solution B (10 pM, 22.7 U / ml) was prepared by mixing 50 μl of standard stock solution A with 200 μl of toxin medium at room temperature. Standard stock solution C (2 pM, 4.54 U / ml) was prepared by mixing 50 μl of standard stock solution B with 200 μl of toxin medium at room temperature.

[0130] Preparation Example 3. Plate coating for neuronal cell culture

[0131] Coat culture plates with gelatin solution (0.1% in 1x PBS), type VI collagen (0.1 mg / ml), or poly-D-lysine (PDL) (50 μg / ml) for three hours or overnight. Rinse the culture plates twice with 15 ml of 1x DPBS and air dry in a tissue culture hood.

[0132] Collagen for plate coating was reconstituted in deionized water to a final concentration of 0.1 mg / ml. Poly-D-lysine was prepared by dissolving 5 mg of the powder in 0.1 M borate buffer (pH 8.5), and was therefore used in the coating.

[0133] Preparation Example 4. Neuron Cells and Culture Medium

[0134] To screen for neurons with high sensitivity to BoNT / A, as shown in Table 1 below, 13 neurons were obtained from 5 organs. These 13 neurons were maintained and multiplied in a culture medium containing the components recommended by the institutions.

[0135] For N2-42F cells cloned from Neuro-2a cells, mycoplasma contamination was monitored every 4 passages, or for SiMa cells every 10 passages, using a PCR Mycoplasma Detection Kit (Takara Bio Inc. 6601). PCR experiments were performed according to the manufacturer's recommended procedure. Briefly, cell culture supernatant was collected and incubated for 3 or 4 days. 3 μl of culture supernatant and 50 μl of a mixture containing 1x PCR buffer, dNTP mixture, MCGp F1 / R2 primers, and TaKaRa EX Taq were used. TM PCR was performed using a mixed solution of (TakaraBio RR001) (50 μl). Then, 10 μl of each PCR product was electrophoresed on a 1% agarose gel and stained with ethidium to confirm whether mycoplasma contamination occurred.

[0136] Table 1

[0137]

[0138]

[0139] Preparation Example 5: Preparation of Antibody Preparation Materials

[0140] The materials used in the preparation of antibodies for this invention are described below.

[0141] 2-Mercaptoethanol (Sigma M3148), 4-iodophenylboronic acid (Sigma 471933), 10x TBS (BIO-RAD 170-6435), 10x Tris / glycine / SDS buffer (Bio-Rad 161-0772), 12% TGX TM(Bio-Rad 456-1046), Acetic acid (Merck 100063), Alkaline phosphatase (Sigma P0114), AMICON Ultra-15, Ultracel 30K (Millipore UFC903024), 100x antibiotic antifungal solution (AA) (Sigma A5955), Bromophenol blue (Sigma B0126), DMEM (Gibco™ 11995065), DMSO (Sigma D2650), DMSO (Sigma 472301), EZ-link NHS-PEG4-Biotin (Thermo Fisher Scientific 21329), Ethylene glycol (Sigma 324558), Fetal bovine serum (FBS; YI Frontier US-FBS-500), Glycerol (Affymetrix USB 16374), Glycine (Bioshop GLN001), Glutaraldehyde solution (Sigma) G7651), horseradish peroxidase (HRP; Sigma P6782), hydrogen peroxide solution (Sigma 216763), luminol (Sigma 123072), magnesium chloride (Sigma M8266), 100x MEM non-essential amino acids (Gibco) TM 11140050), Methanol (Merck 106009), TUBE (Axygen PCR-0208-CP-C), polyvinylidene fluoride (PVDF) membrane (Millipore ISEQ00010), potassium chloride (Sigma P9333), Precision plus protein TM Two-color standard solution (Bio-Rad 1610374), 20% (w / v) SDS solution (Bio-Rad 161-0418), skim milk (BD Difco) TM Sodium acetate (Sigma W302406), sodium bicarbonate (Sigma S6014), sodium borohydride (Sigma 452882), sodium chloride (Merck 106404), sodium periodate (sodium metaperiodate) (Sigma S1878), sodium dihydrogen phosphate (Sigma S5011), disodium hydrogen phosphate (Sigma S7907), sodium stannate trihydrate (Sigma 336262), tetrabutylammonium borohydride (Sigma 230170), T175 (SPL 74175), T75 flask (SPL 70375), Tris (Bioshop TRS001), and zinc chloride (Sigma 229997).

[0142] Preparation Example 6: Setting up the antibody production method

[0143] Preparation Example 6-1. Production of polyclonal and monoclonal antibodies using synthetic peptides

[0144] like Figure 8 As shown, a peptide was synthesized by conjugating keyhole limpet hemocyanin (KHL) at the C- or N-terminus. For this purpose, rabbits were first immunized with the peptide antigen to prepare polyclonal serum. Rabbits were administered the drug regularly for 6 weeks after the initial injection. The reactivity and specificity of the rabbit serum were detected by Western blot analysis and ELISA. The serum was stored at -80°C before use. Next, monoclonal antibodies were prepared by injecting the peptide antigen into four mice. Then, the antibody-producing spleen cells were collected and fused with myeloma cells to form hybridomas, followed by three rounds of single-cell clonal selection. Figure 9 As shown. In short, hybridomas were first screened using peptide antigens via ELISA, and then recombinant SNAP25 protein (GST-SNAP25) was used. FL and GST-SNAP25 197 Total cell lysates, either from neurons or SiMa, were subjected to Western blot analysis, and finally, the total cell lysates were screened by sandwich ELISA. As described below, antibody-producing hybridomas were amplified and stored in the gas phase of liquid nitrogen until they were recovered for antibody production.

[0145] Preparation Example 6-2. Preparation of SNAP25 Affinity Column

[0146] Recombinant SNAP25 197 Protein concentration was concentrated to 10 mg / ml using an AMICO Ultra-15 by repeated centrifugation at 1,000 × g for 10 minutes at 4 °C, while protein concentration was measured using a nanospectrophotometer (Drawell Scientific Instruments Co., Ltd., Shanghai). In the AMICO Ultra-15, aliquots (3 ml) of SNAP25 were... 197 Mix with 12 ml of coupling buffer (0.1 M HEPES, pH 7.5, 0.1 M NaCl) and concentrate by centrifugation at 1,000 x g. Repeat this buffer exchange 6 times, then use SANP25. 197 The concentrate was added to 1 ml of Affi-Gel 15 (Bio-Rad) slurry in deionized water. After incubating for 1 hour at room temperature on a shaker incubator, Affi-Gel 15 was centrifuged at 1,000 × g for 10 minutes at 4 °C. SNAP25 197- The conjugated Affi-Gel 15 (SNAP25-AffiGel) was resuspended in 10 ml of 10 mM ethanolamine hydrochloride and incubated at room temperature for 1 hour at pH 8.0. After washing with 1X PBS, SNAP25-AffiGel was stored in 1×PBS containing 0.2% sodium azide before use.

[0147] Preparation Example 6-3. Purification of Polyclonal Antibodies

[0148] Rabbit serum was diluted 10-fold with 10 mM Tris-HCl, pH 7.5, and centrifuged at 10,000 × g for 10 minutes at 4°C. The serum was then passed through a 0.45 μm microporous filter (Nalgene). TM After filtration, the clear serum dilution was passed through a SNAP25-AffGel three times. The SNAP25-AffGel was then washed with 20 CV of 10 mM Tris-HCl, pH 7.5, and 0.5 M NaCl. Bound proteins were eluted sequentially with 12 CV of 0.1 M sodium acetate, pH 5.5, 0.1 M glycine, pH 4.0, 0.1 M glycine, pH 2.5, and 0.1 M triethylamine, pH 11.5. During elution, protein samples were collected in tubes containing 0.1 mL of 1 M Tris-HCl, pH 8.0. The protein-containing peak fractions were combined and concentrated to 1 mL using an AMICONUltra-15. After dialyzing four times every 90 minutes with 1x PBS and 0.5 L of 10% glycerol, the samples were analyzed by SDS-PAGE and ELISA.

[0149] Preparation Example 6-4. Antibody conjugation with horseradish peroxidase (HRP)

[0150] For HRP conjugation, purified B4 or C16 IgG was concentrated to 10 mg / ml using an AMICON Ultra-15 centrifuge and repeatedly centrifuged at 1,000 x g for approximately 15 minutes at 4°C, with excess conjugation buffer (0.1 M NaHCO3, pH 9.5, 0.9% NaCl) provided at the end of each centrifugation. HRP (5 mg) was dissolved in 1.2 ml of deionized water and mixed with 0.3 ml of 0.1 M sodium periodate in 10 mM sodium phosphate (pH 7.0). After incubation at room temperature for 20 minutes, the HRP solution was dialyzed four times with 1 mM sodium acetate (pH 4.0) at 4°C for 6 hours. The concentrated antibody (5 mg) and activated HRP (5 mg) were mixed together in a microcentrifuge tube and incubated at room temperature for 2 hours in the dark. The conjugation reaction was terminated by adding 0.1 ml of sodium borohydride (4 mg / ml in deionized water). The HRP antibody conjugate was dialyzed using PUR-A-Lyzer MAXI 50000 at 4°C, with three 1×PBS dialysis cycles per hour followed by one 1×PBS / 50% glycerol dialysis cycle. Before use, the HRP antibody conjugate was stored at 4°C or -80°C for long-term preservation.

[0151] Preparation Examples 6-5. Antibody-Conjugated Biotin

[0152] A15 IgG (1 mg / ml) was transferred to a Pur-A-Lyzer Maxi 20000 and dialyzed four times every hour at 4°C with a reaction buffer containing 0.1 M phosphate, pH 7.2, and 0.15 M NaCl. Activated biotin (10 mM in reaction buffer) (EZ-Link NHS-PEG4-Biotin) was mixed with 50 μg aliquots of A15 IgG to final concentrations of 0.1 mM, 0.25 mM, or 0.5 mM. The mixture was adjusted to 100 μl with reaction buffer and incubated at 4°C for 2 hours in the dark. After adding 2 μl of 0.1 M glycine, the reaction mixture was dialyzed three times with PBS and once with 1xPBS / 50% glycerol every hour at 4°C, and stored in microcentrifuge tubes at -20°C. The degree of biotinylation of IgG was assessed using the Pierce Biotin Quantitative Kit (Thermo Fisher Scientific 28005), and the reactivity and specificity of biotinylated IgG were determined by sandwich ELISA.

[0153] Preparation Example 6-6. Crosslinking of AP and Antibody

[0154] By adding glutaraldehyde to 0.25% (20 The mixture contained reaction buffer (0.1 M sodium phosphate, pH 6.8), 50 μg AP (2.2 mg / ml) (Sigma-Aldrich P0114-10KU), and 100 μg purified IgG, such as monoclonal antibody A15, polyclonal antibody rA15 IgG, and polyclonal anti-SNAP25 IgG (Sigma-Aldrich S9684), to initiate alkaline phosphatase (AP) co-occurrence with the antibody. After incubation on ice in the dark for 1 hour, 1 L of 1 M ethanolamine of an aliquot of the sample was added to the reaction solution, and the mixture was incubated at room temperature in the dark for 1 hour. At 4°C, the AP-IgG conjugate was dialyzed three times with 1x PBS and once with storage buffer (25 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 0.1 mM ZnCl2, and 50% glycerol). The antigen-binding specificity and AP activity of the AP-IgG conjugate were analyzed by direct ELISA as described below.

[0155] Preparation Examples 6-7. OCTET RED96 Measurement of K D

[0156] To perform kinetic analysis of the monoclonal antibody, it was used at 30°C according to the manufacturer's recommended procedure. The Red96 instrument was used for biolayer interference (BLI) measurements. In short, recombinant GST-SNAP25 was... FL Or SNAP25 197 Dilute to 125 or 250 nM in 1x kinetic buffer (1x KB) / 1x PBS, then sequentially dilute the analyte (i.e., purified IgG) to 3.9, 7.8, 15.6, 31.25, and 62.5 in 1x kinetic buffer to achieve concentrations of 125 and 250 nM. After equilibration for 1 minute in 1x kinetic buffer, load GST-SANP25 with the anti-GST probe (Forté Bio18-5096). FL Or GST-SNAP25 197 After 30 minutes, immerse it in 1x kinetic buffer for 10 minutes. After 10 minutes each for analyte binding and dissociation, obtain the kinetic profiles and use... Analysis software estimates K D (Equilibrium dissociation constant).

[0157] As an alternative kinetic analysis, an antibody-loaded anti-mouse IgG Fc capture (AMC) biosensor was used for binding / dissociation with serially diluted GST-SNAP25. In short, purified IgG was diluted to 100 or 200 nm, while GST-SNAP25... Fl Or SNAP25197 Serial dilutions were performed to 1.56, 3.125, 6.25, 12.5, 25, 50, and 100 nM. After equilibration in 1x kinetic buffer for 1 min, the AMC biosensor was loaded with IgG for 10 min, followed by immersion in 1x kinetic buffer for 10 min. Assay binding and dissociation were each performed for 10 min, and K was estimated as described above. D .

[0158] Preparation Examples 6-8. Direct ELISA Analysis

[0159] Use 1μg GST-SNAP25 FL Or GST-SNAP25 197 Immunoplates (Thermo Fisher Scientific A71125) were coated at 37°C for 2 hours in 0.1M carbonate buffer (pH 9.5). After washing with 1x PBS, the immunoplates were incubated with 300 μl of blocking buffer (5% skim milk in 1x PBS) at room temperature for 15 minutes. After washing three times with 1x PBST (1x PBS / 0.05% Tween-20), 100 μl of hybridoma cell culture supernatant (1:20–10000 dilution) or ascites fluid (1:1000–312500 dilution) was added to the microplates and incubated at room temperature for 1 hour. After washing three times with 1x PBST, aliquots (100 μl per well) of goat anti-mouse IgG-HRP conjugate (1:1000 dilution) (Ab Frontier LF-SA8001) were added to each well. After incubating at room temperature for 1 hour, the microplate was washed three times with 1x PBST and then flushed with 50 μL of 1-Step PBS. TM The Ultra TMB-ELISA (Thermo Fisher Scientific 34028) was used to perform the HRP reaction for 3–25 minutes at room temperature. The HRP reaction was terminated by adding 50 μL of 1M H2SO4, and the ELISA signal was estimated at 450 nm using Bio-Tek SynergyNeo2.

[0160] Preparation Examples 6-9. Western Blot Analysis

[0161] Reconstituted GST-SNAP25 (per well) Separate the cells using 10% or 12% SDS-PAGE with total cell lysate (15 μg per well) and precise protein standards (3 μl per well). After soaking in transfer buffer containing 48 mM Tris, 38.9 mM glycine, 20% methanol, and 0.05% SDS for 5 minutes, use... (Bio-Rad 170-3940) Protein was transferred onto a PVDF membrane and incubated at 25 V for 45 minutes. The PVDF membrane was briefly washed with 1x TBST (1x TBS / 0.05% Tween 20) and then incubated with blocking buffer (5% skim milk in 1x TBST) at room temperature for 15 minutes. Subsequently, the PVDF membrane was incubated with either hybridoma culture supernatant (diluted 1:100 in blocking buffer) at room temperature for 45 minutes. Polyclonal anti-SNAP25 IgG (Sigma S9684) (1:8,000 dilution) and anti-SNAP25197 IgG (R&D MC6050) (1:100 dilution) were used as positive controls. After washing three times with 1x TBST for 15 minutes, the PVDF membrane was incubated at room temperature for 45 minutes with either goat anti-rabbit IgG-HRP conjugate (1:10000 dilution) or goat anti-mouse IgG-HRP conjugate (1:10000 dilution). After washing three times with 1x TBST, recombinant GST-SNAP25 or endogenous SNAP25 was detected using ECL solution (see below) and Bio-Rad ChemiDoc. TM Quantitative analysis was performed using the Bio-Rad Universal Hood III imaging system.

[0162] Before use, mix solutions A and B in a 1:1 ratio to prepare the ECL working solution. Solution A consists of 0.1 M Tris-HCl (pH 8.8), 2.5 mM luminol in DMSO, 4 mM 4-iodophenylboronic acid, 0.2 mM tetrabutylborohydride, 2% ethylene glycol, and 0.02% Triton X-100. Solution B consists of 0.1 M Tris-HCl (pH 8.8), 10.6 mM hydrogen peroxide, and 0.012% sodium tartrate.

[0163] Example 1: Screening for neurons sensitive to BoNT / A

[0164] Example 1-1. Screening of BoNT / A-sensitive neurons based on BoNT / A cleavage of SNAP25 protein.

[0165] Neurons at 2x10 5Cells were seeded at a density of 10 cells / well in 24-well plates. Cells were cultured for 24 hours, then treated with 2 nM BoNT / A medium and cultured for 3 days. Medium suitable for each cell type was used. Whole-cell lysates were obtained by lysing cells using 1x RIPA buffer (abcam, ab156034). 3.5 μg of protein was then added to 12% SDS-PAGE and electrophoresis was performed. After electrophoresis, the SDS-PAGE was immersed in a transfer buffer consisting of 48 mM Tris, 38.9 mM glycine, 20% methanol, and 0.05% SDS for 5 minutes, and then... Semi-Dry (Bio-Rad 170-3940) was used to transfer proteins from SDS-PAGE to a PVDF membrane. After rinsing the PVDF membrane with 1x TBST (1x TBS / 0.05% Tween 20), the membrane was placed in blocking buffer (5% skim milk powder in 1x TBST) and incubated at room temperature for 15 minutes.

[0166] Then, the membrane was incubated at room temperature for 45 minutes with SNAP25FL (polyclonal anti-SNAP25 IgG (Sigma, S9684)) antibody diluted 1:8000 in blocking buffer and SNAP25197 (anti-SNAP25197 IgG (R&D, MC6050)) antibody diluted 1:100 in blocking buffer. Subsequently, the PVDF membrane was washed three times with 1x TBST for 15 minutes each time and incubated at room temperature for 45 minutes with a diluent containing HRP-conjugated IgG. The PVDF membrane was washed three times with 1x TBST and then incubated with ECL solution and ChemiDoc. TM Imaging and quantification were performed using the MP imaging system (Bio-Rad). The ECL solution was formulated and optimized by the inventors by mixing SolA and SolB in a 1:1 ratio before use. The specific compositions of the SolA and SolB solutions constituting the ECL solution are shown in Table 2 below. The sensitivity to BoNT / A was determined based on the degree of SNAP25 fragmentation, and the results are shown in Table 3 below.

[0167] Table 2

[0168]

[0169] Table 3

[0170]

[0171]

[0172] As shown in Table 3, apart from SiMa cells, it is known that 13 types of nerve cells are sensitive to BoNT / A, and only Neuro-2a and KP-N-RT-BM1 (K-BM1) cells produce detectable levels of SNAP25 protein cleavage.

[0173] Examples 1-2. Screening for BoNT / A-sensitive cells

[0174] Under the same conditions as the materials and methods, Neuro-2a, SiMa, and KP-N-RT-BM-1 cells were cultured in 24-well plates, and the cleavage of SNAP25 protein was analyzed by Western blotting. Results are as follows: Figure 1 As shown.

[0175] For Neuro-2a, SiMa, and K-BM1 cells, the estimated degree of SNAP25 lysis was approximately 33%, 66%, and 29%, respectively.

[0176] Neuro-2a underwent further clonal selection via K-BM1 for the following reasons. First, the in vivo potency of BoNT / A, typically measured by mouse lethality (i.e., mouse LD50), is better summarized using mouse cell lines. Indeed, neuro-2A is a mouse cell line, while K-BM1 is a human cell line. Second, K-BM1 grows much more slowly than Neuro-2a. It should be noted that the population doubling time of the human neuroblastoma cell line SiMa has been reported to be 34 to 100 hours (DSMZ ACC-164). Third, under a microscope, neuro-2a cells appear to be a mixed population of several cell types. Therefore, it has been determined that cells highly sensitive to BoNT / A may exist within the heterogeneous Neruo-2a cell population.

[0177] Example 2: Screening for BoNT / A-sensitive clones

[0178] A method was used to select clones highly sensitive to BoNT / A from the Neuro-2a cell population selected in Example 1.

[0179] Example 2-1. Clonal culture of neuro-2a cells

[0180] Neuro-2a cells were supplemented with 10% fetal bovine serum (FBS), 1x non-essential amino acid (1x NEAA), 1x sodium pyruvate, and 1x GlutaMAX. TM Cultured in MEM containing 1x antibiotic antifungal agent (1x AA).

[0181] When cell confluence reaches approximately 80-90%, use 1x TrypLE (Gibco) TMCells were prepared into a single-cell suspension using a treatment (12604021). After determining the viable cell count using a hemocytometer and trypan blue, the cells were diluted to a density of 10 cells per ml of culture medium. Then, 100 μl of the diluted single-cell suspension was added to a 96-well plate. Colony growth was observed periodically under a microscope. When the single-cell density reached 60%, the cells were transferred to a 24-well plate as described above. Afterward, the cells were divided in half, one half stored in liquid nitrogen, and the other half tested for susceptibility to BoNT / A toxin.

[0182] A total of 672 cell clones were passaged from 24-well plates to 7 x 96-well microplates for BoNT / A toxin assay. On the second day, the culture medium was replaced with PRMI 1640 supplemented with 2 mM L-alanyl-L-glutamine, 1 x B27, 1 x N2, and 1 x NEAA (differentiation medium) to induce neuronal differentiation. On day 4 of induced differentiation, GT1b (diluted in 1 x non-toxin medium) was added to the differentiation medium to a final concentration of 25 μg / ml, and the cells were cultured for 24 hours. Subsequently, the culture medium was replaced with toxin medium containing BoNT / A (0.1 nM), and the cells were incubated for another 2 days. Western blot analysis was performed in the same manner as described in Example 1 to confirm the extent of SNAP25 cleavage, and a primary clonal selection process was performed. This clonal selection process was repeated three times, and the results confirmed in each clonal selection process are as follows: Figure 2 As shown.

[0183] Following Allergan's optimized clone selection procedure for SiMa cells with slight modifications, the sensitivity of 142 clones to BoNT / A was tested. The results are as follows: Figure 2 As shown, a total of 19 positive clones were identified, and these clones were significantly more sensitive to BoNT / A than the parental neuro-2a. Among the positive clones selected through multiple rounds of single-cell clone selection, only clone 42 consistently showed higher BoNT / A sensitivity than the parental neuro-2a. Following the nomenclature recommended by Rust and Pollok (2011) and Sarntivijai et al. (2008), clone 42, finally selected through this step, was named "N2-42F".

[0184] Example 2-2. Recovery, proliferation and storage of frozen cells

[0185] The recovery of frozen cells is as follows.

[0186] The cryogenic storage vials, removed from the liquid nitrogen freezer, were gently stirred in a 37°C water bath and rapidly thawed within 2 minutes. The cell storage vials were then purified by spraying with 70% ethanol. The top of the vial was unscrewed in a laminar flow tissue culture hood. The contents of the vial were then transferred to a sterile 15ml conical tube containing 9ml of preheated complete culture medium. The 15ml tube was gently centrifuged (125xg (gravity)) for 10 minutes, the supernatant was aspirated, and the cells were added to 2ml of complete culture medium. The cells were resuspended by gently pipetting to loosen the precipitate. The cell suspension was then transferred to a T75 flask containing 25ml of complete culture medium and coated according to the method described in the preparation examples. The cell suspension was incubated at 37°C in a CO2 incubator.

[0187] Cell culture was performed as follows.

[0188] When the cells in the flask reach approximately 90% confluence, aspirate the culture medium and rinse the inside of the flask once with 1x DPBS. Add 1 ml of 0.25% trypsin-EDTA to the cells and incubate at 37°C for 5 minutes. Then, add 9 ml of complete culture medium to the cell pellet and disrupt it into single cells by repeated pipetting. Transfer the single-cell suspension to a 15 ml conical tube and centrifuge at 800 x g (gravity) for 5 minutes. Then, completely aspirate the supernatant. Add 10 ml of complete culture medium to the flask and resuspend the cell pellet in it. Assess cell viability using a hemocytometer. Then, transfer 3 ml of the cell suspension to a new T75 flask. Culture the cells for 3 days until they reach approximately 90% confluence.

[0189] Cell cryopreservation for cell bank establishment is performed as follows.

[0190] Cells were cultured in 2 to 8 T75 flasks until approximately 90% confluence was reached. When the cells reached late growth (approximately 2.0 x 10⁷ cells / flask), the culture medium was aspirated, and the cells were trypsinized into single cells and centrifuged in the same manner as in the cell culture procedure. Then, the cells were frozen in medium at 5 x 10⁷ cells / flask. 6 Resuspend the cell pellet at a density of cells / ml. Transfer 1 ml of the cell suspension to a cryopreservation vial, which is then transferred to a cryopreservation container filled with isopropanol. Store the cryopreservation container overnight at -80°C. The next day, remove only the cryopreservation vial and store it in a liquid nitrogen freezer.

[0191] Example 3: Identification of N2-42F characteristics and culture environment

[0192] Example 3-1. Confirming the doubling time

[0193] The division time of Neuro-2a cells and N2-42F confirmed that the cleavage pattern of the SNAP25 protein was detectable. Each cell and clone was plotted at 1.5 × 10⁶ cells per well. 5 Each cell was divided into equal wells in a 6-well plate. The total number of viable cells was counted daily using a hemocytometer and trypan blue staining for 6 consecutive days. The division time was calculated using the number of viable cells obtained during the exponential growth phase, as shown in Table 5 (Table 4). Figure 3 As shown in Table 4 below, T is the incubation time, and Xe and Xb are the cell numbers at the beginning and end of the culture time, respectively.

[0194] Table 4

[0195] Splitting (doubling) time = T × ln2 / ln(Xe / Xb)

[0196] Table 5111

[0197] cell Split (Doubling) Time (Hours) Neuro-2a 24±4.7 N2-42F 24±2.9

[0198] As shown in Table 5 and Figure 3 As shown, the splitting time was 24 ± 4.7 hours for Neuro-2a and 24 ± 2.9 hours for N2-42F, so there was no significant difference.

[0199] Example 3-2. Morphological Confirmation of N2-42F

[0200] Allergan reported the use of a BoNT / A-sensitive clone (H1) isolated from SiMa cells as the parental cell (Fernandez-Salas et al., 2012). Based on this report, it was expected that SiMa cells, like neuro-2a, would represent a heterogeneous cell population with mixed cell types, but their subclones (e.g., H1 and N2-42F) were expected to be homogeneous cell types. Therefore, to confirm that N2-42F was of the same cell type, parental Neuro-2a, N2-42F, and SiMa cells were examined under a microscope, and the results were as follows: Figure 4 As shown.

[0201] like Figure 4 As shown, under the microscope, neuro-2a and SiMa cells, which are considered to be mixed cell types, both exhibit heterogeneous morphology, but N2-42F cells exhibit a very uniform morphology.

[0202] The results above show that N2-42F corresponds to a homogeneous cell type, serving as a single clone among the multiple clones that constitute Neuro-2a.

[0203] Example 3-3. Confirmation of the culture environment

[0204] To confirm the optimized culture environment for N2-42F cells in the BoNT / A toxicity assay, N2-42F cells were cultured in culture plates coated with type IV collagen, gel, and poly-D-lysine, respectively. The culture conditions were measured under a microscope, and the results are as follows: Figure 5 As shown.

[0205] like Figure 5 As shown, parental Neuro-2a cells have been confirmed to proliferate successfully on uncoated plates, while N2-42F cells have proliferated successfully on poly-D-lysine-coated plates. Furthermore, neither uncoated plates nor plates coated with type IV collagen (collagen IV) or gelatin supported effective growth of N2-42F cells. On those uncoated poly-D-lysine-coated plates, N2-42F cells appeared unhealthy, loosely attached to the plates. In contrast, on poly-L-lysine-coated plates, N2-42F cells were confirmed to adhere firmly and be evenly distributed.

[0206] The results above demonstrate that the N2-42F cells of this invention exhibit an optimized culture environment on poly-D-lysine-coated plates. Therefore, in this invention, poly-D-lysine-coated culture plates are used to measure the BoNT / A toxicity of N2-42F cells.

[0207] Example 4: Confirmation of the sensitivity of N2-42F to botulinum neurotoxin

[0208] SiMa cells were used as a control. 1x toxin medium containing different concentrations of BoNT / A was added to 96-well plates cultured with N2-42F cells and coated with poly-D-lysine. BoNT / A from N2-42F cells was then added, and sensitivity was measured.

[0209] Specifically, after culturing N2-42F cells for 4 days under the above conditions, the cells were treated with 50 μl of 1x SDS sample buffer. Western blot analysis was then performed on the cells, and the results are shown in Figure 6a. This experiment was repeated three times.

[0210] As shown in Figure 6a, it was confirmed that 0.93 pM or less of BoNT / A did not significantly cleave endogenous SNAP25 in SiMa cells and N2-42F. After treatment with 2.78 to 25 pM of BoNT / A, the degree of SNAP25 cleavage was confirmed to be 25% to 75% in N2-42F and 33% to 75% in SiMa cells.

[0211] The results above show that the N2-42F of the present invention is as sensitive to BoNT / A as SiMa cells.

[0212] Including BoNT / A, there are seven different serotypes of botulinum neurotoxin, from BoNT / A to BoNT / G. Similar to BoNT / A, Bont / B has also been approved for pharmaceutical use, for example... or Therefore, this study explored whether N2-42F cells could be used for any cell-based efficacy analysis against different botulinum neurotoxin serotypes. To this end, the sensitivity of differentiated N2-42F and Neuro-2a cells to different neurotoxin complexes was compared (Metaboologics CO). ).

[0213] As shown in Figure 6b, 25 pM Metabiologics BoNT / A (M-BoNT / A) saturated SNAP25 lysis in N2-42F cells. Under the same conditions, approximately 44% SNAP25 lysis was observed in Neuro-2a cells. The higher sensitivity of N2-42F cells to M-BoNT / A is consistent with their sensitivity to BoNT / A prepared by HUGEL (i.e., Botulax). N2-42F cells also exhibited significantly higher sensitivity to M-BoNT / B. Poisoning with 2 nM M-BoNT / B resulted in near-complete lysis of Vamp2 in N2-42F cells, compared to only approximately 50% lysis in Neuro-2a. Despite the smaller extent, N2-42F cells showed higher sensitivity to M-BoNT / C and M-BoNT / F. However, for M-BoNT / D (5 ​​pM or 200 pM), there was no significant difference in sensitivity between N2-42F and Neuro-2a cells (data not shown). They also did not show any... or Sensitivity at any detectable level. Our results indicate that N2-42F cells can be used as a host for cell-based BoNT / A, BoNT / B, BoNT / C, and BoNT / F efficacy assays.

[0214] Example 5: Confirmation of the spectral stability of N2-42F

[0215] Lineage stability of neurons is a crucial factor in cell-based detection platforms. Based on this, the lineage stability of N2-42F was confirmed.

[0216] N2-42F cells were continuously cultured and passaged multiple times. A master cell bank was prepared using N2-42F cells from early passages. Cells were also cryopreserved in liquid nitrogen every five passages to maintain clonal stability. Briefly, N2-42F cells stored at passage 5 (P5) and passage 15 (P15) were thawed, and when the N2-42F cell density reached approximately 90%, their sensitivity to BoNT / A was tested according to the method described in Example 4. The results are shown in Figure 7.

[0217] The experiment was repeated three times using SiMa cells as a control.

[0218] As shown in Figure 7, the SNAP25 fragmentation caused by BoNT / A in the 5th generation N2-42F (N2-42F(P5)) is 63% (lane 6), 66% (lane 7) and 68% (lane 8), while in the 15th generation N2-42F (N2-42F(P15)) it is 63% (lane 6), 73% (lane 7) and 71% (lane 8).

[0219] These results indicate that, similar to the H1 clone of SiMa cells constructed by Allergan, the N2-42F clone obtained from the parent Neuro-2a maintains its sensitivity to BoNT / A not only in the 5th generation but also in the 15th generation. Therefore, it can be seen that N2-42F is a highly efficient cell detection platform clone, second only to the H1 clone of SiMa cells.

[0220] Example 6: Experimental procedures for activity determination based on N2-42F

[0221] Example 6-1. Experimental Method

[0222] The 96-well plates were coated as described in the above preparation examples. While the 96-well culture plates were air-dried, they were coated with 5.5 x 10⁻⁶ microspheres. 5 Prepare a total of 7 ml of N2-42F cell suspension at a cell / ml density. Use N2-42F cells at 90% density obtained from a T75 flask. Transfer the cell suspension to a sterile buffer reservoir and dispense aliquots (100 μl) into each well using a multichannel pipette. Incubate the 96-well plate in a CO2 incubator. The outer wells of the 96-well plate should be filled with aliquots (100 μl) of 1x AA solution (1x antibiotic antifungal solution in sterile ddH2O) to avoid severe edge effects.

[0223] The day after cell allocation, all cell culture medium was removed from 96-well plates using a multichannel pipette, followed by washing with 100 μl of RPMI 1640. BoNT / A toxicity medium was then added to each 96-well plate and incubated at 37°C and 5% CO2 for 4 days. Additionally, 7 ml of capture antibody B4 IgG was prepared, aliquoted into 50 μl portions for ELISA plates, and stored overnight at 4°C.

[0224] For measurements, BoNT / A toxic medium was removed from the 96-well plates using a multichannel pipette, and each well was treated with 60 μl of lysis buffer (pH 7.5, 20 mM HEPES, 1% Triton-200 mM NaCl, 1 mM EGTA, and 5 mM EDTA, with proteolytic inhibitors added immediately before use) and incubated at 4 °C for 20 min at 500 rpm. Afterward, the lysis buffer contained in each well was obtained and centrifuged at 4000 rpm for 20 min at 4 °C.

[0225] Wash the ELISA plate three times with washing buffer, add 300 μl of blocking buffer to each well, incubate at room temperature for 15 minutes, and wash twice with washing buffer after removing the blocking buffer.

[0226] Transfer 50 μl of TCL aliquots from the 96-well culture plate to an ELISA plate coated with capture antibody (B4), and incubate the ELISA plate on a microplate shaker at 200 rpm at 4°C for 4 hours. Finally, wash three times with washing buffer.

[0227] For SNAP25 197 For detection, the detection antibody was added to an ELISA plate (50 μl per well), and the plate was incubated at room temperature for 1 hour on a heated shaker (200 rpm). The plate was then washed three times with wash buffer, and 50 μl of 1-Step™ Ultra TMB-ELISA aliquots were added to the plate. After 5 minutes, the HRP reaction was terminated by adding 2M sulfuric acid (50 μl / well). The HRP reaction was measured at 450 nm. 450 The value at this location can represent SNAP25. 197 The relative quantity.

[0228] Example 6-2. Preparation of standard curve and determination of BoNT / A titer

[0229] Calculate the mean A450 value for the control wells of the sandwich ELISA performed without BoNT / A treatment (i.e., 0 pM). Subtract the mean control A450 value from the test A450 value, and then calculate the normalized mean test A450 value. Then, plot the normalized mean A450 value on the Y-axis against the BoNT / A potency on the X-axis using Prism 5.0 (GraphPad Software, La Jolla, CA). Analyze the plot by selecting “Analyze,” “Nonlinear Regression (Curve Fitting),” and then “S-shaped Dose Response,” which will produce the EC50 value. Plot a standard curve using the normalized A450 values ​​of the test wells treated with the BoNT / A standard reference (see Appendix 2, Section D). And use R... 2 A standard curve equation with a value of 0.95 or higher determines the BoNT / A efficacy of the test sample.

[0230] Example 7: Production of a monoclonal antibody specifically targeting SNAP25

[0231] Allergan used the 13-amino acid (AA) residual peptide N-CDSNKTRIDEANQ-C to generate antibodies. This peptide was engineered to react with SNAP25 generated after digestion of BoNT / A. 197 The C-termini are the same. Because SNAP25 FL They also have the same amino acid sequence, so it is reasonable to assume that SNAP25 is the key to identification. 197 The specificity of the monoclonal antibody can be attributed to SNAP25. 197 The features not identified are not its main amino acid sequence. SNAP25 consists of 206 AA residues. Figure 8 ) Through the SNARE diagram motif, it forms a stable ternary complex with synaptic fusion protein 1A and small synaptic vesicle protein 2 (VAMP2). SNAP25 197 It forms a less stable, non-functional ternary complex, while SNAP 180 The formation of a ternary complex was unsuccessful. They found that the 9AA at the C-terminus of SNAP25 is crucial for in vivo function, and the formation of a stable ternary complex indicates that BONT / A-induced cleavage at the 197-AA site did not reveal any structural alteration, particularly in the C-terminus and the second SNARE domain. Therefore, SNAP25... Fl and SNAP25 197 The assumed structural differences between them can produce specificity for SNAP25. 197 Monoclonal antibodies.

[0232] Ten peptide antigens were designed to meet the following two criteria. First, α-helical regions exhibiting low antigenicity were excluded. Second, the peptide sequences were non-redundant and unique, and their properties were essential for reducing antibody cross-reactivity. The ten peptide antigen sequences designed according to the above criteria are shown in Table 6 below.

[0233] Table 6

[0234]

[0235]

[0236] Of the 10 peptides described in Table 6, peptides M and N were used to generate a peptide capable of simultaneously detecting SNAP25. FL and SNAP25 197 Monoclonal antibodies. For the C peptide, three targets are expected to be obtained against (1)SNAP25. FL (2)SNAP25 197 and (3)SNAP25 FL and SNAP25 197 Monoclonal antibodies with binding specificity were used. Throughout our study, two commercially available antibodies were used as controls for ELISA and Western blot analysis. These included rabbit polyclonal anti-SNAP25 IgG (Sigma-Aldrich) and MC6050 (R&D). The former recognizes SNAP25. FL and SNAP25 197 However, the latter is SNAP25 197 Monoclonal antibodies with specificity.

[0237] Each synthetic peptide was injected into two rabbits to obtain polyclonal antibody serum, and injected into four mice to obtain monoclonal antibody serum. The process of establishing hybridoma cells that produce monoclonal antibodies is as follows: Figure 9As shown in Figure 10, four mice were immunized with each peptide antigen to induce antibody production. After ELISA screening of mouse serum, spleen cells were isolated from ELISA-positive mice and fused with SP2 myeloma cells. Hybridoma cells were then seeded as single cells into 96-well plates for each peptide antigen. For selection of positive hybridoma cells, the 24 clones with the highest ELISA reactivity were screened in 7 x 96-well plates, and then subjected to ELISA and Western blot reactions with recombinant SNAP25, from which the 5 clones with the highest reactivity and sensitivity were selected. To isolate single cells from the 5 positive clones, the 5 clones were seeded as single cells into 96-well plates, and 10 clones were randomly selected by ELISA to evaluate their activity in detecting the peptide antigen. Subsequently, when all 10 clones were confirmed positive, their reactivity and sensitivity to recombinant SNAP25 were reassessed by ELISA and Western blot. Representative results of the initial hybridoma screening are shown in Figure 10. Culture supernatants from hybridoma cells (clones 4, B4) grown in 7×96-well plates were collected, and their reactivity was evaluated by direct ELISA. Approximately 25% of the clones were found to be ELISA positive (Fig. 10a and 10b). Twenty-four clones showing strong positivity were selected, and their reactivity to endogenous SNAP25 was assessed using a sandwich ELISA with cell lysates (Fig. 10c). Ten clones were selected for single-cell clone selection.

[0238] For single-cell clone selection, hybridoma clones were seeded at a single-cell density in a 96-well plate. Four days later, the reactivity of the culture supernatant to recombinant SNAP25 was tested by direct ELISA, and five clones producing relatively strong ELISA signals were selected for further analysis. For example, for clone 4, more than 80% of the subclones grown in the 96-well plate were detected as positive by direct ELISA (Fig. 11a). Twelve subclones produced ELISA signals lower than the negative control, including A8-A10, B11, C10, D4, D8, D9, E5, E11, F10, and G7 (Fig. 11a). As described in Materials and Methods, five subclones were also analyzed by sandwich ELISA (Fig. 11b) and Western blot (Fig. 11c) using total cell lysate (TCL). It should be noted that the ELISA signal of the subclone with clone 4 was very consistent with the Western blot signal. Given that TCL underwent denaturation during the protein blot analysis, this result indicates that the monoclonal antibodies generated by hybridoma clone 4 reacted with both undenatured (i.e., ELISA) and denatured SNAP25.

[0239] Single-cell clone selection was repeated twice, as shown in Figures 12 and 13. After the second round of selection, clones 6 and 8 were removed from further screening because none of the culture supernatants in the 96-well plates produced ELISA signals significantly higher than the negative control. Through a series of single-cell clone selections, a total of 12 hybridoma clones were obtained, which produced ELISA signals against SNAP25. FL SNAP25 197 Or monoclonal antibodies specific to both.

[0240] Example 8: Production and purification of monoclonal antibodies

[0241] Hybridoma cells were expanded in T175 flasks and used to prepare a progenitor cell bank. To generate monoclonal antibodies, hybridoma cells were recovered from a stock vial stored in liquid nitrogen gas phase. When the cells reached approximately 90% confluence, they were transferred to multiple T175 flasks according to... Subculture was performed. After incubation for 3-4 days, cells were collected by centrifugation and cultured at 1.0 × 10⁻⁶ cells / mL. 6 Cells / ml were resuspended in serum-free medium. After 4 days of incubation, the culture supernatant was collected and column cultured using a Protein G column (…). IgG was purified using a protein G HP column, as described in Materials and Methods. The yield of monoclonal antibodies purified using culture supernatant varied between different batches of hybridoma cultures, and also for individual hybridoma clones. Different batches of culture supernatant were used... After more than ten purifications, approximately C16 IgG (Table 7).

[0242] Table 7

[0243]

[0244]

[0245] The purification results of other polyclonal antibodies are summarized in Table 8.

[0246] Table 8

[0247]

[0248] On average, approximately 1-2 mg of IgG was obtained from 100 ml of culture supernatant. Although no data are shown, the purity of the IgG and its antigen-binding specificity were verified by SDS-PAGE and ELISA.

[0249] Example 9: Production and purification of polyclonal antibodies

[0250] As described in the preparation examples above, polyclonal antibodies were purified from rabbit serum using SNAP25-AffiGel. Table 9 summarizes the relative distribution of serum proteins in the SNAP25-AffiGel fractions obtained from four different batches of rA15 serum.

[0251] Table 9

[0252]

[0253] Although each serum sample exhibited a distinct protein distribution pattern in the SNAP25-AffiGel fractions (Table 9), IgG was detected as the major component of all fractions by 10% SDS-PAGE (Fig. 14a). However, reactivity to endogenous SNAP25 was detected only at pH 4.0 when tested in a sandwich ELISA. The high signal-to-noise ratio (>25) (Fig. 14b) indicates that IgG in the pH 4.0 fraction is highly specific for endogenous SNAP25. Since similar results were obtained using other rabbit sera, only the pH 4.0 fraction was used throughout our study employing polyclonal sera, unless otherwise stated.

[0254] exist Figure 14A In the diagram, lane M indicates a size marker; lane 1 represents flow; lane 2 represents an IgG elution pool at pH 5.5; lane 3 represents an IgG elution pool at pH 4.0; lane 4 represents an IgG elution pool at pH 2.5; and lane 5 represents an IgG elution pool at pH 11.5.

[0255] Example 10: Sequence Analysis of Monoclonal Antibodies

[0256] Total RNA extracted from hybridoma cells was reverse transcribed into cDNA using oligo-dT antisense primers or gene-specific (mouse IgG1 CH and kappa CL) antisense primers. The cDNA was then amplified by PCR using specific mouse constant-domain primers to determine the antibody isotype. Degenerate V H and V L Primers were used to amplify the variable domain from cDNA. For 5' RACE, a homopolymer [dC] tail was added to the 3' end of the cDNA. The heavy and light chains were then amplified using oligonucleotide [dG] sense primers and gene-specific (CH / KC) antisense primers. The PCR products were cloned into blunt-ended or TA vectors for sequencing. The sequencing results were compared with V... H and V L Chain alignment is used to determine common sequences.

[0257] The CDR sequence can be summarized as: (1) SNAP25 FL- Specific IgG (Table 10), (2) SNAP25 197 Specific IgG (Table 11), and (3) with SNAP25 FL and SNAP25 197 Specific IgG (Table 12).

[0258] Table 10

[0259]

[0260] Table 11

[0261]

[0262]

[0263] Table 12

[0264]

[0265]

[0266] Table 13 summarizes the V values ​​of the antibodies produced in this invention. H and V L Sequence of structural domains.

[0267] Table 13

[0268]

[0269]

[0270]

[0271]

[0272] It is worth noting that V L The CDR3 sequence is derived from SNP25. 197 Specific IgGs are shared, such as C4, C7, C14, C15, and C16 IgGs, while their V... H The sequences are often IgG specific (Table 11). Sequence alignment analysis showed that the individual CDR sequences of IgG listed in Tables 10-12 are consistent with previously reported V sequences of IgG. L and V HThe CDR sequences do not overlap (US Patent US8198034B2). This can be attributed to a more stringent screening strategy using positive hybridoma cells in this study. Specifically, in multiple rounds of screening, only triple-positive hybridoma clones were selected using direct ELISA with peptide antigens, sandwich ELISA with TCL, and Western blot analysis with TCL. This stringent screening strategy helped to significantly reduce the K+ IgG obtained in this study. D value).

[0273] Example 11: Kinetic Analysis of Monoclonal Antibodies

[0274] As detailed in the "Preparation Examples Above", follow the manufacturer's recommended steps using Forté The Red96 instrument performs kinetic analysis of monoclonal antibodies via BLI analysis. First, it uses a device loaded with recombinant GST-SNAP25. 197 (125nm or 250nm) GST-resistant biosensors for SNAP25 197 Specific IgG was subjected to kinetic analysis. Figure 15 shows a set of kinetic curves obtained using C4, C7, C16, and C24 as an example of this study. In short, GST-SNAP25 was loaded... 197 The anti-GST biosensor was sequentially immersed in 1x kinetic buffer, serially diluted IgG samples (7.8, 15.6, 31.2, 62.5, 125, 250, 500 nM) (analyte binding), and 1x kinetic buffer (analyte dissociation). Raw kinetic data were obtained 10 minutes after each analyte binding and dissociation (Fig. 15a). The kinetic curves were then calibrated by subtracting the baseline BLI signal (Fig. 15b) using... Analysis software estimates K D .

[0275] In Figure 15, the IgGs loaded on the AMC biosensor include C16 IgG (I), C24 IgG (II), C4 IgG (III), and C7 IgG (IV). Furthermore, "a" represents antibody loading, "b" represents washing, "c" represents antigen binding, and "d" represents antigen dissociation.

[0276] The analysis results are shown in Table 14.

[0277] Table 14

[0278]

[0279] Considering K DFor values ​​in the nM range, kinetic analysis generally appears to be possible. However, when using immersion in lower concentrations of GST-SNAP25... 197 When performing kinetic analysis using a (125 nM) biosensor, K D The values ​​decreased proportionally, mainly due to changes in the dissociation rate constant Kdis (Table 14). Equally unusual was the decrease in Kdis values ​​for monoclonal antibodies with similar antigen specificity. Compared to previously reported values One or two orders of magnitude lower (US Patent US8198034B2).

[0280] For relatively high K D The Kdis value suggests two plausible reasons. The first is the use of an inappropriate assay buffer, and the second is the inherent limitation of using the anti-GST biosensor for the entire kinetic assay (FortéBio Application Note 14: Biomolecular Binding Kinetic Assays on the Octet Platform). Comparison of various assay buffers used in kinetic analysis indicates that 1x kinetic buffer (data not shown) is the most suitable among all test buffers. As an alternative kinetic assay, the anti-mouse IgG Fc capture (AMC) biosensor is directly loaded with antibody and associated / dissociated with serially diluted GST-SNAP25. In short, purified IgG is diluted to 100 or 200 nm, while GST-SNAP25... Fl Or SNAP25 197 Serial dilutions were performed to 1.56, 3.125, 6.25, 12.5, 25, 50, and 100 nM. After equilibration in 1x kinetic buffer for 1 min, the AMC biosensor was loaded with IgG for 10 min, followed by immersion in 1x kinetic buffer for 10 min. Associativity and dissociation of the analyte were each performed for 10 min, and K was estimated as described above. D The raw data for the alternating dynamics curves can be found in... Figure 16A Through K D The estimated and compiled data is shown in Figure 16b. In Figure 16, the IgGs loaded on the AMC biosensor include C14(I), C24(II), D2(III), D6(IV), E6(V), and A15(VI). Furthermore, "a" represents antibody loading, "b" represents antigen binding, and "c" represents antigen dissociation.

[0281] use K estimated by analysis software D The values ​​are listed in Table 15.

[0282] Table 15

[0283]

[0284]

[0285] The most noteworthy value is the Kdis value (i.e. (Table 15) This is two to three orders of magnitude lower than that obtained using anti-GST biosensors (Table 14). In fact, the dissociation rate is too low to be used with Forté For precise measurement, therefore, a minimum Kdis value of 1.0 x 10⁻⁶ for the six IgGs is tentatively given. -7 As shown in Table 15. These provisional dissociation rate constants are used to estimate K. D Value is It should be noted that the load has SNAP25. 197 AMC biosensor for specific IgG up to 1 μM of GST-SNAP25 FL The combination of these factors was not statistically significant. Similarly, GST-SNAP25... 197 With load SNAP25 FL The AMC biosensor did not bind. These binding specificities of IgG are highly consistent with its reactivity in ELISA.

[0286] Example 12: Confirmation of antigen-binding specificity of monoclonal antibodies

[0287] The monoclonal antibody against SNAP25 was compared and examined using direct ELISA and Western blot analysis. FL and SNAP25 197 The reactivity. First, as described in Materials and Methods, in the coating GST-SNAP25 FL Or GST-SNAP25 197 Direct ELISA was performed on microplates using purified IgG (50 ng per well). The HRP reaction was carried out for 5 minutes, and the results were measured using Bio-Tek SynergyNeo2. 450 Determine the extent of HRP activity. Subtract background A. 450 Later obtained A 450 The value was measured without using IgG, and the SNAP25 was calculated. 197 A450 and SNAP25 FL The ratio of A450 is expressed as Ratio in Table 16. 197 / 206 Consistent with results obtained through BLI analysis, the 197 / 206 ratio of bispecific IgGs (e.g., A15, B4, and B23) was close to 1.0. SNAP 197 Ratio of specific IgGs (e.g., C7, C14, C16, and C24)197 / 206 The value exceeds 950, but SNAP25 FL Specific IgG production Ratio 197 / 206 .

[0288] Table 16

[0289]

[0290]

[0291] Western blot analysis was performed using hybridoma culture supernatant (1:100 dilution) as the primary antibody source, with GST-SNAP25 as the primary antibody. FL and GST-SNAP25 197 Aliquots (0.5 μg) were dissolved on a denaturing gel and then transferred to a PVDF membrane as antigens for testing. Monoclonal antibodies reacted with the denatured SNAP25 antigen with the same specificity as in ELISA analysis. Figure 17 ).exist Figure 17 In the middle, lane 1 represents SNAP25. FL Lane 2 indicates GST-SNAP25 197 .

[0292] Example 13: Conjugation of horseradish peroxidase (HRP) to antibody

[0293] Among the monoclonal antibodies tested, C16 IgG exhibited the most reproducible antigen affinity and specificity retention upon HRP atresia (data not shown). In a typical HRP atresia reaction, as described in Materials and Methods, C16 IgG (5 mg) was incubated with activated HRP (5 mg) at room temperature in the dark for 2 hours. After the addition of 0.1 ml sodium borohydride (4 mg / ml), the HRP-antibody atresia was dialyzed against 1x PBS and 1x PBS / 50% glycerol at 4 °C (Figure 18).

[0294] like Figure 18AAs shown, activated HRP binds very effectively to C16 IgG, and the formation of C16 IgG-HRP conjugates can be detected even without incubation (compare lanes 1-3). Incomplete conjugation of C16 IgG is reflected in free IgG and HRP (lanes 4-6), suggesting that relatively high concentrations of free HRP and IgG are required in the reaction mixture for effective binding. In Figure 18a, lane M indicates size marking; lane 1 represents unconjugated C16 IgG (9 mg); lane 2 represents activated HRP (4 mg); lane 3 represents the C16 IgG / HRP mixture (C16 IgG-HRP) before incubation (4.5 mg); lane 4 represents C16 IgG-HRP after incubation (4.5 mg); lane 5 represents C16 IgG-HRP after blocking (4.3 mg); and lane 6 represents C16 IgG-HRP (4.3 mg) after removal of free HRP by dialysis. In addition, "a" represents the C16 IgG-HRP stop compound.

[0295] Two different C16 IgG-HRP conjugates were examined for their effects on SNAP25 in a direct ELISA. 197 The reactivity of the 96-well microplate was determined by coating it with 0-200 pg of GST-SNAP25. 197 The lysis rate of endogenous SNAP25 in cells grown on microplates was the highest, reaching [percentage missing]. When using 50 μl of 1-Step TM When measured by Ultra TMB-ELISA for 30 minutes, C16 IgG-HRP adducts in aliquots (200 ng) were able to detect as low as 50 pg of GST-SNAP25. 197 (Figure 18b). Additionally, with the addition of GST-SNAP25 197 The quantity proportionally obtained a higher A 450 Values. Based on these results, the C16-HRP stop compound was specifically used for antibody detection in an optimized sandwich ELISA.

[0296] Example 14: Biotin-antibody conjugation

[0297] The sandwich ELISA developed by Allergan utilizes two antibodies: SNAP25 197Specific IgG was used as the capture antibody and polyclonal SNAP25-specific IgG as the detection antibody. In contrast, the sandwich ELISA invented in this study utilizes two monoclonal antibodies as either the capture or detection antibody, making quality control more feasible and easier. This novel sandwich ELISA exhibits high reproducibility, reproducibility, and accuracy. However, adding a second detection antibody (e.g., IgG conjugated with alkaline phosphatase or biotin) can further improve the accuracy of the sandwich ELISA by standardizing the SNAP25 captured in each well.

[0298] As a first attempt, the bispecific monoclonal antibody A15 was conjugated with biotin, as described in Materials and Methods. As shown in Figure 19, the higher the concentration of biotin provided, the more biotin molecules were conjugated per mole of IgG. Quantitative analysis of biotin conjugation using the HABA / avidin premix (Thermo Scientific) showed that approximately 8 moles of biotin were conjugated per mole of IgG in a reaction provided with 0.25 mM biotin. Consistently, the IgG heavy chain conjugated with 0.5 mM biotin migrated significantly slower on SDS-PAGE. Figure 19A and 19B In Figures 19A and 19B, lane M indicates size marking; lane 1 represents A15 IgG; lane 2 represents A15 IgG conjugated with 0.1 mM biotin; lane 3 represents A15 IgG conjugated with 0.25 mM biotin; and lane 4 represents A15 IgG conjugated with 0.5 mM biotin.

[0299] The reactivity of A15 IgG conjugated with 0.1 mM biotin was tested in a sandwich ELISA. Briefly, TCL (60 μl) was prepared from N2-42F cells treated with a specified concentration of BoNT / A. The microwell layer coated with B4 IgG was incubated aliquoted with 50 μL of TCL. Endogenous SNAP25 captured by B4 IgG was detected by incubation with the A15-biotin conjugate and the streptavidin-AP conjugate (diluted 1:500 in 1x TBS). 197 As a bispecific monoclonal antibody, A15 IgG has been characterized to possess considerable affinity and specificity for binding SNAP25. Fl and SNAP25 197 (see Figure 17 For standardization purposes, these properties are the criteria for selecting A15 IgG as a potential second detection antibody. Therefore, it is expected that the binding of A15 to SNAP25 will remain unaffected by the degree of SNAP25 cleavage. However, conversely, the AP activity reflecting SNAP25 binding of biotinylated A15 IgG increases proportionally to the BoNT / A concentration. Figure 19C The change in antigen specificity of IgG light and heavy chains after biotinylation may explain this result (Figure 19b).

[0300] Example 15: Direct cross-linking of alkaline phosphatase (AP) with antibodies

[0301] As an alternative method for detecting antibodies, purified IgG was directly cross-linked with AP using glutaraldehyde, as the degree of cross-linking can be adjusted by the glutaraldehyde concentration. Two polyclonal antibodies and three monoclonal antibodies were cross-linked with AP using glutaraldehyde. Except for C16, they were all cross-linked with SNAP25. FL and SNAP25 197 The reaction is a bispecific antibody, a key characteristic of the second detection antibody used for standardization. C16 IgG was used as a control because the HPR conjugation was effective and did not affect the antigen specificity of C16 (Figure 18).

[0302] To find the optimal conditions for obtaining AP-IgG conjugates that retain their antigen specificity and reactivity, AP and IgG were cross-linked under different incubation conditions and with different IgG to AP ratios (Table 17). The AP-IgG conjugates obtained after glutaraldehyde cross-linking were analyzed by SDS-PAGE.

[0303] Table 17

[0304]

[0305] After glutaraldehyde crosslinking, the resulting AP-IgG conjugates were analyzed by SDS-PAGE. Representative results show the electrophoretic separation of the AP-IgG conjugates, subsequently visualized by Coomassie Brilliant Blue staining in Figure 20. In summary, polyclonal anti-SNAP25 IgG (Sigma) was crosslinked to AP in a reaction provided with 0.2% glutaraldehyde. AP crosslinking was highly efficient, resulting in all provided IgG forming high molecular weight complexes with AP. The resulting AP-IgG conjugates migrated much more slowly on the SDS gel, with some failing to penetrate the stacking gel portion (Figure 20a). Similar banding patterns were obtained for all IgGs tested (data not shown). In Figure 20a, lane M represents size markers, lane 1 represents unconjugated IgG, lane 2 represents AP, and lane 3 represents AP-IgG conjugates. Furthermore, "a" represents the concentrated gel portion of the polyacrylamide gel, and "b" represents the AP-IgG conjugate.

[0306] After confirming cross-linking by SDS-PAGE analysis, 2 ng samples containing different concentrations of GST-SNAP25 were used in a direct ELISA assay. 197The antigen reactivity and specificity of GST-SNAP25 mixture-coated microplates were compared to those of AP-rA15 IgG and AP-Sigma IgG conjugates (100 ng per well). The AP-Sigma IgG conjugate did not produce any ELISA signal, while the AP-rA15 IgG conjugate produced results similar to those of biotinylated A15 IgG (compare Figures 19 and 20b). With GST-SNAP25... 197 With increased concentrations, the use of AP-rA15 IgG stoppers resulted in a higher ELISA signal. The results in Figures 19 and 20 indicate that SNAP25... FL In the case of a reactive antibody, whether as a monoclonal or polyclonal antibody, both the heavy and light chains of IgG are effectively capsulated to activated biotin or cross-linked with AP via glutaraldehyde.

[0307] Example 16: Optimization of N2-42F cell culture and treatment with BoNT / A toxin

[0308] Key reagents, such as highly sensitive neuronal cells to BoNT / A and monoclonal antibodies specific to SNAP25, have been obtained, and a series of experiments have been conducted to optimize all steps of the cell-based titer assay, including the toxin culture medium, sensitizer, BoNT / A treatment time, and capture / detection antibody pairing. Therefore, all steps of the botulinum toxin cell assay can be optimized.

[0309] Example 16-1. Optimization of toxicity time

[0310] First, the BoNT / A treatment time (toxinization time) was optimized. Protocol A (Figure 21a) is a standardized CBPA procedure optimized for SiMa. To examine the BoNT / A sensitivity of Protocol A, N2-42F cells were cultured at 5.6 x 10⁻⁶. 5 Cells / well were seeded in 12-well plates. The next day, the medium was replaced with 1x toxic medium without GT / 1b. Two days later, GT1b (25 mg / ml) was added to the medium, and after one more day of culture, 1x toxic medium containing BoNT / A was added, followed by two more days of culture. Protocol B (Figure 21b) is the CBPA procedure developed in this study. In short, N2-42F cells were seeded at 5.6 × 10⁶ cells / well. 5Cells were seeded per well in 12-well plates and replaced with 1× toxic medium containing 25 pM BoNT / A the following day. Cell lysates were prepared on the designated day by adding 1× SDS sample buffer (200 μl per well) and stored at -20°C before use. Samples (12 μl) were subjected to 12% SDS-PAGE, and SNAP25 was detected by Western blot using polyclonal anti-SNAP25 IgG (Sigma S9684, 1:8,000 dilution) and goat anti-rabbit IgG Fc-HRP (AbFrontier LF-SA8002, 1:8,000 dilution). FL and SNAP25 197 The extent of SNAP25 fragmentation was quantified using Image Lab software (Bio-Rad).

[0311] As described above, to establish the optimal toxicity time for N2-42F cells, protocol A was modified by extending the cell culture time in either 1x toxic medium without GT1b or in 1x toxic medium containing BoNT / A. These changes did not improve the degree of SNAP25 lysis; furthermore, N2-42F cells grown in 1x toxic medium for more than 4 days appeared very unhealthy under a microscope (data not shown). Based on this observation, a new protocol B was established by shortening the culture time in 1x toxic medium supplemented with GT1b and BoNT / A (Fig. 21b). The degree of SNAP25 cleavage in N2-42F cells was analyzed by Western blot comparison starting from day 3 (d4) post-seeding. As shown in Fig. 21c, the estimated SNAP25 lysis rate was less than 20% at d4, but in toxic medium supplemented with GT1b and BoNT / A at d4, extended culture of N2-42F cells after d4 resulted in a significant increase in SNAP25 lysis, reaching 64% by d7. Since the N2-42F cells on day 7 appeared unhealthy under the microscope, day 6 was determined as the day for N2-42 cell harvest and sandwich ELISA analysis to measure BoNT / A potency.

[0312] Example 16-2. Optimization of toxin culture medium

[0313] Osmotic pressure and temperature affect Bocell TMThe BoNT / A sensitivity of NG108-15 cells was improved (US Patent US9526345B2). Furthermore, optimizing the neural differentiation medium significantly enhanced the BoNT / A sensitivity of NG108-15 cells (J Biomol Screen. 2016 Jan; 21(1):65-73). Since BoNT / A sensitivity reflects the extent of BoNT / A uptake via two independent receptors on the cell surface, the polysialic acid ganglioside (PSG) receptor and the protein receptor (SV2) (J Neurochem. 2009 Jun; 109(6)):1584-95), enhancing BoNT / A sensitivity through optimized medium or higher temperatures promotes cellular uptake of BoNT / A.

[0314] Therefore, the sensitivity of N2-42F cells to BoNT / A was tested in three different culture media: RPMI 1640, Neurobasalt, and RPMI 1640. TM Both MEM and MEM were supplemented with 1X N2, 1X B27 and 1X GT1b. When cultured in the specified medium, N2-42F cells were treated with different concentrations (0.93–25 pM) of BoNT / A cells according to Protocol B.

[0315] When measured by Western spectroscopy, the BoNT / A sensitivity of N2-42F cells was highest when measured with RPMI 1640. Figure 22 ). Using Neurobasal TM The BoNT / A sensitivity measured in MEM was 25% or 50% lower than that in RPMI 1640. In all media, the KCl concentration was typically 5.33 mM, but in RPMI 1640, the NaCl concentration was 103 mM, in MEM it was 117 mM, and in Neurobasal™ it was 52 mM. Despite this difference, the osmotic pressure of most vertebrate cells is known to remain within a narrow range of 260 mOsm / kg to 320 mOsm / kg (ATCC Guidelines for Cultured Cells). However, the BoNT / A sensitivity of N2-24F cells in RPMI 1640 may be due to unidentified media components other than osmotic pressure.

[0316] exist Figure 22 Under the conditions described, approximately 48% of endogenous SNAP25 was lysed in N2-42F cells at 8.33 pM BoNT / A, equivalent to a potency of approximately 10 units / ml. Since the culture volume in a 96-well plate is 0.1 ml, the EC50 of the biopotency per well (BoNT / A) on the microplate is estimated to be [missing value]. Therefore, the BoNT / A sensitivity measured using N2-42F cells according to protocol B is sensitive enough to determine the biopotency of BoNT / A by mouse LD50 bioassay.

[0317] Example 16-3. Optimization of sensitizer

[0318] Sensitizers for compounds affecting neural survival or differentiation were optimized, including arginine-ATP (Fields and Stevens, 2000), creatine (Andres et al., 2005), and lipoic acid (Grasso et al., 2014), which are known to affect BoNT / A sensitivity. N2-42F cells were treated with BoNT / A at concentrations ranging from 0.03 to 5.5 pM and analyzed using a sandwich ELISA protocol B with a SNAP25-specific monoclonal antibody. The results are shown in Figure 23. The results indicated that the addition of 1 mM creatine or 5 mM arginine to 1x toxin medium significantly enhanced BoNT / A sensitivity, resulting in a decrease in EC50 values ​​from 2.51 pM to 2.13 to 2.03 pM, respectively. In contrast, ATP and lipoic acid acted as highly effective inhibitors, and therefore SNAP25 cleavage was not detected in N2-42F cells even at a BoNT / A concentration of 25 pM.

[0319] In studies optimizing toxic culture media, it is believed that the respiratory sensitivity of N2-24F cells is influenced by factors other than osmotic pressure. This is because the sensitivity in RPMI 1640 medium is higher than in Neurobasal. TM Or MEM medium. Regarding the arginine content in the medium composition, it is 1.15 mM in RPMI 1640, 0.6 mM in MEM, and in Neurobasal... TM The content is 0.4 mM. Since arginine is a precursor amino acid to creatine, 2x arginine is used. The effect of arginine on BoNT / A sensitivity was further examined using a toxin-containing medium. As shown in Figure 23b, BoNT / A sensitivity gradually increased with increasing arginine concentration up to 5 mM, reflected by a decrease in the EC50 value. Although still higher than the control (EC50 = 2.94 pM), the BoNT / A sensitivity using 10 mM arginine (EC50 = 2.34 pM) was lower than that using 5 mM arginine (EC50 = 1.65 pM). Based on these results, although the mechanism of action remains to be understood, the optimized standard protocol for CBPA uses a toxin-containing medium containing 5 mM arginine.

[0320] In 2002, Schengrund and colleagues first provided experimental evidence that in neuro-2a cells, effective cleavage of SNAP25 by BoNT / A required GT1b in DMEM to be higher than 25 μg / ml. Since N2-42F cells are derived from neuro-2a, Western blot analysis was performed using DMEM containing 25-75 μg / ml of GT1b. The requirement of GT1b for BoNT / A activity was assessed using 1x toxic medium containing GT1b. Without GT1b, 8.3 pM BoNT / A resulted in approximately 18% SNAP25 cleavage (Fig. 24a). In N2-42F cells, the addition of 1x or 3x GT1b resulted in 10% and 18% increases in SNAP25 cleavage at 8.3 pM BoNT / A, respectively (Fig. 24a). To optimize the GT1b concentration, the BoNT / A sensitivity of N2-42F cells was tested in 2x toxic medium containing 25 pM BoNT / A, with the GT1b concentration increased from 1x to 5x. When measured by Western blot analysis, the addition of 1x or 2x GT1b significantly enhanced SNAP25 cleavage, but when GT1b exceeded 2x, SNAP25 cleavage increased slightly, from 63%, 65%, 68% to 70% (Fig. 24b). Parallel assays were performed using an optimized sandwich ELISA. Considering the sensitivity of sandwich ELISA, N2-42F cells were treated with 0.93 pM BoNT / A in 2x toxic medium according to protocol B. Sandwich ELISA more clearly demonstrated the BoNT / A activity requirement for GT1b. In short, when N2-42F cells were treated with BoNT / A in a GT1b-deficient toxic medium, a relatively low BoNT / A level was obtained in sandwich ELISA. 450 Value (Figure 24c). In contrast, the addition of GT1b to the toxin culture medium resulted in A 450 The significantly increased value indicates that, up to 2x GT1b, A 450 The stable increase in values ​​likely reflects an effective and stable three-molecule interaction between BoNT / A, GT1b, and the polysialic acid ganglioside (PSG) receptor. Therefore, the saturation A450 value obtained with 4x GT1b, and even the decreased A450 value with 5x GT5b, can be explained by PSG receptor saturation and / or molar excess of free GT1b competing with the BoNT / A-GT1b complex for the PSG receptor. Based on this reasoning, when measuring BoNT / A activity using N2-42F cells, 2x, or 50 μg / ml, was chosen as the optimal concentration of GT1b.

[0321] N2 (N2 supplement, Thermo Fisher Scientific 17502048) / B27 (B27) were tested.TM Effects of serum-free supplements (Thermo Fisher Scientific 17504-044) on BoNT / A sensitivity in neuronal cultures. B27 containing all trans-retinol (0.1 mg / L) supports motor neuron differentiation of neural progenitor cells (J Cell Biochem. 2008 Oct 15; 105(3): 633-40). N2 contains a subset of the components of B27, including insulin, and is known to promote (1) differentiation of human embryonic stem cells and (2) proliferation / survival of neural progenitor cells (J Cell Biochem. 2008 Oct 15; 105(3): 633-40). According to protocol B, BoNT / A sensitivity of N2-42F cells was assessed by adding 1x GT1b, 8.3 pMBoNT / A, and the specified concentration of N2 / B27 to RPMI 1640. SNAP25 lysis was quantitatively analyzed by Western blot. As shown in Figure 25a, total SNP25 (SNAP25) was quantified. FL +SNAP25 197 Intracellular levels of SNAP25 increased proportionally to N2 / B27 concentration, while SNAP25 levels remained relatively constant, even decreasing in the presence of 4x or 5x N2 / B27. A recent study reported that N2 and B27 work together to protect neurons from cell death following glucose depletion by limiting glycolysis (Front Mol Neurosci. 2017 Sep 29; 10: 305). SNAP25 synthesis was also identified as an increase in promoting cell proliferation and survival in serum-free RPMI 1640 medium containing low glucose concentrations, as part of its function in promoting cell proliferation and survival.

[0322] Furthermore, the effect of N2 / B27 on BoNT / A activity was further examined using RPMI1640 supplemented with 3x GT1b and 5mM arginine. Despite a significant increase in total intracellular SNAP25 levels, the degree of SNAP25 cleavage in N2-42F cells treated with 8.3pM BoNT / A increased from 13% to 56% and 69% in 1x and 2x N2 / B27, respectively (Fig. 25b). Based on this observation, 2x N2 / B27 was added to the optimized toxin medium.

[0323] Example 17: Optimization of Sandwich Elisa

[0324] Example 17-1. Buffer Optimization

[0325] To optimize sandwich ELISA conditions, 11 different cell lysis buffers were compared in ELISA, and the optimal conditions are shown in Tables 18 and 19. Table 18

[0326]

[0327]

[0328] Table 19

[0329]

[0330]

[0331] Example 17-2. Optimization of Capture Antibody Treatment

[0332] Secondly, the functions of three bispecific antibodies, A15, B4, and B23, as capture antibodies in sandwich ELISA were compared. TCL was prepared from N2-42F cells treated with different concentrations (0-25 pM) of BoNT / A in 1x toxic medium. TCL was added to microplates coated with the specified antibody (400 ng per well). After incubation at 4°C for 4 hours, SNAP25 captured by the indicated antibody was detected and quantified using a C16 IgG-HRP conjugate following the procedures described in "Materials and Methods". 197 The amount. One exception is that the HRP reaction is performed for 30 minutes until all test groups produce a positive ELISA signal. As shown in Figure 26a, the estimated EC50 values ​​for A15, B4, and B23 IgG are 15 pM, 0.7 pM, and 5.5 pM, respectively. This result is consistent with SNAP25. FL and SNAP25 197 K D The values ​​are consistent.

[0333] According to protocol B, the optimal amount of B4 IgG was explored in TCLs prepared from N2-42F cells incubated in 2x toxicity medium containing a specified concentration of BoNT / A, using cells coated with an increased amount of B4 IgG (per well). The microplate was used for sandwich ELISA. After incubation at 4°C for 4 hours, the captured SNAP25 was detected as described above. 197 Quantification was performed using the C16 IgG-HRP conjugate. It should be noted that the HRP reaction was carried out for 15 minutes. As shown in Figure 26b, the EC50 value steadily decreased as the amount of B4 IgG increased from 100 ng to 300 ng. The effect of 400 ng of B4 IgG on EC50 did not show any further statistically significant improvement. In conclusion, 300 ng of B4 IgG was used as the standard amount of capture antibody in the optimized sandwich ELISA.

[0334] Example 17-3. Optimization of antibody treatment for detection

[0335] TCLs prepared from SiMa cells treated with 8.33 or 25 pM BoNT / A in 1x toxic medium were incubated in microplates under specified conditions. Subsequently, the captured SNAP25 was detected and quantified using a C16 IgG-HRP conjugate. 197 As shown in Figure 27a, when the microplate was incubated at 4°C for 4 hours, SNAP25 197 The ELISA signal was highest at 37°C, but became very weak after 1 hour of incubation. Although no data were shown, similar results were obtained using TCL from N2-42F cells, and the ELISA signal did not change significantly after more than 4 hours of incubation. Therefore, in the optimized sandwich ELISA, TCL was incubated at 4°C for 4 hours.

[0336] The optimal incubation conditions for antibody detection were explored using direct ELISA. In short, microplates were incubated with different amounts (10 pg to 1 ng) of recombinant GST-SNAP25. 197 The coating represents 0.05% to 50% of endogenous SNAP25 cleavage in standard CBPA. After adding C16 IgG-HRP distillate (200 ng per well), the microplate is incubated under specified conditions. In the conditional assay, incubation of the Cg16 IgG-HRP distillate for 1 hour at room temperature resulted in the detection of all GST-SNAP25 cleavages. 197 The highest ELISA signal was produced within the range, but acceptable levels of ELISA signal could be produced under other conditions. Considering that the subsequent HRP reaction was carried out at room temperature, the C16-HRP complex was incubated at room temperature for 1 hour in the optimized sandwich ELISA.

[0337] Example 17-4. Optimization of C16 IgG-HRP conjugate detection

[0338] Conventional TMB substrates (1-Step) are typically used. TM Ultra TMB-ELISA was used to measure HRP activity. A drawback of using TMB substrates is that the EC50 value is easily affected by the HRP reaction time (Figures 26 and 29). Longer HRP reactions produce lower EC50 values. As an alternative detection method, a fluorescence assay was compared in a sandwich ELISA. Except for the provided HRP reaction substrate, colorimetric and fluorescence assays were performed in essentially the same parallel manner throughout the sandwich ELISA. The EC50 values ​​produced by fluorescence and colorimetric measurements were 0.66 and 0.78 pM, respectively. Figure 28Furthermore, both EC50 values ​​were also affected by the HRP reaction time (data not shown). In summary, although fluorescence assays of the HRP reaction require the use of black microplates and microplate readers with built-in fluorometers, both measurements are accurate and sensitive enough to detect the activity of C16 IgG-HRP conjugates in optimized ELISA.

[0339] Example 18: Validation of CBPA

[0340] Example 18-1. Optimized CBPA timeline inspection

[0341] Figure 29 This is a schematic diagram of the sandwich ELISA method used to determine the botulinum toxin activity of the present invention. Figure 29 As shown, the sandwich ELISA method for botulinum toxin activity assay of the present invention can be completed in just 3 working days. In short, cells are inoculated on day one, and the culture medium containing BoNT / A (0.1-10 U / mL, or less than 4 pM) is changed on day two. On day 6 after cell inoculation, one day is needed for cell lysis and sandwich ELISA analysis. Furthermore, since the working time on the first and second days is only 1-2 hours, the sandwich ELISA method for botulinum toxin activity assay of the present invention is very time-saving. In addition, since the sandwich ELISA of the present invention uses monoclonal antibodies as both capture and detection antibodies, antibody management is much easier than when using polyclonal antibodies, thereby further consolidating the reliability of CBPA.

[0342] Example 18-2. Accuracy and linearity check of CBPA

[0343] Following standard scheme B, the three operators performed a total of 18 CBPAs.

[0344] Eighteen CBPA reactions were performed by three operators according to Protocol B. N2-42F cells were treated with either 2x toxic medium containing different concentrations (0, 0.03, 0.1, 0.2, 0.31, 0.62, 0.93, 2.78, 5.55, 8.33 pM) of BoNT / A (Fig. 30a) or 2x toxic medium containing 3x GT1b (Fig. 30b and Fig. 30c). The HRP reaction was performed for 5 minutes (Fig. 30a and Fig. 30b) or 9 minutes (Fig. 30c), and the EC50 was determined using Gen5 software. The limits of detection (DL) and quantitation (QL) were calculated by linear regression analysis of the A450 values ​​obtained from treating N2-42F cells with 0.1 to 0.93 pM BoNT / A, and the results are shown in Fig. 30.

[0345] The first series of CBPA produced an EC50 of 1.39 pM, with a limit of detection (DL) of 1.5 fM (3.4 mU relative power / ml) and a limit of quantitation (QL) of 4.6 fM. Since 1.39 pM BoNT / A corresponds to [missing value] per assay... The relative potency of the first CBPA was sufficient to measure the biopotency of BoNT / A in place of the mouse LD50 assay. A slightly lower EC50 value of 1.24 pM was obtained by performing a prolonged HRP reaction in the second series of CBPAs (Figure 30b). The effect of HRP reaction time on EC50 has been previously noted. Figure 29 The third series of CBPA assays using toxic medium containing 3x GT1b showed an EC50 as low as 1.09 pM, consistent with the results in Figure 27. These results indicate that the quantification capability and detection limit of CBPA in this study can be adjusted by modifying the HRP reaction time or changing the concentration of GT1b in the toxic medium. Linear regression analysis of ELISA signals from BoNT / A-treated N2-42F cells showed an excellent linear correlation between the relative potency of BoNT / A and the ELISA signal in all three series of CBPA (Figure 30). This result indicates that CBPA is not only highly sensitive but also highly accurate in measuring the relative potency of BoNT / A between 6.8 mU and 0.2 U in each assay.

[0346] Example 18-3. Measurement of biopotency in BoNT / A samples

[0347] BoNT / A( The potency in Hugel (Korea) was determined by mouse LD50. To measure this biopotency using CBPA, Two different batches (HUB 18009 and HUB18011) of 200U each were dissolved in toxic medium (substrate a), deionized water (substrate b), or 5mM arginine at pH 6.0 (substrate c). After incubation at room temperature for 10 minutes, the solutions in the medium were... The samples were sequentially diluted to 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15, and 50 U / ml, while others were diluted to 0.23, 0.48, 0.70, 0.98, 1.41, 2.11, 4.20, 6.32, and 12.6 U / ml in optimized toxicity media. Three different CBPA assays were performed using these samples according to standard protocol B. EC50 values ​​were determined. As shown in Figure 31, the EC50 values ​​for the two batches were 10.6 ± 0.12 and 10.3 ± 0.52 U / ml in matrix a, 4.6 ± 0.04 and 4.6 ± 0.08 U / ml in matrix b, and 4.5 ± 0.11 and 4.4 ± 0.22 U / ml for matrix c. These results indicate that CBPA has sufficient sensitivity and accuracy to measure... The biopotency is high, and its sensitivity (0.4-0.5 U per well) is equivalent to or better than that of mouse bioassays.

[0348] Although this disclosure has been described in detail with reference to specific features, it will be apparent to those skilled in the art that this description is merely a preferred embodiment and does not limit the scope of the invention. Therefore, the essential scope of the invention will be defined by the appended claims and their equivalents.

[0349] [Login ID]

[0350] Accession number KCTC13712BP

[0351] Deposit date: November 13, 2018

[0352] The Korean Center for Type Culture Collection (KCTC) is the depositary institution for this purpose.

[0353] Storage address: South Korea

[0354] Classification and naming: N2-42F

[0355] [Industrial Applicability]

[0356] This invention aims to overcome the limitations of conventional CPBA and develop a more effective CBPA. Currently, in the field of botulinum toxin potency measurement, there is a need to develop a cell-based potency assay (CBPA) to replace the mouse LD50 bioassay (mLD50). The cells and antibodies used to determine botulinum toxin activity according to this invention are cells and antibodies used to replace CPBA for mLD50. CBPA using the cells and antibodies of this invention exhibits high reliability and reproducibility, and therefore holds promise for positive applications in the pharmaceutical and cosmetic fields.

[0357] [Sequence List]

[0358] SEQ ID NO:1-MAEDADMRNELEE

[0359] SEQ ID NO:2-MAEDADMRNELEE

[0360] SEQ ID NO:3-DQLADESLESTRRMLQLVEE

[0361] SEQ ID NO:4-DEQGEQLERIEEGMDQINKD

[0362] SEQ ID NO:5-DEREQMAISGGFIRR

[0363] SEQ ID NO:6-EIDTQNRQIDRIMEK

[0364] SEQ ID NO:7-RIMEKADSNKTRIDE

[0365] SEQ ID NO:8-RIMEKADSNKTRIDEANQ

[0366] SEQ ID NO:9-DSNKTRIDEANQ

[0367] SEQ ID NO:10-KTRIDEANQPATK

[0368] SEQ ID NO:11-GYSITSGYY

[0369] SEQ ID NO:12-GYTFTDYN

[0370] SEQ ID NO:13-GYTFTNYG

[0371] SEQ ID NO:14-IRYDGSN

[0372] SEQ ID NO:15-IYPYNGDT

[0373] SEQ ID NO:16-INTYTGEP

[0374] SEQ ID NO:17-ARDRDSSYYFDY

[0375] SEQ ID NO:18-VRSGDY

[0376] SEQ ID NO:19-ARGYYDY

[0377] SEQ ID NO:20-DHINNW

[0378] SEQ ID NO:21-QSLLDSNGKTY

[0379] SEQ ID NO:22-QSLLDSDGKTY

[0380] SEQ ID NO:23-DTT

[0381] SEQ ID NO:24-LVS

[0382] SEQ ID NO:25-QQYWSAPPT

[0383] SEQ ID NO:26-WQGTLFPYT

[0384] SEQ ID NO:27-WQGTHFPRT

[0385] SEQ ID NO:28-GYSITSDYA

[0386] SEQ ID NO:29-GFTFNTNA

[0387] SEQ ID NO:30-GYTFTNYT

[0388] SEQ ID NO:31-GYTFNTYA

[0389] SEQ ID NO:32-GFTFSNYG

[0390] SEQ ID NO:33-GFTFNTYA

[0391] SEQ ID NO:34-ISYSVGT

[0392] SEQ ID NO:35-IRSKSNNYAT

[0393] SEQ ID NO:36-IRSKSDNYAT

[0394] SEQ ID NO:37-INPSSDYT

[0395] SEQ ID NO:38-IRSKSNNYTT

[0396] SEQ ID NO:39-INSNGGTT

[0397] SEQ ID NO:40-ARKGEYGFAY

[0398] SEQ ID NO:41-VYGRSYGGLSY

[0399] SEQ ID NO:42-VYGRSYGGLGY

[0400] SEQ ID NO:43-VRQVTTAVGGFAY

[0401] SEQ ID NO:44-ARRIFYNGRTYAAMDY

[0402] SEQ ID NO:45-VGQILYYYVGSPAWFAY

[0403] SEQ ID NO:46-ARDRDAMDY

[0404] SEQ ID NO:47-KSVSTSGYSY

[0405] SEQ ID NO:48-KSVSSSGYSY

[0406] SEQ ID NO:49-QSIVNSHGNTY

[0407] SEQ ID NO:50-LAS

[0408] SEQ ID NO:51-KVS

[0409] SEQ ID NO:52-QHSRELPLT

[0410] SEQ ID NO:53-QHSRELPWT

[0411] SEQ ID NO:54-FQGSHVPWT

[0412] SEQ ID NO:55-GFTFSNYG

[0413] SEQ ID NO:56-GINIKDYY

[0414] SEQ ID NO:57-ISSGGSYT

[0415] SEQ ID NO:58-IDPGNGDA

[0416] SEQ ID NO:59-ARHEGGGNPYFDY

[0417] SEQ ID NO:60-NEIAY

[0418] SEQ ID NO:61-QSLVHSNGNTY

[0419] SEQ ID NO:62-QSLLDSDGKTY

[0420] SEQ ID NO:63-KVS

[0421] SEQ ID NO:64-LVS

[0422] SEQ ID NO:65-SQNTLVPWT

[0423] SEQ ID NO:66-WQGTHFPFT

[0424] SEQ ID NO:67- EVKLVESGGGLVKPGGSLKLSCAASGFTFSNYGMSWVRQTPEKRLEWVATISSGGSYTYYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYYCARHEGGGNPYFDYWG QGTTLTVSS

[0425] SEQ ID NO:68- DVLMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVS NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQNTLVPWTFGGGTKLEIK

[0426] SEQ ID NO:69- EVQLQQSGAELVRPGASVKLSCTASGINIKDYYMHWMKQRPEQDLEWIGWIDPGNGDAEYAPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNEIAYWGQGTLVTV SA

[0427] SEQ ID NO:70- DIVMTQSPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPFTFGSGTKLEIK

[0428] SEQ ID NO:71- DVKLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYISYSVGTRYNPSLKSRISITRDTSKNQFFLLLKSVTNEDTATYFCARKGEYGFAYWGQGTL VTVSA

[0429] SEQ ID NO:72- DIVMTQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPLTFGAGTKLELK

[0430] SEQ ID NO:73- QVQLVETGGGLVQPKGSLKLSCAASGFTFNTNAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQSLLYLQMNNLKTEDTAMYYCVYGRSYGGLSY WGQGTLVTVSA

[0431] SEQ ID NO:74- DIVMTQSPASLAVSLGQRATISCRASKSVSSSGYSYMHWYQQKPGQPPKLLIYLAS NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPLTFGAGTKLELK

[0432] SEQ ID NO:75- EVKLVESGGGLVQPKGSLKLSCAASGFTFNTNAMNWVRQAPGKGLEWVARIRSKSDNYATYYADSVKDRFTISRDDSPSMLYLQMNNLKTEDTAMYYCVYGRSYGGLGY WGQGTLVTVSA

[0433] SEQ ID NO:76- DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYVHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPLTFGAGTKLELK

[0434] SEQ ID NO:77- EVKLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVRQVTTAVGGF AYWGQGTLVTVSE

[0435] SEQ ID NO:78- DIVMTQSPASLAVSLGQRTTISCRASKSVSSSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPLTFGAGTKLELR

[0436] SEQ ID NO:79- EVQLQQSGAELARPGASVQMSCKAFGYTFTNYTMHWVRQRPGQGLEWIGFINPSSDYTNYNQKFKDKATLSADKSSSTAYMQLSSLTSEDSAVYYCARRIFYNGRTYAAMDYWGQGTSVTVSS

[0437] SEQ ID NO:80- DIVMTQSPASLAVSLGQRATISCRASKSVSSSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPLTFGAGTKLELK

[0438] SEQ ID NO:81- EVKLVESGGGLVQPKGSLKLSCAASGYTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYTTYYADSVKDRFTISRDDSQSMLYLQINNLKTEDTAMYYCVGQILYYYVGSP AWFAYWGQGTLVTVSA

[0439] SEQ ID NO:82- DIVMTQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQPPKLLIFLASN LESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPWTFGGGTKLEIK

[0440] SEQ ID NO:83- DVKLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYIRYDGSNNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTASYYCARDRDSSYYFDYWGQG TALTVSS

[0441] SEQ ID NO:84- DIVMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISDTTSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSAPPTFGGGTKLEIK

[0442] SEQ ID NO:85- EVQLEESGGGLVQPGGSLKLSCAASGFTFSNYGMSWVRQTPDKRLELVATINSNGGTTYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDSAMYYCARDRDAMDYWGQGT SVTVSS

[0443] SEQ ID NO:86- DVLMTQTPLSLPVSLGDQASISCRSSQSIVNSHGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIK

[0444] SEQ ID NO:87- EVQLQQSGPELVKPGASVKISCKASGYTFTDYNMHWVKQSHGKSLEWIGYIYPYNGDTGYNQKFKSKATLTVDNSSSTAYMELRSLTSEDSAVYYCVRSGDYWGQGTTLT VSS

[0445] SEQ ID NO:88- DVLMTQTPLTLSVTIGQPASISCKSSQSLLDSNGKTYLNWLLQRPGQSPSRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTLFPYTFGGGTKLEIK

[0446] SEQ ID NO:89- QIQLAQSGPELKKPGETVKISCKASGYTFTNYGMSWVKQAPGKGLKWMGWINTYTGEPTYAADFKGRFAFSLETSASTAFLQINNLKNEDTATYFCARGYYDYWGQGTTLT VSS

[0447] SEQ ID NO:90- DVLMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVS KLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPRTFGGGTKLEIK <110> Xiujie Corporation <120> Cell-based methods for determining botulinum toxin activity <130> POPB187383 <160> 90 <170> PatentIn version 3.5 <210> 1 <211> 13 <212> PRT <213> Clostridium botulinum <400> 1 Met Ala Glu Asp Ala Asp Met Arg Asn Glu Leu Glu Glu 1 5 10 <210> 2 <211> 13 <212> PRT <213> Clostridium botulinum <400> 2 Met Ala Glu Asp Ala Asp Met Arg Asn Glu Leu Glu Glu 1 5 10 <210> 3 <211> 20 <212> PRT <213> Clostridium botulinum <400> 3 Asp Gln Leu Ala Asp Glu Ser Leu Glu Ser Thr Arg Arg Met Leu Gln 1 5 10 15 Leu Val Glu Glu 20 <210> 4 <211> 20 <212> PRT <213> Clostridium botulinum <400> 4 Asp Glu Gln Gly Glu Gln Leu Glu Arg Ile Glu Glu Gly Met Asp Gln 1 5 10 15 Ile Asn Lys Asp 20 <210> 5 <211> 15 <212> PRT <213> Clostridium botulinum <400> 5 Asp Glu Arg Glu Gln Met Ala Ile Ser Gly Gly Phe Ile Arg Arg 1 5 10 15 <210> 6 <211> 15 <212> PRT <213> Clostridium botulinum <400> 6 Glu Ile Asp Thr Gln Asn Arg Gln Ile Asp Arg Ile Met Glu Lys 1 5 10 15 <210> 7 <211> 15 <212> PRT <213> Clostridium botulinum <400> 7 Arg Ile Met Glu Lys Ala Asp Ser Asn Lys Thr Arg Ile Asp Glu 1 5 10 15 <210> 8 <211> 18 <212> PRT <213> Clostridium botulinum <400> 8 Arg Ile Met Glu Lys Ala Asp Ser Asn Lys Thr Arg Ile Asp Glu Ala 1 5 10 15 Asn Gln <210> 9 <211> 12 <212> PRT <213> Clostridium botulinum <400> 9 Asp Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn Gln 1 5 10 <210> 10 <211> 13 <212> PRT <213> Clostridium botulinum <400> 10 Lys Thr Arg Ile Asp Glu Ala Asn Gln Pro Ala Thr Lys 1 5 10 <210> 11 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 11 Gly Tyr Ser Ile Thr Ser Gly Tyr Tyr 1 5 <210> 12 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 12 Gly Tyr Thr Phe Thr Asp Tyr Asn 1 5 <210> 13 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 13 Gly Tyr Thr Phe Thr Asn Tyr Gly 1 5 <210> 14 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 14 Ile Arg Tyr Asp Gly Ser Asn 1 5 <210> 15 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 15 Ile Tyr Pro Tyr Asn Gly Asp Thr 1 5 <210> 16 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 16 Ile Asn Thr Tyr Thr Gly Glu Pro 1 5 <210> 17 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 17 Ala Arg Asp Arg Asp Ser Ser Tyr Tyr Phe Asp Tyr 1 5 10 <210> 18 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 18 Val Arg Ser Gly Asp Tyr 1 5 <210> 19 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 19 Ala Arg Gly Tyr Tyr Asp Tyr 1 5 <210> 20 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 20 Asp His Ile Asn Asn Trp 1 5 <210> twenty one <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> twenty one Gln Ser Leu Leu Asp Ser Asn Gly Lys Thr Tyr 1 5 10 <210> twenty two <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> twenty two Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr 1 5 10 <210> twenty three <211> 3 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> twenty three Asp Thr Thr 1 <210> twenty four <211> 3 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> twenty four Leu Val Ser 1 <210> 25 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 25 Gln Gln Tyr Trp Ser Ala Pro Pro Thr 1 5 <210> 26 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 26 Trp Gln Gly Thr Leu Phe Pro Tyr Thr 1 5 <210> 27 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 27 Trp Gln Gly Thr His Phe Pro Arg Thr 1 5 <210> 28 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 28 Gly Tyr Ser Ile Thr Ser Asp Tyr Ala 1 5 <210> 29 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 29 Gly Phe Thr Phe Asn Thr Asn Ala 1 5 <210> 30 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 30 Gly Tyr Thr Phe Thr Asn Tyr Thr 1 5 <210> 31 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 31 Gly Tyr Thr Phe Asn Thr Tyr Ala 1 5 <210> 32 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 32 Gly Phe Thr Phe Ser Asn Tyr Gly 1 5 <210> 33 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 33 Gly Phe Thr Phe Asn Thr Tyr Ala 1 5 <210> 34 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 34 Ile Ser Tyr Ser Val Gly Thr 1 5 <210> 35 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 35 Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr 1 5 10 <210> 36 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 36 Ile Arg Ser Lys Ser Asp Asn Tyr Ala Thr 1 5 10 <210> 37 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 37 Ile Asn Pro Ser Ser Asp Tyr Thr 1 5 <210> 38 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 38 Ile Arg Ser Lys Ser Asn Asn Tyr Thr Thr 1 5 10 <210> 39 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 39 Ile Asn Ser Asn Gly Gly Thr Thr 1 5 <210> 40 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 40 Ala Arg Lys Gly Glu Tyr Gly Phe Ala Tyr 1 5 10 <210> 41 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 41 Val Tyr Gly Arg Ser Tyr Gly Gly Leu Ser Tyr 1 5 10 <210> 42 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 42 Val Tyr Gly Arg Ser Tyr Gly Gly Leu Gly Tyr 1 5 10 <210> 43 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 43 Val Arg Gln Val Thr Thr Ala Val Gly Gly Phe Ala Tyr 1 5 10 <210> 44 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 44 Ala Arg Arg Ile Phe Tyr Asn Gly Arg Thr Tyr Ala Ala Met Asp Tyr 1 5 10 15 <210> 45 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 45 Val Gly Gln Ile Leu Tyr Tyr Tyr Tyr Val Gly Ser Pro Ala Trp Phe Ala 1 5 10 15 Tyr <210> 46 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 46 Ala Arg Asp Arg Asp Ala Met Asp Tyr 1 5 <210> 47 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 47 Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr 1 5 10 <210> 48 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 48 Lys Ser Val Ser Ser Ser Gly Tyr Ser Tyr 1 5 10 <210> 49 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 49 Gln Ser Ile Val Asn Ser His Gly Asn Thr Tyr 1 5 10 <210> 50 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 50 Leu Ala Ser 1 <210> 51 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 51 Lys Val Ser 1 <210> 52 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 52 Gln His Ser Arg Glu Leu Pro Leu Thr 1 5 <210> 53 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 53 Gln His Ser Arg Glu Leu Pro Trp Thr 1 5 <210> 54 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 54 Phe Gln Gly Ser His Val Pro Trp Thr 1 5 <210> 55 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 55 Gly Phe Thr Phe Ser Asn Tyr Gly 1 5 <210> 56 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 56 Gly Ile Asn Ile Lys Asp Tyr Tyr 1 5 <210> 57 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 57 Ile Ser Ser Gly Gly Ser Tyr Thr 1 5 <210> 58 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 58 Ile Asp Pro Gly Asn Gly Asp Ala 1 5 <210> 59 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 59 Ala Arg His Glu Gly Gly Gly Asn Pro Tyr Phe Asp Tyr 1 5 10 <210> 60 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 60 Asn Glu Ile Ala Tyr 1 5 <210> 61 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 61 Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr 1 5 10 <210> 62 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 62 Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr 1 5 10 <210> 63 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 63 Lys Val Ser 1 <210> 64 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 64 Leu Val Ser 1 <210> 65 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 65 Ser Gln Asn Thr Leu Val Pro Trp Thr 1 5 <210> 66 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR antibody against Clostridium botulinum <400> 66 Trp Gln Gly Thr His Phe Pro Phe Thr 1 5 <210> 67 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Antibodies against Clostridium botulinum <400> 67 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg His Glu Gly Gly Gly Asn Pro Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Leu Thr Val Ser Ser 115 120 <210> 68 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Antibody against Clostridium botulinum <400> 68 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Asn 85 90 95 Thr Leu Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 69 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Antibody against Clostridium botulinum <400> 69 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Ile Asn Ile Lys Asp Tyr 20 25 30 Tyr Met His Trp Met Lys Gln Arg Pro Glu Gln Asp Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asp Pro Gly Asn Gly Asp Ala Glu Tyr Ala Pro Lys Phe 50 55 60 Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Asn Glu Ile Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 100 105 110 <210> 70 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Antibody against Clostridium botulinum <400> 70 Asp Ile Val Met Thr Gln Ser Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 71 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Antibody against Clostridium botulinum <400> 71 Asp Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Ser Val Gly Thr Arg Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Leu Leu Lys Ser Val Thr Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Lys Gly Glu Tyr Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ala 115 <210> 72 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Antibodies against Clostridium botulinum <400> 72 Asp Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 73 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Antibodies against Clostridium botulinum <400> 73 Gln Val Gln Leu Val Glu Thr Gly Gly Gly Leu Val Gln Pro Lys Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Asn 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Leu 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Tyr Gly Arg Ser Tyr Gly Gly Leu Ser Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ala 115 120 <210> 74 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Antibody against Clostridium botulinum <400> 74 Asp Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Ser Ser 20 25 30 Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 75 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Antibody against Clostridium botulinum <400> 75 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Asn 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Ser Asp Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Pro Ser Met 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Tyr Gly Arg Ser Tyr Gly Gly Leu Gly Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ala 115 120 <210> 76 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Antibody against Clostridium botulinum <400> 76 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Val His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 77 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Antibody against Clostridium botulinum <400> 77 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Met 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg Gln Val Thr Thr Ala Val Gly Gly Phe Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Glu 115 120 <210> 78 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Antibody against Clostridium botulinum <400> 78 Asp Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Thr Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Ser Ser 20 25 30 Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Arg 100 105 110 <210> 79 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Antibody against Clostridium botulinum <400> 79 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Gln Met Ser Cys Lys Ala Phe Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Thr Met His Trp Val Arg Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Phe Ile Asn Pro Ser Ser Asp Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Ser Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Ile Phe Tyr Asn Gly Arg Thr Tyr Ala Ala Met Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 80 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Antibody against Clostridium botulinum <400> 80 Asp Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Ser Ser 20 25 30 Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 81 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Antibody against Clostridium botulinum <400> 81 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Thr Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Met 65 70 75 80 Leu Tyr Leu Gln Ile Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Gly Gln Ile Leu Tyr Tyr Tyr Val Gly Ser Pro Ala Trp 100 105 110 Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 115 120 125 <210> 82 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Antibody against Clostridium botulinum <400> 82 Asp Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Phe Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 83 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Antibody against Clostridium botulinum <400> 83 Asp Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Arg Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Ser Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Asp Ser Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ala Leu Thr Val Ser Ser 115 <210> 84 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Antibody against Clostridium botulinum <400> 84 Asp Ile Val Met Thr Gln Ser Ser Ser Tyr Leu Ser Val Ser Leu Gly 1 5 10 15 Gly Arg Val Thr Ile Thr Cys Lys Ala Ser Asp His Ile Asn Asn Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Arg Leu Leu Ile 35 40 45 Ser Asp Thr Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Lys Asp Tyr Thr Leu Ser Ile Thr Ser Leu Gln Thr 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Ala Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 85 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Antibody against Clostridium botulinum <400> 85 Glu Val Gln Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Leu Val 35 40 45 Ala Thr Ile Asn Ser Asn Gly Gly Thr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Ser Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Asp Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val 100 105 110 Thr Val Ser Ser 115 <210> 86 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Antibody against Clostridium botulinum <400> 86 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val Asn Ser 20 25 30 His Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 87 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Antibody against Clostridium botulinum <400> 87 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Pro Tyr Asn Gly Asp Thr Gly Tyr Asn Gln Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Asn Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Gly Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser 100 105 110 Ser <210> 88 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Antibodies against Clostridium botulinum <400> 88 Asp Val Leu Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asn Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Ser Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr Leu Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 89 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> Antibodies against Clostridium botulinum <400> 89 Gln Ile Gln Leu Ala Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Ser Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Phe 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Gly Tyr Tyr Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val 100 105 110 Ser Ser <210> 90 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Antibody against Clostridium botulinum <400> 90 Asp Val Leu Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110

Claims

1. An N2-42F cell line, with the accession number KCTC 13712BP.

2. A method for determining the activity of botulinum toxin, comprising the following steps: (a) Culturing the cells as described in claim 1; (b) Treat the cells with botulinum toxin; and (c) By specifically binding to SNAP25 FL Or SNAP25 197 Antibody assay of SNAP25 in the cells FL Or SNAP25 197 ; The botulinum toxin mentioned is selected from BoNT / A and BoNT / C1.

3. The method of claim 2, wherein the cells are cultured in a solid matrix coated with poly-D-lysine.

4. The method of claim 2, wherein the botulinum toxin is botulinum toxin serotype A.

5. The method of claim 2, wherein SNAP25 is measured. FL Or SNAP25 197 It is used to determine the cleavage of endogenous neurosecretory proteins caused by botulinum toxin.

6. A method for detecting botulinum toxin, comprising the following steps: (a) Culturing the cells as described in claim 1; (b) Treat the cells with botulinum toxin; (c) By specifically binding to SNAP25 197 Antibody assay of SNAP25 in the cells 197 ;and (d) When antibody-SNAP25 is detected 197 When the complex is present, it confirms the presence of botulinum toxin in the sample. The botulinum toxin mentioned is selected from BoNT / A and BoNT / C1.

7. The method of claim 6, wherein the cells are cultured in a solid matrix coated with poly-D-lysine.

8. The method of claim 6, wherein the botulinum toxin is botulinum toxin serotype A.

9. A method for analyzing the potency of a neurotoxin, comprising the following steps: (a) N2-42F cells were cultured, with the accession number KCTC 13712BP; (b) Treat the cells with botulinum toxin; (c) Used with SNAP25 FL and SNAP25 197 The cells or sample obtained from step (b) are treated with a specific binding antibody; (d) Used with SNAP25 197 Specifically binds to but does not bind to SNAP25 FL Antibody treatment of the cells or samples obtained in step (c); The neurotoxin is selected from BoNT / A and BoNT / C1.

10. The method of claim 9, wherein the neurotoxin in step (b) is diluted with a culture medium containing GT1b and used to treat the cells.

11. A method for reducing the detection limit of the method as described in claim 2, comprising: The N2-42F cells as described in claim 1 are treated with a neurotoxin in a cell culture medium containing GT1b; wherein the neurotoxin is BoNT / A.

12. The method of claim 11, wherein the concentration of GT1b in the cell culture medium is 30 to 70 μg / ml.

13. The method of claim 11, further comprising creatine and arginine.

14. The method of claim 11, wherein the creatine concentration in the cell culture medium is 0.1 to 10 mM, and the arginine concentration in the cell culture medium is 0.5 to 50 mM.

15. The method of claim 11, wherein the cell culture medium is RPMI 1640 (Roswell Park Memorial Institute 1640) medium.

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