KR102251096B1 - A cell-based method for determining an activity of botulinum toxin - Google Patents

Abstract

The present invention relates to a method of determining botulinum toxin activity. Currently, in the field of botulinum toxin titer measurement, there is a need to develop a cell-based titer assay (CBPA) to replace the mouse LD50 bioassay (mLD50). The present invention relates to an optimal CBPA assay using a monoclonal antibody having remarkably high binding affinity and specificity for N2-42F cells and SNAP25, and it is possible to measure a titer of 0.5 U or less to botulinum toxin. The CBPA assay using the cells and antibodies of the present invention is expected to be a highly reliable and reproducible cell-based titer assay for botulinum toxin.

Description

A cell-based method for determining an activity of botulinum toxin}

The present invention relates to a method for determining botulinum toxin activity using a neuronal cell line.

Currently, the mouse LD50 bioassay (mLD50) is a generally accepted method for the detection of botulinum toxin (BoNT) present in food, clinical, or environmental samples. In particular, in the pharmaceutical field, mLD50 is used as a standard assay for measuring the titer of botulinum toxin used for aesthetic or medical purposes. However, since the titer measurement of botulinum toxin using mLD50 is highly affected by testers, test institutes, and facilities, it is difficult to accurately and reproducibly quantify the biological efficacy of botulinum toxin. Accordingly, in order to minimize the number and pain of animals used in the experiment and standardize the botulinum toxin titer assay, a ZEBET conference was held to encourage the development of an alternative assay for mLD50 (Altern Lab Anim. 2010 Aug;38(4):315). -30). In various countries, many laboratories and industries have undertaken various studies to develop cell-based titer assays (CBPAs) or cell-based bioassays (CBB) that are satisfactory to replace mLD50 in terms of specificity, sensitivity, and reproducibility. Considerations for the successful construction of CBPA or CBB are: ① _{a monoclonal or polyclonal antibody specific for SNAP25 197} , and ② a neuronal cell line that exhibits high sensitivity to low concentration (~pM) botulinum toxin. In early 2004, Dr. Chapman and his colleagues invented a fluorescence reporter assay using botulinum toxin fused with two fluorescent proteins (Proc Natl Acad Sci USA. 2004 Oct 12;101(41):14701-6), and the BoCell ^TM assay. It was commercialized by naming it as (BioSentinel Inc). However, despite the significance of the BoCell ^TM assay as the first assay capable of detecting the endopeptidase activity of botulinum toxin with animal cells cultured in 98-well culture dishes, there was a problem that the sensitivity was 2-3 times lower than that of the mouse assay. (Appl Environ Microbiol vol. 78,21 (2012): 7687-97).

As described above, efforts around the world are continuing to develop CBPA to replace mLD50, so ultimately the analysis method using animals is expected to be discontinued. Although challenges remain to be solved for effective CPBA development, effective CBPA development facilitates the expansion of the usability of various botulinum toxin products, improves the quality control level, and provides a higher level of trust to consumers, thereby providing botulinum toxin products. Will inspire the competitiveness of the company.

Accordingly, the present inventors have made great efforts to overcome the limitations of the conventional CPBA, thereby completing the present invention. The present invention relates to an optimal CBPA assay using a monoclonal antibody having remarkably high binding affinity and specificity for N2-42F cells and SNAP25, and it is possible to measure a titer of 0.5 U or less to botulinum toxin. The CBPA assay using the cells and antibodies of the present invention is expected to be a highly reliable and reproducible cell-based titer assay for botulinum toxin.

An object of the present invention is to provide a cell-based method for determining botulinum toxin activity.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems that are not mentioned will be clearly understood by those of ordinary skill in the art from the following description.

Hereinafter, various embodiments described herein are described with reference to the drawings. In the following description, for a thorough understanding of the present invention, various specific details, such as specific forms, compositions and processes, etc. are set forth. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well-known processes and manufacturing techniques have not been described in specific details in order not to unnecessarily obscure the present invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the present invention. Thus, the context of “in one embodiment” or “an embodiment” expressed in various places throughout this specification does not necessarily represent the same embodiment of the invention. Additionally, particular features, forms, compositions, or properties may be combined in one or more embodiments in any suitable manner.

Unless otherwise defined in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

Botox® manufacturer Allergan has successfully _{constructed a cell line producing a monoclonal antibody specific to SNAP25 197} and a neuronal cell line highly sensitive to botulinum toxin from a known host cell, human neuroblastoma SiMa (SiMa) (PLoS). One.2012;7(11):e49516). With the above antibodies and cells, Allergan developed a new CBPA assay and was approved by the FDA in 2010 as the first CBPA capable of replacing mLD50 (US8455213B2, and US2010/0204559A1). Another botulinum toxin manufacturer, German MERZ, developed CBA-ELISA in 2014 (WO2014/207109A1) and obtained FDA approval in 2015. CBA-ELISA uses a differentiated nerve cell fixation method (in-situ), penetrates the cell membrane, and immunologically detects endogenous SNAP25. Allergan's CBPA and MERZ's CBA-ELISA are equivalent to the mouse bioassay method, with an EC50 of sub-picomolar concentration (ie <1.0 U/well). It shows excellent sensitivity enough to show a number. The technology of Allergan and MERZ detects SNAP25 under optimal conditions using a common commercial rabbit polyclonal antibody antibody (Sigma S9684). In the case of cells, Allergan's CBPA uses the differentiated human neuroma cell line SiMa exclusively, and MERZ's CBA-ELISA uses human differentiated induced pluripotent stem cells (iPS) as standardized and optimized host cells. SiMa proliferates very slowly and generally requires a population doubling time (PDT) of more than 70 hours to double the number of cells, and iPS generation is also very time consuming, and storage is more difficult. Therefore, there is a need for development of a neuronal cell line for CBPA that is very sensitive to botulinum toxin, has a short PDT, and can be easily stored. In addition, a research group from the University of Wisconsin Dr. David Beebe pointed out that SiMa has a problem with the suitability of SiMa as a cell for CBPA because SiMa does not show motor-like characteristics (J Biomol Screen. 2016 Jan;21(1):65-73 ), an alternative CBPA was developed that uses the motor neuron-like cell line NG108-15, which shows an EC50 level of 7.9 pM or less, as a host cell. However, since the developed CBPAs _{determine the endogenous levels of SNAP25 197} and SNAP25 _FL using Western blotting analysis, they are difficult to use in high-throughput assays.

In one embodiment of the present invention, "Botulinum Toxin" is a neurotoxin protein made by Clostridium botulinum bacteria. There are more than 127 species of the genus Clostridium, and they are classified according to their form and function. The anaerobic, Gram-positive bacterium Clostridium botulinum produces botulinum poison, a potent polypeptide neurotoxin that causes a neuroparalytic disease called botulism in humans and animals. Spores of Clostridium botulinum are found in soil and can be cultured in poorly sterilized, sealed, home canned food containers, which contributes to a lot of botulism. Symptoms of botulism typically appear 18 to 36 hours after eating Clostridium botulinum cultures or spore-infected foods. Botulinum toxin appears to be able to pass through the intestinal lining without reducing its toxicity and has a high affinity for cholinergic motor neurons. Symptoms of botulinum toxin poisoning may progress due to impaired walking, dysphagia and speech, paralysis of the respiratory muscles, and death. Botulinum toxin has been used clinically for the treatment of neuromuscular disorders (ie, motor disorders) characterized by hyperactive skeletal muscle. In 1989, the botulinum toxin type A complex was approved by the U.S. Food and Drug Administration for use in the treatment of essential blepharospasm, strabismus and hemifacial spasm. Subsequently, botulinum toxin type A was also approved by the FDA for the treatment of cervical dystonia and for the treatment of glabellar wrinkles, and botulinum toxin type B was also approved for the treatment of cervical dystonia. Botulinum serotypes other than toxin type A appear to have a lower potency and/or shorter duration in activity when compared to botulinum toxin type A. The clinical effect of botulinum toxin type A injected into the peripheral muscle usually occurs within a week after injection. The typical duration of symptom relief by a single intramuscular injection of botulinum toxin type A is about 3 months on average, but therapeutic activity over a significantly longer period has been reported. Although all botulinum toxin serotypes appear to inhibit the release of the neurotransmitter acetylcholine at the neuromuscular junction, they act on different neurosecretory proteins and cleave this protein at different sites. For example, botulinum types A and E both cleave a 25 kDa synaptosome related protein (SNAP25), but target different amino acid sequences of this protein. Botulinum toxin types B, D, F and G act on vesicle-associated proteins (VAMP, so-called synaptobrebin), and each serotype cleaves a different site of this protein. Finally, botulinum toxin type C1 appears to cleave both syntaxin and SNAP25. This difference in mechanism of action will affect the relative potency and/or duration of the action of the different botulinum toxin serotypes. In particular, substrates of botulinum toxin can be found in several different cell types. In addition, in vitro studies have shown that botulinum toxin inhibits the induced release of potassium cation of acetylcholine and norepinephrine from primary cell cultures of brain stem tissue. In addition, botulinum toxin inhibits the induced release of glycine and glutamate in the primary culture of spinal neurons, and the neurotransmitters acetylcholine, dopamine, norepinephrine, CGRP, substance P (Substance P) and glutamate, respectively It has been reported to inhibit release. Therefore, when used in an appropriate concentration, stimulation-induced release of most neurotransmitters is inhibited by botulinum toxin.

In one embodiment of the present invention, "toxin activity" means 1 U (1 unit) of botulinum toxin as measured by the standard assay method, mouse LD50 bioassay (mLD50), 50% mortality rate of 18-20 g rats It represents the intrinsic potency of the botulinum toxin. The botulinum toxin, especially botulinum toxin serotype A, is the most lethal natural biological agent known to humans.It is 1.8 billion times more than diphtheria, 600 million times more than sodium cyanide, 30 million times more than cobra toxin, and 12 million times more than cholera. Since it is a million times more lethal, a difference in titer of about 20% in botulinum toxin results in a significant difference in effect, such as 380,000,000 times diphtheria, or 2.4 million times cholera.

Botulinum toxin preparations for medical or cosmetic purposes are usually freeze-dried or distributed in liquid formulations. Since botulinum toxin itself is a protein, it is activated by temperature, pH, light, physical shock, or gas (air, nitrogen, oxygen, etc.). There is a very unstable problem. When the titer of the botulinum toxin is reduced in this way, it is difficult to exhibit the expected effect of the botulinum toxin. Therefore, it is necessary to accurately predict the titer of the botulinum toxin at the manufacturing stage or the use stage.

In one embodiment of the present invention, the term "antibody" is a term known in the art and refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to the SNAP25 protein, and such an antibody can be prepared by a conventional method. The antibody of the present invention also includes partial peptides that can be made from the protein, and the partial peptide of the present invention contains at least 7 amino acids, preferably 9 amino acids, and more preferably 12 or more amino acids. The form of the antibody of the present invention is not particularly limited, and a polyclonal antibody, monoclonal antibody, or any one having antigen-binding properties is also included in the antibody of the present invention, and all immunoglobulin antibodies are included. Furthermore, the antibody of the present invention also includes special antibodies such as humanized antibodies. The 　 antibody of the present invention includes not only a complete form having a light chain and a heavy chain, but also a functional fragment of an antibody molecule. A functional fragment of an antibody molecule refers to a fragment that has at least an antigen-binding function, and includes Fab, F(ab'), F(ab')2, and Fv.

In one embodiment of the present invention, "kit" means a set of compositions and accessories necessary for a specific purpose. For the purposes of the present invention, the kit of the present invention _{contains an antibody that specifically binds to SNAP25 FL} or SNAP25 ₁₉₇ in order to measure the activity of botulinum toxin, contains a composition containing the antibody, or is coated with the antibody. Cell culture dishes may be included.

In one embodiment of the present invention, (a) culturing neurons of Neuro-2a origin; (b) treating the neurons with neurotoxins; (c) treating the neuron or a sample obtained from the neuron with an antibody that specifically binds _{to SNAP25 FL} and SNAP25 _197; And (d) _{treating an antibody that specifically binds only to SNAP25 197} without binding _{to SNAP25 FL} to (c); providing a cell-based titer assay method of neurotoxin comprising, and the Neuro-2a origin The cell line of N2-42F (accession number: KCTC 13712BP) provides a cell-based titer analysis method of neurotoxin, characterized in that the cell line, wherein the neurotoxin is a botulinum toxin cell-based titer analysis method of neurotoxin It provides a cell-based titer analysis method of neurotoxin, characterized in that the neurotoxin of step (b) is diluted with a medium containing GT1b (Ganglioside GT1b trisodium salt) and treated.

In the antibody of step (c), the heavy chain CDR1 region consists of SEQ ID NO: 55 or 56, the heavy chain CDR2 region consists of SEQ ID NO: 57 or 58, and the heavy chain CDR3 region consists of SEQ ID NO: 59 or 60. , The light chain CDR1 region consists of SEQ ID NO: 61, or 62, the light chain CDR2 region consists of SEQ ID NO: 63, or 64, and the light chain CDR3 region consists of SEQ ID NO: 65 or 66. Provides a method for titer analysis, and more specifically, the antibody has a heavy chain CDR1 region of SEQ ID NO: 55, a heavy chain CDR2 region of SEQ ID NO: 57, a heavy chain CDR3 region of SEQ ID NO: 59, a light chain CDR1 region of SEQ ID NO: 61, and a light chain CDR2 region. This SEQ ID NO: 63, and the light chain CDR3 region provides a cell-based titer analysis method of a neurotoxin, characterized in that consisting of SEQ ID NO: 65, wherein the antibody has a heavy chain CDR1 region of SEQ ID NO: 56, a heavy chain CDR2 region of SEQ ID NO: 58 Provides a cell-based titer analysis method of neurotoxin, characterized in that the heavy chain CDR3 region consists of SEQ ID NO: 60, the light chain CDR1 region is SEQ ID NO: 62, the light chain CDR2 region consists of SEQ ID NO: 64, and the light chain CDR3 region consists of SEQ ID NO: 66. do.

In addition, the antibody of step (d) is composed of any one selected from the group consisting of the heavy chain CDR1 region consisting of SEQ ID NOs: 28 to 33, and the heavy chain CDR2 region consisting of any one selected from the group consisting of SEQ ID NOs: 34 to 39, , The heavy chain CDR3 region is composed of any one selected from the group consisting of SEQ ID NOs: 40 to 46, the light chain CDR1 region is composed of any one selected from the group consisting of SEQ ID NOs: 47 to 49, and the light chain CDR2 region is SEQ ID NO: 50 It is composed of any one selected from the group consisting of 51, and the light chain CDR3 region provides a cell-based titer analysis method of a neurotoxin, characterized in that consisting of any one selected from the group consisting of SEQ ID NOs: 52 to 54, More specifically, in the antibody, the heavy chain CDR1 region is SEQ ID NO: 28, the heavy chain CDR2 region is SEQ ID NO: 34, the heavy chain CDR3 region is SEQ ID NO: 40, the light chain CDR1 region is SEQ ID NO: 47, the light chain CDR2 region is SEQ ID NO: 50, and the light chain CDR3 It provides a cell-based titer analysis method of neurotoxin, characterized in that the region consists of SEQ ID NO: 52, wherein the antibody has a heavy chain CDR1 region of SEQ ID NO: 29, a heavy chain CDR2 region of SEQ ID NO: 35, and a heavy chain CDR3 region of SEQ ID NO: 41 , Provides a cell-based titer analysis method of neurotoxin, characterized in that the light chain CDR1 region consists of SEQ ID NO: 48, the light chain CDR2 region consists of SEQ ID NO: 50, and the light chain CDR3 region is SEQ ID NO: 52, wherein the antibody is a heavy chain CDR1 region. This SEQ ID NO: 29, the heavy chain CDR2 region is SEQ ID NO: 36, the heavy chain CDR3 region is SEQ ID NO: 42, the light chain CDR1 region is SEQ ID NO: 47, the light chain CDR2 region is SEQ ID NO: 50, and the light chain CDR3 region consists of SEQ ID NO: 52. It provides a cell-based titer analysis method of a neurotoxin, wherein the antibody has a heavy chain CDR1 region of SEQ ID NO: 33, a heavy chain CDR2 region of SEQ ID NO: 35, and It provides a cell-based titer analysis method of neurotoxin, characterized in that the chain CDR3 region consists of SEQ ID NO: 43, the light chain CDR1 region consists of SEQ ID NO: 48, the light chain CDR2 region consists of SEQ ID NO: 50, and the light chain CDR3 region consists of SEQ ID NO: 52, , The antibody has a heavy chain CDR1 region of SEQ ID NO: 30, a heavy chain CDR2 region of SEQ ID NO: 37, a heavy chain CDR3 region of SEQ ID NO: 44, a light chain CDR1 region of SEQ ID NO: 48, a light chain CDR2 region of SEQ ID NO: 50, and a light chain CDR3 region of SEQ ID NO: It provides a cell-based titer analysis method of neurotoxin, characterized in that consisting of number 52, wherein the antibody has a heavy chain CDR1 region of SEQ ID NO: 31, a heavy chain CDR2 region of SEQ ID NO: 38, a heavy chain CDR3 region of SEQ ID NO: 45, and a light chain CDR1 It provides a cell-based titer analysis method of neurotoxin, characterized in that the region consists of SEQ ID NO: 47, the light chain CDR2 region is SEQ ID NO: 50, and the light chain CDR3 region is SEQ ID NO: 53, wherein the antibody has a heavy chain CDR1 region of SEQ ID NO: 32, a heavy chain CDR2 region consisting of SEQ ID NO: 39, a heavy chain CDR3 region consisting of SEQ ID NO: 46, a light chain CDR1 region consisting of SEQ ID NO: 49, a light chain CDR2 region consisting of SEQ ID NO: 51, and a light chain CDR3 region consisting of SEQ ID NO: 54 Provides a method for cell-based titer analysis of toxins.

In another embodiment of the present invention, a cell culture solution for treating neurotoxins for neurons containing GT1b (Ganglioside GT1b trisodium salt) is provided, and the concentration of GT1b in the culture solution is 25 to 75 μg/ml. Provides a cell culture solution for treating neurotoxins for cells, wherein the culture solution provides a cell culture solution for neurotoxin treatment for neurons further comprising creatine and arginine, and the creatine concentration in the culture solution is 0.1 to 10 mM , Arginine concentration is 0.5 to 50 mM to provide a cell culture solution for neurotoxin treatment for neurons, wherein the culture solution is RPMI 1640 (Roswell Park Memorial Institute 1640) medium for nerve cells, characterized in that It provides a cell culture solution for treatment of toxins.

Hereinafter, the present invention will be described in detail step by step.

Recently, the demand for botulinum toxin for medical and cosmetic purposes is rapidly increasing, but there is no cell-based titer analysis method with high stability and reproducibility that can be used to measure the titer of botulinum toxin. Since botulinum toxin is a very powerful neurotoxin protein, the development of a precise cell-based titer measurement method with high reproducibility is required above all.

The present invention relates to an optimal CBPA assay using a monoclonal antibody having remarkably high binding affinity and specificity for N2-42F cells and SNAP25, and it is possible to measure a titer of 0.5 U or less to botulinum toxin. The CBPA assay using the cells and antibodies of the present invention is expected to be a highly reliable and reproducible cell-based titer assay for botulinum toxin.

1 is a Western blotting analysis result of measuring sensitivity to botulinum toxin A (BoNT/A) in neurons according to an embodiment of the present invention. Lane M is a protein size marker, and lane 1 is BoNT. /A total cell lysate without toxin, lane 2 shows the level of expression of the SNAP25 protein of BoNT/A toxinated total cell lysate. In addition, N2a represents Neuro-2a cells, and K-BM1 represents KP-N-RT-BM-1 cells.
Figure 2 is a cut level of SNAP25 illustrates the results verified by Western blotting analysis, a clone selection procedure of step 2 (2 ^nd clonal selection) identified in clone selection procedure of step 3 in accordance with one embodiment of the present invention in significantly of 42 6 clones containing the clone showing the cutting phenomenon of SNAP25 by BoNT / a it was selected only 42 clones, selected in the step 3 clones course (3 ^rd clonal selection) 24 times clone (42F) in Shows the phenomena of cleavage of SNAP25 by BoNT/A consistently in a plurality of identical experiments.
3 shows the results of confirming the degree of cleavage of SNAP25 through Western blotting analysis, which appears when various concentrations of BoNT/A are treated in SiMa and N2-42F cells according to an embodiment of the present invention.
4 shows the results of confirming the phylogenetic stability of N2-42F obtained through the clone selection process of the present invention through Western blotting analysis according to an embodiment of the present invention.
5 shows the results of confirming the antigen binding specificity of the monoclonal antibody prepared in the present invention by Western blotting according to an embodiment of the present invention.
6 shows the results of optimizing the toxin time in the method for determining botulinum toxin activity according to an embodiment of the present invention.
7 shows the results of optimizing a toxin medium in a method for determining botulinum toxin activity according to an embodiment of the present invention.
8 shows the results of optimizing the sensitizer in the method for determining botulinum toxin activity according to an embodiment of the present invention.
9 shows the results of optimizing GT1b in a method for determining botulinum toxin activity according to an embodiment of the present invention.
10 shows the results of optimizing N2/B27 in the method for determining botulinum toxin activity according to an embodiment of the present invention.
11 shows the optimization results for the capture antibody treatment in the method for determining botulinum toxin activity according to an embodiment of the present invention.
12 shows an optimization result for treatment with a detection antibody in a method for determining botulinum toxin activity according to an embodiment of the present invention.
13 shows an optimization result of a method for detecting the activity of an HRP conjugate in a method for determining botulinum toxin activity according to an embodiment of the present invention.
14 is a schematic diagram of a sandwich ELISA method for determining botulinum toxin activity of the present invention according to an embodiment of the present invention.
15 shows the results of confirming the accuracy and linearity of the sandwich ELISA method for determining botulinum toxin activity according to an embodiment of the present invention.
FIG. 16 shows the results of measuring biotiter in a sandwich ELISA method for determining botulinum toxin activity according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example 1. Preparation of BoNT/A-sensitive neurons

Example 1-1. Screening of cells sensitive to BoNT/A

Neurons Neuro-2a, SiMa, and KP-N-RT-BM-1 cells were dispensed into 24-well plates, respectively. Then, by confirming the cleavage phenomenon of the SNAP25 protein to BoNT/A through Western blotting analysis, Neuro-2a (N2a), SiMa and K-BM1 (KP-N-RT-BM-1) cells Confirmed that SNAP25 was cleaved by 33%, 66%, and 29%, respectively, by BoNT/A. The results are shown in FIG. 1.

Based on the above results, Neuro-2a continued to be used for the following reasons for clonal selection. First, the in vivo efficacy of BoNT/A, which is usually measured by the mortality rate of mice (ie, mLD50), can be better expressed in mice cells. Second, Neuro-2a is a mouse cell, but KP-N-RT-BM-1 is a human cell, and KP-N-RT-BM-1 grows at a significantly slower rate compared to Neuro-2a. SiMa cells, which are human neuroblastoma cells, have a doubling time of 34 to 100 hours. Third, in the case of Neuro-2a cells observed under a microscope, it was identified as a mixed population of various cell types. Therefore, it was determined that there is a possibility that cells sensitive to BoNT/A may exist in the heterogeneous Neruo-2a cell population.

Example 1-2. Development of Neuro-2a Clone Sensitive to BoNT/A

Neuro-2a cells contain 10% fetal calf serum, 1x non-essential amino acid (1x NEAA), 1x sodium pyruvate, 1x GlutaMAX ^TM and 1x antibiotic antimycotic (1x antibiotic antimycotic; 1x AA). ) To be cultured in MEM culture medium containing conditions.

When the cell density (confluence) reached 80 to 90%, 1x ^{TrypLE (Gibco™} , 12604021, USA) was treated to make the cells a single cell suspension. Then, the viability of single cells was measured using trypan blue and a hemocytometer, and based on this, the single cell suspension was diluted to a density of 10 cells per 1 ml. Then, 100 µl of the diluted single cell suspension was added to a 96-well plate. Colony growth was periodically observed through microscopic examination. When the single cells reached a density of 60%, they were transferred to a 24-well plate in the same manner as above. Cell clones transferred to a 24-well plate were divided equally by half, and half were tested for sensitivity to BoNT/A toxin, and the other half were stored in a liquid nitrogen freezer.

For the BoNT/A toxin test of the cell clone, a total of 672 cell clones were sub-cultured from a 24 well plate to a 7×96 well microplate. The next day, the culture medium of the cell clone was replaced with RPMI 1640 containing 2 mM L-alanyl-L-glutamine, 1x B27, 1x N2 and 1x NEAA (differentiation medium) to induce neural differentiation. At the 4th day after neural differentiation was induced, GT1b (diluted in 1x non-toxinized medium) was added to the differentiation medium so that the final concentration became 25 µg/ml, and cultured for 24 hours. Thereafter, the medium was replaced with a toxin medium containing 0.1 nM BoNT/A, and further cultured for 2 days. Then, Western blotting analysis was performed in the same manner as described in Example 1 to confirm the degree of cleavage of SNAP25 to perform a primary clone selection process. This clone selection process was repeated 3 times, and the results confirmed in each clone selection process are shown in FIG. 2.

As shown in FIG. 2, sensitivity to BoNT/A was tested according to the clone selection process optimized for Allergan's SiMa cells, which are slightly modified for 142 clones. As a result, it was confirmed that the sensitivity to BoNT/A was remarkably higher in 19 positive clones than in the parent cell Neuro-2a through the clonal selection process 3 times. In particular, among the positive clones selected in the multiple clone selection process, only the 42nd positive clone consistently showed a significantly higher sensitivity to BoNT/A compared to the parent cell, Neuro-2a. Through this process, the positive clone No. 42 finally selected was named N2-42F according to the nomenclature recommended in Rust and Pollok, 2011 and Sarntivijai et al., 2008, and was entrusted with the Korea Research Institute of Bioscience and Biotechnology (accession number: KCTC13712BP, Consignment date: 20181113).

Example 1-3. Confirmation of the cleavage phenomenon of SNAP25 by BoNT/A in N2-42F

Using SiMa cells as a control, 1x toxinization medium containing various concentrations of BoNT/A was added to N2-42F cultured in a 96-well plate coated with poly-D-lysine, and BoNT/A sensitivity of N2-42F Was measured. Specifically, after incubating N2-42F for 4 days under the above conditions, 50 µl of 1x SDS sample buffer was treated with the obtained N2-42F. Then, Western blotting analysis was performed to determine the cleavage of the SNAP25 protein for BoNT/A, and the results are shown in FIG. 3. All of these experiments were repeated three times.

As shown in FIG. 3, the cleavage of endogenous SNAP25 by BoNT/A of 0.93 pM or less was found to be inadequate in both SiMa cells and N2-42F. When 2.78-25 pM of BoNT/A was treated, the degree of cleavage of SNAP25 was found to be 25-75% in N2-42F and 33-75% in SiMa cells. From the above results, it was found that N2-42F according to the present invention was as sensitive to BoNT/A as SiMa cells.

Example 1-4. Checking the system stability of N2-42F

In order to be used as a cell-based assay platform, the lineage stability of neurons can be a very important factor. Thus, the systemic stability of N2-42F was confirmed.

N2-42F continued to be cultured in a number of passages. In order to prepare the master cell bank, the initial passage N2-42F was used, and the clone was stored in a liquid nitrogen freezer every five passages to maintain the stability of the clone. Here, after thawing the N2-42F stored in passages 5 and 15 from liquid nitrogen, when the density of the N2-42F becomes about 90%, the sensitivity to BoNT/A was examined, and the results are shown in FIG. Indicated. All of these experiments were repeated three times, and SiMa cells were used as a control. As shown in Fig. 4, the cleavage of SNAP25 indicated by BoNT/A was 63% (lane 6), 66% (lane 7) and 68% at passage 5 N2-42F (N2-42F(P5)). (Lane 8), and was confirmed to be 63 (lane 6), 73% (lane 7) and 71% (lane 8) at passage 15 of N2-42F (N2-42F (P15)).

Through the above results, it was confirmed that the sensitivity to BoNT/A was maintained not only at passage 5 but also at passage 15 in N2-42F obtained from the parent Neuro-2a, similar to the H1 clone of SiMa cells constructed by Allergan. . Therefore, it can be seen that N2-42F is a very suitable clone that can be used in a cell-based assay platform after the H1 clone of SiMa cells.

Example 2. Preparation of SNAP25 monoclonal antibody

Example 2-1. Preparation and sequence analysis of SNAP25 monoclonal antibody

Polyclonal antibody serum was obtained by injecting the peptide antigens of Table 1 to 2 rabbits, and monoclonal serum was obtained by injection to 4 mice. From this _{, three different types of antibodies having binding specificity for ①SNAP25 FL} , ②SNAP25 ₁₉₇ , or ③SNAP25 _FL , and SNAP25 ₁₉₇ were prepared, and the sequence of the monoclonal antibody was analyzed to determine the CDR sequences, and the V _H , and V of the prepared antibodies. _{The L} domain sequence is shown in Tables 2 to 5. The CDR sequences are summarized as ① SNAP25 _FL specific IgGs (Table 2), ② SNAP25 ₁₉₇ specific IgGs (Table 3), or ③ _{SNAP25 FL} , and SNAP25 ₁₉₇ specific IgGs (Table 4), and the V _{H of the prepared antibody} , And V _L domain sequences are summarized in Table 5 below.

Immunogens/AA Position in SNAP25 Sequence number AA Sequence N peptide 1-13 SEQ ID NO: 1 KLH-C-MAEDADMRNELEE 1-13 SEQ ID NO: 2 MAEDADMRNELEE-C-KLH 19-38 SEQ ID NO: 3 KLH-C-DQLADESLESTRRMLQLVEE 51-70 SEQ ID NO: 4 KLH-C-DEQGEQLERIEEGMDQINKD M peptide 122-136 SEQ ID NO: 5 KLH-C-DEREQMAISGGFIRR C peptide 170-184 SEQ ID NO: 6 KLH-C-EIDTQNRQIDRIMEK 180-194 SEQ ID NO: 7 KLH-C-RIMEKADSNKTRIDE 180-197 SEQ ID NO: 8 KLH-C-RIMEKADSNKTRIDEANQ 186-197 SEQ ID NO: 9 KLH-C-DSNKTRIDEANQ 189-201 SEQ ID NO: 10 KLH-C-KTRIDEANQPATK

CDR Sequence number Sequence Identified In V _H CDR1 SEQ ID NO: 11 GYSITSGYY D2 SEQ ID NO: 12 GYTFTDYN D6 SEQ ID NO: 13 GYTFTNYG E6 V _H CDR2 SEQ ID NO: 14 IRYDGSN D2 SEQ ID NO: 15 IYPYNGDT D6 SEQ ID NO: 16 INTYTGEP E6 V _H CDR3 SEQ ID NO: 17 ARDRDSSYYFDY D2 SEQ ID NO: 18 VRSGDY D6 SEQ ID NO: 19 ARGYYDY E6 V _L CDR1 SEQ ID NO: 20 DHINNW D2 SEQ ID NO: 21 QSLLDSNGKTY D6 SEQ ID NO: 22 QSLLDSDGKTY E6 V _L CDR2 SEQ ID NO: 23 DTT D2 SEQ ID NO: 24 LVS D6, E6 V _L CDR3 SEQ ID NO: 25 QQYWSAPPT D2 SEQ ID NO: 26 WQGTLFPYT D6 SEQ ID NO: 27 WQGTHFPRT E6

CDR Sequence number Sequence Identified In V _H CDR1 SEQ ID NO: 28 GYSITSDYA C4 SEQ ID NO: 29 GFTFNTNA C7, C14 SEQ ID NO: 30 GYTFTNYT C16 SEQ ID NO: 31 GYTFNTYA C24 SEQ ID NO: 32 GFTFSNYG D3 SEQ ID NO: 33 GFTFNTYA C15 V _H CDR2 SEQ ID NO: 34 ISYSVGT C4 SEQ ID NO: 35 IRSKSNNYAT C7, C15 SEQ ID NO: 36 IRSKSDNYAT C14 SEQ ID NO: 37 INPSSDYT C16 SEQ ID NO: 38 IRSKSNNYTT C24 SEQ ID NO: 39 INSNGGTT D3 V _H CDR3 SEQ ID NO: 40 ARKGEYGFAY C4 SEQ ID NO: 41 VYGRSYGGLSY C7 SEQ ID NO: 42 VYGRSYGGLGY C14 SEQ ID NO: 43 VRQVTTAVGGFAY C15 SEQ ID NO: 44 ARRIFYNGRTYAAMDY C16 SEQ ID NO: 45 VGQILYYYVGSPAWFAY C24 SEQ ID NO: 46 ARDRDAMDY D3 V _L CDR1 SEQ ID NO: 47 KSVSTSGYSY C4, C14, C24 SEQ ID NO: 48 KSVSSSGYSY C7, C15, C16 SEQ ID NO: 49 QSIVNSHGNTY D3 V _L CDR2 SEQ ID NO: 50 LAS C4, C7, C14, C15, C16, C24 SEQ ID NO: 51 KVS D3 V _L CDR3 SEQ ID NO: 52 QHSRELPLT C4, C7, C14, C15, C16 SEQ ID NO: 53 QHSRELPWT C24 SEQ ID NO: 54 FQGSHVPWT D3

CDR Sequence number Sequence Identified In V _H CDR1 SEQ ID NO: 55 GFTFSNYG B4 SEQ ID NO: 56 GINIKDYY B23 V _H CDR2 SEQ ID NO: 57 ISSGGSYT B4 SEQ ID NO: 58 IDPGNGDA B23 V _H CDR3 SEQ ID NO: 59 ARHEGGGNPYFDY B4 SEQ ID NO: 60 NEIAY B23 V _L CDR1 SEQ ID NO: 61 QSLVHSNGNTY B4 SEQ ID NO: 62 QSLLDSDGKTY B23 V _L CDR2 SEQ ID NO: 63 KVS B4 SEQ ID NO: 64 LVS B23 V _L CDR3 SEQ ID NO: 65 SQNTLVPWT B4 SEQ ID NO: 66 WQGTHFPFT B23

Antibody Sequence number order B4 V _H SEQ ID NO: 67 EVKLVESGGGLVKPGGSLKLSCAASGFTFSNYGMSWVRQTPEKRLEWVATISSGGSYTYYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYYCARHEGGGNPYFDYWGQGTTLTVSS V _L SEQ ID NO: 68 DVLMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQNTLVPWTFGGGTKLEIK B23 V _H SEQ ID NO: 69 EVQLQQSGAELVRPGASVKLSCTASGINIKDYYMHWMKQRPEQDLEWIGWIDPGNGDAEYAPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNEIAYWGQGTLVTVSA V _L SEQ ID NO: 70 DIVMTQSPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPFTFGSGTKLEIK C4 V _H SEQ ID NO: 71 DVKLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYISYSVGTRYNPSLKSRISITRDTSKNQFFLLLKSVTNEDTATYFCARKGEYGFAYWGQGTLVTVSA V _L SEQ ID NO: 72 DIVMTQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPLTFGAGTKLELK C7 V _H SEQ ID NO: 73 QVQLVETGGGLVQPKGSLKLSCAASGFTFNTNAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQSLLYLQMNNLKTEDTAMYYCVYGRSYGGLSYWGQGTLVTVSA V _L SEQ ID NO: 74 DIVMTQSPASLAVSLGQRATISCRASKSVSSSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPLTFGAGTKLELK C14 V _H SEQ ID NO: 75 EVKLVESGGGLVQPKGSLKLSCAASGFTFNTNAMNWVRQAPGKGLEWVARIRSKSDNYATYYADSVKDRFTISRDDSPSMLYLQMNNLKTEDTAMYYCVYGRSYGGLGYWGQGTLVTVSA V _L SEQ ID NO: 76 DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYVHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPLTFGAGTKLELK C15 V _H SEQ ID NO: 77 EVKLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVRQVTTAVGGFAYWGQGTLVTVSE V _L SEQ ID NO: 78 DIVMTQSPASLAVSLGQRTTISCRASKSVSSSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPLTFGAGTKLELR C16 V _H SEQ ID NO: 79 EVQLQQSGAELARPGASVQMSCKAFGYTFTNYTMHWVRQRPGQGLEWIGFINPSSDYTNYNQKFKDKATLSADKSSSTAYMQLSSLTSEDSAVYYCARRIFYNGRTYAAMDYWGQGTSVTVSS V _L SEQ ID NO: 80 DIVMTQSPASLAVSLGQRATISCRASKSVSSSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPLTFGAGTKLELK C24 V _H SEQ ID NO: 81 EVKLVESGGGLVQPKGSLKLSCAASGYTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYTTYYADSVKDRFTISRDDSQSMLYLQINNLKTEDTAMYYCVGQILYYYVGSPAWFAYWGQGTLVTVSA V _L SEQ ID NO: 82 DIVMTQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQPPKLLIFLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPWTFGGGTKLEIK D2 V _H SEQ ID NO: 83 DVKLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYIRYDGSNNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTASYYCARDRDSSYYFDYWGQGTALTVSS V _L SEQ ID NO: 84 DIVMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISDTTSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSAPPTFGGGTKLEIK D3 V _H SEQ ID NO: 85 EVQLEESGGGLVQPGGSLKLSCAASGFTFSNYGMSWVRQTPDKRLELVATINSNGGTTYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDSAMYYCARDRDAMDYWGQGTSVTVSS V _L SEQ ID NO: 86 DVLMTQTPLSLPVSLGDQASISCRSSQSIVNSHGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIK D6 V _H SEQ ID NO: 87 EVQLQQSGPELVKPGASVKISCKASGYTFTDYNMHWVKQSHGKSLEWIGYIYPYNGDTGYNQKFKSKATLTVDNSSSTAYMELRSLTSEDSAVYYCVRSGDYWGQGTTLTVSS V _L SEQ ID NO: 88 DVLMTQTPLTLSVTIGQPASISCKSSQSLLDSNGKTYLNWLLQRPGQSPSRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTLFPYTFGGGTKLEIK E6 V _H SEQ ID NO: 89 QIQLAQSGPELKKPGETVKISCKASGYTFTNYGMSWVKQAPGKGLKWMGWINTYTGEPTYAADFKGRFAFSLETSASTAFLQINNLKNEDTATYFCARGYYDYWGQGTTLTVSS V _L SEQ ID NO: 90 DVLMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPRTFGGGTKLEIK

The results of the sequence alignment analysis showed that the CDR sequences of the present invention described in Tables 2 to 4 _{did not overlap with the previously published CDR V L} and V _H sequences (US Patent US8198034B2). This is believed to be due to the fact that the antibody sequence of the present invention is due to rigorous multiple screening for hybridoma positive cells, whereby the antibody of the present invention is expected to have a remarkably low K _{D value.}

Example 2-2. Confirmation of antigen binding specificity of monoclonal antibody

The monoclonal antibody prepared in the present invention was tested for reactivity to _{SNAP25 FL} and SNAP25 _{197 by direct ELISA and Western blotting analysis.} First, direct ELISA was performed with IgG (50 ng per well) purified from _{GST-SNAP25 FL} or GST-SNAP25 _{197 coated microplates.} HRP reaction was performed for 5 minutes, and HRP activity was measured on A450 using Bio-Tek SynergyNeo2. _{The A450 measurement value was calculated by subtracting} the A450 value measured without IgG from the measurement value, and the ratio (Ratio 197/206) of the SNAP25 _{197 measurement value to the SNAP25 FL} measurement value is shown in Table 6 below. Ratio _197/206 measurement results were consistent with the results obtained by BLI analysis, approaching 1.0 for dual specific IgGs such as A15, B4, and B23, and SNAP25 ₁₉₇ specific such as C7, C14, C16, and C24. In the case of IgG, it was 950 or higher, and in the case of SNAP25 _FL- specific IgG, it was found to be 0.02 to 0.03.

IgGs A ₄₅₀ with SNAP25 _FL A ₄₅₀ with SNAP25 ₁₉₇ Ratio _197/206 A15 1.383 1.445 1.04 B4 2.163 1.797 0.83 B23 1.901 1.754 0.92 C4 0.001 0.919 919 C7 0.001 1.199 1,037 C14 0.001 0.953 953 C16 0.001 0.996 996 C24 0.001 0.973 973 D3 0.125 1.616 12.93 D2 1.448 0.048 0.03 D6 1.878 0.031 0.02 E6 1.808 0.039 0.02

Western blotting analysis was performed with the hybridoma culture supernatant (1: 100 dilution), which is a source of the primary antibody. GST-SNAP25 _FL , or 0.5 µg of GST-SNAP25 _{197 was} dissolved on a denatured gel and transferred to a PVDF membrane to be used as an antigen. Monoclonal antibodies responded to the denatured SNAP25 antigen with the same sensitivity as in the ELISA test. The results are shown in FIG. 5. In FIG. 5, lane 1 is SNAP25 _FL and lane 2 is GST-SNAP25 ₁₉₇ . From the above results, _{all three different types of antibodies having binding specificity for ①SNAP25 FL} , ②SNAP25 ₁₉₇ , or ③SNAP25 _FL , and SNAP25 ₁₉₇ of the present invention all have high affinity and specificity for SNAP25, and denatured or natural target antigens and It can be seen that it reacts efficiently.

Example 3. Development of a method for determining botulinum toxin activity

Example 3-1. Optimization of N2-42F cell culture, and BoNT/A toxin treatment

By obtaining important materials such as neurons highly sensitive to BoNT/A and monoclonal antibodies specific to SNAP25 from Examples 1 and 2 above, the toxinization medium, sensitizer, BoNT/A treatment time, and detection antibody pair were included. Thus, optimization was possible for all steps of the botulinum toxin cell assay.

Example 3-1-1. Optimization of toxin time

First, the BoNT/A treatment time (toxinization time) was optimized. 6A (Protocol A) is a standard CBPA procedure optimized for SiMa. To investigate BoNT/A susceptibility according to Protocol A, N2-42F cells ^{were seeded at a cell density of 5.6x10 5} per well in a 12-well culture plate, and the following day, the medium was GT1b (Ganglioside GT1b trisodium salt, Enzo ALX-302-011 -M005) was replaced with a removed 1x toxin medium. Two days later, GT1b (25 μg/ml) was added to the medium, and a day later, the culture medium was exchanged with 1× medium containing BoNT/A, and cultured for an additional 2 days. Figure 6B (Protocol B) shows the CBPA procedure developed in the present invention. ^{Specifically, N2-42F cells were seeded at a cell density of 5.6×10 5} per well in a 12-well culture plate, and the next day, the medium was replaced with 1× toxin medium containing 25 pM BoNT/A. Cell lysis was performed by adding 200 µl of 1x SDS buffer to each well, and protein separation with 12% SDS-PAGE gel was performed and polyclonal anti-SNAP25 IgGs (Sigma S9684, 1: 8,000 dilution) and goat anti-rabbit IgG Fc- _{SNAP25 FL} and SNAP25 ₁₉₇ were detected by Western blotting using HRP (AbFrontier LF-SA8002, 1: 8,000 dilution). The degree of SNAP25 cleavage was quantified using Image Lab software (Bio-Rad).

As described above, in the present invention, in order to establish an optimal toxin time for N2-42F cells, by extending the cell culture time in 1x toxinization medium from which GT1b is removed, or 1x toxinization medium containing BoNT/A. Protocol A was modified. These changes did not improve the degree of SNAP25 cleavage, and N2-42F cells cultured for more than 4 days in toxinization medium appeared to be very detrimental under the microscope (data not shown). Based on this observation, a new protocol B with shortened incubation time in 1× toxin medium supplemented with both GT1b and BoNT/A was established. Three days after cell seeding (d4), the extent of SNAP25 cleavage in N2-42F cells was analyzed by Western blotting. As shown in FIG. 6C, it was found that less than 20% of SNAP25 was cleaved at d4, and the cleavage rate increased significantly with time. However, as the state of the cells observed under the microscope was concerned about the harmfulness at d7, d6 was determined as the day of the final N2-42 cell lysis and ELISA analysis.

Example 3-1-2. Optimization for toxinization medium

Osmotic pressure and temperature are known to affect the BoNT/A sensitivity of BoCellTM (US Patent US9526345B2), and the BoNT/A sensitivity of NG108-15 cells was significantly improved by optimizing the neuronal differentiation medium (J Biomol Screen. 2016 Jan; 21(1):65-73). Because BoNT/A sensitivity reflects the extent of BoNT/A uptake through two independent receptor polysialoganglioside (PSG) receptors and protein receptor SV2 on the cell surface (J Neurochem. 2009 Jun;109(6)) ):1584-95), BoNT/A sensitivity of cells is deeply related to temperature in culture medium or BoNT/A reaction.

Thus, for optimization for toxinization medium, N2-42F cells were tested for BoNT/A sensitivity in three different culture media: ^{RPMI1640, Neurobasal™} , or MEM to which 1x N2, 1x B27, and 1x GT1b were added. BoNT/A was treated at a concentration of 0.93 to 25 pM according to Protocol B above.

As a result of the experiment, as shown in FIG. 7, the BoNT/A sensitivity of N2-42F cells was highest when RPMI1640 medium was used. When using Neurobasal ^™ or MEM medium, the BoNT/A sensitivity was 25%-50% lower than that of RPMI1640. In the composition of the media, the KCl content is generally 5.33 mM in all media, but the NaCl concentration differs by 103 mM in RPMI1640, 117 mM in MEM, and 52 mM in ^{Neurobasal™ media.} However, despite these differences, the osmotic pressure of the culture medium for most vertebrate cells is known to be maintained in a narrow range of 260 to 320 mOsm/kg (ATCC Culture Cell Guide), so BoNT/A of N2-24F cells in RPMI1640 The sensitivity is thought to be due to factors other than osmotic pressure.

Also, when using RPMI1640 medium, about 48% of endogenous SNAP25 was cleaved by 8.33 pM BoNT/A, which is equivalent to a titer of about 10 U/ml. Since the culture volume of the 96-well plate is 0.1 ml, the EC50 can be evaluated as a biotiter of 1 BoNT/A per well in the microplate. Therefore, it is judged that the BoNT/A sensitivity measured by N2-42F cells according to Protocol B would be sufficient to replace the biotiter of BoNT/A determined by the mouse LD50 bioassay.

Example 3-1-3. Optimization for sensitizer

Arginine, or ATP (Fields and Stevens, 2000), creatine (Andres et al., 2005), lipoic acid (Grasso et al., 2014), etc., which are known to affect the sensitivity of BoNT/A, to the survival or differentiation of neurons. Optimization was carried out for the compound sensitizer that affects the sensitizer. To this end, N2-42F cells were treated with 0.03 to 5.5 pM of BoNT/A, and the results of analysis by sandwich ELISA protocol B using a monoclonal antibody specific for SNAP25 are shown in FIG. 8. As a result of the experiment, it was found that adding 1 mM creatine or 5 mM arginine to 1x toxin medium significantly improved the BoNT/A sensitivity and lowered the EC50 value from 2.51 pM to 2.13 pM and 2.03 pM, respectively. In contrast, ATP and lipoic acid acted as very effective inhibitors in which SNAP25 of N2-42F cells did not divide even at a concentration of 25 pM BoNT/A.

In the optimization study on the toxinization medium, it was thought that the fact that the BoNT/A sensitivity of N2-24F cells was higher than ^{that of Neurobasal TM or MEM medium in RPMI1640 medium was due to factors other than osmotic pressure.} Arginine in the medium composition was 1.15 mM in RPMI1640, 0.6 mM in MEM, and 0.4 mM in ^{Neurobasal™.} Therefore, the sensitivity of cells to BoNT/A was tested in 2x toxin medium containing 0 to 10 mM arginine. As shown in FIG. 8B, the BoNT/A sensitivity gradually increased as the arginine concentration increased to 5 mM. BoNT/A sensitivity was still higher than the control (EC50 = 2.94 pM) when 10 mM arginine (EC50 = 2.34 pM) was added, but lower than 5 mM arginine (EC50 = 1.65 pM). From these results, it was determined that the optimized standard protocol of CBPA was to use a toxin medium containing 5 mM arginine.

In 2002, Schengrund and his colleagues provided experimental evidence that SNAP25 cleavage by BoNT/A in Neuro-2a cells requires GT1b of at least 25 μg/ml (J Biol Chem. 2002 Sep 6;277(36):32815). -9). Since N2-42F cells were derived from Neuro-2a, the influence of GT1b on BoNT/A activity was investigated by Western blotting using a medium containing 25-75 μg/ml GT1b (1-3× GT1b). The results are shown in FIG. 9. As a result of the experiment, when GT1b was not added, 8.3 pM of BoNT/A induced SNAP25 cleavage by about 18%, and the addition of 1x to 3x GT1b increased SNAP25 cleavage by 10 to 18% (FIG. 9A). For optimization of the GT1b concentration, the BoNT/A sensitivity of N2-42F cells was tested in 25 pM BoNT/A, and 2x toxinization medium containing 1x to 5x GT1b concentrations. As measured by Western blotting analysis, SNAP25 cleavage was significantly improved by the addition of 1x to 2x GT1b, but above 3x GT1b, SNAP25 cleavage was 65 to 70%, slightly increased from 63% of 2x (Fig. 9B). In addition, taking into account the sensitivity of the sandwich ELISA, N2-42F cells were treated with 0.93 pM BoNT/A in 2x toxin medium according to Protocol B. As a result of the experiment, when N2-42F cells were treated with BoNT/A in a GT1b-free toxin medium, a relatively low A450 value was obtained in a sandwich ELISA, but the addition of GT1b to the toxin medium significantly increased the A450 value (Fig. 9C. ). From the above results, when measuring BoNT/A activity using N2-42F cells, the optimal concentration of GT1b in the toxin medium was determined to be 50 µg/ml (2x).

N2 (N2 supplement, Thermo Fisher Scientific 17502048)/B27 (B27 ^TM Serum free supplement, Thermo Fisher Scientific 17504-044) in neuronal cell culture was also tested for its effect on BoNT/A sensitivity. B27 contains all trans-retinol (0.1 mg/L), and is known to help neural progenitor cells differentiate into motor neurons (J Cell Biochem. 2008 Oct 15;105(3):633-40) . N2 contains a subset of the B27 component, including insulin, and is known to promote the differentiation of human embryonic stem cells and the proliferation and survival of neural progenitor cells (J Cell Biochem. 2008 Oct 15;105(3):633). -40). BoNT/A sensitivity of N2-42F cells was analyzed by Western blotting in RPMI1640 to which 1x GT1b, 8.3 pM BoNT/A, and 0-5x N2/B27 concentrations were added according to Protocol B. As shown in Figure 10A, _{the intracellular level of total SNAP25 (SNAP25 FL} + SNAP25 ₁₉₇ ) increased in proportion to the N2/B27 concentration, while _{the level of SNAP25 197} decreased by 3x in the presence of 4x to 5x N2/B27. . Recent studies have shown that N2 and B27 protect cells from death by limiting glycosylation after glucose depletion (Front Mol Neurosci. 2017 Sep 29;10:305). The results of FIG. 10A are also part of the function of promoting cell proliferation and survival in a serum-free RPMI1640 medium containing a low concentration of glucose, and it is believed that SNAP25 synthesis was increased with an increase in the concentration of N2/B27.

In addition, 3x GT1b and RPMI1640 medium to which 5 mM arginine was added were used to further investigate the effect of N2/B27 on BoNT/A activity (FIG. 10B). The degree of SNAP25 cleavage in N2-42F cells treated with 8.3 pM BoNT/A increased from 13% to 56% for 1x and 69% for 2x, despite a significant increase in total intracellular levels of SNAP25. From the above results, when measuring BoNT/A activity using N2-42F cells, the optimal concentration of N2/B27 in the toxin medium was determined to be 2x.

Example 3-2. Optimization of the sandwich ELISA method

Example 3-2-1. Optimization for buffer

For the optimization of sandwich ELISA conditions, 11 different types of lysis buffers were compared in ELISA, and selected optimal conditions are shown in Tables 7 and 8.

Parameters Conditions tested Optimal condition Plate coating matrix 3 matrices Poly-D-lysine Cell density 2 densities 5.5x10 ⁴ cells per well Intoxication medium/sensitizer 3 culture media/
6 sensitizers RPMI1640, 2 mM L-alanyl-L-glutamine, 0.1 mM NEAA, 2x N2, 2x B27, GT1b (50 μg/ml), 5 mM arginine Intoxication time 3 time points 96 hr BoNT/A dose 0.03 pM-1 nM 0.03-8.33 pM Capture/detection antibodies 11 combinations B4 (300 ng/50 mL) + C16-HRP (200 ng/50 mL) Lysis buffers 11 buffers 20 mM Hepes-NaOH, pH 7.5, 0.2 M NaCl, 1% Triton X-100, 1 mM EGTA, 5 mM EDTA Lysate incubation 3 temperatures/
3 time points 4 hr at 4℃ Blocking buffers 46 buffers 1% polyvinyl alcohol (Mw 145,000), 3% skim milk in 1x PBS Incubation of detection antibody 4 temperatures/
4 time points 1 hr at RT

Parameters Condition tested Optimal Lysis buffers 50 mM Tris-HCl, pH 8.0,150 mM NaCl, 1% Triton X-100, 2 mM EGTA, 0.01-0.1% SDS, ± 5 mM EDTA 20 mM Hepes-NaOH, pH 7.5,
150 mM NaCl, 1% Triton X-100,
5 mM EDTA,
2 mM EGTA 20 or 50 mM Hepes-NaOH, pH 7.5, 0-0.4 M NaCl, 1 or 2% Triton X-100, 0-0.1% SDS,
0-1.5 mM MgCl ₂ , 0-5 mM EDTA, 1-2 mM EGTA 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40 Coating buffers 0.5% APTES 0.1 M carbonate buffer, pH 9.6 0.1 M sodium acetate buffer, pH 6.0 0.1 M sodium phosphate buffer, pH 7.2 0.1 M sodium citrate buffer, pH 2.8 0.1 M carbonate buffer, pH 9.6 Washing buffers 1x PBS/0.05% Tween-20 (1x PBST), ±0.2 M NaCl 1x PBST Blocking buffers 0.2-3% Ac-BSA, 3-5% skim in 1x PBS 3% skim, 1% PVA (Mw 145,000) in 1x PBS 1% goat serum in 1x PBS 2-3% skim, 0.05-1% PVA(Mw 47,000 or 145,000) in 1x PBS 5% skim or 0.1-0.5% Ac-BSA, with 10 or 100% SuperBlock ^TM (PBS) ^a 2-5% ECL ^b or ECL Prime ^TM Blocking agent ^c ± 0-5% skim or 0.2% Ac-BSA Western BLoT blocking buffer (protein-free) ^{d a} SuperBlock ^TM (PBS) (Thermo Fisher Scientific 37518)
^b ECL Prime ^TM blocking agent (GE Healthcare UK limited RPN418V)
^c ECL (GE Healthcare UK limited RPN2125V)
^d Western BLoT blocking buffer (protein free) (Takara T7132A)

Example 3-2-2. Optimization for capture antibody treatment

Among the antibodies of Example 2, the functions as capture antibodies of A15, B4, and B23 capable of detecting both _{SNAP25 FL} and SNAP25 _{197 were compared.} Cell lysates were prepared from N2-42F cells treated with 0-25 pM BoNT/A in 1x toxin medium, and cell lysates were added to microplates coated with the antibody at 400 ng per well, and at 4°C. _{After incubation for 4 hours, the amount of SNAP25 197 out} of the total SNAP25 captured by the presented antibody was detected and quantified using the C16 IgG-HRP conjugate. The test was carried out for 30 minutes of HRP reaction until all test groups gave positive ELISA signals. As a result of the experiment, as shown in Fig. 11A, the estimated EC50 values for A15, B4, and B23 IgG were 15 pM, 0.7 pM, and 5.5 pM, respectively. This result was consistent with the kinetic values for _{SNAP25 FL} and SNAP25 _197.

In the above results, according to the best result of B4 IgG, N2 cultured in 2x toxin medium containing 0, 0.03, 0.1, 0.31, 0.93, 1.39, 2.78, or 5.55 pM of BoNT/A by the method of Protocol B. The optimal amount of B4 IgG was investigated with the cell lysate prepared from -42F cells. The amount of B4 IgG coated on the sandwich ELISA was performed at 100 to 400 ng per well. SNAP25 capture and detection proceeded in the same manner as above, but the HRP reaction was treated for 15 minutes. As a result of the experiment, as shown in Fig. 11B, the EC50 value steadily decreased as the amount of B4 IgG increased from 100 ng to 300 ng. The EC50 value of 400 ng B4 IgG was not statistically significant. From the above results, it was determined that the optimum condition for the capture antibody used in the sandwich ELISA was 300 ng of B4 IgG.

Example 3-2-3. Optimization for detection antibody treatment

For optimization of the detection antibody, the cell lysate prepared from SiMa cells treated with 8.33 or 25 pM BoNT/A in 1× toxinization medium by the method of Protocol A _{was captured with SNAP25 197} antibody, and the captured SNAP25 ₁₉₇ was C16 IgG. It was detected and quantified using -HRP conjugate. As shown in Figure 12A, _{the ELISA signal for SNAP25 197} was found to be highest when the microplates were incubated at 4°C for 4 hours and weakest when incubated at 37°C for 1 hour. Similar results were obtained even when experiments with the cell lysate of N2-42F cells were obtained, and the ELISA signal was not significantly changed by incubation for more than 4 hours. From the above results, it was determined that the optimal reaction conditions of the cell lysate used in the sandwich ELISA were incubated at 4° C. for 4 hours.

In addition, the optimal reaction conditions for the detection antibody were investigated by direct ELISA. Specifically, the microplates were coated with 10 to 1 ng of _{recombinant GST-SNAP25 197} showing an endogenous SNAP25 cleavage of 0.05% to 50% in standard CBPA. Then 200 ng of C16 IgG-HRP conjugate was added per well, and the microplates were incubated under the indicated conditions. During the condition test, the Cg16 IgG-HRP conjugate was incubated at room temperature for 1 hour, resulting _{in the highest ELISA signal in all ranges of GST-SNAP25 197} tested, but an acceptable level of ELISA signal was produced under other conditions. . Therefore, in consideration of the subsequent HRP reaction carried out at room temperature, it was determined that the optimal reaction conditions for the C16-HRP conjugate used in the sandwich ELISA were incubated at room temperature for 1 hour.

Example 3-2-4. Optimization for C16 IgG-HRP conjugate detection

HRP activity was measured using a commonly used TMB kit (1-Step TM Ultra TMB-ELISA, Thermo Fisher Scientific 34028). The disadvantage of using the TMB substrate is that the EC50 value is affected by the HRP reaction time, and as an alternative detection method, the fluorescence measurement value was evaluated in a sandwich ELISA. With the exception of the HRP reaction substrate provided, colorimetric and fluorescence measurements were performed in essentially the same way in parallel via sandwich ELISA. As a result of the experiment, fluorescence measurement and colorimetric measurement produced EC50s of 0.66 pM and 0.78 pM, respectively (FIG. 13), and both EC50 values were equally affected by HRP reaction time. In conclusion, both measurements were judged to be accurate and sensitive to detect the activity of the C16 IgG-HRP conjugate in ELISA.

Example 3-3. CBPA validation

Example 3-3-1. CBPA's Optimized Timeline Verification

14 is a schematic diagram of a sandwich ELISA method for determining botulinum toxin activity of the present invention. As shown in Fig. 14, the sandwich ELISA method for determining botulinum toxin activity of the present invention can be analyzed in only 3 working days. More specifically, one day for cell seeding, one day to replace with a culture medium containing BoNT/A (0.1 to 10 U/ml, or less than 4 pM) the next day, and the 6th day for cell seeding, the cells are lysed and sandwiched. One day is required for ELISA analysis. In addition, since the time required for the work of the first day and the second day is 1 to 2 hours, the sandwich ELISA method for determining the botulinum toxin activity of the present invention is very efficient in time. In addition, since the sandwich ELISA of the present invention uses a monoclonal antibody for both the capture antibody and the detection antibody, antibody management is much easier than that using a polyclonal antibody, and thus the reliability of CBPA can be further consolidated.

Example 3-3-2. CBPA accuracy and linearity check

A total of 18 CBPAs were performed by 3 researchers by the method of standard protocol B.

N2-42F cells were at various concentrations (0, 0.03, 0.1, 0.2, 0.31, 0.62, 0.93, 2.78) in 2x toxin medium without 3x GT1b (Figure 15A), or with (Figure 15B, and Figure 15C). , 5.55, or 8.33 pM) of BoNT/A, and the HRP reaction was performed for 5 minutes (FIG. 15A and FIG. 15B), or 9 minutes (FIG. 15C), and the EC50 was measured using Gen5 software. Linear regression analysis was performed with A450 values obtained in N2-42F cells treated with 0.1 to 0.93 pM BoNT/A, to calculate the limit of detection (DL) and the limit of quantification (QL). The results are shown in FIG. 15.

As a result of the experiment, in the first group (Fig. 15A), an EC50 of 1.39 pM and a detection limit of 1.5 fM (3.4 mU relative efficacy/ml), and a quantification limit of 4.6 fM were calculated (Clin Biochem Rev. 2008 Aug; 29 Suppl 1 : S49-52). 1.39 pM BoNT/A is equivalent to ~0.3 U relative efficacy per assay, so the results of the first group mean that the CBPA of the present invention is sensitive enough to replace the mouse LD50 assay in measuring the biotiter of BoNT/A. . When analyzing the second and third groups, a slightly reduced EC50 value was obtained either by including 3x GT1b or by extending the HRP reaction time. The above results indicate that it is possible to adjust the limits of quantitation and detection of CBPA by varying the GT1b concentration in the toxin medium or by adjusting the HRP reaction time. In addition, looking at the results of the linear regression analysis of N2-42F cells treated with 0.03 to 0.93 pM BoNT/A, it shows a good linear correlation between the relative efficacy of BoNT/A and ELISA signals in all groups. This suggests that CBPA is not only very sensitive, but also very accurate in measuring the relative potency of BoNT/A between 6.8 mU and 0.2 U.

Example 3-3-3. Biotiter measurement of BoNT/A samples

The titer of BoNT/A (BOTULAX®, Huzel Co., Ltd., Korea) was determined by mouse LD50. In order to measure the bio-efficacy of CBPA, two BoNT/A (HUB 18009 and HUB 18011, 200 U per vial) with different production lots were each added to 1x toxin medium (Figure 16A), deionized water (Figure 16B), or After dissolving in 5 mM arginine solution (Fig. 16C) at pH 6.0 and incubating for 10 minutes at room temperature, BoNT/A dissolved in 1x toxin medium was added to 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15 or 50 U/ It was diluted sequentially in ml. BoNT/A dissolved in deionized water and arginine solution was sequentially diluted to a concentration of 0.23, 0.48, 0.70, 0.98, 1.41, 2.11, 4.20, 6.32, or 12.6 U/ml with an optimized 2x toxin medium. The samples were analyzed by the method of standard protocol B, but the HRP reaction was performed for 5 minutes, and the EC50 was measured using Gen5 software. The results are shown in FIG. 16. As a result of the experiment, the EC50 values of the two lots BoNT/A dissolved in 1x toxin medium, deionized water and arginine solution were 10.6±0.12 and 10.3±0.52 U/ml (1x toxin medium), 4.6±0.04 and 4.6±0.08 U, respectively. /Ml (deionized water), and 4.5±0.11 and 4.4±0.22 U/ml (arginine solution). The above results indicate that the CBPA of the present invention is sufficiently sensitive and accurate to measure the biotiter of BoNT/A, and its sensitivity (0.4-0.5 U per well) is equal to or better than that of the mouse bioassay.

As described above, specific parts of the present invention have been described in detail, and it is obvious that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto for those of ordinary skill in the art. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Korea Research Institute of Bioscience and Biotechnology KCTC13712BP 20181113

<110> AbBio CO., LTD. <120> A cell-based method for determining an activity of botulinum toxin <130> PDPB187219 <160> 90 <170> KoPatentIn 3.0 <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> antibody CDR of 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> antibody CDR of 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> antibody CDR of 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> antibody CDR of clostridium botulinum <400> 14 Ile Arg Tyr Asp Gly Ser Asn 1 5 <210> 15 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> antibody CDR of 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> antibody CDR of 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> antibody CDR of 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> antibody CDR of clostridium botulinum <400> 18 Val Arg Ser Gly Asp Tyr 1 5 <210> 19 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> antibody CDR of clostridium botulinum <400> 19 Ala Arg Gly Tyr Tyr Asp Tyr 1 5 <210> 20 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> antibody CDR of clostridium botulinum <400> 20 Asp His Ile Asn Asn Trp 1 5 <210> 21 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> antibody CDR of clostridium botulinum <400> 21 Gln Ser Leu Leu Asp Ser Asn Gly Lys Thr Tyr 1 5 10 <210> 22 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> antibody CDR of clostridium botulinum <400> 22 Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr 1 5 10 <210> 23 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> antibody CDR of clostridium botulinum <400> 23 Asp Thr Thr One <210> 24 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> antibody CDR of clostridium botulinum <400> 24 Leu Val Ser One <210> 25 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antibody CDR of 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> antibody CDR of 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> antibody CDR of 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> antibody CDR of 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> antibody CDR of 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> antibody CDR of 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> antibody CDR of 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> antibody CDR of 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> antibody CDR of 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> antibody CDR of clostridium botulinum <400> 34 Ile Ser Tyr Ser Val Gly Thr 1 5 <210> 35 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> antibody CDR of 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> antibody CDR of 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> antibody CDR of 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> antibody CDR of 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> antibody CDR of 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> antibody CDR of 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> antibody CDR of 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> antibody CDR of 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> antibody CDR of 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> antibody CDR of 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> antibody CDR of clostridium botulinum <400> 45 Val Gly Gln Ile Leu 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> antibody CDR of 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> antibody CDR of 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> antibody CDR of 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> antibody CDR of 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> antibody CDR of clostridium botulinum <400> 50 Leu Ala Ser One <210> 51 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> antibody CDR of clostridium botulinum <400> 51 Lys Val Ser One <210> 52 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antibody CDR of 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> antibody CDR of 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> antibody CDR of 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> antibody CDR of 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> antibody CDR of 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> antibody CDR of 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> antibody CDR of 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> antibody CDR of 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> antibody CDR of clostridium botulinum <400> 60 Asn Glu Ile Ala Tyr 1 5 <210> 61 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> antibody CDR of 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> antibody CDR of 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> antibody CDR of clostridium botulinum <400> 63 Lys Val Ser One <210> 64 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> antibody CDR of clostridium botulinum <400> 64 Leu Val Ser One <210> 65 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antibody CDR of 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> antibody CDR of 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> antibody of 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 of 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 of 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 of 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 of 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> antibody of 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> antibody of 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 of 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 of 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 of 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 of 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 of 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 of 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 of 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 of 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 of 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 of 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 of 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 of 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 of 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 of 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> antibody of 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> antibody of 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 of 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 (20)

(a) culturing a nerve cell or a nerve cell line isolated from the individual;
(b) treating the cells with botulinum toxin type A;
(c) treating the cell or a sample obtained from the cell with an antibody that specifically binds _{to SNAP25 FL} and SNAP25 _197; And
(d) treating an antibody that does not bind _{to SNAP25 FL but specifically binds to SNAP25 197} to (c); comprising, a cell-based titer assay method of neurotoxin,
The _{antibodies that specifically bind to SNAP25 FL} and SNAP25 ₁₉₇ are (a) or (b),
The antibody that does not bind to _{SNAP25 FL and specifically binds to SNAP25 197} is any one selected from (c) to (child), a titer analysis method;
(A) the heavy chain CDR1 region is SEQ ID NO: 55, the heavy chain CDR2 region is SEQ ID NO: 57, the heavy chain CDR3 region is SEQ ID NO: 59, the light chain CDR1 region is SEQ ID NO: 61, the light chain CDR2 region is SEQ ID NO: 63, and the light chain CDR3 region is SEQ ID NO: An antibody consisting of 65;
(B) the heavy chain CDR1 region is SEQ ID NO: 56, the heavy chain CDR2 region is SEQ ID NO: 58, the heavy chain CDR3 region is SEQ ID NO: 60, the light chain CDR1 region is SEQ ID NO: 62, the light chain CDR2 region is SEQ ID NO: 64, and the light chain CDR3 region is SEQ ID NO: An antibody consisting of 66;
(C) the heavy chain CDR1 region is SEQ ID NO: 28, the heavy chain CDR2 region is SEQ ID NO: 34, the heavy chain CDR3 region is SEQ ID NO: 40, the light chain CDR1 region is SEQ ID NO: 47, the light chain CDR2 region is SEQ ID NO: 50, and the light chain CDR3 region is SEQ ID NO: An antibody consisting of 52;
(D) heavy chain CDR1 region is SEQ ID NO: 29, heavy chain CDR2 region is SEQ ID NO: 35, heavy chain CDR3 region is SEQ ID NO: 41, light chain CDR1 region is SEQ ID NO: 48, light chain CDR2 region is SEQ ID NO: 50, and light chain CDR3 region is SEQ ID NO: An antibody consisting of 52;
(E) The heavy chain CDR1 region is SEQ ID NO: 29, the heavy chain CDR2 region is SEQ ID NO: 36, the heavy chain CDR3 region is SEQ ID NO: 42, the light chain CDR1 region is SEQ ID NO: 47, the light chain CDR2 region is SEQ ID NO: 50, and the light chain CDR3 region is SEQ ID NO: An antibody consisting of 52;
(F) The heavy chain CDR1 region is SEQ ID NO: 33, the heavy chain CDR2 region is SEQ ID NO: 35, the heavy chain CDR3 region is SEQ ID NO: 43, the light chain CDR1 region is SEQ ID NO: 48, the light chain CDR2 region is SEQ ID NO: 50, and the light chain CDR3 region is SEQ ID NO: An antibody consisting of 52;
(G) The heavy chain CDR1 region is SEQ ID NO: 30, the heavy chain CDR2 region is SEQ ID NO: 37, the heavy chain CDR3 region is SEQ ID NO: 44, the light chain CDR1 region is SEQ ID NO: 48, the light chain CDR2 region is SEQ ID NO: 50, and the light chain CDR3 region is SEQ ID NO: An antibody consisting of 52;
(H) The heavy chain CDR1 region is SEQ ID NO: 31, the heavy chain CDR2 region is SEQ ID NO: 38, the heavy chain CDR3 region is SEQ ID NO: 45, the light chain CDR1 region is SEQ ID NO: 47, the light chain CDR2 region is SEQ ID NO: 50, and the light chain CDR3 region is SEQ ID NO: An antibody consisting of 53;
(I) heavy chain CDR1 region is SEQ ID NO: 32, heavy chain CDR2 region is SEQ ID NO: 39, heavy chain CDR3 region is SEQ ID NO: 46, light chain CDR1 region is SEQ ID NO: 49, light chain CDR2 region is SEQ ID NO: 51, and light chain CDR3 region is SEQ ID NO: An antibody consisting of 54.
The method of claim 1,
The nerve cell line, characterized in that the N2-42F (accession number: KCTC 13712BP) cell line, titer analysis method.
delete The method of claim 1,
Botulinum toxin type A of step (b) is characterized in that the treatment is carried out by diluting with a medium containing GT1b (Ganglioside GT1b trisodium salt).
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