JP6742590B2 - Antibody or antigen-binding fragment thereof and hybridoma - Google Patents

Description

The present invention relates to antibodies or antigen-binding fragments thereof and hybridomas.

Neurotrophic factors are a group of humoral factors that promote differentiation, maturation, survival, and function maintenance of nerve cells. Nerve growth factor (NGF), which was discovered as the first neurotrophic factor, is a protein that acts on the differentiation and growth of sympathetic ganglion and sensory nerve neurons. Following this NGF, brain-derived neurotrophic factor (BDNF) was isolated as a second neurotrophic factor by Barde et al. in the 1980s. Since the beginning of the 1990s when the BDNF gene was cloned, research on signal transduction that causes physiological effects of neurotrophic factors has progressed, and BDNF and its receptor TrkB, and related molecules have been produced and analyzed to produce BDNF genes. The physiological role in the living body is being clarified. Since the pathological conditions found in these mutant mice and BDNF are strongly expressed in the brain, attention has recently been paid to the relationship between BDNF and brain diseases. Depression in which BDNF function declines due to long-term continuous stress exposure, resulting in functional and structural impairment of the hippocampus, which is important for memory and learning, and the prefrontal cortex, which is important for emotion control, resulting in depression. "BDNF hypothesis" has been proposed. BDNF dysfunction seen here means molecular pathologies such as processing disorders and secretion disorders. Since the impairment of the molecular function of BDNF is associated with the onset of brain disease, it is an interesting research target.

In contrast to studies on signal transduction via BDNF receptors, the mechanism of secretion and transport of the BDNF molecule itself has been poorly understood. Elucidation of the spatiotemporal control of neurotrophic factors in complex neural circuits is as important as understanding signal transduction through receptors.

FIG. 1 is a diagram showing the process of BDNF production. BDNF is synthesized as a BDNF precursor in which a prodomain (hereinafter, BDNF prodomain) is bound to the N-terminal side of mature BDNF before it becomes a mature BDNF (hereinafter, mature BDNF) having a physiological action. It has been generally believed that a propeptide (hereinafter referred to as BDNF propeptide) produced by being separated from the main body in the process of processing becomes unnecessary, but in Patent Document 3, such a propeptide also exhibits physiological activity. It was shown for the first time to do.

There are many single-nucleotide polymorphisms (SNPs) in proBDNF, but in the group of Kojima et al., VM type mutation (66th Val→Met mutation) causes memory impairment among many SNPs. That is, it was revealed that it causes an abnormal secretion of mature BDNF.

Patent Documents 1 and 2 disclose a non-cleavable proBDNF and a knockin mouse that highly expresses the non-cleavable proBDNF.

JP, 2006-345787, A JP 2007-306899 A JP2011-246420A

An object of the present invention is to provide an antibody specifically recognizing a BDNF prodomain or a BDNF propeptide, or an antigen-binding fragment thereof, and a hybridoma.

The present invention provides the following antibodies or antigen-binding fragments thereof and hybridomas. An antibody having the following characteristics or an antigen-binding fragment thereof (1) Recognizing the 19th to 128th BDNF prodomain of proBDNF and the 19th to 128th BDNF propeptide of proBDNF shown in SEQ ID NO: 1 Does not recognize BDNF (2) Does not recognize region A of the BDNF prodomain, but specifically recognizes region B and/or region C.

(The underlined part is the common part between the regions A and B, and the underlined part is the common part between the regions B and C.)
Item 2. The antibody or antigen-binding fragment thereof according to Item 1, which does not recognize the region A of the BDNF prodomain but specifically recognizes the region C.
Item 3. Item 3. The antibody or antigen-binding fragment thereof according to Item 1 or 2, which can be produced from a hybridoma obtained by fusing antibody-producing cells immunized with the peptide represented by KVRPNEENNKDADLY and myeloma cells.
Item 4. The antibody or antigen-binding fragment thereof according to Item 1, 2 or 3, wherein the antibody is a monoclonal antibody.
Item 5. Item 4 which reacts with the same BDNF prodomain epitope as the monoclonal antibody produced from the hybridoma deposited at the Patent Microorganism Depositary Center, National Institute for Product Evaluation Technology, under the accession numbers P-02011, P-02012, P-02013. The antibody or the antigen-binding fragment thereof according to 1.
Item 6. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 5, which is capable of histochemically analyzing a neuropathological state caused by proBDNF or BDNF propeptide.
Item 7. The following (1) and (2)
(1) binds to the 19th to 128th BDNF prodomain of proBDNF and the 19th to 128th BDNF propeptide of proBDNF shown in SEQ ID NO: 1 (2) for region A of BDNF prodomain, to region B and / Or Region C is specifically recognized.

(The underlined part is the common part between the regions A and B, and the underlined part is the common part between the regions B and C.)
A hybridoma producing a monoclonal antibody having the characteristics of, which has been deposited under the deposit numbers NITE P-02011, P-02012, P-02013 at the Patent Microorganism Depositary Center, National Institute for Product Evaluation Technology, National Institute of Technology and Evaluation.

The antibody or antigen-binding fragment thereof of the present invention can specifically recognize BDNF prodomain or BDNF propeptide, and is useful as an antibody drug for neuropathology, a reagent for immunohistochemical test, a kit, etc. ..

BDNF production process. “Prodomain” is a BDNF prodomain in a BDNF precursor, and when it is cleaved from mature BDNF by processing, the prodomain becomes a physiologically active substance called BDNF propeptide ([Patent Document 3] JP2011-2011-A1). 246420). The amino acid sequence of proBDNF is shown. Affinity of antibodies #111, #113, and #101 for the BDNF prodomain regions A, B, and C. (A) image diagram showing regions A, B, C of proBDNF, (B) amino acid sequence of regions A, B, C of proBDNF, (C) regions A, B, C of proBDNF analyzed by BIAcore and antibody #111, 113, 101 interaction, (D) dot BD bProBDNF regions A, B, C, BDNF propeptide, mature BDNF and antibody #111, 113, 101 interaction Immunostaining of CA3 region of adult mouse hippocampus Immunoprecipitation of proBDNF using antibodies #111, #113 and #101

In the present specification, BDNF, proBDNF and the like are mammals (human, monkey, cow, horse, sheep, pig, mouse, rat, rabbit, dog, cat, etc.), fish, amphibians, other animals including reptiles and birds. Including those derived from. In a preferred embodiment, BDNF, proBDNF, etc. are of mammalian origin, more preferably of human origin.

The accession number of human proBDNF is CAA62632. The sequence of human proBDNF is shown in SEQ ID NO:1.

The proBDNF is cleaved by furin or tissue plasminogen activator (tPA) to be divided into mature BDNF (129-247 of SEQ ID NO: 1) and BDNF propeptide (19-128 of SEQ ID NO: 1). 1-18 of SEQ ID NO: 1 is a signal peptide.

The “antibody or antigen-binding fragment thereof” of the present invention (hereinafter, sometimes simply referred to as “antibody”) is a BDNF prodomain (19-128) of proBDNF consisting of the amino acid sequence represented by SEQ ID NO: 1. It is characterized by combining. The antibody of the present invention specifically recognizes region B and region C with respect to region A of BDNF propeptide. Region A is the 19th to 65th peptide region of SEQ ID NO: 1, Region B is the 44th to 101st peptide region of SEQ ID NO: 1, and Region C is the 76th to 126th peptide region of SEQ ID NO: 1. Area.

In the above description, the underlined part is the common part between the regions A and B, and the underlined part is the common part between the regions B and C. Italics indicate peptides that can be used to make antibodies.

Here, the antibody of the present invention is, for example, an antibody that can be produced in a mammal immunized with a peptide consisting of KVRPNEENNKDADLY, which is the 76-90th amino acid sequence from the N-terminus of human proBDNF (SEQ ID NO: 1) as an antigen. , BDBD prodomains 19th to 128th of proBDNF and BDNF propeptides 19th to 128th of proBDNF are not particularly limited as long as they are antibodies. The epitope of the antibody of the present invention includes a part of the amino acid sequence represented by KVRPNEENNKDADLY, but even if it is a continuous amino acid sequence or a discontinuous sequence in which each is close in the three-dimensional structure. Good. The number of amino acids constituting the epitope is not particularly limited, but generally, in the amino acid sequence constituting the peptide or protein serving as an antigen, 4 or more, specifically, about 4 to 10 is there. Therefore, in the present invention, the epitope of KVRPNEENNKDADLY includes, for example, a continuous or discontinuous sequence consisting of 4 or more amino acids, specifically 4 to 10 amino acids. One of the reasons for this is that the structure formed by region B and region C is different, suggesting that this antibody is a structure-recognizing antibody.

The antibody of the present invention specifically recognizes the 19th to 128th BDNF prodomains of proBDNF and the 19th to 128th BDNF propeptide of proBDNF, and preferably a monoclonal antibody (#111, #113, #101). ) Or an antigen-binding fragment thereof. Monoclonal antibodies (#111, #113, #101) are produced from the hybridomas deposited at the Patent Microorganism Depositary Center, National Institute of Technology and Evaluation, Product Evaluation Technology Infrastructure, under the accession numbers P-02011, P-02012, P-02013.

The antibody of the present invention does not recognize mature BDNF. By using the antibody of the present invention or its antigen-binding fragment, proBDNF and BDNF propeptide in tissues such as nerves can be detected and/or the abundance thereof can be measured.

The reactivity of the antibody with respect to the antigen can be confirmed by known immunological methods such as Western blotting, ELISA (Enzyme-Linked Immuno Sorbent Assay), radioimmunoassay (RIA), enzyme immunoassay (Enzyme). ImmunoAssay (EIA), Fluorescence Immuno Assay (FIA), Fluorescence Immunoassay (Florescence-Linked Immuno Sorbant Assay; FLISA), Cell Immunostaining, Tissue Immunostaining, Flow Cytometry, Fluorescence Marking It can be performed by a cell sorting method (Fluorescence activated cell sorting; FACS), an ELISPOT method (Enzyme-Linked Immuno-Spot), an immunoprecipitation method, or the like. Others, spectroscopy, Frontal Affinity Chromatography (FAC), parallel dialysis, fluorescence quenching, atomic force microscope (AFM), Quartz Crystal Microbalance (QCM), The reactivity can also be confirmed by a surface plasmon resonance (SPR) method or the like. The antibodies of the present invention are also useful in making kits for performing these methods. From the viewpoint of simplicity of operation and measurement accuracy, ELISA method, QCM method, SPR method and the like are preferably used, but as a method for confirming the specificity of the reactivity of the antigen and the antibody, specifically, the Western blotting method is used. Can be mentioned.

Examples of the antibody of the present invention include an antibody that can be produced from a hybridoma obtained by fusing antibody-producing cells immunized with the peptide having the amino acid sequence represented by KVRPNEENNKDADLY and myeloma cells. Specifically, the deposit number: P-02011,P-02012,P-02013, Japan Patent Evaluation Microorganism Depositary Center, National Institute for Product Evaluation Technology (2-5-8 Kazusa Kamasa, Kisarazu City, Chiba Prefecture 292-0818) Monoclonal antibody that can be produced from the hybridoma deposited on February 27, 2015, and an antibody or antigen-binding fragment thereof that reacts with the same BDNF prodomain and BDNF propeptide epitope as the monoclonal antibody produced from the hybridoma. Can be mentioned.

The monoclonal antibody of the present invention can be produced by methods known to those skilled in the art (hybridoma method, phage display method, etc.).

Specifically, a monoclonal antibody against BDNF prodomain and BDNF propeptide is a chemically synthesized peptide (antigen peptide) consisting of an amino acid sequence represented by KVRPNEENNKDADLY, which is a partial sequence of BDNF prodomain or BDNF propeptide. Can be produced by administering it to a mammal, that is, by administering itself or a carrier or diluent to a site capable of producing an antibody. As the carrier, a carrier protein having antigenic stimulation, such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), bovine thyroglobulin, ovalbumin (OVA), etc. can be used. As for the mixing ratio of the carrier and the antigen peptide, as long as the antibody can efficiently cross the antigen peptide immunized by crosslinking the carrier, any kind may be crosslinked at any ratio, and for example, the carrier may be weighted. A method of binding to the antigen peptide 1 at a ratio of about 0.1 to 20, preferably about 1 to 5 is used. Although various condensing agents can be used for binding the antigen peptide to the carrier, glutaraldehyde, carbodiimide, maleimide active ester, active ester reagent containing thiol group, dithiopyridyl group, etc. can be used.

Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered to enhance the antibody-producing ability during administration. The administration is usually performed once every 2 to 6 weeks, about 2 to 10 times in total. Examples of mammals used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, hamsters, etc., but are selected in consideration of compatibility with myeloma cells used for cell fusion. , And generally mouse, rat, hamster, etc. are preferably used.

In the production of monoclonal antibody-producing cells, mammals immunized with an antigen, for example, an individual with an antibody titer is selected from mice, and 2 to 5 days after the final immunization, the spleen or lymph node is collected and included in them. A monoclonal antibody-producing hybridoma can be prepared by fusing the resulting antibody-producing cells with myeloma cells (myeloma cells). The antibody titer in the antiserum can be measured by a method known to those skilled in the art, such as the ELISA method. For example, antiserum is added to a solid phase (eg, a microplate) on which the antigen peptide used as an immunogen is directly or adsorbed together with a carrier, and then an anti-immunoglobulin antibody labeled with a radioactive substance or an enzyme (for cell fusion is used). When the cell used is a mouse, an anti-mouse immunoglobulin antibody is used) or protein A is added to detect a monoclonal antibody bound to a solid phase. The fusion operation can be performed according to a known method, for example, the method of Koehler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used.

Examples of myeloma include NS-1, P3U1, SP2/0 and the like, and SP2/0 is preferably used. The preferred ratio of the number of antibody-producing cells (spleen cells) to the number of myeloma cells used is about 1:1 to 20:1, and PEG (preferably PEG1000 to PEG6000) is added at a concentration of about 10 to 80%. Incubation at about 20 to 40°C, preferably about 30 to 37°C for about 1 to 10 minutes enables efficient cell fusion.

Although various methods can be used for screening a monoclonal antibody-producing hybridoma, for example, the hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which the antigen peptide used as an immunogen is directly or adsorbed with a carrier, Next, anti-immunoglobulin antibody labeled with a radioactive substance or enzyme (if the cell used for cell fusion is a mouse, anti-mouse immunoglobulin antibody is used) or protein A is added, and the monoclonal antibody bound to the solid phase is detected. Method, a method in which a hybridoma culture supernatant is added to a solid phase on which anti-immunoglobulin antibody or protein A is adsorbed, and an antigen peptide labeled with a radioactive substance or an enzyme is added to detect a monoclonal antibody bound to the solid phase Is mentioned.

The monoclonal antibody can be selected according to a method known per se or a method analogous thereto, but usually it can be carried out in a medium for animal cells or the like to which HAT (hypoxanthine, aminopterin, thymidine) is added. As a medium for selection and breeding, any medium may be used as long as the hybridoma can grow therein. For example, RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum, GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or serum-free for hybridoma culture. A medium (SFM-101, Nissui Pharmaceutical Co., Ltd.), a serum-free medium for hybridoma culture containing 10 to 20% fetal bovine serum (Hybridoma-SFM, Invitrogen) and the like can be used. The culture temperature is usually 20 to 40°C, preferably about 37°C. Cultivation time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. Culturing can usually be performed under 5% carbon dioxide. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the above-described antibody titer measurement in antiserum.

Furthermore, for hybridoma clones that were positive by ELISA for the antigenic peptide, after secondary culture, the ability to produce antibodies that recognize proBDNF or BDNF propeptide using the culture supernatant of individual clones was determined for platelet extracts. Hybridomas that produce a monoclonal antibody that specifically recognizes proBDNF or BDNF propeptide can be selected by evaluating by Western blotting to determine whether or not proBDNF or BDNF propeptide is detected.

Specifically, as a suitable hybridoma that produces a monoclonal antibody that specifically recognizes the BDNF pro domain or BDNF pro peptide, the accession numbers: P-02011, P-02012, and P-02013 are incorporated administrative product evaluation technology bases. The hybridomas #111, #113, and #101 deposited at the Organization Patent Microorganisms Depositary Center are listed.

Separation and purification of the monoclonal antibody is carried out in the same manner as the separation and purification of ordinary polyclonal antibodies, for example, the separation and purification method of immunoglobulin, for example, salting out method, alcohol precipitation method, isoelectric focusing method, electrophoresis method, ion exchanger (eg, DEAE) adsorption/desorption method, ultracentrifugation method, gel filtration method, antigen-binding solid phase or specific purification method to obtain antibody by dissociating the bond by collecting only antibody with an active adsorbent such as protein A or protein G Can be done according to.

A polyclonal antibody against the BDNF pro domain or BDNF pro peptide can be produced by a method known per se or a method analogous thereto. Specifically, similar to the above-described method for producing a monoclonal antibody, a chemically synthesized peptide (antigen peptide) consisting of the amino acid sequence represented by KVRPNEENNKDADLY is used to produce antibodies against mammals or chickens. It can be manufactured by administering itself or a carrier and a diluent to possible sites. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered to enhance the antibody-producing ability during administration. The administration can usually be performed once every about 2 to 6 weeks, about 3 to 10 times in total.

The polyclonal antibody can be collected from blood, ascites, breast milk and the like of mammals immunized by the above method, preferably blood, and in the case of chicken, it can be collected from blood and egg yolk.

The measurement of the polyclonal antibody titer in the antiserum, the evaluation of the reactivity to the BDNF pro domain or the BDNF pro peptide, the separation and purification of the polyclonal antibody, etc. can be carried out according to the above-mentioned method for producing a monoclonal antibody.

The “antigen-binding fragment” of the antibody of the present invention contains a portion of the monoclonal antibody of the present invention, preferably the antigen-binding region or variable region thereof, and has the binding property to the BDNF prodomain or BDNF propeptide. Specific examples of the fragment include, for example, Fv, scFv, Fab, F(ab') ₂ , Fab', Fd, dAb, CDR, scFv-Fc fragment, Nanobody, affibody, diabody, avimer, minibody, Versa body etc. are mentioned.

These fragments can be obtained using methods well known to those skilled in the art, and specifically, the antibody is treated with an enzyme such as papain or pepsin to produce an antibody fragment, or these antibody fragments are A gene encoding the gene may be constructed, introduced into an expression vector, and then expressed in an appropriate host cell. The obtained fragment can be evaluated for reactivity with an antigen, specificity and the like in the same manner as for the antibody of the present invention.

In the present invention, an antibody encoded by an antibody gene cloned from antibody-producing cells can also be used. The cloned antibody gene can be expressed as an antibody by incorporating it into a suitable vector and introducing it into a host. Methods for isolation of antibody genes, introduction into vectors, and transformation of host cells have already been established (eg Vandamme, AM et al., Eur. J. Biochem. (1990) 192, 767). -775).

For example, a cDNA encoding the variable region (V region) of the antibody of the present invention can be obtained from a hybridoma cell that produces the antibody of the present invention. For that purpose, first, total RNA is usually extracted from the hybridoma. As a method for extracting mRNA from cells, for example, guanidine ultracentrifugation method (Chirgwin, JM et al., Biochemistry (1979) 18, 5294-5299), AGPC method (Chomczynski, P. et al., Anal. Biochem (1987) 162, 156-159) and the like can be used. The extracted mRNA can be purified using mRNA Purification Kit (manufactured by GE Healthcare Bioscience). Alternatively, a kit for directly extracting total mRNA from cells, such as QuickPrep mRNA Purification Kit (manufactured by GE Healthcare Bioscience), is commercially available. Total mRNA can also be obtained from hybridomas using such a kit. A cDNA encoding the antibody V region can be synthesized from the obtained mRNA using a reverse transcriptase. cDNA can be synthesized by AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (manufactured by Seikagaku Corporation) and the like. For the synthesis and amplification of cDNA, 5'-Ampli FINDER RACE Kit (manufactured by Clontech) and 5'-RACE method using PCR (Frohman, MA et al., Proc. Natl. Acad. Sci. USA ( 1988) 85, 8998-9002, Belyavsky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932). Furthermore, in the process of synthesizing such a cDNA, appropriate restriction enzyme sites described below can be introduced at both ends of the cDNA.

The desired cDNA fragment is purified from the obtained PCR product and then ligated with vector DNA. After the recombinant vector is prepared in this manner and introduced into Escherichia coli or the like to select a colony, a desired recombinant vector can be prepared from the E. coli forming the colony. Whether or not the recombinant vector has the nucleotide sequence of the desired cDNA can be confirmed by a known method such as the dideoxynucleotide chain termination method.

Hereinafter, the present invention will be described in more detail with reference to Examples, but it goes without saying that the present invention is not limited to these Examples.

Example 1: From production of monoclonal antibody to isolation
Preparation of monoclonal antibody against proBDNF pro domain
Antigen for antibody production
A peptide in the prodomain of proBDNF and an E. coli recombinant BDNF propeptide (Fig. 2, amino acids 19-128: the method of preparation is described in Japanese Patent No. 4457216) were used.

KVRPN EENNKDADLY is the peptide sequence used for the antigen.

Peptide synthesis and carrier cross-linking
1. Fluorenyl-MethOxy-Carbonyl(Fmoc) solid-phase synthesis method was used to synthesize 5 mg of each peptide with a purity of 80% or higher. The purity of the peptide was determined to be 80% or higher by the HPLC method, and it was confirmed by MASS analysis that it was in agreement with the theoretical molecular weight of the target peptide.
2. 1 mg peptide was cross-linked to Keyhole limpet hemocyanin (KLH) for immunization, and another 1 mg was cross-linked to BSA for assay. The method of cross-linking was such that no peptide loss occurred, and the three-dimensional structure was carefully considered. We also attempted to effectively enhance the immunogenicity of the recombinant protein by cross-linking the carrier immediately before immunization with a cross-linking method that does not involve the structure, and used it as an administration antigen together with the original non-cross-linked recombinant antigen.

Animal immunity and cell fusion
1. The antigen used was a mixture of 76-90C:KVR16-KLH 0.6 mg/300 μl and human BDNF propeptide (recombinant protein) 500 μl (0.75 mg/ml), and emulsion with the same amount of Freund's complete adjuvant (Pierce) The cells were allowed to homogenize, divided into equal parts, and immunized with 4 C57BL6 mice. Immunization was performed at an effective location for successful lymph node fusion.
2. Thirteen days after the initial immunization, as many lymph nodes as possible were swollen at multiple sites in the body, and lymphocytes from which antibody-producing cells were derived were obtained.
3. Mouse myeloma cells P3-X63-Ag8-U and lymphocytes obtained from the immunized animal were fused in the presence of 50% polyethylene glycol. The fused cells were cultured on 10 or more 96-well culture plates (960-well candidate group). Furthermore, medium exchange was performed 3 to 4 times with a HAT selective medium prepared with a composition close to serum-free.

Screening and specificity testing
1. Screening was performed according to antigen type. All low molecular weight peptide antigens were set as BSA cross-linked products, and assay antigens (i) MSM20 peptide, (ii) HSD13 peptide, (iii) human proBDNF recombinant protein were diluted with PBS to a concentration of 2 μg/ml each for ELISA. A 96 well plate was prepared. The enzyme-labeled secondary antibody for detection used in the ELISA was expected to detect IgG class in the subclass, and had excellent detection sensitivity and specificity. The presence or absence of antibodies in the antiserum and culture supernatant was detected by the ELISA method. 959 wells were measured for each antigen.
2. Each of the obtained positive strains was subjected to a competitive reaction with various immunogens to confirm the specificity of whether or not the epitope was recognized.

Cloning
1. Colony capture method was performed on the antigen-positive strain obtained by cell fusion (cells producing antibody that reacts with recombinant antigen).
2. The captured cells were immediately subjected to complete cloning by the limiting dilution method.
3. Hybridoma clone cells grown from single cells were obtained from the same line in case of an unexpected situation, and two lines (this line and sub-line) were obtained and expanded and cultured.

Cell freezing storage and restoration test
1. Freeze 7 strains and 3 sub-strains in a cryovial with a volume of 2 ml, and thaw 1 strain of this strain at a later date for reconstitution test. The delivery will be 4 strains and 2 sub strains, and the rest will be stored by the contractor as spare cells to avoid unexpected situations such as during transportation.
2. Store and deliver all the obtained hybridoma culture supernatants as a high-quality antibody-containing solution.

Example 2: Affinity of antibody to regions A, B and C of BDNF pro domain Three regions A, B and C in the BDNF pro domain sequence shown in FIGS. 3A and 3B were expressed and purified in E. coli.
Figure 3A shows the locations of the three regions A, B, C within the BDNF prodomain.
Figure 3B shows the number of amino acids, predicted molecular weight, and amino acid sequence of each. Overlapping amino acid sequences are underlined or double underlined.
Figure 3C Binding of each antibody with regions A, B, and C was examined with Biacore. Recombinant proteins in regions A, B or C were bound to a Biacore sensor chip and each antibody was flown to compare the binding strength.
Fig. 3D dot blot reaction of each antibody with regions A, B, C, BDNF prodomain and mature BDNF. Rb pAb is a rabbit polyclonal antibody against BDNF prodomain, and N20 is a rabbit polyclonal antibody against mature BDNF (off-the-shelf).

Experiment content:
Regions A, B, and C of BDNF propeptide expressed and purified in Biacore E. coli were immobilized on BIAcore sensor chip CM5 using a solvent of 10 mM Acetate, pH 4.7. The measurement parameters are temperature: 20° C., flow rate: 20 μL/min, Injection Volume: 120 μL, Dissociation Time: 300 sec, washing solution: 50 mM NaOH, Injection Volume: 60 μL. Under this condition, the interaction between each antibody and regions A, B, C was analyzed.

Dot blot Regions A, B, C, BDNF propeptide or mature BDNF (100 ng/dot) were allowed to stand on a nitrocellulose membrane in the arrangement shown in FIG. 3D and dried. Incubated with blocking solution for 30 minutes at room temperature. Each antibody was diluted 1000 times and an antibody reaction was performed as a primary antibody. After washing, the secondary antibody reaction was allowed to react for 30 minutes at room temperature. After washing, detection was performed with a detection reagent for HRP.

[Example 3] Immunostaining of CA3 region of adult mouse hippocampus FIG. 4 Immunostaining of hippocampal tissue sections of adult mouse and double staining of nuclear staining (DAPI) were performed using the antibodies #111, 113 and 101. Strong staining was observed in the cell bodies of pyramidal cells in the CA3 region for all the antibodies.

Experiment content:
15.7-week-old wild-type mice were perfusion-fixed with PBS and PBS containing 4% PFA, and the fixed brain tissue was taken out. After overnight post-fixation with PBS containing 4% PFA, it was gradually replaced with PBS containing 30% sucrose. After replacing the solution, the brain tissue was frozen, and a coronal section having a thickness of 30 μm was prepared with a cryostat and immersed in a solution of ethylene glycol:glycerol:PBS=1:1:2 and stored at −30° C. before use.
The following staining operation was performed with the section suspended. The sections were washed 3 times for 10 minutes with PBS, and then 3 times for 10 minutes with PBS containing 0.2% Triton X-100 (PBST). Subsequently, blocking was performed with PBS containing 3% bovine serum albumin at room temperature for 2 hours. Then, the cells were incubated with a primary antibody (diluted with blocking solution 500 times) at 4° C. for 2 nights. After washing 3 times with PBST for 5 minutes, the cells were incubated for 2 hours at room temperature with a secondary antibody solution (Alexa555-conjugated anti mouse IgG diluted 1000 times with blocking solution and containing 1 μg/ml DAPI). After washing 3 times with PBST for 5 minutes, the sections were mounted on a slide glass using the mounting agent PermaFluor. Images were acquired with a confocal laser microscope.

[Example 4] Immunoprecipitation of proBDNF using antibody #111, 113, 101 Using antibody #111, 113, 101, immunoprecipitation was performed from BDNF ^pro/+ adult mouse hippocampal tissue lysate. As a result, it was revealed that proBDNF can be immunoprecipitated with all of #101, 103, and 111 (FIG. 5).
IP, antibody used for immunoprecipitation. Control IgG is commercially available mouse IgG. IB, antibody used for detection by Western blot (off-the-shelf).

Experiment content:
BDNF ^pro/+ adult mouse hippocampal tissue was dissolved in a solubilization solution containing 1% Triton X-100, and the supernatant after centrifugation was used as a hippocampal tissue lysate. To 1 mg of protein in lysate, 1 μg of each antibody and control antibody were mixed and incubated at 4° C. overnight. 10 μl of Protein G Sepharose, which had been previously washed with the solubilization solution, was added and incubated for another hour. Sepharose was washed 4 times with 1 ml of a solubilizing solution, and then SDS sample buffer was added and treated at 100° C. for 5 minutes. These were used as IP samples. Western blotting was performed on the IP sample using SDS-PAGE and a commercially available BDNF prodomain antibody.

Claims (2)

A hybridoma producing a monoclonal antibody having the following characteristics (1) and (2), which has an accession number NITE P-02011, P-02012, or P- Hybridoma deposited as 02013.
(1) binds to the 19th to 128th BDNF prodomain of proBDNF and the 19th to 128th BDNF propeptide of proBDNF shown in SEQ ID NO: 1 (2) does not recognize region A of the BDNF prodomain and It specifically recognizes B and/or region C.
(The underlined part is the common part between the regions A and B, and the underlined part is the common part between the regions B and C.) A monoclonal antibody or an antigen-binding fragment thereof produced by the hybridoma according to claim 1.