Anti-gap 43 antibody, method for producing same, and tissue analysis method and kit for tissue analysis method using same

Abstract

Provided are: an anti-GAP 43 antibody capable of quantitatively identifying and detecting a nerve axon and a growth cone during growth or regeneration in an animal including a primate such as a human; a method for producing the anti-GAP 43 antibody; and a tissue analysis method and tissue analysis kit using the anti-GAP 43 antibody. Provided are: an anti-GAP 43 antibody that reacts with the phosphorylated polypeptide described in SEQ ID NO: 1 and does not react with the polypeptide of SEQ ID NO: 2; a method for producing the anti-GAP 43 antibody; and a tissue analysis method and kit for a tissue analysis method using the anti-GAP 43 antibody.

Description

Anti-GAP43 antibody, method for producing same, tissue analysis method using same, and kit for tissue analysis method

[0001] The present invention relates to an antibody used to evaluate neural development and regeneration in animals, including humans, and an analytical method using the same. This application claims priority to Japanese Patent Application No. 2023-100772, filed on June 20, 2023, the contents of which are incorporated herein by reference.

[0002] Growth-associated protein-43 kDa (hereinafter referred to as "GAP43") is known to be a phosphorylated protein involved in neuroplasticity and neural growth. GAP43 is phosphorylated by protein kinase C at serine 41 in rodents and primates, regulating the binding of calmodulin to GAP43 at presynaptic terminals. GAP43 was discovered because of increased axonal transport following nerve injury. It is known to be localized in growth cones (a growth cone marker), increase in long-term potentiation (LTP) of memory, and is an intrinsically denatured protein with no structure. Regarding its association with disease, suppression of GAP43 (shRNA) in epilepsy model rats has been shown to suppress epilepsy. Furthermore, suppression of GAP43 (shRNA) in glioblastoma (the most malignant brain tumor) has been shown to suppress growth.

[0003] In a study of GAP43 in rodents by the present inventors, phosphoproteomic analysis of growth cones immediately after birth in rodents revealed that mouse GAP43 is significantly phosphorylated at serine residue 96 (S96), serine residue 142 (S142), and threonine residue 172 (T172), demonstrating that these residues are marker molecules for nerve growth and are phosphorylated by the stress-responsive kinase c-jun N-terminal kinase 1 (hereinafter referred to as "JNK1"). Antibodies that detect phosphorylation of GAP43 S96 and T172 (pS96 and pT172 antibodies, respectively) are disclosed in Patent Document 1.

[0004] Patent Document 1 discloses an immunological analysis method for GAP43 for evaluating at least one of neural development and regeneration, comprising: (1) contacting a test sample with at least one anti-GAP43 antibody selected from the group consisting of the following antibodies: an anti-GAP43 antibody that binds to the phosphorylated serine residue at position 96 (pS96) of mouse GAP43, thereby being able to distinguish between the unphosphorylated serine residue at position 96 (S96) and the phosphorylated serine residue at position 96 (pS96), and thus capable of specifically detecting growth cones; and an anti-GAP43 antibody that binds to the phosphorylated threonine residue at position 172 (pT172) of mouse GAP43, thereby being able to distinguish between the unphosphorylated threonine residue at position 172 (T172) and the phosphorylated threonine residue at position 172 (pT172), and thus capable of specifically detecting growth cones; and (2) detecting or quantifying the anti-GAP43 antibody bound to the test sample. This technology aims to quantitatively evaluate neural development and / or regeneration by specifically detecting GAP43 phosphorylated at S96 or T172, and to provide an antibody that can specifically detect growth cones, as well as an immunological analysis method that utilizes the antibody.

[0005] Patent No. 6150395

[0006] GAP43, a nerve growth-associated protein, is expressed specifically in neurons, its expression increases during nerve growth and regeneration, and it is phosphorylated during long-term potentiation, which occurs after high-frequency stimulation of neural synapses. Conventionally known anti-GAP43 antibodies react with the GAP43 protein itself and detect phosphorylated and non-phosphorylated GAP43 without distinguishing between them. Therefore, because they react regardless of the state of GAP43 in neurons, these antibodies cannot specifically and quantitatively detect only the axons of growing neurons or their tip, the growth cones.

[0007] The technology of Patent Document 1 can specifically detect GAP43 phosphorylated at S96 or T172. As mentioned above, the present inventors have also been researching a technology that can specifically detect phosphorylation at S142.

[0008] However, S96 is only conserved in GAP43 in rodents such as mice and rats, and therefore antibodies to S96 cannot be used in primates, including humans. On the other hand, as shown in Patent Document 1, the surrounding sequences including rodent S142 and T172 are conserved in species including rodents and primates (corresponding to S151 and T181, respectively, in primates). However, because these amino acid residues are not specific to primate GAP43, they may not be suitable for detecting phenomena that differ significantly between rodents and primates, including humans. In other words, there are significant differences between rodents and primates, and significant differences are thought to exist in neural tissue, its growth, and function. Therefore, there is a need for antibodies that can detect growing axons and growth cones, which are thought to be specific to neural cells in primates, including humans.

[0009] The present invention has been made in consideration of the above-mentioned circumstances, and its object is to provide an anti-GAP43 antibody that can quantitatively identify and detect growing or regenerating axons and growth cones in primates, including humans, a method for producing the same, a tissue analysis method using the same, and a tissue analysis kit.

[0010] To achieve the above object, the present invention has the following aspects: [1] An anti-GAP43 antibody that reacts with the phosphorylated polypeptide of SEQ ID NO: 1 but does not react with the polypeptide of SEQ ID NO: 2. [2] The anti-GAP43 antibody of claim [1], which can distinguish between the unphosphorylated threonine residue at position 107 (T107) and the phosphorylated threonine residue at position 107 (pT107) by binding to the phosphorylated threonine residue at position 107 (pT107) of GAP43. [3] A method for producing the anti-GAP43 antibody of [1] or [2], which comprises administering the phosphorylated polypeptide of SEQ ID NO: 1 as an immunogen to an animal capable of producing an antibody, collecting serum from the animal, and purifying the antibody from the serum. [4] A tissue analysis method for detecting axons or growth cones during nerve development in a test animal, which comprises contacting a sample collected from tissue derived from the test animal with the anti-GAP43 antibody of [1] or [2]. [5] The tissue analysis method according to [4], wherein the subject animal is a primate. [6] The tissue analysis method according to [4] or [5], comprising a step of detecting or quantifying the anti-GAP43 antibody bound to an antigen contained in the sample by at least one method selected from the group consisting of ELISA, Western blotting, surface plasmon resonance, latex agglutination, and immunohistochemistry. [7] A kit for the tissue analysis method, comprising the anti-GAP43 antibody according to [1] or [2].

[0011] According to the present invention, there are provided an anti-GAP43 antibody capable of quantitatively identifying and detecting growing or regenerating axons and growth cones in animals including primates such as humans, a method for producing the same, a tissue analysis method using the same, and a kit for the tissue analysis method.

[0012]

[0033] Figure 1 is a Western blot photograph showing the results of an experiment to detect the expression of a human GAP43-EGFP fusion protein in this example. (a) Western blot photographs showing the results of examining the changes in expression of the fusion protein in the presence or absence of activation with ATP and JNK1 of the precipitate obtained by immunoprecipitating a human GAP43-EGFP fusion protein with an anti-GFP antibody, and (b) changes in the phosphorylation expression of an anti-GAP43 pT107 antibody in this example.

[0034] Figure 1 is a Western blot photograph showing the results of a cross-reactivity experiment of (a) a commercially available anti-GAP43 antibody and (b) an anti-GAP43 pT107 antibody in mouse brains and common marmoset brains during development.

[0035] Figure 1 is a photomicrograph showing the results of immunohistochemical staining of a mouse fetal brain with (a) a commercially available anti-GAP43 antibody and (b) an anti-GAP43 pT107 antibody.

[0036] Figure 1 is a photomicrograph showing the results of immunohistochemical staining evaluation of an anti-GAP43 pT107 antibody in a newborn common marmoset brain.

[0013] The anti-GAP43 antibody, its production method, tissue analysis method using the same, and tissue analysis kit according to the present invention will be described below with reference to embodiments, although the present invention is not limited to the following embodiments.

[0014] (Anti-GAP43 Antibody) The anti-GAP43 antibody of this embodiment is an anti-GAP43 antibody that reacts with the phosphorylated polypeptide set forth in SEQ ID NO: 1 but does not react with the polypeptide of SEQ ID NO: 2. Here, SEQ ID NO: 1: CGKAGE(pT)PSEEKK SEQ ID NO: 2: CGKAGETPSEEKK. Here, (pT) in SEQ ID NO: 1 indicates phosphorylated threonine. (The lowercase p indicates a phosphorylated amino acid.)

[0015] The above-mentioned SEQ ID NOS: 1 and 2 are amino acid sequences containing T107 (threonine 107), which is a part of the GAP43 protein sequence. SEQ ID NOS: 1 contains phosphorylated T107 (pT107), and SEQ ID NOS: 2 contains unphosphorylated T107. SEQ ID NOS: 1 and 2 each represent a part of GAP43 with C (cysteine) added to the beginning of the sequence, which is commonly used in producing peptide antibodies.

[0016] SEQ ID NO: 1 is a phosphorylated polypeptide, and SEQ ID NO: 2 is a non-phosphorylated polypeptide. Hereinafter, the term "non-phosphorylated polypeptide" refers to a polypeptide that contains only non-phosphorylated amino acid residues, and the term "phosphorylated polypeptide" refers to a polypeptide that contains at least one phosphorylated amino acid residue.

[0017] GAP43 of this embodiment (also known by other names such as GAP-43, Neuromodulin, B-50, F1, and PP46) includes GAP43 from various animals, but does not include GAP43 from animal species in which T107 is not conserved. For example, T107 is not conserved in rodent GAP43, and therefore GAP43 of this embodiment excludes rodent GAP43. GAP43 of this embodiment includes primate GAP43, and examples of primate GAP43 include human GAP43. Human GAP43 is the sequence represented by UniProt ID number P17677-1, or NCBI Reference Sequence: NP_002036.1, or GenBank accession number KAI4031029.1.

[0018] The anti-GAP43 antibody of this embodiment is preferably an antibody that can distinguish between the unphosphorylated threonine residue at position 107 (T107) and the phosphorylated threonine residue at position 107 (pT107) by binding to the phosphorylated threonine residue at position 107 (pT107) of GAP43.

[0019] Whether a target antibody reacts with the phosphorylated polypeptide of SEQ ID NO: 1 but does not react with the polypeptide of SEQ ID NO: 2 can be confirmed by immunological analysis for each antibody. For example, whether or not each antibody binds can be confirmed by cross-reactivity using Western blotting or the like, using the phosphorylated polypeptide and non-phosphorylated polypeptide of SEQ ID NO: 1 or 2 as antigens and the target antibody as the primary antibody. Quantitative confirmation can also be performed by ELISA using the same antigen-antibody. Other immunological analysis methods, such as those described below as analytical methods, can be used as appropriate.

[0020] The present inventors have found through phosphoproteomics analysis using primate brains that the 107th threonine residue of GAP43 (hereinafter referred to as "T107") is significantly phosphorylated in primates. The GAP43 molecule is conserved among mammals, and the amino acid sequence [GKAGET ＊ PSEEKK] (T ＊ The region corresponding to T107 is conserved in mammals (rhesus monkeys, common marmosets, and ferrets) except for rodents (mice).

[0021] The anti-GAP43 antibody of this embodiment detects T107, a GAP43 molecule that is not conserved in rodents but is conserved in mammals, including primates. Furthermore, the GAP43 molecule is known to be expressed specifically in nerve cells, its expression level increases during neural growth and regeneration, and it is phosphorylated during long-term potentiation, which occurs after high-frequency stimulation of neural synapses. The anti-GAP43 antibody of this embodiment can detect phosphorylated pT107, a T107 molecule that is conserved in primates, including humans. Therefore, it is possible to specifically detect axons and growth cones in primates, including humans, and quantitatively evaluate neural growth in these animals.

[0022] The anti-GAP43 antibody of this embodiment may be a polyclonal antibody or a monoclonal antibody. In the case of a polyclonal antibody, it can be produced using the phosphorylated polypeptide of SEQ ID NO: 1 as an antigen, as described below. In the case of a monoclonal antibody, any method for producing an antibody that specifically binds to pT107 can be used as appropriate.

[0023] (Method for producing anti-GAP43 antibody) The GAP43 antibody of this embodiment can be produced by a conventionally known method for producing an antibody that reacts with the phosphorylated polypeptide set forth in SEQ ID NO: 1 but does not react with the polypeptide of SEQ ID NO: 2. The method for producing the anti-GAP43 antibody can be appropriately selected from various methods for producing polyclonal antibodies and various methods for producing monoclonal antibodies.

[0024] A method for producing a polyclonal antibody includes, for example, administering the phosphorylated polypeptide set forth in SEQ ID NO: 1 as an immunogen to an animal capable of producing an antibody, collecting serum from the animal, and purifying the antibody from the serum.

[0025] In a more specific example, the phosphorylated polypeptide is injected as an immunogen into an animal capable of producing antibodies. The antibody-producing animal may be a mammal (e.g., mouse, rat, rabbit, sheep, or goat) or a bird (e.g., chicken). The immunogen may induce a superior immune response when linked to the carrier polymer. The immunogen is preferably injected into the animal host according to a predetermined schedule that includes one or more booster immunizations. The immunogen may be mixed with complete or incomplete Freund's adjuvant or other immunopotentiators and then injected into the animal. Serum (antiserum) may be collected from the antibody-producing animal, and antibodies may be purified from the serum. For example, antibodies may be purified from the serum by affinity chromatography using the polypeptide bound to a suitable solid support. The ability of the produced target antibodies to react with the phosphorylated polypeptide set forth in SEQ ID NO: 1 and not with the polypeptide set forth in SEQ ID NO: 2 can be confirmed by immunological analysis for each antibody, as described above. As mentioned above, cross-reactivity can be confirmed by Western blot or ELISA.

[0026] Monoclonal antibodies can be produced using conventional production methods. For example, a phosphorylated polypeptide antigen is injected into an animal capable of producing antibodies, and antibody-producing cells are extracted from the spleen or other organs and fused with immortalized myeloma cells to produce fused cells (hybridomas) that can survive while maintaining the antibody gene. These hybridomas are then screened for hybridomas that produce monoclonal antibodies with the desired activity. The resulting hybridoma cells are injected into an animal to purify the antibody. The antibody can be purified by injecting the cells into the ascites of the animal, collecting the ascites fluid, and purifying it by chromatography or other methods.

[0027] (Tissue Analysis Method) The tissue analysis method of this embodiment comprises a step of contacting the anti-GAP43 antibody with a sample collected from tissue derived from a subject animal, and detecting axons or growth cones during nerve growth in the subject animal.

[0028] The tissue analysis method aims to obtain information about nerve axons or growth cones during nerve growth, particularly to analyze their presence and growth state, by detecting or quantifying whether phosphorylation has occurred at T107 of the GAP43 protein contained in the tissue.

[0029] The subject animal can be selected appropriately, excluding animal species in which T107 in GAP43 is not conserved as described above. For example, rodents are excluded. Animals that can be selected include the order Primates (primates) and the order Carnivora (e.g., Mustelidae, especially ferrets). Primates are preferred, and primates may include, for example, Cercopithecidae (e.g., rhesus monkeys), Capuchin monkeys (e.g., marmosets), and humans. It has been confirmed that T107 in GAP43 is conserved in humans, rhesus monkeys, marmosets, and ferrets.

[0030] Preferably, the tissue is neural tissue.

[0031] The tissue analysis method of this embodiment preferably uses an immunological analysis method that detects antibody-antigen binding. The immunological analysis method may be a method that detects the presence or absence of binding, or a method that quantitatively analyzes binding. The tissue analysis method may use any immunological analysis method that can quantify or detect the binding of the anti-GAP43 antibody. For example, the tissue analysis method may include a step of detecting or quantifying the anti-GAP43 antibody bound to the antigen contained in the sample by at least one method selected from the group consisting of ELISA, Western blotting, surface plasmon resonance, latex agglutination, and immunohistochemistry.

[0032] The tissue analysis method of this embodiment is applicable to a variety of antibody-based approaches, such as histochemistry, blotting, ELISA, cell staining (fluorescence), pathological specimen staining (frozen sections, paraffin sections), immunoprecipitation, or immunoelectron microscopy, and can therefore be used for the diagnosis of human biopsy specimens.

[0033] (Tissue analysis kit) The tissue analysis kit of this embodiment includes the anti-GAP antibody. The tissue analysis kit is preferably a kit used in the tissue analysis method.

[0034] The tissue analysis kit of this embodiment may contain, in addition to the anti-GAP antibody, other components used in the tissue analysis method. For example, the tissue analysis kit of this embodiment may contain an antibody for use as a negative control. This antibody may react with the non-phosphorylated polypeptide of SEQ ID NO: 2 but not with the phosphorylated polypeptide of SEQ ID NO: 1.

[0035] For example, the kit for the tissue analysis method of this embodiment may include reagents or equipment for immunological detection and quantification of anti-GAP43 antibodies. For example, it may include a control sample of known concentration or amount for preparing a calibration curve for quantification. The control sample may be GAP43, a mutant or polypeptide thereof, or a fusion protein thereof, which contains at least a portion of the amino acid sequence of SEQ ID NO: 1 and in which a specific serine or threonine residue is phosphorylated. The kit for the tissue analysis method of this embodiment may also include a reagent for detecting or quantitating the anti-GAP43 antibody of this embodiment. The reagent may include a reagent appropriately selected depending on the method for detecting or quantitating the anti-GAP43 antibody, a buffer solution such as phosphate buffer as a diluent, a color developer, etc.

[0036] (Effects of this embodiment) The anti-GAP43 antibody of this embodiment, its production method, tissue analysis method using the same, and tissue analysis kit make it possible to quantitatively identify and detect growing or regenerating axons and growth cones in animals, including primates such as humans.

[0037] Research into neural growth and regeneration in animals, including humans, is being conducted to confirm the engraftment of iPS cell-derived neurons and confirm functional recovery. Experimentally, advances in iPS research have made it possible to study human neurogenesis using brain organoids (tissues similar to the brain). This neural research has created a particular need for methods that can evaluate neural outgrowth and development histologically, such as by immunohistochemistry.

[0038] If this method can yield antibodies capable of evaluating neural growth in humans or primates, it could be applied to neural regeneration research in primates. Furthermore, for example, human iPS cells transplanted into the brains of rodents could be distinguished from rodent neurons and their engraftment could be confirmed. It could also be applied to drug screening, for example, to discover drugs that promote optimal human neural growth. For example, these could be analyzed using ELISA to detect and quantify the target antigen (in this case, phosphorylated antigen) in a sample solution.

[0039] Although the factors related to nerve axon elongation and growth have not yet been clearly identified, previous studies in rodents have suggested that GAP43 is involved in nerve outgrowth, and antibodies to GAP43 have been used in research. However, antibodies that detect GAP43 itself have difficulty distinguishing between existing nerves and regenerated nerves. Therefore, it would be useful to be able to detect phosphorylated GAP43, which is thought to be involved in nerve growth and regeneration. However, no antibodies capable of specifically detecting phosphorylated GAP43 in humans or primates have been discovered to date, and antibodies more suitable for assessing nerve growth in humans and primates are desired. This embodiment provides anti-GAP43 antibodies, immunological analysis methods and kits for carrying out these methods, as well as reagents and methods for detecting axons and growth cones during nerve growth. This embodiment is expected to be highly useful in research on brain organoids derived from human ES cells and iPS cells, and in cell transplantation experiments at the site of nerve injury. In particular, although the phosphorylation of this sequence was predicted by bioinformatics, it was actually detected by the inventors in the developing brain of a primate (common marmoset) and a specific antibody was created against it, which corresponds to phosphorylation that does not exist in rodents. Therefore, its usefulness can be enhanced by using it in combination with previously discovered rodent-specific phosphorylation antibodies or phosphorylation antibodies common to both rodents and primates.

[0040] (Other Embodiments) The anti-GAP43 antibody of this embodiment can be used in a method for diagnosing a neurological disease. The anti-GAP43 antibody of this embodiment can be used in a method for treating a neurological disease. The anti-GAP43 antibody of this embodiment can be used in a pharmaceutical composition for treating a neurological disease. The anti-GAP43 antibody of this embodiment can be used for producing a pharmaceutical composition for treating a neurological disease. The method for producing the anti-GAP43 antibody of this embodiment can be used in a method for producing a pharmaceutical composition for treating a neurological disease. The tissue analysis method of this embodiment can be used in a method for diagnosing a neurological disease. The tissue analysis method of this embodiment can be used in a method for treating a neurological disease.

[0041] Examples are shown below, but the present invention is not limited to these examples.

[0042] The following experiments were conducted after being submitted and approved as a genetic modification experiment plan in accordance with the Niigata University Genetic Modification Experiment Safety Management Regulations (approval number SD01747: approved July 22, 2022, and approval number SD01541 (approved July 15, 2021) and confirmation number SI00096, under which recombinant JNK1 (baculovirus-insect cell expression system) was delivered). Animal experiments were also conducted after being approved by the Niigata University Animal Experiment Ethics Committee (approval number SA01137: approved April 28, 2022) and the Nagoya City University Animal Experimentation Committee (approval number H22M-09: approved March 26, 2010, and approval number H29M-08: approved March 23, 2017).

[0043] (Test Example 1: Preparation of anti-GAP43 pT107 antibody) (Polypeptide synthesis) A phosphorylated polypeptide was chemically synthesized, which contained a 13 amino acid sequence represented by SEQ ID NO: 1 starting with cysteine ​​at the terminal and in which the seventh threonine was phosphorylated (hereinafter referred to as "GAP43 pT107"), and a non-phosphorylated polypeptide was chemically synthesized, which contained a 13 amino acid sequence represented by SEQ ID NO: 2 starting with cysteine ​​at the terminal (hereinafter referred to as "GAP43 T107"). Chemical synthesis was performed by conventional solid-phase synthesis.

[0044] (Carrier Protein Binding) Keyhole limpet hemocyanin, a carrier protein, was bound to the phosphorylated polypeptide via the cysteine ​​residue at the end of the sequence.

[0045] (Antigen sensitization) The immunization schedule was a 63-day method, and two rabbits were sensitized with the antigen. The phosphorylated polypeptide GAP43 pT107 was mixed with an adjuvant control and administered to the rabbits. After the first immunization, sensitization was performed five times every 14 days. Blood was collected 35 and 49 days later, and antisera were obtained and measured by enzyme-linked immunosorbent assay (ELISA). After 63 days, whole blood was collected.

[0046] (Purification of anti-GAP43 pT107 antibody) The antiserum was affinity purified by methods well known to those skilled in the art. Briefly, affinity purification was performed using the phosphorylated polypeptide GAP43 pT107 described above to obtain affinity-purified fraction I (before absorption with non-phosphorylated antibody). The affinity-purified fraction I was then absorbed with the non-phosphorylated polypeptide GAP43 T107, and the flow-through fraction was stored as affinity-purified fraction II (after absorption with non-phosphorylated antibody) after the addition of sodium azide (final concentration 0.09% (v / v)).

[0047] (Evaluation of anti-GAP43 pT107 antibody using ELISA) The cross-reactivity of the anti-GAP43 pT107 antibody was examined by ELISA using affinity-purified fraction II (after absorption with non-phosphorylated antibody). Briefly, the phosphorylated polypeptide GAP43 pT107 and the non-phosphorylated polypeptide GAP43 T107 were added to a multi-well plate and immobilized. After diluting and reacting the affinity-purified fraction II (after absorption with non-phosphorylated antibody), a peroxidase-labeled goat anti-rabbit IgG antibody was added as a secondary antibody. After adding the substrate solution, the absorbance was measured using a microplate reader.

[0048] (Evaluation of anti-GAP43 pT107 antibody specificity using ELISA) The ELISA results showed that the anti-GAP43 pT107 antibody obtained as affinity-purified fraction II (after absorption with non-phosphorylated antibody) exhibited cross-reactivity with the phosphorylated polypeptide GAP43 pT107, but not with the non-phosphorylated polypeptide GAP43 T107 (not shown). In other words, it was demonstrated that the anti-GAP43 pT107 antibody can clearly distinguish between the phosphorylated polypeptide GAP43 pT107 and the non-phosphorylated polypeptide GAP43 T107.

[0049] (Test Example 2: Evaluation of specificity of anti-GAP43 pT107 antibody using Western blotting) (Culture method of cultured cells) U-251MG cells (cell number IFO50288, JCRB Cell Bank) were cultured in a commercially available medium (Dulbecco's Modified Eagle Medium) containing fetal bovine serum (Sigma-Aldrich Japan Co., Ltd.) [final concentration 10% (v / v)], Antibiotic-Antimycotic (100X) (catalog number 15240062, Gibco, Thermo Fisher Scientific) [final concentration 1% (v / v)], and Plasmocin® prophylactic (catalog number ant-mpp, InvivoGen) [final concentration 0.02% (v / v)]. The cells were incubated at 37°C, 5% CO using a 100% CO2 medium (catalog number 12430054, Gibco, Thermo Fisher Scientific). ２ and cultured under saturated water vapor atmosphere.

[0050] (Expression of Human GAP43) Adherent U-251MG cells were transfected with a plasmid vector of the target protein using a lipofection reagent (Lipofectamine 3000, catalog number L3000001, Thermo Fisher Scientific) according to the product manual.

[0051] The aforementioned plasmid vectors include a vector expressing a fusion protein (hereinafter referred to as "GAP43-EGFP") in which human GAP43 (GenBank accession number AM392759.1) is inserted into the multiple cloning site of the pEGFP-N1 vector [GenBank accession number U55762] (Catalog number Plasmid #6085-1, CLONTECH Laboratories, Inc.) containing a polynucleotide encoding Enhanced Green Fluorescence Protein (hereinafter referred to as "EGFP"); an expression vector containing a polynucleotide encoding a fusion protein (hereinafter referred to as "GAP43 T107A-EGFP") with a mutant in which the 107th threonine residue (T) of human GAP43 inserted into the pEGFP-N1 vector is replaced with an alanine residue (A); and pcDNA3 An expression vector containing a polynucleotide encoding an expression protein (hereinafter referred to as "MKK7B2Jnk1a1") in which Flag was removed from Flag MKK7B2Jnk1a1 (catalog number Plasmid #19726, Addgene Ltd) was used.

[0052] U-251MG cells were transfected with the expression vector, and cell lysates were prepared 24 to 48 hours later. The cell lysates were prepared in a sample preparation buffer (Tris-HCl buffer (pH 6.8) (catalog number 204-07885, Fujifilm Wako Pure Chemical Corporation) [final concentration 125 mM], glycerin (catalog number 075-00616, Fujifilm Wako Pure Chemical Corporation) [final concentration 20% (v / v)], sodium dodecyl sulfate (SDS) (catalog number 191-07145, Fujifilm Wako Pure Chemical Corporation) [final concentration 5% (w / v)]) containing protease inhibitors (leupeptin (catalog number 4041, Peptide Institute Co., Ltd.) [final concentration 20 μg / mL], pepstatin A (catalog number 4397, Peptide Research Co., Ltd.) [final concentration 20 μg / mL], pepstatin B (catalog number 4397, Peptide Research Co., Ltd.) [final concentration 5% (w / v)]). The medium was prepared by sonicating cells in a solution containing a mixture of 10 μg / mL phenylmethylsulfonyl fluoride (catalog number 27327-81, Nacalai Tesque, Inc.) [final concentration 1.0 mM], 1.0 mM ethylenediaminetetraacetic acid (catalog number 345-01865, Fujifilm Wako Pure Chemical Industries, Ltd.) [final concentration 1.0 mM] and a phosphatase inhibitor {sodium fluoride (catalog number 194-01971, Fujifilm Wako Pure Chemical Industries, Ltd.) [final concentration 1.0 mM], disodium molybdate(VI) dihydrate (catalog number 31621-52, Nacalai Tesque, Inc.) [final concentration 1.0 mM], and sodium orthovanadate (catalog number S6508, Sigma-Aldrich Japan K.K.) [final concentration 1.0 mM]}.

[0053] Protein quantification was performed using a DC™ Protein Assay Kit (catalog number 5000116, Bio-Rad Laboratories, Inc.) The cell lysate was reduced with 2-mercaptoethanol (catalog number M6250, Sigma-Aldrich Japan Co., Ltd.) [final concentration 5% (v / v)] at 95°C for 5 minutes.

[0054] (Verification of the specificity of the anti-GAP43 pT107 antibody by Western blotting) The specificity of the anti-GAP43 pT107 antibody was analyzed by Western blotting. The Western blotting was performed according to standard methods well known to those skilled in the art. Briefly, U-251MG cell lysates containing the expressed protein (2.5 μg / μL, 8 μL) and a protein size marker (Precision Plus Protein Two-Color Standard, Catalog No. 1610374, Bio-Rad Laboratories, Inc.) were electrophoresed on a 10% acrylamide gel (Mini-Protean TGX™ Precast Gel, Catalog Nos. 4561035 and 4561036, Bio-Rad Laboratories, Inc.) and transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon®-P Transfer Membrane, Catalog No. IPVH00010, Merck). After blocking with 4% (w / v) Block Ace powder (catalog number UKB80, KAC Corporation) diluted with 0.1% (v / v) polyoxyethylene (20) sorbitan monolaurate (catalog number 166-21213, Fujifilm Wako Pure Chemical Industries, Ltd.) / Tris-buffered saline (TBS-T) (hereinafter referred to as "4% Block Ace / TBS-T"), the cells were incubated overnight at 4°C with primary antibodies diluted in 4% Block Ace / TBS-T. The primary antibodies used were anti-GAP43 pT107 antibody (final concentration 0.57 μg / mL) and a commercially available anti-GAP43 antibody (catalog number 8945, Cell Signaling Technology Japan, Inc.) diluted 1:1000. For the secondary antibody, horseradish peroxidase (hereinafter referred to as "HRP")-labeled goat anti-rabbit IgG antibody (catalog number 7074, Cell Signaling Technology Japan, Inc.) was diluted 1000-fold in 4% Block Ace / TBS-T and allowed to react at room temperature for 1 hour.Detection was performed using cytiva® Amersham™ ECL™ Prime Western luminescence detection reagent (catalog number RPN2236, Global Life Science Technologies Japan, Inc.) with a high-resolution chemiluminescence detector (GeneGnome-5, SYNGENE), and analysis was performed with image capture software (GeneSys, SYNGENE).

[0055] (Verification of human GAP43 T107 phosphorylation by activation of protein kinase JNK1) Phosphorylation of human GAP43 T107 was performed by methods well known to those skilled in the art, such as an enzymatic activation experiment using immunoprecipitation techniques. Briefly, the adherent U-251MG cells prepared in 1.2 above were transfected with a plasmid vector expressing GAP43-EGFP using a lipofection reagent, and then 24 to 48 hours later, the medium was removed, the cells were washed with PBS, recovered with a cell scraper, and suspended in 1 mL of lysis buffer (described below). After sonication, the cells were centrifuged at 11,500 rpm and the supernatant was collected. After measuring the protein concentration, 10 μL of anti-Green Fluorescent Protein (hereinafter referred to as "GFP") monoclonal antibody (catalog number M048-3, Medical and Biological Laboratories Co., Ltd.) was added to 930 μL of the supernatant and allowed to react. 20 μL of Sepharose magnetic beads (catalog number 28944008, Cytiva® Protein G Mag Sepharose, Global Life Science Technologies Japan Co., Ltd.) were washed with lysis buffer and then allowed to react overnight at 4°C with the cell lysate reacting with the anti-GFP antibody. After centrifugation, the supernatant was removed using a magnetic rack (catalog number 28948964, MagRack 6, GE Healthcare Japan Co., Ltd.), and the magnetic beads were washed with Wash Buffer (described below). 1000 μL of Kinase Assay Buffer (described below) warmed to 30°C was added, and the mixture was dispensed into two tubes in 500 μL amounts. The supernatant was removed using a magnetic rack, and 40 μL of Kinase Assay Buffer was added to each tube. Kinase Assay Buffer and 0.5 μg of recombinant JNK1 (catalog number M33-10G, SignalChem Biotech Inc.) were added only to the JNK reaction tube, while only Kinase Assay Buffer was added to the JNK1 unreacted tube. Each tube was heated to 30°C and reacted for a total of 115 minutes with stirring every 5 minutes. The supernatant was removed using a magnetic rack, and the magnetic beads were dissolved in sample preparation buffer. Western blotting was performed as described in 1.3 above.Briefly, anti-GAP43 pT107 antibody (final concentration 0.57 μg / mL) and anti-HRP-labeled GFP antibody (catalog number 598-7, Medical & Biological Laboratories, Inc.) (1:1000 dilution) were used as primary antibodies. The anti-GAP43 pT107 antibody was diluted with Can Get Signal® Solution 1 (NKB-101, Toyobo Co., Ltd.), and the anti-HRP-labeled GFP antibody was diluted with 5% skim milk / TBS-T and incubated overnight at 4°C. To detect the anti-GAP43 pT107 antibody, HRP-labeled goat anti-rabbit IgG antibody was diluted 1:1000 with Can Get Signal® Solution 2 (NKB-101, Toyobo Co., Ltd.) as the secondary antibody and incubated at room temperature for 2 hours. Each antibody was detected using Cytiva® Amersham™ ECL™ Prime Western luminescence detection reagent with a dedicated high-resolution chemiluminescence instrument.

[0056] The composition of the lysis buffer was: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.4) (HEPES buffer) (catalog number H3375-500G, Sigma-Aldrich Japan Co., Ltd.) [final concentration 20 mM], SDS (catalog number 31607-65, Nacalai Tesque Inc.) [final concentration 0.1% (w / v)], sodium deoxycholate (catalog number 192-08312, Fujifilm Wako Pure Chemical Industries, Ltd.) [final concentration 0.05% (v / v)], NP-40 (catalog number NP40S-500ML, Sigma-Aldrich Japan Co., Ltd.) [final concentration 20 mM], Japan Co., Ltd.) [final concentration 1.0% (v / v)], leupeptin [final concentration 10 μg / mL], (p-amidinophenyl)methanesulfonyl fluoride hydrochloride (p-APMSF) (catalog number 019-26331, Fujifilm Wako Pure Chemical Industries, Ltd.) [final concentration 100 μM], pepstatin A [final concentration 10 μg / mL], sodium chloride [final concentration 150 mM], ethylenediaminetetraacetic acid [final concentration 0.5 mM].

[0057] Composition of the wash buffer: HEPES buffer [final concentration 20 mM], polyethylene glycol mono-4-octylphenyl ether [same components as Triton X-100] (catalog number 12967-45, Nacalai Tesque, Inc.) [final concentration 0.5% (v / v)], sodium chloride (catalog number 191-01665, Fujifilm Wako Pure Chemical Industries, Ltd.) [final concentration 150 mM], ethylenediaminetetraacetic acid [final concentration 0.5 mM], leupeptin [final concentration 10 μg / mL], p-APMSF [final concentration 100 μM], pepstatin A [final concentration 10 μg / mL], sodium fluoride [final concentration 5.0 mM], disodium molybdate(VI) dihydrate [final concentration 1.2 mM], sodium orthovanadate [final concentration 2.0 mM].

[0058] Composition of the Kinase Assay Buffer: adenosine triphosphate [final concentration 200 mM], dithiothreitol (048-29224, Fujifilm Wako Pure Chemical Industries) [final concentration 1 mM], HEPES buffer (final concentration 25 mM), sodium chloride (final concentration 15 mM), glycoletherdiaminetetraacetic acid (catalog number 342-01314, Fujifilm Wako Pure Chemical Corporation) (final concentration 1.5 mM), ethylenediaminetetraacetic acid (final concentration 2.5 mM), magnesium chloride (catalog number 135-00165, Fujifilm Wako Pure Chemical Corporation) (final concentration 7.5 mM), leupeptin (final concentration 10 μg / mL), p-APMSF (final concentration 100 μM), pepstatin A (final concentration 10 μg / mL), sodium fluoride (final concentration 5 mM), disodium molybdate(VI) dihydrate (final concentration 1.2 mM), sodium orthovanadate (final concentration 2.0 mM).

[0059] (Evaluation of fusion protein expression) Figure 1 is a Western blot photograph showing the results of an experiment to detect the expression of human GAP43-EGFP fusion protein. The photograph shows the results of an experiment to detect the reactivity of anti-GAP43 antibody (a) and anti-GAP43 pT107 antibody (b) when human GAP43-EGFP, human GAP43-EGFP / MKK7B2Jnk1a1 co-expression, and human GAP43 T107A-EGFP (phosphorylated inactive form) / MKK7B2Jnk1a1 co-expression in U-251MG cells. The lanes represent: 1. U-251MG cells expressing human GAP43 (EGFP-tagged); 2. U-251MG cells expressing human GAP43 (EGFP-tagged) and MKK7bJNK1a1; 3. U-251MG cells expressing human GAP43 (EGFP-tagged); This is a sample in which human GAP43 T107A (EGFP tagged) and MKK7B2JNK1a1 are expressed in U-251MG cells.

[0060] As shown in Figure 1(a), the expression of GAP43-EGFP in U-251MG cells, as well as the expression of GAP43-EGFP and GAP43 T107A-EGFP in the presence of MKK7B2Jnk1a1, was confirmed using an anti-GAP43 antibody. The expression levels of GAP43 were almost the same in all three samples.

[0061] Figure 2 is a Western blot photograph showing the results of examining the change in expression of a human GAP43-EGFP fusion protein immunoprecipitated with an anti-GFP antibody, depending on whether the precipitate was activated with JNK1 and adenosine triphosphate (hereinafter referred to as "ATP"). The graph shows the results of a reactivity experiment of an anti-GFP antibody immunoprecipitate of a human GAP43-EGFP fusion protein and the change in phosphorylation expression of an HRP-labeled anti-GFP antibody (a) and an anti-GAP43 pT107 antibody (b) when the precipitate was activated with JNK1 and ATP. The lanes represent: 1. A precipitate obtained by immunoprecipitating human GAP43 (EGFP-tagged) expressed in U-251MG cells with an anti-GFP antibody, which was reacted in Kinase Assay Buffer at 30°C for 115 minutes. 2. This sample was prepared by immunoprecipitating human GAP43 (EGFP tagged) expressed in U-251MG cells with an anti-GFP antibody, and the resulting precipitate was added with ATP, recombinant JNK1, and Kinase Assay Buffer, followed by reaction at 30°C for 115 minutes.

[0062] As shown in FIG. 2(a), the results of detection with anti-GFP antibody showed that there was no difference in the expression of human GAP43-EGFP fusion protein between activation by JNK1 and activation by ATP.

[0063] (Evaluation of anti-GAP43 pT107 antibody specificity) As shown in Figure 1(b), a Western blot photograph showing the results of evaluating the specificity of the anti-GAP43 pT107 antibody shows that when human GAP43-EGFP alone or human GAP43-EGFP / MKK7B2Jnk1a1 were co-expressed in U-251MG cells, both showed reactivity with the anti-GAP43 pT107 antibody. The MKK7B2Jnk1a1 vector, which expresses a fusion of MKK7 and JNK1, can activate the MKK7-JNK1 signaling pathway. Co-expression of GAP43-EGFP and MKK7B2Jnk1a was found to enhance phosphorylation of GAP43 T107. On the other hand, even in the presence of MKK7B2Jnk1a, the anti-GAP43 pT107 antibody showed no cross-reactivity with GAP43 T107A-EGFP. That is, the anti-GAP43 pT107 antibody did not react with unphosphorylated GAP43 T107A. As shown in Figure 2(b) above, the anti-GAP43 pT107 antibody reacted only when the precipitate obtained by immunoprecipitating human GAP43-EGFP fusion protein with an anti-GFP antibody was reacted with JNK1 and ATP. That is, it was found that JNK1 specifically phosphorylated human GAP43 T107, and that the anti-GAP43 pT107 antibody could specifically detect the phosphorylation of human GAP43 T107.

[0064] (Test Example 3: Evaluation of the specificity of anti-GAP43 pT107 antibody in mouse brain and common marmoset brain using Western blotting) (Developing fetal brain (mouse)) Brain tissue was isolated by a method known to those skilled in the art. Briefly, pregnant mice (C57BL / 6NCrl (IGS), 15 days of gestation, Jackson Laboratory Japan) were isolated under deep anesthesia with isoflurane, and the brains were isolated under hypothermic anesthesia.

[0065] (Developing Neonatal Brain (Common Marmoset)) Brain tissue from a common marmoset on day 1 after birth was extracted by a method known to those skilled in the art. Briefly, the brain tissue was extracted under deep anesthesia with isoflurane.

[0066] (Evaluation of the specificity of anti-GAP43 pT107 antibody using Western blotting) Tissue lysates (5 μg / μL, 5 μL each) from developing mouse brain (including brainstem and cerebellum) and common marmoset brain (pons) were analyzed by Western blotting. The primary antibodies used were anti-GAP43 pT107 antibody (final concentration 0.57 μg / mL) and a commercially available anti-GAP43 antibody (catalog number 8945, Cell Signaling Technology Japan, Inc.) diluted 1:1000. The remaining steps were the same as those for the Western blotting method described in Test Example 2 above.

[0067] (Evaluation of anti-GAP43 antibody reactivity using Western blotting) Figure 3 is a Western blot photograph showing the results of a cross-reactivity experiment of a commercially available anti-GAP43 antibody with mouse brains during development and common marmoset brains. The results of an experiment to detect cross-reactivity of a commercially available anti-GAP43 antibody (a) and an anti-GAP43 pT107 antibody (b) with mouse brains (7 days after birth) and common marmoset brains (1 day after birth) are shown. Each lane contains samples from: 1. 7-day-old mouse brain (including brainstem and cerebellum) 2. 1-day-old marmoset brain (pons)

[0068] As shown in Figure 3(a), the anti-GAP43 antibody showed reactivity in the developing brains of mice and marmosets.

[0069] (Evaluation of specificity of anti-GAP43 pT107 antibody by Western blotting) Figure 3(b) shows a Western blot photograph showing the results of a cross-reactivity experiment of anti-GAP43 pT107 antibody to mouse brain and common marmoset brain during development. The anti-GAP43 pT107 antibody did not cross-react with GAP43 in mouse brain, but did react with common marmoset brain. This indicates that the anti-GAP43 pT107 antibody does not cross-react with GAP43 from rodents such as mice, but is specifically reactive with GAP43 from primates such as common marmosets.

[0070] (Test Example 4: Immunohistochemical Staining Analysis of Anti-GAP43 pT107 Antibody) (Developing Fetal Brain (Mouse)) Brain tissue was extracted using methods well known to those skilled in the art. Briefly, pregnant mice (C57BL / 6NCrl (IGS), 15 days pregnant, Jackson Laboratory Japan, Inc.) were dissected under deep anesthesia with isoflurane to extract fetuses. The fetuses were perfusion-fixed in 4% (w / v) paraformaldehyde / 0.1 M phosphate buffer (hereinafter referred to as "PFA solution") under low-temperature anesthesia. Separately, pregnant mice (IGS, Jackson Laboratory Japan, Inc.) were purchased and perfusion-fixed using PFA solution under deep anesthesia on day 7 after birth. Both brain tissues after perfusion fixation were also fixed in PFA solution for 12-24 hours.

[0071] (Developing Neonatal Brain (Common Marmoset)) Brain tissue from a common marmoset on day 1 after birth was extracted using a method well known to those skilled in the art. Briefly, under deep anesthesia with isoflurane, the brain tissue was perfused with PBS and then PFA solution. The extracted brain tissue was further postfixed in PFA solution for 12-48 hours.

[0072] (Preparation of Paraffin-Embedded Blocks) Sections were prepared from the paraffin-embedded blocks using methods well known to those skilled in the art. Briefly, the mouse brains and common marmoset brains after post-fixation were replaced with PBS, and then replaced with alcohol, xylene, and paraffin using a paraffin infiltration device to prepare paraffin-embedded blocks. Sections with a thickness of 3 μm were prepared from the prepared paraffin-embedded blocks using a microtome and mounted on silane-coated slide glasses (New Silane III, catalog number 513618, Muto Chemical Co., Ltd.).

[0073] (Immunohistochemical staining of experimental animal brains) Immunohistochemical staining was performed by methods well known to those skilled in the art. Briefly, paraffin-embedded sections were deparaffinized with xylene and ethanol, then immersed in 0.3% (v / v) hydrogen peroxide (catalog number 081-04215, Fujifilm Wako Pure Chemical Corporation) / PBS for 30 minutes to block endogenous peroxidase activity. After washing with PBS, the sections were immersed in citrate buffer [prepared by mixing 9 mL of Solution A (citric acid monohydrate (catalog number 035-03495, Fujifilm Wako Pure Chemical Corporation): 2.1 g in 100 mL of distilled water) with 41 mL of Solution B (trisodium citrate dihydrate (catalog number 191-01785, Fujifilm Wako Pure Chemical Corporation): 14.7 g in 500 mL of distilled water) with 450 mL of distilled water], and then heated in an autoclave at 121°C for 10 minutes for antigen retrieval. After washing with PBS, the membrane was permeabilized with 0.2% (v / v) polyethylene glycol mono-4-octylphenyl ether / PBS (PBST) for 15 minutes, followed by blocking with 4% (w / v) Block Ace powder (catalog no. UKB80, KAC Corporation) / PBST for 60 minutes. For staining, anti-GAP43 pT107 antibody (final concentration 0.285 μg / mL) and a commercially available anti-GAP43 antibody (catalog no. 8945, Cell Signaling Technology Japan, Inc.) were used at a 1:1000 dilution. The sections were incubated with an enzyme-labeled secondary antibody (Histofine Simple Stain MAX-PO®, catalog no. 424141, Nichirei Biosciences Corporation) for 30 minutes, washed with PBS, and then developed with a 3,3'-diaminobenzidine (DAB) substrate kit (catalog no. 425011, Nichirei Biosciences Corporation). If nuclear staining was required at this stage, the specimens were immersed in Mayer's hematoxylin solution (catalog number 131-09665, Fujifilm Wako Pure Chemical Industries, Ltd.) for 3 minutes, followed by tap water for 10 minutes to allow the stain to develop. After dehydration with ethanol and clearing with xylene, the coverslips were mounted with a specimen mounting medium (Excel Mount 220, Pharma Co., Ltd.). The stained specimens were observed under an inverted microscope (BZ-X800, Keyence Corporation).

[0074] (Evaluation of anti-GAP43 pT107 in mouse fetal brain) Figure 4 shows the results of immunohistochemical staining evaluation of anti-GAP43 pT107 antibody in mouse fetal brain. The figure shows micrographs of immunohistochemical staining for cross-reactivity detection with anti-GAP43 antibody staining (co-stained with hematoxylin) and anti-GAP43 pT107 antibody (co-stained with hematoxylin) in a mouse brain (embryonic day 15). These immunohistochemically stained images of a mouse brain (coronal section) on embryonic day 15 are shown. (a) is an image stained with a commercially available anti-GAP43 antibody and co-stained with hematoxylin (scale bar: 500 μm). (b) is an enlarged view of the black square in (a). (c) is an image stained with anti-GAP43 pT107 antibody and co-stained with hematoxylin (scale bar: 500 μm). (d) is an enlarged view of the black square part in (c).

[0075] As shown in Figures 4(a) and 4(b), in the mouse fetal brain on embryonic day 15, the anti-GAP43 antibody reacted with nerve fibers in the thalamocortical tract. On the other hand, as shown in Figures 4(c) and 4(d), in the mouse fetal brain on embryonic day 15, no nerve fibers reactive with the anti-GAP43 pT107 antibody were observed, and only nuclei stained with hematoxylin were visualized. This indicates that the anti-GAP43 pT107 antibody does not cross-react with GAP43 in the mouse fetal brain.

[0076] (Evaluation of anti-GAP43 pT107 in common marmoset neonatal brain) Figure 5 shows the results of immunohistochemical staining evaluation using anti-GAP43 pT107 antibody in common marmoset neonatal brain. The figure shows a micrograph of immunohistochemical staining using anti-GAP43 pT107 antibody (co-stained with hematoxylin) in a common marmoset brain (day 1 after birth). Immunohistochemically stained images of a marmoset brain on day 1 after birth, where (a) shows a sagittal section (brainstem and cerebellum) stained with anti-GAP43 pT107 antibody (scale bar: 1 mm, cerebellum: Cb11, MB: Mid brain). (b) shows an enlarged view of the black square in (a) (scp: superior cerebellar peduncle). (c) shows an anti-GAP43 pT107 antibody-stained circular section (cerebral cortical visual cortex) (scale bar: 1 mm; ca: calcarine sulcus; gc: geniculocalcarine tract; V1: Primary Visual Cortex (VCx); V2m: secondary VCx (medial); V2l: V2 (lateral)). (d) shows an enlarged view of the black square in (C). In each figure, I-VI indicate the six-layer structure of the cerebral cortex.

[0077] Figures 5(a) and (b) are micrographs of sagittal sections of the cerebellum and brainstem, and Figures 5(c) and (d) are micrographs of circular sections of the occipital visual cortex of a 1-day-old common marmoset brain stained with anti-GAP43 pT107 antibody. The anti-GAP43 pT107 antibody reacted with nerve fibers in the superior cerebellar peduncle, designated "scp" in Figure 5(b). Furthermore, Figure 5(d) shows that the anti-GAP43 pT107 antibody reacted with nerve fibers in layers I, IVb, and V of the six-layer structure of the cerebral cortex in the marmoset visual cortex (V1).

[0078] Although several embodiments of the present invention have been described above, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their modifications are included within the scope and spirit of the invention, as well as within the scope of the invention and its equivalents as defined in the claims.

[0079] According to the present invention, there are provided an anti-GAP43 antibody capable of quantitatively identifying and detecting growing or regenerating axons and growth cones in animals including primates such as humans, a method for producing the same, a tissue analysis method using the same, and a kit for the tissue analysis method.

Claims

1. An anti-GAP43 antibody which reacts with the phosphorylated polypeptide set forth in SEQ ID NO:1 but does not react with the polypeptide set forth in SEQ ID NO:

2.

2. The anti-GAP43 antibody of claim 1, which is capable of distinguishing between the non-phosphorylated threonine residue at position 107 (T107) and the phosphorylated threonine residue at position 107 (pT107) of GAP43 by binding to the phosphorylated threonine residue at position 107 (pT107).

3. A method for producing an anti-GAP43 antibody according to claim 1 or 2, comprising administering the phosphorylated polypeptide set forth in SEQ ID NO: 1 as an immunogen to an animal capable of producing an antibody, collecting serum from the animal, and purifying the antibody from the serum.

4. A tissue analysis method for detecting nerve axons or growth cones during nerve growth in a subject animal, comprising the step of contacting a sample taken from tissue derived from the subject animal with the anti-GAP43 antibody according to claim 1 or 2.

5. The tissue analysis method according to claim 4, wherein the subject animal is a primate.

6. The tissue analysis method described in claim 4, comprising a step of detecting or quantifying the anti-GAP43 antibody bound to the antigen contained in the sample by at least one method selected from the group consisting of ELISA, Western blotting, surface plasmon resonance, latex agglutination, and immunohistochemistry.

7. A kit for tissue analysis comprising the anti-GAP43 antibody according to claim 1 or 2.