JPWO2006082798A1 - Monoclonal antibody that specifically recognizes rat type I microglia - Google Patents

Abstract

  An object of the present invention is to provide a monoclonal antibody that specifically recognizes rat type I microglia using microglia as an antigen. According to the present invention, there is provided a monoclonal antibody or a fragment thereof that specifically recognizes rat type I microglia.

Description

  The present invention relates to a monoclonal antibody that specifically recognizes rat type I microglia. More specifically, the present invention relates to a monoclonal antibody that specifically recognizes rat type I microglia, a hybridoma producing the antibody, and a method for detecting rat type I microglia using the antibody.

  The CNS has attracted many people's attention as the last frontier in bioscience. The brain is only one organ of the body, but its function is to collect and process information from the outside world, and after proper judgment, use the effector called muscle to command in the form of vocalization and action. Furthermore, through the sympathetic nervous system and parasympathetic nervous system stretched throughout the body, various regulation of the endocrine system, vascular system, etc. is performed. Such a cranial nervous system is mainly composed of cells with two different properties, neurons and glia. Neurons are thought to play a major role in the flow of information, but recently, glial cells are greatly involved in the expression of brain functions, and disorders of glial cells such as astrocytes, oligodendrocytes and microglia are impaired in brain function. It has also become clear that it causes, and its importance is drawing attention.

  One of the greatest challenges in life science is how to protect the aging brain, as the aging of the world tends to progress more and more due to the remarkable progress of medical technology in the 21st century. It is a problem. Alzheimer's disease (AD) is becoming the center of senile dementia because vascular dementia, which was the main cause of old-age dementia, has decreased due to the development of medical treatment and prevention methods. is there. AD is a refractory neurodegenerative disease that develops with aging of the brain, but since the onset age tends to be younger due to the westernization of Japanese lifestyles in recent years, and it imposes a heavy burden on nursing. , Has become an important issue not only medically but also socially. Given these circumstances, the establishment of AD treatment methods and the development of therapeutic agents are one of the most awaited issues. A decrease in the concentration of acetylcholine, which is a neurotransmitter, has been observed in the brains of AD patients, and acetylcholine-degrading enzyme inhibitors are currently in practical use as AD therapeutic agents. Although these therapeutic agents are effective for clinical symptoms such as cognitive and memory disorders, they are symptomatic drugs and cannot be expected to prevent the progression of AD pathology. For this reason, many studies are currently underway around the world to elucidate the etiology of AD and to establish a curative treatment.

  Pathological findings of AD include atrophy of the brain and reduction of synapses, as well as abnormal structures such as senile plaques and neurofibrillary tangles. Amyloid β peptide (Aβ), which accumulates in the form of senile plaques and vascular amyloid, is an insoluble peptide consisting of 40 to 42 amino acids. Aβ is produced by amyloid precursor protein (APP) undergoing two steps of cleavage by a protease and is secreted extracellularly. Since Aβ deposition is extremely disease-specific for AD and is a change in the earliest stages of AD that precedes the appearance of cell death and neurofibrillary tangles, Aβ deposition is deeply involved in the etiology of AD. The "amyloid hypothesis," which is said to be currently supported by many researchers.

  One of glial cells, "microglia," is a cell of the central nervous system that has macrophage-like properties. Its existence dates back to the discovery of Hortega in 1919, but the function and origin of cells still remain unclear. It has been found that microglia are activated during brain damage, neurodegenerative diseases such as AD and Parkinson's disease, multiple sclerosis, cerebral infarction, viral infection and brain tumors, and their relationship with the pathological conditions. Has been noticed. For example, in AD, microglia are extensively activated and produce and release inflammatory cytokines (IL-1β, IL-6, TNF-α, etc.) and nitric oxide (NO). It is said that these factors act on surrounding cells to further induce inflammation in the brain, promote Aβ production and aggregation, and cause nerve cell degeneration and cell death (Eikelenboom P, Bate C, Van Gool. WA, Hoozemans JJ, Rozemuller JM, Veerhuis R, Williams A (2002) Neuroinflammation in Alzheimer's disease and prion disease. Glia 40:232-239). In addition, recent experiments using culture systems have revealed that Aβ can pathologically activate microglia and, as a result, microglia exert cytotoxicity (Meda L, Cassatella MA, Szendrei GI, Otvos L. , Jr., Baron P, Villalba M, Ferrari D, Rossi F (1995) Activation of microglial cells by beta-amyloid protein and interferon-gamma. Nature 374:647-650; McDonald DR, Brunden KR, Landreth GE (1997). Amyloid fibrils activate tyrosine kinase-dependent signaling and superoxide production in microglia. J Neurosci 17:2284-2294; and Barger SW, Harmon AD (1997) Microglial activation by Alzheimer amyloid precursor protein and modulation by apolipoprotein E. Nature 388:878-881. ). On the other hand, microglia stimulated by insolubilized and aggregated Aβ produce and release chemokine (IL-8, MCP-1 etc.) and complement component (C3) that induce proliferation of M-CSF and chemotaxis, and cell membrane. The expression of the upper complement receptor is increased. In the brain, these molecules cause microglial proliferation and accumulation in senile plaques. It has been previously reported that activated microglia accumulate around senile plaques (McGeer PL, Itagaki S, Boyes BE, McGeer EG (1988) Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson's. and Alzheimer's disease brains. Neurology 38:1285-1291), it is suspected that microglia are deeply involved in the etiology of AD (Giulian D (1999) Microglia and the immune pathology of Alzheimer disease. Am J Hum Genet 65: 13-18). However, it has recently been pointed out that microglia accumulated near senile plaques have activated phagocytosis and may remove aggregated Aβ (Bard F, Cannon C, Barbour R, Burke RL, Games D, Grajeda H, Guido T, Hu K, Huang J, Johnson-Wood K, Khan K, Kholodenko D, Lee M, Lieberburg I, Motter R, Nguyen M, Soriano F, Vasquez N, Weiss K, Welch B, Seubert P, Schenk D, Yednock T (2000) Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease. Nat Med 6:916-919; and Wyss-Coray T, Lin C, Yan. F, Yu GQ, Rohde M, McConlogue L, Masliah E, Mucke L (2001) TGF-beta1 promotes microglial amyloid-beta clearance and reduces plaque burden in transgenic mice. Nat Med 7:612-618).

  The cellular characteristics of activated microglia include the proliferative ability, migration ability, phagocytosis ability, and enhancement of antigen-presenting ability, as well as TGF-β, TNF-α, neurotoxic substances such as active oxygen and NO, cytokines, chemokines, and It is to produce and secrete various physiologically active substances such as neurotrophic factors such as NGF, BDNF, and GDNF (Raivich G, Bohatschek M, Kloss CU, Werner A, Jones LL, Kreutzberg GW (1999) Neuroglial activation repertoire. in the injured brain: graded response, molecular mechanisms and cues to physiological function. Brain Res Brain Res Rev 30:77-105; and Hanisch UK (2002) Microglia as a source and target of cytokines. Glia 40:140-155). Furthermore, it interacts with immune system cells that have infiltrated into the brain from peripheral blood, regulates the immune response and acts on surrounding neurons and glial cells, and plays an important role in inflammation and cell death in lesions or tissue repair. Is believed to be responsible for.

  As described above, brain microglial cells are considered to have dual aspects of neuronal injury and protection, but there are still many unclear points regarding their functions and mechanisms. Although microglia show various actions, the actions have been mainly explained so far by considering microglia as macrophage-like cells. However, microglia are morphologically and functionally differentiated in the brain, and can no longer be explained only by the way of thinking as macrophages. Sawada et al. propose that the dihedrality of microglia is not due to a single microglia but to consist of multiple subpopulations (Kanzawa T, Sawada M, Kato K, Yamamoto K, Mori H, Tanaka R. (2000) Differentiated regulation of allo-antigen presentation by different types of murine microglial cell lines. J Neurosci Res 62:383-388; and Katoh Y, Niimi M, Yamamoto Y, Kawamura T, Morimoto-Ishizuka T, Sawada M, Takemori. H, Yamatodani A (2001) Histamine production by cultured microglial cells of the mouse. Neurosci Lett 305:181-184). That is, it is an idea that heterogeneous cell populations having different properties exhibit respective actions, rather than a single microglia exhibiting two-sidedness. However, little is known about the relationship between the existence of this heterogeneous cell group and the dual function of neurons and the mechanism thereof. Under such circumstances, it is essential to elucidate the functions of not only neurons but also glial cells including microglia.

  By the way, one of the indispensable tools for medical biology research is a monoclonal antibody. Especially for blood cells, the analysis of a subset of them has been dramatically advanced by a monoclonal antibody against the membrane antigen of lymphocytes. Monocytes and macrophages have various cell groups that undergo different maturation stages and different differentiation and activation states, and there are also organ-specifically differentiated macrophages such as Kupffer cells in the liver. That is, the monoclonal antibodies that are said to recognize monocyte/macrophage cells include those that show various reactions, and there are many antibodies that show crossover with cells of other lineages.

  Microglia, unlike other glia, is believed to originate from bone marrow-derived monocytes that invaded the brain during childhood. To support this, it is known to express several marker proteins common to macrophages (Nakajima K, Kohsaka S (1998) [Microglia: function in the pathological state]. No To Shinkei 50:5 -16). However, as mentioned above, there are many cases in which microglia cannot be explained from the same viewpoint as macrophages. Nevertheless, the most commonly used monoclonal antibodies for microglia are those produced using peritoneal macrophages and splenocytes as antigens except for a few cases (Austyn JM, Gordon S (1981 ) F4/80, a monoclonal antibody directed specifically against the mouse macrophage.Eur J Immunol 11:805-815; Springer T, Galfre G, Secher DS, Milstein C (1979) Mac-1: a macrophage differentiation antigen identified by monoclonal antibody Eur J Immunol 9:301-306; and McKnight AJ, Gordon S (1998) Membrane molecules as differentiation antigens of murine macrophages. Adv Immunol 68:271-314). Therefore, in order to analyze the detailed function of microglia, it is necessary to prepare a monoclonal antibody using microglia as an antigen.

  An object of the present invention is to provide a monoclonal antibody that specifically recognizes rat type I microglia using microglia as an antigen. Another object of the present invention is to provide a method for specifically detecting rat type I microglia using the above monoclonal antibody.

  The present inventors have conducted extensive studies to solve the above problems, performed rat primary culture microglia according to the mixed glial culture method developed by Sawada et al. (Suzumura et al., 1987; Sawada et al., 1990), and type- 1, type-2 microglia were separated and antibodies were prepared using these as antigens. The present inventors have further analyzed the properties of the obtained monoclonal antibody by Western blotting and fluorescent staining to investigate whether they can discriminate between two different subtypes of microglia, and to identify an antibody that specifically recognizes rat type I microglia. Succeeded in getting. Furthermore, it was also found that the obtained antibody specifically recognizes human oligodendrocyte and does not react with human glioblastoma (glioblastoma). The present invention has been completed based on these findings.

That is, according to the present invention, the following inventions are provided.
(1) A monoclonal antibody or a fragment thereof that specifically recognizes rat type I microglia.
(2) The monoclonal antibody or fragment thereof according to (1), which does not react with rat type II microglia.

(3) The monoclonal antibody or a fragment thereof according to (1) or (2), which specifically recognizes human oligodendrocyte.
(4) The monoclonal antibody or fragment thereof according to any one of (1) to (3), which does not react with human glioblastoma (glioblastoma).

(5) The monoclonal antibody according to any one of (1) to (4), which has a molecular weight of an antigen to be recognized of about 50 to 70 kDa and further loses reactivity with the antigen in the presence of a reducing agent. An antibody or fragment thereof.
(6) The monoclonal antibody according to any one of (1) to (5), which hardly reacts with rat neurons, rat astrocytes, peripheral macrophages, mouse type I microglia and mouse type II microglia. Or a fragment thereof.
(7) A monoclonal antibody or a fragment thereof produced by a hybridoma having accession number FERM BP-10475.

(8) A hybridoma that produces the monoclonal antibody according to any one of (1) to (7).
(9) The hybridoma according to (8), which is obtained by fusing mammalian immune cells immunized with an immunogen containing rat type I microglia and mammalian myeloma cells.
(10) A hybridoma having the deposit number FERM BP-10475.

(11) In any one of (1) to (7), including a step of culturing the hybridoma according to any one of (8) to (10), and a step of collecting and purifying a monoclonal antibody produced by the hybridoma A method for producing the described monoclonal antibody.

(12) A method for detecting rat type I microglia, which comprises performing an immunoassay using the monoclonal antibody or the fragment thereof according to any one of (1) to (7).
(13) The method for detecting rat type I microglia according to (12), which comprises at least the following steps (a) and (b).
(A) a step of reacting the sample with a monoclonal antibody or a fragment thereof according to any one of (1) to (7); and (b) an antigen-antibody complex formed in step (a), and Reacting with a labeled antibody;

(14) A method for detecting human oligodendrglioma, which comprises performing an immunoassay using the monoclonal antibody or the fragment thereof according to any one of (1) to (7).
(15) The method for detecting human oligodendrocyte glioma according to (14), which comprises at least the following steps (a) and (b).
(A) a step of reacting the sample with a monoclonal antibody or a fragment thereof according to any one of (1) to (7); and (b) an antigen-antibody complex formed in step (a), and Reacting with a labeled antibody;

(16) A kit for detecting rat type I microglia, which comprises the monoclonal antibody or fragment thereof according to any one of (1) to (7).
(17) A kit for detecting a human oligodendrocyte that contains the monoclonal antibody or fragment thereof according to any one of (1) to (7).

  Microglia is one of the glial cells along with astrocytes and oligodendrocytes observed in the central nervous system, and accounts for about 5% of all glial cells (Kreutzberg GW (1996) Microglia: a sensor for pathological events in the CNS). Trends Neurosci 19:312-318.). Its existence dates back to the discovery of Hortega in 1919, but the function and origin of cells still remain unclear. In the normal brain, small cell bodies have long branched projections (ramified form), but when the brain is injured or damaged, quiescent microglia reacts rapidly and greatly changes its properties. This is the activation of microglia. Activated microglia have degenerated protrusions, resulting in large rounded cell bodies, and morphology similar to that of ameboid microglia found in the developing brain. This change is quicker and more dramatic than any other cell in the brain. It is known that activated microglia show strong migratory ability, act as phagocytic cells to remove degenerated cells, and produce nerve growth factors such as BDNF and NGF to show neuroprotective action (Sawada M , Suzumura A, Marunouchi T (1995) Cytokine network in the central nervous system and its roles in growth and differentiation of glial and neuronal cells.Int J Dev Neurosci 13:253-264.; Nakajima K, Kohsaka S (2001) Microglia: activation and their significance in the central nervous system. J Biochem (Tokyo) 130:169-175.; and Salimi K, Moser K, Zassler B, Reindl M, Embacher N, Schermer C, Weis C, Marksteiner J, Sawada M, Humpel C (2002) Glial cell line-derived neurotrophic factor enhances survival of GM-CSF dependent rat GMIR1-microglial cells. Neurosci Res 43:221-229.). On the other hand, activated microglia also release oxygen radicals, TNF-α, IL-1β, and nitric oxide (NO), which are cytotoxic factors, and attack pathogens and tumor cells, which in turn damage the surrounding neurons. Has been suggested (Meda L, Cassatella MA, Szendrei GI, Otvos L, Jr., Baron P, Villalba M, Ferrari D, Rossi F (1995) Activation of microglial cells by beta-amyloid protein and interferon -gamma. Nature 374:647-650.). In recent years, it has been suspected that activated cytoglia having this cytotoxicity may be involved in the etiology of neurological diseases. Thus, microglia are considered to be cells that have the duality of good and evil.

  Sawada et al. propose that the duality of microglia is not due to a single microglia, but because it consists of multiple subpopulations. We established several types of microglial cell lines that proliferate in a GM-CSF-dependent manner but express different proteins.One of these cell lines, 6-3, is a highly cytotoxic microglia (type-1 microglia). ), expresses CD40, which is not observed in other mouse-derived microglial cell lines (Kanzawa T, Sawada M, Kato K, Yamamoto K, Mori H, Tanaka R (2000) Differentiated regulation of allo-antigen presentation by different types of murine microglial cell lines. J Neurosci Res 62:383-388.). Thus, the expression levels of proteins and proteins expressed by each cell line are different, which strongly suggests that microglia is not a single subpopulation but multiple subpopulations. However, little is known about the function of each of the microglial subtypes.

  Therefore, the present inventors separated primary cultured rat microglia into type-1 microglia with strong cytotoxicity and type-2 microglia with strong cytoprotective properties, and produced monoclonal antibodies using them as immunogens, We succeeded in confirming that the obtained antibody can distinguish two subtypes.

Hereinafter, embodiments and methods of the present invention will be described.
1. Monoclonal Antibody of the Present Invention The monoclonal antibody of the present invention is characterized by specifically recognizing rat type I microglia. In the present invention, “specifically recognizing rat type I microglia” means recognizing and reacting with rat type I microglia, but not reacting with rat type II microglia, or reacting with rat type I microglia. Significantly lower reactivity with rat type II microglia compared to sex.

  More preferably, the monoclonal antibody of the present invention is characterized by specifically recognizing human oligodendrogliomas. In the present invention, “specifically recognizing human oligodendrocyte glioma” means recognizing and reacting with human oligodendrocyte glioma, but not reacting with human glioblastoma or human oligodendrocyte. This means that the reactivity with human glioblastoma is significantly lower than that with glioma.

  An example of the monoclonal antibody of the present invention is a monoclonal antibody characterized in that the recognized antigen has a molecular weight of about 50 to 70 kDa and further loses reactivity with the antigen in the presence of a reducing agent.

  Further, examples of the monoclonal antibody of the present invention include a monoclonal antibody characterized by hardly reacting with rat neurons, rat astrocytes, peripheral macrophages, mouse type I microglia, and mouse type II microglia.

  An example of the monoclonal antibody of the present invention is the monoclonal antibody 9F5 described in the examples of the present specification. The hybridoma producing the monoclonal antibody 9F5 has an Accession No. FERM AP-20353 (Accession No. FERM P-20353) as of January 5, 2005, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center. It has been deposited at Chuo No. 6 (Postal Code 305-8566), 1-chome, Higashi 1-1-chome, Tsukuba, Ibaraki, Japan. It was internationally designated as FERM BP-10475 on December 9, 2005. It has been transferred to a deposit.

The term “antibody” as used herein is used to include not only full-length antibodies but also antibody fragments. That is, the present invention provides a fragment of a monoclonal antibody that specifically recognizes rat type I microglia. The antibody fragment is preferably a functional fragment, and examples thereof include F(ab′) ₂ and Fab′.

F(ab′) ₂ and Fab′ are produced by treating immunoglobulin with a proteolytic enzyme (eg, pepsin or papain) and are present between two H chains in the hinge region. Antibody fragment produced by digestion before and after the disulfide bond.
Furthermore, when the term “antibody fragment” is used in the present specification, it also includes a protein containing an antigen-binding site derived from a gene encoding the antibody.

For example, when IgG1 is treated with papain, an L chain consisting of VL (L chain variable region) and CL (L chain constant region) is cleaved upstream of a disulfide bond existing between two H chains in the hinge region, It is possible to produce two homologous antibody fragments in which the H chain fragment consisting of VH (H chain variable region) and CHγ1 (γ1 region in the H chain constant region) is linked by a disulfide bond at the C-terminal region. Each of these two homologous antibody fragments is called Fab'. Further, when IgG is treated with pepsin, it is cleaved downstream of the disulfide bond existing between two H chains in the hinge region to produce an antibody fragment slightly larger than that in which the two Fab's are linked at the hinge region. You can This antibody fragment is called F(ab') ₂ .

  Further, the monoclonal antibody of the present invention can be used as an immobilized antibody immobilized on an insoluble carrier such as a solid phase carrier or as a labeled antibody labeled with a labeling substance. All such immobilized antibodies and labeled antibodies are within the scope of the present invention.

  The immobilized antibody refers to an antibody that is carried on an insoluble carrier by physical adsorption, chemical bonding, or the like. These immobilized antibodies can be used for detecting, quantifying, separating or purifying an antigen contained in a sample (for example, a body fluid sample such as plasma, a culture supernatant or a centrifugation supernatant). Examples of insoluble carriers that can be used for immobilizing antibodies include (1) plastics made of polystyrene resin, polycarbonate resin, silicone resin, nylon resin, etc., and water-insoluble substances typified by glass, etc. Plate, test tube or tube having an internal volume, beads, balls, filters, membranes, and the like, and (2) cellulose-based carrier, agarose-based carrier, polyacrylamide-based carrier, dextran-based carrier, polystyrene-based carrier, Examples thereof include insoluble carriers used for affinity chromatography such as polyvinyl alcohol-based carriers, polyamino acid-based carriers and porous silica-based carriers.

The labeled antibody means an antibody labeled with a labeling substance, and these labeled antibodies are antigens (ie, rat) contained in a sample (for example, body fluid sample such as plasma, culture supernatant or centrifugation supernatant). Antigen specific for type I microglia) can be used for detection or quantification. The labeling substance that can be used in the present invention is not particularly limited as long as it can be detected by binding it to an antibody by a physical bond, a chemical bond or the like. Specific examples of the labeling substance include an enzyme, a fluorescent substance, a chemiluminescent substance, biotin, avidin or a radioisotope, and more specifically, peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose. -Su-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, catalase, apoglucose oxidase, urease, enzymes such as luciferase or acetylcholinesterase, fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelate , A fluorescent substance such as dansyl chloride or tetramethylrhodamine isothiocyanate, a radioactive isotope such as ³ H, ¹⁴ C, ¹²⁵ I or ¹³¹ I, biotin, avidin, or a chemiluminescent substance. A known method such as a glutaraldehyde method, a maleimide method, a pyridyl disulfide method, or a periodic acid method can be used as a method for binding the labeling substance and the antibody.

  Here, the radioisotope and the fluorescent substance can provide a detectable signal by themselves, but the enzyme, chemiluminescent substance, biotin and avidin cannot produce a detectable signal by themselves, and thus one more species is used. A detectable signal is generated by reacting with the above-mentioned other substances. For example, at least a substrate is required in the case of an enzyme, and various substrates are used depending on the method for measuring the enzyme activity (colorimetric method, fluorescence method, bioluminescence method, chemiluminescence method, etc.). In the case of biotin, at least avidin or enzyme-modified avidin is generally reacted. If necessary, various color-developing substances depending on the substrate may be used.

2. Hybridoma Producing Monoclonal Antibody of the Present Invention The present invention also relates to a hybridoma producing a monoclonal antibody that specifically recognizes the rat type I microglia of the present invention described above. The monoclonal antibody of the present invention can be produced using the hybridoma.

  Hereinafter, a method for producing a hybridoma that produces a monoclonal antibody that specifically recognizes rat type I microglia of the present invention will be described.

  First, antibody-producing cells are prepared in an animal by immunizing a mammal with rat type I microglial cells. The type of mammal is not particularly limited, but generally includes mice, rats, cows, rabbits, goats, sheep and the like, preferably rodents such as mice and rats, and more preferably mice. Examples of the mouse include A/J strain, BALB/C strain, DBA/2 strain, C57BL/6 strain, C3H/He strain, SJL strain, NZB strain, CBA/JNCrj strain mouse. The BALB/C strain mouse is preferable because the myeloma-derived cell line of the strain has been established at the time of hybridoma production.

As the rat type I microglial cells used for immunization, cells themselves may be used, or cell extracts may be used.
Prior to immunization, rat type I microglial cells may be mixed with an adjuvant to enhance the immune response. Examples of adjuvants include water-in-oil emulsions (eg, incomplete Freund's adjuvant), water-in-oil emulsions, oil-in-water emulsions, liposomes, aluminum hydroxide gel, silica adjuvants, powdered bentonites, and tapioca adjuvants. In addition, bacterial cells such as BCG and Propionibacterium acnes, cell walls and bacterial cell components such as trehalose dicholate (TDM); lipopolysaccharide (LPS) and lipid A fraction which are endotoxins of Gram-negative bacteria; β-glucan (polysaccharide) Body); muramyl dipeptide (MDP); bestatin; synthetic compounds such as levamisole; proteins or peptide substances derived from biological components such as thymus hormone, thymic hormone humoral factor and tuftsin; and mixtures thereof (eg complete Freund's adjuvant) ) And the like. These adjuvants are effective in enhancing or suppressing the immune response depending on the administration route, dose, administration time and the like. Furthermore, depending on the type of adjuvant, there are differences in the production of antibody in the blood against the antigen, induction of cell-mediated immunity, immunoglobulin class, and the like. Therefore, it is preferable to select an adjuvant appropriately according to the immune response of interest. Methods for treatment with adjuvants are known in the art.

  Immunization of mammals is performed according to methods known in the art. For example, the antigen, rat type I microglial cells, is injected subcutaneously, intradermally, intravenously, or intraperitoneally in a mammal. Since the immune response varies depending on the type and strain of the mammal to be immunized, the immunization schedule is appropriately set according to the animal used. The antigen administration is repeated several times after the first immunization. Boosts can be given, for example, 4 weeks, 6 weeks, and 6 months after the first immunization.

  After immunization, blood is collected from the mammal, and the obtained blood is assayed for the presence of rat type I microglia binding activity to confirm that antibodies to rat type I microglial cells are produced in the body of the mammal. Examples of the assay method include known methods such as enzyme immunoassay (ELISA method), radioimmunoassay method (RIA), and fluorescent antibody method.

  After confirming the production of the rat type I microglia-binding antibody, a boost (additional injection of an immunogen) can be performed to bring immune cells capable of producing a specific antibody into a state suitable for cell fusion. The amount of rat type I microglial cells to be administered by boosting is not particularly limited, but is preferably about 4 to 5 times the amount immunized initially. Boosting can generally be performed with an emulsion of immunogen and Freund's incomplete adjuvant. The route of administration is appropriately selected from subcutaneous, intradermal, intravenous, intraperitoneal and the like.

  After the final immunization, spleen cells are extracted from the immunized mammal and fused with a myeloma-derived cell line. For cell fusion, it is preferable to use a cell line having a high proliferation ability, and it is preferable that the cell line derived from myeloma is compatible with the mammal from which the splenocytes to be fused are derived. Cell lines derived from mouse myeloma include P3X63-Ag8.653, Sp2/O-Ag14, FO.1, S194/5. XX0 BU. 1, P3/NS1/1-Ag4-1 and the like.

Cell fusion is performed according to a method known in the art. Examples of the cell fusion method include a polyethylene glycol method, a method using Sendai virus, and a method using electric current.
The resulting fused cells can be grown according to conditions known in the art. The desired fused cells are selected based on the binding ability of the antibody produced.

  The binding ability of antibodies produced from the fused cells can be assayed based on methods known in the art. In the present invention, in order to obtain a fused cell producing an antibody having a specific binding ability to rat type I microglia and a high binding ability, a target cell line is cloned by utilizing selection based on the binding ability to rat type I microglia. To do. The binding ability of an antibody can be assayed using a method such as an ELISA method, a RIA method, or a fluorescent antibody method, as described above for confirmation of antibody production. The ELISA method is preferable because it is simple and has high sensitivity.

  Cloning of the fused cells can be performed using a method known in the art. Examples of the cloning method include a limiting dilution method and a soft agar method. The limiting dilution method is preferable because it is easy to operate and highly reproducible. In order to efficiently select useful cells from many fused cells obtained by cell fusion, cell selection is preferably performed from the initial stage of cloning. In this way, a fused cell line producing an antibody with the desired binding ability can be finally selected.

  By culturing the monoclonal antibody-producing cell line selected as described above in a large amount, a large amount of a monoclonal antibody specific to rat type I microglia can be produced. Large-scale culture methods for monoclonal antibody-producing cell lines include in vivo and in vitro culture. Examples of large-scale culture in vivo include a method of injecting the fused cells into the abdominal cavity of a mammal, allowing the cells to grow, and producing antibodies in the ascites. For in vitro culture, the fused cells are cultured in medium and the antibody is produced in the medium.

  The monoclonal antibody of the present invention can be purified from the ascites or the culture supernatant obtained by large-scale culture using a method known in the art. For purification, for example, DEAE anion exchange chromatography, affinity chromatography, ammonium sulfate fractionation method, PEG fractionation method, ethanol fractionation method and the like are used in appropriate combination. The antibody of the present invention can be purified so that it is preferably about 90% pure, preferably about 95% pure, more preferably about 98% pure.

3. Detection of rat type I microglia or human oligodendrocyte glioma using the monoclonal antibody of the present invention The present invention further comprises performing immunoassay using the monoclonal antibody or fragment thereof of the present invention, rat type I microglia. Alternatively, it relates to a method for detecting human oligodendrocyte. This method preferably includes at least the following steps (a) and (b).
(A) reacting the monoclonal antibody or fragment thereof of the present invention with a sample; and (b) reacting the antigen-antibody complex formed in step (a) with a labeled antibody for detection;

  Since the monoclonal antibody of the present invention can specifically recognize human oligodendrocyte glioma, it can be used for detecting or diagnosing oligodendrocyte glioma in human.

  The detection and/or quantification method of the present invention may be any method as long as it is an assay using an antibody, that is, an immunoassay, for example, enzyme-linked immunosorbent assay (ELISA), fluorescent immunoassay, radioimmunoassay (RIA). ), luminescent immunoassay, enzyme antibody method, fluorescent antibody method, immunonephelometry, latex agglutination reaction, latex nephelometry, erythrocyte agglutination reaction, particle agglutination reaction or Western blotting method.

  The sample used for the detection and/or quantification method of the present invention is not particularly limited, and is not particularly limited as long as it is a biological sample which may contain rat type I microglia or human oligodendrocyte.

  When the detection method of the present invention is carried out by an immunoassay using a labeled antibody such as an enzyme immunoassay, a fluorescent immunoassay, a radioimmunoassay or a luminescent immunoassay, it should be carried out by a sandwich method or a competitive method. In the case of the sandwich method, at least one of the immobilized antibody and the labeled antibody may be the monoclonal antibody of the present invention.

  As the solid phase carrier, those mentioned above as specific examples of the insoluble carrier in relation to the immobilized antibody can be used. Further, as the labeling substance, those described above in connection with the labeled antibody can be used.

  The operation method of the measurement is a known method (edited by the Japanese Society for Clinical Pathology, "Special Issue on Clinical Pathology, Special Issue No. 53, Immunoassay for Clinical Testing-Technology and Application-", Clinical Pathology Publishing Association, 1983, Eiji Ishikawa et al. Immunoassay", 3rd edition, Medical School, 1987, edited by Kitagawa Tsunehiro et al., "Protein/Nucleic Acid Enzyme Separate Volume No. 31 Enzyme Immunoassay", Kyoritsu Shuppan, 1987).

  For example, the immobilized antibody and the sample are reacted with the labeled antibody at the same time, or the labeled antibody is reacted after washing to form a complex of the immobilized antibody-antigen-labeled antibody. Then, the unbound labeled antibody can be washed and separated, and the amount of the antigen in the sample can be measured from the amount of the bound labeled antibody. Specifically, in the case of enzyme-linked immunosorbent assay (ELISA), a labeled enzyme is reacted with a substrate under the optimum conditions, and the amount of the reaction product is measured by an optical method or the like. In the case of the fluorescent immunoassay, the fluorescence intensity by the fluorescent substance label is measured, and in the case of the radioimmunoassay, the radiation dose by the radioactive substance label is measured. In the case of the luminescent immunoassay, the amount of luminescence by the luminescence reaction system is measured.

  The detection and/or quantification method of the present invention is an immunoturbidimetric method, latex agglutination reaction, latex nephelometry, erythrocyte agglutination reaction, particle agglutination reaction, etc. When measuring by a static method or by a visual measuring method, a phosphate buffer, a glycine buffer, a Tris buffer or a Good's buffer can be used as a solvent, and a reaction such as polyethylene glycol You may include a promoter and a nonspecific reaction inhibitor.

  When the antibody is used by sensitizing it to a solid phase carrier, the solid phase carrier may be polystyrene, styrene-butadiene copolymer, (meth)acrylic acid ester polymer, latex, gelatin, liposome, microcapsule, red blood cell, Particles made of a material such as silica, alumina, carbon black, a metal compound, a metal, a ceramic or a magnetic material can be used.

  As the sensitizing method, a known method such as a physical adsorption method, a chemical bonding method or a combination of these methods can be used. The operation method of the measurement can be carried out by a known method. For example, when the measurement is performed by an optical method, the sample is reacted with the antibody, or the sample and the antibody sensitized to the solid phase carrier are reacted, and the endpoint method is used. Alternatively, transmitted light or scattered light is measured by the rate method.

  In the case of visual measurement, the sample and the antibody sensitized to the solid phase carrier are reacted in a container such as a plate or a microtiter plate, and the state of aggregation is visually determined. Note that the measurement may be performed using a device such as a microplate reader instead of the visual measurement.

4. Kit for Detection of Rat Type I Microglia or Human Oligodendriglioma Using the Monoclonal Antibody of the Present Invention The kit of the present invention is a monoclonal antibody or fragment thereof that specifically recognizes rat type I microglia provided by the present invention. It preferably contains, in addition to rat type I microglia, a monoclonal antibody or a fragment thereof that specifically recognizes human oligodendrocyte glioma. The monoclonal antibody or fragment thereof referred to herein may be an immobilized antibody or a labeled antibody described above in the present specification.

For example, when the monoclonal antibody specifically recognizing rat type I microglia provided by the present invention is used as a primary antibody, the kit of the present invention includes a kit for detecting a complex formed by an antigen-antibody binding reaction. Secondary antibodies may be included. In addition to these antibodies, the kit of the present invention may contain various adjuvants so that the kit can be used efficiently and conveniently. Examples of the auxiliary agent include a solubilizer for dissolving a solid secondary antibody, a detergent used for washing the insolubilized carrier, and an enzyme activity when an enzyme is used as a labeling substance for the antibody. Examples of the kit that are usually used as a kit of immunological measurement reagents such as the substrate and the reaction stopper thereof.
The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the examples.

(A) Experimental method (1) Cell culture (1-1) Preparation method of primary culture rat microglia The preparation method of rat microglia is the method of Sawada et al. (Suzumura A, Mezitis SG, Gonatas NK, Silberberg DH (1987) MHC antigen expression on bulk isolated macrophage-microglia from newborn mouse brain: induction of Ia antigen expression by gamma-interferon. J Neuroimmunol 15:263-278; and Sawada M, Suzumura A, Yamamoto H, Marunouchi T (1990) Activation and proliferation of the isolated microglia by colony stimulating factor-1 and possible involvement of protein kinase C. Brain Res 509:119-124). The detailed procedure is shown below.

(1-1-1) Preparation of rat brain primary culture system Newborn rats (Wistar rats, 0 to 2 days old, 14 to 20 rats) were decapitated with scissors. The head was cut with scissors and the brain was removed. The brain was suspended in cold PBS, the meninges were removed using tapered tweezers under a stereomicroscope, and Mi medium (MEM final concentration of 5 μg/ml insulin, 0.2% glucose, 10% FCS, 100 U/ml penicillin, And a medium containing 100 μg/ml streptomycin). The cells were suspended 10 to 12 times with a 10 ml measuring pipette. After centrifugation at 1,200 rpm at 4°C for 5 minutes, the supernatant was removed by suction. Mi medium 15 ml was added and resuspended 2-3 times. After centrifugation at 1,200 rpm at 4°C for 5 minutes, the supernatant was removed by suction. An appropriate amount of Mi medium was added to obtain a cell suspension. The cell suspension was seeded in a 75 cm ² plastic bottle (Greiner) at 2.5 bottles/brain. Culturing was started at 37°C in an atmosphere containing 5% CO ₂ . The first medium was replaced on the second day, the second on the ninth day, and then every 3 days thereafter.

(1-1-2) Separation and culture of type-1 (type I) microglial cells
The primary culture bottle cultured for 14 to 16 days was shaken (150 rpm, 1 h, 37°C). The culture solution containing floating cells was transferred to a 100 mm dish for bacteria (Falcon 1001) and left standing in a CO ₂ incubator. After 30 minutes, the medium was removed to remove non-adherent cells. Adherent cells were washed once with 10 ml medium. The collected microglia was about 3-4×10 ⁶ cells per bottle, and the purity was almost 100%. Mi medium was used for the culture of microglia. The cells were seeded at 4 to 5×10 ⁶ cells/dish in a 100 mm dish (Falcon 1001) and cultured at 37° C. in the atmosphere containing 5% CO ₂ .

(1-1-3) Separation and culture of type-2 (type II) microglial cells
The supernatant of the primary culture bottle that had been cultured for 16 to 18 days was removed by suction, washed once with PBS, added with 5 mM EDTA/PBS, and allowed to stand for 10 to 20 minutes. All floating cells were collected, centrifuged at 1,200 rpm for 5 minutes, and the supernatant was removed by suction. An appropriate amount of Mi medium was added to make a cell suspension, which was transferred to a 100 mm dish for bacteria (Falcon, 1001) and left standing in a CO ₂ incubator. After 30 minutes, the medium was removed to remove non-adherent cells. Adherent cells were washed 2-3 times with 10 ml of medium. The collected microglia was about 3-4 × 10 ⁶ cells per bottle, and the purity was about 90%. Mi medium was used for the culture of microglia. The cells were seeded at 4 to 5×10 ⁶ cells/dish in a 100 mm dish (Falcon 1001) and cultured at 37° C. in the atmosphere containing 5% CO ₂ .

(1-2) Primary Culture of Rat Peritoneal and Exudative Peritoneal Macrophages Rat peritoneal macrophages were prepared according to the method of Tokunaga et al. (Tokunaga et al., 1997), and male Wistar rats were used. As a stimulant for collecting exudative macrophages, a 4.05% thioglycollate medium was autoclaved and left brown for at least one month at room temperature to turn brown. 7 ml of this thioglycollate medium was injected into the rat peritoneal cavity, and 4 days later, peritoneal macrophages were collected.

After the rats anesthetized with diethyl ether were exsanguinated, cold PBS was injected into the abdominal cavity, the abdomen was rubbed well, and the PBS was collected in a centrifuge tube. Centrifugation was performed at 1,000 rpm for 5 minutes at 4°C, and the cells were seeded so that the number of cells became appropriate. After static culture for 1 hour at 37°C in an atmosphere containing 5% CO ₂ , macrophages were attached. The culture dish was shaken at 80 rpm for 30 seconds to remove the medium and floating cells, and washed with warm PBS. This washing operation was repeated 1 to 3 times until non-adherent cells were removed.

(1-3) Myeloma P3U1 cell culture method Cell culture was carried out at 37° C. in an atmosphere containing 5% CO ₂ . P3U1 cells were cultured in RPMI1640 containing 10% FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin. If necessary, the culture supernatant was sprayed to remove the cells, and the cells were collected and passaged or stored.

(2) Preparation of mouse anti-primary culture rat microglia monoclonal antibody (2-1) Preparation of immune antigen Primary culture rat microglia was used as an immune antigen. That is, type-1 microglia (antigen a) and type-1 microglia (antigen b) and type-2 microglia (antigen c) that were stimulated with LPS (0.1 μg/ml) of type-1 microglia separated above were stimulated. And type-2 are IL-4 activated Type-2 microglia (antigen d) stimulated with IL-4 (5 ng/ml). These cells were washed 3 times with cold PBS and then detached and collected using a cell scraper. The number of cells was counted, the supernatant was removed by centrifugation, and the cells were frozen and stored at -80°C.

(2-2) Immunity A conventional method was used. The antigen obtained in (2-1) above and Freund's complete adjuvant (FCA) were mixed well in a 1:1 amount to give a water-in-oil emulsion. This was injected into the abdominal cavity of (BALB/c, female, 6 weeks old). The immunization amount was the number of cells per animal. The first immunization was 1 × 10 ⁷ cells, and the second and subsequent immunizations were 5 × 10 ⁶ cells every 2 weeks for a total of 5 to 8 immunizations. The second and subsequent immunizations were performed by changing the immunoadjuvant to Freund's incomplete adjuvant (FIA), and the final immunization was performed without the immunoadjuvant.

(2-3) Measurement of antibody titer The antibody titer was measured by an ELISA method using primary culture rat microglia as an antigen. Add 50 μl of antigen (2.5 μg/ml) dissolved in coating buffer (0.1 M sodium carborate, pH 9.6) to each plate (Falcon, 96-well flexible assay plate) and let the plate adsorb at 4°C overnight. It was Blocking was performed with 2% skim milk (100 μl/well) at room temperature for 2 hours. After washing three times with a washing buffer (0.1% Tween20-TBS, 100 µl/well), antiserum (50 µl/well) diluted in each step with the washing buffer was added and reacted at room temperature for 2 hours. After the reaction, the plate was washed 3 times with a washing buffer, HRP-labeled goat anti-rat IgG (50 μl/well) diluted 1000-fold with a washing buffer was added, and the mixture was reacted at room temperature for 2 hours. After washing three times with a washing buffer, an o-phenylenediamin solution (0.4 mg/ml, 0.1% H ₂ O ₂ , citrate/phosphate buffer, pH 5.0, 50 μl/well) was added to develop color. The reaction was stopped by adding 6 NH ₂ SO ₄ (50 μl/well), and the absorbance at 490 nm was measured with a microplate reader.

(2-4) Cell fusion According to the method of Toyama et al. (Sakuji Tomiyama, Tamei Ando (1987) Monoclonal Antibody Experiment Manual, Kodansha, 8-84). Three days after the final immunization, the mouse was anesthetized with diethyl ether, blood was collected with a cardiac needle, the abdomen was opened, and the spleen was extracted. The collected spleen cells were fused with mouse myeloma P3U1 cells using polyethylene glycol in an abundance ratio of 10:1, and seeded in an appropriate number of 96-well plates. One week to 10 days after the fusion, the microglia used as an immunizing antigen was used as an antigen for screening by the ELISA method. For screening, the culture supernatant of the fused antibody-producing hybridoma cells was used as the primary antibody. Wells that showed a positive result in the screening were cloned by the limiting dilution method according to a conventional method. From 10 days to 2 weeks after cloning, screening was performed on type-1 microglial cells, and hybridomas with large differences in reactivity with type-1 microglial cells were cloned.

(3) Western blot analysis (3-1) Extraction of protein After adding 5 volumes of lysis buffer to the cells and incubating at 4°C for 30 minutes, the cells were cooled and centrifuged at 10,000 rpm, 4°C for 5 minutes, and the supernatant was added to the protein. It was recovered as an extract.

Lysis buffer
20 mM Hepes-NaOH (pH 7.4)
150 mM NaCl
1 mM EDTA
1% Triton X-100
0.5% sodium deoxycholate
0.1% SDS
10 mM sodium fluoride
2 mM sodium orthovanadate
1 mM phenylmethylsulfonyl fluoride
1 μg/ml pepstatin A
1 μg/ml leupeptin
10 μg/ml soybean trypsin inhibitor

  The protein quantification was carried out by the Bradford method (Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254) or the BCA method (Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76-85). Bradford protein assay (Bio-Rad, Herculese, USA) or BCA protein assay kit (Pierce Chemical Co.) was used as a reagent. The calibration curve was created using BSA.

(3-2) Western Blotting The protein extract was mixed with sampling buffer at a ratio of 1:1 and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970).

SDS sampling buffer
10 mM Tris-HCl (pH 7.6)
1% (w/v) SDS
20 mM dithiothreitol
4 mM EDTA
2% (w/v) sucrose

After SDS-PAGE, semi-dry blotting equipment (Nippon Aid-NA-1512) was used to electrically (0.9 mA/cm ² , 2.5 hours) the nitrocellulose membrane (Protran) or PVDF membrane (Millipore Corp.). It was transcribed.

  The detection method was performed according to the protocol attached to the ECL kit (Amersham Pharmacia Biotech, Buckinghamshire, USA). That is, the transferred membrane was incubated with a blocking solution (TBS solution containing 2 to 5% non-fat skim milk) for 1 to 2 hours, and then a primary antibody reaction and a secondary antibody reaction were performed. Finally, the reaction was performed with the ECL reagent, which is a chemiluminescent substrate, and the luminescence was captured by a cooled CCD camera and analyzed using LAS 1000 plus (Fuji Photo Film Co.), which can perform quantification.

The antibodies used in Western blotting of each protein and their dilution concentrations are shown below.
β-actin detection primary antibody: Anti-β-actin mouse monoclonal antibody (Sigma; 1:1000 dilution in TBS-Tween20)
Secondary antibody: Horseradish peroxidase-conjugated goat anti-mouse IgG (H+L) (Zymed Laboratories Inc.; 1: 5000 dilution in 0.5% skim milk/TBS-Tween20)

CD40 detection primary antibody: Anti-CD40 goat polyclonal antibody (Santa Cruz; 1:1000 dilution in TBS-Tween20)
Secondary antibody: Horseradish peroxidase-conjugated goat anti-goat IgG (H+L) (Zymed Laboratories Inc.; 1: 5000 dilution in 0.5% skim milk/TBS-Tween20)

Detection of CD86 Primary antibody: Anti-rat B7-2 goat polyclonal antibody (Genzyme Techne; 1:1000 dilution in TBS-Tween20)
Secondary antibody: Horseradish peroxidase-conjugated rabbit anti-goat IgG (H+L) (Zymed Laboratories Inc.; 1: 5000 dilution in 0.5% skim milk/TBS-Tween20)

(4) Immunofluorescent staining of mixed glial cells (Fig. 7)
The microglial cells obtained from the primary culture were seeded on a Lab-Tech II chamber slide (Nunc) coated with poly-L-lysine. The culture supernatant was removed, and the cells were fixed by allowing them to stand in a CO ₂ incubator at 37°C for 10 minutes with 4% paraformaldehyde. After removing 4% paraformaldehyde, it was lightly rinsed with PBS, 0.05% Triton X-100/PBS was added, and the mixture was left at room temperature for 10 minutes. After washing once with PBS, 2.5% BSA/PBS was added and blocking was performed at room temperature for 30 minutes. 2.5% BSA/PBS was removed, and the primary antibody diluted with 2.5% BSA/PBS was reacted for 16 hours in a cold room. After washing twice with PBS, the secondary antibody diluted with 0.05% Triton X-100/PBS was reacted for 1 hour at room temperature. After the reaction, the plate was washed twice with PBS, and the sample was sealed and observed with a confocal laser microscope (FLUOVIEW, Olympus, Tokyo) (Fig. 7).

(5) Tissue staining (Fig. 8)
(I) 1st day after birth (P1) Fixation of rat brain (PFA perfusion)
P1 rats were anesthetized with diethyl ether. The abdomen was peeled off with scissors and tweezers so as not to damage the organs, the ribs were removed, and the heart was exposed. Under a stereomicroscope, a cut hole was made in the vicinity of the right atrium with ophthalmic scissors (it becomes an escape route for blood). A winged needle was inserted into the left ventricle, and 2 ml of 4% paraformaldehyde (PFA) was poured. The brain was decapitated and the brain was taken out and suspended in 4% PFA (4° C., overnight).

(Ii) Cryoprotect The sample fixed with 4% PFA was washed once with PBS. The sample was immersed in 10% sucrose (10% sucrose/PBS) (4°C, 5-6 hours). It was immersed in 20% sucrose (20% sucrose/PBS) (4° C., overnight).

(Iii) Preparation of Tissue Section The cryoprotected tissue sample was washed once with PBS. The OCT compound was put into an appropriate container (aluminum box, etc.) suitable for the size of the sample, and the sample was drained and immersed. It was placed in a -80°C deep freezer and frozen (30 minutes). The sample was placed in a Leica (freezing microtome) set at -20°C to prepare a tissue section, which was attached to a slide.

(Iv) Staining The tissue section attached to the slide was surrounded by DAKOPEN. 4% PFA was mounted on the section and fixed (room temperature, 15 minutes). The 4% PFA was discarded and the plate was washed twice with PBS (5 minutes on a shaker). Blocking buffer (10% normal goat serume, 10 % BSA, 0.1% NaN 3, 0.1% TritonX-100 / PBS) and blocked loaded (at room temperature, 30 minutes). The blocking buffer was discarded and the plate was washed twice with PBS (5 minutes on a shaker). The primary antibody was diluted with an antibody diluent (10% BSA, 0.1% NaN ₃ , 0.1% TritonX-100/PBS) and mounted on a section (in a wet box, 4°C, overnight). The primary antibody was discarded and the plate was washed once with PBS (10 minutes on a shaker). The secondary antibody was diluted with an antibody diluent containing Hoechst 33258 (8 μg/ml) and placed on the section (in a humid box, room temperature, 1 hour, protected from light). The secondary antibody was discarded and the plate was washed once with 0.1% Triton-X/PBS (10 minutes after shaking). It was washed twice with PBS (5 minutes on a shaker). It was washed once with purified water (5 minutes on a shaker). The water was removed, and the Fluorescent mounting medium was placed and the cover glass was placed. It was observed with a fluorescence microscope.

The antibodies used are shown below.
Primary antibody: KM9F5 culture supernatant Secondary antibody: Cy3 conjugated affinity purified anti-mouse IgG (H+L)[goat] (ROCKLAND; in 10% BSA, 0.1% NaN ₃ and 0.1% Triton X-100 in PBS (Diluted 1:200)

Primary antibody: Anti Iba1 rabbit polyclonal antibody (Wako; diluted 1:200 in PBS containing 10% BSA, 0.1% NaN ₃ and 0.1% Triton X-100)
Secondary antibody: Alexa fluor 488 goat anti-rabbit IgG (H+L) (Molecular Probes; diluted 1:400 in PBS containing 10% BSA, 0.1% NaN ₃ and 0.1% Triton X-100)

(6) Tissue staining method (FIGS. 9 and 10)
The tissue specimen of human glioma was obtained from a patient who underwent surgery to remove a brain tumor or undergo biopsy in the field of neurosurgery, Kumamoto University School of Medicine and Pharmacy. After removing the sample, it was suspended in 4% PFA and fixed. After cryoprotection using OCT compound, a 5 μm section was cut out with a microtome and attached to a slide. After inhibiting the endogenous peroxidase activity with 0.3% methanolic hydrogen peroxide, it was reacted with the primary antibody (KM9F5 culture supernatant) (4°C, overnight). Color development was performed using the VECTASTAIN Elite ABC standard kit (Vector) according to the manual. As the substrate, 0.05% diaminobenzine tetrahydrochloride was used. Counterstaining was performed using Hematoxylin.

(B) Experimental results (1) Preparation of immune antigen Primary cultured rat microglia was used as an antigen. According to the method of Sawada et al., a brain cell suspension was prepared from a newborn rat and seeded in a 75 cm ² flask. The cells were cultured at 37°C in an atmosphere containing 5% CO ₂ , and type-1 microglia were separated and collected on the 14th day and the 16th day (Fig. 1). As shown in FIG. 1, although there were differences in the number of cells in the microscopic preparation, no morphological difference between the subtypes was observed.

    To confirm that these microglia are segregated as a subtype, CD40 is expressed only in type-1 microglia, and CD86 is said to have higher expression level in type-1 than in type-2. (Kanzawa T, Sawada M, Kato K, Yamamoto K, Mori H, Tanaka R (2000) Differentiated regulation of allo-antigen presentation by different types of murine microglial cell lines. J Neurosci Res 62:383-388.) Method (Fig. 2). CD40 should be expressed only in type-1 microglia, but it was also detected in type-2. It is considered that this is because the type-1 microglia was slightly mixed with the type-2 microglia. As expected, the expression of CD86 was about twice as high in type-1 microglia. Therefore, the two microglial subtypes were considered to be substantially well separated. Subsequent experiments were performed using the type-1 and type-2 microglia thus isolated.

  After subtype separation, type-1 was treated with inflammatory stimulus LPS for 24 hours, and type-2 was treated with anti-inflammatory cytokine Interleukin-4 (IL-4) for 6 days (Fig. 3).

  Type-1 microglia changed into a slightly round and ameboid-type microglia-like morphology upon LPS stimulation. On the other hand, in the type-2 microglia treated with IL-4, cells proliferated and extended thin and long protrusions as compared to when unstimulated. Based on the observation of these morphological changes, it was considered that type-1 induced toxicity and type-2 induced an activated form with an enhanced protective property. Non-stimulated type-1 and type-2 microglia were quiescent, and four types of cells were used as antigens together with the stimulated and activated type.

(2) Immunization Mice were immunized with the prepared four types of immunizing antigens in the abdominal cavity according to a conventional method. Freund's complete adjuvant and then Freund's incomplete adjuvant were used as immunization adjuvants, and immunization was performed every two weeks. The titer of the antiserum was measured by the ELISA method using microglia (2.5 μg/ml) of each immunogen as an antigen (FIG. 4). As a control, the serum of non-immunized mice was used.

  Mice immunized with Type-1 microglia showed a rapid increase in antibody titer, whereas mice immunized with type-2 microglia did not. Type-1 microglia are cells with strong cytotoxicity, present many MHC class II and complement receptors on the cell membrane, and it is considered that the expression level of proteins such as CD40 and CD86 is high. Therefore, when considered as an immunogen, a large amount of proteins that can be recognized as foreign substances are expressed. On the other hand, type-2 microglia are rather mild cells, and it is considered that the level of expressed protein tends to be lower than that of type-1. Therefore, it is considered that the same microglia had different immune responses. Conversely, type-1 microglia and type-2 microglia are still different cells, which was thought to be a result supporting the hypothesis that microglia consist of multiple subpopulations.

(3) Examination of reactivity of monoclonal antibody by cell fusion and ELISA method The final immunization was performed without an immunoadjuvant. Three days after the final immunization, B cells of the spleen and mouse myeloma P3U1 were subjected to cell fusion using polyethylene glycol and seeded in an appropriate number of 96-well plates. A total of 4 cell fusions were performed, and the fusion rate was approximately 98%. Screening and cloning were carried out according to a conventional method to establish a plurality of monoclonal antibodies that recognize microglia. FIG. 5 shows the reactivity of each clone against 4 types of cells used as immunogens, which was examined by ELISA. Among the obtained monoclonal antibodies, (i) high specificity for type-1 9F5, (ii) strong reactivity for both type-1 and type-2 11F1, (iii) specificity for type-2 Three clones with high 6D5 are considered to be useful antibodies for microglia and subtype analysis.

(4) Analysis of clones by Western blotting As a result of the examination in (3) above, the reactivity of 9F5, which had high specificity for type-1, was analyzed by Western blotting (Fig. 6). As a result, it was found that 9F5 did not react with type-2 microglia at all, but specifically reacted with type-1 microglia (Fig. 6A). Since the antigen is approximately 50 to 70 kDa and loses its reactivity under the condition of the reducing agent DTT(+), it is considered that it recognizes the three-dimensional structure of the antigen protein.

  Subsequently, the reactivity to central nervous system cells such as neurons and astrocytes, peripheral macrophages, and mouse microglial cells 6-3 (type-1 microglia) and Ra2 (type-2 microglia) was examined (FIG. 6B). Interestingly, it was shown that 9F5 responded to not only central neurons and astrocytes but also peripheral macrophages (lanes 6 to 13). That is, it was found that the central type-1 microglia selectively react. In addition, the band of 9F5 antigen is slightly smeared, so it may be modified by sugar chains.

(5) Cell and tissue staining with type I microglia-specific antibody 9F5
Cellular staining of mixed glia and tissue staining of brain were performed with 9F5, and the reactivity of 9F5 antibody to microglia was analyzed as compared with the antibody to Iba1, which is a microglial marker protein.
Cell staining of mixed glia was performed using Type-1 specific antibody 9F5 (Fig. 7). Cells in which 9F5 reacts are shown in red, cells in which Iba1 antibody reacts are shown in green.

  9F5 clearly had less microglia stained than the Iba1 antibody. This is probably because the Iba1 antibody reacts with almost all microglia, whereas 9F5 specifically recognizes only type-1 microglia. However, our results suggest that 9F5-positive microglia are considerably smaller in terms of the total proportion of microglia. It is considered that Type-1 has a lower original abundance than Type-2. Alternatively, it was considered that the antigen protein recognized by 9F5 is expressed only in a part of type-1 microglia. If so, microglia may not be composed of two subtypes, but may exist as more cell subpopulations. FIG. 7B shows an enlarged image of microglia stained with 9F5. What is stained is a granular material around the nucleus, which seems to be a lysosome. That is, the 9F5 antigen may be present in the lysosome.

  Next, tissue staining was performed using a brain section of a newborn rat (1 day after birth) (FIG. 8). Cells that respond to 9F5 are shown in red (A, C, E) and cells that respond to Iba1 are shown in green (B, D, F). In addition, the nuclei are stained blue by Hoechst staining, indicating the region where cells are present.

  Also in the fourth ventricle of the brain tissue and the hippocampus, the number of cells stained with 9F5 was clearly smaller than that with the Iba1 antibody (Fig. 8). It was shown that the number of cells to which 9F5 responds was smaller than that to the cells to which the Iba1 antibody responds, both in the cells of the primary culture system and in the brain tissues shown here. That is, 9F5 reacts only with a part of the cell population of microglia. 9F5 is an antibody that should support the hypothesis of Sawada et al. that microglia is a group of cells from multiple subpopulations, and it selectively recognizes type-1 microglia, which is a useful tool for analysis of microglial subtype Is expected to be.

  In addition, as a result of investigating the reactivity of 9F5 between human oligodendrocyte and human glioblastoma (glioblastoma), 9F5 reacts with human oligodendrocyte (Fig. 9), but human glioblastoma does. It was found to not react with (glioblastoma) (Fig. 10).

(C) Summary of Examples Although microglia have a ramified form with long branched projections in a small cell body in normal brain, quiescent microglia rapidly act upon injury or damage to the brain. And change its nature greatly. Activated microglia are known to have strong migratory ability and act as phagocytes, and also to produce neuronal growth factors such as BDNF and exhibit neuroprotective effects (Sawada M, Suzumura A, Marunouchi T (1995) Cytokine network in the central nervous system and its roles in growth and differentiation of glial and neuronal cells.Int J Dev Neurosci 13:253-264; Nakajima K, Kohsaka S (2001) Microglia: activation and their significance in the central nervous system. Biochem (Tokyo) 130:169-175; and Salimi K, Moser K, Zassler B, Reindl M, Embacher N, Schermer C, Weis C, Marksteiner J, Sawada M, Humpel C (2002) Glial cell line-derived neurotrophic factor. enhances survival of GM-CSF dependent rat GMIR1-microglial cells. Neurosci Res 43:221-229). On the other hand, it has been suggested that cytotoxic factors such as oxygen radicals, IL-1β, and NO may be released to exert a damaging effect on surrounding neurons (Meda L, Cassatella MA, Szendrei GI, Otvos L, Jr. , Baron P, Villalba M, Ferrari D, Rossi F (1995) Activation of microglial cells by beta-amyloid protein and interferon-gamma. Nature 374:647-650). Thus, microglia are considered to be cells that have the duality of good and evil.

  The duality of microglia is advocated by Sawada et al. (Kanzawa T, Sawada M, Kato K, Yamamoto K, Mori H, Tanaka R (2000) Differentiated regulation of allo-antigen presentation by different types of murine microglial cell lines. J Neurosci Res 62:383-388: and Katoh Y, Niimi M, Yamamoto Y, Kawamura T, Morimoto-Ishizuka T, Sawada M, Takemori. H, Yamatodani A (2001) Histamine production by cultured microglial cells of the mouse. Neurosci Lett 305:181-184). That is, it is an idea that heterogeneous cell populations having different properties exhibit respective actions, rather than a single microglia exhibiting two-sidedness. However, little is known about how each of these heterologous cells functions. In order to analyze the dihedrality of microglia in more detail, we considered that an antibody that identifies a microglial subtype is necessary.

  Against this background, the present inventors separated primary cultured rat microglia into cytotoxic type-1 microglia and cytoprotective type-2 microglia, and produced monoclonal antibodies using them as immunogens, It was investigated whether the subtype could be identified. According to a conventional method, a mouse was used to prepare a monoclonal antibody to obtain a plurality of clones that react with microglia. The reactivity of four types of microglia used as immunogens was compared, and 9i5 with high specificity for (i) type-1 was analyzed.

  As a result of Western blot analysis, 9F5 was shown to react specifically with type-1 microglia. Further, 9F5 was further analyzed by cell staining and tissue staining. Antibodies against 9F5 and Iba1, which is a microglial marker protein, were used to compare their reactivity to microglia. The number of cells stained with 9F5 was apparently lower than that of the Iba1 antibody in rat mixed glia cell staining and tissue staining using rat neonatal brain sections. It is considered that 9F5 reacts only with type-1 microglia, whereas Iba1 reacts with microglia and cerebrovascular macrophages.

  The monoclonal antibody 9F5 produced in the present invention is an antibody that supports the hypothesis that microglia consists of multiple subpopulations. 9F5 specifically recognizes type-1 microglia, that is, it does not react with nerve cells in the center, glial cells, or peripheral macrophages. As one of the type-1 microglial marker proteins, there is CD40 which was already revealed by Kanzawa et al. (Kanzawa T, Sawada M, Kato K, Yamamoto K, Mori H, Tanaka R (2000) Differentiated regulation of allo-antigen. presentation by different types of murine microglial cell lines. J Neurosci Res 62:383-388). CD40 is an important molecule expressed on B cells, involved in the close interaction between T cells and B cells, and also expressed on monocytes and macrophages. Therefore, it is considered that CD40 can distinguish the microglial subtype but cannot distinguish it from cerebrovascular macrophages. On the other hand, since 9F5 does not react with peripheral macrophages at all, it is useful for selectively recognizing type-1 microglia in the central nervous system/glial cell line and analyzing the transition of its appearance associated with its localization and functional changes. It is believed that there is.

  By using the monoclonal antibody of the present invention, rat type I microglia and human oligodendriglioma can be specifically identified.

FIG. 1 shows primary rat microglial cells. Figure 2 shows immunoblot analysis of CD40 and CD86 in rat type I and type II microglial cells. FIG. 3 shows type I and type II microglial cells stimulated with LPS and IL-4, respectively. Figure 4 shows titration of antisera against the corresponding antigens by ELISA. Figure 5 shows the reactivity of monoclonal antibodies with type I and type II microglial cells stimulated with LPS and IL-4, respectively. FIG. 6 shows the identification of 9F5 reactive molecules by Western blot. Figure A: 1: Type I microglia stimulated with LPS 2: Type I microglia 3: Type II microglia stimulated with IL-4 4: Type II microglia, B Figure 1: Mouse microglia MG5 2:6-3 (mouse type I) Microglia-like) 3: Ra2 (mouse type II microglia-like) 4: Rat type I microglia 5: Rat type I microglia stimulated with LPS 6: Rat type II microglia 7: Rat type II microglia stimulated with IL-4 8: Rat Neuron 9: Rat astrocyte 10: Mouse macrophage RAW264.7 11: Rat peritoneal macrophage 12: LPS-stimulated rat peritoneal macrophage 13: Thioglycollate-stimulated rat exudative peritoneal macrophage (1-7: microglia 10-13: Macrophage) FIG. 7 shows immunostaining of mixed glial cells with 9F5 and Iba1. FIG. 8 shows immunostaining of rat neonatal brain sections with 9F5 and Iba1. FIG. 9 shows immunostaining of human oligodendrogliomas (case 21 years old) with 9F5. FIG. 10 shows immunostaining of human glioblastoma (glioblastoma) (case 72 years old) with 9F5.

Claims (17)

A monoclonal antibody or a fragment thereof that specifically recognizes rat type I microglia. The monoclonal antibody or fragment thereof according to claim 1, which does not react with rat type II microglia. The monoclonal antibody or a fragment thereof according to claim 1 or 2, which specifically recognizes human oligodendrocyte. The monoclonal antibody or fragment thereof according to any one of claims 1 to 3, which does not react with human glioblastoma. The monoclonal antibody or fragment thereof according to any one of claims 1 to 4, wherein the antigen to be recognized has a molecular weight of about 50 to 70 kDa and further loses reactivity with the antigen in the presence of a reducing agent. The monoclonal antibody or fragment thereof according to any one of claims 1 to 5, which is characterized in that it hardly reacts with rat neurons, rat astrocytes, peripheral macrophages, mouse type I microglia and mouse type II microglia. A monoclonal antibody or fragment thereof produced by a hybridoma having the deposit number FERM BP-10475. A hybridoma producing the monoclonal antibody according to claim 1. The hybridoma according to claim 8, which is obtained by fusing mammalian immune cells immunized with an immunogen containing rat type I microglia and mammalian myeloma cells. A hybridoma having the deposit number FERM BP-10475. A method for producing the monoclonal antibody according to any one of claims 1 to 7, comprising a step of culturing the hybridoma according to any one of claims 8 to 10, and a step of collecting and purifying a monoclonal antibody produced by the hybridoma. .. A method for detecting rat type I microglia, which comprises performing an immunoassay using the monoclonal antibody or a fragment thereof according to any one of claims 1 to 7. The method for detecting rat type I microglia according to claim 12, comprising at least the following steps (a) and (b).
(A) a step of reacting the sample with a monoclonal antibody or a fragment thereof according to any one of claims 1 to 7; and (b) an antigen-antibody complex formed in step (a), and a labeled antibody for detection Reacting with; An immunoassay is carried out using the monoclonal antibody or fragment thereof according to any one of claims 1 to 7, which is a method for detecting human oligodendrogliomas. The method for detecting human oligodendrocyte glioma according to claim 14, comprising at least the following steps (a) and (b).
(A) a step of reacting the sample with a monoclonal antibody or a fragment thereof according to any one of claims 1 to 7; and (b) an antigen-antibody complex formed in step (a), and a labeled antibody for detection Reacting with; A kit for detecting rat type I microglia, which comprises the monoclonal antibody or fragment thereof according to any one of claims 1 to 7. A kit for detecting human oligodendrocyte glioma, which comprises the monoclonal antibody or fragment thereof according to any one of claims 1 to 7.