JP2006038536A - In vitro diagnostic kit and in vitro diagnostic method - Google Patents

Abstract

[PROBLEMS] To use materials that can be collected relatively easily such as blood, urine, cerebrospinal fluid, or peripheral tissues among living body-derived materials in mammals, such as Creutzfeldt-Jakob disease, bovine spongiform encephalopathy , Scrapie, deer chronic debilitating disease, Gerstmann-Straussler-Scheinker syndrome, degenerative neurodegenerative diseases such as fatal familial insomnia can be detected from early stages An in vitro diagnostic kit and an in vitro diagnostic method are provided.
A detection material for a biological component containing an anti-Fas antibody is provided.
[Selection figure] None

Description

  The present invention relates to an in vitro diagnostic kit for prion disease and a method for in vitro diagnosis in mammals, and in particular, can be collected relatively easily from mammalian biomaterials such as blood, urine, cerebrospinal fluid, or peripheral tissues. The present invention relates to an in vitro diagnostic kit and an in vitro diagnostic method that can use various materials and can be detected from an early stage of onset of prion disease.

  In addition to Creutzfeldt-Jakob disease (CJD), mammalian prion diseases include Gerstmann-Strausler-Scheinker Syndrome (GSS), which is hereditary prion disease, in addition to Creutzfeldt-Jakob disease (CJD). , Fatal familial insomnia, Kuru, etc. CJD is sporadic CJD of unknown etiology, hereditary familial CJD, mutant CJD caused by oral infection from bovine spongiform encephalopathy (BSE) (for example, see Non-Patent Document 1), human pituitary extract preparation, It is classified into iatrogenic CJD caused by infection via human dura mater, cornea, etc., but it is common in that abnormal prion protein (PrPSc) accumulates in the central nervous system. On the other hand, in mutant CJD, PrPSc is also found in lymphoid tissues such as tonsils [see, for example, Non-Patent Document 2]. BSE is considered to be caused by infection from a mixed feed contaminated by sheep or goat scrapie organs, and similar to scrapie, degeneration is observed in the central nervous system and the like, and PrPSc is deposited, but PrPSc is deposited in lymphoid tissues. It is different from scrapie in that it is not recognized [see, for example, Non-Patent Document 3].

  As a method for diagnosing prion disease in mammals, in human prion disease, in the case of mutant CJD, immunohistochemical examination has been proposed for lymph tissue biopsy materials such as tonsils [for example, non- See Patent Document 2]. In sporadic CJD, cerebrospinal fluid specifically increases 14-3-3 protein (see, for example, Non-Patent Document 4), and neuron-specific enolase (Neuron-Specific Enolase) is supplementary. Known as a diagnostic marker.

  BSE has been detected using histopathological, clinical, and epidemiological methods. Brain stem (medullary medulla) tissue is collected, and proteinase K (PK) is used for normal prion protein (PrPC). A commercially available test kit for detecting abnormal prions by reacting a fragment called "PrP27-30", which is completely degraded, but remains after degradation with PK with a prion protein (PrPC, PrPSc) -specific antibody. [See, for example, Non-Patent Document 5]. In addition, for example, a diagnostic method characterized by comparing and classifying an infrared spectrum of a specimen with a reference database for a pathological change of a tissue (see, for example, Patent Document 1), a reaction with a monovalent antibody is used. Abnormal prion protein quantification method (see, for example, Patent Document 2), expression of marker proteins such as prion protein, interferon, laminin receptor or laminin precursor in target blood cells concentrated from blood before or after the onset of BSE A method of determining by an immunity test or the like and comparing it with a control [see, for example, Patent Document 3] has been proposed.

  Mammalian prion diseases as described above, that is, scrapie, BSE, CJD, CWD, MTE, and the like are also referred to as infectious spongiform encephalopathy (TSEs) in neurodegenerative diseases. In experiments on scrapie mice infected by inoculation of 87V scrapie in the brain, prior to accumulation of abnormal prion protein, early induction of Fas, which is involved in apoptosis, also called CD95 or APO-1, and caspase 3 (Caspase 3) was observed. [For example, see Non-Patent Document 6]. Furthermore, Fas, Fas ligand (FasL), ERK, MEK, Bcl-2, Bax, N-myc, c-myc, sporadic CJD and olive pons, cerebellar atrophy patient cerebellum by immunohistochemical method. As a result of examining the expression of procaspase-2 and caspase-3, it is suggested that overexpression of these proteins may have a function different from apoptosis in the pathway of cell death in neurodegeneration [for example, non-patent In addition, many studies relating to apoptosis have been conducted on degenerative (progressive) neurodegenerative diseases other than TSE.

  Note that Fas, which is a major apoptosis-inducing factor, was discovered as an antigen against this mouse monoclonal antibody because a monoclonal antibody obtained by immunizing a mouse with human fibroblasts (FS-7) kills the cell [for example, The non-patent document 8] shows that the amino acid sequence has been confirmed from its cDNA cloning and is a type I membrane protein containing a transmembrane portion belonging to the TNF receptor family that has a transmembrane portion and is present on the cell surface layer. [For example, see Non-Patent Document 9, Non-Patent Document 10, and Patent Document 4]. In addition, mouse Fas gene and bovine Fas gene have also been cloned [see, for example, Non-Patent Document 11 and Non-Patent Document 12].

Special table 2003-500168 gazette JP 2003-121448 A JP 2002-543389 US Pat. No. 5,874,546 Will, R.A. G. et al; Lancet Vol. 347, pp921-925 (1996) Hill, A.M. F. et al; Lancet Vol. 349, pp99-100 (1997) O'Rourke, K.M. I. et al; Clin. Microbiol. Vol. 38, pp 3254-3259 (2000) Hsich, G.M. et al; Eng. J. et al. Med. Vol. 335, pp 924-930 (1996) Ken Noda, Hideki Nagai, Midori Hirasawa, Veterinary and Livestock Newspaper, Vol. 56, No. 9, 719-725, 2003 Elizabeth Jamieson et al; Neurochemistry Vol. 12, no. 16, pp 3567-3572 (2001) Puig, B.M. et al; Acta Neuropathol. Vol. 102, pp207-215 (2001) Yonehara, S .; et al; Exp. Med. Vol. 169, pp 1747-1756 (1989) Ito, N .; et al; Cell Vol. 66, pp 233-243 (1991) Nagata, S .; et al; Cell Vol. 88, pp355-365 (1997) Watanabe-Fukunaga R.M. et al; Immunol. Vol. 148, pp 1274-1279 (1992) Yoo, J .; et al; DNA Cell Biol. Vol. 15 (3), pp 227-234 (1996)

  In the mutant CJD, as shown in Non-Patent Document 2, it is possible to make a prenatal diagnosis by collecting lymph tissues such as tonsils and detecting PrPSc, but it is not invasive and practical. In sporadic CJD, the diagnostic method using the 14-3-3 protein as a marker in Non-Patent Document 4 is highly sensitive, but as with other human prion diseases, the brain is supplemented with an MRI examination of the brain. It is only applied as one of spinal fluid examinations, and finally a definitive diagnosis is made by brain biopsy or brain pathology at autopsy. Similarly to the diagnosis of mutant CJD in scrapie, PrPSc distributed in lymphoid tissue can be detected as shown in Non-Patent Document 3 to perform prenatal diagnosis, but there is a problem that it is not practical. As for BSE, most of the examination methods including Non-Patent Document 5, Patent Document 1, Patent Document 2 and the like show that PrPSc is deposited in the central nervous system and is not substantially distributed in blood or peripheral lymphoid tissues. Implementation as a diagnosis was difficult. In addition, there is a method of performing diagnosis before and after the onset using blood as a material as in Patent Document 3, but the operation such as concentration of target blood cells is necessary and the operation is complicated. In addition, PrPSc in urine is detected. Since it is difficult to obtain sufficient sensitivity even in the method of performing prion disease, a diagnostic method and a diagnostic kit for prion diseases, particularly BSE, applicable to large-scale simple screening are desired.

  The present invention has been made in view of the above situation, and uses a material that can be collected relatively easily, such as blood, urine, cerebrospinal fluid, or peripheral tissue, among mammalian biomaterials. It is an object of the present invention to provide an in vitro diagnostic kit and an in vitro diagnostic method that can be detected from an early stage of onset of prion disease.

  In order to achieve the above object, the in vitro diagnostic kit for prion diseases in mammals according to the present invention is characterized by comprising a detection material for a biological component containing an anti-Fas antibody. In such an in vitro diagnostic kit, it is preferable that the biological component is a component contained in blood, and it is preferably used for primary screening for prion disease. It may be selected from cattle, goats, sheep, deer, elk, mink, pigs, monkeys, mice, rats, hamsters, cats, and prion diseases are Creutzfeldt-Jakob disease (CJD), From bovine spongiform encephalopathy (BSE), scrapie, deer chronic wasting disease (CWD), transmissible mink encephalopathy (TME), Gerstmann-Strausler-Scheinker syndrome, lethal familial insomnia It is preferable that one or more are selected. The prion disease is preferably prion disease in a ruminant mammal, and the detection material is a dye-labeling agent, a color-developing labeling agent, a chemiluminescent labeling agent, a bioluminescent labeling agent, Preferably, the detection material can comprise a fluorescent labeling agent, and the detection material further comprises a protein having an amino acid sequence capable of binding to the anti-Fas antibody or a partial peptide thereof. Mammalian Fas protein obtained from the mammalian Fas gene or a partial peptide thereof can be included.

  In order to achieve the above object, the in vitro diagnostic method for prion diseases in mammals according to the present invention is characterized by using a detection material for a biological component containing an anti-Fas antibody. In this in vitro diagnostic method for prion diseases in mammals, the biological component is preferably a component contained in blood, and is preferably used for primary screening for prion diseases. It may be selected from human, cow, goat, sheep, deer, elk, mink, pig, monkey, mouse, rat, hamster, cat, and prion disease is Creutzfeldt-Jakob disease (CJD). ), Bovine spongiform encephalopathy (BSE), scrapie, deer chronic wasting disease (CWD), transmissible mink encephalopathy (TME), Gerstmann-Straussler-Scheinker syndrome, lethal familial insomnia It is preferable that one or more are selected from the symptoms. The prion disease is preferably a prion disease in a ruminant mammal, and the detection material is a dye-labeling agent, a chromogenic labeling agent, a chemiluminescent labeling agent, a bioluminescent labeling agent, or a bioluminescent component, or Preferably, the detection material can comprise a fluorescent labeling agent, and the detection material further comprises a protein having an amino acid sequence capable of binding to the anti-Fas antibody or a partial peptide thereof. Mammalian Fas protein obtained from the mammalian Fas gene or a partial peptide thereof may be included.

  According to the in vitro diagnostic kit and in vitro diagnostic method for prion disease in mammals of the present invention, anti-Fas antibodies appearing in the living body of mammals, particularly in blood, urine, cerebrospinal fluid, peripheral tissues, etc. in the early onset of prion disease. Since a detectable detection material is provided and such a detection material is used, a prenatal diagnosis in the early onset of prion disease is possible. In addition, biomaterials to be diagnosed can be easily collected, and anti-Fas antibodies can be easily detected using serum, plasma, urine, cerebrospinal fluid, etc. as samples, so it is also applicable to large-scale screening for prion diseases. it can.

  In addition, the present invention has confirmed by experiments using mice inoculated with PrPSc that the biologically derived components including anti-Fas antibody appear and increase in blood and the like with the onset of mammalian prion disease. Although the mechanism of action is unclear, prion disease is a neurodegenerative disease involving apoptosis. Therefore, the in vitro diagnostic kit and in vitro diagnosis method of the present invention are useful for screening for degenerative neurodegenerative diseases. Can also be used. Furthermore, by combining with a diagnostic marker specific for a neurodegenerative disease including each prion disease, a prenatal diagnosis for each neurodegenerative disease can be performed.

  Hereinafter, the best mode for carrying out the in vitro diagnostic kit and in vitro diagnostic method for prion diseases in mammals of the present invention will be described in detail with reference to the drawings.

[First Embodiment]
The in vitro diagnostic kit and in vitro diagnostic method for mammalian prion disease according to the first embodiment include a detection material for a biological component containing an anti-Fas antibody, and also uses a detection material for a biological component containing an anti-Fas antibody. It is. The biological component is a component contained in a biological material sample from a mammal diagnosis target individual such as blood, urine, cerebrospinal fluid, and peripheral tissue, and can be easily collected. The blood can be a serum or plasma specimen. In this embodiment, the biological component containing the anti-Fas antibody can be separated from the anti-Fas antibody and other biological components by a separation method using a known separation material, separation device, etc. As a detection material in, as long as a biological component including an anti-Fas antibody can be detected, a staining labeling agent, a coloring labeling agent (including a combination of an enzyme labeling agent and a substrate agent), a chemiluminescent labeling agent, bioluminescence Any detection material such as a labeling agent or a fluorescent labeling agent can be used. Moreover, it is good also as a detection material which combined 2 or more types of these labeling agents.

  Furthermore, it can also be set as the in vitro diagnostic kit which combined the separation material and / or the separation apparatus in addition to the detection material. Here, any known separation method can be used as the separation method of the anti-Fas antibody and other biological components by the separation material and / or separation device. For example, agar, agarose, starch, polyacrylamide gel (PAGE), slab gel electrophoresis such as sodium dodecyl sulfate-added polyacrylamide gel (SDS-PAGE), disk electrophoresis, capillary electrophoresis, isotachophoresis, isoelectric focusing Electrophoresis (IEF), filter paper electrophoresis, cellulose acetate membrane electrophoresis, gel bead electrophoresis, immunoelectrophoresis, and electrophoresis using a gradient gel with a gel density gradient or pH gradient for these gel carriers (separating agents) Further, various known electrophoresis methods such as a combination of two or more electrophoresis methods or a two-dimensional electrophoresis method can be applied. Various detailed conditions for using such an electrophoretic separation method, separation material, and separation apparatus are described in [Laemmli, U.S. Pat. K. et al; Nature London Vol. 227, p680 (1970)], [Davis, B .; et al; Ann. N. Y. Acad. Sci. Vol. 121, p404 (1964)], [Norio Okuyama et al .; Protein Nucleic Acid Enzyme Separate Volume "Isoelectric Focusing and Isotachophoresis" Kyoritsu Shuppan (1978)], [Kohn, J. et al. et al; Clin. Chim. Acta. Vol. 2, p297 (1957)], [Shapiro, A. et al. L. et al; Biochem. Biophys. Res. Commun. Vol. 28, p815 (1967)], [Weber, K. et al. et al; Biol. Chem. Vol. 244, p4406 (1969)], [Manabe et al., “Immune Experiment Manipulation”, published by the Japanese Society for Immunology, Vol. 12, p3807 (1983)], [O'Farrel, P.A. H. et al; Biol. Chem. Vol. 250, p4007 (1975)], [Manabe, T .; et al; Biochem. Vol. 85, p649 (1979)] [edited by the Japanese Biochemical Society, “Basic Biochemical Experimental Method; Volume 3, Protein I”, Tokyo Chemical Doujin (2001)].

  In addition to electrophoresis, ultracentrifugation, ultrafiltration using anisotropic filtration membranes and holofibers, and various chromatographic methods including high performance liquid chromatography (HPLC) [both ion exchange chromatography and gel filtration Size Exclusion (SE) chromatography, Hydrophobic (HIC) chromatography, adsorption chromatography, chromatofocusing (isoelectric point chromatography), affinity chromatography, reversed phase (RP) chromatography Etc.] can also be used. Various detailed conditions when using a separation method such as a chromatograph, a separation material, and a separation apparatus are as follows: [Japan Biochemical Society, “Biochemistry Experiment Course 1: Protein Chemistry”, Tokyo Kagaku Dojin (1976)], [Japan Biochemical Society, "Biochemistry Experiment Course 5; Enzyme Research Method", Tokyo Chemical Doujin (1975)], [Japan Biochemical Society, "Second Biochemistry Experiment Course 2: Protein Chemistry", Tokyo Chemical Doujin (1987) )], [Japan Biochemical Society, “Neochemistry Experiment Course 1; Protein I”, Tokyo Chemical Doujin (1990)], [Japan Biochemical Society, “Basic Biochemistry Experiments; Volume 3, Protein I”, Tokyo Chemical doujin (2001)] and the like.

  Among such separation methods, for example, two different types of separation, such as molecular weight and isoelectric point, molecular weight and affinity or adsorption or hydrophobicity to a specific substance, and isoelectric point and affinity or adsorption to a specific substance Combination of separation methods based on the principle (SDS-PAGE or SE-HPLC and isoelectric focusing or chromatofocusing, SDS-PAGE or SE-HPLC and affinity chromatography or adsorption chromatography or HI or RT-HPLC, pH gradient ( IEF) / SDS-PAGE two-dimensional electrophoresis, etc., and pH gradient (IEF) / SDS-PAGE two-dimensional electrophoresis is preferred in terms of detection operability, sensitivity, and comparison with negative controls after separation. The method is more preferable, and this embodiment employs such a two-dimensional electrophoresis method. Detection after electrophoresis may be performed after transfer to a transfer film such as a nylon film, a nitrocellulose film, or a polyvinylidene difluoride (PVDF) film, or may be detected directly on a gel without being transferred.

  Further, as the detection material in the present embodiment, the detection sensitivity is a level of about ng to μg and is lower than other labeling agents, but a staining labeling material is used because it is widely used and can be easily dyed. To do. As the staining labeling agent, any staining labeling agent can be used as long as it can stain components derived from living bodies such as peptides and proteins including glycoproteins. For example, various known Coomassie Brilliant Blue (CBB: CBB- G250, CBB-R150, CBB-R250, CBB-R350, etc.), water-soluble silver compounds such as ammoniacal silver nitrate, silver colloid, gold colloid, ethidium bromide (ETBr), acid blue, amide black B, eriochrome black T, etc. May be suitably used, and each may be used by appropriately dissolving in a known buffer solution or an organic solvent mixed aqueous solution. In addition, about the staining conditions by such a dye | staining labeling agent, for example, [Wilson, C. M.M. et al; Anal. Biochem. Vol. 96, p236 (1979)], [Oakley, B. et al. R. et al; Anal. Biochem. Vol. 105, p361 (1980)], [Moeremans, M .; et al; Immunol. Methods, Vol. 74, p353 (1984)] and the like.

  When a detection material other than such a staining labeling agent is used, as the coloring labeling agent, a coloring substrate agent or an enzyme (labeling) agent is bound to a biological component such as a peptide or a protein containing a glycoprotein, or physically Any coloring labeling agent may be used as long as it can be detected by adhering or adsorbing and coloring in such a state. In addition, a combination of an enzyme (labeling) agent, a coloring substrate agent, a coloring aid, a coloring enhancer, a pH buffering agent, and the like can be included. As the coloring substrate agent, any coloring substrate agent can be used as long as it develops color with an enzyme agent or a coloring aid. For example, o-phenylenediamine, 5-aminosalicylic acid, p-nitrophenol phosphate, phenol phosphorus Examples include acid, phenol, 4-aminoantipyrine, o-dianisidine, o-nitrophenol-β-D-galacside, 3,3 ′, 5,5′-tetramethylbenzidine (TMB), and the like. Examples of the enzyme (labeling) agent include peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, hexokinase and the like. Appropriate chromogenic substrates and enzyme agents may be combined, for example, peroxidase and o-phenylenediamine, peroxidase and 5-aminosalicylic acid, peroxidase and TMB, alkaline phosphatase and p-nitrophenol phosphate, alkaline phosphatase and phenol phosphorus. Acid, alkaline phosphatase and phenol, 4-aminoantipyrine, β-D-galactosidase and o-nitrophenol-β-D-galacside, and the like. You may add the pH buffer which makes it suitable pH. Furthermore, a coloring labeling agent combining biotin and avidin can also be used. The detailed conditions for using such a coloring labeling agent are as follows: [Japan Biochemical Society, “Biochemistry Experiment Course 5; Enzyme Research Method”, Tokyo Kagaku Dojin (1975)], [Ishikawa Eiji et al. "Measurement Methods" Medical School (1987)], [Japan Biochemical Society, edited by "Neochemistry Experiment Course 1; Protein V", Tokyo Chemical Doujin (1991)], [Japan Biochemical Society, "Neochemistry Experiment Course 12, Molecular Immunity" Science III ", Tokyo Chemical Doujin (1992)], etc.

  As a chemiluminescent labeling agent, a chemiluminescent agent or an enzyme (labeling) agent binds to or physically adheres or adsorbs to a biological component such as a protein including a peptide or glycoprotein, and in this state, an oxidation reaction or the like Any chemiluminescent labeling agent may be used as long as it can be detected by exciting the chemiluminescent agent itself by chemiluminescence. Further, in addition to the chemiluminescent agent, a combination of an enzyme (label) agent and a substrate agent, a chemiluminescence enhancer, a pH buffer agent and the like can be included. Examples of chemiluminescent agents include 2,4,5 triphenylimidazole, 3-methylindole, tetrakisethylene and the like, as well as benzoperylene-1,2-dicarboxylic acid hydrazide and N-alkyl derivatives of isoluminol. Luminol derivatives, acridine compounds such as acridinium salts and lucigenin, adamantyl-1 such as 3- (2′-spiroadamantane) -4-methoxy-4- (3 ″ -phosphoryloxy) phenyl-1,2-dioxetane , 2-dioxetane derivatives, etc. Examples of the enzyme (labeling) agent, substrate agent, and chemiluminescence enhancer that can be combined with the chemiluminescent agent include (micro) peroxidase and N- Acetylglucosamidase, alkaline phosphatase, β-D-galacto Enzyme agents such as dase and glucose oxidase, oxidants such as hydrogen peroxide, permanganate and hypochlorous acid, and chemiluminescence enhancers of metals such as iron, cobalt, copper and manganese Can be selected as appropriate.

  As a bioluminescent labeling agent, a bioluminescent substrate agent or an enzyme (labeling) agent binds to or physically adheres or adsorbs to a biological component such as a peptide or a protein containing a glycoprotein, and bioluminescence is caused in such a state. Any bioluminescent labeling agent may be used as long as it can be detected. In addition to a bioluminescent substrate or an enzyme (label) agent, a combination of other enzyme agents, reaction aids, pH buffering agents, and the like can be included. Examples of bioluminescent substrate agents include firefly (Pintinus pyralis) luciferin, Cypridina luciferin, Renilla reniformis luciferin, luminescent earthworm (Lipiferin), In addition to luciferin derived from a luminescent organism such as luciferin (reduced flavin mononucleotide), aequorin derived from Aequorea jellyfish can be used.

  In addition, as an enzyme (labeling) agent and a reaction auxiliary agent, metals such as luciferase, AMP (adenosine-1-phosphate), ATP (adenosine-3-phosphate), magnesium salt and calcium salt, which are derived from the corresponding organisms, respectively. A salt, an aliphatic aldehyde, etc. can be mentioned, What is necessary is just to select so that it may become a suitable combination suitably. Each detailed condition when using the above-mentioned chemiluminescent labeling agent or bioluminescent labeling agent is described in [VanDyke, K. et al. et al; “Bioluminescence And Chemiluminescence” Vol. I, II, CRC Press Florida (1985)], Kazuhiro Imai, “Bioluminescence and Chemiluminescence” Hirokawa Shoten (1989), [Toshio Goto, “Bioluminescence” Kyoritsu Shuppan (1975)], [Inaba Fumio et al., “Measurement and Application of Latest Luminescence, NTS Publishing (1990)], [Hasting, J. et al. W. et al; Annu. Rev. Microbiol. Vol. 31, p549 (1977)], [Shimmura, O .; et al; Proc. Natl. Acad. Sci. Vol. 227, p680 (1970)], [Schroeder, H .; R. et al; Methods in Enzymol. Vol. 57, p424 (1978)], [Nakano, M .; et al; Anal. Biochem. Vol. 159, p363 (1986)], [Voyta, J. et al. C. et al; Clin. Chem. Vol. 34, p680 (1988)], [Bronstein, I. et al. et al; Biolumin. Chemilumin. Vol. 4, p99 (1989)].

  As a fluorescent labeling agent, a fluorescent substrate agent or an enzyme (labeling) agent binds or physically adheres or adsorbs to a biological component such as a peptide or a protein containing a glycoprotein, and emits fluorescence in such a state. Any fluorescent labeling agent may be used as long as it can be detected by the above method. In addition to the fluorescent substrate agent or the enzyme (labeling) agent, a combination of other enzyme agent, reaction auxiliary agent, pH buffering agent and the like can be included. Examples of the fluorescent substrate agent include aromatic heterocyclic compounds such as fluorescein, acridine, quinoline, coumarin, and resorufin, and polycyclic aromatic compounds such as naphthalene, anthracene, perylene, and pyrene, anthranilic acid, nitrobenzofuran, and stilbene. And the like, and various derivatized compounds of these fluorophore compounds, and any fluorescent substrate agent is applicable. Examples of the combination of the chromogenic substrate agent and the enzyme (labeling) agent include alkaline phosphatase and 4-methylumbelliferyl phosphate, β-D galactosidase and 4-methylumbelliferyl-β-D galactoside, peroxidase and p- Hydroxyphenylacetic acid or p-hydroxyphenylpropionic acid or fluorescein isothiocyanate or tyramine can also be applied. Furthermore, an enzyme agent containing an oxidizing agent such as hydrogen peroxide or a coenzyme as a reaction aid, and a pH buffer that adjusts to the optimum pH for each reaction may be added as necessary. Detailed conditions for using such a fluorescent labeling agent as a detection material are as follows: [Edited by the Japanese Biochemical Society, “Biochemistry Experiment Course 5; Enzyme Research Method”, Tokyo Kagaku Dojin (1975)], [Eiji Ishikawa et al., "Enzyme immunoassay", Medical Shoin (1987)], [The Japan Biochemical Society, edited by "Neurochemistry Experiment Course 1: Protein V", Tokyo Kagaku Dojin (1991)], [Frei, R .; W. et al; “Chemical Derivation In Analytical Chemistry” Vol. I, II, CRC Press (1981, 82)] and the like.

  The anti-Fas antibody contained in the biological component to be detected by the detection material is a type I membrane protein Fas containing a cell membrane penetrating part, a secretory Fas, an extracellular part Fas, an intracellular part Fas, and partial fragments Fas thereof (peptides In this embodiment, the detection material is a detection material specific for any anti-Fas antibody, or any anti-Fas antibody. Even if it exists, it can be set as the universal detection material which can be detected widely. As the detection material specific for any of the above-mentioned anti-Fas antibodies, each of the above-mentioned Fas proteins corresponding to each anti-Fas antibody can be preferably used, and each of these proteins is labeled with each of the above-mentioned labeling agents. May be. These Fas proteins have different molecular weights depending on the animal species from which they are derived. For example, the homology in the amino acid sequence of the type I membrane protein Fas containing a transmembrane portion is 50% in humans and mice, and in humans and cows. It is known to be 57% (see Patent Document 4 and Non-Patent Documents 8 to 12). The amino acid sequences for human Fas (sequence 1) and bovine Fas (sequence 2) are shown below.

  Sequence 1 (human Fas): MLGIWTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNLEGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFSSKCRRCRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCTKCEHGIIKECTLTSNTKCKEEGSRSNLGWLCLLLLPIPLIVWVKRKEVQKTCRKHRKENQGSHESPTLNPETVAINLSDVDLSKYITTIAGVMTLSQVKGFVRKNGVNEAKIDEIKNDNVQDTAEQKVQLLRNWHQLHGKKEAYDTLIKDLKKANLCTLAEKIQTIILKDITSDS NSNFRNEIQSLV (Patent Document 4, Non-Patent Document 8; see 335 amino acids)

  SEQ ID NO: 2 (bovine Fas): MSGIWVHLSLIFISVSGPLSKGENAHMAGINSEGLKLNITEANSCQEGLYREHQFCCQPCPPGKRKNGDCKRDGDTPECVLCSEGNEYTDKSHHSDKCIRCSICDEEHGLEVEQNCTRTRNTKCRCKSNFFCNSSPCEHCNPCTTCEHGIIEKCTPTSNTKCKGSRSHANSLWALLILLIPIVLIIYKVVKSRERNKKNDYCNSAASNDEGRQLNLTDVDLGKYIPSIAEQMRITEVKEFVRKNGMEEAKIDDIMHDNVHETAEQKVQLLRNWYQSHGKKNAYCTLTKSLPKALAEKICDIVMKDITNERENANLQNENENL (Non-Patent Document 12; see 323 amino acids)

  In addition, the amino acid sequences of secreted human Fas, extracellular membrane portion human Fas (soluble Fas), and intracellular membrane portion human Fas are signal sequences corresponding to the 1st to 16th amino acid sequences in Sequence 1, respectively. Sequence 3 excluding the region and the transmembrane region corresponding to the amino acid sequence of Nos. 174 to 190, sequence 4 corresponding to the amino acid sequence of Nos. 17 to 173 in Sequence 1, and Nos. 191 to It has been reported that it is sequence 5 corresponding to the amino acid sequence of No. 335 [Seiichi Kobayashi et al .; Japanese Clinical Vol. 54 (7), pp1741-1746 (1996)].

  Sequence 3 (secreted human Fas): RLSSKSVNAQVTDINSKGLELRKTVTTVETQNLEGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFSSKCRRCRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCTKCEHGIIKECTLTSNTKCKEEGSRSNKRKEVQKTCRKHRKENQGSHESPTLNPETVAINLSDVDLSKYITTIAGVMTLSQVKGFVRKNGVNEAKIDEIKNDNVQDTAEQKVQLLRNWHQLHGKKEAYDTLIKDLKKANLCTLAEKIQTIILKDITSDSENSNFRNEIQSLV

  Sequence 4 (extracellular part human Fas): RLSSKSVNAQVTDINSKGLELRKTVTTTVETQNLEGHLHHDGQFCHKPCPPGERKARDDCTVNGDEDCVPCQEGKEYYTDKAHFSSKCRRCTCRCKTCNGTCNTKTC

  Sequence 5 (partial cell membrane human Fas): KRKEVQKTRCHRHKENQGSSHSTPLNPETVAINLSDVDLSKYITTIAGVMMTLSQVGFNFDSQKINKDIQ

[Second Embodiment]
The in vitro diagnostic kit and in vitro diagnostic method for mammalian prion disease according to the second embodiment include a detection material for a biological component containing an anti-Fas antibody as in the first embodiment, and also includes a living body containing an anti-Fas antibody. A detection material for a derived component is used. It differs from the first embodiment mainly in that a detection material applied to the enzyme immunoassay is used. The biological component is a component contained in a biological material sample from a mammal diagnosis target individual such as blood, urine, and peripheral tissue and can be easily collected, and the blood can be a serum or plasma sample. In the second embodiment, the anti-Fas antibody and other biological components are separated from the biological component including the anti-Fas antibody by a separation method using the same separation material and separation device as in the first embodiment. Although it can be in a separated state, it is not necessary to separate in advance, and the anti-Fas antibody can be specifically detected directly by an enzyme immunoassay.

  Enzyme immunoassays (including EIA: ELISA) are broadly classified into competitive methods and non-competitive methods, and antigen-antibody reaction conjugate (B: Bound Form) and unbound unreacted (F: Free Form), respectively. Is divided into a heterogeneous method (heterogeneous method: separation method) for separating BF from BF and a homogeneous method (homogeneous method: non-separation method) without BF separation. The heterogeneous method includes a liquid phase method in which an antigen / antibody reaction is performed in a liquid phase, and a solid phase method in which a solid phase and a liquid phase are performed. In the case of directly measuring anti-Fas antibody using serum or plasma as a biological material specimen, it is substantially difficult to use the homogeneous method, and the same separation material, separation apparatus, and the like as in the first embodiment It is desirable to use, as a specimen, a fraction containing an anti-Fas antibody obtained by previously separating an anti-Fas antibody and other biological components (particularly non-specific immunoglobulin) by a separation method using. Although both competitive and non-competitive methods can be applied and are not limited, it is preferable to apply the non-competitive method over the competitive method in terms of excellent detection sensitivity. It is preferable to apply. In the second embodiment, the heterogeneous-solid phase method is adopted. The solid phase may be in any form such as a tube, microplate, bead, strip, microchip, and the material is for enzyme immunoassay such as polystyrene, glass, latex, cellulose, sepharose, filter paper, etc. As long as the antigen or antibody is insolubilized and supported by physical adsorption, covalent bond, etc., any solid phase of any material can be applied, and magnetic particles may be used.

  As the antigen for enzyme immunoassay in the second embodiment, that is, the antigen that can be bound to the anti-Fas antibody contained in the biological component to be detected by the detection material, The type I membrane protein human Fas (amino acid sequence 1), secretory human Fas (amino acid sequence 3), intracellular membrane portion human Fas (amino acid sequence 4), and extracellular membrane portion human Fas (amino acid sequence 4) including the transmembrane portion described in the embodiments. In addition to the amino acid sequence 5) and partial fragment Fas (peptide) thereof, human Fas ligand conjugate human Fas and the like can be used. These human Fas and Fas-related proteins are disclosed in Patent Document 1, Non-Patent Document 8, [Pamphlet of International Patent Publication WO95-13293], [Sambrook, et al; “Molecular Cloning, A Laboratory Manual 2nd Edition” Cold Spring. (Harbor Laboratory (1989))] and the like. In addition, the amino acid sequence of the anti-human Fas antibody has also been clarified (Japanese Patent Application Laid-Open No. 11-171900), and the protein or partial peptide thereof having an amino acid sequence that can bind to the variable region in the Fab region of the anti-Fas antibody The partial fragment Fas (peptide) can also be designed, and the peptide may be prepared using a known synthesis method or synthesis apparatus. A combination of a protein having an amino acid sequence that can bind to a fixed specific sequence of a Fas antibody Fc region of a mammal to be diagnosed or an antigen containing a partial peptide thereof is also possible.

  Further, even when the mammal to be diagnosed is a bovine, it is possible to apply the bovine Fas protein (amino acid sequence 2) containing the cell membrane penetrating portion as described in the first embodiment as an antigen for enzyme immunoassay. For example, it can be prepared according to Non-Patent Document 12, [Sambrook, et al; “Molecular Cloning, A Laboratory Manual 2nd Edition”, Cold Spring Harbor Laboratory (1989)]. In the second embodiment, mouse Fas protein and bovine Fas protein obtained by the method described later as mouse Fas cloning and bovine Fas cloning can be used as antigens for enzyme immunoassay. When mammals other than humans, cows, and mice are to be diagnosed, Fas proteins of each mammal can be similarly prepared and used as antigens for enzyme immunoassay. In addition, the secretory Fas, the intracellular membrane portion Fas, the extracellular membrane portion Fas, and the partial fragment Fas (peptide) thereof in each mammal also have the above-described human secreted Fas, intracellular membrane portion Fas, and extracellular membrane portion, respectively. Fas and their partial fragments Fas (peptides) can be prepared in the same manner and applied as antigens for enzyme immunoassay.

  Examples of the antibody for enzyme immunoassay that can be included as a part of the detection material in the second embodiment include the antigens of the respective mammals to be diagnosed described above (Fas including a transmembrane portion, secretory Fas, A polyclonal antibody purified from an antiserum obtained by sensitizing and immunizing an animal for immunization such as a rabbit or goat using a partial Fas, an extracellular part Fas, and a partial fragment Fas thereof, Fas ligand conjugate Fas, etc., Alternatively, a monoclonal antibody prepared using a hybridoma clone that produces a desired antibody cloned from a hybridoma obtained by cell fusion of antibody-producing cells such as the spleen of an immunized animal and myeloma, and selective culture in a HAT culture solution, Monoclonal produced by introducing Fas monoclonal antibody gene into E. coli, yeast, etc. Body like, it can be applied by any of various antibodies produced by any known methods of generating antibodies. The conditions for producing such an antibody include, for example, [Non-patent Documents 8 to 12], [Japanese Patent Laid-Open No. 11-171900], [Harlow, H. et al. et al; “Antibodies, A Laboratory Manual” Cold Spring Harbor Laboratory (1988)], edited by the Japanese Biochemical Society, “Neurochemistry Experiment Course 1; Protein IV”, Tokyo Chemical Dojin (1992)], “Toshio Kuroki et al.,” It can be prepared in accordance with “Experimental Medicine Separate Volume Cell Engineering Handbook”, Yodosha (1992)].

  In the second embodiment, the enzyme (label) agent, color developing substrate agent, and the like that can be included as a part of the detection material include the enzyme (label) agent and color development shown in the color labeling agent of the first embodiment. In addition to the substrate agent, coloring aids, pH buffering agents and the like can also be used. The enzyme (labeling) agent or chromogenic substrate agent can be labeled on the above-described antigen or antibody for enzyme immunoassay. Examples of the labeling method include periodate method, carbodiimide method, glutaraldehyde method, maleimide The labeling can be carried out by any known labeling method such as the method, pyridyl disulfide method. Such detailed conditions of the labeling method are described in [Eiji Ishikawa et al., “Enzyme Immunoassay”, Medical School (1987)], [Avrameas, S. et al. et al "Development of Immunology" Elsevier Vol. 18, (1983)], [Butt, W. et al. R. et al “Practical Immunoassay” Marcel Dekker (1984)] and the like.

In the in vitro diagnostic kit of the second embodiment, an anti-Fas antibody appropriately selected from the above-mentioned antibodies for enzyme immunoassay can be provided as an anti-Fas antibody standard in addition to the above-described detection material. By creating a calibration curve by the above, it becomes possible to quantify or semi-quantify the anti-Fas antibody in the biological material specimen. Such an anti-Fas antibody standard is desirably a Fas antibody of a mammal to be diagnosed. As described above, as an in vitro diagnostic kit and in vitro diagnostic method according to the second embodiment, examples of enzyme immunoassay methods have been described, but in addition to or in place of the enzyme (label) agent and the chromogenic substrate agent as described above, By using the chemiluminescent labeling agent, bioluminescent labeling agent, and fluorescent labeling agent in the first embodiment, chemiluminescence (enzyme) immunoassay, bioluminescence (enzyme) immunoassay, and fluorescence (enzyme) immunoassay, respectively. In vitro diagnostic kits and in vitro diagnostic methods can be obtained. In addition, by using a radioisotope labeling agent such as ¹²⁵ I, ¹³¹ I, ³ H, and ¹⁴ C instead of an enzyme (labeling) agent and a chromogenic substrate agent, an in vitro diagnostic kit and an in vitro diagnostic method using a radioimmunoassay method It is also possible to do.

  Hereinafter, the in vitro diagnostic kit and in vitro diagnostic method for prion disease in mammals of the present invention will be specifically described with reference to test examples and examples, but the present invention is not limited thereto.

[Test Example 1]: (Collecting biological material from a mammal with prion disease)
[T1-A]: (PrPSc-infected mouse production)
The production method is described in Tg7 mice lacking mouse prion protein and expressing hamster prion protein ([Priola, SA et al; Science Vol. 287, p1503 (2000)], etc .: male n = 30 , Female n = 30), and hamster scrapie (PrPSc) 263K was injected into the brain of each mouse to produce PrPSc-infected mice. Preliminary breeding and breeding conditions after the injection of PrPSc were carried out under an SPF (specific pathogen free) environment, and were infected by injection at 8 weeks of age. The injection conditions of PrPSc were 1% brain emulsion prepared using physiological saline from the 263K diseased end stage Tg7 mouse brain, 20 μl, and injected into the right parietal brain at the injection site. As a control (Negative Control), instead of PrPSc, a 1% brain emulsion of normal Tg7 mice was prepared with physiological saline, and 20 μL of this was injected into the Tg7 mouse brain in the same manner as in the case of PrPSc-infected mice.

[T1-B]: (Blood collection)
About 200 μL of blood was collected from the heart under deep anesthesia on days 35 and 55 (just before death in infected mice) for each PrPSc-infected mouse and control mouse, and blood cells were removed by centrifugation. Each plasma specimen was obtained as a biological material.
[T1-C]: (Brain pathological analysis)
Each PrPSc-infected mouse was euthanized every week after injection, immediately dissected and subjected to pathological examination of brain tissue. As a result, it was confirmed that abnormal prion protein deposition was observed in the brain from around 35 days after injection (after infection).

[Example 1]: (polyacrylamide gel two-dimensional electrophoresis)
Each plasma sample as a biological material obtained in Test Example 1 (T1-B) was diluted 10-fold with distilled water, frozen with liquid nitrogen, and stored in a freezer at −30 ° C. The solution was re-melted before use and used as each sample plasma dilution or control plasma dilution. In this example, two-dimensional electrophoresis in the first embodiment: IEF using a pH gradient (gradient) polyacrylamide gel in the first dimension and SDS-PAGE in the second dimension) Were separated using a detection material containing CBB-R250 as a staining marker. Detailed implementation conditions were as shown below.

[E1-A]: (Preparation of first dimension IEF gel)
As a first dimension IEF gel, a dry type that is swollen with a swelling liquid at the time of use, a strip-shaped (elongated plate-shaped) immobilized pH gradient (IPG) gel, in this case, a pH of 3 to 10 An IPG strip having a pH gradient range and a length of 11 cm (Immobiline ^™ Dry Strips pH 3-10, 11 cm; manufactured by Amersham Biosciences) was used. To each sample plasma dilution or control plasma dilution 20 μL, a swelling solution [1.5 M Tris-HCl (pH 8.8; manufactured by Kanto Chemical Co., Inc.), 8 M urea (Urea; Kanto) 2% CHAPS (Cyclohexylaminoprone sulfonic acid; manufactured by Wako Pure Chemical Industries, Ltd.), BPB (trace amount, Bromophenol Blue; manufactured by Kanto Chemical Co., Inc.), DTT (Dithiothreitol, manufactured by Hanai Pharmaceutical Co., Ltd.) 180 μl of 5% IPG buffer (IPG Buffer pH 3-10; manufactured by Amersham Pharmacia Biotech)] was mixed, reacted at room temperature for 30 minutes, and then used as each swelling solution. The swelling liquid was added to each of the swelling trays, and the first-dimensional IEF gel (IPG strip) surface was placed on the liquid surface. In order to prevent the gel from drying, it was covered with silicon oil (KF96L-1.5CS; manufactured by Shin-Etsu Chemical Co., Ltd.) and swollen for about 18 hours.

[E1-B]: (first dimension electrophoresis)
Each IPG swollen in [E1-A] was set in advance to a constant temperature circulator (Multiphor II, manufactured by Pharmacia Biotech) at 20 ° C. to prevent Urea crystal formation on the gel surface during electrophoresis. After washing the surface of the strip with double distilled water, the IPG strip was set in a constant temperature circulator, and electrophoresis was performed by applying current at 300 V for 1 minute, 1000 V for 1 hour 30 minutes, and 3000 V for 2 hours 35 minutes. The IPG strip subjected to the first dimension electrophoresis was stored at −30 ° C. when the second dimension electrophoresis (SDS-PAGE) was not performed.

[E1-C]: (second dimension electrophoresis)
A constant temperature circulator (Multiphor III; manufactured by Pharmacia Biotech) was set at 15 ° C. in advance, and the gel was equilibrated in order to introduce a reagent necessary for the second-dimensional electrophoresis into the gel. SDS equilibration buffer [50 mM 1.5 M Tris-HCl (pH 8.8; manufactured by Kanto Chemical Co.), 30% glycerol (Glycerol; manufactured by Kishida Chemical Co., Ltd.), 2% SDS (manufactured by Amersham Biosciences), 6M urea (Urea; Kanto Chemical Co., Inc.), BPB (trace amount, Bromophenol Blue; manufactured by Kanto Chemical Co., Ltd.)] The first equilibration solution obtained by [E1-B] was added to the first equilibration solution to which 100 mg of DTT was added per 10 mL. Of IPG strips and shaken for 30 minutes. Further, the IPG strip was transferred to a second equilibration solution to which 250 mg of iodoacetamide was added per 10 ml of SDS equilibration buffer, and shaken for 30 minutes. The IPG strip was then placed on moistened filter paper to remove excess equilibration solution. A constant temperature circulator (Multiphor III; manufactured by Pharmacia Biotech) was set at 15 ° C. in advance, and a gel for the second dimension (gradient SDS-PAGE) (Excel Gel ^™ SDS Gradient 8-18; manufactured by Amersham Biosciences) was used. Place in the circulator and place the gel surface of the equilibrated IPG strip in direct contact with the surface of the gel for the second dimension. After electrophoresis at 20 mA for 30-35 minutes, remove the IPG strip and remove at 50 mA. It was energized for 1 hour and electrophoresed. This was dyed with a CBB-R250 (Nacalai Tesque) dyeing solution (0.1% CBB-R250, 50% methanol, 10% acetic acid), and then with a decolorizing solution (10% methanol, 7% acetic acid). Decolorized. In addition, the molecular weight of each spot detected was as follows: phosphorylase b (97 kDa), albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa), α-lactalbumin ( 14.4 kDa) was used to measure the molecular weight marker (Low Molecular Weight Calibration Kit for Electrophoresis; manufactured by Amersham Biosciences).

[E1-D]: (Specific protein fractionation / trypsin digestion)
For each plasma sample obtained from the PrPSc-infected mouse (40 days after intracerebral injection) and the control mouse (40 days after intracerebral injection) in Test Example 1, the polyps were obtained from [E1-A] to [E1-C]. FIG. 1 shows an electrophoregram when acrylamide gel two-dimensional electrophoresis is performed. Also, in FIG. 1, among the spots specific to PrPSc-infected mice, the vicinity of three spots with arrows (from pI neutral side to Spot1 to Spot3: molecular weight of about 30 kDa, pI of about 5 to 6) is partially enlarged. This is shown in FIG. Although not shown, it was confirmed that the amount (staining concentration and size) of each of the specific spots of Spot1 to Spot3 slightly increased compared to the plasma samples in PrPSc-infected mice on the 35th day after intracerebral injection. It was done. The gels of the three specific spot portions were individually cut out with a cutter (razor) and collected. Each excised specific spot gel was further shredded and crushed, washed with 200 μL of 50% acetonitrile aqueous solution for 15 min, and then with the same amount of acetonitrile (100%) for 15 min. I let you. Next, each dried specific spot gel was swollen by immersing it in 50 mM ammonium bicarbonate buffer containing 2.5 ng / μL trypsin (sequence grade modified trypsin; Promega) and 5 mM calcium chloride for 20 minutes. The excess liquid was removed without infiltrating the gel, 50 mM ammonium bicarbonate buffer was added, and the mixture was allowed to react at 37 ° C. for 15 hours to perform in-gel trypsin digestion. Each supernatant liquid was subjected to mass spectrometry: (nano) ESI (Electrospray Ionization) -MS / MS.

[E1-E]: (ESI-MS / MS test and analysis of specific protein)
Each supernatant obtained in [E1-D] was adsorbed onto an in-chip column (ZipTip _C18 ; manufactured by Millipore) having a reverse phase carrier supported on the tip of a micropipette tip, and 60% acetonitrile / 0.00. Elution was performed with a 1% formic acid solution to remove salts from each supernatant. Mass analysis was performed using a Q filter-time-of-flight MS / MS mass spectrometer (Q-TOF Ultimate Global; manufactured by Micromass), and analysis of the results was performed from the NCBInr database (The National Center for Biotechnology Information). Search and analysis was performed using a search program (MASCOT; Matrix Science) (consigned by Gene World Co., Ltd.). From this analysis result, it was confirmed that the specific protein corresponding to each specific spot; Spot 1 to Spot 3 has a peptide fragment that matches the amino acid sequence of the Fab portion of the anti-Fas antibody shown in FIG. 3 and contains the anti-Fas antibody. It was. Therefore, in the in vitro diagnostic kit and the in vitro diagnostic method according to the first embodiment, for example, a test is performed on a predetermined amount of anti-Fas antibody (Fas antibody standard for each target mammal, etc.) as in the case of each plasma sample. From the comparison with the test results (molecular weight, pI, retention time, measurement amount (concentration), etc.) of this anti-Fas antibody and control (Negative Control), the anti-Fas antibody contained in each plasma sample is qualitatively or quantitatively determined. By detecting this in advance, a prenatal diagnosis of prion disease is possible without performing mass spectrometry.

[Example 2]: (ELISA)
This example is an example of an in vitro diagnostic kit and in vitro diagnostic method for mammalian prion disease by heterogeneous-solid phase method (ELISA) in the enzyme immunoassay method of the second embodiment. (96-well microplate for ELISA; Disposable Sterile ELISA Plate 25801, manufactured by Corning) As a detection material, human Fas protein (recombinant soluble human Fas protein; Recombinant Soluble Human Fas, BD Biosciences) as an antigen for enzyme immunoassay ), As a coloring labeling agent, a goat anti-mouse IgG antibody (HRP-labeled goat anti-mouse IgG antibody; manufactured by PIERCE) labeled by applying peroxidase (horseradish peroxidase: HRP) as an enzyme labeling agent And 3,3 ′, 5,5′-tetramethylbenzidine (3,3 ′, 5,5′-tetramethyl-benzidine (TMB) Liquid Substrate System for ELISA; manufactured by SIGMA) as a coloring (substrate) agent The measurement system configuration included. In addition, a mouse anti-human Fas antibody (purified mouse anti-human Fas protein antibody; Purified Mouse Anti-human Fas (CD95) Monoclonal Antibody, manufactured by BD Biosciences) was used as an anti-Fas antibody standard for preparing a calibration curve. The anti-Fas antibody concentration (detection amount) was measured for each plasma specimen as a biological material obtained in 1 (T1-B) using normal mouse plasma as a blank. Detailed implementation conditions were as shown below.

[E2-A]: (Preparation of immobilized microplate)
Human Fas antigen for enzyme immunoassay was diluted to 100 ng / mL with PBS (Phosphate Buffered Saline), and 100 μL was dispensed into each well of the microplate and incubated at 4 ° C. overnight to be immobilized. . The plate is blocked by incubating at room temperature for 1 hour 30 minutes using a blocking solution (Block Ace; manufactured by Dainippon Pharmaceutical Co., Ltd.). For washing the plate, polysorbate 20 (Tween 20; manufactured by Wako Pure Chemical Industries, Ltd.) is used in PBS. ) Was added 0.05% (V / V).

[E2-B]: (calibration curve creation and anti-Fas antibody concentration measurement)
The anti-Fas antibody standard was diluted to 10 ng / mL, 1 ng / mL, 0.1 ng / mL, and 0.05 ng / mL with normal mouse plasma dilution (normal mouse plasma diluted 400-fold with PBS), and each of the calibration curves An anti-Fas antibody standard solution was prepared. Each PrPSc-infected mouse as a biological material obtained in Test Example 1 (T1-B), each plasma specimen for the control mouse was diluted 400 times with PBS, and each PrPSc-infected mouse plasma diluted sample solution, A control mouse plasma diluted sample solution was used. The blank was similarly diluted 400 times with PBS to obtain a blank solution. Each anti-Fas antibody standard solution, each PrPSc-infected mouse plasma diluted sample solution, each control mouse plasma diluted sample solution, and blank solution were dispensed at 100 μL / well onto the solid-phased microplate prepared by [E2-A]. (Triple measurement: 3 wells were used for each standard solution, each sample solution, and blank solution), incubated at 37 ° C. for 1 hour, and then washed with plate washing solution B (PBS supplemented with 0.5% Tween 20). Subsequently, 100 μL / well of enzyme labeling agent diluted 10-fold with a blocking solution for enzyme labeling agent was dispensed at 100 μL / well to each well and incubated at 4 ° C. for 1 hour, and then the plate washing solution A And washed. Next, 100 μL / well of the color former was dispensed into each well and incubated at room temperature for 30 minutes while being shielded from light. Then, 0.5 M sulfuric acid was dispensed into each well at 100 μL / well and stirred at 450 nm. Absorbance was measured with a microplate reader.

  In Test Example 1, PrPSc-infected mice at the 35th day after intracerebral injection (35-13) and at the end of infection (T-1, T-5: 2 mice just before death) and control mice (C-2, C -7: Table 1 shows the results of measuring anti-Fas antibody concentrations by [E2-A] and [E2-B] for each plasma sample obtained from 2 mice on day 55 after intracerebral injection) It was. The calibration curve is as shown in FIG. From the results of this example (Table 1), the detected amount of anti-Fas antibody in the plasma sample of PrPSc-infected mice on the 35th day after intracerebral injection was about 5 times the detected amount in the plasma samples of control mice, and the end stage of infection. It was confirmed that the anti-Fas antibody increased in the mouse plasma specimen by about 25 times. Therefore, prenatal diagnosis of mammalian prion disease is possible by the in vitro diagnostic kit and in vitro diagnostic method in the second embodiment.

  The in vitro diagnostic kit and in vitro diagnosis method of the present invention include, in addition to mammalian prion disease, bulbar spinal muscular atrophy, Huntington's disease, spinocerebellar degeneration, dentate nucleus red nucleus pallidal Louis atrophy, Parkinson's disease, Lewy body dementia, multisystem atrophy, Alzheimer's disease, Down syndrome, familial amyotrophic lateral sclerosis, FTDP-17, progressive nuclear epithelial palsy It is also applicable to screening for degenerative neurodegenerative diseases such as cortical basal ganglia degeneration, Pick disease, and familial dementia. In addition, by combining with a diagnostic marker specific for a neurodegenerative disease including each prion disease, a prenatal diagnosis for each neurodegenerative disease can be performed.

FIG. 5 is a drawing-substituting photograph showing an electrophoregram when two-dimensional electrophoresis is performed according to Example 1 for each plasma specimen obtained from a PrPSc-infected mouse and a control (Negative Control) mouse in Test Example 1. FIG. FIG. 2 is a drawing-substituting photograph in which the vicinity of three spots (Spot 1 to Spot 3) with arrows among the spots specific to the PrPSc-infected mouse in FIG. 1 is partially enlarged. It is a figure which shows the amino acid sequence of the peptide fragment contained in each trypsin digest about 3 specific spots in FIG.1 and FIG.2, and corresponds to the amino acid sequence of an anti- Fas antibody. FIG. 3 shows a calibration curve of anti-Fas antibody in the enzyme immunoassay system (ELISA) of Example 2.

Claims (18)

  An in vitro diagnostic kit for a prion disease in a mammal provided with a detection material for a biological component containing an anti-Fas antibody.   The in vitro diagnostic kit according to claim 1, wherein the biological component is a component contained in blood.   The in vitro diagnostic kit according to claim 1 or 2, wherein the kit is used for primary screening of the prion disease.   The prion diseases include Creutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy (BSE), scrapie, deer chronic wasting disease (CWD), transmissible mink encephalopathy (TME), Gerstmann Stroisler Scheinker (Gerstmann) The in vitro diagnostic kit according to any one of claims 1 to 3, wherein the kit is selected from one or more of-Straussler-Scheinker syndrome and lethal familial insomnia.   5. The mammal according to claim 1, wherein the mammal is selected from human, cow, goat, sheep, deer, elk, mink, pig, monkey, mouse, rat, hamster and cat. An in vitro diagnostic kit according to 1.   The in vitro diagnostic kit according to any one of claims 1 to 5, wherein the prion disease is a prion disease in a ruminant mammal.   7. The detection material according to any one of claims 1 to 6, wherein the detection material comprises a staining labeling agent for a biological component, a coloring labeling agent, a chemiluminescent labeling agent, a bioluminescent labeling agent, or a fluorescent labeling agent. An in vitro diagnostic kit according to 1.   The in vitro diagnostic kit according to any one of claims 1 to 7, wherein the detection material comprises a protein having an amino acid sequence capable of binding to the anti-Fas antibody or a partial peptide thereof.   The in vitro diagnostic kit according to any one of claims 1 to 8, wherein the detection material comprises a mammalian Fas protein obtained from a cloned mammalian Fas gene or a partial peptide thereof.   An in vitro diagnostic method for prion disease in a mammal using a detection material for a biological component containing an anti-Fas antibody.   The in-vitro diagnostic method according to claim 10, wherein the biological component is a component contained in blood.   The in vitro diagnosis method according to claim 10 or 11, wherein the method is used for primary screening of the prion disease.   The prion diseases include Creutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy (BSE), scrapie, deer chronic wasting disease (CWD), transmissible mink encephalopathy (TME), Gerstmann Stroisler Scheinker (Gerstmann) The in-vitro diagnostic method according to any one of claims 10 to 12, wherein the method is one or more selected from Straussler-Scheinker syndrome and lethal familial insomnia.   14. The mammal according to claim 10, wherein the mammal is selected from human, cow, goat, sheep, deer, elk, mink, pig, monkey, mouse, rat, hamster, and cat. The in vitro diagnostic method according to 1.   The in vitro diagnostic method according to claim 10, wherein the prion disease is a prion disease in a ruminant mammal.   16. The detection material according to any one of claims 10 to 15, wherein the detection material comprises a staining labeling agent of a biological component, a coloring labeling agent, a chemiluminescent labeling agent, a bioluminescent labeling agent, or a fluorescent labeling agent. The in vitro diagnostic method according to 1.   The in vitro diagnostic method according to any one of claims 10 to 16, wherein the detection material comprises a protein having an amino acid sequence capable of binding to the anti-Fas antibody or a partial peptide thereof.   18. The in vitro diagnostic method according to any one of claims 10 to 17, wherein the detection material comprises a mammalian Fas protein obtained from a cloned mammalian Fas gene or a partial peptide thereof.