JP2009052933A - Detecting method and diagnosing kit of prion disease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a detection method and a diagnosing kit of prion disease capable of confirming early onset of the prion disease, and performing antemortem diagnosis during the early stage. <P>SOLUTION: In the detection method, an anti-glial fibrillary acidic protein (GFAP) antibody or GFAP in a test sample originated in a living body of a mammal is analyzed. The diagnosing kit includes GFAP or its antigen fragment or an anti-GFAP antibody. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a detection method and a diagnostic kit for prion diseases in mammals.

  Prion disease is sporadic, contagious or partly hereditary and is a progressive neurodegenerative disease that affects humans and many animals. Prion protein that is expressed abundantly in nerve cells is insolubilized and aggregated and accumulates in nerve tissue as the main factor, leading to death due to destruction of nerve cells such as inducing apoptosis. Abnormal prion protein is considered the source of infection and is the only infectious agent that does not contain nucleic acids. However, it is not known how aggregated prion protein damages neurons.

  On the other hand, among prion diseases, bovine spongiform encephalopathy (BSE) has a great impact on the economy and society. It has been said that since 1972 cattle developed BSE in the UK, the number of affected animals has exceeded 1.84 million. In addition, Creutzfeldt-Jakob disease (CJD), which is a human prion disease that has been mainly limited to elderly people, has occurred in large numbers among young people in their twenties, and there is an unprecedented variant CJD with a large outbreak of BSE Suspected to be related. Meat, milk, and the like produced from cattle are used as raw materials for food and industrial products. Therefore, in order to provide a safe food supply, it is required that cattle suspected of being BSE can be eliminated at the prenatal stage.

  In order to enable prenatal diagnosis, it is effective if a component appearing in blood after BSE infection can be identified and detected. In 1982, Aoki et al. Detected autoantibodies against neurofilaments in prion disease serum (Non-patent Document 1). In 1985, Toh et al. Detected autoantibodies against neurofilaments (200 kDa, 150 kDa, 70 kDa) (55-78%) at a high rate in sera of Kuru, CJD, and other neurological diseases. However, 28-67% was also found in the serum of healthy people, and this result alone could not be said to be effective for serodiagnosis of prion disease (Non-patent Documents 2 and 3).

  Cells constituting the central nervous system include glial cells as well as nerve cells. In general, glial cells are classified into three types: astrocytes (astrocytes), oligodendrocytes (oligodendrocytes) and microglia. Among them, the most astrocytes are known to perform the uptake of neurotransmitter leaked from the synaptic cleft and the regulation of extracellular ion concentration at the synapse during the synaptic transmission process of the nerve cell. In addition, astrocytes play important roles such as the construction of the nervous system and the formation of the blood-brain barrier. In this astrocyte, there is a glial fibrillary acidic protein (GFAP) that is specifically expressed. This GFAP is an intermediate filament protein that supports the strength of nerve cells. Many researchers have shown that astrocytes are activated and GFAP is overexpressed in the brains of naturally infected scrapie sheep and experimentally infected mice (eg, Non-patent Document 4). . It is also known that many abnormal prions are accumulated in astrocytes in the scrapie mouse brain (Non-patent Document 5). In 2006, Gray et al. Found that GFAP-degraded fragments accumulated in hippocampal tissues of scrapie-infected mice (Non-patent Document 6).

  As a method for diagnosing prion disease, a method for detecting the presence of abnormal isoform prion protein in urine (Patent Document 1) or a method for diagnosing prion by forming a complex with a specific antibody (Patent Document 2) ) Etc. are known. In addition, there is a method in which diagnosis is performed by detecting biological components that are not directly detected from prions but are denatured due to the effects of prion disease and leak into blood or the like. For example, a method of diagnosis using an anti-Fas antibody is known (Patent Document 3).

Special table 2004-511809 gazette JP 2004-340924 A JP 2006-38536 A “Infection and Immunity” (USA), 1982, 38, p. 316-324, Aoki, T., Gibbs, CJ, Jr., Sotelo, J. and Gajdusek, DC , Heterogeneic Autoantibody Against Neurofilament Protein in the Sera of Animals with Experimental kuru and Creutzfeldt-Jakob Disease and Natural Scrapie Infection. “Proceedings of the National Academy of Sciences of the United States of America” (USA), 1985 82, p. 3485-3489, Toh, BH, Gibbs, CJ, Jr., Gajdusek, DC, Goudsmit, J. and Dahl, D., The 200- and 150-kDa neurofilament proteins react with IgG autoantibodies from patients with kuru, Creutzfeldt-Jakob disease, and other neurologic disease. “Proceedings of the National Academy of Sciences of the United States of America” (USA), 1985 82, p. 3894-3896, Toh, BH, Gibbs, CJ, Jr., Gajdusek, DC, David, DT and Dahl, D., The 200- and 150-kDa neurofilament proteins react with IgG autoantibodies from chimpanzees with kuru or Creutzfeldt-Jakob disease; a 62kDa neurofilament-associated protein reacts with sera from sheep with natural scrapie. “The bulletin of the French Academy of Sciences (Comptes Rendus de P Accademie des Sciences (Paris))” (France), 1981, Vol. 293, p. 53-56, Dormont, D., Delpech, B., Delpech, A ., Courcel, MN, Viret, J., Markovits, P. And Court, L., Hyperproduction de protein gliofibrillaire acide (GFAP) au cours de l'evolution de la tremblante experimentale de la souris. "Proceedings of the National Academy of Sciences of the United States of America" (USA), 1991 88, p. 375-379, Diedrich, JF, Bendheim, PE, Kim, YS, Carp, RI and Haase, AT, Scrapie-associated prion protein accumulates in astrocytes during scrapie infection. “Biochemical Society Transactions” (UK), 2006, Volume 34, p. 51-54, Gray, BC, Skippt, P., O'Connor, VM and Perry VH, Increased expression of glial fibrillary acidic protein fragments and μ-calpain activation within the hippocampus of prion-infected mice.

  However, it cannot be said that the method for diagnosing the onset of prion disease at an early stage and in a prenatal state is necessarily a sufficient method. Therefore, a definitive diagnosis is finally made by brain biopsy or brain pathological examination at autopsy. Therefore, it is desired to develop a highly practical method for diagnosing prion disease that can easily find a mammal suffering from prion disease at the prenatal stage.

  Thus, the present inventors have conducted extensive studies to enable prenatal diagnosis for prion disease, and as a result, have found a kit and a detection method for diagnosing prion disease by detecting anti-GFAP antibody. It is. In addition, the present invention has found a kit and a detection method capable of diagnosing the disease of prion disease by detecting GFAP in the blood of a mammal that has developed prion disease.

That is, the present invention
[1] A method for detecting prion disease, comprising analyzing an anti-glial fiber acidic protein antibody or glial fiber acidic protein in a test sample derived from a living body of a mammal,
[2] The detection method of [1], wherein the test sample is a blood-derived sample,
[3] A diagnostic kit for prion diseases, comprising a glial fibrillary acidic protein or an antigenic fragment thereof, or an anti-glial fibrillary acidic protein antibody.

  With the diagnostic kit or detection method for prion diseases of the present invention, it is possible to confirm early onset of prion diseases and to perform prenatal diagnosis at an early stage.

  The detection method of the present invention is a method for diagnosing the onset of prion disease by analyzing anti-GFAP antibody against GFAP (glial fibrillary acidic protein) appearing in the biological component of a mammal that has developed prion disease, or GFAP. is there. The diagnostic kit of the present invention can detect anti-GFAP antibody or GFAP in a biological component serving as a diagnostic marker for prion disease, and includes GFAP or an antigen fragment thereof, or anti-GFAP antibody.

  In this specification, “analysis” means “detection” for determining the presence or absence of an analyte (for example, anti-GFAP antibody or GFAP), and quantitative or semi-quantitative analysis of the amount (concentration) of the analyte. And “measurement” to be determined. Examples of the mammal include human, cow, goat, sheep, deer, elk, mink, pig, monkey, mouse, rat, hamster, cat, rabbit and the like.

  In the present specification, prion disease means Creutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy (BSE), scrapie, deer chronic wasting disease (CWD), transmissible mink encephalopathy (TME), Gerstman Streisler・ Sinker (Gerstmann-Straussler-Scheinker) syndrome, meaning lethal familial insomnia.

  The test sample that can be used in the present invention is a biological sample collected from a mammal to be diagnosed (whether prenatal or postmortem, but preferably prenatal) and taken out of the living body or a processed product thereof. As long as it is not particularly limited, for example, biological fluid (for example, blood, plasma, serum, urine, bone marrow fluid, saliva, tears), cell or tissue disruption fluid, etc., blood, plasma, Serum, urine, saliva and tears are preferred.

  The GFAP analyzed in the present invention is an intermediate filament protein (astrocyte) that is specifically expressed in a normal state in an astrocyte (astrocyte) that is one of the glial cells that constitute the central nervous system together with the nerve cell. The molecular weight is about 50 kDa).

  In the present invention, when an anti-GFAP antibody is detected in a test sample (especially in blood) or when it shows a high value compared with the normal value range of a healthy individual, it is determined that the disease is prion disease. can do. Moreover, in this invention, when GFAP is detected in a test sample, or when a high value is shown compared with the normal value range of a healthy individual, it can determine with prion disease. The normal value range of a healthy individual varies depending on the type of animal to be diagnosed. However, those skilled in the art can determine the normal value range and / or threshold value by measuring the value of a healthy individual in advance and performing statistical processing. Can be easily determined.

  Detection of the presence or absence of a diagnostic marker or measurement of its amount in the present invention can be performed by a known method (for example, a method using an antigen-antibody reaction). Hereinafter, specific analysis techniques of the present invention will be described based on an aspect of analyzing an anti-GFAP antibody, which is one of diagnostic markers, using GFAP as an antigen. Thus, it will be easily understood that GFAP, which is one of diagnostic markers, can be analyzed using an anti-GFAP antibody. Examples of known methods utilizing antigen-antibody reaction include immunological measurement methods, mass spectrometry methods, electrophoresis methods, liquid chromatography (LC) methods, etc., and these methods are generally used in combination. be able to. Among them, the most suitable method is to use a combination of immunological measurement and electrophoresis.

  The anti-GFAP antibody detected in the present invention can be measured by an immunoassay based on an antigen-antibody reaction with GFAP, which is an antigen in a biological component, or an antigen fragment thereof. As used herein, “antigen fragment of GFAP” means a fragment of GFAP that can react with an anti-GFAP antibody. As the immunological measurement method, a known method can be used. Specific examples include enzyme immunoassay (EIA method), immunoturbidimetric assay (TIA method), latex immunoaggregation method (LATEX method), electrochemiluminescence method, and fluorescence method. In this case, an ELISA method is suitable for quantitative analysis. Further, a method of performing quantitative analysis using image analysis software or the like such as an image obtained by Western blotting or the like is also a general technique and can be used for the analysis of the present invention.

  Enzyme immunoassays (EIA methods) are broadly classified into those based on competitive binding and those based on non-competitive binding based on the measurement principle. Competitive binding refers to a method of measurement using the fact that a labeled substance and an unlabeled substance bind competitively to a certain amount of antibody. Non-competitive binding is a method of measurement by so-called sandwich binding in which a substance to be measured binds to a sufficient amount of immobilized antibody and is recognized by a labeled antibody.

Examples of the labeling substance include enzymes, luminescent substances, and fluorescent substances. Further, the luminescent materials are classified into chemiluminescence and bioluminescence.
Examples of the enzyme labeling substance include β-glucuronidase, peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, hexokinase and the like. To these enzymes, an oxidizing agent such as hydrogen peroxide or a pH buffer solution can be appropriately added so that an enzyme reaction can be performed at an optimum pH. Further, a coloring labeling substance such as biotin and 2,4-dinitrophenol can be used. In addition, as a labeling substance for a luminescent substance, any chemiluminescent labeling substance can be used as long as it can be detected by chemiluminescence by binding to or adsorbing to proteins, peptides, etc., and the chemiluminescent labeling substance itself being excited by a chemical reaction. May be. Chemiluminescent labeling substances include 2,4,5 triphenylimidazole, 3-methylindole, tetrakisethylene and the like, as well as luminol such as benzoperylene-1,2-dicarboxylic acid hydrazide and N-alkyl derivatives of isoluminol. 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, A 2-dioxetane derivative, etc. can be used As a bioluminescent labeling substance, luciferin possessed by fireflies, sea fireflies, and the like can be used.

The labeling substance of the fluorescent substance refers to a substance that emits fluorescence when irradiated with ultraviolet rays. Aromatic heterocyclic compounds such as fluorescein, acridine, quinoline, coumarin, resorufin, polycyclic aromatic compounds such as naphthalene, anthracene, perylene, pyrene, compounds that become fluorophores such as anthranilic acid, nitrobenzofuran, stilbene, etc. Can be used. The above labeling substances can be used in combination with each other.
There is also a staining method as one of the detection methods. General Coomassie Brilliant Blue (CBB) staining or silver staining can be used.

  An example of a method for separating the antigen GFAP from the biological component is electrophoresis. Examples include polyacrylamide gel electrophoresis and two-dimensional electrophoresis. Of these, two-dimensional electrophoresis is most often used. Specifically, it is separated by isoelectric focusing (first dimension), and then separated by SDS-polyacrylamide gel electrophoresis (second dimension). In the first dimension of the two-dimensional electrophoresis method, separation is performed using the isoelectric point of the protein (isoelectric focusing). Although proteins are amphoteric molecules, the charge state changes depending on the pH of the solvent, and the change varies depending on the type of protein, so that the proteins can be separated from each other. In this isoelectric focusing, proteins can be separated by the difference in isoelectric point by using a gel with a pH gradient. Examples of gels that can be used include Immobilized pH Gradient gels. A trade name of Immobiline DryStrip [manufactured by GE Healthcare Biosciences] can be used for this IPG gel. Since this is a dried precast gel, it is necessary to change the composition of the solution according to the properties of the sample and swell the gel before use. As swelling liquids, denaturing agents such as urea and thiourea, reducing agents such as dithiothreitol (DTT), and interfaces such as CHAPS (3-[(3-cholamido-propyl) dimethyl-ammonio] -1-propane sulfonate) A solution containing the active agent can be used. In isoelectric focusing, agarose or acrylamide can generally be used as a gel support.

  In the second dimension of the two-dimensional electrophoresis method, the protein can be separated by the difference in molecular weight using SDS-PAGE. Immobiline DryStrip [manufactured by GE Healthcare Bioscience Co., Ltd.] after the completion of the first-dimensional electrophoresis is equilibrated with SDS, and then the second-dimensional electrophoresis is performed. By this SDS equilibration treatment, proteins in the IPG gel can be separated into distinct spots by reduction and alkylation.

The anti-GFAP antibody detected in the present invention is not particularly limited as long as it is an antibody against mammalian GFAP. Among them, an anti-GFAP antibody having a molecular weight of about 50 kDa is preferable.
Examples of the method for detecting an anti-GFAP antibody include Western blotting and ELISA using the GFAP prepared by the above method as an antigen.
In the present invention, bovine GFAP (SEQ ID NO: 1), human GFAP (SEQ ID NO: 2), mouse GFAP (SEQ ID NO: 3), rat GFAP (SEQ ID NO: 4), sheep GFAP (SEQ ID NO: 5; Partial sequence), zebrafish GFAP (SEQ ID NO: 6), and the like can be used. When diagnosing mammals other than these animals, various GFAPs prepared by cloning by the method described later can be used as antigens for enzyme immunoassay.

Examples of antibodies that can be used for the enzyme immunoassay include polyclonal and monoclonal antibodies prepared by a method that has already been widely used. These antibodies can be obtained, for example, by using GFAP purified from brain homogenate as an antigen. These proteins may be obtained by purification from blood, but may also be obtained by chemical synthesis using a known peptide synthesis technique. A protein produced from cultured cells or the like can also be used as an antigen. Furthermore, it is also possible to use a full-length recombinant protein produced by genetic engineering, a variant thereof, or a part thereof.
The monoclonal antibody is a hybridoma obtained by immunizing an animal using the above-described various antigens, that is, a protein serving as a marker as an immunogen, and fusing an antibody-producing cell derived from the spleen and the like with a bone marrow tumor cell. Can also be produced.

  The hybridoma can be obtained by the following method. That is, the antigen obtained as described above, that is, the marker protein, is mixed together using known Freund's complete, incomplete adjuvant, aluminum hydroxide adjuvant, pertussis adjuvant, etc. The mouse is immunized by dividing it into several doses by intraperitoneal subcutaneous or tail vein administration every 1 to 3 weeks. The amount of sensitizing antigen is usually between 1 μg and 100 mg, but generally about 50 μg is preferred. The number of immunizations is generally 2 to 7, but various methods are known. Next, antibody-producing cells derived from the spleen and the like, bone marrow tumor cells (myeloma cells) and the like are fused in vitro. As a fusion method, for example, fusion can be performed by using polyethylene glycol (PEG) according to a known method of Kohler and Milstein (Nature. 256, 495.1975). Fusion can also be performed by Sendai virus or electrofusion.

  A method for selecting a hybridoma that produces an antibody that recognizes GFAP as a marker from fused cells can be performed as follows. That is, hybridomas are selected from the colonies produced by the cells surviving in the HAT medium and HT medium from the fused cells by the limiting dilution method. If the colony culture supernatant made of fused cells in 96-wells contains antibodies to the marker protein, the antibody is immobilized on the assay plate with the marker protein immobilized on the plate. A monoclonal antibody-producing clone against a protein serving as a marker can be selected by ELISA method in which a secondary labeled antibody such as an anti-mouse immunoglobulin-horseradish peroxidase (HRP) labeled antibody is reacted after the reaction. In addition to HRP, enzymes such as alkaline phosphatase, fluorescent substances, radioactive substances and the like can be used as the labeling substance of the labeled antibody. Further, as a control, an ELISA using an assay plate bound only with bovine serum albumin (BSA), which is a blocking agent, can be performed simultaneously to screen for specific antibodies against each protein serving as a marker. In other words, clones that are positive in any of the protein plates that serve as markers and that are negative by BSA ELISA can be selected.

  The hybridoma can be cultured in a medium usually used for cell culture, such as α-MEM, RPMI1640, ASF, S-clone, and the monoclonal antibody can be recovered from the culture supernatant. An animal from which a hybridoma is derived and a nude mouse are treated in advance with pristane, and ascites can be retained by intraperitoneal injection of cells into the animal, and a monoclonal antibody can be recovered from the ascites. As a method for recovering the monoclonal antibody from the supernatant or ascites, a usual method can be used. For example, salting-out method using ammonium sulfate, sodium sulfate or the like, chromatography, ion exchange chromatography, affinity chromatography using protein A, etc. may be mentioned.

Proteins in serum can be accurately measured by the monoclonal antibody purified by the above method. As a method for measuring proteins in serum by the EIA method, known methods can be used. As the antibody, one or more monoclonal antibodies against a marker protein can be used. More specifically, monoclonals for proteins that serve as markers by using physical bonds, chemical bonds, or affinity directly or indirectly to a solid phase, which is a known method such as polystyrene, polypropylene, polycarbonate, polyethylene, nylon, and polymethacrylate. Antibodies can be bound. The amount of the sensitizing antibody can be generally used in the range of 1 ng to 100 mg / mL. A sample is added to a monoclonal antibody bound to a solid phase by physical bonding, chemical bonding, affinity, or the like, and allowed to react. After reacting for a certain period of time, the solid phase is washed, and the corresponding secondary labeled antibody is added, followed by further secondary reaction. The solid phase is washed again and reacted with a DAB chromogenic substrate. When HRP is used as the labeling substance, known DAB, TMB, etc. can be used as the substrate, and the labeling substance is not limited to this. For example, not only enzymes but also labeled metals such as colloidal gold, europium, various chemical and biological fluorescence such as FITC (fluorescein isothiocyanate), rhodamine, Texas Red, Alexa, GFP (green fluorescent protein) Substances that can be identified include radioactive substances such as substances and ³² P and ⁵¹ Cr.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated in more detail, this invention is not limited by this.

Example 1
(1) Sample preparation Phosphate buffered saline (PBS; pH 7.0) was added to mouse cerebellum (other than cerebrum), and a homogenate was prepared with a microtube homogenizer (manufactured by Kenis). This homogenate was subjected to cell fractionation using a Proteo Extract ™ cell fractionation kit (Subcellular Proteome Extraction Kit (S-PEK; manufactured by MERCK)) to obtain each fraction.

(2) SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
The protein solution of each fraction was applied to a polyacrylamide gel at 23 μg / lane. SDS-PAGE was performed by the Laemmli method using the following reagents. As a result, proteins in the cytoplasm fraction (lane 1), cell membrane / organelle fraction (lane 2), nuclear fraction (lane 3), and cytoskeleton fraction (lane 4) were electrophoresed.

[reagent]
1. Buffer for SDS-PAGE: 250 mmol / L Trishydroxymethylaminomethane-hydrochloric acid (Tris-HCl), 25% glycerin, 0.01% bromophenol blue (BPB)
2. 2. Swelling solution: 1.5 mol / L Tris-HCl (pH 8.8), 8 mol / L urea, 4% CHAPS, 18 mmol / L DTT, 0.5% IPG buffer SDS equilibration buffer 1: final concentration 50 mmol / L 1.5 mol / L Tris-HCl (pH 8.8), 30% glycerol, 2% sodium dodecyl sulfate (SDS), 6 mol / L urea, BPB (Trace), 64.8mmol / L DTT
4). SDS equilibration buffer 2: final concentration 50 mmol / L 1.5 mol / L Tris-HCl (pH 8.8), 30% glycerol, 2% SDS, 6 mol / L urea, BPB (Trace), 135 mmol / L iodoacetamide

(3) Western blotting (WB)
The protein of each fraction separated by SDS-PAGE was blotted onto a nitrocellulose membrane using a semi-dry blotting system (manufactured by Scientific). The blotted membrane was blocked with PBS containing 4% skim milk [Morinaga Milk Industry Co., Ltd.] for 30 minutes, and then washed with PBS. Next, BSE-affected bovine serum (1000 times) diluted with PBS as a primary antibody or BSE-uninfected bovine serum (1000 times) was reacted at room temperature for 1 hour, and then HRP-labeled anti-antibody diluted with PBS as a secondary antibody. Bovine IgG antibody (2000 times) was reacted for 1 hour. Both used serums from the Institute of Veterinary Medicine, UK.
As a molecular weight marker, a commercially available marker (Presteined Protein Marker; manufactured by New England BioLabs) was used. Further, incubation was performed for 5 minutes with ECL (trade name) Western Blotting Detection Reagents [manufactured by GE Healthcare Biosciences] as a detection reagent. Detection was performed by exposing an X-ray film (manufactured by Fuji Film Co., Ltd.) with ECL (Enhanced chemiluminscence).

The results are shown in FIG. 1 (M represents a molecular weight marker). A strong signal was observed in the cytoskeletal fraction (lane 4) of BSE-affected bovine serum (BSE +).
That is, as indicated by the arrow in FIG. 1, a positive band (50 to 52 kDa) was observed. It was shown that antibodies against mouse cerebellar cytoskeleton protein (near 50 kDa) exist in BSE-affected bovine serum (BSE +). On the other hand, no positive band was observed in BSE-uninfected bovine serum (BSE-).
From this, it was confirmed that antibodies against proteins in the cytoskeleton of neural tissue cells exist in the serum.

Example 2
(1) Sample preparation Mouse cerebellum homogenate obtained in the same manner as in Example 1 (1) was subjected to cell fractionation with S-PEK.

(2) Two-dimensional electrophoresis 50 μg of cytoskeletal protein solution obtained in (1) above was used as a sample. This sample was swollen with IPG buffer [pH 3-10, 11 cm; manufactured by GE Healthcare Biosciences] IPG gel [Immobiline ™ Dry Strips pH 3-10, 11 cm (IPG strip); GE Healthcare The first-dimensional isoelectric focusing (IEF) was performed using Bioscience Co., Ltd.]. As the electrophoresis apparatus, Multiphor II [manufactured by GE Healthcare Biosciences] was used. Other devices manufactured by GE Healthcare Biosciences were used.
Subsequently, the IPG strip was equilibrated and then subjected to second-dimensional electrophoresis (SDS-PAGE). Thereafter, the gel was subjected to CBB staining to visualize the protein in the gel.

(3) Western blotting (WB)
As in (3) of Example 1, WB was blotted on a PVDF (polyvinylidene difluoride) membrane after separation by SDS-PAGE. The blotted membrane was blocked with PBS containing 4% skim milk [Morinaga Milk Industry Co., Ltd.] for 30 minutes, and then washed with PBS. Next, BSE-affected bovine serum (1000 times) diluted with PBS as a primary antibody or BSE-uninfected bovine serum (1000 times) was reacted at room temperature for 1 hour, and then HRP-labeled anti-antibody diluted with PBS as a secondary antibody. Bovine IgG antibody (2000 times) was reacted for 1 hour. Both used serums from the Institute of Veterinary Medicine, UK. As a molecular weight marker, a commercially available marker (Presteined Protein Marker; manufactured by New England BioLabs) was used. Moreover, incubation was performed for 5 minutes with ECL Western Blotting Detection Reagents [GE Healthcare Bioscience Co., Ltd.] as a detection reagent. Detection was performed by exposing the X-ray film to light by ECL (Enhanced chemiluminscence).

  The results using BSE-affected bovine serum are shown in FIG. 2, and the results using BSE-uninfected bovine serum are shown in FIG. As a result, spots with a strong signal were observed in BSE-affected bovine serum (arrows in FIG. 2). However, no clear positive spots were detected in BSE-uninfected bovine serum (FIG. 3).

(4) In-gel digestion with trypsin In order to identify a protein whose antigen is an antibody appearing in serum by two-dimensional electrophoresis with WB, a target spot to be confirmed was cut out from the gel and made fine with a scalpel.
It was decolored by immersing it in a CBB decolorization solution (50% methanol, 50 mmol / L NH ₄ HCO ₃ ). Thereafter, a reducing solution [10 mmol / L dithiothreitol (DTT), 25 mmol / L NH ₄ HCO ₃ ] was added, reacted at 56 ° C. for 1 hour, and washed with pure water. Next, an alkylating solution (55 mmol / L iodoacetamide, 25 mmol / L NH ₄ HCO ₃ ) was added, shaken for 45 minutes, and washed with pure water. Then, 100% acetonitrile was added to the gel to dry it.

Next, trypsin solution (10 ng / mL, 25 mmol / L NH ₄ HCO ₃ ) was added and stored on ice. Thereto, 20 μL of 100 mmol / L Tris buffer was added, and the enzyme was digested overnight at 37 ° C.
Next, in order to extract the peptide from the solution after enzymatic digestion, add 50 μL of the extract (50% acetonitrile / 5% trifluoroacetic acid mixture (TFA)), and after centrifugation, take care not to suck the gel. The supernatant was collected with a pipette. This step was performed twice, and 100% acetonitrile was added to recover all the eluted supernatant. The obtained supernatant was extracted with a peptide solution concentrated by a centrifugal evaporator.

(5) Identification of protein by MALDI-TOF MS The concentrated peptide solution was desalted and concentrated with a pipette tip for concentration (ZipTip C ₁₈ ; manufactured by Millipore). This solution was dropped onto a metal plate together with a Matrix solution [a saturated solution in which about 1 mg of α-CHCA (α-Cyano-4-Hydoroxycinnamic Acid) was dissolved in 100 μL of 50% acetonitrile and 0.1% TFA] and dried.
As a result of analyzing this by MALDI-TOF MS (matrix-assisted laser desorption ionization time-of-flight mass spectrometry) by the PMF method (Peptide mass fingerprinting), a peptide map having a plurality of peptide signals was obtained. Each signal peak is a peptide fragment obtained by digesting a target protein spot.
This peptide fragment was subjected to Mascot search (MASCOT search site www.matrixscience.com; MATRIX SCIENCE) to identify the target protein. As a result, the sizes of GFAP and the peptide fragment matched, and it was identified that this protein was GFAP. From this, it was confirmed that the anti-GFAP antibody specifically expressed in the astrocytes of nerve cells was present in the serum of BSE-affected cows.

Example 3
(1) Sample preparation The protein of bovine medulla was fractionated by the same method as in (1) of Example 1, and each fraction was obtained.

(2) SDS-PAGE
The protein solution of each of the above fractions was applied to a polyacrylamide gel at 15 μg / lane. SDS-PAGE was performed by the Laemmli method.

(3) Western blotting (WB)
Proteins of each fraction separated by SDS-PAGE were blotted on a nitrocellulose membrane. In all other cases, proteins were detected by ECL in the same manner as described in Example 1, (3).
The results are shown in FIG. 4 (M represents a molecular weight marker). A band was shown around 50 kDa, and it was confirmed to be positive. That is, it was shown that an antibody against a protein of the bovine medullary cytoskeleton is present in the serum of BSE-affected bovine serum.

Example 4
(1) Sample preparation A sample prepared by diluting 4 samples each of BSE-affected bovine serum and BSE-uninfected bovine serum 5 times with PBS, which was distributed from the Institute of Veterinary Medicine, Webridge, England.

(2) SDS-PAGE
Each of the above samples was applied to a polyacrylamide gel at 10 μg / lane. SDS-PAGE was performed by the Laemmli method using the reagents used in Example 1.

(3) Western blotting (WB)
Proteins separated by SDS-PAGE were blotted onto a PVDF membrane using a semi-drive blotting system (Scientific). The blotted membrane was blocked with PBS containing 4% skim milk [Morinaga Milk Industry Co., Ltd.] and washed with PBS. Thereafter, an anti-human medullary GFAP polyclonal antibody (MP Biomedical; 1000 times) diluted with PBS as a primary antibody was reacted at room temperature for 30 minutes, and then an HRP-labeled anti-rabbit IgG antibody (1000) diluted with PBS as a secondary antibody. Times) for 30 minutes. As a molecular weight marker, a commercially available marker (Presteined Protein Marker; manufactured by New England BioLabs) was used. Moreover, ECL Western Blotting Detection Reagents [manufactured by GE Healthcare Biosciences] was used as a detection reagent. Detection was performed by exposing the X-ray film to light by ECL (Enhanced chemiluminscence).

The results are shown in FIG. 5 (M represents a molecular weight marker).
In lanes 10, 11 and 12 of BSE-affected bovine serum (BSE +), a clear band was observed around 50 kDa.
From this, it was confirmed that GFAP specifically expressed in neuronal astrocytes was present in the serum of BSE-affected cows. That is, it was recognized that GFAP, an intermediate filament that forms the cytoskeleton in astrocyte cells, leaked from the cells and deviated into the peripheral blood.

Example 5
As an antigen for enzyme immunoassay, bovine GFAP antigen was diluted to 1.0 mg / mL with PBS, dispensed 100 μL into each well of a microplate, and incubated at 37 ° C. for 1 hour. The plate was blocked with a blocking solution (trade name: Block Ace; manufactured by Dainippon Sumitomo) and incubated at 37 ° C. for 1.5 hours. Anti-GFAP antibody standard (trade name: Polyclonal Anti-GFAP; manufactured by MP BIOCHEMICALS) is diluted 10,000 times with a diluted solution of fetal bovine serum (FBS) 400 times with PBS (hereinafter referred to as FBS diluted solution). The solutions were diluted 30,000 times, 50,000 times, 100,000 times, 300,000 times, 500,000 times, and 1 million times, respectively, and used as standard solutions of anti-GFAP antibodies for preparing a calibration curve. Samples obtained by diluting the BSE-affected bovine serum and BSE-uninfected bovine serum used in Example 4 400 times with an FBS diluent were used. The microplate was dispensed at 100 μL / well, incubated at 37 ° C. for 1 hour, and washed with a washing solution (PBS supplemented with 0.5% Tween 20). Next, an HRP-labeled anti-bovine IgG antibody diluted 10,000 times with a blocking solution (trade name: Block Ace; manufactured by Dainippon Sumitomo) was dispensed at 100 μL / well into each well, and then at room temperature for 1 hour. After incubation, the plate was washed with a washing solution. Next, tetramethylbenzidine (TMB; manufactured by Sigma) was dispensed at 100 μL / well in each well, incubated for 30 minutes at room temperature in the dark, and then 0.5 mol / L sulfuric acid was added at 100 μL / well in each well. After dispensing and stirring, the absorbance at 450 nm with respect to the absorbance at 620 nm was measured with a microplate reader.

The results are shown in FIG. 6 together with the average value ± average error (mean ± SE).
BSE-uninfected cattle (BSE-) showed low values, and BSE-affected cattle (BSE +) exceeded the 0.02 value in all cattle. Therefore, it was found that the measurement conditions of this example can diagnose that the anti-GFAP antibody is suffering from BSE if the anti-GFAP antibody has a value of 0.02 or more in terms of absorbance.

  The diagnostic kit and diagnostic method of the present invention can be used for prenatal diagnosis of prion diseases.

It is the photograph which replaces drawing which shows the analysis result of the electrophoresis of a mouse | mouth cerebellum protein using BSE serum [BSE diseased bovine serum (BSE +) or BSE non-infected bovine serum (BSE-)) as a primary antibody. It is the photograph which replaces drawing which shows the analysis result of the electrophoresis of a mouse | mouth cerebellar cytoskeletal protein using BSE disease bovine serum as a primary antibody, and a Western blotting. It is the photograph which replaces drawing which shows the analysis result of the electrophoresis of a mouse | mouth cerebellar cytoskeletal protein which used BSE non-infected bovine serum as a primary antibody, and Western blotting. It is the photograph which replaces drawing which shows the analysis result of the electrophoresis of the bovine medulla protein which used BSE disease bovine serum as a primary antibody, and a Western blotting. It is the photograph which replaces drawing which shows the analysis result of the electrophoresis of a bovine serum protein by an anti- GFAP antibody, and a Western blotting. It is a graph which shows the result of having measured the anti- GFAP antibody in bovine serum [BSE diseased bovine serum (BSE +) or BSE non-infected bovine serum (BSE-)] by ELISA.

Claims (3)

  A method for detecting prion disease, comprising analyzing an anti-glial fiber acidic protein antibody or glial fiber acidic protein in a test sample derived from a living body of a mammal.   The detection method according to claim 1, wherein the test sample is a blood-derived sample.   A diagnostic kit for prion diseases, comprising glial fibrillary acidic protein or an antigenic fragment thereof, or anti-glia fibrillary acidic protein antibody.