JP5653725B2 - Amyotrophic lateral sclerosis marker and use thereof - Google Patents

Description

  The present invention relates to an amyotrophic lateral sclerosis marker useful for testing amyotrophic lateral sclerosis. Specifically, the present invention relates to a biomarker that can be used to determine the possibility of developing amyotrophic lateral sclerosis, a method for testing amyotrophic lateral sclerosis, and the like.

  Amyotrophic lateral sclerosis (ALS) is a chronic progressive degenerative disease that almost selectively damages upper and lower motor neurons. Muscle weakness due to motor neuron departure is the main symptom of ALS, but sensory neurons are normal and the body is not free. The glutamate antagonist riluzole is the only therapeutic agent used, but no satisfactory effect has been obtained. ALS is a neurological intractable disease for which no effective treatment can be found even though many years of intensive research activities, and it is necessary to elucidate the onset mechanism and develop a therapeutic drug as soon as possible.

JP-T-2006-514620 Special table 2009-528059 gazette Special table 2007-528487 gazette

Serum proteome analysis for profiling protein markers associated with carcinogenesis and lymph node metastasis in nasopharyngeal carcinoma., Clin Exp Metastasis., 2008. APPLYING PROTEOMICS TO THE DIAGNOSIS AND TREATMENT OF ALS AND RELATED DISEASES., Muscle Nerve., 2009. Plasma and Cerebrospinal Fluid-Based Protein Biomarkers for Motor Neuron Disease., Mol Diag Ther., 2006.

Although many ALS disease state-related molecules have been identified in many years of research (see, for example, Patent Documents 1 to 3 and Non-Patent Documents 1 to 3), the cause of ALS remains unclear. With current technology, it is not easy to diagnose ALS as well as to detect ALS onset (or possibility of onset) at an early stage. In many cases, there is no objective index useful for diagnosing ALS, and therapeutic intervention is delayed, resulting in intractable cases.
Accordingly, an object of the present invention is to provide an ALS-specific biomarker and its use, that is, a technique useful for testing ALS, in order to enable early diagnosis of ALS, highly reliable diagnosis, and the like. Specifically, an object of the present invention is to provide a biomarker useful for testing ALS, a test method using the biomarker, a test reagent used for the test method, and the like.

In order to find an ALS-specific biomarker, the present inventors conducted research using ALS model rats (SOD1 ^H46R rats). As a result of proteomic analysis using SOD1 ^H46R rat serum as a sample, inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) and glutathione peroxidase 3 (Gpx3) are biomolecule candidate molecules. Was identified as As a result of further investigation, it was ^found that in the SOD1 ^H46R rat serum, the amount of ITIH4 increased and Gpx3 decreased as the disease progressed. In addition, an increased amount of truncated ITIH4 was observed at the end of the disease state. On the other hand, as a result of analyzing ITIH4 expression level and GPX3 expression level in serum by ALS patients, Alzheimer's disease (AD) patients, Parkinson's disease (PD) patients, and disease patients without neurodegeneration, ALS patients It was found that the amount of cleaved ITIH4 increased in a serum-specific manner and the amount of GPX3 decreased. That is, ITIH4 and GPX3 were shown to be extremely useful clinically as ALS biomarkers. The present invention described below is mainly based on the above results.
[1] An amyotrophic lateral sclerosis marker comprising a protein molecule selected from the group consisting of inter-α trypsin inhibitor heavy chain 4, truncated inter-α trypsin inhibitor heavy chain 4 and glutathione peroxidase 3.
[2] Using in-sample levels of one or more protein molecules selected from the group consisting of inter-α trypsin inhibitor heavy chain 4, cleaved inter-α trypsin inhibitor heavy chain 4 and glutathione peroxidase 3 as an index And amyotrophic lateral sclerosis testing method.
[3] The amyotrophic lateral sclerosis test method according to [2], comprising the following steps (1) to (3):
(1) a step of preparing a subject-derived specimen;
(2) detecting one or more of the protein molecules in the specimen; and (3) determining a current or future possibility of developing amyotrophic lateral sclerosis based on the detection result.
[4] For inter-α trypsin inhibitor heavy chain 4 and cleaved inter-α trypsin inhibitor heavy chain 4, in accordance with the criterion that the detection probability is high if the detection value is high, or the criterion that the detection probability is high if detection is possible ,
For glutathione peroxidase 3, the determination according to step (3) is performed according to the criterion that the probability of onset is high when the detection value is low, or the criterion that the probability of onset is high if the detection value is not detected. Amyotrophic lateral sclerosis test.
[5] Amyotrophic lateral sclerosis according to [3] or [4], wherein the determination in step (3) is performed based on a comparison between the detection value obtained in step (2) and the detection value of the control sample. Inspection method.
[6] The determination in step (3) is performed based on a comparison between the detection value obtained in step (2) and the detection value in a sample collected in the past from the same subject, [3] or [ 4] The method for testing amyotrophic lateral sclerosis.
[7] The method for testing amyotrophic lateral sclerosis according to any one of [2] to [6], wherein the specimen is blood, plasma, serum, bone marrow, or cerebrospinal fluid.
[8] A test reagent for amyotrophic lateral sclerosis comprising a substance exhibiting specific binding to the amyotrophic lateral sclerosis marker according to [1].
[9] The amyotrophic lateral sclerosis test reagent according to [8], wherein the substance is an antibody.
[10] A kit for testing amyotrophic lateral sclerosis comprising the reagent for testing amyotrophic lateral sclerosis according to [8] or [9].

It is a two-dimensional electrophoresis image of SOD1 ^H46R before onset and wild type (WT) rat serum protein. 1 indicates Gpx3 and 2 indicates ITIH4 spots. It is a two-dimensional electrophoresis image of SOD1 ^H46R and wild type (WT) rat serum protein after onset. 1 indicates Gpx3 and 2 indicates ITIH4 spots. It is a table | surface which shows the protein identified by MALDI-TOF MS or LC-MS / MS analysis of the 2 types of focused spots. The NCBInr database (http://www.ncbi.nlm.nih.gov/) was used for the search. Gi number, molecular weight (MW), percentage of matching peptides, number of matching peptides and Mascot score are shown. Molecular weight and pI (isoelectric point) are theoretical values registered in the database. It is a graph which shows transition of the Gpx3 spot volume of the two-dimensional electrophoresis image applied before and after the onset of SOD1 ^H46R and wild type (WT) rats. Data are expressed as mean ± standard error. ** P <0.01, † P <0.05 vs. WT of the same week or SOD1 ^H46R after onset (Student's t test), ## P <0.01 vs. WT (two-way repeated measure ANOVA )). It is a graph which shows transition of the ITIH4 spot volume of the two-dimensional electrophoresis image applied before and after onset of SOD1 ^H46R and wild type (WT) rats. Data are expressed as mean ± standard error. * P <0.05, † P <0.05 vs. WT of the same week or SOD1 ^H46R after onset (Student's t test); #P <0.05 vs. WT (two-way repeated measure ANOVA) . It is the figure which showed ^GOD3 in SOD1 ^H46R and wild type (WT) rat serum before onset, after onset, and the end of a disease state by the western blotting method. It is a graph which shows the change of the amount of Gpx3 in a rat serum accompanying ALS disease state progression. Data are expressed as mean ± standard error. ** P <0.01 vs. WT (Student t test). It is the table | surface which put together the background of 50 subjects who collected the serum sample. Data for age and period (months) from decision to vote are expressed as mean ± standard deviation. It is a figure which shows the amount of GPX3 in serum of an arcuate ALS patient, an Alzheimer's disease patient, a Parkinson's disease patient, and a macular hole patient (non-neurodegenerative disease control). Data are expressed as mean ± standard error. * P <0.05, #P <0.05, † P <0.05 vs. Control, AD patient, or PD patient (Student's t test). It is the figure which showed ^ITIH4 in the SOD1 ^H46R and WT rat serum before onset, after onset, and the end stage of a disease state by the Western blotting method. A signal specific to SOD1 ^H46R rat was observed around 85 kDa. It is a graph which shows the change of the amount of 120 kDa ITIH4 in serum accompanying ALS disease state progression. Data are expressed as mean ± standard error. * P <0.05, ** P <0.01 vs. WT (Student t test). It is a graph which shows the change of the 85 kDa ITIH4 amount in serum accompanying ALS disease state progression. Data are expressed as mean ± standard error. * P <0.05 vs. WT (Student t test). It is the figure which showed ITIH4 in the serum of an arcuate ALS patient, an Alzheimer's disease patient, a Parkinson's disease patient, and a macular hole patient (non-neurodegenerative disease control) by the Western blotting method. An arcuate ALS patient-specific signal was observed around 85 kDa. It is a graph which shows the change of the amount of 120 kDa ITIH4 in serum of an arcuate ALS patient, an Alzheimer's disease patient, a Parkinson's disease patient, and a macular hole patient (non-neurodegenerative disease control). Data are expressed as mean ± standard error. It is a graph which shows the change of the 85 kDa ITIH4 amount in serum of a patient with Alzheimer's disease, a patient with Parkinson's disease and a macular hole (non-neurodegenerative disease control). Data are expressed as mean ± standard error. ** P <0.01 vs. control; ## P <0.01 vs. AD; †† P <0.01 vs. PD (Student t test). It is a graph which shows the amount of cleaved plasma kallidinogenases in the sera of arcuate ALS patients and macular hole patients (non-neurodegenerative disease control). Data are expressed as mean ± standard error. ** P <0.01 vs. control (Student t test).

(First aspect of the present invention: Biomarker of amyotrophic lateral sclerosis (ALS))
The first aspect of the present invention relates to an ALS biomarker (hereinafter also referred to as “the biomarker of the present invention”). The biomarker of the present invention is a useful index for evaluating the possibility of current or future onset of ALS. The “current possibility of onset” represents whether or not ALS has developed at the time of examination, or the probability of developing it. On the other hand, “probability of future onset” represents the possibility (risk) of developing ALS in the future.

  The biomarker of the present invention comprises a protein molecule that has been correlated with ALS as a result of the study by the present inventors, that is, inter-α trypsin inhibitor heavy chain 4, cleaved inter-α trypsin inhibitor heavy chain 4 or glutathione peroxidase 3. . In the following description, according to the convention, the inter-α trypsin inhibitor heavy chain 4 and the truncated inter-α trypsin inhibitor heavy chain 4 are represented by the gene symbol “ITIH4”, and the gene for glutathione peroxidase 3 is also represented. The symbol “GPX3” is used to represent the molecule.

  ITIH4 is a kind of serum glycoprotein and exhibits inhibitory activity against serine endopeptidase. It has been reported that the ITIH family is involved in inflammation, wound healing, cancer metastasis, and the like. In addition, ITIH4 is an acute ischemic stroke (Clin Chim Acta. 2009 Apr; 402 (1-2): 160-3.), Breast cancer (Oncol Rep. 2009 Jul; 22 (1): 205-13.) Or It is expected as a marker for lung cancer (Proteomics. 2007 Dec; 7 (23): 4292-302.). However, there is no previous report on the relationship between ITIH4 and ALS. There are two transcription variants of ITIH4: isoform 1 consisting of 930 amino acids and isoform 2 consisting of 900 amino acids. The amino acid sequence of isoform 1 of ITIH4 and the nucleotide sequence encoding it are shown in SEQ ID NO: 1 in the sequence listing (NCBI ACCESSION: NP_002209, DEFINITION: inter-alpha-trypsin inhibitor heavy chain H4 isoform 1 precursor [Homo sapiens].) And SEQ ID NO: 2 (NCBI ACCESSION: NM_002218, DEFINITION: Homo sapiens inter-alpha (globulin) inhibitor H4 (plasma Kallikrein-sensitive glycoprotein) (ITIH4), transcript variant 1, mRNA.). In the amino acid sequence of SEQ ID NO: 1, amino acids 1 to 28 are signal peptides, and the mature protein is composed of amino acids 29 to 930.

  Similarly, the amino acid sequence of isoform 2 of ITIH4 and the nucleotide sequence encoding it are shown in SEQ ID NO: 3 in the sequence listing (NCBI ACCESSION: NP_001159921, DEFINITION: inter-alpha-trypsin inhibitor heavy chain H4 isoform 2 precursor [Homo sapiens] .) And SEQ ID NO: 4 (NCBI ACCESSION NM_001166449, DEFINITION: Homo sapiens inter-alpha (globulin) inhibitor H4 (plasma Kallikrein-sensitive glycoprotein) (ITIH4), transcript variant 2, mRNA.). In the amino acid sequence of SEQ ID NO: 3, amino acids 1 to 28 are signal peptides, and the mature protein is composed of amino acids 29 to 900.

  Cleaved ITIH4 is an N-terminal fragment with a molecular weight of 85 kDa, and is produced by cleavage of 120 kDa ITIH4 by plasma kallidinogenase. Cleaved ITIH4 is further cleaved into an N-terminal fragment with a molecular weight of 57 kDa and a C-terminal fragment with a molecular weight of 28 kDa. (Song J Fau-Patel, M., et al. Quantification of fragments of human serum inter-alpha-trypsin inhibitor heavy chain 4 by a surface-enhanced laser desorption / ionization-based immunoassay. Clin Chem. 2006 Jun; 52 (6 ): 1045-53.Epub 2006 Mar 30 .; Pu XP, Iwamoto, A., Nishimura, H., Nagasawa, S. Purification and characterization of a novel substrate for plasma kallikrein (PK-120) in human plasma.Biochim Biophys Acta. 1994 Oct 19; 1208 (2): 338-43 .; Nishimura H. Kakizaki, I., et al. CDNA and deduced amino acid sequence of human PK-120, a plasma kallikrein-sensitive glycoprotein. FEBS Lett. 1995 Jan 3; 357 (2): 207-11.).

  GPX3 belongs to the glutathione peroxidase family and is an enzyme having an antioxidant action. There is selenium / cysteine in the active center. GPX3 is known as an antioxidant marker, and its expression level has been reported to increase in various diseases such as asthma, leukemia and HIV. The amount of mRNA is large in the liver, kidney, heart and lung and is secreted into serum. Although there are various reports, there is no unified view on the relationship between glutathione peroxidase and ALS. In addition, there is no report that mentions GPX3, which is one of glutathione peroxidases, and mentions the relationship with ALS. The amino acid sequence of GPX3 and the nucleotide sequence encoding it are shown in SEQ ID NO: 5 (NCBI ACCESSION: NP_002075, DEFINITION: glutathione peroxidase 3 precursor [Homo sapiens].) And SEQ ID NO: 6 (NCBI ACCESSION: NM_002084, DEFINITION : Homo sapiens glutathione peroxidase 3 (plasma) (GPX3), mRNA. In the amino acid sequence of SEQ ID NO: 5, amino acids 1 to 20 are signal peptides, and the mature protein is composed of amino acids 21 to 211.

(Second aspect of the present invention: ALS inspection method)
The second aspect of the present invention relates to the use of the above-described biomarker of the present invention, and provides a method for examining the current or future onset possibility of ALS (hereinafter also referred to as “test method of the present invention”). The test method of the present invention is useful as a means for determining whether ALS is presently occurring or as a means for determining the possibility of developing ALS in the future. That is, the test method of the present invention provides useful information for diagnosing ALS. According to the test method of the present invention, it is possible to easily and objectively determine the possibility of developing ALS.

  In the test method of the present invention, the level of the biomarker of the present invention in a specimen derived from a subject is used as an index. Here, “level” typically means “amount” or “concentration”. However, the term “level” is also used to indicate whether or not a molecule to be detected can be detected (ie, the presence or absence of an apparent presence) in accordance with common practice and common technical knowledge. Preferably, two or more types of biomarkers selected from the above-mentioned three types (two types of genes) are used in combination as an index to obtain a test result. Basically, the more types of biomarkers used in combination, the better the inspection accuracy and reliability. Therefore, preferably two or more, more preferably all three are used in combination. Note that the number and type of biomarkers to be employed may be determined in consideration of the required accuracy and the ease of inspection.

In the inspection method of the present invention, the following steps are performed.
(1) A step of preparing a specimen derived from a subject (2) A step of detecting one or more of the protein molecules (that is, one or more biomarkers employed as an index) in the specimen (3) Determining the current or future likelihood of developing amyotrophic lateral sclerosis (ALS) based on the detection results

  In step (1), a specimen derived from the subject is prepared. As the specimen, blood, plasma, serum, bone marrow, cerebrospinal fluid, etc. of the subject can be used. The subject is not particularly limited. In other words, it is necessary to determine the current or future possibility of developing ALS (that is, whether there is a possibility of developing ALS, the degree of possibility of developing ALS, the degree of possibility of developing ALS in the future) The present invention can be widely applied to such a person. For example, when the present invention is applied to a patient diagnosed as having ALS by a doctor's inquiry or the like, whether or not the diagnosis is appropriate can be determined based on an objective index called an expression level. That is, according to the inspection method of the present invention, information that assists or supports the conventional diagnosis can be obtained. The information is useful for determining a more appropriate treatment policy, and promotes improvement of the treatment effect and improvement of the patient's QOL (Quality of Life). On the other hand, the present invention can be used for monitoring the diseased state to prevent intractable disease, seriousness, recurrence and the like.

  Those who are presumed to have a high risk of suffering from ALS based on their family background (high-risk subjects) are also suitable subjects. Applying the present invention before the symptoms of ALS appear in such subjects enables prevention or delay of onset or early treatment intervention. The present invention is also useful for identifying those who are at high risk of suffering from ALS. Such identification enables a reduction in the possibility of onset (possibility of morbidity) due to, for example, preventive measures or improvement of lifestyle habits. A person who cannot determine whether or not it is ALS by conventional diagnosis, such as a person who has no subjective symptoms, is also a suitable subject of the present invention. In addition, you may decide to implement this invention as one item of a health check.

  In step (2), a biomarker in the sample is detected. It is not essential to strictly quantify the level of the biomarker. That is, the level of the biomarker may be detected to the extent that the possibility of developing ALS can be determined in the subsequent step (3). For example, detection can be performed so that it can be determined whether or not the level of the biomarker in the sample exceeds a predetermined reference value.

  The method for detecting the biomarker is not particularly limited, but preferably an immunological technique is used. According to immunological techniques, rapid and sensitive detection is possible. Also, the operation is simple. In measurement by an immunological technique, a substance having specific binding property to the biomarker to be used is used. As the substance, an antibody is usually used. However, any substance can be used as long as it has a specific binding property to the biomarker and can measure the binding amount. In addition, not only a commercially available antibody but the antibody newly prepared using the immunological method, the phage display method, the ribosome display method etc. may be used.

  Examples of the measurement method include latex agglutination method, fluorescence immunoassay method (FIA method), enzyme immunoassay method (EIA method), radioimmunoassay method (RIA method), and Western blot method. Preferable measurement methods include FIA method and EIA method (including ELISA method). According to these methods, it is possible to detect with high sensitivity, quick and simple. In the FIA method, a fluorescently labeled antibody is used, and an antigen-antibody complex (immune complex) is detected using fluorescence as a signal. On the other hand, in the EIA method, an enzyme-labeled antibody is used, and an immune complex is detected using color development or luminescence based on an enzyme reaction as a signal.

  The ELISA method has many advantages such as high detection sensitivity, high specificity, excellent quantitativeness, simple operation, and suitability for simultaneous processing of multiple samples. An example of a specific operation method when using the ELISA method is shown below. First, an anti-biomarker antibody is immobilized on an insoluble support. Specifically, for example, the surface of the microplate is sensitized (coated) with an anti-biomarker monoclonal antibody. The specimen is brought into contact with the antibody thus immobilized. As a result of this operation, an immune complex is formed if an antigen to the immobilized anti-biomarker antibody (protein molecule that is a biomarker) is present in the specimen. After removing non-specific binding components by washing operation, an immune complex is labeled by adding an antibody conjugated with an enzyme, and then a color is developed by reacting the substrate of the enzyme. Then, immune complexes are detected using the color development amount as an index. The details of the ELISA method are described in many books and papers, and can be referred to when setting the experimental procedure and experimental conditions of each method. In addition, you may decide to use not only a non-competitive method but the competing method (The method of making it compete with an antigen added with a test substance). Further, a method of directly detecting a biomarker in a specimen with a labeled antibody may be employed, or a sandwich method may be employed. In the sandwich method, two types of antibodies having different epitopes (capture antibody and detection antibody) are used.

  Means capable of simultaneously detecting a large number of specimens such as a protein array and a protein chip may be used. For example, a target biomarker-specific antibody is used as the probe.

  In step (3), the current or future onset possibility of ALS is determined based on the detection result. In order to enable accurate determination, it is preferable to perform the determination after comparing the detection value obtained in step (2) with the detection value of the control sample (control). Determination of the possibility of onset may be qualitative or quantitative. It should be noted that the determination here can be automatically / mechanically performed without depending on the determination of a person having specialized knowledge such as a doctor or a laboratory technician, as is apparent from the determination criteria.

  In the present invention, the following criteria are typically adopted for each biomarker. Needless to say, the criterion “the detection probability is high when the detection value is low” is synonymous with the criterion “the detection probability is low when the detection value is high” (the same applies to other criteria).

  Biomarkers that adopt the criteria of “High detection value indicates high probability of onset” or “High probability of detection detectable”: ITIH4, truncated ITIH4

  Biomarker that adopts the criterion of “high probability of onset if detection value is low” or “high probability of onset if not detected”: GPX3

  Specific examples of qualitative judgment and quantitative judgment are shown below. For convenience of explanation, there are two types of biomarkers: a biomarker (ITIH4 or truncated ITIH4) whose increase in expression level correlates with the onset of ALS, and a biomarker (GPX3) whose decrease in expression level correlates with the onset of ALS. The case of using was taken as an example. In other embodiments in which the number of biomarkers used is different, the determination criteria can be set according to this example.

(Example 1 of qualitative judgment)
When the detection value of biomarker 1 (level in sample) is higher than the reference value and the detection value of biomarker 2 (level in sample) is lower than the reference value, it is determined that “the likelihood of onset is high”, and the reference When the detection value (in-specimen level) of biomarker 1 is lower than the value and the detection value (in-specimen level) of biomarker 2 is higher than the reference value, it is determined that “the possibility of onset is low”.
(Example 2 of qualitative judgment)
When biomarker 1 can be detected (reactivity is recognized) and biomarker 2 cannot be detected (reactivity is not recognized), it is determined that the “probability of onset is high”, biomarker 1 cannot be detected, and biomarker When marker 2 can be detected, it is determined that “the possibility of onset is low”.

(Example of quantitative judgment)
As shown below, the possibility of onset (%) is set in advance for each range of detection values, and the possibility of onset (%) is determined from the detection values.
Detection value of biomarker 1 is a to b, detection value of biomarker 2 is a ′ to b ′: probability of onset is 10% or less detection value b to c of biomarker 1, detection value b ′ to biomarker 2 c ′: the probability of onset is 10% to 30%
Detection values c to d of biomarker 1 and detection values c ′ to d ′ of biomarker 2: the likelihood of onset is 30% to 50%
Detection values de to e of biomarker 1 and detection values d ′ to e ′ of biomarker 2: probability of onset is 50% to 70%
Detection values e to f of biomarker 1 and detection values e ′ to f ′ of biomarker 2: probability of onset is 70% to 90%

When two or more types of biomarkers are used in combination, the following determination method (1) or (2) may be employed.
(1) When all of the biomarkers included in the combination are positive (cutoff value or higher), it is determined as positive (for example, 50% or more likely to develop), and otherwise the case is negative (for example, 50% likely to develop) Less than).
(2) Even if one of the biomarkers included in the combination is positive (cutoff value or higher), it is determined as positive (for example, 50% or more likelihood of onset), and other cases are negative (for example, 50% of possibility of onset) Less than).

  Usually, diagnosis (detection) sensitivity and diagnosis (detection) specificity vary depending on the type and number of biomarkers to be combined. Therefore, an optimal combination of biomarkers may be selected according to the purpose. For example, combinations with high diagnostic sensitivity are suitable for screening tests. In contrast, a combination with a high diagnostic specificity is suitable for a test that requires a more reliable determination (for example, a secondary test or a tertiary test). By combining determination methods with different balances of diagnostic sensitivity and diagnostic specificity, it is possible to improve efficiency and improve accuracy or reliability. For example, after narrowing down positive subjects using a combination of biomarkers giving high diagnostic sensitivity (primary test, screening test), a final determination is made using a combination of biomarkers giving high diagnostic specificity (secondary Inspection). Not only such a two-step determination but also a three-step or higher determination can be performed.

  The number of determination categories, the level of the biomarker associated with each determination category, and the determination results can be arbitrarily set through preliminary experiments or the like without being limited to the above example. For example, the “threshold” when determining the likelihood of onset using a predetermined threshold as a boundary, and the “biomarker level range” related to the category related to the high or low possibility of onset are statistical values using a large number of specimens. It can be determined by analysis. In the case of analyzing using statistical processing, it is generally effective to set a high risk group and a low risk group. For example, the high risk group corresponds to a group of ALS patients or a family with many ALS patients, and the low risk group corresponds to a group of healthy persons or a family with no ALS patients.

  In determining the likelihood of developing ALS, other indicators useful for ALS testing may also be used. That is, the possibility of developing ALS may be determined using the biomarker of the present invention in combination with another index.

  In one embodiment of the present invention, for the same subject, the level of a biomarker measured at a certain time point is compared with the level of a biomarker measured in the past, Or examine the degree of increase or decrease. The resulting data on the change in the expression level of the biomarker is useful information for monitoring the possibility of ALS onset, grasping the therapeutic effect, or estimating the prognosis. Specifically, for example, based on the fluctuation of the biomarker level, it can be determined that the possibility of onset has increased or decreased or has not changed between the previous test and the current test. If such an evaluation is performed in parallel with the treatment of ALS, the therapeutic effect can be confirmed, and signs of recurrence of ALS can be grasped in advance. This makes it possible to determine a more appropriate treatment policy. Thus, the present invention can make a great contribution to maximizing the therapeutic effect and improving the patient's QOL (quality of life).

(Third Aspect of the Present Invention: Reagent and Kit for Examining ALS Probability)
The present invention further provides reagents and kits for examining the possibility of developing ALS. The reagent of the present invention comprises a substance (hereinafter referred to as “binding molecule”) that exhibits specific binding to the biomarker of the present invention. Examples of binding molecules include antibodies, nucleic acid aptamers and peptide aptamers that specifically recognize biomarkers. The type and origin of the binding molecule are not particularly limited as long as it has specific binding properties for the biomarker employed. In the case of an antibody, any of a polyclonal antibody, an oligoclonal antibody (a mixture of several to several tens of antibodies), and a monoclonal antibody may be used. As a polyclonal antibody or an oligoclonal antibody, an anti-serum-derived IgG fraction obtained by animal immunization, or an affinity-purified antibody using an antigen can be used. Antibody fragments such as Fab, Fab ′, F (ab ′) ₂ , scFv, and dsFv antibodies may also be used.

  The binding molecule may be prepared by a conventional method. If a commercial item is available, the commercial item may be used. For example, antibodies can be prepared using immunological techniques, phage display methods, ribosome display methods, and the like. Preparation of a polyclonal antibody by an immunological technique can be performed by the following procedure. An antigen (biomarker or a part thereof) is prepared, and an animal such as a mouse, rat or rabbit is immunized using the antigen. An antigen can be obtained by purifying a biological sample. A recombinant antigen can also be used. A recombinant antigen can be obtained by, for example, introducing a gene encoding a biomarker (which may be a part of a gene) into a suitable host using a vector and expressing the gene in a recombinant cell obtained. Can be prepared.

  In order to enhance the immunity-inducing action, an antigen bound with a carrier protein may be used. As the carrier protein, KLH (Keyhole Limpet Hemocyanin), BSA (Bovine Serum Albumin), OVA (Ovalbumin) and the like are used. The carbodiimide method, the glutaraldehyde method, the diazo condensation method, the MBS (maleimidobenzoyloxysuccinimide) method, etc. can be used for the coupling | bonding of carrier protein. On the other hand, an antigen in which a biomarker (or part thereof) is expressed as a fusion protein with GST, β-galactosidase, maltose-binding protein, histidine (His) tag or the like can also be used. Such a fusion protein can be easily purified by a general method.

  Immunization is repeated as necessary, and blood is collected when the antibody titer sufficiently increases, and serum is obtained by centrifugation or the like. The obtained antiserum is affinity purified to obtain a polyclonal antibody.

  On the other hand, a monoclonal antibody can be prepared by the following procedure. First, an immunization operation is performed in the same procedure as described above. Immunization is repeated as necessary, and antibody-producing cells are removed from the immunized animal when the antibody titer sufficiently increases. Next, the obtained antibody-producing cells and myeloma cells are fused to obtain a hybridoma. Subsequently, after this hybridoma is monoclonalized, a clone that produces an antibody having high specificity for the target protein is selected. The target antibody can be obtained by purifying the culture medium of the selected clone. On the other hand, the desired antibody can be obtained by growing the hybridoma to a desired number or more, then transplanting it into the abdominal cavity of an animal (for example, a mouse), growing it in ascites, and purifying the ascites. For purification of the culture medium or ascites, affinity chromatography using protein G, protein A or the like is preferably used. Alternatively, affinity chromatography in which an antigen is immobilized may be used. Furthermore, methods such as ion exchange chromatography, gel filtration chromatography, ammonium sulfate fractionation, and centrifugation can also be used. These methods can be used alone or in any combination.

  Various modifications can be made to the obtained antibody on condition that the specific binding property to the biomarker is maintained. Such a modified antibody may be used as the reagent of the present invention.

  If a labeled antibody is used as a specific binding molecule, it is possible to directly detect the amount of bound antibody using the amount of label as an index. Therefore, a simpler inspection method can be constructed. On the other hand, in addition to the necessity of preparing an antibody to which a labeling substance is bound, there is a problem that detection sensitivity is generally lowered. Therefore, it is preferable to use an indirect detection method such as a method using a secondary antibody to which a labeling substance is bound or a method using a polymer to which a secondary antibody and a labeling substance are bound. The secondary antibody here is an antibody having a specific binding property to an antibody specific to the biomarker employed. For example, when an antibody specific for a biomarker is prepared as a rabbit antibody, an anti-rabbit IgG antibody can be used as a secondary antibody. Labeled secondary antibodies that can be used against various types of antibodies such as rabbits, goats, and mice are commercially available (for example, Funakoshi Co., Ltd., Cosmo Bio Co., Ltd., etc.) and are appropriate for the reagents of the present invention. Can be appropriately selected and used.

Examples of labeling substances include peroxidase, microperoxidase, horseradish peroxidase (HRP), alkaline phosphatase, β-D-galactosidase, enzymes such as glucose oxidase and glucose-6-phosphate dehydrogenase, fluorescein isothiocyanate (FITC), Fluorescent materials such as tetramethylrhodamine isothiocyanate (TRITC) and europium, chemiluminescent materials such as luminol, isoluminol and acridinium derivatives, coenzymes such as NAD, biotin, and radioactive materials such as ¹³¹ I and ¹²⁵ I.

  In one embodiment, the reagent of the present invention is immobilized on the basis of its use. The insoluble support used for the solid phase is not particularly limited. For example, a resin such as polystyrene resin, polycarbonate resin, silicon resin, nylon resin, or an insoluble support made of a water-insoluble substance such as glass can be used. The antibody can be supported on the insoluble support by physical adsorption or chemical adsorption.

  The kit of the present invention contains the reagent of the present invention as a main component. Other reagents (buffers, blocking reagents, enzyme substrates, coloring reagents, etc.) and / or devices or instruments (containers, reaction devices, fluorescent readers, etc.) used in performing the test method may be included in the kit. Good. Moreover, it is preferable to include the biomarker molecule of the present invention or a fragment thereof as a standard sample in a kit. Usually, an instruction manual is attached to the kit of the present invention.

The following experiments were conducted with the aim of identifying ALS-related molecules.
1. Materials and methods
(1) Human Serum Sample This test was conducted in accordance with the Declaration of Helsinki after obtaining the deliberation and approval of the Gifu University Ethics Committee. In addition, informed consent was given to all subjects, and the name and seal on the consent form for the experiment were obtained. The subjects were 13 arctic ALS patients, 10 Alzheimer's disease patients, 10 Parkinson's disease patients and 17 non-neurological patients (macular hole). All tests using human serum samples were conducted at Gifu University School of Medicine. Age, sex, and serum collection time are shown in FIG. Serum samples were stored at -80 ° C until testing.

(2) Experimental animals
SOD1 ^H46R rats and wild-type (WT) rats [SD-Tg (SOD1H46R-4)] were purchased from SLS Japan (Shizuoka). SOD1 ^H46R and WT rats were tested in three groups: pre-onset (12 weeks of age, n = 3-7), post-onset (26 weeks of age, n = 3-7), and end-stage (30 Age, n = 3-7). In this study, the time when the hind limb began to be dragged was considered as ALS onset. All rats have set temperature: 23 ° C (tolerance: 20-26 ° C), set humidity: 55% (tolerance: 40-70%), light and darkness for 12 hours each (lighting: 8:00 am to 8:00 pm) ) Was kept in the University's animal breeding house maintained in In conducting this study, an application for animal experiment approval was submitted to the Gifu Pharmaceutical University Animal House Steering Committee, and permission was granted.

(3) Rat serum sample Before and after onset, SOD1 ^H46R and WT rats were deeply anesthetized by intraperitoneal administration of Nembutal 80 mg / kg, and blood was collected from the inferior vena cava. The blood was collected in an Eppendorf tube and allowed to stand at room temperature for 30 minutes, and then centrifuged at 1,500 × g for 20 minutes at 4 ° C. to separate serum. The collected serum was stored at -80 ° C.

(4) Two-dimensional electrophoresis Two-dimensional electrophoresis, MALDI-TOF MS analysis and LC-MS / MS analysis were commissioned to Apro Science (Tokushima). A 20 μL rat serum sample was treated with Vivapure Anti-HAS / IgG Kit (Satorius AG, Frankfurt, Germany), and then protein was precipitated with cold acetone. The resulting precipitate was added to a swelling solution (7 M urea, 2% Thiourea, 20 mM dithiothreitol [DTT], 2 mM Tris- (2-cyanoethyl) phosphine, 2% 3- (3-cholamidepropyl) dimethylammonio-1-propanesulphonate [CHAPS ], 0.2% [v / v] BioLyte 3-10, and trace of bromophenol blue), and after quantifying the amount of protein from the centrifuged supernatant by the Bradford method, the required amount is mixed with the sample for two-dimensional electrophoresis. did.

  Based on the quantification results from these prepared two-dimensional electrophoresis samples, each 120 μg protein equivalent was mixed with a swelling solution containing Bromophenol Blue, and IPG ReadyStrip gel (17 cm, pH 3-10NL, BIO-RAD) was used for 12 hours or longer. Swelled. This gel was run with the prescribed program (first dimension: isoelectric focusing), and then the IPG gel was equilibrated with buffer A (50 mM Tris-HCl [pH 8.5], 6 M urea, 30% glycerol, 2 % SDS, 1% DTT, and 0.005% bromophenol blue) for 15 minutes, followed by equilibration buffer B (50 mM Tris-HCl [pH 8.5], 6 M Urea, 30% Glycerol, 2% SDS, 4.5% iodacetamide , and 0.005% bromophenol blue) for 15 minutes. Thereafter, the equilibrated IPG gel was set on a 10/16% gradient gel (19 × 17 cm), and the second dimensional migration was performed (second dimensional: SDS-PAGE). The gel after electrophoresis was stained with SYPRO Ruby (Invitrogen, Carlsbad, CA, USA), and the image was captured with Molecular Imager FX (Bio-Rad Laboratories, Philadelphia, PA, USA). Thereafter, a comparative analysis of spot patterns between gels was performed.

(5) Image analysis After generating average data of sample groups using image analysis software Progenesis SameSpot (Nonlinear Dynamics, Newcastle upon Tyne, UK), pre-onset SOD1 ^H46R : WT, post-onset SOD1 ^H46R : WT and pre-onset SOD1 ^H46R : Comparative analysis was performed on SOD1 ^H46R after onset. Further, in the comparative analysis, spots where the Norm. Volume value of the spots (value calculated by Progenesis SameSpot) was 1.5 times or more and P <0.05 were selected between each group.

(6) MALDI-TOF MS analysis
The target spot was cut out from the SDS polyacrylamide gel. After decolorizing the gel piece, Tris buffer (pH 8.0) containing trypsin was added and treated at 35 ° C. for 20 hours. Thereafter, the sample solution was treated with ZipTip C18 (Millipore Corporation, Bedford, MA, USA), eluted into a matrix solution, and spotted on a plate. After natural drying, it was subjected to MALDI-TOF MS analysis. The raw data obtained by applying external calibration (Raw Data) is internally calibrated with trypsin-derived peaks, and the resulting monoisotopic mass is Mascot (http: Searched NCBInr database at http://www.matrixscience.com/, MatrixSicence Ltd., London, UK) (http://www.ncbi.nlm.nih.gov/). Search conditions are (i) Taxonomy: Rattus norvegicus (Rat), (ii) Peptide Mass Tolerance: ± 20 ppm, (iii) Max Missed Cleavages: 1, (iv) Variable modifications: Carbamidomethyl and Oxidation. A protein score (Protein score) of 61 or more was considered significant (P <0.05).

(7) LC-MS / MS analysis
The target spot was cut out from the SDS polyacrylamide gel. After decolorizing the gel piece, Tris buffer (pH 8.0) containing trypsin was added and treated at 35 ° C. for 20 hours. Thereafter, the entire sample solution was subjected to LC-MS / MS analysis. The obtained mass spectrum was searched for NCBInr database by Mascot (http://www.matrixscience.com/, MatrixScience Ltd., London, UK) (http://www.ncbi.nlm.nih.gov/). An ion score of 53 or more was considered significant (P <0.05).

(8) Western blotting Human and rat serum samples were dissolved in RIPA [containing 1/100 protease inhibitor cocktail, 1/100 phosphatase inhibitor cocktail 1 and 1/100 phosphatase inhibitor cocktail 2], and the solution was bicinchoninic acid ( The protein concentration was quantified by BCA) method. Thereafter, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with an equal amount of protein. Western blotting was performed by transferring the protein in the gel to a polyvinylidene difluoride (PVDF) membrane. After blocking the PVDF membrane with Block-One P (Nacalai Tesque), each primary antibody [anti-ITIH4 antibody and Gpx3 antibody] was reacted overnight at 4 ° C, and the HRP-conjugated goat as the secondary antibody. The mixture was reacted with an anti-rabbit or rabbit anti-goat antibody for 1 hour at room temperature. Color and visualize the band using Super Signal West Femto Maximum Sensitivity Substrate, and brightness of the band using Luminescent image analyzer LAS-4000 UV mini (Fujifilm Corporation) and Multi Gauge Ver.3.0 (Fujifilm Corporation). Measured.

(9) Statistical analysis The experimental results are shown as mean ± standard error or standard deviation. Statistical comparisons were made by two-way analysis of variance or Student's t-test using STAT VIEW (SAS institute, Cary, NC, USA). A risk rate of less than 5% was considered significant.

2. result
(1) Rat serum proteome analysis SOD1 ^H46R rat serum proteome analysis was performed before and after the onset to search for pathological proteins in the serum. As a result of separating proteins in serum by two-dimensional electrophoresis and staining the proteins using CYPRO Ruby, electrophoretic images were obtained before (Fig. 1) and after (Fig. 2). Before the onset, 69 out of 1,397 spots were significantly (P <0.05) between SOD1 ^H46R and WT rats, with 40 out of 1,412 spots after the onset. Among these spots, MALDI-TOF MS or LC-MS / MS analysis was performed focusing on two spots where the spots were clear and changed before and after onset. The two proteins identified are shown in FIG. In pre-onset SOD1 ^H46R rat serum, the% volume value of Gpx3 spot increased 1.6-fold (P <0.05) compared to the same-week-old WT (Fig. 4). In addition, the amount of Gpx3 in SOD1 ^H46R rat serum after onset decreased to 68.5 ± 5.1% (P <0.05) compared to before onset (Fig. 4). On the other hand, the amount of ITIH4 in SOD1 ^H46R rat serum after the onset increased 1.6 times (P <0.05) compared to the same-week-old WT, and 1.9 times (P <0.05) compared to before the onset (Fig. 5). Changes in these two proteins in WT serum were not observed (FIGS. 4 and 5). As described above, two types of proteins (Gpx3, ITIH4) that are correlated with the onset of ALS were identified.

(2) Changes in the amount of Gpx3 in rat serum accompanying the progression of ALS pathology Based on the results of two-dimensional electrophoresis, the amount of Gpx3 in rat serum was examined by Western blotting (FIG. 6). In pre-onset SOD1 ^H46R rat serum, Gpx3 levels were increased 1.3-fold (P <0.01) compared to same-week-old WT. In addition, Gpx3 content decreased with the progression of the disease state, and decreased to 74.3 ± 2.9% (P <0.05) compared with WT at the end of the disease state (Fig. 7). Thus, it was shown that the amount of serum Gpx3 decreases with the onset of ALS and the progression of disease state.

(3) Gpx3 in the serum of arcuate ALS patients
Serum Gpx3 levels in arcuate ALS patients, Alzheimer's disease patients, Parkinson's disease patients, and macular hole patients (non-neurodegenerative disease control) were examined by Western blotting. The patient background is shown in FIG. A total of 13 arctic ALS patients (11 men, 2 women), 10 Alzheimer's disease patients (6 men, 4 women), and 10 Parkinson disease patients (3 men, 7 women) And a total of 17 patients (3 males and 14 females). The age of patients with arcing ALS, Alzheimer's disease, Parkinson's disease, and macular hole was 64.8 ± 3.4 years, 74.7 ± 7.7 years, 67.5 ± 5.6 years, and 64.6 ± 4.5 years, respectively. The amount of GPX3 in arcuate ALS serum was 62.9 ± 6.4% (P <0.05), 59.2 ± 6.1% (P <0.05), and 71.8 ± 7.4% (P <0.05), respectively, compared with Alzheimer's disease, Parkinson's disease and control. 0.05) (Fig. 9). Thus, it was found that the amount of serum GPX3 decreased specifically in ALS patients. That is, it was confirmed that the amount of GPX3 became an ALS-specific marker even in clinical specimens. In addition, the ability to detect such changes in serum is extremely important for the clinical application of the molecule.

(4) Changes in rat serum ITIH4 levels as ALS progresses
Similar to Gpx3, the amount of ITIH4 in rat serum was examined by Western blotting (FIG. 10). In the post-onset and end-stage SOD1 ^H46R rat serum, the 120 kDa ITIH4 level increased 2.5-fold (P <0.01) and 3.3-fold (P <0.05), respectively, compared with the same-week-old WT (Fig. 11). Similar to the results of two-dimensional electrophoresis, there was no difference in the amount of 120 kDa ITIH4 before onset. Furthermore, a truncated 85 kDa ITIH4 produced by the cleavage of plasma kallidinogenase was observed in the end-stage SOD1H46R rat serum (FIG. 12). Thus, it was shown that the amount of serum ITIH4 increases with the onset. In addition, there was a correlation between the progression of the disease state and the increased amount of truncated ITIH4.

(5) ITIH4 in the serum of arcuate ALS patients
Serum ITIH4 levels in arcuate ALS patients, Alzheimer's disease patients, Parkinson's disease patients, and macular hole patients (non-neurodegenerative disease control) were examined by Western blotting (FIG. 13). The patient background is shown in FIG. A total of 13 arctic ALS patients (11 men, 2 women), 10 Alzheimer's disease patients (6 men, 4 women), and 10 Parkinson disease patients (3 men, 7 women) And a total of 17 patients (3 males and 14 females). Truncated 85 kDa ITIH4 is found specifically in arcuate ALS serum and is expressed 2.1-fold (P <0.01), 2.7-fold (P <0.01), and Alzheimer's disease, Parkinson's disease and controls It increased 3.3 times (P <0.01) (Fig. 15). On the other hand, there was no difference between the groups regarding the amount of 120 kDa ITIH4 (FIG. 14). In addition, the amount of activated kallidinogenase present in arcuate ALS serum increased 1.7-fold (P <0.01) compared to the control (FIG. 16). As described above, serum-cut ITIH4 was detected specifically in ALS patients. That is, it was confirmed that serum-cut ITIH4 is clinically useful as an ALS-specific marker. In addition, the ability to detect such changes in serum is extremely important for the clinical application of the molecule.

  The test method of the present invention makes it possible to easily and objectively determine the possibility of developing ALS. The test method of the present invention is useful as a means for determining whether or not ALS has developed. It is also expected to be used as a means for grasping the possibility of future onset. Early detection and early treatment using the test method of the present invention is expected to prevent intractable, serious (progressive disease), recurrence, etc. of ALS. It is also assumed that the inspection method of the present invention can be used as a means for estimating the prognosis.

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

Claims (10)

  An amyotrophic lateral sclerosis marker comprising a protein molecule selected from the group consisting of inter-α trypsin inhibitor heavy chain 4, truncated inter-α trypsin inhibitor heavy chain 4 and glutathione peroxidase 3. In order to examine amyotrophic lateral sclerosis, one or more protein molecules selected from the group consisting of inter-α trypsin inhibitor heavy chain 4, cleaved inter-α trypsin inhibitor heavy chain 4 and glutathione peroxidase 3 are used. A method for measuring levels in a specimen. The method according to claim 2, comprising the following steps (1) to (3):
(1) a step of preparing a subject-derived specimen;
(2) detecting one or more of the protein molecules in the specimen; and (3) determining a current or future possibility of developing amyotrophic lateral sclerosis based on the detection result. For the inter-α trypsin inhibitor heavy chain 4 and the truncated inter-α trypsin inhibitor heavy chain 4, according to the criterion that the detection probability is high if the detection value is high, or the criterion that the detection probability is high if it can be detected,
The glutathione peroxidase 3, in accordance with the standards of the high possibility of onset with a reference, or can not be detected with high possibility of onset and the detected value is lower, it is determined in step (3) The method according to claim 3 . The method according to claim 3 or 4, wherein the determination in step (3) is performed based on a comparison between the detection value obtained in step (2) and the detection value of the control sample. The determination in step (3) is performed based on a comparison between the detection value obtained in step (2) and the detection value in a sample collected in the past from the same subject. Way . The method according to any one of claims 2 to 6, wherein the specimen is blood, plasma, serum, spinal cord, or cerebrospinal fluid.   A test reagent for amyotrophic lateral sclerosis comprising a substance exhibiting specific binding to the amyotrophic lateral sclerosis marker according to claim 1.   The reagent for examining amyotrophic lateral sclerosis according to claim 8, wherein the substance is an antibody.   A kit for testing amyotrophic lateral sclerosis comprising the reagent for testing amyotrophic lateral sclerosis according to claim 8 or 9.

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