Biomarker for mental disorders, including cognitive disorders, and method of using said biomarker fo

专利摘要:

公开号:ES2617918T9
申请号:ES10777546.2T
申请日:2010-05-17
公开日:2018-02-16
发明作者:Kazuhiko Uchida；Takashi Ishii；Kohji Meno；Hideaki Suzuki
申请人:MCBI Inc；
IPC主号:

专利说明:

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DESCRIPTION
Biomarker for mental disorders, including cognitive disorders, and method of using said biomarker for the detection of mental disorders, including cognitive disorders
The present invention relates to new biomarkers of cognitive impairment, including mild cognitive impairment and Alzheimer's disease and methods of detecting cognitive impairment using said biomarkers. Likewise, the present invention relates to new biomarkers for non-dementia neurological diseases such as depression, schizophrenia, etc., and to methods for the detection of non-dementia neurological diseases using said biomarkers.
Prior art
The means commonly used to differentiate between the normal and non-normal states of a human subject using its biological materials are mainly those that have been used in the field of diagnosis. The most frequently used methods are those that present as biomarker targets in blood. In the present field, a comparative measurement of the amount of a specific protein or peptide having a molecular weight of less than 10,000 or, in the case of an enzymatic protein, the enzymatic activities in samples of normal (healthy) subjects and the from sick individuals to help diagnosis. Here, before testing real samples, measurements are made on a fixed number of samples from healthy controls and from patients with certain diseases with respect to the amount or amounts or activity (activities) of individual specific proteins or peptides. or multiple and the respective ranges of abnormal and normal values are determined. Next, the sample to be evaluated by the same method is analyzed and the resulting value is evaluated with respect to whether it is in the normal range or in an abnormal range.
In actual measurements, the amount or amounts of the protein or proteins or peptide or peptides specified in the test samples, without modification or after dilution, are determined by using an enzyme-linked immunosorption assay (ELISA) that uses a primary or secondary antibody labeled with an enzyme that reacts with a substrate that yields a color with the reaction, a chemiluminescent immunoassay (CLIA), a radioimmunoassay (RIA), which uses a primary or secondary antibody labeled with a radioactive isotope and, in the case that the protein is an enzyme, the measurement of the activity of the enzyme by the addition of the substrate thereof and the determination of the intensity of the color produced, etc. These antibody-based methods are called enzymatic, fluorescent or radioisotopic labeling methods, respectively. In addition, there is a method in which an enzymatic reaction product derived from the corresponding substrate is determined by high performance liquid chromatography (HPLC). Additionally, there is a method in which HPLC is combined with a mass spectrometer, called LC-MS / MS, and there is a method called selected reaction monitoring (SRM) / multiple reaction monitoring ( MRM, which uses LC-MS / MS. In another method to determine the concentration in a sample, it is subjected to an appropriate pretreatment and the separation of the proteins or peptide is carried out by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and the target protein or peptide is determined by silver staining, Coomassie blue staining or immunological staining (western blot) that uses a target antibody in the protein or peptide. Still further, there is a method that uses mass spectrometry to determine the amount of protein or target peptide in fractionated samples by column chromatography. Instead of column chromatography, protein chips and magnetic beads can also be used for pretreatment.
In addition, said inventors have developed an immuno-EM method in which a target protein or peptide is captured in beads (including magnetic beads) with antibody bound to the protein or peptide, eluted from the beads and determined by mass spectrometry. In addition, the analysis of intact proteins has been reported by mass spectrometry using the methods indicated above after trypsin digestion, etc. (PTL 1). In this method, intact target proteins are selected by fractionation or by adsorption to a specific adsorbent thereof and then determined by mass spectrometry.
The number of patients suffering from cognitive impairment, such as Alzheimer's disease, is increasing rapidly as well as the increase in the elderly population in Japan. It is estimated that the number of patients was 1.3 million in 1995 and 1.9 million in 2005 and that it will reach approximately 3.0 million in 2020. It is reported that 60% to 90% of cases of cognitive impairment They are due to Alzheimer's disease. Because the manifestation of Alzheimer's disease is not only memory loss but several alterations in daily work, the increase in patients with this disease is becoming an important social issue that must be resolved. In Japan, because donepezil hydrochloride, an anti-acetylcholine esterase inhibitor, has been available for the medical treatment of Alzheimer's disease since 1999, it has been possible to efficiently 'slow down' the progress of cognitive impairment in these patients in cases diagnosed in an early stage. Thus, in the medication of Alzheimer's disease, the most important issue is the 'early diagnosis' to effectively treat patients with current drugs and future drugs.
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The main diagnostic criteria for Alzheimer's disease described in DSM IV, which has been published by the American Psychiatric Association (NPL 1), are provided below.
A. The development of multiple cognitive deficits manifested in both:
(1) memory impairment (impairment of the ability to learn new information or to remember previously learned information)
(2) one (or more) of the following cognitive disorders:
a) aphasia (language impairment)
b) apraxia (impaired ability to perform motor activities despite presenting an intact motor function)
c) agnosia (failure to recognize or identify objects despite presenting an intact sensory function)
d) disturbances of executive functioning (ie, planning, organization, sequencing and abstraction).
B. The cognitive deficits in criteria A1 and A2 cause a significant deterioration of social or occupational functioning and represent a significant decline with respect to a previous level of functioning.
There are several types of neurological disorder related to Alzheimer's disease (AD). Because cognitive dysfunction gradually appears in dementia, also in AD, there is a pre-stage of dementia similar to the disease state. This stage is called mild cognitive impairment (MCI). In the United States, 10% of the DCL develops in EA in 1 year and 50% of the DCL develops in EA in 4 years. The DCL is defined as a condition characterized by a newly acquired cognitive decline that manifests itself in a measure that exceeds that expected by age or level of training, but that does not cause significant functional impairment and that does not entail alterations in daily life. Frontotemporal dementia (frontotemporal lobular degeneration (DLF)) manifests itself in loss of personal consciousness, loss of social consciousness, hyperorality and stereotyped persevering behavior. These clinical characteristics are different from those of AD. DLF includes Pick's disease, which is characterized microscopically by Pick bodies usually present in the limbic, paralympic and ventral temporal lobe cortex. Dementia with Lewy bodies (DCL) is characterized as a progressive disease and psychiatric symptoms include anxiety, depression, hallucinations (usually visual) and delusions (false beliefs). It is believed that DCL is the second most common subtype and that 10% to 30% of dementias are DCL. The symptoms of DCL are caused by the histological accumulation of Lewy bodies. DLF and DCL belong to the dementia neurological diseases class since memory loss, problem-solving capacity and the ability to maintain emotional control also occur (NPL 1). In the description of the present patent, cognitive impairment includes AD, MCI and dementia neurological diseases.
The widely used dementia screening trials are the revised Hasegawa dementia scale (HDS-R) and the mental state miniexamen (MMSE). In these screening trials, the evaluator asks several questions and evaluates the level of cognitive impairment of each subject through scores. HDS-R is a revised version of the HDS published in 1991. In the HDS-R, the essay consists of 9 questions to analyze orientation, memory, calculation, retention and recall, and common sense. The total score is 30 and a person with a score below 23 is suspected of presenting dementia. MMSE was developed in the United States to screen and diagnose dementia and to analyze global cognitive function, with items that assess orientation, word recall, attention and calculation, linguistic abilities and visuospatial ability (drawing ability) . This essay consists of 11 questions and the maximum score is 30 and a person with a score below 23 is suspected of presenting dementia. The results of the HDS-R and the MMSE coincide. Both are used for screening, not for diagnosis and for staging the progress of dementia disease (NPL 1).
Neurological imaging techniques for dementia are computerized tomography (CT) and magnetic resonance imaging (MRI), which assess morphological changes such as cerebral atrophy and ventricular dilation, and tomography Computerized single photon emission (SPECT), which analyzes blood flow in regions of the brain and PET (for its acronym in English), which shows brain metabolism from the measurement of oxygen consumption and sugar. SPECT and PET nuclear imaging techniques can identify neuronal dysfunction at the preclinical stage (NPL 1). However, these neuroimaging techniques cannot be widely used in hospitals because they require special facilities to carry out nuclear imaging techniques and neuroimaging may not be an objective trial since diagnostic imaging is completely dependent on Skills of the doctor who analyzes the images.
In this way, the present methods of screening and diagnosis of dementia, including AD, depend on tests that are not objective and that depend on expensive instruments, so it is very difficult to use the currently available tests for stage screening Early cognitive impairment. In the case of
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If we had blood biomarkers (serum / plasma) of cognitive impairment, which allowed an objective trial using easy-to-obtain specimens, we could identify cognitive impairment at an early (preclinical) stage by using a blood test with that biomarker. The present patent provides new biomarkers and a powerful new diagnostic method for cognitive impairment through the use of said biomarkers and biomarkers described herein. In addition, the present patent provides a diagnostic method and new biomarkers for non-dementia neurological diseases such as depression, schizophrenia, etc.
Reference List
Patent literature
LTP 1, JP-A-2004-333274 LTP 2, JP-A-2006-308533
Non-patent literature
LNP 1, "The better understanding of Alzheimer's disease", edited by Imaharu Nakano and HIdehiro Mizusawa, Nagai Shoten Co., Ltd., 2004 (in Japanese)
LNP 2, Benkirane N. et al., J. Biol. Chem. Vol. 268, 26279-26285, 1993.
Document No. WO03087768 (A2) discloses the provision of mitochondrial targets for drug screening tests and for therapeutic intervention in the treatment of diseases associated with altered mitochondrial function. Complete amino acid sequences [SEQ ID No. 1 to 3025] of polypeptides comprising the mitochondrial proteome of the human heart are provided using fractionated proteins derived from highly purified mitochondrial preparations in order to identify previously unidentified mitochondrial molecular components.
Summary Description of the Invention
Technical problem
The present invention aims to present methods for the detection of cognitive impairment, including mild cognitive impairment and Alzheimer's disease, using a protein or partial peptide thereof that differs in the presence or absence thereof, or in the amount of it in subjects with non-cognitive impairment (including healthy people; human subjects that may be affected by any disease and not affected by cognitive impairment) and patients with cognitive impairment, and has as an additional objective to present biomarkers that comprise said protein and said partial peptide for use in the detection of cognitive impairment, including mild cognitive impairment and Alzheimer's disease. Likewise, the present invention aims to present new biomarkers for non-dementia neurological diseases such as depression, schizophrenia, etc., in addition to methods for the detection of cognitive impairment through the use of said biomarkers.
Solution to the problem
The present inventors have investigated in order to find means to detect cognitive impairment and have found a peptide capable of detecting cognitive impairment and psychiatric diseases, including mild cognitive impairment and Alzheimer's disease, in the serum. Said peptides found in the present invention are significant as a biomarker for detection in the case of serum, not only in other biological materials such as blood, plasma, cerebrospinal fluid and urine. Likewise, the protein or peptide that is the origin of said peptides (hereinafter referred to as intact proteins or peptides) is also significant as a biomarker. The present invention provides a peptide or protein as defined in claim 1, an in vitro method for the detection of psychiatric diseases or cognitive impairment as defined in claim 5, and a kit as defined in claim 7 or 8. Advantageous embodiments are disclosed in the respective dependent claims.
Specifically, said inventors have found a biomarker comprising at least one protein selected from the group consisting of a precursor of neurexin-2-beta consisting of the amino acid sequence expressed by SEQ ID NO: 1, a prothrombin precursor consisting of the amino acid sequence expressed by SEQ ID No. 3, pendrin consisting of the amino acid sequence expressed by SEQ ID No. 6, zeta-1 subunit coatomer consisting of the amino acid sequence expressed by SEQ ID. No. 8, precursor of retinoic acid receptor 2 responding protein consisting of the amino acid sequence expressed by SEQ ID No. 1.0, gelsolin precursor consisting of the amino acid sequence expressed by SEQ ID No. 13 , a clusterin precursor consisting of the amino acid sequence expressed by SEQ ID No. 15, subunit of the eukaryotic translation initiation factor 3 ca consisting of the amino acid sequence expressed by SEQ ID No. 18, and protein 27 that
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Contains leucine-rich repeats consisting of the amino acid sequence expressed by SEQ ID No. 20, could be used as biomarkers for the detection of psychiatric disease or cognitive impairment.
In addition, said inventors have found a biomarker comprising at least one peptide selected from the group consisting of NRX2B peptide derived from neurexin-2-beta precursor consisting of the amino acid sequence expressed by SEQ ID NO: 2, a prothrombin precursor derived peptide, THRB (R-), which consists of the amino acid sequence expressed by SEQ ID No. 4, a prothrombin precursor derived peptide, THRB (R +), consisting of the amino acid sequence expressed by SEQ ID No. 5, the pendrin-derived S26A4 peptide consisting of the amino acid sequence expressed by SEQ ID No. 7, COPZ1 peptide derived from the zeta-1 subunit coatomer consisting of the amino acid sequence expressed by the SEQ ID No. 9, RARR2 (S-) peptide derived from the retinoic acid receptor 2 responding protein precursor consisting of the amino acid sequence expressed by SEQ ID n 11, RARR2 (S +) peptide derived from the retinoic acid receptor 2 responding protein precursor consisting of the amino acid sequence expressed by SEQ ID No. 12, the gelsolin precursor derived GELS peptide consisting of the sequence of amino acids expressed by SEQ ID No. 14, the CLUS peptide (SDVP N-term.) derived from a clusterin precursor consisting of the amino acid sequence expressed by SEQ ID No. 16, the CLUS peptide (RFFT N-term. ) Clusterin precursor derivative consisting of the amino acid sequence expressed by SEQ ID No. 17, EIF3J peptide derived from subunit J of eukaryotic translation initiation factor 3 consisting of the amino acid sequence expressed by SEQ ID No. 19, and the LRC27 peptide derived from protein 27 containing leucine-rich repeats consisting of the amino acid sequence expressed by SEQ ID No. 21, could use Assist as biomarkers for the detection of psychiatric disease or cognitive impairment.
These inventors have completed the present invention by achieving simultaneous determination of this multitude of proteins and partial peptides thereof by using the two-dimensional high performance liquid chromatography method-MALDI TOF-EM and the immuno-EM method.
[1] A biomarker for the detection of psychiatric diseases or cognitive impairment comprising a fragment of protein or peptide not less than 5 amino acid residues that arises from at least one protein or peptide selected from the group consisting of a precursor of neurexin- 2-beta consisting of the amino acid sequence expressed in SEQ ID No. 1, a prothrombin precursor consisting of the amino acid sequence expressed by SEQ ID No. 3, pendrin consisting of the amino acid sequence expressed by SEQ. ID No. 6, zeta-1 subunit coatomer consisting of the amino acid sequence expressed by SEQ ID No. 8, precursor of retinoic acid receptor 2 responding protein consisting of the amino acid sequence expressed by SEQ ID No. 10, gelsolin precursor consisting of the amino acid sequence expressed by SEQ ID No. 13, a clusterin precursor consisting of the sequence Amino acidity expressed by SEQ ID No. 15, subunit J of the eukaryotic translation initiation factor 3 consisting of the amino acid sequence expressed by SEQ ID No. 18, and protein 27 containing leucine-rich repeats consisting of the amino acid sequence expressed by SEQ ID No. 20.
[2] A biomarker for the detection of psychiatric diseases comprising at least one peptide selected from the group consisting of the NRX2B peptide derived from a neurexin-2-beta precursor consisting of the amino acid sequence expressed by SEQ ID No 2, a prothrombin precursor derived peptide, THRB (R-), which consists of the amino acid sequence expressed by SEQ ID No. 4, a prothrombin precursor derived peptide, THRB (R +), consisting of the amino acid sequence expressed by SEQ ID No. 5, the pendrin-derived S26A4 peptide consisting of the amino acid sequence expressed by SEQ ID No. 7, COPZ1 peptide derived from the zeta-1 subunit coatomer consisting of the amino acid sequence expressed by SEQ ID No. 9, RARR2 (S-) peptide derived from the retinoic acid receptor 2 responding protein precursor consisting of the amino acid sequence expressed by SEQ ID No. 11, RARR2 (S +) peptide derived from the retinoic acid receptor 2 responsive protein precursor consisting of the amino acid sequence expressed by SEQ ID No. 12, the gelsolin precursor derived GELS peptide which consists of the amino acid sequence expressed by SEQ ID No. 14, CLUS peptide (SDVP N-term.) derived from clusterin precursor consisting of the amino acid sequence expressed by SEQ ID No. 16, the CLUS peptide (RFFT N-term.) Clusterin precursor derivative consisting of the amino acid sequence expressed by SEQ ID No. 17, EIF3J peptide derived from the J subunit of eukaryotic translation initiation factor 3, consisting of the expressed amino acid sequence by SEQ ID No. 19 and the LRC27 peptide derived from protein 27 containing leucine-rich repeats, consisting of the amino acid sequence expressed by SEQ ID No. 21.
[3] A biomarker for the detection of cognitive impairment comprising at least one peptide selected from the group consisting of the NRX2B peptide derived from neurexin-2-beta precursor consisting of the amino acid sequence expressed by SEQ ID n 2, a prothrombin precursor derived peptide, THRB (R-), which consists of the amino acid sequence expressed by SEQ ID No. 4, a prothrombin precursor derived peptide, THRB (R +), consisting of the amino acid sequence
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expressed by SEQ ID No. 5, the pendrin-derived S26A4 peptide consisting of the amino acid sequence expressed by SEQ ID No. 7, COPZ1 peptide derived from the zeta-1 subunit coatomer consisting of the amino acid sequence expressed by the SEQ ID No. 9, RARR2 (S-) peptide derived from the retinoic acid receptor 2 responding protein precursor consisting of the amino acid sequence expressed by SEQ ID No. 11, RARR2 (S +) peptide derived from the protein precursor Retinoic acid receptor 2 responder consisting of the amino acid sequence expressed by SEQ ID No. 12, the gelsolin precursor-derived GELS peptide consisting of the amino acid sequence expressed by SEQ ID No. 14, the peptide CLUS (SDVP N-term.) Derived from clusterin precursor consisting of the amino acid sequence expressed by SEQ ID No. 16, the CLUS peptide (RFFT N-term.) Derived from precur Sister of clusterin consisting of the amino acid sequence expressed by SEQ ID No. 17, EIF3J peptide derived from the J subunit of the eukaryotic translation initiation factor 3 consisting of the amino acid sequence expressed by SEQ ID No. 19 , and the LRC27 peptide derived from protein 27 containing leucine-rich repeats consisting of the amino acid sequence expressed by SEQ ID No. 21.
[4] A biomarker of cognitive impairment comprising the peptides selected from the group consisting of the amino acid sequence expressed by SEQ ID NO. 2, 5, 7, 9, 11, 12, 14 and 16 that is present or increased in biological material from patients with cognitive impairment compared to biological material from subjects who do not suffer from a psychiatric illness.
[5] A biomarker of cognitive impairment comprising the peptides selected from the group consisting of the amino acid sequence expressed by sEc ID No. 4, 17, 19 and 21, which is not present or is reduced in the Biological material from patients with cognitive impairment compared to biological material from subjects who do not suffer from a psychiatric illness.
[6] A biomarker of Alzheimer's disease comprising the peptides selected from the group consisting of the amino acid sequence expressed by SEQ ID No. 2 that is present or increased in biological material from Alzheimer's disease patients in Comparison with biological material of subjects who do not suffer from a neurological disease not dementia.
[7] An Alzheimer's disease biomarker comprising the peptides selected from the group consisting of the amino acid sequence expressed by SEQ ID No. 4 that is not present or is reduced in biological material from disease patients of Alzheimer's disease compared to biological material of subjects who do not suffer from a neurological disease not dementia.
[8] Method for the detection of psychiatric illness that involves the determination in biological material of at least one biomarker of psychiatric illness as indicated in [1] or [2].
[9] Method for the detection of cognitive impairment that involves the determination in biological material of at least one biomarker of cognitive impairment as indicated in [1] or [3].
[10] Method for the detection of cognitive impairment in which it is evaluated whether the patient suffers from cognitive impairment in the event that, after the determination in biological material of at least one biomarker of cognitive impairment as indicated in [4 ], the presence of said biomarker is detected in a higher quantity than in subjects who do not suffer from a psychiatric illness.
[11] Method for the detection of cognitive impairment in which it is evaluated that the patient suffers from cognitive impairment in the event that, after the determination in biological material of at least one biomarker of cognitive impairment as indicated in [ 5], the presence of said biomarker is detected in lower quantity than in subjects who do not suffer from a psychiatric disease.
[12] Method for the detection of a psychiatric disease as indicated in [8], in which the detection is carried out by an immunoblotting procedure, western blotting, a method of enzymatic, fluorescent or radioisotopically labeled antibodies, spectrometry mass, an immuno-MS method or a surface plasmon resonance method.
[13] Method for the detection of cognitive impairment as indicated in [9] to [11], in which the detection is carried out by an immunoblotting procedure, western blotting, a method of enzymatic, fluorescent or labeled antibodies radioisotopically, mass spectrometry, an immuno-MS method or a surface plasmon resonance method.
[14] A kit for the detection of a psychiatric illness to determine at least one biomarker as indicated in [1] or [2].
[15] A kit for the detection of cognitive impairment to determine at least one biomarker as indicated in [1], [3] to [5].
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[16] A kit for the detection of a psychiatric illness that contains an antibody or aptamer against at least one biomarker as indicated in [1] or [2].
[17] A kit for the detection of a psychiatric disease containing an antibody or aptamer against at least one biomarker as indicated in claims [1], [3] to [5].
[18] A kit for detection as indicated in [16] or [17], in which the antibody or aptamer solidifies on a plate or plates.
Beneficial Effects of the Invention
According to the present invention, it is possible to diagnose a subject as to whether he suffers a psychiatric illness or cognitive impairment by determining in biological material obtained from said subject the type and amount of at least one peptide selected from the group consisting of the NRX2B peptide neurexin-2-beta precursor derivative consisting of the amino acid sequence expressed in SEQ ID No. 2, THRB (R-) peptide derived from prothrombin precursor, consisting of the amino acid sequence expressed by SEQ ID No. 4, the THRB (R +) peptide derived from prothrombin precursor, consisting of the amino acid sequence expressed by SEQ ID No. 5, pendrin-derived S26A4 peptide, consisting of the amino acid sequence expressed by SEQ ID No. 7, COPZ1 peptide derived from the zeta-1 subunit of coatomer, consisting of the sequence of ammonia acids expressed by SEQ ID No. 9, peptide RARR2 (S-) deri Ford Retinoic Acid Receptor 2 Protein Precursor, consisting of the amino acid sequence expressed by SEQ ID No. 11, RARR2 (S +) peptide derived from Retinoic Receptor Receptor 2 Protein Precursor, consisting of the amino acid sequence expressed by SEQ ID No. 12, GELS peptide derived from gelsolin precursor, consisting of the amino acid sequence expressed by SEQ ID No. 14, the CLUS peptide (SDVP N-term.) derived from precursor of clusterin consisting of the amino acid sequence expressed by SEQ ID No. 16, the CLUS peptide (RFFT N-term.) derived from clusterin precursor consisting of the amino acid sequence expressed by SEQ ID No. 17, peptide EIF3J derived from the J subunit of the eukaryotic translation start factor 3 consisting of the amino acid sequence expressed by SEQ ID No. 19, and the LRC27 peptide derived from the prote na 27 that contains leucine-rich repetitions consisting of the amino acid sequence expressed by sEc ID No. 21. In addition, it is possible to diagnose a subject as to whether he suffers from Alzheimer's disease by comparing with the increase in the biological material of Non-dementia neurological disease patients by determining the amount of peptide consisting of the amino acid sequence expressed by SEQ ID No. 2 and it is possible to diagnose a subject as to whether he suffers from Alzheimer's disease by comparing it to the reduction in The biological material of patients with non-dementia neurological disease by determining the amount of peptide consisting of the amino acid sequence expressed by SEQ ID No. 4.
The present invention presents a diagnostic system that has high precision and specificity. The present invention allows a highly accurate diagnosis of cognitive impairment to be obtained in which no specific test methods are available for biological materials such as blood. In addition, the biomarkers disclosed in the present invention are highly useful for evaluating the efficacy of the drugs.
Brief description of the drawings
[Fig. 1] Figure 1 illustrates the isolation of Alzheimer's disease serum by the CL-MALDI TOF 2D-EM method (Example 1).
[Fig. 2] Figure 2 illustrates the case of Marker A, which is an example of the result of the differential analysis. As shown in Figure 3, Marker A is the NRX2B peptide derived precursor of neurexin-2-beta. Figure 2A illustrates a comparison between DNA, DCL and eA, and Figure 2B illustrates a comparison between DNA, EA, ENtodas, ENdem and ENnon. For each sample the average value (divided by 1,000) and the (SD) (divided by 1,000) are the following: A) DNA 0.1 (0.1); DCL 45.8 (42.2); EA 41.7 (22.2). B) 0.1 DNA (0.2); EA 34.0 (27.8); N All 19.2 (15.8); NDdem 24.3 (20.8); NDnon 14.0 (6.1). c), D) and E) illustrate, respectively, the COR curve of the DCL vs. comparisons. DNA, Ea vs. DNA and EA vs. ENnon (Example 1).
[Fig. 3] Figure 3 illustrates the MS / MS spectrum of Marker A (SEQ ID No. 2, NRX2B) obtained by using the TOF / TOF mass spectrometer (Example 1).
[Fig. 4] Figure 4 illustrates the comparison between subjects of non-psychiatric disease (DNA) and patients of psychiatric disease, including dementia, for THRB (R-) (A) and (B) of Figure 4) and THRB ( R +) (C) and (D) of Figure 4) in serum (Example 1).
[Fig. 5] Figure 5A illustrates the comparison of each individual between subjects of non-psychiatric disease (DNA) and
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cognitive impairment patients (DCL, EA) for THRB (R-) and THRB (R +) in serum. Figure 5B illustrates the COR curve of the comparison of EA vs. ENnon for THRB (R-) in serum. (Example 1).
[Fig. 6] Figure 6 illustrates the comparison (A) and (B) of Figure 6 between subjects of non-psychiatric disease (DNA) and patients of psychiatric disease, including dementia, for serum S264A, and the comparison of (C) and (D) of Figure 6 between subjects of non-psychiatric disease (DNA) and patients of psychiatric disease, including dementia, for serum COPZ1 (Example 1).
Fig. 7] Figure 7 illustrates the comparison of each individual between subjects of non-psychiatric disease (DNA) and patients of cognitive impairment (DCL, EA) for PARR2 (S-) (A) of Figure 7 and PARR2 (S +) (B) of Figure 7 in serum (Example 1).
[Fig. 8] Figure 8 illustrates the comparison (A) and (B) of Figure 8 between subjects of non-psychiatric disease (DNA) and patients of psychiatric disease, including dementia, for serum GELS (Example 1).
[Fig. 9] Figure 9 illustrates the comparison (A) and (B) of Figure 9 between subjects of non-psychiatric disease (DNA) and patients of psychiatric disease, including dementia, for CLUS (SDVP N-term.) In serum, and the comparison of (C) and (D) of Figure 9 between subjects of non-psychiatric disease (DNA) and patients of psychiatric disease, including dementia, for CLUS (RFFT N-term.) in serum (Example 1).
[Fig. 10] Figure 10 illustrates the comparison (A) and (B) of Figure 10 between subjects of non-psychiatric disease (DNA) and patients of psychiatric disease, including dementia, for serum EIF3J, and the comparison of (C) and (D) of Figure 10 between subjects of non-psychiatric disease (DNA) and cognitive impairment patients (DCL, Ea) for serum LRC27 (Example 1).
[Fig. 11] Figure 11 illustrates the mass spectrum of the NRX2B peptide captured and detected by the immuno-EM method using a serum-specific NRX2B antibody from EA and DCL patients. The figure on the right is an enlarged view of the parts indicated by arrows in the figure on the left. The endogenous NRX2B peptide (continuous arrows) and the isotopically labeled synthetic NRX2B peptide (dashed arrows) are shown for each peak (Example 4).
Description of realizations
The present invention is a method for determining the type and amount of intact protein and / or partial peptide thereof in the event that the test subject suffers from cognitive impairment as well as for the diagnosis of whether the test subject suffers from deterioration. cognitive and in the event that the test subject has been diagnosed with a psychiatric illness. It is generally said of a peptide that is a chemical entity constituted by polymerization of amino acids, of less than 10,000 molecular weight, or by polymerization of several and up to about 50 amino acid residues. Although in the present invention a partial peptide of an intact protein can be used as a biomarker for the detection of cognitive impairment, said partial peptide is defined as a peptide of less than 10,000 molecular weight consisting of a part of the amino acid sequence of the intact protein Said peptide may appear as a partial peptide during expression by transcription followed by synthesis by translation before maturation in an intact protein or as a peptide produced by enzymatic digestion in the body after synthesis of the intact protein. It is possible that, in the event that the body is in an abnormal state in which it suffers from a disease such as cognitive impairment, the mechanism for protein synthesis and regulation is deregulated. In other words, the present invention is also a method for determining if the test subject is in a normal state or if he suffers from cognitive impairment, by utilizing the degree of protein synthesis and / or protein digestion by way of indicator. The detection of cognitive impairment in the present invention refers to the evaluation and differentiation, that is, the diagnosis of whether the test subject suffers from cognitive impairment. The present invention may also include the evaluation of the patient's risk of suffering from a more serious cognitive impairment.
Specifically, in one method, examples of intact protein that can be used for cognitive impairment include the precursor of neurexin-2-beta, which consists of the amino acid sequence expressed in SEQ ID No. 1, a precursor of prothrombin consisting of the amino acid sequence expressed by SEQ ID No. 3, pendrin consisting of the amino acid sequence expressed by SEQ ID No. 6, zeta-1 subunit coatomer consisting of the amino acid sequence expressed by SEQ. ID No. 8, precursor of retinoic acid receptor 2 responding protein consisting of the amino acid sequence expressed by SEQ ID No. 10, gelsolin precursor consisting of the amino acid sequence expressed by SEQ ID No. 13, a clusterin precursor consisting of the amino acid sequence expressed by SEQ ID No. 15, subunit J of the eukaryotic translation initiation factor 3 consisting of d e the amino acid sequence expressed by SEQ ID No. 18, and protein 27 containing leucine-rich repeats consisting of the amino acid sequence expressed by SEQ ID No. 20, and the peptide fragments can also be used for the same purpose. which comprise partial peptides of not less than 5 amino acid residues of said intact proteins.
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Still further, an example of biomarkers for cognitive impairment includes partial peptides consisting of the amino acid sequence expressed in SEQ ID No. 2 of the NRX2B peptide derived from the precursor of neurexin-2-beta, SEQ ID No. 4 of the THRB (R-) peptide derived from the prothrombin precursor, SEC iD No. 5 of the THRB peptide (R +) derived from the prothrombin precursor, SEQ ID No. 7 of the S26A4 peptide derived from pendrin, SEQ ID No. 9 of the COPZ 1 peptide derived from the zeta-1 subunit of the coatomer, SEQ ID No. 11 of the RARR2 (S-) peptide derived from the retinoic acid receptor 2 responsive protein precursor, SEQ ID No. 12 of the RARR2 (S +) peptide derived from the precursor of Retinoic acid receptor responsive protein 2, SEQ ID No. 14 of the GELS peptide derived from gelsolin, SEQ ID No. 16 of the CLUS peptide (SDVP N-term.) derived from the clusterin precursor, SEQ ID No. 17 of the CLUS peptide (RFFT N-term.) Derived from the cluster precursor rina, SEQ ID No. 19 of the EIF3J peptide derived from the J subunit of the eukaryotic translation initiation factor 3, SEQ ID No. 21 of the LRC27 peptide derived from protein 27 containing leucine-rich repeats. In the present invention, proteins and peptides consisting of the amino acid sequences obtained from SEQ ID No. 1 or 2 by deletion, exchange and / or addition of one or a few amino acids can be used as biomarkers and are comprised within the present invention. The term "one or a few" herein refers to "one or three," preferably "one or two," or "one." In addition, partial peptides that can be used as biomarkers include those peptide fragments consisting of not less than 5 amino acid residues that appear respectively from SEQ ID No. 1 to 21. The basis of the limitation of the peptide fragments which consist of not less than 5 amino acid residues is provided in the description, subsequently, in non-patent document No. 2. The document reports that an antibody obtained by using the IRGERA peptide as an immunogen, which was the C-terminus terminal (130-135) of histone H3, recognizes the IKGERA peptide derived by exchanging K for R and the CGGGERA peptide that was derived by IR deletion followed by the addition of CGG. The foregoing demonstrates that immunogenicity (antigenicity) is recognized by a peptide of not less than 4 amino acid residues. In order to expand said result to other peptides apart from the C-terminal end of histone H3, the number of amino acid residues is defined as not less than 5 instead of 4. To construct said low molecular weight peptide it is important that the Detection and differentiation method uses immunological means, including immunoblotting, ELISA and immuno-MS.
It should be noted that there are cases in which one or more saccharide chains have been added to an intact protein or a partial peptide thereof to form glycosylated entities. Partial proteins and peptides in glycosylated form can also be used as biomarkers for the detection of cognitive impairment.
It should also be noted that, in the present invention, the biomarker can be quantified or its presence or absence qualitatively determined.
Two-dimensional electrophoresis (E2-D) or two-dimensional chromatography (C2-D) can be used in the present invention to separate biomarkers in biological materials, including serum. Known chromatographic methods can be selected from ion exchange chromatography, reverse phase chromatography and gel filtration chromatography. It is also possible to quantify the SRM / MRM method in the CL-EM / EM technology. In addition, the immuno-MS method developed by the present inventors, in which the target protein or peptide is captured with beads (including magnetic beads) with antibody bound to the protein or peptide, is eluted from the beads and determined by Mass spectrometry, allows the convenient determination of the presence or absence or amount of target protein, protein fragment or peptide without using E2-D or chromatography.
It is possible to use the method disclosed in the present invention for the evaluation in the stage of mild cognitive dysfunction of the test subject and therefore could be useful in prophylactic medicine. In addition, the administration of psychotherapy and / or drug therapy to patients with cognitive impairment is reflected in the amount of proteins and partial peptides in biological materials such as serum in the event that the progression of the disorder has been inhibited. Therefore, by measuring said proteins and partial peptides it is possible to evaluate and determine the therapeutic effect.
The type and quantity of a protein in biological materials can be determined by various methods. In the event that the target protein has been characterized (including a protein fragment and a partial peptide) and in the case that an antibody (primary antibody) thereof has already been obtained, the following methods can be used:
1. Immunoblot
This is one of the simplest methods. The test serum in a fixed amount (approximately 1 microliter) after stepwise dilution is applied on an appropriate membrane, such as nitrocellulose, and air dried. The membrane is treated with a blocking solution containing a protein, such as BSA, washed, reacted with primary antibody and washed. Next, the membrane is reacted with labeled secondary antibody to detect the primary antibody. The membrane is washed and the label is visualized to measure its density.
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2. Western transfer
After separation by one-dimensional or two-dimensional electrophoresis involving the isoelectric or SDS-PAGE approach, the proteins are transferred onto an appropriate membrane, such as nitrocellulose, and the amounts thereof are determined, such as in the aforementioned immunoblot, using primary antibody and labeled secondary antibody.
3. ELISA
The antibody against the protein or partial peptide thereof is fixed on said plate in the form of a chemically modified microtiter plate. Appropriate amounts of the samples are applied to the plate after stepwise dilution and incubated. Uncaptured proteins and peptides are removed by washing. Next, the plate is incubated with secondary antibody labeled with a fluorescent or chemiluminescent substance or enzyme. After the addition of the respective substrate, fluorescence, chemiluminescence or visible light due to the enzymatic reaction is measured, for evaluation and decision.
Further examples of methods are illustrated below (see LTP 2), although the invention is not limited to such examples.
4. Methods that use microarrays (microchips)
A microarray is a general term for devices in which solidified materials with affinity for target substances are applied neatly on a solid support (plate). In the present invention, antibodies or aptamers of proteins and partial peptides are neatly applied. A sample of biological material is applied on the microarray for the fixation of proteins or partial target peptides and then the microarray is incubated with secondary antibody labeled with fluorescent or chemiluminescent substance or enzyme. After the addition of the respective substrate, fluorescence, chemiluminescence or visible light due to the enzymatic reaction is measured.
5. Mass spectrometry
In mass spectrometry, for example, the antibody of a specified partial protein or peptide binds to chemically modified microbeads or plaque (protein chip). The microbeads can be magnetic beads. There are no requirements for the plate material. The antibody to be used could be: (1) an antibody that recognizes the full length form of the specified protein only, (2) an antibody that recognizes only a partial peptide, (3) all antibodies that recognize both the specified protein as its partial peptide, or a combination of (1) and (2), (1) and (3) or (2) and (3). Samples after stepwise dilution with original solvent or buffer are added to the microbeads or carrier plate of the antibody or antibodies and incubated. Uncaptured partial proteins and peptides are removed by washing. The protein or partial peptide captured with microbeads or plaque is eluted and analyzed by mass spectrometry with MALDI-TOF-EM, SELDI-TOF-EM, etc. Measurements are made with respect to the mass and intensity of the peak due to the protein, protein fragment or partial peptide. Before the measurements, a fixed amount of a substance that serves as an internal standard is added to the original biological material and the intensity of the peak thereof is also measured. The concentration of the target in the original biological material can be calculated from the ratio of intensities of the peak of the target and the peak of the internal standard. The above is called the immuno-MS method. In addition, quantification is possible, after diluting the sample with the original solvent or buffer, or after removing part of the proteins, by HPLC separation followed by mass spectrometry using the electrospray ionization method (IEP). In this way the SRM / MRM method can be used for absolute quantification using an internal standard peptide isotopically labeled.
In addition, in addition to the methods indicated above, it is possible to analyze proteins and partial peptides by using 2-DE, surface plasmon resonance, etc.
The present invention includes the method of detecting cognitive impairment from the presence or absence or amount of the biomarker indicated above after applying the biological material obtained from the test subject to 2- ED or surface plasmon resonance.
Example 1
Discovery of a marker peptide for the detection of cognitive impairment using two-dimensional liquid chromatography (2D-CL) -MALDI TOF-EM.
(1) Serum samples
Next, the characters in parentheses are an abbreviation.
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Serums obtained from 20 patients of AD (Alzheimer's disease), 20 DNA patients (subjects who do not suffer from psychiatric disease and patients of corresponding age and sex with AD, "N" means normal) and 20 patients from EN (all neurological disease) . All consists of 10 ENdem patients (neurological disease dementia) and 10 ENnon (neurological disease non-dementia). In addition, ENdem consists of dementia with Lewy bodies and frontotemporal dementia, each consisting of 5 cases and EDnon consists of schizophrenia and depression, consisting of each of 5 cases.
(2) Methods
After the addition of 475 pl of 0.1% trifluoroacetic acid (TFA) to each of the 25 pl of serum, the samples were boiled for 15 min. at 100 degrees. Next, in order to recover peptides of a molecular weight of 10,000 or less, ultrafiltration was carried out using a YM-10 filtration unit (Millipore Corp.). Next, the analysis using 2D-CL-MALDI TOF-EM was carried out as follows. In other words, the recovered samples were fractionated into 1,146 fractions per sample using two-dimensional HPLC (SCX cation exchange column and C18 reverse phase column). All fractionated samples were spotted on a MALDI target plate for MALDI TOF / TOF mass spectrometer (ultraflex TOF / TOF, Bruker Daltonics) and matrix solution (alpha-cyano-hydroxycinnamic acid, CHCA) and mixed and mixed. they crystallized, and the mass and peak area of the mass were automatically measured in refrectron mode by irradiating the crystallized sample by laser. The peak area was normalized with 250 fmoles for each well of bradykinin fragment 1-7 that was added to the matrix solution previously. In other words, the value of the area was calculated as 10,000 times the value by dividing the peak area in terms of specific sample mass by the peak area obtained from 250 fmoles of bradykinin fragment 17. Said area value corresponds to 25 pl of sample serum. The detection of the difference in serum peptide abundance between groups (called differential analysis) was carried out using the DeView multigroup statistical analysis software developed by the present inventors. The amino acid sequence of the peptides in which differences in abundance were observed by EM / MS analysis by ultraflex TOF / TOF were directly determined and intact proteins or peptides of origin were identified.
(3) Results
Figure 1 shows the result obtained from the serum of an EA patient case that was applied to 2D-CL-MALDI TOF-EM. The samples were fractionated into 6 fractions by SCX cation exchange column in the first dimension, and then each of the fractions were fractionated into 191 fractions by C18 reverse phase column. Mass spectra of 191 fractions were obtained by MALDI TOF-EM measurements. Because the horizontal axis is m / z and the vertical axis are the reverse phase column chromatography fractions; Figure 1 was visualized by Deview. SCX 1 shows the eluted fractions; SCX 2 shows the eluted fractions with a salt concentration of 10%; SCX 3 shows the eluted fractions with a saline concentration of 20%; SCX 4 shows the eluted fractions with a saline concentration of 30%; SCX 5 shows the eluted fractions with a saline concentration of 50%; SCX 6 shows the eluted fractions with a salt concentration of 100%. As seen in Figure 1, many peptides are present in many sera of SCX 1, SCX 3, SCX 4 and SCX 5. The total number of peptides fractionated by 2D-CL and detected by MALDI TOF-EM was approximately 4,000
As an example of the results of the differential analysis, Figure 2 shows the case of marker A. The marker A, as shown below, in Figure 3, was the NRX2B peptide derived from a neurexin-2 beta precursor. A) of Figure 2 shows a comparison between DNA, DCL and EA. A) and B) show the result of experiments carried out separately; DNA and EA were used in the same samples in both experiments (ie, for DNA and EA, the measurement results would indicate reproducibility). In A) of Figure 2 it was found that marker A was increased in patients with DCL and AD compared to DNA patients. In B) of Figure 2 it was found that marker A was increased in patients with AD, EN, all, ENdem and ENnon compared with DNA patients. In particular, in the comparison between EA and ENnon, in EA it was significantly higher than in ENnon (t test, p = 0.035). From these results it was found that marker A was useful to distinguish between patients with cognitive impairment (DCL, EA, ENdem) and patients with non-dementia neurological disease (ENnon).
From the results of A) and B) in Figure 2, in order to assess the degree to which marker A is useful as a biomarker, the analysis was carried out using the receiver operating characteristic curve (COR ). C), D) and E) in Figure 2 show, respectively, the COR curve of the DCL vs. comparisons. DNA, EA vs. DNA and EA vs. ENnon In the event that the value of the area (hereinafter referred to as the COR value) under the COR curve is close to 1, the utility as a biomarker of marker A will be higher. In C), D) and E) of Figure 2, the typical values of sensitivity and specificity are the values of the point (white squares in the figure) of the coordinate in the COR curve at which the distance is minimized when drawing a straight line to the COR curve from the 100% point on the y axis. The cut-off value provided by said point becomes a useful threshold to distinguish between the different groups, and the sensitivity and specificity values at that time (that is, higher than the typical values) becomes an indicator of the utility of the biomarkers together with the COR values. In C) of Figure 2, as typical values in DCL vs. DNA, the sensitivity was 90%, the specificity was 100% and the
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COR value was 0.99. In D) of Figure 2, as typical values in EA vs. DNA, the sensitivity was 100%, the specificity was 100% and the COR value was 1. In E) of Figure 2, with the comparison EA vs. CCC, the sensitivity was 100%, the specificity was 50% and the COR value was 0.710. Thus, it was revealed that marker A (NRX2B) was useful for distinguishing DCL and EA from DNA. And in addition, it was revealed that marker A was useful for distinguishing AD from non-dementia neurological disease (ENnon). In particular, because DCL is the state of the previous stage of AD, marker A (NRX2B) is considered to be an extremely useful marker for detecting DCL for the early diagnosis of subjects who will potentially migrate to AD.
Figure 3, for marker A, illustrates the results of the EM / MS spectrum using ultraflex TOF / TOF. The signals showing the ions and the b ions appeared in sufficient degree and the amino acid sequence could be easily identified. A Mascot search was carried out with said result and the source protein or the peptide (hereinafter referred to as intact proteins or peptides) was the precursor of neurexin-2-beta and the detected peptide was found to have the sequence RSGgNaTLQvDsWP ( SEQ ID No. 2). NRX2B is the name of the Swiss-Prot entry of the precursor of neurexin-2-beta as an abbreviation of the name of the peptide. In addition, for the other peptides that were detected, the Swiss-Prot entry names used as abbreviations of the peptide names in the description below.
Including the A marker, peptides that showed differences in abundance between groups in the serum were measured in the MS / MS spectra using ultraflex TOF / TOF and in addition to determining the amino acid sequence, the results identified intact proteins or peptides shown below. For peptides other than marker A, the signals showing ions and b ions appeared to a sufficient degree and the amino acid sequence could be easily identified. The following amino acid sequence showing a group of two sequences, the entire sequence of the first sequence shows the amino acid sequence of intact proteins or peptides. The peptide comprising the underlined part of the first sequence and the second sequence is the peptide detected by 2D-CL-MALDI TOF-EM. 001 represents the N-terminal end. Regarding the peptide that was sequenced with the oxidation state of methionine in the peptides consisting of the second sequence, it was indicated as (oxidation + (M)) at the end of the amino acid sequence. For the protein with amino acid mutation by gene mutation, the applicable amino acid residue is indicated as (X).
(1) NRX2B peptide derived from the precursor of neurexin-2-beta
NRX2B shown as SEQ ID No. 2 was not detected in the DNA patient and was detected in the patients of DCL, EA, ENtodas, ENdem and ENnon. In addition, in the comparison between EA and ENnon, the EA showed a higher value than ENnon, and it was shown that NRX2B showed the ability to distinguish (previously described in Figure 2).
Intact protein / peptide
001 MPPGGSGPGGCPRRPPALAG PLPPPPPPPP PPLLPLLPLL LLLLLGAAEG 051 ARVSSSLSTT HHVHHFHSKH GTVPIAINRM PFLTRGGHAG TTYIFGKGGA 101 LITYTWPPND RPSTRMDRLA VGFSTHQRSA VLVRVDSASG LGDYLQLHID 151 QGTVGVIFNV GTDDITIDEP NAIVSDGKYH WRFTRSGGN ATLOVDSWPV 201 NERYPAGNFD NERLAIARQR IPYRLGRWD EWLLDKGRQL TIFNSQAAIK 251 IGGRDQGRPF QGQVSGLYYN GLKVLALAAE SDPNVRTEGH LRLVGEGPSV 301 LVASAECPSD DEDLEECEPS TGGELILPII TEDSLDPPPV ATRSPFVPPP 351 PTFYPFLTGV GATQDTLPPP AARRPPSGGP CQAERDDSDC EEPIEASGFA 401 SGEVFDSSLP PTDDEDFYTT FPLVTDRTTL LSPRKPAPRP NLRTDGATGA 451 PGVLFAPSAP APNLPAGKMN HRDPLQPLLE NPPLGPGAPT SFEPRRPPPL 501 RPGVTSAPGF PHLPTANPTG PGERGPPGAV EV1RESSSTT GMWGIVAAA 551 ALCÍLILLYA MYKYRNRDEG SYQVDQSRNY ISNSAQSNGA WKEKAPAAP 601 KTPSKAKKNK DKEYYV (SEQ ID No. 1)
NRX2B peptide derived from the precursor of neurexin-2-beta
RSGGNATLQVDSWP (SEQ ID No. 2)
(2) THRB (R-) peptide derived from the prothrombin precursor
Prothrombin precursor derived peptides are of two types and (R-) refers to the peptide that does not have R (arginine residue) at the C-terminal end. THRB (R-), shown as SEQ ID No. 4, was specifically detected in the DNA patient and an extremely low value was detected in the patients of DCL, EA, ENtodas, ENdem and ENnon. The THRB (R-) and THRB (R +) diagrams are shown side by side in Figures 4 and 5. Figure 4 shows a scatter plot. Figure 5A shows that the appearance of THRB (R-) and THRB (R +) is different in each individual of DNA, DCL and EA. In the same individual, THRB (R-) appeared very high in DNA; THRB (R +) appeared
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to a very high degree in DCL and EA. It can be said that both peptides are extremely useful markers for determining DCL and DNA. Figure 5B shows the COR curve comparing EA and ENnon with THRB (R-). The COR value indicated a high value of 0.815. The value in the EA was lower than in the ENnon. In other words, THRB (R-), as well as NRX2B, was found as a useful marker to distinguish between patients with cognitive impairment (DCL, EA and ENdem) and patients with non-dementia neurological disease (ENnon).
Intact protein / peptide
001 MAHVRGLQLP GCLALAALCS LVHSQHVFLA PQQARSLLQR VRRANTFLEE 051 VRKGNLEREC VEETCSYEEA FEALESSTAT DVFWAKYTAC ETARTPRDKL 101 AACLEGNCAE GLGTNYRGHV NITRSGIECQ LWRSRYPHKP EINSTTHPGA 151 DLQENFCRNP DSSTTGPWCY TTDPTVRRQE CSIPVCGQDQ VTVAMTPRSE 201 GSSVNLSPPL EQCVPDRGQQ YQGRLAVTTH GLPCLAWASA QAKALSKHQD 251 FNSAVQLVEN FCRNPDGDEE GVWCYVAGKP GDFGYCDLNY CEEAVEEETG 301 DGLDEDSDRA IEGRTATSEY QTFFNPRTFG SGEADCGLRP LFEKKSLEDK 351 TERELLESYI DGRIVEGSDA EIGMSPWQVM LFRKSPQELL CGASLISDRW 401 VLTAAHCLLY PPWDKNFTEN DLLVRIGKHS RTRYERNIEK ISMLEKIYIH 451 PRYNWRENLD RDIALMKLKK PVAFSDYIHP VCLPDRETAA SLLQAGYKGR 501 VTGWGNLKET WTANVGKGQP SVLQWNLPI VERPVCKDST RIRITDNMFC 551 AGYKPDEGKR GDACEGDSGG PFVMKSPFNN RWYQMGIVSW GEGCDRDGKY GFYTHVFRLK KWIQKVIDQF GE 601 (SEQ ID No 3)
THRB (R-) peptide derived from the prothrombin precursor GLDEDSDRAIEG (SEQ ID No. 4)
(3) Prothrombin precursor derived peptide (THRB (R +))
THRB (R-), shown as SEQ ID No. 5, DNA was not detected in the patient and was detected in patients with DCL, EA, ENtodas, ENdem and ENnon (Figure 4). (R +) refers to the fact that the peptide has R (arginine residue) at the C-terminal end. For an explanation, see (2) prothrombin precursor derived peptide (THRB (R-)).
Intact protein / peptide
001 MAHVRGLQLP GCLALAALCS LVHSQHVFLA PQQARSLLQR VRRANTFLEE 051 VRKGNLEREC VEETCSYEEA FEALESSTAT DVFWAKYTAC ETARTPRDKL 101 AACLEGNCAE GLGTNYRGHV NITRSGIECQ LWRSRYPHKP EINSTTHPGA 151 DLQENFCRNP DSSTTGPWCY TTDPTVRRQE CSIPVCGQDQ VTVAMTPRSE 201 GSSVNLSPPL EQCVPDRGQQ YQGRLAVTTH GLPCLAWASA QAKALSKHQD 251 FNSAVQLVEN FCRNPDGDEE GVWCYVAGKP GDFGYCDLNY CEEAVEEETG 301 DGLDEDSDRA IEGRTATSEY QTFFNPRTFG SGEADCGLRP LFEKKSLEDK 351 TERELLESYI DGRIVEGSDA EIGMSPWQVM LFRKSPQELL CGASLISDRW 401 VLTAAHCLLY PPWDKNFTEN DLLVRIGKHS RTRYERNIEK ISMLEKIYIH 451 PRYNWRENLD RDIALMKLKK PVAFSDYIHP VCLPDRETAA SLLQAGYKGR 501 VTGWGNLKET WTANVGKGQP SVLQWNLPI VERPVCKDST RIRITDNMFC 551 AGYKPDEGKR GDACEGDSGG PFVMKSPFNN RWYQMGIVSW GEGCDRDGKY GFYTHVFRLK KWIQKVIDQF GE 601 (SEQ ID No 3)
THRB (R +) peptide derived from the prothrombin precursor GLDEDSDRAIEGR (SEQ ID No. 5)
(4) Pendrin derived peptide (S26A4)
S26A4, shown as SEQ ID No. 7, DNA was not detected in the patient and was detected in patients with DCL, EA, ENtodas, ENdem and ENnon (Figure 6).
Intact protein / peptide
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001 MAAPGGRSEP PQLPEYSCSY MVSRPVYSEL AFQQQHERRL QERKTLRES 051 AKCCSCSRKR AFGVLKTLVP ILEWLPKYRV KEWLLSDVIS GVSTGLVATL 101 QGMAYALLAA VPVGYGLYSA FFPILTYFIF GTSRHISVGP FPWSLMVGS 151 WLSMAPDEH FLVSSSNGTV LNTTMIDTAA RDTARVLIAS ALTLLVGIIQ 201 LIFGGLQIGF IVRYLADPLV GGFTTAAAFQ VLVSQLKIVL NVSTKNYNGV 251 LSIIYTLVEI FQNIGDTNLA DFTAGLLTIV VCMAVKELND RFRHKIPVPI 301 PIEVIVTIIA TAISYGANLE KNYNAGIVKS IPRGFLPPEL PPVSLFSEML 351 AASFSIAWA YAIAVSVGKV YATKYDYTID GNQEFIAFGI SNIFSGFFSC 401 FVATTALSRT AVQESTGGKT QVAGIISAAI VMIAILALGK LLEPLQKSVL 451 AAWIANLKG MFMQLCDIPR LWRQNKIDAV IWVFTCIVSIILGLDLGLLA 501 GLIFGLLTW LRVQFPSWNG LGSIPSTDIY KSTKNYKNIE EPQGVKILR 551 SSPIFYGNVD GFKKCIKSTV GFDAIRVYNK RLKALRKIQK LIKSGQLRAT 601 KNGIISDAVS TNNAFEPDED IEDLEELDIP TKEIEIQVDW NSELPVKVNV 651 PKVPIHSLVL DCGAISFLDV VGVRSLRVIV KEFQRIDVNV YFASLQDYV 701 EKLEQCGFFD DNIRKDTFFL TVHDAILYLQ NQVKSQEGQG SILETITLIQ 751 DCKDTLELIE TELTEEELDV QDEAMRTLAS (SEQ ID No 6)
Pendulum S26A4 peptide derived from LAGLIFGLLTVVLR (SEQ ID No. 7)
(5) Peptide derived from zeta-1 subunit of coatomer (COPZ1)
COPZ1, shown as SEQ ID No. 9, showed a low value in the DNA patient and a high value in the patients of DCL, EA and ENdem (Figure 6).
Intact protein / peptide
001 MEALILEPSL YTVKAILILD NDGDRLFAKY YDDTYPSVKE QKAFEKNIFN 051 KTHRTDSEIA LLEGLTWYK SSIDLYFYVI GSSYENELML MAVLNCLFDS 101 LSQMLRKNVE KRALLENMEG LFLAVDEIVD GGVILESDPQ QWHRVALRG 151 EDVPLTEQTV SQVLQSAKEQ IKWSLLR {SEQ ID No. 8)
Peptide derived from zeta-1 subunit of coatomer (COPZ1)
AILILDNDGDRLFAKYYDD (SEQ ID No. 9)
(6) Peptide derived from the precursor of retinoic acid receptor responsive protein 2 (RARR2 (S-))
RARR2 (S-), shown as SEQ ID No. 11, DNA was not detected in the patient and was detected in the AD and DCL patients (Figure 7). Peptides derived from retinoic acid receptor responsive protein 2 precursor and (S-) refers to the peptide that does not have S (serine residue) at the C-terminal end.
Intact protein / peptide
001 MRRLLIPLAL WLGAVGVGVA ELTEAQRRGL QVALEEFHKH PPVQWAFQET 051 SVESAVDTPF PAGIFVRLEF KLQQTSCRKR DWKKPECKVR PNGRKRKCLA 101 CIKLGSEDKV LGRLVHCPIE TQVLEDFQQQFQQFQQQFQQFQQQFQQQFQQQFQQGQQGQQQFQQQGQGQQQGQQQGQQGQGGQGQQQQQQQKDGQQQGQGQGQGQGQQKQQQQKDGQQQGQGQGQGQGQGQQKQQQQFQGQG
Peptide derived from the precursor of retinoic acid receptor responsive protein 2 (RARR2 (S-)) PHSFYFPGQFAFSKALPR (SEQ ID No. 11)
(7) Peptide derived from the precursor of retinoic acid receptor responsive protein 2 (RARR2 (S +))
RARR2 (S +), shown as SEQ ID No. 12, no DNA was detected in the patient as well as RARR2 (S-), and it was detected in patients with AD and DCL (Figure 7). (S +) refers to the fact that the peptide has S (serine residue) at the C-terminal end.
Intact protein / peptide
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Peptide derived from the precursor of retinoic acid receptor responsive protein 2 (RARR2 (S +)) PHSFYFPGQFAFSKALPRS (SEQ ID No. 12)
(8) Gelsolin precursor derived peptide (GELS)
GELS, shown as SEQ ID No. 14, showed a low value in the DNA patient and a relatively high value in the patients of DCL and EA (Figure 8).
Intact protein / peptide
OO1 MAPHRPAPAL LCALSLALCA LSLPVRAATA SRGASQAGAP QGRVPEARPN 051 SMWEHPEFL KAGKEPGLQI WRVEKFDLVP VPTNLYGDFF TGDAYVILKT 101 VQLRNGNLQY DLHYWLGNEC SQDESGAAAI FTVQLDDYLN GRAVQHREVQ 151 GFESATFLGY FKSGLKYKKG GVASGFKHW PNEVWQRLF QVKGRRWRA 201 TEVPVSWESF NNGDCFILDL GNNIHQWCGS NSNRYERLKA TQVSKGIRDN 251 ERSGRARVHV SEEGTEPEAM LQVLGPKPAL PAGTEDTAKE DAANRKLAKL 301 YKVSNGAGTM SVSLVADENP FAQGALKSED CFILDHGKDG KIFVWKGKQA 351 NTEERKAALK TASDFITKMD YPKQTQVSVL PEGGETPLFK QFFKNWRDPD 401 QTDGLGLSYL SSHIANVERV PFDAATLHTS TAMAAQHGMD DDGTGQKQIW 451 RIEGSNKVPV DPATYGQFYG GDSYIILYNY RHGGRQGQII YNWQGAQSTQ 501 DEVAASAILT AQLDEELGGT PVQSRWQGK EPAHLMSLFG GKPMIIYKGG 551 TSREGGQTAP ASTRLFQVRA NSAGATRAVE VLPKAGALNS NDAFVLKTPS 601 AAYLWVGTGA SEAEKTGAQE LLRVLRAQPV QVAEGSEPDG FWEALGGKAA 651 YRTSPRLKDK KMDAHPPRLF ACSNKIGRFV IEE TGELMQ EDLATDDVML 701 LDTWDQVFVW VGKDSQEEEK TEALTSAKRY IETDPANRDR RTPITWKQG 751 FEPPSFVGWF LGWDDDYWSV DPLDRAMAEL AA (SEQ ID No. 13)
Peptide derived from gelsolin precursor (GELS)
PVRAATASRGAS (SEQ ID No. 14)
(9) Peptide derived from the clusterin precursor (CLUS (SDVP N-term.))
CLUS (SDVP N-term.), Shown as SEQ ID No. 16, showed a low value in the DNA patient and a relatively high value in the patients of DCL and EA (Figure 9). Clusterin precursor derived peptides are of two types and (N-termSDVP) refers to the amino acid sequence of the N-terminal end of the peptide being SDVP.
Intact protein / peptide
001 MMKTLLLFVG LLLTWESGQV LGDQTVSDNE LQEMSNQGSK YVNKEIQNAV
051 NGVKQIKTLI EKTNEERKTL LSNLEEAKKK KEDALNETRE SETKLKELPG
101 VCNETMMALW EECKPCLKQT CMKFYARVCR SGSGLVGRQL EEFLNQSSPF
151 YFWMNGDRID SLLENDRQQT HMLDVMQDHF SRASSIIDEL FQDRFFTREP
201 QDTYHYLPFS LPHRRPHFFF PKSRIVRSLM PFSPYEPLNF HAMFQPFLEM
251 IHEAQQAMDI HFHSPAFQHP PTEFIREGDD DRTVCREIRH NSTGCLRMKD
301 QCDKCREILS VDCSTNNPSQ AKLRRELDES LQVAERLTRK YNELLKSYQW
351 KMLNTSSLLE QLNEQFNWVS RLANLTQGED QYYLRVTTVA SHTSDSDVPS
401 GVTEWVKLF DSDPITVTVP VEVSRKNPKF METVAEKALQ EYRKKHREE (SEQ ID
n ° 15)
CLUS clusterin precursor derived peptide (SDVP N-term.)
SDVPSGVTEVVVKLFDS (SEQ ID No. 16)
(10) Peptide derived from the clusterin precursor (CLUS (RFFT N-term.))
CLUS (RFFT N-term.), Shown as SEQ ID No. 17, DNA was not detected in the patient and was not fully detected in AD patients (Figure 9). (RFFT N-term.) Refers to the amino acid sequence of the N-terminal end of the peptide is RFFT.
Intact protein / peptide
001 MMKTLLLFVG LLLTWESGQV LGDQTVSDNE LQEMSNQGSK YVNKEIQNAV
051 NGVKQIKTL1 EKTNEERKTL LSNLEEAKKK KEDALNETRE SETKLKELPG
101 VCNETMMALW EECKPCLKQT CMKFYARVCR SGSGLVGRQL EEFLNQSSPF
151 YFWMNGDRID SLLENDRQQT HMLDVMQDHF SRASSIIDEL FQDRFFTREP
201 QDTYHYLPFS LPHRRPHFFF PKSRIVRSLM PFSPYEPLNF HAMFQPFLEM
251 IHEAQQAMDI HFHSPAFQHP PTEFIREGDD DRTVCREIRH NSTGCLRMKD
301 QCDKCREILS VDCSTNNPSQ AKLRRELDES LQVAERLTRK YNELLKSYQW
351 KMLNTSSLLE QLNEQFNWVS RLANLTQGED QYYLRVTTVA SHTSDSDVPS
401 GVTEVWKLF DSDPITVTVP VEVSRKNPKF METVAEKALQ EYRKKHREE (SEQ ID
n ° 15)
5 Peptide derived from the CLUS clusterin precursor (RFFT N-term.)
RFFTREPQDTYHYLPFSLPH (SEQ ID No. 17)
(11) Peptide derived from subunit J of eukaryotic translation initiation factor 3 (EIF3J)
10
EIF3J, shown as SEQ ID No. 19, was not detected in the patient DNA and was not detected at all or practically was not detected in the patients of DCL, EA, ENtodas, ENdem and ENnon (Figure 10).
Intact protein / peptide 15
001 MAÁAAAAAGD SDSWDADAFS VEDPVRKVGG GGTAGGDRWE GEDEDEDVKD 051 NWDDDDDEKK EEAEVKPEVK ISEKKKIAEK IKEKERQQKK RQEEIKKRLE 101 EPEEPKVLTP EEQLADKLRL KKLQEESDLE LAKETFGVNN AVYGIDAMNP 151 SSRDDFTEFG KLLKDKITQY EKSLYYASFL EVLVRDVCIS LEIDDLKKIT 201 NSLTVLCSEK QKQEKQSKAK KKKKGWPGG GLKATMKDDL ADYGGYDGGY VQDYEDFM 251 (SEQ ID No. 18)
twenty
Peptide derived from subunit J of eukaryotic translation initiation factor 3 (EIF3J) GVVPGGGLKATMKDDLADYGGYDGG + oxidation (M) (SEQ ID No. 19)
(12) Protein 27 derived peptide containing leucine rich repeats (LRC27)
25 LRC27, shown as SEQ ID No. 21, DNA was not detected in the patient and was not fully detected in AD patients (Figure 10).
30
Intact protein / peptide
001 MEGSSSYEVP SVAAADLEEG AGQTRSLPAT PSKDVHKGVG GIIFSSSPIL 051 DLSESGLCRL EEVFRIPSLO QLHLQRNALC VIPQDFFQLL PNLTWLDLRY 101 NRIKALPSGI GAHQHLKTLL LERNPIKMLP VELGSVTTLK ALNLRHCPLE 151 FPPQLWQKG LVAIQRFLRM WAVEHSLPRN PTSQEAPPVR EMTLRDLPSP 201 GLELSGDHAS NQGAVNAQDP EGAVMKEKAS FLPPVEKPDL SELRKSADSS 251 ENWPSEEEIR RFWKLRQEIV EHVKADVLGD QLLTRELPPN LKAALNIEKE 301 LPKPRHVFRR KTASSRSILP DLLSPYQMAI RAKRLEESRA AALRELQEKQ 351 ALMEQQRREK RALQEWRERA QRMRKRKEEL SKLLPPRRSM VASKIPSATD 401 LIDNRKVPLN PPGKMKPSKE KSPQASKEMS ALQERNLEEK IKQHVLQMRE 451 QRRFHGQAPL EEMRKAAEDL EIATELQDEV LKLKLGLTLN KDRRRAALTG 501 NLSLGLPAAQ PQNTFFNTKY GESGNVRRYQ ID 20)
Peptide derived from protein 27 containing leucine-rich repeats (LRC27)
SSPILDLSESGLCRLEEVFRIPS (SEQ ID No. 21)
35
Although they have already been cited, for the peptides of SEQ ID No. 2 (NRX2B) to SEQ ID No. 21 (LRC27), the scatter plots of the comparisons between the DNA, DCL and EA patients, and the scatter plot of the comparison between the patients of DNA, AD, all, ENdem and ENnon, and the p-value of the t-test of each comparison are shown in Figures 2, 4 and 6 to 10.
5
10
fifteen
twenty
25
30
35
40
Four. Five
fifty
Table 1 shows the list of COR values of the DCL vs. comparisons. DNA and EA vs. DNA with respect to the 12 marker peptides shown above.
[Table 1]
 Marker peptides  DCL vs. EA DNA vs. DNA
 Swiss-Prot entry  Sequence No. COR value DCL was COR value In the EA:
 NRX2B  2 0.99 increase 1 increase
 THRB (R-)  4 0.854 reduction 0.841 reduction
 THRB (R +)  5 0.94 increase 0.985 increase
 S26A4  7 0.925 increase 0.95 increase
 COPZ1  9 0.786 increase 0.767 increase
 RARR2 (S-)  11 0.885 increase 0.914 increase
 RARR2 (S +)  12 0.95 increase 0.919 increase
 GELS  14 0.716 increase 0.762 increase
 CLUS (SDVP N-term.)  16 0.739 increase 0.717 increase
 CLUS (RFFT N-term.)  17 0.675 reduction 0.75 reduction
 EIF3J  19 0.748 reduction 0.775 reduction
 LRC27  21 0.699 reduction 0.755 reduction
Table 1 shows the usefulness of each marker peptide in the detection of cognitive impairment (DCL and EA). By using said labeled peptides, individually or in combination, using or not liquid chromatography and / or any other suitable separation methods, directly measuring serum abundance using other methods, such as mass spectrometry or immunological methods or enzymatic methods It is possible to distinguish between types of non-dementia and dementia in neurological disease and diagnose cognitive impairment, such as AD and MCI. The marker peptide that is not detected in the DNA patient and that is detected in the patients of DCL, Ea, ENtodas, ENdem and ENnon, or vice versa, the marker peptide that is detected in the DNA patient and that is not detected in The patients of DCL, EA, ENtodas, ENdem and ENnon, are also useful for the detection of psychiatric diseases.
Example 2
Example 2. Synthesis of a marker peptide and preparation of a polyclonal antibody specific for marker peptide
The antigenic peptide was synthesized in order to prepare the specific antibody that recognizes the NRX2B peptide derived from the neurexin-2-beta precursor of SEQ ID No. 2. To the synthetic peptide for coupling to a carrier protein was added the residue of cysteine (called C or Cys) at the C-terminal end. The peptide that was combined with the carrier protein (RSGGNATC-KLH, see below) was mixed with an adjuvant and the mixture was used to immunize rabbits. A total of eight immunizations were carried out every 1-2 weeks and the test blood was extracted twice every 4 weeks and the antibody titers were measured by enzymatic immunoassay (ElA). After three months from the beginning of the immunization, the whole blood of the rabbits was collected and the antiserum was obtained; in addition, purification of the specific antibody was carried out using the peptide column in which the antigenic peptide was bound as a ligand.
The sequence of the synthetic antigenic peptide for the preparation of the peptide specific antibody is shown below.
RSGGNAT + Cys (SEQ ID No. 22)
Example 3
Example 3. Preparation of antibody beads (1) Method
(1-1) Preparation of antibody and binding to magnetic beads
The antibody solution: 1 mg of the antibody (anti-NRX2B antibody, rabbit IgG) that specifically recognized the amino acid sequence peptide expressed by SEQ ID No. 22 was dissolved with 3 ml of 0.1 M MES. After washing 1 ml (10 mg of beads) of the magnetic beads (Magnosphere MS300 / carboxyl, JSR Corporation) by using 0.1 M MES, the magnetic beads were mixed with the antibody solution and
5
10
fifteen
twenty
25
30
35
40
Four. Five
fifty
55
60
65
stirred gently for 30 min. at room temperature.
(1-2) Cross-linking of antibody and magnetic beads
400 μl of EDC solution (10 mg / ml of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride in 0.1 M MES) was added to antibody-pearl solution and gently suspended for 3 hours for antibody binding to beads by covalent bond.
(1-3) Lock
1 ml of 200 mM ethanolamine (pH 8.0) was added to wash the beads and an additional 1 ml of 200 mM ethanolamine (pH 8.0) was added and gently stirred for 1 h at room temperature to block the amine groups.
(1-4) Wash
After removal of 200 mM ethanolamine (pH 8.0) the beads were washed three times with 1 ml of TBST solution (25 mM Tris-HCl (pH 7.2) containing 0.15 M NaCl and 0-Tween-20 , 05%).
(1-5) Storage
After suspending the beads by adding 1 ml of TBST solution, they were stored at 4 ° C.
Example 4
Example 4. Proof by the method of immuno-MS that the peak of m / z 1,488 in patient serum detected by 2D-CL-MALDI-TOF-EM is NRX2B.
(1) Methods
As a control for comparison, the stable isotope labeled NRX2B synthetic peptide (12C and 13C of V was replaced by 15N and 14N) of mass greater than NRX2B. The mass difference between NRX2B and the stable isotope peptide was 6 u. Both the endogenous peptide and the stable isotope-labeled peptide were captured with anti-NRX2B antibody. 1 pl of 200 fmoles / pl of synthetic isotope-labeled NRX2B synthetic peptide was added to 25 pl of each of the EA and DCL patient sera, and incubated for 10 min. at 4 ° C. Then, 475 pl of 0.1% trifluoroacetic acid (TFA) was added and boiled for 5 min. at 100 ° C. After centrifugation for 15 min. at 14,000xg, 500 µl of 100 mM Tris-Hcl buffer (pH 7.5) containing 0.3 M NaCl and 0.2% n-octyl glycoside were added to the supernatant. 20 pl of anti-NRX2B antibody beads in Example 3 were added to peptide solutions and gently stirred for 2 hours. Then, after resting for 1 min. on the magnetic support, the supernatant was removed. 1 ml of 50 mM Tris-HCl buffer (pH 7.5) (TBS) containing 0.15 M NaCl and 0.1% n-octyl glucoside was added and gently stirred for 10 min. After standing for 1 min. on the magnetic support, the supernatant was removed. In addition, after adding 500 pl of TBS and let stand for 1 min. on the magnetic support, the supernatant was removed. This procedure was repeated three times. In addition, after adding 500 µl of 50 mM ammonium carbonate and let stand for 1 min. on the magnetic support, the supernatant was removed. This procedure was repeated three times. 50 pl of 2-propanol: H2O: formic acid (4: 4: 1) was added and allowed to stand for 10 min. and then, after resting for 1 min. on the magnetic support, the filtrate was recovered. This procedure was repeated twice. The filtrates were dried completely using a vacuum centrifuge. Then, 20 µl of 0.095% TFA containing 5% acetonitrile was added and redissolved by sonication. The peptides were concentrated using a C18 pipette tip (PerfectPure C-18 tip, Eppendorf) and applied on points on the MALDI target plate (AnchorChip ™ 600/384 MTP plate, Bruker Daltonics) by elution from the tip of C18 pipette and then the peptides were analyzed using a MALDI TOF mass spectrometer (AXiMa CFRplus, Shimadzu).
(2) Results
Figure 11 shows the result of the mass spectrometry of the NRX2B peptide detected in the serum of EA and DCL patients using the method indicated above. Figure 11A shows the global mass spectrum and Figure 11B shows an enlarged view of the parts with arrows in Figure 11A. The signal indicated by the dashed arrows in Figure 11B is the synthetic NRX2B peptide labeled with stable isotope added and the signal indicated by the continuous arrows was endogenous NRX2B peptide. The observed mass value was within the error of measurement of the expected value. And in addition the mass difference between the endogenous NRX2B peptide and the stable isotope-labeled peptide was 6 u. Therefore, it could be shown that the trapped peptide was NRX2B.
In the present experiment, NRX2B, which was the peptide marker detected in the serum by using the immuno-MS method originally developed by the present inventors, could be shown to result
18
5
10
fifteen
twenty
25
30
35
40
It is possible to distinguish between EA and DCL patients with respect to DNA patients. In addition, in the present experiment, it has also been shown that the specific antibody against NRX2B is useful in the detection of its peptide marker. Additionally, it also demonstrates that the immunological detection method could be effective against the peptide or protein comprised in the amino acid sequence of NRX2B using the specific antibody against NRX2B. In addition, in the present experiment the determination was made through the use of specific antibodies that recognized a peptide marker, but the combination of biomarker-specific antibodies that recognize other peptides found in Example 1, is expected to further increase the accuracy of the diagnosis of pathologies.
Industrial applicability
Cognitive impairment, including mild cognitive impairment and Alzheimer's disease and cognitive impairment and non-psychiatric illnesses, can be detected by using the biomarkers disclosed in the present invention. The invention is applicable to the use in the field of medical diagnosis, including that of diagnostic agents.
Sequence listing
09P01007_Sequence.txt LIST OF SEQUENCES
<110> MCBI Inc.
<120> New biomarkers for the detection of Alzheimer's disease <130> 09P01007 <160> 25
<170> PatentIn version 3.5
<210> 1 <211> 616 <212> PRT <213> Homo sapiens
<400> 1

权利要求:
Claims (10)
[1]
1. Peptide or protein for use as a biomarker for the detection of psychiatric diseases or cognitive impairment, in which
5 Protein is a precursor of neurexin-2-beta consisting of the amino acid sequence
expressed by SEQ ID No. 1 or the protein is a protein consisting of the amino acid sequence obtained from SEQ ID No. 1 by deletion, exchange and / or addition of one to three amino acids, and
The peptide is the NRX2B peptide derived from a neurexin-2-beta precursor consisting of the
10 amino acid sequence expressed by SEQ ID No. 2 or the peptide is a peptide consisting
of an amino acid sequence derived from SEQ ID No. 2 by deletion, exchange and / or addition of one to three amino acids.
[2]
2. Peptide or protein according to claim 1, wherein the peptide or protein is a peptide for use
15 as a biomarker of cognitive impairment consisting of the expressed amino acid sequence
by SEQ ID No. 2 that is present or increased in biological material from patients with cognitive impairment compared to biological material of subjects who do not suffer from a psychiatric illness.
3. Peptide or protein according to claim 1, wherein the peptide or protein is a peptide for use
as a biomarker of Alzheimer's disease consisting of the amino acid sequence expressed by SEQ ID No. 2 that is present or increased in biological material from patients with Alzheimer's disease compared to the biological material of subjects who do not suffer from Neurological disease not dementia.
25
[4]
4. Peptide or protein according to any one of claims 1 to 3, wherein the peptide has a molecular weight of less than 10,000 or less than 50 amino acids.
[5]
5. In vitro method for the detection of a psychiatric illness or cognitive impairment that involves
Determination in biological material of at least one peptide or protein according to claim 1.
[6]
6. In vitro method according to claim 5 for the detection of cognitive impairment in which it is evaluated that the patient suffers from cognitive impairment in the event that, after the determination in biological material of the peptide as a biomarker of cognitive impairment according to claim 2 , it is observed that said peptide is
35 is present in a higher quantity than in a subject that does not suffer from a disease
Psychiatric
[7]
7. Kit for the detection of a psychiatric disease containing a specific antibody or aptamer for at least one peptide or protein according to claim 1, for the determination of at least one
40 peptide or protein according to claim 1.
[8]
8. Kit for the detection of cognitive impairment, which contains a specific antibody or aptamer for at least one peptide or protein according to claim 1, for the determination of at least one peptide or protein according to claim 1 or 2.
Four. Five
image 1
Z / LU
OR)
Area
Fig 2
A Marker A = NRX2B
P ® 0.707
image2
image3
image4
100-Specificity
DCL vs. DNA
Sensitivity
image5
100-Specificity EA vs. DNA
g
image6
100-Specificity EA vs. ENnon
s: breeding
Abs. Int * 1000
in
■ fs.
image7
‘‘
CO
Area
Fig. 4
TO
THRB
R-
P = 0.0000029
r
P «0.75
1 P = 0.00000358
image8
Area
P = 0.17155 ^ ^
¡-: ------- 1 P = 0.12676
| ----------------------- 1 P ~ 0.00431
I ---------'—------- 1
P - 0.43779
p-2jw ^ r P. „1194M ''
{----------- í ----------- j P = 0.00414
r
i p =
image9
image10
image11
image12
Sensitivity
B THRB
R-
image13
Area
TO
Fig. 6
9x105 8x105 7xt05 6x105 5x105 4x105 3x105
2x10s
P <0.05
P = 0.30854
I ------------ 1
1 P <0.05
 »•    *
 *    •
 * *
 ►    ♦
 F    1 * i
 Go    * i * •;
 ■ W f    m m i
 ...... Jl ........... ... r ....................
 DNA DCL EA
image14
co
<D
P = 0.25609
r
i p
r
P ® 0.00475
------------------------ j
P »0.40523
r
one
P = 4
r
i
P = 8,24438E-7
 - _ .4  I ---------- —11 3 = 1.3378C
 2.0x10  r * 1 l
 1.8x10 *  m am 9
 1.6x10®  «*
 1.4x10®
 1.2X10 1.0X10®
 8.0x10 * 6.0x10s  * * # #:
 4.0x10 *  : 1 _ * 1 ♦ • * * •
 0.0  i | t t X
EA ENOD ENdem ENnon
cu
0
OR
p m *
one
T P = 0.06287
L

 -
 *

 *  *
 * <9      «
 0
 -  | *
 T
 ♦ *:
 i * »A * -
 --one................... %
DCL
EA
D
you
(D
2,400 2,100 1,800 1.
0
1 p-
r
P = 0.35 í ----------- | P
r
i
P - 0.40 I ------ 1
1 P ® 0.79
one
 - *
 * ■  • * «
 p.    * m
 #
 ’*  • • *
 „*  one" •
 * • A * í *! * «4 s •: X
 • .......... i, ............  ..................... _i_
EA ENOD ENdem ENnon
RARR2
T-
s
or
i
or
or
R
or
II
I
00
s
Yes
or
o '»
or
n
UL
•
-H
h-
d)
<y>«><*>
ooooo
■ »......
BK ^ "P *
x XXXX
CO h- CD in ^
B0JV
3x103 2x103 1x103
<
LU
image15
THE
co
Q
<
image16
image17
image18
Area
image19
CLUS
SDVP N-term.
co
<D
P = 0.954757
J ----------- 1 P- 0.435567
l ----------------------- f P = 0.271468
í ---------------------------------- 1
P “0.532365
p ------- 1 P = 0.572272
P = 0.171892
------------------ j
5x102 4x102 3x102
1x102
r
r
i p = 0.86919 ---------- 1 P = 0.257674

 *  t
 w      «»
 *  • • •
 m m¡  •
 -    S * jfc • m a
 <w # W «w *
 r      T m t
 -  I $ l 1 ”W” "«
 **  * ♦
 ___l_______ t * t _______ l— ^
EA ENOD ENdem ENnon
image20
image21
image22
Area
image23
Area
image24
P - 0.273066
P = 0.311711 | ---------'-------- 1
| ----------------- 1 P = 0,175414
image25
CT>
TO
DCL sample
EA sample
image26
image27
t
Mass / Load
Fig. 11
image28
1489.2
r 090406-E4-lp48-2 0001, 0904Ü6-F4-p48-2 0001
Kratos PCAxíma CFRplus V2.4.0
% lnt 5.1mV 7.0mV
[1488]
1488.5
Sample
[1479]
1479.4
from DCL
[1495]
1495.1
Sample
from EA
Mass / Load

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引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

AU783760B2|1999-08-13|2005-12-01|David E. Berg|Method for detecting, treating, and monitoring conditions associated with activation of the coagulation response|

---

US20020037528A1|1999-08-13|2002-03-28|Berg David E.|Method for detecting procoagulant genetic and metabolic conditions associated with, and potentially predispositional for, activation of the coagulation response|

---

WO2002088322A2|2001-05-02|2002-11-07|Incyte Genomics, Inc.|Cell adhesion and extracellular matrix proteins|

---

WO2003087768A2|2002-04-12|2003-10-23|Mitokor|Targets for therapeutic intervention identified in the mitochondrial proteome|

---

AU2003253014A1|2002-06-21|2004-01-06|Innogenetics N.V.|Method for the diagnosis and differential diagnosis of neurological diseases|

---

DK2221620T3|2002-08-23|2014-01-06|Bayer Pharma AG|Polypeptide biomarkers for the diagnosis of Alzheimer's disease|

---

JP4170139B2|2003-05-07|2008-10-22|財団法人癌研究会|Method for detecting change in serum apolipoprotein A-II amount|

---

PT2211183E|2003-11-19|2013-05-31|Satoris Inc|Method for diagnosis and monitoring of alzheimer`s disease|

---

US7972601B2|2004-05-11|2011-07-05|The Regents Of The University Of California|Method of promoting delivery of an antioxidant agent to a cell expression neuroligin|

---

GB0421639D0|2004-09-29|2004-10-27|Proteome Sciences Plc|Methods and compositions relating to alzheimer's disease|

---

JP4801930B2|2005-05-02|2011-10-26|株式会社Ｍｃｂｉ|Novel liver cancer biomarker and method for detecting liver cancer using the biomarker|

---

WO2006121952A2|2005-05-05|2006-11-16|The Regents Of The University Of California|Diagnostic biomarkers for neurodevelopmental disorders|

---

EP1949123A2|2005-10-18|2008-07-30|Cambridge Enterprise Limited|Methods and biomarkers for diagnosing and monitoring psychotic disorders|

---

KR101393946B1|2005-10-21|2014-05-12|카탈리스트 바이오사이언시즈, 인코포레이티드|Modified proteases that inhibit complement activation|

---

US20080199426A1|2007-01-11|2008-08-21|Sukhatme Vikas P|Methods and compositions for the treatment and diagnosis of vascular inflammatory disorders or endothelial cell disorders|

---

GB0701626D0|2007-01-22|2007-03-07|Cambridge Entpr Ltd|Methods and biomarkers for diagnosing and monitoring psychotic disorders|

---

JP2010271078A|2009-05-19|2010-12-02|Mcbi:Kk|Biomarker of mental disorder containing cognitive disorder, and method of detecting mental disorder containing cognitive disorder using biomarker|JP2010271078A|2009-05-19|2010-12-02|Mcbi:Kk|Biomarker of mental disorder containing cognitive disorder, and method of detecting mental disorder containing cognitive disorder using biomarker|

---

JP6012923B2|2010-12-22|2016-10-25|株式会社Ｍｃｂｉ|Biomarker for cognitive dysfunction disease and method for detecting cognitive dysfunction disease using the biomarker|

---

GB201310150D0|2013-06-07|2013-07-24|Electrophoretics Ltd|Methods and compositions relating to alzheimer's disease|

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SG11201510701QA|2013-06-28|2016-01-28|Mcbi Inc|Biomarkers for cognitive dysfunction diseases and method for detecting cognitive dysfunction disease using biomarkers|

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CN105067817B|2015-07-08|2017-05-10|上海清流生物医药科技有限公司|Methods and devices for acquiring signals and tracking cells by adopting light sensitive chips|

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JP2019020206A|2017-07-14|2019-02-07|株式会社島津製作所|Cognitive disorder biomarker quantification method using mass analysis and mass spectroscope|

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法律状态:

优先权:

---

申请号 | 申请日 | 专利标题

---

JP2009121226A|JP2010271078A|2009-05-19|2009-05-19|Biomarker of mental disorder containing cognitive disorder, and method of detecting mental disorder containing cognitive disorder using biomarker|

---

JP2009121226|2009-05-19|

---

PCT/JP2010/003295|WO2010134308A1|2009-05-19|2010-05-17|Biomarker for mental disorders including cognitive disorders, and method using said biomarker to detect mental disorders including cognitive disorders|

---