Biomarker for mental disorders including cognitive disorders, and method using said biomarker to detect mental disorders including cognitive disorders

Abstract

Methods are provided that detect cognitive impairment including mild cognitive impairment and Alzheimer disease by using a protein or its partial peptide that differs in presence or absence. Novel biomarkers are also provided for cognitive impairment and non-psychiatric disease, as well as methods for detecting cognitive impairment using such biomarkers. Specifically, a biomarker for diagnosis is provided that comprises a protein fragment or peptide of not less than 5 amino acid residues arising from at least one protein or peptide selected from the group of proteins consisting of an amino acid sequence expressed by SEQ ID NO: 1, 3, 6, 8, 10, 13, 15, 18, or 20 and selected from the group of partial peptide in these proteins consisting of an amino acid sequence expressed by SEQ ID NO: 2, 4, 5, 7, 9, 11, 12, 14, 16, 17, 19, or 21.

Claims

1. A method for detecting a biomarker for mild cognitive impairment or Alzheimer's disease in a human subject at a preclinical stage, comprising: (a) obtaining a serum sample from the human subject; (b) measuring at least one biomarker in the serum sample, using a detection method selected from the group consisting of two-dimensional liquid chromatography (2D-LC)-MALDI TOF-MS and LC-MS/MS, wherein the serum sample of said (b) is an acid-treated sample or an ultrafiltered sample after acid-treatment, wherein the at least one biomarker is at least one peptide selected from the group consisting of: prothrombin precursor derived peptide THRB(R−) consisting of the amino acid sequence of SEQ ID NO: 4, and prothrombin precursor-derived peptide THRB(R+) consisting of the amino acid sequence of SEQ ID NO: 5; and (c) diagnosing the human subject with a mild cognitive impairment or Alzheimer's disease based on the level of the at least one biomarker, if: (i) the peptide having the amino acid sequence of SEQ ID NO:4 is absent or decreased in the serum sample, as compared to the amount of the peptide having the amino acid sequence of SEQ ID NO:4 in biological material of subjects not suffering from mild cognitive impairment or Alzheimer's disease; and/or (ii) the peptide having the amino acid sequence of SEQ ID NO:5 is present or increased in the serum sample, as compared to the amount of the peptide having the amino acid sequence of SEQ ID NO:5 in biological material of subjects not suffering from mild cognitive impairment or Alzheimer's disease.

2. The method according to claim 1, using a detection method that is two-dimensional liquid chromatography (2D-LC)-MALDI TOF-MS.

3. The method according to claim 1, using a detection method that is LC-MS/MS.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 illustrates the isolation of serum of Alzheimer's disease by 2D-LC-MALDI TOF-MS method. (Example 1)

(2) FIGS. 2A)-2E) illustrate the case of Marker A that is the one example of the result of differential analysis. As shown in FIG. 3, Marker A is Neurexin-2-beta precursor-derived peptides NRX2B. FIG. 2A) illustrates the comparison between ADN, MCI and AD, and FIG. 2B) illustrates the comparison between ADN, AD, NDall, NDdem and NDnon. For each samples, the average value (divided by 1,000) and (SD) (divided by 1,000) are as follows. FIG. 2A) ADN 0.1 (0.1); MCI 45.8 (42.2); AD 41.7 (22.2). FIG. 2B) ADN 0.1 (0.2); AD 34.0 (27.8); NDall 19.2 (15.8); NDdem 24.3 (20.8); NDnon 14.0 (6.1). FIGS. 2C), 2D), and 2E), respectively, the ROC curve of the comparison of MCI vs. ADN, AD vs. ADN, and AD vs. NDnon. (Example 1)

(3) Abbreviations used are; AD (Alzheimer's disease), ADN (subjects not suffering from psychiatric disease and age and sex-matched patients with AD, “N” means normal), NDall (neurological disease), NDdem (demented neurological disease), NDnon (non-demented neurological disease).

(4) FIG. 3 illustrates the MS/MS spectrum of Marker A (SEQ ID NO: 2, NRX2B) obtained by using TOF/TOF mass spectrometer. (Example 1)

(5) FIGS. 4A)-4D) illustrate the comparison between non-psychiatric disease subjects (ADN) and patients of psychiatric disease including sementia for THRB(R−) FIGS. 4A) and 4B), and THRB(R+) FIGS. 4C) and 4D) in serum. (Example 1)

(6) FIG. 5A) illustrates the each individual comparison between non-psychiatric disease subjects (ADN) and patients of cognitive impairment (MCI, AD) patient for THRB(R−) and THRB(R+) in serum. FIG. 5B) illustrates the ROC curve of the comparison of AD vs. NDdon for THRB(R−) in serum. (Example 1)

(7) FIGS. 6A) and 6B) illustrate the comparison between non-psychiatric disease subjects (ADN) and patients of psychiatric disease including dementia for S264A in serum, and FIGS. 6C) and 6D) illustrate the comparison between non-psychiatric disease subjects (ADN) and patients of psychiatric disease including dementia for COPZ1 in serum. (Example 1)

(8) FIGS. 7A) and 7B) illustrate each individual comparison between non-psychiatric disease subjects (ADN) and patients of cognitive impairment (MCI, AD) patient for PARR2(S−) and PARR2(S+) 7B)) in serum. (Example 1)

(9) FIGS. 8A) and 8B) illustrate the comparison between non-psychiatric disease subjects (ADN) and patients of psychiatric disease including dementia for GELS in serum. (Example 1) FIGS. 9A) and 9B) illustrate the comparison between non-psychiatric disease subjects (ADN) and patients of psychiatric disease including dementia for CLUS(N-term SDVP) in serum, and FIGS. 9C) and 9D) illustrate the comparison between non-psychiatric disease subjects (ADN) and patients of psychiatric disease including dementia for CLUS(N-term RFFT) in serum. (Example 1)

(10) FIGS. 10A) and 10B) illustrate the comparison between non-psychiatric disease subjects (ADN) and patients of psychiatric disease including dementia for EIF3J in serum, and FIG. 10C) illustrates the comparison between non-psychiatric disease subjects (ADN) and patients of cognitive impairment (MCI, AD) patient for LRC27 in serum. (Example 1)

(11) FIGS. 11A) and 11B) illustrate the mass spectrum of NRX2B peptide that captured and detected by immunoMS method using NRX2B-specific antibody from the serum of AD and MCI patients. FIG. 11A) is the enlarged view of arrow parts in FIG. 11B). Endogenous NRX2B peptide (solid arrows) and stable isotope labeled-NRX2B synthetic peptide NRX2B (dashed arrows) are shown for each peak FIG. 11A). (Example 4)

DESCRIPTION OF EMBODIMENTS

(12) The present invention is a method for determining the kind and the amount of intact protein and/or its partial peptide when test subject is suffering from cognitive impairment as well as for diagnosing whether test subject is suffering from cognitive impairment and, whether test subject is diagnosed to be suffering from psychiatric disease. A peptide is generally said to be a chemical entity, made by polymerizing a number of amino acids, of less than 10,000 in molecular weight or by polymerizing several to less than about 50 amino acid residues. While in the present invention a partial peptide of an intact protein can be used as a biomarker for detection of cognitive impairment, such partial peptide is defined as a peptide of less than 10,000 in molecular weight consisting of a part of the amino acid sequence of the intact protein. Such peptide may arise as a partial peptide during the expression by transcription followed by synthesis by translation before maturing into an intact protein or as a peptide produced by enzyme digestion in the body after the intact protein has been synthesized. It is possible that, when the body is in abnormal state suffering from such disease as cognitive impairment, the mechanism for protein synthesis and regulation is de-regulated. In other words, the present invention is also a method for determining if test subject is in normal state or is suffering from cognitive impairment by using the degree of protein synthesis and/or protein digestion as an indicator. The detection of cognitive impairment in the present invention means evaluation and differentiation, i.e., diagnosis of test subject as to whether the subject is suffering from cognitive impairment. The present invention can also include the evaluation of patient's risk of suffering from more serious cognitive impairment.

(13) Specifically, in the method of the present invention, the examples of intact protein that can be used as a cognitive impairment include Neurexin-2-beta precursor consisting of amino acid sequence expressed by SEQ ID NO: 1, Prothrombin precursor consisting of amino acid sequence expressed by SEQ ID NO: 3, Pendrin consisting of amino acid sequence expressed by SEQ ID NO: 6, Coatomer subunit zeta-1 consisting of amino acid sequence expressed by SEQ ID NO: 8, Retinoic acid receptor responder protein 2 precursor consisting of amino acid sequence expressed by SEQ ID NO: 10, Gelsolin precursor consisting of amino acid sequence expressed by SEQ ID NO: 13, Clusterin precursor consisting of amino acid sequence expressed by SEQ ID NO: 15, Eukaryotic translation initiation factor 3 subunit J consisting of amino acid sequence expressed by SEQ ID NO: 18, and Leucine-rich repeat-containing protein 27 consisting of amino acid sequence expressed by SEQ ID NO: 20, and further, the peptide fragments that comprise of partial peptides of not less than 5 amino acid residues of these intact proteins can be used as same purpose.

(14) Still further, an example of biomarkers for cognitive impairment of the present invention includes the partial peptides consisting of amino acid sequence expressed by SEQ ID NO: 2 of Neurexin-2-beta precursor-derived peptide NRX2B, SEQ ID NO: 4 of Prothrombin precursor-derived peptide THRB(R−), SEQ ID NO: 5 of Prothrombin precursor-derived peptide THRB(R+), SEQ ID NO: 7 of Pendrin-derived peptide S26A4, SEQ ID NO: 9 of Coatomer subunit zeta-1-derived peptide COPZ1, SEQ ID NO: 11 of Retinoic acid receptor responder protein 2 precursor-derived peptide RARR2(S−), SEQ ID NO: 12 of Retinoic acid receptor responder protein 2 precursor-derived peptide RARR2(S+), SEQ ID NO: 14 of Gelsolin precursor-derived peptide GELS, SEQ ID NO: 16 of Clusterin precursor-derived peptide CLUS(N-term SDVP), SEQ ID NO: 17 of Clusterin precursor-derived peptide CLUS(N-term RFFT), SEQ ID NO: 19 of Eukaryotic translation initiation factor 3 subunit J-derived peptide EIF3J, SEQ ID NO: 21 of Leucine-rich repeat-containing protein 27-derived peptide LRC27. In the present invention, proteins and peptides consisting of amino acid sequences derived from SEQ ID NO: 1 to 21 by deletion, exchange, and/or addition of one or a few amino acids can be used as biomarkers and are included in the present invention. ‘”One or a few” herein means “one or three,” “one or two,” or “one.” Furthermore, the partial peptides that can be used as biomarkers in the present invention include those peptide fragments consisting of not less than 5 amino acid residues arising respectively from SEQ ID NO: 1 to 21. The basis for the limitation of peptide fragments consisting of not less than 5 amino acid residues is in the description below in Non-patent Document 2. The document reported that an antibody obtained by using the peptide IRGERA (SEQ ID NO: 23) as immunogen, which was the C-terminus (130-135) of histone H3, recognized the peptide IKGERA (SEQ ID NO: 24) derived by exchange of K for R and the peptide CGGGERA (SEQ ID NO: 25) which was derived by deletion of IR followed by addition of CGG. This demonstrates that the immunogenicity (antigenicity) is recognized by a peptide of not less than 4 amino acid residues. In order to expand this finding to other peptides than the C-terminus of histone H3, the number of amino acid residue is defined as not less than 5 instead of 4 in the present invention. To make such a low molecular weight peptide as the subject of the present invention is important when the method of detection and differentiation uses immunological means including immunoblot, ELISA and immunoMS.

(15) It is to be noted that there are cases where a sugar chain or sugar chains have been added to an intact protein or its partial peptide to form glycated entities. Proteins and partial peptides in glycated form can also be used as biomarkers for detection of cognitive impairment.

(16) It is also to be noted that, in the present invention, biomarker can be quantified or its presence or absence can be determined qualitatively.

(17) Two-dimensional electrophoresis (2-DE) or 2-dimensional chromatography (2-DC) 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 make quantification with the SRM/MRM method in LC-MS/MS technology. Furthermore, the immunoMS method which these inventors have developed, where target protein or peptide is captured by beads (including magnetic ones) with antibody linked to the protein or peptide, eluted from the beads, and determined by mass spectrometry enables convenient determination of presence or absence or the amount of target protein, protein fragment or peptide without the use of 2-DE or chromatography.

(18) It is possible with the use of the method disclosed in the present invention to evaluate at the stage of mild of cognitive dysfunction in test subject and therefore it can be useful in prophylactic medicine. Further, when psychotherapy and/or drug therapy is given to patients with cognitive impairment, it is reflected in the amount of proteins and partial peptides in biological materials such as serum if the progression of the disorder has been inhibited. Therefore, by measuring these proteins and partial peptides, it is possible to evaluate and determine therapeutic effect.

(19) The kind and amount of a protein in biological materials can be determined by various methods. If target protein (including protein fragment and partial peptide) has been characterized and when an antibody (primary antibody) to it has already been obtained, the following methods can be used:

(20) 1. Immunoblot

(21) This is one of the simplest methods. Test serum in a fixed amount (about 1 microliter) after stepwise dilution is dropped onto an appropriate membrane such as of nitrocellulose and dried in air. The membrane is treated with a blocking solution containing a protein such as BSA, washed, reacted with primary antibody, and washed. Thereafter, 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.

(22) 2. Western Blotting

(23) After separation with one-dimensional or two-dimensional electrophoresis involving isoelectric focusing or SDS-PAGE, proteins are transferred onto such an appropriate membrane as of nitrocellulose and their amounts are determined, as in above-mentioned immunoblot, using primary antibody and labeled secondary antibody.

(24) 3. ELISA

(25) Antibody to protein or its partial peptide is fixed to such a plate as a chemically modified microtiter plate. Appropriate amounts of samples after stepwise dilution are applied to the plate and incubated. Proteins and peptides not captured are removed by washing. Next, the plate is incubated with secondary antibody labeled with fluorescent or chemiluminescent substance or enzyme. After addition of respective substrate, fluorescence, chemiluminescence or visible light due to enzyme reaction is measured for evaluation and judgment.

(26) Additional examples of methods are illustrated below (see PTL 2) but the invention is not limited by these examples.

(27) 4. Methods that Use Microarray (Microchip)

(28) A microarray is a general term for devices where solidified materials with affinity for target substances are arrayed on solid support (plate). In the present invention, antibodies or aptamers to proteins and partial peptides are arrayed. A sample of biological material is placed on the microarray for fixation of target proteins or partial peptides and the microarray is then incubated with secondary antibody labeled with fluorescent or chemiluminescent substance or enzyme. After addition of respective substrate, fluorescence, chemiluminescence or visible light due to enzyme reaction is measured.

(29) 5. Mass Spectrometry

(30) In mass spectrometry, for example, antibody to a specified protein or partial peptide is attached to chemically modified microbeads or plate (protein chip). The microbeads could be magnetic beads. There are no requirements for the material of the plate. The antibody to be used could be (1) an antibody which recognizes the full length form of the specified protein only, (2) an antibody which recognizes a partial peptide only, (3) all of antibodies which recognizes both the specified protein and 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 plate carrying antibody or antibodies and incubated. Those proteins and partial peptides not captured are removed by washing. The protein or partial peptide captured by microbeads or plate is eluted, and analyzed by mass spectrometry with MALDI-TOF-MS, SELDI-TOF-MS, etc. Measurements are made with respect to the mass and intensity of the peak due to the protein, protein fragment or partial peptide. Prior to the measurements a fixed amount of substance serving as the internal standard is added to the original biological material and the intensity of its peak is also measured. The concentration of the target in the original biological material can be calculated from the ratio of peak intensity of the target to the peak intensity of the internal standard. This is called immunoMS method. Further, it is possible to make quantification, after the sample is diluted with original solvent or buffer, or after part of proteins are removed, by separation with HPLC followed by mass spectrometry with electrospray ionization (ESI) method. Therein the SRM/MRM method can be utilized for absolute quantification with the use of an isotope-labeled internal standard peptide.

(31) Furthermore, in addition to the above-mentioned methods, it is possible to analyze proteins and partial peptides by using 2-DE, surface plasmon resonance, etc.

(32) The present invention includes the method to detect cognitive impairment from the presence or absence or amount of the above-mentioned biomarker after applying biological material obtained from test subject to 2-DE or surface plasmon resonance.

Example 1

(33) Discovery of a marker peptide for detection of cognitive impairment using two-dimensional liquid chromatography (2D-LC)-MALDI TOF-MS.

(34) (1) Serum Samples.

(35) Followings, the characters before the parenthesis are an abbreviation.

(36) A sera obtained from 20 AD (Alzheimer's disease), 20 ADN (subjects not suffering from psychiatric disease and age and sex-matched patients with AD, “N” means normal), and 20 NDall (neurological disease) were used. NDall consists of 10 NDdem (demented neurological disease) and 10 NDnon (non-demented neurological disease). Furthermore, NDdem consists of dementia with Lewy body and frontotemporal dementia each consisting of 5 cases, and NDnon consists of schizophrenia and depression each consisting of 5 cases.

(37) (2) Methods

(38) After 475 μl of 0.1% trifluoroacetic acid (TFA) were added in each of 25 μl of sera, samples were boiled for 15 min at 100 degrees. Subsequently, in order to recover peptides of molecular weight of 10,000 or less, ultrafiltration were performed by using YM-10 filter unit (Millipore Corp.). Then the analysis using 2D-LC-MALDI TOF-MS were performed as follows. In other words, recovering samples were fractionated to 1,146 fractions per sample by using two-dimensional HPLC (SCX cation exchange column and C18 reverse-phase column). All fractionated samples were spotted on MALDI target plate for MALDI TOF/TOF mass spectrometer (ultraflex TOF/TOF, Bruker Daltonics), and matrix solution (alpha-cyano-hydroxycinnamic acid, CHCA) were mixed and crystallized, and the mass and the peak area of the mass were measured automatically in refrectron mode by irradiating to crystallised sample by laser. Peak area was normalized with 250 fmole of per each well of bradykinin 1-7 fragment that was added into matrix solution in advance. In other words, the area value was calculated in 10,000 times of the value dividing the peak area in specific mass of sample by the peak area obtained from 250 fmole of bradykinin1-7 fragment. This area value is corresponding in 25 μl of sample serum. Detection of difference in abundance of peptides in serum between groups (called differential analysis) was performed using multi-group statistical analysis software DeView developed by us. Peptide that was observed to difference in abundance was directly determined amino acid sequence in MS/MS analysis by ultraflex TOF/TOF, and intact proteins or peptides of their origin were identified.

(39) (3) Results

(40) FIG. 1 shows the result that was obtained from serum of one case of AD patient that was applied to 2D-LC-MALDI TOF-MS. Sample was fractionated into 6 fractions by SCX cation exchange column in the first dimension, then each of fractions were fractionated into 191 fractions by C18 reverse-phase column. Mass spectra of 191 fractions were obtained by MALDI TOF-MS measuring. As the horizontal axis is the m/z and the vertical axis is the fractions of reverse-phase column chromatography, FIG. 1 was visualized by Deview software developed by present inventors. SCX 1 shows flow-through fractions, SCX 2 shows fractions eluted in 10% salt concentration, SCX 3 shows fractions eluted in 20% salt concentration, SCX 4 shows fractions eluted in 30% salt concentration, SCX 5 shows fractions eluted in 50% salt concentration, SCX 6 shows fractions eluted in 100% salt concentration. As seen in FIG. 1, many peptides in many sera are present in fractions of SCX1, SCX 3, SCX 4, and SCX 5. Total numbers of peptides fractionated by 2D-LC and detected by MALDI TOF-MS were about 4,000.

(41) As one example of the results of differential analysis, FIG. 2 shows the case of Marker A. Marker A, as shown later in FIG. 3, is Neurexin-2-beta precursor-derived peptide NRX2B. A) of FIG. 2 shows a comparison between ADN, MCI, AD. A) and B) shows the result of experiments carried out separately, ADN and AD were used the same samples in both experiments (i.e., for ADN and AD, the measurement results would indicate the reproducibility). In A) of FIG. 2, it was found that Marker A is increased in MCI and AD patients than ADN patient. In B) of FIG. 2, it was found that Marker A is increased in AD, NDall, NDdem, and NDnon patients than ADN patient. In particular, in comparison of AD and NDnon, AD was significantly higher than NDnon (t-test, p=0.035). From these results, it was found that Marker A was useful to distinguish between cognitive impairment (MCI, AD, NDdem) patient and non-demented neurological disease (NDnon).

(42) From the results of A) and B) in FIG. 2, in order to evaluate the extent to which the Marker A is useful as biomarker, the analysis by receiver operating characteristic (ROC) curve was performed. C), D) and E) in FIG. 2 shows respectively the ROC curve of the comparison of MCI vs. ADN, AD vs. ADN, and AD vs. NDnon. If the area value (hereinafter referred to as the ROC value) of under the ROC curve is close to 1, the usefulness as biomarker of Marker A will be higher. In C), D), E) of FIG. 2, the typical values of sensitivity and specificity are the values of the point (open square in the figure) of the coordinate on ROC curve that the distance is minimized when a straight line was drawn to ROC curve from the point of 100% on y-axis. The value of cut-off giving this point becomes a useful threshold to distinguish between the different groups, and the values of sensitivity and specificity at that time (i.e., above the typical values) becomes an indicator of the usefulness of biomarkers together with ROC values. In C) of FIG. 2, as typical values in MCI vs. ADN, the sensitivity was 90%, the specificity was 100%, and the ROC value was 0.99. In D) of FIG. 2, as typical values in AD vs. ADN, the sensitivity was 100%, the specificity was 100%, and the ROC value was 1. In E) of FIG. 2, it was comparing between AD vs. CCC, the sensitivity was 100%, the specificity was 50%, and the ROC value was 0.710. Thus, it was revealed that Marker A (NRX2B) was useful to distinguish MCI and AD with ADN. And also, it was revealed that Marker A was useful to distinguish AD with non-demented neurological disease (NDnon). In particular, since MCI is the state of previous stage of AD, Marker A (NRX2B) is considered to be a extremely useful marker to detect MCI for early diagnosis of potential subjects to migrate to AD.

(43) FIG. 3, for Marker A, illustrates the results of MS/MS spectrum using ultraflex TOF/TOF. The signals that show y-ions and b-ions have enough appeared, and the amino acid sequence could be readily identified. Mascot search was performed on this result, and the protein of origin or the peptide (hereinafter referred to as intact proteins or peptides) is Neurexin-2-beta precursor, and the detected peptide was found that the sequence is RSGGNATLQVDSWP (SEQ ID NO: 2). NRX2B of entry name of Swiss-Prot against Neurexin-2-beta precursor will use as an abbreviation of the peptide name. Also as for other peptides that were detected, the entry names of Swiss-Prot will be used as abbreviations of the peptide names in the following descriptions.

(44) Including the Marker A, the peptides that have difference in abundance between the groups in serum were measured MS/MS spectra using ultraflex TOF/TOF, and in addition to determining the amino acid sequence, the results identified intact proteins or peptides were shown below. For peptides other than Marker A, the signals that show y-ions and b-ions has enough appeared, and the amino acid sequence could be readily identified. The following amino acid sequence that shows a set of two sequences, the entire sequence of the first sequence shows the amino acid sequence of intact proteins or peptides. The peptide comprising of the underlined portion of the first sequence and the second sequence is peptide detected by 2D-LC-MALDI TOF-MS. 001 represents N-terminus. The peptide which has oxidation of methionine was indicated as (+Oxidation (M)) at the end of the amino acid sequence. For the protein with mutation of amino acid by gene mutation, applicable amino acid residue was expressed with (X).

(45) (1) Neurexin-2-beta Precursor-Derived Peptide NRX2B

(46) NRX2B shown as SEQ ID NO: 2 was not detected in ADN patient, and was detected in MCI, AD, NDall, NDdem, and NDnon patients. Furthermore, in comparison of AD and NDnon, AD shown higher value than NDnon, NRX2B was shown distinction ability (previously described in FIG. 2).

(47) TABLE-US-00001 Intact protein/peptide (SEQ ID NO: 1) 001 MPPGGSGPGG CPRRPPALAG PLPPPPPPPP PPLLPLLPLL     LLLLLGAAEG 051 ARVSSSLSTT HHVHHFHSKH GTVPIAINRM PFLTRGGHAG     TTYIFGKGGA 101 LITYTWPPND RPSTRMDRLA VGFSTHQRSA VLVRVDSASG     LGDYLQLHID 151 QGTVGVIFNV GTDDITIDEP NAIVSDGKYH VVRFTRSGGN     ATLQVDSWPV 201 NERYPAGNFD NERLAIARQR IPYRLGRVVD 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 EVIRESSSTT     GMVVGIVAAA 551 ALCILILLYA MYKYRNRDEG SYQVDQSRNY ISNSAQSNGA     VVKEKAPAAP 601 KTPSKAKKNK DKEYYV
Neurexin-2-beta Precursor-Derived Peptide NRX2B

(48) TABLE-US-00002 (SEQ ID NO: 2) RSGGNATLQVDSWP
(2) Prothrombin Precursor-Derived Peptide (THRB(R−))

(49) Prothrombin precursor-derived peptides are two types, and (R−) means the peptide lacking of R (Arginine residue) of C-terminus. THRB(R−) shown as SEQ ID NO: 4 was detected specifically in ADN patient, and was detected extremely low value in MCI, AD, NDall, NDdem, NDnon patients. Diagrams of THRB(R−) and THRB(R+) showed side by side in FIG. 4 and FIG. 5. FIG. 4 shows scatter plot. FIG. 5A) shows that the appearance of THRB(R−) and THRB(R+) how is different in every individual of ADN, MCI and AD. In the same individual, THRB(R−) has appeared overwhelmingly in ADN, THRB(R+) has appeared overwhelmingly in MCI and AD. It can be said that both peptides are extremely useful markers determining MCI and ADN. FIG. 5B) shows ROC curve comparing AD and NDnon of THRB(R−). ROC value indicated a high value of 0.815. the value in AD was lower compared to NDnon. In other words, it has been found that THRB(R−) as well as NRX2B is useful marker to distinguish between patients of cognitive impairment (MCI, AD, NDdem) and patients of non-demented neurological disease (NDnon).

(50) TABLE-US-00003 Intact protein/peptide (SEQ ID NO: 3) 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 SVLQVVNLPI VERPVCKDST     RIRITDNMFC 551 AGYKPDEGKR GDACEGDSGG PFVMKSPFNN RWYQMGIVSW     GEGCDRDGKY 601 GFYTHVFRLK KWIQKVIDQF GE
Prothrombin Precursor-Derived Peptide THRB(R−)

(51) TABLE-US-00004 (SEQ ID NO: 4) GLDEDSDRAIEG
(3) Prothrombin Precursor-Derived Peptide (THRB(R+))

(52) THRB(R+) shows as SEQ ID NO: 5 was not detected in ADN patient, and was detected in MCI, AD, NDall, NDdem, and NDnon patients (FIG. 4). (R+) means that the peptide having of R (Arginine residue) of C-terminus. For explanation, refer to (2) Prothrombin precursor-derived peptide (THRB(R−)).

(53) TABLE-US-00005 Intact protein/peptide (SEQ ID NO: 3) 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 SVLQVVNLPI VERPVCKDST     RIRITDNMFC 551 AGYKPDEGKR GDACEGDSGG PFVMKSPFNN RWYQMGIVSW     GEGCDRDGKY 601 GFYTHVFRLK KWIQKVIDQF GE
Prothrombin Precursor-Derived Peptide THRB(R+)

(54) TABLE-US-00006 (SEQ ID NO: 5) GLDEDSDRAIEGR
(4) Pendrin-Derived Peptide (S26A4)

(55) S26A4 shows as SEQ ID NO: 7 was not detected in ADN patient, and was detected in MCI, AD, NDall, NDdem, and NDnon patients (FIG. 6).

(56) TABLE-US-00007 Intact protein/peptide (SEQ ID NO: 6) 001 MAAPGGRSEP PQLPEYSCSY MVSRPVYSEL AFQQQHERRL     QERKTLRES 051 AKCCSCSRKR AFGVLKTLVP ILEWLPKYRV KEWLLSDVIS     GVSTGLVATL 101 QGMAYALLAA VPVGYGLYSA FFPILTYFIF GTSRHISVGP     FPVVSLMVGS 151 VVLSMAPDEH FLVSSSNGTV LNTTMIDTAA RDTARVLIAS     ALTLLVGIIQ 201 LIFGGLQIGF IVRYLADPLV GGFTTAAAFQ VLVSQLKIVL     NVSTKNYNGV 251 LSIIYTLVEI FQNIGDTNLA DFTAGLLTIV VCMAVKELND     RFRHKIPVPI 301 PIEVIVTIIA TAISYGANLE KNYNAGIVKS IPRGFLPPEL     PPVSLFSEML 351 AASFSIAVVA YAIAVSVGKV YATKYDYTID GNQEFIAFGI     SNIFSGFFSC 401 FVATTALSRT AVQESTGGKT QVAGIISAAI VMIAILALGK     LLEPLQKSVL 451 AAVVIANLKG MFMQLCDIPR LWRQNKIDAV IWVFTCIVSI     ILGLDLGLLA 501 GLIFGLLTVV 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 
Pendrin-Derived Peptide S26A4

(57) TABLE-US-00008 (SEQ ID NO: 7) LAGLIFGLLTVVLR
(5) Coatomer Subunit Zeta-1-Derived Peptide (COPZ1)

(58) COPZ1 shows as SEQ ID NO: 9 was shown low value in ADN patient, was shown high value in MCI, AD, and NDdem patients (FIG. 6).

(59) TABLE-US-00009 Intact protein/peptide (SEQ ID NO: 8) 001 MEALILEPSL YTVKAILILD NDGDRLFAKY YDDTYPSVKE     QKAFEKNIFN 051 KTHRTDSEIA LLEGLTVVYK SSIDLYFYVI GSSYENELML     MAVLNCLFDS 101 LSQMLRKNVE KRALLENMEG LFLAVDEIVD GGVILESDPQ     QVVHRVALRG 151 EDVPLTEQTV SQVLQSAKEQ IKWSLLR
Coatomer Subunit Zeta-1-Derived Peptide COPZ1

(60) TABLE-US-00010 (SEQ ID NO: 9) AILILDNDGDRLFAKYYDD
(6) Retinoic Acid Receptor Responder Protein 2 Precursor-Derived Peptide (RARR2(S−))

(61) RARR2(S−) shows as SEQ ID NO: 11 was not detected in ADN patient, and was detected in AD and MCI patients (FIG. 7). Retinoic acid receptor responder protein 2 precursor-derived peptides are two types, and (S−) means the peptide lacking of S (Serine residue) of C-terminus.

(62) TABLE-US-00011 Intact protein/peptide (SEQ ID NO: 10) 001 MRRLLIPLAL WLGAVGVGVA ELTEAQRRGL QVALEEFHKH      PPVQWAFQET 051 SVESAVDTPF PAGIFVRLEF KLQQTSCRKR DWKKPECKVR      PNGRKRKCLA 101 CIKLGSEDKV LGRLVHCPIE TQVLREAEEH QETQCLRVQR      AGEDPHSFYF 151 PGQFAFSKAL PRS
Retinoic Acid Receptor Responder Protein 2 Precursor-Derived Peptide RARR2(S−)

(63) TABLE-US-00012 (SEQ ID NO: 11) PHSFYFPGQFAFSKALPR
(7) Retinoic Acid Receptor Responder Protein 2 Precursor-Derived Peptide (RARR2(S+))

(64) RARR2(S+) shows as SEQ ID NO: 12 was not detected in ADN patient as well as RARR2(S−), and was detected in AD and MCI patients (FIG. 7). (S+) means that the peptide having of S (Serine residue) of C-terminus.

(65) TABLE-US-00013 Intact protein/peptide (SEQ ID NO: 10) 001 MRRLLIPLAL WLGAVGVGVA ELTEAQRRGL QVALEEFHKH      PPVQWAFQET 051 SVESAVDTPF PAGIFVRLEF KLQQTSCRKR DWKKPECKVR      PNGRKRKCLA 101 CIKLGSEDKV LGRLVHCPIE TQVLREAEEH QETQCLRVQR      AGEDPHSFYF 151 PGQFAFSKAL PRS
Retinoic Acid Receptor Responder Protein 2 Precursor-Derived Peptide RARR2(S+)

(66) TABLE-US-00014 (SEQ ID NO: 12) PHSFYFPGQFAFSKALPRS
(8) Gelsolin Precursor-Derived Peptide (GELS)

(67) GELS shows as SEQ ID NO: 14 was shown low value in ADN patient, and was shown relatively high value in MCI and AD patients (FIG. 8).

(68) TABLE-US-00015 Intact protein/peptide (SEQ ID NO: 13) 001 MAPHRPAPAL LCALSLALCA LSLPVRAATA SRGASQAGAP      QGRVPEARPN 051 SMVVEHPEFL KAGKEPGLQI WRVEKFDLVP VPTNLYGDFF      TGDAYVILKT 101 VQLRNGNLQY DLHYWLGNEC SQDESGAAAI FTVQLDDYLN      GRAVQHREVQ 151 GFESATFLGY FKSGLKYKKG GVASGFKHVV PNEVVVQRLF      QVKGRRVVRA 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 PVQSRVVQGK EPAHLMSLFG      GKPMIIYKGG 551 TSREGGQTAP ASTRLFQVRA NSAGATRAVE VLPKAGALNS      NDAFVLKTPS 601 AAYLWVGTGA SEAEKTGAQE LLRVLRAQPV QVAEGSEPDG      FWEALGGKAA 651 YRTSPRLKDK KMDAHPPRLF ACSNKIGRFV IEEVPGELMQ      EDLATDDVML 701 LDTWDQVFVW VGKDSQEEEK TEALTSAKRY IETDPANRDR      RTPITVVKQG 751 FEPPSFVGWF LGWDDDYWSV DPLDRAMAEL AA
Gelsolin Precursor-Derived Peptide GELS

(69) TABLE-US-00016 (SEQ ID NO: 14) PVRAATASRGAS
(9) Clusterin Precursor-Derived Peptide (CLUS(N-Term SDVP))

(70) CLUS(N-term SDVP) shows as SEQ ID NO: 16 was shown low value in ADN patient, and was shown relatively high value in MCI and AD patients (FIG. 9). Clusterin precursor-derived peptides are two types, and (N-termSDVP) means that the amino acid sequence of N-terminus in peptide is SDVP.

(71) TABLE-US-00017 Intact protein/peptide (SEQ ID NO: 15) 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 GVTEVVVKLF DSDPITVTVP VEVSRKNPKF METVAEKALQ      EYRKKHREE
Clusterin Precursor-Derived Peptide CLUS(N-Term SDVP)

(72) TABLE-US-00018 (SEQ ID NO: 16) SDVPSGVTEVVVKLFDS
(10) Clusterin Precursor-Derived Peptide (CLUS(N-Term RFFT))

(73) CLUS(N-term RFFT) shows as SEQ ID NO: 17 was detected in ADN patient, and was not completely detected in AD patient (FIG. 9). (N-term RFFT) means that the amino acid sequence of N-terminus in peptide is RFFT.

(74) TABLE-US-00019 Intact protein/peptide (SEQ ID NO: 15) 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 GVTEVVVKLF DSDPITVTVP VEVSRKNPKF METVAEKALQ      EYRKKHREE
Clusterin Precursor-Derived Peptide CLUS(N-Term RFFT)

(75) TABLE-US-00020 (SEQ ID NO: 17) RFFTREPQDTYHYLPFSLPH
(11) Eukaryotic Translation Initiation Factor 3 Subunit J-Derived Peptide (EIF3J)

(76) EIF3J shows as SEQ ID NO: 19 was detected in ADN patient, and was not at all or almost detected in MCI, AD, NDall, NDdem, and NDnon patients (FIG. 10).

(77) TABLE-US-00021 Intact protein/peptide (SEQ ID NO: 18) 001 MAAAAAAAGD 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 KKKKGVVPGG GLKATMKDDL      ADYGGYDGGY 251 VQDYEDFM
Eukaryotic Translation Initiation Factor 3 Subunit J-Derived Peptide EIF3J

(78) TABLE-US-00022 (SEQ ID NO: 19) GVVPGGGLKATMKDDLADYGGYDGG + Oxidation (M)
(12) Leucine-Rich Repeat-Containing Protein 27-Derived Peptide (LRC27)

(79) LRC27 shows as SEQ ID NO: 21 was detected in ADN patient, and was not completely detected in AD patient (FIG. 10).

(80) TABLE-US-00023 Intact protein/peptide (SEQ ID NO: 20) 001 MEGSSSYEVP SVAAADLEEG AGQTRSLPAT PSKDVHKGVG      GIIFSSSPIL 051 DLSESGLCRL EEVFRIPSLQ QLHLQRNALC VIPQDFFQLL      PNLTWLDLRY 101 NRIKALPSGI GAHQHLKTLL LERNPIKMLP VELGSVTTLK      ALNLRHCPLE 151 FPPQLVVQKG 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
Leucine-Rich Repeat-Containing Protein 27-Derived Peptide LRC27

(81) TABLE-US-00024 (SEQ ID NO: 21) SSPILDLSESGLCRLEEVFRIPS

(82) There have been already quoted, but for the peptides of SEQ ID NO: 2 (NRX2B) to SEQ ID NO: 21 (LRC27), the scatter plots of comparison between ADN, MCI and AD patients, and the scatter plot of comparison between ADN, AD, NDall, NDdem and NDnon patients, and the p-value of t-test in each comparison were showed in FIG. 2, FIG. 4 and FIG. 6 through FIG. 10.

(83) Table 1 shows the list of 12 marker peptides described above and their ROC values for comparison of MCI vs. ADN and AD vs. ADN.

(84) TABLE-US-00025 TABLE 1 Marker peptides MCI vs. ADN AD vs. ADN Sequence ROC MCI ROC AD Swiss-Prot Entry No. value was value was NRX2B 2 0.99 up 1 up THRB (R−) 4 0.854 down 0.841 down THRB (R+) 5 0.94 up 0.985 up S26A4 7 0.925 up 0.95 up COPZ1 9 0.786 up 0.767 up RARR2 (S−) 11 0.885 up 0.914 up RARR2 (S+) 12 0.95 up 0.919 up GELS 14 0.716 up 0.762 up CLUS (N-term SDVP) 16 0.739 up 0.717 up GLUS (N-term RFFT) 17 0.675 down 0.75 down EIF3J 19 0.748 down 0.775 down LRC27 21 0.699 down 0.755 down

(85) Table 1 shows the usefulness of each marker peptide in detection of cognitive impairment (MCI and AD). Using these marker peptides in singly or in combination, using or without using liquid chromatography and/or any other suitable separation methods, directly measuring the abundance in serum using other methods such as mass spectrometry or immunological methods or enzymatic methods, it is possible to distinguish between non-dementia and dementia in neurological disease and diagnose cognitive impairment like AD and MCI. The marker peptide that is not detected in ADN and is detected in MCI, AD, NDall, NDdem and NDnon patients, or vice versa, the marker peptide that is detected in ADN patient and is not detected in MCI, AD, NDall, NDdem and NDnon patients, are also useful for the detection of psychiatric diseases.

Example 2

Example 2. Synthesis of a Marker Peptide, and Preparation of a Marker Peptide Specific Polyclonal Antibody

(86) The antigenic peptide was synthesized to prepare the specific antibody that recognizes Neurexin-2-beta precursor-derived peptide NRX2B of SEQ ID NO: 2. The synthetic peptide for coupling to a carrier protein was added the cysteine residue (labeled as C or Cys) in C-terminus. The peptide that was combined with carrier protein (RSGGNATC(SEQ ID NO: 22)-KLH carrier protein, see below) was mixed with an adjuvant, and the mixture was immunized in rabbit. Total eight times immunizations is performed every 1-2 weeks, and the test blood collection performed twice every 4 weeks, and the antibody titers were measured by enzyme immunoassay (EIA). After three months from the start of the immunization, the whole blood was collected from rabbit and the antiserum was obtained, furthermore the purification of the specific antibody was performed using the peptide column that antigen peptide was bound as ligand.

(87) The sequence of synthetic antigen peptide for preparation of peptide specific antibody is shown below.

(88) TABLE-US-00026 (SEQ ID NO: 22) RSGGNAT + Cys

Example 3

Example 3. Preparation of Antibody-Beads

(89) (1) Method

(90) (1-1) Preparation of Antibody, and Binding to Magnetic-Beads

(91) The antibody solution, 1 mg of the antibody (anti-NRX2B antibody, Rabbit IgG) that specifically recognizes the peptide of amino acid sequence expressed by SEQ ID NO: 22 was dissolved with 3 ml of 0.1 M MES. After washing 1 ml (10 mg 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 were gently shaken for 30 min at room temperature.

(92) (1-2) Cross-Linking of Antibody and Magnetic-Beads

(93) 400 μl of EDC solution (10 mg/ml 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride in 0.1M MES) was added in antibody-beads solution and was suspended gently for 3 hours to bind antibody with beads by covalent bond.

(94) (1-3) Blocking

(95) 1 ml of 200 mM ethanolamine (pH8.0) was added to wash beads, and further 1 ml of 200 mM ethanolamine (pH8.0) was added and was shaken gently for 1 hours at room temperature to block amine groups.

(96) (1-4) Washing

(97) After removal of 200 mM 200m M ethanolamine (pH8.0), the beads were washed three times by 1 ml of TBST solution (25 mM Tris-HCl (pH7.2) containing 0.15M NaCl and 0.05% Tween 20).

(98) (1-5) Storage

(99) After suspending the beads by adding with 1 ml of TBST solution, and stored at 4° C.

Example 4

Example 4. The Proof by immunoMS Method that the Peak of m/z 1,488 in Patient Serum Detected by 2D-LC-MALDI-TOF-MS is NRX2B

(100) (1) Methods

(101) As the control for comparison, stable isotope-labeled NRX2B synthetic peptide (12C and 13C5 of V has been replaced by 15N and 14N) greater than mass of NRX2B was used. The mass difference between NRX2B and its stable isotope peptide is 6 u. Both the endogenous peptide and the stable isotope-labeled peptide are captured by anti-NRX2B antibody. 1 μl of 200 fmol/μl stable isotope-labeled NRX2B synthetic peptide was added in 25 μl of each of patient serum in AD and MCI, and incubated for 10 min at 4° C. Then, 475 μl of 0.1% trifluoroacetic acid (TFA) was added and boiled for 5 min at 100° C. After centrifugation for 15 min at 14,000×g, 500 μl of 100 mM Tris-HCl buffer (pH 7.5) containing 0.3 M NaCl and 0.2% n-octyl glycoside was added in supernatant as peptide solutions. 20 μl of anti-NRX2B antibody-beads created in Example 3 was added in the peptide solutions, and was shaken gently for 2 hours. Then, after standing for 1 min on the magnetic stand, 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 glycoside was added, and was shaken gently for 10 min. After standing for 1 min on the magnetic stand, the supernatant was removed. In addition, after adding 500 μl of TBS, and standing for 1 min on the magnetic stand, the supernatant was removed. This procedure was repeated three times. Furthermore, after adding 500 μl of 50 mM ammonium carbonate, and standing for 1 min on the magnetic stand, the supernatant was removed. This procedure was repeated three times. 50 μl of 2-propanol: H2O: formic acid (4:4:1) solution was added, and was stood for 10 min, and then after standing for 1 min on the magnetic stand, the filtrate was recovered. This procedure was repeated twice. The filtrates were completely dried using vacuum centrifuge. Then, 20 μl of 0.095 TFA containing 5% acetonitrile was added and was re-dissolved by sonication. The peptides were concentrated using C18 pipette tip (PerfectPure C-18 Tip, Eppendorf), and were spotted on MALDI target plate (MTP AnchorChip™ 600/384 plate, BRUKER DALTONICS) by eluting from C18 pipette tip, and then the peptides were analyzed using MALDI TOF mass spectrometer (AXIMA CFRplus, SHIMADZU).

(102) (2) Results

(103) FIG. 11 shows the result of the mass spectrum of NRX2B peptide detected from the patient serum in AD and MCI using the above method. FIG. 11 A) shows the overall mass spectrum, and FIG. 11 B) shows the enlarged view of the arrow parts in FIG. 11 A). The signal indicated by dashed arrows in FIG. 11 B) is stable isotope-labeled NRX2B synthetic peptide that was spiked, and the signal indicated by solid arrows is endogenous NRX2B peptide. The observed mass value was within the measurement error of the expected value. And also the mass difference between endogenous NRX2B peptide and its stable isotope-labeled peptide was 6 u. Therefore, it was demonstrated that the trapped peptide is NRX2B.

(104) In this experiment, NRX2B which is the peptide marker was detected from serum by using immunoMS method that developed originally by present inventors, and it could be shown that it is possible to distinguish between AD and MCI patients from ADN. At the same time, in this experiment, it has also shown that the specific antibody against NRX2B is useful in detecting its peptide marker. In addition, it also shows that 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 this experiment, it was determined by using the specific antibodies that recognize one peptide marker, but the combination of biomarkers specific antibodies that recognize other peptides that were found in Example 1, is expected to further increase the accuracy of diagnosis of the pathosis.

INDUSTRIAL APPLICABILITY

(105) As cognitive impairment including mild cognitive impairment and Alzheimer disease and cognitive impairment and non-psychiatric disease 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

(106) 09P01007_Sequence.txt