JP5892169B2 - Diagnostic kit, diagnostic marker and detection method for Alzheimer type dementia by measuring sugar chain of complement C3 protein - Google Patents

Description

  The present invention relates to early detection of Alzheimer-type dementia by measuring the sugar chain of complement C3 protein.

  In Japan, in recent years, dementia in the elderly has become a major social problem as the population ages. Currently, 1 in 10 people over the age of 65 has dementia, and there are currently 1.3 million elderly people with cognitive impairment, and is expected to reach 3 million by 2035. Most of the dementia is cerebrovascular dementia and Alzheimer's disease (AD), and about half of them are Alzheimer's disease. The former has made medical treatments such as blood pressure management and recurrence prevention possible, but Alzheimer's disease is still unclear with regard to its cause, treatment, and prevention, and an immediate medical solution is required. In addition to dementia other than Alzheimer's disease, in addition to the above-mentioned cerebrovascular dementia, especially dementia collectively called tauopathy (frontotemporal lobar degeneration, Lewy body dementia, corticobasal degeneration, Progressive supranuclear palsy, etc.) also exist, and these are dementia that is difficult to differentiate from Alzheimer's disease because they exhibit symptoms similar to Alzheimer's disease. If the differential diagnosis of Alzheimer's disease can be differentiated from other dementias before the appearance of disturbing cognitive symptoms, the currently attempted treatment can be started earlier and the progression of dementia will be delayed. Therefore, the establishment of an early diagnosis method for Alzheimer's disease is a social urgent matter.

  In patients with Alzheimer's disease, cortical atrophy is observed, and pathologically, in addition to severe neuronal loss, characteristic lesions such as senile plaques and neurofibrillary tangles are observed. Of these, senile plaques appear the earliest and are characteristic changes in Alzheimer's disease, and are considered the most important changes in Alzheimer's disease. The main component of senile plaques is amyloid β protein (Aβ), and there is a mutation in amyloid precursor protein (APP), which is the precursor gene of Aβ in familial Alzheimer's disease showing autosomal dominant inheritance. It is thought that abnormal production or degradation of selenium is deeply related to the onset and progression of Alzheimer's disease. Aβ is produced from amyloid precursor protein through two cleavages by β-secretase belonging to aspartic protease and γ-secretase (presenilin complex).

  As therapeutic agents for Alzheimer's disease, acetylcholinesterase inhibitors that act to compensate for deficient acetylcholine are known, and various other signal transduction target drugs based on the above mechanism are being developed.

  Diagnosis of Alzheimer's disease (Alzheimer-type dementia) is diagnosed by whether or not various diagnostic criteria are clinically satisfied. For this purpose, various examinations such as clinical symptoms, neuropsychological examinations, physiological examinations, and brain function image examinations and detailed interrogation are performed. However, Alzheimer's disease in the early stages of the disease has few specific abnormal test findings, and requires thorough interviews with the person and family and exclusion diagnosis by medical examination. In fact, in the early stages of Alzheimer's disease, symptoms such as personality changes, difficulty in speaking, memory loss, misplacement of objects, forgetting of names, etc. are observed. It is very difficult to accurately distinguish forgetfulness (physiological forgetfulness). This is also a major problem that makes it difficult to detect Alzheimer's disease early.

  Currently, the diagnosis of Alzheimer's disease is comprehensively performed by neuropsychological examination, imaging diagnosis, neurophysiological examination, biological examination and the like. Examples of neuropsychological examinations include mini-mental state examination (MMSE), revised Hasegawa simple cognitive function evaluation scale (HDS-R), and Alzheimer ’s disease assessment scale (ADAS-cog). Examples of diagnostic imaging include computed tomography (CT), magnetic resonance imaging (MRI), single photon emission computed tomography (SPECT), and positron emission tomography (PET). In Alzheimer's disease, cerebral blood flow decreased in the posterior cingulate gyrus, oxygen metabolism and glucose metabolism can be confirmed from early in SPECT and PET, and cerebral blood flow and glucose metabolism in the temporal lobe and parietal lobe decreased with progress. To go.

  For the purpose of screening a large number of people at an early stage, a biological diagnostic marker that can easily and reliably detect Alzheimer's disease is considered to be most effective. Establishing diagnostic markers for Alzheimer's disease will enable early diagnosis and pre-diagnostic diagnosis, planning medications and treatments based on definitive diagnoses, and further paving the way for Alzheimer's disease prevention . At the same time, these early diagnoses can also reduce significant costs required for medical care and care for Alzheimer patients. Therefore, the invention and development of a diagnostic marker that can easily and reliably detect Alzheimer's disease is strongly desired.

  In addition to reflecting the pathology of Alzheimer's disease, the diagnostic marker must have high sensitivity and specificity for detecting Alzheimer's disease. To date, various diagnostic markers for Alzheimer's disease have been proposed by many groups in Japan and overseas. For example, amyloid β protein, glycosylated acetylcholinesterase, interleukin-6, substance-P, cystatin C, serum apolipoprotein, serum homocysteine, APP isoform, interleukin-6, α1-antichymotrypsin (ACT), oxygenase Measurement of -1,24S-hydroxycholesterol, acetylcholine, somatostatin, vasopressin, acetylcholine transferase activity, acetylcholinesterase activity, etc. has been proposed as a diagnostic marker for Alzheimer's disease.

  Non-Patent Document 1 describes quantitative changes in WGA-binding protein in Alzheimer's disease. Non-Patent Document 2 describes quantitative changes in complement C3 protein in cerebrospinal fluid.

  However, many of these many reported diagnostic markers have unclear relationships with pathological changes, and diagnostic values have not necessarily been established. At present, only two cerebrospinal fluid tau protein, phosphorylated tau protein and amyloid β42 are recognized as diagnostic markers for Alzheimer's disease that have been confirmed to be clinically useful.

  Tau protein is a causative agent of pathological changes called neurofibrillary tangles occurring in the brain of Alzheimer's disease patients. In cerebrospinal fluid of Alzheimer's disease, phosphorylated tau protein (or total tau protein) is increased, and it is said that measuring this protein will aid in the diagnosis of Alzheimer's disease.

  The amyloid β protein used as a diagnostic marker is mainly Aβ42, which is a kind of amyloid β peptide consisting of 42 amino acids, and is a causative substance of pathological changes called senile plaques occurring in the brain of Alzheimer's disease patients. In the brain of Alzheimer's disease patients, amyloid β protein aggregates to form senile plaques, so Aβ42 in the cerebrospinal fluid is decreased, and it is said that measuring this helps to diagnose Alzheimer's disease. Yes.

Lisa R. Fodero, Javier Saez-Valero, Maria-Sagrario Barquero, Alberto Marcos, Catriona A. McLean and David H. Small; “Wheat germ agglutinin-binding glycoproteins are decreased in Alzheimer's disease cerebrospinal fluid.”, Journal of Neurochemistry, 2001 , 79, 1022-1026 William TH et al., Biomarker discovery for Alzheimer ’s disease, frontotemporal lobar degeneration, and Parkinson ’s disease. Acta Neuropathol., 2010

  Regarding the diagnosis of Alzheimer's disease using tau protein, dementia due to an increase in phosphorylated tau protein accompanied by neurofibrillary tangles is not limited to Alzheimer's disease, so distinguishing them from other dementias (tauopathy) has been an issue. It was. In addition, neuronal cell death had already progressed at the time when phosphorylated tau was rising, and it was one of the issues that complete recovery of nerve function could not be expected even if treatment was started at this time .

  In addition, amyloid β protein used as a diagnostic marker correlates with the severity of late stage Alzheimer's disease, but there are large individual differences and it does not mean that Alzheimer's disease is uniformly reduced. Moreover, the reduction | decrease of amyloid (beta) protein had the subject that it is not Alzheimer's disease specific.

  In addition, sialic acid modified in glycoprotein contained in cerebrospinal fluid (CSF) of Alzheimer's disease patients was found to decrease (Non-Patent Document 1) and is expected as a new marker for diagnosis of Alzheimer's disease. However, it has been pointed out that the protein has not been identified and that sialic acid becomes unstable upon repeated freeze-thawing. In addition, the amount of complement C3 protein in the CSF of Alzheimer's disease patients was found to change (Non-patent Document 2), and it is expected as a new marker for the diagnosis of Alzheimer's disease. Since the quantitative change of C3 protein is not specific to Alzheimer's disease and is a change found in various diseases, it was considered that the possibility of using it for diagnosis of Alzheimer's disease is low.

  As an ideal marker, it can be diagnosed before the death of nerve cells, preferably it can be diagnosed before the onset of Alzheimer's disease, and it can also be differentially diagnosed with normal brain and other dementia, And what can screen many easily is desirable. However, as mentioned above, all the markers proposed so far are far from ideal markers and are insufficient for definitive diagnosis of Alzheimer's disease. Has not been found.

  The present invention has been made in view of such a situation, and an object thereof is to develop a new diagnostic kit, diagnostic marker, and detection method having high effectiveness for the diagnosis of Alzheimer's disease.

  The present invention is a diagnostic kit for diagnosing Alzheimer's disease, and for detecting quantitatively the amount of sugar chain derived from complement C3 protein in a body fluid sample of a subject mammal Diagnostic kits having means are provided. It is demonstrated in the examples described later that the amount of sugar chain derived from complement C3 protein in body fluid is changed in patients suffering from Alzheimer's disease. Therefore, by using this diagnostic kit, early diagnosis of Alzheimer's disease and differential diagnosis from other dementia exhibiting symptoms similar to Alzheimer's disease become possible.

  In addition, according to the present invention, there is provided a diagnostic marker for diagnosing Alzheimer's disease, the diagnostic marker comprising the amount of sugar chain derived from complement C3 protein in a body fluid sample of a subject mammal Is done. It is demonstrated in the examples described later that the amount of sugar chain derived from complement C3 protein in body fluid is changed in patients suffering from Alzheimer's disease. Therefore, this diagnostic marker can serve as a marker that enables early diagnosis of Alzheimer's disease and differential diagnosis from other dementia exhibiting symptoms similar to Alzheimer's disease.

  Further, according to the present invention, there is provided a method for detecting a disease state index of Alzheimer's disease, comprising a step of quantitatively detecting the amount of sugar chain derived from complement C3 protein in a body fluid sample of a subject mammal. Including detection methods are provided. It is demonstrated in the examples described later that the amount of sugar chain derived from complement C3 protein in body fluid is changed in patients suffering from Alzheimer's disease. Therefore, by using this detection method, early detection of Alzheimer's disease and other dementia exhibiting symptoms similar to Alzheimer's disease can be distinguished and detected.

  The above-described diagnostic kit, diagnostic marker, and detection method are merely one embodiment of the present invention, and according to the present invention, a diagnostic method for Alzheimer's disease using these is also provided.

  The present invention makes it possible to make a diagnosis highly correlated with the Alzheimer's disease state.

FIG. 1 is a graph showing the results of measuring the concentration of complement C3 protein in a control group and an Alzheimer's disease patient (AD) group in CSF and blood. FIG. 2 is a graph showing the results of measuring the amount of sugar chains derived from complement C3 protein in the control group and AD patient group in CSF. FIG. 3 is a graph showing the results of measuring the amount of sugar chains derived from complement C3 protein in the control group and AD patient group in blood. FIG. 4 is a diagram showing the results of measuring the WGA binding amount and ConA binding amount of the sugar chain of complement C3 protein in the control group and AD patient group in CSF, and multiplying them respectively. FIG. 5 is a diagram showing the results of measuring the WGA binding amount and ConA binding amount of the sugar chain of complement C3 protein in the control group and AD patient group in blood and multiplying them respectively. FIG. 6 is a diagram showing the results of WGA binding amount of sugar chains of complement C3 protein in the control group and AD patient group in CSF and blood analyzed by clinical dementia scale (CDR). FIG. 7 is a graph showing the results of ConA binding amounts of sugar chains of complement C3 protein in the control group and AD patient group in CSF and blood analyzed by CDR. FIG. 8 is a diagram showing the results of measuring the WGA binding amount and the ConA binding amount of the sugar chain of the complement C3 protein in the control group and AD patient group in the CSF, analyzed by CDR, and multiplying them respectively. FIG. 9 is a diagram showing the results of measuring the WGA binding amount and the ConA binding amount of the sugar chain of the complement C3 protein in the control group and AD patient group in blood, analyzed by CDR, and multiplying them respectively. FIG. 10 is a correlation diagram of the amount of amyloid β protein 42 of AD patient group in CSF and the amount of WGA binding or ConA binding of complement C3 protein. FIG. 11 is a correlation diagram between the amount of amyloid β protein 42 in the CSF of the AD patient group and the amount of WGA binding of complement C3 protein in the blood. FIG. 12 is a correlation diagram between the amount of WGA binding and the amount of ConA binding of complement C3 protein in the control group and AD patient group in CSF. FIG. 13 is a correlation diagram between the amount of WGA binding of complement C3 protein in CSF and the amount of WGA binding of complement C3 protein in blood in AD patients.

  As a result of intensive studies, the present inventors have found that it is possible to reliably diagnose Alzheimer's disease by quantitatively detecting the amount of sugar chains derived from complement C3 protein in body fluids. . In addition, the present inventors have found that a combination of known Alzheimer's disease diagnosis markers enables complex diagnosis of Alzheimer's disease. Furthermore, the present inventors have quantitatively detected the amount of mannose and the amount of N-acetylglucosamine in the sugar chain, and have found that simple and rapid diagnosis of Alzheimer's disease can be made from the quantitative ratio.

  Hereinafter, embodiments of the present invention will be described in detail. In addition, about the same content, in order to avoid the complexity of repetition, description of omission is abbreviate | omitted.

<Diagnostic kit>
One embodiment of the present invention is a diagnostic kit for diagnosing Alzheimer's disease, for quantitatively detecting the amount of sugar chain derived from complement C3 protein in a body fluid sample of a subject mammal This diagnostic kit has an early diagnosis of Alzheimer's disease and a differential diagnosis from other dementia exhibiting symptoms similar to Alzheimer's disease.

The “complement C3 protein” in the present embodiment is one component of a group of proteins that mediate an immune response contained in a body fluid. Complement C3 protein is a glycoprotein composed of a dimer of 116 kDa α chain and 70 kDa β chain. As for the α chain, the N-linked sugar chain of Man _8-9 GlcNAc ₂ is modified at the 268th asparagine, and the N-linked sugar chain of Man _5-6 GlcNAc ₂ is modified at the 63rd asparagine. (Crispin MD et al., (2004) Monoglucosylated glycans in the secreted human complement component C3: implications for protein biosynthesis and structure.FEBS Lett 566, 270-274 and Hase S et al., (1985) Structures of sugar chains of the third component of human complement. J Biochem 98, 863-874.). Complement C3 protein is usually an inactive precursor form, but is decomposed into C3a (molecular weight 9,000) and C3b (molecular weight 185,000) to become an active form when assisting the elimination of pathogens. Complement C3 protein has been suggested to cause various diseases, and tests for measuring complement C3 protein in blood have already been put to practical use. However, various studies have been conducted on the relationship between Alzheimer's disease and complement C3 protein from the viewpoint of its amount and activity, but regarding the relationship between complement C3 protein and its derived sugar chain, It was not revealed until.

  As demonstrated in the examples described later, the amount of sugar chains derived from complement C3 protein in body fluids is changed in patients suffering from Alzheimer's disease. Therefore, for example, when the amount of the sugar chain is quantitatively detected and a change is confirmed, it is diagnosed that the possibility of Alzheimer's disease is high. However, the present invention is not limited to this, and other symptoms / conditions related to Alzheimer's disease are also measured by daily methods well known to doctors in this technical field, and their disease state indices are determined using the diagnostic kit according to the present embodiment. It is easy to get.

  In the present embodiment, “Alzheimer's disease” refers to dementia in which atrophy of the cerebral cortex is observed, and pathologically, in addition to severe neuronal loss, characteristic lesions such as senile plaques and neurofibrillary tangles are observed. Dementia caused by Alzheimer's disease is called Alzheimer-type dementia. Of these lesions, senile plaques appear the earliest and are characteristic changes in Alzheimer's disease, and are considered the most important changes in Alzheimer's disease. The main component of senile plaques is amyloid β protein (Aβ), and there is a mutation in amyloid precursor protein (APP), which is the precursor gene of Aβ in familial Alzheimer's disease showing autosomal dominant inheritance. It is thought that abnormal production or degradation of selenium is deeply related to the onset and progression of Alzheimer's disease. Aβ is produced from amyloid precursor protein through two cleavages by β-secretase belonging to aspartic protease and γ-secretase (presenilin complex). Alzheimer's disease is diagnosed based on whether it meets various diagnostic criteria from clinical symptoms, neuropsychological examination, physiological examination, and brain function imaging examination.

  The “mammal” in the present embodiment refers to any mammal, and includes humans, domestic animals, pet animals, zoo animals, or sports animals such as dogs, cats, cows, pigs, horses, sheep. Including rabbits. The mammal is preferably a human.

  “Quantitative detection” in the present embodiment means detection based on numerical data such as concentration, intensity, mobility, mobility, and the like. In another embodiment, “quantitative detection” means detection based on the presence or absence of numerical data, for example, by adjusting the concentration, adjusting the detection sensitivity, setting a threshold value, or the like.

  In the present embodiment, the “sugar chain detection means” may be a plurality of sugar chain detection means. The plurality of sugar chain detection means are, for example, different sugar chain detection means for multifaceted analysis, for example, the same or similar sugar chain detection means for the purpose of obtaining statistically reliable data, or a combination thereof. It may be.

  Further, in a further embodiment of the present invention, the amount of the sugar chain of the subject of the same species as the mammal is shown as a disease state index of Alzheimer's disease relative to the amount of the sugar chain in a healthy mammal. This is a diagnostic kit. By employing the amount of the sugar chain in a healthy mammal as a control, Alzheimer's disease can be reliably diagnosed.

  For example, when it is observed that the amount of the sugar chain of the complement C3 protein in the subject is significantly lower than the amount of the sugar chain in a healthy mammal of the same species as the subject, Diagnosed as having a high possibility of Alzheimer's disease. However, the present invention is not limited to this, and other symptoms / conditions related to Alzheimer's disease are also measured by daily methods well known to doctors in this technical field, and their disease state indices are determined using the diagnostic kit according to the present embodiment. It is easy to get.

  The “pathological index of Alzheimer's disease” in the present embodiment refers to an index relating to the state of Alzheimer's disease including, for example, the onset of Alzheimer's disease or Alzheimer-type dementia. Here, the pathological index of Alzheimer's disease is, for example, prediction of the risk of developing Alzheimer's disease, early diagnosis / prediction of Alzheimer's disease, estimation of the degree of cerebral dysfunction, understanding of the disease state, prediction of the course, treatment results Includes indicators such as observation / evaluation and prognosis prediction. A plurality of Alzheimer's disease state indices can be combined. Thereby, a more reliable diagnosis result can be obtained. The above symptoms and parameters representing the symptoms can be easily measured by routine techniques well known to physicians in this technical field.

  “Significant” in the present embodiment means that the p-value (U test, two-sided test) is preferably <0.05, more preferably <0.01, and most preferably <0.001.

  The “amount of the sugar chain in a healthy mammal” in the present embodiment may be based on data measured in advance. The measurement method and conditions performed on a healthy mammal are not particularly limited, but the measurement method and conditions performed on a subject are preferably the same. In addition, within the scope of the present embodiment, the measurement method and conditions performed on healthy mammals are different from the measurement method and conditions performed on the subject as long as the obtained diagnostic results are not affected. is there.

  Furthermore, a further embodiment of the present invention is the above-described diagnostic kit, wherein the amount of the sugar chain is a value obtained by combining the amount of mannose and the amount of N-acetylglucosamine in the sugar chain. By using the value obtained by totaling the amount of mannose and N-acetylglucosamine in the sugar chain as a disease state index of Alzheimer's disease, the difference between a healthy mammal as a control and the subject is clarified and reliable. It becomes possible to diagnose Alzheimer's disease. The total value obtained above is a value obtained by performing commonly used statistical processing, and is preferably the statistical processing described above so that the difference from the healthy control body is clear. There is no limit to statistical processing.

  Moreover, the further embodiment of this invention is the above-mentioned diagnostic kit whose value obtained by the said synthesis | combination is a value obtained by multiplication. By using the value obtained by the above multiplication as a disease state index of Alzheimer's disease, a difference from a healthy body as a control becomes clear by easy statistical processing, and it becomes possible to diagnose Alzheimer's disease with certainty.

  Moreover, the further embodiment of this invention is the above-mentioned diagnostic kit which further has a known marker detection means which detects quantitatively the quantity of the known Alzheimer's disease diagnostic marker. By combining the amount of the sugar chain and the amount of the known marker, it becomes possible to diagnose Alzheimer's disease with certainty.

  Examples of the known markers include amyloid β protein, glycosylated acetylcholinesterase, interleukin-6, substance-P, cystatin C, serum apolipoprotein, serum homocysteine, APP isoform, interleukin-6, α1-antichymotrypsin ( ACT), oxygenase-1, 24S-hydroxycholesterol, acetylcholine, somatostatin, vasopressin and the like. The amount of the known marker may be an active amount. Examples thereof include acetylcholine transferase activity and acetylcholinesterase activity. However, as long as it is possible to diagnose Alzheimer's disease by combining with the amount of the sugar chain, any of them may be adopted. The known marker is not limited to Alzheimer's disease specific marker. This is because it is possible to reliably diagnose Alzheimer's disease by combining with the amount of the sugar chain.

  In addition, a plurality of known markers can be adopted as the known marker, and a plurality of known marker detection means matched to each known marker can be prepared. Examples of the detection means include an enzyme immunoassay using a specific marker-specific antibody, a Western blot, an antibody array, and the like, and a plurality of combinations can also be used.

  Furthermore, a further embodiment of the present invention is the above-described diagnostic kit, which shows a quantitative ratio between the amount of the sugar chain and the amount of the known marker in a subject that is a mammal as a disease state index of Alzheimer's disease. Alzheimer's disease can be diagnosed in a complex manner by using the ratio of the amount of the sugar chain and the amount of the known marker as a disease state index of Alzheimer's disease.

  For example, in this diagnostic kit, when there is a positive correlation between the amount of the sugar chain in the subject and the amount of the known marker, the possibility of Alzheimer's disease is high as a result based on complex detection. Is diagnosed. However, the present invention is not limited to this, and other symptoms / conditions related to Alzheimer's disease are also measured by daily methods well known to doctors in this technical field, and their disease state indices are determined using the diagnostic kit according to the present embodiment. It is easy to get.

  Moreover, the further embodiment of this invention is the above-mentioned diagnostic kit whose said known marker is amyloid beta protein. Amyloid β protein has been confirmed to be clinically useful as a diagnostic marker for Alzheimer's disease. Therefore, it is possible to diagnose Alzheimer's disease with high reliability and accuracy by using the quantitative ratio between the amount of amyloid β protein and the amount of sugar chain derived from complement C3 protein as a disease state index of Alzheimer's disease. Become.

  Furthermore, a further embodiment of the present invention is the above-described diagnostic kit, which shows the quantitative ratio of the mannose amount of the sugar chain and the N-acetylglucosamine amount in a subject that is a mammal as an indicator of Alzheimer's disease. . Alzheimer's disease can be diagnosed easily and quickly by using the above-mentioned ratio of the amount of mannose and the amount of N-acetylglucosamine as a disease state index of Alzheimer's disease.

  For example, in this diagnostic kit, the decrease in the ratio of the mannose amount of the sugar chain to the N-acetylglucosamine amount of the sugar chain of the complement C3 protein is compared to the ratio obtained from the same species of mammal as the subject. If it is observed, it is diagnosed that the possibility of Alzheimer's disease is high. However, the present invention is not limited to this, and other symptoms / conditions related to Alzheimer's disease are also measured by daily methods well known to doctors in this technical field, and their disease state indices are determined using the diagnostic kit according to the present embodiment. It is easy to get. In addition, either the N-acetylglucosamine amount or the mannose amount may be detected first. Furthermore, it is also possible to detect continuously by a high throughput method.

  Furthermore, a further embodiment of the present invention is the above-described diagnostic kit using cerebrospinal fluid or blood as a body fluid. Since collection of cerebrospinal fluid or blood has been established, it is possible to make a diagnosis with low risk to the patient. In addition, since cerebrospinal fluid is close to the brain, it is possible to obtain a diagnosis result having a higher correlation with brain symptoms. In addition, since blood is a body fluid that can be collected from a patient more quickly and easily, it is possible to make a very convenient diagnosis for the patient.

  Cerebrospinal fluid is not limited to ventricular cerebrospinal fluid, and lumbar cerebrospinal fluid can also be used. When lumbar spinal fluid is used, the risk is lower in living subjects. Diagnosis can be performed. Regarding cerebrospinal fluid collection, a cerebrospinal fluid collection method usually performed, such as cerebral puncture, lumbar puncture, ventricular puncture, and spinal puncture, can be used depending on the situation.

  Here, with respect to blood, it is also possible to use a serum that has been subjected to an appropriate centrifugation operation and reduced to a negligible amount of lymphocytes. In the present embodiment, it is possible to react a detection reagent with the serum.

  Furthermore, diagnosis using the above-described diagnostic kit using a blood sample, which is faster and simpler and more convenient for the patient, is performed as a primary screening, and as a secondary diagnosis, diagnosis using the above-described diagnostic kit using cerebrospinal fluid is performed. Can also be performed, and is included in this embodiment. In that case, a diagnostic index that also includes the amount of the sugar chain of complement C3 protein used as the primary screening in the secondary diagnosis can be used.

  Here, with respect to the sample, one or a plurality of arbitrary processing steps may be added to the obtained sample such as cerebrospinal fluid, blood, and serum. Examples of such treatment steps include removal of impurities by various columns and immunoprecipitation methods, purification of target substances, protein fragmentation by trypsin, etc., separation of sugar chains by enzyme treatment or chemical treatment, other enzymes Treatment, various chemical modifications, and the like, but are not limited thereto.

  Moreover, the further embodiment of this invention is the above-mentioned diagnostic kit whose said subject is a subject whose diagnosis result by dementia severity evaluation is very mild or mild. By combining the diagnosis results of Alzheimer's disease based on the evaluation of the severity of dementia, it becomes possible to diagnose Alzheimer's disease with certainty. Moreover, it is advantageous for pre-diagnosis diagnosis and early diagnosis of Alzheimer's disease, and enables effective prevention and treatment. Furthermore, even if the diagnosis result of the dementia severity assessment is extremely mild, it can be combined, so that early diagnosis of Alzheimer's disease becomes possible. Dementia severity tests include, for example, the Clinical Dementia Scale (CDR), the Elderly Depression Scale (GDS), the revised Hasegawa Simple Intelligence Rating Scale (HDS-R), the Mini Mental State (MMSE) test, Wexler adults Intelligence test (WAIS-R), Alzheimer's disease evaluation scale (ADAS), etc. can be mentioned.

  The diagnostic kit according to the present embodiment may further include a dementia severity evaluation means. By having the evaluation means, detection of the amount of the sugar chain and the evaluation can be performed in parallel or in succession.

  Further, either the detection of the sugar chain amount of the complement C3 protein or the diagnosis based on the evaluation of the severity of dementia may be performed first. Preferably, the dementia severity assessment is performed first with a low burden on the subject. This is because the burden of collecting body fluid on a subject can be avoided when it is diagnosed that the possibility of Alzheimer's disease is low at the stage of the diagnosis of dementia severity.

  Moreover, the further embodiment of this invention is the above-mentioned diagnostic kit whose said dementia severity evaluation is a clinical dementia scale (CDR). CDR is a dementia evaluation method widely used internationally, and focuses on family information. The evaluation is a five-level evaluation, with 0 being normal, 0.5 being extremely mild, 1 being mild, 2 being moderate, and 3 being severe. By combining the diagnosis results by CDR, it becomes possible to diagnose Alzheimer's disease with certainty. The diagnostic kit according to the present invention enables early diagnosis of Alzheimer's disease even with CDR 0.5 (very mild) or CDR 1 (mild).

  Further, a further embodiment of the present invention is the above-described diagnostic kit in which the sugar chain is a WGA- and ConA-binding sugar chain, and according to this diagnostic kit, the pathological condition of Alzheimer's disease can be grasped more accurately. It becomes. As WGA and ConA-binding sugar chains derived from complement C3 protein are observed with more certain quantitative fluctuations in patients with Alzheimer's disease, they can be used for more reliable diagnosis.

  Further, a further embodiment of the present invention is to derive a complement C3 protein by performing a lectin enzyme immunoassay using a lectin as a sugar chain detection means or an enzyme immunoassay using a sugar chain recognition antibody. It is the above-mentioned diagnostic kit that detects the sugar chain. This diagnostic kit is a well-established technology that is used in the clinical practice of enzyme immunoassay, or by performing lectin enzyme immunoassay, which is a modified method using lectins in enzyme immunoassay. It enables simple, reliable and inexpensive numerical measurement and can process a large amount of samples.

  Here, the lectin used in the embodiment of the present invention is not particularly limited as long as it is WGA-binding and recognizes a ConA-binding sugar chain. For example, lectins that recognize mannose include Canavalia ensiformis agglutinin (ConA), Lens culinaris agglutinin (LCA), Pisum sativum agglutinin (PSA), Bowringia milbraedii agglutinin (BMA), Dolichos lablab agglutinin (DLA), Glisinal (DLA), ), Gerardia savaglia lectin (GSL), Machaerium biovulatum agglutinin (MBA), Macharium lunatus agglutinin (MLA), Narcissus pseudonarcissus agglutinin (NPA), Epipactis helleborine agglutinin (EHA), Listera ovataAgglutinin (EHA), Listera ovataagglutinin (EHA) Narcissus lobularis agglutinin (NLA), Vigna racemosa agglutinin (VRA), Pterocarpus rhorii agglutinin (PRA), and the like. It is also possible to measure the amount of mannose from the amount of lectin bound to the sugar chain, preferably a method of measuring the amount of mannose from the amount of ConA bound, which has a high binding affinity for mannose. Also, lectins that recognize N-acetylglucosamine include Wheat germ agglutinin (WGA), Datura stramonium agglutinin (DSA), Oryza sativa agglutinin (OSA), Phytolacca americana agglutinin (PWM), Urtica dioica agglutinin (UDA), Solanum tuberosum Examples include lectin (STL), Lycipersicon esculentum agglutinin (LEA), Datura stramonium agglutinin (DSA), Pokeweed mitogen (PWM) and its isoforms (PL-D1 and PL-D2) and Ym1. It is also possible to measure the amount of N-acetylglucosamine from the amount of lectin bound to the sugar chain. Preferably, the amount of N-acetyl has a binding affinity for N-acetylglucosamine and is easily available. This is a method for measuring the amount of glucosamine.

  The lectin used is not limited to a purified natural lectin, and may be, for example, a lectin that is expressed and purified in a host such as Escherichia coli. In addition, a sequence other than the sugar recognition site (base sequence or amino acid sequence) may be modified (for example, mutated, deleted, inserted, added) using standard molecular biological techniques. Even a part may be modified to keep its function at a minimum, improve it, or give a new function.

  In the embodiment of the present invention, in order to detect or measure an antibody or a lectin, a labeled substance capable of generating a signal, a labeled antibody and a labeled lectin in which the antibody and the lectin itself are bound may be used. In that case, in addition to those directly bound, it is also possible to use an antibody or a lectin and a labeling substance bound by an avidin-biotin or streptavidin-biotin system or a secondary antibody. Is included in the technical scope. As the secondary antibody used here, a primary antibody or an antibody capable of binding to a lectin can be used.

  When using an enzyme as a label, for example, horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase, glucoamylase, carbonic acid dehydrogenase, acetylcholinesterase, lysozyme, malonate dehydrogenase, glucose-6-phosphate Dehydrogenase or the like can be used as a label. As a method for labeling with these enzymes, the sugar chain of the enzyme is oxidized with periodic acid, and an amino acid of an antibody or lectin is bound to the generated aldehyde group, or a maleimide group or a pyridyl sulfide group is introduced into the enzyme. And a method of binding to a thiol group present in an antibody Fab ′ fragment or lectin.

  When using an enzyme as a label, after incubating the test sample and the labeled antibody or lectin, the released labeled antibody or lectin is washed away and then reacted with the above-mentioned labeled enzyme substrate to react with color, etc. The labeled antibody or lectin can be detected by measuring. For example, when labeled with peroxidase, brown or yellow is produced in combination with hydrogen peroxide as a substrate and diaminobenzidine or O-phenylenediamine as a coloring reagent. In the case of labeling with glucose oxidase, for example, 2, 2′-acid-di- (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) can be used as a substrate.

  When a fluorescent dye is used as the label, for example, the antibody or lectin can be labeled with a fluorescent dye such as FITC (fluorescein isothiocyanate) or TRITC (tetramethylrhodamine B isothiocyanate). Binding of the antibody or lectin and the fluorescent dye can be performed by a conventional method.

  When using a colored labeling substance as a label, for example, colloidal metal and colored latex can be used as the label. Typical examples of colloidal metals include metal colloidal particles that are dispersed particles of gold sol, silver sol, selenium sol, tellurium sol, platinum sol, and the like. The size of the colloidal metal particles is usually preferably about 3 to 60 nm in diameter. Moreover, as a representative example of colored latex, synthetic latex such as polystyrene latex colored with respective colorants such as red and blue can be mentioned. Natural latex such as natural rubber latex may be used as the latex. The size of the colored latex can be selected from a diameter of several tens to several hundreds of nanometers. Commercially available products can be used as they are for these color labeling substances, but they can be further processed or produced by methods known per se.

  The binding between the antibody or lectin and the color labeling substance can be performed by a conventional method. For example, in the case of colloidal gold particles in which the color labeling substance is a dispersed particle of gold sol, it is usually possible to physically bond the two by mixing the antibody and the gold sol at room temperature. .

In addition to the above, a radioisotope label (for example, ¹²⁵ I, ¹³¹ I, ³ H, ¹⁴ C, etc.) can be used as the label, and is included in the scope of the present invention.

  As a sugar chain recognition antibody, for example, a 2G12 antibody that recognizes terminal α1,2 mannose on HIV-1 env gp120, which is a glycoprotein, may be used. Moreover, you may use the antibody with respect to the sugar chain which added the appropriate compound. For example, an antibody against acetylglucosamine labeled with 2-aminopyridine (see Biochemistry Vol. 75 No. 8 (2003)) can also be used. Furthermore, it is possible to prepare and use a sugar chain recognition antibody. For example, it can be produced by the following method.

  When preparing a sugar chain-recognizing polyclonal antibody, for example, a sugar chain or a part thereof, a complement C3 protein or a partial peptide to which a sugar chain is added, or a complex of these and another carrier molecule or carrier protein is heated. It can be prepared by immunizing a blood animal, collecting blood containing a polyclonal antibody from the immunized animal, and performing antibody separation and purification.

  An example of a manufacturing method is shown below. The kind of carrier protein and the mixing ratio of the carrier and the antigen may be any conditions as long as the antibody can be efficiently produced against the antigen immunized by crosslinking with the carrier. For example, bovine serum albumin, bovine thyroglobulin, hemocyanin And the like are bound to antigen 1 at a weight ratio of about 0.1 to 20, preferably about 1 to 5. In addition, various condensing agents can be used for coupling the antigen and the carrier. For example, glutaraldehyde, carbodiimide, maleimide active ester, thiol group, active ester reagent containing dithiobilidyl group and the like are used. When a small molecule is used as an antigen, it is particularly preferable to use a carrier protein.

  The antigen or antigen-carrier complex is administered to a warm-blooded animal per se or together with a suitable carrier, buffer, or diluent at a site where antibody production is possible. In order to increase the antibody production ability upon administration, for example, complete Freund's adjuvant, incomplete Freund's adjuvant, RAS (MPL (Monophosphoryl Lipid A) + TDM (Synthetic Trehalose Dicorynomycolate) + CWS (Cell Wall Skeleton) adjuvant system), aluminum hydroxide, etc. Ordinarily used adjuvants may be administered. These adjuvants and antigens may be administered in the form of a suspension or emulsion using a diluent. As used herein, an adjuvant refers to a substance that nonspecifically enhances the immune response to an antigen when administered together with the antigen.

  As the warm-blooded animal to be immunized, mammals such as rabbits, mice, hamsters, guinea pigs, chickens, rats, dogs, goats, sheep and cows can be used. For example, the immunization method can be performed by administering an antigen one or more times using a normal immunization method known to those skilled in the art. Specifically, antigen administration can usually be performed about once every 6 weeks, about 2 to 10 times in total. For example, the dosage may be about 0.05 to 2 mg of antigen per administration. The administration route is not particularly limited, and for example, subcutaneous administration, intradermal administration, intraperitoneal administration, intravenous administration, intramuscular administration, etc. can be selected as appropriate, and intravenous, intraperitoneal or subcutaneous injection is performed. Is preferably administered. After raising the immunized mammal for 0.5 to 4 months, a small sample of the mammal's serum can be collected from the ear vein or the like, and the antibody titer can be measured. When the antibody titer rises, the antigen is administered as appropriate depending on the situation. For example, booster immunization can be performed using 10 μg to 1000 μg of antigen. The antibody titer can be measured, for example, by reacting the labeled protein with antiserum and then measuring the activity of the labeling agent bound to the antibody.

  Blood or ascites is collected from mammals immunized 1-2 months after the last dose by normal methods, treated at 56 ° C for 30 minutes to inactivate the complement system, and then specific by affinity chromatography The antibody is separated and purified to obtain a polyclonal antibody. As the affinity carrier, for example, an antigen peptide immobilized on Affigel or the like can be used. Alternatively, the obtained blood may be separated and purified by ordinary methods such as centrifugation, precipitation using ammonium sulfate or polyethylene glycol, gel filtration chromatography, ion exchange chromatography and the like.

  When preparing a sugar chain-recognizing monoclonal antibody, for example, a sugar chain or a part thereof, a complement C3 protein to which a sugar chain is added or a partial peptide thereof, or a complex of these and another carrier molecule or carrier protein is used. Immunize a warm-blooded animal, collect spleen cells, lymph node cells, B lymphocytes, etc. from the immunized animal, and obtain a hybridoma that produces a monoclonal antibody by cell fusion of the obtained cells with a myeloma cell line, It can be produced by isolating a monoclonal antibody from the hybridoma.

  An example of a manufacturing method is shown below. Up to the immunization step of the warm-blooded animal can be carried out by the same method as the production of the polyclonal antibody described above. Examples of the warm-blooded animal to be immunized include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats and chickens. Preferably, mice or rats are used. The immunization method can be performed by, for example, administering an antigen one or more times using a normal immunization method known to those skilled in the art. Specifically, antigen administration can usually be performed about once every 6 weeks, about 2-10 times in total. For example, the dosage may be about 0.05 to 2 mg of antigen per administration. The administration route is not particularly limited, and for example, subcutaneous administration, intradermal administration, intraperitoneal administration, intravenous administration, intramuscular administration, etc. can be selected as appropriate, and intravenous, intraperitoneal or subcutaneous injection is performed. Is preferably administered.

  Individuals with antibody titers are selected from warm-blooded animals immunized with the antigen, and the spleen or lymph nodes are collected 2 to 5 days after the final immunization, and the spleen cells, lymph node cells, and B lymphocytes contained therein Obtain antibody-producing cells. The antibody titer in the antiserum can be measured, for example, by reacting the labeled protein with the antiserum and then measuring the activity of the labeling agent bound to the antibody.

  Subsequently, these antibody-producing cells are fused with myeloma (myeloma cells), and the fusion operation is performed by a known method such as the method of Kohler and Milstein (G. Kohler et al., Nature, 1975, 495, 256). ). Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but preferably PEG is used. Examples of myeloma include NS-1, P3U1, SP2 / 0, AP-1, and the like, but P3U1, SP2 / 0, and P3X63Ag8 are particularly preferably used for mice. The preferred ratio of the number of antibody-producing cells used and the number of myeloma is about 1: 1-20: 1, PEG (preferably PEG1000-PEG6000) is added at a concentration of about 10-80%, 20-40 ° C., Preferably, cell fusion can be carried out efficiently by culturing at 30 to 37 ° C. for 1 to 10 minutes.

  Selective culture that also serves as a hybridoma screening can be carried out in an animal cell medium supplemented with HAT (hypoxanthine, aminopterin, thymidine). As a breeding medium, any medium may be used as long as it can grow a hybridoma. For example, RPMI-1640 medium containing 1-20% fetal bovine serum, preferably 10-20% fetal bovine serum, GIT medium containing 1-10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or hybridoma culture A serum medium (SFM-101, Nissui Pharmaceutical Co., Ltd.) or the like can be used. The culture temperature is usually 20-40 ° C, preferably about 37 ° C. The culture time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. Culturing can usually be performed under 5% carbon dioxide gas. Moreover, the hybridoma obtained by cell fusion can be cloned by a limiting dilution method or the like.

  Various methods can be used for screening monoclonal antibodies produced by each hybridoma.For example, the hybridoma culture supernatant is added to a solid phase (such as a microplate) on which an antigen is adsorbed directly or together with a carrier. Next, the anti-immunoglobulin antibody labeled with a radioactive substance or enzyme (an antibody that reacts with the same type of immunoglobulin as the antibody-producing cells used for cell fusion), protein A or protein G, and monoclonal bound to the solid phase Antibody detection method, Immunoglobulin antibody, Hybridoma culture supernatant added to solid phase adsorbed with protein A or protein G, antigen labeled with radioactive substance or enzyme etc., added monoclonal antibody bound to solid phase A detection method or the like is used.

  In order to produce a target monoclonal antibody from the hybridoma thus selected, the hybridoma is cultured by a normal cell culture method or ascites formation method, and the monoclonal antibody is purified from the culture supernatant or ascites. Purification of the monoclonal antibody from the culture supernatant or ascites can be performed by a conventional method. For example, immunoglobulin separation and purification methods (salting out method, ammonium sulfate fractionation, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, adsorption / desorption method by ion exchanger (DEAE etc.), chromatography, ultracentrifugation method, Gel filtration method, affinity chromatography, antigen-binding solid phase or specific purification method in which only antibody is collected by an active adsorbent such as protein A or protein G, and the antibody is obtained by dissociating the binding.

  In addition, as a monoclonal antibody against a sugar chain, an IgM antibody is often obtained, and the obtained IgM antibody can be used as it is, or it can be used after being modified to an IgG antibody by a genetic engineering technique well known to those skilled in the art. Is also possible.

  The above is an exemplification, and those skilled in the art can easily make various design changes / modifications and adjustments of various conditions in order to obtain a target sugar chain-recognizing antibody in accordance with an ordinary antibody production method.

  Further, in a further embodiment of the present invention, the diagnosis described above detects a sugar chain derived from complement C3 protein by performing lectin affinity electrophoresis using a gel containing lectin as the sugar chain detection means. It is a kit. In electrophoresis on gels containing lectins, proteins with a sugar chain that binds to the target lectin have a lower mobility from the point of application as the lectin affinity (binding force) is stronger. It is possible to clearly separate complement C3 protein having a small amount (weak lectin binding ability) and complement C3 protein having a high sugar chain and high affinity.

  Further, in a further embodiment of the present invention, a sugar chain derived from complement C3 protein is obtained by performing lectin blot analysis using lectin as the sugar chain detection means or Western blot analysis using a sugar chain recognition antibody. It is the above-mentioned diagnostic kit which detects. By blotting with a lectin or an antibody that specifically binds to a sugar chain, it is possible to identify a sugar chain derived from complement C3 protein according to the state of the sugar chain.

  Further, in a further embodiment of the present invention, the sugar chain derived from complement C3 protein is detected by performing thin-layer chromatography using a lectin or a sugar chain recognition antibody as the sugar chain detection means. This is a diagnostic kit. By using thin-layer chromatography that can develop the target sample in a short time, it is possible to separate complement C3 protein from other contaminating proteins, and recognize lectins or sugar chains easily and quickly. Sugar chains can be detected by antibodies. Also, this embodiment includes changing the mobility of complement C3 protein using an antibody against complement C3 protein or a carrier to which the antibody is bound.

  Further, in a further embodiment of the present invention, a lectin array using a lectin array chip provided with a lectin as the sugar chain detecting means or an antibody array using an antibody array chip provided with a sugar chain recognition antibody It is the above-mentioned diagnostic kit which detects the sugar chain derived from complement C3 protein by performing. By using a lectin array or an antibody array, a multifaceted simultaneous analysis including many factors such as other proteins and a simple analysis are possible. In particular, by combining an antibody that recognizes other factors with a lectin or sugar chain recognition antibody, it becomes possible to measure the sugar chain addition status of many factors at once, allowing more detailed analysis of pathophysiology and pathogenesis. It becomes possible.

  Furthermore, a further embodiment of the present invention is the above-described diagnostic kit for detecting a sugar chain derived from complement C3 protein using WGA and / or ConA as the lectin. By using WGA and / or ConA as a lectin, it is possible to reliably detect the sugar chain state of complement C3 protein.

  Further, a further embodiment of the present invention is a diagnostic kit for detecting a sugar chain cleaved from complement C3 protein by performing liquid chromatography as a detection means. By fluorescently labeling the cleaved sugar chain with, for example, 2-aminopyridine, it is possible to detect even a picomolar sugar chain amount with a fluorescence detector. Therefore, it is possible to detect the sugar chain state of complement C3 protein even with a small amount of body fluid. In addition, the present embodiment also includes cleaving the sugar chain after purifying complement C3 protein. The method for cleaving the sugar chain from the complement C3 protein is not particularly limited, but is preferably a hydrazine decomposition method using anhydrous hydrazine in which the protein is degraded but the sugar chain is not degraded.

<Diagnostic marker>
Another embodiment of the present invention is a diagnostic marker for diagnosing Alzheimer's disease, comprising a quantity of sugar chains derived from complement C3 protein in a body fluid sample of a subject mammal It is a marker, and this diagnostic marker can serve as a marker that enables early diagnosis of Alzheimer's disease and differential diagnosis from other dementia exhibiting symptoms similar to Alzheimer's disease.

  As demonstrated in the examples described later, the amount of sugar chain derived from complement C3 protein in body fluid is changed in patients suffering from Alzheimer's disease. Therefore, for example, by using the amount of the sugar chain derived from the complement C3 protein as a diagnostic marker, it plays a role as a marker indicating that there is a high possibility of Alzheimer's disease from the quantitative change of the sugar chain. It is possible to diagnose Alzheimer's disease based on the quantitative change of the sugar chain. However, the present invention is not limited to this, and other symptoms / conditions related to Alzheimer's disease are also measured by daily methods well known to doctors in this technical field, and diagnosed using the diagnostic marker according to the present embodiment. It is possible easily.

  Further, in a further embodiment of the present invention, the amount of the sugar chain is the amount of the sugar chain of the subject of the same species as the mammal relative to the amount of the sugar chain in a healthy mammal. It is a diagnostic marker. By adopting the amount of the sugar chain in a healthy mammal as a control, it becomes a diagnostic marker that can be used for reliable Alzheimer's disease diagnosis.

  A further embodiment of the present invention is also a diagnostic marker as described above further comprising an amount of a known Alzheimer's disease diagnostic marker. By combining the amount of the sugar chain and the amount of the known marker, it becomes a diagnostic marker that can be used for more reliable diagnosis of Alzheimer's disease.

  Moreover, the further embodiment of this invention is the above-mentioned diagnostic marker containing the quantity ratio of the quantity of the said sugar_chain | carbohydrate, and the quantity of the said known marker. By using the quantitative ratio between the amount of the sugar chain and the amount of the known marker as a disease state index of Alzheimer's disease, it becomes a diagnostic marker that can be used for multifaceted and highly accurate diagnosis of Alzheimer's disease.

  For example, according to this diagnostic marker, when there is a positive correlation between the amount of the sugar chain and the amount of the marker in the subject, it plays a role as a marker indicating that the possibility of Alzheimer's disease is high Based on this correlation, it is possible to reliably diagnose Alzheimer's disease. However, the present invention is not limited to this, and other symptoms / conditions related to Alzheimer's disease are also measured by daily methods well known to doctors in this technical field, and diagnosed using the diagnostic marker according to the present embodiment. It is possible easily.

  Furthermore, a further embodiment of the present invention is the above-mentioned diagnostic marker, wherein the amount of the sugar chain is a quantitative ratio between the amount of mannose and the amount of N-acetylglucosamine in the sugar chain. By using the quantitative ratio between the amount of mannose and the amount of N-acetylglucosamine as a disease state index of Alzheimer's disease, it becomes a diagnostic marker that can be used for simple and rapid diagnosis of Alzheimer's disease.

  For example, according to this diagnostic marker, a decrease in the ratio of the amount of mannose in the sugar chain to the amount of N-acetylglucosamine in the sugar chain is observed relative to the ratio obtained from a mammal of the same species as the subject. It is possible to serve as a marker indicating that the possibility of Alzheimer's disease is high, and it is possible to reliably diagnose Alzheimer's disease based on the decrease in this ratio. However, the present invention is not limited to this, and other symptoms / conditions related to Alzheimer's disease are also measured by daily methods well known to doctors in this technical field, and diagnosed using the diagnostic marker according to the present embodiment. It is possible easily.

<Detection method>
Another embodiment of the present invention is a method for detecting a disease state index of Alzheimer's disease, which quantitatively detects the amount of a sugar chain derived from complement C3 protein in a body fluid sample of a subject mammal. It is a detection method that includes a step of performing early detection of Alzheimer's disease and other dementia that exhibits symptoms similar to Alzheimer's disease.

  As demonstrated in the examples described later, the amount of sugar chain derived from complement C3 protein in body fluid is changed in patients suffering from Alzheimer's disease. Therefore, for example, by quantitatively detecting the amount of the sugar chain, a change in the amount can be detected. Based on the result, a material judged to have a high possibility of Alzheimer's disease is obtained. However, the present invention is not limited to this, and other symptoms / conditions related to Alzheimer's disease are also measured by a daily method well known to doctors in this technical field, and their judgment is made using the detection method according to the present embodiment. It is easy to do.

  Further, in a further embodiment of the present invention, whether or not the amount of the sugar chain of the subject of the same species as the mammal is significantly reduced relative to the amount of the sugar chain in a healthy mammal. It is the above-mentioned detection method further including the process of determining. For example, when the amount of the sugar chain of the subject is significantly reduced relative to the amount of the sugar chain in a healthy mammal employed as a control, the material is considered to have a high possibility of Alzheimer's disease It becomes. Therefore, Alzheimer's disease can be reliably diagnosed by using this detection method.

  Moreover, the further embodiment of this invention is the above-mentioned detection method further including the process of detecting quantitatively the quantity of the known Alzheimer's disease diagnostic marker substance. By combining the amount of the sugar chain and the amount of the known marker, it is possible to obtain a material that is more reliably determined to have a high possibility of Alzheimer's disease.

  Furthermore, a further embodiment of the present invention is the above detection method further comprising a step of calculating a quantitative ratio between the amount of the sugar chain and the amount of the known marker. By using the quantitative ratio between the amount of the sugar chain and the amount of the known marker as a disease state index of Alzheimer's disease, it becomes possible to obtain a material that is judged to have a high possibility of Alzheimer's disease as a composite result. .

  For example, according to this detection method, when there is a positive correlation between the amount of the sugar chain and the amount of the known marker in the subject, it is determined that the possibility of Alzheimer's disease is high as a combined detection result. Material. However, the present invention is not limited to this, and other symptoms / conditions related to Alzheimer's disease are also measured by a daily method well known to doctors in this technical field, and their judgment is made using the detection method according to the present embodiment. It is easy to do.

  Further, a further embodiment of the present invention is the above-described detection method, wherein the step of quantitatively detecting the amount of sugar chain is a step of quantitatively detecting the amount of mannose and the amount of N-acetylglucosamine. By detecting the amount of mannose and the amount of N-acetylglucosamine, it becomes possible to detect more detailed and reliable Alzheimer's disease.

  Moreover, the further embodiment of this invention is the above-mentioned detection method further including the process of calculating the quantitative ratio of the said mannose amount and the said N-acetylglucosamine amount. By using the quantitative ratio between the amount of mannose and the amount of N-acetylglucosamine as a disease state index of Alzheimer's disease, it is possible to obtain a material that is judged to be highly likely to have Alzheimer's disease simply and quickly.

  For example, according to this detection method, the decrease in the ratio of the amount of mannose in the sugar chain to the amount of N-acetylglucosamine in the sugar chain of the complement C3 protein is compared to the ratio obtained from a mammal of the same species as the subject. If it is observed, the material is considered to be highly likely to have Alzheimer's disease. However, the present invention is not limited to this, and other symptoms / conditions related to Alzheimer's disease are also measured by a daily method well known to doctors in this technical field, and their judgment is made using the detection method according to the present embodiment. It is easy to do.

  Note that the diagnostic kit, diagnostic marker, and detection method described by the above embodiment do not limit the present invention, but are disclosed for the purpose of illustration. The technical scope of the present invention is defined by the description of the scope of claims, and those skilled in the art can make various design changes within the technical scope of the invention described in the scope of claims. For example, detection of sugar chains derived from complement C3 protein may be achieved by various immunochemical techniques, slot or dot blot assays, or various modified electrophoresis methods such as two-dimensional electrophoresis, Needless to say, such a diagnostic kit, diagnostic marker, and detection method are included in the technical scope of the present invention.

  Examples of the various immunochemical methods described above include, for example, a labeled antigen in which an antigen in a sample solution and a labeled antigen are competitively reacted with an antibody and then bound to an unreacted labeled antigen (F) and the antibody. There is a method of separating (B) (B / F separation), measuring the labeled amount of either B or F, and quantifying the amount of antigen in the sample solution (competitive method). As the B / F separation method, a soluble antibody is used as an antibody, and a B / F separation is performed using a polymer liquid phase (polyethylene glycol, etc.) and a secondary antibody against the soluble antibody. Examples of the antibody include a solid-phased antibody, or a solid-phase method using a secondary antibody solid-phased with a soluble primary antibody. As another immunochemical method, the antigen in the sample solution and the solid phased antigen are competitively reacted with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated, or the sample is separated. After reacting the antigen in the solution with an excess amount of the labeled antibody, and then adding the immobilized antigen to bind the unreacted labeled antibody to the solid phase, the solid phase and the liquid phase are separated. There is also a method of measuring the amount of labeling in this phase and quantifying the amount of antigen in the sample solution (immunometric method). Further, there is a method of measuring the amount of insoluble precipitate produced as a result of the antigen-antibody reaction in the gel or in the solution (nephrometry method). Even when the amount of the antigen in the test solution is small and only a small amount of precipitate can be obtained, laser nephelometry or the like utilizing laser light scattering can be used.

  Furthermore, the embodiment according to the present invention is a manufactured product including the diagnostic kit according to the present invention. The article of manufacture includes a container and a label or package insert attached to the container. Containers include, for example, bottles, vials, syringes, etc., and are selected from suitable materials such as glass or plastic. The label or package insert indicates that the diagnostic kit according to the present invention is used for Alzheimer's disease diagnosis. The label or package insert further includes precautionary notes for use in diagnosis. The article of manufacture may further comprise additional containers containing control samples, various buffers, diluents, filters, needles, syringes, bacteriostatic water (BWFI) for injection, and the like.

  In the diagnostic kit contained in the above-mentioned manufactured product, the antibody or lectin may be preliminarily immobilized or may be labeled in advance, depending on the embodiment. The solid phase that can be used in the diagnostic kit according to the embodiment of the present invention is not particularly limited. For example, insoluble carriers such as polymers such as polystyrene, glass beads, magnetic particles, microplates, filter paper for immunochromatography, and glass filters are used. Can be mentioned. The above-mentioned manufactured article can further contain other optional components. Examples of other optional components include, but are not limited to, enzymes used for labeling, substrates thereof, radioisotopes, luminescent materials, fluorescent materials, colored materials, buffers, and plates. .

  Furthermore, the above-described diagnostic kit, diagnostic marker, and detection method are merely one embodiment of the present invention, and according to the present invention, a diagnostic method for Alzheimer's disease using the same is also provided. It is also possible to make a diagnosis highly correlated with the pathological condition of

  A method for screening a therapeutic agent or malignant substance for Alzheimer's disease or a method for analyzing a disease state in humans or mammals using the diagnostic kit, diagnostic marker, detection method and diagnostic method described in the above embodiment Etc. are also included in the technical scope of the present invention.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this is an example and this invention is not limited to these. The commercially available reagents mentioned in the examples were used according to the manufacturer's instructions unless otherwise indicated.

Example 1
<Subject and sample>
We fully explain the purpose of the study to the patients and their families as subjects and obtain informed consent. The study was also approved by Tottori University's ethical review and all subjects were diagnosed by specialists. The diagnosis included medical history and family history, internal and neurological examination, clinical examination, neuropsychological examination, and imaging (CT and / or MRI and SPECT).

  There are 58 AD patients (16 males, 42 females, mean age 76.1 ± 3.6 years) diagnosed based on the diagnostic criteria of DSM-IV and NINCDS-ADRDA. As a control without dementia, patients with cognitive dysfunction and other diseases without autoimmune diseases (motor neuron disease, spinocerebellar degeneration, hyperlipidemia, Parkinson's disease, cerebrovascular disease, etc.) were used. The control group consisted of 32 patients who collected CSF (16 men, 16 women, mean age 76.2 ± 7.5 years), and 31 patients who collected blood (9 men, 22 women, mean age 75.2 ±) 3.0 years old).

  CSF was collected from 93 patients by lumbar puncture and blood was collected from 89 patients and immediately stored at -80 ° C. In the control group, CSF and blood data were different, and in the AD group, CSF and blood data were from the same patient. There was no significant difference between the two groups according to age.

Example 2
<Measurement of CSF and blood complement C3 concentrations using ELISA (Enzyme-Linked Immunosorbent Assay)>
The concentrations of complement C3 protein in CSF and complement C3 protein in serum were measured using Assay Max Human Complement C3 ELISA Kit (Assaypro, USA). 25 μl sample or standard (Human Complement C3 Standard, 30 μg / ml) diluted with Mix Diluent and Biotinylated complement C3 protein on a plate on which anti-human complement C3 protein antibody is immobilized Were mixed in 25 μl, and incubated at 25 ° C. for 2 hours. After the reaction, each well was washed 5 times with 200 μl of Wash Solution, 50 μl of Streptavidin-Peroxidase Conjugate was added and incubated at 25 ° C. for 2 hours. After the reaction, each well was washed 5 times with 200 μl of Wash Solution, and 50 μl of Chromogen Substrate was added. After color development at 25 ° C. for 8 minutes, 50 μl of Stop Solition (0.5N hydroxychloric acid) was added to stop the reaction. Absorbance at 450 nm was measured with a microplate reader, and a calibration curve was prepared with the absorbance of complement C3 protein standard. Based on the calibration curve, the complement C3 protein concentration of the specimen was calculated.

  FIG. 1 represents the concentration of complement C3 protein in cerebrospinal fluid and blood. In cerebrospinal fluid, AD patients showed higher levels of complement C3 protein than controls (p <0.001). On the other hand, in blood, there was no significant difference between control and AD patients.

  From the above results, it was revealed that the concentration of complement C3 protein in cerebrospinal fluid was higher than that of controls in AD patients. On the other hand, it was shown that the complement C3 protein in blood exists at a constant concentration regardless of control-AD patients.

Example 3
<Measurement of sugar chain amount of complement C3 protein in CSF and blood using lectin enzyme immunoassay>
The amount of sugar chain of complement C3 protein in CSF and complement C3 protein in serum was measured by partially modifying Assay Max Human Complement C3 ELISA Kit (Assaypro, USA). Add 50 μl each of the sample or standard diluted with Mix Diluent (Human Complement C3 Standard, 30 μg / ml) to a plate on which anti-complement C3 protein antibody (polyclonal antibody against human complement C3) has been immobilized, at 25 ° C. Incubated for 2 hours. After the reaction, each well was washed 5 times with 200 μl of Wash Solution, 50 μl of biotin-labeled WGA or biotin-labeled Con A (J-Oil Mills, Japan) was added and incubated at 25 ° C. for 1 hour. After the reaction, each well was washed 5 times with 200 μl of Wash Solution, 50 μl of Streptavidin-HRP (GE Healthcare, USA) was added and incubated at 25 ° C. for 1 hour. After the reaction, each well was washed 5 times with 200 μl of Wash Solution, and 50 μl of TMB Solution (Wako, Japan) was added. After color development at 25 ° C. for 5 minutes, 50 μl of 2M H ₂ SO ₄ was added to stop the reaction. Absorbance at 450 nm was measured with a microplate reader, and a calibration curve was prepared with the absorbance of complement C3 protein standard. Based on the calibration curve, the amount of WGA-linked sugar chain of complement C3 protein and the amount of Con A-linked sugar chain of complement C3 protein in the sample were calculated. The amount of each sugar chain is expressed as fluorescence intensity per 1 mg of complement C3 protein.

  FIG. 2 shows the amount of WGA binding and ConA binding of complement C3 protein contained in cerebrospinal fluid. Comparing the amount of WGA binding between control and AD patients, the amount of WGA binding in AD patients was significantly less than the amount of WGA binding in controls (p <0.001). Regarding the discrimination between the control group and the AD group, it was revealed that when the cut-off value was set to 9.59, the sensitivity was 88.2% and the specificity was 75.0%.

  Further, when a similar experiment was performed using ConA, the amount of ConA bound in AD patients was significantly reduced compared to the amount of ConA bound in the control (p <0.001). Regarding the discrimination between the control group and the AD group, it was revealed that when the cutoff value was set to 1.70, the sensitivity was 82.0% and the specificity was 83.3%.

  From the above results, it was revealed that in AD patients, the sugar chain of complement C3 protein in cerebrospinal fluid has decreased both WGA-binding sugar chains and ConA-binding sugar chains.

  FIG. 3 shows the WGA binding amount and ConA binding amount of complement C3 protein contained in blood. Comparing the amount of WGA binding between control and AD patients, the amount of WGA binding in AD patients was significantly less than the amount of WGA binding in controls (p <0.001). Regarding the discrimination between the control group and the AD group, it was revealed that when the cutoff value was set to 3.51, the sensitivity was 86.0% and the specificity was 75.0%. In addition, when a similar experiment was performed using ConA, the amount of ConA bound in AD patients was significantly reduced compared to the amount of ConA bound in the control (p <0.05).

  From the above results, it was found that in AD patients, the sugar chain of complement C3 protein in blood has decreased WGA-binding and ConA-binding sugar chains. Therefore, it was revealed that a decrease in the sugar chain amount of complement C3 protein can be detected even in blood.

Example 4
<Results of WGA binding amount x ConA binding amount of complement C3 protein in CSF and blood>
FIG. 4 is a comparison based on a value (WGA binding amount × ConA binding amount) obtained by multiplying the WGA binding amount of the complement C3 protein in CSF and the ConA binding amount. Comparing the amount of WGA binding x ConA binding in control and AD patients, the amount of WGA binding x ConA binding in AD patients was significantly less than the amount of WGA binding x ConA in control (p <0.001) . Regarding the discrimination between the control group and the AD patient group, it was revealed that when the cut-off value was set to 18.07, the sensitivity was 89.8% and the specificity was 83.3%.

  From the above results, by multiplying the amount of WGA binding and ConA binding of complement C3 protein in CSF, it is possible to show a higher ratio of both sensitivity and specificity than in the case of each alone Became clear.

  FIG. 5 is a comparison based on the amount of WGA binding x ConA binding of complement C3 protein in blood. Comparing the amount of WGA binding x ConA binding in control and AD patients, the amount of WGA binding x ConA binding in AD patients was significantly less than the amount of WGA binding x ConA in control (p <0.001) . Regarding the discrimination between the control group and the AD patient group, it was found that when the cutoff value was set to 9.2, the sensitivity was 83.3% and the specificity was 78.6%.

  From the above results, it was revealed that it is possible to show a high ratio of both sensitivity and specificity by multiplying the complement C3 protein WGA binding amount and ConA binding amount in blood.

Example 5
<WGA binding amount or Con A binding amount of complement C3 protein in CSF and blood and clinical dementia scale>
Explain the purpose of the study to the patients and their families as subjects, obtain informed consent, and establish a clinical dementia scale (CDR) to assess the severity of dementia for the subjects. It carried out in accordance with a conventional method. Subjects were categorized by evaluation (very mild: CDR 0.5, mild: CDR 1, moderate: CDR 2, severity: CDR 3), and the WGA binding level or ConA binding level was controlled for each evaluation. Compared with.

  FIG. 6 shows the amount of WGA binding of complement C3 protein contained in CSF and blood analyzed by CDR. As described above, there is a significant difference between the WGA binding amount of the control and the WGA binding amount of the AD patient group. Table 1 summarizes the cut-off value, sensitivity, and specificity for each CDR evaluation (excluding CDR 3) regarding the discrimination between the control group and the AD patient group in cerebrospinal fluid. It was found that sensitivity was 93.3% and specificity was 62.9% when the cut-off value was set to 11.93 in a very mild CDR 0.5 patient. In the case of mild (CDR 1), it was revealed that when the cutoff value was set to 8.80, the sensitivity was 92.9% and the specificity was 75.3%.

  Table 2 is a table summarizing the cut-off value, sensitivity, and specificity for each CDR evaluation (excluding CDR 3) regarding the discrimination between the control group and the AD patient group in blood. It was found that sensitivity was 82.4% and specificity was 71.4% when the cut-off value was set to 3.72 in patients with very mild CDR 0.5. In the case of mild (CDR 1), it was revealed that when the cutoff value is set to 3.55, the sensitivity is 88.2% and the specificity is 71.4%.

  From the above results, it is clear that the WGA-binding sugar chain of complement C3 protein in CSF and blood is also decreased in extremely mild (CDR 0.5) and mild (CDR 1) dementia. became.

  FIG. 7 shows the ConA binding amount of complement C3 protein contained in CSF and blood analyzed by CDR. As described above, there is a significant difference between the control ConA binding amount and the AD patient group ConA binding amount. Table 3 summarizes the cut-off value, sensitivity, and specificity for each CDR evaluation (excluding CDR 3) regarding the discrimination between the control group and the AD patient group in CSF. It was found that sensitivity was 93.8% and specificity was 56.2% when the cut-off value was set to 2.38 in patients with very mild CDR 0.5. In the case of mild (CDR 1), it was revealed that when the cutoff value was set to 1.50, the sensitivity was 92.9% and the specificity was 87.5%.

  On the other hand, the right figure of FIG. 7 is the figure put together for every evaluation of CDR regarding the ConA binding amount of the complement C3 protein contained in blood. As shown in the results of FIG. 3, since there is a significant difference between the ConA binding amount in the control group and the ConA binding amount in the AD patient group in blood, there is a significant difference even when classified for each CDR evaluation. I can say that. Here, it was revealed that when the cutoff value for CDR 1 is set to 2.45, the sensitivity is 84.6% and the specificity is 67.9%.

  From the above results, it was clarified that the ConA-binding sugar chain of complement C3 protein in CSF was also reduced even in extremely mild (CDR 0.5) or mild (CDR 1) dementia. It was also clarified that the ConA-binding sugar chain of complement C3 protein in blood also decreased at least in the mild (CDR 1) stage.

Example 6
<WGA binding amount of complement C3 protein in CSF and blood x Con A binding amount and clinical dementia scale>
FIG. 8 shows WGA binding amount × Con A binding amount of complement C3 protein contained in CSF analyzed by CDR. As described above, there is a significant difference between the control WGA binding amount × Con A binding amount and the AD patient WGA binding amount × Con A binding amount. Table 4 summarizes the cut-off value, sensitivity, and specificity for each CDR evaluation (excluding CDR 3) regarding the discrimination between the control group and AD patient group in cerebrospinal fluid. It was found that sensitivity was 94.4% and specificity was 70.0% when the cut-off value was set to 27.41 in patients with very mild CDR 0.5. In the case of mild (CDR 1), it was revealed that when the cutoff value was set to 12.57, the sensitivity was 100% and the specificity was 90.0%.

  FIG. 9 shows WGA binding amount × Con A binding amount of complement C3 protein contained in blood analyzed by CDR. As described above, there is a significant difference between the control WGA binding amount × Con A binding amount and the AD patient WGA binding amount × Con A binding amount. Table 5 is a table summarizing the cut-off value, sensitivity, and specificity for each CDR evaluation (excluding CDR 3) regarding the discrimination between the control group and the AD patient group in blood. It was found that sensitivity was 90.9% and specificity was 46.7% when the cut-off value was set to 13.25 in patients with very mild CDR 0.5. In the case of mild (CDR 1), it was revealed that when the cut-off value was set to 6.88, the sensitivity was 88.9% and the specificity was 90.0%.

  From the above results, even in extremely mild (CDR 0.5) and mild (CDR 1) dementia, by multiplying the amount of WGA binding and the amount of ConA in complement C3 protein in CSF and blood, It became clear that both sensitivity and specificity can show a high percentage.

Example 7
<Measurement of amyloid β protein 42 of CSF using ELISA method>
Amyloid β protein 42 (Aβ42) in CSF and blood was measured using Aβ42 sandwich ELISA kit (Human Amyloid β (1-42) Assay kit (Immune Biological, Japan)). Place 100 μl of specimen or standard diluted with dilution buffer (Human Aβ42 Standard, 1,600 pg / ml) on a plate with immobilized anti-Aβ42 antibody (human Aβ42) at 4 ° C overnight. Incubated. After the reaction, each well was washed seven times with 300 μl of a washing solution, and 100 μl of HRP-labeled anti-Aβ42 antibody (monoclonal antibody against human Aβ42) diluted with a lysing solution for labeled antibody was added and incubated at 4 ° C. for 1 hour. After the reaction, each well was washed 9 times with 300 μl of washing solution, and 100 μl of TMB substrate solution was added. After color development at 25 ° C. for 30 minutes, 100 μl of a reaction stop solution (1N H ₂ SO ₄ ) was added to stop the reaction. Absorbance at 450 nm was measured with a microplate reader, and a calibration curve was prepared with the absorbance of Aβ42 standard. Based on the calibration curve, the amount of Aβ42 in the sample was calculated.

<Correlation between Aβ42 and WGA-binding sugar chain or ConA-binding sugar chain>
FIG. 10 is a diagram showing the correlation between the amount of Aβ42 in CSF and the amount of the WGA-binding sugar chain or ConA-binding sugar chain of complement C3 protein. As a result, both the WGA-binding sugar chain and the ConA-binding sugar chain showed a positive correlation with Aβ42, and each showed a significant correlation of p <0.01.

  While Aβ42 is decreased in the CSF of AD patients, it has already been reported that this decrease is not Alzheimer's disease specific. However, the results in FIG. 10 reveal that there is a positive correlation between the decrease in Aβ42 and the decrease in the amount of sugar chain of complement C3 protein (both WGA and ConA-binding sugar chains) in AD patients. . Therefore, it was clarified that Alzheimer's disease can be detected from the quantitative ratio by measuring the amount of sugar chains of amyloid β protein and complement C3 protein (the amount of both WGA and ConA-binding sugar chains) in cerebrospinal fluid.

  FIG. 11 is a diagram showing the correlation between the amount of Aβ42 in CSF and the amount of WGA-binding sugar chain of complement C3 protein in blood. As a result, the amount of WGA-binding sugar chain and the amount of amyloid β protein 42 showed a positive correlation, showing a significant correlation of p <0.05.

  As shown in FIG. 11, Aβ42 in the CSF of AD patients decreases to such a degree that it shows a positive correlation (significant correlation of P <0.05) with the amount of WGA-binding sugar chain of complement C3 protein in blood. It became clear. This makes it possible to derive a decrease in the amount of Aβ42 in CSF from the decrease in the amount of WGA-binding sugar chain of complement C3 protein in blood. Therefore, it was clarified that Alzheimer's disease can be detected by measuring the amount of WGA-binding sugar chain of complement C3 protein in blood.

Example 8
<Quantity ratio of ConA binding amount and WGA binding amount>
FIG. 12 shows the correlation between the amount of ConA binding and the amount of WGA binding of complement C3 protein in CSF. The control showed a weak correlation (correlation coefficient r = 0.29), whereas AD patients showed a moderate correlation (r = 0.66).

  From the above results, it is clear that the sugar chain of complement C3 protein present in the CSF of AD patients has decreased ConA-binding sugar chains compared to WGA-binding sugar chains compared to controls. became.

Example 9
<Amount ratio between the amount of WGA bound in CSF and the amount of WGA bound in blood>
FIG. 13 shows the correlation between the amount of WGA binding of complement C3 protein in CSF and the amount of WGA binding of complement C3 protein in blood in AD patients. As a result, the amount of WGA binding of complement C3 protein in CSF and the amount of WGA binding of complement C3 protein in blood showed a positive correlation, and showed a significant correlation of p <0.05.

  As a result, it was clarified that when the amount of WGA binding of complement C3 protein in blood decreases in AD patients, the amount of WGA binding of complement C3 protein in CSF also decreases. When the amount of WGA binding of complement C3 protein in the blood in AD patients is reduced relative to the amount of WGA binding of complement C3 protein in blood in healthy patients, the amount of complement C3 protein in CSF in AD patients The amount of WGA binding has also decreased. The same can be said for the reverse case. Therefore, considering the results of FIG. 2 and FIG. 3, it is clear that Alzheimer's disease can be detected only by observing a decrease in the amount of WGA binding to complement C3 protein in either CSF or blood. became.

  The statistical analysis used in each example is the Mann-Whitney U test (two-sided test). The correlation between each variable was evaluated using Pearson's product moment correlation coefficient. Receiver operating characteristic (ROC) analysis was performed to calculate sensitivity, specificity, and cutoff values.

<Discussion>
From the results of this Example, Alzheimer's disease can be diagnosed by quantitatively detecting the amount of sugar chains derived from complement C3 protein in body fluids. In addition, by quantitatively detecting the amount of the sugar chain of complement C3 protein in body fluids, unlike conventional markers, early diagnosis of Alzheimer's disease and other dementia exhibiting symptoms similar to Alzheimer's disease Differential diagnosis is possible, and treatment of Alzheimer's disease can be started at an extremely mild or mild stage.

  Further, by using a product of the WGA binding amount and the ConA binding amount, it becomes possible to diagnose Alzheimer's disease in which the difference from the control becomes clearer. Furthermore, Alzheimer's disease can be diagnosed more easily and quickly from the quantitative ratio between the amount of mannose and the amount of N-acetylglucosamine in the sugar chain. In addition, a reliable diagnosis of Alzheimer's disease can be made by performing a combined diagnosis combining the evaluation results of Aβ42 and CDR.

  In the above, this invention was demonstrated based on the Example. It is to be understood by those skilled in the art that this embodiment is merely an example, and that various modifications are possible and that such modifications are within the scope of the present invention.

Claims (24)

A diagnostic kit for diagnosing Alzheimer's disease,
A diagnostic kit comprising a sugar chain detection means for quantitatively detecting the amount of a sugar chain derived from complement C3 protein in a body fluid sample of a mammalian subject.   The diagnostic kit according to claim 1, wherein the amount of the sugar chain of the subject of the same species as the healthy mammal is indicated as a pathological index of Alzheimer's disease with respect to the amount of the sugar chain in the healthy mammal.   The diagnostic kit according to claim 1 or 2, wherein the amount of the sugar chain is a value obtained by combining the amount of mannose and the amount of N-acetylglucosamine in the sugar chain.   The diagnostic kit according to claim 3, wherein the total value obtained is a value obtained by multiplication.   The diagnostic kit according to any one of claims 1 to 4, further comprising a known marker detection means for quantitatively detecting the amount of a known Alzheimer's disease diagnostic marker.   The diagnostic kit according to claim 5, wherein a quantitative ratio between the amount of the sugar chain and the amount of the known marker in a subject that is a mammal is indicated as a disease state index of Alzheimer's disease.   The diagnostic kit according to claim 5 or 6, wherein the known marker is amyloid β protein.   The diagnostic kit according to any one of claims 1 to 7, wherein a quantitative ratio between the amount of mannose in the sugar chain and the amount of N-acetylglucosamine in a subject that is a mammal is indicated as a disease state index of Alzheimer's disease.   The diagnostic kit according to any one of claims 1 to 8, wherein the body fluid is cerebrospinal fluid or blood.   The diagnostic kit according to any one of claims 1 to 9, wherein the subject is a subject having a very mild or mild diagnostic result by dementia severity assessment.   The diagnostic kit of claim 10, wherein the dementia severity assessment is a clinical dementia scale.   The diagnostic kit according to any one of claims 1 to 11, wherein the sugar chain is a WGA- and ConA-binding sugar chain.   The diagnostic kit according to any one of claims 1 to 12, wherein the sugar chain detection means comprises a lectin for a lectin enzyme immunoassay or a sugar chain recognition antibody for an enzyme immunoassay.   The diagnostic kit according to any one of claims 1 to 12, wherein the sugar chain detection means includes a lectin for lectin affinity electrophoresis.   The diagnostic kit according to any one of claims 1 to 12, wherein the sugar chain detection means comprises a lectin for lectin blot analysis or a sugar chain recognition antibody for Western blot analysis.   The diagnostic kit according to any one of claims 1 to 12, wherein the sugar chain detection means comprises a lectin for thin layer chromatography or a sugar chain recognition antibody.   The diagnostic kit according to any one of claims 1 to 12, wherein the sugar chain detection means includes a lectin array chip or an antibody array chip using a sugar chain recognition antibody.   The diagnostic kit according to any one of claims 13 to 17, wherein the lectin is WGA and / or ConA.   The diagnostic kit according to any one of claims 1 to 12, wherein the sugar chain detection means includes liquid chromatography for detecting a sugar chain cleaved from complement C3 protein. A method for detecting a disease state index of Alzheimer's disease,
Quantitatively detecting the amount of sugar chain derived from complement C3 protein in a body fluid sample of a subject mammal ;
A detection method comprising a step of determining whether or not the amount of the sugar chain of the subject of the same species as the mammal is significantly changed relative to the amount of the sugar chain in a healthy mammal. 21. The detection method according to claim 20 , further comprising the step of quantitatively detecting the amount of a known Alzheimer's disease diagnostic marker substance. The method according to claim 21 , further comprising a step of calculating a quantitative ratio between the amount of the sugar chain and the amount of the known marker. The detection method according to any one of claims 20 to 22 , wherein the step of quantitatively detecting the amount of the sugar chain is a step of quantitatively detecting the amount of mannose and the amount of N-acetylglucosamine. The detection method according to claim 23 , further comprising a step of calculating a quantitative ratio between the amount of mannose and the amount of N-acetylglucosamine.