TWI542877B - Diagnostic kit, diagnostic marker, and detection method for Alzheimer's type dementia based on sugar chain determination of complement C3 protein - Google Patents

Description

Diagnostic kit, diagnostic marker and detection method for Alzheimer's type dementia based on sugar chain of complement C3 protein

The present invention relates to the early detection of Alzheimer's dementia based on the sugar chain of the complement C3 protein.

In Japan, Alzheimer's disease has become a major social problem in recent years as the population ages. Now, one out of every 10 people aged 65 or older is demented, and there are currently 1.3 million elderly people with cognitive impairment, and it is expected to reach 3 million in 2035. Most of the dementia are cerebrovascular dementia and Alzheimer's disease (Alzheimer's disease), and Alzheimer's disease accounts for about half of them. For cerebral vascular dementia, medical treatment such as blood pressure management and recurrence prevention can be performed, and the cause, treatment, and prevention method for Alzheimer's disease are not clear, and it is urgent to be medically resolved. In addition to dementia other than Alzheimer's disease, in addition to the above-mentioned cerebrovascular dementia, there is a dementia (fronto-temporal degeneration, Lewy body dementia, which is generally called a tauopathy). Cortical basal ganglia degeneration, progressive supranuclear palsy, etc., which are symptoms similar to Alzheimer's disease, are dementias that are difficult to distinguish from Alzheimer's disease. If it is possible to distinguish between Alzheimer's disease and other dementias before the onset of dementia symptoms, it is possible to start the currently tried treatment at an earlier stage, and to delay the deterioration of dementia. Establishing an early diagnosis of Alzheimer's disease is a top priority in today's society.

In patients with Alzheimer's disease, atrophy of the cerebral cortex can be found. In addition to the high degree of neuronal cell shedding, pathologically, lesions such as senile plaques and neurofibrillary changes can be observed. Among them, because the senile plaque first appeared, which belongs to the most characteristic change of Alzheimer's disease, it is considered that the appearance of senile plaque is the most important change of Alzheimer's disease. The main component of senile plaques is amyloid beta protein (Aβ), which is a starchy precursor protein that is a precursor gene of Aβ in familial Alzheimer's disease that shows autosomal dominant inheritance. (APP), etc., so it is considered that the abnormality of the production or decomposition of Aβ is closely related to the complications and progression of Alzheimer's disease. Aβ is produced by two-time cleavage of the amyloid precursor protein, which utilizes β-secreting 酶 and γ-secreting &#37238, which are aspartic acid proteins 酶 (Presenilin complex) is produced by cutting.

As a therapeutic drug for Alzheimer's disease, an acetylcholine ester 酶 inhibitor is known, which functions to supplement insufficient acetylcholine, and other mechanisms under development are based on the above mechanism. The various signals convey the target drug.

For the diagnosis of Alzheimer's disease (Alzheimer's type dementia), whether the diagnosis satisfies various diagnostic criteria clinically. Therefore, detailed examinations such as clinical symptoms, neuropsychological examination, physiological examination, and brain function image examination are performed as follows. However, there is almost no abnormal abnormality in the early stage of Alzheimer's disease. Because it is necessary to conduct a thorough diagnosis based on the consultation and examination of the person and the family, the current status is that the judgment of the disease depends largely on the specialized doctor. . In fact, in the early days of Alzheimer's disease, you can find changes in personality, difficulty speaking, memory loss, forgetting objects, forgetting names, etc., but correctly distinguishing the "forgetfulness" of the disease at the beginning of the disease and the increase due to age. Forgetfulness (physiological forgetfulness) is very difficult. This is also a major problem in the early detection of Alzheimer's disease.

Now, diagnosis of Alzheimer's disease is comprehensively performed by neuropsychological examination, image diagnosis, neurophysiological examination, and biological examination. As a neuropsychological examination, there are a mini-mental state examination (MMSE), a revised version of the Hasegawa-style simple cognitive function rating scale (HDS-R), and an Alzheimer's disease assessment scale (ADAS-cog). As image diagnosis, there are computed tomography (CT), magnetic resonance imaging (MRI), singlephoton emission computed tomography (SPECT), positronemission tomography (PET), and the like. For Alzheimer's disease, it is possible to use SPECT and PET to confirm the reduction of cerebral blood flow, oxygen metabolism, and glucose metabolism from the early stage, and to confirm the temporal lobe and the parietal lobe. Blood flow and sugar metabolism are gradually reduced.

In the early days, for the purpose of screening multiple people, it was considered that the most effective is the ability to easily and reliably detect a biological diagnostic marker for Alzheimer's disease. If an early diagnosis or pre-morbid diagnosis can be performed by establishing a diagnostic marker for Alzheimer's disease, it is possible to make a plan for administration and treatment based on the confirmed diagnosis, and also to prevent Alzheimer's disease. Open a road. At the same time, with these early diagnoses, it is also possible to control the large cost of medical and nursing care for Alzheimer's patients. Therefore, it is highly desirable to invent or develop a diagnostic marker capable of detecting Alzheimer's disease simply and reliably.

The diagnostic marker is a symptom that reflects Alzheimer's disease, and it is necessary to make a marker that detects the sensitivity and specificity of Alzheimer's disease. As a diagnostic marker for Alzheimer's disease, many research groups at home and abroad have proposed various methods. As a diagnostic marker for Alzheimer's disease, for example, amyloid beta protein, glycosylated acetylcholine ester 酶 interleukin-6, P-substance, cystatin C, serum-loaded Lipoprotein, serum homocysteine, APP subtype, interleukin-6, α1-anti-prion protein 酶 (ACT), oxygen 酶 -1, 24S-hydroxycholesterol, acetylcholine , somatostatin, vasopressin, acetylcholine transfer 酶 activity, acetylcholine ester 酶 assay of activity, etc.

Non-Patent Document 1 describes a change in the amount of WGA-bonded protein of Alzheimer's disease. Further, Non-Patent Document 2 describes a change in the amount of the complement C3 protein in the spinal fluid.

However, the relationship between most of these diagnostic markers and pathological changes is not clear and may not establish the value of the diagnosis. At present, as a diagnostic marker for Alzheimer's disease, which is clinically useful, it is considered that only the Tau protein, the phosphorylated Tau protein, and the amyloid β42 are contained in the cerebrospinal fluid.

The Tau protein is a substance that causes the pathological changes in neurofibrillar changes in the brain of Alzheimer's patients. In the spinal fluid of Alzheimer's disease, phosphorylated Tau protein (or total Tau protein) is increased, and by measuring the protein, it can contribute to the diagnosis of Alzheimer's disease.

The amyloid beta protein used as a diagnostic marker is mainly Aβ42, which is a type of amyloid β peptide (Aβ) formed by 42 amino acids, which is produced in the brain of patients with Alzheimer's disease. The cause of this pathological change in age spots. Since the amyloid β protein in the brain of Alzheimer's disease is aggregated to form senile plaques, Aβ42 in the spinal fluid is instead reduced, and by measuring it, it can contribute to the diagnosis of Alzheimer's disease.

[Non-Patent Document 1] Lisa R. Fodero, Javier Saez-Valero, Maria-Sagrario Barquero, Alberto Marcos, Catriona A. McLean and David H. Small; "Wash germ agglutinin-binding glycoproteins are decreased in Alzheimer's disease cerebrospinal fluid.", Journal of Neurochemistry , 2001, 79, 1022-1026 [Non-Patent Document 2] 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 caused by an increase in phosphorylated Tau protein accompanied by changes in neurofibrils is not limited to Alzheimer's disease, so it is necessary to be associated with other dementias ( Tau disease (tauopathy) is distinguished. Further, as another problem, since the nerve cells have continued to die at the time when the phosphorylated protein rises, it is impossible to restore complete nerve function even if treatment is started at this period.

In addition, although the amyloid β protein used as a diagnostic marker is associated with the severity of Alzheimer's disease in the late stage, there is a large difference between individuals, and it is not uniformly reduced by Alzheimer's disease. In addition, the reduction in amyloid beta protein is not necessarily related to Alzheimer's disease specificity.

In addition, it has been found that the glycoprotein-modified sialic acid contained in the spinal fluid (CSF) of Alzheimer's disease patients is reduced (Non-Patent Document 1), and it is expected to be a new marker for the diagnosis of Alzheimer's disease. However, there are problems such as the fact that the protein is not identified, and the sialic acid becomes unstable when repeatedly thawed. In addition, the amount of the complement C3 protein in the CSF of the Alzheimer's disease patient can be found to change (Non-Patent Document 2), and it is expected to be a new marker for the diagnosis of Alzheimer's disease, but due to the The change in the amount of the complement C3 protein is a change which can be found in various diseases, and does not have a special change in Alzheimer's disease, and therefore it is considered that the possibility of diagnosis for Alzheimer's disease is low.

As an ideal marker, it is possible to diagnose before the death of nerve cells, to diagnose the disease before Alzheimer's disease, to differentiate it from normal brain and other dementias, and to easily screen multiple Better. However, as mentioned above, the markers currently proposed are far from the ideal markers, the definitive diagnosis of Alzheimer's disease is inadequate, and no decisive markers for the diagnosis of Alzheimer's disease have yet been found.

The present invention has been made in view of the above circumstances, and an object thereof is to develop a new diagnostic kit, a diagnostic marker, and a detection method which are highly effective for diagnosis of Alzheimer's disease.

The invention provides a diagnostic kit for diagnosing a diagnosis kit for Alzheimer's disease, wherein a sugar chain detecting unit and a sugar chain detecting unit are used for quantitatively detecting the amount of a sugar chain, wherein the sugar chain is derived from Complement C3 protein in a mammalian body fluid sample as a subject. For patients with Alzheimer's disease, the amount of sugar chain derived from the complement C3 protein in body fluids is changed, which is confirmed in the examples described later. Therefore, by using the diagnostic kit, it is possible to perform early diagnosis of Alzheimer's disease, differential diagnosis of Alzheimer's disease and other dementias exhibiting similar symptoms.

Further, according to the present invention, there is provided a diagnostic marker for diagnosing a diagnostic marker for Alzheimer's disease, comprising an amount of a sugar chain derived from a sample of a mammalian body fluid as a subject Complement C3 protein. For patients with Alzheimer's disease, the amount of sugar chain derived from the complement C3 protein in body fluids is changed, which is confirmed in the examples described later. Therefore, the diagnostic marker can have a function as a marker as described below: an early diagnosis of Alzheimer's disease, a differential diagnosis of Alzheimer's disease and other dementias exhibiting similar symptoms.

Further, according to the present invention, there is provided a method for detecting a symptom index of Alzheimer's disease, comprising the step of quantitatively detecting the amount of a sugar chain derived from a mammalian body fluid as a subject Complement C3 protein in the sample. For patients with Alzheimer's disease, the amount of sugar chain derived from the complement C3 protein in body fluids is changed, which is confirmed in the examples described later. Therefore, by using this detection method, early detection of Alzheimer's disease can be performed, and detection of differentiation from other dementias exhibiting symptoms similar to Alzheimer's disease can be performed.

The above diagnostic kit, diagnostic marker, and detection method are merely one embodiment of the present invention, and according to the present invention, a method for diagnosing Alzheimer's disease using the same is also provided. [effect of the invention]

The present invention enables diagnosis with high correlation with symptoms of Alzheimer's.

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 a sugar chain derived from a complement C3 protein in a body fluid. It has also been found that Alzheimer's disease can be comprehensively diagnosed by combining known Alzheimer's disease diagnostic markers. Further, it has been found that the amount of mannose and the amount of N-acetylglucosamine in the above-mentioned sugar chain are quantitatively detected, and Alzheimer's disease can be easily and rapidly diagnosed by the ratio thereof.

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

<Diagnostic kit> A diagnostic kit related to an embodiment of the present invention is a diagnostic kit for diagnosing Alzheimer's disease, the diagnostic kit having a sugar chain detecting unit for quantifying Detecting an amount of a sugar chain derived from a complement C3 protein in a mammalian body fluid sample as a test subject, according to which the early diagnosis of Alzheimer's disease can be performed, or A differential diagnosis between Alzheimer's disease and other dementias with similar symptoms can be performed.

The "complement C3 protein" in the present embodiment refers to one component of a group of proteins involved in an immune reaction contained in a body fluid. The complement C3 protein is a glycoprotein composed of a 116 kDa alpha chain and a 70 kDa beta strand diploid. For the α chain, the asparagine of SEQ ID NO: 268 modifies the N-bonded sugar chain of Man8-9GlcNAc2, and for the β chain, the N-bond of the asparagine modified Man5-6GlcNAc2 of SEQ ID NO: 63 Crispin MD et al., (2004) Monoglucosylated glycans in the secreted human complement component C3: implications for protein biosynthesis and structure. FEBS Lett566, 270-274 and Hase S et al., (1985) Structures of sugar chains Of the third component of humancomplement. J Biochem 98, 863-874.). The complement C3 protein is usually an inactive precursor type, but when it is excluded from the auxiliary pathogen, it is decomposed into C3a (molecular weight 9000) and C3b (molecular weight 185,000) to become active. The complement C3 protein is known to be a cause of various diseases, and examination for measuring complement C3 protein in blood has been put into practical use. From the viewpoint of its content and activity, various studies have been conducted on the correlation between Alzheimer's disease and the complement C3 protein, but the correlation between the complement C3 protein and the sugar chain derived therefrom has not yet been clarified.

As demonstrated in the examples described later, the amount of the sugar chain derived from the complement C3 protein in the body fluid was changed for the patient suffering from Alzheimer's disease. Therefore, for example, when the amount of the above-mentioned sugar chain is quantitatively detected and the change can be confirmed, the possibility of diagnosis of Alzheimer's disease is high. However, it is not limited thereto, and even if it involves other symptoms or states related to Alzheimer's disease, it can be measured by a daily method well known to a physician in the technical field, and the diagnostic kit of the present embodiment can be easily used. Get these symptom indicators.

The "Alzheimer's disease" in the present embodiment can be said to be a dementia in which atrophy of the cerebral cortex, pathologically high neuronal cell shedding, and lesions capable of observing senile plaques and neurofibrillary changes are observed. Dementia caused by Alzheimer's disease is called Alzheimer's type dementia. Since senile plaques appear in the earliest stages of these lesions, and this phenomenon is a characteristic change in Alzheimer's disease, it is also considered to be the most important change in Alzheimer's disease. The main component of senile plaques is amyloid beta protein (Aβ), which is mutated into a starchy precursor protein (APP) which is a precursor gene of Aβ in familial Alzheimer's disease showing autosomal dominantness. Therefore, it is considered that the abnormality of the production or decomposition of Aβ is closely related to the complications and progression of Alzheimer's disease. Aβ is produced from the amyloid precursor protein by two times due to β-secretion of aspartic acid protein &#37238; and γ-secreting &#37238; (presenilin complex) of. The diagnosis of Alzheimer's disease is diagnosed by satisfying various diagnostic criteria based on clinical symptoms, neuropsychological examination, physiological examination, and brain function image examination.

The term "mammal" in the present embodiment means any mammal, including a human, a domestic animal, a pet animal, a zoo animal, or a sports animal, and includes, for example, a dog, a cat, a cow, a pig, a horse, a sheep, and a rabbit. Wait. The mammal is preferably a human.

The "quantitative detection" in the present embodiment means detection based on numerical data such as concentration, intensity, mobility, and mobility. In addition, in the other embodiment, "quantitative detection" means detection based on the presence or absence of numerical data obtained by adjustment of a density, adjustment of detection sensitivity, setting of a threshold, etc., for example.

In the present embodiment, the "sugar chain detecting unit" may be a plurality of sugar chain detecting units. The plurality of sugar chain detecting units may be, for example, sugar chain detecting units different from each other for performing multifaceted analysis, or the same or similar sugar chain detecting units for the purpose of statistically reliable data, or a combination thereof.

Further, in the diagnostic kit according to another embodiment of the present invention, the amount of the sugar chain of the test subject of the same kind as the mammal is expressed as the amount of the sugar chain of the healthy mammal as Symptoms of Alzheimer's disease. Alzheimer's disease can be reliably diagnosed by using the amount of the aforementioned sugar chain in a healthy mammal as a control.

For example, in the case where the amount of the sugar chain of the above-described complement C3 protein of the subject is intentionally reduced as compared with the amount of the above-mentioned sugar chain of the same type of healthy mammal as the above-mentioned subject, it is diagnosed as Alzheimer. The possibility of disease is high. However, it is not limited thereto, and even if other symptoms or states related to Alzheimer's disease are involved, it can be measured by a daily method well known to a physician in the technical field, and the diagnostic kit of the present embodiment can be easily used. Get these symptom indicators.

The "symptom index of Alzheimer's disease" in the present embodiment refers to a state involving Alzheimer's disease including, for example, complications of Alzheimer's disease or Alzheimer's type dementia. mark. Here, the symptomatic marker of Alzheimer's disease refers to, for example, the prediction of the risk of complications of Alzheimer's type dementia, the early diagnosis or early diagnosis of Alzheimer's disease, and the degree of brain dysfunction. Markings such as inference, symptom grasping, prediction of progress, observation or evaluation of treatment results, and prediction after treatment. In addition, a plurality of symptom markers of Alzheimer's disease may be combined. Thereby, a more reliable diagnosis result can be obtained. The above symptoms, parameters indicating the symptoms, and the like can be easily measured by a physician in the technical field by a well-known daily method.

"Intentional" 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 aforementioned sugar chain of a healthy mammal" in the present embodiment can be obtained from data measured in advance. The measurement method and conditions for the healthy mammal are not particularly limited, but are preferably the same as the measurement methods and conditions applied to the subject. In addition, it is within the scope of the present embodiment that the measurement methods and conditions applied to the healthy mammal are different from the measurement methods and conditions applied to the subject, as long as they are within the range that does not affect the obtained diagnosis result.

Further, in the above diagnostic kit according to another embodiment of the present invention, the amount of the sugar chain is a value obtained by combining the amount of mannose in the sugar chain and the amount of N-acetyl glucosamine. A value obtained by integrating the amount of mannose and the amount of N-acetylglucosamine in the above sugar chain is used as a symptom index of Alzheimer's disease, thereby clarifying the healthy mammal and the subject as a control. The difference can reliably diagnose Alzheimer's disease. It is preferable that the value obtained by the above-mentioned synthesis is a value obtained by performing statistical processing that is generally used, and the above-described statistical processing makes the difference with the healthy body as a control clear, but is not limited to a specific statistical process.

Further, as a diagnostic kit according to another embodiment of the present invention, the value obtained by the above combination is a value obtained by multiplication. By using the value obtained by the above multiplication as a symptom index of Alzheimer's disease, it is possible to make a clearer difference between the healthy body and the control body by simple statistical processing, and to reliably diagnose Alzheimer's disease. Disease.

Further, the above diagnostic kit according to another embodiment of the present invention further has a detection unit for quantitatively detecting a known marker of Alzheimer's disease. Alzheimer's disease can be reliably diagnosed by combining the amount of the aforementioned sugar chain with the above-described known amount of labeling.

As the above-mentioned known label, amyloid β protein, glycosylated acetylcholine ester &#37238; interleukin-6, substance P, and cystatin C can be exemplified. , apolipoprotein, serum homocysteine, APP isoform, interleukin-6, α1-anti-protein &#37238; (ACT), oxygen &#37238; -1,24S-hydroxycholesterol, acetylcholine, somatostatin, vasopressin, and the like. Further, the amount of the above known label may be an amount of activity. For example, it may be acetylcholine transfer &#37238; activity, acetylcholine ester &#37238; activity, and the like. However, any one can be used as long as it can diagnose Alzheimer's disease by combining the amount of the above-mentioned sugar chain. In addition, the above-mentioned known markers are not limited to Alzheimer's disease abnormally labeled. Therefore, it is possible to reliably diagnose Alzheimer's disease by combining with the amount of the above-mentioned sugar chain.

In addition, the above known marks may also employ a plurality of known marks, or a plurality of known mark detecting units that combine the respective known marks may be prepared. Examples of the detection unit include &#37238; immunoassay, immunoblotting, and antibody array (Antibody Array) using known labeled abnormal antibodies, and a plurality of combinations may be used.

Further, in the above diagnostic kit according to another embodiment of the present invention, the amount of the above-mentioned sugar chain in the subject as a mammal is compared with the amount of the above-mentioned known marker as a symptom of Alzheimer's disease. Indicators are displayed. Alzheimer's disease can be compounded and diagnosed by using the ratio of the amount of the above-mentioned sugar chain to the amount of the above-mentioned known label as a symptom index of Alzheimer's disease.

For example, in the diagnostic kit, in the case where there is a positive correlation between the amount of the above-mentioned sugar chain of the subject and the amount of the above known marker, the possibility of diagnosis of Alzheimer's disease is diagnosed based on the result of the composite test. High sex. However, it is not limited thereto, and even if it relates to other symptoms or states related to Alzheimer's disease, it can be measured by a daily method well known to a physician in the technical field, and the diagnostic kit of the present embodiment can be easily used. Get these symptom indicators.

Further, in the above diagnostic kit of another embodiment of the present invention, the above-mentioned known label is amyloid β protein. It is confirmed that the amyloid β protein is clinically useful as a diagnostic marker for Alzheimer's disease. Therefore, by using the ratio of the amount of the amyloid β protein to the amount of the sugar chain derived from the complement C3 protein as a symptom index of Alzheimer's disease, the Alzheim can be diagnosed with high reliability and high accuracy. Mohs disease.

Further, in the above diagnostic kit according to another embodiment of the present invention, the ratio of the amount of mannose of the sugar chain and the amount of N-acetylglucosamine in the subject as a mammal is regarded as Alzheimer's. Symptoms of the disease show. Alzheimer's disease can be easily and rapidly diagnosed by using the ratio of the amount of the above mannose to the amount of N-acetylglucosamine as a symptom marker of Alzheimer's disease.

For example, in the diagnostic kit, the ratio of the amount of mannose in the sugar chain of the above-mentioned complement C3 protein to the amount of N-acetylglucosamine was observed, and it was observed that it was obtained from a mammal of the same kind as the subject. In the case where the ratio is decreased, the possibility of being able to diagnose Alzheimer's disease is high. However, it is not limited thereto, and even if it relates to other symptoms or states related to Alzheimer's disease, it can be measured by a daily method well known to a physician in the technical field, and the diagnostic kit of the present embodiment can be easily used. Get these symptom indicators. Further, the amount of the above N-acetylglucosamine and the amount of mannose may be detected first. Further, continuous detection can also be carried out by a high-throughput screening method.

Further, in the above diagnostic kit of another embodiment of the present invention, spinal fluid or blood is used as the body fluid. Since the method of taking spinal fluid or blood has been established, it is possible to diagnose a patient with a low risk. In addition, since the spinal fluid is close to the brain, it is possible to obtain a diagnosis with a higher correlation with brain symptoms. Further, since blood is a body fluid that can be taken from a patient more quickly and easily, it is possible to perform a highly convenient diagnosis on a patient.

Here, the spinal fluid is not limited to the ventricle cerebrospinal fluid, and the lumbar spinal fluid can also be used. When the lumbar spinal fluid is used, the surviving subject can be diagnosed with a lower risk. Regarding the taking of the spinal fluid, depending on the situation, a usual method of taking the spinal fluid such as cerebrospinal puncture, lumbar puncture, ventricular puncture, or spinal puncture can be used.

Here, regarding the blood, an appropriate centrifugation operation may be performed, and serum in which the presence of lymphocytes is reduced to a negligible amount may also be used. In the present embodiment, the detection reagent can also be reacted with the serum.

Furthermore, in the present embodiment, the diagnosis of the above-described diagnostic kit using a blood sample is performed more quickly and easily, and the patient is more convenient, and as a secondary diagnosis, The diagnosis of the above diagnostic kit using spinal fluid can be performed. At this time, at the time of the second diagnosis, a diagnostic index combining the amount of the sugar chain of the complement C3 protein used as one screening can also be used.

Here, for the sample, one or a plurality of arbitrary processing steps may be added to the obtained samples of the spinal fluid, blood, serum, and the like. Examples of such a treatment step include various columns, removal of impurities by immunoprecipitation, purification of a target substance, and fragmentation of proteins according to trypsin &#37238; according to &#37238; Chemically treated sugar chain separation, other &#37238; treatment, various chemical modifications, etc., but are not limited thereto.

Further, in the above diagnostic kit according to another embodiment of the present invention, the subject is an extremely mild or mild subject according to a diagnosis result of the severity of dementia. The diagnosis result of the combined Alzheimer's disease is evaluated based on the severity of dementia, whereby Alzheimer's disease can be reliably diagnosed. In addition, it is advantageous for pre-morbid diagnosis and initial diagnosis of Alzheimer's disease, and can be effectively prevented and treated. Further, even if the diagnosis result of the dementia severity evaluation is extremely slight, the combination can be performed, and thus the early diagnosis of Alzheimer's disease can be performed. As a dementia severity test, for example, a clinical dementia rating scale (CDR), an elderly depression scale (GDS), a modified Hasegawa-style simple intelligence rating scale (HDS-R), and a simple mental state amount are mentioned. Table (MMSE) examination, Webster's Adult Intelligence Scale (WAIS-R), Alzheimer's Disease Rating Scale (ADAS), etc.

The diagnostic kit of the present embodiment may further have a dementia severity evaluation unit. By having the above evaluation means, the detection of the amount of the above-mentioned sugar chain can be carried out in parallel or continuously with the above evaluation.

In addition, the detection of the amount of the sugar chain of the complement C3 protein and the diagnosis based on the evaluation of the severity of dementia may be performed first, and it is preferable to first perform the evaluation of the severity of dementia with little burden on the subject. This is because when the possibility of diagnosis of Alzheimer's disease is low in the diagnosis result of the severity of dementia, it is possible to prevent the body fluid from taking a burden on the subject.

Further, in the above diagnostic kit according to another embodiment of the present invention, the severity of the dementia is evaluated as a clinical dementia rating scale (CDR). The CDR is an evaluation method for dementia that is widely used internationally, focusing on family information. The evaluation was divided into the following five stages: 0 was normal, 0.5 was extremely mild, 1 was mild, 2 was moderate, and 3 was severe. Alzheimer's disease can be reliably diagnosed by combining the diagnosis results based on the CDRs. Even if the diagnostic kit of the present invention is CDR 0.5 (very light) or CDR 1 (mild), early diagnosis of Alzheimer's disease can be performed.

Further, in the diagnostic kit according to another embodiment of the present invention, the sugar chain is a WGA- and Con A-bonded sugar chain, and according to the diagnostic kit, the symptoms of Alzheimer's disease can be more accurately grasped. . In the case of Alzheimer's disease, the amount of WGA and Con A-bonded sugar derived from the complement C3 protein can be more reliably observed. Therefore, by using these, it is possible to diagnose more reliably.

Further, in the above-described diagnostic kit according to another embodiment of the present invention, as the sugar chain detecting unit, a lectin using a lectin &#37238; an immunoassay or a recognition antibody using a sugar chain is used. #37238; Immunoassay to detect sugar chains derived from the complement C3 protein. This diagnostic kit is an established technique and is performed by the actual clinically widely used &lt;37238; immunoassay or as a lectin using the &lt;37238; immunoassay lectin modification method&#37238 According to the immunoassay, it is possible to perform numerical measurement simply, reliably, and inexpensively, and it is also possible to perform processing of a large number of samples.

Here, the lectin used in the embodiment of the present invention is WGA-bonding property, and is not particularly limited as long as it is a lectin which can recognize a sugar chain of Con A bonding property. For example, as a lectin for recognizing mannose, Canavaliaensiformis agglutinin (Con A), Lens culinarisagglutinin (LCA), Pisum sativum agglutinin (PSA), Bowringia milbraedii agglutinin (BMA), Dolichoslablab agglutinin (DLA), Galanthus nivalisagglutinin (GNA), Gerardia savaglia lectin (GSL), Machaerium biovulatum agglutinin (MBA), Machariumlunatus agglutinin (MLA), Narcissus pseudonarcissusagglutinin (NPA), Epipactis helleborine agglutinin (EHA), Listera ovata agglutinin (LOA), Lablab nigeragglutinin (LNA), Narcissus lobularis agglutinin ( NLA), Vigna racemosa agglutinin (VRA), and Pterocarpusrhorii agglutinin (PRA). The amount of mannose may be measured from the amount of bonding between the lectin and the sugar chain, and a method of measuring the amount of mannose from a bonding amount of Con A having a high bonding affinity with mannose is preferable. Further, as a lectin for recognizing N-acetylglucosamine, examples are abbreviations: germ agglutinin (WGA), Datura stramonium agglutinin (DSA), Oryza sativa agglutinin (OSA), Phytolacca americana agglutinin (PWM), Urticadioica agglutinin (UDA), Solanum tuberosum lectin (STL), Lycipersicon esculentum agglutinin (LEA), Daturastramonium agglutinin (DSA), Pokeweed mitogen (PWM) and subtypes thereof (PL-D1 and PL-D2) and Ym1. The amount of N-acetylglucosamine may also be determined by the amount of binding of the above lectin to the sugar chain, preferably by a bonding affinity with N-acetylglucosamine and a readily available WGA. A method of measuring the amount of N-acetylglucosamine.

The lectin to be used is not limited to a purified natural lectin, and may be, for example, a lectin which is found and purified in a host such as coliform. In addition, standard molecular biology methods can be used to change (eg, mutate, remove, insert, add) arrangements other than sugar recognition sites (thiol arrangement or amino acid arrangement), even for sugar recognition sites. Keep its functionality minimal, improve, or give changes to new features.

In the embodiment of the present invention, in order to detect or measure an antibody or a lectin, a marker antibody and a marker lectin obtained by binding a marker substance capable of generating a signal and an antibody and a lectin itself may be used. In this case, in addition to direct binding, a substance obtained by binding an antibody or a lectin to a labeling substance by avidin-biotin or streptavidin-biotin or a secondary antibody may be used. Within the technical scope of the present invention. As the secondary antibody used herein, an antibody that binds to a primary antibody or a lectin can be used.

When using &#37238; as the identification, for example, horseradish peroxide &#37238;, alkaline phosphate &#37238;, glucose oxidation &#37238;, β-galactose &#33527;&#37238; , glucose starch &#37238;, dehydrogenation of carbonic acid &#37238;, acetylcholine ester &#37238;, lytic &#37238;, malonate dehydrogenation &#37238;, glucose-6-phosphate Hydrogen &#37238; etc. as the logo. As a method of labeling these &#37238;, the method of oxidizing the sugar chain of the #37238; and the amino acid bond of the produced aldehyde group and an antibody or a lectin by periodic acid, and the method of &#37238 A method in which a maleic imine group or a &lt;21537; pyridinedithio group is bonded to a thio group of a Fab' fragment of an antibody or a lectin.

In the case of using &#37238; as a marker, after the test sample is incubated with the labeled antibody or lectin, the free labeled antibody or lectin is removed by washing, and then the substrate of the above-mentioned label &#37238; The reaction is measured, whereby the labeled antibody or lectin can be detected. For example, in the case of the peroxide &#37238;, hydrogen peroxide is used as a matrix, and diaminobenzidine or O-phenylenediamine is combined as a coloring reagent to form a brown or yellow color. In the case of labeling with glucose oxidation &#37238; as a substrate, for example, 2,2'-diazide-bis(3-ethylbenzo&#22139; azole &#21833;-6-sulfonic acid) can be used ( ABTS) and so on.

When a fluorescent dye is used as the label, for example, an antibody or a lectin can be labeled with a fluorescent dye such as FITC (fluorescein isothiocyanate) or TRITC (tetramethylrhodamine B isothiocyanate). The binding of the antibody or lectin to the fluorescent pigment can be carried out by an ordinary method.

In the case where a chromogenic labeling substance is used as the label, for example, a colloidal metal, a colored latex, or the like can be used as the label. Typical examples of the colloidal metal include metal colloid particles in which gold sol, silver sol, selenium sol, cerium sol or platinum sol are dispersed particles. The size of the particles of the colloidal metal is usually preferably about 3 to 60 nm in diameter. In addition, as a representative example of the colored latex, a synthetic latex such as polystyrene latex which is colored with each coloring material such as red or blue is used. As the latex, a natural latex such as natural rubber latex can be used. The size of the colored latex can be selected from a diameter of several tens of nm to several hundreds of nm. These chromogenic labeling materials can be directly used as a commercial product, or can be processed or produced by a method known per se.

The binding of the antibody or lectin to the chromogenic labeling substance can be carried out by an existing method. For example, when the color-developing labeling substance is a gold colloidal particle of a dispersed particle of a gold sol, an antibody and a gold sol are usually mixed at room temperature, and the two can be physically bonded.

Incidentally, as the marker, in addition to the above, a radioactive isotope marker (for example, 125I, 131I, 3H, 14C, etc.) or the like may be used and included in the scope of the present invention.

As the recognition antibody of the sugar chain, for example, a 2G12 antibody which recognizes terminal α1 and 2 mannose on HIV-1 envgp120 which is a glycoprotein can be used. Alternatively, an antibody to a sugar chain to which an appropriate compound is added may be used. For example, an antibody against acetylglucosamine identified by 2-amino &#21537; pyridine can also be used (see Biochemical Vol. 75 No. 8 (2003)). It can also be used as a recognition antibody for sugar chains. For example, it can also be produced by the following method.

When the sugar chain recognition polyclonal antibody is prepared, for example, a sugar chain or a part thereof, or a complement C3 protein or a part of a sugar chain added thereto, or a complex thereof with other carrier molecules or carrier proteins is thermostated. The animal is immunized, and the polyclonal antibody-containing blood is taken from the immunized animal to separate and purify the antibody.

An example of the production method is shown below. The type of the carrier protein and the mixing ratio of the carrier to the antigen may be any conditions as long as the antibody to the immunized antigen can be efficiently produced by cross-linking with the carrier, and for example, one antigen can be used with respect to the weight ratio. The ratio of 0.1 to 20 is preferably a method of bonding bovine serum albumin, bovine thyroglobulin, hemocyanin or the like in a ratio of about 1 to 5. Further, for the coupling of the antigen and the carrier, various condensing agents may be used, and for example, glutaraldehyde, carbodiimide, maleic imide active ester, thiol, dithio & cation, pyridine, etc. may be used. Active ester reagent. In the case of using a small molecule as an antigen, it is particularly preferable to use a carrier protein.

For a warm-blooded animal, the antigen or antigen&apos;s carrier complex itself or it is administered together with a suitable carrier, buffer, diluent to the site where the antibody can be produced. In order to increase the antibody production ability at the time of 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 may be administered. System], an adjuvant such as aluminum hydroxide or the like which can be usually used. These adjuvants and antigens can be administered in the form of a suspension or emulsion using a diluent. Here, the adjuvant refers to a substance which does not particularly enhance the immune response to the antigen when administered together with the antigen.

As the warm-blooded animal to be immunized, for example, a mammal such as a rabbit, a mouse, a hamster, a guinea pig, a chicken, a rat, a dog, a goat, a sheep, or a cow can be used. For example, as a method of immunosensitization, a general immunosensitization method known to those skilled in the art can be used, for example, by administering one or more antigens. Specifically, the antigen administration is usually performed once in about 1 to 6 weeks, and is about 2 to 10 times. For each dose, for example, the antigen is based on about 0.05 to 2 mg. The administration route is not particularly limited. For example, subcutaneous administration, intradermal administration, intraperitoneal administration, intravenous administration, intramuscular administration, and the like can be appropriately selected, but it is preferably carried out by intravenous, intraperitoneal or subcutaneous injection. Cast. After incubating the immunosensitized mammal for 0.5 to 4 months, the mammalian serum can be taken from a small sample such as the ear vein to measure the antibody levels. If the antibody level rises, an appropriate number of antigen administrations are performed depending on the situation. For example, 10 μg to 1000 μg of antigen can be used for booster immunization. The measurement of the antibody level is carried out, for example, by reacting the labeled protein with an antiserum and measuring the activity of the labeling agent bound to the antibody.

Blood or ascites is taken from a mammal that has been immunosensitized one to two months after the last administration by a usual method, and the complement system is inactivated at 56 ° C for 30 minutes, and then subjected to affinity chromatography. The specific antibody is isolated and purified to obtain a polyclonal antibody. As the affinity carrier, for example, a substance which is formed by immobilizing the antigen &#32957; in Affigel or the like can be used. Alternatively, the obtained blood may be isolated and purified by a usual method such as centrifugation, precipitation using ammonium sulfate or polyethylene glycol, gel filtration chromatography, or ion exchange chromatography.

When the sugar chain recognition monoclonal antibody is prepared, for example, a sugar chain or a part thereof, or a complement C3 protein or a part of a sugar chain added thereto, or a complex thereof with other carrier molecules or carrier proteins in a constant temperature animal Immunization is carried out, and spleen cells, lymph node cells, B lymphocytes, and the like are taken from the immunized animal, and the obtained cells are fused with cells of the myeloma cell line to obtain a hybridoma cell producing a monoclonal antibody, from which the hybridization is performed. Tumor cells are isolated by isolating monoclonal antibodies.

An example of the production method is shown below. The immunization step up to the warm-blooded animal can be carried out in the same manner as the preparation of the above polyclonal antibody. As the warm-blooded animal to be immunized, for example, monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, chickens can be taken, but mice or rats can be preferably used. As a method of immunosensitization, a general immunosensitization method known to those skilled in the art can be used, for example, by administering one or more antigens. Specifically, the antigen administration is usually performed once in about 1 to 6 weeks, and is about 2 to 10 times. For each dose, for example, the antigen is based on about 0.05 to 2 mg. The administration route is not particularly limited. For example, subcutaneous administration, intradermal administration, intraperitoneal administration, intravenous administration, intramuscular administration, and the like can be appropriately selected, but it is preferably carried out by intravenous, intraperitoneal or subcutaneous injection. Cast.

An individual who can select an antibody level after immunization with an antigen takes an spleen or a lymph node 2 to 5 days after the final immunization, and obtains antibodies of splenocytes, lymph node cells, B lymphocytes, and the like contained therein. Produce cells. The measurement of the antibody level in the antiserum is carried out, for example, by reacting the labeled protein with an antiserum and measuring the activity of the labeling agent bound to the antibody.

Next, fusion of these antibody-producing cells with myeloma (myeloma cells) is carried out, but the fusion operation can be carried out according to known methods, for example, the Kohler and Milstein methods (G. Kohler et al., Nature, 1975, 495, 256). To implement. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used. Examples of the myeloma include NS-1, P3U1, SP2/0, and AP-1. However, in mice, P3U1, SP2/0, and P3X63Ag8 are particularly preferably used. The preferred ratio of the number of antibody-producing cells to the number of myeloma used is about 1:1 to 20:1, and PEG (preferably PEG1000 to PEG6000) is added at a concentration of about 10 to 80%, preferably at 20 to 40 ° C. Incubation at 30 to 37 ° C for 1 to 10 minutes enables efficient cell fusion.

The selective culture for screening for hybridoma cells can usually be carried out using a culture medium for animal cells to which HAT (hypoxanthine, ampicillin, thorax &#33527;) is added. Further, as the culture medium for breeding, any medium can be used as long as the hybridoma cells can be produced. For example, RPMI-1640 medium containing 1 to 20%, preferably 10 to 20%, of bovine fetal serum, GIT medium (Wako Pure Chemical Industries, Ltd.) or hybridoma containing 1 to 10% of bovine fetal serum can be used. A serum-free medium for cell culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) or the like. The culture temperature is usually from 20 to 40 ° C, preferably about 37 ° C. The culture time is usually from 5 days to 3 weeks, preferably from 1 week to 2 weeks. The cultivation can usually be carried out under 5% carbonic acid gas. Further, hybridoma cells obtained by cell fusion can be cloned by a limiting dilution method or the like.

Various methods can be used for screening for monoclonal antibodies producing hybridoma cells. As an example, a method of adding a hybridoma to a solid phase (microplate or the like) obtained by directly or adsorbing an antigen together with a carrier can be used. The cell culture supernatant is followed by the addition of an anti-immunoglobulin antibody (an antibody reactive with an immunoglobulin of the same kind as the antibody-producing cell used in cell fusion) identified by a radioactive substance or the like, protein A or protein G. a method for detecting a monoclonal antibody bound to a solid phase; adding a culture supernatant of a hybridoma cell to a solid phase to which an immunoglobulin antibody, protein A or protein G is adsorbed, and adding a radioactive substance fire &#37238 A method of identifying an antigen, detecting a monoclonal antibody bound to a solid phase, and the like.

In order to produce a monoclonal antibody of interest from the hybridoma cells thus selected, the hybridoma cells are cultured by a usual cell culture method or ascites formation method, and the monoclonal antibody can be purified from the culture supernatant or ascites. Purification of monoclonal antibodies by culture supernatant or ascites can be carried out by an ordinary method. For example, it can be separated and purified according to immunoglobulin [salting method, ammonium sulfate fractionation, ethanol precipitation method, isoelectric precipitation method, electrophoresis method, ion exchange (DEAE, etc.) adsorption desorption method &#65381 Chromatography, ultracentrifugation, gel filtration, affinity chromatography, active adsorption by antigen-bonded solid phase, or protein A or protein G, only adopting antibodies, dissociating bonds to obtain special purification methods for antibodies, etc. ] Come and proceed.

Further, as the monoclonal antibody to the sugar chain, an IgM antibody is often obtained, and the obtained IgM antibody can be used as it is, or can be used as an IgG antibody by a genetic engineering method well known to those skilled in the art.

As described above, as long as it is a person skilled in the art, various design changes, changes, and adjustment of various conditions can be easily performed in order to obtain the target sugar chain-recognizing antibody according to a general antibody production method.

Further, in the above-described diagnostic kit according to another embodiment of the present invention, the sugar chain detecting unit performs lectin affinity electrophoresis using a gel containing a lectin to detect a sugar chain derived from a complement C3 protein. In the electrophoresis of a gel containing a lectin, a protein having a sugar chain bonded to a target lectin has a stronger lectin affinity (bonding force), and the mobility from the point of implementation is lower, so that the protein can be clearly separated. A complement C3 protein having a small affinity for a small sugar chain (weak binding ability of a lectin) and a high affinity for a C3 protein and a sugar chain.

Further, in the above-described diagnostic kit according to another embodiment of the present invention, as the sugar chain detecting unit, a lectin blot analysis using a lectin or an immunoblot analysis using a recognition antibody of a sugar chain is performed to detect a complement derived from complement. The sugar chain of the C3 protein. By performing blotting with a lectin or an antibody that is abnormally bonded to a sugar chain, the sugar chain derived from the complement C3 protein can be recognized by the state of the sugar chain.

Further, in the above-described diagnostic kit according to another embodiment of the present invention, as the sugar chain detecting means, thin layer chromatography using a recognition antibody of a lectin or a sugar chain is performed to detect a sugar chain derived from a complement C3 protein. By using thin layer chromatography capable of developing a target sample in a short time, the complement C3 protein can be separated from other contaminating proteins, and the detection of the sugar chain using the recognition antibody of the lectin or the sugar chain can be performed easily and rapidly. Further, the case where the mobility of the complement C3 protein is changed by using an antibody against the complement C3 protein or a vector bound to the antibody is also included in the present embodiment.

Further, in the above diagnostic kit according to another embodiment of the present invention, the glycochain detecting unit performs a lectin array or an antibody array to detect a sugar chain derived from a complement C3 protein, wherein the lectin array is provided with A lectin array for a lectin array using an antibody array wafer having a recognition antibody having a sugar chain. By using a lectin array or an antibody array, simultaneous analysis and simple analysis of multiple faces including a plurality of factors such as other proteins can be performed. In particular, by combining an antibody recognizing other factors with a recognition antibody of a lectin or a sugar chain, it is possible to detect the addition state of a plurality of factors of a sugar chain at a time, and to analyze a more detailed symptom or cause.

Further, in the above diagnostic kit according to another embodiment of the present invention, WGA and/or Con A is used as the lectin to detect a sugar chain derived from a complement C3 protein. By using WGA and/or Con A as a lectin, the state of the sugar chain of the complement C3 protein can be reliably detected.

Further, in the diagnostic kit according to another embodiment of the present invention, by performing liquid chromatography as a detecting unit, a sugar chain cut by the complement C3 protein is detected. By fluorescently labeling the cut sugar chain with, for example, 2-amino&#21537; pyridine or the like, even a picomolar sugar chain amount can be detected by a fluorescent detector. Therefore, the state of the sugar chain of the complement C3 protein can be detected even with a small amount of body fluid. Further, the purification of the complement C3 protein and the cleavage of the sugar chain are also included in the present embodiment. The method of cutting the sugar chain from the complement C3 protein is not particularly limited. However, it is preferred that the protein is decomposed and the sugar chain is not decomposed using the &#32956; (hydrazineanhydrous) &#32956; decomposition method.

<Diagnostic Marker> Further, the diagnostic marker according to another embodiment of the present invention is for diagnosing a diagnostic marker for Alzheimer's disease, the diagnostic marker containing an amount of a sugar chain derived from a subject The complement C3 protein in a mammalian body fluid sample, the diagnostic marker can perform the following functions: early diagnosis of Alzheimer's disease, differential diagnosis of Alzheimer's disease and other dementias with similar symptoms .

As demonstrated in the examples described later, the amount of the sugar chain derived from the complement C3 protein in the body fluid changes for a patient 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 is possible to exhibit a marker which is highly likely to exhibit Alzheimer's disease depending on the change in the amount of the sugar chain. Function, it is possible to diagnose Alzheimer's disease according to the change in the amount of the sugar chain. However, it is not limited thereto, and even if it relates to other symptoms or states related to Alzheimer's disease, it can be measured by a daily method well known to a physician in the technical field, and is easily used using the diagnostic marker of the present embodiment. Diagnose them.

Further, in the diagnostic marker according to another embodiment of the present invention, the amount of the sugar chain is the amount of the sugar chain of the same type of the subject of the mammal as the amount of the sugar chain of the healthy mammal. By using the amount of the aforementioned sugar chain of a healthy mammal as a control, a diagnostic marker for reliably diagnosing Alzheimer's disease can be obtained.

Further, the diagnostic marker of another embodiment of the present invention further contains a known amount of the Alzheimer's disease diagnostic marker. By combining the amount of the aforementioned sugar chain with the amount of the above-mentioned known label, a diagnostic marker for more reliable diagnosis of Alzheimer's disease can be obtained.

Furthermore, in another embodiment of the present invention, the diagnostic marker includes an amount ratio of the amount of the sugar chain to the amount of the known label. By using the ratio of the amount of the above-mentioned sugar chain to the amount of the above-mentioned known label as a symptom index of Alzheimer's disease, a diagnostic marker for diagnosing Alzheimer's disease in a multi-faceted and highly accurate manner can be obtained.

For example, when the amount of the sugar chain of the subject is positively correlated with the amount of the marker, the diagnostic marker can function as a marker that is highly likely to display Alzheimer's disease. This correlation enables reliable diagnosis of Alzheimer's disease. However, it is not limited thereto, and even if it relates to other symptoms or states related to Alzheimer's disease, it can be measured by a daily method well known to a physician in the technical field, and the diagnostic marker of the present embodiment is easily used. They are diagnosed.

Further, in the diagnostic marker according to another embodiment of the present invention, the amount of the sugar chain is an amount ratio of the amount of mannose of the sugar chain to the amount of N-acetylglucosamine. By using the ratio of the amount of mannose and the amount of N-acetylglucosamine as a symptom marker of Alzheimer's disease, it is possible to obtain a diagnostic index which is simple and rapid for diagnosis of Alzheimer's disease.

For example, according to the diagnostic marker, the ratio of the amount of mannose in the sugar chain to the amount of N-acetylglucosamine is observed, and the ratio obtained with respect to the mammal having the same species from the subject is observed. In the case of a decrease, it is possible to exhibit a function as a marker which is highly likely to show Alzheimer's disease, and it is possible to reliably diagnose Alzheimer's disease based on the decrease in the ratio. However, it is not limited thereto, and even if it relates to other symptoms or states related to Alzheimer's disease, it can be measured by a daily method well known to a physician in the technical field, and the diagnostic marker of the present embodiment is easily used. They are diagnosed.

<Detection method> The detection method according to another embodiment of the present invention is a method for detecting a symptom index of Alzheimer's disease, and includes a step of quantitatively detecting the amount of a sugar chain derived from The complement C3 protein in the mammalian body fluid sample of the test is capable of early detection of Alzheimer's disease, or differential detection of Alzheimer's disease and other dementias exhibiting similar symptoms.

As demonstrated in the examples described later, the amount of the sugar chain derived from the complement C3 protein in the body fluid changes for a patient suffering from Alzheimer's disease. Therefore, for example, by quantitatively detecting the amount of the above sugar chain, it is possible to detect a change in the amount thereof. According to this result, a material which is highly likely to be able to determine Alzheimer's disease can be obtained. However, the present invention is not limited thereto, and even if it relates to other symptoms or states related to Alzheimer's disease, it can be measured by a daily method well known to a physician in the technical field, and the detection method of the present embodiment is easily used. They judge.

Furthermore, the detection method according to another embodiment of the present invention, further comprising: determining whether the amount of the sugar chain relative to the healthy mammal is intentionally reduced by the amount of the sugar chain of the subject of the same kind as the mammal A step of. For example, when the amount of the above-mentioned sugar chain of the healthy mammal to be used as a control is intended to reduce the amount of the sugar chain of the subject, it is a material which is highly likely to be determined to be Alzheimer's disease. . Therefore, by using this detection method, Alzheimer's disease can be reliably diagnosed.

Further, the above detection method according to another embodiment of the present invention, further comprising quantitatively detecting the amount of the known Alzheimer's disease diagnostic marker. By combining the amount of the above-mentioned sugar chain and the amount of the above-mentioned known label, it is possible to obtain a material which is highly likely to be more reliable in determining Alzheimer's disease.

Furthermore, the above detection method according to another embodiment of the present invention, further comprising the step of calculating the amount ratio of the amount of the sugar chain and the amount of the known label. By using the ratio of the amount of the above-mentioned sugar chain to the amount of the above-mentioned known label as a symptom index of Alzheimer's disease, it is possible to obtain a material which is highly likely to be used as a comprehensive result for judging Alzheimer's disease.

For example, according to the detection method, when there is a positive correlation between the amount of the sugar chain of the subject and the amount of the known marker, the possibility of being able to determine Alzheimer's disease as a comprehensive detection result can be obtained. High material. However, the present invention is not limited thereto, and even if it relates to other symptoms or states related to Alzheimer's disease, it can be measured by a daily method well known to a physician in the technical field, and the detection method of the present embodiment is easily used. They judge.

Further, in the above detection method according to another embodiment of the present invention, 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. Alzheimer's disease can be detected in more detail and reliably by detecting the amount of mannose and the amount of N-acetylglucosamine, respectively.

Further, the above detection method according to another embodiment of the present invention, further comprising the step of calculating the amount ratio of the amount of the mannose to the amount of the N-acetylglucosamine. By using the ratio of the amount of mannose and the amount of N-acetylglucosamine as a symptom index of Alzheimer's disease, it is possible to obtain a material which is highly likely to be easily and rapidly determined to be Alzheimer's disease. .

For example, according to the detection method, the ratio of the amount of mannose in the sugar chain of the complement C3 protein to the amount of N-acetylglucosamine is observed, and the ratio obtained with respect to the mammal of the same type from the subject is observed. When the ratio is decreased, it can be used as a basis for determining the possibility of Alzheimer's disease. However, the present invention is not limited thereto, and even if it relates to other symptoms or states related to Alzheimer's disease, it can be measured by a daily method well known to a physician in the technical field, and the detection method of the present embodiment is easily used. They judge.

It should be noted that the diagnostic kit, the diagnostic marker, and the detection method described by the above embodiments are not limited to the present invention, and the examples are intended to be disclosed. The scope of the present invention is defined by the scope of the claims, and various modifications can be made by those skilled in the art within the scope of the invention described in the scope of the claims. For example, various types of immunochemical methods, slit or dot blotting, or two-dimensional electrophoresis can be used to detect the sugar chain derived from the complement C3 protein. Diagnostic kits and diagnostic markers in this manner can be used. And the detection method is of course also included in the technical scope of the present invention.

Examples of the various methods of immunochemistry include, for example, an unreacted labeled antigen (F) and a labeled antigen bound to an antibody after the antigen in the sample solution and the labeled antigen compete with each other for the antibody ( B) Separation (B/F separation), which measures the amount of B or F and quantifies the amount of antigen in the sample solution (competition method). As a method of B/F separation, a liquid phase method using B-F separation using a soluble antibody as an antibody, a polymer liquid phase (such as polyethylene glycol), and a secondary antibody to a soluble antibody, and the like are used. A solid phase method for a secondary antibody in which a primary antibody is immobilized or a secondary antibody that is solid-phased with a soluble primary antibody. Further, as another method of immunochemistry, a solid phase and a liquid phase obtained by subjecting an antigen and a solid phase antigen in a sample solution to a competitive reaction with a predetermined amount of the labeled antibody may be separated or may be The antigen in the sample solution reacts with an excessive amount of the labeled antibody, and then the solid phase antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated, and the labeled amount of any phase is determined. A method for quantifying the amount of antigen in the sample solution (immunoassay). Further, a method (nephelometry) for measuring the amount of insoluble precipitate produced as a result of an antigen-antibody reaction in a gel or in a solution is also mentioned. When the amount of the antigen in the test liquid is small and only a small amount of precipitate is obtained, a laser scattering turbidity or the like using a scattering of a laser may be used.

Further, an embodiment of the present invention is an article of manufacture including the diagnostic kit of the present invention. The article of manufacture includes a label or package illustration of the container and container. The container may, for example, comprise a bottle, a vial, a syringe or the like, and is selected from suitable materials such as glass or plastic. The label or package illustration indicates that the diagnostic kit of the present invention is used for the diagnosis of Alzheimer's disease. The label or package illustration also includes a note description when used in the diagnosis. The manufactured product may further include an additional container containing a control sample, various buffers, a diluent, a filter, a needle, a syringe, and bacteriostatic water (BWFI) for injection.

In the diagnostic kit included in the above-described article of manufacture, depending on the embodiment, the antibody or lectin may be pre-phased or pre-labeled. The solid phase which can be used in the diagnostic kit of the embodiment of the present invention is not particularly limited, and examples thereof include a polymer such as polystyrene, glass beads, magnetic particles, a microplate, a filter paper for immunochromatography, and glass filtration. Insoluble carrier such as a device. The above-mentioned manufactured product may further contain other optional components. Examples of other optional components include, but are not limited to, &lt;37238;; a substrate, a radioactive isotope element, a luminescent substance, a fluorescent substance, a coloring substance, a buffer, a plate, and the like.

Further, the above diagnostic kit, diagnostic marker, and detection method are merely one of the embodiments of the present invention, and according to the present invention, a diagnosis method using Alzheimer's disease is also provided, and can be performed by the diagnostic method A diagnosis that is highly correlated with the symptoms of Alzheimer's.

In the case of using a diagnostic kit, a diagnostic marker, a detection method, a diagnosis method, and the like described in the above embodiments, a method for screening a therapeutic drug or a malignant substance for Alzheimer's disease in a human or a mammal is used. Methods for analyzing symptoms and the like are also included in the scope of the technology of the present invention. 【&#23454;Example】

Hereinafter, the present invention will be specifically described by way of examples, which are merely examples, and the present invention is not limited to the examples. It should be noted that the commercially available reagents mentioned in the examples are used according to the manufacturer's instructions for use unless otherwise specified.

Example 1 <Subject and sample> The subject of the study was sufficiently explained to the patient and the family as the subject, and the informed consent was obtained. In addition, the study was approved by the Tottori University's ethical review, and all subjects were diagnosed by a professional doctor. The diagnosis includes medical records, family medical records, internal medicine, neurological examinations, clinical examinations, neuropsychological examinations, and image examinations (CT and/or MRI and SPECT).

AD patients were 58 patients diagnosed according to the diagnostic criteria of DSM-IV and NINCDS-ADRDA (16 males, 42 females, mean age 76.1 ± 3.6 years). As a non-dementia control, patients with other diseases without cognitive dysfunction and autoimmune diseases (motor neuron disease &#65381; ridge &#39620; cerebellar degeneration &#65381; hyperlipidemia &#65381; Jinsen's disease &#65381; cerebrovascular disease, etc.). The control group had 32 patients with CSF (16 males, 16 females, mean age 76.2±7.5 years), and 31 patients with blood (9 males, 22 females, average age 75.2) ±3.0 years old).

CSF was taken from 93 patients by lumbar puncture, and blood was taken from 89 patients and immediately stored at -80 °C. The data of CSF and blood in the control group were studied separately for different patients, and the data of CSF and blood of AD group were tested in the same patient. In addition, there was no significant difference in age between the 2 groups.

Example 2 <CSF using ELISA method (Enzyme-Linked Immunosorbent Assay), Determination of complement C3 concentration in blood> The concentration of complement C3 protein in CSF and the level of complement C3 protein in serum using Assay Max Human Complement C3 ELISA Kit ( Assaypro, USA) was assayed. 25 μl of a 25 μl sample or standard diluted with Mix Diluent (Human Complement C3 Standard, 30 μg/ml) was added to each plate obtained by solid-phase anti-human complement C3 protein antibody (polyclonal antibody against human complement C3). The cells were mixed with Biotinylated complement C3 protein 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 Streptavidin-Peroxidase Conjugate was added for incubation for 2 hours at 25 °C. After the reaction, the cells were washed 5 times with 200 μl of Wash Solution, and 50 μl of each Chromogen Substrate was added. After coloring at 25 ° C for 8 minutes, 50 μl of each of Stop Solition (0.5 N hydroxychloric acid) was added to stop the reaction. The absorbance at 450 nm was measured with a &#37238; and the standard curve was prepared with the absorbance of the complement C3 protein standard. The complement C3 protein concentration of the sample was calculated from its standard curve.

Figure 1 shows the concentration of complement C3 protein in spinal fluid and blood. In the spinal fluid, one of the AD patients showed a higher concentration of complement C3 protein than the control (p < 0.001). On the other hand, in the blood, no significant difference was seen between the control and AD patients.

From the above results, the concentration of complement C3 protein in the spinal fluid was higher in AD patients than in the control. On the other hand, both the control and AD patients have a fixed concentration of complement C3 protein in the blood.

Example 3 <Measurement of sugar chain amount of CSF using a lectin&#37238; immunoassay and complement C3 protein in blood> For the amount of glycochain of the complement C3 protein in the CSF and the complement C3 protein in the serum, The assay was performed by changing the Assay Max Human Complement C3 ELISA Kit (Assaypro, USA). 50 μl of a sample diluted with Mix Diluent or a standard (Human Complement C3 Standard, 30 μg/ml) was added to each of the plates obtained by immobilizing the anti-complement C3 protein antibody (polyclonal antibody against human complement C3) at 25 Incubate for 2 hours at °C. After the reaction, each volume was washed 5 times with WashSolution 200 μl, and 50 μl of biotin-labeled WGA or biotin label Con A (J-Oil Mills, Japan) was added thereto, and incubated at 25 ° C for 1 hour. After the reaction, each volume was washed 5 times with 200 μl of Wash Solution, and 50 μl of each Streptavidin-HRP (GE Healthcare, USA) was added thereto, and incubated at 25 ° C for 1 hour. After the reaction, each of the cells was washed 5 times with 200 μl of WashSolution, and 50 μl of TMB Solution (Wako, Japan) was added to each. After 5 minutes of color development at 25 ° C, 50 μl of 2M H 2 SO 4 was added to stop the reaction. The absorbance at 450 nm was measured with a &#37238; and the standard curve was prepared with the absorbance of the complement C3 protein standard. The amount of WGA-bonded sugar chain of the complement C3 protein in the sample and the amount of ConA-bonded sugar chain of the complement C3 protein were calculated from the standard curve. The amount of each sugar chain is expressed by the fluorescence intensity per 1 mg of the complement C3 protein.

Fig. 2 is a graph showing the amount of WGA bonding and the amount of Con A bonding of the complement C3 protein contained in the spinal fluid. If the WGA bonding amount of the control and AD patients was compared, the WGA bonding amount of the AD patients was significantly reduced compared to the WGA bonding amount of the control (p < 0.001). For the identification of the control group and the AD group, it was found that if the boundary value was set to 9.59, the sensitivity was 88.2% and the specificity was 75.0%.

In addition, the same experiment was performed using Con A, and as a result, the ConA bonding amount of the AD patient was significantly reduced (p < 0.001) compared to the Con A bonding amount of the control. For the identification of the control group and the AD group, it was found that if the boundary value was set to 1.70, the sensitivity was 82.0% and the specificity was 83.3%.

From the above results, it was found that in the AD patients, both the WGA-bonded sugar chain and the Con A-bonded sugar chain were reduced in the sugar chain of the complement C3 protein in the spinal fluid.

Fig. 3 is a graph showing the amount of WGA bonding and the amount of Con A bonding of the complement C3 protein contained in blood. If the WGA binding amount of the control and AD patients was compared, the WGA bonding amount of the AD patients was significantly reduced compared to the WGA bonding amount of the control (p < 0.001). For the identification of the control group and the AD group, it was found that if the boundary value was set to 3.51, the sensitivity was 86.0% and the specificity was 75.0%. In addition, the same experiment was performed using Con A, and as a result, the ConA bonding amount of the AD patient was significantly reduced (p < 0.05) compared to the Con A bonding amount of the control.

From the above results, it was found that in the AD patients, the sugar chains of the complement C3 protein in the blood, the WGA-bonding property and the Con A-bonding sugar chain were all decreased. Therefore, it can be seen that the amount of the sugar chain of the complement C3 protein can be detected even in the blood.

Example 4 <WGA Binding Amount of Complement C3 Protein in CSF and Blood × Results of Con A Bonding Amount> FIG. 4 is obtained by multiplying the WGA bonding amount of the complement C3 protein in the CSF by the Con A bonding amount. A value obtained by comparing the value (WGA bonding amount × Con A bonding amount). If the WGA bonding amount × Con A bonding amount of the control and AD patients is compared, the WGA bonding amount × Con A bonding amount of the AD patient is significantly reduced compared with the control WGA bonding amount × Con A bonding amount. (p < 0.001). For the identification of the control group and the AD patient group, it was found that if the boundary value was set to 18.07, the sensitivity was 89.8% and the specificity was 83.3%.

From the above results, it is understood that by multiplying the WGA bonding amount of the complement C3 protein in the CSF by the Con A bonding amount, both the sensitivity and the specificity are displayed as compared with the case of the respective bonding amounts. High ratio.

Fig. 5 is a graph obtained by comparing the WGA binding amount of the complement C3 protein in the blood to the Con A bonding amount. If the WGA bonding amount × Con A bonding amount of the control and AD patients is compared, the WGA bonding amount × Con A bonding amount of the AD patient is significantly reduced compared to the control WGA bonding amount × Con A bonding amount ( p < 0.001). For the identification of the control group and the AD patient group, it was found that if the boundary value was set to 9.2, the sensitivity was 83.3% and the specificity was 78.6%.

From the above results, it is understood that by multiplying the amount of WGA bonding of the complement C3 protein in the blood by the amount of Con A bonding, it is possible to display a high ratio of both sensitivity and specificity.

Example 5 <WGA binding amount of the complement C3 protein in the CSF and the blood, and the amount of the Con A bonding and the clinical dementia rating scale> The subject of the study and the family of the subject are fully explained, and the subject is obtained. On the basis of the informed consent, the clinical dementia rating scale (CDR) for evaluating the severity of dementia is administered to the subject according to an ordinary method. The subjects were classified according to the evaluation (very mild: CDR 0.5, mild: CDR 1, moderate: CDR 2, severity: CDR 3), and the WGA bonding amount or Con A bonding was compared with the respective evaluations. The amount is compared with the control.

Fig. 6 is a graph showing the WGA binding amount of the complement C3 protein contained in the blood in the CSF analyzed by the CDR. The amount of WGA binding of the control was significantly different from the amount of WGA bonding of the AD patient population as described above. Table 1 shows the identification of the control group and the AD patient group in the spinal fluid, and the boundary value, sensitivity, and specificity are summarized in a table in which the CDRs are evaluated (excluding the CDR 3). For patients with a very mild CDR 0.5, it is known that if the boundary value is set to 11.93, the sensitivity is 93.3% and the specificity is 62.9%. Further, in the case of mild (CDR 1), it was found that when the boundary value was set to 8.80, the sensitivity was 92.9%, and the specificity was 75.3%.

【Table 1】

Table 2 is a table for identifying the control group and the AD patient group in blood, and summarizing the boundary value, sensitivity, and specificity in each evaluation of the CDR (excluding CDR 3). For patients with a very mild CDR 0.5, it was found that if the boundary value was set to 3.72, the sensitivity was 82.4% and the specificity was 71.4%. Further, in the case of mild (CDR 1), it was found that when the boundary value was set to 3.55, the sensitivity was 88.2%, and the specificity was 71.4%.

【Table 2】

From the above results, it was found that the WGA-bonded sugar chain of the complement C3 protein in the CSF and in the blood was reduced even in the case of extremely mild (CDR 0.5) or mild (CDR 1) dementia.

Fig. 7 is a graph showing the amount of Con A binding of the complement C3 protein contained in the CSF and the blood by CDR analysis. The amount of ConA binding of the control was significantly different from the amount of Con A binding of the patient population of AD as described above. Table 3 shows the identification of the control group and the AD patient group in the CSF, and the boundary value, the sensitivity, and the specificity are summarized in a table in which the CDRs are evaluated (excluding the CDR 3). For patients with a very mild CDR 0.5, it was found that if the boundary value was set to 2.38, the sensitivity was 93.8% and the specificity was 56.2%. Further, in the case of mild (CDR 1), it was found that when the boundary value was set to 1.50, the sensitivity was 92.9%, and the specificity was 87.5%.

【table 3】

On the other hand, Fig. 7 is a diagram in which the ConA bonding amount of the complement C3 protein contained in blood is summarized in units of CDR evaluation. As shown in the results of FIG. 3, since the Con A binding amount of the control group in the blood is significantly different from the Con A bonding amount of the AD patient group, even in the case of classifying by the evaluation of the CDR, It can also be considered that there are significant differences. Here, for CDR 1, it is understood that if the boundary value is set to 2.45, the sensitivity is 84.6% and the specificity is 67.9%.

From the above results, it was found that the Con A-bonded sugar chain of the complement C3 protein in CSF was reduced even in the case of extremely mild (CDR 0.5) and mild (CDR 1) dementia. In addition, it was found that the Con A-bonded sugar chain of the complement C3 protein in the blood was also reduced at least at the stage of mild (CDR 1).

Example 6 <WGA bonding amount of complement C3 protein in CSF and blood × Con A bonding amount and clinical dementia rating scale> Fig. 8 is a WGA bond showing complement C3 protein contained in CSF by CDR analysis A graph of the amount × Con A bonding amount. The amount of WGA bonding × Con A bonding of the control was significantly different from the amount of WGA bonding × Con A bonding of AD patients, as described above. Table 4 shows the identification of the control group and the AD patient group for the spinal fluid, and the boundary value, sensitivity, and specificity are summarized in the table in which the CDRs are evaluated (excluding the CDR 3). For patients with a very mild CDR 0.5, it was found that if the boundary value was set to 27.41, the sensitivity was 94.4% and the specificity was 70.0%. Further, in the case of mild (CDR 1), it was found that when the boundary value was set to 12.57, the sensitivity was 100% and the specificity was 90.0%.

【Table 4】

Fig. 9 is a graph showing the amount of WGA bonding × Con A bonding amount of the complement C3 protein contained in blood analyzed by CDR. The amount of WGA bonding × Con A bonding of the control was significantly different from the amount of WGA bonding of the AD patient x Con A bonding amount as described above. Table 5 summarizes the boundary value, sensitivity, and specificity for the control group and the AD patient group in blood in units of evaluation of CDRs (excluding CDR 3). For patients with a very mild CDR 0.5, it was found that if the boundary value was set to 13.25, the sensitivity was 90.9% and the specificity was 46.7%. Further, in the case of mild (CDR 1), it was found that when the boundary value was set to 6.88, the sensitivity was 88.9% and the specificity was 90.0%.

【table 5】

From the above results, even in the extremely mild (CDR 0.5) and mild (CDR 1) dementia, the amount of WGA bonding of the complement C3 protein in the CSF neutralizing blood was multiplied by the Con A bonding amount. It is also possible to show a high ratio of sensitivity and specificity.

Example 7 <Measurement of amyloid β protein 42 of CSF using ELISA method> Amyloid β protein 42 (Aβ42) in CSF neutralizing blood using Aβ42 sandwich ELISA kit (Human Amyloid β(1-42) Assay kit ( ImmuneBiological, Japan)) to determine. To a plate obtained by immobilizing an anti-Aβ42 antibody (polyclonal antibody against human Aβ42), 100 μl of each sample diluted with a buffer or a standard (Human Aβ42 Standard, 1,600 pg/ml) was added, and the plate was incubated at 4° C. One &#26217;. After the reaction, each of the cells was washed 7 times with 300 μl of the washing solution, and 100 μl of each of the HRP-labeled anti-Aβ42 antibody (monoclonal antibody against human Aβ42) diluted with the lysate was added, and incubated at 4 ° C for 1 hour. After the reaction, each of the cells was washed 9 times with 300 μl of the washing solution, and 100 μl of each TMB substrate solution was added. After coloring at 25 ° C for 30 minutes, the reaction was stopped after adding 100 μl of each reaction stop solution (1N H 2 SO 4 ). The absorbance at 450 nm was measured with a &#37238; and the standard curve was prepared with the absorbance of the Aβ42 standard. The amount of A?42 in the specimen was calculated based on the standard curve.

<Correlation between Aβ42 and WGA-bonded sugar chain or Con A-bonded sugar chain> Fig. 10 shows the amount of Aβ42 in CSF and the amount of WGA-bonded sugar chain or ConA-bonded sugar chain of complement C3 protein Related diagrams. The results showed that both WGA-bonded sugar chains and ConA-bonded sugar chains were positively correlated with Aβ42, and were significantly correlated with p < 0.01.

Aβ42 has been reported to decrease in CSF in patients with AD, but the reduction does not cause abnormalities in Alzheimer's disease. However, a decrease in the Aβ42 in the AD patient and a decrease in the amount of the sugar chain of the complement C3 protein (the amount of both the WGA and the Con A-binding sugar chain) showed a positive correlation as is apparent from the results of FIG. Thus, the amount of the sugar chain of the amyloid β protein and the complement C3 protein in the spinal fluid (the amount of both the WGA and the Con A bonded sugar chain) was measured, and the Alzheimer's disease was detected by the amount ratio.

Fig. 11 is a graph showing the amount of A?42 in CSF correlated with the amount of WGA-bonded sugar chain of complement C3 protein in blood. The results showed that the amount of the WGA-bonded sugar chain was positively correlated with the amount of the amyloid β protein 42 and was a significant correlation of p < 0.05.

As can be seen from the results of Fig. 11, Aβ42 in the CSF of AD patients was reduced to the extent that it was positively correlated with the amount of WGA-bonded sugar chains of complement C3 protein in the blood (P < 0.05 was significantly correlated). This is because the decrease in the amount of Aβ42 in the CSF can be derived from the decrease in the amount of the WGA-bonded sugar chain of the complement C3 protein in the blood. From this, it was found that the amount of the WGA-bonded sugar chain of the complement C3 protein in the blood was measured, and the Alzheimer's disease was detected by the decrease in the amount.

Example 8 <Amount Ratio of ConA Bonding Amount to WGA Bonding Amount> FIG. 12 is a graph showing the correlation between the amount of Con A bonding of the complement C3 protein in CSF and the amount of WGA bonding. It can be seen that the control was moderately correlated (r = 0.66) with respect to the weak correlation (correlation coefficient r = 0.29).

From the above results, it was found that the sugar chain of the complement C3 protein present in the CSF of AD patients was reduced in comparison with the control, and the Con A-bonded sugar chain was reduced with respect to the WGA-bonded sugar chain.

Example 9 <Amount ratio of WGA bonding amount in CSF to WGA bonding amount in blood> Fig. 13 is a WGA binding amount of complement C3 protein in CSF of AD patients and WGA of complement C3 protein in blood A graph related to the amount of bonding. The results showed that the WGA binding amount of the complement C3 protein in CSF was positively correlated with the WGA binding amount of the complement C3 protein in the blood, showing a significant correlation of p < 0.05.

From the above results, it is understood that in AD patients, if the WGA binding amount of the complement C3 protein in the blood is decreased, the WGA binding amount of the complement C3 protein in the CSF is also reduced. If the WGA binding amount of the complement C3 protein in the blood of the AD patient is reduced relative to the WGA binding amount of the complement C3 protein in the blood of the healthy patient, the WGA binding amount of the complement C3 protein in the CSF of the AD patient is also reduced. It can be said that the opposite is true for this case. Therefore, considering the results of FIG. 2 and FIG. 3, it is only possible to observe that the amount of WGA in any of CSF or blood is reduced relative to the complement C3 protein, and Alzheimer's disease can be detected. .

The statistical analysis used in the examples was the Mann-Whitney U test (two-sided test). The correlation between the variables was evaluated using Pearson's product difference correlation coefficient. Receiver operatingcharacteristic (ROC) analysis is used to calculate sensitivity, specificity, and boundary values.

<Investigation> As a result of the present example, the diagnosis of Alzheimer's disease can be performed by quantitatively detecting the amount of the sugar chain derived from the complement C3 protein in the body fluid. In addition, by quantitatively detecting the amount of the sugar chain of the complement C3 protein in the body fluid, it is possible to perform early diagnosis of Alzheimer's disease, Alzheimer's disease, and other symptoms similar to those of the conventional marker. The differential diagnosis of dementia begins the treatment of Alzheimer's disease in a very mild or mild phase.

Further, by using the multiplication value of the WGA bonding amount and the Con A bonding amount, it is possible to diagnose the Alzheimer's disease in which the difference from the control is more clear. Further, the ratio of the amount of mannose in the sugar chain to the amount of N-acetylglucosamine can more easily and quickly diagnose Alzheimer's disease. In addition, by performing a composite diagnosis in which the evaluation results of Aβ42 and CDR are combined, it is possible to reliably diagnose Alzheimer's disease.

Hereinabove, the present invention has been described based on the embodiments. This embodiment is merely illustrative, and various modifications can be made, and such modifications are also within the scope of the invention, as will be understood by those skilled in the art.

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. Fig. 2 is a graph showing the results of measuring the amount of a sugar chain derived from the complement C3 protein of the control group and the AD patient group in CSF. Fig. 3 is a graph showing the results of measuring the amount of a sugar chain derived from the complement C3 protein of the control group and the AD patient group in the blood. 4 is a graph showing the amount of WGA bonding and the amount of Con A bonding of a sugar chain of a complement C3 protein in a control group and an AD patient group in CSF, and multiplying the amount of WGA bonding and the amount of Con A in each group. The result of the graph. 5 is a graph showing the amount of WGA bonding and the amount of Con A bonding of a sugar chain of a complement C3 protein in a control group and an AD patient group in blood, and multiplying the amount of WGA bonding and the amount of Con A in each group. The result of the graph. Fig. 6 is a graph showing the results of WGA bonding amount of a sugar chain of a complement C3 protein in a control group and an AD patient group in CSF and analyzed in a clinical dementia rating scale (CDR). Fig. 7 is a graph showing the results of the Con A bonding amount of the sugar chain of the complement C3 protein of the control group and the AD patient group in the CSF analyzed by the CDR. Fig. 8 is a graph showing the amount of WGA bonding and the amount of Con A bonding of the sugar chain of the complement C3 protein of the control group and the AD patient group in the CSF analyzed by CDR analysis, and the WGA bonding amount and the Con A bond in the respective groups. A graph of the results of the multiplication. Fig. 9 is a graph showing the amount of WGA bonding and the amount of Con A bonding of the sugar chain of the complement C3 protein in the control group and the AD patient group in the blood analyzed by CDR analysis, and the WGA bonding amount and the Con A bond in the respective groups. A graph of the results of the multiplication. Fig. 10 is a correlation diagram between the amount of amyloid β protein 42 in the AD patient group in CSF and the WGA binding amount or Con A bonding amount of the complement C3 protein. Fig. 11 is a graph showing the correlation between the amount of amyloid β protein 42 in the CSF of the AD patient group and the WGA binding amount of the complement C3 protein in the blood. Fig. 12 is a correlation diagram between the WGA binding amount of the complement C3 protein and the Con A bonding amount in the control group and the AD patient group in the CSF. Figure 13 is a graph showing the correlation between the WGA binding amount of the complement C3 protein in the CSF of AD patients and the WGA binding amount of the complement C3 protein in the blood.

Claims (25)

A diagnostic kit for diagnosing Alzheimer's disease, characterized in that the diagnostic kit has a sugar chain detecting unit for quantitatively detecting the amount of a sugar chain, wherein the sugar chain source From the complement C3 protein in a mammalian body fluid sample as a subject. The diagnostic kit of claim 1, wherein the sugar chain of the subject of the same species as the healthy mammal is relative to the amount of the sugar chain in a healthy mammal. The amount is expressed as a symptom indicator of Alzheimer's disease. The diagnostic kit of claim 1, 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 of claim 3, wherein the combined value is a value obtained by multiplication. The diagnostic kit of claim 1, further comprising a known label detecting unit that quantitatively detects the amount of the known Alzheimer's disease diagnostic marker. The diagnostic kit of claim 5, wherein a positive correlation between the amount of the sugar chain in the subject of the mammal and the amount of the known marker is expressed as Alzheim Symptoms of Mohs disease. The diagnostic kit of claim 5, wherein the known marker is amyloid beta protein. The diagnostic kit according to any one of claims 1 to 6, wherein the amount of mannose and the amount of N-acetylglucosamine of the sugar chain in the subject to be a mammal are The ratio is expressed as a symptom indicator of Alzheimer's disease. The diagnostic kit of any one of claims 1 to 6, wherein the body fluid is spinal fluid or blood. The diagnostic kit according to any one of claims 1 to 6, wherein the subject is an extremely mild or mild subject according to a diagnosis result of the severity of dementia. The diagnostic kit according to claim 10, wherein the dementia severity is evaluated as a clinical dementia rating scale. The diagnostic kit according to any one of claims 1 to 6, wherein the sugar chain is a sugar chain in which WGA and Con A are bonded. The diagnostic kit according to claim 1, wherein the sugar chain detecting unit comprises a lectin for a lectinase immunoassay or a recognition antibody for a sugar chain for an enzyme immunoassay. The diagnostic kit of claim 1, wherein the sugar chain detecting unit comprises a lectin for lectin affinity electrophoresis. The diagnostic kit of claim 1, wherein the sugar chain detecting unit comprises a lectin for lectin blot analysis or a recognition antibody for a sugar chain for immunoblot analysis. The diagnostic kit of claim 1, wherein the sugar chain detecting unit comprises a recognition antibody for a lectin or a sugar chain for thin layer chromatography. The diagnostic kit of claim 1, wherein the sugar chain detecting unit comprises a lectin array wafer or an antibody array wafer using a sugar chain recognition antibody. The diagnostic kit of any one of claims 13 to 17, wherein the lectin is WGA and/or Con A. The diagnostic kit of any one of claims 1 to 6, wherein the sugar chain detecting unit comprises a liquid chromatography for detecting a sugar chain cleaved from the complement C3 protein. A method for detecting a symptom index of Alzheimer's disease, comprising: a step of quantitatively detecting an amount of a sugar chain derived from a sample of a mammalian body fluid as a subject Complement C3 protein. The method of detecting according to claim 20, further comprising: determining whether the amount of the sugar chain of the same species as the mammal is the same as the amount of the sugar chain relative to the mammal Significantly changing steps. The method of detecting according to claim 20, further comprising the step of quantitatively detecting the amount of the known Alzheimer's disease diagnostic marker. The method of claim 22, further comprising the step of calculating a ratio of the amount of the sugar chain to the amount of the known label. The detection method according to any one of claims 20 to 23, 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 method of detecting according to claim 24, further comprising the step of calculating the amount ratio of the amount of mannose and the amount of the N-acetylglucosamine.