KR20110122958A - A method for the diagnosis of Alzheimers disease from determination of PGCP concentration in human plasma - Google Patents

Abstract

PURPOSE: A method for diagnosing dementia through measurement of PGCP concentrate in plasma is provided to simply diagnose dementia. CONSTITUTION: A kit for diagnosing dementia contains anti-PGCP(plasma glutamate carboxypeptidase) antibody. The anti-PGCP antibody is conjugated on a ligand. The ligand is biotin or biotin derivative which binds to avidin or streptavidin. The kit additionally comprises a visualization conjugate conjugated with chromogenic enzyme, chromogenic material or fluorescent molecule. The chromogenic enzyme is HRP(horseradish peroxidase) or alkaline phosphatase. The chromogenic material is colloid gold. The fluorescent molecule is FITC(poly L-lysine-fluorescein isothiocyanate) and RITC(rhodamine-Bisothiocyanate).

Description

A method for the diagnosis of Alzheimer's disease from determination of PGCP concentration in human plasma}

It relates to a dementia diagnostic kit by measuring the concentration of PGCP in plasma, and a method for diagnosing dementia using the same.

Alzheimer's Disease (AD) is a progressive brain disease that is a serious condition in which memory, language, and the ability to recognize time and space gradually decrease and eventually become unable to care for themselves. . First described by German doctor Alois Alzheimer in 1906, it was initially thought to be a very rare disease that only occurs in young people, but nowadays it is the most common disease that causes loss of intellectual ability in people over 65 years old. Turned out. Alzheimer's disease can occur in people in their 30s, 40s and 50s, but it occurs in less than 10% of all Alzheimer's patients and most patients are in their 60s or older. Alzheimer's disease, which occurs before age 65, is called premature Alzheimer's disease, and has a stronger genetic tendency than it does after age 65. But as you age, your chances of getting sick increase. Since the incidence of Alzheimer's disease increases with age, it has been recognized as a disease that occurs naturally with age, but Alzheimer's disease is known to be caused by pathological degenerative neuronal changes unlike normal aging.

Senile plaque and neurofibrillary tangles observed in the brains of patients who died from AD appear to be a pathological feature of AD. The senile plaque is formed by accumulation of proteins and dead cells outside the cell, and its main component is a peptide called amyloid-beta (hereinafter referred to as "Aβ") (Hardy, J. etal, Nat Neurosci). 1: 355-358, 1998). The gradual loss of cognitive function, a major feature of AD patients, appears to be caused by abnormally accumulated Aβ, which is produced through proteolysis from amyloid precursor protein ("APP"). do. The precursor APP is degraded by β-secretase (BSCE) and γ-secretase, resulting in Aβ production (Craven, R., Nat Rev. Neurosci. 2: 533, 2001; David, HS et al , Nat Rev. Neurosci. 2: 595-598, 2001; Yankner, BA, Neuron 16: 921-932, 1996; Selkoe, DJ, Nature 399: A23-A31, 1999).

Alzheimer's dementia is the most common form, accounting for 50-70% of dementia, a serious disease affecting 4 million Americans and nearly 15 million people worldwide. Alzheimer's dementia inflicts tremendous harm and suffering personally, but also causes many problems socially, especially in later stages of the disease. Significant economic costs will be spent through supportive care and hospitalization. The rapid increase in the proportion of the elderly population in society means a rapid increase in the number of patients with AD, which is why early detection and treatment of AD have become an important issue worldwide. However, the diagnosis process to determine the exact cause of Alzheimer's disease caused by various etiologies is very difficult, and it is also difficult to distinguish between AD and other types of dementia by current tests.

Currently, diagnostic tests for dementia include psychological tests such as Mini Mental State Examination (MMSE) and neuropsychological tests such as CDR (clinical dementia rating).

MMSE, a simple mental health test, is a test to determine whether cognitive decline is caused by a disease such as dementia or alcoholism.It is a tool for measuring orientation, recall, short-term memory, concentration, constructive behavior, and language ability. If the score is 18 or less, it is judged as definite dementia (clear cognitive impairment) if it is less than 18 points, suspected dementia (mild cognitive impairment) if it is 19 ~ 23, and normal (cognitive impairment of cognitive impairment) more than 24 points None). While it is very simple and time-consuming, it is convenient, but other tests must be performed together to obtain accurate information on which function is compromised.

CDR, a clinical determinant of dementia, divides the stages of dementia into six indicators (memory, persistence, judgment and problem solving, social activities, family life and hobbies and hygiene and grooming). , 0.5 for mild cognitive impairment, 1 for mild dementia, 2 for severe dementia, 3 for severe dementia, 4 for severe dementia, and 5 for late dementia.

According to the diagnostic criteria, Alzheimer's disease patients can be divided into mild cognitive impairment, mild dementia (MILD AD), moderate dementia and severe AD. Normal people also suffer from some degree of memory impairment as they age, but symptoms such as personality changes that are specific to Alzheimer's patients do not appear. This is called mild cognitive impairment (MCI). Mild cognitive impairment is considered a prognostic symptom of Alzheimer's disease and is characterized by short-term memory loss, spatial memory loss, and emotional imbalance, which are further categorized in several stages. The MCI associated with double memory loss is called amnestic MCI, which has a 1 to 3% chance that a 65-year-old normal person has been converted to Alzheimer's within a certain period of time, compared to 8 out of 10 people with forgetful MCI. It has been shown that life-threatening patients with Alzheimer's disease have a high possibility of developing Alzheimer's dementia if they have forgetful mild cognitive impairment.

Compared with other disease areas, early diagnosis is more important in the field of dementia. This is because there is no effective treatment available at present. The ultimate goal of Alzheimer's disease treatment is to reverse and cure the disease itself and to reduce and eliminate the cognitive impairments, mental disorders and abnormal behavior manifested by dementia. Although many drugs are currently being used to treat Alzheimer's disease, most of the medicines are still being tested for efficacy, and moreover, all the medicines so far may slow the progression of Alzheimer's disease or cause symptoms of Alzheimer's disease. It is designed to treat the disease, but none of them are designed and made to cure the underlying disease of Alzheimer's disease. Thus, if a simple diagnostic technique is provided to distinguish Alzheimer's patients early, rapid and appropriate treatment, such as the administration of drugs in the early stages of the disease, can alleviate the symptoms and weaken the extent of the onset of dementia. Diagnosis studies are very important in the treatment of dementia.

To date, many studies have been conducted to diagnose Alzheimer's disease early, but there are no markers effective for diagnosis. Recent studies have shown that levels of Aβ-40 / -42 in plasma and various signal-regulating proteins can be used to distinguish Alzheimer's dementia from controls, and in several proteome-based studies, compared to AD plasma. It has been found that α2-macroglobulin (α2M), complement factor H (CFH), and α1-antitrypsin (AIAT) are present at high levels, thereby making it possible to utilize them as markers applicable to the diagnosis of AD. Expected. However, the sensitivity and specificity of the proteins using individual proteins or combinations thereof are insufficient for the initial diagnosis of MCI and AD, and the development of new clinically useful biomarkers for the initial diagnosis of MCI and AD is needed.

Plasma glutamate carboxypeptidase (PGCP) in human blood is a metalloprotease that exhibits high sequence homology with the active site of glutamate carboxypeptidase II (GCP II). GCP II is known as N-acetylated α-linked acidic dipeptidase (NAALADase) or prostate-specific membrane antigen (PSMA). However, despite the fact that PGCP is a glycoprotein commonly expressed in human tissue and found primarily in serum, little is known about the biological function of PGCP and the exact physiological substrate of PGCP.

Therefore, the present inventors have developed a novel antibody that recognizes PGCP, using the enzyme-linked immunosorbent assay (ELISA) to determine the concentration of PGCP in the plasma of normal, mild cognitive impairment group and dementia patient group As a result, the concentration of PGCP in plasma was significantly higher in dementia patients with cognitive impairment by measuring MMSE and CDR. Using PGCP protein as a marker, Alzheimer's disease was difficult to diagnose due to the absence of special signs and symptoms. The present invention has been completed by revealing that it can be usefully used for the diagnosis of sexual dementia.

It is an object of the present invention to provide a dementia diagnostic kit or immunochromatography strip using PGCP as a marker in plasma.

Still another object of the present invention is to provide a method for diagnosing dementia from blood using the kit or immunochromatography strip.

In order to achieve the above object, the present invention provides a diagnostic kit for dementia containing an anti-PGCP (plasma glutamate carboxypepridase) antibody.

In addition,

1) an adhesive support;

2) a sample pad attached to a test sample to be analyzed, which is attached to an upper surface of the adhesive plastic support;

3) a conjugate pad linked to the sample pad and containing a conjugate that specifically binds to the PGCP protein contained in the sample to be tested;

4) a signal detection pad interlocked with the conjugate pad and comprising a test line in which a protein binding to the conjugate is linearly immobilized and a control line in which human antibody immunoglobulin is immobilized ; And

5) The present invention provides an immunochromatography strip for diagnosing dementia, comprising an absorbent pad positioned downstream of the signal detecting pad, which absorbs the test sample after the signal detecting reaction is completed.

In addition,

1) treating the subject-derived blood on a solid substrate and then washing it;

2) treating the substrate with an anti-PGCP antibody and then washing the substrate; And

3) providing a protein detection method for providing information for diagnosing dementia, comprising determining a subject having an increased degree of attachment of an anti-PGCP antibody on the substrate as a subject having or at risk of dementia. do.

In addition,

1) treating the subject-derived blood on a solid substrate to which the anti-PGCP antibody is attached and then washing the blood;

2) treating the substrate with an anti-PGCP antibody and then washing the substrate; And

3) providing a protein detection method for providing information for diagnosing dementia, comprising determining a subject having an increased degree of attachment of an anti-PGCP antibody on the substrate as a subject having or at risk of dementia. do.

The present invention provides a method for diagnosing dementia using the PGCP as a marker by confirming that the concentration of plasma glutamate carboxypeptidase (PGCP) in plasma is significantly increased in patients with dementia compared to normal and mild cognitive impairment patients. Therefore, it is possible to diagnose dementia easily and simply by specifically detecting PGCP present in the blood derived from the subject.

1 is a graph showing the distribution of PGCP concentrations in plasma of normal, mild cognitive impairment and dementia patients by diagnosis group and sex:
A-result measured by ELISA Figure;
B-Western blot and quantification thereof Figure;
C-dot blot result plot;
Picture of quantification of D-dot blot results;
Control-normal population;
MCI-Mild Cognitive Impairment Group; And
AD-group of Alzheimer's dementia.
FIG. 2 is a plot showing the concentration of PGCP in plasma of normal, mild cognitive impairment patients and dementia patients according to age and MMSE results:
Control-normal population;
MCI-Mild Cognitive Impairment Group; And
AD-group of Alzheimer's dementia.

Hereinafter, the present invention will be described in detail.

The present invention provides a kit for diagnosing dementia containing an anti-PGCP (plasma glutamate carboxypeptidase) antibody.

The anti-PGCP antibody may be prepared by the injection of a PGCP protein or commercially available, all of which is available, the plasma glutamate carboxypeptidase (PGCP) is preferably human PGCP, amino acid sequence described in SEQ ID NO: 1 It is more preferable to have. In addition, the antibodies include polyclonal antibodies, monoclonal antibodies, fragments capable of binding epitopes, and the like.

Polyclonal antibodies can be produced by conventional methods of injecting the PGCP protein into an animal and collecting blood from the animal to obtain serum containing the antibody. Such polyclonal antibodies can be purified by any method known in the art and can be made from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, cattle, dogs, and the like, and host rabbits. Preferably, but is not limited thereto.

Monoclonal antibodies can be prepared using any technique that provides for the production of antibody molecules through the culture of continuous cell lines. Such techniques include, but are not limited to, hybridoma technology, human B-cell hybridoma technology, and EBV-hybridoma technology (Kohler G et. al ., Nature 256: 495-497, 1975; Kozbor D et al . , J Immunol Methods 81: 31-42, 1985; Cote RJ et al . , Proc Natl Acad Sci 80: 2026-2030, 1983; And Cole SP et al ., Mol Cell Biol 62: 109-120, 1984).

In addition, antibody fragments containing specific binding sites for the PGCP proteins can be prepared. For example, but not limited to, F (ab ') 2 fragments can be prepared by digesting antibody molecules with pepsin, and Fab fragments can be prepared by reducing the disulfide bridges of F (ab') 2 fragments. Alternatively, the Fab expression library can be made smaller to quickly and easily identify monoclonal Fab fragments with the desired specificity (Huse WD et. al . , Science 254: 1275-1281, 1989).

The antibody can be bound to a solid substrate to facilitate subsequent steps such as washing or separation of the complex. Solid substrates include synthetic resins, nitrocellulose, glass substrates, metal substrates, glass fibers, microspheres and microbeads. In addition, the synthetic resins include polyester, polyvinyl chloride, polystyrene, polypropylene, PVDF and nylon.

In a specific embodiment of the present invention, mRNA isolated from HEK293 cells was subjected to RT-PCR as a template to amplify PGCP cDNA, and inserted it into pENTR3C-PGCP, a recombinant PGCP baculovirus delivery vector, and with baculovirus DNA. The transfection vector was co-transduced into insect cells, and then a large amount of expression was induced to produce a large amount of recombinant PGCP protein. Thereafter, the recombinant PGCP protein was purified using DEAE-Sepharose resin, and electrophoresed on an SDS-polyacrylamide gel, and then the PGCP protein band was obtained. After cutting and injecting into rabbits, rabbit anti-serum immunoglobulin proteins were purified using proteinine A-agarose to prepare anti-PGCP polyclonal antibodies.

In addition, in order to measure the concentration of PGCP protein in plasma using the prepared anti-PGCP antibody, enzyme immunoassay (ELISA), dot blot (Western blot) and Western blot (Western blot) analysis was performed, as a result Compared with the control group, the patients with Alzheimer's dementia had lower MMSE-K values, higher CDR scores, and lower plasma albumin and total protein concentrations in the dementia group than in the normal and mild cognitive impairment groups. In contrast, the concentration of PGCP in plasma measured by ELISA was 126.0 ± 25.2 ug / ml in the normal group, 123.5 ± 26.0 ug / ml in the mild cognitive impairment group, and 135.2 ± 35.9 ug / ml in the dementia group. Was found to be significantly higher than the PGCP concentration of the normal and mild cognitive impairment group (A of FIG. 1). Western blot (see FIG. 1B) and dot blot analysis (see C and D of FIG. 1) showed a statistically significant difference between normal and dementia patients in the order of normal <mild cognitive impairment <dementia. When the dot blot results were divided into female and male, the difference was similar to that of the whole sample. It was confirmed that the concentration of PGCP in plasma was higher in the mild cognitive impairment group than in the normal control group, especially in Alzheimer's dementia patients.

In addition, as the age increased, the concentration of PGCP also increased ( p <0.01, A in FIG. 2), which was inversely related to the MMSE score, which showed a statistically significant negative correlation in men. (R = 0.31, p <0.001, see B of FIG. 2).

The correlation between MMSE and CDR scores, which are currently used as diagnostic tests for dementia, and plasma concentrations of PGCP, age, and sex were analyzed. As a result, PGCP concentrations increased with increasing MMSE score. Decreased (r = 0.147, p = <0.003). In addition, the MMSE score and the plasma concentration of PGCP were statistically significant in all subjects and the male group, which remained significant even after adjusting for age, sex, and education. A significant correlation was also seen in the CDR scores (see Table 2).

Therefore, the concentration of PGCP in plasma is inversely correlated with the MMSE score and is proportionally correlated with the CDR score. Therefore, the concentration of PGCP in plasma is increased when cognitive impairment is reduced, that is, the incidence of dementia is high. In addition, it was confirmed that the increase significantly.

In addition, we examined the correlation between depression and PGCP in elderly patients to determine whether increasing plasma concentrations of PGCP were specific to dementia. The concentrations of PGCP in plasma according to the presence of depressive symptoms in normal and mild cognitive impairment groups were compared. As a result, the concentration of PGCP in plasma was significantly increased in patients with depressive symptoms in both normal and mild cognitive impairment groups. It was reduced ( p <0.001), and it was confirmed that the patients with depressive symptoms showed significantly lower concentrations of PGCP in plasma than those without depressive symptoms regardless of cognitive abilities (see Table 3).

Therefore, when the results are summarized, it can be seen that the significantly high PGCP in the plasma of dementia patients is a phenomenon that is sensitively changed as the cognitive function decreases, and thus it is easy to diagnose dementia. have.

In the dementia diagnostic kit of the present invention, it is preferable to use an anti-PGCP antibody bound to a ligand which is a biotin or a biotin derivative having essentially the same binding action as biotin to avidin or streptavidin. It is preferable to use a binding conjugated with the ligand to a visualization conjugate to which a chromophore, a chromophore, or a fluorescent molecule bound to an appropriate binding molecule is bound, but is not limited thereto.

The color development enzyme is preferably HRP (horseradish peroxidase) or basic dephosphatase (alkaline phosphatase), the color development material is preferably colloidal gold (coloid gold), the fluorescent molecule is a FITC (poly L-lysine-fluorescein isothiocyanate ) And RITC (rhodamine-B-isothiocyanate), but is not limited thereto.

The dementia diagnostic kit of the present invention can diagnose dementia by quantitatively or qualitatively analyzing the binding reaction through an antigen-antibody binding reaction or a protein-ligand binding reaction, and the binding reaction is a conventional enzyme-linked immunoassay (ELISA). , Radioimmunoassay (RIA), sandwich assay, western blotting, immunoprecipitation, immunohistochemical staining, fluorescence immunoassay, enzyme substrate coloration, antigen-antibody aggregation, etc. Can be measured.

The dementia diagnostic kit of the present invention may be used as a support, a well plate synthesized from a nitrocellulose membrane, a PVDF membrane, a polyvinyl resin or a polystyrene resin, a slide glass made of glass, or the like.

The dementia diagnostic kit of the present invention is a chromophore, chromophores or fluorescent molecules as a label, a conventional chromophore is preferred, the color development reaction, HRP (horseradish peroxidase), basic dephosphatase (alkaline phosphatase), colloidal gold (coloid gold) , Fluorescent materials such as poly L-lysine-fluorescein isothiocyanate (FITC), rhodamine-B-isothiocyanate (RITC), dyes, and the like may be used.

Dementia diagnostic kit of the present invention preferably comprises a phosphate buffer, NaCl and Tween 20 as a wash solution, but is not limited thereto.

The present invention also provides a immunochromatographic strip for diagnosing dementia comprising a conjugate specifically binding to a PGCP protein and a protein binding to the conjugate.

The immunochromatographic strip according to the present invention preferably comprises an adhesive plastic support, and a sample pad, a conjugate pad, a signal detection pad, and an absorption pad attached to the adhesive plastic support, but are not limited thereto.

Specifically, the immunochromatography strip preferably has the following configuration, but is not limited thereto.

1) an adhesive support;

2) a sample pad attached to a test sample to be analyzed, which is attached to an upper surface of the adhesive plastic support;

3) a conjugate pad linked to the sample pad and containing a conjugate that specifically binds to the PGCP protein contained in the sample to be tested;

4) a signal detection pad interlocked with the conjugate pad and comprising a test line in which a protein binding to the conjugate is linearly immobilized and a control line in which human antibody immunoglobulin is immobilized ; And

5) an absorption pad positioned downstream of the signal detection pad, the sample being absorbed after the signal detection reaction is completed.

In the above configuration, the color developer of step 3) is preferably colloidal gold particles, but is not limited thereto.

In the above configuration, the signal detection pad of step 4) is preferably made of a nitrocellulose film, but is not limited thereto.

In the above configuration, the absorbent pad of step 5) preferably includes a porous support and an absorbent dispersed in pores of the porous support or adsorbed or coated on the fiber yarn of the porous support. It is preferable to further include a porous film layer attached to the upper surface, but is not limited thereto. The absorbent is preferably selected from the group consisting of calcium chloride, magnesium chloride, diatomaceous earth, bentonite, dolomite, gypsum, silica gel and mixtures thereof, but is not limited thereto.

The immunochromatography strip according to the present invention is characterized in that a test sample is determined as a positive sample of dementia when colored lines appear in the control line and the detection line on the immunochromatography strip.

In the immunochromatography strip according to the present invention, plasma, which is a test sample, is first supplied to the immunochromatography strip through the sample pad. The sample pad may further have a function of filtering in order to further improve the selectivity for the analyte or to minimize the influence of the interference material which may be included in the test sample. If necessary, an auxiliary pad may be further provided upstream of the sample pad containing a substance that can increase the reaction between the analyte and the conjugate or eliminate the influence by the interference. Blood introduced through the sample pad is transferred to a conjugate pad located upstream of the sample pad through chromatographic movement. The conjugate pad contains a conjugate that specifically binds to the PGCP protein contained in the blood. The conjugate is labeled with gold particles, latex particles, fluorescent materials, enzymes and the like. The test sample passing through the conjugate pad moves to the signal detection pad. The signal detection pad includes a detection line for detecting whether an analyte is present in the test sample, and a control line for confirming whether the assay kit is normally operated regardless of the presence or absence of the analyte. To this end, the detection line is coated with a substance (or signal detection material) that selectively and specifically binds to a binding product between the analyte and the conjugate contained in the conjugate pad, and the control line is coated with the conjugate pad. It is preferred that the material specifically binding to the conjugate contained in the coating. The signal detection pad is composed of a porous membrane pad, it may be made of nitrocellulose, cellulose, polyethylene, polyethersulfone, nylon and the like.

The present invention also provides a method for diagnosing dementia using a composition for diagnosing dementia or a dementia diagnostic kit comprising an anti-PGCP antibody.

The method for diagnosing dementia is preferably performed in the following steps, but is not limited thereto.

1) treating the subject-derived blood on a solid substrate and then washing it;

2) treating the substrate with an anti-PGCP antibody and then washing the substrate; And

3) Determining that the subject with increased degree of adhesion of anti-PGCP antibody on the substrate as a subject having dementia or risk of getting dementia.

The method for diagnosing dementia is preferably performed in the following steps, but is not limited thereto.

1) treating the subject-derived blood on a solid substrate to which the anti-PGCP antibody is attached and then washing the blood;

2) treating the substrate with an anti-PGCP antibody and then washing the substrate; And

3) Determining that the subject with increased degree of adhesion of anti-PGCP antibody on the substrate as a subject having dementia or risk of getting dementia.

In the method, the subject of step 1) is a vertebrate, including human, preferably a mammal, more preferably human, ape, bovine, pig, rat, rabbit, guinea peak, hamster, dog or Cats can be used, but are not limited to these.

In the above method, the solid substrate of step 1) may be any one selected from the group consisting of an NC (nitrocellulose) film, a polyvinylidene fluoride (PVDF) film, a microplate, a glass substrate, a polystyrene, a silicon substrate, and a metal plate. It doesn't work.

In the above method, the anti-PGCP antibody of step 2) is bound to a ligand, the ligand is bound to a specific binding molecule, it is preferable to use a binding enzyme or a chromophore or fluorescent molecules bound to the binding molecule, but is not limited thereto. It doesn't work.

In the method, the binding degree of step 3) is Western blotting method, Enzyme-linked immunosorbent assay (ELISA), immunohistochemical staining, immunoprecipitation And immunofluorescence (immunofluorescence) is preferably measured by any one selected from the group consisting of, but not limited to.

It will be described below in detail by Examples and Experimental Examples of the present invention.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the present invention is not limited to the following Examples and Experimental Examples.

< Example  1> Anti- PGCP Polyclonal  Preparation of antibodies

To prepare anti-PGCP polyclonal antibodies, mRNA isolated from HEK293 cells was used as a template, and RT-PCR was performed to amplify the full-length PGCP cDNA, which was cloned into pQE30 vector. In order to express the PGCP in insect cells, were prepared for viral vector delivery of pENTR3C-PGCP PGCP a recombinant baculovirus, each Kpn I and Xho I restriction enzyme sites with primer [Forward 5'-CGGGGTACCATGAAATTCCTTATC-3 '(SEQ ID No. For this 2) and reverse 5′-CCCTC GAGATGGACCTAGGCAG-3 (SEQ ID NO: 3)] were used to determine whether PGCP was inserted into the pENTR3C vector through sequencing. The baculovirus DNA and the delivery vector were then cotransformed into Sf 9 insect cells using the BaculoDirect transfecftion kit (available from Invitrogen). Recombinant PGCP virus stock was prepared through two infections of insect cells, which were then infected with insect cells to mass express recombinant PGCP (rPGCP). Sf 9 cells were cultured using spinner flasks in Sf900II-Sf medium (purchased from Invitrogen) with monolayer culture and 10% fetal bovine serum (FBS) and 1% anti-biotic solution (purchased from GIBCO). Incubated at 27 ° C. Then, in order to optimize the expression of rPGCP, Sf 9 cells were cultured in large quantities, and then rPGCP virus was infected. After culturing virus infected insect cells, the medium was collected and concentrated using a Centricon concenctrator (using a molecular weight 10 KDa cutoff membrane, purchased from Amicon), followed by centrifugation at 13,000 rpm for 20 minutes. Then, to purify the rPGCP, the concentrated PGCP was reacted with DEAE-Sepharose resin overnight, and then the binding buffer solution (pH 8.0) of 10 times the volume for the resin was used. The original resin was washed and eluted sequentially using a binding buffer solution containing 0.1, 0.25, 0.5 and 1 molar sodium chloride, respectively. After collecting all the eluted fractions from the eluted fractions, they were precipitated with acetone, electrophoresed on SDS-polyacrylamide gels, and only the PGCP protein bands were cut out to immunize rabbits for PGCP antibody production. (Manufactured by LabFrontier). Thereafter, rabbit anti-serum immunoglobulin proteins were purified using protein A-agarose, thereby obtaining anti-PGCP polyclonal antibodies.

< Example  2> normal group, Mild cognitive impairment group  And Dementia  Plasma separation

Patients with Alzheimer's dementia (AD) were collected from the Department of Psychiatry, Seoul Samsung Hospital, Seoul, Korea. Healthy controls and patients with MCI (Middy Cognitive Impairment) were collected from the Community Ansan Coin Cohort at the Korea Institute of Geriatric Health in Ansan, Korea. Collected through). All AD patients were diagnosed using NINCDS-ADRDA criteria, and mild cognitive impairment (MCI) was diagnosed based on the Mayo clinic criteria. Cognitive function and memory impairment were assessed using the Korean Simple Mental State Test (MMSE-K) and Neuropsychological Test [CERAD-K (N)]. Depression symptoms were 30-item-Geriatric Depression Scale; GDS-K), and depression when the GDS-K score was 18 or more.

Blood samples were obtained using standard venipuncture with ethylenediamine tetraacetic acid (EDTA), then plasma was separated by centrifugation at 1,300 μg for 10 minutes, and the separated plasma was stored at −80 ° C. for analysis.

< Experimental Example  1> normal group, Mild cognitive impairment group  And Dementia  PGCP in plasma ( plasma glutamate  carboxypeptidase) concentration comparison

In order to compare the concentrations of PGCP in plasma of the normal group, the mild cognitive impairment group and the dementia group, enzyme immunoassay (ELISA), dot blot analysis and Western blot analysis were performed.

<1.1> Concentration Comparison by Enzyme Immunoassay ( ELISA )

Enzyme immunoassay (ELISA) was performed on 96 well plates (Nunc) with 100 ul of plasma samples diluted 1: 30,000 using dimethyl sulfoxide (DMSO) buffer solution (PBS containing 1% DMSO and 0.02% NaN3). Maxisorp) was performed by coating proteins in plasma in 96 wells through incubation overnight at 4 ° C. PGCP purified in HEK293T cell medium was used as a calibration standard (dilution to 0-8 pg / ml with DMSO buffer solution). The plates were washed three times with 200 ul of PBST (PBS containing 0.05% Tween 20) buffer and 1 at room temperature with PBS buffer containing 100 ul of 3% bovine albumin serum (BSA, purchased from GIBCO). It was left standing for hours and blocked. Thereafter, the rabbit anti-PGCP polyclonal antibody prepared in Example 1 was diluted 1: 5000 with PBS buffer containing 1% BSA, added to each well at 100 ul / well, and then PBST buffer solution. After washing three times, the mixture was allowed to stand at room temperature for 2 hours, and then washed three times with PBST. Thereafter, the plate was treated with 100 ul of an anti-rabbit IgG peroxidase conjugate specific to an Fc fragment diluted 1: 5000 with PBS buffer containing 1% BSA as a secondary antibody, and then allowed to stand at room temperature for 1 hour. Wash with PBST buffer. Thereafter, the plate was treated with 3,3 ', 5,5'-tetramethylbenzidine (TMB, purchased from amresco) at 50 ul / well, and reacted for 15 minutes at room temperature under light blocking conditions, and then 50 ul / The reaction was terminated by treatment of the stop solution (1 mol / l hydrochloric acid solution) of the well, and the absorbance was measured at 450 nm.

186 normal controls (mean age 69.2 ± 4.3 years), 89 subjects with mild cognitive impairment (mean age 73.7 ± 5.5 years) and 137 Alzheimer's dementia patients (mean age 74.6 ± 6.7) The PGCP concentration in plasma was measured by the ELISA method, and a one way ANOVA test was used for statistical analysis between various values. Individual correlations between age or MMSE values and PGCP concentrations were calculated for the entire sample and three study groups (control, MCI and AD), and the samples were analyzed by the correlation between sex and test values and the analyzed PGCP concentrations. The analysis of covariance was covariates, where PGCP concentrations across all three diagnostic groups, including age, gender, and degree of education, were performed using ANOVA, chi-square test, Student's T-test. black (student's t-test) and multivariate logistic regression analysis (multiple logistic regression analysis) was performed using the SAS 9.1 program, p value (p value) was significantly determined that, the statistical when below 0.05, and The results are shown in the following [Table 1].

General Characteristics and PGCP Concentrations in Subject Groups Normal control Mild Cognitive Impairment (MCI) Alzheimer's Dementia (AD) Sample Count 186 89 137 Age (years) 69.2 ± 4.3 73.7 ± 5.5 74.6 ± 6.7 Gender (male / female) 116/70 36/53 48/89 Education age 11.4 ± 4.1 7.3 ± 4.1 7.9 ± 5.3 MMSE Score 27.8 ± 1.6 26.0 ± 2.5 15.9 ± 6.9 CDR overall score 0.00 ± 0.04 0.06 ± 0.20 1.28 ± 8.7 * Albumin in serum (g / dl) 4.4 ± 0.2 4.4 ± 0.2 4.0 ± 0.6 Total serum protein (g / dl) 7.2 ± 0.4 7.2 ± 0.4 6.8 ± 1.0 ** ApoE-ε4 allele (%) 17.2 14.6 34.6 PGCP concentration (ug / ml) 126.0 ± 25.2 123.5 ± 26.0 135.2 ± 35.9

Values calculated using Student's T-Test are expressed as ± standard deviation.

* Results from 125 samples

** Analysis of 114 samples

As shown in Table 1, Alzheimer's patients were significantly older, less educated, and had fewer men (26% AD and 62% control) than the control group, and a total of 137 Alzheimer's patients The analysis was based on the scores of 125 patients (91%) with moderate MMSE score and CDR score values.

As a result, the MMSE value of Alzheimer's dementia patients was low, the overall CDR score was high, and the plasma albumin and total protein concentrations were lower in the dementia group than in the normal and mild cognitive impairment groups. Plasma concentrations of PGCP measured by ELISA were 126.0 ± 25.2 μg / ml in the normal group, 123.5 ± 26.0 μg / ml in the mild cognitive impairment group, and 135.2 ± 35.9 μg / ml in the dementia group. It was confirmed that the cognitive impaired group was significantly higher than the PGCP concentration (A of FIG. 1).

<1.2> dot Blot ( Dot blot ) And Weston Blot ( Western blot Comparison of concentrations through analysis

Dot blot analysis was performed by dropping 3 ul of plasma sample diluted 1: 10 in DMSO buffer solution onto a nitrocellulose (NC) membrane and drying at room temperature for 30 minutes. The membrane was blocked with PBST buffer containing 5% skim milk powder, reacted with PGCP polyclonal antibody, and washed three times with PBST buffer. The membrane was then treated with 100 ul of a secondary anti-rabbit IgG peroxidase conjugate specific to the Fc fragment diluted 1: 5000 with PBST buffer containing 3% skim milk powder, and allowed to stand at room temperature for 1 hour, followed by PBST buffer Were washed and the protein spots were visualized by treatment with ECL solution (purchased from PIECE) according to the manufacturer's instructions.

In addition, Western blot analysis was performed by separating 3 ul of the plasma sample diluted 1: 10 through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% gel, and then transferring it to the NC membrane. The membrane was confirmed by reacting with a PGCP antibody, followed by reacting a goat-rabbit or mouse IgG antibody (purchased from PIERCE) conjugated with horseradish peroxidase, and following the manufacturer's instructions, an ECL solution ( Protein bands were processed to visualize the protein bands and then quantified by scanning the images of the visualized PGCP spots and bands.

As a result, there was a statistically significant difference between normal and dementia patients in Western blot (B of FIG. 1) and dot blot analysis (C and D of FIG. 1) in the order of normal <mild cognitive impairment <dementia. When the dot blot results were divided into female and male, the difference was similar to that of the whole sample.

Therefore, it was confirmed that the concentration of PGCP in plasma was higher in the mild cognitive impairment group than in the normal control group, especially in Alzheimer's dementia patients.

< Experimental Example  2> in plasma PGCP ( plasma glutamate carboxypeptidase Correlation between the concentration of) and cognitive test scores

Correlation between MMSE and CDR scores, plasma concentrations of PGCP, age and sex, which are currently used as diagnostic tests for dementia, were analyzed.

The MMSE score is 30 points, usually 18 points or less, deterministic dementia (obvious cognitive impairment), 19 to 23 points suspected dementia (mild cognitive impairment), and more than 24 points normal (cognitive function) No cognitive impairment of disability), the lower the score, the less cognitive function was determined. In addition, the CDR score is 0 because it is not dementia, 0.5 is mild cognitive impairment, 1 is mild dementia, 2 is severe dementia, 3 is severe dementia, 4 is deep dementia and 5 is late dementia. The higher the value, the more severe the dementia.

As a result, the concentration of PGCP increased with age ( p <0.01, A in FIG. 2), which was inversely related to the MMSE score, which was statistically significant in males. It was confirmed (r = 0.31, p <0.001, B of FIG. 2).

In addition, as shown in Table 2 below, PGCP concentration decreased as the MMSE score increased (r = 0.147, p = <0.003). In addition, the MMSE score and plasma concentrations of PGCP were statistically significant in all subjects and the male group, which remained significant even after adjusting for age, gender, and education. The CDR scores also showed a significant correlation.

Total patients (530) Male (245 people) Female (285 people) r p r p r p MMSE Score Before correction -0.159 0.0002 -0.237 0.0002 -0.101 0.090 After calibration * -0.148 0.0007 -0.246 0.0001 -0.086 0.149 CDR score Before correction 0.186 <0.0001 0.217 0.0006 0.162 0.006 After calibration * 0.162 0.0002 0.217 0.0006 0.109 0.070

r : Pearson correlation coefficient

* Correction based on age, gender and training period

Therefore, the concentration of PGCP in plasma is inversely correlated with the MMSE score and is proportionally correlated with the CDR score. Therefore, the concentration of PGCP in plasma is increased when cognitive impairment is reduced, that is, the incidence of dementia is high. In addition, it was confirmed that the increase significantly.

< Experimental Example  3> normal group and In mild cognitive impairment group  Plasma PGCP (Plasma) According to Depression Symptoms glutamate carboxypeptiddase Comparison of concentrations of

In patients with dementia, increased plasma levels of PGCP were identified as dementia-specific phenomena, and mild cognitive impairment was associated with depression. To determine the correlation between plasma PGCP and depression, we compared plasma levels of PGCP with and without depression in normal and mild cognitive impairment groups.

Specifically, plasma PGCP levels of 186 cognitively normal, no-depressive, 50 cognitively normal and 66 MCI patients with depression were compared and analyzed.

As a result, as shown in Table 3 below, PGCP concentration in plasma was significantly decreased in patients with depressive symptoms in both normal and mild cognitive impairment groups ( p <0.001).

Cognitively Normal Controls Mild Cognitive Impairment Group (MCI) No depression
(186 people) Depression metaphor
(50 people) p No depression
(89 people) Depression metaphor
(66 people) p Age (years) 69.3 ± 4.5 69.0 ± 5.6 0.716 73.6 ± 5.4 70.9 ± 5.6 0.04 Education age (years) 11.4 ± 4.1 10.3 ± 4.1 0.091 7.3 ± 4.1 4.5 ± 4.3 <.0001 Albumin in serum (g / dl) 4.4 ± 0.2 * 4.4 ± 0.3 ** 0.921 4.4 ± 0.2 4.3 ± 0.2 0.999 Total serum protein (g / dl) 7.2 ± 0.4 * 7.2 ± 0.5 ** 0.645 7.2 ± 0.4 7.2 ± 0.4 0.424 MMSE Score 27.8 ± 1.6 27.3 ± 2.2 0.143 26.0 ± 2.5 24.6 ± 3.1 0.002 PGCP concentration (ug / ml) 126.0 ± 25.2 88.1 ± 39.3 *** <.0001 123.5 ± 26.0 85.1 ± 39.9 0.0002

Values calculated using Student's T-Test are expressed as ± standard deviation.

* 185 specimens

** Results from 38 samples

*** Results from 48 samples

Therefore, it was confirmed that the patients with depressive symptoms showed significantly lower concentrations of PGCP in plasma than those without depressive symptoms regardless of cognition. This shows that the concentration of PGCP in plasma can be used in differential diagnosis of elderly depression on screening of dementia.

As can be seen from the above, the present invention can be usefully used for the development of dementia diagnosis compositions and diagnostic kits, and the development of dementia diagnosis methods and treatments for dementia.

<110> Korea Center for Disease Control and Prevention <120> A method for the diagnosis of Alzheimer's disease from          determination of PGCP concentration in humnan plasma <130> 10p-02-14 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 472 <212> PRT <213> Homo sapiens <400> 1 Met Lys Phe Leu Ile Phe Ala Phe Gly Gly Val His Leu Leu Ser   1 5 10 15 Leu Cys Ser Gly Lys Ala Ile Cys Lys Asn Gly Ile Ser Lys Arg Thr              20 25 30 Phe Glu Glu Ile Lys Glu Glu Ile Ala Ser Cys Gly Asp Val Ala Lys          35 40 45 Ala Ile Ile Asn Leu Ala Val Tyr Gly Lys Ala Gln Asn Arg Ser Tyr      50 55 60 Glu Arg Leu Ala Leu Leu Val Asp Thr Val Gly Pro Arg Leu Ser Gly  65 70 75 80 Ser Lys Asn Leu Glu Lys Ala Ile Gln Ile Met Tyr Gln Asn Leu Gln                  85 90 95 Gln Asp Gly Leu Glu Lys Val His Leu Glu Pro Val Arg Ile Pro His             100 105 110 Trp Glu Arg Gly Glu Glu Ser Ala Val Met Leu Glu Pro Arg Ile His         115 120 125 Lys Ile Ala Ile Leu Gly Leu Gly Ser Ser Ile Gly Thr Pro Pro Glu     130 135 140 Gly Ile Thr Ala Glu Val Leu Val Val Thr Ser Phe Asp Glu Leu Gln 145 150 155 160 Arg Arg Ala Ser Glu Ala Arg Gly Lys Ile Val Val Tyr Asn Gln Pro                 165 170 175 Tyr Ile Asn Tyr Ser Arg Thr Val Gln Tyr Arg Thr Gln Gly Ala Val             180 185 190 Glu Ala Ala Lys Val Gly Ala Leu Ala Ser Leu Ile Arg Ser Val Ala         195 200 205 Ser Phe Ser Ile Tyr Ser Pro His Thr Gly Ile Gln Glu Tyr Gln Asp     210 215 220 Gly Val Pro Lys Ile Pro Thr Ala Cys Ile Thr Val Glu Asp Ala Glu 225 230 235 240 Met Met Ser Arg Met Ala Ser His Gly Ile Lys Ile Val Ile Gln Leu                 245 250 255 Lys Met Gly Ala Lys Thr Tyr Pro Asp Thr Asp Ser Phe Asn Thr Val             260 265 270 Ala Glu Ile Thr Gly Ser Lys Tyr Pro Glu Gln Val Val Leu Val Ser         275 280 285 Gly His Leu Asp Ser Trp Asp Val Gly Gln Gly Ala Met Asp Asp Gly     290 295 300 Gly Gly Ala Phe Ile Ser Trp Glu Ala Leu Ser Leu Ile Lys Asp Leu 305 310 315 320 Gly Leu Arg Pro Lys Arg Thr Leu Arg Leu Val Leu Trp Thr Ala Glu                 325 330 335 Glu Gln Gly Gly Val Gly Ala Phe Gln Tyr Tyr Gln Leu His Lys Val             340 345 350 Asn Ile Ser Asn Tyr Ser Leu Val Met Glu Ser Asp Ala Gly Thr Phe         355 360 365 Leu Pro Thr Gly Leu Gln Phe Thr Gly Ser Glu Lys Ala Arg Ala Ile     370 375 380 Met Glu Glu Val Met Ser Leu Leu Gln Pro Leu Asn Ile Thr Gln Val 385 390 395 400 Leu Ser His Gly Glu Gly Thr Asp Ile Asn Phe Trp Ile Gln Ala Gly                 405 410 415 Val Pro Gly Ala Ser Leu Leu Asp Asp Leu Tyr Lys Tyr Phe Phe Phe             420 425 430 His His Ser His Gly Asp Thr Met Thr Val Met Asp Pro Lys Gln Met         435 440 445 Asn Val Ala Ala Ala Val Trp Ala Val Val Ser Tyr Val Val Ala Asp     450 455 460 Met Glu Glu Met Leu Pro Arg Ser 465 470 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PGCP forward primer <400> 2 cggggtacca tgaaattcct tatc 24 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PGCP Reverse primer <400> 3 ccctcgagat ggacctaggc ag 22

Claims (14)

Dementia diagnostic kit containing an anti-PGCP (plasma glutamate carboxypeptidase) antibody.
The kit of claim 1, wherein the anti-PGCP antibody is bound to a ligand.
3. The kit of claim 2, wherein the ligand is a biotin or a biotin derivative having essentially the same binding action as biotin to avidin or streptavidin.
The kit of claim 3, further comprising a visualization conjugate in which a chromophore, a chromophore, or a fluorescent molecule bound to a ligand specific binding molecule is bound.
The kit according to claim 4, wherein the chromophore is horseradish peroxidase (HRP) or alkaline phosphatase.
The kit as claimed in claim 4, wherein the coloring material is colloidal gold.
The kit of claim 4, wherein the fluorescent molecules are poly L-lysine-fluorescein isothiocyanate (FITC) and rhodamine-B-isothiocyanate (RITC).
1) an adhesive support;
2) a sample pad attached to a test sample to be analyzed, which is attached to an upper surface of the adhesive plastic support;
3) a conjugate pad linked to the sample pad and containing a conjugate that specifically binds to the PGCP protein contained in the sample to be tested;
4) a signal detection pad interlocked with the conjugate pad and comprising a test line in which a protein binding to the conjugate is linearly immobilized and a control line in which human antibody immunoglobulin is immobilized ; And
5) An immunochromatography strip for diagnosing dementia comprising an absorbent pad located downstream of the signal detecting pad, the absorbing test sample having completed the signal detecting reaction.
9. The immunochromatographic strip of claim 8, wherein the colorant of step 3) is a colloidal gold particle.
The immunochromatography strip according to claim 8, wherein the signal detection pad of step 4) is any one selected from the group consisting of nitrocellulose, cellulose, polyethylene, polyethersulfone and nylon.
9. The immunochromatography according to claim 8, wherein the absorbent pad of step 5) comprises a porous support and an absorbent dispersed in a pore of the porous support or adsorbed or coated on the fiber yarn of the porous support. strip.
The immunochromatographic strip according to claim 8, wherein when a colored line appears in the control line and the detection line on the immunochromatography strip, the test sample is determined as a positive sample of dementia.
1) treating the subject-derived blood on a solid substrate and then washing it;
2) treating the substrate with an anti-PGCP antibody and then washing the substrate; And
3) A method for detecting a protein for providing information on diagnosis of dementia, comprising determining a subject having an increased degree of attachment of an anti-PGCP antibody on the substrate as a subject having or at risk of dementia.
1) treating the subject-derived blood on a solid substrate to which the anti-PGCP antibody is attached and then washing the blood;
2) treating the substrate with an anti-PGCP antibody and then washing the substrate; And
3) A method for detecting a protein for providing information on diagnosis of dementia, comprising determining a subject having an increased degree of attachment of an anti-PGCP antibody on the substrate as a subject having or at risk of dementia.

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