KR20020007432A - Diagnostic Agent for Alzheimer's Disease Which Comprises Monoclonal Antibody against Melanoma Transferrin as an Active Ingredient - Google Patents
Diagnostic Agent for Alzheimer's Disease Which Comprises Monoclonal Antibody against Melanoma Transferrin as an Active Ingredient Download PDFInfo
- Publication number
- KR20020007432A KR20020007432A KR1020000040102A KR20000040102A KR20020007432A KR 20020007432 A KR20020007432 A KR 20020007432A KR 1020000040102 A KR1020000040102 A KR 1020000040102A KR 20000040102 A KR20000040102 A KR 20000040102A KR 20020007432 A KR20020007432 A KR 20020007432A
- Authority
- KR
- South Korea
- Prior art keywords
- alzheimer
- disease
- melanotransferrin
- monoclonal antibody
- patients
- Prior art date
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
- G01N33/6857—Antibody fragments
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5002—Partitioning blood components
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5047—Cells of the immune system
- G01N33/505—Cells of the immune system involving T-cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2814—Dementia; Cognitive disorders
- G01N2800/2821—Alzheimer
Abstract
Description
본 발명은 멜라노트랜스페린(melanotransferrin, p97)에 대한 단클론 항체를 유효성분으로 포함하는 알츠하이머병의 진단시약에 관한 것이다. 좀 더 구체적으로, 본 발명은 알츠하이머병에 특이적인 멜라노트랜스페린을 검출하여 알츠하이머병을 판별·진단하기 위한, 멜라노트랜스페린에 대한 단클론 항체를 유효성분으로 포함하는, 민감성·특이성·정확성이 향상된 진단시약에 관한 것이다.The present invention relates to a diagnostic reagent for Alzheimer's disease comprising a monoclonal antibody against melanotransferrin (p97) as an active ingredient. More specifically, the present invention relates to a diagnostic reagent having improved sensitivity, specificity, and accuracy, comprising a monoclonal antibody against melanotransferin as an active ingredient for detecting and diagnosing Alzheimer's disease by detecting melanotransferin specific to Alzheimer's disease. It is about.
알츠하이머병(Alzheimer's disease)은 뇌의 진행성 퇴행성 질환으로서, 전세계적으로 치매환자 중 70%의 상당한 부분을 차지하는데, 아밀로이드(amyloid) 소체를 둘러싼 축삭말단(telodentron) 및 수상돌기(dendrite)로 구성된 노인성판(senile plaque)으로 불리는 뚜렷한 병리조직학적 변화를 동반하며, 대뇌피질 전반에 걸친 미만성 위축(diffuse cerebral atrophy)과 세포내 신경원 섬유덩어리(neurofibrillary tangle)를 특징으로 한다. 특히, 알츠하이머병은 질환이 진행됨에 따라 신경세포의 다량 손실이 동반되지만 초기의 임상적인 변화를 관찰하기 어려우므로, 다른 질병보다도 진단을 통하여 병을 조기에 발견하는 것이 상당히 중요하다(참조: Nagy, Z.et al.,Dement. Geriatr. Cogn. Disord., 10, 109-114, 1999; Varma, A.R.et al.,J. Neurol. Neurosurg. Psychiatry, 66, 184-188, 1999).Alzheimer's disease is a progressive degenerative disease of the brain and accounts for a significant portion of 70% of dementia patients worldwide, with senile aging consisting of the axon terminal and dendrite surrounding the amyloid body. It is accompanied by a distinct pathological change called the senile plaque and is characterized by diffuse cerebral atrophy and intracellular neurofibrillary tangles throughout the cerebral cortex. In particular, Alzheimer's disease is accompanied by massive loss of nerve cells as the disease progresses, but it is difficult to observe early clinical changes, so it is more important to detect the disease early than other diseases (see Nagy, Z. et al ., Dement.Geriatr. Cogn. Disord ., 10, 109-114, 1999; Varma, AR et al ., J. Neurol. Neurosurg. Psychiatry , 66, 184-188, 1999).
현재까지 베타-아밀로이드단백질(Aβ, β-amyloid), 타우단백질(Tau), 아포리포단백질 E(ApoE, apolipoprotein E), α1-안티키모트립신(α1-antichymotrypsin) 및 케모카인(chemokine)이 알츠하이머병을 진단할 수 있는 물질로 제시되었으나(참조: Geldmacher, D.S.et al.,New Engl. J. Med.,335, 330-336, 1996), 이들은 알츠하이머병을 초기에 효과적으로 진단할 수 없는 단점이 있어, 알츠하이머병에 대한 생화학적 표지자 및 진단시약의 개발이 시급히 요구되었다.Current to the beta-amyloid protein (Aβ, β-amyloid), tau protein (Tau), apo lipoprotein E (ApoE, apolipoprotein E), α 1-anti-chymotrypsin (α 1 -antichymotrypsin) and chemokines (chemokine) Alzheimer's Although it has been suggested as a substance capable of diagnosing the disease (see Geldmacher, DS et al. , New Engl. J. Med., 335, 330-336, 1996), they are not able to effectively diagnose Alzheimer's disease at an early stage . Therefore, the development of biochemical markers and diagnostic reagents for Alzheimer's disease is urgently needed.
한편, 멜라노트랜스페린(melanotransferrin, p97)은 세포에서 고농도로 발견되고 철이온의 섭취(iron uptake) 및 철의 운반에 관여하며 97kDa의 크기를 가지며, 최근에는 뇌조직의 아밀로이드판(amyloid plaque)의 축적과 관련이 있다고 알려졌다. 이에, 알츠하이머병 환자의 뇌척수액(cerebrospinal fluid, CSF) 및 혈청에 대하여 멜라노트랜스페린에 대한 항체를 사용함으로써 알츠하이머병을 진단하고자 하는 시도가 있었으나(참조: Kennard et al.,Nature Med., 2, 1230-1235, 1996), 알츠하이머병 환자와 정상인의 뇌척수액에서의 멜라노트랜스페린의 양의 변화는 현저히 차이를 보였지만 선택성이 낮고, 더우기 혈청의 멜라노트랜스페린에 대한 민감성, 정확성이 낮으며, 알츠하이머병 환자의 뇌척수액이나 혈청에서조차 멜라노트랜스페린이 검출되지 않는 특이성의 문제점이 있어서, 효과적인 진단시약으로 사용될 수 없는 단점이 있었다.On the other hand, melanotransferrin (p97) is found in cells at high concentrations, is involved in iron uptake and iron transport, has a size of 97 kDa, and recently, the accumulation of amyloid plaque in brain tissue. It is said to be related. Therefore, there have been attempts to diagnose Alzheimer's disease by using antibodies against melanotransferrin in cerebrospinal fluid (CSF) and serum of Alzheimer's disease patients (Kennard et al., Nature Med. , 2, 1230-). 1235, 1996), but there was a significant difference in the amount of melanotransferin in cerebrospinal fluid between Alzheimer's disease patients and normal subjects, but the selectivity was low, and the serum sensitivity and accuracy of melanotransferrin was lower. Even in the melanotransferrin has a problem of specificity not detected, there was a disadvantage that can not be used as an effective diagnostic reagent.
이에, 본 발명자들은 알츠하이머병을 진단할 수 있는 효과적인 진단시약을 개발하고자 예의 연구 노력한 결과, 마우스의 T 세포와 사람의 암세포가 융합된 하이브리도마 세포주를 제조하는데 성공하였으며, 이로부터 멜라노트랜스페린에 대한 단클론 항체를 수득하여, 전기 수득된 항체를 유효성분으로 하는 진단시약으로 사용하여 혈청의 멜라노트랜스페린을 검출할 경우, 멜라노트랜스페린이 알츠하이머병 환자에서 민감성(sensitivity), 특이성(selectivity) 및 정확성(accuracy)이 높게 측정되어, 전기 멜라노트랜스페린에 대한 단클론 항체를 진단시약의 유효성분으로사용할 수 있음을 확인하고, 본 발명을 완성하게 되었다.Accordingly, the present inventors have made efforts to develop an effective diagnostic reagent for diagnosing Alzheimer's disease. As a result, the present inventors have succeeded in producing a hybridoma cell line in which a mouse T cell and a human cancer cell are fused. When monoclonal antibodies are obtained and the melanotransferrin in serum is detected using the previously obtained antibody as an active ingredient, melanotransferrin is sensitive, specific, and accurate in patients with Alzheimer's disease. This high measurement confirmed that the monoclonal antibody against melanotransferrin could be used as an active ingredient of a diagnostic reagent, thereby completing the present invention.
결국, 본 발명의 주된 목적은 멜라노트랜스페린에 대한 단클론 항체를 유효성분으로 포함하는 알츠하이머병의 진단시약을 제공하는 것이다.After all, the main object of the present invention is to provide a diagnostic reagent for Alzheimer's disease comprising a monoclonal antibody against melanotransferrin as an active ingredient.
도 1은 알츠하이머병 환자, 비알츠하이머 치매환자 또는 정상인의 혈청 멜라노트랜스페린의 농도를 비교한 그래프이다.1 is a graph comparing the concentration of serum melanotransferrin in patients with Alzheimer's disease, non-Alzheimer's dementia, or normal persons.
도 2는 알츠하이머병 환자의 혈청 멜라노트랜스페린의 농도 대 연령의 곡선을 나타낸 그래프이다.Figure 2 is a graph showing the curve of the concentration of serum melanotransferrin versus age in Alzheimer's disease patients.
도 3은 알츠하이머병 환자의 혈청 멜라노트랜스페린의 농도에 의한 ROC 커브를 나타낸 그래프이다.Figure 3 is a graph showing the ROC curve by the concentration of serum melanotransferrin in Alzheimer's disease patients.
이하, 본 발명의 멜라노트랜스페린에 대한 단클론 항체를 유효성분으로 포함하는 알츠하이머병의 진단시약을 구체적으로 설명하고자 한다.Hereinafter, a diagnostic reagent for Alzheimer's disease comprising a monoclonal antibody against melanotransferrin of the present invention as an active ingredient will be described in detail.
우선, 본 발명자들은 모세혈관내피(capillary endothelium)에서 발현되며, 알츠하이머병 환자 뇌조직의 소신경교세포(microglia)에 존재하는 아밀로이드판(amyloid plaque)내에 함께 나타나는 멜라노트랜스페린과 알츠하이머병과의 연관성을 알아보았다: 즉, 멜라노마 세포주로부터 수득한 97kDa의 멜라노트랜스페린과, 뇌손상, 알콜중독 및 의약품남용의 현상이 나타나지 않는 알츠하이머병 환자, 비알츠하이머-치매(non-Alzheimer's Disease dementia)의 일종인 혈관성 치매(vascular dementia)와 파킨슨병(Parkinson's disease) 환자 또는 정상인으로부터 수득된 혈청에 멜라노트랜스페린에 대한 단클론 항체와 반응시켜, 멜라노트랜스페린이 알츠하이머병 환자들에서만 고농도로 존재함을 확인하였다.First, the present inventors examined the association between melanotransferrin and Alzheimer's disease, which are expressed in capillary endothelium and coexist in amyloid plaques present in microglia of brain tissue of Alzheimer's disease patients. : In other words, 97kDa melanotransferin obtained from a melanoma cell line, vascular dementia, a type of non-Alzheimer's Disease dementia, a patient with Alzheimer's disease without brain injury, alcoholism and drug abuse Serum obtained from patients with dementia and Parkinson's disease or normal subjects was reacted with monoclonal antibodies against melanotransferin to confirm that melanotransferrin was present in high concentrations only in Alzheimer's disease patients.
이어, 초기와 후기 단계별 알츠하이머병 환자, 비알츠하이머-치매환자 또는 정상인을 대상으로 멜라노트랜스페린의 양을 상기 항체를 반응시켜 측정해 본 결과, 초기단계 및 후기단계의 알츠하이머병 환자에서의 멜라노트랜스페린의 수치에는 큰 차이를 나타내지 않았다. 이에 반하여, 비알츠하이머-치매환자는 정상인과 같은 수치를 나타냈는 바, 멜라노트랜스페린이 알츠하이머병에만 특이적으로 나타남을 확인할 수 있었다.In the early and late stages of Alzheimer's disease, non-Alzheimer's dementia patients or normal people, the amount of melanotransferin was measured by reacting the antibody, and as a result, the melanotransferin levels in early and late stage Alzheimer's disease patients. Did not show a significant difference. In contrast, non-Alzheimer's-dementia patients showed the same level as normal, indicating that melanotransferin was specific to Alzheimer's disease only.
아울러, 전기의 결과로부터 수득된 멜라노트랜스페린양의 수치로부터 ROC(receiver operating characteristic curve)를 작성한 결과, 놀라울 정도로 진단의 민감성, 특이성 및 정확성이 높게 확인되었다. 따라서, 본 발명의 멜라노트랜스페린에 대한 항체를 환자 또는 정상인으로부터 수득된 혈청에 처리하여, 멜라노트랜스페린에 대한 항체와 반응하는 멜라노트랜스페린의 증가된 양으로 알츠하이머병임을 판별할 수 있으므로, 혈청의 멜라노트랜스페린의 양을 측정할 수 있는 물질을 사용하여 알츠하이머병의 초기 발병을 특이적으로 진단할 수 있음을 확인할 수 있었다.In addition, as a result of creating a receiver operating characteristic curve (ROC) from the melanotransferrin amount obtained from the above results, the sensitivity, specificity and accuracy of the diagnosis were surprisingly high. Thus, the antibody against melanotransferrin of the present invention can be treated with serum obtained from a patient or a normal person to determine that it is Alzheimer's disease with an increased amount of melanotransferrin reacting with the antibody to melanotransferrin. It was confirmed that the early detection of Alzheimer's disease can be specifically diagnosed by using a quantity-quantifying substance.
본 발명의 알츠하이머병의 진단시약의 유효성분으로 포함되는 멜라노트랜스페린에 대한 항체는 단클론 항체, 다클론 항체를 사용할 수 있으며, 바람직하게는 마우스 T 세포 및 사람의 암세포주가 융합된 하이브리도마 세포로부터 분리된 단클론 항체를 사용할 수 있다. 예를 들어, 마우스 비장세포 및 사람의 멜라노마 세포주가 융합된 하이브리도마 세포주(ATCC HB8446)으로부터 수득된 단클론 항체를 사용할 수 있으나, 본 발명이 이에 한정되는 것은 아니다.The antibody against melanotransferrin included as an active ingredient of the diagnostic reagent of Alzheimer's disease of the present invention may be a monoclonal antibody or a polyclonal antibody, preferably isolated from hybridoma cells fused with mouse T cells and human cancer cell lines. Monoclonal antibodies can be used. For example, monoclonal antibodies obtained from a hybridoma cell line (ATCC HB8446) fused with mouse splenocytes and human melanoma cell lines may be used, but the present invention is not limited thereto.
이하, 실시예를 통하여 본 발명을 보다 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .
실시예 1: 알츠하이머병과 멜라노트랜스페린과의 상관관계 Example 1 Correlation between Alzheimer's Disease and Melanotransferin
알츠하이머병 환자, 혈관성 치매 환자, 파킨슨병 환자 또는 정상인의 그룹을 선발하기 위하여 진단학적인 측면에서 알츠하이머병의 척도는 맥칸 등의 방법(참조: McKhann , G.et al., Neurology, 34, 939-944, 1984)을 사용하였고, 비알츠하이머-치매인 혈관성 치매 및 파킨슨병의 척도는 각각 로만 등의 방법(참조: Romann, G.C.et al.,Neurology, 43, 250-260, 1993) 및 휴 등(참조: Hughes, A.J.et al., Neurol. Neurosurg. Psychiatry, 55, 181-184, 1992)의 방법이 사용되었다. 아울러, 전기 환자 또는 정상인들은 전기 치매이외에 다른 치매의 가능성을 배제시키는 조건하에서, 뇌의 MRI(brain magnetic resonance imaging) 및 뇌파검사법(electroencephalography)의 신경정신적 테스트가 수행되었으며, 뇌손상(head trauma), 알콜중독 및 의약품 남용의 병력이 없는 알츠하이머병 환자 71명, 비알츠하이머-치매환자 56명 및 정상인 84명 그룹이 선발되었다. 전기 선발된 환자 또는 정상인의 혈액을 채취한 다음, 혈청 및 DNA를 수득하여 4℃에 보관하면서 24시간이내에 사용하였다. 대조군으로는 사람 멜라노마 세포주(SK-MEL 28, ATCC, U.S.A.)로부터 멜라노트랜스페린을 분리하여 사용하였다. 사람 멜라노마 세포주는 56℃에서 30분간 열로 불활성화된 소의 자궁내에 있는 송아지혈청(fetalbovine serum), 100mU/㎖의 포타슘 페니실린G, 2mM 글루타민 및 20mM 중탄산나트륨이 첨가된 둘베코의 변형된 배지(Dulbecco's modified essential medium, DMEM)로 포화시켜 75cm3의 플라스크내에서 37℃, 5%의 이산화탄소와 95%의 습도조건의 배양기에서 3일동안 배양하였다. 하기 표 1은 진단하려는 환자의 그룹과 상태를 나타낸다.Patients with Alzheimer's disease, vascular dementia, Parkinson's disease, or normal For screening, in terms of diagnosis, Alzheimer's disease is measured by McCann et al. (McKhann, G.et al., Neurology, 34, 939-944, 1984), and measures of vascular dementia and Parkinson's disease, which are non-Alzheimer's dementia, were measured by Roman et al. (Romann, G.C.et al.,Neurology, 43, 250-260, 1993) and Hugh et al. (Hughes, A.J.et al., Neurol. Neurosurg. Psychiatry, 55, 181-184, 1992). In addition, electrical patients or normal people underwent neuropsychological tests of brain magnetic resonance imaging (MRI) and electroencephalography under conditions that exclude the possibility of dementia other than electrical dementia, brain trauma, A group of 71 patients with Alzheimer's disease, 56 patients with non-Alzheimer's dementia and 84 healthy subjects were selected without a history of alcoholism and drug abuse. Blood was collected from the electroselected patients or normal subjects, and serum and DNA were obtained and used within 24 hours while stored at 4 ° C. Melanotransferrin was isolated from human melanoma cell lines (SK-MEL 28, ATCC, U.S.A.) as a control. Human melanoma cell line Dulbecco's modified medium (Dulbecco's) with fetal bovine serum, 100 mM / ml potassium penicillin G, 2 mM glutamine and 20 mM sodium bicarbonate added to heat inactivated bovine for 30 minutes at 56 ° C. saturated essential medium (DMEM)3The flask was incubated for 3 days in an incubator at 37 ° C., 5% carbon dioxide and 95% humidity. Table 1 below shows the groups and conditions of patients to be diagnosed.
* : 임상적인 치매 확률(Clinical Dementia Rating)*: Clinical Dementia Rating
†: 신경상태검사(Mini-mental State Examination)의 한국형†: Korean version of Mini-mental State Examination
전기 수득한 각각의 혈청 1㎕과 대조군인 사람 멜라노마 세포에서 분리된 멜라노트랜스페린을 나이트로셀룰로스막(nitrocellulose membrane, 이하 'NC'라 함)에 적하하고, 완전히 건조시켰다. 이어, 건조된 NC를 상온에서 1시간 동안 차단용액(blocking solution)에 적시고, 멜라노트랜스페린에 대한 단클론 항체인L235(ATCC-HB 8446 L235(H-19))로 4℃에서 밤새 반응시킨 후, 0.01%의 트윈-20(Tween 20)이 용해된 PBS로 3회 세척하고, 이차항체로 상온에서 1시간동안 반응시켰다. 이어, 0.01% 트윈-20이 용해된 PBS로 3회 이상 세척하고, 퍼옥시다제 기질 용액(peroxidase substrate solution, LumiGLO, New England Biolabs,Inc.)을 사용하여 상온에서 1분동안 반응시킨 후, X-레이 필름상에서 1분동안 노출시켜, 멜라노트랜스페린의 양을 대조군과 비교하였다.Melanotransferrin isolated from 1 μl of each obtained serum and human melanoma cells as a control group was added dropwise to a nitrocellulose membrane (hereinafter referred to as 'NC') and completely dried. Subsequently, the dried NC was soaked in a blocking solution for 1 hour at room temperature, and reacted overnight at 4 ° C. with L235 (ATCC-HB 8446 L235 (H-19)), a monoclonal antibody to melanotransferin, and then 0.01 Tween 20 was washed three times with dissolved PBS and reacted with a secondary antibody at room temperature for 1 hour. Subsequently, washed three times with 0.01% Tween-20 dissolved PBS and reacted at room temperature for 1 minute using a peroxidase substrate solution (LumiGLO, New England Biolabs, Inc.), followed by X Exposed for 1 minute on the ray film, the amount of melanotransferin was compared to the control.
도 1은 알츠하이머병 환자, 비알츠하이머 치매환자 또는 정상인의 혈청 멜라노트랜스페린의 농도를 비교한 그래프이다. 도 1에서 보듯이, 멜라노트랜스페린은 알츠하이머병 환자들의 경우 10.20 내지 17.00pg/㎕(중앙값: 15.00pg/㎕)로 나타났는데 비하여, 비알츠하이머-치매환자의 경우 1.93 내지 7.15pg/㎕(중앙값: 2.85pg/㎕), 정상인의 경우 2.55 내지 3.95pg/㎕(중앙값: 3.20pg/㎕)로 나타났는 바, 알츠하이머병 환자는 비알츠하이머-치매환자 또는 정상인과는 멜라노트랜스페린 수치에서 상당한 차이를 보이며, 알츠하이머병 환자의 경우에 멜라노트랜스페린의 수치가 3배 내지 5배 이상 높은 것을 확인하였다.1 is a graph comparing the concentration of serum melanotransferrin in patients with Alzheimer's disease, non-Alzheimer's dementia, or normal persons. As shown in FIG. 1, melanotransferin was found to be 10.20 to 17.00 pg / μl (median: 15.00 pg / μl) in Alzheimer's disease patients, compared to 1.93 to 7.15 pg / μl in non-Alzheimer's dementia patients (median: 2.85). pg / μl), and 2.55-3.95pg / μl (median: 3.20pg / μl) for normal subjects, and patients with Alzheimer's disease show significant differences in melanotransferin levels from non-Alzheimer-demented patients or normal subjects. In the case of sick patients, it was confirmed that the level of melanotransferin was 3 to 5 times higher.
아울러, 전기 환자와 정상과는 연령 차이가 있으므로, 연령 차이가 전기 결과에 영향을 줄 수 있는지 여부를 확인하고자, 50세 이상의 정상인과 가장 나이가 적은 환자에 대해서도 동일하게 멜라노트랜스페린의 수치를 측정하였다.In addition, since there is an age difference between the previous patient and the normal, in order to determine whether the age difference may affect the electrical results, melanotransferrin levels were measured in the same manner for the normal persons 50 years or older and the youngest patients.
도 2는 알츠하이머병 환자의 혈청 멜라노트랜스페린의 농도 대 연령의 곡선을 나타낸 그래프이다. 도 2에서 보듯이, 멜라노트랜스페린의 양과 연령에는 상관관계가 없음을 확인하였고, 성별 차이 역시 이러한 결과에 영향을 주지 못함을 확인하였다.Figure 2 is a graph showing the curve of the concentration of serum melanotransferrin versus age in Alzheimer's disease patients. As shown in Figure 2, it was confirmed that there is no correlation between the amount and age of the melanotransferrin, it was confirmed that gender differences do not affect these results.
실시예 2: 초기 및 후기단계별 알츠하이머병에서 멜라노트랜스페린의 양적 변화 Example 2 Quantitative Changes of Melanotransferrin in Early and Late Stage Alzheimer's Disease
임상적인 치매확률(CDR)이 0.5 또는 1.0인 초기단계의 알츠하이머병 환자 38명, 임상적인 치매확률이 2.0 또는 3.0인 후기단계의 알츠하이머병 환자 33명 및 비알츠하이머-치매환자와 정상인 140명을 대상으로 수득한 혈청에 대하여, 멜라노트랜스페린의 양을 전기 실시예 1과 동일한 방법으로 수행하였다.Thirty eight patients with early-stage Alzheimer's disease with a clinical dementia probability of 0.5 or 1.0, 33 patients with late-stage Alzheimer's disease with a clinical dementia probability of 2.0 or 3.0, and 140 non-Alzheimer-demented patients For the serum obtained by, the amount of melanotransferrin was carried out in the same manner as in Example 1 above.
그 결과, 초기단계의 알츠하이머병 환자들은 멜라노트랜스페린이 10.20 내지 17.00pg/㎕(중앙값: 14.45pg/㎕)이며, 후기 알츠하이머병 환자 33명의 경우에는 멜라노트랜스페린이 12.05 내지 17.05pg/㎕(중앙값: 15.00pg/㎕)로서 초기단계 및 후기단계는 차이를 보이지 않았다. 그에 비해, 비알츠하이머-치매환자와 정상인의 멜라노트랜스페린의 수치는 2.10 내지 4.50pg/㎕(중앙값: 3.15pg/㎕)을 나타났는 바, 알츠하이머병의 발병초기의 멜라노트랜스페린의 양은 알츠하이머병이 진행되는 과정에서 더 이상 증가하지 않음을 확인하였다.As a result, patients with early stage Alzheimer's disease had melanotransferin between 10.20 and 17.00 pg / μl (median: 14.45 pg / μl), and those with 33 patients with late Alzheimer's disease had melanotransferrin between 12.05 and 17.05 pg / μl (median: 15.00 pg / μl) did not show any difference between the early and late stages. In contrast, the levels of melanotransferin in non-Alzheimer-demented patients and normal subjects ranged from 2.10 to 4.50 pg / μl (median: 3.15 pg / μl), suggesting that the amount of melanotransferrin in the early stage of Alzheimer's disease is associated with Alzheimer's disease. It was confirmed that no further increase in the process.
실시예 3: 멜라노트랜스페린에 대한 항체를 이용한 알츠하이머병의 진단 Example 3 Diagnosis of Alzheimer's Disease Using Antibodies to Melanotransferin
전기 실시예 1 및 2의 혈청의 멜라노트랜스페린의 수치로부터 x축을 위양성율(위양성율=1-특이성)로 하고, y축을 민감성(sensitivity)으로 하여 ROC 곡선(receiver operating characteristic curve)를 작성하였다. 아울러, 통계분석에는 SAS 소프트웨어 (version 6.12)가 사용되었다.From the numerical values of melanotransferrin in the serum of Examples 1 and 2 described above, a ROC curve was prepared with the x-axis as the false positive rate (false positive rate = 1-specificity) and the y axis as the sensitivity. In addition, SAS software (version 6.12) was used for statistical analysis.
도 3은 알츠하이머병 환자의 혈청 멜라노트랜스페린의 농도에 의한 ROC 커브를 나타낸 그래프이다: 이때, 그래프 상에서 상부의 점일 수록 민감성이 크고, 좌측의 점일수록 위양성율이 감소되어 특이성이 증가함을 나타내며, 점선은 멜라노트랜스페린의 양이 역치(cut-off level)임을 나타낸다. 도 3에서 보듯이, 멜라노트랜스페린의 양이 10.0pg/㎕일 때, 알츠하이머병으로 진단할 수 있는 민감성, 특이성 및 정확성은 각각 0.915, 0.914 및 0.915로 나타났으며, 50세 이상의 환자나 정상인으로부터 알츠하이머병을 진단할 수 있는 민감성, 특이성 및 정확성은 각각 0.901, 0.888 및 0.894임을 알 수 있었다. 아울러, 알츠하이머병 환자와 비알츠하이머-치매 환자를 구별할 수 있는 멜라노트랜스페린의 양도 10.0pg/㎕이었으며, 이때의 민감성, 특이성 및 정확성은 각각 0.901, 0.857 및 0.882로 나타났다. 또한, 알츠하이머병 환자와 50세 이상의 정상인을 구별할 수 있는 멜라노트랜스페린의 양은 9.0pg/㎕로 나타났고, 이때 0.915, 0.917 및 0.916의 민감성, 특이성 및 정확성을 가짐을 확인하였다.Figure 3 is a graph showing the ROC curve by the concentration of serum melanotransferrin in patients with Alzheimer's disease: where the upper point is more sensitive, the left point is false positive rate is increased, the specificity is increased, the dotted line is The amount of melanotransferin is the cut-off level. As shown in FIG. 3, when the amount of melanotransferin was 10.0 pg / μl, the sensitivity, specificity, and accuracy that can be diagnosed with Alzheimer's disease were 0.915, 0.914, and 0.915, respectively. The sensitivity, specificity, and accuracy for diagnosing the disease were 0.901, 0.888, and 0.894, respectively. In addition, the amount of melanotransferin that can distinguish between Alzheimer's disease and non-Alzheimer's-dementia patients was 10.0 pg / μl, and the sensitivity, specificity, and accuracy were 0.901, 0.857, and 0.882, respectively. In addition, the amount of melanotransferin that can distinguish patients with Alzheimer's disease and normal people over 50 was 9.0 pg / μl, and it was confirmed that the sensitivity, specificity, and accuracy of 0.915, 0.917, and 0.916.
상술한 바와 같이, 알츠하이머병 환자를 다른 비알츠하이머-치매환자와 정상인으로부터 구별할 수 있는 멜라노트랜스페린의 역치값은 10pg/㎕이며, 멜라노트랜스페린에 대한 항체를 사용하여 알츠하이머병을 진단할 경우 민감성·특이성·정확성이 높게 나타났는 바, 본 발명의 멜라노트랜스페린에 대한 단클론 항체를 진단시약의 유효성분으로 이용하여 환자로부터 수득된 혈청에 처리할 경우 알츠하이머병을 특이적으로 진단할 수 있음을 확인하였다.As described above, the threshold value of melanotransferin, which can distinguish Alzheimer's disease patients from other non-Alzheimer-dementia patients, is 10 pg / μl, and the sensitivity and specificity when diagnosing Alzheimer's disease using an antibody against melanotransferrin As the accuracy was high, it was confirmed that Alzheimer's disease can be specifically diagnosed when the monoclonal antibody against melanotransferrin of the present invention is used as an active ingredient of a diagnostic reagent and treated with serum obtained from a patient.
이상에서 상세히 설명하고 입증하였듯이, 본 발명은 멜라노트랜스페린(melanotransferrin, p97)에 대한 단클론 항체를 유효성분으로 포함하는 알츠하이머병의 진단시약을 제공한다. 본 발명의 알츠하이머병의 진단시약은 마우스의 T 세포와 사람의 암세포가 융합된 하이브리도마 세포주로부터 분리된 멜라노트랜스페린에 대한 단클론 항체를 유효성분으로 하는 바, 환자로부터 수득된 혈청에 처리되어 면역반응으로 검출된 멜라노트랜스페린의 양을 정상인의 그것과 비교하고, 정상인에 비하여 증가된 멜라노트랜스페린의 양을 기준으로 알츠하이머병임을 판별·진단할 수 있으며, 민감성·특이성·정확성이 높아 특히 진단이 어려운 초기단계의 알츠하이머병의 진단에 유용하다.As described and demonstrated in detail above, the present invention provides a diagnostic reagent for Alzheimer's disease comprising a monoclonal antibody against melanotransferrin (p97) as an active ingredient. The diagnostic reagent of Alzheimer's disease of the present invention is a monoclonal antibody against melanotransferrin isolated from a hybridoma cell line in which T cells of a mouse and human cancer cells are fused. Compared with those of normal people, the amount of melanotransferrin detected by the control group can be compared with that of normal people, and it is possible to discriminate and diagnose Alzheimer's disease based on the increased amount of melanotransferin compared to normal people, and it is particularly difficult to diagnose due to its high sensitivity, specificity, and accuracy. Useful for the diagnosis of Alzheimer's disease.
Claims (3)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020000040102A KR20020007432A (en) | 2000-07-13 | 2000-07-13 | Diagnostic Agent for Alzheimer's Disease Which Comprises Monoclonal Antibody against Melanoma Transferrin as an Active Ingredient |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020000040102A KR20020007432A (en) | 2000-07-13 | 2000-07-13 | Diagnostic Agent for Alzheimer's Disease Which Comprises Monoclonal Antibody against Melanoma Transferrin as an Active Ingredient |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| KR20020007432A true KR20020007432A (en) | 2002-01-29 |
Family
ID=19677759
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020000040102A KR20020007432A (en) | 2000-07-13 | 2000-07-13 | Diagnostic Agent for Alzheimer's Disease Which Comprises Monoclonal Antibody against Melanoma Transferrin as an Active Ingredient |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR20020007432A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101038346B1 (en) * | 2010-06-23 | 2011-05-31 | (주)해바람에너지 | Electronic breaking apparatus using armature reaction of wind power generator |
| KR101311717B1 (en) * | 2011-07-05 | 2013-09-25 | 한국과학기술연구원 | Protein marker melanotransferrin for colon cancer diagnosis and diagnosis kit for colon cancer using antibodies against the same |
| EP3189079A4 (en) * | 2014-09-05 | 2018-08-08 | Abbvie Stemcentrx LLC | Novel anti-mfi2 antibodies and methods of use |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5578451A (en) * | 1993-02-18 | 1996-11-26 | The General Hospital Corporation | Methods and systems for screening potential alzheimer's disease therapeutics |
| WO1997037228A1 (en) * | 1996-03-29 | 1997-10-09 | The Trustees Of Boston University | Methods for diagnosing and treating alzheimer's disease |
| JPH1169981A (en) * | 1996-12-14 | 1999-03-16 | Smithkline Beecham Corp | Aspi |
-
2000
- 2000-07-13 KR KR1020000040102A patent/KR20020007432A/en not_active Application Discontinuation
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5578451A (en) * | 1993-02-18 | 1996-11-26 | The General Hospital Corporation | Methods and systems for screening potential alzheimer's disease therapeutics |
| WO1997037228A1 (en) * | 1996-03-29 | 1997-10-09 | The Trustees Of Boston University | Methods for diagnosing and treating alzheimer's disease |
| JPH1169981A (en) * | 1996-12-14 | 1999-03-16 | Smithkline Beecham Corp | Aspi |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101038346B1 (en) * | 2010-06-23 | 2011-05-31 | (주)해바람에너지 | Electronic breaking apparatus using armature reaction of wind power generator |
| KR101311717B1 (en) * | 2011-07-05 | 2013-09-25 | 한국과학기술연구원 | Protein marker melanotransferrin for colon cancer diagnosis and diagnosis kit for colon cancer using antibodies against the same |
| EP3189079A4 (en) * | 2014-09-05 | 2018-08-08 | Abbvie Stemcentrx LLC | Novel anti-mfi2 antibodies and methods of use |
| US10428156B2 (en) | 2014-09-05 | 2019-10-01 | Abbvie Stemcentrx Llc | Anti-MFI2 antibodies and methods of use |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2230372C (en) | Quantitation of p97 to diagnose and monitor alzheimer's disease | |
| Hampel et al. | Core biological marker candidates of Alzheimer’s disease–perspectives for diagnosis, prediction of outcome and reflection of biological activity | |
| US6811988B2 (en) | Compositions and methods for diagnosing Alzheimer's disease | |
| US5270165A (en) | Method of diagnosis of amyloidoses | |
| Southwick et al. | Assessment of amyloid β protein in cerebrospinal fluid as an aid in the diagnosis of Alzheimer's disease | |
| JP4908514B2 (en) | Change in ERK1 / ERK2 phosphorylation ratio specific for Alzheimer's disease-Molecular biomarker specific for Alzheimer's disease (ADSMB) | |
| Ghanbari et al. | Biochemical assay for AD7C‐NTP in urine as an Alzheimer's disease marker | |
| Bush et al. | An abnormality of plasma amyloid protein precursor in Alzheimer's disease | |
| WO2005011599A2 (en) | Antibodies specific for toxic amyloid beta protein oligomers | |
| JP2002519702A (en) | Differential diagnosis of neurodegeneration | |
| Frölich et al. | Integrity of the blood-CSF barrier in dementia of Alzheimer type: CSF/serum ratios of albumin and IgG | |
| US9377472B2 (en) | Biological complex specific for Alzheimer's disease detection in vitro and use thereof | |
| US20080076140A1 (en) | Biomarkers of Alzheimer's Disease | |
| US20020172676A1 (en) | Method of treatment of alzheimer's disease and device therefor | |
| KR20020007432A (en) | Diagnostic Agent for Alzheimer's Disease Which Comprises Monoclonal Antibody against Melanoma Transferrin as an Active Ingredient | |
| JP3752219B2 (en) | Identification method of dementia disease | |
| JP2010511159A (en) | Method for diagnosis and early diagnosis of neurodegenerative diseases in vitro | |
| US9618522B2 (en) | Diagnostic testing in dementia and methods related thereto | |
| US20220308073A1 (en) | Biomarker for alzheimer's disease | |
| Mehta | Amyloid beta and tau proteins in Alzheimer’s disease and Down syndrome | |
| KR20230148764A (en) | Diagnostic composition of Alzheimer's disease or mild cognitive impairment comprising hydrolase and diagnostic method using the same | |
| KR20240002810A (en) | Method for Early Diagnosis and Staging of Alzheimer's Disease and Screening Cerebral Amyloid Deposition Using Phosphorylated Peptide Derived from Alzheimer's Disease Related Protein | |
| JP2024064487A (en) | Diagnostic markers for dementia | |
| KR20200077779A (en) | A composition for predicting a risk of neurodegenerative diseases and a method for predicting neurodegenerative diseases using the same | |
| MXPA98001641A (en) | Quantification of p97 to diagnose and monitor alzhei's disease |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A201 | Request for examination | ||
| E902 | Notification of reason for refusal | ||
| E601 | Decision to refuse application |