JP4264469B2 - Testing method for dementia - Google Patents
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- JP4264469B2 JP4264469B2 JP2003043452A JP2003043452A JP4264469B2 JP 4264469 B2 JP4264469 B2 JP 4264469B2 JP 2003043452 A JP2003043452 A JP 2003043452A JP 2003043452 A JP2003043452 A JP 2003043452A JP 4264469 B2 JP4264469 B2 JP 4264469B2
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- Prior art keywords
- annexin
- dementia
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Description
【0001】
【発明の属する技術分野】
本発明は臨床検査の分野で用いられ、とりわけ痴呆症の検査方法として用いられる。
【0002】
【従来の技術】
アルツハイマー病(AD)はおもに老年期に発病する進行性の神経変性疾患で認知機能障害を特徴とする。ADにおける痴呆症の程度はAD患者の大脳皮質に見出される老人班(SP)の数に関連づけられてきた (例えば非特許文献1)。そして組織学的には、SPは退化性神経突起及び活性の星状膠細胞に囲まれ、また生化学的には、SPはβアミロイドと呼ばれる小分子の不溶性繊維状タンパク質を含んでいる(例えば非特許文献2) 。ADの原因は不明であり、ADの治療法も乏しいのが現状である。ADの診断は診断基準によるところの除外診断および脳の画像診断を主とし、それ以外の客観的方法はほとんどない。すなわち、発症前或いは発症後の患者におけるAD鑑別診断のための簡便な検査法は存在しない。特許文献1によれば脳脊髄液中の分子量33000の蛋白質を免疫学的に測定する方法が開示されているが、侵襲性の高い脳脊髄液を用いなければならないといった問題がある。脳血管障害及びAD患者の脳において数種のアネキシンが多量に発現している報告もある(例えば、非特許文献3)。さらにアルコール依存症の患者の脳にはアネキシンIVが正常群よりも多く発現していることも報告されている(非特許文献4)。以上の例はすべて死後患者から採取された脳脊髄液や脳組織の分析が行われているが、これらは診断治療に用いることができる方法ではない。非特許文献4に示されているように、アルコール依存症患者において血中の抗アネキシンIV抗体量がアルコール依存症の診断に有効であるという報告は、簡便な検査方法といえる。しかしながら、前にも述べたようにADの簡便な診断検査の方法は実用化されておらず、侵襲性が低くかつ簡単に患者より採取できる血液等の体液を用いるADの簡便な検査方法の開発が望まれている。
【0003】
【特許文献1】
特開平7−253429号。
【特許文献2】
特開平7−72147号。
【非特許文献1】
Nature、1966、209巻、109頁。
【非特許文献2】
Biochem Biophys
Res Commun. 120巻、885-890頁(1984)
【非特許文献3】
Am J Pathol,
145, 640-649
(1994)
【非特許文献4】
Alcohol Clin
Exp Res, 25, 55S-58S (2001)
【0004】
【発明が解決しようとする課題】
本発明の課題は痴呆症の検査方法であって、血液等の簡単に採取できる体液を用いて簡便に検査できる方法を提供することである。
【0005】
【課題を決するための手段】
本発明者らは、痴呆症の簡便な検査方法を鋭意研究した結果、血液中のアネキシン類、とりわけアネキシンVの濃度が痴呆の発症と深く関連することから簡便な痴呆症の検査が可能であることを見出し、本発明を完成させた。
【0006】
本発明は以下の構成からなる。
1、血液中のアネキシンV濃度を測定することを特徴とする痴呆症の検査方法。
2、血液が血漿である前記1に記載の方法。
3、前記1〜2に記載の方法を用いる試薬又はキット。
【0007】
【発明の実施の形態】
本発明は、痴呆症の検査に関するものであり、体液中のアネキシン類、とりわけ
アネキシンVの濃度を測定することによる痴呆症の検査方法に関するものである。本発明は血液、より好ましくは血漿中のアネキシンVを特異的な抗体(抗体全分子又は抗体活性を有する部分断片を含む)を用いて免疫学的に測定することにより実施される。用いる抗体は、ポリクローナル抗体及びモノクローナル抗体のいずれも用いることが可能であり、特に限定されるものではない。免疫学的測定法は、EIA、RIA、ラテックス凝集反応、免疫沈降法、ウエスタンブロット、ドットブロット、イムノクロマト法等公知の方法が適用される。アネキシンV測定のためのEIA法を例にとれば、固相に固定化された被検試料中のアネキシンVを反応させ、さらに酵素標識した抗アネキシンV抗体を反応させ、固相に捕獲された標識抗体の量を検出することにより試料中のアネキシンVの濃度を測定することができる。
【0008】
またアネキシンVの生物学的性質を利用して、例えばリン脂質、好ましくはホスファチジルセリンを固定化した固相に被検試料中のアネキシンVを捕獲した後、標識抗アネキシンV抗体を反応させ、その反応量を検出することによっても測定できる。
【0009】
本発明は、血液中のアネキシンVと他のアネキシン類、例えばアネキシンIVの濃度を組み合わせて検査してもよい。例えば両者の比を算出すること、両方の測定値について、それぞれ閾値を設定しておき、その閾値を両方が超える、どちらか片方が超える、どちらも超えない等の判定結果から、その診断的有用性を検討することにより行われる。さらにアネキシンVとアネキシンIVの測定は、血液中のアネキシンVが測定されていれば、必ずしも同一被検試料である必要はなく、例えば血清と血漿、脳脊髄液と血漿等の異なる試料の組合せでも可能である。
【0010】
アネキシン分子種は、痴呆やアルコール依存症などの神経精神疾患で発現変化することが報告されている(例えば、非特許文献2、3)。前述のようにアネキシンVとアネキシンIVを組み合わせて用いることにより、アネキシンV濃度の上昇が痴呆症によるものか、あるいは他の疾患によるものかを的確に鑑別できる。逆にアネキシンIVの上昇がアルコール依存性か否かを検証するのにもアネキシンVとの組み合わせが有効である。以上のように、アネキシンV濃度及びアネキシンIV濃度を組み合わせて検査することにより特異的な痴呆症の検査が期待できる。
【0011】
アネキシンVとアネキシンIVの測定を組み合わせて行う方法は、前述のようにそれぞれ個々に構築された免疫学的手法、例えばEIA、ラテックス比濁法等を用いてそれぞれの濃度を測定することはもちろんのことであるが、それ以外にも、それぞれの抗体(そのフラグメントを含む)をひとつの反応容器に固定化しておき、そして被検試料を反応させ、ひとつの反応容器内にアネキシンVとアネキシンIVの2種類の分子を捕獲し、それらを、それぞれの分子に対する抗体(そのフラグメントを含む)を異なる標識物質で標識した標識抗体で検出することによる方法も可能である。さらにはイムノクロマト法を利用して、同時に2種類の抗原を検出することも可能である(図1)。
【0012】
【実施例】
本発明の実施例を以下に示すが、本発明は実施例に限定されるものではない。
【実施例1】
(アネキシンV濃度測定用ELISAの構築)
ヒト胎盤より精製したアネキシンVを免疫原としてマウスモノクローナル抗体を調製した。調製したモノクローナル抗体(クローンNo.23)を96穴のELISA用マイクロタイタープレートに固相化し、抗アネキシンV抗体固相プレートを調製した。固相プレートに用いたクローンと異なるモノクローナル抗体(クローンNo.49)をビオチン標識して標識抗体を調製した。これらを組み合わせてアネキシンV測定用ELISA試薬を構築した。
【0013】
【実施例2】
(ウエスタンブロットでの検討)
痴呆患者11名(アルツハイマー病患者:男性3名、女性6名、脳血管性痴呆患者:女性2名)及びコントロール10名(男性5名、女性5名)の血漿中のアネキシンをウエスタンブロットで検出した。各患者は予め長谷川式簡易知能評価スケールにて知能得点を調べた。各血漿1mLをPBS緩衝液で10倍に希釈し、ホスファチジルセリンとホスファチジルコリンの混合比を1:9(モル比)のリポソーム100μgを加え、さらに終濃度で6mM塩化カルシウムを加え15分間インキュベートした。その後遠心操作により沈殿画分を集め、洗浄後SDS−PAGEを行い、PVDF膜に転写して、ウサギ由来の抗アネキシンV抗体でウエスタンブロットを行い、血液中のアネキシンVの存在と痴呆の関連を調べた。コントロール群ではアネキシンVのいずれも検出されなかったのに対して、痴呆症ではアネキシンVは11例中10例(感度90.9%)において検出され血液中のアネキシンVの検出又は測定が痴呆症の検査に有用であることがわかった。
【0014】
【実施例3】
(酵素免疫測定法での検討)
実施例2で検討した検体中のアネキシンV濃度を実施例1で調製したELISA法で測定した。その結果を表1に示した。
【0015】
【表1】
【0016】
ELISA法で測定において、痴呆症の患者血漿中のアネキシンV濃度はコントロール群に比べて有意に高い値を示し、血漿中のアネキシンV濃度の測定が痴呆症の検査に有用であることがわかった。
【0017】
【発明の効果】
本発明は、血液を用いて痴呆症の検査を簡単に行うことができる方法を提供する。
【0018】
【図面の簡単な説明】
【図1】 イムノクロマト法による本発明の実施の1例図である。
【図2】 ウエスタンブロット法によるアネキシンVの検出結果である。
【符号の説明】
1、試料添加部
2、標識抗体保持部
3、展開部
4、抗アネキシンV抗体(抗アネキシンV抗体)固定部
5、抗アネキシンIV抗体(抗アネキシンV抗体)抗体固定部
6、吸収パッド[0001]
BACKGROUND OF THE INVENTION
The present invention is used in the field of clinical examination, and in particular as a method for examining dementia.
[0002]
[Prior art]
Alzheimer's disease (AD) is primarily a cognitive dysfunction characterized by progressive neurodegenerative disease onset in old age. The degree of dementia in AD has been linked to the number of senile plaques (SP) found in the cerebral cortex of AD patients (eg, Non-Patent Document 1). And histologically, SP is surrounded by degenerative neurites and active astrocytes, and biochemically, SP contains a small insoluble fibrous protein called β-amyloid (eg, Non-patent document 2). The cause of AD is unknown, and there are currently few treatments for AD. Diagnosis of AD mainly includes exclusion diagnosis based on diagnostic criteria and brain image diagnosis, and there are few other objective methods . That is, there is no simple test method for differential AD diagnosis in patients before onset or after onset. Although
[0003]
[Patent Document 1]
JP-A-7-253429.
[Patent Document 2]
JP-A-7-72147.
[Non-Patent Document 1]
Nature, 1966, 209, 109.
[Non-Patent Document 2]
Biochem Biophys
Res Commun. 120, 885-890 (1984)
[Non-Patent Document 3]
Am J Pathol,
145, 640-649
(1994)
[Non-Patent Document 4]
Alcohol Clin
Exp Res, 25, 55S-58S (2001)
[0004]
[Problems to be solved by the invention]
An object of the present invention is to provide a method for testing dementia, which can be easily tested using body fluids such as blood that can be easily collected.
[0005]
[Means for determining the problem]
As a result of intensive studies on a simple test method for dementia, the present inventors can easily test for dementia because the concentration of annexins in blood, especially annexin V, is closely related to the onset of dementia. As a result , the present invention has been completed.
[0006]
The present invention has the following configuration.
1. A method for examining dementia, comprising measuring annexin V concentration in blood .
2. The method according to 1 above, wherein the blood is plasma .
3. A reagent or kit using the method according to 1-2 above.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a test for dementia, and annexins in body fluids, especially
The present invention relates to a test method for dementia by measuring the concentration of annexin V. The present invention is carried out by immunologically measuring annexin V in blood, more preferably plasma, using specific antibodies (including whole antibodies or partial fragments having antibody activity). As the antibody to be used, either a polyclonal antibody or a monoclonal antibody can be used, and it is not particularly limited. As the immunological measurement method, known methods such as EIA, RIA, latex agglutination, immunoprecipitation, Western blot, dot blot, and immunochromatography are applied. Taking the EIA method for annexin V measurement as an example, annexin V in a test sample immobilized on a solid phase was reacted, and an enzyme-labeled anti-annexin V antibody was further reacted to be captured on the solid phase. By detecting the amount of labeled antibody, the concentration of annexin V in the sample can be measured.
[0008]
Further, using the biological properties of annexin V, for example, after capturing annexin V in a test sample on a solid phase on which phospholipid, preferably phosphatidylserine is immobilized, the labeled anti-annexin V antibody is reacted, It can also be measured by detecting the reaction amount.
[0009]
The present invention may be tested by combining the concentrations of Annexin V in the blood and other annexins such as Annexin IV. For example, calculating the ratio between the two, setting a threshold value for both measured values, and determining the diagnostic usefulness based on the determination result that both threshold values are exceeded, either one is exceeded, or neither is exceeded It is done by examining sex. Further, the measurement of annexin V and annexin IV is not necessarily the same test sample as long as the annexin V in the blood is measured . For example, a combination of different samples such as serum and plasma, cerebrospinal fluid and plasma, etc. Is possible.
[0010]
Annexin molecular species have been reported to change in expression in neuropsychiatric diseases such as dementia and alcoholism (for example, Non-
[0011]
The method of performing the measurement of annexin V and annexin IV in combination is of course the measurement of each concentration using an immunological technique constructed individually as described above, such as EIA, latex turbidimetry, etc. In addition to this, each antibody (including its fragment) is immobilized in one reaction vessel, and the test sample is allowed to react, and annexin V and annexin IV are contained in one reaction vessel. A method is also possible by capturing two kinds of molecules and detecting them with a labeled antibody in which an antibody against each molecule (including a fragment thereof) is labeled with a different labeling substance. Furthermore, it is also possible to detect two types of antigens simultaneously using an immunochromatography method (FIG. 1).
[0012]
【Example】
Examples of the present invention are shown below, but the present invention is not limited to the examples.
[Example 1]
(Construction of an annexin V concentration measurement ELISA)
A mouse monoclonal antibody was prepared using annexin V purified from human placenta as an immunogen. The prepared monoclonal antibody (clone No. 23) was immobilized on a 96-well ELISA microtiter plate to prepare an anti-annexin V antibody solid phase plate. A monoclonal antibody (clone No. 49) different from the clone used for the solid phase plate was labeled with biotin to prepare a labeled antibody. These were combined to construct an annexin V assay ELISA reagent.
[0013]
[Example 2]
(Examination by Western blot)
Annexin detection in plasma of 11 patients with dementia (Alzheimer's disease: 3 males, 6 females, cerebrovascular dementia: 2 females) and 10 controls (5 males, 5 females) by Western blot did. Each patient examined the intelligence score in advance on the Hasegawa simple intelligence evaluation scale. 1 mL of each plasma was diluted 10-fold with PBS buffer, 100 μg of liposome having a mixing ratio of phosphatidylserine and phosphatidylcholine of 1: 9 (molar ratio) was added, 6 mM calcium chloride was further added at a final concentration, and the mixture was incubated for 15 minutes. Thereafter, the precipitate fraction was collected by centrifugation, washed and then subjected to SDS-PAGE, transferred to a PVDF membrane , Western blotted with a rabbit-derived anti-annexin V antibody, and the relationship between the presence of annexin V in the blood and dementia was observed. Examined. None of the annexin V was detected in the control group, whereas in dementia, annexin V was detected in 10 of 11 cases (sensitivity 90.9%), and detection or measurement of annexin V in the blood was dementia It was found to be useful for the examination.
[0014]
[Example 3]
(Examination with enzyme immunoassay)
The annexin V concentration in the specimen examined in Example 2 was measured by the ELISA method prepared in Example 1. The results are shown in Table 1.
[0015]
[Table 1]
[0016]
In the measurement by ELISA method, the annexin V concentration in the plasma of patients with dementia was significantly higher than that in the control group, and it was found that the measurement of the annexin V concentration in plasma was useful for testing dementia. .
[0017]
【The invention's effect】
The present invention provides a method capable of easily performing a test for dementia using blood.
[0018]
[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is an example of an embodiment of the present invention by immunochromatography.
FIG. 2 shows annexin V detection results by Western blotting.
[Explanation of symbols]
1,
Claims (2)
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