JP7495106B2 - Methods for detecting cancer and neurodegenerative diseases - Google Patents
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Description
本発明は、対象におけるがん及び神経変性疾患の検出方法及び診断キットに関する。より詳しくは、対象から採取された血液試料中の細胞外小胞の量を指標としてがん及び神経変性疾患を検出する方法等に関する。 The present invention relates to a method and a diagnostic kit for detecting cancer and neurodegenerative diseases in a subject. More specifically, the present invention relates to a method for detecting cancer and neurodegenerative diseases using the amount of extracellular vesicles in a blood sample collected from a subject as an indicator.
血中に出現する疾患特異的な細胞外小胞（エクソソーム）の検出による診断技術が開発されている。例えば、非特許文献１には、血中のCD147/CD9陽性エクソソームの検出によ
る大腸がんの診断技術が報告されている。また、非特許文献２は、前立腺特異的エクソソームを報告している。
Diagnostic techniques have been developed that detect disease-specific extracellular vesicles (exosomes) that appear in the blood. For example, Non-Patent Document 1 reports a diagnostic technique for colon cancer by detecting CD147/CD9-positive exosomes in the blood. Non-Patent Document 2 also reports prostate-specific exosomes.
本発明は、血中エクソソームの検出に基づく新規診断技術を提供することを主な目的とする。 The main objective of the present invention is to provide a new diagnostic technology based on the detection of exosomes in blood.
上記課題解決のため、本発明は、以下の［１］－［１３］を提供する。
［１］ 対象におけるがん又は神経変性疾患を検出する方法であって、
対象から採取された血液試料中の、Ｔｉｍ４結合性であり、かつ、ＣＤ９、ＣＤ４１、ＣＤ６１及びＣＤ６３からなる群から選択されるいずれか１以上が陽性である細胞外小胞の量を測定する手順と、
細胞外小胞量が所定の閾値以上である場合に、対象におけるがん又は神経変性疾患を検出する手順と、
を含む方法。
［２］ 前記細胞外小胞は、ＣＤ９、ＣＤ４１、ＣＤ６１及びＣＤ６３がいずれも陽性である、［１］の方法。
［３］ 前記神経変性疾患が、アルツハイマー病、うつ又は統合失調症である、［１］又は［２］の方法。
［４］ 前記がんが、結腸がん、膀胱がん、乳がん、非小細胞肺がん、胃がん、膵管腺がん、非ホジキンリンパ腫、子宮頸がん、直腸腺がん、肝細胞がん、甲状腺がん、前立腺がん、子宮体がん、食道がん、卵巣がん、胆管がん、膵がん、黒色腫及び慢性骨髄性白血病からなる群から選択される１以上である、［１］又は［２］の方法。
In order to solve the above problems, the present invention provides the following [1] to [13].
[1] A method for detecting cancer or a neurodegenerative disease in a subject, comprising:
Measuring the amount of extracellular vesicles that bind to Tim4 and are positive for any one or more selected from the group consisting of CD9, CD41, CD61, and CD63 in a blood sample collected from a subject;
Detecting cancer or a neurodegenerative disease in a subject when the amount of extracellular vesicles is equal to or greater than a predetermined threshold;
The method includes:
[2] The method according to [1], wherein the extracellular vesicles are positive for CD9, CD41, CD61 and CD63.
[3] The method according to [1] or [2], wherein the neurodegenerative disease is Alzheimer's disease, depression or schizophrenia.
[4] The method of [1] or [2], wherein the cancer is one or more selected from the group consisting of colon cancer, bladder cancer, breast cancer, non-small cell lung cancer, gastric cancer, pancreatic ductal adenocarcinoma, non-Hodgkin's lymphoma, cervical cancer, rectal adenocarcinoma, hepatocellular carcinoma, thyroid cancer, prostate cancer, uterine cancer, esophageal cancer, ovarian cancer, bile duct cancer, pancreatic cancer, melanoma, and chronic myeloid leukemia.
［５］ 対象におけるがん又は神経変性疾患を検出する方法であって、
対象から採取された血液試料を、第一のプローブが固相化された支持体表面に接触させる手順と、
第二のプローブを、支持体表面に接触させる手順と、
第一のプローブと第二のプローブを含んでなる複合体の量を測定する手順と、
測定値が所定の閾値以上である場合に、対象におけるがん又は神経変性を検出する手順と、を含み、
第一のプローブ及び第二のプローブが、Ｔｉｍ４、抗ＣＤ９抗体、抗ＣＤ４１抗体、抗ＣＤ６１抗体、抗ＣＤ６３抗体、ＴｘＬＣ１レクチン及びＤＳＡレクチンからなる群よりそれぞれ選択される、方法。
［６］ 前記神経変性疾患が、アルツハイマー病、うつ又は統合失調症である、［５］の方法。
［７］ 前記がんが、結腸がん、膀胱がん、乳がん、非小細胞肺がん、胃がん、膵管腺がん、非ホジキンリンパ腫、子宮頸がん、直腸腺がん、肝細胞がん、甲状腺がん、前立腺がん、子宮体がん、食道がん、卵巣がん、胆管がん、膵がん、黒色腫及び慢性骨髄性白血病からなる群から選択される１以上である、［５］又は［６］の方法。
[5] A method for detecting cancer or a neurodegenerative disease in a subject, comprising:
A step of contacting a blood sample collected from a subject with a support surface having a first probe immobilized thereon;
contacting a second probe with the support surface;
measuring the amount of a complex comprising the first probe and the second probe;
detecting cancer or neurodegeneration in the subject if the measured value is equal to or greater than a predetermined threshold;
The method, wherein the first probe and the second probe are each selected from the group consisting of Tim4, anti-CD9 antibody, anti-CD41 antibody, anti-CD61 antibody, anti-CD63 antibody, TxLC1 lectin and DSA lectin.
[6] The method according to [5], wherein the neurodegenerative disease is Alzheimer's disease, depression or schizophrenia.
[7] The method of [5] or [6], wherein the cancer is one or more selected from the group consisting of colon cancer, bladder cancer, breast cancer, non-small cell lung cancer, gastric cancer, pancreatic ductal adenocarcinoma, non-Hodgkin's lymphoma, cervical cancer, rectal adenocarcinoma, hepatocellular carcinoma, thyroid cancer, prostate cancer, uterine cancer, esophageal cancer, ovarian cancer, bile duct cancer, pancreatic cancer, melanoma, and chronic myeloid leukemia.
［８］ がん又は神経変性疾患の診断キットであって、
支持体表面に固相化される第一のプローブと、
第二のプローブと、を含んでなり、
第一のプローブ及び第二のプローブが、Ｔｉｍ４、抗ＣＤ９抗体、抗ＣＤ４１抗体、抗ＣＤ６１抗体、抗ＣＤ６３抗体、ＴｘＬＣ１レクチン及びＤＳＡレクチンからなる群よりそれぞれ選択される、キット。
［９］ 前記神経変性疾患が、アルツハイマー病、うつ又は統合失調症である、［５］のキット。
［１０］ 前記がんが、結腸がん、膀胱がん、乳がん、非小細胞肺がん、胃がん、膵管腺がん、非ホジキンリンパ腫、子宮頸がん、直腸腺がん、肝細胞がん、甲状腺がん、前立腺がん、子宮体がん、食道がん、卵巣がん、胆管がん、膵がん、黒色腫及び慢性骨髄性白血病からなる群から選択される１以上である、［５］又は［６］のキット。
[8] A diagnostic kit for cancer or a neurodegenerative disease, comprising:
A first probe immobilized on a support surface;
a second probe,
A kit, wherein the first probe and the second probe are each selected from the group consisting of Tim4, anti-CD9 antibody, anti-CD41 antibody, anti-CD61 antibody, anti-CD63 antibody, TxLC1 lectin, and DSA lectin.
[9] The kit according to [5], wherein the neurodegenerative disease is Alzheimer's disease, depression, or schizophrenia.
[10] The kit according to [5] or [6], wherein the cancer is one or more selected from the group consisting of colon cancer, bladder cancer, breast cancer, non-small cell lung cancer, gastric cancer, pancreatic ductal adenocarcinoma, non-Hodgkin's lymphoma, cervical cancer, rectal adenocarcinoma, hepatocellular carcinoma, thyroid cancer, prostate cancer, uterine cancer, esophageal cancer, ovarian cancer, bile duct cancer, pancreatic cancer, melanoma, and chronic myeloid leukemia.
［１１］ Ｔｉｍ４結合性であり、かつ、ＣＤ９、ＣＤ４１、ＣＤ６１及びＣＤ６３からなる群から選択されるいずれか１以上が陽性である細胞外小胞の、対象におけるがん又は神経変性疾患を検出するための使用。
［１２］ Ｔｉｍ４、抗ＣＤ９抗体、抗ＣＤ４１抗体、抗ＣＤ６１抗体、抗ＣＤ６３抗体、ＴｘＬＣ１レクチン又はＤＳＡレクチンの、対象におけるがん又は神経変性疾患を検出するための使用。
［１３］ Ｔｉｍ４、抗ＣＤ９抗体、抗ＣＤ４１抗体、抗ＣＤ６１抗体、抗ＣＤ６３抗体、ＴｘＬＣ１レクチン又はＤＳＡレクチンの、対象におけるがん又は神経変性疾患の診断キットの製造のための使用。
[11] Use of extracellular vesicles that are Tim4-binding and positive for any one or more selected from the group consisting of CD9, CD41, CD61, and CD63, for detecting cancer or a neurodegenerative disease in a subject.
[12] Use of Tim4, anti-CD9 antibody, anti-CD41 antibody, anti-CD61 antibody, anti-CD63 antibody, TxLC1 lectin or DSA lectin for detecting cancer or a neurodegenerative disease in a subject.
[13] Use of Tim4, an anti-CD9 antibody, an anti-CD41 antibody, an anti-CD61 antibody, an anti-CD63 antibody, TxLC1 lectin, or DSA lectin for the manufacture of a diagnostic kit for cancer or a neurodegenerative disease in a subject.
本発明により、血中エクソソームの検出に基づく新規診断技術が提供される。 The present invention provides a new diagnostic technique based on the detection of exosomes in blood.
以下、本発明を実施するための好適な形態について説明する。なお、以下に説明する実施形態は、本発明の代表的な実施形態の一例を示したものであり、これにより本発明の範囲が狭く解釈されることはない。 The following describes a preferred embodiment of the present invention. Note that the embodiment described below is an example of a typical embodiment of the present invention, and the scope of the present invention should not be interpreted narrowly based on this.
１．がん又は神経変性疾患の検出方法
本発明に係るがん又は神経変性疾患の検出方法は、以下の手順（１）及び（２）を含む。
（１）対象から採取された血液試料中の、Ｔｉｍ４結合性であり、かつ、ＣＤ９、ＣＤ４１、ＣＤ６１及びＣＤ６３からなる群から選択されるいずれか１以上が陽性である細胞外小胞の量を測定する手順。
（２）細胞外小胞量が所定の閾値以上である場合に、対象におけるがん又は神経変性疾患を検出する手順。
以下、細胞外小胞を「エクソソーム」とも称し、Ｔｉｍ４結合性であり、かつ、ＣＤ９、ＣＤ４１、ＣＤ６１及びＣＤ６３からなる群から選択されるいずれか１以上が陽性である細胞外小胞を「マーカーエクソソーム」とも称する。
1. Method for detecting cancer or neurodegenerative disease The method for detecting cancer or neurodegenerative disease according to the present invention includes the following steps (1) and (2).
(1) A procedure for measuring the amount of extracellular vesicles in a blood sample taken from a subject that are Tim4-binding and positive for one or more selected from the group consisting of CD9, CD41, CD61, and CD63.
(2) A procedure for detecting cancer or a neurodegenerative disease in a subject when the amount of extracellular vesicles is equal to or greater than a predetermined threshold.
Hereinafter, extracellular vesicles are also referred to as "exosomes", and extracellular vesicles that are Tim4-binding and positive for one or more selected from the group consisting of CD9, CD41, CD61, and CD63 are also referred to as "marker exosomes".
１－１．測定手順
対象は、特に限定されず、ヒトを含む哺乳類あるいは鳥類であり、好ましくはヒトである。
血液試料は、全血、血清及び血漿であってよく、好ましくは血清である。
1-1. Measurement Procedure The subject is not particularly limited and may be a mammal, including a human, or a bird, preferably a human.
The blood sample may be whole blood, serum or plasma, preferably serum.
マーカーエクソソームの量の測定は、血液試料からエクソソームを分離した後に行っても、分離を行うことなく行ってもよい。エクソソームの分離は、従来公知の手法によって行えばよく、例えばエクソソーム結合性分子を固相化した磁気ビーズを用いた手法を採用できる。エクソソーム結合性分子を固相化した磁気ビーズとしては、Ｔｉｍ４（T-cell immunoglobulin domain and mucin domain-containing protein 4）を固相化した磁気ビーズが市販されている（MagCaptureTM Exosome Isolation Kit PS、富士フイルム和光純薬
、293-77601）。磁気ビーズには、エクソソーム結合性分子として、Ｔｉｍ４に替えて、抗ＣＤ９抗体、抗ＣＤ４１抗体、抗ＣＤ６１抗体、抗ＣＤ６３抗体、ＴｘＬＣ１レクチン及びＤＳＡレクチンのいずれか１以上を固相化してもよい。
The amount of marker exosomes may be measured after exosomes are separated from a blood sample, or may be measured without separation. Separation of exosomes may be performed by a conventional method, for example, a method using magnetic beads on which exosome-binding molecules are immobilized can be adopted. As magnetic beads on which exosome-binding molecules are immobilized, magnetic beads on which Tim4 (T-cell immunoglobulin domain and mucin domain-containing protein 4) is immobilized are commercially available (MagCapture TM Exosome Isolation Kit PS, Fujifilm Wako Pure Chemical Industries, 293-77601). On the magnetic beads, one or more of anti-CD9 antibody, anti-CD41 antibody, anti-CD61 antibody, anti-CD63 antibody, TxLC1 lectin, and DSA lectin may be immobilized as an exosome-binding molecule instead of Tim4.
マーカーエクソソームの量の測定は、従来公知の手法によって行えばよく、例えばウェスタンブロット、ＥＬＩＳＡ及びフローサイトメトリー等を採用できる。
ＥＬＩＳＡは、一般的に以下の手順によって行うことができる。
（Ａ）対象から採取された血液試料を、第一のプローブ（捕捉プローブ）が固相化された支持体表面に接触させ、捕捉プローブとマーカーエクソソームを含んでなる複合体を形成させる手順。
（Ｂ）第二のプローブ（検出プローブ）を、支持体表面に接触させて、捕捉プローブとマーカーエクソソームと検出プローブとを含んでなる複合体を形成させる手順。
（Ｃ）第一のプローブとマーカーエクソソームと第二のプローブとを含んでなる複合体の量を測定する手順。
The amount of marker exosomes may be measured by a conventionally known method, for example, Western blot, ELISA, flow cytometry, etc.
ELISA can generally be performed by the following procedure.
(A) A procedure in which a blood sample collected from a subject is contacted with a support surface on which a first probe (capture probe) is immobilized, and a complex comprising the capture probe and a marker exosome is formed.
(B) A procedure in which a second probe (detection probe) is contacted with the surface of the support to form a complex comprising the capture probe, marker exosomes, and the detection probe.
(C) A procedure for measuring the amount of a complex comprising a first probe, a marker exosome, and a second probe.
本発明において、捕捉プローブには、Ｔｉｍ４、抗ＣＤ９抗体、抗ＣＤ４１抗体、抗ＣＤ６１抗体、抗ＣＤ６３抗体、ＴｘＬＣ１レクチン及びＤＳＡレクチンから選択されるいずれか一以上が用いられる。また、検出プローブにも、Ｔｉｍ４、抗ＣＤ９抗体、抗ＣＤ４１抗体、抗ＣＤ６１抗体、抗ＣＤ６３抗体、ＴｘＬＣ１レクチン（配列番号１）及びＤＳＡレクチン（配列番号２）から選択されるいずれか一以上を用いることができる。
捕捉プローブと検出プローブには、互いに異なるものを用いることが好ましい。
特に限定されないが、例えば、捕捉プローブにＴｉｍ４を、検出プローブに抗ＣＤ９抗体、抗ＣＤ４１抗体、抗ＣＤ６１抗体及び抗ＣＤ６３抗体のいずれかを用いることができる。あるいは、捕捉プローブに抗ＣＤ４１抗体を、検出プローブにＴｘＬＣ１レクチン及びＤＳＡレクチンを用いることができる。
In the present invention, the capture probe may be any one or more selected from Tim4, anti-CD9 antibody, anti-CD41 antibody, anti-CD61 antibody, anti-CD63 antibody, TxLC1 lectin, and DSA lectin. The detection probe may be any one or more selected from Tim4, anti-CD9 antibody, anti-CD41 antibody, anti-CD61 antibody, anti-CD63 antibody, TxLC1 lectin (SEQ ID NO: 1), and DSA lectin (SEQ ID NO: 2).
It is preferable that the capture probe and the detection probe are different from each other.
For example, but not limited to, Tim4 can be used as the capture probe, and any one of anti-CD9 antibody, anti-CD41 antibody, anti-CD61 antibody, and anti-CD63 antibody can be used as the detection probe. Alternatively, anti-CD41 antibody can be used as the capture probe, and TxLC1 lectin and DSA lectin can be used as the detection probe.
捕捉プローブが固相化される支持体は、特に限定されないが、プラスチック製のマイクロウェルプレートが好適に用いられ得る。捕捉プローブを固相化したマイクロウェルプレートとして、Ｔｉｍ４を固相化したマイクロウェルプレートが市販されている（Tim4-Fc固定化プレート、富士フイルム和光純薬）。 The support on which the capture probe is immobilized is not particularly limited, but a plastic microwell plate can be suitably used. As a microwell plate on which the capture probe is immobilized, a microwell plate on which Tim4 is immobilized is commercially available (Tim4-Fc immobilized plate, Fujifilm Wako Pure Chemical Industries).
血液試料を支持体表面に接触させ、捕捉プローブとマーカーエクソソームを含んでなる複合体を形成させる。その後、必要に応じて支持体表面を洗浄した後、検出プローブを支持体表面に接触させ、捕捉プローブとマーカーエクソソームと検出プローブとを含んでなる複合体を形成させる。この後、必要に応じて支持体表面の洗浄をさらに行ってもよい。 The blood sample is brought into contact with the support surface to form a complex containing the capture probe and the marker exosomes. Thereafter, the support surface is washed as necessary, and then the detection probe is brought into contact with the support surface to form a complex containing the capture probe, the marker exosomes, and the detection probe. After this, the support surface may be further washed as necessary.
捕捉プローブとマーカーエクソソームと検出プローブとを含んでなる複合体の量は、検出プローブに標識された物質からの信号に基づいて光学的に測定することができる。あるいは、検出プローブが抗体である場合には、複合体中の検出抗体にさらに２次抗体を結合させて、この２次抗体に標識された物質からの信号に基づいて、複合体の量を測定してもよい。
標識物質には、従来公知の酵素や蛍光タンパク質を用いればよく、例えばペルオキシダーゼを用いることができる。
また、２次抗体には、検出プローブに用いられる抗体の由来動物種に応じて、当該動物種のイムノグロブリンに対する抗体が用いられる。
The amount of the complex comprising the capture probe, the marker exosome, and the detection probe can be optically measured based on a signal from a substance labeled with the detection probe. Alternatively, when the detection probe is an antibody, a secondary antibody may be further bound to the detection antibody in the complex, and the amount of the complex may be measured based on a signal from a substance labeled with the secondary antibody.
The labeling substance may be a conventionally known enzyme or fluorescent protein, for example, peroxidase.
As the secondary antibody, an antibody against the immunoglobulin of the animal species from which the antibody used in the detection probe is derived is used according to the animal species.
１－２．検出手順
捕捉プローブとマーカーエクソソームと検出プローブとを含んでなる複合体の量（すなわち、マーカーエクソソームの量）が所定の閾値以上である場合、対象におけるがん又は神経変性疾患が検出される。
マーカーエクソソームは、Ｔｉｍ４結合性であり、かつ、ＣＤ９、ＣＤ４１、ＣＤ６１及びＣＤ６３からなる群から選択されるいずれか１以上が陽性である。マーカーエクソソームは、ＣＤ９、ＣＤ４１、ＣＤ６１及びＣＤ６３からなる群から選択されるいずれか２以上が陽性であることが好ましく、より好ましくは３以上、さらに好ましくは全てが陽性である。
1-2. Detection Procedure When the amount of a complex comprising a capture probe, a marker exosome, and a detection probe (i.e., the amount of marker exosome) is equal to or greater than a predetermined threshold, cancer or a neurodegenerative disease in a subject is detected.
The marker exosome is Tim4-binding and positive for any one or more selected from the group consisting of CD9, CD41, CD61, and CD63. The marker exosome is preferably positive for any two or more selected from the group consisting of CD9, CD41, CD61, and CD63, more preferably positive for three or more, and even more preferably positive for all.
神経変性疾患は、アルツハイマー病、うつ又は統合失調症であり、睡眠障害、双極性障害、てんかん、発達障害、PTSD、摂食障害及び適応障害が含まれ得る。神経変性疾患は、特に好ましくはアルツハイマー病である。 The neurodegenerative disease is Alzheimer's disease, depression or schizophrenia, and may include sleep disorders, bipolar disorder, epilepsy, developmental disorders, PTSD, eating disorders and adjustment disorders. The neurodegenerative disease is particularly preferably Alzheimer's disease.
がんは、結腸がん、膀胱がん、乳がん、非小細胞肺がん、胃がん、膵管腺がん、非ホジキンリンパ腫、子宮頸がん、直腸腺がん、肝細胞がん、甲状腺がん、前立腺がん、子宮体がん、食道がん、卵巣がん、胆管がん、膵がん、黒色腫及び慢性骨髄性白血病からなる群から選択される１以上である。 The cancer is one or more selected from the group consisting of colon cancer, bladder cancer, breast cancer, non-small cell lung cancer, gastric cancer, pancreatic ductal adenocarcinoma, non-Hodgkin's lymphoma, cervical cancer, rectal adenocarcinoma, hepatocellular carcinoma, thyroid cancer, prostate cancer, uterine cancer, esophageal cancer, ovarian cancer, bile duct cancer, pancreatic cancer, melanoma, and chronic myeloid leukemia.
閾値は、血液試料のマーカーエクソソームの量に基づいて健常者と患者とを区別可能な値として、検出系毎及び具体的な疾患毎に適宜設定され得るものである。
例えば閾値は、健常者の血液試料中のマーカーエクソソーム量の平均値、中央値又は最頻値の１．５倍、１．８倍、２倍、３倍、４倍、５倍、好ましくは６倍、７倍、８倍、９倍、１０倍、より好ましくは２０倍、３０倍、４０倍、５０倍である。あるいは、閾値は、健常者の血液試料中のマーカーエクソソーム量の最大値、又はその１．１倍、１．２倍、１．３倍、１．４倍、１．５倍、２倍、好ましくは３倍、４倍、５倍、６倍、７倍、より好ましくは８倍、９倍、１０倍、２０倍である。
より具体的には、検出系として捕捉プローブにＴｉｍ４を、検出プローブに抗ＣＤ６３抗体を用いたＥＬＩＳＡ（Ｔｉｍ４－抗ＣＤ６３抗体サンドイッチＥＬＩＳＡ）を用いる場合、健常者のマーカーエクソソームの量は545-2800 ng/mLであり、がん又は神経変性疾患のマーカーエクソソームの量はいずれも4400 ng/mL以上を示した。したがって、閾値は、例えば健常者の最大値の１．５倍である4200 ng/ml、好ましくは１．８倍である5040 ng/ml、より好ましくは２倍である5600 ng/mlに設定され得る。
検出系として捕捉プローブにＴｉｍ４を、検出プローブに抗ＣＤ９抗体を用いたＥＬＩＳＡ（Ｔｉｍ４－抗ＣＤ９抗体サンドイッチＥＬＩＳＡ）を用いる場合、健常者のマーカーエクソソームの量は20,100 ng/mL未満であった。したがって、閾値は、例えば20,100 ng/mLである。
The threshold value can be set appropriately for each detection system and each specific disease as a value capable of distinguishing between healthy individuals and patients based on the amount of marker exosomes in a blood sample.
For example, the threshold value is 1.5 times, 1.8 times, 2 times, 3 times, 4 times, 5 times, preferably 6 times, 7 times, 8 times, 9 times, 10 times, more preferably 20 times, 30 times, 40 times, 50 times the average, median, or mode of the amount of marker exosomes in blood samples from healthy individuals. Alternatively, the threshold value is the maximum amount of marker exosomes in blood samples from healthy individuals, or 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, preferably 3 times, 4 times, 5 times, 6 times, 7 times, more preferably 8 times, 9 times, 10 times, 20 times.
More specifically, when an ELISA (Tim4-anti-CD63 antibody sandwich ELISA) using Tim4 as a capture probe and an anti-CD63 antibody as a detection probe was used as a detection system, the amount of marker exosomes in healthy subjects was 545-2800 ng/mL, while the amount of marker exosomes in cancer or neurodegenerative disease was 4400 ng/mL or more. Therefore, the threshold value can be set to, for example, 4200 ng/ml, which is 1.5 times the maximum value in healthy subjects, preferably 5040 ng/ml, which is 1.8 times, and more preferably 5600 ng/ml, which is twice the maximum value.
When an ELISA (Tim4-anti-CD9 antibody sandwich ELISA) using Tim4 as a capture probe and an anti-CD9 antibody as a detection probe was used as a detection system, the amount of marker exosomes in healthy individuals was less than 20,100 ng/mL. Therefore, the threshold value is, for example, 20,100 ng/mL.
また、検出系として捕捉プローブにＴｉｍ４を、検出プローブに抗ＣＤ４１抗体を用いたＥＬＩＳＡ（Ｔｉｍ４－抗ＣＤ４１抗体サンドイッチＥＬＩＳＡ）を用いた場合、ＥＬＩＳＡでの吸光強度（OD450/620）は、健常者で0.24未満であった。したがって、閾値は、例えば吸光強度で0.24である。
検出系として捕捉プローブにＴｉｍ４を、検出プローブに抗ＣＤ６１抗体を用いたＥＬＩＳＡ（Ｔｉｍ４－抗ＣＤ６１抗体サンドイッチＥＬＩＳＡ）を用いた場合、ＥＬＩＳＡでの吸光強度（OD450/620）は、健常者で0.4未満であった。したがって、閾値は、例えば吸光強度で0.4である。
検出系として捕捉プローブに抗ＣＤ４１抗体を、検出プローブにＴｘＬＣ１レクチンを用いたＥＬＩＳＡ（抗ＣＤ４１抗体－ＴｘＬＣ１レクチンサンドイッチＥＬＩＳＡ）を用いた場合、ＥＬＩＳＡでの吸光強度（OD450/620）は、健常者で0.18未満であった。したがって、閾値は、例えば吸光強度で0.18である。
検出系として捕捉プローブに抗ＣＤ４１抗体を、検出プローブにＤＳＡレクチンを用いたＥＬＩＳＡ（抗ＣＤ４１抗体－ＤＳＡレクチンサンドイッチＥＬＩＳＡ）を用いた場合、ＥＬＩＳＡでの吸光強度（OD450/620）は、健常者で0.06未満であった。したがって、閾値は、例えば吸光強度で0.06である。
In addition, when an ELISA (Tim4-anti-CD41 antibody sandwich ELISA) was used as a detection system using Tim4 as a capture probe and an anti-CD41 antibody as a detection probe, the absorbance intensity (OD450/620) in the ELISA was less than 0.24 in healthy subjects. Therefore, the threshold value is, for example, 0.24 in terms of absorbance intensity.
When an ELISA (Tim4-anti-CD61 antibody sandwich ELISA) was used as a detection system using Tim4 as a capture probe and an anti-CD61 antibody as a detection probe, the absorbance intensity (OD450/620) in the ELISA was less than 0.4 in healthy subjects. Therefore, the threshold value is, for example, 0.4 in terms of absorbance intensity.
When an ELISA (anti-CD41 antibody-TxLC1 lectin sandwich ELISA) was used as a detection system using an anti-CD41 antibody as a capture probe and TxLC1 lectin as a detection probe, the absorbance intensity (OD450/620) in the ELISA was less than 0.18 in healthy subjects. Therefore, the threshold value is, for example, 0.18 in terms of absorbance intensity.
When an ELISA (anti-CD41 antibody-DSA lectin sandwich ELISA) was used as a detection system using an anti-CD41 antibody as a capture probe and DSA lectin as a detection probe, the absorbance intensity (OD450/620) in the ELISA was less than 0.06 in healthy subjects. Therefore, the threshold value is, for example, 0.06 in terms of absorbance intensity.
２．がん又は神経変性疾患の診断キット
本発明は、上述した、第一のプローブ（捕捉プローブ）と、第二のプローブ（検出プローブ）と、を含んでなるがん又は神経変性疾患の診断キットをも提供する。
本発明に係る診断キットには、捕捉プローブと検出プローブに加えて、捕捉プローブが固相化される支持体、検出プローブ（抗体）に結合させる標識２次抗体及び標識物質（酵素）の基質などが含まれていてもよい。
2. Diagnostic Kit for Cancer or Neurodegenerative Disease The present invention also provides a diagnostic kit for cancer or neurodegenerative disease, comprising the first probe (capture probe) and the second probe (detection probe) described above.
In addition to the capture probe and detection probe, the diagnostic kit of the present invention may also include a support on which the capture probe is immobilized, a labeled secondary antibody to be bound to the detection probe (antibody), and a substrate for the labeling substance (enzyme).
１．材料と方法
［血清および細胞外小胞］
健常者及び患者の血清は、BioreclamationIVTから購入した。
T-cell immunoglobulin domain and mucin domain-containing protein 4（Tim4）タンパク質を固相化した磁気ビーズを用いて、血清から細胞外小胞を分離した。細胞外小胞の分離は、キット（MagCaptureTM Exosome Isolation Kit PS、富士フイルム和光純薬、293-77601）に添付の指示書に従って行った。分離した細胞外小胞は、吸着防止剤（Exocap Ultracentrifugation Storage Booster、MBLサイエンス、MEK-USB）を添加して-80℃で保存した。
1. Materials and Methods [Serum and Extracellular Vesicles]
Sera from healthy subjects and patients were purchased from BioreclamationIVT.
Extracellular vesicles were isolated from serum using magnetic beads immobilized with T-cell immunoglobulin domain and mucin domain-containing protein 4 (Tim4). The isolation of extracellular vesicles was performed according to the instructions attached to the kit (MagCapture TM Exosome Isolation Kit PS, Fujifilm Wako Pure Chemical Industries, Ltd., 293-77601). The isolated extracellular vesicles were stored at -80°C with the addition of an anti-adsorption agent (Exocap Ultracentrifugation Storage Booster, MBL Science, MEK-USB).
［フローサイトメトリー］
細胞外小胞を結合したTim4磁気ビーズを、それぞれ蛍光標識した抗ＣＤ９抗体（Invitrogen、10626D）、抗ＣＤ４１抗体（R&D、MAB7616）、抗ＣＤ６１抗体（R&D、MAB2266）及び抗ＣＤ６３抗体（富士フイルム和光純薬、297-79201、又は、コスモバイオ、SHI-EXO-MO2）で染色し、フローサイトメトリー（CytoFLEX, Beckman）により検出した。フローサイトメトリーによる検出は、市販のキット（PS Captur Exosome Flow Cytometry Kit, 富士フイルム和光純薬）を用いて、キット添付の指示書に従って行った。
[Flow cytometry]
The Tim4 magnetic beads bound to extracellular vesicles were stained with fluorescently labeled anti-CD9 antibody (Invitrogen, 10626D), anti-CD41 antibody (R&D, MAB7616), anti-CD61 antibody (R&D, MAB2266), and anti-CD63 antibody (Fujifilm Wako Pure Chemical, 297-79201, or Cosmo Bio, SHI-EXO-MO2), and detected by flow cytometry (CytoFLEX, Beckman). Detection by flow cytometry was performed using a commercially available kit (PS Captur Exosome Flow Cytometry Kit, Fujifilm Wako Pure Chemical) according to the instructions attached to the kit.
［ウェスタンブロット］
細胞外小胞0.15 μg/laneを5-20%グラジエントゲル（DRC、NTH-671HP）で電気泳動後、PVDF膜（BIO-RAD、162-0176）にブロットした。１次抗体を４℃一晩、さらにペルオキシダーゼ標識２次抗体を室温１時間反応させた後、Western Lighting Plus（PerkinElmer、NEL104001EA）を用いて検出を行った。
[Western Blot]
Extracellular vesicles (0.15 μg/lane) were electrophoresed on a 5-20% gradient gel (DRC, NTH-671HP) and then blotted onto a PVDF membrane (BIO-RAD, 162-0176). The primary antibody was incubated overnight at 4°C, and the peroxidase-labeled secondary antibody was incubated for 1 hour at room temperature, after which detection was performed using Western Lighting Plus (PerkinElmer, NEL104001EA).
［Ｔｉｍ４－抗体サンドイッチＥＬＩＳＡ］
1%BSAを添加した反応/洗浄液（富士フイルム和光純薬）で100倍希釈した血清100 μL/wellをTim4-Fc固定化プレート（富士フイルム和光純薬）にアプライした。約500 rpmで攪拌しながら室温2時間反応させた。
反応/洗浄液で洗浄後、1%BSAを添加した反応/洗浄液で希釈した１次抗体を100 μL/wellアプライした。約500 rpmで攪拌しながら室温１時間反応させた。
反応/洗浄液で洗浄後、1%BSAを添加した反応/洗浄液で希釈したペルオキシダーゼ標識２次抗体100 μL/wellをアプライした。室温１時間反応させた。２次抗体には、Peroxidase-conjugated Affinipure Goat Anti-Mouse IgG (H+L) (JIR、115-035-003)又はPeroxidase-conjugated AffiniPure Donkey Anti-Goat IgG (H+L) (JIR、705-035-003)を用いた。
反応/洗浄液で洗浄後、TMB Solution（富士フイルム和光純薬）100 μL/wellをアプライした。室温30分反応させた。
Stop Solution（富士フイルム和光純薬）100 μL/wellをアプライして、励起/検出波長450/620 nmで測定した。
[Tim4-antibody sandwich ELISA]
100 μL/well of serum diluted 100-fold with reaction/washing solution (Fujifilm Wako Pure Chemical Industries, Ltd.) containing 1% BSA was applied to a Tim4-Fc-immobilized plate (Fujifilm Wako Pure Chemical Industries, Ltd.). The reaction was carried out at room temperature for 2 hours while stirring at approximately 500 rpm.
After washing with the reaction/washing solution, 100 μL/well of the primary antibody diluted with the reaction/washing solution containing 1% BSA was applied and reacted at room temperature for 1 hour while stirring at about 500 rpm.
After washing with reaction/washing solution, 100 μL/well of peroxidase-labeled secondary antibody diluted with reaction/washing solution containing 1% BSA was applied. The reaction was allowed to proceed at room temperature for 1 hour. The secondary antibody used was Peroxidase-conjugated Affinipure Goat Anti-Mouse IgG (H+L) (JIR, 115-035-003) or Peroxidase-conjugated AffiniPure Donkey Anti-Goat IgG (H+L) (JIR, 705-035-003).
After washing with the reaction/washing solution, 100 μL/well of TMB Solution (FUJIFILM Wako Pure Chemical Industries, Ltd.) was applied and reacted at room temperature for 30 minutes.
Stop Solution (FUJIFILM Wako Pure Chemical Industries, Ltd.) was applied at 100 μL/well, and measurements were performed at excitation/detection wavelengths of 450/620 nm.
［抗ＣＤ４１抗体－レクチンサンドイッチＥＬＩＳＡ］
アビジンプレート（住友ベークライト）にビオチン化抗CD41抗体（1 μg/mL）50 μL/wellをアプライした。室温１時間インキュベートした。
PBS/0.1% Tween20で洗浄後、100倍希釈した血清50 μL/wellをアプライした。室温１時間反応させた。
PBS/0.1% Tween20で洗浄後、ペルオキシダーゼ標識したTxLC1レクチン（1 μg/mL）又はDSAレクチン（2 μg/mL）50 uLをアプライした。室温１時間反応させた。
PBS/0.1% Tween20で洗浄後、TMB Solution（富士フイルム和光純薬製）50 μL/wellをアプライした。室温30分反応させた。
1N HCL 50 μLで反応を停止させ、励起/検出波長450/620 nmで測定した。
[Anti-CD41 antibody-lectin sandwich ELISA]
Biotinylated anti-CD41 antibody (1 μg/mL) was applied to an avidin plate (Sumitomo Bakelite) at 50 μL/well and incubated at room temperature for 1 hour.
After washing with PBS/0.1% Tween 20, 50 μL/well of 100-fold diluted serum was applied and incubated at room temperature for 1 hour.
After washing with PBS/0.1% Tween 20, 50 uL of peroxidase-labeled TxLC1 lectin (1 μg/mL) or DSA lectin (2 μg/mL) was applied and incubated at room temperature for 1 hour.
After washing with PBS/0.1% Tween 20, 50 μL/well of TMB Solution (FUJIFILM Wako Pure Chemical Industries, Ltd.) was applied and incubated at room temperature for 30 minutes.
The reaction was stopped with 50 μL of 1N HCL and measured at excitation/detection wavelengths of 450/620 nm.
２．結果
［ウェスタンブロットによる細胞外小胞の検出］
Tim4磁気ビーズを用いて血清から分離した細胞外小胞に対して、抗ＣＤ９抗体（Invitrogen、10626D）、抗ＣＤ４１抗体（R&D、MAB7616）、抗ＣＤ６１抗体（R&D、MAB2266）及び抗ＣＤ６３抗体（富士フイルム和光純薬、297-79201、又は、コスモバイオ、SHI-EXO-MO2）を一次抗体に用いてウェスタンブロットを行った。また、細胞外小胞を結合したTim4磁気ビーズを、蛍光標識した上記抗体で染色し、フローサイトメトリーにより検出した。
結果を図１－４に示す。
アルツハイマー病、うつ及び統合失調症の患者の血清において、Ｔｉｍ４結合性であり、かつ、ＣＤ９陽性、ＣＤ４１陽性、ＣＤ６１陽性又はＣＤ６３陽性である細胞外小胞（マーカーエクソソーム）が、健常者の血清に比して高いレベルで検出された（図１、４参照）。
さらに、上記細胞外小胞は、各種がんの患者血清でも検出された（図２－４参照）。
この結果から、血清中のＴｉｍ４結合性であり、かつ、ＣＤ９陽性、ＣＤ４１陽性、ＣＤ６１陽性又はＣＤ６３陽性である細胞外小胞が、アルツハイマー病、うつ及び統合失調症等の特定の疾患のマーカーとなり得ることが明らかとなった。
2. Results [Detection of extracellular vesicles by Western blot]
Extracellular vesicles isolated from serum using Tim4 magnetic beads were subjected to Western blotting using anti-CD9 antibody (Invitrogen, 10626D), anti-CD41 antibody (R&D, MAB7616), anti-CD61 antibody (R&D, MAB2266) and anti-CD63 antibody (FUJIFILM Wako Pure Chemical, 297-79201, or COSMO BIO, SHI-EXO-MO2) as primary antibodies. Tim4 magnetic beads bound to extracellular vesicles were stained with the above-mentioned fluorescently labeled antibodies and detected by flow cytometry.
The results are shown in Figures 1-4.
In the sera of patients with Alzheimer's disease, depression, and schizophrenia, extracellular vesicles (marker exosomes) that bind to Tim4 and are CD9-, CD41-, CD61-, or CD63-positive were detected at higher levels than in the sera of healthy individuals (see Figures 1 and 4).
Furthermore, the above-mentioned extracellular vesicles were also detected in the sera of patients with various cancers (see Figures 2-4).
These results revealed that Tim4-binding and CD9-, CD41-, CD61-, or CD63-positive extracellular vesicles in serum could be markers for certain diseases such as Alzheimer's disease, depression, and schizophrenia.
［Ｔｉｍ４－抗体サンドイッチＥＬＩＳＡによる細胞外小胞の検出］
抗ＣＤ９抗体（Invitrogen、10626D）、抗ＣＤ４１抗体（R&D、MAB7616）、抗ＣＤ６１抗体（R&D、MAB2266）及び抗ＣＤ６３抗体（富士フイルム和光純薬、297-79201、又はコスモバイオ、SHI-EXO-MO2）を一次抗体に用いてサンドイッチELISAを行った。
結果を図５－８に示す。
Ｔｉｍ４結合性であり、かつ、ＣＤ９陽性、ＣＤ４１陽性、ＣＤ６１陽性又はＣＤ６３陽性である細胞外小胞が、がん、アルツハイマー病、うつ及び統合失調症の患者血清において、健常者の血清に比して高いレベルで検出された。
[Detection of extracellular vesicles by Tim4-antibody sandwich ELISA]
Sandwich ELISA was performed using anti-CD9 antibody (Invitrogen, 10626D), anti-CD41 antibody (R&D, MAB7616), anti-CD61 antibody (R&D, MAB2266), and anti-CD63 antibody (Fujifilm Wako Pure Chemical Industries, 297-79201, or Cosmo Bio, SHI-EXO-MO2) as primary antibodies.
The results are shown in Figures 5-8.
Extracellular vesicles that bind to Tim4 and are CD9-positive, CD41-positive, CD61-positive or CD63-positive were detected at higher levels in the sera of patients with cancer, Alzheimer's disease, depression and schizophrenia compared to the sera of healthy individuals.
ＣＤ９、ＣＤ４１、ＣＤ６１、ＣＤ６３及びＣＤ８１に対する一次抗体をそれぞれ用いたELISAで得られた検出強度間の相関係数を「表１」に示す。ＣＤ９、ＣＤ４１、ＣＤ６１及びＣＤ６３に対する抗体によるELISAで得られた検出強度は互いに高い相関（相関係数＞0.7）を示した。一方、ＣＤ８１に対する抗体（コスモバイオ、SHI-EXO-MO3）によるELISAで得られた検出強度には、このような相関は認められなかった。この結果から、ＣＤ９、ＣＤ４１、ＣＤ６１及びＣＤ６３に対する抗体をそれぞれ用いたELISAで検出されているエクソソームは、ＣＤ９＋／ＣＤ４１＋／ＣＤ６１＋／ＣＤ６３＋のエクソソームであることが示唆された。 The correlation coefficients between the detection intensities obtained by ELISA using primary antibodies against CD9, CD41, CD61, CD63, and CD81 are shown in Table 1. The detection intensities obtained by ELISA using antibodies against CD9, CD41, CD61, and CD63 showed a high correlation with each other (correlation coefficient >0.7). On the other hand, no such correlation was observed in the detection intensities obtained by ELISA using an antibody against CD81 (Cosmobio, SHI-EXO-MO3). These results suggest that the exosomes detected by ELISA using antibodies against CD9, CD41, CD61, and CD63 are CD9+/CD41+/CD61+/CD63+ exosomes.
［種々の神経変性疾患における検討］
アルツハイマー病、うつ及び統合失調症以外の神経変性疾患の患者血清についてもTim4－CD63抗体サンドイッチELISAによる検討を行った。
結果を図９に示す。
睡眠障害、双極性障害、てんかん、発達障害、PTSD、摂食障害及び適応障害の患者血清においても、Ｔｉｍ４結合性であり、かつ、ＣＤ９陽性、ＣＤ４１陽性、ＣＤ６１陽性又はＣＤ６３陽性である細胞外小胞が検出され、該細胞外小胞がこれらの神経変性疾患のマーカーにもなり得ることが明らかとなった。
[Study on various neurodegenerative diseases]
Sera from patients with neurodegenerative diseases other than Alzheimer's disease, depression, and schizophrenia were also examined by Tim4-CD63 antibody sandwich ELISA.
The results are shown in Figure 9.
Tim4-binding, CD9-, CD41-, CD61-, or CD63-positive extracellular vesicles were also detected in the sera of patients with sleep disorders, bipolar disorders, epilepsy, developmental disorders, PTSD, eating disorders, and adjustment disorders, demonstrating that the extracellular vesicles may also be markers for these neurodegenerative diseases.
［抗ＣＤ４１抗体－レクチンサンドイッチＥＬＩＳＡによる細胞外小胞の検出］
健常者、神経変性疾患患者及びがん患者の血清に対して、抗CD41抗体と、TxLC1レクチン又はDSAレクチンと、を用いてサンドイッチELISAを行った。
結果を図１０及び図１１に示す。
TxLC1レクチン及びDSAレクチンを用いても、マーカーエクソソームを、がん、アルツハイマー病、うつ及び統合失調症の患者血清中で検出できることが明らかになった。
[Detection of extracellular vesicles by anti-CD41 antibody-lectin sandwich ELISA]
Sandwich ELISA was performed on the sera of healthy subjects, patients with neurodegenerative diseases, and patients with cancer using anti-CD41 antibody and TxLC1 lectin or DSA lectin.
The results are shown in Figures 10 and 11.
It was also revealed that marker exosomes could be detected in the sera of patients with cancer, Alzheimer's disease, depression, and schizophrenia using TxLC1 lectin and DSA lectin.
さらに、神経変性疾患患者について、アルツハイマー病、うつ及び統合失調症の患者の検体数を増やすとともに、睡眠障害、双極性障害、てんかん、発達障害、PTSD、摂食障害及び適応障害の患者を加えて、同様に血清に対するサンドイッチELISAを行った。
結果を図１２に示す。
アルツハイマー病、うつ、統合失調症睡眠障害、双極性障害、てんかん、発達障害、PTSD、摂食障害及び適応障害の患者血清においても、Ｔｉｍ４結合性であり、かつ、ＣＤ９陽性、ＣＤ４１陽性、ＣＤ６１陽性又はＣＤ６３陽性である細胞外小胞が検出され、該細胞外小胞がこれらの神経変性疾患のマーカーにもなり得ることが明らかとなった（図１２（Ａ）参照）。
特にアルツハイマー病患者血清では、健常者血清に比してマーカーエクソソームの量が有意に高かった（図１２（Ｂ）参照）。アルツハイマー病患者では、血清中のマーカーエクソソームの量が吸光強度（主波長450nm, 副波長620nm）で約0.5を超える患者が多く認められ、吸光強度の最大値は1.83であった。一方、健常者血清中のマーカーエクソソームの量は吸光強度で最大で0.11までの範囲であった。したがって、アルツハイマー病患者と健常者を鑑別するための閾値は、吸光強度で1.1を超える値、あるいは0.11－0.5の範囲内の値（例えば、0.2、0.3又は0.4）に設定され得ることが明らかとなった。
Furthermore, we increased the number of samples from patients with neurodegenerative diseases, including Alzheimer's disease, depression, and schizophrenia, and also performed sandwich ELISA on sera from patients with sleep disorders, bipolar disorder, epilepsy, developmental disorders, PTSD, eating disorders, and adjustment disorders.
The results are shown in Figure 12.
Tim4-binding, CD9-, CD41-, CD61-, or CD63-positive extracellular vesicles were also detected in the sera of patients with Alzheimer's disease, depression, schizophrenia-induced sleep disorder, bipolar disorder, epilepsy, developmental disorder, PTSD, eating disorder, and adjustment disorder, demonstrating that the extracellular vesicles may also be markers for these neurodegenerative diseases (see FIG. 12(A)).
In particular, the amount of marker exosomes in the serum of Alzheimer's disease patients was significantly higher than that in the serum of healthy subjects (see FIG. 12(B)). Many Alzheimer's disease patients had an amount of marker exosomes in their serum that exceeded approximately 0.5 in terms of absorbance intensity (main wavelength 450 nm, sub-wavelength 620 nm), with the maximum absorbance intensity being 1.83. On the other hand, the amount of marker exosomes in the serum of healthy subjects was in the range of up to 0.11 in terms of absorbance intensity. Therefore, it was revealed that the threshold for distinguishing between Alzheimer's disease patients and healthy subjects can be set to a value exceeding 1.1 in terms of absorbance intensity, or a value within the range of 0.11-0.5 (e.g., 0.2, 0.3, or 0.4).
各アルツハイマー病患者の性、年齢、人種及び認知症スクリーニング検査結果（Mini Mental State Examination: ＭＭＳＥスコア）を「表２」に示す。一般に軽度とされるスコア２６－２０の患者においてもマーカーエクソソーム量の顕著な上昇が認められたことから、マーカーエクソソームがアルツハイマー病の早期診断のために有用である可能性が示された。 The gender, age, race, and dementia screening test results (Mini Mental State Examination: MMSE score) of each Alzheimer's disease patient are shown in Table 2. A significant increase in the amount of marker exosomes was observed even in patients with scores of 26-20, which are generally considered to be mild, indicating that marker exosomes may be useful for the early diagnosis of Alzheimer's disease.
配列番号１：ＴｘＬＣ１レクチンのアミノ酸配列
配列番号２：ＤＳＡレクチンのアミノ酸配列
SEQ ID NO: 1: Amino acid sequence of TxLC1 lectin SEQ ID NO: 2: Amino acid sequence of DSA lectin
Claims (5)
対象から採取された血液試料中の、Ｔｉｍ４結合性であり、かつ、ＣＤ９、ＣＤ４１、ＣＤ６１及びＣＤ６３からなる群から選択されるいずれか１以上が陽性である細胞外小胞の量を測定する手順と、
細胞外小胞量が所定の閾値以上である場合に、対象における神経変性疾患を検出する手順と、
を含む方法。 1. A method for detecting a neurodegenerative disease in a subject, comprising:
Measuring the amount of extracellular vesicles that bind to Tim4 and are positive for any one or more selected from the group consisting of CD9, CD41, CD61, and CD63 in a blood sample collected from a subject;
Detecting a neurodegenerative disease in a subject when the amount of extracellular vesicles is equal to or greater than a predetermined threshold;
The method includes:
対象から採取された、血液試料を、第一のプローブが固相化された支持体表面に接触させる手順と、
第二のプローブを、支持体表面に接触させる手順と、
第一のプローブと第二のプローブを含んでなる複合体の量を測定する手順と、
測定値が所定の閾値以上である場合に、対象における神経変性を検出する手順と、を含み、
第一のプローブがＴｉｍ４であり、第二のプローブが抗ＣＤ９抗体、抗ＣＤ４１抗体、抗ＣＤ６１抗体及び抗ＣＤ６３抗体からなる群より選択される、方法。 1. A method for detecting a neurodegenerative disease in a subject, comprising:
A step of contacting a blood sample taken from a subject with a support surface having a first probe immobilized thereon;
contacting a second probe with the support surface;
measuring the amount of a complex comprising the first probe and the second probe;
detecting neurodegeneration in the subject if the measurement is equal to or greater than a predetermined threshold;
The method, wherein the first probe is Tim4 and the second probe is selected from the group consisting of an anti- CD9 antibody, an anti-CD41 antibody, an anti-CD61 antibody and an anti-CD63 antibody .
支持体表面に固相化される第一のプローブと、
第二のプローブと、を含んでなり、
第一のプローブがＴｉｍ４であり、第二のプローブが抗ＣＤ９抗体、抗ＣＤ４１抗体、抗ＣＤ６１抗体及び抗ＣＤ６３抗体からなる群より選択される、キット。 A diagnostic kit for a neurodegenerative disease, comprising:
A first probe immobilized on a support surface;
a second probe,
A kit, wherein the first probe is Tim4 and the second probe is selected from the group consisting of an anti- CD9 antibody, an anti-CD41 antibody, an anti-CD61 antibody and an anti-CD63 antibody .
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