WO2010134567A1 - Method for detection of free nucleic acid in blood - Google Patents
Method for detection of free nucleic acid in blood Download PDFInfo
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- WO2010134567A1 WO2010134567A1 PCT/JP2010/058503 JP2010058503W WO2010134567A1 WO 2010134567 A1 WO2010134567 A1 WO 2010134567A1 JP 2010058503 W JP2010058503 W JP 2010058503W WO 2010134567 A1 WO2010134567 A1 WO 2010134567A1
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- G—PHYSICS
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- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- 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
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/5432—Liposomes or microcapsules
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- 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
- G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
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- the present invention relates to a blood test method for free nucleic acids in blood.
- Non-Patent Document 1 Non-Patent Document 1
- glioblastoma glioblastoma
- glioblastoma glioblastoma
- brain microvascular endothelial cells and the like brain microvascular endothelial cells and the like.
- mRNA mutations and miRNA characteristics found in glioma are detected in microvesicles in the serum of patients with glioblastoma. It has been suggested that vesicles are useful for diagnosis and treatment of cancer patients (Non-patent Document 2).
- An object of the present invention is to provide means for enabling a blood test superior to the conventional one from such a viewpoint.
- Microvesicles in plasma or serum samples are generally collected by size separation by ultracentrifugation or the like, and used for measurement of blood free nucleic acid level.
- the inventor has included platelet-derived microvesicles in blood-derived samples, which are released in large amounts from platelets activated during or after blood collection, and may be present in some of the blood before that. Therefore, in conventional analysis of free nucleic acids in blood, not only endogenous microvesicles that contain nucleic acids that serve as diagnostic markers, but also such platelet-derived microvesicles have been collected. I noticed that there was room for improvement in the reliability of the free nucleic acid measurements. Then, it was found that the blood level of endogenous microvesicles can be measured more accurately by removing the platelet-derived microvesicles from the blood-derived sample, and the present invention has been completed.
- the method for analyzing blood free nucleic acid contained in a blood-derived sample comprises a step of removing platelet-derived microvesicles in a plasma or serum sample (hereinafter referred to as “blood-derived sample”).
- blood-derived sample a plasma or serum sample
- the blood free nucleic acid is encapsulated in an endogenous microvesicle in a blood-derived sample.
- the platelet-derived microvesicles in the blood-derived sample are preferably removed by using an antibody that binds to an epitope that specifically exists on the surface thereof.
- the epitope is, for example, in a glycoprotein, and examples of the glycoprotein include at least one selected from the group consisting of GpIb, GpIX, GpIa, GpIIa, GpIIb, GpIIIa, and GpIIIb, or a complex thereof.
- the platelet-derived microvesicle in the blood-derived sample is immobilized on the carrier after contacting with the antibody immobilized on the carrier or after forming a complex of the platelet-derived microvesicle and the antibody. It can be removed by a method of contacting with a secondary antibody against the antibody.
- the influence of platelet-derived microvesicles mixed in a sample is suppressed to a minimum, and the blood level of nucleic acid contained in an endogenous microvesicle reflecting the clinical state of a patient is more accurately measured. It becomes possible to measure.
- the measurement results obtained in this way are useful information for diagnosing, preventing, treating, etc. disease more accurately than before.
- the graph which shows the ratio of the microvesicle classified by cell origin measured about each of the sample 1 (immediately after blood collection), the sample 2 (after 24 hours), and the sample 3 (after platelet-derived microvesicle removal) in Example 2.
- FIG. 1 The graph which shows the ratio of the microvesicle classified by cell origin measured about each of the sample 1 (immediately after blood collection), the sample 2 (after 24 hours), and the sample 3 (after platelet-derived microvesicle removal) in Example 2.
- the method for analyzing blood free nucleic acid contained in a blood-derived sample includes a step of removing platelet-derived microvesicles in a plasma or serum sample (blood-derived sample), and other steps as appropriate. Can be combined.
- a plasma or serum sample used for analysis of free nucleic acid in blood may be prepared according to known methods.
- plasma is obtained by removing blood cell components by cooling and centrifugation or membrane filtration of blood to which an anticoagulant (such as EDTA) has been added, and the collected blood is allowed to stand and precipitate ( Serum is obtained by removing the clot).
- an anticoagulant such as EDTA
- the free nucleic acid in blood originally targeted by the analysis method of the present invention is usually encapsulated in a stable form in “endogenous microvesicles” contained in blood or plasma or serum samples prepared as described above.
- endogenous microvesicles contained in blood or plasma or serum samples prepared as described above.
- platelet-derived microvesicles since the same kind of blood free nucleic acid may be encapsulated in “platelet-derived microvesicles”, the above-described process characterizing the present invention is useful.
- the microvesicles released from platelets activated during or after blood collection and the microvesicles previously released from platelets contained in plasma or serum samples are referred to as “platelet-derived microvesicles”.
- platelet-derived microvesicles Collectively.
- all of those other than platelet-derived microvesicles correspond to “endogenous microvesicles”.
- blood cell-derived microvesicles such as T cells, B cells, neutrophils and monocytes
- microvesicles such as epithelial cells correspond to endogenous microvesicles.
- blood free nucleic acids are DNA molecules and RNA that are free in the blood in the form of being encapsulated in endogenous microvesicles (possibly encapsulated in mixed platelet-derived microvesicles). It refers to a molecule (mRNA, miRNA, etc.), and its type is not particularly limited.
- the method using an antibody (X) that binds to an epitope is preferred.
- the antibody (X) may be either a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody.
- epitope examples include those located on glycoproteins such as GpIb, GpIX, GpIa, GpIIa, GpIIb, GpIIIa, GpIIIb, or complexes thereof (eg, GPIIb / IIIa complex, GPIb / IX complex). Is mentioned.
- platelet-derived microvesicles can be removed by carrying (immobilizing) antibody (X) on a carrier such as an affinity chromatography column and passing a blood-derived sample through the column filled with this carrier.
- a carrier such as an affinity chromatography column
- a secondary antibody (Y) against the antibody (X) is separately prepared, and a carrier on which the secondary antibody (Y) is immobilized after a complex of platelet-derived microvesicles and the antibody (X) is formed. Even when a sample containing the complex is passed through a packed column, platelet-derived microvesicles can be removed.
- an antibody is added to whole blood, plasma, or serum to form an antigen-antibody reaction with a surface antigen on a platelet-derived microvesicle, and a secondary antibody (Y).
- the platelet-derived microvesicles can be removed by forming aggregates of platelet-derived microvesicles and latex particles by adding latex particles to which () is immobilized, and then precipitating the aggregates by centrifugation or the like.
- platelet-derived microvesicles are excluded as much as possible, and it is excellent as a sample for analyzing nucleic acids encapsulated in endogenous microvesicles.
- Other steps relating to analysis of free nucleic acid in blood that is, a step of recovering endogenous microvesicles, a step of extracting nucleic acid encapsulated in the recovered endogenous microvesicles, a step of quantifying the nucleic acid, etc. This is the same as the conventional one.
- Example 1 (Preparation of sample 1) Peripheral blood samples were collected from veins into EDTA anticoagulant-containing test tubes and immediately centrifuged at 2000 G for 10 minutes to prepare plasma.
- This sample was passed through an affinity column (SulfoLink Immobilization Kit, manufactured by PIERCE, trade name) on which a goat anti-mouse IgG polyclonal antibody (Acris Antibodies) was immobilized, and platelet-derived microvesicles were removed.
- affinity column SulfoLink Immobilization Kit, manufactured by PIERCE, trade name
- microvesicle-derived microRNA extraction (Microvesicle-derived microRNA extraction, reverse transcription and real-time PCR) Each of the samples 1 to 3 was subjected to ultracentrifugation at 160,000 ⁇ g for 4 hours at 4 ° C., and the supernatant was removed to collect microvesicles in the plasma sample. From the collected microvesicles, total microRNAs derived from microvesicles were extracted using mirVana miRNA Isolation Kit (Ambion, trade name). The extracted microRNA was reverse transcribed using Megaplex primer pools (Applied Biosystems), the obtained cDNA was applied to microRNA TaqMan (R) Array (Applied Biosystems), and the Ct value was measured by real-time PCR. .
- Example 2 In order to show that platelet-derived microvesicles have been removed, the microvesicles in each sample are labeled with an antibody against a cell-specific surface antigen, and the ratio of microvesicles by cell origin is determined by flow cytometry (BD Biosciences). It was measured.
- endogenous microvesicles include neutrophil-derived (CD66b-FITC, manufactured by BD Biosciences), epithelial cell-derived (CD202b (Tie2) -PE, manufactured by R & D Systems), macrophage-derived (CD206 PE- Cy5, BD Biosciences), B cell (CD79a-APC, BD Biosciences), monocyte (CD14 Pe-Cy7, BD Biosciences), T cell (CD3-Alexa 610, BD Biosciences) Each of which was labeled with each of the above antibodies, while the platelet-derived microvesicles were labeled with CD41a-PE-Cy5 (BD Biosciences).
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Abstract
Disclosed is an analysis method in which the influence of a free nucleic acid that is irrelevant to a disease such as a tumor or the like can be eliminated to measure only the blood level of an exactly desired free nucleic acid (which has been employed as a diagnostic marker for the disease) as accurately as possible in the measurement of the blood level of the desired free nucleic acid. Specifically disclosed is a method for analyzing a free blood nucleic acid contained in a blood-derived sample, which is characterized by comprising a step of removing platelet-derived microvesicles from a plasma or serum sample (i.e., the blood-derived sample). Preferably, the platelet-derived microvesicles in the blood-derived sample are removed using an antibody capable of binding to epitopes existing specifically on the surfaces of the microvesicles.
Description
本発明は、血中遊離核酸等を対象とする血液検査の方法に関する。
The present invention relates to a blood test method for free nucleic acids in blood.
近年、血液（血漿または血清試料）中に遊離している核酸を疾患の診断や治療のために利用する手法に関して、盛んに研究開発が進められている。
たとえば、しばしばがん細胞で調節不全に陥るｍｉＲＮＡは、がんの分類や診断のためのマーカーとして有望視されている。ｍｉＲＮＡは内在性のＲＮａｓｅに対して極めて安定的な形態で血漿中に存在し、たとえば前立腺がんで発現するｍｉＲ－１４１のヒト血清中のレベルを測定することにより、前立腺がん患者と正常人とを区別できることが示唆されている（非特許文献１）。 In recent years, research and development have been actively conducted on techniques for utilizing nucleic acids released in blood (plasma or serum samples) for diagnosis and treatment of diseases.
For example, miRNAs that are often dysregulated in cancer cells are promising as markers for cancer classification and diagnosis. miRNAs are present in plasma in a very stable form against endogenous RNase and, for example, by measuring the level of miR-141 expressed in prostate cancer in human serum, prostate cancer patients and normal individuals It is suggested that can be distinguished (Non-Patent Document 1).
たとえば、しばしばがん細胞で調節不全に陥るｍｉＲＮＡは、がんの分類や診断のためのマーカーとして有望視されている。ｍｉＲＮＡは内在性のＲＮａｓｅに対して極めて安定的な形態で血漿中に存在し、たとえば前立腺がんで発現するｍｉＲ－１４１のヒト血清中のレベルを測定することにより、前立腺がん患者と正常人とを区別できることが示唆されている（非特許文献１）。 In recent years, research and development have been actively conducted on techniques for utilizing nucleic acids released in blood (plasma or serum samples) for diagnosis and treatment of diseases.
For example, miRNAs that are often dysregulated in cancer cells are promising as markers for cancer classification and diagnosis. miRNAs are present in plasma in a very stable form against endogenous RNase and, for example, by measuring the level of miR-141 expressed in prostate cancer in human serum, prostate cancer patients and normal individuals It is suggested that can be distinguished (Non-Patent Document 1).
また、グリオブラストーマ（神経膠芽細胞腫）の腫瘍細胞がｍＲＮＡ、ｍｉＲＮＡおよび血管新生に関するタンパク質を内包するマイクロベシクルを放出し、脳微小血管の内皮細胞等がこれを受容しｍＲＮＡがその細胞内で翻訳されることや、グリオブラストーマ患者の血清中のマイクロベシクルにはグリオーマ（神経膠腫）にみられるｍＲＮＡ変異、ｍｉＲＮＡ特性が検出されることが報告され、したがってそのような腫瘍由来のマイクロベシクルががん患者に対する診断、治療のために有用であることが示唆されている（非特許文献２）。
In addition, tumor cells of glioblastoma (glioblastoma) release microvesicles that contain mRNA, miRNA and angiogenesis proteins, and these are received by brain microvascular endothelial cells and the like. It is reported that mRNA mutations and miRNA characteristics found in glioma (glioma) are detected in microvesicles in the serum of patients with glioblastoma. It has been suggested that vesicles are useful for diagnosis and treatment of cancer patients (Non-patent Document 2).
腫瘍等の疾患の診断マーカーなどとして利用される血中遊離核酸の血中レベルを測定する際には、その疾患とは無関係の血中遊離核酸の影響を排除し、真に対象としたい血中遊離核酸の血中レベルのみを極力正確に測定する必要がある。本発明は、そのような観点から従来よりも優れた血液検査を可能とするための手段を提供することを課題とする。
When measuring the level of blood free nucleic acid used as a diagnostic marker for diseases such as tumors, the effect of blood free nucleic acid unrelated to the disease is eliminated, and the blood that is truly targeted Only the blood level of free nucleic acid needs to be measured as accurately as possible. An object of the present invention is to provide means for enabling a blood test superior to the conventional one from such a viewpoint.
血漿または血清試料中のマイクロベシクルは、一般的に超遠心などによるサイズ分離によって回収され、血中遊離核酸レベルの測定に供される。本発明者は、採血時または採血後に活性化した血小板から多量に血液中に放出され、それ以前の血液中にもいくらか存在することがある、血小板に由来するマイクロベシクルが血液由来試料に含まれていること、したがって従来の血中遊離核酸の分析においては、診断マーカーとなる核酸を内包する内在性マイクロベシクルのみならず、そのような血小板由来マイクロベシクルも回収してしまっているため、血中遊離核酸の測定値の信頼性に改善の余地があることに気付いた。そして、血液由来試料中の血小板由来マイクロベシクルを除去すれば、内在性マイクロベシクルの血中レベルをより正確に測定することができることを見出し、本発明を完成させるに至った。
Microvesicles in plasma or serum samples are generally collected by size separation by ultracentrifugation or the like, and used for measurement of blood free nucleic acid level. The inventor has included platelet-derived microvesicles in blood-derived samples, which are released in large amounts from platelets activated during or after blood collection, and may be present in some of the blood before that. Therefore, in conventional analysis of free nucleic acids in blood, not only endogenous microvesicles that contain nucleic acids that serve as diagnostic markers, but also such platelet-derived microvesicles have been collected. I noticed that there was room for improvement in the reliability of the free nucleic acid measurements. Then, it was found that the blood level of endogenous microvesicles can be measured more accurately by removing the platelet-derived microvesicles from the blood-derived sample, and the present invention has been completed.
すなわち、本発明に係る血液由来試料に含まれる血中遊離核酸の分析方法は、血漿または血清試料（以下「血液由来試料」という。）中の血小板由来マイクロベシクルを除去する工程を含むことを特徴とする。
That is, the method for analyzing blood free nucleic acid contained in a blood-derived sample according to the present invention comprises a step of removing platelet-derived microvesicles in a plasma or serum sample (hereinafter referred to as “blood-derived sample”). And
前記血中遊離核酸は、より具体的には、血液由来試料中の内在性マイクロベシクルに内包されるものである。
前記血液由来試料中の血小板由来マイクロベシクルは、その表面に特異的に存在するエピトープと結合する抗体を用いることにより除去することが好適である。前記エピトープは、たとえば糖タンパク質にあるものであり、その糖タンパク質としては、GpIb，GpIX，GpIa，GpIIa，GpIIb，GpIIIaおよびGpIIIbからなる群より選ばれる少なくとも１種またはそれらの複合体が挙げられる。 More specifically, the blood free nucleic acid is encapsulated in an endogenous microvesicle in a blood-derived sample.
The platelet-derived microvesicles in the blood-derived sample are preferably removed by using an antibody that binds to an epitope that specifically exists on the surface thereof. The epitope is, for example, in a glycoprotein, and examples of the glycoprotein include at least one selected from the group consisting of GpIb, GpIX, GpIa, GpIIa, GpIIb, GpIIIa, and GpIIIb, or a complex thereof.
前記血液由来試料中の血小板由来マイクロベシクルは、その表面に特異的に存在するエピトープと結合する抗体を用いることにより除去することが好適である。前記エピトープは、たとえば糖タンパク質にあるものであり、その糖タンパク質としては、GpIb，GpIX，GpIa，GpIIa，GpIIb，GpIIIaおよびGpIIIbからなる群より選ばれる少なくとも１種またはそれらの複合体が挙げられる。 More specifically, the blood free nucleic acid is encapsulated in an endogenous microvesicle in a blood-derived sample.
The platelet-derived microvesicles in the blood-derived sample are preferably removed by using an antibody that binds to an epitope that specifically exists on the surface thereof. The epitope is, for example, in a glycoprotein, and examples of the glycoprotein include at least one selected from the group consisting of GpIb, GpIX, GpIa, GpIIa, GpIIb, GpIIIa, and GpIIIb, or a complex thereof.
前記血液由来試料中の血小板由来マイクロベシクルは、担体上に固定化された前記抗体と接触させる方法、または血小板由来マイクロベシクルと前記抗体との複合体を形成させた後に、担体上に固定化された前記抗体に対する二次抗体と接触させる方法により除去することができる。
The platelet-derived microvesicle in the blood-derived sample is immobilized on the carrier after contacting with the antibody immobilized on the carrier or after forming a complex of the platelet-derived microvesicle and the antibody. It can be removed by a method of contacting with a secondary antibody against the antibody.
本発明の方法を用いれば、試料中に混入した血小板由来マイクロベシクルの影響を最小限に抑制し、患者の臨床状態を反映した内在性マイクロベシクルに内包される核酸の血中レベルをより正確に測定できるようになる。このようにして得られた測定結果は、従来よりも的確に疾患の診断、予防、治療等を行うために有用な情報となる。
By using the method of the present invention, the influence of platelet-derived microvesicles mixed in a sample is suppressed to a minimum, and the blood level of nucleic acid contained in an endogenous microvesicle reflecting the clinical state of a patient is more accurately measured. It becomes possible to measure. The measurement results obtained in this way are useful information for diagnosing, preventing, treating, etc. disease more accurately than before.
本発明に係る、血液由来試料に含まれる血中遊離核酸の分析方法は、血漿または血清試料（血液由来試料）中の血小板由来マイクロベシクルを除去する工程を含み、必要に応じて適宜その他の工程を組み合わせることができる。
The method for analyzing blood free nucleic acid contained in a blood-derived sample according to the present invention includes a step of removing platelet-derived microvesicles in a plasma or serum sample (blood-derived sample), and other steps as appropriate. Can be combined.
血中遊離核酸の分析に供する血漿または血清試料は公知の従って調製すればよい。一般的には、抗凝固剤（ＥＤＴＡ等）が添加された血液を冷却遠心分離または膜濾過して血球成分を除去することにより血漿が得られ、また採取した血液を静置して沈殿物（血餅）を除去することにより血清が得られる。
A plasma or serum sample used for analysis of free nucleic acid in blood may be prepared according to known methods. In general, plasma is obtained by removing blood cell components by cooling and centrifugation or membrane filtration of blood to which an anticoagulant (such as EDTA) has been added, and the collected blood is allowed to stand and precipitate ( Serum is obtained by removing the clot).
本発明の分析方法が本来対象とする血中遊離核酸は通常、血液ないし上記のようにして調製した血漿または血清試料に含まれる「内在性マイクロベシクル」に、安定的な形態で内包されている。しかしながら、同種の血中遊離核酸は「血小板由来マイクロベシクル」に内包されている可能性もあるため、本発明を特徴付ける上記の工程が有用になる。
The free nucleic acid in blood originally targeted by the analysis method of the present invention is usually encapsulated in a stable form in “endogenous microvesicles” contained in blood or plasma or serum samples prepared as described above. . However, since the same kind of blood free nucleic acid may be encapsulated in “platelet-derived microvesicles”, the above-described process characterizing the present invention is useful.
ここで、本発明においては、血漿または血清試料に含まれる、採血時または採血後に活性化した血小板から放出されたマイクロベシクルおよびそれ以前に血小板から放出されていたマイクロベシクルを「血小板由来マイクロベシクル」と総称する。換言すれば、血漿または血清試料に含まれるマイクロベシクルのうち、血小板由来マイクロベシクル以外のものはすべて「内在性マイクロベシクル」に該当する。具体的には、Ｔ細胞、Ｂ細胞、好中球、単球などの血球由来マイクロベシクルや、上皮細胞由来等のマイクロベシクルが内在性マイクロベシクルに該当する。
Here, in the present invention, the microvesicles released from platelets activated during or after blood collection and the microvesicles previously released from platelets contained in plasma or serum samples are referred to as “platelet-derived microvesicles”. Collectively. In other words, among the microvesicles contained in the plasma or serum sample, all of those other than platelet-derived microvesicles correspond to “endogenous microvesicles”. Specifically, blood cell-derived microvesicles such as T cells, B cells, neutrophils and monocytes, and microvesicles such as epithelial cells correspond to endogenous microvesicles.
また、血中遊離核酸とは、内在性マイクロベシクルに内包されたような形態で血中に遊離している（混入した血小板由来マイクロベシクルにも内包されている可能性のある）ＤＮＡ分子およびＲＮＡ分子（ｍＲＮＡ、ｍｉＲＮＡ等）をいい、その種類は特に限定されるものではない。
In addition, blood free nucleic acids are DNA molecules and RNA that are free in the blood in the form of being encapsulated in endogenous microvesicles (possibly encapsulated in mixed platelet-derived microvesicles). It refers to a molecule (mRNA, miRNA, etc.), and its type is not particularly limited.
血液由来試料中に含まれる内在性マイクロベシクルと血小板由来マイクロベシクルのうち後者のマイクロベシクルのみを除去するためには、血小板由来マイクロベシクルの表面に特異的に存在する（内在性マイクロベシクルの表面には存在しない）エピトープと結合する抗体（Ｘ）を用いる方法が好適である。抗体（Ｘ）は、モノクローナル抗体、ポリクローナル抗体いずれであってもよいが、望ましくはモノクローナル抗体である。
In order to remove only the latter microvesicles from the endogenous microvesicles and platelet-derived microvesicles contained in the blood-derived sample, they exist specifically on the surface of the platelet-derived microvesicles (on the surface of the endogenous microvesicles). The method using an antibody (X) that binds to an epitope is preferred. The antibody (X) may be either a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody.
上記のエピトープとしては、たとえば、GpIb，GpIX，GpIa，GpIIa，GpIIb，GpIIIa，GpIIIbなどの糖タンパク質あるいはこれらの複合体（例：GPIIb/IIIa複合体、GPIb/IX複合体）上に位置するものが挙げられる。
Examples of the epitope include those located on glycoproteins such as GpIb, GpIX, GpIa, GpIIa, GpIIb, GpIIIa, GpIIIb, or complexes thereof (eg, GPIIb / IIIa complex, GPIb / IX complex). Is mentioned.
抗体（Ｘ）を用いて血小板由来マイクロベシクルを除去するためには、抗原抗体反応を利用した公知の各種の方法を適用することができ、抗体（Ｘ）の使用態様は特に限定されるものではない。
In order to remove platelet-derived microvesicles using the antibody (X), various known methods utilizing antigen-antibody reaction can be applied, and the usage mode of the antibody (X) is not particularly limited. Absent.
たとえば、アフィニティクロマトグラフィ用カラムなどの担体に抗体（Ｘ）を担持（固定化）し、この担体が充填されたカラムに血液由来試料を通過させることにより、血小板由来マイクロベシクルを除去することができる。
For example, platelet-derived microvesicles can be removed by carrying (immobilizing) antibody (X) on a carrier such as an affinity chromatography column and passing a blood-derived sample through the column filled with this carrier.
あるいは、抗体（Ｘ）に対する二次抗体（Ｙ）を別途用意し、血小板由来マイクロベシクルと抗体（Ｘ）との複合体を形成させた後に、二次抗体（Ｙ）が固定化された担体が充填されたカラムにその複合体を含む試料を通過させても、血小板由来マイクロベシクルを除去することができる。また、免疫凝集の原理を応用し、全血、血漿、または血清中に抗体（Ｘ）を添加することで血小板由来マイクロベシクル上の表面抗原と抗原抗体反応を形成し、更に二次抗体（Ｙ）が固定化されたラテックス粒子を添加することにより血小板由来マイクロベシクルとラテックス粒子の凝集体を形成した後、遠心分離などにより凝集体を沈殿させることで血小板由来マイクロベシクルを除去することができる。
Alternatively, a secondary antibody (Y) against the antibody (X) is separately prepared, and a carrier on which the secondary antibody (Y) is immobilized after a complex of platelet-derived microvesicles and the antibody (X) is formed. Even when a sample containing the complex is passed through a packed column, platelet-derived microvesicles can be removed. In addition, by applying the principle of immunoagglutination, an antibody (X) is added to whole blood, plasma, or serum to form an antigen-antibody reaction with a surface antigen on a platelet-derived microvesicle, and a secondary antibody (Y The platelet-derived microvesicles can be removed by forming aggregates of platelet-derived microvesicles and latex particles by adding latex particles to which () is immobilized, and then precipitating the aggregates by centrifugation or the like.
以上のような本発明の方法により得られた試料からは、血小板由来マイクロベシクルが極力排除されており、内在性マイクロベシクルに内包されている核酸を分析するための試料として優れている。血中遊離核酸の分析に関するこれ以外の工程、すなわち内在性マイクロベシクルを回収する工程、回収された内在性マイクロベシクルに内包されている核酸を抽出する工程、その核酸を定量する工程などの態様は、従来と同様である。
From the sample obtained by the method of the present invention as described above, platelet-derived microvesicles are excluded as much as possible, and it is excellent as a sample for analyzing nucleic acids encapsulated in endogenous microvesicles. Other steps relating to analysis of free nucleic acid in blood, that is, a step of recovering endogenous microvesicles, a step of extracting nucleic acid encapsulated in the recovered endogenous microvesicles, a step of quantifying the nucleic acid, etc. This is the same as the conventional one.
実施例１
（サンプル１の調製）
末梢血試料を静脈よりＥＤＴＡ凝固防止剤含有試験管中に採取し、直ちに２０００Ｇ、１０分間の遠心分離にかけ、血漿を調製した。 Example 1
(Preparation of sample 1)
Peripheral blood samples were collected from veins into EDTA anticoagulant-containing test tubes and immediately centrifuged at 2000 G for 10 minutes to prepare plasma.
（サンプル１の調製）
末梢血試料を静脈よりＥＤＴＡ凝固防止剤含有試験管中に採取し、直ちに２０００Ｇ、１０分間の遠心分離にかけ、血漿を調製した。 Example 1
(Preparation of sample 1)
Peripheral blood samples were collected from veins into EDTA anticoagulant-containing test tubes and immediately centrifuged at 2000 G for 10 minutes to prepare plasma.
（サンプル２の調製）
末梢血試料を静脈よりＥＤＴＡ凝固防止剤含有試験管中に採取し、２４時間後に２０００Ｇ、１０分間の遠心分離にかけ、血漿を調製した。 (Preparation of sample 2)
Peripheral blood samples were collected from veins into EDTA anticoagulant-containing test tubes, and after 24 hours, centrifuged at 2000 G for 10 minutes to prepare plasma.
末梢血試料を静脈よりＥＤＴＡ凝固防止剤含有試験管中に採取し、２４時間後に２０００Ｇ、１０分間の遠心分離にかけ、血漿を調製した。 (Preparation of sample 2)
Peripheral blood samples were collected from veins into EDTA anticoagulant-containing test tubes, and after 24 hours, centrifuged at 2000 G for 10 minutes to prepare plasma.
（サンプル３の調製）
末梢血試料を静脈よりＥＤＴＡ凝固防止剤含有試験管中に採取し、２４時間後に２０００Ｇ、１０分間の遠心分離にかけ、血漿を調製した。この血漿を生理食塩水で５倍希釈して調製した５０００マイクロリットルのサンプル中に、１０マイクログラム／ミリリッターに調製した抗ヒトＧｐＩｂ-マウスモノクローナル抗体（Exbio Praha a.s.社製）を１００分の１容量添加し、血小板由来マイクロベシクルとの抗原抗体反応物を形成させた。このサンプルをヤギ抗マウスＩｇＧポリクローナル抗体（Acris Antibodies社製）が固定化されたアフィニティーカラム（SulfoLink Immobilization Kit, PIERCE社製、商品名）に通し、血小板由来マイクロベシクルを除去した。 (Preparation of sample 3)
Peripheral blood samples were collected from veins into EDTA anticoagulant-containing test tubes, and after 24 hours, centrifuged at 2000 G for 10 minutes to prepare plasma. An anti-human GpIb-mouse monoclonal antibody (Exbio Praha as) prepared at 10 microgram / milliliter was diluted to 1/100 in a 5000 microliter sample prepared by diluting this plasma 5 times with physiological saline. A volume was added to form an antigen-antibody reaction with platelet-derived microvesicles. This sample was passed through an affinity column (SulfoLink Immobilization Kit, manufactured by PIERCE, trade name) on which a goat anti-mouse IgG polyclonal antibody (Acris Antibodies) was immobilized, and platelet-derived microvesicles were removed.
末梢血試料を静脈よりＥＤＴＡ凝固防止剤含有試験管中に採取し、２４時間後に２０００Ｇ、１０分間の遠心分離にかけ、血漿を調製した。この血漿を生理食塩水で５倍希釈して調製した５０００マイクロリットルのサンプル中に、１０マイクログラム／ミリリッターに調製した抗ヒトＧｐＩｂ-マウスモノクローナル抗体（Exbio Praha a.s.社製）を１００分の１容量添加し、血小板由来マイクロベシクルとの抗原抗体反応物を形成させた。このサンプルをヤギ抗マウスＩｇＧポリクローナル抗体（Acris Antibodies社製）が固定化されたアフィニティーカラム（SulfoLink Immobilization Kit, PIERCE社製、商品名）に通し、血小板由来マイクロベシクルを除去した。 (Preparation of sample 3)
Peripheral blood samples were collected from veins into EDTA anticoagulant-containing test tubes, and after 24 hours, centrifuged at 2000 G for 10 minutes to prepare plasma. An anti-human GpIb-mouse monoclonal antibody (Exbio Praha as) prepared at 10 microgram / milliliter was diluted to 1/100 in a 5000 microliter sample prepared by diluting this plasma 5 times with physiological saline. A volume was added to form an antigen-antibody reaction with platelet-derived microvesicles. This sample was passed through an affinity column (SulfoLink Immobilization Kit, manufactured by PIERCE, trade name) on which a goat anti-mouse IgG polyclonal antibody (Acris Antibodies) was immobilized, and platelet-derived microvesicles were removed.
（マイクロベシクル由来microRNAの抽出、逆転写およびリアルタイムPCR）
上記サンプル１～３それぞれについて、160,000×ｇ、４℃で一時間の超遠心分離を行い、上清を除去することにより、血漿サンプル中のマイクロベシクルを回収した。回収されたマイクロベシクルから、mirVana miRNA Isolation Kit （Ambion社製、商品名）を用いてマイクロベシクル由来の全microRNAを抽出した。抽出されたmicroRNAをMegaplex primer pools （アプライドバイオシステムス社）を用いて逆転写し、得られたcDNAをmicroRNA TaqMan (R) Array（アプライドバイオシステム社）にアプライし、リアルタイムＰＣＲによりＣｔ値を測定した。 (Microvesicle-derived microRNA extraction, reverse transcription and real-time PCR)
Each of the samples 1 to 3 was subjected to ultracentrifugation at 160,000 × g for 4 hours at 4 ° C., and the supernatant was removed to collect microvesicles in the plasma sample. From the collected microvesicles, total microRNAs derived from microvesicles were extracted using mirVana miRNA Isolation Kit (Ambion, trade name). The extracted microRNA was reverse transcribed using Megaplex primer pools (Applied Biosystems), the obtained cDNA was applied to microRNA TaqMan (R) Array (Applied Biosystems), and the Ct value was measured by real-time PCR. .
上記サンプル１～３それぞれについて、160,000×ｇ、４℃で一時間の超遠心分離を行い、上清を除去することにより、血漿サンプル中のマイクロベシクルを回収した。回収されたマイクロベシクルから、mirVana miRNA Isolation Kit （Ambion社製、商品名）を用いてマイクロベシクル由来の全microRNAを抽出した。抽出されたmicroRNAをMegaplex primer pools （アプライドバイオシステムス社）を用いて逆転写し、得られたcDNAをmicroRNA TaqMan (R) Array（アプライドバイオシステム社）にアプライし、リアルタイムＰＣＲによりＣｔ値を測定した。 (Microvesicle-derived microRNA extraction, reverse transcription and real-time PCR)
Each of the samples 1 to 3 was subjected to ultracentrifugation at 160,000 × g for 4 hours at 4 ° C., and the supernatant was removed to collect microvesicles in the plasma sample. From the collected microvesicles, total microRNAs derived from microvesicles were extracted using mirVana miRNA Isolation Kit (Ambion, trade name). The extracted microRNA was reverse transcribed using Megaplex primer pools (Applied Biosystems), the obtained cDNA was applied to microRNA TaqMan (R) Array (Applied Biosystems), and the Ct value was measured by real-time PCR. .
（結果および考察）
結果を下記表に示す。サンプル２（２４時間後）から検出されたmicroRNA（ここではＣｔ値が３５サイクル未満のものをいう。）の種類数は、サンプル１（採血直後）から検出されたmicroRNAの種類数とほとんど変わらないが、それらのmicroRNAの平均Ｃｔ値はサンプル２の方が小さい。これは、採血後に活性化した血小板から血小板由来マイクロベシクルが放出され、血漿中のmicroRNAの量（コピー数）が全体的に増加したことを示している。それに対し、サンプル３（血小板由来マイクロベシクル除去後）は、サンプル１および２のどちらと比較しても、検出されたmicroRNAの種類数は少なく、平均Ｃｔ値は大きい。これは、サンプル中から血小板由来マイクロベシクル由来のmicroRNAが除去され、内在性マイクロベシクル由来のmicroRNAが検出されていることを示している。 (Results and Discussion)
The results are shown in the table below. The number of microRNAs detected from sample 2 (24 hours later) (here, Ct value is less than 35 cycles) is almost the same as the number of microRNAs detected from sample 1 (immediately after blood collection). However, the average Ct value of those microRNAs is smaller in sample 2. This indicates that platelet-derived microvesicles were released from platelets activated after blood collection, and the amount (copy number) of microRNA in plasma increased overall. On the other hand, sample 3 (after removing platelet-derived microvesicles) has a smaller number of types of detected microRNAs and a larger average Ct value than both samples 1 and 2. This indicates that microRNA derived from platelet-derived microvesicles was removed from the sample, and microRNA derived from endogenous microvesicles was detected.
結果を下記表に示す。サンプル２（２４時間後）から検出されたmicroRNA（ここではＣｔ値が３５サイクル未満のものをいう。）の種類数は、サンプル１（採血直後）から検出されたmicroRNAの種類数とほとんど変わらないが、それらのmicroRNAの平均Ｃｔ値はサンプル２の方が小さい。これは、採血後に活性化した血小板から血小板由来マイクロベシクルが放出され、血漿中のmicroRNAの量（コピー数）が全体的に増加したことを示している。それに対し、サンプル３（血小板由来マイクロベシクル除去後）は、サンプル１および２のどちらと比較しても、検出されたmicroRNAの種類数は少なく、平均Ｃｔ値は大きい。これは、サンプル中から血小板由来マイクロベシクル由来のmicroRNAが除去され、内在性マイクロベシクル由来のmicroRNAが検出されていることを示している。 (Results and Discussion)
The results are shown in the table below. The number of microRNAs detected from sample 2 (24 hours later) (here, Ct value is less than 35 cycles) is almost the same as the number of microRNAs detected from sample 1 (immediately after blood collection). However, the average Ct value of those microRNAs is smaller in sample 2. This indicates that platelet-derived microvesicles were released from platelets activated after blood collection, and the amount (copy number) of microRNA in plasma increased overall. On the other hand, sample 3 (after removing platelet-derived microvesicles) has a smaller number of types of detected microRNAs and a larger average Ct value than both samples 1 and 2. This indicates that microRNA derived from platelet-derived microvesicles was removed from the sample, and microRNA derived from endogenous microvesicles was detected.
実施例２
血小板由来マイクロベシクルが除去されたことを示すために、各サンプル中のマイクロベシクルを細胞特異的表面抗原に対する抗体で標識化し、フローサイトメトリー（BD Biosciences社製）で細胞由来別マイクロベシクルの比率を測定した。マイクロベシクルのうち、内在性マイクロベシクルには、好中球由来（CD66b-FITC, BD Biosciences社製）、上皮細胞由来（CD202b (Tie2)-PE, Ｒ＆Ｄシステムズ社製）、マクロファージ由来（CD206 PE-Cy5, BD Biosciences社製）、Ｂ細胞由来（CD79a-APC, BD Biosciences社製）、単球由来（CD14 Pe-Cy7, BD Biosciences社製）、Ｔ細胞由来（CD3-Alexa 610, BD Biosciences社製）のものが含まれ、それぞれ上記の各抗体で標識化し、一方血小板由来マイクロベシクルはCD41a-PE-Cy5（BD Biosciences社製）で標識化した。 Example 2
In order to show that platelet-derived microvesicles have been removed, the microvesicles in each sample are labeled with an antibody against a cell-specific surface antigen, and the ratio of microvesicles by cell origin is determined by flow cytometry (BD Biosciences). It was measured. Among microvesicles, endogenous microvesicles include neutrophil-derived (CD66b-FITC, manufactured by BD Biosciences), epithelial cell-derived (CD202b (Tie2) -PE, manufactured by R & D Systems), macrophage-derived (CD206 PE- Cy5, BD Biosciences), B cell (CD79a-APC, BD Biosciences), monocyte (CD14 Pe-Cy7, BD Biosciences), T cell (CD3-Alexa 610, BD Biosciences) Each of which was labeled with each of the above antibodies, while the platelet-derived microvesicles were labeled with CD41a-PE-Cy5 (BD Biosciences).
血小板由来マイクロベシクルが除去されたことを示すために、各サンプル中のマイクロベシクルを細胞特異的表面抗原に対する抗体で標識化し、フローサイトメトリー（BD Biosciences社製）で細胞由来別マイクロベシクルの比率を測定した。マイクロベシクルのうち、内在性マイクロベシクルには、好中球由来（CD66b-FITC, BD Biosciences社製）、上皮細胞由来（CD202b (Tie2)-PE, Ｒ＆Ｄシステムズ社製）、マクロファージ由来（CD206 PE-Cy5, BD Biosciences社製）、Ｂ細胞由来（CD79a-APC, BD Biosciences社製）、単球由来（CD14 Pe-Cy7, BD Biosciences社製）、Ｔ細胞由来（CD3-Alexa 610, BD Biosciences社製）のものが含まれ、それぞれ上記の各抗体で標識化し、一方血小板由来マイクロベシクルはCD41a-PE-Cy5（BD Biosciences社製）で標識化した。 Example 2
In order to show that platelet-derived microvesicles have been removed, the microvesicles in each sample are labeled with an antibody against a cell-specific surface antigen, and the ratio of microvesicles by cell origin is determined by flow cytometry (BD Biosciences). It was measured. Among microvesicles, endogenous microvesicles include neutrophil-derived (CD66b-FITC, manufactured by BD Biosciences), epithelial cell-derived (CD202b (Tie2) -PE, manufactured by R & D Systems), macrophage-derived (CD206 PE- Cy5, BD Biosciences), B cell (CD79a-APC, BD Biosciences), monocyte (CD14 Pe-Cy7, BD Biosciences), T cell (CD3-Alexa 610, BD Biosciences) Each of which was labeled with each of the above antibodies, while the platelet-derived microvesicles were labeled with CD41a-PE-Cy5 (BD Biosciences).
結果を図１に示す。サンプル２（２４時間後）の内在性マイクロベシクルの比率はサンプル１（採血直後）よりも減少しており、活性化した血小板から血小板由来マイクロベシクルが放出されていることを示している。一方、サンプル３（血小板由来マイクロベシクル除去後）では内在性マイクロベシクルの比率が大幅に増加することから、血漿サンプルに本発明の処理を施すことで、より正確に内在性マイクロベシクル由来のmicroRNAを測定できることを示している。
The results are shown in FIG. The ratio of endogenous microvesicles in sample 2 (after 24 hours) is lower than that in sample 1 (immediately after blood collection), indicating that platelet-derived microvesicles are released from activated platelets. On the other hand, in sample 3 (after removing platelet-derived microvesicles), the ratio of endogenous microvesicles is greatly increased. Therefore, by applying the treatment of the present invention to a plasma sample, microRNA derived from endogenous microvesicles can be more accurately obtained. It shows that it can be measured.
Claims (6)
- 血漿または血清試料（以下「血液由来試料」という。）中の血小板由来マイクロベシクルを除去する工程を含むことを特徴とする、血液由来試料に含まれる血中遊離核酸の分析方法。 A method for analyzing blood free nucleic acid contained in a blood-derived sample, comprising a step of removing platelet-derived microvesicles in a plasma or serum sample (hereinafter referred to as “blood-derived sample”).
- 前記血中遊離核酸が血液由来試料中の内在性マイクロベシクルに内包されるものである、請求項１に記載の分析方法。 The analysis method according to claim 1, wherein the blood free nucleic acid is encapsulated in an endogenous microvesicle in a blood-derived sample.
- 前記血液由来試料中の血小板由来マイクロベシクルを、その表面に特異的に存在するエピトープと結合する抗体を用いることにより除去することを特徴とする、請求項１または２に記載の分析方法。 3. The analysis method according to claim 1, wherein the platelet-derived microvesicle in the blood-derived sample is removed by using an antibody that binds to an epitope that specifically exists on the surface thereof.
- 前記エピトープが糖タンパク質にあるものである、請求項３に記載の分析方法。 The analysis method according to claim 3, wherein the epitope is in a glycoprotein.
- 前記糖タンパク質がGpIb，GpIX，GpIa，GpIIa，GpIIb，GpIIIaおよびGpIIIbからなる群より選ばれる少なくとも１種またはそれらの複合体である、請求項４に記載の分析方法。 The analysis method according to claim 4, wherein the glycoprotein is at least one selected from the group consisting of GpIb, GpIX, GpIa, GpIIa, GpIIb, GpIIIa and GpIIIb or a complex thereof.
- 前記血液由来試料中の血小板由来マイクロベシクルを、担体上に固定化された前記抗体と接触させる方法、または血小板由来マイクロベシクルと前記抗体との複合体を形成させた後に、担体上に固定化された前記抗体に対する二次抗体と接触させる方法により除去する、請求項３～５のいずれかに記載の分析方法。 A method of contacting platelet-derived microvesicles in the blood-derived sample with the antibody immobilized on a carrier, or a complex of platelet-derived microvesicles and the antibody is formed and then immobilized on the carrier. The analysis method according to any one of claims 3 to 5, which is removed by a method of contacting with a secondary antibody against the antibody.
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