JP2017181381A - Method of detecting severity of arterial stenosis - Google Patents
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Abstract
Description
本発明は、アテローム血栓症のイベントリスクの判定や、アテローム血栓症に対する治療効果の判定などを行うために有用な、動脈狭窄の程度を検知する方法に関する。 The present invention relates to a method for detecting the degree of arterial stenosis, which is useful for determining the event risk of atherothrombosis and the therapeutic effect on atherothrombosis.
脳梗塞などの脳血管疾患や心筋梗塞などの心血管疾患の原因となるアテローム血栓症（ＡＴＩＳ：Ａｔｈｅｒｏｔｈｒｏｍｂｏｓｉｓ）は、動脈壁肥厚が進行した結果、血管内部に動脈硬化性のプラーク（粥腫）が形成され、内皮の損傷やプラークの破綻などによって血小板が活性化されて血栓が形成されるに至った状態であり、近年、死亡原因となる疾患として著しく増加していることは一般にもよく知られた事実である。従って、そのイベントリスクの判定を正確に行い、リスクが高い患者に対して適切な治療を行うことは、生命の危険や重篤な機能障害の発生を回避する上で非常に重要である。現在、アテローム血栓症の診断は、超音波（エコー）、血管造影、ＭＲＩなどの画像に基づいて行われている。しかしながら、これらの画像による診断は、画像の良し悪しが撮影者の技術によって左右されるため、いずれもゴールドスタンダードになり得ない。こうした点に鑑み、アテローム血栓症のイベントリスクの判定や、アテローム血栓症に対する治療効果の判定などを行うために有用な、動脈壁肥厚の程度を検知するためのバイオマーカーとして、Ｃ３ａ−ｄｅｓＡｒｇ（アルギニン欠損型補体成分Ｃ３ａ）が特許文献１において提案されている。Ｃ３ａ−ｄｅｓＡｒｇは、巨大な補体成分であるＣ３が分解されて産生される分子量が約９０００のＣ３ａのＣ末端のアルギニンが切除されることで生成する物質であることは、当業者に周知の通りである。 Atherothrombosis (ATIS), which is a cause of cerebrovascular diseases such as cerebral infarction and cardiovascular diseases such as myocardial infarction, is caused by the progression of arterial wall thickening, resulting in atherosclerotic plaque (atheroma) inside the blood vessel. It is a well-known fact that it has increased significantly as a disease causing death in recent years because platelets are activated and blood clots are formed due to endothelial damage and plaque failure. It is a fact. Therefore, it is very important to accurately determine the event risk and to appropriately treat a high-risk patient in order to avoid the risk of life and the occurrence of serious dysfunction. At present, diagnosis of atherothrombosis is performed based on images such as ultrasound (echo), angiography, and MRI. However, the diagnosis based on these images cannot be the gold standard because the quality of the image depends on the technique of the photographer. In view of these points, C3a-desArg (arginine) is a biomarker for detecting the degree of arterial wall thickening, which is useful for determining the risk of atherothrombotic events and determining the therapeutic effect on atherothrombosis. A defective complement component C3a) is proposed in US Pat. It is well known to those skilled in the art that C3a-desArg is a substance produced by excision of C3a C-terminal arginine having a molecular weight of about 9000 produced by degradation of C3, which is a large complement component. Street.
アテローム血栓症のイベントリスクの判定や、アテローム血栓症に対する治療効果の判定などを行うために、動脈狭窄の程度を検知することは、動脈壁肥厚の程度を検知することと同様に有用である。特許文献１の教示によれば、Ｃ３ａ−ｄｅｓＡｒｇは、動脈狭窄の程度を検知するためのバイオマーカーとしても用いることができることが期待される。しかしながら、被験者由来の血液検体（血清）中のＣ３ａ−ｄｅｓＡｒｇ量は、採血してから血清を分離するまでの時間や、血清を分離してからＣ３ａ−ｄｅｓＡｒｇ量の測定を開始するまでの時間の経過に伴って増加するため、その血中濃度の定量性を担保することができないことが知られている（必要であれば例えば特許文献２を参照のこと）。従って、被験者由来の血清中のＣ３ａ−ｄｅｓＡｒｇ量が多いことが、被験者のＣ３ａ−ｄｅｓＡｒｇの血中濃度が高いことを必ずしも意味しないことから、Ｃ３ａ−ｄｅｓＡｒｇが動脈狭窄の程度を検知するためのバイオマーカーとして用いることができたとしても、例えば血清中のＣ３ａ−ｄｅｓＡｒｇ量が多いので動脈狭窄の程度が強いといったような判定は一概にすることができない点において改善の余地がある。 Detecting the degree of arterial stenosis in order to determine the event risk of atherothrombosis and the therapeutic effect on atherothrombosis is useful as well as detecting the degree of arterial wall thickening. According to the teaching of Patent Document 1, it is expected that C3a-desArg can also be used as a biomarker for detecting the degree of arterial stenosis. However, the amount of C3a-desArg in the blood sample (serum) derived from the subject is the time from blood collection to separation of the serum and the time from separation of the serum to the start of measurement of the amount of C3a-desArg. Since it increases with progress, it is known that the quantitativeness of the blood concentration cannot be ensured (see, for example, Patent Document 2 if necessary). Therefore, since a large amount of C3a-desArg in the serum derived from the subject does not necessarily mean that the blood concentration of C3a-desArg in the subject is high, a bio for C3a-desArg to detect the degree of arterial stenosis. Even if it can be used as a marker, there is room for improvement in that it is impossible to unify such a determination that the degree of arterial stenosis is strong because the amount of C3a-desArg in the serum is large.
そこで本発明は、アテローム血栓症のイベントリスクの判定や、アテローム血栓症に対する治療効果の判定などを行うために有用な、定量性に優れた動脈狭窄の程度を検知する方法を提供することを目的とする。 Therefore, the present invention has an object to provide a method for detecting the degree of arterial stenosis excellent in quantitativeness, which is useful for determining the event risk of atherothrombosis and determining the therapeutic effect on atherothrombosis. And
本発明者らは上記の点に鑑みて鋭意検討を行った結果、ＥＤＴＡ（エチレンジアミン四酢酸）血漿検体に、２価の金属イオンとしてカルシウムイオンと、抗凝固剤としてヘパリンを加えた検体が、動脈狭窄の程度を検知するための検体として有用であること、この検体中の、Ｃ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識する抗体で検出される物質の存在量が、動脈狭窄の程度を検知するための定量性に優れた指標となることを見出した。 As a result of intensive studies in view of the above points, the present inventors have found that a specimen obtained by adding calcium ion as a divalent metal ion and heparin as an anticoagulant to an EDTA (ethylenediaminetetraacetic acid) plasma specimen is an artery. It is useful as a specimen for detecting the degree of stenosis, and the amount of a substance detected by an antibody that recognizes both C3a and C3a-desArg in this specimen is used to detect the degree of arterial stenosis. It was found that the index is excellent in quantitativeness.
上記の知見に基づいてなされた本発明の動脈狭窄の程度を検知する方法は、請求項１記載の通り、ＥＤＴＡ血漿検体に、２価の金属イオンと、抗凝固剤としてヘパリンまたはその塩を加えた検体中の、Ｃ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識する抗体で検出される物質の存在量を指標とすることによる。
また、請求項２記載の方法は、請求項１記載の方法において、２価の金属イオンがカルシウムイオンである。
また、本発明の動脈狭窄の程度を検知するための検体を調製する方法は、請求項３記載の通り、ＥＤＴＡ血漿検体に、２価の金属イオンと、抗凝固剤としてヘパリンまたはその塩を加えることによる。
また、本発明は、請求項４記載の通り、ＥＤＴＡ血漿検体に、２価の金属イオンと、抗凝固剤としてヘパリンまたはその塩を加えた検体の、動脈狭窄の程度を検知するための検体としての使用である。
The method for detecting the degree of arterial stenosis of the present invention based on the above findings is as described in claim 1 in which divalent metal ions and heparin or a salt thereof as an anticoagulant are added to an EDTA plasma specimen. By using as an index the amount of a substance detected by an antibody that recognizes both C3a and C3a-desArg in the sample.
The method according to claim 2 is the method according to claim 1, wherein the divalent metal ion is a calcium ion.
The method for preparing a specimen for detecting the degree of arterial stenosis according to the present invention comprises adding a divalent metal ion and heparin or a salt thereof as an anticoagulant to an EDTA plasma specimen as described in claim 3. It depends.
The present invention also provides a sample for detecting the degree of arterial stenosis of a sample obtained by adding a divalent metal ion and heparin or a salt thereof as an anticoagulant to an EDTA plasma sample as described in claim 4. Is the use of.
本発明によれば、アテローム血栓症のイベントリスクの判定や、アテローム血栓症に対する治療効果の判定などを行うために有用な、定量性に優れた動脈狭窄の程度を検知する方法を提供することができる。 According to the present invention, it is possible to provide a method for detecting the degree of arterial stenosis with excellent quantitativeness, which is useful for determining the event risk of atherothrombosis and determining the therapeutic effect on atherothrombosis. it can.
本発明の動脈狭窄の程度を検知する方法は、ＥＤＴＡ血漿検体に、２価の金属イオンと、抗凝固剤としてヘパリンまたはその塩を加えた検体中の、Ｃ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識する抗体で検出される物質の存在量を指標とすることによるものである。以下、本発明の動脈狭窄の程度を検知する方法を、順を追って説明する。 The method for detecting the degree of arterial stenosis according to the present invention recognizes both C3a and C3a-desArg in a sample obtained by adding a divalent metal ion and heparin or a salt thereof as an anticoagulant to an EDTA plasma sample. This is because the amount of the substance detected by the antibody is used as an index. Hereinafter, the method for detecting the degree of arterial stenosis of the present invention will be described in order.
本発明の動脈狭窄の程度を検知する方法においては、まず、ＥＤＴＡ血漿検体に、２価の金属イオンと、抗凝固剤としてヘパリンやその塩を加える。ＥＤＴＡ血漿検体は、例えば市販のＥＤＴＡ２ナトリウム入りの採血管を用いて採血した被験者の血液から調製される、一般的な血液検査を行うためのものであってよい。 In the method for detecting the degree of arterial stenosis of the present invention, first, divalent metal ions and heparin or a salt thereof as an anticoagulant are added to an EDTA plasma specimen. The EDTA plasma specimen may be for performing a general blood test prepared from blood of a subject collected using, for example, a commercially available blood collection tube containing disodium EDTA.
ＥＤＴＡ血漿検体に加える２価の金属イオンは、血漿中のカルシウムイオンに対するＥＤＴＡによるキレート化によってＣ３カスケードの進行が阻止されている状態を解除することで、検体中においてＣ３カスケードを進行させて、検体中にＣ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識する抗体で検出される物質を生成させるためのものである。こうしてＣ３カスケードを進行させることによって生成する物質の存在量が、動脈狭窄の程度を検知するための指標となる。２価の金属イオンとしては、例えばカルシウムイオンが挙げられるが、マグネシウムイオンや亜鉛イオンなどであってもよい。ＥＤＴＡによってＣ３カスケードの進行が阻止されている状態を解除するためには、２価の金属イオンの添加量は、検体に含まれるＥＤＴＡ１モルに対して１モル以上が望ましく２モル以上がより望ましい。 The divalent metal ion added to the EDTA plasma specimen advances the C3 cascade in the specimen by releasing the state in which the progress of the C3 cascade is blocked by chelation by EDTA to calcium ions in the plasma, It is for generating the substance detected with the antibody which recognizes both C3a and C3a-desArg inside. Thus, the abundance of the substance generated by advancing the C3 cascade serves as an index for detecting the degree of arterial stenosis. Examples of the divalent metal ions include calcium ions, but may be magnesium ions or zinc ions. In order to cancel the state in which the progress of the C3 cascade is blocked by EDTA, the amount of the divalent metal ion added is preferably 1 mol or more and more preferably 2 mol or more with respect to 1 mol of EDTA contained in the specimen.
ＥＤＴＡ血漿検体に加える抗凝固剤は、ヘパリンやその塩（ナトリウム塩など）であり、検体に２価の金属イオンを加えることにより、ＥＤＴＡが血漿中のカルシウムイオンをキレート化することによる抗凝固作用が解除されることで検体中に不溶性の血液凝固物が生成することを阻止し、血液凝固物が、検体中のＣ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識する抗体で検出される物質の存在量の測定に悪影響（ピペッティング操作の妨げなど）を及ぼさないようにするためのものである。抗凝固剤の添加量は、検体中に血液凝固物が生成することを効果的に阻止するために、検体中の濃度が１Ｕｎｉｔ／ｍｌ以上になるように加えることが望ましい（上限は例えば１２Ｕｎｉｔ／ｍｌである）。検体中に血液凝固物が生成することを効果的に阻止するためには、抗凝固剤は２価の金属イオンと同時に検体に加えることが望ましい。 Anticoagulants added to EDTA plasma specimens are heparin and its salts (sodium salts, etc.), and by adding divalent metal ions to the specimen, EDTA chelates calcium ions in the plasma, thereby causing anticoagulant action. Is released to prevent the formation of insoluble blood coagulum in the sample, and the amount of the substance detected by the antibody that recognizes both C3a and C3a-desArg in the sample. This is to prevent adverse effects on the measurement (such as hindering pipetting operations). The addition amount of the anticoagulant is desirably added so that the concentration in the sample becomes 1 Unit / ml or more in order to effectively prevent the formation of blood coagulum in the sample (the upper limit is, for example, 12 Unit / ml). ml). In order to effectively prevent the formation of blood clots in the specimen, it is desirable to add the anticoagulant to the specimen simultaneously with the divalent metal ions.
ＥＤＴＡ血漿検体に２価の金属イオンと抗凝固剤を加えた検体中の、Ｃ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識する抗体で検出される物質の存在量を測定するために用いる、Ｃ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識する抗体は、モノクローナル抗体であってもよいし、ポリクローナル抗体であってもよい。また、市販の抗体や公知の抗体であってもよいし、自家作製した抗体であってもよい。抗体は、容易かつ簡便に動脈狭窄の程度を検知することができるように、洗浄液などとともにキット化してもよい。Ｃ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識する抗体で検出される物質の存在量の測定は、抗原抗体反応を利用する免疫学的手法、具体的には、ＥＬＩＳＡ（Ｅｎｚｙｍｅ−Ｌｉｎｋｅｄ Ｉｍｍｕｎｏｓｏｒｂｅｎｔ Ａｓｓａｙ）法、ウエスタンブロッティング法、ラテックス凝集比濁法、フローサイトメトリー法などにより、それぞれの方法の標準的なプロトコルに従って行うことができる。その測定は、検体中においてＣ３カスケードを進行させて、検体中にＣ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識する抗体で検出される物質を生成させるため、ＥＤＴＡ血漿検体を例えばｐＨ５．０〜７．０に調整した後、２価の金属イオンを加えてから（同時に抗凝固剤を加えることが望ましいことは上述の通りである）、３５〜４０℃で３０分間以上保温した後に行うことが望ましい（検体の調製から測定に至るまでの時間と測定に要する時間の合計時間を考慮すれば保温時間は２時間以下が望ましい）。 C3a and C3a- used for measuring the amount of a substance detected by an antibody recognizing both C3a and C3a-desArg in a sample obtained by adding a divalent metal ion and an anticoagulant to an EDTA plasma sample The antibody that recognizes both desArg may be a monoclonal antibody or a polyclonal antibody. Moreover, a commercially available antibody, a well-known antibody may be sufficient, and the antibody produced in-house may be sufficient. The antibody may be kitted together with a washing solution so that the degree of arterial stenosis can be detected easily and simply. The amount of a substance detected by an antibody recognizing both C3a and C3a-desArg is measured by an immunological technique using an antigen-antibody reaction, specifically, an ELISA (Enzyme-Linked Immunosorbent Assay) method, Western Blotting, latex agglutination turbidimetry, flow cytometry, and the like can be performed according to the standard protocol of each method. The measurement proceeds with the C3 cascade in the sample to generate a substance that is detected by an antibody that recognizes both C3a and C3a-desArg in the sample, so that the EDTA plasma sample has a pH of 5.0 to 7.0, for example. It is desirable to carry out after maintaining the temperature at 35 to 40 ° C. for 30 minutes or more after adding the divalent metal ion (the addition of the anticoagulant is desirable as described above). Taking into account the total time from preparation to measurement and the time required for measurement, the incubation time is preferably 2 hours or less).
本発明の動脈狭窄の程度を検知する方法において検知対象とする動脈は、狭窄が起こりうる動脈であれば体内のどの部位に存在する動脈であってもよいが、検知対象とする好適な動脈としては頸動脈が挙げられる。検知対象とする動脈が頸動脈の場合、本発明の方法は、例えば次のようにして利用することができる。健康診断などにおいて頸動脈におけるアテローム血栓症のイベントリスクの判定を行うために、頸動脈狭窄（ＣＡＳ：ｃａｒｏｔｉｄ ａｒｔｅｒｙ ｓｔｅｎｏｓｉｓ）の程度を知ることを目的として、被験者由来のＥＤＴＡ血漿検体に２価の金属イオンと抗凝固剤を加えた検体中の、Ｃ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識する抗体で検出される物質の存在量を測定し、頸動脈狭窄の程度が軽度（例えばＥＣＳＴ法やＮＡＳＣＥＴ法による狭窄率（ＳＲ：ｓｔｅｎｏｓｉｓ ｒａｔｅ）が３０％より大きく６０％より小さい）と重度（ＳＲが６０％より大きい）のそれぞれの場合の予め定めた標準存在量と比較する。比較の結果、被験者の存在量が、頸動脈狭窄の程度が軽度の場合の標準存在量よりも少なければ治療の必要なし、軽度の場合の標準存在量に相当すれば薬物療法を実施する、重度の場合の標準存在量に相当すれば頸動脈内膜剥離術（ＣＥＡ）を施行するといったように、治療の必要性の有無を判断したり、治療方針を策定したりする。また、頸動脈内膜剥離術を施行する必要性の有無を判断するための測定値の基準値（カットオフ値）を予め定めておけば、健常者やアテローム血栓症患者と頸動脈内膜剥離術対象患者を区別することができる。こうした治療の必要性の有無の判断や治療方針の策定を行う際、現在、アテローム血栓症の診断に利用されている、超音波（エコー）、血管造影、ＭＲＩなどの画像による診断の結果を参考にしてもよい。また、本発明の方法は、所定の治療を行った患者を被験者として、そのＥＤＴＡ血漿検体に２価の金属イオンと抗凝固剤を加えた検体中の、Ｃ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識する抗体で検出される物質の存在量を測定し、治療前の存在量と比較することで、頸動脈におけるアテローム血栓症に対する治療効果の判定などにも利用することもできる。 In the method for detecting the degree of arterial stenosis according to the present invention, the artery to be detected may be an artery present in any part of the body as long as the artery can cause stenosis. Includes the carotid artery. When the artery to be detected is the carotid artery, the method of the present invention can be used as follows, for example. In order to determine the risk of carotid artery stenosis (CAS) in order to determine the event risk of atherothrombosis in the carotid artery in medical examinations, etc., a divalent metal is added to the subject EDTA plasma specimen. The amount of a substance detected by an antibody that recognizes both C3a and C3a-desArg in a sample to which ions and an anticoagulant are added is measured, and the degree of carotid artery stenosis is mild (for example, by the ECST method or NASCET method). Comparison is made with a predetermined standard abundance in each case of stenosis rate (SR: greater than 30% and less than 60%) and severe (SR is greater than 60%). As a result of comparison, if the abundance of the subject is less than the standard abundance when the degree of carotid artery stenosis is mild, no treatment is necessary, and if the abundance is equivalent to the standard abundance in the mild case, drug therapy is performed. If it corresponds to the standard abundance in the above case, carotid endarterectomy (CEA) is performed, and the necessity of treatment is judged, or a treatment policy is formulated. In addition, if a reference value (cut-off value) for determining the necessity of performing carotid endarterectomy is determined in advance, normal patients and patients with atherothrombosis and carotid endarterectomy The target patient can be distinguished. When determining the necessity of such treatment and formulating a treatment policy, refer to the results of diagnosis using images such as ultrasound (echo), angiography, and MRI, which are currently used for the diagnosis of atherothrombosis. It may be. In addition, the method of the present invention recognizes both C3a and C3a-desArg in a sample obtained by adding a divalent metal ion and an anticoagulant to the EDTA plasma sample, using a patient who has undergone predetermined treatment as a subject. By measuring the abundance of the substance detected by the antibody and comparing it with the abundance before treatment, it can also be used to determine the therapeutic effect on atherothrombosis in the carotid artery.
以下、本発明を実施例によって詳細に説明するが、本発明は以下の記載に限定して解釈されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is limited to the following description and is not interpreted.
参考例１：Ｃ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識するモノクローナル抗体の作製（その１）
（Ａ）ハイブリドーマの作製
Ｃ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識するモノクローナル抗体を作製するため、マウスへの免疫を以下のようにして行った。免疫抗原はＣ３ａ−ｄｅｓＡｒｇのＣ末端１１アミノ酸ペプチド（ＱＨＡＲＡＳＨＬＧＬＡ：配列番号１）のＫＬＨコンジュゲートとし、１０μｇ（ペプチド相当）をアジュバントであるＴｉｔｅｒＭａｘ Ｇｏｌｄ（ＴｉｔｅｒＭａｘ ＵＳＡ）とともにＢＡＬＢ／ｃマウスに腹腔内投与した。２〜４週間ごとに同様の追加免疫を行い、さらにＰＢＳに溶解したペプチド１０μｇを最終免疫として尾静脈内に投与した。
Reference Example 1: Preparation of monoclonal antibody that recognizes both C3a and C3a-desArg (Part 1)
(A) Production of hybridoma To produce a monoclonal antibody that recognizes both C3a and C3a-desArg, immunization of mice was performed as follows. The immunizing antigen was KLH conjugate of C3a-desArg C-terminal 11 amino acid peptide (QHARASHLGLA: SEQ ID NO: 1), and 10 μg (equivalent to peptide) was intraperitoneally administered to BALB / c mice together with TiterMax Gold (TiterMax USA) as an adjuvant. . The same boosting was performed every 2 to 4 weeks, and 10 μg of peptide dissolved in PBS was administered into the tail vein as the final immunization.
最終免疫後３日目にマウスから脾臓を無菌的に摘出し、ハサミおよび金属メッシュを用いて脾臓を個々の細胞にほぐし、ＲＰＭＩ−１６４０培地で３回洗浄した。対数増殖期のマウス骨髄腫細胞株Ｓｐ２／０−Ａｇ１４をＲＰＭＩ−１６４０培地で３回洗浄後、この細胞と脾臓細胞を１：５の細胞数比で混合した。２００×ｇで５分間遠心した後、上清を除去し、細胞隗を緩やかに混合しながら５０％ポリエチレングリコール（ＰＥＧ）４０００（Ｍｅｒｃｋ）を含むＲＰＭＩ−１６４０培地１ｍｌをゆっくりと加え、さらにＲＰＭＩ−１６４０培地１０ｍｌを加えて細胞融合させた。 Three days after the final immunization, the spleen was aseptically removed from the mouse, and the spleen was loosened into individual cells using scissors and a metal mesh, and washed three times with RPMI-1640 medium. The mouse myeloma cell line Sp2 / 0-Ag14 in the logarithmic growth phase was washed three times with RPMI-1640 medium, and the cells and spleen cells were mixed at a cell number ratio of 1: 5. After centrifugation at 200 × g for 5 minutes, the supernatant was removed, and 1 ml of RPMI-1640 medium containing 50% polyethylene glycol (PEG) 4000 (Merck) was slowly added while gently mixing the cell sputum, and further RPMI− Cell fusion was performed by adding 10 ml of 1640 medium.
得られた融合細胞を、２００×ｇで５分間遠心してＰＥＧを除いた後、１０％ウシ胎児血清およびヒポキサンチン・アミノプテリン・チミジン（ＨＡＴ）を含むＲＰＭＩ−１６４０培地に懸濁し、９６ウエル細胞培養プレートに播種した。約１０日間培養してハイブリドーマのみを増殖させた後、免疫抗原に対して特異的に反応するモノクローナル抗体を産生するクローンをＥＬＩＳＡ法により検索し、所望のハイブリドーマを得、限界希釈法により単一クローン化を行い、ハイブリドーマを樹立した。 The obtained fused cells were centrifuged at 200 × g for 5 minutes to remove PEG, and then suspended in RPMI-1640 medium containing 10% fetal bovine serum and hypoxanthine / aminopterin / thymidine (HAT) to obtain 96-well cells. Inoculated on culture plates. After culturing for about 10 days and growing only the hybridoma, a clone producing a monoclonal antibody that reacts specifically with the immunizing antigen is searched by ELISA to obtain the desired hybridoma, and a single clone is obtained by limiting dilution. And a hybridoma was established.
（Ｂ）Ｃ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識するモノクローナル抗体の作製および解析
（Ａ）で樹立したハイブリドーマの１つ（クローン名：Ｃ３ａ−１９）を、無血清培地（Ｈｙｂｒｉｄｏｍａ−ＳＦＭ、ＧＩＢＣＯ）を用い、３７℃にて５％炭酸ガス中において７２〜９６時間培養した。培地中に産生されたモノクローナル抗体を精製するため、その培養液を、プロテインＡセファロースカラムを用いたアフィニティークロマトグラフィーにかけた。
(B) Production and analysis of monoclonal antibody that recognizes both C3a and C3a-desArg One of the hybridomas established in (A) (clone name: C3a-19) was treated with serum-free medium (Hybridoma-SFM, GIBCO). The culture was performed at 37 ° C. in 5% carbon dioxide gas for 72 to 96 hours. In order to purify the monoclonal antibody produced in the medium, the culture solution was subjected to affinity chromatography using a protein A sepharose column.
Ｃ３ａ−１９が産生するモノクローナル抗体について、抗マウスＩｇ各アイソタイプ抗体を用いたアイソタイプタイピングキット（Ｚｙｍｅｄ）により、サブクラスを同定した結果、ＩｇＧ１であることがわかった。また、Ｃ３ａ−１９が産生するモノクローナル抗体は、市販のヒトＣ３ａ精製物（Ｃａｌｂｉｏｃｈｅｍ）と市販のヒトＣ３ａ−ｄｅｓＡｒｇ精製物（Ｃａｌｂｉｏｃｈｅｍ）の両方に反応することがわかった。 As a result of identifying the subclass of the monoclonal antibody produced by C3a-19 with an isotype typing kit (Zymed) using anti-mouse Ig isotype antibodies, it was found to be IgG1. Moreover, it was found that the monoclonal antibody produced by C3a-19 reacts with both a commercially available human C3a purified product (Calbiochem) and a commercially available human C3a-desArg purified product (Calbiochem).
参考例２：Ｃ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識するモノクローナル抗体の作製（その２）
免疫抗原としてＣ３ａ−ｄｅｓＡｒｇのＮ端側１６アミノ酸ペプチド（ＫＲＭＤＫＶＧＫＹＰＫＥＬＲＫＣ：配列番号２）のＫＬＨコンジュゲートを用いること以外は参考例１と同様にして、所望のハイブリドーマを樹立した。その１つ（クローン名：Ｃ３Ｎ−６）が産生するモノクローナル抗体について、参考例１と同様にしてサブクラスを同定した結果、ＩｇＧ１であることがわかった。また、Ｃ３Ｎ−６が産生するモノクローナル抗体は、市販のヒトＣ３ａ精製物と市販のヒトＣ３ａ−ｄｅｓＡｒｇ精製物の両方に反応することがわかった。
Reference Example 2: Preparation of monoclonal antibody that recognizes both C3a and C3a-desArg (Part 2)
A desired hybridoma was established in the same manner as in Reference Example 1 except that the KLH conjugate of the N-
実施例１：本発明の方法による頚動脈狭窄の程度の検知
（実験方法）
頚動脈狭窄の程度が軽度（ＥＣＳＴ法によるＳＲが６０％未満）で血管イベントの既往がない４８例（ＣＡＳ ＳＲ＜６０％ ｗ／ｏ ｅｖｅｎｔｓ群）と、頸動脈狭窄の程度が重度（ＥＣＳＴ法によるＳＲが６０％以上）であることから頸動脈内膜剥離術の施行が必要と判断された７５例（ＣＡＳ ＳＲ≧６０％群）について、それぞれの患者から同意を得てＥＤＴＡ血漿検体を採取した。検体をリン酸緩衝液によりｐＨ６．０に調整した後、ＣａＣｌ２とへパリンＮａ（ＷＡＫＯ）を最終濃度がそれぞれ１０ｍＭと８Ｕｎｉｔ／ｍｌになるように同時に加え（ＣａＣｌ２の添加量はＥＤＴＡのモル数の２倍モル量）、３７℃で１時間保温してから、後述するプロトコルに従ってＥＬＩＳＡを行い、Ｃ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識する抗体で検出される物質の存在量の測定を行った。なお、採血してから血漿を分離するまでの時間や、血漿を分離してからＣ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識する抗体で検出される物質の存在量の測定を開始するまでの時間の経過の違いにより、Ｃ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識する抗体で検出される物質の存在量が変化することはほとんどないことを予め確認していたので、これらの時間は特に定めなかった。
Example 1: Detection of the degree of carotid artery stenosis by the method of the present invention (experimental method)
48 cases with mild carotid artery stenosis (less than 60% SR by ECST) and no history of vascular events (CAS SR <60% w / o events group), severe carotid artery stenosis (by ECST method) With regard to 75 cases (CAS SR ≧ 60% group) judged to require carotid endarterectomy, EDTA plasma samples were obtained from each patient with consent. . After adjusting the specimen to pH 6.0 with phosphate buffer, CaCl 2 and heparin Na (WAKO) were added simultaneously so that the final concentrations were 10 mM and 8 Unit / ml, respectively (the amount of CaCl 2 added was the molar amount of EDTA). (2 times the molar amount), and incubated at 37 ° C. for 1 hour, followed by ELISA according to the protocol described later, and the amount of substance detected by the antibody recognizing both C3a and C3a-desArg was measured. . The time from blood collection to plasma separation, and the time elapsed from the start of plasma separation until the start of measurement of the amount of a substance detected by an antibody that recognizes both C3a and C3a-desArg Since it was previously confirmed that the abundance of the substance detected by the antibody recognizing both C3a and C3a-desArg was hardly changed by these differences, these times were not particularly defined.
（実験結果）
ＣＡＳ ＳＲ＜６０％ ｗ／ｏ ｅｖｅｎｔｓ群の４８例とＣＡＳ ＳＲ≧６０％群の７５例の両群の、Ｃ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識する抗体で検出される物質の存在量の測定結果を、Ｍａｎｎ−Ｗｈｉｔｎｅｙ検定により比較したところ、ｐ＜０．０００１で両群を区分することができた（図１）。また、両群について、Ｃ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識する抗体で検出される物質の存在量から予測したＲＯＣ曲線のＡＵＣ（曲線下面積）は０．８９６３であり、頸動脈狭窄の程度の違いによって両群を区別する上において、Ｃ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識する抗体で検出される物質の存在量を測定することの有用性を確認することができた（図２）。ＲＯＣ解析の結果から、暫定的に設定したカットオフ値（４０．０ｎｍｏｌ／ｌ）を用いて両群を見極めることの可能性を評価すると、表１に示すように、感度８４．０％（６３／７５）、特異度８１．３％（３９／４８）、正診率８２．９％（１０２／１０３）となり、頸動脈狭窄の程度の違いによる両群の見極めが可能であると考えられた。検体にＣａＣｌ２を加えるのと同時にヘパリンＮａを加えたことで、例えばピペッティング操作の妨げとなるような血液凝固物の生成は認められなかった。
(Experimental result)
Measurement results of the abundance of substances detected by antibodies recognizing both C3a and C3a-desArg in 48 groups of CAS SR <60% w / o events group and 75 groups of CAS SR ≧ 60% group Were compared by Mann-Whitney test, both groups could be distinguished by p <0.0001 (FIG. 1). In both groups, the ROC curve AUC (area under the curve) predicted from the amount of the substance detected by the antibody recognizing both C3a and C3a-desArg is 0.8963, indicating the degree of carotid artery stenosis. In distinguishing both groups by difference, the usefulness of measuring the abundance of a substance detected by an antibody recognizing both C3a and C3a-desArg could be confirmed (FIG. 2). From the results of the ROC analysis, when the possibility of discriminating both groups using a provisionally set cutoff value (40.0 nmol / l) was evaluated, as shown in Table 1, the sensitivity was 84.0% (63 / 75), specificity 81.3% (39/48), correct diagnosis rate 82.9% (102/103), and it was thought that it was possible to identify both groups depending on the degree of carotid artery stenosis . When heparin Na was added to the specimen at the same time as CaCl 2 was added, for example, the formation of blood coagulation that hindered the pipetting operation was not observed.
なお、ＥＤＴＡ血漿検体をリン酸緩衝液によりｐＨ６．０に調整した後、ＣａＣｌ２とへパリンＮａを同時に加えてからの３７℃での保温時間と、Ｃ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識する抗体で検出される物質の存在量の関係を、ＣＡＳ ＳＲ≧６０％群のＥＤＴＡ血漿検体２例について表２に示す（いずれも保温時間が長くなると存在量は増加する）。 An antibody that recognizes both C3a and C3a-desArg after maintaining the incubation time at 37 ° C. after adding CaCl 2 and heparin Na simultaneously after adjusting EDTA plasma specimen to pH 6.0 with phosphate buffer Table 2 shows the relationship of the abundance of substances detected in Table 2 for two EDTA plasma samples in the CAS SR ≧ 60% group (both of them increase as the incubation time becomes longer).
（Ｃ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識する抗体で検出される物質の存在量を測定するためのＥＬＩＳＡ法のプロトコル）
参考例２で作製したモノクローナル抗体（Ｃ３Ｎ−６）を、終濃度が５μｇ／ｍｌになるように０．１５Ｍ ＮａＣｌを含む１０ｍＭリン酸ナトリウム緩衝液（ｐＨ７．３）で希釈し、９６ウエルマイクロプレート（ヌンク社）１ウエルにつき１００μｌづつ分注した。４℃で一晩静置した後、０．１５Ｍ ＮａＣｌを含む１０ｍＭリン酸ナトリウム緩衝液（ｐＨ７．３）０．３５ｍｌを用いてそれぞれのウエルを２回洗浄した。その後、０．５％カゼイン−Ｎａを含む１０ｍＭリン酸ナトリウム緩衝液（ｐＨ７．３）（以下「ブロッキング液」）０．３５ｍｌをそれぞれのウエルに添加し、さらに室温で４時間静置した。ブロッキング液を除去した後、０．２Ｍ ＮａＣｌと１％ＢＳＡと０．０７％カゼイン−Ｎａを含む１００ｍＭリン酸ナトリウム緩衝液（ｐＨ７．３）９０μｌと、検体または段階的に希釈した標準物質（特許文献１に記載の方法で調製したＴｒｐＥ−Ｃ３ａ−ｄｅｓＡｒｇ融合抗原）１０μｌを、それぞれのウエルに添加して１００μｌとし、室温で１時間反応させた。反応後、０．１５Ｍ ＮａＣｌと０．０５％ Ｔｗｅｅｎ２０を含む１０ｍＭリン酸ナトリウム緩衝液（ｐＨ７．３）（以下「洗浄液」）０．３５ｍｌを用いてそれぞれのウエルを５回洗浄した。その後、参考例１で作製したモノクローナル抗体（Ｃ３ａ−１９）をペルオキシダーゼ（ＰＯＤ）標識して調製した標識抗体液（０．２Ｍ ＮａＣｌと１％ＢＳＡと０．０７％カゼイン−Ｎａを含む１００ｍＭリン酸ナトリウム緩衝液（ｐＨ７．３））１００μｌをそれぞれのウエルに添加し、室温で３０分間反応させた。洗浄液０．３５ｍｌを用いてそれぞれのウエルを５回洗浄した後、基質（オルトフェニレンジアミン）溶液１００μｌを添加し、室温暗所で３０分間反応させた。反応後、２Ｎ硫酸溶液１００μｌをそれぞれのウエルに添加し、波長６３０ｎｍの吸光度を対照として波長４９０ｎｍにおける吸光度（ＯＤ４９０）を測定することで、Ｃ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識する抗体で検出される物質の存在量を測定した。
(ELISA protocol protocol for measuring the abundance of substances detected by antibodies recognizing both C3a and C3a-desArg)
The monoclonal antibody (C3N-6) prepared in Reference Example 2 was diluted with 10 mM sodium phosphate buffer (pH 7.3) containing 0.15 M NaCl so that the final concentration was 5 μg / ml, and a 96-well microplate. (Nunk) 100 μl was dispensed per well. After standing overnight at 4 ° C., each well was washed twice with 0.35 ml of 10 mM sodium phosphate buffer (pH 7.3) containing 0.15 M NaCl. Thereafter, 0.35 ml of 10 mM sodium phosphate buffer (pH 7.3) (hereinafter “blocking solution”) containing 0.5% casein-Na was added to each well, and the mixture was allowed to stand at room temperature for 4 hours. After removing the blocking solution, 90 μl of 100 mM sodium phosphate buffer (pH 7.3) containing 0.2 M NaCl, 1% BSA and 0.07% casein-Na, and the sample or serially diluted standard (patented) 10 μl of TrpE-C3a-desArg fusion antigen prepared by the method described in Document 1 was added to each well to make 100 μl, and reacted at room temperature for 1 hour. After the reaction, each well was washed 5 times with 0.35 ml of 10 mM sodium phosphate buffer (pH 7.3) (hereinafter “washing solution”) containing 0.15 M NaCl and 0.05% Tween20. Thereafter, a labeled antibody solution prepared by labeling the monoclonal antibody (C3a-19) prepared in Reference Example 1 with peroxidase (POD) (100 mM phosphoric acid containing 0.2 M NaCl, 1% BSA, and 0.07% casein-Na). 100 μl of sodium buffer (pH 7.3)) was added to each well and allowed to react for 30 minutes at room temperature. Each well was washed 5 times with 0.35 ml of the washing solution, and then 100 μl of a substrate (orthophenylenediamine) solution was added and allowed to react for 30 minutes in the dark at room temperature. After the reaction, 100 μl of 2N sulfuric acid solution is added to each well, and the absorbance at a wavelength of 490 nm (OD490) is measured using the absorbance at a wavelength of 630 nm as a control, so that an antibody that recognizes both C3a and C3a-desArg is detected. The amount of substance present was measured.
実施例１の結果からのＣ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識する抗体で検出される物質に関する考察
Ｒｏｄｒｉｇｕｅｚらの方法（ＪＢＣ ２９０，２３３４−２３５０，２０１５）に従い、市販のヒトＣ３精製物（Ｃａｌｂｉｏｃｈｅｍ）を、２００ｍＭヒドラジンと０．１５Ｍ ＮａＣｌを含む１０ｍＭリン酸ナトリウム緩衝液（ｐＨ６．０）中、３７℃で２時間反応させ、Ｃ３の加水化物であるＣ３（Ｈ２Ｏ）を産生させた。反応終了後の試料について、実施例１に記載したプロトコルに従ってＥＬＩＳＡを行ったところ、Ｃ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識する抗体で検出される物質の存在量は７７５．８ｎｍｏｌ／ｌであった。これに対し、ヒドラジンを加えないこと以外は同じ条件で実験を行って得た試料のＣ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識する抗体で検出される物質の存在量は７５．３ｎｍｏｌ／ｌであったことから、Ｃ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識する抗体によってＣ３の加水化物であるＣ３（Ｈ２Ｏ）が検出されることがわかった。血液中にはＣ３（Ｈ２Ｏ）とともにＣ３ａ−ｄｅｓＡｒｇが存在し、Ｃ３ａ−ｄｅｓＡｒｇも、Ｃ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識する抗体で検出されることと、血液中にＣ３ａが存在したとしてもすぐさまカルボキシペプチダーゼＮによってＣ３ａ−ｄｅｓＡｒｇに変換されることを考えれば、本発明の方法においてＣ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識する抗体で検出される血液中の物質は、Ｃ３（Ｈ２Ｏ）とＣ３ａ−ｄｅｓＡｒｇの２者であり、Ｃ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識する抗体で検出される物質の存在量は、両者の存在量の合計量であると推察された。Ｃ３（Ｈ２Ｏ）とＣ３ａ−ｄｅｓＡｒｇの存在量の合計量の違いが、動脈狭窄の程度の違いを反映するのは、Ｃ３カスケードの第２経路の進行に関与する酵素、とりわけ、Ｃ３が加水化されることによるＣ３（Ｈ２Ｏ）への構造変換に関与する酵素の量や活性の違いによるものではないかと本発明者らは考察している。
Consideration about the substance detected with the antibody which recognizes both C3a and C3a-desArg from the result of Example 1 According to the method of Rodriguez et al. (JBC 290, 2334-2350, 2015), commercially available human C3 purified product (Calbiochem) Was reacted in 10 mM sodium phosphate buffer (pH 6.0) containing 200 mM hydrazine and 0.15 M NaCl for 2 hours at 37 ° C. to produce C3 (H 2 O), which is a hydrolyzate of C3. When the sample after the reaction was subjected to ELISA according to the protocol described in Example 1, the amount of the substance detected by the antibody recognizing both C3a and C3a-desArg was 775.8 nmol / l. In contrast, the amount of the substance detected by the antibody recognizing both C3a and C3a-desArg in the sample obtained by conducting the experiment under the same conditions except that hydrazine was not added was 75.3 nmol / l. Therefore, it was found that C3 (H 2 O), which is a hydrolyzate of C3, was detected by an antibody that recognizes both C3a and C3a-desArg. C3a-desArg is present in the blood together with C3 (H 2 O), and C3a-desArg is detected by an antibody that recognizes both C3a and C3a-desArg, and even if C3a is present in the blood Given that carboxypeptidase N immediately converts to C3a-desArg, the substance in the blood detected by the antibody that recognizes both C3a and C3a-desArg in the method of the present invention is C3 (H 2 O). It was speculated that the abundance of substances detected by antibodies that recognize both C3a and C3a-desArg, which are two of C3a-desArg, is the total of both abundances. The difference in the total amount of C3 (H 2 O) and C3a-desArg presents the difference in the degree of arterial stenosis because enzymes involved in the progression of the second pathway of the C3 cascade, particularly C3, are hydrolyzed. The present inventors consider that it may be due to the difference in the amount and activity of the enzyme involved in the structural conversion to C3 (H 2 O) by being converted to C3.
参考例３：ＥＤＴＡ血漿検体にＣａＣｌ２とへパリンＮａを同時に加えることの効果
ＥＤＴＡ血漿検体をリン酸緩衝液によりｐＨ６．０に調整した後、ＣａＣｌ２とへパリンＮａを最終濃度がそれぞれ１０ｍＭと１，４，８，１２Ｕｎｉｔ／ｍｌになるように同時に加え、３７℃で１時間保温してから、実施例１と同様にしてＥＬＩＳＡを行い、Ｃ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識する抗体で検出される物質の存在量の測定を行った。ＣＡＳ ＳＲ≧６０％群のＥＤＴＡ血漿検体３例についての結果を表３に示す。表３には、ＣａＣｌ２とへパリンＮａを加えない、リン酸緩衝液によりｐＨ６．０に調整したＥＤＴＡ血漿検体について、実施例１と同様にしてＥＬＩＳＡを行い、Ｃ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識する抗体で検出される物質の存在量の測定を行った結果をあわせて示す（「無処理」のカラム）。表３から明らかなように、ＥＤＴＡ血漿検体にＣａＣｌ２とへパリンＮａを同時に加えて３７℃で１時間保温することで、Ｃ３ａとＣ３ａ−ｄｅｓＡｒｇの両方を認識する抗体で検出される物質の存在量が増加した。へパリンＮａの最終濃度が１Ｕｎｉｔ／ｍｌの場合でも、例えばピペッティング操作の妨げとなるような血液凝固物の生成は認められなかった。検体にＣａＣｌ２だけを加えてヘパリンＮａを加えなかった場合、例えばピペッティング操作の妨げとなるような血液凝固物の生成が認められた。
Reference Example 3: Effect of simultaneous addition of CaCl 2 and heparin Na to EDTA plasma sample After adjusting the EDTA plasma sample to pH 6.0 with phosphate buffer, the final concentration of CaCl 2 and heparin Na was 10 mM respectively. Add to 1, 4, 8, 12 Unit / ml at the same time, incubate at 37 ° C for 1 hour, perform ELISA as in Example 1, and detect with antibody recognizing both C3a and C3a-desArg The abundance of substances to be measured was measured. The results for 3 EDTA plasma specimens in the CAS SR ≧ 60% group are shown in Table 3. Table 3 shows that EDTA plasma samples adjusted to pH 6.0 with phosphate buffer without adding CaCl 2 and heparin Na were subjected to ELISA in the same manner as in Example 1, and both C3a and C3a-desArg were determined. The result of measuring the amount of the substance detected by the antibody to be recognized is also shown (“no treatment” column). As is apparent from Table 3, the presence of a substance detected by an antibody that recognizes both C3a and C3a-desArg by simultaneously adding CaCl 2 and heparin Na to an EDTA plasma specimen and incubating at 37 ° C. for 1 hour. The amount increased. Even when the final concentration of heparin Na was 1 Unit / ml, for example, the formation of blood clots that hindered the pipetting operation was not observed. When only CaCl 2 was added to the specimen and no heparin Na was added, for example, the formation of blood clots that hindered the pipetting operation was observed.
本発明は、アテローム血栓症のイベントリスクの判定や、アテローム血栓症に対する治療効果の判定などを行うために有用な、定量性に優れた動脈狭窄の程度を検知する方法を提供することができる点において産業上の利用可能性を有する。 The present invention can provide a method for detecting the degree of arterial stenosis excellent in quantitativeness, which is useful for determining the event risk of atherothrombosis and the therapeutic effect on atherothrombosis. Has industrial applicability.
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