JP2020153832A - Method and reagent for detecting neuropathic pain through autotaxin measurement - Google Patents
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Abstract
Description
本発明は、ヒト脳脊髄液中のオートタキシン濃度を測定することにより、画像診断が困難な神経障害性疼痛を検出する方法および検出試薬に関する。 The present invention relates to a method and a detection reagent for detecting neuropathic pain for which diagnostic imaging is difficult by measuring the autotaxin concentration in human cerebrospinal fluid.
痛みの種類は、その原因によって大きく３種類に分類される。即ち、
１.怪我等による炎症や刺激を原因とする侵害受容性疼痛、
２.疾病を原因とし神経が障害される事による神経障害性疼痛、さらには
３.ストレス等が原因となる心因性疼痛
がある。
このうち神経障害性疼痛は、障害部分や炎症部分が表在しておらず、また原因が多岐にわたるため特定が容易ではない。脊柱管狭窄症の様に物理的な神経障害は、画像所見が認められるため、画像により確定診断が可能であるが、その一方で、帯状疱疹後神経痛、腰部癒着性くも膜炎などは画像所見が認められないため診断が困難な場合が多く、さらに心因性疼痛との弁別も困難な場合がある。ここで言う画像所見が認められる神経障害性疼痛、画像所見が認められない神経障害性疼痛を言い換えれば、それぞれ脊柱管狭窄症による神経障害性疼痛と脊柱管狭窄症によらない神経障害性疼痛と言い換える事もできる。このように画像所見が認められない神経障害性疼痛の診断は、その治療方針決定において重要だが、確度の高い検査がなく、多くの検査を繰り返し実施するため患者負担が大きいため、診断能の高い診断マーカーの開発が望まれている。
The types of pain are roughly classified into three types according to the cause. That is,
1. Nociceptive pain caused by inflammation and irritation caused by injury, etc.
2. There is neuropathic pain caused by nerve damage caused by illness, and 3. psychogenic pain caused by stress.
Of these, neuropathic pain is not easy to identify because the damaged part and the inflamed part are not superficial and the causes are diverse. Physical neuropathy such as spinal stenosis can be confirmed by imaging because imaging findings are observed, but on the other hand, imaging findings such as postherpetic neuralgia and lumbar adhesive arachnoiditis are observed. Diagnosis is often difficult because it is not observed, and it may be difficult to distinguish it from psychogenic pain. In other words, neuropathic pain with imaging findings and neuropathic pain without imaging findings are neuropathic pain due to spinal stenosis and neuropathic pain not due to spinal stenosis, respectively. In other words, it can be paraphrased. Diagnosis of neuropathic pain without imaging findings is important in deciding the treatment policy, but it is highly diagnostic because there is no highly accurate test and the burden on the patient is heavy because many tests are repeated. Development of diagnostic markers is desired.
ヒトオートタキシンは、１９９２年Ｍ．Ｌ．ＳｔｒａｃｋｅらによってＡ２０５８ヒト黒色腫細胞培養培地から細胞運動性を惹起する物質として単離された分子量約１２５ＫＤａの糖蛋白質である。オートタキシンは、そのリゾホスホリパーゼＤ活性によりリゾホスファチジルコリンを基質としリゾホスファチジン酸（ＬＰＡ）を産生する酵素であり、脳脊髄液中のオートタキシン濃度が測定可能であること（特許文献１、非特許文献１）は既に知られている。
Human autotaxin was introduced in 1992 by M. et al. L. It is a glycoprotein having a molecular weight of about 125 kDa isolated from A2058 human melanoma cell culture medium as a substance that induces cell motility by Stracke et al. Autotaxin is an enzyme that produces lysophosphatidic acid (LPA) using lysophosphatidylcholine as a substrate by its lysophospholipase D activity, and its autotaxin concentration in cerebrospinal fluid can be measured (
上記のとおりオートタキシンが脳脊髄液中に存在し、血清濃度に比較し高濃度に存在していることが明らかにされているが、その濃度変動と疼痛との関連性は明らかにされていなかった。 As described above, it has been clarified that autotaxin is present in cerebrospinal fluid and is present at a high concentration compared to the serum concentration, but the relationship between the concentration fluctuation and pain has not been clarified. It was.
これまでにオートタキシンの血液中の濃度が様々な疾病により変動することが報告されているが、脳脊髄液中のオートタキシン濃度と疾患との関連性に関しては報告が少なく、神経障害性疼痛との関連性に関しては症状との関連性がない事が報告されているのみである（非特許文献２）。本発明の目的は脳脊髄液中のオートタキシン濃度を測定することにより、画像所見が認められない神経障害を原因とする神経障害性疼痛を検出する方法を提供することにある。 It has been reported that the blood concentration of autotaxin varies depending on various diseases, but there are few reports on the relationship between the autotaxin concentration in cerebrospinal fluid and the disease, and it is called neuropathic pain. It has only been reported that there is no relationship with the symptoms regarding the relationship between the above (Non-Patent Document 2). An object of the present invention is to provide a method for detecting neuropathic pain caused by a neurological disorder in which no imaging findings are observed by measuring the autotaxin concentration in cerebrospinal fluid.
本発明者らは、様々な疾患での脳脊髄液中のオートタキシン濃度を測定し、鋭意検討を重ねた結果、画像所見が認められない神経障害性疼痛患者において脳脊髄液中のオートタキシン濃度が高値を示すことを見いだし、本発明に到達した。即ち本発明は下記の発明を包含する： The present inventors have measured the autotaxin concentration in the cerebrospinal fluid in various diseases, and as a result of intensive studies, the autotaxin concentration in the cerebrospinal fluid in a neuropathic pain patient with no imaging findings. We found that the value was high and reached the present invention. That is, the present invention includes the following inventions:
＜１＞ヒト脳脊髄液中のオートタキシン濃度を測定することを特徴とする、神経障害性疼痛を検出する方法。
＜２＞神経障害性疼痛が画像所見が認められない神経障害を原因とする疼痛である、＜１＞に記載の方法。
＜３＞画像所見が認められない神経障害が、帯状疱疹後神経痛、腰部癒着性くも膜炎、胸部脊髄炎、腰椎神経根障害、頸椎症性脊髄症、化学療法誘発性末梢神経障害、栄養代謝性多発神経障害、又は頚髄軟化症からなる、＜２＞に記載の方法。
＜４＞神経障害性疼痛を有さない陰性と画像所見が認められない神経障害性疼痛とを弁別する、＜１＞から＜３＞のいずれかの１項に記載の方法。
＜５＞画像所見が認められる神経障害性疼痛と画像所見が認められない神経障害性疼痛とを弁別する、＜１＞から＜３＞のいずれか１項に記載の検出方法。
＜６＞オートタキシンを特異的に認識する抗体を用いた免疫化学的方法により測定する、＜１＞から＜５＞のいずれか１項に記載の方法。
＜７＞オートタキシンを特異的に認識する抗体を含有することを特徴とする、神経障害性疼痛の検出に使用するための試薬。
<1> A method for detecting neuropathic pain, which comprises measuring the concentration of autotaxin in human cerebrospinal fluid.
<2> The method according to <1>, wherein the neuropathic pain is pain caused by a neurological disorder in which no imaging findings are observed.
<3> Neuropathies with no imaging findings include post-herpes zoster neuropathy, lumbar adhesive spider membrane inflammation, thoracic myelitis, lumbar radiculopathy, cervical spondylotic myelopathy, chemotherapy-induced peripheral neuropathy, and nutritional metabolism. The method according to <2>, which comprises multiple neuropathy or cervical myelopathy.
<4> The method according to any one of <1> to <3>, which distinguishes between negative without neuropathic pain and neuropathic pain with no imaging findings.
<5> The detection method according to any one of <1> to <3>, which distinguishes between neuropathic pain in which imaging findings are observed and neuropathic pain in which no imaging findings are observed.
<6> The method according to any one of <1> to <5>, which is measured by an immunochemical method using an antibody that specifically recognizes autotaxin.
<7> A reagent for use in the detection of neuropathic pain, which comprises an antibody that specifically recognizes autotaxin.
本発明によれば、ヒト脳脊髄液検体中のオートタキシンを測定することにより、画像所見が認められない神経障害を原因とする神経障害性疼痛を検出することが可能である。特許文献１に記載の免疫学的定量試薬を用い測定を実施すれば、検体中に含まれる内在性の測定妨害因子や競合酵素の影響を受けることなく、かつ短時間でヒトオートタキシンを定量可能であり、簡便、低コストで診断可能な画像所見が認められない神経障害を原因とする神経障害性疼痛を検出する試薬を提供することが可能である。
According to the present invention, it is possible to detect neuropathic pain caused by neuropathy in which no imaging findings are observed by measuring autotaxin in a human cerebrospinal fluid sample. If the measurement is carried out using the immunological quantification reagent described in
本発明においてオートタキシンを測定する方法には特に限定はないが、例えばオートタキシンを特異的に認識する抗体を用いた免疫化学的方法があげられる。抗体を用いたヒトオートタキシン定量方法は、ヒトオートタキシンを特異的に捕捉し、その結果、生成した抗体−ヒトオートタキシン複合体が検出可能な方法であれば手法を選ばない。好ましくは、イムノアッセイで汎用されている標識抗原と検体中のヒトオートタキシンの抗体に対する競合を利用した競合法、エピトープの異なる２抗体を用いてヒトオートタキシンとの３者の複合体を形成させるサンドイッチ法が簡便かつ汎用しやすい。特異性、感度、汎用性などの点から、エピトープの異なる２抗体サンドイッチ免疫測定方法が優れている。好ましくは、オートタキシンを特異的に認識する抗体を含有する神経障害性疼痛検出試薬を用いて行うことができる。このとき、例えば測定対象のオートタキシンを特異的に認識する固相化抗体と、それとは異なる部位で測定対象のオートタキシンを特異的に認識する標識化抗体とを含む試薬を用いてサンドイッチ法により行うことが好ましい。 In the present invention, the method for measuring autotaxine is not particularly limited, and examples thereof include an immunochemical method using an antibody that specifically recognizes autotaxine. The method for quantifying human autotaxine using an antibody can be any method as long as it specifically captures human autotaxine and the resulting antibody-human autotaxin complex can be detected. Preferably, a competition method utilizing competition between a labeled antigen and a human autotaxin antibody in a sample, which is widely used in immunoassays, and a sandwich in which two antibodies having different epitopes are used to form a three-way complex with human autotaxin. The method is simple and easy to use. The two-antibody sandwich immunoassay method having different epitopes is excellent in terms of specificity, sensitivity, versatility, and the like. Preferably, it can be carried out using a neuropathic pain detection reagent containing an antibody that specifically recognizes autotaxin. At this time, for example, a reagent containing a immobilized antibody that specifically recognizes the autotaxin to be measured and a labeled antibody that specifically recognizes the autotaxin to be measured at a site different from the immobilized antibody is used by a sandwich method. It is preferable to do so.
例えば、ヒトオートタキシン定量試薬に用いる抗ヒトオートタキシンモノクローナル抗体は、WO2008/016186記載の方法により取得可能である。具体的には、ｃＤＮＡライブラリーよりクローニングしたヒトオートタキシンｃＤＮＡを用い、昆虫細胞などの蛋白質発現系を用い蛋白質発現を行う。ｃＤＮＡライブラリーは、公知の方法を利用して組織からＲＮＡを単離することにより容易に調製可能であり、また市販のものを利用してもかまわない。発現蛋白質は、ポリヒスチジンタグやｃ−ｍｙｃタグなどを付加し発現を行えば、それらに特異的なアフィニティーカラムなどにより精製が可能である。これらの方法は標準的であり充分技術確立されている。 For example, the anti-human autotaxin monoclonal antibody used for the human autotaxin quantification reagent can be obtained by the method described in WO2008 / 016186. Specifically, human autotaxin cDNA cloned from a cDNA library is used to express proteins using a protein expression system such as insect cells. The cDNA library can be easily prepared by isolating RNA from tissues using a known method, or a commercially available one may be used. If the expressed protein is expressed by adding a polyhistidine tag, a c-myc tag, or the like, it can be purified by an affinity column or the like specific to them. These methods are standard and well-established.
抗体は上記精製抗原を動物に免疫することで取得が可能であり、例えばラットを用いる場合、精製ヒトオートタキシン抗原とフロイント完全アジュバントのエマルジョンを足底球（ｆｏｏｔｐａｄ）に免疫する。必要に応じ、精製抗原とフロイント不完全アジュバントにより繰り返し追加免疫を行ない、細胞融合の数日前に、最終免疫としてアジュバントとエマルジョン化することなく抗原のみを動物に投与する。ラットリンパ節を回収しミエローマ細胞と融合することにより、ハイブリドーマ細胞の作製が可能であり、これらの技術は充分確立されている。 The antibody can be obtained by immunizing an animal with the purified antigen. For example, when a rat is used, an emulsion of the purified human autotaxin antigen and Freund's complete adjuvant is immunized on the footpad. If necessary, repeated booster immunization with purified antigen and Freund's incomplete adjuvant is performed, and only the antigen is administered to the animal as final immunization several days before cell fusion without emulsification with the adjuvant. Hybridoma cells can be produced by collecting rat lymph nodes and fusing them with myeloma cells, and these techniques are well established.
上記方法により樹立したハイブリドーマ細胞産生抗体を、必要に応じて硫酸アンモニウム沈殿により濃縮した後、プロテインＡやプロテインＧ固相化担体を用いたアフィニティークロマトグラフィーにより、モノクローナル抗体の精製を行うことが可能である。また、精製した抗体は、ビオチンあるいはアルカリ性ホスファターゼ等の酵素により標識を施すことにより、ヒトオートタキシン２抗体サンドイッチ免疫測定系構築の検証に使用することが可能であり、これらの方法は標準的であり充分技術確立されている。 The hybridoma cell-producing antibody established by the above method can be concentrated by ammonium sulfate precipitation if necessary, and then the monoclonal antibody can be purified by affinity chromatography using a protein A or protein G immobilization carrier. .. In addition, the purified antibody can be used for verification of human autotaxin 2 antibody sandwich immunoassay system construction by labeling with an enzyme such as biotin or alkaline phosphatase, and these methods are standard. The technology is well established.
２０１１年国際疼痛学会による神経障害性疼痛の定義は「体性感覚神経系の病変や疾患によって引き起こされる疼痛」とされており、末梢神経から大脳に至るまでの侵害情報伝達経路のいずれかに病変や疾患が存在する際に生じる複数の発症機序を基盤として様々な症状や徴候により構成される症候群である。本発明において、画像所見が認められない神経障害とは、例えば脊柱管狭窄症の様な物理的な狭窄や変形といった変質を伴わず、画像所見を得ることができない、あるいは得にくい神経障害をいい、特に限定されるものではないが、例えば帯状疱疹後神経痛、腰部癒着性くも膜炎、胸部脊髄炎、腰椎神経根障害、頸椎症性脊髄症、化学療法誘発性末梢神経障害、栄養代謝性多発神経障害、又は頚髄軟化症等があげられる。 The 2011 International Pain Society defines neuropathic pain as "pain caused by lesions or diseases of the somatosensory nervous system" and lesions in any of the nociceptive signaling pathways from the peripheral nerves to the cerebral. It is a syndrome composed of various symptoms and signs based on multiple pathogenic mechanisms that occur when a disease or disease is present. In the present invention, the neuropathy in which no imaging findings are observed refers to a neuropathy in which imaging findings cannot be obtained or is difficult to obtain without alteration such as physical stenosis or deformation such as spinal stenosis. , But not particularly limited, for example, postherpetic neuralgia, lumbar adhesion spider membrane inflammation, thoracic myelitis, lumbar radiculopathy, cervical spondylotic myelopathy, chemotherapy-induced peripheral neuropathy, nutritional polyneuropathy. Disorders, cervical myelopathy, etc. may be mentioned.
また本発明の方法によれば、神経障害性疼痛を有さない陰性と画像所見が認められない神経障害性疼痛とを弁別することができ、また画像所見が認められる神経障害性疼痛と画像所見が認められない神経障害性疼痛とを弁別することができる。 Further, according to the method of the present invention, it is possible to distinguish between negative without neuropathic pain and neuropathic pain without imaging findings, and neuropathic pain with imaging findings and imaging findings. Can be distinguished from neuropathic pain in which is not observed.
以下に実施例を示すが、本発明は実施例に記載された例に限られるものではない。以下の実験を行うに当たっては、各施設の研究倫理委員会での承認のもと実施した。オートタキシン濃度測定は、自動免疫測定装置ＡＩＡシリーズ（東ソー社製）及び特許文献１記載のサンドイッチＥＬＩＳＡ測定法の試薬を用い実施した。
Examples are shown below, but the present invention is not limited to the examples described in the examples. The following experiments were conducted with the approval of the Research Ethics Committee of each institution. The autotaxin concentration measurement was carried out using an automatic immunoassay device AIA series (manufactured by Tosoh Corporation) and a reagent of the sandwich ELISA measurement method described in
実施例１：患者分類とオートタキシン濃度の測定
対象者は泌尿器科手術時の脊椎麻酔時に脳脊髄液を採取した患者をコントロール群、脊柱管狭窄症患者を画像所見が認められる神経障害性疼痛群（画像診断疼痛）、画像所見が認められない神経障害性疼痛群（画像診断不可疼痛）である。画像診断不可疼痛の原因疾患ならびに各群のオートタキシン濃度は表１の通りである。脳脊髄液検体はサイトスピン後の上清をサンプルとしオートタキシン濃度を測定した。平均値±標準偏差を表１に示す。また本対象者における各分類でのオートタキシン濃度の分布を図１に示す。
実施例２：有意差検定
実施例１の対象３群間のオートタキシン濃度の有意差検定をＭａｎｎ−Ｗｈｉｔｎｅｙ検定法を用い行った結果を表２に示す。検定の結果、画像診断不可疼痛患者のオートタキシン濃度は、コントロール群ならびに画像診断疼痛群のオートタキシン濃度と有意差をもって差異がある事が明らかとなった。
実施例３：ＲＯＣ解析
実施例１の対象群において、コントロール対画像診断不可疼痛、画像診断疼痛対画像診断不可疼痛、コントロールと画像診断疼痛対画像診断不可疼痛、のＲＯＣ解析を行い、曲線下面積、カットオフ値、感度、特異度、陽性適中率、陰性適中率を算出し、結果を表３に示す。本結果より画像診断不可疼痛は、コントロール群、画像診断疼痛群と高い感度、特異度をもって弁別できる事が示された。なお表中括弧内は９５％信頼区間を示す。
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