JP5625155B2 - Test method and test agent for systemic lupus erythematosus by measuring phosphatidylserine-specific phospholipase A1 - Google Patents
Test method and test agent for systemic lupus erythematosus by measuring phosphatidylserine-specific phospholipase A1 Download PDFInfo
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Description
本発明はヒト検体中のホスファチジルセリン特異的ホスホリパーゼＡ１濃度の測定による全身性エリテマトーデスの検査方法および検査薬に関する。 The present invention relates to a method and a test agent for systemic lupus erythematosus by measuring phosphatidylserine-specific phospholipase A1 concentration in a human specimen.
全身性エリテマトーデス（全身性紅斑性狼瘡）（以下、ＳＬＥ）は、全身の臓器に原因不明の炎症が起こる自己免疫疾患の一種であり、膠原病の１つとして分類されている。 Systemic lupus erythematosus (systemic lupus erythematosus) (hereinafter, SLE) is a type of autoimmune disease in which inflammation of unknown cause occurs in organs throughout the body, and is classified as one of collagen diseases.
ＳＬＥは、以下の：
（１）顔面紅斑、
（２）円板状皮疹、
（３）光線過敏症、
（４）口腔内潰瘍、
（５）関節炎（２関節以上で非破壊性）、
（６）漿膜炎（胸膜炎または心膜炎）、
（７）腎病変（０．５ｇ／日以上の持続的蛋白尿か細胞性円柱の出現）、
（８）神経学的病変（痙攣発作あるいは精神障害）、
（９）血液学的異常（溶血性貧血、白血球減少、リンパ球減少または血小板減少）、
（１０）免疫学的異常（抗２本鎖ＤＮＡ抗体陽性、抗Ｓｍ抗体陽性又は抗リン脂質抗体陽性、抗カルジオリピン抗体陽性、ループスアンチコアグラント、梅毒反応偽陽性）、
（１１）抗核抗体陽性、
の１１項目のうち４項目以上を満たす場合に、ＳＬＥと診断される。
SLE has the following:
(1) facial erythema,
(2) discoid eruption,
(3) photosensitivity,
(4) oral ulcers,
(5) Arthritis (non-destructive with 2 or more joints),
(6) serositis (pleurisy or pericarditis),
(7) Renal lesions (appearance of continuous proteinuria or cellular casts of 0.5 g / day or more),
(8) neurological lesions (convulsive seizures or mental disorders),
(9) Hematological abnormalities (hemolytic anemia, leukopenia, lymphopenia or thrombocytopenia),
(10) Immunological abnormalities (anti-double-stranded DNA antibody positive, anti-Sm antibody positive or anti-phospholipid antibody positive, anti-cardiolipin antibody positive, lupus anticoagulant, syphilis false positive),
(11) antinuclear antibody positive,
SLE is diagnosed when 4 or more of 11 items are satisfied.
ＳＬＥの病態を把握するため、従来から疾患活動性の指標であるＢＩＬＡＧやＳＬＥＤＡＩが用いられている。ＢＩＬＡＧは一般全身症状、粘膜皮膚症状、神経系症状、筋骨格系症状、心血管系および呼吸器系症状、血管炎、腎症などのそれぞれ細分化された症状、並びに血液検査の結果をスコアリングし、ＢＩＬＡＧ総スコアで病態を診断する方法である。例えば一般全身性症状としては、発熱、体重減少、リンパ節腫脹／脾腫、リンパ節腫脹、易疲労／全身倦怠／脱力、食欲低下／嘔気／嘔吐などに細分化され、それら各診断を総合し病態をカテゴリー分類する（非特許文献１および２）。ＳＬＥＤＡＩも同様に、痙攣、精神症状、器質的脳障害、視力障害、脳神経障害、ループス頭痛、脳血管障害、血管炎、関節炎、筋炎、尿円柱、血尿、蛋白尿、膿尿、新たな皮疹、脱毛、粘膜潰瘍、胸膜炎、心膜炎、低補体血症、抗ＤＮＡ抗体上昇、発熱、血小板減少、白血球減少、の診断項目に加重をかけスコアリングし病態把握を行う（非特許文献３）。 Conventionally, BILAG and SLEDAI, which are indicators of disease activity, have been used to grasp the pathological condition of SLE. BILAG scores generalized symptoms, mucocutaneous symptoms, nervous system symptoms, musculoskeletal symptoms, cardiovascular and respiratory symptoms, vasculitis, nephropathy, etc., and blood test results In addition, this is a method for diagnosing a disease state based on the BILAG total score. For example, general systemic symptoms are subdivided into fever, weight loss, lymphadenopathy / splenomegaly, lymphadenopathy, fatigue / systemic malaise / weakness, decreased appetite / nausea / vomiting, etc. Are classified into categories (Non-Patent Documents 1 and 2). Similarly for SLEDAI, convulsions, psychiatric symptoms, organic brain disorders, visual impairment, cranial nerve disorders, lupus headache, cerebrovascular disorders, vasculitis, arthritis, myositis, urinary cast, hematuria, proteinuria, pyouria, new skin rash, hair loss In addition, the diagnosis items such as mucosal ulcer, pleurisy, pericarditis, hypocomplementemia, anti-DNA antibody elevation, fever, thrombocytopenia, and leukopenia are weighted and scored to grasp the pathological condition (Non-patent Document 3).
以上に示すように、従来ＳＬＥの病態把握は非常に多くの診断項目が必要であり、かつ定性的な診断項目も多く含まれるため、ＳＬＥの病態を把握できる簡便なマーカーが期待されていた。 As described above, grasping the pathological condition of conventional SLE requires a large number of diagnostic items and includes many qualitative diagnostic items, and therefore, a simple marker that can grasp the pathological condition of SLE has been expected.
前述したように、全身性エリテマトーデス（ＳＬＥ）患者においてはその発症する病態は多様であり、従来の病態把握方法では定性的な診断基準を含んでいることから、更なる独立した診断マーカーが必要とされていた。そこで、本願発明の課題はＳＬＥの病態を反映する新たなマーカーを提供し、それを用いたＳＬＥの検査方法および検査薬を提供することにある。 As described above, pathological conditions that develop in patients with systemic lupus erythematosus (SLE) are diverse, and the conventional pathological condition grasping method includes qualitative diagnostic criteria. Therefore, further independent diagnostic markers are required. It had been. Then, the subject of this invention is providing the new marker which reflects the pathological condition of SLE, and providing the inspection method and test | inspection agent of SLE using it.
前記課題を鑑み発明者が鋭意検討した結果、ホスファチジルセリン特異的ホスホリパーゼＡ１（以下、ＰＳ−ＰＬＡ１）の生体内濃度が全身性エリテマトーデス（以下、ＳＬＥ）患者において濃度の上昇が認められることから、ＰＳ−ＰＬＡ１濃度がＳＬＥの病態把握に有効であることを見いだし、本発明を完成するに至った。 As a result of intensive studies by the inventor in view of the above problems, an increase in the in vivo concentration of phosphatidylserine-specific phospholipase A1 (hereinafter referred to as PS-PLA1) is observed in systemic lupus erythematosus (hereinafter referred to as SLE) patients. -It was found that the PLA1 concentration is effective for understanding the pathological condition of SLE, and the present invention has been completed.
詳しくは、本願は下記の発明を包含する：
（１）ヒト検体中のホスファチジルセリン特異的ホスホリパーゼＡ１を測定することによる全身性エリテマトーデスの検査方法。
（２）ホスファチジルセリン特異的ホスホリパーゼＡ１を測定し、その値が健常者測定値から算出したカットオフ値に対し高値を示した場合に全身性エリテマトーデスを罹患する、とすることを特徴とする、（１）に記載の検査方法。
（３）ホスファチジルセリン特異的ホスホリパーゼＡ１を測定し、全身性エリテマトーデス罹患患者の疾患活動期を判断することを特徴とする、（１）に記載の検査方法。
（４）上記測定が、ヒト検体中のホスファチジルセリン特異的ホスホリパーゼＡ１の濃度又は活性を測定することによる（１）から（３）のいずれかに記載の検査方法。
（５）少なくとも一つの既存の全身性エリテマトーデス検査方法と（１）から（４）のいずれかに記載の検査方法とを組み合わせた全身性エリテマトーデスの検査方法。
（６）既存の全身性エリテマトーデス検査方法が顔面紅斑、円板状皮疹、光線過敏症、口腔内潰瘍、関節炎、漿膜炎、腎病変などの身体的病変検査である（５）に記載の検査方法。
（７）既存の全身性エリテマトーデス検査方法が痙攣発作、精神障害などの神経学的病変検査である（５）に記載の検査方法。
（８）既存の全身性エリテマトーデス検査方法が溶血性貧血検査、白血球検査、リンパ球検査、血小板検査などの血液学的検査である（５）に記載の検査方法。
（９）既存の全身性エリテマトーデス検査方法が抗２本鎖ＤＮＡ抗体検査，抗Ｓｍ抗体検査、抗リン脂質抗体検査、抗核抗体検査などの免疫学的検査である（５）に記載の検査方法。
（１０）ヒト検体が、全血、血球、血清もしくは血漿などのヒト血液成分またはヒト細胞もしくはヒト組織の抽出液である（１）から（９）のいずれかに記載の検査方法。
（１１）ホスファチジルセリン特異的ホスホリパーゼＡ１の測定が抗体を用いた免疫化学的方法により行なわれる（１）から（１０）のいずれかに記載の検査方法。
（１２）全身性エリテマトーデスの治療効果を検査する（１）から（１１）のいずれかに記載の検査方法。
（１３）（１）から（１２）のいずれかに記載の検査方法を利用した全身性エリテマトーデスの検査薬。
Specifically, this application includes the following inventions:
(1) A method for examining systemic lupus erythematosus by measuring phosphatidylserine-specific phospholipase A1 in a human specimen.
(2) phosphatidylserine-specific phospholipase A1 is measured, and systemic lupus erythematosus is affected when the value shows a high value relative to the cut-off value calculated from the healthy subject measurement value, The inspection method according to 1).
(3) The test method according to (1), wherein phosphatidylserine-specific phospholipase A1 is measured to determine a disease active period of a patient with systemic lupus erythematosus.
(4) The test method according to any one of (1) to (3), wherein the measurement is performed by measuring the concentration or activity of phosphatidylserine-specific phospholipase A1 in a human specimen.
(5) A systemic lupus erythematosus test method combining at least one existing systemic lupus erythematosus test method and the test method according to any one of (1) to (4).
(6) The existing systemic lupus erythematosus test method is a physical lesion test such as facial erythema, discoid eruption, photosensitivity, oral ulcer, arthritis, serositis, renal lesion, etc. .
(7) The test method according to (5), wherein the existing systemic lupus erythematosus test method is a neurological lesion test such as seizures and psychiatric disorders.
(8) The test method according to (5), wherein the existing systemic lupus erythematosus test method is a hematological test such as hemolytic anemia test, leukocyte test, lymphocyte test, and platelet test.
(9) The existing systemic lupus erythematosus test method is an immunological test such as an anti-double-stranded DNA antibody test, an anti-Sm antibody test, an antiphospholipid antibody test, an antinuclear antibody test, etc. .
(10) The test method according to any one of (1) to (9), wherein the human specimen is a human blood component such as whole blood, blood cells, serum or plasma, or an extract of human cells or human tissues.
(11) The test method according to any one of (1) to (10), wherein the measurement of phosphatidylserine-specific phospholipase A1 is performed by an immunochemical method using an antibody.
(12) The test method according to any one of (1) to (11), wherein the therapeutic effect of systemic lupus erythematosus is tested.
(13) A test agent for systemic lupus erythematosus using the test method according to any one of (1) to (12).
本発明によれば、ヒト検体中のホスファチジルセリン特異的ホスホリパーゼＡ１（ＰＳ−ＰＬＡ１）を測定することにより全身性エリテマトーデス（ＳＬＥ）の検査およびＳＬＥ検査の補助を可能とし、さらにＳＬＥの病態把握をすることが可能である。ＰＳ−ＰＬＡ１を測定する際、特許文献１に記載の方法を原理とする免疫学的定量試薬を用いて測定を実施することにより、短時間でＰＳ−ＰＬＡ１を定量可能である。そのため、本発明により、小規模医療施設においても簡便かつ低コストでＳＬＥ検査が可能な検査薬を提供することが可能である。 According to the present invention, by measuring phosphatidylserine-specific phospholipase A1 (PS-PLA1) in a human specimen, it is possible to assist systemic lupus erythematosus (SLE) and SLE examination, and to further understand the pathology of SLE It is possible. When PS-PLA1 is measured, PS-PLA1 can be quantified in a short time by performing measurement using an immunological quantification reagent based on the method described in Patent Document 1. Therefore, according to the present invention, it is possible to provide a test drug capable of performing an SLE test easily and at low cost even in a small-scale medical facility.
また、少なくとも一つの既存のＳＬＥ検査（例えば、身体的病変検査、神経学的病変検査、血液学的検査、免疫学的検査）とＰＳ−ＰＬＡ１測定とを組み合わせることにより、既存のＳＬＥ検査およびＰＳ−ＰＬＡ１を単独マーカーとしたＳＬＥ検査と比較し、より高い診断精度でＳＬＥ検査やその治療効果を判定することが可能となる。
既存のＳＬＥ検査は全て自己免疫疾患による免疫応答の結果発症する自己抗体、またはそれを原因とする全身症状である。一方、ＰＳ−ＰＬＡ１は脂質代謝酵素であり既存のＳＬＥ検査項目とは異なる機序により濃度変動が誘導されることより、単独での検査のみならず既存ＳＬＥ検査との組み合わせることによる検査の意義は大きい。
ＳＬＥ患者はその疾患活動期を長期間にわたり定期的にモニター検査することが必要であるが、ＢＩＬＡＧやＳＬＥＤＡＩなどの指標は複数検査項目の診断の総合点として疾患活動期を判定すること、その個々の診断項目には定性的な判定項目も含まれるため専門医が必要であり、診断の施設間差は否定できない。一方、ＰＳ−ＰＬＡ１は単一の非侵襲的定量的検査項目であるため、疾患活動期の診断が容易であり施設間差を考慮する必要はない利点がある。
Also, by combining at least one existing SLE test (eg, physical lesion test, neurological lesion test, hematology test, immunological test) and PS-PLA1 measurement, an existing SLE test and PS -Compared with the SLE test using PLA1 as a single marker, it becomes possible to determine the SLE test and its therapeutic effect with higher diagnostic accuracy.
All existing SLE tests are autoantibodies that develop as a result of an immune response due to an autoimmune disease, or systemic symptoms caused by it. On the other hand, PS-PLA1 is a lipid-metabolizing enzyme, and the concentration variation is induced by a mechanism different from that of the existing SLE test items. Therefore, the significance of the test not only by the single test but also by combining with the existing SLE test is large.
SLE patients are required to regularly monitor the disease activity period over a long period of time, but indicators such as BILAG and SLEDAI determine the disease activity period as a comprehensive point of diagnosis of multiple test items, These diagnostic items include qualitative judgment items, so a specialist is required, and the difference between diagnostic facilities cannot be denied. On the other hand, since PS-PLA1 is a single non-invasive quantitative test item, there is an advantage that it is easy to diagnose a disease active period and it is not necessary to consider differences between facilities.
以下、本発明を詳細に説明する。
ＰＳ−ＰＬＡ１は１９９７年に同定されたリパーゼファミリーに属する酵素であり（非特許文献４）、極性頭部にセリン残基を有したセリンリン脂質であるホスファチジルセリンやリゾホスファチジルセリンを特異的基質としてｓｎ−１位のアシル基を加水分解する酵素である。本酵素は活性化血小板やマクロファージより主に産生される事が知られているが、その機能は未だ明らかになっていない。しかし、本酵素の活性から予測される機能として生体内からのホスファチジルセリンの消去、２−アシル−１−リゾホスファチジルセリンの産生により何らかの生体機能の調節に関与していることが予想される。ホスファチジルセリンは血液凝固の際、血小板細胞表面に露出することにより血液凝固の場を提供していることが知られており、ホスファチジルセリンを加水分解し消去することにより血液凝固の阻害制御を行なっていることや、アポトーシス細胞表面にもホスファチジルセリンが露出することから死細胞処理の制御に関与していることも示唆される。また、リゾホスファチジルセリン産生活性から想定される機能として、肥満細胞活性化による炎症反応への関与、神経細胞活性化への関与などが示唆されている。
Hereinafter, the present invention will be described in detail.
PS-PLA1 is an enzyme belonging to the lipase family identified in 1997 (Non-Patent Document 4), and phosphatidylserine and lysophosphatidylserine, which are serine phospholipids having a serine residue in the polar head, are used as specific substrates. It is an enzyme that hydrolyzes the acyl group at position -1. This enzyme is known to be produced mainly from activated platelets and macrophages, but its function has not been clarified yet. However, the functions predicted from the activity of this enzyme are expected to be involved in the regulation of some biological functions by elimination of phosphatidylserine from the living body and production of 2-acyl-1-lysophosphatidylserine. Phosphatidylserine is known to provide a place for blood coagulation by being exposed on the surface of platelet cells during blood coagulation, and it inhibits blood coagulation by hydrolyzing and eliminating phosphatidylserine. It is also suggested that phosphatidylserine is also exposed on the surface of apoptotic cells and is involved in the control of dead cell treatment. In addition, as functions assumed from lysophosphatidylserine production activity, involvement in inflammatory reaction by mast cell activation, involvement in nerve cell activation, and the like have been suggested.
ＰＳ−ＰＬＡ１はこれまで定量測定系がなかったため、ＰＳ−ＰＬＡ１と様々な疾病との関連の解析はなされていなかったが、２００６年、発明者によりＰＳ−ＰＬＡ１の定量測定方法が確立された（特許文献１）。特許文献１に記載の定量測定方法は血清などのヒト検体中のＰＳ−ＰＬＡ１濃度を前処理を必要とすることなく簡便、短時間、かつ信頼性高く定量可能な測定方法である。そこで特許文献１に記載の測定方法を用いて鋭意検討を重ねた結果、血液中のＰＳ−ＰＬＡ１濃度がＳＬＥ患者にて高値を示すこと、さらにＳＬＥの病態に比例して上昇することを見出した。また、ＰＳ−ＰＬＡ２は単独マーカーとして従来のＳＬＥ病態把握法であるＢＩＬＡＧ（非特許文献１および２）やＳＬＥＤＡＩ（非特許文献３）と正の相関性を示すことが見いだされた。以上より、ＰＳ−ＰＬＡ１がＳＬＥの病態を反映するマーカーとなりうることを見いだされた。 Since PS-PLA1 has no quantitative measurement system so far, the analysis of the relationship between PS-PLA1 and various diseases has not been made. However, in 2006, the inventors established a quantitative measurement method for PS-PLA1 ( Patent Document 1). The quantitative measurement method described in Patent Document 1 is a measurement method capable of quantitatively measuring PS-PLA1 concentration in a human specimen such as serum easily, in a short time, and with high reliability without requiring pretreatment. Thus, as a result of intensive studies using the measurement method described in Patent Document 1, it was found that the PS-PLA1 concentration in blood shows a high value in SLE patients and further increases in proportion to the pathological condition of SLE. . In addition, it was found that PS-PLA2 shows a positive correlation with BILAG (Non-patent Documents 1 and 2) and SLEDAI (Non-patent Document 3), which are conventional SLE pathologic condition grasp methods, as a single marker. From the above, it was found that PS-PLA1 can be a marker reflecting the pathological condition of SLE.
なお、ＰＳ−ＰＬＡ１の血液中での濃度上昇の機序は明らかでないが、少なくとも炎症性疾患に起因した病態や自己免疫疾患に起因した病態とは異なる機序でＰＳ−ＰＬＡ１の濃度上昇が認められていることが予想される。 Although the mechanism of the increase in the PS-PLA1 concentration in the blood is not clear, at least an increase in the concentration of PS-PLA1 was observed by a mechanism different from the pathological condition caused by the inflammatory disease or the pathological condition caused by the autoimmune disease. It is expected that
本発明に係る検査方法は、ヒト検体中のホスファチジルセリン特異的ホスホリパーゼＡ１を測定し、その値が健常者測定値からなる正常値範囲より算出したカットオフ値以上の値を示した場合に全身性エリテマトーデスを罹患する、とする。また、全身性エリテマトーデス罹患者の疾患活動期をモニターするのにも使用でき、ホスファチジルセリン特異的ホスホリパーゼＡ１濃度が正常値範囲に低下した場合、非活動期と判断する。健常者血清としては医師によりエリテマトーデス、並びにエリテマトーデスに関与する病態、例えば顔面紅斑、円板状皮疹、光線過敏症、口腔内潰瘍、関節炎、漿膜炎、腎病変などを患っていないと診断された者（以下、単に「健常者」と称す）の血清を使用した。このような正常値範囲は、当業者であれば健常者及びエリテマトーデス患者におけるホスファチジルセリン特異的ホスホリパーゼＡ１の濃度の分布を参照して任意に決定することができる。正常範囲の決定法は、例えば（健常者血清中のホスファチジルセリン特異的ホスホリパーゼＡ１濃度測定値の中央値＋３×標準偏差）を上限カットオフ値に設定することや、健常者測定値の９５パーセンタイル値上限をカットオフ値に設定することや、健常者測定値並びに全身性エリテマトーデス罹患者群を用いたＲＯＣ曲線を用いた統計的解析によりカットオフ値の設定が可能である。 The test method according to the present invention measures phosphatidylserine-specific phospholipase A1 in a human sample, and when the value shows a value equal to or higher than the cut-off value calculated from a normal value range consisting of a healthy subject measurement value, Suppose you have lupus erythematosus. It can also be used to monitor the disease active phase of individuals with systemic lupus erythematosus. When the phosphatidylserine-specific phospholipase A1 concentration falls to the normal value range, it is determined as the inactive phase. Serum of healthy subjects diagnosed by a doctor as not suffering from lupus erythematosus and pathological conditions related to lupus erythematosus, such as facial erythema, discoid rash, photosensitivity, oral ulcer, arthritis, serositis, renal lesions, etc. Serum (hereinafter simply referred to as “healthy person”) was used. Such a normal value range can be arbitrarily determined by those skilled in the art with reference to the distribution of the concentration of phosphatidylserine-specific phospholipase A1 in healthy subjects and lupus erythematosus patients. The normal range is determined by, for example, setting (the median value of phosphatidylserine-specific phospholipase A1 concentration measured in serum of normal subjects + 3 × standard deviation) to the upper cutoff value, or the 95th percentile value of values measured in healthy subjects The upper limit can be set as a cutoff value, or the cutoff value can be set by statistical analysis using a healthy subject measurement value and a ROC curve using a systemic lupus erythematosus affected group.
ＰＳ−ＰＬＡ１は既存ＳＬＥ診断マーカーとは独立した機序により変動する因子である。そのため、ＰＳ−ＰＬＡ１を単独マーカーとしてＳＬＥを検査してもよいし、既存のＳＬＥ検査方法や検査基準とを組み合わせてＳＬＥを検査してもよい。特に後者の検査方法はさらに高精度にＳＬＥの検査および病態把握が可能であるため好ましい。既存のＳＬＥ検査方法としては、
（Ａ）顔面紅斑、円板状皮疹、光線過敏症、口腔内潰瘍、関節炎、漿膜炎、腎病変などの身体的病変検査、
（Ｂ）痙攣発作、精神障害などの神経学的病変検査、
（Ｃ）溶血性貧血検査、白血球検査、リンパ球検査、血小板検査などの血液学的検査、
（Ｄ）抗２本鎖ＤＮＡ抗体検査、抗Ｓｍ抗体検査、抗リン脂質抗体検査、抗核抗体検査などの免疫学的検査、
が例示でき、これらの検査の中から少なくとも一つを選択すればよい。
PS-PLA1 is a factor that varies by a mechanism independent of existing SLE diagnostic markers. Therefore, SLE may be inspected using PS-PLA1 as a single marker, or SLE may be inspected in combination with existing SLE inspection methods and inspection standards. In particular, the latter inspection method is preferable because it enables SLE inspection and disease state grasping with higher accuracy. As an existing SLE inspection method,
(A) Physical lesion examinations such as facial erythema, discoid eruption, photosensitivity, oral ulcer, arthritis, serositis, renal lesions,
(B) Examination of neurological lesions such as seizures, mental disorders,
(C) Hematological tests such as hemolytic anemia test, leukocyte test, lymphocyte test, platelet test,
(D) immunological tests such as anti-double-stranded DNA antibody test, anti-Sm antibody test, anti-phospholipid antibody test, anti-nuclear antibody test,
And at least one of these tests may be selected.
本発明に開示するホスファチジルセリン特異的ホスホリパーゼＡ１測定方法は、ホスファチジルセリン特異的ホスホリパーゼＡ１、抗体との複合体を検出可能な方法であれば検出方法を選ばない。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ホスファチジルセリン特異的ホスホリパーゼＡ１の抗ホスファチジルセリン特異的ホスホリパーゼＡ１抗体に対する競合を利用した競合法、あるいはホスファチジルセリン特異的ホスホリパーゼＡ１上の異なるエピトープを認識する２種の抗体を用い、ホスファチジルセリン特異的ホスホリパーゼＡ１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。抗体を担体に結合させる場合、担体としてはイムノプレート、ラテックス粒子、磁性微粒子、ニトロセルロース膜、ＰＶＤＦ膜などイムノアッセイで使用されるものであれば特に担体を選ばない。担体を用いる場合、担体に固定化した抗体により捕捉したホスファチジルセリン特異的ホスホリパーゼＡ１の酵素活性を検出する方法、抗体を固定化したチップに検体を接触させてホスファチジルセリン特異的ホスホリパーゼＡ１結合依存的なシグナルを検出する表面プラズモン共鳴などの方法、あるいは抗体を固定化したチップに検体を接触させてホスファチジルセリン特異的ホスホリパーゼＡ１結合させた後、抗原抗体結合を乖離させる溶媒にて抗原を遊離させ、遊離した抗原をマススペクトロメーターなどで定量する方法等でホスファチジルセリン特異的ホスホリパーゼＡ１の測定が可能である。また、蛍光標識した抗体がホスファチジルセリン特異的ホスホリパーゼＡ１と結合することによる蛍光偏光を検出するようなホモジニアス測定方法においてもホスファチジルセリン特異的ホスホリパーゼＡ１の定量は可能である。これら試薬、装置は十分技術確立されている。 The method for measuring phosphatidylserine-specific phospholipase A1 disclosed in the present invention is not limited as long as it can detect a complex with phosphatidylserine-specific phospholipase A1 and an antibody. Preferably, a competitive antigen-based competitive method using a labeled antigen and a phosphatidylserine-specific phospholipase A1 in a sample to compete with an anti-phosphatidylserine-specific phospholipase A1 antibody, or a different epitope on a phosphatidylserine-specific phospholipase A1 The sandwich method of forming a ternary complex with phosphatidylserine-specific phospholipase A1 using the two types of antibodies is easy and versatile. When the antibody is bound to a carrier, the carrier is not particularly selected as long as it is used in an immunoassay such as an immunoplate, latex particles, magnetic fine particles, a nitrocellulose membrane, and a PVDF membrane. When using a carrier, a method for detecting the enzyme activity of phosphatidylserine-specific phospholipase A1 captured by the antibody immobilized on the carrier, contacting the sample with the chip on which the antibody is immobilized, and depending on the phosphatidylserine-specific phospholipase A1 binding A method such as surface plasmon resonance for detecting a signal, or a specimen is brought into contact with a chip on which an antibody is immobilized to bind phosphatidylserine-specific phospholipase A1, and then the antigen is released with a solvent that dissociates the antigen-antibody binding. The phosphatidylserine-specific phospholipase A1 can be measured by, for example, a method of quantifying the obtained antigen with a mass spectrometer or the like. The phosphatidylserine-specific phospholipase A1 can also be quantified in a homogeneous measurement method in which fluorescence polarization is detected by binding of a fluorescently labeled antibody to the phosphatidylserine-specific phospholipase A1. These reagents and devices are well established.
前記の測定方法において特異性、感度、汎用性など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。本測定系構築が可能な抗ホスファチジルセリン特異的ホスホリパーゼＡ１モノクローナル抗体の組み合わせの選択は、精製した抗体群とその標識抗体群を用い、組換えヒトホスファチジルセリン特異的ホスホリパーゼＡ１をサンプルとしてサンドイッチ測定系が構築可能であるかの検証にて行なう。具体的には、精製した未標識の抗ホスファチジルセリン特異的ホスホリパーゼＡ１モノクローナル抗体をイムノプレートに結合させ、ウシ血清アルブミンなどを含む緩衝液でイムノプレート表面のブロッキング処理を行う。続いて、組換えホスファチジルセリン特異的ホスホリパーゼＡ１を含む緩衝液をイムノプレートに添加し、固相抗体に捕捉させる。未反応物質を除去するためＰＢＳなどの緩衝液でイムノプレートを洗浄後、標識を施したホスファチジルセリン特異的ホスホリパーゼＡ１に対する抗体を反応させ２種類の抗体でのホスファチジルセリン特異的ホスホリパーゼＡ１のサンドイッチ複合体を形成させる。未反応物質を除去するためＰＢＳなどの緩衝液でイムノプレートを洗浄後、抗体標識に酵素標識を行った場合は基質の添加を行い、ビオチン標識などさらに反応が必要な場合は酵素標識ストレプトアビジンなどを反応させる。最終的にホスファチジルセリン特異的ホスホリパーゼＡ１を介して固相表面に結合した酵素を発色、蛍光、化学発光基質等を用い組換えホスファチジルセリン特異的ホスホリパーゼＡ１の検出を行なう。以上の実験を組換えホスファチジルセリン特異的ホスホリパーゼＡ１を含まない緩衝液で同様に行いバックグラウンド値として用いる。組換えホスファチジルセリン特異的ホスホリパーゼＡ１を用いた測定値がバックグラウンド低値でありかつ測定値に対し有意に高い組み合わせをホスファチジルセリン特異的ホスホリパーゼＡ１免疫測定系候補とする。 Among the measurement methods described above, the two-antibody sandwich immunoassay method with different epitopes is superior in terms of specificity, sensitivity, versatility and the like. Selection of a combination of anti-phosphatidylserine-specific phospholipase A1 monoclonal antibodies capable of constructing this measurement system is performed using a purified antibody group and its labeled antibody group, and a sandwich measurement system using recombinant human phosphatidylserine-specific phospholipase A1 as a sample. This is done by verifying whether it can be constructed. Specifically, purified unlabeled anti-phosphatidylserine-specific phospholipase A1 monoclonal antibody is bound to an immunoplate, and the immunoplate surface is blocked with a buffer solution containing bovine serum albumin or the like. Subsequently, a buffer solution containing recombinant phosphatidylserine-specific phospholipase A1 is added to the immunoplate and captured by the solid phase antibody. After washing the immunoplate with a buffer solution such as PBS to remove unreacted substances, the antibody against the labeled phosphatidylserine-specific phospholipase A1 is reacted to form a sandwich complex of phosphatidylserine-specific phospholipase A1 with two types of antibodies. To form. After removing the immunoreactor with a buffer solution such as PBS to remove unreacted substances, add enzyme to the antibody label and add a substrate. If further reaction such as biotin labeling is required, enzyme labeled streptavidin, etc. React. Finally, the enzyme bound to the solid phase surface through phosphatidylserine-specific phospholipase A1 is colored, fluorescent, chemiluminescent substrate, etc. are used to detect recombinant phosphatidylserine-specific phospholipase A1. The above experiment is carried out in the same manner with a buffer solution not containing recombinant phosphatidylserine-specific phospholipase A1 and used as a background value. A combination of a measured value using a recombinant phosphatidylserine-specific phospholipase A1 having a low background value and significantly higher than the measured value is set as a phosphatidylserine-specific phospholipase A1 immunoassay candidate.
前記の通り選択したホスファチジルセリン特異的ホスホリパーゼＡ１の２抗体サンドイッチ免疫測定系候補の信頼性を検証するため組換ホスファチジルセリン特異的ホスホリパーゼＡ１およびヒト血清の希釈系列サンプルを準備し、希釈倍率依存的に反応性が認められるか検証を行い測定法として選択する。特許文献１に記載の手法により得られた２抗体サンドイッチ測定系を用いることにより未知検体の濃度定量が可能である。すなわち、最終的に基質から得られる発色、蛍光、化学発光シグナルの強度はホスファチジルセリン特異的ホスホリパーゼＡ１を介して固相に結合した酵素量に依存すること、すなわちホスファチジルセリン特異的ホスホリパーゼＡ１量に依存することから、既知濃度のホスファチジルセリン特異的ホスホリパーゼＡ１を用いた標準曲線から未知濃度の検体中のホスファチジルセリン特異的ホスホリパーゼＡ１濃度が定量可能である。 A dilution series sample of recombinant phosphatidylserine-specific phospholipase A1 and human serum was prepared in order to verify the reliability of the phosphatidylserine-specific phospholipase A1 2-antibody sandwich immunoassay candidate selected as described above. Verify if reactivity is observed and select as the measurement method. By using the 2-antibody sandwich measurement system obtained by the method described in Patent Document 1, the concentration of an unknown sample can be determined. That is, the intensity of the color, fluorescence, and chemiluminescence signals finally obtained from the substrate depends on the amount of enzyme bound to the solid phase via phosphatidylserine-specific phospholipase A1, that is, depends on the amount of phosphatidylserine-specific phospholipase A1. Therefore, the phosphatidylserine-specific phospholipase A1 concentration in the sample at an unknown concentration can be quantified from a standard curve using a known concentration of phosphatidylserine-specific phospholipase A1.
選択した２種の抗体組み合わせを用い、ホスファチジルセリン特異的ホスホリパーゼＡ１測定試薬の調製を行う。２ステップサンドイッチ測定試薬の場合、２種の抗体の一方をイムノプレート、磁性粒子などＢ／Ｆ分離可能な担体に結合させる。結合方法は、疎水結合を利用した物理的結合、２物質間を架橋可能なリンカー試薬などを用いた化学的結合いずれでもかまわない。非特異的結合を避けるため担体表面を牛血清アルブミン、スキムミルクあるいは市販のイムノアッセイ用ブロッキング剤などでブロッキング処理を行ない１次試薬とする。２次試薬として標識を施したもう一方の抗体を含む溶液を準備する。抗体標識はペルオキシダーゼ、アルカリ性ホスファターゼなどの酵素、蛍光物質、化学発光物質、ラジオアイソトープなどの検出可能な物質、ビオチン、アビジンなどの特異的結合パートナーが存在する物質などでの標識が好ましい。また、２次試薬溶液は抗原抗体反応が良好に行える緩衝液、例えばリン酸緩衝液、Ｔｒｉｓ−ＨＣｌ緩衝液などが基本となる中性付近の緩衝液が好ましい。実検体の測定は、１次試薬と実検体を一定時間、一定温度のもと接触させる。反応条件は４から４０℃の温度で５分から３時間の反応が好ましい。未反応物質をＢ／Ｆ分離により除去し、続いて２次試薬と一定時間、一定温度のもと接触させサンドイッチ複合体を形成させる。反応条件に関しては４から４０℃の温度で５分から３時間の反応が好ましい。未反応物質をＢ／Ｆ分離により除去し、標識抗体の標識物質を定量し、既知濃度ホスファチジルセリン特異的ホスホリパーゼＡ１を標準とし作成した検量線により、実検体中のホスファチジルセリン特異的ホスホリパーゼＡ１濃度を定量する。１ステップサンドイッチ測定試薬の場合、２ステップサンドイッチ測定試薬と同様、担体に抗体を結合させブロッキング処理を行ったものを準備する。本抗体固相化担体に標識抗体を含む緩衝液を添加し試薬とする。必要に応じ、試薬を凍結乾燥品とすることも可能である。１ステップ試薬では抗原−抗体の使用量バランスにより抗原あるいは抗体の過不足が生じ測定系構築が困難であることが多い。本発明ではＥＬＩＳＡ法の場合、担体に結合させる抗体量を９６穴イムノプレート１ウェルあたり５から５００ｎｇ、好ましくは５０ｎｇ、標識抗体１から１００ｎｇ、好ましくは５０ｎｇを使用することにより良好な結果が得られる。測定に用いるヒト検体は、血清、血漿、尿、精漿、脳脊髄液などがあるが、用いる検体の希釈倍率は無希釈から１００希釈での使用が好ましく、特に血清、血漿においては４０倍希釈検体を５０μＬを用いることにより良好な結果が得られる。 A phosphatidylserine-specific phospholipase A1 measuring reagent is prepared using the selected two antibody combinations. In the case of a two-step sandwich measurement reagent, one of the two types of antibodies is bound to a B / F-separable carrier such as an immunoplate or magnetic particles. The bonding method may be either physical bonding using a hydrophobic bond or chemical bonding using a linker reagent capable of crosslinking between two substances. In order to avoid non-specific binding, the surface of the carrier is blocked with bovine serum albumin, skim milk, or a commercially available blocking agent for immunoassay to make a primary reagent. Prepare a solution containing the other labeled antibody as the secondary reagent. The antibody label is preferably labeled with an enzyme such as peroxidase or alkaline phosphatase, a fluorescent substance, a chemiluminescent substance, a detectable substance such as a radioisotope, or a substance having a specific binding partner such as biotin or avidin. In addition, the secondary reagent solution is preferably a buffer solution capable of satisfactorily performing an antigen-antibody reaction, for example, a neutral buffer solution based on a phosphate buffer solution, a Tris-HCl buffer solution, or the like. In the measurement of the real sample, the primary reagent and the real sample are brought into contact with each other at a constant temperature for a fixed time. The reaction conditions are preferably 4 to 40 ° C. and 5 minutes to 3 hours. Unreacted substances are removed by B / F separation, followed by contact with a secondary reagent for a certain period of time at a certain temperature to form a sandwich complex. Regarding the reaction conditions, a reaction of 4 to 40 ° C. for 5 minutes to 3 hours is preferable. The unreacted substance is removed by B / F separation, the labeled substance of the labeled antibody is quantified, and the concentration of phosphatidylserine-specific phospholipase A1 in the actual sample is determined by using a calibration curve prepared using phosphatidylserine-specific phospholipase A1 as a standard. Quantify. In the case of a one-step sandwich measurement reagent, as in the two-step sandwich measurement reagent, a reagent prepared by binding an antibody to a carrier and performing a blocking treatment is prepared. A buffer containing the labeled antibody is added to the antibody-immobilized carrier to obtain a reagent. If necessary, the reagent can be a freeze-dried product. In one-step reagents, it is often difficult to construct a measurement system due to excess or deficiency of antigen or antibody due to the balance of the amount of antigen-antibody used. In the present invention, in the case of the ELISA method, good results can be obtained by using an antibody amount of 5 to 500 ng, preferably 50 ng, and labeled antibody 1 to 100 ng, preferably 50 ng per well of a 96-well immunoplate in the case of ELISA. . Human specimens used for measurement include serum, plasma, urine, seminal plasma, cerebrospinal fluid, etc. The specimen to be used is preferably diluted from undiluted to 100-diluted, and in particular, serum and plasma are diluted 40-fold. Good results are obtained by using 50 μL of specimen.
以下に本発明をさらに詳細に説明するために実施例を示すが、これら実施例は本発明の一例を示すものであり、本発明は実施例に限定されるものではない。なお、実施例で使用した血清検体の採取および測定は東京大学医学部附属病院および東京医科大学病院で実施し、研究倫理について東京大学大学院医学研究科倫理委員会および東京医科大学病院での承認のもと実施した。また、ホスファチジルセリン特異的ホスホリパーゼＡ１（ＰＳ−ＰＬＡ１）濃度の測定は、特許文献１の方法に基づき、自動免疫測定装置ＡＩＡシリーズ（東ソー社製）を用い実施した。 Examples will be shown below to describe the present invention in more detail. However, these examples show examples of the present invention, and the present invention is not limited to the examples. The serum samples used in the examples were collected and measured at the University of Tokyo Hospital and the Tokyo Medical University Hospital, and research ethics were approved by the University of Tokyo Graduate School of Medicine Ethics Committee and the Tokyo Medical University Hospital. And implemented. Moreover, the measurement of the phosphatidylserine specific phospholipase A1 (PS-PLA1) density | concentration was implemented using the automatic immunoassay apparatus AIA series (made by Tosoh Corporation) based on the method of patent document 1. FIG.
実施例１：健常者検体中のＰＳ−ＰＬＡ１測定
患者検体測定に先立ち、コントロール群として健常者血清中のＰＳ−ＰＬＡ１濃度を測定した。血液は採血７日前より投薬のない健常な成人ボランティアからインフォームドコンセントを得た後、肘前中静脈より採取し、室温１５分放置後、１５００×ｇにて５分間遠心分離することにより血清を取得し測定を実施した。健常者１９０名（男性１１３名、女性７７名）の血清中のＰＳ−ＰＬＡ１濃度測定値は３３．２±１４．５μｇ／Ｌ（算術平均値±標準偏差）であり、中央値２９．８μｇ／Ｌ、９５パーセンタイルからなる参考基準範囲は１６．１μｇ／Ｌから６７．４μｇ／Ｌであった。本測定値から正常値を算出した場合、（中央値＋３×標準偏差）では７３．１μｇ／Ｌ、健常者測定値の９５パーセンタイル値上限では６７．４μｇ／Ｌである。
Example 1: PS-PLA1 measurement in healthy subject specimen Prior to patient specimen measurement, PS-PLA1 concentration in healthy subject serum was measured as a control group. After obtaining informed consent from healthy adult volunteers who have not taken medication 7 days before blood collection, blood is collected from the middle antecubital vein, left at room temperature for 15 minutes, and then centrifuged at 1500 × g for 5 minutes to obtain serum. Acquired and measured. The measured value of PS-PLA1 concentration in the serum of 190 healthy persons (113 males and 77 females) was 33.2 ± 14.5 μg / L (arithmetic mean ± standard deviation), with a median of 29.8 μg / The reference standard range consisting of L and 95th percentile was 16.1 μg / L to 67.4 μg / L. When a normal value is calculated from this measurement value, it is 73.1 μg / L for (median value + 3 × standard deviation), and 67.4 μg / L for the upper limit of the 95th percentile value of the healthy person measurement value.
実施例２：全身性エリテマトーデス（ＳＬＥ）患者検体中のＰＳ−ＰＬＡ１測定
男性ＳＬＥ患者９名、女性ＳＬＥ患者１７名、計２６名のＳＬＥ患者について血清中のＰＳ−ＰＬＡ１濃度を測定した。２６名のＰＳ−ＰＬＡ１濃度測定値は９９．８±７１．１μｇ／Ｌ（算術平均値±標準偏差）であり、Ｍａｎｎ−Ｗｈｉｔｎｅｙの有意差検定において健常者測定値に対して有意差ｐ＜０．０００１を示した（図１）。また診断率をＲＯＣ曲線による統計学的方法にて評価した結果、ＲＯＣ曲線のＡＵＣ値（ａｒｅａ ｕｎｄｅｒ ｃｕｒｖｅ）値は０．８６８であり、その際の感度は８０．８％、特異度は７６．８％と非常に良好な診断効率を示した（図２）。
Example 2 PS-PLA1 Measurement in Systemic Lupus Erythematosus (SLE) Patient Specimens Serum PS-PLA1 concentration was measured for 26 male SLE patients and 17 female SLE patients, for a total of 26 SLE patients. The measured value of PS-PLA1 concentration of 26 persons is 99.8 ± 71.1 μg / L (arithmetic mean ± standard deviation), and a significant difference p <0 with respect to the healthy person measured value in the Mann-Whitney significant difference test .0001 (FIG. 1). Moreover, as a result of evaluating the diagnosis rate by a statistical method using the ROC curve, the AUC value (area under curve) value of the ROC curve is 0.868, the sensitivity at that time is 80.8%, and the specificity is 76. The diagnostic efficiency was very good at 8% (FIG. 2).
実施例３：関節リウマチ、全身性硬化症患者検体中のＰＳ−ＰＬＡ１測定
ＳＬＥおよび他の膠原病である関節リウマチ（ＲＡ）４０例と全身性硬化症（ＳＳｃ）７例について血清中のＰＳ−ＰＬＡ１濃度を測定した。ＲＡおよびＳＳｃのＰＳ−ＰＬＡ１測定値はそれぞれ３８．１±１６．５μｇ／Ｌ、４０．０±２７．６μｇ／Ｌ（算術平均値±標準偏差）であり、Ｍａｎｎ−Ｗｈｉｔｎｅｙの有意差検定において健常者測定値と有意差は認められずＰＳ−ＰＬＡ１濃度測定によりＳＬＥとＲＡ、ＳＳｃとの判別が可能である結果であった（図３）。
Example 3: PS-PLA1 Measurement in Rheumatoid Arthritis, Systemic Sclerosis Patient Specimens Serum PS- for 40 cases of SLE and other collagenous rheumatoid arthritis (RA) and 7 cases of systemic sclerosis (SSc) PLA1 concentration was measured. The measured values of PS-PLA1 for RA and SSc are 38.1 ± 16.5 μg / L, 40.0 ± 27.6 μg / L (arithmetic mean ± standard deviation), respectively, and healthy in the Mann-Whitney significance test There was no significant difference from the measured value of the person, and it was a result that discrimination between SLE, RA, and SSc was possible by PS-PLA1 concentration measurement (FIG. 3).
実施例４：ＰＳ−ＰＬＡ１測定値とＳＬＥ疾患マーカーとの相関性
ＳＬＥ患者血清中のＰＳ−ＰＬＡ１濃度と発熱、関節痛、皮膚症状、漿膜炎、腎症、中枢神経ループスの有無との関係を解析した。その結果、ＰＳ−ＰＬＡ１濃度はこれら因子の有無で分類した際の各グループ間に有意差は認められず、これら因子に依存しないことが明らかとなった（図４）。また、ＰＳ−ＰＬＡ１濃度とリンパ球数、白血球数、ＣＲＰ値、ＧＯＴ値、Ｃ１ｑ結合性免疫複合体値との間にも相関性は認められなかった（図５）。以上の結果より、ＰＳ−ＰＬＡ１濃度は既存のＳＬＥ診断マーカーとは独立した因子であることが明らかとなった。
Example 4: Correlation between PS-PLA1 measured value and SLE disease marker Relationship between PS-PLA1 concentration in serum of SLE patient and presence of fever, joint pain, skin symptoms, serositis, nephropathy, central nervous lupus Analyzed. As a result, it was revealed that PS-PLA1 concentration was not dependent on these factors because no significant difference was observed between the groups when classified according to the presence or absence of these factors (FIG. 4). In addition, no correlation was observed between the PS-PLA1 concentration and the lymphocyte count, leukocyte count, CRP value, GOT value, and C1q-binding immune complex value (FIG. 5). From the above results, it was revealed that the PS-PLA1 concentration is a factor independent of existing SLE diagnostic markers.
実施例５：ＰＳ−ＰＬＡ１測定値とＢＩＬＡＧおよびＳＬＥＤＡＩの相関性
ＳＬＥ患者血清中のＰＳ−ＰＬＡ１濃度と各患者のＢＩＬＡＧおよびＳＬＥＤＡＩ値との相関性を検証した。ＰＳ−ＰＬＡ１濃度とＢＩＬＡＧの相関係数はｒ＝０．４６３（図６Ａ）、ＳＬＥＤＡＩとの相関係数はｒ＝０．５５６（図６Ｂ）であり、ＰＳ−ＰＬＡ１を測定することによりＳＬＥの病態把握、またはその補助因子となりうることを示している。
Example 5: Correlation between measured values of PS-PLA1 and BILAG and SLEDAI Correlation between PS-PLA1 concentration in serum of SLE patients and BILAG and SLEDAI values of each patient was examined. The correlation coefficient between PS-PLA1 concentration and BILAG is r = 0.463 (FIG. 6A), and the correlation coefficient with SLEDAI is r = 0.556 (FIG. 6B). By measuring PS-PLA1, the SLE This indicates that it can be used as a cognitive factor for understanding the disease state.
実施例６：治療によるＳＬＥ患者血清中のＰＳ−ＰＬＡ１濃度変化
治療前後におけるＳＬＥ患者血清中のＰＳ−ＰＬＡ１濃度変化を検証した。その結果、ほとんどのＳＬＥ患者において治療により血清中のＰＳ−ＰＬＡ１濃度が減少していた（図７）。前記結果は、ＳＬＥ患者血清中のＰＳ−ＰＬＡ１濃度変化をモニターすることで、ＳＬＥ病態および治療効果の把握が可能なことを示している。
Example 6: Change in PS-PLA1 concentration in SLE patient serum due to treatment The change in PS-PLA1 concentration in SLE patient serum before and after treatment was verified. As a result, in most SLE patients, the PS-PLA1 concentration in serum was decreased by treatment (FIG. 7). The above results indicate that it is possible to grasp the SLE disease state and the therapeutic effect by monitoring the change in PS-PLA1 concentration in the serum of SLE patients.
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