WO2001048479A1 - Method of judging the possibility of abortion/premature birth and reagent therefor - Google Patents

Method of judging the possibility of abortion/premature birth and reagent therefor Download PDF

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Publication number
WO2001048479A1
WO2001048479A1 PCT/JP2000/009301 JP0009301W WO0148479A1 WO 2001048479 A1 WO2001048479 A1 WO 2001048479A1 JP 0009301 W JP0009301 W JP 0009301W WO 0148479 A1 WO0148479 A1 WO 0148479A1
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premature birth
possibility
antibody
abortion
concentration
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PCT/JP2000/009301
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French (fr)
Japanese (ja)
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Takaji Kurosu
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Fuji Chemical Industries, Ltd.
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Priority to AU22272/01A priority Critical patent/AU2227201A/en
Publication of WO2001048479A1 publication Critical patent/WO2001048479A1/en

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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads

Definitions

  • the present invention relates to a method for determining the possibility of premature birth and a reagent therefor.
  • a PL is a general term for autoantibodies to phospholipids such as anti-cardiolipin antibody (aGL) ⁇ anti-phosphatidylserine antibody (aPS) and lupus anticoagulant (LA).
  • aGL anti-cardiolipin antibody
  • aPS anti-phosphatidylserine antibody
  • LA lupus anticoagulant
  • APS antiphospholipid antibody syndrome
  • a PL is recognized antigen is not a phospholipid itself, yS 2 -glycoprotein I (S 2 GP I) and proteinC that caused structural changes to form a phospholipid complexes, prote INS, such AnexinV ( Since all reported as cof actors, studies on this cofactor have been promoted, and many reports have been published on aPL associated with habitual miscarriage and thrombosis and cofactor involvement. . However, a conclusion has not yet been reached as to how PL causes blood clots in living organisms.
  • TM thrompomodulin
  • the present invention provides a method for determining the possibility of abortion prematurely, which comprises measuring the TM in a body fluid.
  • the present invention also provides a reagent for determining the possibility of abortion prematurely, which comprises an antithrombomodulin antibody.
  • the present invention a method has been provided which can more accurately determine the possibility of premature birth than the conventional method. If the potential for miscarriage is accurately demonstrated, precautionary measures can be taken to reduce miscarriages. Therefore, the present invention is expected to greatly contribute to safe childbirth.
  • FIG. 2 is a diagram showing the time-dependent change in serum TM concentration of aPL-negative pregnant women at the first consultation and the change in aPL-positive negative.
  • TM is a coagulation inhibitory factor present on vascular endothelial cells, and is released into the blood when injuries such as inflammation occur in blood vessels.
  • TM released into the blood binds to thrombin and has the anticoagulant effect of losing thrombin clotting activity.
  • this thrombin ⁇ TM complex activates protein C and activates protein S.
  • Blood TM has been conventionally used as a marker for determining whether blood vessels have inflammation. However, the use of blood TM as a marker for determining the possibility of premature birth has not been known or suggested.
  • the body fluid used in the method of the present invention examples include, but are not limited to, blood (including serum and plasma), urine, vaginal secretion, and cervical secretion. Among them, it is preferable to use blood, especially serum or plasma.
  • the TM concentration in a body fluid is measured, and the value is compared with a normal value. That is, as specifically shown in the following examples, when premature birth occurs, the TM concentration in the body fluid increases several weeks before (particularly about 4 to 6 weeks before).
  • TM in body fluids of normal pregnant women pregnant women who reached term delivery without any signs of urgency or pregnancy toxicity
  • SD standard deviation
  • TM Panacera trade name, manufactured by Fuji Pharmaceutical
  • the threshold value is not necessarily limited to the above value or the above-mentioned average + 2 SD value, and can be set as appropriate according to the purpose of the determination (for example, whether it is a screening purpose or a definite diagnosis purpose). . For example, if you want to determine as few possible pregnant women as to have a premature birth for screening purposes, the threshold may be set, for example, in the range of the average of normal pregnant women + 1 SD to the average + 2 SD, or almost certainly.
  • the threshold may be set, for example, in the range of +2 SD to +4 SD for normal pregnant women.
  • the threshold in the case of serum TM concentration measured using "Panasera" (trade name) manufactured by Fuji Pharmaceutical Co., Ltd. is lower than 3.8 FU / ml, for example, in the range of 3.0 FU / ml to 3.7 FU / ml. It is also possible to set within a higher range, for example, from 3.9 FU / ml to 5.0 FU / ml.
  • the determination may be made based on whether the TM concentration in the sample is statistically significantly higher than that of a normal pregnant woman.
  • the method of the present invention has higher accuracy than the conventional aPL-based diagnostic method.
  • kits for TM measurement using sandwich ELISA are commercially available, in which the antibody sensitized with an anti-TM monoclonal antibody is used as the first antibody, and the enzyme-labeled anti-TM monoclonal antibody is used as the second antibody.
  • concentration of TM in body fluid can be easily measured according to the package insert of "Panasera” (trade name) manufactured by Fuji Pharma Co., Ltd.
  • the TM concentration in the body fluid may be measured by any method, and the measuring method is not limited at all.
  • the immunoassay consists of an anti-thrombomodulin antibody (either polyclonal or monoclonal antibody) It measures the concentration of thrombomodulin in body fluids using an antigen-antibody reaction with jurin.
  • the immunoassay that can be used is not limited to the sandwich ELISA described above, and any known immunoassay can be employed. That is, when classified based on the measurement principle, there are a San German method, a competitive method, an agglutination method, etc., and any of these methods can be adopted.
  • the method for determining the possibility of miscarriage of the present invention may be performed in combination with another diagnostic method, and in some cases, the accuracy may be further improved. For example, if the aPL in blood changes from negative to positive in addition to the TM in the body fluid, and if the aPL in the blood changes from negative to positive, and the TM concentration in the body fluid rises rapidly, the risk of premature birth is high Can be determined to be very high.
  • the present invention further provides a reagent for determining the possibility of abortion prematurely, which comprises an anti-TM antibody.
  • This reagent can be used for the various well-known immunoassays described above by a well-known method.
  • the blood TM concentrations of 43 pregnant women were measured over time. The measurement was performed using 50 juI of serum collected from each pregnant woman as a sample and using the TM Panacera TM (trade name) manufactured by Fuji Pharmaceutical Co., Ltd., which is a commercial TM measurement kit for sandwich ELISA. It was performed by sandwich ELISA according to the usual method described.
  • aPL anti-cardiolipin antibody (aCL) and anti-phosphatidylserine antibody (aPS) were measured. When at least one of them was positive, aPL was determined to be positive.
  • aCL and aPS are measured by sandwich ELISA using microplate did.
  • 100 I of protamine sulfate was dispensed into each well of the microplate and allowed to stand at room temperature for 30 minutes.
  • the heart-derived cardiolipin solution manufactured by SI GMA
  • concentration: 20 ⁇ gZmI or L- ⁇
  • -phosphatidyl-L-serine solution manufactured by SIGM A
  • concentration: 20 u gZml was dispensed at 10 OI, left overnight at 4 ° C, and washed three times with PBST.
  • test serum or standard serum diluted 50-fold was dispensed, left at room temperature for 1.5 hours, and washed three times with PBST.
  • a peroxidase-labeled anti-human IgG antibody manufactured by SIGMA
  • a peroxidase-labeled anti-human IgM antibody manufactured by SIGMA
  • a peroxidase-labeled anti-human IgM antibody manufactured by SIGMA
  • diluted to an appropriate concentration were dispensed in 1 OO ju I each, and 1 ml at room temperature. After standing for 5 hours, the plate was washed three times with PBST.
  • 0.4 mM 0-phenylenediamine 100 / iI was dispensed, and allowed to stand at room temperature for 30 minutes.
  • Figures 1 and 2 show the results of 15 representative cases among the 43 cases.
  • Figure 1 shows the results for pregnant women who were aPL-positive at the first visit
  • Figure 2 shows the results for pregnant women who were aPL-negative at the first visit.
  • Cases 1, 2, 9, and 10 were premature births, and the other cases were term births.
  • the first and second pregnancies with positive aPL and premature birth in cases 1 and 2 and cases 3 and 4 showing signs of urgency showed a rapid rise in blood TM level, but the symptoms recovered after treatment In cases 3 and 4, the ascent curve thereafter stopped.
  • TM2 continues to rise.
  • the M-value increase in Case 1 that resulted in miscarriage shortly after treatment was rapid.
  • the TM value increased before the occurrence of miscarriage or imminent miscarriage. Since no increase was observed in normal pregnant women, TM measurement was easy to distinguish between normal and abnormal.

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Abstract

A method of judging the possibility of abortion/premature birth whereby the possibility of abortion/premature birth can be exactly judged compared with the existing methods; and a reagent to be used in this method. This method comprises measuring thrombomodulin in a bodily fluid. A significantly higher thrombomodulin concentration in the bodily fluid than normal subjects indicates the higher possibility of abortion/premature birth than normal subjects.

Description

明細書  Specification
流早産の可能性の判定方法及びそのための試薬  Method for determining the possibility of miscarriage and reagents therefor
技術分野  Technical field
本発明は、 流早産の可能性の判定方法及びそのための試薬に関する。  The present invention relates to a method for determining the possibility of premature birth and a reagent therefor.
背景技術  Background art
流早産を起こす原因には多くの原因が知られているが、 近年は抗リン脂質抗体 (aPL) による習慣流産がその頻度が高く特に注目されている。  There are many known causes of miscarriage, but in recent years, habitual miscarriage due to anti-phospholipid antibody (aPL) has been observed with high frequency and has received special attention.
a P Lは抗カルジォリピン抗体 （aGL) ゃ抗フォスファチジルセリン抗体 （aPS ) ， ループスアンチコアグラント （LA) 等のリン脂質に対する自己抗体の総称で あるが、 習慣流産のほか動静脈血栓症， 血小板減少症などの臨床症状を呈する抗 リン脂質抗体症候群 （APS) の診断に欠かすことのできない検査である。 しかし 近年、 a P Lが認識する抗原はリン脂質そのものではなく、 リン脂質と複合体を 形成し構造変化を起こした yS2-glycoprotein I ( S 2GP I ) や proteinC, prote inS, anexinV などの （いずれも血液凝固を制御する蛋白） cof actorである と報告されてからは、 この cofactorに関する研究が進められ、 習慣流産や血栓 症と cofactor関与の a P Lについての報告が多く出されるようになった。 しか し a P Lが生体にどのような機序をもって血栓を生じさせるのかについては未だ 結論が出ていない。 a PL is a general term for autoantibodies to phospholipids such as anti-cardiolipin antibody (aGL) ゃ anti-phosphatidylserine antibody (aPS) and lupus anticoagulant (LA). In addition to habitual miscarriage, arteriovenous thrombosis, platelet It is an indispensable test for the diagnosis of antiphospholipid antibody syndrome (APS), which presents with clinical symptoms such as hypotension. However, in recent years, a PL is recognized antigen is not a phospholipid itself, yS 2 -glycoprotein I (S 2 GP I) and proteinC that caused structural changes to form a phospholipid complexes, prote INS, such AnexinV ( Since all reported as cof actors, studies on this cofactor have been promoted, and many reports have been published on aPL associated with habitual miscarriage and thrombosis and cofactor involvement. . However, a conclusion has not yet been reached as to how PL causes blood clots in living organisms.
北海道社会事業協会病院の島野らは 1993年よリ感染症が起因する切迫流早産、 流早産を防止するという観点から妊娠初診時に細菌性膣症 （bacterial vagi nasi a： BV) ， Chlamydia trachomatis感染を診断し、 治療してきた。 しかし、 感染 症が起因ではない切迫流早産が相当数存在することから、 種々の検査を実施し、 それらの多くが A P S類似であることを確認した。  In 1993, Shimano et al. Of the Hokkaido Social Welfare Association Hospital developed bacterial vaginosis (bacterial vagi nasi a: BV) and Chlamydia trachomatis infection at the first consultation of pregnancy from the perspective of preventing imminent premature birth caused by infectious disease and premature birth. Diagnosed and treated. However, due to the considerable number of threatened premature births not due to infectious diseases, various tests were performed to confirm that many of them were similar to APS.
このようなことから、 島野らは改めて妊娠初診時に BV治療を行い、 さらに切 迫徴候出現時に a P Lの検査を行い、 A PSを疑う切迫流早産症例に対してプレ ドニゾロン （PSL) および低用量アスピリン （low dose aspirin： LDA) 療法など の治療を施したところ、 後期流産， 早産の著しい減少を得る事ができた。  Based on these findings, Shimano et al. Performed BV treatment again at the first consultation of pregnancy and performed aPL tests at the onset of imminent signs, and showed prednisolone (PSL) and low-dose Treatment with aspirin (low dose aspirin: LDA) therapy resulted in a significant decrease in late abortion and premature birth.
し力、し、 A PS疑いの流早産は未だ若干例存在している。 これらは妊娠中に a P Lが産生され、 この a P Lにより発症したと思われる acute type と、 A P S の治療中にもかかわらず急激に流早産に至った acute change に区別できるが、 いずれも切迫症状が急激に出現し、 かつ短期間に進行したため、 来院した時には 流早産が避けられない状態であった症例である。 ちなみに acute type は、 定期 健診時の検査では予測することができず、 流早産後の原因調査で a P Lによる流 早産と判明している。 There are still some cases of premature birth of suspected APS. These are during pregnancy a PL is produced, and it can be distinguished into acute type, which seems to be caused by this a PL, and acute change, which led to sudden premature birth despite treatment of APS. This was a case where premature birth was unavoidable at the time of hospital visit due to progression in a short period of time. By the way, acute type cannot be predicted by examination at regular medical check-up, and it has been found by a PL that abortion is premature birth due to cause investigation after abortion.
一方、 a P Lが陽性でありながら、 全く症状を現わさずに経過し、 正期産とな つた症例も多数存在する。 よって、 a P L陽性ということだけで、 治療に入るこ とは決して薦められるものではない。  On the other hand, there are a number of cases in which aPL is positive but the disease has passed without any symptoms, resulting in term delivery. Thus, it is by no means recommended to go into treatment just because of aPL positive.
このようなことから、 一般的に行われている A P Sの検査以外で、 流早産の可 能性を予知できる方法が求められている。  For this reason, there is a need for a method that can predict the possibility of premature birth, other than the general APS inspection.
発明の開示  Disclosure of the invention
従って、 本発明の目的は、 流早産の可能性を従来法よりも的確に判定すること が可能な、 流早産の可能性を判定する新規な方法、 及び該方法に用いられる試薬 を提供することである。  Accordingly, it is an object of the present invention to provide a novel method for determining the possibility of premature birth, which can more accurately determine the possibility of premature birth than the conventional method, and a reagent used in the method. It is.
本願発明者は、 鋭意研究の結果、 体液中トロンポモジュリン （T M) 濃度が切 迫徴候を示す約 4週間前ないし約 6週間前から増加していることを見出し、 体液 中 T M濃度を測定することにより流早産の可能性を的確に判定できることを見出 して本発明を完成した。  As a result of intensive studies, the present inventor has found that the concentration of thrompomodulin (TM) in body fluid has increased from about 4 weeks to about 6 weeks before the onset of imminent signs, and measures the TM concentration in body fluid. Thus, the present inventors have found that the possibility of premature birth can be accurately determined, and thus completed the present invention.
すなわち、 本発明は、 体液中の T Mを測定することを含む流早産の可能性の判 定方法を提供する。 また、 本発明は、 抗トロンボモジュリン抗体を含む、 流早産 の可能性の判定用試薬を提供する。  That is, the present invention provides a method for determining the possibility of abortion prematurely, which comprises measuring the TM in a body fluid. The present invention also provides a reagent for determining the possibility of abortion prematurely, which comprises an antithrombomodulin antibody.
本発明により、 流早産の可能性を従来法よりも的確に判定できる方法が提供さ れた。 流早産の可能性が的確に示されれば、 予防処置を採ることができるので、 流早産を減らすことができる。 従って、 本発明は安全な出産に大いに寄与するも のと期待される。  According to the present invention, a method has been provided which can more accurately determine the possibility of premature birth than the conventional method. If the potential for miscarriage is accurately demonstrated, precautionary measures can be taken to reduce miscarriages. Therefore, the present invention is expected to greatly contribute to safe childbirth.
図面の簡単な説明  BRIEF DESCRIPTION OF THE FIGURES
Ιί¾1 1は、 初診時 aPL陽性妊産婦の血清 Τ Μ濃度の経時変化及び aPL陽性陰性の変 化を示す図である。 Ιί¾11 Changes in serum Τ Μ concentration of aPL-positive pregnant women over time and changes in aPL-positive FIG.
図 2は、 初診時 aPL陰性妊産婦の血清 TM濃度の経時変化及び aPL陽性陰性の変 化を示す図である。  FIG. 2 is a diagram showing the time-dependent change in serum TM concentration of aPL-negative pregnant women at the first consultation and the change in aPL-positive negative.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
TMとは血管内皮細胞上に存在する凝固阻害因子で、 血管などに炎症等の障害 が生じると血中に放出される。 血中に放出された TMはトロンビンと結合し、 ト ロンビンの凝固活性を失わせるという抗凝固作用を有するほかに、 このトロンビ ン■ TM複合体が proteinCを活性化させ、 さらに prote i n Sを活性化、 活性化凝 固第 V因子および第 VI I I因子を不活性化させるという抗凝固作用を有する。血中 T Mは、 従来より、 血管に炎症があるか否かのマーカーとして利用されている。 し かしながら、 流早産の可能性を判定するためのマーカーとして血中 TMを用いる ことは知られておらず、 示唆もされていない。  TM is a coagulation inhibitory factor present on vascular endothelial cells, and is released into the blood when injuries such as inflammation occur in blood vessels. TM released into the blood binds to thrombin and has the anticoagulant effect of losing thrombin clotting activity.In addition, this thrombin ■ TM complex activates protein C and activates protein S. Has the anticoagulant effect of inactivating factor V and factor VIII. Blood TM has been conventionally used as a marker for determining whether blood vessels have inflammation. However, the use of blood TM as a marker for determining the possibility of premature birth has not been known or suggested.
本発明の方法に供される体液としては、 血液 （血清及び血漿を包含する） 、 尿、 膣分泌液、 子宮頸部分泌液等を挙げることができるがこれらに限定されるもので はない。 これらのうち、 血液、 とりわけ血清又は血漿を用いることが好ましい。 本発明の方法では、 体液中の TM濃度を測定し、 その値を正常値と比較する。 すなわち、 下記実施例において具体的に示すように、 流早産が起きる場合には、 その数週間前 （特に約 4〜 6週間前） に体液中 TM濃度が上昇する。 体液中 TM 濃度が正常妊婦 （切迫徴候及び妊娠中毒なしに正期産に至った妊婦） の平均 +2 SD (SDは標準偏差） 程度 （例えば、 富士薬品工業社製 「TMパナセラ」 （商 品名） を用いて測定した血清中 TM濃度の場合には 3· 8FU/ml)以下であれば、 流 早産の可能性はほとんどない。 一方、 この値 （以下、 「閾値」 ） を超えると流早 産の可能性がかなりある。 よって、 体液中 TM濃度を測定することにより、 流早 産の可能性を判定することが可能である。 また、 流早産が起きる場合には、 その 数週間前 （特に約 4〜 6週間前） に体液中 TM濃度が上昇するので、 体液中 TM 濃度の測定は 4週間毎位に経時的に行うことが好ましく、 体液中丁 M濃度が急上 昇した場合には、 流早産となる可能性が高い。 流早産になる可能性がある場合に は、 プレドニゾロンゃ低用量ァスピリン投与等の治療ないし予防処置を採ること ができ、 流早産の危険性を低減することができる。 なお、 閾値は、 上記値又は上 記した平均 + 2 S Dの値に必ずしも限定されるものではなく、 判定の目的 （例え ばスクリーニング目的か確定診断目的か等） に応じて適宜設定することができる。 例えば、 スクリーニング目的で流早産の可能性のある妊婦をできるだけもれなく 判定したい場合には、 閾値を例えば正常妊婦の平均 + 1 S Dないし平均 +2 S D の範囲に設定してもよいし、 ほぼ確実に流早産が起きる場合のみを判定したい場 合には、 閾値を例えば正常妊婦の平均 +2 SDないし平均 +4 S Dの範囲に設定 してもよい。 また、 同様に、 富士薬品工業社製 ΓΤΜパナセラ」 （商品名） を用 いて測定した血清中 TM濃度の場合の閾値を 3.8 FU/mlよりも低い例えば 3.0 FU/ ml〜3.7 FU/mlの範囲内で設定したり、 より高い例えば 3.9 FU/ml〜5.0 FU/mlの 範囲内で設定することも可能である。 あるいは、 検体中の TM濃度を正常妊婦の 測定値と比較して、 統計学的に有意に高いか否かによつて判定してもよい。 Examples of the body fluid used in the method of the present invention include, but are not limited to, blood (including serum and plasma), urine, vaginal secretion, and cervical secretion. Among them, it is preferable to use blood, especially serum or plasma. In the method of the present invention, the TM concentration in a body fluid is measured, and the value is compared with a normal value. That is, as specifically shown in the following examples, when premature birth occurs, the TM concentration in the body fluid increases several weeks before (particularly about 4 to 6 weeks before). Average of TM in body fluids of normal pregnant women (pregnant women who reached term delivery without any signs of urgency or pregnancy toxicity) +2 SD (SD is standard deviation) (eg, “TM Panacera” (trade name, manufactured by Fuji Pharmaceutical) If the TM concentration in serum measured using) is less than 3.8FU / ml), there is little possibility of premature birth. On the other hand, if this value (hereinafter referred to as “threshold”) is exceeded, there is considerable possibility of premature birth. Therefore, the possibility of premature birth can be determined by measuring the TM concentration in the body fluid. Also, when premature birth occurs, the TM concentration in body fluids increases several weeks before (particularly about 4 to 6 weeks before), so the TM concentration in body fluids should be measured every 4 weeks. If the concentration of M in body fluids rises sharply, there is a high possibility of premature birth. If there is a possibility of premature birth, take therapeutic or preventive measures such as administration of prednisolone or low-dose aspirin. The risk of premature birth can be reduced. Note that the threshold value is not necessarily limited to the above value or the above-mentioned average + 2 SD value, and can be set as appropriate according to the purpose of the determination (for example, whether it is a screening purpose or a definite diagnosis purpose). . For example, if you want to determine as few possible pregnant women as to have a premature birth for screening purposes, the threshold may be set, for example, in the range of the average of normal pregnant women + 1 SD to the average + 2 SD, or almost certainly. If it is desired to determine only when abortion occurs, the threshold may be set, for example, in the range of +2 SD to +4 SD for normal pregnant women. Similarly, the threshold in the case of serum TM concentration measured using "Panasera" (trade name) manufactured by Fuji Pharmaceutical Co., Ltd. is lower than 3.8 FU / ml, for example, in the range of 3.0 FU / ml to 3.7 FU / ml. It is also possible to set within a higher range, for example, from 3.9 FU / ml to 5.0 FU / ml. Alternatively, the determination may be made based on whether the TM concentration in the sample is statistically significantly higher than that of a normal pregnant woman.
下記実施例において具体的に示すように、 初診時に aPLが陽性でも、 体液中 T M濃度が閾値以下で推移する場合には流早産はほとんど起きない。 一方、 しが 陽性か陰性かに関わらず、 体液中 TM濃度が急上昇している場合には、 流早産と なる可能性が高い。 よって、 本発明の方法は、 従来の aPLに基づく診断方法より も精度が高いと言える。  As will be specifically shown in the following examples, even if aPL is positive at the first consultation, premature birth hardly occurs when the body fluid TM concentration changes below the threshold. On the other hand, if the TM concentration in body fluids rises sharply, regardless of whether the pest is positive or negative, it is highly likely that premature birth will occur. Therefore, it can be said that the method of the present invention has higher accuracy than the conventional aPL-based diagnostic method.
体液中 TM濃度の測定方法自体は周知であり、 例えば、 血清又は血漿を検体と して用いたサンドイッチ E L I SA等の免疫測定法により容易に実施することが できる。 担体ビーズ上に、 抗 TMモノクローナル抗体を感作したものを第 1抗体 とし、 酵素標識抗 TMモノクローナル抗体を第 2抗体とする、 サンドイッチ E L I SAによる TM測定用キッ卜が市販されているので （例えば富士薬品工業社製 ΓΤΜパナセラ」 （商品名） ） 、 このような市販のキットを用い、 その添付文書 に従って容易に体液中 TM濃度を測定することができる。  The method of measuring the TM concentration in a body fluid itself is well known, and can be easily carried out, for example, by an immunoassay such as sandwich ELISA using serum or plasma as a sample. Kits for TM measurement using sandwich ELISA are commercially available, in which the antibody sensitized with an anti-TM monoclonal antibody is used as the first antibody, and the enzyme-labeled anti-TM monoclonal antibody is used as the second antibody. Using such a commercially available kit, the concentration of TM in body fluid can be easily measured according to the package insert of "Panasera" (trade name) manufactured by Fuji Pharma Co., Ltd.
なお、 本発明の方法では、 いずれかの方法により体液中の TM濃度を測定すれ ばよいのであり、 その測定方法は何ら限定されない。 通常、 TM濃度は、 免疫測 定法により測定することが好ましい。 免疫測定法は、 抗トロンボモジュリン抗体 (ポリクローナル抗体でもモノクローナル抗体でもよい） と体液中のト口ンボモ ジュリンとの抗原抗体反応を利用して、 体液中のトロンボモジュリンの濃度を測 定するものである。 この場合、 用いることができる免疫測定法は、 上記したサン ドイッチ E L I S Aに限定されるものではなく、 公知のいずれの免疫測定法をも 採用することができる。 すなわち、 測定原理に基づいて分類すると、 サンドイツ チ法、 競合法、 凝集法等があるがこれらのいずれをも採用することができる。 ま た、 用いる標識に基づいて分類すると、 酵素免疫測定、 放射免疫測定、 蛍光免疫 測定、 ビォチン免疫測定等があるがこれらのいずれをも採用することができる。 さらに、 これらの各免疫測定は、 さらに種々の測定方法に細分化されるが、 それ らのいずれをも採用することができる。 In the method of the present invention, the TM concentration in the body fluid may be measured by any method, and the measuring method is not limited at all. Usually, it is preferable to measure the TM concentration by an immunoassay. The immunoassay consists of an anti-thrombomodulin antibody (either polyclonal or monoclonal antibody) It measures the concentration of thrombomodulin in body fluids using an antigen-antibody reaction with jurin. In this case, the immunoassay that can be used is not limited to the sandwich ELISA described above, and any known immunoassay can be employed. That is, when classified based on the measurement principle, there are a San German method, a competitive method, an agglutination method, etc., and any of these methods can be adopted. When classified based on the label used, there are enzyme immunoassay, radioimmunoassay, fluorescence immunoassay, biotin immunoassay and the like, and any of these can be adopted. Further, each of these immunoassays is further subdivided into various measurement methods, and any of them can be adopted.
本発明の流早産の可能性の判定方法は、 他の診断方法と組み合わせて行っても よく、 それによつて、 より精度を高めることが可能な場合がある。 例えば、 体液 中 T M濃度に加え、 血中 aPLが陽性か陰性かを調べ、 血中 aPLが陰性から陽性に変 わり、 かつ、 体液中 T M濃度が急上昇している場合には、 流早産の危険性が非常 に高いと判定できる。  The method for determining the possibility of miscarriage of the present invention may be performed in combination with another diagnostic method, and in some cases, the accuracy may be further improved. For example, if the aPL in blood changes from negative to positive in addition to the TM in the body fluid, and if the aPL in the blood changes from negative to positive, and the TM concentration in the body fluid rises rapidly, the risk of premature birth is high Can be determined to be very high.
本発明は、 さらに、 抗 T M抗体を含む、 流早産の可能性の判定用試薬をも提供 する。 この試薬は、 上記した周知の各種免疫測定に、 周知の方法により用いるこ とができる。  The present invention further provides a reagent for determining the possibility of abortion prematurely, which comprises an anti-TM antibody. This reagent can be used for the various well-known immunoassays described above by a well-known method.
実施例 Example
以下、 本発明を実施例に基づきより具体的に説明する。  Hereinafter, the present invention will be described more specifically based on examples.
(1 ) 妊産婦の血中 T M濃度の測定 (1) Measurement of blood TM concentration in pregnant women
4 3 3例の妊産婦の血中 T M濃度を経時的に測定した。 測定は、 各妊産婦から 採取した血清 5 0 ju I を検体とし、 サンドイッチ E L I S Aによる市販の T M測 定用キットである富士薬品工業社製 「T Mパナセラ」 （商品名） を用い、 その指 示書に記載された常法に従って、 サンドイッチ E L I S Aにより行った。  The blood TM concentrations of 43 pregnant women were measured over time. The measurement was performed using 50 juI of serum collected from each pregnant woman as a sample and using the TM Panacera ™ (trade name) manufactured by Fuji Pharmaceutical Co., Ltd., which is a commercial TM measurement kit for sandwich ELISA. It was performed by sandwich ELISA according to the usual method described.
(2) 妊産婦の血中 aPLの測定 (2) Measurement of blood aPL in pregnant women
aPLとしては、 抗カルジオリピン抗体（aCL)及び抗フォスファチジルセリン抗体 (aPS)を測定し、 少なくともいずれか一方が陽性の場合、 aPL陽性とした。  As the aPL, anti-cardiolipin antibody (aCL) and anti-phosphatidylserine antibody (aPS) were measured. When at least one of them was positive, aPL was determined to be positive.
aCL及び aPSは、 マイクロプレートを用いたサンドイッチ E L I S Aにより測定 した。 マイクロプレートの各ゥエルに硫酸プロタミンを 1 00 I分注し、 室温 で 30分間静置した。 P B Sで 3回洗浄後、 ゥシ心臓由来カルジォリピン液 （SI GMA社製） （濃度 20 μ gZm I ) 又は L- α; -フォスファチジル- L-セリン液（SIGM A社製） （濃度20 u gZm l ) を 1 0 O I分注し、 4°Cで一昼夜放置後、 P B S Tで 3回洗浄した。 次いで 50倍希釈した被検血清又は標準血清を 1 00 I分注し、 室温で 1.5時間静置後、 P BS Tで 3回洗浄した。 次いで適当な濃度 に希釈したペルォキシダーゼ標識抗ヒト I g G抗体 （SIGMA社製） 及びペルォキ シダ一ゼ標識抗ヒト I g M抗体 （SIGMA社製） を各 1 O O ju I分注し、 室温で 1, 5 時間静置後、 P B S Tで 3回洗浄した。 次いで 0.4 mM 0-フエ二レンジァミン 1 00 /i I を分注し、 室温で 30分間静置後、 2.5N硫酸を分注して反応を停止した。 次いでィムノリーダーにて波長 490 nmの吸光度を測定した。 吸光度が標準血清の 測定値以上の場合を陽性と判定した。 aCL and aPS are measured by sandwich ELISA using microplate did. 100 I of protamine sulfate was dispensed into each well of the microplate and allowed to stand at room temperature for 30 minutes. After washing three times with PBS, the heart-derived cardiolipin solution (manufactured by SI GMA) (concentration: 20 μgZmI) or L-α; -phosphatidyl-L-serine solution (manufactured by SIGM A) (concentration: 20 u gZml) was dispensed at 10 OI, left overnight at 4 ° C, and washed three times with PBST. Then, 100 I of test serum or standard serum diluted 50-fold was dispensed, left at room temperature for 1.5 hours, and washed three times with PBST. Next, a peroxidase-labeled anti-human IgG antibody (manufactured by SIGMA) and a peroxidase-labeled anti-human IgM antibody (manufactured by SIGMA) diluted to an appropriate concentration were dispensed in 1 OO ju I each, and 1 ml at room temperature. After standing for 5 hours, the plate was washed three times with PBST. Next, 0.4 mM 0-phenylenediamine 100 / iI was dispensed, and allowed to stand at room temperature for 30 minutes. Thereafter, 2.5N sulfuric acid was dispensed to stop the reaction. Next, the absorbance at a wavelength of 490 nm was measured using an Imno reader. When the absorbance was equal to or higher than the measured value of the standard serum, it was determined to be positive.
(3) 結果 (3) Result
43 3例中の代表的な 1 5例の結果を図 1及び図 2に示す。 図 1には、 初診時 aPL陽性であった妊産婦についての結果を、 図 2には、 初診時 aPL陰性であった 妊産婦についての結果を示す。 なお、 症例 1、 2、 9、 1 0が流早産に終わった 症例であり、 これら以外の症例は正期産であった。 妊娠初診時に a P Lが陽性で、 流早産となった症例 1， 2 および切迫徴候を示した症例 3， 4はいずれも血中 TM値の急速な上昇が見られるが、 治療により症状が回復した症例 3， 4は、 そ の後の上昇カーブが止まっている。 治療中であるにもかかわらず、 早産となった 症例 2は TM値が上昇し続けている。 治療する間もなく流産に至った症例 1の丁 M値上昇は急激である。 a P Lが陽性であるにもかかわらず、 何の症状もなく正 期産となった症例 5〜 8の TM値は、 症例 5の 36W以降の上昇を除き、 たえず 3.8 FU/ml以内で推移していた。 妊娠初診時の検査で a P L陰性であるにもか かわらず妊娠中に a P Lが産生され、 早産になつたり切迫徴候が見られた症例 9 〜11 も、 TM値の急速な上昇が見られ、 TM値上昇後に a P Lを検出、 そして、 切迫徴候が出現するという特徴が見られた。  Figures 1 and 2 show the results of 15 representative cases among the 43 cases. Figure 1 shows the results for pregnant women who were aPL-positive at the first visit, and Figure 2 shows the results for pregnant women who were aPL-negative at the first visit. Cases 1, 2, 9, and 10 were premature births, and the other cases were term births. The first and second pregnancies with positive aPL and premature birth in cases 1 and 2 and cases 3 and 4 showing signs of urgency showed a rapid rise in blood TM level, but the symptoms recovered after treatment In cases 3 and 4, the ascent curve thereafter stopped. In premature birth, despite treatment, TM2 continues to rise. The M-value increase in Case 1 that resulted in miscarriage shortly after treatment was rapid. a The TM value of Cases 5 to 8 that had a term delivery without any symptoms despite positive PL was constantly within 3.8 FU / ml, except for the rise after 36 W in Case 5. I was Even in cases 9 to 11 where aPL was produced during pregnancy despite pregnancy and aPL was negative at the first pregnancy examination, and premature birth or signs of imminence were observed, TM values also increased rapidly. The aPL was detected after the TM value increased, and the signs of urgency appeared.
このように、 T M値は流早産や切迫流早産を起こす以前からその値が上昇して おり、 正常妊婦には値の上昇がみられないため、 TMの測定は正常 '異常の区別 がっきやすい結果となった。 Thus, the TM value increased before the occurrence of miscarriage or imminent miscarriage. Since no increase was observed in normal pregnant women, TM measurement was easy to distinguish between normal and abnormal.

Claims

請求の範囲 The scope of the claims
1 . 体液中のト口ンボモジュリンを測定することを含む流早産の可能性の判定 方法。  1. A method for determining the likelihood of premature birth, including measuring tombo modulin in bodily fluids.
2 . トロンボモジユリンを経時的に複数回測定し、 各測定値を比較する請求項 1記載の方法。  2. The method according to claim 1, wherein thrombomodulin is measured a plurality of times over time, and the measured values are compared.
3 . 血中トロンボモジュリンを測定する請求項 1又は 2記載の方法。  3. The method according to claim 1 or 2, wherein blood thrombomodulin is measured.
4 . 血清又は血漿中のトロンポモジュリンを測定する請求項 3記載の方法。  4. The method according to claim 3, wherein the tropomodulin in serum or plasma is measured.
5 . 血中リン脂質抗体濃度を測定することをさらに含む請求項 1ないし 4のい ずれか 1項に記載の方法。  5. The method according to any one of claims 1 to 4, further comprising measuring a blood phospholipid antibody concentration.
6 . 抗トロンボモジュリン抗体と トロンボモジュリンとの抗原抗体反応を利用 した免疫測定方法により トロンポモジュリンを測定する請求項 1ないし 5のいず れか 1項に記載の方法。  6. The method according to any one of claims 1 to 5, wherein thrompomodulin is measured by an immunoassay using an antigen-antibody reaction between an antithrombomodulin antibody and thrombomodulin.
フ. 抗トロンボモジュリン抗体を含む、 流早産の可能性の判定用試薬。 F. A reagent for determining the possibility of premature birth, including an antithrombomodulin antibody.
8 . 抗トロンボモジュリン抗体の、 流早産の可能性の判定用試薬としての用途。  8. Use of anti-thrombomodulin antibody as a reagent to determine the possibility of premature birth.
PCT/JP2000/009301 1999-12-28 2000-12-27 Method of judging the possibility of abortion/premature birth and reagent therefor WO2001048479A1 (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6447391A (en) * 1987-04-01 1989-02-21 Mitsubishi Gas Chemical Co Monoclonal antibody and use thereof
WO1991006857A1 (en) * 1989-11-02 1991-05-16 Teijin Limited Immunoassay of human thrombomodulin, and reagent and kit therefor

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6447391A (en) * 1987-04-01 1989-02-21 Mitsubishi Gas Chemical Co Monoclonal antibody and use thereof
WO1991006857A1 (en) * 1989-11-02 1991-05-16 Teijin Limited Immunoassay of human thrombomodulin, and reagent and kit therefor

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