JP4207283B2 - How to distinguish fetal chromosomal abnormalities - Google Patents
How to distinguish fetal chromosomal abnormalities Download PDFInfo
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- JP4207283B2 JP4207283B2 JP02532099A JP2532099A JP4207283B2 JP 4207283 B2 JP4207283 B2 JP 4207283B2 JP 02532099 A JP02532099 A JP 02532099A JP 2532099 A JP2532099 A JP 2532099A JP 4207283 B2 JP4207283 B2 JP 4207283B2
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- sugar chain
- lectin
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Description
【０００１】
【発明の属する技術分野】
本発明は、より簡便な胎児染色体異常の判別方法、特に胎児ダウン症候群の簡便な判別方法を提供することにある。
【０００２】
【従来の技術】
ダウン症候群は、染色体異常症候群の一つとして広く知られている。ダウン症候群に於いてみられる染色体異常は常染色体異常であり、２１番染色体が３つあるトリソミー２１型が95％を占め、４％が２１番染色体の転移型、残り１％がモザイク型である。臨床症状としては典型的な特有の顔貌、身体発育のおくれ、重度の知的障害が多い。
【０００３】
ダウン症候群の出生前診断としては、例えば受胎10〜20週の羊水穿刺して、細胞の染色体を調べることにより胎児が２１トリソミーであるかどうかを検査する方法が一般的である。しかしながら、羊水穿刺は、診断効率は高いが、0.5〜１％の確率で流産を引き起こすという問題がある。また、超音波診断法、絨毛検査等の方法もあるがそれぞれ信頼度が低い、胎児に障害を与える可能性がある、等の問題点を有している。その他、母体の血液成分の測定に基づいて行う種々のスクリーニング法が開発されており、特に母親の年齢と、母体血中のα-フェトプロテイン(AFP)、ヒト絨毛性ゴナドトロピン(hCG)及び非抱合エストリオール(uE3)の濃度とから危険率を算出する方法が広く使用されている（Wald NJ, Densem JW, Smith D, Klee GG: Four-Marker Serum Screening For Down's Syndrome. Prenat. Diagn, 1994;14：707-716）。その他にも、例えば特表平8-500181号公報には、妊婦の母体血液を乾燥処理した後、hCGレベルを基準値と比較する方法、及びＡＦＰレベルの値も併用する方法が記載されている。また、特許第2525474号公報には、母体の血清サンプルのuE3、プロゲステロン、16α-ヒドロキシデヒドロエピアンドロステロン等の含有値を測定する方法に於いて、ＡＦＰの測定も併せて行う方法が開示されている。また、特開平2-5895号公報には、妊婦の血清、羊水等の細胞外体液中のSOD-1濃度を測定する方法等が記載されている。しかしながら、これらの方法は、測定結果の解析が煩雑なこと、感度が６０〜７０％と依然低いこと、体重、糖尿病の有無、喫煙の習慣の有無等母体条件の影響を受けやすいこと等の問題点を有している。
【０００４】
【発明が解決しようとする課題】
本発明は、上記した如き状況に鑑みなされたもので、母体及び胎児に対する安全性に優れ、検査結果の信頼性も高い胎児染色体異常の判別方法、及びこれに用いられる試薬又はキットを提供することを目的とする。
【０００５】
【課題を解決するための手段】
本発明は、妊婦の体液中に存在するＡＦＰ類を糖鎖構造の違いに基づいて分画した後、特定糖鎖構造を有するＡＦＰ類の一つ又はそれ以上を測定し、その測定結果に基づいて判別することを特徴とする、胎児染色体異常の判別のためのデータを得るための方法の発明である。
また、本発明は、ＡＦＰ類の少なくとも一つの特定糖鎖構造を認識し得るタンパク質を含有して成る、胎児染色体異常の判別用試薬の発明である。
更に、本発明は、妊婦の体液中に存在するＡＦＰ類を糖鎖構造の違いに基づいて分画した後、総ＡＦＰ量中の、特定糖鎖構造を有するＡＦＰ類の一つ又はそれ以上の割合を測定し、その測定結果に基づいて判別することを特徴とする、胎児染色体異常の判別のためのデータを得るための方法の発明である。
更にまた、本発明は、(1)ＡＦＰ類の少なくとも一つの特定糖鎖構造を認識し得るレクチン、及び(2)抗ＡＦＰ抗体、を含んで成る、胎児染色体異常判別用キットの発明である。
また、本発明は、(1)ＡＦＰ類の少なくとも一つの特定糖鎖構造を認識し得るレクチン、(2)当該レクチンの結合の有無に関わらず全てのＡＦＰ類に結合し得る抗ＡＦＰ抗体及び(3)当該レクチンが結合したＡＦＰ類には反応性が低いが、当該レクチンが結合していないＡＦＰ類には反応性が高い抗ＡＦＰ抗体、を含んで成る、胎児染色体異常判別用キットの発明である。
【０００６】
即ち、本発明者等は、母体及び胎児に対する影響がなく、検査結果の信頼性も高く、且つ簡便な胎児染色体異常の判別方法を確立すべく鋭意研究の結果、妊婦の体液中に存在する特定の糖鎖構造を有するＡＦＰ類を測定することにより、胎児染色体異常の危険度を信頼性高く判別することができることを見出し、本発明を完成するに到った。
【０００７】
ＡＦＰは、胎児血清或いは肝細胞癌、ヨークサック腫瘍等の悪性腫瘍で発現される癌胎児性糖タンパク質であり、各種肝疾患診断の重要な指標の一つとして良く用いられている。
【０００８】
ＡＦＰは、一般には１分子あたり１本のアスパラギン結合型（N-グリコシド型）の二本鎖複合型糖鎖（コンプレックス二本鎖）を有する。そのため、ＡＦＰは、オリゴ糖に対する特異性が異なるレクチンを用いた親和電気泳動で分画されたＡＦＰバンドを抗体親和転写法で検出する方法により異なる糖鎖を持つＡＦＰに分画することができる。例えばコンカナバリンＡ（ConA）を用いた場合、ＡＦＰはその糖鎖の種類によりAFP-C1、AFP-C2に分画される。レンズマメレクチン（LCA）を用いた場合は、AFP-L1、AFP-L2、AFP-L3に分画される。インゲンマメレクチンＥ４（PHA-E4）を用いると、AFP-P1〜AFP-P5に分画され、AFP-P3よりも移動度が速いAFP-P3fも出現する。カブトムシ幼虫レクチン（alloA）では、AFP-A1、AFP-A2、AFP-A3に分画され、AFP-A1より移動度が遅いAFP-A1sが出現することもある。ヒママメレクチン（RCA-120）を用いた場合、AFP-R1、AFP-R2、AFP-R3に分画され、AFP-R1より移動度が速いAFP-R1fが出現することもある。ダツラレクチン（DSA）では、AFP-D1〜AFP-D6に分画される。ヒイロチャワンタケレクチン（AAL）では、AFP-AA1〜AFP-AA4に分画される（武田和久,「臨床検査」vol.39,No.1,p.66-70(1995)、Shimizu K.,Clin.Chim.Acta.,254,23-40(1996)）。
【０００９】
ＡＦＰは、妊娠時に羊水及び母体血清で高値を示すことが知られている。これは未成熟の胎児肝臓でＡＦＰが生産されるためである。即ち、胎生期の初期には肝臓でコアのN-アセチルグルコサミン（GlcNAc）がフコシル化されたもの、及びマンノース（Man）α１→６分岐側のガラクトース（Gal）が露出したモノシアロＡＦＰが比較的多く作られる。また、妊娠初期の羊水中にはバイセクト（bisect）型のGlcNAcを有するＡＦＰが出現する。胎児が成長し肝臓が成熟するにつれ、ＡＦＰの生成は低下する。一方、成人では通常血清ＡＦＰ濃度は10ng／ml以下であるので、妊娠時に母体血清中に出現するＡＦＰの大部分は胎児に由来していると考えられている。
【００１０】
本発明に係るＡＦＰ類としては、特定の糖鎖構造を有するものが好ましく挙げられ、特定の糖鎖構造としては、例えば以下のようなものが挙げられる。
１．下記式１で示されるコンプレックス２本鎖
【００１１】
【式１】
２．コンプレックス２本鎖が変異したもの
▲１▼糖が付加したもの
ｉ）還元末端のGlcNAcへフコースがα１→６結合で付加したもの
具体的には、例えば下記式２で示されるもの等が挙げられる。
【００１３】
【式２】
ii）α１→６,α１→３及びβ１→４結合を有するManへGlcNAcがβ１→４結合で付加したもの
【００１５】
具体的には、例えば下記式３で示されるもの等が挙げられる。
【式３】
iii）β１→２及びα１→６結合を有するMan又は／及びβ１→２及びα１→３結合を有するManへ糖鎖が付加して３本鎖、４本鎖構造となったもの
【００１７】
▲２▼糖が脱離したもの
ｉ）末端シアル酸が欠如したもの
具体的には、例えば下記式４〜７で示されるもの等が挙げられる。
【００１８】
【式４】
【式５】
【式６】
【式７】
ii）非還元末端側Galが欠如したもの
具体的には、例えば下記式８で示されるもの等が挙げられる。
【００２３】
【式８】
▲３▼糖が付加及び脱離したもの
具体的には、例えば下記式９で示されるもの等が挙げられる
【００２５】
【式９】
▲４▼末端シアル酸の結合様式が変異したもの（通常はα２→６結合で付加、変異はα２→３結合で付加）
具体的には、例えば下記式１０で示されるもの等が挙げられる。
【式１０】
また、本発明に係る特定の糖鎖構造を有するＡＦＰ類には、各種レクチンによって分画されるものも含まれ、具体的には、例えばＣonＡを用いて分画されるAFP-C1，AFP-C2、ＬＣＡを用いて分画されるAFP-L1，AFP-L2，AFP-L3、ＰＨＡ―Ｅ４を用いて分画されるAFP-P1，AFP-P2，AFP-P3，AFP-P4，AFP-P5，AFP-P3f、alloＡを用いて分画されるAFP-A1，AFP-A2，AFP-A3，AFP-A1s、ＲＣＡ−１２０を用いて分画されるAFP-R1，AFP-R2，AFP-R3，AFP-R1f，AFP-R3f、ＤＳＡを用いて分画されるAFP-D1，AFP-D2，AFP-D3，AFP-D4，AFP-D5，AFP-D6，AFP-D7、ＡＡＬを用いて分画されるAFP-AA1，AFP-AA2，AFP-AA3，AFP-AA4等が挙げられるが、これらに限定されない。また、用いられるレクチン、及びＡＦＰ類もこれらに限定されない。
【００２８】
尚、式１で示されるコンプレックス２本鎖の糖鎖構造を有するＡＦＰ類は、レクチンを用いて分画した場合、例えばAFP-C2，AFP-L2，AFP-P2，AFP-A3，AFP-R1等に分画される。式２で示される糖鎖構造を有するＡＦＰ類はAFP-C2，AFP-L3，AFP-P2，AFP-A3，AFP-R1等に分画される。式３、式４及び式９で示される糖鎖構造を有するＡＦＰ類はAFP-C1，AFP-P5，AFP-A3，AFP-R3等に分画される。式５で示される糖鎖構造を有するＡＦＰ類はAFP-C2，AFP-P4，AFP-A3，AFP-R2等に分画される。式６で示される糖鎖構造を有するＡＦＰ類はAFP-C2，AFP-P4等に分画される。式７及び式８で示される糖鎖構造を有するＡＦＰ類はAFP-C2，AFP-P3，AFP-A3等に分画される。式１０で示される糖鎖構造を有するＡＦＰ類はAFP-C2，AFP-P5，AFP-A3，AFP-R1等に分画される。また、上記以外のレクチンを用いて分画すれば、これらＡＦＰ類は、使用したレクチンの性質に従って、種々の画分に分画される。
【００２９】
また、特定の糖鎖構造を有するＡＦＰ類を測定するに当たっては、ＡＦＰ類を予め、例えばシアリダーゼ、N-アセチルヘキソサミニダーゼ、ガラクトシダーゼ、フコシダーゼ等の糖加水分解酵素により処理してから、測定に供してもよい。例えば１８トリソミー、２１トリソミー（＝胎児ダウン症候群）等の常染色体異常を判別する場合には、これら特定の糖鎖構造を有するＡＦＰ類の中でも、例えばＬＣＡ、ＰＨＡ―Ｅ４、ＣonＡ等のレクチンで分画されるものが好ましく挙げられ、より好ましくはAFP-L2、AFP-L3、AFP-P4、AFP-P5、AFP-C1等が挙げられる。また、性染色体異常を判別する場合には、例えばＬＣＡ等で分画されるＡＦＰ類が好ましく挙げられ、より好ましくはAFP-L2、AFP-L3等が挙げられる。
【００３０】
本発明に係る測定用試料として用いられる母体液としては、羊水、血漿、又は血清等が挙げられる。また、母体液としては、妊娠週数１０〜２０週の範囲にある妊婦から採取されたものが通常用いられる。
【００３１】
本発明に係る特定の糖鎖構造を有するＡＦＰ類の糖鎖構造或いはその近傍を認識することができるタンパク質としては、例えばレクチン類、抗体等が挙げられる。このようなレクチン類としては、例えばＣonＡ，ＬＣＡ，alloＡ，RCA−120，ＤＳＡ，ＡＡＬ，ＰＨＡ−Ｅ４，エンドウマメレクチン，ピーナッツレクチン，小麦胚芽レクチン等が挙げられ、測定しようとするＡＦＰ類の特定の糖鎖構造に合わせて適宜選択して用いられる。中でも、ＣonＡ，ＬＣＡ及びＰＨＡ−Ｅ４が好ましく用いられる。
【００３２】
抗体としては、ＡＦＰ類の特定の糖鎖構造或いはその近傍に対する抗体であれば何れにてもよく、特に限定されない。例えばポリクローナル抗体でもモノクローナル抗体でも何れにてもよく、これらを単独で或はこれらを適宜組み合わせて用いる等は任意である。尚、均一の性質を有する抗体の特異性を考慮すると、ポリクローナル抗体よりもモノクローナル抗体の方が好ましい。また、これら抗体は、要すればペプシン，パパイン等の酵素を用いて消化してF(ab')2、Fab'、或はFabとして使用してもよい。さらに抗体には種々の物質、例えば酵素免疫測定法（EIA）に於いて用いられるアルカリホスファターゼ，β-ガラクトシダーゼ，ペルオキシダーゼ，マイクロペルオキシダーゼ，グルコースオキシダーゼ，グルコース-6-リン酸脱水素酵素，アセチルコリンエステラーゼ，リンゴ酸脱水素酵素，ルシフェラーゼ等の酵素類、例えば放射免疫測定法（RIA）で用いられる99mＴc，131Ｉ，125Ｉ，14Ｃ，3Ｈ等の放射性同位元素、例えば蛍光免疫測定法（FIA）で用いられるフルオレセイン，ダンシル，フルオレスカミン，クマリン，ナフチルアミン或はこれらの誘導体等の蛍光性物質、例えばルシフェリン，イソルミノール，ルミノール，ビス(2,4,6-トリフロロフェニル)オキザレート等の発光性物質、例えばフェノール，ナフトール，アントラセン或はこれらの誘導体等の紫外部に吸収を有する物質、例えば4-アミノ-2,2,6,6-テトラメチルピペリジン-1-オキシル，3-アミノ-2,2,5,5-テトラメチルピロリジン-1-オキシル，2,6-ジ-t-ブチル-α-(3,5-ジ-t-ブチル-4-オキソ-2,5-シクロヘキサジエン-1-イリデン)-p-トリルオキシル等のオキシル基を有する化合物に代表されるスピンラベル化剤としての性質を有する物質等の標識物質、高速液体クロマトグラフィー（HPLC）等を用いて抗原抗体複合物を分離する際に当該複合体の例えば分子量、疎水性、等電点等の性質を変化させて、当該複合体とその他の成分とをより明確に分離させ得る性質を有する物質（以下、分離向上物質と略記する。）、具体的には例えばα−キモトリプシノーゲン，β−ガラクトシダーゼ，リゾチーム，チトクロームｃ，トリプシンインヒビター等のタンパク質、例えばフェニルアラニン，プロリン，アルギニン，リジン，アスパラギン酸，グルタミン酸等のアミノ酸を含むペプチド、例えば臭素，塩素，沃素等のハロゲン原子、例えばポリエチレングリコール等の合成高分子、例えばポリグルタミン酸，ポリアスパラギン酸，ポリリジン，ポリアルギニン，ポリフェニルアラニン，ポリチロシン等のポリアミノ酸、例えば特開平9-301995号公報に開示された、例えば硫酸、リン酸等の強酸に由来する残基を少なくとも３個有するポリペプチド、炭素数３〜10のアルキル鎖、例えばパルミチン酸，オレイン酸，ステアリン酸等の脂肪酸、例えばN-(ε-マレイミドカプロイルオキシ)スクシンイミド［N-(ε-maleimidocaproyloxy)succinimide］（EMCS）,N-スクシンイミヂル-6-マレイミドヘキサノエイト（N-Succinimidyl-6-maleimidohexanoate），ビスマレイミドヘキサン（Bismaleimidohexane）（BMH），オクチルアミン等の第１抗体に結合し得る反応基を有し且つ疎水性若しくはイオン性を有する化学物質等を導入して使用してもよい。
【００３３】
本発明に係る特定の糖鎖構造を有するＡＦＰ類の測定方法の具体例としては、例えばＡＦＰ類の特定の糖鎖構造を認識し得るタンパク質（例えばレクチン類や抗体等）を用いる方法等が挙げられる。より具体的には、自体公知の、抗ＡＦＰ抗体やＡＦＰの特定の糖鎖構造を認識し得るタンパク質としてレクチンを用いるレクチン親和電気泳動法、レクチンアフィニティーカラムクロマトグラフィーを用いる方法、高速液体クロマトグラフィー（HPLC）を用いる方法等が挙げられる。これら自体公知の方法に関しては、各種キットが市販されているので、それを用いて行ってもよい。
【００３４】
レクチン親和電気泳動法は、例えば以下のようにして行えばよい。
即ち、先ず試料をＡＦＰ類の特定糖鎖構造を認識し得るレクチンを含有するゲルで電気泳動し、ＡＦＰ類の画分を得る。次いで抗ＡＦＰ抗体、酵素で標識された、抗ＡＦＰ抗体に対する抗体で処理し、ゲルを洗浄後、酵素基質を含有する溶液で処理して酵素反応により発色させ、次いでデンシトメーター等で各画分の吸収を測定する。総ＡＦＰ面積に対するＡＦＰ類画分のピーク面積の百分率を、ＡＦＰ類の分画比（％）として求める。
【００３５】
ＨＰＬＣを用いる方法は、例えば以下のようにして行えばよい。即ち、レクチンと、酵素等の標識物質又は分離向上物質を結合させた、ＡＦＰ類に対する認識部位の異なる二種の抗体を用いる。まず試料を、少なくとも一つのＡＦＰ類の特定糖鎖構造を認識し得るレクチンと、当該レクチンの結合の有無に関わらず全てのＡＦＰ類に結合し得る標識物質結合抗体と反応させる。次いで、当該レクチンが結合したＡＦＰ類には反応性が低いが当該レクチンが結合していないＡＦＰ類には反応性が高い分離向上物質結合抗体と更に反応させた後、ＨＰＬＣにより、ＡＦＰ類を分画する。各画分中の標識物質を測定し、その結果に基づいて総ＡＦＰ面積に対するＡＦＰ類画分のピーク面積の百分率を、ＡＦＰ類の分画比（％）として求める。
【００３６】
レクチン親和電気泳動法による測定方法を、例えばＡＦＰ―Ｌ３測定用キットであるＡＦＰ−Ｌ３テストワコー（和光純薬工業(株)製）を用いて、AFP-L3を測定する方法を例にとって具体的に説明すると、概略以下の如くである。
即ち、先ず試料を例えばレクチンとしてＬＣＡを含有するアガロースゲル等で電気泳動し、ＡＦＰ類の夫々の画分を得る。該アガロースゲルに、モノクローナル抗ヒトＡＦＰマウス抗体が結合した抗体結合膜をかぶせて抗原抗体反応を行わせる。これによりＡＦＰ画分は、抗体結合膜上に移る。アガロースゲルから抗体結合膜をはがし、洗浄する。次いでウサギ抗ヒトＡＦＰ抗体溶液に浸し、免疫反応を起こさせる。次いで洗浄後、ペルオキシダーゼ（POD）標識抗ウサギIgGヤギ抗体溶液に浸し、反応後、洗浄し、次いで該抗体結合膜を発色試液に浸して染色する。染色、洗浄後、測定波長550〜650nmで、デンシトメーターで吸収を測定する。AFP―L3分画比（％）は、デンシトグラムから各ピークの面積を算出し、総ＡＦＰ面積に対するAFP-L3画分のピーク面積の百分率として求める。
【００３７】
ＨＰＬＣを用いる測定方法は、具体的には例えば特開平9-301995号公報に記載された方法に準じて行えば良い。即ち、AFP-L3を測定する方法を例にとって説明すると、ＡＦＰ類の認識部位が異なる３種類の抗ＡＦＰモノクローナル抗体から得られたFab'に、例えばPOD等の酵素等の標識物質、例えば4-(p-マレイミドフェニル）ブチリルAla-(Tyr(SO3H))5、4-(p-マレイミドフェニル）ブチリルAla-(Tyr(SO3H))8等の分離向上物質を結合させてFab'-1-POD、Fab'-2-Ala-(Tyr(S03H))5、Fab'-3-Ala-(Tyr(SO3H))8とする。試料とFab'-1-PODとＬＣＡとを反応させた後、Fab'-2-Ala-(Tyr(S03H))5、Fab'-3-Ala-(Tyr(SO3H))8をさらに反応させる。これらのうち、Fab'-3はＬＣＡが結合したＡＦＰ類に対しては反応性が低いが、ＬＣＡが親和性を有さない（結合しない）ＡＦＰ類に対しては反応性が高いという性質を有している。また、Fab'-1及びFab'-2は、ＬＣＡが結合しているか否かに関わらず、全てのＡＦＰ類に結合する性質を有している。そのため反応液中に２種類の免疫複合体、即ち、ＬＣＡが親和性を有するＡＦＰ類とFab'-2-Ala-(Tyr(S03H))5とFab'-1ーPODの免疫複合体と、ＬＣＡが親和性を有さない（結合しない）ＡＦＰ類とFab'-2-Ala-(Tyr(S03H))5とFab'-3-Ala-(Tyr(SO3H))8とFab'-1-PODの免疫複合体が生成する。次に、陰イオン交換クロマトグラフィーを用いて免疫複合体中の硫酸残基の個数により、この２種の複合体を分離し、各々のピークのPOD活性を測定する。２種の免疫複合体ピークの合計面積は総ＡＦＰ量を示すので、ＬＣＡが親和性を有するＡＦＰピーク面積を総ＡＦＰのピーク面積で割ることにより、AFP-L3分画比の値（％）を求めることができる。
【００３８】
尚、測定するＡＦＰ類は、一つであっても、また２つ以上であっても何れにてもよい。
【００３９】
胎児染色体異常の判別方法としては、まず検査対象の妊婦と実質的に年齢及び妊娠週数が一致し、且つ羊水検査により染色体異常を有さない胎児を持つことが確認された妊婦（正常）から得られた試料について、本発明の方法を用いて、測定を行う。即ち、妊婦の体液中に存在するＡＦＰ類を糖鎖構造の違いに基づいて分画し、特定糖鎖構造を有するＡＦＰ類の一つ又はそれ以上を測定する。このようにして得られた特定のＡＦＰ類の量を基準値として用い、上記と同様の方法で試料から得られた特定のＡＦＰ類の量を、正常値と比較する。これらの量の違いに有意差が見られる場合を陽性［胎児染色体異常の可能性有り（危険度が高い）］と判断する。
【００４０】
また、例えば年齢及び妊娠週数がほぼ一致し、羊水穿刺検査等により胎児が染色体異常ではないことが確認された母体（正常母体）から得られた試料について、本願発明の方法を用いて、妊婦の体液中に存在するＡＦＰ類を糖鎖構造の違いに基づいて分画し、特定糖鎖構造を有する一つ又はそれ以上のＡＦＰ類量の、総ＡＦＰ量に対する比を求め、得られた所定のＡＦＰ類の分画比の値を基準として、診断すべき試料から同様にして得られた所定のＡＦＰ類の分画比の値を比較し、基準値との有意差が認められれば、陽性と判断する等してもよい。ＡＦＰ類の分画比の値が基準値と比較して高い場合を陽性とするか、低い場合を陽性とするかは、測定対象のＡＦＰ類の種類、試料の種類によって異なる。例えば胎児ダウン症候群の診断をAFP(L2＋L3)分画比に基づいて行う場合、血清及び羊水中の値が正常群に比べて有意に高値となるし、AFP-C1分画比に基づいて行う場合、血清中の値が正常群に比較して有意に低値を示す。また、AFP(P4＋P5)分画比、AFPーP4分画比に基づいて行う場合、羊水中の値が正常群に比較して有意に低値を示す。性染色体異常の診断を、AFP(L2＋L3)分画比に基づいて行う場合、羊水中の値が正常群に比較して有意に低値を示す。
【００４１】
本発明の胎児染色体異常判別用試薬は、ＡＦＰ類の特定の糖鎖構造を認識し得るタンパク質、例えば抗体、レクチン等を含有するものであり、その構成要素の好ましい態様と具体例は上で述べたとおりである。
【００４２】
本発明の胎児染色体異常判別用キットは、少なくとも一つのＡＦＰ類の特定の糖鎖を認識し得るレクチンと、抗ＡＦＰ抗体を含有するものである。
【００４３】
また、本願発明に係る胎児染色体異常判別用キットは、ＡＦＰ類の少なくとも一つの特定糖鎖構造を認識し得るレクチン、当該レクチンの結合の有無に関わらず全てのＡＦＰ類に結合し得る抗ＡＦＰ抗体及び当該レクチンが結合したＡＦＰ類には反応性が低いが、当該レクチンが結合していないＡＦＰ類には反応性が高い抗ＡＦＰ抗体、を含有していても良い。
【００４４】
より具体的には、例えば所定のレクチンを含有するアガロースゲル，抗ヒトＡＦＰ抗体が結合した抗体結合膜，抗ＡＦＰ抗体（第２抗ＡＦＰ抗体）を含有する溶液，POD等の酵素で標識された、第２抗ヒトＡＦＰ抗体に対する二次抗体溶液，発色試液［例えば標識酵素がPODの場合はβ−ニコチンアミドアデニンジヌクレオチド還元型及びニトロテトラゾリウムブルー(Nitro-TB)等］，過酸化水素水，洗浄液等を含有するレクチン親和電気泳動法用キット、例えばＬＣＡ等のレクチン，例えばAla-(Tyr(SO3H))5、Ala-(Tyr(SO3H))8等の分離向上物質が結合した抗ＡＦＰ抗体―１，POD等の酵素で標識された抗ＡＦＰ抗体―２，抗ＡＦＰ抗体―１に結合させたものとは異なる分離向上物質が結合した抗ＡＦＰ抗体―３，基質液（PODを抗体の標識酵素として用いる場合は4-アセトアミドフェノール，過酸化水素水等を含有。）等を含有するカラムを用いるＡＦＰ類測定法用キット等が挙げられる。ここで、抗ＡＦＰ抗体―１と抗ＡＦＰ抗体―２は、当該レクチンの結合の有無に関わらず全てのＡＦＰ類に結合し得る抗体であって、抗ＡＦＰ抗体―３は、当該レクチンが結合したＡＦＰ類には反応性が低いが、当該レクチンが結合していないＡＦＰ類には反応性が高い抗体である。
また、これらの構成要素の好ましい態様と具体例は、上で述べた通りである。
【００４５】
これらキットに含まれる試薬中には、通常この分野で用いられる試薬類、例えば緩衝剤、反応促進剤、糖類、タンパク質、塩類、界面活性剤等の安定化剤、防腐剤等であって、共存する試薬等の安定性を阻害したり、ＡＦＰ類とレクチン類（又は抗体）との反応を阻害しないものが含まれていてもよい。またその濃度も、通常この分野で通常用いられる濃度範囲から適宜選択すればよい。
また、マグネシウム等の金属イオンがレクチン活性や安定性に影響を与えることはよく知られており、これらを含んでいてもよい。
【００４６】
本発明の試薬やキットに於いて用いることのできる緩衝剤としては、例えばトリス緩衝剤、リン酸緩衝剤、ベロナール緩衝剤、ホウ酸緩衝剤、グッド緩衝剤等通常免疫比濁法、免疫比ろう法、ラジオイムノアッセイ、酵素免疫測定法に用いられている緩衝剤は全て挙げられ、測定反応時のｐＨとしては抗原抗体反応を抑制しない範囲であれば特に限定されないが、通常６〜１０の範囲から好ましく選択される。
【００４７】
以下に実施例を挙げて本発明を更に詳細に説明するが、本発明はこれら実施例により何等制限されるものではない。
【００４８】
【実施例】
実施例 １．胎児ダウン症候群における母体血清中および羊水中のＡＦＰ類についての検討
（１）検体
羊水検査によりダウン症候群罹患胎児を持つことが確認された２７〜４４歳の妊婦１７例の、妊娠１５〜１９週の血清および羊水を使用した。
また、該罹患症例と年齢および妊娠週数が一致し、且つ羊水検査により染色体異常を有さない胎児を持つことが確認された正常妊婦２０例の血清および羊水を対照として用いた。
（２）総ＡＦＰ量の測定
アボット社のIMx AFPアッセイシステムを使用し、添付文書に記載の標準操作法に従って、測定した。
（３）ＡＦＰ分画比の測定
２μｌの血清検体あるいは総ＡＦＰ濃度が50ng／ml前後になるように生理食塩水で希釈した羊水検体２μlを、所定レクチンを所定濃度含む厚さ１mmの１％アガロースゲルに塗布し、以下の条件で電気泳動を行い、特定の糖鎖構造を有するＡＦＰ（以下、マーカーと略記する。）を分離した。尚、レクチンとしてDSA又はRCA-120を使用する際は、検体をシアリダーゼで処理した後に、アガロース膜に塗布した。
【００４９】
分離された各ＡＦＰ画分をモノクローナル抗ＡＦＰ抗体（マウス）を塗布したニトロセルロース膜に転写し、洗浄処理後、該膜上で更にポリクローナル抗ＡＦＰ抗体（ウサギ）とPOD標識抗ウサギIgG抗体（ヤギ）とを反応させた。洗浄処理後、これにニトロテトラゾリウムブルー、NADH、フェノールおよび過酸化水素を反応させ、各ＡＦＰ画分を発色させた。各画分のピーク面積をデンシトメーター（580nm）により測定し、分画比を次の式により算出した。
分画比（％）＝（各画分のピーク面積／各画分のピーク面積の総和）×１００
（４）結果
それぞれのマーカーについて、胎児ダウン症候群と正常群の平均値±ＳＤと２群間の有意差を表２に示した。尚、有意差検定にはｔ検定（Student's t）を使用した。
【００５１】
表 ２
表２から明らかな如く、胎児ダウン症候群は正常群と比較すると、血清中総ＡＦＰ量で有意に低値を示し、AFP(L2＋L3)分画比で有意に高値を示した。また、AFP(L2＋L3)分画比は、総ＡＦＰ量よりも、正常群に対し有意差が大きかった。更に、AFP-C1分画比は、検体数が少ないため有意差は出なかったが、正常群に対し低値を示す傾向が強く示唆された。
同様に、羊水ではAFP(L2＋L3)分画比で有意に高値を、AFP(P4＋P5)分画比で有意に低値を、AFPーP4分画比で有意に低値を示した。また、AFP(L2＋L3)分画比、ＡＦＰ(P4＋P5)分画比、AFP-P4分画比共に、総ＡＦＰ量よりも正常群に対し有意差が大きかった。
【００５３】
表２で有意差のあったものについて、平均値＋２ＳＤ又は平均値―２ＳＤをカットオフ値とし、感度、特異性及び診断効率を求めた結果を表３に示す。
【００５４】
表 ３
表３から明らかな如く、血清中および羊水中のAFP(L2＋L3)画分の診断効率が73％と一番高く、胎児のダウン症診断において有用性が高いことが確認された。言い換えれば、糖鎖の還元末端GlcNAcにフコースがα１→６付加したＡＦＰ類が、胎児のダウン症候群診断に有効であることが確認された。
【００５６】
実施例 ２．胎児染色体異常群と正常群との、母体血清および羊水中のAFP(L2＋L3)分画比の検討
（１）検体
羊水検査により胎児の２１トリソミー及び１８トリソミーの常染色体異常、あるいは性染色体異常が確認された24〜44歳の妊婦の妊娠週数15〜19週の血清および羊水を使用した。
また、該罹患症例と年齢および妊娠週数が一致し、且つ羊水検査により染色体異常を有さない胎児を持つことが確認された正常妊婦の血清およ羊水を対照として用いた。
（２）ＡＦＰ(L2＋L3)分画比の測定
ＡＦＰ−Ｌ３テストワコー（和光純薬(株)製）を用い、添付文書の標準操作法に従って、各画分を分離し、各画分のピーク面積をデンシトメーター（580nm）により測定して、AFP(L2＋L3)分画比を実施例１と同様に算出した。
（３）結果
それぞれの群について平均値±ＳＤと正常群に対する有意差を表４に示した。尚、有意差検定にはｔ検定（Student's t）を使用した。
【００５７】
表 ４
表４から明らかな如く、２１トリソミー群で血清および羊水中のAFP(L2＋L3)分画比が有意に高かった。また、性染色体異常群では羊水中のAFP(L2＋L3)分画比が有意に低かった。
【００５９】
次に有意差のあったものについて、診断効率が最大となる値をカットオフ値とした場合の、感度、特異性および診断効率を求めた結果を表５に示す。
【００６０】
表 ５
表５から明らかな如く、２１トリソミーあるいは性染色体異常診断において、血清および羊水中のAFP(L2＋L3)分画比の有用性、言い換えれば糖鎖の還元末端GlcNAcにフコースがα１→６付加したＡＦＰ類の有用性が確認された。
【００６２】
実施例 ３．胎児ダウン症候群における母体血清中の総ＡＦＰ量およびAFP(L2＋L3)分画比の検討
（１）検体
羊水検査によりダウン症候群罹患胎児を持つことが確認された27〜44歳の妊婦18名の、妊娠15〜19週の血清を使用した。
尚、該罹患症例と年齢および妊娠週数が一致し、且つ羊水検査により染色体異常を有さない胎児を持つことが確認された正常妊婦70名の血清を対照として用いた。
（２）総ＡＦＰ量の測定
アボット社のIMx AFPアッセイシステムを使用し、添付文書の標準操作法に従って測定した。
（３）AFP(L2＋L3)分画比の測定
ＡＦＰ−Ｌ３テストワコー（和光純薬工業(株)製）を用い、添付文書の標準操作法に従って、分離した各ＡＦＰ画分のピーク面積をデンシトメーター（580nm）で測定し、実施例１と同様の方法でAFP(L2＋L3)を算出した。尚、有意差検定にはｔ検定（student's t）を使用した。
【００６３】
（４）結果
正常群と胎児ダウン症候群に於ける母血清中の総ＡＦＰ量の分布を図１（ａ）に、AFP(L2＋L3)分画比分布を図１（ｂ）に夫々示す。
これらの結果から、総ＡＦＰ量及びAFP(L2＋L3)分画比は、何れも胎児ダウン症候群および正常群間で有意差が認められた。
次いで総ＡＦＰ量のカットオフ値を24ng／ml、AFP(L2＋L3)分画比のカットオフ値を42％とした場合の、それぞれの感度、特異性および診断効率を表６に示した。
【００６４】
また、総ＡＦＰ量及びAFP(L2＋L3)分画比の受信者動作特性（Receiver Operating Characteristic : ＲＯＣ）曲線を図２（ａ），（ｂ）に夫々示す。図２（ａ），（ｂ）をもとに、ＡＵＣ（area under the curve）値を算出したところ、総ＡＦＰ量の場合のＡＵＣ値は0.700，AFP(L2＋L3)分画比の場合のＡＦＰ値は0.835であった。これらのことより、ダウン症診断において、母体血清中のAFP(L2＋L3)分画比は総ＡＦＰ量よりも有用であり、より正しい診断ができることが判る。
【００６６】
【発明の効果】
本発明は、母体及び胎児に対する影響がなく、検査結果の信頼性も高く、且つ簡便な胎児染色体異常の判別方法、試薬及びキットを提供するもので、斯業に貢献するところ極めて大なる発明である。
【００６７】
【図面の簡単な説明】
【図１】図１は、実施例３で得られた結果を示し（ａ）は正常母体又は胎児ダウン症候群罹患胎児を持つことが確認された母体の、各血清検体の総ＡＦＰ量の分布を、（ｂ）はAFP(L2+L3)分画比分布を夫々示す。
【図２】図２は、実施例３で得られた結果に基づく受信者動作特性曲線を示し、（ａ）は、総ＡＦＰ量を測定した結果に基づく受信者動作特性曲線、（ｂ）は、AFP(L2+L3)分画比を測定した結果に基づく受信者動作特性曲線を夫々示す。[0001]
BACKGROUND OF THE INVENTION
It is an object of the present invention to provide a simpler method for discriminating fetal chromosomal abnormalities, in particular, a simple discriminating method for fetal down syndrome.
[0002]
[Prior art]
Down's syndrome is widely known as a chromosomal abnormality syndrome. Chromosomal abnormalities seen in Down's syndrome are autosomal abnormalities, with 95% being trisomy type 21 with 3 chromosomes, 4% being metastatic type of chromosome 21, and the remaining 1% being mosaic type . Clinical symptoms include typical unique facial features, physical growth, and severe intellectual disabilities.
[0003]
As a prenatal diagnosis of Down's syndrome, for example, a method of examining whether a fetus is trisomy 21 by puncturing the amnion at 10 to 20 weeks of conception and examining the chromosome of the cell is common. However, although amniocentesis has high diagnostic efficiency, it has a problem of causing miscarriage with a probability of 0.5 to 1%. In addition, there are methods such as an ultrasonic diagnostic method and a villus examination, but each has problems such as low reliability and possible damage to the fetus. In addition, various screening methods based on the measurement of maternal blood components have been developed, particularly maternal age, maternal blood α-fetoprotein (AFP), human chorionic gonadotropin (hCG) and unconjugated Es The method of calculating the risk factor from the concentration of triol (uE3) is widely used (Wald NJ, Densem JW, Smith D, Klee GG: Four-Marker Serum Screening For Down's Syndrome. Prenat. Diagn, 1994; 14: 707-716). In addition, for example, Japanese translation of PCT publication No. 8-500181 describes a method of comparing maternal blood of a pregnant woman and then comparing the hCG level with a reference value, and a method of using an AFP level value in combination. . In addition, Japanese Patent No. 2525474 discloses a method for measuring AFP in a method for measuring the content of uE3, progesterone, 16α-hydroxydehydroepiandrosterone, etc. in a maternal serum sample. Yes. Japanese Patent Application Laid-Open No. 2-5895 describes a method for measuring SOD-1 concentration in extracellular body fluids such as serum and amniotic fluid of pregnant women. However, these methods have problems such as complicated analysis of measurement results, sensitivity still as low as 60 to 70%, and being easily affected by maternal conditions such as weight, presence of diabetes, presence of smoking habits, etc. Has a point.
[0004]
[Problems to be solved by the invention]
The present invention has been made in view of the situation as described above, and provides a method for discriminating fetal chromosomal abnormalities excellent in safety for a mother and a fetus and having high reliability of a test result, and a reagent or kit used therefor. With the goal.
[0005]
[Means for Solving the Problems]
In the present invention, after fractionating AFPs present in the body fluid of pregnant women based on the difference in sugar chain structure, one or more AFPs having a specific sugar chain structure are measured, and based on the measurement results Discriminating fetal chromosomal abnormalities, characterized by For getting data for It is a method invention.
Further, the present invention is an invention of a reagent for discriminating a fetal chromosomal abnormality, comprising a protein capable of recognizing at least one specific sugar chain structure of AFPs.
Furthermore, the present invention relates to one or more AFPs having a specific sugar chain structure in the total amount of AFP after fractionating AFPs present in the body fluid of pregnant women based on the difference in sugar chain structure. Discrimination of fetal chromosomal abnormalities, characterized by measuring the proportion and discriminating based on the measurement result For getting data for It is a method invention.
Furthermore, the present invention provides: (1) A lectin capable of recognizing at least one specific sugar chain structure of AFPs, and (2) It is an invention of a kit for discriminating fetal chromosome abnormality, comprising an anti-AFP antibody.
The present invention also provides: (1) A lectin capable of recognizing at least one specific sugar chain structure of AFPs, (2) An anti-AFP antibody capable of binding to all AFPs with or without binding of the lectin, and (3) It is an invention of a kit for discriminating fetal chromosome abnormality comprising an anti-AFP antibody having low reactivity to AFPs bound to the lectin but having high reactivity to AFPs not bound to the lectin.
[0006]
That is, the present inventors have determined that there is no influence on the mother and the fetus, the reliability of the test result is high, and a simple method for determining fetal chromosomal aberrations. By measuring AFPs having the following sugar chain structure, it was found that the risk of fetal chromosomal abnormality can be determined with high reliability, and the present invention has been completed.
[0007]
AFP is an oncofetal glycoprotein expressed in fetal serum or malignant tumors such as hepatocellular carcinoma and Yorksack tumor, and is often used as one of the important indicators for various liver disease diagnosis.
[0008]
AFP generally has one asparagine-linked (N-glycoside) double-stranded complex type sugar chain (complex double chain) per molecule. Therefore, AFP can be fractionated into AFPs having different sugar chains by a method in which AFP bands fractionated by affinity electrophoresis using lectins having different specificities for oligosaccharides are detected by antibody affinity transcription. For example, when Concanavalin A (ConA) is used, AFP is fractionated into AFP-C1 and AFP-C2 depending on the type of sugar chain. When lentil lectin (LCA) is used, it is fractionated into AFP-L1, AFP-L2, and AFP-L3. When kidney bean lectin E4 (PHA-E4) is used, AFP-P3f which is fractionated into AFP-P1 to AFP-P5 and has a higher mobility than AFP-P3 also appears. In the beetle larva lectin (alloA), AFP-A1, AFP-A2, and AFP-A3 may be fractionated, and AFP-A1s having a slower mobility than AFP-A1 may appear. When castor lectin (RCA-120) is used, AFP-R1f, which is fractionated into AFP-R1, AFP-R2, and AFP-R3 and has a higher mobility than AFP-R1, may appear. Datsura lectin (DSA) is fractionated into AFP-D1 to AFP-D6. In herochawantake lectin (AAL), it is fractionated into AFP-AA1 to AFP-AA4 (Kazuhisa Takeda, “Clinical Laboratory” vol.39, No.1, p.66-70 (1995), Shimizu K., Clin .Chim.Acta., 254 , 23-40 (1996)).
[0009]
AFP is known to show high levels in amniotic fluid and maternal serum during pregnancy. This is because AFP is produced in immature fetal liver. That is, there are relatively many monosialo AFPs in which the core N-acetylglucosamine (GlcNAc) is fucosylated in the early embryonic period and mannose (Man) α1 → 6 branch side galactose (Gal) is exposed. Made. In addition, AFP having bisect type GlcNAc appears in amniotic fluid in early pregnancy. As the fetus grows and the liver matures, the production of AFP decreases. On the other hand, since the serum AFP concentration is usually 10 ng / ml or less in adults, most of AFP appearing in maternal serum during pregnancy is considered to be derived from the fetus.
[0010]
As the AFPs according to the present invention, those having a specific sugar chain structure are preferably mentioned, and examples of the specific sugar chain structure include the following.
1. Complex duplex represented by the following formula 1
[0011]
[Formula 1]
2. Complex double strand mutated
(1) Sugar added
i) Fucose added to GlcNAc at the reducing end with an α1 → 6 bond
Specifically, what is shown by following formula 2, etc. are mentioned, for example.
[0013]
[Formula 2]
ii) GlcNAc added to Man with α1 → 6, α1 → 3 and β1 → 4 bonds by β1 → 4 bonds
[0015]
Specifically, for example, one represented by the following formula 3 can be mentioned.
[Formula 3]
iii) A sugar chain added to Man having β1 → 2 and α1 → 6 bonds and / or Man having β1 → 2 and α1 → 3 bonds to form a three-stranded or four-stranded structure
[0017]
(2) The sugar is removed
i) lack of terminal sialic acid
Specific examples include those represented by the following formulas 4 to 7.
[0018]
[Formula 4]
[Formula 5]
[Formula 6]
[Formula 7]
ii) lack of non-reducing terminal Gal
Specifically, for example, those represented by the following
[0023]
[Formula 8]
(3) Added and desorbed sugar
Specifically, for example, those represented by the following
[0025]
[Formula 9]
(4) Mutation of terminal sialic acid binding mode (usually added by α2 → 6 bond, mutation added by α2 → 3 bond)
Specifically, for example, those represented by the following formula 10 can be mentioned.
[Formula 10]
Further, AFPs having a specific sugar chain structure according to the present invention include those fractionated by various lectins. Specifically, for example, AFP-C1, AFP- fractionated using ConA, for example. CFP, AFP-L1, AFP-L2, AFP-L3, AFP-P1, AFP-P2, AFP-P3, AFP-P4, AFP- fractionated using PHA-E4 AFP-A1, AFP-A2, AFP-A3, AFP-A1s, AFP-R2, AFP-R2, and AFP- fractionated using P5, AFP-P3f, and alloA Using AFP-D1, AFP-D2, AFP-D3, AFP-D4, AFP-D5, AFP-D6, AFP-D7, AAL fractionated using R3, AFP-R1f, AFP-R3f, DSA Examples include, but are not limited to, fractionated AFP-AA1, AFP-AA2, AFP-AA3, AFP-AA4, and the like. Moreover, the lectin and AFP used are not limited to these.
[0028]
In addition, AFPs having a complex double-chain sugar chain structure represented by Formula 1 are, for example, AFP-C2, AFP-L2, AFP-P2, AFP-A3, and AFP-R1 when fractionated using lectins. Etc. AFPs having a sugar chain structure represented by Formula 2 are fractionated into AFP-C2, AFP-L3, AFP-P2, AFP-A3, AFP-R1, and the like. AFPs having a sugar chain structure represented by Formula 3, Formula 4 and
[0029]
In measuring AFPs having a specific sugar chain structure, the AFPs are treated in advance with a sugar hydrolase such as sialidase, N-acetylhexosaminidase, galactosidase, fucosidase, etc. before measurement. May be provided. For example, when autosomal abnormalities such as
[0030]
Examples of the mother fluid used as the measurement sample according to the present invention include amniotic fluid, plasma, and serum. Moreover, as mother fluid, those collected from pregnant women in the range of 10 to 20 weeks of gestation are usually used.
[0031]
Examples of the protein capable of recognizing the sugar chain structure of AFPs having a specific sugar chain structure or the vicinity thereof according to the present invention include lectins and antibodies. Examples of such lectins include ConA, LCA, alloA, RCA-120, DSA, AAL, PHA-E4, pea lectin, peanut lectin, wheat germ lectin, and the like. Are appropriately selected according to the sugar chain structure. Of these, ConA, LCA and PHA-E4 are preferably used.
[0032]
The antibody is not particularly limited as long as it is an antibody against a specific sugar chain structure of AFPs or the vicinity thereof. For example, either a polyclonal antibody or a monoclonal antibody may be used, and these may be used alone or in appropriate combination. In view of the specificity of the antibody having uniform properties, the monoclonal antibody is preferable to the polyclonal antibody. In addition, these antibodies are digested with enzymes such as pepsin and papain if necessary, and F (ab ') 2 , Fab ′, or Fab. In addition, antibodies include various substances such as alkaline phosphatase, β-galactosidase, peroxidase, microperoxidase, glucose oxidase, glucose-6-phosphate dehydrogenase, acetylcholinesterase, apple used in enzyme immunoassay (EIA). Used in enzymes such as acid dehydrogenase and luciferase, such as radioimmunoassay (RIA) 99 mTc, 131 I, 125 I, 14 C, Three Radioisotopes such as H, for example, fluorescent substances such as fluorescein, dansyl, fluorescamine, coumarin, naphthylamine or their derivatives used in fluorescence immunoassay (FIA), such as luciferin, isoluminol, luminol, bis ( 2,4,6-trifluorophenyl) oxalate and other luminescent substances such as phenol, naphthol, anthracene or derivatives thereof having absorption in the ultraviolet region such as 4-amino-2,2,6,6 -Tetramethylpiperidine-1-oxyl, 3-amino-2,2,5,5-tetramethylpyrrolidine-1-oxyl, 2,6-di-t-butyl-α- (3,5-di-t- Labeling substances such as butyl-4-oxo-2,5-cyclohexadiene-1-ylidene) -p-tolyloxyl and other compounds having a property as a spin labeling agent typified by a compound having an oxyl group, high-speed liquid Chromatography (HPLC When separating an antigen-antibody complex using, for example, the properties of the complex such as molecular weight, hydrophobicity, isoelectric point, etc., so that the complex and other components can be separated more clearly. Substances (hereinafter abbreviated as separation enhancing substances), specifically, for example, proteins such as α-chymotrypsinogen, β-galactosidase, lysozyme, cytochrome c, trypsin inhibitor, such as phenylalanine, proline, arginine, lysine, asparagine Peptides containing amino acids such as acid and glutamic acid, for example, halogen atoms such as bromine, chlorine and iodine, synthetic polymers such as polyethylene glycol, such as polyglutamic acid, polyaspartic acid, polylysine, polyarginine, polyphenylalanine and polytyrosine Polyamino acids, such as JP 9-301995 A polypeptide having at least three residues derived from a strong acid such as sulfuric acid or phosphoric acid, an alkyl chain having 3 to 10 carbon atoms, such as fatty acids such as palmitic acid, oleic acid, stearic acid, etc. For example, N- (ε-maleimidocaproyloxy) succinimide [N- (ε-maleimidocaproyloxy) succinimide] (EMCS), N-Succinimidyl-6-maleimidohexanoate, bismaleimide hexane ( Bismaleimidohexane) (BMH), a chemical group having a reactive group capable of binding to the first antibody such as octylamine and having hydrophobicity or ionicity may be used.
[0033]
Specific examples of the method for measuring AFPs having a specific sugar chain structure according to the present invention include, for example, a method using a protein (for example, lectins, antibodies, etc.) capable of recognizing a specific sugar chain structure of AFPs. It is done. More specifically, known per se anti-AFP antibodies and lectin affinity electrophoresis using lectin as a protein capable of recognizing a specific sugar chain structure of AFP, a method using lectin affinity column chromatography, high performance liquid chromatography ( HPLC). Regarding these methods known per se, various kits are commercially available and may be used.
[0034]
The lectin affinity electrophoresis may be performed as follows, for example.
That is, first, a sample is electrophoresed on a gel containing a lectin capable of recognizing a specific sugar chain structure of AFPs to obtain a fraction of AFPs. Next, treatment with an anti-AFP antibody and an antibody against an anti-AFP antibody labeled with an enzyme, and after washing the gel, it is treated with a solution containing an enzyme substrate to develop a color by an enzymatic reaction, and then each fraction is analyzed with a densitometer or the like. Measure the absorption. The percentage of the peak area of the AFP fraction relative to the total AFP area is determined as the fraction ratio (%) of the AFP.
[0035]
The method using HPLC may be performed as follows, for example. That is, two types of antibodies having different recognition sites for AFPs, in which a lectin is bound to a labeling substance such as an enzyme or a separation enhancing substance, are used. First, a sample is reacted with a lectin capable of recognizing a specific sugar chain structure of at least one AFP and a labeled substance-bound antibody capable of binding to all AFPs regardless of the presence or absence of the lectin binding. Next, after further reacting with the separation-enhancing substance-bound antibody having low reactivity to the AFPs bound to the lectin but not highly bound to the lectin, the AFPs are separated by HPLC. Draw. The labeling substance in each fraction is measured, and based on the result, the percentage of the peak area of the AFP class relative to the total AFP area is determined as the fraction ratio (%) of the AFPs.
[0036]
Specific measurement method using lectin affinity electrophoresis, for example, using AFP-L3 Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.), which is an AFP-L3 measurement kit, as a specific example The outline is as follows.
That is, first, the sample is electrophoresed on, for example, an agarose gel containing LCA as a lectin to obtain respective fractions of AFPs. The agarose gel is covered with an antibody binding membrane to which a monoclonal anti-human AFP mouse antibody is bound to cause an antigen-antibody reaction. As a result, the AFP fraction moves onto the antibody-binding membrane. Peel off the antibody binding membrane from the agarose gel and wash. Then, it is immersed in a rabbit anti-human AFP antibody solution to cause an immune reaction. Next, after washing, the plate is immersed in a peroxidase (POD) -labeled anti-rabbit IgG goat antibody solution. After the reaction, the plate is washed, and then the antibody-bound membrane is immersed in a coloring reagent and stained. After staining and washing, the absorption is measured with a densitometer at a measurement wavelength of 550 to 650 nm. The AFP-L3 fraction ratio (%) is obtained as a percentage of the peak area of the AFP-L3 fraction with respect to the total AFP area by calculating the area of each peak from the densitogram.
[0037]
Specifically, the measurement method using HPLC may be performed according to the method described in JP-A-9-301995, for example. That is, taking a method for measuring AFP-L3 as an example, Fab ′ obtained from three types of anti-AFP monoclonal antibodies having different recognition sites for AFPs is labeled with a labeling substance such as an enzyme such as POD, for example, 4- (p-Maleimidophenyl) butyryl Ala- (Tyr (SO Three H)) Five 4- (p-maleimidophenyl) butyryl Ala- (Tyr (SO Three H)) 8 Fab'-1-POD, Fab'-2-Ala- (Tyr (S0 Three H)) Five , Fab'-3-Ala- (Tyr (SO Three H)) 8 And After reacting the sample with Fab′-1-POD and LCA, Fab′-2-Ala- (Tyr (S0 Three H)) Five , Fab'-3-Ala- (Tyr (SO Three H)) 8 Is further reacted. Of these, Fab'-3 has a low reactivity to AFPs to which LCA is bound, but has a high reactivity to AFPs to which LCA has no affinity (does not bind). Have. Further, Fab′-1 and Fab′-2 have a property of binding to all AFPs regardless of whether LCA is bound or not. Therefore, two types of immune complexes, namely AFPs having an affinity for LCA and Fab'-2-Ala- (Tyr (S0 Three H)) Five And Fab'-1-POD immune complex, LCA has no affinity (does not bind) AFPs and Fab'-2-Ala- (Tyr (S0 Three H)) Five And Fab'-3-Ala- (Tyr (SO Three H)) 8 And Fab'-1-POD immune complex is formed. Next, the two types of complexes are separated by the number of sulfate residues in the immune complex using anion exchange chromatography, and the POD activity of each peak is measured. Since the total area of the two immune complex peaks indicates the total amount of AFP, the AFP-L3 fraction ratio value (%) is obtained by dividing the AFP peak area where LCA has affinity by the peak area of total AFP. Can be sought.
[0038]
The number of AFPs to be measured may be one, or two or more.
[0039]
As a method of distinguishing fetal chromosomal abnormalities, first from a pregnant woman (normal) that has been confirmed to have a fetus that has substantially the same age and gestational age as the test subject and that has no chromosomal abnormality by amniotic fluid test The obtained sample is measured using the method of the present invention. That is, AFPs present in the body fluid of pregnant women are fractionated based on the difference in sugar chain structure, and one or more AFPs having a specific sugar chain structure are measured. Using the amount of the specific AFPs thus obtained as a reference value, the amount of the specific AFPs obtained from the sample in the same manner as described above is compared with the normal value. If there is a significant difference between these amounts, it is judged as positive [possible fetal chromosomal abnormality (high risk)].
[0040]
For example, for a sample obtained from a mother (normal mother) whose age and gestational age are almost the same and the fetus is confirmed not to have a chromosomal abnormality by amniocentesis, etc., AFPs present in the body fluid are fractionated on the basis of the difference in sugar chain structure, and the ratio of the amount of one or more AFPs having a specific sugar chain structure to the total AFP amount is determined, and the obtained predetermined Compare the fraction ratio values of the predetermined AFPs obtained in the same manner from the sample to be diagnosed with the fraction ratio value of the AFPs as a reference, and if a significant difference from the reference value is found, it is positive It may be judged. Whether the value of the fraction ratio of AFPs is higher than the reference value is positive or low is positive depending on the type of AFPs to be measured and the type of sample. For example, when fetal Down syndrome is diagnosed based on the AFP (L2 + L3) fraction ratio, serum and amniotic fluid levels are significantly higher than in the normal group, and based on the AFP-C1 fraction ratio Serum values are significantly lower than those in the normal group. Moreover, when it performs based on AFP (P4 + P5) fraction ratio and AFP-P4 fraction ratio, the value in amniotic fluid shows a significantly low value compared with a normal group. When diagnosis of sex chromosome abnormality is performed based on the AFP (L2 + L3) fraction ratio, the value in amniotic fluid is significantly lower than that in the normal group.
[0041]
The reagent for discriminating fetal chromosome abnormality of the present invention contains a protein capable of recognizing a specific sugar chain structure of AFPs, for example, an antibody, a lectin and the like. Preferred embodiments and specific examples of the constituent elements are described above. That's right.
[0042]
The kit for discriminating fetal chromosome abnormality of the present invention contains a lectin capable of recognizing a specific sugar chain of at least one AFP and an anti-AFP antibody.
[0043]
The kit for discriminating fetal chromosome abnormality according to the present invention is a lectin that can recognize at least one specific sugar chain structure of AFPs, and an anti-AFP antibody that can bind to all AFPs regardless of whether or not the lectin is bound. In addition, the AFPs to which the lectin is bound may have low reactivity, but the AFPs not bound to the lectin may contain an anti-AFP antibody having high reactivity.
[0044]
More specifically, for example, agarose gel containing a predetermined lectin, an antibody-binding membrane bound with an anti-human AFP antibody, a solution containing an anti-AFP antibody (second anti-AFP antibody), and an enzyme such as POD Secondary antibody solution against the second anti-human AFP antibody, coloring reagent [for example, β-nicotinamide adenine dinucleotide reduced type and nitrotetrazolium blue (Nitro-TB) etc. when the labeling enzyme is POD], hydrogen peroxide solution, A lectin affinity electrophoresis kit containing a washing solution, such as a lectin such as LCA, such as Ala- (Tyr (SO Three H)) Five , Ala- (Tyr (SO Three H)) 8 Anti-AFP antibody bound to a separation enhancing substance such as 1, anti-AFP antibody labeled with an enzyme such as POD-2, anti-AFP antibody bound to a separation enhancing substance different from that bound to anti-AFP antibody-1 (3) AFP measurement method kits using a column containing a substrate solution (when POD is used as an antibody labeling enzyme, 4-acetamidophenol, hydrogen peroxide solution, etc.) are included. Here, anti-AFP antibody-1 and anti-AFP antibody-2 are antibodies that can bind to all AFPs regardless of the presence or absence of binding of the lectin, and anti-AFP antibody-3 was bound to the lectin. It is an antibody having low reactivity to AFPs but high reactivity to AFPs to which the lectin is not bound.
Moreover, the preferable aspect and specific example of these components are as having described above.
[0045]
Among the reagents included in these kits are reagents usually used in this field, such as buffers, reaction accelerators, saccharides, proteins, salts, surfactants and other stabilizers, preservatives, etc. A reagent that does not inhibit the stability of the reagent or the like that does not inhibit the reaction between the AFPs and the lectins (or antibodies) may be included. Also, the concentration may be appropriately selected from the concentration range usually used in this field.
Further, it is well known that metal ions such as magnesium affect lectin activity and stability, and these may be included.
[0046]
Examples of the buffer that can be used in the reagent or kit of the present invention include Tris buffer, phosphate buffer, veronal buffer, borate buffer, Good buffer, etc. Examples include buffering agents used in the method, radioimmunoassay, and enzyme immunoassay, and the pH during the measurement reaction is not particularly limited as long as it does not suppress the antigen-antibody reaction, but usually from the range of 6-10. Preferably selected.
[0047]
The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.
[0048]
【Example】
Example 1. Study on AFPs in maternal serum and amniotic fluid in fetal Down syndrome
(1) Sample
Serum and amniotic fluid from 15 to 19 weeks of gestation of 17 pregnant women aged 27 to 44 years who were confirmed to have fetuses with Down syndrome by amniotic fluid testing were used.
In addition, sera and amniotic fluid from 20 normal pregnant women whose age and gestational age coincided with the affected cases and confirmed to have fetuses having no chromosomal abnormality by amniotic fluid examination were used as controls.
(2) Measurement of total AFP amount
Measurements were made using an Abbott IMx AFP assay system according to standard operating procedures described in the package insert.
(3) Measurement of AFP fraction ratio
2 μl of 2 μl serum sample or 2 μl of amniotic fluid sample diluted with physiological saline so that the total AFP concentration is around 50 ng / ml is applied to a 1% agarose gel of 1 mm thickness containing a predetermined concentration of lectin under the following conditions: Electrophoresis was performed to separate AFP having a specific sugar chain structure (hereinafter abbreviated as a marker). When DSA or RCA-120 was used as the lectin, the sample was treated with sialidase and then applied to the agarose film.
[0049]
Each separated AFP fraction was transferred to a nitrocellulose membrane coated with a monoclonal anti-AFP antibody (mouse), washed, and then further treated with a polyclonal anti-AFP antibody (rabbit) and a POD-labeled anti-rabbit IgG antibody (goat). ). After the washing treatment, this was reacted with nitrotetrazolium blue, NADH, phenol and hydrogen peroxide to develop each AFP fraction. The peak area of each fraction was measured with a densitometer (580 nm), and the fraction ratio was calculated by the following formula.
Fraction ratio (%) = (peak area of each fraction / total sum of peak areas of each fraction) × 100
(4) Results
For each marker, the mean value ± SD of fetal Down syndrome and normal group and the significant difference between the two groups are shown in Table 2. In addition, t test (Student's t) was used for the significant difference test.
[0051]
Table 2
As is clear from Table 2, fetal Down's syndrome was significantly lower in serum total AFP level and significantly higher in AFP (L2 + L3) fraction ratio than in the normal group. Further, the AFP (L2 + L3) fraction ratio was significantly different from the normal group than the total AFP amount. Furthermore, the AFP-C1 fraction ratio was not significantly different due to the small number of specimens, but strongly suggested a tendency to be lower than the normal group.
Similarly, in amniotic fluid, the AFP (L2 + L3) fraction ratio was significantly higher, the AFP (P4 + P5) fraction ratio was significantly lower, and the AFP-P4 fraction ratio was significantly lower. In addition, the AFP (L2 + L3) fraction ratio, the AFP (P4 + P5) fraction ratio, and the AFP-P4 fraction ratio were significantly different from the normal group than the total AFP amount.
[0053]
Table 3 shows the results of determining sensitivity, specificity, and diagnostic efficiency with the average value + 2SD or the average value −2SD as the cutoff value for those having significant differences in Table 2.
[0054]
Table 3
As is clear from Table 3, the diagnostic efficiency of the AFP (L2 + L3) fraction in serum and amniotic fluid was the highest at 73%, confirming that it was highly useful in fetal Down syndrome diagnosis. In other words, it was confirmed that AFPs in which fucose is α1 → 6 added to the reducing terminal GlcNAc of the sugar chain is effective for fetal Down syndrome diagnosis.
[0056]
Example 2. Examination of AFP (L2 + L3) fraction ratio in maternal serum and amniotic fluid between fetal chromosomal abnormality group and normal group
(1) Sample
Serum and amniotic fluid of a 15- to 19-week gestational age of a 24-44-year-old pregnant woman whose fetal 21 and 18 trisomy autosomal abnormalities or sex chromosome abnormalities were confirmed by amniotic fluid examination were used.
Moreover, the serum and amniotic fluid of normal pregnant women whose age and gestational age coincided with the affected cases and confirmed to have fetuses without chromosomal abnormality by amniotic fluid examination were used as controls.
(2) Measurement of AFP (L2 + L3) fraction ratio
Using AFP-L3 Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.), according to the standard operating method of the package insert, each fraction was separated, and the peak area of each fraction was measured with a densitometer (580 nm), The AFP (L2 + L3) fraction ratio was calculated in the same manner as in Example 1.
(3) Results
Table 4 shows the mean ± SD and the significant difference from the normal group for each group. In addition, t test (Student's t) was used for the significant difference test.
[0057]
Table 4
As is clear from Table 4, the AFP (L2 + L3) fraction ratio in serum and amniotic fluid was significantly higher in the 21 trisomy group. In the sex chromosome abnormality group, the AFP (L2 + L3) fraction ratio in amniotic fluid was significantly low.
[0059]
Next, Table 5 shows the results of determining the sensitivity, specificity, and diagnostic efficiency when there is a significant difference and the value that maximizes the diagnostic efficiency is the cut-off value.
[0060]
Table 5
As is apparent from Table 5, the usefulness of the fraction of AFP (L2 + L3) in serum and amniotic fluid in trisomy 21 or sex chromosome abnormality diagnosis, in other words, AFPs with fucose α1 → 6 added to the reducing end GlcNAc of sugar chain The usefulness of was confirmed.
[0062]
Example 3. Examination of total AFP and maternal serum AFP (L2 + L3) fraction ratio in fetal Down syndrome
(1) Sample
Sera from 15 to 19 weeks of gestation of 18 pregnant women aged 27 to 44 years who were confirmed to have fetuses with Down syndrome by amniotic fluid testing were used.
Sera from 70 normal pregnant women whose age and gestational age were the same as those affected and confirmed to have fetuses without chromosomal abnormalities by amniotic fluid examination were used as controls.
(2) Measurement of total AFP amount
Measurements were made using the Abbott IMx AFP assay system according to the standard operating procedure in the package insert.
(3) Measurement of AFP (L2 + L3) fraction ratio
Using AFP-L3 Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.), the peak area of each separated AFP fraction was measured with a densitometer (580 nm) according to the standard operating method of the package insert. AFP (L2 + L3) was calculated in the same manner. In addition, t test (student's t) was used for the significant difference test.
[0063]
(4) Results
FIG. 1 (a) shows the distribution of the total AFP amount in the mother serum in the normal group and fetal Down syndrome, and FIG. 1 (b) shows the AFP (L2 + L3) fraction ratio distribution.
From these results, both the total AFP amount and the AFP (L2 + L3) fraction ratio were significantly different between the fetal Down syndrome and the normal group.
Next, Table 6 shows the sensitivity, specificity, and diagnostic efficiency of each when the cutoff value of the total AFP amount is 24 ng / ml and the cutoff value of the AFP (L2 + L3) fraction ratio is 42%.
[0064]
Also, receiver operating characteristic (ROC) curves of the total AFP amount and the AFP (L2 + L3) fraction ratio are shown in FIGS. 2 (a) and 2 (b), respectively. When the AUC (area under the curve) value was calculated based on FIGS. 2A and 2B, the AUC value in the case of the total AFP amount was 0.700, and the AFP value in the case of the AFP (L2 + L3) fraction ratio. Was 0.835. From these, it can be seen that in the diagnosis of Down's syndrome, the AFP (L2 + L3) fraction ratio in maternal serum is more useful than the total AFP amount, and a more accurate diagnosis can be made.
[0066]
【The invention's effect】
The present invention provides a simple determination method of fetal chromosomal abnormality, reagent and kit which has no influence on the mother and the fetus, has high reliability of the test result, and contributes to this business. is there.
[0067]
[Brief description of the drawings]
FIG. 1 shows the results obtained in Example 3, wherein (a) shows the distribution of the total AFP amount in each serum sample of a normal mother or a mother who has been confirmed to have a fetus with a fetus Down syndrome. , (B) shows the AFP (L2 + L3) fraction ratio distribution, respectively.
FIG. 2 shows a receiver operating characteristic curve based on the result obtained in Example 3, (a) shows a receiver operating characteristic curve based on a result of measuring the total AFP amount, and (b) shows The receiver operating characteristic curves based on the measurement results of the AFP (L2 + L3) fraction ratio are shown.
Claims (22)
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