JP2024062278A - Method for measuring core fucosylated human immunoglobulin G and reagent for measuring same - Google Patents
Method for measuring core fucosylated human immunoglobulin G and reagent for measuring same Download PDFInfo
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- fucosylated human
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Abstract
【課題】コアフコシル化ヒト免疫グロブリンＧを測定する方法及び測定試薬の提供。【解決手段】試料中のコアフコシル化ヒト免疫グロブリンＧを抗コアフコシル化ヒト免疫グロブリンＧ抗体又はその抗体断片が結合したラテックス粒子と反応させることを備える、ラテックス凝集法によって試料中のコアフコシル化ヒト免疫グロブリンＧを測定する方法であって、上記抗コアフコシル化ヒト免疫グロブリンＧ抗体又はその抗体断片は、コアフコシル化ヒト免疫グロブリンＧに結合し、かつ、アコアフコシル化ヒト免疫グロブリンＧに結合しない抗体又はその抗体断片である、方法、又はその方法に用いる試薬。【選択図】図６[Problem] To provide a method and a measuring reagent for measuring core-fucosylated human immunoglobulin G. [Solution] A method for measuring core-fucosylated human immunoglobulin G in a sample by a latex agglutination method, comprising reacting core-fucosylated human immunoglobulin G in the sample with latex particles bound to an anti-core fucosylated human immunoglobulin G antibody or an antibody fragment thereof, wherein the anti-core fucosylated human immunoglobulin G antibody or antibody fragment thereof is an antibody or antibody fragment thereof that binds to core-fucosylated human immunoglobulin G and does not bind to a-core fucosylated human immunoglobulin G, or a reagent used in the method. [Selected Figure] Figure 6
Description
本開示は、コアフコシル化ヒト免疫グロブリンＧを測定する方法及びその測定試薬に関する。 The present disclosure relates to a method for measuring core fucosylated human immunoglobulin G and a measurement reagent therefor.
タンパク質の翻訳後修飾の１つに糖鎖修飾があり、近年、様々な疾患マーカーとして、糖鎖修飾されたタンパク質（糖タンパク質）の研究が盛んに行われている。糖タンパク質を臨床検査の指標とする場合、糖タンパク質の量的変化に加えて、質的変化、すなわち修飾された糖鎖の構造（糖鎖構造）の変化も重要な指標になり得る。例えば、糖タンパク質α－フェトプロテイン（ＡＦＰ）のＮ結合型糖鎖にフコースが付加されたα－フェトプロテインＬ３（ＡＦＰ－Ｌ３）に関して、全ＡＦＰに占めるＡＦＰ－Ｌ３の割合（ＡＦＰ－Ｌ３％）が、肝細胞がんの診断の指標として有用であることが報告されている（非特許文献１）。 Glycosylation is one of the post-translational modifications of proteins, and in recent years, glycosylated proteins (glycoproteins) have been actively studied as markers for various diseases. When glycoproteins are used as indicators in clinical tests, in addition to quantitative changes in glycoproteins, qualitative changes, i.e., changes in the structure of modified glycans (glycostructures), can also be important indicators. For example, it has been reported that the proportion of AFP-L3 (AFP-L3), which is an N-linked glycan of the glycoprotein α-fetoprotein (AFP), with fucose added to it, in total AFP, (AFP-L3%), is useful as an indicator for diagnosing hepatocellular carcinoma (Non-Patent Document 1).
ヒト免疫グロブリンＧ（以下では、「ヒトＩｇＧ」とも記載する。）も糖タンパク質であり、様々な糖鎖構造を有するヒトＩｇＧが存在する。その中でも、疾患に関連した質的変化を生ずるものとして、ヒトＩｇＧのＦｃ領域に含まれる重鎖２９７番目のアスパラギンに結合したＮ－アセチルグルコサミンの６位と、フコースの１位とがα１－６結合した糖鎖構造を有するヒトＩｇＧ（以下では、「コアフコシル化ヒトＩｇＧ」とも記載する。）が知られている（非特許文献２）。コアフコシル化ヒトＩｇＧに含まれる該フコースは、コアフコースと呼ばれる。 Human immunoglobulin G (hereinafter also referred to as "human IgG") is also a glycoprotein, and there are human IgGs with various glycan structures. Among them, human IgGs with a glycan structure in which the 6th position of N-acetylglucosamine bound to asparagine at position 297 of the heavy chain contained in the Fc region of human IgG is α1-6 linked to the 1st position of fucose (hereinafter also referred to as "core fucosylated human IgG") are known to cause disease-related qualitative changes (Non-Patent Document 2). The fucose contained in core fucosylated human IgG is called core fucose.
特許文献１には、コアフコースが結合しているＮ結合型糖鎖をＦｃ領域に含むヒトＩｇＧ（コアフコシル化ヒトＩｇＧ）に結合し、かつ、コアフコースが結合していないＮ結合型糖鎖をＦｃ領域に含むヒトＩｇＧには結合しない抗コアフコシル化ヒトＩｇＧ抗体が開示されている。当該抗体と、抗ヒトＩｇＧ抗体とを用いるコアフコシル化ヒトＩｇＧのサンドイッチＥＬＩＳＡ法であって、不溶性担体としてマイクロタイタープレートを用いる方法も報告されている。 Patent Document 1 discloses an anti-core fucosylated human IgG antibody that binds to human IgG containing an N-linked glycan bound to core fucose in the Fc region (core fucosylated human IgG) and does not bind to human IgG containing an N-linked glycan not bound to core fucose in the Fc region. A sandwich ELISA method for core fucosylated human IgG using the antibody and an anti-human IgG antibody, in which a microtiter plate is used as an insoluble carrier, has also been reported.
特許文献１に開示されたＥＬＩＳＡ法による測定方法を用いて、生体試料などの高濃度でコアフコシル化ヒトＩｇＧを含有する試料中のコアフコシル化ヒトＩｇＧを測定する場合、ＥＬＩＳＡ法は高感度な測定法であるため、試料を高倍率で希釈しなければ有意な測定を行うことができず、希釈誤差が生じてしまう。 When measuring core fucosylated human IgG in a sample containing core fucosylated human IgG at high concentrations, such as a biological sample, using the ELISA measurement method disclosed in Patent Document 1, the ELISA method is a highly sensitive measurement method, so unless the sample is highly diluted, a meaningful measurement cannot be performed, resulting in a dilution error.
そこで、本開示の課題は、希釈誤差が抑制された、試料中のコアフコシル化ヒトＩｇＧを測定する方法を提供することである。 The objective of the present disclosure is to provide a method for measuring core fucosylated human IgG in a sample with reduced dilution error.
本発明者らは、上記課題を解決すべく鋭意検討を行った結果、抗コアフコシル化ヒトＩｇＧ選択的抗体を用いたラテックス凝集法によると、ＥＬＩＳＡ法のような過度な試料の希釈を行うことなく、試料中のコアフコシル化ヒトＩｇＧが測定可能であることを見いだした。 As a result of intensive research conducted by the present inventors to solve the above problems, they discovered that the latex agglutination method using an anti-core fucosylated human IgG selective antibody makes it possible to measure core fucosylated human IgG in a sample without excessively diluting the sample as in the ELISA method.
さらに、本発明者らは、試料としてヒトＩｇＧに占めるコアフコシル化ヒトＩｇＧの割合の変動が生じる疾患に罹患した被験者由来の生体試料を用いた場合、抗コアフコシル化ヒトＩｇＧ選択的抗体を用いたラテックス凝集法によれば、ＥＬＩＳＡ法と比較して、従来法であるヒトＩｇＧに占めるコアフコシル化ヒトＩｇＧの存在割合を指標とした評価により近く、かつ統計的有意差をもって罹患の有無に応じたコアフコシル化ヒトＩｇＧ量の変動を評価することができることを見出し、本開示を完成させた。 Furthermore, the present inventors have discovered that when a biological sample from a subject suffering from a disease in which the proportion of core fucosylated human IgG in human IgG changes is used as a sample, the latex agglutination method using an anti-core fucosylated human IgG selective antibody is able to evaluate the variation in the amount of core fucosylated human IgG depending on the presence or absence of the disease with a statistically significant difference, closer to the conventional evaluation using the proportion of core fucosylated human IgG as an index, compared to the ELISA method, and thus completed the present disclosure.
すなわち、本開示は以下の［１］～［３６］に関する。
［１］試料中のコアフコシル化ヒト免疫グロブリンＧを抗コアフコシル化ヒト免疫グロブリンＧ抗体又はその抗体断片が結合したラテックス粒子と反応させることを備える、ラテックス凝集法によって試料中のコアフコシル化ヒト免疫グロブリンＧを測定する方法であって、上記抗コアフコシル化ヒト免疫グロブリンＧ抗体又はその抗体断片は、コアフコシル化ヒト免疫グロブリンＧに結合し、かつ、アコアフコシル化ヒト免疫グロブリンＧに結合しない抗体又はその抗体断片である、方法。
［２］上記反応に用いる抗コアフコシル化ヒト免疫グロブリンＧ抗体は、１種類の抗コアフコシル化ヒト免疫グロブリンＧ抗体又はその抗体断片である、［１］に記載の方法。
［３］上記反応に用いる抗コアフコシル化ヒト免疫グロブリンＧ抗体又はその抗体断片は、コアフコシル化ヒト免疫グロブリンＧに対するモノクローナル抗体である、［１］又は［２］に記載の方法。
［４］上記コアフコシル化ヒト免疫グロブリンＧに含まれる、フコースが還元末端ＧｌｃＮＡｃとα１－６結合しているＮ結合型糖鎖は、下記式（Ｉ）：
で表される糖鎖を含むＮ結合型糖鎖である、［１］～［３］のいずれか１つに記載の方法。
［５］上記反応が、抗アコアフコシル化ヒト免疫グロブリンＧ抗体及びその抗体断片の非存在下で行われる、［１］～［４］のいずれか１つに記載の方法。
［６］上記ラテックス粒子の平均粒径が１００ｎｍ～３００ｎｍである、［１］～［５］のいずれか１つに記載の方法。
［７］抗コアフコシル化ヒト免疫グロブリンＧ抗体又はその抗体断片と、ラテックス粒子との結合が化学結合である、［１］～［６］のいずれか１つに記載の方法。
［８］上記反応が緩衝液を含む水性媒体中で行われる、［１］～［７］のいずれか１つに記載の方法。
［９］抗コアフコシル化ヒト免疫グロブリンＧ抗体又はその抗体断片が結合したラテックス粒子を含む、ラテックス凝集法によって試料中のコアフコシル化ヒト免疫グロブリンＧを測定するための試薬であって、上記抗コアフコシル化ヒト免疫グロブリンＧ抗体又はその抗体断片は、コアフコシル化ヒト免疫グロブリンＧに結合し、かつ、アコアフコシル化ヒト免疫グロブリンＧに結合しない抗体又はその抗体断片である、試薬。
［１０］上記反応に用いる抗コアフコシル化ヒト免疫グロブリンＧ抗体又はその抗体断片は、１種類の抗コアフコシル化ヒト免疫グロブリンＧ抗体又はその抗体断片である、［９］に記載の試薬。
［１１］上記反応に用いる抗コアフコシル化ヒト免疫グロブリンＧ抗体又はその抗体断片は、コアフコシル化ヒト免疫グロブリンＧに対するモノクローナル抗体である、［９］又は［１０］に記載の方法。
［１２］上記コアフコシル化ヒト免疫グロブリンＧに含まれる、フコースが還元末端ＧｌｃＮＡｃとα１－６結合しているＮ結合型糖鎖は、下記式（Ｉ）：
で表される糖鎖を含むＮ結合型糖鎖である、［９］～［１１］のいずれか１つに記載の試薬。
［１３］抗アコアフコシル化ヒト免疫グロブリンＧ抗体及びその抗体断片を含まない、［９］～［１２］のいずれか１つに記載の試薬。
［１４］上記ラテックス粒子の平均粒径が１００ｎｍ～３００ｎｍである、［９］～［１３］のいずれか１つに記載の試薬。
［１５］さらに緩衝液を含む、［９］～［１４］のいずれか１つに記載の試薬。
［１６］被験者の生体試料中のコアフコシル化ヒト免疫グロブリンＧを測定することを備える、上記被験者が、炎症を伴う肺疾患に罹患している否かを判定するためのデータを収集する方法。
［１７］被験者の生体試料中のコアフコシル化ヒト免疫グロブリンＧを測定すること、上記生体試料中のヒト免疫グロブリンＧを測定すること並びに上記生体試料中のヒト免疫グロブリンＧに占めるコアフコシル化ヒト免疫グロブリンＧ及び／又はアコアフコシル化ヒト免疫グロブリンＧの割合を算出することを備える、上記被験者が、炎症を伴う肺疾患に罹患している否かを判定するためのデータを収集する方法。
［１８］被験者の生体試料中のコアフコシル化ヒト免疫グロブリンＧを測定することを備える、炎症を伴う肺疾患の診断方法又は診断を補助する方法。
［１９］被験者の生体試料中のコアフコシル化ヒト免疫グロブリンＧを測定すること、及び、得られたコアフコシル化ヒト免疫グロブリンＧの測定値が基準値以下である場合には、被験者が炎症を伴う肺疾患に罹患していると判定し、上記測定値が基準値より大きい場合には、被験者が炎症を伴う肺疾患に罹患していないと判定すること、を備える、炎症を伴う肺疾患の診断方法。
［２０］被験者の生体試料中のコアフコシル化ヒト免疫グロブリンＧを測定すること、及び、得られたコアフコシル化ヒト免疫グロブリンＧの測定値が基準値以下である場合には、被験者が炎症を伴う肺疾患に罹患している可能性が高いと判定し、上記測定値が基準値より大きい場合には、被験者が炎症を伴う肺疾患に罹患していない可能性が高いと判定すること、を備える、炎症を伴う肺疾患の炎症を伴う肺疾患に罹患している否かを判定するためのデータを収集する方法又は炎症を伴う肺疾患の診断を補助する方法。
［２１］被験者の生体試料中のコアフコシル化ヒト免疫グロブリンＧを測定すること、上記生体試料中のヒト免疫グロブリンＧを測定すること、得られたコアフコシル化ヒト免疫グロブリンＧ及びヒト免疫グロブリンＧの測定値を用いて、上記生体試料中のヒト免疫グロブリンＧに占めるコアフコシル化ヒト免疫グロブリンＧの割合を算出すること、及び、得られたコアフコシル化ヒト免疫グロブリンＧの割合が基準値以下である場合には、被験者が炎症を伴う肺疾患に罹患していると判定し、上記測定値が基準値より大きい場合には、被験者が炎症を伴う肺疾患に罹患していないと判定すること、を備える、炎症を伴う肺疾患の診断方法。
［２２］被験者の生体試料中のコアフコシル化ヒト免疫グロブリンＧを測定すること、上記生体試料中のヒト免疫グロブリンＧを測定すること、得られたコアフコシル化ヒト免疫グロブリンＧ及びヒト免疫グロブリンＧの測定値を用いて、上記生体試料中のヒト免疫グロブリンＧに占めるコアフコシル化ヒト免疫グロブリンＧの割合を算出すること、及び、得られたコアフコシル化ヒト免疫グロブリンＧの割合が基準値以下である場合には、被験者が炎症を伴う肺疾患に罹患している可能性が高いと判定し、上記測定値が基準値より大きい場合には、被験者が炎症を伴う肺疾患に罹患していない可能性が高いと判定すること、を備える、炎症を伴う肺疾患に罹患している否かを判定するためのデータを収集する方法又は炎症を伴う肺疾患の診断を補助する方法。
［２３］被験者の生体試料中のコアフコシル化ヒト免疫グロブリンＧを測定すること、上記生体試料中のヒト免疫グロブリンＧを測定すること、得られたコアフコシル化ヒト免疫グロブリンＧ及びヒト免疫グロブリンＧの測定値を用いて、上記生体試料中のヒト免疫グロブリンＧに占めるアコアフコシル化ヒト免疫グロブリンＧの割合を算出すること、及び、得られたアコアフコシル化ヒト免疫グロブリンＧの割合が基準値以上である場合には、被験者が炎症を伴う肺疾患に罹患していると判定し、上記測定値が基準値より小さい場合には、被験者が炎症を伴う肺疾患に罹患していないと判定すること、を備える、炎症を伴う肺疾患の診断方法。
［２４］被験者の生体試料中のコアフコシル化ヒト免疫グロブリンＧを測定すること、上記生体試料中のヒト免疫グロブリンＧを測定すること、得られたコアフコシル化ヒト免疫グロブリンＧ及びヒト免疫グロブリンＧの測定値を用いて、上記生体試料中のヒト免疫グロブリンＧに占めるアコアフコシル化ヒト免疫グロブリンＧの割合を算出すること、及び、得られたアコアフコシル化ヒト免疫グロブリンＧの割合が基準値以上である場合には、被験者が炎症を伴う肺疾患に罹患している可能性が高いと判定し、上記測定値が基準値より小さい場合には、被験者が炎症を伴う肺疾患に罹患していない可能性が高いと判定すること、を備える、炎症を伴う肺疾患に罹患している否かを判定するためのデータを収集する方法又は炎症を伴う肺疾患の診断を補助する方法。
［２５］コアフコシル化ヒト免疫グロブリンＧの測定において、コアフコシル化ヒト免疫グロブリンＧに結合し、かつ、アコアフコシル化ヒト免疫グロブリンＧには結合しない抗コアフコシル化ヒトグロブリンＧ抗体又はその抗体断片を用いる、［１６］～［２４］のいずれか１つに記載の方法。
［２６］上記抗体又はその抗体断片がラテックス粒子と結合している、［２５］に記載の方法。
［２７］コアフコシル化ヒト免疫グロブリンＧの測定がラテックス凝集法により行われる、［２６］に記載の方法。
［２８］（ａ）コアフコシル化ヒト免疫グロブリンＧに結合し、かつ、アコアフコシル化ヒト免疫グロブリンＧには結合しない抗コアフコシル化ヒトグロブリンＧ抗体又はその抗体断片、及び（ｂ）抗ヒト免疫グロブリンＧ抗体を含む、［１７］及び［２１］～［２７］のいずれか１つに記載の方法を行うための、キット。
［２９］（ａ）コアフコシル化ヒト免疫グロブリンＧに結合し、かつ、アコアフコシル化ヒト免疫グロブリンＧには結合しない抗コアフコシル化ヒトグロブリンＧ抗体又はその抗体断片、及び（ｂ）コアフコシル化ヒトＩｇＧの測定値が基準値以下である場合には、被験者が、炎症を伴う肺疾患に罹患していると判定し、コアフコシル化ヒトＩｇＧの測定値が基準値より大きい場合には、被験者が、炎症を伴う肺疾患に罹患していないという判定する、という基準に従って判定を行う旨が説明された添付文書、を含む、［１６］～［２８］のいずれか１つに記載の方法を行うための、キット。
［３０］コアフコシル化ヒト免疫グロブリンＧに結合し、かつ、アコアフコシル化ヒト免疫グロブリンＧには結合しない抗コアフコシル化ヒトグロブリンＧ抗体又はその抗体断片を含む、炎症を伴う肺疾患の診断薬。
［３１］炎症を伴う肺疾患の診断薬を製造するための、コアフコシル化ヒト免疫グロブリンＧに結合し、かつ、アコアフコシル化ヒト免疫グロブリンＧには結合しない抗コアフコシル化ヒトグロブリンＧ抗体又はその抗体断片の使用。
［３２］炎症を伴う肺疾患の診断に用いる、コアフコシル化ヒト免疫グロブリンＧに結合し、かつ、アコアフコシル化ヒト免疫グロブリンＧには結合しない抗コアフコシル化ヒトグロブリンＧ抗体又はその抗体断片。
［３３］上記抗体又はその抗体断片がラテックス粒子と結合している、［２８］～［３２］のいずれか１つに記載のキット、診断薬、使用又は抗体若しくはその抗体断片。
［３４］コアフコシル化ヒト免疫グロブリンＧの測定がラテックス凝集法により行われる、又は、コアフコシル化ヒト免疫グロブリンＧの測定をラテックス凝集法により行うための、［２８］～［３３］のいずれか１つに記載のキット、診断薬、使用又は抗体若しくはその抗体断片。
［３５］上記コアフコシル化ヒト免疫グロブリンＧに含まれる、フコースが還元末端ＧｌｃＮＡｃとα１－６結合しているＮ結合型糖鎖は、下記式（Ｉ）：
で表される糖鎖を含むＮ結合型糖鎖である、［２５］～［３４］のいずれか１つに記載の方法、キット、診断薬、使用又は抗体若しくはその抗体断片。
［３６］上記炎症を伴う肺疾患が、間質性肺炎、肺がん及び慢性閉塞性肺疾患からなる群より選ばれる少なくとも１つである、［１６］～［３５］のいずれか１つに記載の方法、キット、診断薬、使用又は抗体若しくはその抗体断片。
That is, the present disclosure relates to the following [1] to [36].
[1] A method for measuring core fucosylated human immunoglobulin G in a sample by a latex agglutination method, comprising reacting core fucosylated human immunoglobulin G in the sample with latex particles to which an anti-core fucosylated human immunoglobulin G antibody or an antibody fragment thereof is bound, wherein the anti-core fucosylated human immunoglobulin G antibody or antibody fragment thereof is an antibody or antibody fragment thereof that binds to core fucosylated human immunoglobulin G and does not bind to a-core fucosylated human immunoglobulin G.
[2] The method according to [1], wherein the anti-core fucosylated human immunoglobulin G antibody used in the reaction is one type of anti-core fucosylated human immunoglobulin G antibody or an antibody fragment thereof.
[3] The method according to [1] or [2], wherein the anti-core fucosylated human immunoglobulin G antibody or its antibody fragment used in the reaction is a monoclonal antibody against core fucosylated human immunoglobulin G.
[4] The N-linked glycan in which fucose is linked to reducing terminal GlcNAc via an α1-6 bond contained in the core fucosylated human immunoglobulin G is represented by the following formula (I):
The method according to any one of [1] to [3], wherein the N-linked glycan comprises a glycan represented by the formula:
[5] The method according to any one of [1] to [4], wherein the reaction is carried out in the absence of an anti-aco-fucosylated human immunoglobulin G antibody or an antibody fragment thereof.
[6] The method according to any one of [1] to [5], wherein the average particle size of the latex particles is 100 nm to 300 nm.
[7] The method according to any one of [1] to [6], wherein the anti-core fucosylated human immunoglobulin G antibody or its antibody fragment is bonded to the latex particles by chemical bonding.
[8] The method according to any one of [1] to [7], wherein the reaction is carried out in an aqueous medium containing a buffer.
[9] A reagent for measuring core fucosylated human immunoglobulin G in a sample by a latex agglutination method, comprising latex particles to which an anti-core fucosylated human immunoglobulin G antibody or an antibody fragment thereof is bound, wherein the anti-core fucosylated human immunoglobulin G antibody or antibody fragment thereof is an antibody or antibody fragment thereof that binds to core fucosylated human immunoglobulin G and does not bind to aco-fucosylated human immunoglobulin G.
[10] The reagent described in [9], wherein the anti-core fucosylated human immunoglobulin G antibody or its antibody fragment used in the above reaction is one type of anti-core fucosylated human immunoglobulin G antibody or its antibody fragment.
[11] The method according to [9] or [10], wherein the anti-core fucosylated human immunoglobulin G antibody or an antibody fragment thereof used in the reaction is a monoclonal antibody against core fucosylated human immunoglobulin G.
[12] The N-linked glycan in which fucose is linked to reducing terminal GlcNAc via α1-6 bond contained in the above-mentioned core fucosylated human immunoglobulin G is represented by the following formula (I):
The reagent according to any one of [9] to [11], wherein the N-linked glycan contains a glycan represented by the formula:
[13] The reagent according to any one of [9] to [12], which does not contain an anti-aco-fucosylated human immunoglobulin G antibody or an antibody fragment thereof.
[14] The reagent according to any one of [9] to [13], wherein the average particle size of the latex particles is 100 nm to 300 nm.
[15] The reagent according to any one of [9] to [14], further comprising a buffer solution.
[16] A method for collecting data for determining whether a subject is suffering from a lung disease associated with inflammation, comprising measuring core fucosylated human immunoglobulin G in a biological sample from the subject.
[17] A method for collecting data for determining whether or not a subject is suffering from a lung disease accompanied by inflammation, comprising: measuring core-fucosylated human immunoglobulin G in a biological sample from the subject; measuring human immunoglobulin G in the biological sample; and calculating a proportion of core-fucosylated human immunoglobulin G and/or aco-fucosylated human immunoglobulin G in the human immunoglobulin G in the biological sample.
[18] A method for diagnosing or assisting in the diagnosis of a lung disease associated with inflammation, comprising measuring core fucosylated human immunoglobulin G in a biological sample from a subject.
[19] A method for diagnosing an inflammatory pulmonary disease, comprising: measuring core-fucosylated human immunoglobulin G in a biological sample from a subject; and, if the measured value of the obtained core-fucosylated human immunoglobulin G is equal to or lower than a reference value, determining that the subject is suffering from an inflammatory pulmonary disease; and, if the measured value is greater than the reference value, determining that the subject is not suffering from an inflammatory pulmonary disease.
[20] A method for collecting data for determining whether or not a subject is suffering from an inflammatory pulmonary disease or a method for assisting in the diagnosis of an inflammatory pulmonary disease, comprising: measuring core-fucosylated human immunoglobulin G in a biological sample from a subject; and, if the measured value of core-fucosylated human immunoglobulin G obtained is below a standard value, determining that the subject is likely to be suffering from an inflammatory pulmonary disease, and, if the measured value is greater than the standard value, determining that the subject is likely not suffering from an inflammatory pulmonary disease.
[21] A method for diagnosing a pulmonary disease associated with inflammation, comprising: measuring core-fucosylated human immunoglobulin G in a biological sample from a subject; measuring human immunoglobulin G in the biological sample; calculating a proportion of core-fucosylated human immunoglobulin G in the human immunoglobulin G in the biological sample using the obtained measured values of core-fucosylated human immunoglobulin G and human immunoglobulin G; and determining that the subject is suffering from a pulmonary disease associated with inflammation if the obtained proportion of core-fucosylated human immunoglobulin G is equal to or lower than a standard value, and determining that the subject is not suffering from a pulmonary disease associated with inflammation if the measured value is greater than the standard value.
[22] A method for collecting data for determining whether or not a subject is suffering from an inflammatory pulmonary disease or a method for assisting in the diagnosis of an inflammatory pulmonary disease, comprising: measuring core-fucosylated human immunoglobulin G in a biological sample from the subject; measuring human immunoglobulin G in the biological sample; calculating a ratio of core-fucosylated human immunoglobulin G to human immunoglobulin G in the biological sample using the obtained measured values of core-fucosylated human immunoglobulin G and human immunoglobulin G; and determining that the subject is likely to be suffering from an inflammatory pulmonary disease if the obtained ratio of core-fucosylated human immunoglobulin G is equal to or lower than a standard value, and determining that the subject is likely not suffering from an inflammatory pulmonary disease if the measured value is greater than the standard value.
[23] A method for diagnosing a pulmonary disease associated with inflammation, comprising: measuring core-fucosylated human immunoglobulin G in a biological sample from a subject; measuring human immunoglobulin G in the biological sample; calculating a ratio of aco-fucosylated human immunoglobulin G to human immunoglobulin G in the biological sample using the obtained measured values of core-fucosylated human immunoglobulin G and human immunoglobulin G; and determining that the subject is suffering from a pulmonary disease associated with inflammation if the obtained ratio of aco-fucosylated human immunoglobulin G is equal to or greater than a reference value, and determining that the subject is not suffering from a pulmonary disease associated with inflammation if the measured value is less than the reference value.
[24] A method for collecting data for determining whether or not a subject is suffering from an inflammatory pulmonary disease or a method for assisting in the diagnosis of an inflammatory pulmonary disease, comprising: measuring core-fucosylated human immunoglobulin G in a biological sample from a subject; measuring human immunoglobulin G in the biological sample; calculating a ratio of aco-fucosylated human immunoglobulin G to human immunoglobulin G in the biological sample using the obtained measured values of core-fucosylated human immunoglobulin G and human immunoglobulin G; and determining that the subject is highly likely to be suffering from an inflammatory pulmonary disease if the obtained ratio of aco-fucosylated human immunoglobulin G is equal to or higher than a reference value, and determining that the subject is highly likely not suffering from an inflammatory pulmonary disease if the measured value is lower than the reference value.
[25] The method according to any one of [16] to [24], wherein the measurement of core fucosylated human immunoglobulin G uses an anti-core fucosylated human immunoglobulin G antibody or an antibody fragment thereof that binds to core fucosylated human immunoglobulin G but does not bind to aco-fucosylated human immunoglobulin G.
[26] The method according to [25], wherein the antibody or antibody fragment thereof is bound to latex particles.
[27] The method according to [26], wherein the measurement of core fucosylated human immunoglobulin G is carried out by latex agglutination.
[28] A kit for carrying out the method according to any one of [17] and [21] to [27], comprising: (a) an anti-core fucosylated human immunoglobulin G antibody or an antibody fragment thereof that binds to core fucosylated human immunoglobulin G and does not bind to aco-fucosylated human immunoglobulin G; and (b) an anti-human immunoglobulin G antibody.
[29] A kit for performing the method according to any one of [16] to [28], comprising: (a) an anti-core fucosylated human immunoglobulin G antibody or antibody fragment thereof that binds to core fucosylated human immunoglobulin G but does not bind to aco-fucosylated human immunoglobulin G; and (b) a package insert explaining that the determination is made in accordance with the following criteria: if the measured value of core fucosylated human IgG is equal to or lower than a reference value, the subject is determined to be suffering from a pulmonary disease accompanied by inflammation; and if the measured value of core fucosylated human IgG is higher than the reference value, the subject is determined to not be suffering from a pulmonary disease accompanied by inflammation.
[30] A diagnostic agent for a lung disease accompanied by inflammation, comprising an anti-core fucosylated human immunoglobulin G antibody or an antibody fragment thereof that binds to core fucosylated human immunoglobulin G but does not bind to aco-fucosylated human immunoglobulin G.
[31] Use of an anti-core fucosylated human immunoglobulin G antibody or an antibody fragment thereof that binds to core fucosylated human immunoglobulin G but does not bind to aco-fucosylated human immunoglobulin G, for the manufacture of a diagnostic agent for a lung disease accompanied by inflammation.
[32] An anti-core fucosylated human immunoglobulin G antibody or an antibody fragment thereof that binds to core fucosylated human immunoglobulin G but does not bind to aco-fucosylated human immunoglobulin G, for use in the diagnosis of a lung disease accompanied by inflammation.
[33] The kit, diagnostic agent, use, or antibody or antibody fragment thereof according to any one of [28] to [32], wherein the antibody or antibody fragment thereof is bound to latex particles.
[34] The kit, diagnostic agent, use, or antibody or antibody fragment thereof according to any one of [28] to [33], for measuring core fucosylated human immunoglobulin G by a latex agglutination method, or for measuring core fucosylated human immunoglobulin G by a latex agglutination method.
[35] The N-linked glycan in which fucose is linked to reducing terminal GlcNAc via α1-6 bond contained in the above-mentioned core fucosylated human immunoglobulin G is represented by the following formula (I):
The method, kit, diagnostic agent, use, or antibody or antibody fragment thereof according to any one of [25] to [34], wherein the N-linked glycan comprises a glycan represented by the formula:
[36] The method, kit, diagnostic agent, use, or antibody or antibody fragment thereof according to any one of [16] to [35], wherein the pulmonary disease accompanied by inflammation is at least one selected from the group consisting of interstitial pneumonia, lung cancer, and chronic obstructive pulmonary disease.
本開示によれば、抗コアフコシル化ヒトＩｇＧ選択的抗体を用いたラテックス凝集法によって、ＥＬＩＳＡ法のような過度な試料の希釈を行うことなく、試料中のコアフコシル化ヒトＩｇＧを測定することができる。 According to the present disclosure, the latex agglutination method using an anti-core fucosylated human IgG selective antibody makes it possible to measure core fucosylated human IgG in a sample without excessive sample dilution as in the ELISA method.
さらに、試料として、ヒトＩｇＧに占めるコアフコシル化ヒトＩｇＧの存在割合の変動が生じる疾患に罹患した被験者由来の生体試料を用いた場合、抗コアフコシル化ヒトＩｇＧ選択的抗体を用いたラテックス凝集法によれば、ＥＬＩＳＡ法と比較して、従来法であるヒトＩｇＧに占めるコアフコシル化ヒトＩｇＧの存在割合を指標とした評価により近く、かつ統計的有意差をもって罹患の有無に応じたコアフコシル化ヒトＩｇＧ量の変動を評価することができる。 Furthermore, when a biological sample from a subject suffering from a disease in which the proportion of core fucosylated human IgG in human IgG changes is used as a sample, the latex agglutination method using an anti-core fucosylated human IgG selective antibody is closer to the conventional method of evaluation using the proportion of core fucosylated human IgG as an index compared to the ELISA method, and is capable of evaluating the change in the amount of core fucosylated human IgG depending on the presence or absence of the disease with a statistically significant difference.
さらに、試料として、炎症を伴う肺疾患に罹患した患者由来の生体試料を用いた場合、抗コアフコシル化ヒトＩｇＧ選択的抗体を用いたラテックス凝集法によれば、統計的有意差をもって、健常人と、炎症を伴う肺疾患患者との鑑別診断を行うことができる。 Furthermore, when a biological sample from a patient suffering from pulmonary disease accompanied by inflammation is used as a sample, the latex agglutination method using an anti-core fucosylated human IgG selective antibody can perform a differential diagnosis between healthy subjects and patients with pulmonary disease accompanied by inflammation with a statistically significant difference.
以下、本開示を実施するための形態について詳細に説明するが、本開示は以下の実施形態に限定されるものではない。 The following describes in detail the form for implementing this disclosure, but the disclosure is not limited to the following embodiment.
本開示において、コアフコースとは、ヒト免疫グロブリンＧ（以下では、「ヒトｌｇＧ」とも記載する。）のＦｃ領域の２９７番目のアスパラギンに結合しているＮ結合型糖鎖に含まれる単糖のうち、該アスパラギンに直接結合しているＮ－アセチルグルコサミン、すなわち、還元末端ＧｌｃＮＡｃ（還元末端Ｎ－アセチルグルコサミン）の６位の炭素と、α１－６結合したフコースをいう。Ｎ結合型糖鎖は、還元末端Ｎ－アセチルグルコサミンに各種の単糖がグリコシド結合によって繋がった化合物であれば特に限定されず、単糖としては、例えばグルコース、ガラクトース、マンノース、Ｎ－アセチルグルコサミン、Ｎ－アセチルガラクトサミン、フコース、キシロース及びシアル酸等が挙げられる。グリコシド結合とは、単糖のヘミアセタールと他の単糖のヒドロキシル基との間の結合をいい、例えば、２つのＮ－アセチルグルコサミンの間の結合、２つのマンノースの間の結合、マンノースとＮ－アセチルグルコサミンとの間の結合及びＮ－アセチルグルコサミンとフコースとの間の結合等が挙げられ、これらの結合様式として、例えばα１－３結合、α１－６結合及びβ１－４結合等が挙げられる。 In the present disclosure, core fucose refers to the N-acetylglucosamine directly bound to the asparagine at position 297 of the monosaccharides contained in the N-linked glycan bound to the Fc region of human immunoglobulin G (hereinafter also referred to as "human IgG"), that is, fucose bound to the carbon at position 6 of reducing end GlcNAc (reducing end N-acetylglucosamine) by α1-6 bond. The N-linked glycan is not particularly limited as long as it is a compound in which various monosaccharides are linked to reducing end N-acetylglucosamine by glycosidic bonds, and examples of monosaccharides include glucose, galactose, mannose, N-acetylglucosamine, N-acetylgalactosamine, fucose, xylose, and sialic acid. A glycosidic bond refers to a bond between a hemiacetal of a monosaccharide and a hydroxyl group of another monosaccharide, and examples of such bonds include a bond between two N-acetylglucosamines, a bond between two mannose, a bond between mannose and N-acetylglucosamine, and a bond between N-acetylglucosamine and fucose. Examples of such bond types include an α1-3 bond, an α1-6 bond, and a β1-4 bond.
本開示において、コアフコシル化ヒト免疫グロブリンＧ（以下では、「コアフコシル化ヒトＩｇＧ」とも記載する。）とは、コアフコースが結合しているＮ結合型糖鎖をＦｃ領域に含むヒトｌｇＧである。コアフコシル化ヒトＩｇＧは、少なくとも１つのＦｃ領域にコアフコースが結合しているＮ結合型糖鎖が含まれていればよく、２つのＦｃ領域にコアフコースが結合しているＮ結合型糖鎖が含まれていてもよい。コアフコースが結合しているＮ結合型糖鎖としては、例えば以下の式に示すＮ結合型糖鎖を挙げることができる。
本開示において、アコアフコシル化ヒト免疫グロブリンＧ（以下では、「アコアフコシル化ヒトＩｇＧ」とも記載する。）とは、ヒトｌｇＧの２つのＦｃ領域の２９７番目のアスパラギンに結合しているＮ結合型糖鎖に含まれる還元末端ＧｌｃＮＡｃのいずれにもコアフコースが結合していないヒトｌｇＧである。アコアフコシル化ヒトＩｇＧに含まれるＮ結合型糖鎖、すなわちコアフコースが結合していないＮ結合型糖鎖としては、例えば以下の式に示すＮ結合型糖鎖を挙げることができる。
本開示に係るコアフコシル化ヒトＩｇＧ及びアコアフコシル化ヒトＩｇＧが結合する抗原の種類は特に限定されず、例えば、自己抗原、同種抗原(ａｌｌｏａｎｔｉｇｅｎ)、ウイルス由来抗原及び細菌由来抗原等が挙げられる。自己抗原としては細胞核内抗原等が挙げられ、同種抗原としては主要組織適合遺伝子複合体抗原(ＭＨＣ抗原）等が挙げられ、ウイルス由来抗原としてはＢ型肝炎ウイルスのＨＢｓ抗原、Ｃ型肝炎ウイルスのコア抗原等が挙げられ、細菌由来抗原としてはグラム陰性菌外膜のＯ抗原、百日咳菌の百日咳毒素等が挙げられる。 The type of antigen to which the core-fucosylated human IgG and aco-fucosylated human IgG of the present disclosure bind is not particularly limited, and examples include autoantigens, alloantigens, virus-derived antigens, and bacteria-derived antigens. Autoantigens include intranuclear antigens, alloantigens include major histocompatibility complex antigens (MHC antigens), virus-derived antigens include HBs antigens of hepatitis B virus and core antigens of hepatitis C virus, and bacteria-derived antigens include O antigens of the outer membrane of gram-negative bacteria and pertussis toxin of Bordetella pertussis.
本開示において、抗コアフコシル化ヒト免疫グロブリンＧ抗体（以下では、「抗コアフコシル化ヒトＩｇＧ抗体」とも記載する。）とは、コアフコシル化ヒトＩｇＧに結合する抗体の総称である。すなわち、図１に示すように、抗コアフコシル化ヒトＩｇＧ抗体には、コアフコシル化ヒトＩｇＧ及びアコアフコシル化ヒトＩｇＧの両方に結合する抗体（以下では、「抗ヒトＩｇＧ抗体」とも記載する。）並びにコアフコシル化ヒトＩｇＧに結合し、かつ、アコアフコシル化ヒトＩｇＧに結合しない抗体（以下では、「抗コアフコシル化ヒトＩｇＧ選択的抗体」とも記載する。）の両者が包含される。 In the present disclosure, anti-core fucosylated human immunoglobulin G antibody (hereinafter also referred to as "anti-core fucosylated human IgG antibody") is a general term for antibodies that bind to core fucosylated human IgG. That is, as shown in FIG. 1, anti-core fucosylated human IgG antibody includes both an antibody that binds to both core fucosylated human IgG and acore fucosylated human IgG (hereinafter also referred to as "anti-human IgG antibody") and an antibody that binds to core fucosylated human IgG but does not bind to acore fucosylated human IgG (hereinafter also referred to as "anti-core fucosylated human IgG selective antibody").
本開示において、抗アコアフコシル化ヒト免疫グロブリンＧ抗体（以下では、「抗アコアフコシル化ヒトＩｇＧ抗体」とも記載する。）とは、アコアフコシル化ヒトＩｇＧに結合する抗体の総称である。すなわち、図１に示すように、抗アコアフコシル化ヒトＩｇＧ抗体には、コアフコシル化ヒトＩｇＧ及びアコアフコシル化ヒトＩｇＧの両方に結合する抗体（抗ヒトＩｇＧ抗体）並びにコアフコシル化ヒトＩｇＧに結合せず、かつ、アコアフコシル化ヒトＩｇＧに結合する抗体（以下では、「抗アコアフコシル化ヒトＩｇＧ選択的抗体」とも記載する。）の両者が包含される。 In the present disclosure, anti-aco-fucosylated human immunoglobulin G antibody (hereinafter also referred to as "anti-aco-fucosylated human IgG antibody") is a general term for antibodies that bind to aco-fucosylated human IgG. That is, as shown in FIG. 1, anti-aco-fucosylated human IgG antibody includes both antibodies that bind to both core-fucosylated human IgG and aco-fucosylated human IgG (anti-human IgG antibody) and antibodies that do not bind to core-fucosylated human IgG and bind to aco-fucosylated human IgG (hereinafter also referred to as "anti-aco-fucosylated human IgG selective antibody").
本開示の一側面は、試料中のコアフコシル化ヒト免疫グロブリンＧを抗コアフコシル化ヒト免疫グロブリンＧ抗体又はその抗体断片が結合したラテックス粒子と反応させることを備える、ラテックス凝集法によって試料中のコアフコシル化ヒト免疫グロブリンＧを測定する方法であって、上記抗コアフコシル化ヒト免疫グロブリンＧ抗体又はその抗体断片は、コアフコシル化ヒト免疫グロブリンＧに結合し、かつ、アコアフコシル化ヒト免疫グロブリンＧに結合しない抗体又はその抗体断片である、方法である。すなわち、本開示の一側面は、試料中のコアフコシル化ヒトＩｇＧを抗コアフコシル化ヒトＩｇＧ選択的抗体又はその抗体断片が結合したラテックス粒子と反応させることを備える、ラテックス凝集法によって試料中のコアフコシル化ヒトＩｇＧを測定する方法である。 One aspect of the present disclosure is a method for measuring core fucosylated human immunoglobulin G in a sample by a latex agglutination method, comprising reacting core fucosylated human immunoglobulin G in the sample with latex particles bound to an anti-core fucosylated human immunoglobulin G antibody or an antibody fragment thereof, wherein the anti-core fucosylated human immunoglobulin G antibody or antibody fragment thereof is an antibody or antibody fragment thereof that binds to core fucosylated human immunoglobulin G and does not bind to acore fucosylated human immunoglobulin G. That is, one aspect of the present disclosure is a method for measuring core fucosylated human IgG in a sample by a latex agglutination method, comprising reacting core fucosylated human IgG in the sample with latex particles bound to an anti-core fucosylated human IgG selective antibody or an antibody fragment thereof.
本開示において、試料は、コアフコシル化ヒトＩｇＧを含む可能性がある試料であれば特に制限はなく、例えば、全血、血漿、血清、尿、腹水、髄液、唾液、羊水、尿、汗及び膵液等の生体試料並びに遺伝子組換え微生物、遺伝子組換え動物細胞若しくは培養癌細胞の細胞可溶化物若しくは培養上清等を挙げることができる。微生物としては、例えば酵母が挙げられ、動物細胞としては、例えばチャイニーズハムスター卵巣細胞（ＣＨＯ細胞）が挙げられる。培養癌細胞としては、例えば、培養膵癌細胞、培養大腸癌細胞、培養肝癌細胞、培養胆道癌細胞、培養乳癌細胞及び培養卵巣癌細胞等が挙げられる。 In the present disclosure, the sample is not particularly limited as long as it is a sample that may contain core-fucosylated human IgG, and examples thereof include biological samples such as whole blood, plasma, serum, urine, ascites, cerebrospinal fluid, saliva, amniotic fluid, urine, sweat, and pancreatic juice, as well as cell lysates or culture supernatants of genetically modified microorganisms, genetically modified animal cells, or cultured cancer cells. Examples of microorganisms include yeast, and examples of animal cells include Chinese hamster ovary cells (CHO cells). Examples of cultured cancer cells include cultured pancreatic cancer cells, cultured colon cancer cells, cultured hepatic cancer cells, cultured biliary tract cancer cells, cultured breast cancer cells, and cultured ovarian cancer cells.
本開示に係る抗コアフコシル化ヒトＩｇＧ選択的抗体は、ポリクローナル抗体又はモノクローナル抗体のいずれでもよいが、モノクローナル抗体が好ましい。モノクローナル抗体としては、ハイブリドーマにより生産されるモノクローナル抗体、抗体遺伝子を含む発現ベクターで形質転換した形質転換体により生産される遺伝子組換えモノクローナル抗体等が挙げられる。ハイブリドーマにより生産されるモノクローナル抗体としては、例えば、受託番号ＮＩＴＥ ＢＰ－０２３４２として寄託されているハイブリドーマにより生産されるモノクローナル抗体等が挙げられる。 The anti-core fucosylated human IgG selective antibody according to the present disclosure may be either a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody. Examples of monoclonal antibodies include monoclonal antibodies produced by hybridomas, and recombinant monoclonal antibodies produced by transformants transformed with an expression vector containing an antibody gene. Examples of monoclonal antibodies produced by hybridomas include monoclonal antibodies produced by hybridomas deposited under accession number NITE BP-02342.
本発明のハイブリドーマとしては、本発明の抗コアフコシル化ヒトｌｇＧモノクローナル抗体を生産するハイブリドーマであれば特に制限はなく、例えば、２０１６年８月３０日付で、独立行政法人製品評価技術基盤機構特許微生物寄託センター（日本国千葉県木更津市かずさ鎌足２－５－８）に、受託番号ＮＩＴＥ ＢＰ－０２３４２として寄託されているハイブリドーマ株１８－３Ｄ１１が挙げられる。 The hybridoma of the present invention is not particularly limited as long as it is a hybridoma that produces the anti-core fucosylated human IgG monoclonal antibody of the present invention, and an example thereof is hybridoma strain 18-3D11, deposited on August 30, 2016 at the National Institute of Technology and Evaluation, Patent Microorganisms Deposit Center (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under deposit number NITE BP-02342.
本発明のハイブリドーマは、公知の方法、例えば、「モノクローナル抗体」（羊土社、１９９６年）等に記載されている方法等で製造することができる。 The hybridomas of the present invention can be produced by known methods, such as those described in "Monoclonal Antibodies" (Yodosha, 1996).
本開示において、抗体断片とは、抗原を認識する抗体の一部であって、抗体の可変ドメインを含むか、少なくとも抗原結合領域を含むものをいう。本開示における抗体断片としては、例えば抗体をパパイン処理することにより得られるＦａｂ、抗体をペプシン処理することにより得られるＦ（ａｂ’）２、抗体をペプシン処理一還元処理することにより得られるＦａｂ’、抗体軽鎖の可変領域(ＶＬ領域）と抗体重鎖の可変領域(ＶＨ領域）を人工的に１本のポリペプチドとして繋いだ１本鎖抗体(ｓｃＦｖ)、ラクダ科動物等が保有する抗体軽鎖を持たない１本鎖抗体の抗体重鎖の可変領域(ＶＨＨ領域）断片等が挙げられる。 In the present disclosure, an antibody fragment refers to a part of an antibody that recognizes an antigen, and includes the variable domain of the antibody or at least the antigen-binding region. Examples of antibody fragments in the present disclosure include Fab obtained by treating an antibody with papain, F(ab') 2 obtained by treating an antibody with pepsin, Fab' obtained by treating an antibody with pepsin and reducing it, single-chain antibodies (scFv) in which the variable region of the antibody light chain ( VL region) and the variable region of the antibody heavy chain ( VH region) are artificially linked as a single polypeptide, and fragments of the variable region of the antibody heavy chain ( VHH region) of single-chain antibodies that do not have an antibody light chain, such as those possessed by camelids.
本開示に係る抗コアフコシル化ヒトＩｇＧ選択的抗体及びその抗体断片の製造方法は、抗コアフコシル化ヒトＩｇＧ選択的抗体及びその抗体断片を製造し得る方法であれば特に限定されず、例えば、受託番号ＮＩＴＥ ＢＰ－０２３４２として寄託されているハイブリドーマの培養上清から、プロテインＡ、プロテインＧ又はヒトｌｇＧのＦｃ領域に結合する抗体等の抗体に結合する分子を用いて採取することにより、得ることができる。より詳細には、例えば特許文献１に記載の方法により得ることができる。 The method for producing the anti-core fucosylated human IgG selective antibody and antibody fragment thereof according to the present disclosure is not particularly limited as long as it is a method capable of producing the anti-core fucosylated human IgG selective antibody and antibody fragment thereof, and can be obtained, for example, by collecting the antibody from the culture supernatant of the hybridoma deposited under accession number NITE BP-02342 using a molecule that binds to the antibody, such as protein A, protein G, or an antibody that binds to the Fc region of human IgG. More specifically, the antibody can be obtained, for example, by the method described in Patent Document 1.
本開示に係るラテックス粒子は、ラテックス凝集法によって試料中のコアフコシル化ヒトＩｇＧを測定することができるものであれば特に限定されず、例えば、有機高分子物質の微粒子若しくは無機酸化物の微粒子又は核となる上記微粒子の表面を有機物等で表面処理した微粒子等であってよい。具体的には、例えば、ポリスチレン、スチレンを主成分とする共重合体、ポリ塩化ビニル、ポリプロピレン、（メタ）アクリル樹脂、ポリメチルメタクリレート等の合成樹脂等が挙げられる。上記ラテックス粒子は、１種類を単独で使用してもよいし、２種類以上を併用してもよい。 The latex particles according to the present disclosure are not particularly limited as long as they can measure core-fucosylated human IgG in a sample by the latex agglutination method, and may be, for example, fine particles of organic polymeric substances or fine particles of inorganic oxides, or fine particles in which the surface of the above-mentioned core fine particles has been surface-treated with an organic substance or the like. Specific examples include synthetic resins such as polystyrene, copolymers mainly composed of styrene, polyvinyl chloride, polypropylene, (meth)acrylic resin, and polymethyl methacrylate. The above-mentioned latex particles may be used alone or in combination of two or more types.
本開示に係るラテックス粒子の平均粒径は、ラテックス凝集法によって試料中のコアフコシル化ヒトＩｇＧを測定することができるものであれば特に限定されず、例えば、１００ｎｍ～３００ｎｍ、１００ｎｍ～１５０ｎｍ、１５０ｎｍ～２００ｎｍ、２００ｎｍ～２５０ｎｍ又は２５０ｎｍ～３００ｎｍであってもよい。平均粒径（Ｄ５０）は、例えば、レーザー回折式粒度分布計にて測定することができる。平均粒径（Ｄ５０）は、粒度分布における積算値５０％（体積基準）での粒径を平均粒径とする。ラテックス粒子の形状としては、特に制限はなく、例えば、球状、楕円上、凹凸形状等が挙げられる。 The average particle size of the latex particles according to the present disclosure is not particularly limited as long as it allows the measurement of core fucosylated human IgG in a sample by the latex agglutination method, and may be, for example, 100 nm to 300 nm, 100 nm to 150 nm, 150 nm to 200 nm, 200 nm to 250 nm, or 250 nm to 300 nm. The average particle size (D50) can be measured, for example, by a laser diffraction particle size distribution analyzer. The average particle size (D50) is defined as the particle size at an integrated value of 50% (volume basis) in the particle size distribution. The shape of the latex particles is not particularly limited, and examples include spherical, elliptical, and irregular shapes.
本開示に係るラテックス粒子と抗コアフコシル化ヒトＩｇＧ選択的抗体又はその抗体断片の結合様式は、抗コアフコシル化ヒトＩｇＧ選択的抗体又はその抗体断片のコアフコシル化ヒトＩｇＧへの結合性を損なわないものであれば特に限定されず、例えば、物理吸着による結合、化学結合による結合等であってよい。物理吸着としては、静電的結合、水素結合、疎水結合等が挙げられる。化学結合としては、共有結合、配位結合等が挙げられる。 The binding mode between the latex particles according to the present disclosure and the anti-core fucosylated human IgG selective antibody or its antibody fragment is not particularly limited as long as it does not impair the binding ability of the anti-core fucosylated human IgG selective antibody or its antibody fragment to core fucosylated human IgG, and may be, for example, binding by physical adsorption, binding by chemical bond, etc. Examples of physical adsorption include electrostatic bonds, hydrogen bonds, hydrophobic bonds, etc. Examples of chemical bonds include covalent bonds, coordinate bonds, etc.
ラテックス粒子と抗コアフコシル化ヒトＩｇＧ選択的抗体又はその抗体断片は、直接結合していてもよく、間接的に結合していてもよい。間接的に結合する方法としては、例えば、ビオチンとアビジン類（アビジン、ストレプトアビジン、ニュートラアビジン等）等の一組の親和性物質の特異的結合を利用する方法及びリンカーを介した共有結合によりラテックス粒子に結合する方法等が挙げられる。 The anti-core fucosylated human IgG selective antibody or its antibody fragment may be directly or indirectly bound to the latex particle. Examples of indirect binding methods include a method that utilizes the specific binding of a pair of affinity substances such as biotin and avidins (avidin, streptavidin, neutravidin, etc.) and a method that binds to the latex particle by a covalent bond via a linker.
上記間接的に結合する方法として一組の親和性物質を利用する方法を用いる場合、抗コアフコシル化ヒトＩｇＧ選択的抗体又はその抗体断片が結合したラテックス粒子は、一組の親和性物質の一方（Ａ）が結合した抗コアフコシル化ヒトＩｇＧ選択的抗体又はその抗体断片と、一組の親和性物質の他方（ａ）が結合したラテックス粒子とを結合させることにより得ることができる。このとき、抗コアフコシル化ヒトＩｇＧ選択的抗体又はその抗体断片が結合したラテックス粒子は、抗原抗体反応の反応液中で生成されてもよい。 When the above-mentioned indirect binding method uses a pair of affinity substances, latex particles bound to an anti-core fucosylated human IgG selective antibody or its antibody fragment can be obtained by binding an anti-core fucosylated human IgG selective antibody or its antibody fragment bound to one of the pair of affinity substances (A) with latex particles bound to the other of the pair of affinity substances (a). In this case, the latex particles bound to an anti-core fucosylated human IgG selective antibody or its antibody fragment may be produced in the reaction solution of the antigen-antibody reaction.
一組の親和性物質Ａ－ａの組み合わせとしては、例えば以下の組み合わせを挙げることができる。
・ビオチンとアビジン類（アビジン、ニュートラアビジン、ストレプトアビジン等）との組み合わせ；
・アビジン類（アビジン、ニュートラアビジン、ストレプトアビジン等）とビオチンとの組み合わせ；
Examples of a combination of affinity substances Aa include the following combinations.
Combinations of biotin and avidins (avidin, neutravidin, streptavidin, etc.);
- combination of avidins (avidin, neutravidin, streptavidin, etc.) with biotin;
上記間接的に結合する方法としてリンカーを介した共有結合によりラテックス粒子に結合する方法を用いる場合、リンカーとしては、ラテックス粒子表面の官能基と、抗コアフコシル化ヒトＩｇＧ選択的抗体又はその抗体断片が有する官能基の両者を共有結合できる分子等を用いることができ、例えば、抗コアフコシル化ヒトＩｇＧ選択的抗体又はその抗体断片が有する官能基と反応することができる第１の反応活性基と、ラテックス粒子表面の官能基と反応することができる第２の反応活性基とを同時に持つ分子であって、第１の反応活性基と第２の反応活性基とが異なる基である分子が好ましく用いられる。 When the indirect binding method involves binding to latex particles by covalent bonding via a linker, the linker can be a molecule capable of covalently bonding both the functional group on the surface of the latex particle and the functional group possessed by the anti-core fucosylated human IgG selective antibody or its antibody fragment, and for example, a molecule that simultaneously has a first reactive group capable of reacting with the functional group possessed by the anti-core fucosylated human IgG selective antibody or its antibody fragment, and a second reactive group capable of reacting with the functional group on the surface of the latex particle, where the first reactive group and the second reactive group are different groups, is preferably used.
抗コアフコシル化ヒトＩｇＧ選択的抗体又はその抗体断片が有する官能基、及びラテックス粒子がその表面に保持している官能基としては、カルボキシル基、アミノ基、グリシジル基、スルフヒドリル基、水酸基、アミド基、イミノ基、Ｎ－ヒドロキシサクシニル基、マレイミド基等が挙げられる。リンカーにおける反応活性基としては、アリルアジド、カルボジイミド、ヒドラジド、アルデヒド、ヒドロキシメチルホスフィン、イミドエステル、イソシアネート、マレイミド、Ｎ－ヒドロキシスクシンイミド（ＮＨＳ）エステル、ペンタフルオロフェニル（ＰＦＰ）エステル、ソラレン、ピリジルジスルフィド、ビニルスルホン等の基が挙げられる。 Functional groups possessed by the anti-core fucosylated human IgG selective antibody or its antibody fragment, and functional groups retained on the surface of latex particles include carboxyl groups, amino groups, glycidyl groups, sulfhydryl groups, hydroxyl groups, amide groups, imino groups, N-hydroxysuccinyl groups, maleimide groups, etc. Reactive groups in the linker include allyl azide, carbodiimide, hydrazide, aldehyde, hydroxymethylphosphine, imide ester, isocyanate, maleimide, N-hydroxysuccinimide (NHS) ester, pentafluorophenyl (PFP) ester, psoralen, pyridyl disulfide, vinyl sulfone, etc.
ラテックス粒子に結合させる抗コアフコシル化ヒトＩｇＧ選択的抗体又はその抗体断片の量は、ラテックス粒子に上記抗体又はその抗体断片を結合させる溶液の含有量を基準として、ラテックス粒子１００質量部に対して例えば１００質量部以上２００００質量部以下であってよく、２００質量部以上１５０００質量部以下であってもよく、３００質量部以上１１０００質量部以下であってもよく、４００質量部以上８０００質量部以下であってもよく、５００質量部以上５０００質量部以下であってもよい。 The amount of anti-core fucosylated human IgG selective antibody or its antibody fragment to be bound to latex particles may be, for example, 100 parts by mass or more and 20,000 parts by mass or less, 200 parts by mass or more and 15,000 parts by mass or less, 300 parts by mass or more and 11,000 parts by mass or less, 400 parts by mass or more and 8,000 parts by mass or less, or 500 parts by mass or more and 5,000 parts by mass or less, relative to 100 parts by mass of latex particles, based on the content of the solution in which the antibody or its antibody fragment is bound to the latex particles.
本開示に係る抗コアフコシル化ヒトＩｇＧ選択的抗体又はその抗体断片とコアフコシル化ヒトＩｇＧの反応は、抗原抗体反応である。すなわち、上記反応は、ラテックス粒子の表面に結合した抗コアフコシル化ヒトＩｇＧ選択的抗体又はその抗体断片と、試料中のコアフコシル化ヒトＩｇＧが抗原抗体複合体を形成する反応である。上記反応の反応温度としては、抗原抗体反応が生じる反応温度であれば特に制限はなく、通常、０～５０℃であり、４～４０℃が好ましい。反応時間としては、上記抗原抗体反応をラテックス凝集法により検出可能な反応時間であれば特に制限はなく、通常、１分間～７２時間であり、５分間～２０時間が好ましい。 The reaction between the anti-core fucosylated human IgG selective antibody or antibody fragment thereof according to the present disclosure and core fucosylated human IgG is an antigen-antibody reaction. That is, the above reaction is a reaction in which the anti-core fucosylated human IgG selective antibody or antibody fragment thereof bound to the surface of the latex particle and the core fucosylated human IgG in the sample form an antigen-antibody complex. The reaction temperature of the above reaction is not particularly limited as long as it is a reaction temperature at which an antigen-antibody reaction occurs, and is usually 0 to 50°C, preferably 4 to 40°C. The reaction time is not particularly limited as long as it is a reaction time at which the above antigen-antibody reaction can be detected by the latex agglutination method, and is usually 1 minute to 72 hours, and preferably 5 minutes to 20 hours.
上記反応は、通常、水性媒体中で行われる。水性媒体としては、例えば脱イオン水、蒸留水、緩衝液等が挙げられ、緩衝液を含む水性媒体が好ましい。緩衝液の濃度は測定に適した濃度であれば特に制限はされないが、０．００１～２．０ｍｏｌ／Ｌが好ましく、０．００５～１．０ｍｏｌ／Ｌがより好ましく、０．０１～０．１ｍｏｌ／Ｌが特に好ましい。緩衝液の調製に使用される緩衝剤としては、緩衝能を有するものならば特に限定されないが、ｐＨ１～１１の例えば乳酸緩衝剤、クエン酸緩衝剤、酢酸緩衝剤、コハク酸緩衝剤、フタル酸緩衝剤、リン酸緩衝剤、トリエタノールアミン緩衝剤、ジエタノールアミン緩衝剤、リジン緩衝剤、バルビツール緩衝剤、イミダゾール緩衝剤、リンゴ酸緩衝剤、シュウ酸緩衝剤、グリシン緩衝剤、ホウ酸緩衝剤、炭酸緩衝剤、グリシン緩衝剤、トリス緩衝剤、グッド緩衝剤等が挙げられる。緩衝剤は、１種類を単独で使用してもよいし、２種類以上を併用してもよい。 The above reaction is usually carried out in an aqueous medium. Examples of the aqueous medium include deionized water, distilled water, and buffer solutions, and an aqueous medium containing a buffer solution is preferred. The concentration of the buffer solution is not particularly limited as long as it is suitable for the measurement, but is preferably 0.001 to 2.0 mol/L, more preferably 0.005 to 1.0 mol/L, and particularly preferably 0.01 to 0.1 mol/L. The buffer used to prepare the buffer solution is not particularly limited as long as it has buffering capacity, and examples of the buffer used to prepare the buffer solution include lactate buffer, citrate buffer, acetate buffer, succinate buffer, phthalate buffer, phosphate buffer, triethanolamine buffer, diethanolamine buffer, lysine buffer, barbiturate buffer, imidazole buffer, malate buffer, oxalate buffer, glycine buffer, borate buffer, carbonate buffer, glycine buffer, Tris buffer, Good's buffer, and the like, each having a pH of 1 to 11. The buffer solution may be used alone or in combination of two or more types.
上記グッド緩衝剤としては、例えば２－モルホリノエタンスルホン酸（ＭＥＳ）緩衝剤、ビス（２－ヒドロキシエチル）イミノトリス（ヒドロキシメチル）メタン（Ｂｉｓ－Ｔｒｉｓ）緩衝剤、トリス（ヒドロキシメチル）アミノメタン（Ｔｒｉｓ）緩衝剤、Ｎ－（２－アセトアミド）イミノ二酢酸（ＡＤＡ）緩衝剤、ピペラジン－Ｎ，Ｎ’－ビス（２－エタンスルホン酸）（ＰＩＰＥＳ）緩衝剤、２－［Ｎ－（２－アセトアミド）アミノ］エタンスルホン酸（ＡＣＥＳ）緩衝剤、３－モルホリノ－２－ヒドロキシプロパンスルホン酸（ＭＯＰＳＯ）緩衝剤、２－［Ｎ，Ｎ－ビス（２－ヒドロキシエチル）アミノ］エタンスルホン酸（ＢＥＳ）緩衝剤、３－モルホリノプロパンスルホン酸（ＭＯＰＳ）緩衝剤、２－｛Ｎ－［トリス（ヒドロキシメチル）メチル］アミノ｝エタンスルホン酸（ＴＥＳ）緩衝剤、Ｎ－（２－ヒドロキシエチル）－Ｎ’－（２－スルホエチル）ピペラジン（ＨＥＰＥＳ）緩衝剤、３－［Ｎ，Ｎ－ビス（２－ヒドロキシエチル）アミノ］－２－ヒドロキシプロパンスルホン酸（ＤＩＰＳＯ）緩衝剤、２－ヒドロキシ－３－｛［Ｎ－トリス（ヒドロキシメチル）メチル］アミノ｝プロパンスルホン酸（ＴＡＰＳＯ）緩衝剤、ピペラジン－Ｎ，Ｎ’－ビス（２－ヒドロキシプロパン－３－スルホン酸）（ＰＯＰＳＯ）緩衝剤、Ｎ－（２－ヒドロキシエチル）－Ｎ’－（２－ヒドロキシ－３－スルホプロピル）ピペラジン（ＨＥＰＰＳＯ）緩衝剤、Ｎ－（２－ヒドロキシエチル）－Ｎ’－（３－スルホプロピル）ピペラジン（ＥＰＰＳ）緩衝剤、トリシン［Ｎ－トリス（ヒドロキシメチル）メチルグリシン］緩衝剤、ビシン［Ｎ，Ｎ－ビス（２－ヒドロキシエチル）グリシン］緩衝剤、３－［Ｎ－トリス（ヒドロキシメチル）メチル］アミノプロパンスルホン酸（ＴＡＰＳ）緩衝剤、２－（Ｎ－シクロヘキシルアミノ）エタンスルホン酸（ＣＨＥＳ）緩衝剤、３－（Ｎ－シクロヘキシルアミノ）－２－ヒドロキシプロパンスルホン酸（ＣＡＰＳＯ）緩衝剤、３－（Ｎ－シクロヘキシルアミノ）プロパンスルホン酸（ＣＡＰＳ）緩衝剤等が挙げられる。 Examples of the Good buffer include 2-morpholinoethanesulfonic acid (MES) buffer, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris) buffer, tris(hydroxymethyl)aminomethane (Tris) buffer, N-(2-acetamido)iminodiacetic acid (ADA) buffer, piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) buffer, 2-[N-(2-acetamido)amino]ethanesulfonic acid (ACES) buffer, 3-morpholinoethanesulfonic acid (3-morpholinoethanesulfonic acid) ... 2-[N,N-bis(2-hydroxyethyl)amino]ethanesulfonic acid (BES) buffer, 3-morpholinopropanesulfonic acid (MOPS) buffer, 2-{N-[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES) buffer, N-(2-hydroxyethyl)-N'-(2-sulfoethyl)piperazine (HEPES) buffer, 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (MOPS) buffer, diphenyl ether sulfonic acid (DIPSO) buffer, 2-hydroxy-3-{[N-tris(hydroxymethyl)methyl]amino}propanesulfonic acid (TAPSO) buffer, piperazine-N,N'-bis(2-hydroxypropane-3-sulfonic acid) (POPSO) buffer, N-(2-hydroxyethyl)-N'-(2-hydroxy-3-sulfopropyl)piperazine (HEPPSO) buffer, N-(2-hydroxyethyl)-N'-(3-sulfopropyl)piperazine (EPPS) buffer, tricine [N -tris(hydroxymethyl)methylglycine] buffer, bicine [N,N-bis(2-hydroxyethyl)glycine] buffer, 3-[N-tris(hydroxymethyl)methyl]aminopropanesulfonic acid (TAPS) buffer, 2-(N-cyclohexylamino)ethanesulfonic acid (CHES) buffer, 3-(N-cyclohexylamino)-2-hydroxypropanesulfonic acid (CAPSO) buffer, 3-(N-cyclohexylamino)propanesulfonic acid (CAPS) buffer, etc.
本開示に係る水性媒体は、さらに、金属イオン、塩類、糖類、防腐剤、蛋白質、蛋白質安定化剤等を含んでもよい。金属イオンとしては、例えばマグネシウムイオン、マンガンイオン、亜鉛イオン等が挙げられる。塩類としては、例えば塩化ナトリウム、塩化カリウム等が挙げられる。糖類としては、例えばマンニトール、ソルビトール等が挙げられる。防腐剤としては、例えばアジ化ナトリウム、抗生物質（ストレプトマイシン、ペニシリン、ゲンタマイシン等）、バイオエース、プロクリン３００、プロキセル（Ｐｒｏｘｅｌ）ＧＸＬ等が挙げられる。蛋白質としては、例えばウシ血清アルブミン（ＢＳＡ）、ウシ胎児血清（ＦＢＳ）、カゼイン、ブロックエース（株式会社ケー・エー・シー社製）等が挙げられる。蛋白質安定化剤としては、例えばペルオキシダーゼ安定化緩衝液［Ｐｅｒｏｘｉｄａｓｅ Ｓｔａｂｉｌｉｚｉｎｇ Ｂｕｆｆｅｒ、ダコサイトメーション（ＤａｋｏＣｙｔｏｍａｔｉｏｎ）社製］等が挙げられる。 The aqueous medium according to the present disclosure may further include metal ions, salts, sugars, preservatives, proteins, protein stabilizers, and the like. Examples of metal ions include magnesium ions, manganese ions, zinc ions, and the like. Examples of salts include sodium chloride, potassium chloride, and the like. Examples of sugars include mannitol, sorbitol, and the like. Examples of preservatives include sodium azide, antibiotics (streptomycin, penicillin, gentamicin, and the like), Bioace, Proclin 300, Proxel GXL, and the like. Examples of proteins include bovine serum albumin (BSA), fetal bovine serum (FBS), casein, Block Ace (manufactured by KAC Co., Ltd.), and the like. Examples of protein stabilizers include peroxidase stabilizing buffer [Peroxidase Stabilizing Buffer, manufactured by DakoCytomation], and the like.
一般に、コアフコシル化ヒトＩｇＧにおいては、ヒトＩｇＧが有する２つのＮ結合型糖鎖がコアフコシル化されているため、１分子のコアフコシル化ヒトＩｇＧに対し、最大２分子の抗コアフコシル化ヒトＩｇＧ選択的抗体又はその抗体断片が結合することができる。したがって、上記反応において、コアフコシル化ヒトＩｇＧは２価抗原として振る舞うことができる。 In general, in core fucosylated human IgG, two N-linked glycans of human IgG are core fucosylated, so that up to two molecules of anti-core fucosylated human IgG selective antibody or antibody fragment thereof can bind to one molecule of core fucosylated human IgG. Therefore, in the above reaction, core fucosylated human IgG can behave as a bivalent antigen.
本開示に係る、試料中のコアフコシル化ヒトＩｇＧを測定する方法によれば、試料中のコアフコシル化ヒトＩｇＧ量を得ることができる。本開示における「コアフコシル化ヒトＩｇＧ量」は、コアフコシル化ヒトＩｇＧ濃度の他、測定方法に応じて、単位体積当たりのコアフコシル化ヒトＩｇＧの存在量に基づくシグナル強度（例えば吸光度）としても定量化され表示され得る。コアフコシル化ヒトＩｇＧ濃度は、試料の単位量当たりのコアフコシル化ヒトＩｇＧの存在量を意味し、典型的には試料の単位体積当たりのコアフコシル化ヒトＩｇＧの質量又はモル数として表される。また、コアフコシル化ヒトＩｇＧ濃度は、必ずしも試料の単位体積当たりのコアフコシル化ヒトＩｇＧ量として定量化されるとは限らず、例えば試料の単位重量当たりのコアフコシル化ヒトＩｇＧ量でもあり得る。 According to the method for measuring core fucosylated human IgG in a sample of the present disclosure, the amount of core fucosylated human IgG in the sample can be obtained. In addition to the core fucosylated human IgG concentration, the "amount of core fucosylated human IgG" in the present disclosure can be quantified and displayed as a signal intensity (e.g., absorbance) based on the amount of core fucosylated human IgG present per unit volume depending on the measurement method. The core fucosylated human IgG concentration means the amount of core fucosylated human IgG present per unit amount of sample, and is typically expressed as the mass or moles of core fucosylated human IgG per unit volume of sample. In addition, the core fucosylated human IgG concentration is not necessarily quantified as the amount of core fucosylated human IgG per unit volume of sample, and can also be, for example, the amount of core fucosylated human IgG per unit weight of sample.
本開示において、ラテックス凝集法とは、試料中の多価抗原と、ラテックス粒子に結合させた抗体又はその抗体断片との抗原抗体反応によって生じるラテックス粒子の凝集の程度を指標として、試料中の抗原を検出及び／又は定量する方法である。上記ラテックス粒子の凝集の程度の評価方法は特に限定されず、例えば、吸光度、散乱光強度又は透過光強度等を、当業者が通常行う方法で測定することにより評価することができる。通常、吸光度、散乱光強度又は透過光強度等の測定は、上記抗原抗体反応を生じさせた反応液又はその希釈液を測定することにより行われる。 In the present disclosure, the latex agglutination method is a method for detecting and/or quantifying antigens in a sample using as an index the degree of agglutination of latex particles caused by an antigen-antibody reaction between a polyvalent antigen in the sample and an antibody or an antibody fragment thereof bound to latex particles. The method for evaluating the degree of agglutination of the latex particles is not particularly limited, and can be evaluated, for example, by measuring absorbance, scattered light intensity, transmitted light intensity, etc. by a method normally performed by a person skilled in the art. Usually, the measurement of absorbance, scattered light intensity, transmitted light intensity, etc. is performed by measuring the reaction solution in which the antigen-antibody reaction has occurred or a diluted solution thereof.
一実施形態において、上記反応は、抗コアフコシル化ヒトＩｇＧ選択的抗体又はその抗体断片が結合したラテックス粒子を含む水性媒体と、希釈していない試料を混和することにより調製した反応液中で行ってもよく、かつ、上記測定は、上記反応液を希釈することなく行ってもよい。希釈工程を備えないことで、希釈誤差を抑制することができる。また、上記反応は、抗コアフコシル化ヒトＩｇＧ選択的抗体又はその抗体断片が結合したラテックス粒子を含む水性媒体と、低い希釈倍率で希釈した試料を混和することにより調製した反応液中で行ってもよく、かつ、上記測定は、上記反応液を希釈することなく行ってもよい。低い希釈倍率で試料を希釈するため、希釈誤差を抑制することができる。低い希釈倍率は、１．５倍～３０倍であってよく、２．０倍～２０倍であってよく、２．０倍～１０倍であってよい。一般に、ＥＬＩＳＡ法に代表される抗原抗体反応を簡便に評価可能な測定方法は、蛍光又は発光等によるシグナル増幅を伴うため高感度であり、結果として有意な測定には、高い希釈倍率で試料を希釈することが必要であるため、希釈誤差が生じてしまう。 In one embodiment, the reaction may be carried out in a reaction solution prepared by mixing an aqueous medium containing latex particles to which an anti-core fucosylated human IgG selective antibody or an antibody fragment thereof is bound with an undiluted sample, and the measurement may be carried out without diluting the reaction solution. By not providing a dilution step, dilution errors can be suppressed. The reaction may be carried out in a reaction solution prepared by mixing an aqueous medium containing latex particles to which an anti-core fucosylated human IgG selective antibody or an antibody fragment thereof is bound with a sample diluted at a low dilution ratio, and the measurement may be carried out without diluting the reaction solution. Since the sample is diluted at a low dilution ratio, dilution errors can be suppressed. The low dilution ratio may be 1.5 to 30 times, 2.0 to 20 times, or 2.0 to 10 times. In general, measurement methods that can easily evaluate antigen-antibody reactions, such as ELISA, are highly sensitive because they involve signal amplification by fluorescence or luminescence, and as a result, in order to obtain meaningful measurements, it is necessary to dilute the sample at a high dilution ratio, which can lead to dilution errors.
同様の観点から、試料に対する上記反応及び／又は測定に用いる溶液の総量を抑えることによって、希釈誤差を抑制することができる。試料に対する上記反応及び／又は測定に用いる溶液の総量は、測定時において、体積基準で試料の１００００倍以下であってよく、５０００倍以下であってもよく、１０００倍以下であってよく、５００倍以下であってもよく、１００倍以下であってもよく、５０倍以下であってもよく、３０倍以下であってもよい。 From a similar perspective, dilution errors can be suppressed by reducing the total amount of solution used in the above reaction and/or measurement of the sample. The total amount of solution used in the above reaction and/or measurement of the sample may be 10,000 times or less, 5,000 times or less, 1,000 times or less, 500 times or less, 100 times or less, 50 times or less, or 30 times or less of the sample on a volume basis at the time of measurement.
上記反応における抗コアフコシル化ヒトＩｇＧ選択的抗体又はその抗体断片が結合したラテックス粒子の濃度は、上記反応をラテックス凝集法により検出可能な濃度であれば特に制限はなく、例えば、０．００１％（ｗ／ｖ）以上、０．００３％（ｗ／ｖ）以上、０．００５％（ｗ／ｖ）以上、０．０１％（ｗ／ｖ）以上、０．０１１％（ｗ／ｖ）以上、０．０１２％（ｗ／ｖ）以上、又は０．０１３％（ｗ／ｖ）以上であってよい。また、上記反応における抗コアフコシル化ヒトＩｇＧ選択的抗体又はその抗体断片が結合したラテックス粒子の濃度は、例えば、１．０％（ｗ／ｖ）以下、０．８％（ｗ／ｖ）以下、０．６％（ｗ／ｖ）以下、０．４％（ｗ／ｖ）以下、０．２％（ｗ／ｖ）以下、０．１％（ｗ／ｖ）以下、０．０７５％（ｗ／ｖ）以下、０．０５％（ｗ／ｖ）以下、又は０．０２５％（ｗ／ｖ）以下であってよい。上記の数値は自由に組み合わせることができる。 The concentration of latex particles to which anti-core fucosylated human IgG selective antibody or its antibody fragment is bound in the above reaction is not particularly limited as long as the concentration is such that the above reaction can be detected by latex agglutination, and may be, for example, 0.001% (w/v) or more, 0.003% (w/v) or more, 0.005% (w/v) or more, 0.01% (w/v) or more, 0.011% (w/v) or more, 0.012% (w/v) or more, or 0.013% (w/v) or more. In addition, the concentration of the latex particles bound to the anti-core fucosylated human IgG selective antibody or its antibody fragment in the above reaction may be, for example, 1.0% (w/v) or less, 0.8% (w/v) or less, 0.6% (w/v) or less, 0.4% (w/v) or less, 0.2% (w/v) or less, 0.1% (w/v) or less, 0.075% (w/v) or less, 0.05% (w/v) or less, or 0.025% (w/v) or less. The above values can be freely combined.
一実施形態において、上記反応は、抗アコアフコシル化ヒトＩｇＧ抗体及びその抗体断片の非存在下で行われてもよい。すわわち、一実施形態において、上記反応は、抗ヒトＩｇＧ抗体若しくは抗アコアフコシル化ヒトＩｇＧ選択的抗体及びそれらの抗体断片の非存在下で行われてもよい。試料としてヒトＩｇＧに占めるコアフコシル化ヒトＩｇＧの割合の変動が生じる疾患に罹患した被験者由来の生体試料を用いた場合、生体試料中のコアフコシル化ヒトＩｇＧ量は、生体試料中の全ヒトＩｇＧ量に依存して変動し得るという課題があり、ある個体の生体試料中のコアフコシル化ヒトＩｇＧ量が高いことが確認された場合でも、当該個体の生体試料中の全ヒトＩｇＧ量がもともと高くそれに依存してコアフコシル化ヒトＩｇＧ量が高いのか、それとも、当該個体の生体試料中の全ヒトＩｇＧ量はあまり高くないが、コアフコシル化ヒトＩｇＧ量だけが高いのか、判断することができない。そのため、コアフコシル化ヒトＩｇＧによる生体試料の評価は、通常、コアフコシル化ヒトＩｇＧに加えて全ヒトＩｇＧも測定し、全ヒトＩｇＧ量に占めるコアフコシル化ヒトＩｇＧ量（以下、「コアフコシル化ヒトＩｇＧ／全ヒトＩｇＧ比」とも記載する。）に基づいて評価される。これに対し、一実施形態に係る抗コアフコシル化ヒトＩｇＧ選択的抗体を用いたラテックス凝集法によれば、ＥＬＩＳＡ法と比較して、従来法であるヒトＩｇＧに占めるコアフコシル化ヒトＩｇＧの存在割合を指標とした評価により近く、かつ統計的有意差をもって罹患の有無に応じたコアフコシル化ヒトＩｇＧ量の変動を評価することができる。したがって、一実施形態に係る抗コアフコシル化ヒトＩｇＧ選択的抗体を用いたラテックス凝集法によれば、コアフコシル化ヒトＩｇＧ量の変動が生じる疾患を診断又はその補助をする場合において、全ヒトＩｇＧ量及びアコアフコシル化ヒトＩｇＧ量を測定することなく、簡便に罹患の有無に応じたコアフコシル化ヒトＩｇＧ量の変動を評価することができる。 In one embodiment, the reaction may be performed in the absence of anti-aco-fucosylated human IgG antibodies and antibody fragments thereof. That is, in one embodiment, the reaction may be performed in the absence of anti-human IgG antibodies or anti-aco-fucosylated human IgG selective antibodies and antibody fragments thereof. When a biological sample from a subject suffering from a disease in which the ratio of core fucosylated human IgG to human IgG fluctuates is used as a sample, there is a problem that the amount of core fucosylated human IgG in the biological sample may fluctuate depending on the amount of total human IgG in the biological sample. Even if the amount of core fucosylated human IgG in a biological sample of an individual is confirmed to be high, it is not possible to determine whether the amount of total human IgG in the biological sample of the individual is originally high and the amount of core fucosylated human IgG is high depending on the amount of total human IgG in the biological sample of the individual, or whether the amount of total human IgG in the biological sample of the individual is not very high but only the amount of core fucosylated human IgG is high. Therefore, in the evaluation of a biological sample using core fucosylated human IgG, in addition to core fucosylated human IgG, total human IgG is usually measured, and the evaluation is based on the amount of core fucosylated human IgG in the amount of total human IgG (hereinafter also referred to as "core fucosylated human IgG/total human IgG ratio"). In contrast, the latex agglutination method using an anti-core fucosylated human IgG selective antibody according to one embodiment is closer to the evaluation using the proportion of core fucosylated human IgG in human IgG as an index, as compared with the ELISA method, and is capable of evaluating the fluctuation in the amount of core fucosylated human IgG depending on the presence or absence of disease with a statistically significant difference. Therefore, according to the latex agglutination method using an anti-core fucosylated human IgG selective antibody of one embodiment, when diagnosing or assisting in the diagnosis of a disease in which the amount of core fucosylated human IgG changes, it is possible to easily evaluate the change in the amount of core fucosylated human IgG depending on the presence or absence of a disease without measuring the amount of total human IgG and the amount of acore fucosylated human IgG.
上記ラテックス凝集法は、ホールピペット等の溶液分注用の器具、抗原抗体反応のための加温用の恒温槽、吸光度又は散乱光の測定機器などを組み合わせて使用する、いわゆる用手法で実施してもよく、機器上で一連の溶液分注、加温、吸光度又は散乱光の測定を行う、いわゆる自動分析装置を使用して実施してもよい。上記自動分析装置は、特に制限はないが、例えば、試料、測定用試薬又はそれらの混合液の攪拌のため、上記混合液等が入った反応セルへ棒又はヘラ状の攪拌用部材を差し込み、その後に、上記攪拌用部材を前後若しくは左右に移動させる機構、又は、上記攪拌用部材を回転させる機構（以下、ヘラ攪拌機構と記載する）を備えた自動分析装置であってもよく、試料、測定用試薬又はそれらの混合液の攪拌のため、上記混合液等が入った反応セルの外側から超音波をあてる機構（以下、超音波攪拌機構と記載する）を備えた自動分析器であってもよい。 The latex agglutination method may be performed by a so-called manual method using a combination of a solution dispensing tool such as a volumetric pipette, a thermostatic bath for heating for antigen-antibody reaction, and an absorbance or scattered light measuring device, or may be performed using a so-called automatic analyzer that performs a series of solution dispensing, heating, and absorbance or scattered light measurements on the device. The automatic analyzer is not particularly limited, but may be, for example, an automatic analyzer equipped with a mechanism for inserting a rod or spatula-shaped stirring member into a reaction cell containing the mixed liquid or the like in order to stir the sample, measurement reagent, or a mixture thereof, and then moving the stirring member back and forth or left and right, or a mechanism for rotating the stirring member (hereinafter referred to as a spatula stirring mechanism), or an automatic analyzer equipped with a mechanism for applying ultrasonic waves from the outside of a reaction cell containing the mixed liquid or the like in order to stir the sample, measurement reagent, or a mixture thereof (hereinafter referred to as an ultrasonic stirring mechanism).
上記ヘラ攪拌機構を備えた自動分析装置としては、特に制限はないが、例えば、自動分析装置３５００、自動分析装置７１８０（以上、日立ハイテク社製）、ＪＣＡ－ＺＳ０５０自動分析装置クリナライザ ＢｉｏＭａｊｅｓｔｙ（登録商標）ＺＥＲＯ、ＪＣＡ－ＢＭ９１３０自動分析装置クリナライザ ＢｉｏＭａｊｅｓｔｙ（以上、日本電子社製）、ＴＢＡ（登録商標）－ｃ１６０００、ＴＢＡ－１２０ＦＲ（以上、キヤノンメディカルシステムズ社製）、自動分析装置ＡＵ５８００、及び自動分析装置ＡＵ６８０（以上、ベックマンコールター社製）が挙げられる。上記超音波攪拌機構を備えた自動分析装置としては、特に制限はないが、例えば、自動分析装置ＬＡＢＯＳＰＥＣＴ（登録商標）００３、自動分析装置ＬＡＢＯＳＰＥＣＴ（登録商標） ００６、及び自動分析装置ＬＡＢＯＳＰＥＣＴ（登録商標） ００８α（以上、日立ハイテク社製）が挙げられる。 There are no particular limitations on the automatic analyzer equipped with the spatula stirring mechanism, but examples include the automatic analyzer 3500, the automatic analyzer 7180 (all manufactured by Hitachi High-Tech Corporation), the JCA-ZS050 automatic analyzer Cleanerizer BioMajesty (registered trademark) ZERO, the JCA-BM9130 automatic analyzer Cleanerizer BioMajesty (all manufactured by JEOL Ltd.), the TBA (registered trademark)-c16000, the TBA-120FR (all manufactured by Canon Medical Systems Corporation), the automatic analyzer AU5800, and the automatic analyzer AU680 (all manufactured by Beckman Coulter, Inc.). The automatic analyzer equipped with the ultrasonic stirring mechanism is not particularly limited, but examples include the automatic analyzer LABOSPECT (registered trademark) 003, the automatic analyzer LABOSPECT (registered trademark) 006, and the automatic analyzer LABOSPECT (registered trademark) 008α (all manufactured by Hitachi High-Tech Corporation).
本開示におけるラテックス凝集法は、感度が高くなるという観点から、試料を超音波で処理すること（超音波処理）を含んでいてもよい。「超音波で処理する」とは、上記のとおり超音波で攪拌することであってもよい。超音波処理は、超音波処理することのできる任意の装置によって行うことができ、例えば、上記の装置が挙げられる。超音波処理の時間は、本開示の測定方法が可能な限り制限されないが、例えば、３０秒間以下、２０秒間以下、又は１０秒間以下であってよい。超音波処理の時間は、例えば、５秒間以上、３秒間以上、１秒間以上、又は０．５秒間以上であってよい。上記の数値は自由に組み合わせることができる。 The latex agglutination method of the present disclosure may include treating the sample with ultrasound (ultrasonic treatment) in order to increase sensitivity. "Treatment with ultrasound" may mean stirring with ultrasound as described above. Ultrasonic treatment may be performed by any device capable of ultrasonic treatment, such as the devices described above. The duration of ultrasonic treatment is not limited as far as the measurement method of the present disclosure can be used, but may be, for example, 30 seconds or less, 20 seconds or less, or 10 seconds or less. The duration of ultrasonic treatment may be, for example, 5 seconds or more, 3 seconds or more, 1 second or more, or 0.5 seconds or more. The above values may be freely combined.
ラテックス粒子の凝集の程度を吸光度に基づいて評価する場合、吸光度の測定波長は、通常は３４０ｎｍ～１０００ｎｍ、好ましくは５００ｎｍ～９００ｎｍで測定すればよい。ラテックス凝集反応を測光する時間は、ラテックス凝集反応が生じている時間を時間当たりの変化速度、あるいは一定時間の変化量によって測光することができる。例えば、吸光度を測定する場合、ラテックス凝集反応が始まってから３０秒後から５分後の時間当たりの吸光度変化速度、あるいは一定時間の吸光度変化量によって測光することができる。反応温度は１０～５０℃であることが好ましく、２０～４０℃であることがより好ましい。反応時間は適宜決定することができ、例えば汎用自動分析機では５～１５分間の反応時間で測定することができる。 When the degree of agglutination of latex particles is evaluated based on absorbance, the wavelength for measuring absorbance is usually 340 nm to 1000 nm, preferably 500 nm to 900 nm. The time for measuring the latex agglutination reaction can be measured based on the rate of change per unit time during which the latex agglutination reaction is occurring, or the amount of change over a fixed period of time. For example, when measuring absorbance, it can be measured based on the rate of change in absorbance per unit time from 30 seconds to 5 minutes after the start of the latex agglutination reaction, or the amount of change in absorbance over a fixed period of time. The reaction temperature is preferably 10 to 50°C, and more preferably 20 to 40°C. The reaction time can be determined as appropriate; for example, a reaction time of 5 to 15 minutes can be used for measurement with a general-purpose automatic analyzer.
一実施形態において、ラテックス凝集法によって試料中のコアフコシル化ヒトＩｇＧを測定する方法は、既知濃度のコアフコシル化ヒトＩｇＧを用いて作成されたコアフコシル化ヒトＩｇＧ濃度と測定値との関係を表す検量線を作成すること、及び作成された検量線と試料の測定値とから試料中のコアフコシル化ヒトＩｇＧ濃度を決定することを含んでもよい。 In one embodiment, the method for measuring core fucosylated human IgG in a sample by latex agglutination may include creating a calibration curve showing the relationship between the core fucosylated human IgG concentration and the measured value, which is created using core fucosylated human IgG of known concentrations, and determining the core fucosylated human IgG concentration in the sample from the created calibration curve and the measured value of the sample.
検量線の作成に用いる既知濃度のコアフコシル化ヒトＩｇＧは特に限定されず、例えば特許文献１に記載された方法により取得することができる。既知濃度のコアフコシル化ヒトＩｇＧについて、複数の濃度を用意してもよい。 The core fucosylated human IgG of known concentration used to create the calibration curve is not particularly limited, and can be obtained, for example, by the method described in Patent Document 1. Multiple concentrations of core fucosylated human IgG of known concentration may be prepared.
本開示の他の一側面は、抗コアフコシル化ヒト免疫グロブリンＧ抗体又はその抗体断片が結合したラテックス粒子を含む、ラテックス凝集法によって試料中のコアフコシル化ヒト免疫グロブリンＧを測定するための試薬であって、上記抗コアフコシル化ヒト免疫グロブリンＧ抗体又はその抗体断片は、コアフコシル化ヒト免疫グロブリンＧに結合し、かつ、アコアフコシル化ヒト免疫グロブリンＧに結合しない抗体又はその抗体断片である、試薬である。すなわち、本開示の他の一側面は、抗コアフコシル化ヒトＩｇＧ選択的抗体又はその抗体断片が結合したラテックス粒子を含む、ラテックス凝集法によって試料中のコアフコシル化ヒトＩｇＧを測定するための試薬である。言い換えれば、本開示の他の一側面に係る試薬は、本開示の一側面に係るラテックス凝集法によって試料中のコアフコシル化ヒトＩｇＧを測定する方法に用いる試薬であって、抗コアフコシル化ヒト免疫グロブリンＧ抗体又はその抗体断片が結合したラテックス粒子を含む、試薬である。本開示の一側面に係る試薬における抗コアフコシル化ヒトＩｇＧ選択的抗体及びその抗体断片が結合したラテックス粒子は、上述の本開示の一側面に係るラテックス凝集法によって試料中のコアフコシル化ヒトＩｇＧを測定する方法において説明したものを用いることができ、また上述した方法により製造することができる。また、本開示の一側面に係る試薬は、例えば上述の本開示の一側面に係るラテックス凝集法によって試料中のコアフコシル化ヒトＩｇＧを測定する方法において説明した方法により使用することができる。 Another aspect of the present disclosure is a reagent for measuring core fucosylated human immunoglobulin G in a sample by a latex agglutination method, comprising latex particles to which an anti-core fucosylated human immunoglobulin G antibody or an antibody fragment thereof is bound, wherein the anti-core fucosylated human immunoglobulin G antibody or an antibody fragment thereof is an antibody or an antibody fragment thereof that binds to core fucosylated human immunoglobulin G and does not bind to acore fucosylated human immunoglobulin G. That is, another aspect of the present disclosure is a reagent for measuring core fucosylated human IgG in a sample by a latex agglutination method, comprising latex particles to which an anti-core fucosylated human IgG selective antibody or an antibody fragment thereof is bound. In other words, the reagent according to another aspect of the present disclosure is a reagent used in a method for measuring core fucosylated human IgG in a sample by a latex agglutination method according to one aspect of the present disclosure, comprising latex particles to which an anti-core fucosylated human immunoglobulin G antibody or an antibody fragment thereof is bound. The latex particles to which the anti-core fucosylated human IgG selective antibody and its antibody fragment are bound in the reagent according to one aspect of the present disclosure can be those described in the method for measuring core fucosylated human IgG in a sample by the latex agglutination method according to one aspect of the present disclosure above, and can be produced by the method described above. In addition, the reagent according to one aspect of the present disclosure can be used, for example, by the method described in the method for measuring core fucosylated human IgG in a sample by the latex agglutination method according to one aspect of the present disclosure above.
一実施形態において、上記試薬は、抗アコアフコシル化ヒトＩｇＧ抗体及びその抗体断片を含まない。上述したとおり、試料としてヒトＩｇＧに占めるコアフコシル化ヒトＩｇＧの割合の変動が生じる疾患に罹患した被験者由来の生体試料を用いた場合、本開示の一実施形態に係る抗コアフコシル化ヒトＩｇＧ選択的抗体を用いたラテックス凝集法によれば、ＥＬＩＳＡ法と比較して、従来法であるヒトＩｇＧに占めるコアフコシル化ヒトＩｇＧの存在割合を指標とした評価により近く、かつ統計的有意差をもって罹患の有無に応じたコアフコシル化ヒトＩｇＧ量の変動を評価することができる。したがって、一実施形態に係る試薬は、全ヒトＩｇＧ量及び／又はアコアフコシル化ヒトＩｇＧ量を測定するための抗体を含有しなくても、簡便に罹患の有無に応じたコアフコシル化ヒトＩｇＧ量の変動を評価するために用いることができる。 In one embodiment, the reagent does not contain an anti-aco-fucosylated human IgG antibody or an antibody fragment thereof. As described above, when a biological sample from a subject suffering from a disease in which the proportion of core-fucosylated human IgG in human IgG changes is used as a sample, the latex agglutination method using an anti-core-fucosylated human IgG selective antibody according to one embodiment of the present disclosure can evaluate the variation in the amount of core-fucosylated human IgG depending on the presence or absence of disease more closely than the conventional evaluation using the proportion of core-fucosylated human IgG in human IgG as an index, and with a statistically significant difference, compared to the ELISA method. Therefore, the reagent according to one embodiment can be used to easily evaluate the variation in the amount of core-fucosylated human IgG depending on the presence or absence of disease, even if it does not contain an antibody for measuring the amount of total human IgG and/or the amount of aco-fucosylated human IgG.
本開示の他の一側面は、被験者の生体試料中のコアフコシル化ヒト免疫グロブリンＧを測定することを備える、上記被験者が、炎症を伴う肺疾患に罹患している否かを判定するためのデータを収集する方法であって、上記測定が、コアフコシル化ヒト免疫グロブリンＧに結合し、かつ、アコアフコシル化ヒト免疫グロブリンＧには結合しない抗コアフコシル化ヒトグロブリンＧ抗体又はその抗体断片が結合したラテックス粒子を用いたラテックス凝集法により行われる、方法である。すなわち、本開示の他の一側面は、被験者の生体試料中のコアフコシル化ヒト免疫グロブリンＧを測定することを備える、上記被験者が、炎症を伴う肺疾患に罹患している否かを判定するためのデータを収集する方法であって、上記測定が、抗コアフコシル化ヒトＩｇＧ選択的抗体又はその抗体断片が結合したラテックス粒子を用いたラテックス凝集法により行われる、方法（以下では、「データ収集方法」とも記載する。）である。 Another aspect of the present disclosure is a method for collecting data for determining whether or not a subject suffers from a pulmonary disease accompanied by inflammation, comprising measuring core fucosylated human immunoglobulin G in a biological sample from the subject, the measurement being performed by a latex agglutination method using latex particles to which an anti-core fucosylated human globulin G antibody or an antibody fragment thereof that binds to core fucosylated human immunoglobulin G and does not bind to acore fucosylated human immunoglobulin G is bound. That is, another aspect of the present disclosure is a method for collecting data for determining whether or not a subject suffers from a pulmonary disease accompanied by inflammation, comprising measuring core fucosylated human immunoglobulin G in a biological sample from the subject, the measurement being performed by a latex agglutination method using latex particles to which an anti-core fucosylated human IgG selective antibody or an antibody fragment thereof is bound (hereinafter also referred to as a "data collection method").
本開示の他の一側面は、被験者の生体試料中のコアフコシル化ヒト免疫グロブリンＧを測定することを備える、炎症を伴う肺疾患の診断方法又は診断を補助する方法であって、前記測定が、コアフコシル化ヒト免疫グロブリンＧに結合し、かつ、アコアフコシル化ヒト免疫グロブリンＧには結合しない抗コアフコシル化ヒトグロブリンＧ抗体又はその抗体断片が結合したラテックス粒子を用いたラテックス凝集法により行われる、方法である。すなわち、本開示の他の一側面は、被験者の生体試料中のコアフコシル化ヒト免疫グロブリンＧを測定することを備える、炎症を伴う肺疾患の診断方法又は診断を補助する方法であって、前記測定が、抗コアフコシル化ヒトＩｇＧ選択的抗体又はその抗体断片が結合したラテックス粒子を用いたラテックス凝集法により行われる、方法（以下では、それぞれ「診断方法」「診断補助方法」とも記載する。）である。 Another aspect of the present disclosure is a method for diagnosing or assisting in the diagnosis of pulmonary disease accompanied by inflammation, comprising measuring core fucosylated human immunoglobulin G in a biological sample from a subject, the measurement being performed by a latex agglutination method using latex particles to which an anti-core fucosylated human immunoglobulin G antibody or an antibody fragment thereof that binds to core fucosylated human immunoglobulin G and does not bind to acore fucosylated human immunoglobulin G is bound. That is, another aspect of the present disclosure is a method for diagnosing or assisting in the diagnosis of pulmonary disease accompanied by inflammation, comprising measuring core fucosylated human immunoglobulin G in a biological sample from a subject, the measurement being performed by a latex agglutination method using latex particles to which an anti-core fucosylated human IgG selective antibody or an antibody fragment thereof is bound (hereinafter, also referred to as a "diagnostic method" and a "diagnostic assisting method", respectively).
本開示において、被験者とは、炎症を伴う肺疾患に罹患しているヒト及び炎症を伴う肺疾患に罹患していないヒトの両方を含む概念であり、炎症を伴う肺疾患に罹患している可能性があるヒトであってもよい。すなわち、一実施形態に係るデータ収集方法、診断方法又は診断補助方法は、例えば炎症を伴う肺疾患への罹患が疑われる症状を示すヒトについての判定又は診断に用いられる方法であってもよく、健康診断等において炎症を伴う肺疾患に罹患している可能性があるヒトを発見（スクリーニング）するために用いられる方法であってもよく、炎症を伴う肺疾患に罹患しているヒトについて、該肺疾患からの回復をモニタリングするために用いられる方法であってもよい。 In the present disclosure, the subject is a concept including both humans suffering from pulmonary disease accompanied by inflammation and humans not suffering from pulmonary disease accompanied by inflammation, and may be a human who may be suffering from pulmonary disease accompanied by inflammation. That is, the data collection method, diagnosis method, or diagnostic support method according to one embodiment may be, for example, a method used to determine or diagnose a human who shows symptoms suspected of suffering from pulmonary disease accompanied by inflammation, a method used to discover (screen) a human who may be suffering from pulmonary disease accompanied by inflammation in a health checkup, or a method used to monitor the recovery of a human suffering from pulmonary disease accompanied by inflammation.
本開示において、生体試料とは、試料のうち、コアフコシル化ヒトＩｇＧを含有しうるヒト由来の試料であれば特に限定されず、例えばヒト由来の全血、血漿、血清、尿、腹水、髄液、唾液、羊水、尿、汗及び膵液等を挙げることができる。 In the present disclosure, a biological sample is not particularly limited as long as it is a sample derived from a human that can contain core fucosylated human IgG, and examples of such a sample include whole blood, plasma, serum, urine, ascites, cerebrospinal fluid, saliva, amniotic fluid, urine, sweat, and pancreatic juice derived from a human.
本開示に係る炎症を伴う肺疾患は、肺疾患のうち炎症を伴うものであれば特に限定されず、例えば肺炎、肺がん、慢性閉塞性肺疾患（ＣＯＰＤ）又は肺結核等であってよい。また、炎症を伴う肺疾患が肺炎である場合、肺炎の種類は特に限定されず、例えば市中肺炎（ＣＡＰ）、医療・介護関連肺炎（ＮＨＣＡＰ）又は院内肺炎（ＨＡＰ）のいずれであってもよく、肺胞性肺炎又は間質性肺炎のいずれであってもよく、またその原因となる細菌又はウイルス等も特に限定されない。一実施形態において、炎症を伴う肺疾患は、間質性肺炎、肺がん及び慢性閉塞性肺疾患からなる群より選ばれる少なくとも１つであってもよい。 The pulmonary disease accompanied by inflammation according to the present disclosure is not particularly limited as long as it is a pulmonary disease accompanied by inflammation, and may be, for example, pneumonia, lung cancer, chronic obstructive pulmonary disease (COPD), or pulmonary tuberculosis. In addition, when the pulmonary disease accompanied by inflammation is pneumonia, the type of pneumonia is not particularly limited, and may be, for example, any of community-acquired pneumonia (CAP), healthcare-associated pneumonia (NHCAP), or hospital-acquired pneumonia (HAP), and may be any of alveolar pneumonia or interstitial pneumonia, and the bacteria or viruses that cause it are not particularly limited. In one embodiment, the pulmonary disease accompanied by inflammation may be at least one selected from the group consisting of interstitial pneumonia, lung cancer, and chronic obstructive pulmonary disease.
一実施形態において、データ収集方法、診断方法又は診断補助方法は、さらに、生体試料中のヒト免疫グロブリンＧを測定すること及び前記生体試料中のヒト免疫グロブリンＧに占めるコアフコシル化ヒト免疫グロブリンＧ及び／又はアコアフコシル化ヒト免疫グロブリンＧの割合を算出することを備えてもよい。 In one embodiment, the data collection method, diagnostic method, or diagnostic support method may further comprise measuring human immunoglobulin G in a biological sample and calculating the proportion of core-fucosylated human immunoglobulin G and/or aco-fucosylated human immunoglobulin G in the human immunoglobulin G in the biological sample.
ヒト免疫グロブリンＧを測定する方法は、ヒト免疫グロブリンＧ量を得ることができる方法であれば特に限定されず、例えば、抗ヒト免疫グロブリンＧ抗体又はその標識体を用いた、酵素結合免疫吸着法（ＥＬＩＳＡ法）等の酵素免疫測定法（ＥＩＡ法）、蛍光抗体法又はイムノクロマトグラフィー法等であってもよい。 The method for measuring human immunoglobulin G is not particularly limited as long as it is a method that can obtain the amount of human immunoglobulin G, and may be, for example, an enzyme immunoassay (EIA method) such as an enzyme-linked immunosorbent assay (ELISA method) using an anti-human immunoglobulin G antibody or a labeled form thereof, a fluorescent antibody method, or an immunochromatography method.
本開示に係る抗ヒト免疫グロブリンＧ抗体は、コアフコシル化ヒトＩｇＧ及びアコアフコシル化ヒトＩｇＧの両方に結合する抗体であれば、その動物種の由来等は特に限定されない。抗ヒト免疫グロブリンＧ抗体及びその標識体は、例えば市販のものを利用することができる。 The anti-human immunoglobulin G antibody according to the present disclosure is not particularly limited in terms of the animal species of origin, so long as it is an antibody that binds to both core-fucosylated human IgG and aco-fucosylated human IgG. The anti-human immunoglobulin G antibody and its labeled form may be, for example, a commercially available product.
本開示における「ヒト免疫グロブリンＧ量」は、ヒト免疫グロブリンＧ濃度の他、測定方法に応じて、単位体積当たりのヒト免疫グロブリンＧの存在量に基づくシグナル強度（例えば吸光度）としても定量化され表示され得る。すなわち、一実施形態におけるヒト免疫グロブリンＧに占めるコアフコシル化ヒト免疫グロブリンＧ及びアコアフコシル化ヒト免疫グロブリンＧの割合は、ヒト免疫グロブリンＧにおけるシグナル強度に対するコアフコシル化ヒト免疫グロブリンＧ及びアコアフコシル化ヒト免疫グロブリンＧにおけるシグナル強度の割合としても表示され得る。 In the present disclosure, the "amount of human immunoglobulin G" can be quantified and displayed not only as the concentration of human immunoglobulin G, but also as a signal intensity (e.g., absorbance) based on the amount of human immunoglobulin G present per unit volume depending on the measurement method. That is, the proportion of core-fucosylated human immunoglobulin G and aco-fucosylated human immunoglobulin G in human immunoglobulin G in one embodiment can also be displayed as the ratio of the signal intensity of core-fucosylated human immunoglobulin G and aco-fucosylated human immunoglobulin G to the signal intensity of human immunoglobulin G.
ヒト免疫グロブリンＧ濃度は、試料の単位量当たりのヒト免疫グロブリンＧの存在量を意味し、典型的には試料の単位体積当たりのヒト免疫グロブリンＧの質量又はモル数として表される。また、ヒト免疫グロブリンＧ濃度は、必ずしも試料の単位体積当たりのヒト免疫グロブリンＧ量として定量化されるとは限らず、例えば試料の単位重量当たりのヒト免疫グロブリンＧ量でもあり得る。ヒト免疫グロブリンＧ濃度は、例えば、既知濃度のヒト免疫グロブリンＧを用いて作成されたヒト免疫グロブリンＧと測定値との関係を表す検量線を作成すること、及び作成された検量線と試料の測定値とから試料中のヒト免疫グロブリンＧ濃度を決定すること等により算出することができる。 Human immunoglobulin G concentration means the amount of human immunoglobulin G present per unit amount of sample, and is typically expressed as the mass or moles of human immunoglobulin G per unit volume of sample. Furthermore, human immunoglobulin G concentration is not necessarily quantified as the amount of human immunoglobulin G per unit volume of sample, but may be, for example, the amount of human immunoglobulin G per unit weight of sample. Human immunoglobulin G concentration can be calculated, for example, by creating a calibration curve showing the relationship between human immunoglobulin G and the measured value, which is created using human immunoglobulin G of known concentration, and determining the human immunoglobulin G concentration in the sample from the created calibration curve and the measured value of the sample.
一実施形態において、データ収集方法、診断方法又は診断補助方法は、上述した生体試料中のヒト免疫グロブリンＧに占めるコアフコシル化ヒト免疫グロブリンＧの割合を算出することに加えて、さらに、得られたコアフコシル化ヒト免疫グロブリンＧの測定値が基準値以下である場合には、被験者が炎症を伴う肺疾患に罹患していると判定し、上記測定値が基準値より大きい場合には、被験者が炎症を伴う肺疾患に罹患していないと判定すること、を備えてもよい。 In one embodiment, the data collection method, diagnostic method, or diagnostic support method may, in addition to calculating the proportion of core fucosylated human immunoglobulin G in the human immunoglobulin G in the biological sample described above, further comprise determining that the subject is suffering from a pulmonary disease accompanied by inflammation if the measured value of the obtained core fucosylated human immunoglobulin G is equal to or lower than a reference value, and determining that the subject is not suffering from a pulmonary disease accompanied by inflammation if the measured value is greater than the reference value.
一実施形態において、データ収集方法、診断方法又は診断補助方法は、上述した生体試料中のヒト免疫グロブリンＧに占めるアコアフコシル化ヒト免疫グロブリンＧの割合を算出することに加えて、さらに、得られたアコアフコシル化ヒト免疫グロブリンＧの割合が基準値以上である場合には、被験者が炎症を伴う肺疾患に罹患していると判定し、上記測定値が基準値より小さい場合には、被験者が炎症を伴う肺疾患に罹患していないと判定すること、を備えてもよい。 In one embodiment, the data collection method, diagnostic method, or diagnostic support method may, in addition to calculating the proportion of aco-fucosylated human immunoglobulin G in the human immunoglobulin G in the biological sample described above, further comprise determining that the subject is suffering from a pulmonary disease accompanied by inflammation if the obtained proportion of aco-fucosylated human immunoglobulin G is equal to or greater than a reference value, and determining that the subject is not suffering from a pulmonary disease accompanied by inflammation if the measured value is less than the reference value.
本開示に係る、データ収集方法、診断方法又は診断補助方法は、例えば、被験者の生体試料中のコアフコシル化ヒト免疫グロブリンＧの測定、又は、コアフコシル化ヒト免疫グロブリンＧ及びヒト免疫グロブリンＧ及びの測定により得られる、コアフコシル化ヒト免疫グロブリンＧ量、又は、ヒト免疫グロブリンＧに占めるコアフコシル化ヒト免疫グロブリンＧ若しくはアコアフコシル化ヒト免疫グロブリンＧの割合と、基準値と比較することで判定を行う。すなわち、本開示に係る、データ収集方法又は診断補助方法は、例えば、コアフコシル化ヒト免疫グロブリンＧ量、又は、ヒト免疫グロブリンＧに占めるコアフコシル化ヒト免疫グロブリンＧの割合が基準値と比べて低下している場合に、被験者が炎症を伴う肺疾患に罹患している可能性が高い、と判定し、上記測定値が基準値と比べて上昇している場合には、被験者が炎症を伴う肺疾患に罹患していない可能性が高いと判定する。また、本開示に係る、データ収集方法又は診断補助方法は、例えば、ヒト免疫グロブリンＧに占めるアコアフコシル化ヒト免疫グロブリンＧの割合が基準値と比べて上昇している場合に、被験者が炎症を伴う肺疾患に罹患している可能性が高い、と判定し、上記測定値が基準値と比べて低下している場合には、被験者が炎症を伴う肺疾患に罹患していない可能性が高いと判定する。また、本開示に係る診断方法は、例えば、コアフコシル化ヒト免疫グロブリンＧ量、又は、ヒト免疫グロブリンＧに占めるコアフコシル化ヒト免疫グロブリンＧ若しくはアコアフコシル化ヒト免疫グロブリンＧの割合が基準値と比べて低下している場合に、被験者が炎症を伴う肺疾患に罹患している、と判定し、上記測定値が基準値と比べて上昇している場合には、被験者が炎症を伴う肺疾患に罹患していない、と判定する。また、本開示に係る診断方法は、例えば、ヒト免疫グロブリンＧに占めるアコアフコシル化ヒト免疫グロブリンＧの割合が基準値と比べて上昇している場合に、被験者が炎症を伴う肺疾患に罹患している、と判定し、上記測定値が基準値と比べて低下している場合には、被験者が炎症を伴う肺疾患に罹患していない、と判定する。 The data collection method, diagnostic method, or diagnostic support method according to the present disclosure, for example, measures core fucosylated human immunoglobulin G in a biological sample of a subject, or measures core fucosylated human immunoglobulin G and human immunoglobulin G, and compares the amount of core fucosylated human immunoglobulin G or the proportion of core fucosylated human immunoglobulin G or acoa fucosylated human immunoglobulin G in human immunoglobulin G obtained by the measurement, with a reference value to make a judgment. That is, the data collection method or diagnostic support method according to the present disclosure, for example, determines that the subject is highly likely to suffer from a pulmonary disease accompanied by inflammation when the amount of core fucosylated human immunoglobulin G or the proportion of core fucosylated human immunoglobulin G in human immunoglobulin G is decreased compared to the reference value, and determines that the subject is highly likely not to suffer from a pulmonary disease accompanied by inflammation when the measured value is increased compared to the reference value. In addition, the data collection method or diagnostic support method according to the present disclosure, for example, judges that the subject is highly likely to suffer from a pulmonary disease accompanied by inflammation when the ratio of aco-fucosylated human immunoglobulin G in human immunoglobulin G is increased compared to a reference value, and judges that the subject is highly likely not to suffer from a pulmonary disease accompanied by inflammation when the above measured value is decreased compared to the reference value. In addition, the diagnostic method according to the present disclosure, for example, judges that the subject is suffering from a pulmonary disease accompanied by inflammation when the amount of core-fucosylated human immunoglobulin G or the ratio of core-fucosylated human immunoglobulin G or aco-fucosylated human immunoglobulin G in human immunoglobulin G is decreased compared to a reference value, and judges that the subject is not suffering from a pulmonary disease accompanied by inflammation when the above measured value is increased compared to the reference value. Furthermore, the diagnostic method according to the present disclosure determines, for example, that a subject is suffering from a pulmonary disease accompanied by inflammation when the ratio of acoafucosylated human immunoglobulin G in human immunoglobulin G is elevated compared to a reference value, and determines that a subject is not suffering from a pulmonary disease accompanied by inflammation when the above measured value is decreased compared to the reference value.
本開示のデータ収集方法、診断方法又は診断補助方法に係る基準値は、健常人及び炎症を伴う肺疾患に罹患していると確定診断された患者におけるコアフコシル化ヒト免疫グロブリンＧ量、又は、ヒト免疫グロブリンＧに占めるコアフコシル化ヒト免疫グロブリンＧ若しくはアコアフコシル化ヒト免疫グロブリンＧの割合を参照することにより定めることができる。 The reference value for the data collection method, diagnostic method, or diagnostic support method of the present disclosure can be determined by referring to the amount of core-fucosylated human immunoglobulin G in healthy individuals and patients who have been definitively diagnosed as suffering from a lung disease accompanied by inflammation, or the proportion of core-fucosylated human immunoglobulin G or aco-fucosylated human immunoglobulin G in human immunoglobulin G.
本開示の他の一側面は、コアフコシル化ヒト免疫グロブリンＧに結合し、かつ、アコアフコシル化ヒト免疫グロブリンＧには結合しない抗コアフコシル化ヒトグロブリンＧ抗体又はその抗体断片が結合したラテックス粒子を含む、炎症を伴う肺疾患の診断薬である。 Another aspect of the present disclosure is a diagnostic agent for pulmonary diseases accompanied by inflammation, comprising latex particles to which an anti-core fucosylated human immunoglobulin G antibody or an antibody fragment thereof is bound, the anti-core fucosylated human immunoglobulin G antibody or an antibody fragment thereof that binds to core fucosylated human immunoglobulin G and does not bind to acofucosylated human immunoglobulin G.
本開示の他の一側面は、コアフコシル化ヒト免疫グロブリンＧに結合し、かつ、アコアフコシル化ヒト免疫グロブリンＧには結合しない抗コアフコシル化ヒトグロブリンＧ抗体又はその抗体断片が結合したラテックス粒子、及び、抗ヒト免疫グロブリンＧ抗体を含む、本開示の一側面に係るデータ収集方法、診断方法又は診断補助方法を行うための、キットである。 Another aspect of the present disclosure is a kit for carrying out a data collection method, a diagnostic method, or a diagnostic support method according to an aspect of the present disclosure, comprising latex particles to which an anti-core fucosylated human immunoglobulin G antibody or an antibody fragment thereof that binds to core fucosylated human immunoglobulin G but does not bind to aco-fucosylated human immunoglobulin G is bound, and an anti-human immunoglobulin G antibody.
本開示の他の一側面は、コアフコシル化ヒト免疫グロブリンＧに結合し、かつ、アコアフコシル化ヒト免疫グロブリンＧには結合しない抗コアフコシル化ヒトグロブリンＧ抗体又はその抗体断片が結合したラテックス粒子、及び、コアフコシル化ヒトＩｇＧの測定値が基準値以下である場合には、被験者が、炎症を伴う肺疾患に罹患していると判定し、コアフコシル化ヒトＩｇＧの測定値が基準値より大きい場合には、被験者が、炎症を伴う肺疾患に罹患していないという判定する、という基準に従って判定を行う旨が説明された添付文書を含む、本開示の一側面に係るデータ収集方法、診断方法又は診断補助方法を行うための、キットである。 Another aspect of the present disclosure is a kit for carrying out the data collection method, diagnostic method, or diagnostic assistance method according to one aspect of the present disclosure, comprising latex particles bound to an anti-core fucosylated human globulin G antibody or an antibody fragment thereof that binds to core fucosylated human immunoglobulin G but not to aco-fucosylated human immunoglobulin G, and a package insert that explains that the determination is made according to the following criteria: if the measured value of core fucosylated human IgG is equal to or less than a reference value, the subject is determined to have a pulmonary disease accompanied by inflammation, and if the measured value of core fucosylated human IgG is greater than the reference value, the subject is determined to not have a pulmonary disease accompanied by inflammation.
本開示の一側面に係る診断薬及びキットにおける抗コアフコシル化ヒトＩｇＧ選択的抗体及びその抗体断片が結合したラテックス粒子は、上述の本開示の一側面に係るラテックス凝集法によって試料中のコアフコシル化ヒトＩｇＧを測定する方法において説明したものを用いることができ、また上述した方法により製造することができる。また、抗ヒト免疫グロブリンＧ抗体は、上述した方法により取得することができる。また、本開示の一側面に係る診断薬及びキットは、例えば上述の本開示の一側面に係るデータ収集方法、診断方法及び診断補助方法において説明した方法により使用することができる。 The latex particles to which the anti-core fucosylated human IgG selective antibody and its antibody fragment are bound in the diagnostic agent and kit according to one aspect of the present disclosure can be those described in the method for measuring core fucosylated human IgG in a sample by the latex agglutination method according to one aspect of the present disclosure above, and can be produced by the method described above. In addition, the anti-human immunoglobulin G antibody can be obtained by the method described above. In addition, the diagnostic agent and kit according to one aspect of the present disclosure can be used, for example, by the methods described in the data collection method, diagnostic method, and diagnostic support method according to one aspect of the present disclosure above.
以下、本開示を実施例により、更に詳細に説明するが、これらは本開示の範囲を何ら制限するものではない。なお、以下の実施例においては、次のメーカーの試薬類を使用した。 The present disclosure will be explained in more detail below with reference to examples, but these are not intended to limit the scope of the present disclosure in any way. In the following examples, reagents from the following manufacturers were used.
カルボキシル基修飾ポリスチレンラテックス（Ｃ２００：平均粒径２００ｎｍ）（藤倉化成社製）、Ｗａｔｅｒ Ｓｏｌｕｂｌｅ Ｃａｒｂｏｄｉｉｍｉｄｅ（ＷＳＣ）（同仁化学研究所社製）、２－［４－（２－ヒドロキシエチル）－１－ピペラジニル］エタンスルホン酸（ＨＥＰＥＳ）（同仁化学研究所社製）、リン酸水素二ナトリウム（無水）（関東化学株式会社製）、リン酸二水素ナトリウム（無水）（関東化学株式会社製）、塩化ナトリウム（富士フイルム和光純薬株式会社製）、ポリオキシエチレン（２０）ソルビタンモノラウレート（以下、Ｔｗｅｅｎ２０（登録商標）と記す）（富士フイルム和光純薬社製）、ウシ血清アルブミン（ＢＳＡ）（シグマアルドリッチ社製）、抗コアフコシル化ヒトＩｇＧ選択的抗体（受託番号ＮＩＴＥ ＢＰ－０２３４２として寄託されているハイブリドーマにより産生される抗体）、抗ヒトＩｇＧポリクローナル抗体（サーモフィッシャーサイエンティフィック社製）、ペルオキシダーゼ標識抗ヒトＩｇＧ抗体溶液（ＧＥヘルスケア・ジャパン社製）、Ｈｕｍａｎ ＩｇＧ ＨＲＰ－ｌｉｎｋｅｄ ｗｈｏｌｅ Ａｂ ｓｈｅｅｐ（グローバルライフサイエンステクノロジーズジャパン社製）を使用した。 Carboxyl group-modified polystyrene latex (C200: average particle size 200 nm) (manufactured by Fujikura Chemical Industries, Ltd.), Water Soluble Carbodiimide (WSC) (manufactured by Dojindo Laboratories, Ltd.), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) (manufactured by Dojindo Laboratories, Ltd.), disodium hydrogen phosphate (anhydrous) (manufactured by Kanto Chemical Co., Ltd.), sodium dihydrogen phosphate (anhydrous) (manufactured by Kanto Chemical Co., Ltd.), sodium chloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), polyoxyethylene (20) sorbitan monolaurate (hereinafter referred to as Tween 20 (registered trademark)) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), bovine serum albumin (BSA) (manufactured by Sigma-Aldrich Co., Ltd.), anti-core fucosylated human IgG selective antibody (accession number NITE Antibodies produced by the hybridoma deposited under the designation BP-02342), anti-human IgG polyclonal antibody (manufactured by Thermo Fisher Scientific), peroxidase-labeled anti-human IgG antibody solution (manufactured by GE Healthcare Japan), and Human IgG HRP-linked whole Ab sheep (manufactured by Global Life Science Technologies Japan) were used.
［参考例１：肺疾患患者における、全ヒトＩｇＧ量に占めるコアフコシル化ヒトＩｇＧ量の割合の測定］
まず、ラテックス凝集法によって試料中のコアフコシル化ヒトＩｇＧを測定する前に、実施例においてコアフコシル化ヒトＩｇＧを測定する試料として用いる健常人から採取した血清（９検体）、肺がん患者から採取した血清（２９検体）、ＣＯＰＤ患者から採取した血清（３１検体）及び間質性肺炎患者から採取した血清（１５検体）について、炎症を伴う肺疾患の罹患により血清中のコアフコシル化ヒトＩｇＧ量の変動が見られるかをＥＬＩＳＡ法により検討した。
[Reference Example 1: Measurement of the ratio of core fucosylated human IgG to total human IgG in patients with lung disease]
First, before measuring core-fucosylated human IgG in samples by latex agglutination, sera collected from healthy individuals (9 samples), sera collected from lung cancer patients (29 samples), sera collected from COPD patients (31 samples), and sera collected from interstitial pneumonia patients (15 samples) used as samples for measuring core-fucosylated human IgG in the Examples were examined by ELISA to determine whether the amount of core-fucosylated human IgG in serum was altered due to the presence of pulmonary diseases accompanied by inflammation.
［実験方法１：ＥＬＩＳＡ法によるコアフコシル化ヒトＩｇＧの測定］
（１－１）抗コアフコシル化ヒトＩｇＧ選択的抗体固相化プレートの作製
９６ウェルのＥＩＡ用プレート（住友ベークライト社製）に、１００ｍｍｏｌ／Ｌ 炭酸ナトリウム緩衝液（ｐＨ９．６）で５μｇ／ｍＬに希釈した抗コアフコシル化ヒトＩｇＧ選択的抗体を１００μＬ／ウェルずつ分注し、４℃で１６時間静置して抗体をプレートに固相した。抗体を含む緩衝液を除去後、ブロッキング溶液［５％ ＢＳＡ及び１５０ｍｍｏｌ／Ｌ 塩化ナトリウムを含有する１０ｍｍｏｌ／Ｌ リン酸緩衝液（ｐＨ７．４）］を分注し、２５℃で１時間静置してプレートをブロッキングした。ブロッキング溶液を除去後、コアフコシル化ヒトＩｇＧの測定に使用した。
[Experimental method 1: Measurement of core fucosylated human IgG by ELISA method]
(1-1) Preparation of anti-core fucosylated human IgG selective antibody immobilized plate 100 μL/well of anti-core fucosylated human IgG selective antibody diluted to 5 μg/mL with 100 mmol/L sodium carbonate buffer (pH 9.6) was dispensed and allowed to stand at 4 ° C for 16 hours to immobilize the antibody on the plate. After removing the buffer containing the antibody, blocking solution [10 mmol/L phosphate buffer (pH 7.4) containing 5% BSA and 150 mmol/L sodium chloride] was dispensed and allowed to stand at 25 ° C for 1 hour to block the plate. After removing the blocking solution, the plate was used for measuring core fucosylated human IgG.
（１－２）検量線の作成
（１－１）で調製した抗コアフコシル化ヒトＩｇＧ選択的抗体固相化プレートに、コアフコシル化ヒトＩｇＧ濃度を値付けされたヒトＩｇＧ（既知濃度のヒトＩｇＧに、健常者由来のヒトＩｇＧにおけるコアフコシル化率を乗じて、コアフコシル化ヒトＩｇＧ濃度を算出した）をＰＢＳで希釈した標準試料を１００μＬ／ウェルずつ分注した。２５℃で２時間静置した後、検体希釈液を除去し、洗浄液（０．００５％ Ｔｗｅｅｎ２０を含むＰＢＳ溶液）で５回ウェルを洗浄した。次に、ペルオキシダーゼ標識抗ヒトＩｇＧ抗体溶液をＰＢＳで２，０００倍に希釈し、この溶液（以下では「標識抗体溶液」とも記載する。）を１００μＬ／ウェルずつ分注した。２５℃で１時間静置した後、標識抗体溶液を除去し、洗浄液で５回ウェルを洗浄した。そこに、基質液［０．２６ｍｇ／ｍＬ ｏ－フェニレンジアミン及び０．００９％ 過酸化水素を含む１００ｍｍｏｌ／Ｌ クエン酸緩衝液（ｐＨ ５．０）］を添加し、２５℃で１０分間静置（暗所）した後、プレートリーダーを用いて各ウェルの吸光度を主波長４９２ｎｍで測定した。上記結果より、コアフコシル化ヒトＩｇＧ濃度と吸光度との関係を表す検量線を作成した。
(1-2) Preparation of a calibration curve On the anti-core fucosylated human IgG selective antibody solid-phase plate prepared in (1-1), human IgG with a valued core fucosylated human IgG concentration (the core fucosylated human IgG concentration was calculated by multiplying the known concentration of human IgG by the core fucosylation rate in human IgG derived from a healthy subject) was diluted with PBS to prepare a standard sample, which was dispensed at 100 μL/well. After standing at 25 ° C. for 2 hours, the sample dilution solution was removed, and the wells were washed five times with a washing solution (PBS solution containing 0.005% Tween 20). Next, the peroxidase-labeled anti-human IgG antibody solution was diluted 2,000 times with PBS, and this solution (hereinafter also referred to as the "labeled antibody solution") was dispensed at 100 μL/well. After standing at 25 ° C. for 1 hour, the labeled antibody solution was removed, and the wells were washed five times with a washing solution. A substrate solution [100 mmol/L citrate buffer (pH 5.0) containing 0.26 mg/mL o-phenylenediamine and 0.009% hydrogen peroxide] was added thereto, and the plate was left to stand (in the dark) for 10 minutes at 25° C., after which the absorbance of each well was measured at a dominant wavelength of 492 nm using a plate reader. From the above results, a calibration curve showing the relationship between the core fucosylated human IgG concentration and absorbance was created.
（１－３）ＥＩＡ用プレートを用いたコアフコシル化ヒトＩｇＧの測定
上記（１－２）の標準試料の代わりに、ＰＢＳで２００倍に希釈したヒト血清検体を用いる以外は、（１－２）と同様の方法により、当該ヒト血清検体に対する吸光度を測定した。各検体の測定で得られた吸光度と、（１－２）で作成した検量線とから、ヒト血清検体中のコアフコシル化ヒトＩｇＧ濃度を決定した。
(1-3) Measurement of core-fucosylated human IgG using EIA plate The absorbance of the human serum specimen was measured in the same manner as in (1-2) except that a human serum specimen diluted 200-fold with PBS was used instead of the standard sample in (1-2) above. The concentration of core-fucosylated human IgG in the human serum specimen was determined from the absorbance obtained by measuring each specimen and the calibration curve prepared in (1-2).
［実験方法２：全ヒトＩｇＧの測定］
（２－１）検量線の作成
９６ウェルのＥＩＡ用プレート（住友ベークライト社製）に、既知濃度のヒトＩｇＧをＰＢＳで希釈した標準試料を１００μＬ／ウェルずつ分注し、４℃で１６時間静置した。洗浄液（０．００５％ Ｔｗｅｅｎ２０を含むＰＢＳ溶液）で３回洗浄した後、ブロッキング液［５％ ＢＳＡ、１５０ｍｍｏｌ／Ｌ 塩化ナトリウムを含有する、１０ｍｍｏｌ／Ｌ リン酸緩衝液（ｐＨ７．４）］を用いてプレートをブロッキングした（２５℃で１時間）。洗浄液で３回洗浄した後、Ｈｕｍａｎ ＩｇＧ， ＨＲＰ－ｌｉｎｋｅｄ ｗｈｏｌｅ Ａｂ ｓｈｅｅｐを希釈液（１％ ＢＳＡを含むＰＢＳ溶液）で２，０００倍に希釈し、この標識抗体溶液を１００μＬ／ウェルずつ分注した。２５℃で１時間静置した後、標識抗体溶液を除去し、洗浄液で５回ウェルを洗浄した。基質液［０．２６ｍｇ／ｍＬ ｏ－フェニレンジアミン、０．００９％ 過酸化水素を含む、１００ｍｍｏｌ／Ｌ クエン酸緩衝液（ｐＨ ５．６）］を添加し、２５℃で１０分間静置（暗所）した後、プレートリーダーを用いて各ウェルの吸光度を主波長４９２ｎｍで測定した。上記結果より、ヒトＩｇＧ濃度と吸光度との関係を表す検量線を作成した。
[Experimental method 2: Measurement of total human IgG]
(2-1) Preparation of a calibration curve A standard sample prepared by diluting human IgG of known concentration with PBS was dispensed into a 96-well EIA plate (manufactured by Sumitomo Bakelite Co., Ltd.) at 100 μL/well and allowed to stand at 4° C. for 16 hours. After washing three times with a washing solution (PBS solution containing 0.005% Tween 20), the plate was blocked (1 hour at 25° C.) using a blocking solution [10 mmol/L phosphate buffer (pH 7.4) containing 5% BSA and 150 mmol/L sodium chloride]. After washing three times with a washing solution, Human IgG, HRP-linked whole Ab sheep was diluted 2,000-fold with a diluent (PBS solution containing 1% BSA), and this labeled antibody solution was dispensed at 100 μL/well. After standing at 25°C for 1 hour, the labeled antibody solution was removed and the wells were washed five times with washing solution. Substrate solution [100 mmol/L citrate buffer (pH 5.6) containing 0.26 mg/mL o-phenylenediamine and 0.009% hydrogen peroxide] was added and the wells were left to stand at 25°C for 10 minutes (in the dark), after which the absorbance of each well was measured at a dominant wavelength of 492 nm using a plate reader. From the above results, a calibration curve showing the relationship between human IgG concentration and absorbance was created.
（２－２）ヒト血清検体の測定とヒトＩｇＧ濃度の決定
上記（２－１）の標準試料の代わりに、ＰＢＳで４００，０００倍に希釈したヒト血清検体を用いる以外は、（２－１）と同様の方法により、当該ヒト血清検体に対する吸光度を測定した。各検体の測定で得られた吸光度と、（２－１）で作成した検量線とから、ヒト血清検体中のヒトＩｇＧ濃度を決定した。
(2-2) Measurement of human serum samples and determination of human IgG concentration The absorbance of the human serum samples was measured in the same manner as in (2-1) except that a human serum sample diluted 400,000 times with PBS was used instead of the standard sample in (2-1) above. The human IgG concentration in the human serum samples was determined from the absorbance obtained by measuring each sample and the calibration curve prepared in (2-1).
［肺疾患患者における、全ヒトＩｇＧ量に占めるコアフコシル化ヒトＩｇＧ量の割合の算出］
健常人から採取した血清（９検体）、肺がん患者から採取した血清（２９検体）、ＣＯＰＤ患者から採取した血清（３１検体）及び間質性肺炎患者から採取した血清（１５検体）について、実験方法１に従って決定したコアフコシル化ヒトＩｇＧ濃度及び実験方法２に従って決定したヒトＩｇＧ濃度を用いて、以下の式に従って、全ヒトＩｇＧに占めるコアフコシル化ヒトＩｇＧの割合（以下、「コアフコシル化ヒトＩｇＧ／全ヒトＩｇＧ比」とも記載する。）を算出した。
コアフコシル化ヒトＩｇＧ／全ヒトＩｇＧ比＝（コアフコシル化ヒトＩｇＧ濃度）÷（全ヒトＩｇＧ濃度）
[Calculation of the ratio of core fucosylated human IgG to total human IgG in patients with lung disease]
For sera collected from healthy individuals (9 samples), sera collected from lung cancer patients (29 samples), sera collected from COPD patients (31 samples), and sera collected from interstitial pneumonia patients (15 samples), the ratio of core fucosylated human IgG to total human IgG (hereinafter also referred to as "core fucosylated human IgG/total human IgG ratio") was calculated according to the following formula, using the core fucosylated human IgG concentrations determined according to Experimental Method 1 and the human IgG concentrations determined according to Experimental Method 2.
Core fucosylated human IgG/total human IgG ratio=(core fucosylated human IgG concentration)÷(total human IgG concentration)
各群のヒト血清検体について、算出された割合を図２に示す。また、多群間解析（ｏｎｅ－ｗａｙ ＡＮＯＶＡ）の解析結果を表１に示し、対照群（健常人）に対する多重比較（Ｄｕｎｎｅｔｔ’ｓ ｍｕｌｔｉｐｌｅ ｃｏｍｐａｒｉｓｏｎｓ ｔｅｓｔ）の解析結果を表２に示す。なお、p値が０．０００１未満の場合は、「＜０．０００１」と記載した。 The calculated ratios for the human serum samples from each group are shown in Figure 2. The results of the one-way ANOVA are shown in Table 1, and the results of the Dunnett's multiple comparisons test against the control group (healthy individuals) are shown in Table 2. Note that when the p-value was less than 0.0001, it was recorded as "<0.0001."
図２、表１及び表２の結果より、対照群（健常人）におけるコアフコシル化ヒトＩｇＧ／全ヒトＩｇＧ比と比較して、肺がん患者、ＣＯＰＤ患者及び間質性肺炎患者におけるコアフコシル化ヒトＩｇＧ／全ヒトＩｇＧ比が統計学的に有意に低下していた。 The results of Figure 2, Tables 1 and 2 show that the core-fucosylated human IgG/total human IgG ratio was statistically significantly lower in lung cancer patients, COPD patients and interstitial pneumonia patients compared to the core-fucosylated human IgG/total human IgG ratio in the control group (healthy individuals).
［実施例１：抗コアフコシル化ヒトＩｇＧ選択的抗体が結合したラテックス粒子溶液の作製］
カルボキシル基修飾ポリスチレンラテックス（Ｃ２００）を含むラテックス懸濁液（１０ｗｔ％）を、１０ｍｍｏｌ／Ｌ ＨＥＰＥＳ水溶液（ｐＨ ７．０）で２０倍に希釈し、０．５ｗｔ％ ラテックス懸濁液を調製した。０．５ｗｔ％ カルボキシル基修飾ポリスチレンラテックス（Ｃ２００）の懸濁液（１ｍＬ）に、１ｍｇ／ｍＬ Ｗａｔｅｒ Ｓｏｌｕｂｌｅ Ｃａｒｂｏｄｉｉｍｉｄｅ（ＷＳＣ）を含む１０ｍＭ ＨＥＰＥＳ溶液（ｐＨ ７．０）（３００μＬ）を添加し、ローテーション攪拌を行なった（２５℃、２０分間）。ローテーション攪拌後の溶液に、抗コアフコシル化ヒトＩｇＧ選択的抗体（１００μｇ）を加え、ローテーション攪拌を行なった（２５℃、６０分間）。ローテーション攪拌後の溶液に、１０％ Ｔｗｅｅｎ２０溶液（１５μＬ）を添加した後、超音波処理を行った（２５℃、１０分間）。超音波処理した溶液を遠心分離した（２５℃、１５，０００ｒｐｍ、３０分間）後、上清を除去した。上清除去後の沈殿物に、１％ ＢＳＡ溶液（１ｍＬ）を添加し、超音波処理を行った（２５℃、１０分間）。その後、溶液をローテーション攪拌した（２５℃、６０分間）。ローテーション攪拌後の溶液を遠心分離した（２５℃、１５，０００ｒｐｍ、３０分間）後、上清を除去した。上清除去後の沈殿物に、０．１％ ＢＳＡを含む２０ｍｍｏｌ／Ｌ ＨＥＰＥＳ水溶液（ｐＨ ７．０）（１ｍＬ）を添加し、懸濁液を調製した。該懸濁液（２００μＬ）を、０．１％ ＢＳＡを含む２０ｍｍｏｌ／Ｌ ＨＥＰＥＳ水溶液（ｐＨ ７．０）（７５０μＬ）で希釈し、抗コアフコシル化ヒトＩｇＧ選択的抗体が結合したラテックス粒子溶液を調製した。
[Example 1: Preparation of latex particle solution bound to anti-core fucosylated human IgG selective antibody]
A latex suspension (10 wt%) containing carboxyl group-modified polystyrene latex (C200) was diluted 20 times with 10 mmol/L HEPES aqueous solution (pH 7.0) to prepare a 0.5 wt% latex suspension. A 10 mM HEPES solution (pH 7.0) (300 μL) containing 1 mg/mL Water Soluble Carbodiimide (WSC) was added to a suspension (1 mL) of 0.5 wt% carboxyl group-modified polystyrene latex (C200), and rotational stirring was performed (25 ° C, 20 minutes). An anti-core fucosylated human IgG selective antibody (100 μg) was added to the solution after rotational stirring, and rotational stirring was performed (25 ° C, 60 minutes). After the rotational stirring, 10% Tween 20 solution (15 μL) was added to the solution, and then ultrasonic treatment was performed (25° C., 10 minutes). The ultrasonicated solution was centrifuged (25° C., 15,000 rpm, 30 minutes), and the supernatant was removed. After the supernatant was removed, 1% BSA solution (1 mL) was added to the precipitate, and ultrasonic treatment was performed (25° C., 10 minutes). Then, the solution was rotated and stirred (25° C., 60 minutes). After the rotational stirring, the solution was centrifuged (25° C., 15,000 rpm, 30 minutes), and the supernatant was removed. After the supernatant was removed, 20 mmol/L HEPES aqueous solution (pH 7.0) (1 mL) containing 0.1% BSA was added to the precipitate to prepare a suspension. The suspension (200 μL) was diluted with 750 μL of a 20 mmol/L aqueous HEPES solution (pH 7.0) containing 0.1% BSA to prepare a solution of latex particles bound with anti-core fucosylated human IgG selective antibodies.
［実験方法３：ラテックス凝集法による試料中のコアフコシル化ヒトＩｇＧの測定］
反応容器中の、実施例１において調製した抗コアフコシル化ヒトＩｇＧ選択的抗体が結合したラテックス粒子溶液（９５０μＬ）に、ヒト血清（５０μＬ）を添加し、反応液を調製した。当該反応液を転倒混和した後、６６０ｎｍでの吸光度を測定した。当該反応液をローテーション攪拌（２５℃、１６時間）した後、６６０ｎｍでの吸光度を測定した。ローテーション攪拌前後における吸光度の増加量を、各試料における測定値（コアフコシル化ヒトＩｇＧ量）とした。
[Experimental method 3: Measurement of core fucosylated human IgG in samples by latex agglutination method]
Human serum (50 μL) was added to the latex particle solution (950 μL) bound to the anti-core fucosylated human IgG selective antibody prepared in Example 1 in a reaction vessel to prepare a reaction solution. The reaction solution was mixed by inversion, and then the absorbance at 660 nm was measured. The reaction solution was subjected to rotational stirring (25° C., 16 hours), and then the absorbance at 660 nm was measured. The increase in absorbance before and after rotational stirring was taken as the measured value (amount of core fucosylated human IgG) for each sample.
［実施例２：コアフコシル化ヒトＩｇＧをラテックス凝集法で測定した場合と、ＥＬＩＳＡ法で測定した場合の、日差再現性試験］
測定精度を評価する方法の１つとして日差再現性試験がある。日差再現性試験とは、測定日を変えて同一検体を測定したときの測定値のばらつきを評価する試験であり、変動係数（Ｃｏｅｆｆｉｃｉｅｎｔ ｏｆ Ｖａｒｉａｔｉｏｎ：ＣＶ（％）＝（標準偏差）／（平均値）×１００）で評価することができる。変動係数が小さいほど、測定日ごとの測定値のばらつきが小さく、測定対象物を正確に測定できていると評価することができる。そこで、実験方法３（ラテックス凝集法）及び実験方法１（ＥＬＩＳＡ法）に従ってコアフコシル化ヒトＩｇＧを測定した場合の、日差再現性試験の結果に比較した。
[Example 2: Day-to-day reproducibility test when measuring core fucosylated human IgG by latex agglutination method and by ELISA method]
One method for evaluating measurement accuracy is the daily reproducibility test. The daily reproducibility test is a test to evaluate the variation in the measured values when the same sample is measured on different measurement days, and can be evaluated by the coefficient of variation (CV (%) = (standard deviation) / (average value) x 100). The smaller the coefficient of variation, the smaller the variation in the measured values for each measurement day, and the more accurately the measured object can be measured. Therefore, the results were compared with the results of the daily reproducibility test when core fucosylated human IgG was measured according to Experimental Method 3 (latex agglutination method) and Experimental Method 1 (ELISA method).
実験方法３及び１に従って、健常人の血清（検体１）を３日間測定し、コアフコシル化ヒトＩｇＧ量の変動係数を算出した。変動係数（％）を表３に示す。 According to Experimental Methods 3 and 1, serum from a healthy subject (sample 1) was measured for three days, and the coefficient of variation of the amount of core-fucosylated human IgG was calculated. The coefficient of variation (%) is shown in Table 3.
表３の結果より、ラテックス凝集法を用いた日差再現性試験は変動係数が２０％未満となり、ＥＬＩＳＡ法よりも測定値のばらつきを抑制して検体中のコアフコシル化ヒトＩｇＧを測定することができた。一因として、ラテックス凝集法によるコアフコシル化ヒトＩｇＧの測定においては検体の希釈を行う必要がなかったため、希釈誤差が抑制されたことが考えられた。 From the results in Table 3, the coefficient of variation in the day-to-day repeatability test using the latex agglutination method was less than 20%, and it was possible to measure core-fucosylated human IgG in samples with less variability in the measurement values than the ELISA method. One reason for this is thought to be that there was no need to dilute the sample when measuring core-fucosylated human IgG using the latex agglutination method, which reduced dilution errors.
［実施例３：健常人及び間質性肺炎患者由来血清を用いた、コアフコシル化ヒトＩｇＧ測定］
参考例１において検体中のコアフコシル化ヒトＩｇＧ／全ヒトＩｇＧ比の有意な低下が見られた間質性肺炎に関し、ラテックス凝集法又はＥＬＩＳＡ法を用いたコアフコシル化ヒトＩｇＧの測定によって、全ヒトＩｇＧの測定を行うことなくコアフコシル化ヒトＩｇＧ量の変動を評価できるかを検討した。比較のために、参考例１と同様に、ＥＬＩＳＡ法を用いたコアフコシル化ヒトＩｇＧ／全ヒトＩｇＧ比の算出も行った。
[Example 3: Measurement of core fucosylated human IgG using sera from healthy subjects and patients with interstitial pneumonia]
Regarding interstitial pneumonia in which a significant decrease in the core fucosylated human IgG/total human IgG ratio in the samples was observed in Reference Example 1, it was examined whether it is possible to evaluate the change in the amount of core fucosylated human IgG by measuring core fucosylated human IgG using the latex agglutination method or the ELISA method without measuring total human IgG. For comparison, the core fucosylated human IgG/total human IgG ratio was also calculated using the ELISA method in the same manner as in Reference Example 1.
健常人から採取した血清（１３例）と間質性肺炎患者から採取した血清（１５例）について、実験方法３（ラテックス凝集法）及び実験方法１（ＥＬＩＳＡ法）に従って、コアフコシル化ヒトＩｇＧを測定した。また、同じ血清について、実験方法１及び２に従ってコアフコシル化ヒトＩｇＧ及び全ヒトＩｇＧをＥＬＩＳＡ法で測定し、測定値を用いて参考例１と同様の方法でコアフコシル化ヒトＩｇＧ／全ヒトＩｇＧ比を算出した。実験方法３（ラテックス凝集法）によるコアフコシル化ヒトＩｇＧの測定結果を図３に、実験方法１（ＥＬＩＳＡ法）によるコアフコシル化ヒトＩｇＧの測定結果を図４に、実験方法１、実験方法２及び参考例１の方法によるコアフコシル化ヒトＩｇＧ／全ヒトＩｇＧ比の算出結果を図５に、それぞれ示す。 Sera collected from healthy subjects (13 cases) and sera collected from patients with interstitial pneumonia (15 cases) were measured for core fucosylated human IgG according to Experimental Method 3 (latex agglutination) and Experimental Method 1 (ELISA). In addition, for the same sera, core fucosylated human IgG and total human IgG were measured by ELISA according to Experimental Methods 1 and 2, and the core fucosylated human IgG/total human IgG ratio was calculated using the measured values in the same manner as in Reference Example 1. The measurement results of core fucosylated human IgG by Experimental Method 3 (latex agglutination) are shown in Figure 3, the measurement results of core fucosylated human IgG by Experimental Method 1 (ELISA) are shown in Figure 4, and the calculation results of the core fucosylated human IgG/total human IgG ratio by the methods of Experimental Method 1, Experimental Method 2, and Reference Example 1 are shown in Figure 5.
図４によれば、ＥＬＩＳＡ法を用いたコアフコシル化ヒトＩｇＧの測定においては、健常人と間質性肺炎患者の間に測定値の有意な差は見られなかった。これに対し、図３によれば、ラテックス凝集法を用いたコアフコシル化ヒトＩｇＧの測定においては、健常人と間質性肺炎患者の間に測定値の有意な差が見られ、図５に示したコアフコシル化ヒトＩｇＧ／全ヒトＩｇＧ比を指標とした評価の結果により近い結果となった。以上から、ラテックス凝集法を用いて血清中のコアフコシル化ヒトＩｇＧ量のみを測定することで、血清中の全ヒトＩｇＧ量を測定することなく、統計的有意差をもって罹患の有無に応じたコアフコシル化ヒトＩｇＧ量の変動を評価することができた。 As shown in Figure 4, no significant difference in the measured values was observed between healthy subjects and patients with interstitial pneumonia when core fucosylated human IgG was measured using the ELISA method. In contrast, as shown in Figure 3, a significant difference in the measured values was observed between healthy subjects and patients with interstitial pneumonia when core fucosylated human IgG was measured using the latex agglutination method, and the results were closer to the results of the evaluation using the core fucosylated human IgG/total human IgG ratio as an index shown in Figure 5. From the above, by measuring only the amount of core fucosylated human IgG in serum using the latex agglutination method, it was possible to evaluate the fluctuation in the amount of core fucosylated human IgG depending on the presence or absence of disease with a statistically significant difference, without measuring the amount of total human IgG in serum.
［実施例４：ラテックス凝集法による、健常人、肺がん患者、ＣＯＰＤ患者及び間質性肺炎患者由来血清中のコアフコシル化ヒトＩｇＧの測定］
参考例１で評価したヒト血清検体について、実験方法３（ラテックス凝集法）に従って、コアフコシル化ヒトＩｇＧを測定した。実験方法３（ラテックス凝集法）によるコアフコシル化ヒトＩｇＧの測定結果を図６に示す。また、多群間解析（ｏｎｅ－ｗａｙ ＡＮＯＶＡ）の解析結果を表４に示し、対照群（健常人）に対する多重比較（Ｄｕｎｎｅｔｔ’ｓ ｍｕｌｔｉｐｌｅ ｃｏｍｐａｒｉｓｏｎｓ ｔｅｓｔ）の解析結果を表５に示す。
[Example 4: Measurement of core fucosylated human IgG in sera from healthy subjects, lung cancer patients, COPD patients, and interstitial pneumonia patients by latex agglutination method]
Core fucosylated human IgG was measured for the human serum samples evaluated in Reference Example 1 according to Experimental Method 3 (latex agglutination method). The results of measuring core fucosylated human IgG by Experimental Method 3 (latex agglutination method) are shown in Figure 6. The results of the one-way ANOVA analysis are shown in Table 4, and the results of the Dunnett's multiple comparisons test against the control group (healthy subjects) are shown in Table 5.
図６、表４及び表５の結果より、対照群（健常人）におけるコアフコシル化ヒトＩｇＧ量と比較して、肺がん患者、ＣＯＰＤ患者及び間質性肺炎患者におけるコアフコシル化ヒトＩｇＧ量が統計学的に有意に低下していた。
以上の結果より、ラテックス凝集法を用いて、炎症を伴う肺疾患患者におけるコアフコシル化ヒトＩｇＧ量を測定することにより、健常人と、炎症を伴う肺疾患患者との鑑別診断が可能であることが判明した。
The results of Figure 6, Table 4 and Table 5 show that the amount of core-fucosylated human IgG in the lung cancer patients, COPD patients and interstitial pneumonia patients was statistically significantly reduced compared to the amount of core-fucosylated human IgG in the control group (healthy individuals).
From the above results, it was found that by using the latex agglutination method to measure the amount of core-fucosylated human IgG in patients with pulmonary disease accompanied by inflammation, it is possible to make a differential diagnosis between healthy individuals and patients with pulmonary disease accompanied by inflammation.
Claims (21)
前記抗コアフコシル化ヒト免疫グロブリンＧ抗体又はその抗体断片は、コアフコシル化ヒト免疫グロブリンＧに結合し、かつ、アコアフコシル化ヒト免疫グロブリンＧに結合しない抗体又はその抗体断片である、
方法。 A method for measuring core fucosylated human immunoglobulin G in a sample by a latex agglutination method, comprising reacting core fucosylated human immunoglobulin G in the sample with latex particles to which an anti-core fucosylated human immunoglobulin G antibody or an antibody fragment thereof is bound,
The anti-core fucosylated human immunoglobulin G antibody or antibody fragment thereof is an antibody or antibody fragment thereof that binds to core fucosylated human immunoglobulin G and does not bind to a-core fucosylated human immunoglobulin G.
Method.
で表される糖鎖を含むＮ結合型糖鎖である、請求項１に記載の方法。 The N-linked glycan in which fucose is linked to reducing terminal GlcNAc by α1-6 bond, which is contained in the core fucosylated human immunoglobulin G, is represented by the following formula (I):
The method according to claim 1, wherein the N-linked glycan comprises a glycan represented by the formula:
前記抗コアフコシル化ヒト免疫グロブリンＧ抗体又はその抗体断片は、コアフコシル化ヒト免疫グロブリンＧに結合し、かつ、アコアフコシル化ヒト免疫グロブリンＧに結合しない抗体又はその抗体断片である、
試薬。 A reagent for measuring core fucosylated human immunoglobulin G in a sample by a latex agglutination method, comprising latex particles to which an anti-core fucosylated human immunoglobulin G antibody or an antibody fragment thereof is bound,
The anti-core fucosylated human immunoglobulin G antibody or antibody fragment thereof is an antibody or antibody fragment thereof that binds to core fucosylated human immunoglobulin G and does not bind to a-core fucosylated human immunoglobulin G.
reagent.
で表される糖鎖を含むＮ結合型糖鎖である、請求項６に記載の試薬。 The N-linked glycan in which fucose is linked to reducing terminal GlcNAc by α1-6 bond, which is contained in the core fucosylated human immunoglobulin G, is represented by the following formula (I):
The reagent according to claim 6, which is an N-linked glycan comprising a glycan represented by the formula:
前記測定が、コアフコシル化ヒト免疫グロブリンＧに結合し、かつ、アコアフコシル化ヒト免疫グロブリンＧには結合しない抗コアフコシル化ヒトグロブリンＧ抗体又はその抗体断片が結合したラテックス粒子を用いたラテックス凝集法により行われる、
方法。 1. A method for collecting data for determining whether a subject suffers from a lung disease associated with inflammation, comprising measuring core fucosylated human immunoglobulin G in a biological sample from the subject,
The measurement is carried out by a latex agglutination method using latex particles to which an anti-core fucosylated human immunoglobulin G antibody or an antibody fragment thereof that binds to core fucosylated human immunoglobulin G and does not bind to aco-fucosylated human immunoglobulin G is bound.
Method.
前記測定が、コアフコシル化ヒト免疫グロブリンＧに結合し、かつ、アコアフコシル化ヒト免疫グロブリンＧには結合しない抗コアフコシル化ヒトグロブリンＧ抗体又はその抗体断片が結合したラテックス粒子を用いたラテックス凝集法により行われる、
方法。 1. A method for aiding in the diagnosis of a lung disease associated with inflammation, comprising measuring core fucosylated human immunoglobulin G in a biological sample from a subject,
The measurement is carried out by a latex agglutination method using latex particles to which an anti-core fucosylated human immunoglobulin G antibody or an antibody fragment thereof that binds to core fucosylated human immunoglobulin G and does not bind to aco-fucosylated human immunoglobulin G is bound.
Method.
で表される糖鎖を含むＮ結合型糖鎖である、請求項１１又は１２に記載の方法。 The N-linked glycan in which fucose is linked to reducing terminal GlcNAc by α1-6 bond, which is contained in the core fucosylated human immunoglobulin G, is represented by the following formula (I):
The method according to claim 11 or 12, wherein the N-linked glycan comprises a glycan represented by the formula:
で表される糖鎖を含むＮ結合型糖鎖である、請求項１６又は１７に記載の診断薬。 The N-linked glycan in which fucose is linked to reducing terminal GlcNAc by α1-6 bond, which is contained in the core fucosylated human immunoglobulin G, is represented by the following formula (I):
The diagnostic agent according to claim 16 or 17, which is an N-linked glycan comprising a glycan represented by the formula:
抗ヒト免疫グロブリンＧ抗体
を含む、請求項１１又は１２に記載の方法を行うための、キット。 A kit for carrying out the method according to claim 11 or 12, comprising latex particles to which an anti-core fucosylated human immunoglobulin G antibody or an antibody fragment thereof that binds to core fucosylated human immunoglobulin G and does not bind to aco-fucosylated human immunoglobulin G is bound, and an anti-human immunoglobulin G antibody.
で表される糖鎖を含むＮ結合型糖鎖である、請求項１９に記載のキット。 The N-linked glycan in which fucose is linked to reducing terminal GlcNAc by α1-6 bond, which is contained in the core fucosylated human immunoglobulin G, is represented by the following formula (I):
The kit according to claim 19, wherein the N-linked glycan comprises a glycan represented by the formula:
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