JP5747315B2 - Antibody recognizing sugar chain-deficient human IgA1 hinge region and use thereof - Google Patents
Antibody recognizing sugar chain-deficient human IgA1 hinge region and use thereof Download PDFInfo
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Description
本発明はIgA腎症の検査に有用な抗体に関する。詳しくは、糖鎖不全ヒトIgA1ヒンジ部を認識する抗体及び当該抗体を利用したIgA腎症の検査法などに関する。 The present invention relates to an antibody useful for testing for IgA nephropathy. Specifically, the present invention relates to an antibody recognizing a sugar chain-deficient human IgA1 hinge part, a test method for IgA nephropathy using the antibody, and the like.
IgA腎症は我が国では慢性腎炎の約40%を占め、未治療ではその約40%が末期腎不全に陥る。我が国の慢性維持透析患者は増加の一途をたどり2008年には約27万人あまりとなった。そのうち本症を原疾患とした透析患者は推定数万人とされ、本症への治療法・予防法の開発は透析医療費の抑制という医療経済的見地からも、重要課題の一つと考えられる。 IgA nephropathy accounts for about 40% of chronic nephritis in Japan, and about 40% of those suffering from end stage renal failure without treatment. The number of chronic maintenance dialysis patients in Japan continued to increase, reaching about 270,000 in 2008. Among them, the estimated number of dialysis patients with this disease as the primary disease is estimated to be tens of thousands, and the development of treatment and prevention methods for this disease is considered to be one of the important issues from the medical economic viewpoint of reducing dialysis medical expenses. .
現在のところ、IgA腎症の検査は生検のみであり、簡易で精度の高い検査法の確立が望まれている。実際、いくつかの研究グループによって、IgA腎症の検査法を確立する目的の下、精力的な研究が行われている。その一つとして、Moldoveanuら（非特許文献１）及び本発明者らの研究グループ（非特許文献２）は、IgA1とN-アセチルガラクトサミン（以下、「GalNAc」ともいう）を認識するレクチン（helix aspersa:HAA）との結合度を検討し、IgA腎症患者で有意な増加が観察されることを報告した。しかしながら、ROC曲線による解析の結果は、「HAAとの結合度」が本症診断のマーカーとして臨床応用される可能性を否定するものであった。また、臨床病理学的重症度や、扁摘パルス治療と「HAAとの結合度」の間に関連は認めず、疾患の進行度、治療効果の判定指標として有効でないことが示唆された。尚、IgA腎症の検査法（診断法）に関する先行特許文献を以下に開示する（特許文献１〜３）。特許文献１ではIgA1分子間の結合能の差を利用してIgA腎症を診断する方法、特許文献２ではIgA1ヒンジ部に対する抗体を利用してIgA腎症を診断する方法、特許文献３ではIgA1ヒンジ部に結合する糖鎖数を指標としてIgA腎症を診断する方法がそれぞれ提案されている。
At present, the only test for IgA nephropathy is biopsy, and the establishment of a simple and highly accurate test method is desired. In fact, several research groups are actively researching with the goal of establishing a test for IgA nephropathy. As one of them, Moldoveanu et al. (Non-patent Document 1) and our research group (Non-patent Document 2) have reported that a lectin (helix) that recognizes IgA1 and N-acetylgalactosamine (hereinafter also referred to as “GalNAc”). aspersa: HAA) and reported that a significant increase was observed in patients with IgA nephropathy. However, the results of the ROC curve analysis denied the possibility of clinical application of “degree of binding to HAA” as a marker for diagnosis of this disease. In addition, there was no association between the clinicopathological severity or the ablation pulse treatment and the "degree of binding with HAA", suggesting that it is not effective as an indicator of the degree of disease progression or therapeutic effect. In addition, the prior patent document regarding the test | inspection method (diagnosis method) of IgA nephropathy is disclosed below (patent documents 1-3). In
以上の背景の下、本発明は、IgA腎症を簡便に且つ低侵襲的に検査する方法及び当該方法に有用な試薬やキットなどを提供することを課題とする。 In view of the above background, an object of the present invention is to provide a method for simply and minimally inspecting IgA nephropathy, and a reagent or kit useful for the method.
上記課題に鑑みて本発明者らは鋭意検討した。即ち、一般にレクチンよりもエピトープを認識する親和性が格段に高いと考えられている抗体に注目し、IgA腎症の検査に有効な抗体の創出を目指した。具体的には、O結合型糖鎖が高頻度に存在しているといわれる５箇所の部位にGalNAcを付加した、IgA1抗体のヒンジ部に相当する糖ペプチドを合成し、これに対する抗体の作製を試みた。得られた抗体（ポリクローナル抗体）の性能を評価したところ、当該抗体が、ヒンジ部のガラクトース含量の低下した糖鎖不全IgA1を認識することが示された。また、当該抗体の血清IgA1への結合度が健常者や他の腎疾患患者に比べIgA腎症患者群で有意に高いことが示された。さらには、同一血清を用いた比較検討によって、当該抗体の血清IgA1への結合度がHAAの血清IgA1への結合度と高い相関を示す一方で、検出感度は格段に高いことが判明した。このように、取得に成功した抗体がIgA腎症の検査に極めて有用であることが確認された。 In view of the above problems, the present inventors have intensively studied. That is, attention was focused on antibodies that are generally considered to have much higher affinity for recognizing epitopes than lectins, and the aim was to create antibodies that are effective in testing for IgA nephropathy. Specifically, a glycopeptide corresponding to the hinge part of IgA1 antibody, in which GalNAc is added at five sites where O-linked sugar chains are said to exist at high frequency, is synthesized, and an antibody against this is prepared. Tried. When the performance of the obtained antibody (polyclonal antibody) was evaluated, it was shown that the antibody recognizes sugar chain-deficient IgA1 having a reduced galactose content in the hinge region. In addition, it was shown that the degree of binding of the antibody to serum IgA1 was significantly higher in the IgA nephropathy patient group than in healthy subjects and other renal disease patients. Furthermore, a comparative study using the same serum revealed that the binding degree of the antibody to serum IgA1 was highly correlated with the binding degree of HAA to serum IgA1, while the detection sensitivity was remarkably high. As described above, it was confirmed that the successfully obtained antibody is extremely useful for the examination of IgA nephropathy.
更に検討を進めた結果、取得した抗体から、IgA1ヒンジ上のペプチド部分を認識する抗体を分離除去することに成功した。 As a result of further investigation, the antibody that recognizes the peptide portion on the IgA1 hinge was successfully separated and removed from the obtained antibody.
一方、ヒンジ部における上記５箇所の糖鎖結合部位の内、ガラクトースの欠落が最も高頻度で認められ、IgA腎症との関係において重要と考えられる部位を特異的に認識する抗体の作製を試みた。得られた抗体の特性を調べた結果、当該抗体が、先に取得した抗体と同様に、IgA1のヒンジ部に対して高い特異性を示すことが判明した。 On the other hand, of the above five sugar chain binding sites in the hinge region, we tried to produce an antibody that specifically recognizes the site where galactose loss was most frequently observed and considered important in relation to IgA nephropathy. It was. As a result of examining the characteristics of the obtained antibody, it was found that the antibody showed high specificity for the hinge part of IgA1, as with the previously obtained antibody.
以下に示す本発明は主として以上の成果・知見に基づく。
［１］ガラクトース含量の低下した糖鎖不全ヒトIgA1ヒンジ部を特異的に認識する抗体。
［２］シアル酸及びガラクトースを含まない糖鎖が結合したヒトIgA1ヒンジ部を特異的に認識する抗体である、［１］に記載の抗体。
［３］ポリクローナル抗体である、［１］又は［２］に記載の抗体。
［４］糖鎖が結合していないヒトIgA1ヒンジ部を認識する抗体を含まない、［３］に記載の抗体。
［５］以下の配列：VPST(GalNAc)PPT(GalNAc)PS(GalNAc)PS(GalNAc)TPPT(GalNAc)PSPS（配列番号１）（但し、Vはバリンを、Pはプロリンを、Sはセリンを、Tはスレオニンを、括弧内のGalNacは直前のアミノ酸残基に結合したO結合型N-アセチルガラクトサミンをそれぞれ表す）、からなる合成ヒンジ部糖ペプチドに対する抗体として調製された抗体である、［３］に記載の抗体。
［６］以下の配列：TPPT(GalNAc)PSPS（配列番号２）（但し、Tはスレオニンを、Pはプロリンを、Sはセリンを、括弧内のGalNacは直前のアミノ酸残基に結合したO結合型N-アセチルガラクトサミンをそれぞれ表す）、からなる合成ヒンジ部糖ペプチドに対する抗体として調製された抗体である、［３］に記載の抗体。
［７］［１］〜［６］のいずれか一項に記載の抗体によって、ガラクトース含量の低下した糖鎖不全ヒトIgA1を検出することを特徴とする、IgA腎症の検査法。
［８］以下のステップ(i)〜(iii)を含む、［７］に記載のIgA腎症の検査法：
(i)シアリダーゼ処理後のヒトIgA1検体と、［１］〜［６］のいずれか一項に記載の抗体とを接触させるステップ；
(ii)ステップ(i)によって生じた免疫複合体を検出するステップ；
(iii)検出結果に基づき、IgA腎症の罹患ないし発症の有無若しくは可能性、又はIgA腎症を罹患ないし発症する可能性を判定するステップ。
［９］ヒトIgA1がヒト血清IgA1である、［８］に記載のIgA腎症の検査法。
［１０］以下のステップ(1)〜(5)を含む、［７］に記載のIgA腎症の検査法：
(1)固相化した抗ヒトIgA抗体にヒト検体を接触させるステップ；
(2)非特異的結合成分を除去した後、シアリダーゼで処理するステップ；
(3)［１］〜［６］のいずれか一項に記載の抗体を接触させるステップ；
(4)非特異的結合成分を除去した後、特異的に結合した前記抗体を検出するステップ；
(5)検出結果に基づき、IgA腎症の罹患ないし発症の有無若しくは可能性、又はIgA腎症を罹患ないし発症する可能性を判定するステップ。
［１１］ステップ(4)が、以下のステップ(4-1)及び(4-2)を含む、［１０］に記載のIgA腎症の検査法：
(4-1)非特異的結合成分を除去した後、ステップ(3)で使用する前記抗体に対する標識化抗体を接触させるステップ；
(4-2)非特異的結合成分を除去した後、特異的に結合した前記標識化抗体を検出するステップ。
［１２］以下のステップ(1)’〜(4)’を含む、［７］に記載のIgA腎症の検査法：
(1)’固相化した抗ヒトIgA抗体と、シアリダーゼ処理後のヒト検体とを接触させるステップ；
(2)’非特異的結合成分を除去した後、［１］〜［６］のいずれか一項に記載の抗体を接触させるステップ；
(3)’非特異的結合成分を除去した後、特異的に結合した前記抗体を検出するステップ；
(4)’検出結果に基づき、IgA腎症の罹患ないし発症の有無若しくは可能性、又はIgA腎症を罹患ないし発症する可能性を判定するステップ。
［１３］ヒト検体がヒト血清検体である、［１０］〜［１２］のいずれか一項に記載のIgA腎症の検査法。
［１４］［１］〜［６］のいずれか一項に記載の抗体を含む、IgA腎症検査用試薬。
［１５］［１４］に記載のIgA腎症検査用試薬を含む、IgA腎症検査用キット。
［１６］IgA腎症検査用試薬に対する標識化抗体を更に含む、［１５］に記載のIgA腎症検査用キット。
The present invention described below is mainly based on the above results and knowledge.
[1] An antibody that specifically recognizes a sugar chain-deficient human IgA1 hinge part having a reduced galactose content.
[2] The antibody according to [1], which is an antibody that specifically recognizes a human IgA1 hinge portion to which a sugar chain not containing sialic acid and galactose is bound.
[3] The antibody according to [1] or [2], which is a polyclonal antibody.
[4] The antibody according to [3], which does not include an antibody that recognizes a human IgA1 hinge part to which no sugar chain is bound.
[5] The following sequence: VPST (GalNAc) PPT (GalNAc) PS (GalNAc) PS (GalNAc) TPPT (GalNAc) PSPS (SEQ ID NO: 1) (where V is valine, P is proline, S is serine) , T represents threonine, and GalNac in parentheses represents O-linked N-acetylgalactosamine bound to the immediately preceding amino acid residue), and was prepared as an antibody against a synthetic hinge glycopeptide [3 ].
[6] The following sequence: TPPT (GalNAc) PSPS (SEQ ID NO: 2) (where T is threonine, P is proline, S is serine, and GalNac in parentheses is an O bond linked to the immediately preceding amino acid residue. The antibody according to [3], which is an antibody prepared as an antibody against a synthetic hinge glycopeptide consisting of N-acetylgalactosamine.
[7] A method for testing IgA nephropathy, characterized by detecting sugar chain-deficient human IgA1 having a reduced galactose content by the antibody according to any one of [1] to [6].
[8] The IgA nephropathy testing method according to [7], comprising the following steps (i) to (iii):
(i) contacting the human IgA1 specimen after sialidase treatment with the antibody according to any one of [1] to [6];
(ii) detecting the immune complex produced by step (i);
(iii) A step of determining, based on the detection result, whether or not IgA nephropathy is affected or present or possible, or whether or not IgA nephropathy is affected or developed.
[9] The test method for IgA nephropathy according to [8], wherein the human IgA1 is human serum IgA1.
[10] The method for testing IgA nephropathy according to [7], comprising the following steps (1) to (5):
(1) contacting a human specimen with an immobilized anti-human IgA antibody;
(2) removing non-specific binding components and then treating with sialidase;
(3) The step of contacting the antibody according to any one of [1] to [6];
(4) detecting the antibody specifically bound after removing the non-specific binding component;
(5) A step of determining, based on the detection result, whether or not IgA nephropathy is affected or present, or the possibility of developing or causing IgA nephropathy.
[11] The test method for IgA nephropathy according to [10], wherein step (4) includes the following steps (4-1) and (4-2):
(4-1) After removing the non-specific binding component, contacting the labeled antibody to the antibody used in step (3);
(4-2) A step of detecting the labeled antibody specifically bound after removing the non-specific binding component.
[12] The examination method for IgA nephropathy according to [7], comprising the following steps (1) ′ to (4) ′:
(1) 'contacting the solid-phased anti-human IgA antibody with a sialidase-treated human specimen;
(2) After removing the non-specific binding component, the step of contacting the antibody according to any one of [1] to [6];
(3) detecting the antibody specifically bound after removing the non-specific binding component;
(4) 'Determining whether or not IgA nephropathy is affected or present or based on the detection result, or whether or not IgA nephropathy is affected or developed.
[13] The IgA nephropathy testing method according to any one of [10] to [12], wherein the human specimen is a human serum specimen.
[14] A reagent for testing IgA nephropathy, comprising the antibody according to any one of [1] to [6].
[15] A kit for testing IgA nephropathy, comprising the reagent for testing IgA nephropathy according to [14].
[16] The kit for testing IgA nephropathy according to [15], further comprising a labeled antibody against a reagent for testing IgA nephropathy.
本発明の第１の局面は、ガラクトース含量の低下した糖鎖不全ヒトIgA1ヒンジ部を特異的に認識する抗体に関する。特に言及しない限り、本明細書における抗体は単離された状態である。「単離された状態の抗体」には、天然であって且つ何ら外的操作（人為的操作）が施されていない抗体、即ちあるヒト体内で産生され、そこに留まっている状態の抗体は含まれない。尚、単離された状態の抗体は、複数種類の抗体が混在した状態（即ちポリクローナル）であっても、単一の抗体又はその集合（即ち、モノクローナル）であってもよい。また、抗体以外の成分（例えば精製水、緩衝液、保存液、保護剤）を含んでいてもよい。 The first aspect of the present invention relates to an antibody that specifically recognizes a sugar chain-deficient human IgA1 hinge region having a reduced galactose content. Unless stated otherwise, the antibodies herein are in an isolated state. An “isolated antibody” includes an antibody that is natural and has not been subjected to any external manipulation (artificial manipulation), that is, an antibody that has been produced in a human body and remains there. Not included. The isolated antibody may be a state in which a plurality of types of antibodies are mixed (that is, polyclonal), or may be a single antibody or an assembly thereof (that is, monoclonal). In addition, components other than antibodies (for example, purified water, buffer solution, preservative solution, protective agent) may be included.
本発明の抗体は、その特徴として、ガラクトース含量の低下した糖鎖不全ヒトIgA1ヒンジ部を特異的に認識する。ヒトIgAにはIgA1とIgA2の二つのサブタイプが存在する。IgA腎症での糸球体沈着IgAはIgA1サブタイプが優位である。IgA1はヒンジ部に糖鎖が付加された特徴的な構造を有する。IgA1ヒンジ部にはO結合型糖鎖が結合できるSer（セリン）とThr（スレオニン）が存在する。O結合型糖鎖は一般に内側からN-アセチルガラクトサミン（GalNAc）とガラクトース（Gal）により構成され、更にその外側にシアル酸（NeuAc）が結合し得る。本明細書において「ガラクトース含量の低下した」とは、ヒンジ部に結合した糖鎖のGal含量が健常者の場合よりも低いことを意味する。健常者のIgA1のヒンジ部と区別するため、このような特徴のヒンジ部を本明細書では便宜上「糖鎖不全ヒトIgA1ヒンジ部」と表現する。質量分析による検討の結果からは、「糖鎖不全ヒトIgA1ヒンジ部」のGal含量は、ヒンジ部全体の糖鎖を構成する糖残基に占めるGalの数で比較して、健常者の場合（糖鎖）の約80〜90％と推定される。 The antibody of the present invention specifically recognizes a sugar chain-deficient human IgA1 hinge region having a reduced galactose content. There are two subtypes of human IgA, IgA1 and IgA2. The IgA1 subtype is predominant in glomerular IgA in IgA nephropathy. IgA1 has a characteristic structure in which a sugar chain is added to the hinge part. There are Ser (serine) and Thr (threonine) capable of binding O-linked sugar chains at the IgA1 hinge. O-linked sugar chains are generally composed of N-acetylgalactosamine (GalNAc) and galactose (Gal) from the inside, and sialic acid (NeuAc) can be bound to the outside. In the present specification, “decreased galactose content” means that the Gal content of the sugar chain bound to the hinge part is lower than that of a healthy person. In order to distinguish from the hinge part of a healthy person's IgA1, the hinge part of such a feature is expressed as a “sugar chain-deficient human IgA1 hinge part” for convenience in this specification. From the results of the mass spectrometric analysis, the Gal content of the “glycan-deficient human IgA1 hinge part” is compared with the number of Gals in the sugar residues constituting the sugar chain of the entire hinge part. Sugar chain) is estimated to be about 80-90%.
本発明の抗体が認識する糖鎖不全ヒトIgA1ヒンジ部に結合する糖鎖の数は特に限定されないが、例えば４〜５である。 The number of sugar chains that bind to the sugar chain-deficient human IgA1 hinge region recognized by the antibody of the present invention is not particularly limited, but is 4 to 5, for example.
一態様において本発明の抗体は、シアル酸及びガラクトースを含まない糖鎖が結合したヒトIgA1ヒンジ部を特異的に認識する。「シアル酸及びガラクトースを含まない糖鎖」とは、GalNAc残基からなる糖鎖のことをいう。 In one embodiment, the antibody of the present invention specifically recognizes a human IgA1 hinge region to which a sugar chain not containing sialic acid and galactose is bound. The “sugar chain not containing sialic acid and galactose” refers to a sugar chain composed of GalNAc residues.
一態様において本発明の抗体はポリクローナル抗体である。ポリクローナル抗体とは、上記の通り、複数種類の抗体が混在した状態の抗体である。本発明の特徴、即ち、ガラクトース含量の低下した糖鎖不全ヒトIgA1ヒンジ部を特異的に認識するという特徴を備える抗体を含有する限りにおいて、ポリクローナル抗体を構成する各抗体の特性（例えばエピトープ）は特に限定されない。好ましくは、本発明のポリクローナル抗体は、糖鎖が結合していないヒトIgA1ヒンジ部を認識する抗体を含まない。即ち、この態様のポリクローナル抗体は、ヒトIgA1ヒンジ部を構成するペプチド部分をエピトープとする抗体を含まない。当該特徴によって糖鎖不全ヒトIgA1ヒンジ部に対する特異性が高まり、IgA腎症検査用抗体として一層好ましいものとなる。 In one embodiment, the antibody of the present invention is a polyclonal antibody. A polyclonal antibody is an antibody in which a plurality of types of antibodies are mixed as described above. As long as the antibody has the characteristics of the present invention, that is, the antibody having the characteristic of specifically recognizing a sugar chain-deficient human IgA1 hinge part having a reduced galactose content, the characteristics (for example, epitope) of each antibody constituting the polyclonal antibody are as follows: There is no particular limitation. Preferably, the polyclonal antibody of the present invention does not include an antibody that recognizes a human IgA1 hinge region to which no sugar chain is bound. That is, the polyclonal antibody of this embodiment does not include an antibody having a peptide part constituting the human IgA1 hinge part as an epitope. This feature increases the specificity for the sugar chain-deficient human IgA1 hinge region, making it more preferable as an antibody for testing IgA nephropathy.
本発明の抗体の調製法は特に限定されないが、典型的には免疫学的手法によって本発明の抗体を調製することができる。ポリクローナル抗体であれば例えば次の手順で調製することができる。まず、抗原を調製し、これを用いてウサギ、ヤギ、ヒツジ、マウス、ラット、モルモット等の動物に免疫を施す。抗原としては、以下の配列からなる糖ペプチドを用いるとよい。尚、Vはバリンを、Pはプロリンを、Sはセリンを、Tはスレオニンを、括弧内のGalNacは直前のアミノ酸残基に結合したO結合型N-アセチルガラクトサミンをそれぞれ表す。
VPST(GalNAc)PPT(GalNAc)PS(GalNAc)PS(GalNAc)TPPT(GalNAc)PSPS（配列番号１）
The method for preparing the antibody of the present invention is not particularly limited. Typically, the antibody of the present invention can be prepared by an immunological technique. For example, a polyclonal antibody can be prepared by the following procedure. First, an antigen is prepared and used to immunize animals such as rabbits, goats, sheep, mice, rats, and guinea pigs. As an antigen, a glycopeptide having the following sequence may be used. V represents valine, P represents proline, S represents serine, T represents threonine, and GalNac in parentheses represents O-linked N-acetylgalactosamine bound to the immediately preceding amino acid residue.
VPST (GalNAc) PPT (GalNAc) PS (GalNAc) PS (GalNAc) TPPT (GalNAc) PSPS (SEQ ID NO: 1)
上掲の抗原の例はIgA1抗体のヒンジ部に相当する糖ペプチドであり、５箇所にGalNAcが付加されている。当該抗原では、ヒトIgA1１ヒンジ部と同様、O結合型糖鎖が結合できる部位が合計９箇所存在する。この９箇所の内、１箇所ないし９箇所（例えば１箇所、２箇所、３箇所、４箇所）にGalNAcを付加した抗原を採用することも可能である。GalNAcの付加部位が５個以下の場合には、上掲の抗原の例でGalNAcを付加した５箇所の部位の中から付加部位を選択することが好ましい。当該５箇所の部位は、ヒトIgA1において糖鎖の付加が高頻度に観察される部位に相当する。尚、抗原は公知のペプチド合成法（例えば固相合成法、液相合成法）を利用して調製することができる。 An example of the above-mentioned antigen is a glycopeptide corresponding to the hinge part of IgA1 antibody, and GalNAc is added at five positions. In the antigen, there are a total of nine sites to which O-linked sugar chains can bind, as in the human IgA11 hinge region. It is also possible to employ an antigen to which GalNAc has been added at one to nine (for example, one, two, three, four) of these nine places. When the number of GalNAc addition sites is 5 or less, it is preferable to select an addition site from among the 5 sites to which GalNAc has been added in the above example of antigen. The five sites correspond to sites where sugar chain addition is frequently observed in human IgA1. The antigen can be prepared by using a known peptide synthesis method (for example, solid phase synthesis method, liquid phase synthesis method).
一方、以下の配列からなる糖ペプチドを抗原に用いることもできる。
TPPT(GalNAc)PSPS（配列番号２）
当該抗原は、IgA1抗体のヒンジ部の一部に相当する糖ペプチドである。この部位に結合する糖鎖では、ガラクトースの欠落が最も高頻度で認められる。この点において、当該部位を抗原とすれば、IgA腎症の検査に特に有用な抗体が取得できるといえる。
On the other hand, glycopeptides having the following sequences can also be used as antigens.
TPPT (GalNAc) PSPS (SEQ ID NO: 2)
The antigen is a glycopeptide corresponding to a part of the hinge part of IgA1 antibody. In sugar chains that bind to this site, galactose loss is most frequently observed. In this respect, if the site is used as an antigen, it can be said that an antibody particularly useful for examination of IgA nephropathy can be obtained.
免疫惹起作用を増強するために、キャリアタンパク質を結合させた抗原を用いることが好ましい。キャリアタンパク質としてはKLH（Keyhole Limpet Hemocyanin）、BSA（Bovine Serum Albumin）、OVA（Ovalbumin）などが使用される。キャリアタンパク質の結合にはカルボジイミド法、グルタルアルデヒド法、ジアゾ縮合法、MBS（マレイミドベンゾイルオキシコハク酸イミド）法などを使用できる。一方、GST、βガラクトシダーゼ、マルトース結合タンパク、又はヒスチジン（His）タグ等との融合タンパク質として発現させた抗原を用いることもできる。このような融合タンパク質は、汎用的な方法により簡便に精製することができる。 In order to enhance the immunity-inducing action, it is preferable to use an antigen to which a carrier protein is bound. As the carrier protein, KLH (Keyhole Limpet Hemocyanin), BSA (Bovine Serum Albumin), OVA (Ovalbumin) and the like are used. The carbodiimide method, the glutaraldehyde method, the diazo condensation method, the MBS (maleimidobenzoyloxysuccinimide) method, etc. can be used for the coupling | bonding of carrier protein. On the other hand, an antigen expressed as a fusion protein with GST, β-galactosidase, maltose-binding protein, histidine (His) tag or the like can also be used. Such a fusion protein can be easily purified by a general method.
本発明の第２の局面は、本発明の抗体の用途の一つである、IgA腎症の検査法に関する。本発明の検査法は、ガラクトース含量の低下した糖鎖不全ヒトIgA1（以下、説明の便宜上、「ガラクトース含量の低下した糖鎖不全ヒトIgA1」を「糖鎖不全IgA1」と略称する）を本発明の抗体によって検出することを特徴とする。上記の通り、本発明の抗体は糖鎖不全IgA1ヒンジ部を特異的に認識する。従って、本発明の抗体を用いた検出法によれば、検体中の糖鎖不全IgA1を特異的に検出することができ、検出結果はIgA腎症の罹患ないし発症の有無若しくは可能性、又はIgA腎症を将来、罹患ないし発症する可能性を知る上で有益な情報となる。例えば、IgA腎症の患者を対象とした場合には、当該患者の病態の評価ないし把握、治療効果の評価などに有益な情報を得ることができる。このように本発明の検査法を治療効果のモニターに利用することも可能である。一方、患者以外の者、即ちIgA腎症が認定されていない者を対象とした場合には早期診断のための有益な情報が得られ、予防又は早期の治療介入が可能となる。さらに本症の成因は複数の可能性もあり、この糖鎖不全IgA1を成因として発症したIgA腎症が本手法によりサブタイプとして識別できる可能性もある。 The second aspect of the present invention relates to a test method for IgA nephropathy, which is one of the uses of the antibody of the present invention. According to the test method of the present invention, a sugar chain-deficient human IgA1 having a decreased galactose content (hereinafter, “glycan-deficient human IgA1 having a decreased galactose content” is abbreviated as “sugar chain-deficient IgA1” for convenience of explanation). It detects with the antibody of. As described above, the antibody of the present invention specifically recognizes the sugar chain-deficient IgA1 hinge region. Therefore, according to the detection method using the antibody of the present invention, sugar chain-deficient IgA1 in a specimen can be specifically detected, and the detection result is presence or absence or possibility of IgA nephropathy, or IgA It is useful information to know the possibility of developing or developing nephropathy in the future. For example, when targeting a patient with IgA nephropathy, it is possible to obtain information useful for evaluation or grasping of the patient's disease state, evaluation of therapeutic effect, and the like. Thus, the test method of the present invention can be used for monitoring the therapeutic effect. On the other hand, when the subject is a person other than the patient, that is, a person who is not certified with IgA nephropathy, useful information for early diagnosis is obtained, and prevention or early treatment intervention is possible. Furthermore, the cause of this disease may be multiple, and IgA nephropathy that develops due to this sugar chain-deficient IgA1 may be identified as a subtype by this technique.
検出法としては、例えば、ELISA（Enzyme-Linked ImmunoSorbent Assay）法、ラジオイムノアッセイ、FACS（fluorescence activated cell sorting）、免疫沈降法、イムノブロッティング等の定性的又は定量的な方法が挙げられる。免疫組織化学的染色法を採用することもできる。中でもELISA法（サンドイッチELISAや競合ELISA等）を利用して糖鎖不全IgA1を検出することが好ましい。ELISA法は検出感度が高いことや特異性が高いこと、定量性に優れること、操作が簡便であること、多検体の同時処理に適することなど、多くの利点を有する。 Examples of the detection method include qualitative or quantitative methods such as ELISA (Enzyme-Linked ImmunoSorbent Assay), radioimmunoassay, FACS (fluorescence activated cell sorting), immunoprecipitation, and immunoblotting. An immunohistochemical staining method can also be employed. Among them, it is preferable to detect sugar chain-deficient IgA1 using ELISA methods (sandwich ELISA, competitive ELISA, etc.). The ELISA method has many advantages such as high detection sensitivity, high specificity, excellent quantitativeness, simple operation, and suitability for simultaneous processing of multiple samples.
本発明の検査法の一態様では、以下のステップ(i)〜(iii)を実施する。
(i)シアリダーゼ処理後のヒトIgA1検体と、本発明の抗体とを接触させるステップ
(ii)ステップ(i)によって生じた免疫複合体を検出するステップ
(iii)検出結果に基づき、IgA腎症の罹患ないし発症の有無若しくは可能性、又はIgA腎症を罹患ないし発症する可能性を判定するステップ
In one aspect of the inspection method of the present invention, the following steps (i) to (iii) are performed.
(i) a step of contacting a human IgA1 specimen after sialidase treatment with the antibody of the present invention
(ii) detecting the immune complex produced by step (i)
(iii) A step of determining whether or not IgA nephropathy is affected or present or based on the detection result, or whether or not IgA nephropathy is affected
ステップ(i)ではまず、シアリダーゼ処理後のヒトIgA1検体を用意する。即ち、ヒンジ部の糖鎖からシアル酸を予め除去したヒトIgA1を検体とする。続いて、本発明の抗体と検体とを接触させる。この接触操作は典型的にはウェル内で行われるが、これに限られるものではない。ヒトIgA1は対象（IgA腎症の患者又は健常者）の血清、血漿、尿、髄液、腹水、胸水等の生体液から調製すればよい。好ましくは血清から調製したヒト血清IgA1を検体とする。シアリダーゼは市販のもの（例えばベーリンガーマンハイム社、ロッシュ・ダイアグノスティックス株式会社）を用いればよい。 In step (i), first, a human IgA1 sample after sialidase treatment is prepared. That is, human IgA1 from which sialic acid has been previously removed from the sugar chain of the hinge portion is used as a specimen. Subsequently, the antibody of the present invention is brought into contact with the specimen. This contact operation is typically performed in a well, but is not limited thereto. Human IgA1 may be prepared from a biological fluid such as serum, plasma, urine, spinal fluid, ascites, pleural effusion of the subject (IgA nephropathy patient or healthy subject). Preferably, human serum IgA1 prepared from serum is used as a specimen. A commercially available sialidase (for example, Boehringer Mannheim, Roche Diagnostics) may be used.
用意したヒトIgA1が、ガラクトース含量の低下した糖鎖不全ヒトIgA1であった場合には、ステップ(i)の結果、本発明の抗体による特異的結合が生じ、免疫複合体（本発明の抗体と糖鎖不全IgA1との複合体）が形成される。ステップ(ii)では、当該免疫複合体を検出する。 When the prepared human IgA1 is a sugar chain-deficient human IgA1 having a reduced galactose content, as a result of step (i), specific binding by the antibody of the present invention occurs, and an immune complex (the antibody of the present invention and A complex with sugar chain deficient IgA1). In step (ii), the immune complex is detected.
ステップ(iii)では、ステップ(ii)の検出結果に基づき、IgA腎症の罹患ないし発症の有無若しくは可能性、又はIgA腎症を罹患ないし発症する可能性を判定する。「IgA腎症を罹患ないし発症する可能性」を判定することは、即ち「IgA腎症の罹患ないし発症の予測」である。このように本発明は、IgA腎症の罹患ないし発症の予測にも利用できる。さらに本症の成因は複数の可能性もあり、この糖鎖不全IgA1を成因として発症したIgA腎症が本手法によりサブタイプとして識別できる可能性もある。 In step (iii), based on the detection result of step (ii), the presence or absence or possibility of IgA nephropathy, or the possibility of developing or developing IgA nephropathy is determined. Determining “possibility to develop or develop IgA nephropathy” is “prediction of the occurrence or development of IgA nephropathy”. Thus, the present invention can also be used for predicting the morbidity or development of IgA nephropathy. Furthermore, the cause of this disease may be multiple, and IgA nephropathy that develops due to this sugar chain-deficient IgA1 may be identified as a subtype by this technique.
ここでの判定は定性的、定量的のいずれであってもよい。定性的判定と定量的判定の例を以下に示す。尚、ここでの判定は、その判定基準から明らかな通り、医師や検査技師など専門知識を有する者の判断によらずとも自動的／機械的に行うことができる。
（定性的判定の例１）
基準値よりも検出値が高いときに「IgA腎症に罹患している」、「IgA腎症に罹患している可能性が高い」、又は「将来、IgA腎症に罹患する可能性が高い」などと判定し、基準値よりも検出値が低いときに「IgA腎症に罹患していない」、「IgA腎症に罹患していない可能性が高い」、又は「将来、IgA腎症に罹患する可能性が低い」などと判定する。
（定性的判定の例２）
検出されたとき（即ち陽性のとき）に「IgA腎症に罹患している」、「IgA腎症に罹患している可能性が高い」、又は「将来、IgA腎症に罹患する可能性が高い」などと判定し、検出されないとき（即ち陰性のとき）に「IgA腎症に罹患していない」、「IgA腎症に罹患していない可能性が高い」、又は「将来、IgA腎症に罹患する可能性が低い」などと判定する。
The determination here may be either qualitative or quantitative. Examples of qualitative judgment and quantitative judgment are shown below. It should be noted that the determination here can be automatically / mechanically performed without depending on the determination of a person having specialized knowledge such as a doctor or a laboratory technician, as is apparent from the determination criteria.
(Example 1 of qualitative judgment)
When the detected value is higher than the reference value, “I am suffering from IgA nephropathy”, “I am likely to be suffering from IgA nephropathy”, or “I am likely to be suffering from IgA nephropathy in the future” When the detected value is lower than the reference value, "I do not suffer from IgA nephropathy", "It is highly possible that I do not have IgA nephropathy", or " It is determined that the possibility of being affected is low.
(Example 2 of qualitative judgment)
When detected (ie, positive) “I am suffering from IgA nephropathy”, “I am likely to be suffering from IgA nephropathy”, or “I am likely to suffer from IgA nephropathy in the future When it is judged as “high” and is not detected (ie, when it is negative), it is “not likely to have IgA nephropathy”, “highly likely not to have IgA nephropathy”, or “in the future, IgA nephropathy” Is less likely to be affected ”.
（定量的判定の例１）
以下に示すように検出値の範囲毎に「IgA腎症に罹患している可能性（％）」を予め設定しておき、検出値から「IgA腎症に罹患している可能性（％）」を判定する。
検出値ａ〜ｂ：IgA腎症に罹患している可能性は１０％以下
検出値ｂ〜ｃ：IgA腎症に罹患している可能性は１０％〜３０％
検出値ｃ〜ｄ：IgA腎症に罹患している可能性は３０％〜５０％
検出値ｄ〜ｅ：IgA腎症に罹患している可能性は５０％〜７０％
検出値ｅ〜ｆ：IgA腎症に罹患している可能性は７０％〜９０％
(Example 1 of quantitative determination)
As shown below, “Possibility of suffering from IgA nephropathy (%)” is preset for each detection value range, and “Possibility of suffering from IgA nephropathy (%) Is determined.
Detection value ab: The possibility of having IgA nephropathy is 10% or less Detection value bc: The possibility of having IgA nephropathy is 10% to 30%
Detection values cd: 30% to 50% likely to have IgA nephropathy
Detection values d to e: Possibility of suffering from IgA nephropathy 50% to 70%
Detection values ef: 70% to 90% likely to have IgA nephropathy
（定量的判定の例２）
以下に示すように検出値の範囲毎に「将来、IgA腎症に罹患する可能性（％）」を予め設定しておき、検出値から「将来、IgA腎症に罹患する可能性（％）」を予測する。
検出値ａ〜ｂ：将来、IgA腎症に罹患する可能性は１０％以下
検出値ｂ〜ｃ：将来、IgA腎症に罹患する可能性は１０％〜３０％
検出値ｃ〜ｄ：将来、IgA腎症に罹患する可能性は３０％〜５０％
検出値ｄ〜ｅ：将来、IgA腎症に罹患する可能性は５０％〜７０％
検出値ｅ〜ｆ：将来、IgA腎症に罹患する可能性は７０％〜９０％
(Example 2 of quantitative determination)
As shown below, “Possibility of suffering from IgA nephropathy in the future (%)” is preset for each range of detection values, and from the detection value “Possibility of suffering from IgA nephropathy in the future (%) Is predicted.
Detected values a to b: 10% or less likely to have IgA nephropathy in the future Detected values bc to 10 to 30% likely to have IgA nephropathy in the future
Detection values cd: 30% to 50% likely to have IgA nephropathy in the future
Detection value de: 50% to 70% possibility of suffering from IgA nephropathy in the future
Detected values ef: 70% to 90% likely to have IgA nephropathy in the future
本発明の検査法の他の一態様では、以下のステップ(1)〜(5)を実施する。
(1)固相化した抗ヒトIgA抗体にヒト検体を接触させるステップ
(2)非特異的結合成分を除去した後、シアリダーゼで処理するステップ
(3)本発明の抗体を接触させるステップ
(4)非特異的結合成分を除去した後、特異的に結合した前記抗体を検出するステップ
(5)検出結果に基づき、IgA腎症の罹患ないし発症の有無若しくは可能性、又はIgA腎症を罹患ないし発症する可能性を判定するステップ
In another aspect of the inspection method of the present invention, the following steps (1) to (5) are performed.
(1) A step of bringing a human specimen into contact with an immobilized anti-human IgA antibody
(2) Step of removing with non-specific binding component and then treating with sialidase
(3) Step of contacting the antibody of the present invention
(4) detecting the antibody specifically bound after removing the non-specific binding component
(5) Based on the detection result, determining whether or not IgA nephropathy is present or present, or the possibility of developing or causing IgA nephropathy
ステップ(1)ではまず、固相化した抗ヒトIgA抗体、即ち不溶性支持体に固定した抗ヒトIgA抗体（以下、「固相化抗体」と呼ぶ）を用意する。不溶性支持体としては例えばポリスチレン樹脂、ポリカーボネート樹脂、シリコン樹脂、ナイロン樹脂等の樹脂や、ガラス等の水に不溶性の物質を用いることができる。抗ヒトIgA抗体は例えば市販のもの（例えばJackson Immuno Reseach Labs）を用いればよい。抗ヒトIgA抗体として好ましくは抗IgA1抗体を用いる。モノクローナル抗IgA1抗体が特に好ましいが、血清中ではIgA1がIgA全体の90％を占めるので、汎用性を考慮するとポリクローナル抗IgA抗体であってもよい。 In step (1), an anti-human IgA antibody immobilized on solid phase, that is, an anti-human IgA antibody immobilized on an insoluble support (hereinafter referred to as “solid-phase antibody”) is prepared. As the insoluble support, for example, a resin such as polystyrene resin, polycarbonate resin, silicon resin, nylon resin, or a water-insoluble substance such as glass can be used. For example, a commercially available anti-human IgA antibody (for example, Jackson Immuno Reseach Labs) may be used. An anti-IgA1 antibody is preferably used as the anti-human IgA antibody. Monoclonal anti-IgA1 antibody is particularly preferred, but since anti-IgA1 occupies 90% of the total IgA in the serum, polyclonal anti-IgA antibody may be used in consideration of versatility.
不溶性支持体の形態は特に限定されない。不溶性支持体の形態の例はウェル、プレートである。 The form of the insoluble support is not particularly limited. Examples of forms of insoluble supports are wells, plates.
次に、固相化抗体にヒト検体を接触させる。この操作によって、ヒト検体中のIgA（IgA1抗体を含む）が固相化抗体にトラップされる（固相化抗体として抗ヒトIgA1抗体を使用した場合には、IgA1抗体のみをトラップすることができる）。ヒト検体としては、対象（IgA腎症の患者又は健常者）の血清、血漿、尿、髄液、腹水、胸水等の生体液が用いられる。好ましくは血清が用いられる。血清を用いれば簡便な測定が可能である。 Next, the human specimen is brought into contact with the immobilized antibody. By this operation, IgA (including IgA1 antibody) in the human specimen is trapped by the immobilized antibody (when the anti-human IgA1 antibody is used as the immobilized antibody, only the IgA1 antibody can be trapped. ). As the human specimen, biological fluids such as serum, plasma, urine, spinal fluid, ascites, pleural effusion of the subject (IgA nephropathy patient or healthy person) are used. Serum is preferably used. If serum is used, simple measurement is possible.
続くステップ(2)では、非特異的結合成分を洗浄除去した後、シアリダーゼで処理する。この処理によって、固相化抗体にトラップされたIgA1のヒンジ部に結合した糖鎖からシアル酸が除去され、Gal残基及び／又はGalNAc残基が露出する。 In the subsequent step (2), the non-specific binding component is washed away and then treated with sialidase. By this treatment, sialic acid is removed from the sugar chain bound to the hinge part of IgA1 trapped in the immobilized antibody, and the Gal residue and / or GalNAc residue are exposed.
次に、本発明の抗体を接触させる（ステップ(3)）。その結果、固相抗体にトラップされたIgA1が糖鎖不全IgA1であれば、本発明の抗体が特異的に結合し、免疫複合体が形成される。他方、固相抗体にトラップされたIgA1が正常なIgA1（Gal含量が多いIgA1）の場合、本発明の抗体による特異的結合は生じない。或いは、生じたとしても非常に少なくなる。このように、ヒト検体中のIgA1の状態に応じて免疫複合体の形成量に差が生まれる。本発明ではこの差をIgA腎症の判定に利用する。 Next, the antibody of the present invention is contacted (step (3)). As a result, if the IgA1 trapped in the solid phase antibody is a sugar chain-deficient IgA1, the antibody of the present invention specifically binds to form an immune complex. On the other hand, when IgA1 trapped in the solid phase antibody is normal IgA1 (IgA1 having a high Gal content), specific binding by the antibody of the present invention does not occur. Or, if it occurs, it is very little. Thus, a difference is produced in the amount of immune complex formed depending on the state of IgA1 in the human specimen. In the present invention, this difference is used to determine IgA nephropathy.
ステップ(4)では、非特異的結合成分を洗浄除去した後、特異的に結合した抗体（換言すれば免疫複合体）を検出する。例えば、ステップ(3)に使用する抗体を予め標識化しておけば、非特異的結合成分を洗浄除去した後に標識量を直接検出すればよい。一方、いわゆる二次抗体を利用して検出することもできる。この場合、非特異的結合成分を除去した後、ステップ(3)で使用する抗体に対する標識化抗体を接触させるステップ(ステップ(4-1))と、非特異的結合成分を除去した後、特異的に結合した標識化抗体を検出するステップ(ステップ(4-2))を実施する。尚、ステップ(3)に使用する抗体や二次抗体の標識化には例えばフルオレセイン、ローダミン、テキサスレッド、オレゴングリーン等の蛍光色素、ホースラディッシュペルオキシダーゼ、マイクロペルオキシダーゼ、アルカリ性ホスファターゼ、β−Ｄ−ガラクトシダーゼ等の酵素、ルミノール、アクリジン色素等の化学又は生物発光化合物、32P、131I、125I等の放射性同位体、及びビオチン等を用いることができる。二次抗体として使用可能な数多くの標識化抗体（例えば標識化抗ウサギIgG抗体）が市販されており、当該標識化抗体を利用することもできる。 In step (4), the non-specific binding component is washed away, and then the specifically bound antibody (in other words, immune complex) is detected. For example, if the antibody used in step (3) is labeled in advance, the amount of label may be directly detected after washing away nonspecific binding components. On the other hand, it is also possible to detect using a so-called secondary antibody. In this case, after removing the non-specific binding component, contacting the labeled antibody against the antibody used in step (3) (step (4-1)), after removing the non-specific binding component, A step (step (4-2)) of detecting the labeled antibody bound thereto is carried out. For labeling the antibody or secondary antibody used in step (3), for example, fluorescent dyes such as fluorescein, rhodamine, Texas red, oregon green, horseradish peroxidase, microperoxidase, alkaline phosphatase, β-D-galactosidase, etc. Enzymes, chemical or bioluminescent compounds such as luminol and acridine dyes, radioisotopes such as 32 P, 131 I, and 125 I, biotin, and the like can be used. Many labeled antibodies (for example, labeled anti-rabbit IgG antibodies) that can be used as secondary antibodies are commercially available, and the labeled antibodies can also be used.
ステップ(5)では、検出結果に基づき、IgA腎症の罹患ないし発症の有無若しくは可能性、又はIgA腎症を罹患ないし発症する可能性を判定する。このステップは、上記態様におけるステップ(iii)と同様であるのでその説明を省略する。 In step (5), based on the detection result, the presence or absence or possibility of IgA nephropathy, or the possibility of developing or developing IgA nephropathy is determined. Since this step is the same as step (iii) in the above embodiment, its description is omitted.
本発明の更に別の態様では、以下のステップ(1)’〜(4)’を実施する。
(1)’固相化した抗ヒトIgA抗体と、シアリダーゼ処理後のヒト検体とを接触させるステップ
(2)’非特異的結合成分を除去した後、本発明の抗体を接触させるステップ
(3)’非特異的結合成分を除去した後、特異的に結合した前記抗体を検出するステップ
(4)’検出結果に基づき、IgA腎症の罹患ないし発症の有無若しくは可能性、又はIgA腎症を罹患ないし発症する可能性を判定するステップ
In still another aspect of the present invention, the following steps (1) ′ to (4) ′ are performed.
(1) Step of bringing the immobilized anti-human IgA antibody into contact with a human sample after sialidase treatment
(2) Step of contacting the antibody of the present invention after removing the non-specific binding component
(3) 'detecting the specifically bound antibody after removing non-specific binding components
(4) 'Determining whether or not IgA nephropathy is present or present based on the detection result, or the possibility of developing or developing IgA nephropathy
この態様では、予めシアリダーゼ処理した検体を使用する。その他の構成は上記態様と同様であり、ステップ(1)’及び(2)’が上記態様のステップ(1)〜(3)に対応する。また、ステップ(3)’が上記態様のステップ(4)に相当し、ステップ(4)’が上記対応のステップ(5)に相当する。尚、上記態様と同様に、標識化した二次抗体を利用した検出を行うことにしてもよい。 In this embodiment, a specimen that has been previously treated with sialidase is used. Other configurations are the same as those in the above embodiment, and steps (1) 'and (2)' correspond to steps (1) to (3) in the above embodiment. Further, step (3) 'corresponds to step (4) of the above aspect, and step (4)' corresponds to the corresponding step (5). In addition, you may decide to detect using the labeled secondary antibody similarly to the said aspect.
本発明の第３の局面はIgA腎症検査用試薬に関する。本発明の試薬は本発明の検査法に好適に使用され得るが、IgA腎症の発症メカニズムや症状の進展メカニズムなどを研究するための実験ツールとしても使用され得る。さらに本症の成因は複数の可能性もあり、この糖鎖不全IgA1を成因として発症したIgA腎症が本手法により「糖鎖不全型IgA腎症」といったサブタイプとして識別できる可能性もある。一方、O結合型糖鎖上のGalNAc残基を認識するという特性に鑑みれば、粘膜やムチン等の研究用ツールとしての使用も想定される。本発明の試薬は本発明の抗体を含む。不溶性支持体に固定化された状態（固相化試薬）で本発明の試薬が提供されてもよい。また、標識化された状態（蛍光標識、酵素標識など）で提供されてもよい。 The third aspect of the present invention relates to a reagent for testing IgA nephropathy. The reagent of the present invention can be suitably used in the test method of the present invention, but can also be used as an experimental tool for studying the onset mechanism of IgA nephropathy and the mechanism of symptom progression. Furthermore, the cause of this disease may be multiple, and IgA nephropathy that develops due to this sugar chain-deficient IgA1 may be identified as a subtype such as “sugar chain-deficient IgA nephropathy” by this method. On the other hand, in view of the property of recognizing a GalNAc residue on an O-linked sugar chain, it can be used as a research tool for mucous membranes, mucins and the like. The reagent of the present invention contains the antibody of the present invention. The reagent of the present invention may be provided in a state immobilized on an insoluble support (solid phase reagent). Alternatively, it may be provided in a labeled state (fluorescent label, enzyme label, etc.).
本発明の第４の局面は、本発明の方法に使用可能なキット（IgA腎症検査用キット）を提供する。本発明のキットは本発明の試薬を含んで構成される。本発明の方法の実施に必要な反応用試薬、希釈液、反応容器などをキットに含めることができる。尚、本発明のキットには通常、使用説明書が添付される。キットを用いることによって、本発明の方法をより簡便に且つより短時間で実施することが可能となる。 The fourth aspect of the present invention provides a kit (IgA nephropathy test kit) that can be used in the method of the present invention. The kit of the present invention comprises the reagent of the present invention. Reagents, diluents, reaction vessels and the like necessary for carrying out the method of the present invention can be included in the kit. The kit of the present invention usually includes instructions for use. By using the kit, the method of the present invention can be carried out more easily and in a shorter time.
好ましい一態様では、本発明の試薬に加えて二次抗体（本発明の試薬に対する標識化抗体）が含まれる。二次抗体を含むキットによれば、本発明の試薬と検体との接触による免疫複合体の形成、免疫複合体への標識化抗体の結合、結合した標識量の測定といった一連の操作を実施可能となる。 In a preferred embodiment, a secondary antibody (labeled antibody against the reagent of the present invention) is included in addition to the reagent of the present invention. With a kit containing a secondary antibody, a series of operations such as formation of an immune complex by contacting the reagent of the present invention with a specimen, binding of a labeled antibody to the immune complex, and measurement of the amount of bound label can be performed. It becomes.
本発明のキットに標準試料を含めてもよい。ここでの標準試料は、本発明のキットを使用した検出結果を評価するための基準となる。例えば、糖鎖不全IgA1又は糖鎖不全IgA1ヒンジ部ペプチド若しくはその一部を標準試料とする。標準試料は合成によって或いはIgA腎症患者の血清からの分離精製によって調製することができる。 A standard sample may be included in the kit of the present invention. The standard sample here serves as a reference for evaluating the detection result using the kit of the present invention. For example, a sugar chain-deficient IgA1 or sugar chain-deficient IgA1 hinge peptide or a part thereof is used as a standard sample. Standard samples can be prepared by synthesis or by separation and purification from the serum of patients with IgA nephropathy.
以前、本発明者らはGalNAc残基を認識するレクチンHAA (Helix aspersa)を用いたELISA法によって、本症患者血清IgA1に対するレクチンHAAの結合度が健常者や他の腎疾患患者に比べ有意に増加していることを観察した(非特許文献２）。しかし、ROC曲線を用いその臨床応用の可能性を検討したところ、他の腎疾患群を対照としたときのAUC（Area under the curve）は0.774にとどまり、臨床応用は難しいと考えられた。一般に、抗体はレクチンよりもエピトープを認識する親和性が10〜100倍高いといわれている。そこで、より親和性の高い抗体に注目し、本症患者に特異的なマーカーの作製を目指して検討を進めた。 Previously, the present inventors showed that the binding degree of lectin HAA to serum IgA1 in patients with this disease was significantly higher than that of healthy subjects and patients with other renal diseases by ELISA using lectin HAA (Helix aspersa) that recognizes GalNAc residues. The increase was observed (Non-patent Document 2). However, when the possibility of clinical application was examined using the ROC curve, the AUC (Area under the curve) was 0.774 when the other kidney disease groups were controlled, and clinical application was considered difficult. In general, it is said that antibodies have an affinity for recognizing epitopes 10 to 100 times higher than lectins. Therefore, we focused on antibodies with higher affinity, and proceeded with studies aimed at creating markers specific to patients with this disease.
１．ヒトIgA1ヒンジ糖ペプチド合成
保護ペプチド樹脂は自動合成機を使用し、tert-butoxycarbonyl法（Boc法）により後述するヒトIgA1ヒンジ部に相当する糖ペプチド(synthetic human IgA1 hinge glycopeptide, 以下sHGPと略す)を合成した。得られた保護糖ペプチド樹脂を無水フッ化水素で処理し、この粗糖ペプチドを逆相HPLCにて精製して目的のsHGPを高純度で得た。sHGPはヒトIgA1ヒンジが持つ19merのアミノ酸配列に、O-グリカン側鎖が高頻度に存在しているといわれる５箇所の部位にGalNAcを付加した以下の構造である。
VPST(GalNAc)PPT(GalNAc)PS(GalNAc)PS(GalNAc)TPPT(GalNAc)PSPS-NH2（配列番号１）
この合成ヒンジに対するウサギポリクローナル抗体を以下のように作製した。
1. Human IgA1 hinge glycopeptide synthesis Protected peptide resin uses an automatic synthesizer, and glycan peptide corresponding to human IgA1 hinge part (synthetic human IgA1 hinge glycopeptide, hereinafter abbreviated as sHGP) is described by tert-butoxycarbonyl method (Boc method) Synthesized. The obtained protected glycopeptide resin was treated with anhydrous hydrogen fluoride, and the crude glycopeptide was purified by reverse phase HPLC to obtain the desired sHGP with high purity. sHGP has the following structure in which GalNAc is added to five sites that are said to have a high frequency of O-glycan side chains in the 19-mer amino acid sequence of the human IgA1 hinge.
VPST (GalNAc) PPT (GalNAc) PS (GalNAc) PS (GalNAc) TPPT (GalNAc) PSPS-NH 2 (SEQ ID NO: 1)
A rabbit polyclonal antibody against this synthetic hinge was prepared as follows.
２．KLHコンジュゲートの作製
sHGPをリン酸緩衝液（pH 8.0）中で、KLH(Keyhole limpet hemocyanin, SIGMA社製)と混合し、グルタルアルデヒドで結合させた。Tris-HCl(pH 4)にて反応停止させ、透析にて脱塩をした。得られたKLHコンジュゲートsHGPの一部をアミノ酸分析し、sHGPの含量を算出した。コンジュゲート1mgに含有するペプチドは約49〜51 nmolであった。
2. Preparation of KLH conjugate
sHGP was mixed with KLH (Keyhole limpet hemocyanin, manufactured by SIGMA) in phosphate buffer (pH 8.0) and bound with glutaraldehyde. The reaction was stopped with Tris-HCl (pH 4) and desalted by dialysis. A part of the obtained KLH conjugate sHGP was subjected to amino acid analysis, and the content of sHGP was calculated. The peptide contained in 1 mg of the conjugate was about 49-51 nmol.
３．抗血清作製
作製したKLH-sHGPを生理食塩水に懸濁し、フロイントアジュバントを混合乳化し、国産雑種白色ウサギに注射した。初回免疫には完全フロイントアジュバントを使用し、追加免疫は三回行い、不完全フロイントアジュバントを使用した。初回免疫から７週目に全採血を施行し、ウサギ抗血清を得た。免疫後の抗血清のsHGPに対する抗体価をELISAで検討し、合成(糖)ペプチドと容量依存的に結合することを確認した(図１)。
3. Preparation of antiserum The prepared KLH-sHGP was suspended in physiological saline, mixed and emulsified with Freund's adjuvant, and injected into a domestic white rabbit. Complete Freund's adjuvant was used for the first immunization, booster was performed three times, and incomplete Freund's adjuvant was used. On the 7th week after the first immunization, whole blood was collected to obtain rabbit antiserum. The antibody titer against sHGP of the antiserum after immunization was examined by ELISA, and it was confirmed that it bound to the synthetic (sugar) peptide in a dose-dependent manner (FIG. 1).
４．ウサギ抗血清からのIgG分画の精製
精製は4℃低温室で行った。得られたウサギ抗血清を0.02 M リン酸緩衝生理食塩水（phosphate buffered saline;以下PBSと略す）(pH 7.0)で一晩（overnight；以下O/Nと略す）透析し、得られたサンプル5ml毎にPBS 5mlを加え、硫酸アンモニウム5mlを滴下して、33%硫安沈殿法を施行した。4℃で30分安置後、2,000rpm、10分間、遠心分離した。沈殿物にPBSを加えて溶解した。その後、0.02M PBSにO/N透析を施行し、サンプルを0.45μmのフィルターに通した後、プロテインGカラム（HiTrap Protein G HP 5ml、GE Healthcare UK Ltd. Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, England）に供し、IgG分画を吸着させた。ろ液の吸光度（ABS値）0.02未満になるまでプロテインGカラムを0.02 M PBS(pH 7.0)で洗浄した。その後、0.1 MグリシンHCl(pH 2.7)を用いてIgG分画を溶出した。得られたサンプルは1 M Tris(pH 9.0)を加えて中性に戻し、さらに脱塩目的で、0.01 M PBS(pH 7.4)にてO/N透析し、抗sHGP抗体を精製した。得られた抗体（約100ml）の濃度をローリー法で測定した結果、25mg/mlであった。
4). Purification of IgG fraction from rabbit antiserum Purification was performed in a cold room at 4 ° C. The obtained rabbit antiserum was dialyzed overnight with 0.02 M phosphate buffered saline (hereinafter abbreviated as PBS) (pH 7.0) (overnight; hereinafter abbreviated as O / N). Each time, 5 ml of PBS was added and 5 ml of ammonium sulfate was added dropwise to carry out 33% ammonium sulfate precipitation. After incubating at 4 ° C. for 30 minutes, the mixture was centrifuged at 2,000 rpm for 10 minutes. PBS was added to the precipitate and dissolved. Thereafter, O / N dialysis was performed on 0.02M PBS, and the sample was passed through a 0.45 μm filter. Then, a protein G column (HiTrap
５．ヒト血清IgAと抗sHGP抗体との反応性及び至適血清希釈率の設定
図２は、後で詳述する間接ELISA法で得られた、ヒト血清IgAと抗sHP抗体の用量反応曲線（Dose-response curve）である。X軸にヒト血清量を5倍希釈系列で示し、Y軸は吸光度である。抗sHGP抗体は合成糖ペプチドだけでなくヒトIgAにも容量依存的に結合した。血清希釈が1/100から1/500の間では曲線はほぼ水平（plateau）になり、キャプチャー抗体に結合するIgA量は飽和していると考えられた。従って、以降の実験では全てのサンプル血清について1/500希釈の条件下で検討することにした。
5. Reactivity of Human Serum IgA with Anti-sHGP Antibody and Setting of Optimal Serum Dilution Ratio FIG. 2 is a dose response curve (Dose-) of human serum IgA and anti-sHP antibody obtained by an indirect ELISA method described in detail later. response curve). The X-axis shows the amount of human serum in a 5-fold dilution series, and the Y-axis is absorbance. The anti-sHGP antibody bound not only to the synthetic glycopeptide but also to human IgA in a dose-dependent manner. When the serum dilution was between 1/100 and 1/500, the curve was almost horizontal (plateau), and the amount of IgA bound to the capture antibody was considered to be saturated. Therefore, in subsequent experiments, all sample sera were examined under the condition of 1/500 dilution.
６．抗sHGP抗体と各種グリコシダーゼ連続処理後のIgA1との反応性の検討
健常者プール血清をジャカリンカラム（Vector Laboratories社）で精製し、IgA1画分を得た。得られたIgA1画分より、以下の試料、即ち、(1)精製した未処理IgA1(native IgA1)；(2)シアリダーゼ処理を行ったIgA1（desialo(DeS） IgA1)；(3)シアリダーゼ処理に加えてガラクトシダーゼ処理を行ったIgA1（desialo/degalacto(DeS/DeG） IgA1)；(4)シアリダーゼ処理とガラクトシダーゼ処理に加えてO-グリコシダーゼ処理を行ったIgA1 (deglycosylated（naked） IgA1)を調製した。(1)〜(4)について予め免疫比濁法でIgA値を測定した。各試料と上記４．で得られた抗sHGP抗体との結合度を、試料のIgA1濃度が0(negative control)、1μg/ml及び5μg/mlの3点において、間接ELISA法で比較検討した。その結果、抗sHGP抗体は、native IgA1及び DeS IgA1と比較し、DeS/DeG IgA1に対して著しく強く結合し、またnaked IgA1とも強い結合をみた（図３）。以上より、抗sHGP抗体はIgA1ヒンジ上のGalNAc残基を認識する抗体を含むこと、及びペプチド部分を認識する抗体が混在していることが明らかとなった。IgA1ヒンジ上のGalNAc残基を認識する抗体を含むことは、抗sHGP抗体が、Gal含量の低下した糖鎖不全IgA1を認識できることを意味する。尚、ペプチド部分を認識する抗体については、例えばペプチド部分を担持させたアフィニティーカラムなどによって、容易に分離除去できる。
6). Examination of reactivity between anti-sHGP antibody and IgA1 after continuous treatment with various glycosidases Healthy patient pool serum was purified with a Jakarin column (Vector Laboratories) to obtain an IgA1 fraction. From the obtained IgA1 fraction, the following samples were obtained: (1) purified untreated IgA1 (native IgA1); (2) IgA1 treated with sialidase (desialo (DeS) IgA1); (3) treated with sialidase In addition, IgA1 (desialo / degalacto (DeS / DeG) IgA1) treated with galactosidase was prepared; (4) IgA1 (deglycosylated (naked) IgA1) treated with O-glycosidase in addition to sialidase treatment and galactosidase treatment was prepared. Regarding (1) to (4), IgA values were measured in advance by immunoturbidimetry. Each sample and 4. The degree of binding to the anti-sHGP antibody obtained in the above was compared by an indirect ELISA method at three points where the IgA1 concentration of the sample was 0 (negative control), 1 μg / ml and 5 μg / ml. As a result, the anti-sHGP antibody bound to DeS / DeG IgA1 remarkably stronger than native IgA1 and DeS IgA1, and also showed strong binding to naked IgA1 (FIG. 3). From the above, it was revealed that the anti-sHGP antibody contains an antibody that recognizes the GalNAc residue on the IgA1 hinge and that an antibody that recognizes the peptide portion is mixed. Inclusion of an antibody that recognizes a GalNAc residue on the IgA1 hinge means that the anti-sHGP antibody can recognize sugar chain-deficient IgA1 having a reduced Gal content. The antibody recognizing the peptide portion can be easily separated and removed by, for example, an affinity column carrying the peptide portion.
７．IgA腎症、健常者、他の腎疾患患者間での抗sHGP抗体のIgA1結合度の比較（本実験）
IgA腎症患者（IgA-N群；n=39）と健常者（HC群；n=37）、他の腎疾患患者（OKD群；n=36）を対象に、500倍希釈した各血清IgA1と抗sHGP抗体との結合度を間接ELISA法で比較検討した。ポリスチレン・マイクロタイタープレート(Corning、NY、USA)を使用し、以下の手順で間接ELISAを施行した。
(1)キャプチャー抗体としてF(ab)'2 Fragment Goat Anti-Human Serum IgA ab(Jackson Immuno Reseach Labs、West Grove、PA、USA)を使用した。希釈液（0.05 M重炭酸塩緩衝液（pH 9.6））で2.6μg/mlの濃度に希釈したキャプチャー抗体でプレートをコートし(0.13μg/well)、4℃下にてO/N静置した。
(2) 0.05 % TWEEN（登録商標）20（SIGMA-ALDRICH、St. Louis、MO、USA）を含む0.01 M PBS(pH7.4)を洗浄液（以下PBS-TWEENと略す）として用いた。マイクロプレートウォッシャー（model1575 BIO RAD）を使用し、1回500μl/wellで3回ずつプレート洗浄を施行した。
(3)ウシ血清アルブミン(BSA；SIGMA-ALDRICH、St. Louis、MO、USA)を0.01 M PBS(pH 7.4)に溶解し、各ウェルに添加し（100μl/well）室温下に2時間静置してブロッキングし、PBS-TWEENで同様に3回洗浄した。
(4)BSAをPBS-TWEENに溶解したサンプル希釈液（1% BSA/PBS-TWEEN）を用いて500倍希釈したヒト血清IgA1サンプルを各ウェルに加え（50μl/well）、モイストチャンバー内（室温）で2時間静置した。
(5)シアリダーゼ（ロッシュ・ダイアグノスティックス株式会社）を酢酸緩衝液(pH 5.0)で17 mU/mlに希釈し、各ウェルに添加（100μl/well）した後、37℃下に3時間静置してシアリダーゼ処理を行い、PBS-TWEENで同様に3回洗浄した。
(6)抗sHGP抗体を各ウェルに添加（50μl/well）し、モイストチャンバー内で静置した（4℃、O/N）。
(7)PBS-TWEENで同様に3回洗浄後、二次標識抗体としてペルオキシダーゼ標識ヤギ抗ウサギIgG抗体(Jackson Immuno Research Labs、West Grove、PA、USA)を1：10,000に希釈して各ウェルに添加（50μl/well）した。室温下に1時間静置した後、PBS-TWEENで5回洗浄した。
(8)0.1 Mクエン酸緩衝液（pH 5.0）に溶解したO-フェニレンジアミン(OPD)-H2O2（Pierce）を発色液とし、各ウェルに100μl/wellずつ加え発色させた。1M硫酸を各ウェルに100μl/wellずつ添加して発色停止後、比色計で490nmの吸光度を計測した。
(9)3群間の比較にはボンフェローニ補正マンホイットニー検定(Mann-Whitney U-test with Bonferroni correction）を使用した。
7). Comparison of IgA1 binding degree of anti-sHGP antibody among patients with IgA nephropathy, healthy subjects, and other renal diseases (this experiment)
Serum IgA1 diluted 500-fold for patients with IgA nephropathy (IgA-N group; n = 39), healthy subjects (HC group; n = 37), and other renal disease patients (OKD group; n = 36) The indirect ELISA method was used to compare the degree of binding between the anti-sHGP antibody and the anti-sHGP antibody. Using a polystyrene microtiter plate (Corning, NY, USA), indirect ELISA was performed according to the following procedure.
(1) F (ab) ′ 2 Fragment Goat Anti-Human Serum IgA ab (Jackson Immuno Reseach Labs, West Grove, PA, USA) was used as a capture antibody. The plate was coated with a capture antibody diluted to a concentration of 2.6 μg / ml with a diluted solution (0.05 M bicarbonate buffer (pH 9.6)) (0.13 μg / well), and O / N was left to stand at 4 ° C. .
(2) 0.01 M PBS (pH 7.4) containing 0.05% TWEEN (registered trademark) 20 (SIGMA-ALDRICH, St. Louis, MO, USA) was used as a washing solution (hereinafter abbreviated as PBS-TWEEN). Using a microplate washer (model1575 BIO RAD), the plate was washed 3 times at 500 μl / well.
(3) Bovine serum albumin (BSA; SIGMA-ALDRICH, St. Louis, MO, USA) was dissolved in 0.01 M PBS (pH 7.4), added to each well (100 μl / well), and allowed to stand at room temperature for 2 hours. Then, it was blocked and washed three times with PBS-TWEEN.
(4) A human serum IgA1 sample diluted 500-fold with a sample diluent (1% BSA / PBS-TWEEN) in which BSA is dissolved in PBS-TWEEN is added to each well (50 μl / well) in the moist chamber (room temperature ) For 2 hours.
(5) Dilute sialidase (Roche Diagnostics Co., Ltd.) to 17 mU / ml with acetate buffer (pH 5.0), add to each well (100 μl / well), and then allow to stand at 37 ° C for 3 hours. Then, the mixture was treated with sialidase and washed with PBS-TWEEN three times in the same manner.
(6) An anti-sHGP antibody was added to each well (50 μl / well) and allowed to stand in a moist chamber (4 ° C., O / N).
(7) After washing with PBS-TWEEN three times in the same manner, a peroxidase-labeled goat anti-rabbit IgG antibody (Jackson Immuno Research Labs, West Grove, PA, USA) was diluted 1: 10,000 as a secondary labeled antibody to each well. Added (50 μl / well). After leaving still at room temperature for 1 hour, it wash | cleaned 5 times by PBS-TWEEN.
(8) O-phenylenediamine (OPD) -H 2 O 2 (Pierce) dissolved in 0.1 M citrate buffer (pH 5.0) was used as a color developing solution, and 100 μl / well was added to each well for color development. After 1 M sulfuric acid was added to each well at 100 μl / well to stop color development, the absorbance at 490 nm was measured with a colorimeter.
(9) For comparison between the three groups, the Mann-Whitney U-test with Bonferroni correction was used.
間接ELISA法による比較検討の結果を図４に示す。抗sHGP抗体とヒト血清IgA1との結合度はHC群(p=0.008)、OKD群(p=0.049)に比しIgA-N群で有意に高値を示した。 The result of the comparative study by the indirect ELISA method is shown in FIG. The degree of binding between the anti-sHGP antibody and human serum IgA1 was significantly higher in the IgA-N group than in the HC group (p = 0.008) and OKD group (p = 0.049).
８．HAAのIgA1結合度と抗sHGP抗体のIgA1結合度との、同一対象者間における相関性の検討
以前HAA ELISA法の検討に用いた同一者血清85例(IgA腎症34例、健常者24例、その他の腎疾患27例)について、HAA-IgA1結合度と、抗sHGP抗体-IgA1結合度の相関性の有無を検討した。その結果、両者の間に高い相関性を認めた（図５）。この結果は、IgA腎症患者の血清IgA1は糖鎖不全であることを裏付けるとともに、抗sHGP抗体の結合度がIgA腎症の診断マーカー（検査の指標）として有効であることを示す。一方、抗sHGP抗体の結合度の検出感度は、HAAのそれに比して二桁オーダーで高い。このことは、抗sHGP抗体の結合度がIgA腎症の診断ないし検査に極めて有用であることを示す。
8). Examination of the correlation between IgA1 binding degree of HAA and IgA1 binding degree of anti-sHGP antibody among the
９．抗IgA1GalNAc抗体の単離
上記の通り（６．を参照）、抗sHGP抗体はIgA1ヒンジのペプチド部分を認識する抗体（抗sHP抗体）とヒンジ上のGalNAc残基を認識する抗体（抗IgA1GalNAc抗体）を含むものであった。そこで、これらの抗体を選別することを試みた。具体的には、抗sHGP抗体をsHPカラムにかけ抗sHP抗体を吸収させることにした。その結果、抗IgA1GalNAc抗体の単離に成功した（図６）。
9. Isolation of anti-IgA1GalNAc antibody As described above (see 6.), anti-sHGP antibody is an antibody that recognizes the peptide part of IgA1 hinge (anti-sHP antibody) and an antibody that recognizes GalNAc residue on hinge (anti-IgA1GalNAc antibody) Was included. Therefore, an attempt was made to select these antibodies. Specifically, the anti-sHGP antibody was applied to the sHP column to absorb the anti-sHP antibody. As a result, the anti-IgA1GalNAc antibody was successfully isolated (FIG. 6).
１０．ヒトIgA1ヒンジ糖ペプチドの特定部位を抗原とした抗体作製
IgA1ヒンジ部に結合する糖鎖の中で最もN末端側に位置する糖鎖では、ガラクトースの欠落が最も高頻度で認められる。即ち、最も安定性が低く、IgA腎症との関係において重要と考えられる。そこで、当該部位に対応する以下の糖ペプチドを抗原として用い、当該部位を特異的に認識する抗体の作製を試みた。尚、抗体作製のための各操作は上記１．〜４．に準じた。
TPPT(GalNAc)PSPS-NH2（配列番号２）
10. Antibody production using a specific site of human IgA1 hinge glycopeptide as an antigen
Of the sugar chains that bind to the IgA1 hinge part, galactose loss is most frequently observed in the sugar chain that is located on the most N-terminal side. That is, it has the lowest stability and is considered important in relation to IgA nephropathy. Therefore, an attempt was made to produce an antibody that specifically recognizes the site using the following glycopeptide corresponding to the site as an antigen. In addition, each operation for antibody preparation is as described in 1. above. ~ 4. According to
TPPT (GalNAc) PSPS-NH 2 (SEQ ID NO: 2)
取得に成功した抗体（ウサギポリクローナル抗体）の特性を、先に取得した抗sHGP抗体と比較評価した。その結果、先に取得した抗sHGP抗体と同様に、IgA1のヒンジ部に対して高い特異性を示すことが判明した（図７）。 The characteristics of the successfully acquired antibody (rabbit polyclonal antibody) were compared with the previously acquired anti-sHGP antibody. As a result, it was found that, like the previously obtained anti-sHGP antibody, it showed high specificity for the hinge part of IgA1 (FIG. 7).
本発明の抗体はIgA腎症の検査法に利用される。本発明の抗体を用いればIgA腎症を簡便且つ低侵襲的に検査することが可能となる。 The antibody of the present invention is used in a test method for IgA nephropathy. By using the antibody of the present invention, IgA nephropathy can be examined easily and minimally invasively.
この発明は、上記発明の実施の形態及び実施例の説明に何ら限定されるものではない。特許請求の範囲の記載を逸脱せず、当業者が容易に想到できる範囲で種々の変形態様もこの発明に含まれる。
本明細書の中で明示した論文、公開特許公報、及び特許公報などの内容は、その全ての内容を援用によって引用することとする。
The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.
Claims (14)
(i)シアリダーゼ処理後のヒトIgA1検体と、請求項１〜４のいずれか一項に記載の抗体とを接触させるステップ；
(ii)ステップ(i)によって生じた免疫複合体を検出するステップ。 The method for assisting the examination of IgA nephropathy according to claim 5, comprising the following steps (i) to (ii) :
(i) contacting the human IgA1 specimen after sialidase treatment with the antibody according to any one of claims 1 to 4;
(ii) Step for detecting the immune complexes produced by step (i).
(1)固相化した抗ヒトIgA抗体にヒト検体を接触させるステップ；
(2)非特異的結合成分を除去した後、シアリダーゼで処理するステップ；
(3)請求項１〜４のいずれか一項に記載の抗体を接触させるステップ；
(4)非特異的結合成分を除去した後、特異的に結合した前記抗体を検出するステップ。 The method for assisting the examination of IgA nephropathy according to claim 5, comprising the following steps (1) to (4) :
(1) contacting a human specimen with an immobilized anti-human IgA antibody;
(2) removing non-specific binding components and then treating with sialidase;
(3) contacting the antibody according to any one of claims 1 to 4;
(4) After removing non-specific binding components, steps of detecting the antibodies specifically bind.
(4-1)非特異的結合成分を除去した後、ステップ(3)で使用する前記抗体に対する標識化抗体を接触させるステップ；
(4-2)非特異的結合成分を除去した後、特異的に結合した前記標識化抗体を検出するステップ。 The method for assisting the examination of IgA nephropathy according to claim 8, wherein step (4) comprises the following steps (4-1) and (4-2):
(4-1) After removing the non-specific binding component, contacting the labeled antibody to the antibody used in step (3);
(4-2) A step of detecting the labeled antibody specifically bound after removing the non-specific binding component.
(1)’固相化した抗ヒトIgA抗体と、シアリダーゼ処理後のヒト検体とを接触させるステップ；
(2)’非特異的結合成分を除去した後、請求項１〜４のいずれか一項に記載の抗体を接触させるステップ；
(3)’非特異的結合成分を除去した後、特異的に結合した前記抗体を検出するステップ。 The method for assisting the examination of IgA nephropathy according to claim 5, comprising the following steps (1) 'to (3)' :
(1) 'contacting the solid-phased anti-human IgA antibody with a sialidase-treated human specimen;
(2) 'contacting the antibody according to any one of claims 1 to 4 after removing the non-specific binding component;
(3) 'after removal of nonspecifically bound components, steps of detecting the antibodies specifically bind.
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