JP6935184B2 - Monoclonal antibodies that react with glycopeptides and their uses - Google Patents
Monoclonal antibodies that react with glycopeptides and their uses Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/471—Pregnancy proteins, e.g. placenta proteins, alpha-feto-protein, pregnancy specific beta glycoprotein
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
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Description
本発明は、糖ペプチドと反応するモノクローナル抗体およびその用途に関する。 The present invention relates to monoclonal antibodies that react with glycopeptides and their uses.
肝炎・肝硬変から肝臓がんへ移行する際、生体試料中にレンズ豆レクチン（LCA）に結合するα−フェトプロテイン（AFP）（LCA結合性AFP）が増加すると言われている。特許文献1には、LCA結合性AFPを検出するための抗体が記載されている。特許文献1の抗体は、結合性AFPに反応性を示し、LCA非結合性AFPには反応性を示さない旨記載されている。
It is said that α-fetoprotein (AFP) (LCA-binding AFP) that binds to lentil lectins (LCA) increases in biological samples when transitioning from hepatitis / cirrhosis to liver cancer.
特許文献1には、LCAが結合するAFPの糖鎖は、フコース（Fucose）が存在する、と記載されている。生体試料中のレンズ豆レクチンに結合するAFPの画分は、AFP-L3画分と呼ばれる。AFP-L3画分は、フコシル化AFP（AFPのアスパラギン残基にコアフコース（N型糖鎖の還元末端のN-アセチルグルコサミン（GlcNAｃ）にα1-6結合しているフコース）が付加したAFP）により構成される。
特許文献１の実施例では、LCA結合性AFP（AFP-LCA-R）に結合し、LCA非結合性AFP（AFP-LCA-NR）に結合しない抗体が取得されている（実施例1、表1、図1参照）。しかし、この抗体のエピトープは不明である。実施例では、LCA結合性と非結合性は上述の通りフコースの有無に起因しているとされており、抗原との結合性はペプチド部分の配列に依存せずフコース部分に依存する可能性がある。その場合、AFPだけでなく、それ以外のフコースを有するタンパク質にも非特異的に結合する可能性がある。そのため、糖ペプチドのフコース部分とペプチド部分のアミノ酸の両方をエピトープとするモノクローナル抗体の開発が望まれる。
In the examples of
本発明は、糖ペプチドと反応するモノクローナル抗体であって、前記糖ペプチドが、コアフコースと、糖鎖付加アスパラギンからC末側に連続した4残基以上のアミノ酸を含み、前記糖ペプチドのコアフコースと、糖ペプチドの糖鎖付加アスパラギンからC末側に3残基以上離れたアミノ酸の両方をエピトープとする、前記抗体を提供する。 The present invention is a monoclonal antibody that reacts with a glycopeptide, wherein the glycopeptide contains a core fucose and amino acids having 4 or more residues continuous from the glycosylated asparagine to the C-terminal side, and the core fucose of the glycopeptide. Provided are the above-mentioned antibody having both amino acids having 3 or more residues separated from the glycosylated asparagine of the glycopeptide on the C-terminal side as epitopes.
本発明は、上記抗体を生産する方法であって、コアフコースと、糖鎖付加アスパラギンからC末側に連続した4残基以上のアミノ酸とを含む糖ペプチド抗原を動物に免疫する工程を含む、前記方法を提供する。 The present invention is a method for producing the above-mentioned antibody, which comprises a step of immunizing an animal with a glycopeptide antigen containing core fucose and amino acids having 4 or more residues continuous from sugar chain-added asparagine to the C-terminal side. Provide a method.
本発明は、上記抗体を産生するハイブリドーマを生産する方法であって、コアフコースと、糖鎖付加アスパラギンからC末側に連続した4残基以上のアミノ酸を含む糖ペプチド抗原を動物に免疫する工程を含む、前記方法を提供する。 The present invention is a method for producing a hybridoma that produces the above-mentioned antibody, which comprises immunizing an animal with a glycopeptide antigen containing core fucose and a glycopeptide antigen containing 4 or more consecutive amino acids on the C-terminal side from glycosylated asparagine. The method is provided, including.
本発明は、以下の糖ペプチドに反応し、
本発明は、上記抗体を生産する方法であって、以下の構造を含む糖ペプチド抗原を動物に免疫する工程を含む、
（Xは、任意の糖鎖である）
前記方法を提供する。
The present invention is a method for producing the above antibody, which comprises a step of immunizing an animal with a glycopeptide antigen containing the following structure.
(X is any sugar chain)
The method is provided.
本発明は、上記抗体を産生するハイブリドーマを生産する方法であって、以下の構造を含む糖ペプチド抗原を動物に免疫する工程を含む、
前記方法を提供する。
The present invention is a method for producing a hybridoma that produces the above antibody, which comprises a step of immunizing an animal with a glycopeptide antigen containing the following structure.
The method is provided.
本発明は、上記抗体を用いる、フコシル化AFPの測定方法を提供する。 The present invention provides a method for measuring fucosylated AFP using the above antibody.
本発明は、フコシル化AFPの捕捉用抗体、フコシル化AFPの検出用抗体、固相を含み、前記捕捉用抗体または前記検出用抗体が上記抗体である、フコシル化AFP検出用キットを提供する。 The present invention provides a fucosylated AFP detection kit comprising a capture antibody for fucosylated AFP, a detection antibody for fucosylated AFP, and a solid phase, wherein the capture antibody or the detection antibody is the antibody.
本発明は、国際寄託番号がNITE BP-02263またはNITE BP-02264のハイブリドーマを提供する。 The present invention provides a hybridoma having an international deposit number of NITE BP-02263 or NITE BP-02264.
本発明は、糖ペプチドのフコース部分とペプチド部分のアミノ酸の両方をエピトープとするモノクローナル抗体を提供する。 The present invention provides a monoclonal antibody in which both the fucose portion of the glycopeptide and the amino acid of the peptide portion are epitopes.
本実施形態の抗体は、本実施形態の抗体が使用される生体試料等を用いた測定系で特異性を発揮すればよい。例えば、生体試料に含まれない物質と非特異的に結合したとしても、本実施形態の抗体が通常使用される環境で特異性を発揮すれば、本発明の作用効果を奏する。具体的には、全血、血清、血漿などの血液試料中の物質をELISAにより検出する場合、ELISA測定系において特異性を示せばよく、血液試料やELISA試薬に通常含まれない物質に結合してもよい。 The antibody of the present embodiment may exhibit specificity in a measurement system using a biological sample or the like in which the antibody of the present embodiment is used. For example, even if it binds non-specifically to a substance not contained in a biological sample, the action and effect of the present invention can be obtained if the antibody of the present embodiment exhibits specificity in an environment where it is usually used. Specifically, when a substance in a blood sample such as whole blood, serum, or plasma is detected by ELISA, it is sufficient to show specificity in the ELISA measurement system, and the substance is bound to a substance that is not normally contained in the blood sample or the ELISA reagent. You may.
本実施形態の抗体は、糖ペプチドと反応するモノクローナル抗体であって、前記糖ペプチドが、コアフコースと、糖鎖付加アスパラギンからC末側に連続した4残基以上のアミノ酸を含み、前記糖ペプチドのコアフコースと、糖ペプチドの糖鎖付加アスパラギンからC末側に3残基以上離れたアミノ酸の両方をエピトープとする。 The antibody of the present embodiment is a monoclonal antibody that reacts with a glycopeptide, wherein the glycopeptide contains core fucose and amino acids having 4 or more residues continuous from the glycosylated asparagine to the C-terminal side, and is the glycopeptide. Both core fucose and amino acids separated by 3 or more residues on the C-terminal side from glycosylated asparagine of glycopeptides are used as epitopes.
本実施形態の抗体が反応する糖ペプチドは、コアフコースと、糖鎖付加アスパラギンからC末側に連続した4残基以上のアミノ酸を含むものであればよい。 The glycopeptide with which the antibody of the present embodiment reacts may contain core fucose and amino acids having 4 or more residues continuous from the glycosylated asparagine to the C-terminal side.
本実施形態の抗体は、コアフコースと、糖鎖付加アスパラギンからC末側に連続した4残基以上のアミノ酸とを含む糖ペプチド抗原を動物に免疫する工程を含む方法で生産される。 The antibody of the present embodiment is produced by a method including a step of immunizing an animal with a glycopeptide antigen containing core fucose and amino acids having 4 or more residues contiguously on the C-terminal side from glycosylated asparagine.
糖ペプチド抗原のN末には、KLH、BSA等の生体高分子を、PEG等を介して結合させてもよい。また、これら糖ペプチド抗原のC末はアミド化等してもよい。N末に生体高分子を結合する方法は、公知の方法を利用できる。また、C末のアミド化も公知の方法を利用できる。 A biopolymer such as KLH or BSA may be bound to the N-terminal of the glycopeptide antigen via PEG or the like. Further, the C-terminal of these glycopeptide antigens may be amidated or the like. As a method for binding a biopolymer to the N-terminal, a known method can be used. Also, a known method can be used for amidation of C-terminal.
糖ペプチド抗原を動物に免疫する工程は、公知のモノクローナル抗体の生産方法における動物に免疫する工程を利用することができる。モノクローナル抗体の生産方法として、例えば、マウス脾臓法、マウス腸骨リンパ節法（特許第4098796号公報参照）等が挙げられる。 As a step of immunizing an animal with a glycopeptide antigen, a step of immunizing an animal in a known method for producing a monoclonal antibody can be utilized. Examples of the method for producing a monoclonal antibody include a mouse spleen method and a mouse iliac lymph node method (see Japanese Patent No. 4098796).
免疫される動物は特に限定されることはなく、非ヒト動物の中からモノクローナル抗体の生産方法にあわせて適宜選択すればよい。 The animal to be immunized is not particularly limited, and may be appropriately selected from non-human animals according to the method for producing the monoclonal antibody.
具体的に、モノクローナル抗体の生産方法としてマウス腸骨リンパ節法を用いる場合、糖ペプチドをKLHなどのキャリアータンパク質とコンジュゲートし、フロイントアジュバントなどと混合したエマルジョンを、糖ペプチド溶液（（2.5mg/mL）キャリアータンパク質換算）0.06mL/匹の量で少なくとも1回マウスの尾根部に免疫すればよい。 Specifically, when the mouse iliac lymph node method is used as a method for producing a monoclonal antibody, an emulsion obtained by conjugating a glycopeptide with a carrier protein such as KLH and mixing it with Freund's adjuvant is used as a glycopeptide solution ((2.5 mg /)). mL) Carrier protein equivalent) 0.06 mL / animal may be immunized at least once on the ridge of the mouse.
動物に免疫した後は、公知の方法に従って、ハイブリドーマの作製、選別等を行い、本実施形態の抗体を得ればよい。 After immunizing an animal, a hybridoma may be prepared, sorted, or the like according to a known method to obtain an antibody of the present embodiment.
ハイブリドーマの選別の際には、抗原とした糖ペプチド、抗原とした糖ペプチドからコアフコースを除去したもの、抗原とした糖ペプチドと同じ糖鎖構造を持ち異なるペプチド鎖を持つもの、を陽性抗原または陰性抗原として適宜利用すればよい。前記抗原とした糖ペプチドのアミノ酸残基を短くしたものは、例えば、糖ペプチドのC末側を、糖鎖付加アスパラギンから連続した3残基にしたものが好ましい。 When selecting hybridomas, a glycopeptide used as an antigen, a glycopeptide obtained by removing core fucose from the glycopeptide used as an antigen, or a peptide having the same sugar chain structure as the glycopeptide used as an antigen and having a different peptide chain are positive or negative. It may be appropriately used as an antigen. As for the shortened amino acid residues of the glycopeptide used as the antigen, for example, it is preferable that the C-terminal side of the glycopeptide has three consecutive residues from the glycosylated asparagine.
ハイブリドーマの選別の基準は、例えば、ELISAを用いる場合であれば、陽性抗原と陰性抗原とのOD450値の差が0.05以上であり、かつ陰性抗原のOD450値が0.05以下である。 The criteria for selecting hybridomas are, for example, when ELISA is used, the difference in OD450 value between the positive antigen and the negative antigen is 0.05 or more, and the OD450 value of the negative antigen is 0.05 or less.
本実施形態の抗体のアイソタイプは特に限定されない。また、本実施形態の抗体には、F(ab’)2、Fab’、Fab、CDRを含むペプチド等の断片も含まれる。 The isotype of the antibody of this embodiment is not particularly limited. The antibody of the present embodiment also includes fragments of peptides and the like containing F (ab') 2, Fab', Fab, and CDR.
本実施形態の抗体は、ビオチン化、ALP化等の標識化をしてもよい。 The antibody of the present embodiment may be labeled such as biotinylated and ALPylated.
以上説明した本実施形態の抗体は、上記性質を有するため、例えば、コアフコースと、糖鎖付加アスパラギンからC末側に連続した4残基以上のアミノ酸を部分配列に含む糖タンパクと特異的に反応する。 Since the antibody of the present embodiment described above has the above-mentioned properties, it specifically reacts with, for example, core fucose and a glycoprotein whose partial sequence contains amino acids having 4 or more residues continuous from the glycosylated asparagine to the C-terminal side. do.
本実施形態の抗体は、ELISA、ウェスタンブロット、免疫組織染色、免疫沈降等に利用することができる。 The antibody of this embodiment can be used for ELISA, Western blotting, immunohistochemical staining, immunoprecipitation and the like.
別の本実施形態のモノクローナル抗体は、以下の糖ペプチド（配列番号13）に特異的に反応する。
このモノクローナル抗体は、以下の糖ペプチド（配列番号14）に反応せず、
即ち、本実施形態の抗体は、コアフコースを有さず且つアミノ酸「EIQ」を有する抗原（上記（２））に反応せず、コアフコースを有し且つアミノ酸配列「EIQ」を有さない抗原（上記（３））に反応しない。このことから、本実施形態の抗体のエピトープは、コアフコースおよびアミノ酸配列「EIQ」の両方を含むと考えられる。 That is, the antibody of the present embodiment does not react with the antigen having no core fucose and having the amino acid "EIQ" (the above (2)), and has the core fucose and does not have the amino acid sequence "EIQ" (the above). Does not react to (3)). From this, it is considered that the epitope of the antibody of the present embodiment contains both core fucose and the amino acid sequence “EIQ”.
なお、本明細書において、本実施形態の抗体が「糖ペプチドと反応する」とは、抗原抗体反応により糖ペプチドと抗体とが結合することをいう。 In addition, in this specification, "reacting with a glycopeptide" by an antibody of this embodiment means that a glycopeptide and an antibody bind to each other by an antigen-antibody reaction.
更に、上記抗体は、以下の糖ペプチド（配列番号16）に反応しないことが好ましい。
更に、上記抗体は、フコシル化AFPに反応するものが好ましい。 Further, the antibody is preferably one that reacts with fucosylated AFP.
更に、上記抗体は、SDSなどの変性剤や熱などで前処理した変性フコシル化AFPと結合し得る。前処理としてSDSを含む溶液を用いる場合の、フコシル化AFPを十分に変性させるためのSDS濃度（以下、これを「前処理時SDS濃度」という）は、特に限定されないが、0.03質量／質量％（以下、単に「％」という）以上であることが好ましい。一方、抗原抗体反応時は変性剤が過剰に含まれると、抗体も変性して抗原抗体反応に好ましくない影響が生じる可能性があるため、希釈などによって変性剤の濃度を低下させることが好ましい。変性剤としてSDSを含む溶液を用いる場合、抗原抗体反応時のSDS濃度（以下、これを「最終SDS濃度」という）は、特に限定されないが、0.025％以下であることが好ましい。 Furthermore, the antibody can bind to a denaturing agent such as SDS or a modified fucosylated AFP pretreated with heat or the like. When a solution containing SDS is used as the pretreatment, the SDS concentration for sufficiently modifying the fucosylated AFP (hereinafter, this is referred to as “pretreatment SDS concentration”) is not particularly limited, but is 0.03% by mass / mass%. (Hereinafter, simply referred to as "%") or more is preferable. On the other hand, if an excessive amount of the denaturing agent is contained during the antigen-antibody reaction, the antibody may also be denatured and an unfavorable effect on the antigen-antibody reaction may occur. Therefore, it is preferable to reduce the concentration of the denaturing agent by dilution or the like. When a solution containing SDS is used as the denaturing agent, the SDS concentration at the time of the antigen-antibody reaction (hereinafter, this is referred to as "final SDS concentration") is not particularly limited, but is preferably 0.025% or less.
なお、本明細書において、本実施形態の抗体が「フコシル化AFPと反応する」とは、抗原抗体反応によりフコシル化AFPと抗体とが結合することをいう。フコシル化AFPは、組換え体、天然の何れでもよい。天然のフコシル化AFPは、たとえば、ヒト血中に存在するAFPである。ヒトAFPの配列は、たとえば、GenBank Accession No. NM_001134に登録されており、配列番号25のアミノ酸配列を有する。 In addition, in this specification, "reacting with fucosylated AFP" by the antibody of this embodiment means that fucosylated AFP and an antibody bind to each other by an antigen-antibody reaction. Fucosylated AFP may be recombinant or natural. Natural fucosylated AFP is, for example, AFP present in human blood. The sequence of human AFP is registered in, for example, GenBank Accession No. NM_001134 and has the amino acid sequence of SEQ ID NO: 25.
更に、上記抗体は、SDSおよびDTTの存在下で変性したフコシル化AFPに反応するものが好ましい。変性の条件は、2％のSDS、50mMのDTT存在下、常温（25℃）での反応である。 Further, the antibody is preferably one that reacts with fucosylated AFP denatured in the presence of SDS and DTT. The conditions for denaturation are the reaction at room temperature (25 ° C) in the presence of 2% SDS and 50 mM DTT.
更に、上記抗体は、SDSおよびDTTの存在下で変性した非フコシル化AFPに反応しないものが好ましい。変性の条件は、2％のSDS、50mMのDTT存在下、常温（25℃）での反応である。 Furthermore, the antibody is preferably one that does not react with the denatured non-fucosylated AFP in the presence of SDS and DTT. The conditions for denaturation are the reaction at room temperature (25 ° C) in the presence of 2% SDS and 50 mM DTT.
本実施形態の抗体のCDRとしては、例えば、以下のものが挙げられる。 Examples of the CDR of the antibody of this embodiment include the following.
＜CDR-A＞
重鎖のCDRが、配列番号1で示されるアミノ酸配列、配列番号2で示されるアミノ酸配列、配列番号3で示されるアミノ酸配列を含む。
軽鎖のCDRが、配列番号4で示されるアミノ酸配列、配列番号5で示されるアミノ酸配列、配列番号6で示されるアミノ酸配列を含む。
<CDR-A>
The CDR of the heavy chain contains the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence shown in SEQ ID NO: 2, and the amino acid sequence shown in SEQ ID NO: 3.
The CDR of the light chain comprises the amino acid sequence set forth in SEQ ID NO: 4, the amino acid sequence set forth in SEQ ID NO: 5, and the amino acid sequence set forth in SEQ ID NO: 6.
＜CDR-B＞
重鎖のCDRが、配列番号7で示されるアミノ酸配列、配列番号8で示されるアミノ酸配列、配列番号9で示されるアミノ酸配列を含む。
軽鎖のCDRが、配列番号10で示されるアミノ酸配列、配列番号11で示されるアミノ酸配列、配列番号12で示されるアミノ酸配列を含む。
<CDR-B>
The heavy chain CDR comprises the amino acid sequence set forth in SEQ ID NO: 7, the amino acid sequence set forth in SEQ ID NO: 8, and the amino acid sequence set forth in SEQ ID NO: 9.
The CDR of the light chain comprises the amino acid sequence set forth in SEQ ID NO: 10, the amino acid sequence set forth in SEQ ID NO: 11, and the amino acid sequence set forth in SEQ ID NO: 12.
CDRとして、上記CDR-A、CDR-Bを有する本実施形態の抗体を産生するハイブリドーマは、それぞれI2-1F8、I2-2F11と名付け、2016年5月20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（〒292-0818 日本国千葉県木更津市かずさ鎌足2丁目5番地8 122号室）にNITE BP-02264、NITE BP-02263として国際寄託した。 The hybridomas that produce the antibodies of this embodiment having the above CDR-A and CDR-B as CDRs were named I2-1F8 and I2-2F11, respectively, and were named I2-1F8 and I2-2F11 on May 20, 2016 by the National Institute of Technology and Evaluation. International deposits were made as NITE BP-02264 and NITE BP-02263 at the Patent Microorganisms Depositary Center (Room 2-5-8 122, Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818).
本実施形態の抗体は、以下の構造を含む糖ペプチド抗原（配列番号17）を動物に免疫する工程を含む方法により得られる。
糖ペプチド抗原（配列番号18）としては、以下のものがより好ましい。
糖ペプチド抗原のN末には、KLH等の生体高分子を、PEG等を介して結合させてもよい。また、これら糖ペプチド抗原のC末はアミド化等してもよい。N末に生体高分子を結合する方法は、公知の方法を利用できる。また、C末のアミド化も公知の方法を利用できる。 A biopolymer such as KLH may be bound to the N-terminal of the glycopeptide antigen via PEG or the like. Further, the C-terminal of these glycopeptide antigens may be amidated or the like. As a method for binding a biopolymer to the N-terminal, a known method can be used. Also, a known method can be used for amidation of C-terminal.
糖ペプチド抗原を動物に免疫する工程は、公知のモノクローナル抗体の生産方法における動物に免疫する工程を利用することができる。モノクローナル抗体の生産方法として、例えば、マウス脾臓法、マウス腸骨リンパ節法（特許第4098796号公報参照）等が挙げられる。 As a step of immunizing an animal with a glycopeptide antigen, a step of immunizing an animal in a known method for producing a monoclonal antibody can be utilized. Examples of the method for producing a monoclonal antibody include a mouse spleen method and a mouse iliac lymph node method (see Japanese Patent No. 4098796).
免疫される動物は特に限定されることはなく、非ヒト動物の中からモノクローナル抗体の生産方法にあわせて適宜選択すればよい。 The animal to be immunized is not particularly limited, and may be appropriately selected from non-human animals according to the method for producing the monoclonal antibody.
具体的に、モノクローナル抗体の生産方法としてマウス腸骨リンパ節法を用いる場合、糖ペプチドをKLHなどのキャリアータンパク質とコンジュゲートし、フロイントアジュバントなどと混合したエマルジョンを、糖ペプチド溶液（（2.5mg/mL）キャリアータンパク質換算）0.06mL/匹の量で少なくとも1回マウスの尾根部に免疫すればよい。 Specifically, when the mouse iliac lymph node method is used as a method for producing a monoclonal antibody, an emulsion obtained by conjugating a glycopeptide with a carrier protein such as KLH and mixing it with Freund's adjuvant is used as a glycopeptide solution ((2.5 mg /)). mL) Carrier protein equivalent) 0.06 mL / animal may be immunized at least once on the ridge of the mouse.
動物に免疫した後は、公知の方法に従って、ハイブリドーマの作製、選別等を行い、本実施形態の抗体を得ればよい。 After immunizing an animal, a hybridoma may be prepared, sorted, or the like according to a known method to obtain an antibody of the present embodiment.
ハイブリドーマの選別の際には、抗原とした糖ペプチド、抗原とした糖ペプチドからコアフコースを除去したもの、抗原とした糖ペプチドのアミノ酸残基を短くしたもの、SDSおよびDTTの存在下で変性したフコシル化AFP、SDSおよびDTTの存在下で変性した非フコシル化AFP、コアフコースを有し且つAFPとは異なるアミノ酸配列のポリペプチドを有する糖ペプチドまたは糖タンパク質（例えば、フコシル化ALPなど）等を陽性抗原または陰性抗原として適宜利用すればよい。前記抗原とした糖ペプチドのアミノ酸残基を短くしたものは、例えば、糖ペプチドのC末側を、糖鎖付加アスパラギンから連続した3残基にしたものが好ましい。 When selecting hybridomas, glycopeptides used as antigens, those in which core fucose was removed from glycopeptides used as antigens, those in which the amino acid residues of glycopeptides used as antigens were shortened, and fucosyl modified in the presence of SDS and DTT. Non-fucosylated AFP denatured in the presence of AFP, SDS and DTT, glycopeptides or glycoproteins having a core fucose and a polypeptide having an amino acid sequence different from that of AFP (for example, fucosylated ALP) and the like as positive antigens. Alternatively, it may be appropriately used as a negative antigen. As for the shortened amino acid residues of the glycopeptide used as the antigen, for example, it is preferable that the C-terminal side of the glycopeptide has three consecutive residues from the glycosylated asparagine.
ハイブリドーマの選別の基準は、例えば、ELISAを用いる場合であれば、陽性抗原と陰性抗原とのOD450値の差が0.05以上であり、かつ陰性抗原のOD450値が0.05以下である。 The criteria for selecting hybridomas are, for example, when ELISA is used, the difference in OD450 value between the positive antigen and the negative antigen is 0.05 or more, and the OD450 value of the negative antigen is 0.05 or less.
本実施形態の抗体のアイソタイプは特に限定されない。また、本実施形態の抗体には、F(ab’)2、Fab’、Fab、CDRを含むペプチド等の断片も含まれる。 The isotype of the antibody of this embodiment is not particularly limited. The antibody of the present embodiment also includes fragments of peptides and the like containing F (ab') 2, Fab', Fab, and CDR.
本実施形態の抗体は、ビオチン化、ALP化等の標識化をしてもよい。 The antibody of the present embodiment may be labeled such as biotinylated and ALPylated.
本実施形態の抗体は、上記性質を有するため、フコシル化AFPと反応し、非フコシル化AFPと反応しない。そのため、本実施形態の抗体は、例えば、生体試料中のフコシル化AFPを測定することができる。 Since the antibody of the present embodiment has the above-mentioned properties, it reacts with fucosylated AFP and does not react with non-fucosylated AFP. Therefore, the antibody of the present embodiment can measure, for example, fucosylated AFP in a biological sample.
生体試料としては、例えば、被験者から採取した全血、血清、血漿等が挙げられる。この生体試料は、遠心分離、変性処理等の前処理を行ってもよい。生体試料は変性処理を行うことが好ましい。変性処理の条件は、2％のSDS、50mMのDTT存在下、常温（25℃）の反応である。 Examples of the biological sample include whole blood, serum, plasma and the like collected from the subject. This biological sample may be subjected to pretreatment such as centrifugation or denaturation. The biological sample is preferably denatured. The conditions for the denaturation treatment are a reaction at room temperature (25 ° C) in the presence of 2% SDS and 50 mM DTT.
本実施形態の抗体を用いて、フコシル化AFPを測定するには、公知の免疫学的測定方法が利用できる。免疫学的測定法としては、例えば、酵素結合免疫吸着法（ＥＬＩＳＡ法）、免疫複合体転移測定方法（特開平1-254868号公報参照）、免疫比濁法、イムノクロマト法、ラテックス凝集法等が挙げられる。測定工程の一例として、サンドイッチELISA法により生体試料中のフコシル化AFP濃度を測定する場合を以下に説明する。 A known immunological measurement method can be used to measure fucosylated AFP using the antibody of the present embodiment. Examples of the immunological measurement method include an enzyme-linked immunosorbent assay (ELISA method), an immune complex transfer measurement method (see JP-A 1-254868), an immunoturbidimetry method, an immunochromatography method, and a latex aggregation method. Can be mentioned. As an example of the measurement process, the case where the fucosylated AFP concentration in the biological sample is measured by the sandwich ELISA method will be described below.
まず、生体試料中のフコシル化AFPを捕捉するための抗体(以下、「捕捉用抗体」ともいう)と、フコシル化AFPを検出するための抗体(以下、「検出用抗体」ともいう)と、フコシル化AFPとを含む複合体を固相上に形成させる。この複合体は、生体試料にフコシル化AFPが含まれる場合は、生体試料と、捕捉用抗体と、検出用抗体とを混合することにより形成できる。そして、複合体を含む溶液を、捕捉用抗体を捕捉できる固相と接触させることにより、上記の複合体を固相上に形成させることができる。あるいは、捕捉用抗体をあらかじめ固定した固相を用いてもよい。すなわち、捕捉用抗体を固定した固相と、生体試料と、検出用抗体とを接触させることにより、上記の複合体を固相上に形成させることができる。本実施形態の抗体は、捕捉用抗体および検出用抗体の少なくとも一方に用いることができる。 First, an antibody for capturing fucosylated AFP in a biological sample (hereinafter, also referred to as “capture antibody”) and an antibody for detecting fucosylated AFP (hereinafter, also referred to as “detection antibody”). A complex containing fucosylated AFP is formed on the solid phase. When the biological sample contains fucosylated AFP, this complex can be formed by mixing the biological sample, the capture antibody, and the detection antibody. Then, by contacting the solution containing the complex with a solid phase capable of capturing the capture antibody, the above complex can be formed on the solid phase. Alternatively, a solid phase in which a capture antibody is immobilized in advance may be used. That is, the above complex can be formed on the solid phase by contacting the solid phase on which the capture antibody is immobilized, the biological sample, and the detection antibody. The antibody of the present embodiment can be used for at least one of a capture antibody and a detection antibody.
捕捉用抗体の固相への固定の態様は特に限定されない。例えば、捕捉用抗体と固相とを直接結合させてもよいし、捕捉用抗体と固相とを別の物質を介して間接的に結合させてもよい。直接の結合としては、例えば、物理的吸着等が挙げられる。間接的な結合としては、例えば、ビオチンと、アビジンまたはストレプトアビジン（以下、「アビジン類」ともいう）との組み合わせを介した結合が挙げられる。この場合、捕捉用抗体をあらかじめビオチンで修飾し、固相にアビジン類をあらかじめ結合させておくことにより、ビオチンとアビジン類との結合を介して、捕捉用抗体と固相とを間接的に結合させることができる。 The mode of fixing the capture antibody to the solid phase is not particularly limited. For example, the capture antibody and the solid phase may be directly bound, or the capture antibody and the solid phase may be indirectly bound via another substance. Examples of the direct bond include physical adsorption and the like. Examples of the indirect binding include binding via a combination of biotin and avidin or streptavidin (hereinafter, also referred to as "avidins"). In this case, by modifying the capture antibody with biotin in advance and binding the avidins to the solid phase in advance, the capture antibody and the solid phase are indirectly bound via the binding between biotin and the avidins. Can be made to.
固相の素材は特に限定されず、例えば、有機高分子化合物、無機化合物、生体高分子等から選択できる。有機高分子化合物としては、ラテックス、ポリスチレン、ポリプロピレン等が挙げられる。無機化合物としては、磁性体(酸化鉄、酸化クロムおよびフェライト等)、シリカ、アルミナ、ガラス等が挙げられる。生体高分子としては、不溶性アガロース、不溶性デキストラン、ゼラチン、セルロース等が挙げられる。これらのうちの2種以上を組み合わせて用いてもよい。固相の形状は特に限定されず、例えば、粒子、膜、マイクロプレート、マイクロチューブ、試験管等が挙げられる。 The material of the solid phase is not particularly limited, and can be selected from, for example, an organic polymer compound, an inorganic compound, a biopolymer and the like. Examples of the organic polymer compound include latex, polystyrene, polypropylene and the like. Examples of the inorganic compound include magnetic substances (iron oxide, chromium oxide, ferrite, etc.), silica, alumina, glass, and the like. Examples of the biopolymer include insoluble agarose, insoluble dextran, gelatin, cellulose and the like. Two or more of these may be used in combination. The shape of the solid phase is not particularly limited, and examples thereof include particles, membranes, microplates, microtubes, and test tubes.
固相上に形成された複合体を、当該技術において公知の方法で検出することにより、生体試料におけるフコシル化AFPの測定値を取得できる。例えば、検出用抗体として、標識物質で標識した抗体を用いた場合は、その標識物質により生じるシグナルを検出することにより、フコシル化AFPの測定値を取得できる。あるいは、検出用抗体に対する標識二次抗体を用いた場合も、同様にしてフコシル化AFPの測定値を取得できる。 By detecting the complex formed on the solid phase by a method known in the art, the measured value of fucosylated AFP in the biological sample can be obtained. For example, when an antibody labeled with a labeling substance is used as the detection antibody, the measured value of fucosylated AFP can be obtained by detecting the signal generated by the labeling substance. Alternatively, when a labeled secondary antibody against the detection antibody is used, the measured value of fucosylated AFP can be obtained in the same manner.
本実施形態では、フコシル化AFPを、上記のようにして前処理することが好ましい。前処理としてSDSを含む溶液を用いる場合の、前処理時SDS濃度は、特に限定されないが、0.03％以上であることが好ましい。抗原抗体反応時は、上記の通り、希釈などによって変性剤の濃度を低下させることが好ましい。変性剤としてSDSを含む溶液を用いる場合、最終SDS濃度は、特に限定されないが、0.025％以下であることが好ましい。本実施形態では、このような処理をして、前記抗体と前記フコシル化AFPとを反応させてフコシル化AFPの測定値を取得することが好ましい。 In this embodiment, the fucosylated AFP is preferably pretreated as described above. When a solution containing SDS is used as the pretreatment, the SDS concentration during the pretreatment is not particularly limited, but is preferably 0.03% or more. At the time of the antigen-antibody reaction, it is preferable to reduce the concentration of the denaturing agent by dilution or the like as described above. When a solution containing SDS is used as the denaturing agent, the final SDS concentration is not particularly limited, but is preferably 0.025% or less. In the present embodiment, it is preferable to carry out such a treatment to react the antibody with the fucosylated AFP to obtain a measured value of the fucosylated AFP.
本実施形態では、複合体の形成工程と複合体の検出工程との間に、複合体を形成していない未反応の遊離成分を除去するB/F(Bound/Free)分離を行ってもよい。未反応の遊離成分とは、複合体を構成しない成分をいう。例えば、フコシル化AFPと結合しなかった抗体、生体試料中のフコシル化AFP以外の物質（夾雑物質）等が挙げられる。B/F分離の手段は特に限定されないが、固相が粒子であれば、遠心分離により、複合体を捕捉した固相だけを回収することによりB/F分離ができる。固相がマイクロプレートやマイクロチューブ等の容器であれば、未反応の遊離成分を含む液を除去することによりB/F分離ができる。また、固相が磁性粒子の場合は、磁石で磁性粒子を磁気的に拘束した状態でノズルによって未反応の遊離成分を含む液を吸引除去することによりB/F分離ができる。未反応の遊離成分を除去した後、複合体を捕捉した固相をPBS等の適切な水性媒体で洗浄してもよい。 In the present embodiment, B / F (Bound / Free) separation for removing unreacted free components that do not form a complex may be performed between the complex formation step and the complex detection step. .. The unreacted free component refers to a component that does not form a complex. For example, an antibody that does not bind to fucosylated AFP, a substance other than fucosylated AFP (contaminant substance) in a biological sample, and the like can be mentioned. The means for B / F separation is not particularly limited, but if the solid phase is particles, B / F separation can be performed by recovering only the solid phase in which the complex is captured by centrifugation. If the solid phase is a container such as a microplate or microtube, B / F separation can be performed by removing the liquid containing unreacted free components. When the solid phase is magnetic particles, B / F separation can be performed by suctioning and removing the liquid containing the unreacted free component with a nozzle while the magnetic particles are magnetically constrained by a magnet. After removing the unreacted free components, the solid phase trapped in the complex may be washed with a suitable aqueous medium such as PBS.
本明細書において、「シグナルを検出する」とは、シグナルの有無を定性的に検出すること、シグナル強度を定量すること、および、シグナルの強度を半定量的に検出することを含む。半定量的な検出とは、シグナルの強度を、「シグナル発生せず」、「弱」、「中」、「強」等のように段階的に示すことをいう。本実施形態では、シグナルの強度を定量的または半定量的に検出することが好ましい。 As used herein, "detecting a signal" includes qualitatively detecting the presence or absence of a signal, quantifying the signal intensity, and semi-quantitatively detecting the signal intensity. Semi-quantitative detection means that the signal intensity is indicated stepwise, such as "no signal generated", "weak", "medium", "strong", and the like. In this embodiment, it is preferable to detect the signal intensity quantitatively or semi-quantitatively.
標識物質は、検出可能なシグナルが生じるかぎり特に限定されない。例えば、それ自体がシグナルを発生する物質（以下、「シグナル発生物質」ともいう）であってもよいし、他の物質の反応を触媒してシグナルを発生させる物質であってもよい。シグナル発生物質としては、例えば、蛍光物質、放射性同位元素等が挙げられる。他の物質の反応を触媒して検出可能なシグナルを発生させる物質としては、例えば、酵素等が挙げられる。酵素としては、アルカリホスファターゼ、ペルオキシダーゼ、β−ガラクトシダーゼ、ルシフェラーゼ等が挙げられる。蛍光物質としては、フルオレセインイソチオシアネート(FITC)、ローダミン、Alexa Fluor（登録商標）等の蛍光色素、GFP等の蛍光タンパク質等が挙げられる。放射性同位元素としては、125I、14C、32P等が挙げられる。それらの中でも、標識物質として、酵素が好ましく、アルカリホスファターゼおよびペルオキシダーゼが特に好ましい。 The labeling substance is not particularly limited as long as a detectable signal is generated. For example, it may be a substance that generates a signal by itself (hereinafter, also referred to as a “signal generating substance”), or may be a substance that catalyzes the reaction of another substance to generate a signal. Examples of the signal generating substance include a fluorescent substance, a radioisotope and the like. Examples of substances that catalyze the reaction of other substances to generate a detectable signal include enzymes and the like. Examples of the enzyme include alkaline phosphatase, peroxidase, β-galactosidase, luciferase and the like. Examples of the fluorescent substance include fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine and Alexa Fluor (registered trademark), and fluorescent proteins such as GFP. Radioisotopes include 125 I, 14 C, 32 P and the like. Among them, enzymes are preferable as labeling substances, and alkaline phosphatase and peroxidase are particularly preferable.
シグナルを検出する方法自体は、当該技術において公知である。本実施形態では、上記の標識物質に由来するシグナルの種類に応じた測定方法を適宜選択すればよい。例えば、標識物質が酵素である場合、該酵素に対する基質を反応させることによって発生する光、色等のシグナルを、分光光度計等の公知の装置を用いて測定することにより行うことができる。 The method itself for detecting a signal is known in the art. In the present embodiment, the measuring method according to the type of the signal derived from the above-mentioned labeling substance may be appropriately selected. For example, when the labeling substance is an enzyme, signals such as light and color generated by reacting a substrate with the enzyme can be measured using a known device such as a spectrophotometer.
酵素の基質は、該酵素の種類に応じて公知の基質から適宜選択できる。例えば、酵素としてアルカリホスファターゼを用いる場合、基質として、CDP-Star(登録商標)(4-クロロ-3-(メトキシスピロ[1, 2-ジオキセタン-3, 2'-(5'-クロロ)トリクシロ[3. 3. 1. 13, 7]デカン]-4-イル)フェニルリン酸２ナトリウム)、CSPD(登録商標)(3-(4-メトキシスピロ[1, 2-ジオキセタン-3, 2-(5'-クロロ)トリシクロ[3. 3. 1. 13, 7]デカン]-4-イル)フェニルリン酸2ナトリウム)等の化学発光基質、5-ブロモ-4-クロロ-3-インドリルリン酸(BCIP)、5-ブロモ-6-クロロ−インドリルリン酸2ナトリウム、p-ニトロフェニルリン酸等の発色基質が挙げられる。また、酵素としてペルオキシダーゼを用いる場合、基質としては、ルミノールおよびその誘導体等の化学発光基質、2, 2'-アジノビス(3-エチルベンゾチアゾリン-6-スルホン酸アンモニウム)(ABTS)、1, 2-フェニレンジアミン(OPD)、3, 3', 5, 5'-テトラメチルベンジジン(TMB)等の発色基質が挙げられる。 The substrate of the enzyme can be appropriately selected from known substrates according to the type of the enzyme. For example, when alkaline phosphatase is used as the enzyme, CDP-Star® (4-chloro-3- (methoxyspiro [1, 2-dioxetane-3, 2'-(5'-chloro) trixhiro] [ 3. 3. 1. 13, 7] Decane] -4-yl) disodium phenylphosphate), CSPD® (3- (4-methoxyspiro [1, 2-dioxetane-3, 2- (5) Chemically luminescent substrates such as'-chloro) tricyclo [3. 3. 1. 13, 7] decane] -4-yl) disodium phenylphosphate), 5-bromo-4-chloro-3-indrill phosphate ( BCIP), 2-sodium 5-bromo-6-chloro-indrill phosphate, p-nitrophenyl phosphate and other color-developing substrates can be mentioned. When peroxidase is used as the enzyme, the substrate is a chemiluminescent substrate such as luminol and its derivatives, 2, 2'-azinobis (3-ethylbenzothiazolin-6-ammonium sulfonate) (ABTS), 1, 2- Color-developing substrates such as phenylenediamine (OPD), 3, 3', 5, 5'-tetramethylbenzidine (TMB) can be mentioned.
標識物質が放射性同位体である場合は、シグナルとしての放射線を、シンチレーションカウンター等の公知の装置を用いて測定できる。また、標識物質が蛍光物質である場合は、シグナルとしての蛍光を、蛍光マイクロプレートリーダー等の公知の装置を用いて測定できる。なお、励起波長および蛍光波長は、用いた蛍光物質の種類に応じて適宜決定できる。 When the labeling substance is a radioisotope, the radiation as a signal can be measured using a known device such as a scintillation counter. When the labeling substance is a fluorescent substance, the fluorescence as a signal can be measured using a known device such as a fluorescent microplate reader. The excitation wavelength and the fluorescence wavelength can be appropriately determined according to the type of the fluorescent substance used.
シグナルの検出結果は、フコシル化AFPの測定値として用いてもよい。例えば、シグナルの強度を定量的に検出する場合は、シグナル強度の測定値自体または該測定値から取得される値を、フコシル化AFPの測定値として用いることができる。シグナル強度の測定値から取得される値としては、例えば、フコシル化AFPの測定値から陰性対照試料の測定値またはバックグラウンドの値を差し引いた値等が挙げられる。陰性対照試料は適宜選択できるが、例えば、健常者から得た生体試料等が挙げられる。 The signal detection result may be used as a measured value of fucosylated AFP. For example, when the signal intensity is quantitatively detected, the measured value of the signal intensity itself or the value obtained from the measured value can be used as the measured value of fucosylated AFP. Examples of the value obtained from the measured value of the signal intensity include a value obtained by subtracting the measured value of the negative control sample or the background value from the measured value of fucosylated AFP. The negative control sample can be appropriately selected, and examples thereof include biological samples obtained from healthy subjects.
本実施形態では、フコシル化AFP濃度が既知の複数の標準試料についてフコシル化AFPの測定値を取得し、フコシル化AFP濃度とフコシル化AFPの測定値との関係を示す検量線を作成してもよい。生体試料から取得したフコシル化AFPの測定値をこの検量線に当てはめて、生体試料中のフコシル化AFP濃度の値を取得することができる。 In the present embodiment, even if the measured values of fucosylated AFP are acquired for a plurality of standard samples having known fucosylated AFP concentrations and a calibration curve showing the relationship between the fucosylated AFP concentration and the measured values of fucosylated AFP is created. good. By applying the measured value of fucosylated AFP obtained from the biological sample to this calibration curve, the value of the fucosylated AFP concentration in the biological sample can be obtained.
本実施形態では、磁性粒子に固定された捕捉用抗体と、標識物質で標識された検出用抗体とを用いるサンドイッチELISA法により、生体試料中のフコシル化AFP濃度を測定してもよい。この場合、測定は、HISCLシリーズ（シスメックス株式会社製）等の市販の全自動免疫測定装置を用いて行ってもよい。 In the present embodiment, the fucosylated AFP concentration in a biological sample may be measured by a sandwich ELISA method using a capture antibody immobilized on magnetic particles and a detection antibody labeled with a labeling substance. In this case, the measurement may be performed using a commercially available fully automatic immunoassay device such as the HISCL series (manufactured by Sysmex Corporation).
また、本実施形態の抗体は、フコシル化AFP検出用キットに利用することができる。本実施形態のフコシル化AFP検出用キットは、捕捉用抗体、検出用抗体、固相を含む。捕捉用抗体または検出用抗体として、本実施形態の抗体が用いられ得る。サンドイッチイムノアッセイにおいては、捕捉用抗体および検出用抗体のいずれかに本実施形態の抗体を用いることができる。 In addition, the antibody of the present embodiment can be used in a kit for detecting fucosylated AFP. The fucosylated AFP detection kit of the present embodiment includes a capture antibody, a detection antibody, and a solid phase. The antibody of the present embodiment can be used as a capture antibody or a detection antibody. In the sandwich immunoassay, the antibody of the present embodiment can be used as either a capture antibody or a detection antibody.
本実施形態のフコシル化AFP検出用キットは、更に、検出用抗体の標識物質が酵素である場合、本実施形態のフコシル化AFP検出用キットは、酵素に対する基質を更に含んでいてもよい。標識物質および基質の形態は特に限定されず、固体（例えば、粉末、結晶、凍結乾燥品等）であってもよいし、液体（例えば、溶液、懸濁液、乳濁液等）であってもよい。 In the fucosylated AFP detection kit of the present embodiment, when the labeling substance of the detection antibody is an enzyme, the fucosylated AFP detection kit of the present embodiment may further contain a substrate for the enzyme. The form of the labeling substance and the substrate is not particularly limited, and may be a solid (for example, powder, crystal, lyophilized product, etc.) or a liquid (for example, solution, suspension, emulsion, etc.). May be good.
本実施形態のフコシル化AFP検出用キットは、上記したフコシル化AFPを前処理するために、更にSDSを0.03％以上含む前処理試薬を含んでもよい。前記試薬は上記したSDSを0.03％以上含む溶液と同様である。 The fucosylated AFP detection kit of the present embodiment may further contain a pretreatment reagent containing 0.03% or more of SDS in order to pretreat the fucosylated AFP described above. The reagent is the same as the above-mentioned solution containing 0.03% or more of SDS.
本実施形態のフコシル化AFP検出用キットは、更に、生体試料の前処理液、固相の洗浄液、酵素反応の停止剤、キャリブレーター等を適宜含んでいてもよい。 The fucosylated AFP detection kit of the present embodiment may further appropriately contain a pretreatment liquid for a biological sample, a solid phase cleaning liquid, an enzyme reaction terminator, a calibrator and the like.
本実施形態のフコシル化AFP検出用キットは、捕捉用抗体、検出用抗体、固相等をキットの形態にあわせて適宜容器に収容するか、個別に包装すればよい。本実施形態のフコシル化AFP検出用キットにおいて、捕捉用抗体は固相に直接結合させてもよいし、捕捉用抗体と固相とを、別の物質を介して間接的に結合させてもよい。捕捉用抗体と固相とを間接的に結合させる場合は、本実施形態のキットにおいて捕捉用抗体と固相とを別々の容器に収容してもよい。捕捉用抗体と固相とを、例えば、ビオチンとアビジン類を介して間接的に結合させる場合、ビオチンで修飾した捕捉用抗体を1つの容器に収容し、アビジン類を結合した固相を別の容器に収容すればよい。なお、生体試料、捕捉用抗体、検出用抗体、固相等についての詳細は、上記の測定方法の説明で述べたことと同様である。 In the fucosylated AFP detection kit of the present embodiment, the capture antibody, the detection antibody, the solid phase and the like may be appropriately contained in a container or individually packaged according to the form of the kit. In the fucosylated AFP detection kit of the present embodiment, the capture antibody may be directly bound to the solid phase, or the capture antibody and the solid phase may be indirectly bound via another substance. .. When the capture antibody and the solid phase are indirectly bound, the capture antibody and the solid phase may be contained in separate containers in the kit of the present embodiment. When the capture antibody and the solid phase are indirectly bound to each other via, for example, biotin and avidins, the capture antibody modified with biotin is contained in one container, and the solid phase to which the avidins are bound is contained in another container. It may be contained in a container. The details of the biological sample, the capture antibody, the detection antibody, the solid phase, and the like are the same as those described in the above description of the measurement method.
フコシル化AFPは肝臓癌との関連が知られている。そのため、本実施形態のフコシル化AFP検出用キットは、肝臓癌の診断に用いられ得る。 Fucosylated AFP is known to be associated with liver cancer. Therefore, the fucosylated AFP detection kit of the present embodiment can be used for the diagnosis of liver cancer.
以下、本発明を実施例により詳細に説明するが、本発明はこれら実施例に何ら限定されるものではない。 Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples.
実施例１
抗体の取得：
（１）抗体群の取得
マウス腸骨リンパ節法（特許第4098796号）により抗体群を産生するハイブリドーマを得た。具体的には、下記表1に記載の構造の糖ペプチドA（配列番号19）を合成し、これをKLHとコンジュゲートし、フロイントコンプリートアジュバントと2.5mg/mL（キャリアータンパク質換算）となるように混合したエマルジョンを、0.06mL/匹でマウスの尾根部に1回注射することにより行われた。また、免疫17日後に、2.5mg/mL（キャリアータンパク質換算）のKLHコンジュゲート糖ペプチドA溶液を、0.06mL/匹でマウスの尾根部に1回注射することにより追加免疫を行った。更に、追加免疫の4日後、腸骨リンパ節よりリンパ球を単離し、ミエローマと細胞融合させハイブリドーマを得た。
Example 1
Acquisition of antibody:
(1) Acquisition of antibody group A hybridoma producing an antibody group was obtained by the mouse iliac lymph node method (Patent No. 4098796). Specifically, glycopeptide A (SEQ ID NO: 19) having the structure shown in Table 1 below is synthesized and conjugated with KLH so that it becomes 2.5 mg / mL (carrier protein equivalent) with Freund's complete adjuvant. The mixed emulsion was made by injecting 0.06 mL / animal once into the ridge of the mouse. In addition, 17 days after immunization, booster immunization was performed by injecting 2.5 mg / mL (carrier protein equivalent) KLH conjugated glycopeptide A solution at 0.06 mL / animal once into the ridge of the mouse. Furthermore, 4 days after booster immunization, lymphocytes were isolated from the iliac lymph nodes and fused with myeloma to obtain hybridomas.
糖ペプチドのN末は、KLH-PEG4であり、C末はアミド化されている。
The N-terminal of the glycopeptide is KLH-PEG4, and the C-terminal is amidated.
（２）一次スクリーニング
上記（１）で得たハイブリドーマから表2に記載の陽性抗原（糖ペプチドA）または陰性抗原（非フコシル化糖ペプチドA：配列番号20）を用いた抗原固相ELISAにより、陽性抗原に反応を示し、陰性抗原への反応が少ないウェルを選定した。抗原固相ELISAは以下の手法で行った。結果を表3に示した。
(2) Primary screening By antigen solid phase ELISA using the positive antigen (glycopeptide A) or negative antigen (non-fucosylated glycopeptide A: SEQ ID NO: 20) shown in Table 2 from the hybridoma obtained in (1) above. Wells that responded to positive antigens and responded less to negative antigens were selected. Antigen solid phase ELISA was performed by the following method. The results are shown in Table 3.
糖ペプチドのN末は、BSA-PEG4であり、C末はアミド化されている。
The N-terminal of the glycopeptide is BSA-PEG4, and the C-terminal is amidated.
＜手法＞
(1)96穴プレート（nunc Maxisoap/446612）に、各スクリーニング抗原1μg/ml（希釈液10mMリン酸バッファーpH7）を50μl/well添加し、37℃、1hr固相化する。
(2)各ウェルをPBST 300μl/well×5回洗浄する。
(3)各ウェルを1%BSA-PBS 100μl/well、4℃、Overnightブロッキングする。
(4)各ウェルをPBST 300μl/well×5回洗浄する。
(5)抗体培養上清（一次抗体）を1%BSA-PBSで10倍希釈し、50μl/well添加し、RT、1hr反応させる。
(6)各ウェルをPBST 300μl/well×5回洗浄する。
(7)anti mouse IgG-HRP（JIR/715-035-151）およびanti mouse IgG Lchain-HRP（JIR/115-035-174）を1%BSA-PBSで20,000倍希釈し、50μl/well添加し、RT、0.5hr反応させる。
(8)各ウェルをPBST 300μl/well×5回洗浄する。
(9)HRP発色基質を100μl/well添加し、発色させる。
(10)停止液を100μl/well添加し、発色を停止させる。
(11)OD450を測定する。
(12)目視にて陽性抗原に対するシグナルと陰性抗原に対するシグナルの差が大きく、陰性抗原への反応が少ない7ウェルを二次スクリーニングへ選定。
<Method>
(1) To a 96-well plate (nunc Maxisoap / 446612), add 1 μg / ml of each screening antigen (diluted solution 10 mM phosphate buffer pH 7) at 50 μl / well, and solidify at 37 ° C. for 1 hr.
(2) Wash each well with PBST 300 μl / well × 5 times.
(3) Block each well at 1% BSA-PBS 100 μl / well, 4 ° C, Overnight.
(4) Wash each well with PBST 300 μl / well × 5 times.
(5) The antibody culture supernatant (primary antibody) is diluted 10-fold with 1% BSA-PBS, 50 μl / well is added, and RT is reacted for 1 hr.
(6) Wash each well with PBST 300 μl / well × 5 times.
(7) Anti mouse IgG-HRP (JIR / 715-035-151) and anti mouse IgG Lchain-HRP (JIR / 115-035-174) were diluted 20,000 times with 1% BSA-PBS and added 50 μl / well. , RT, 0.5 hr reaction.
(8) Wash each well with PBST 300 μl / well × 5 times.
(9) Add 100 μl / well of HRP color-developing substrate to develop color.
(10) Add 100 μl / well of stop solution to stop color development.
(11) Measure OD450.
(12) 7 wells with a large difference between the signal for the positive antigen and the signal for the negative antigen and less reaction to the negative antigen were selected for the secondary screening.
（３）二次スクリーニング
上記（２）で得たウェルから以下の抗原を用いたウエスタンブロットによりコアフコースとアミノ酸の両方を認識するウェルを選択し、クローニングに進めた。ウエスタンブロットは以下のように行った。
(3) Secondary screening From the wells obtained in (2) above, wells that recognize both core fucose and amino acids were selected by Western blotting using the following antigens and proceeded to cloning. Western blotting was performed as follows.
＜材料＞
・スクリーニング抗原
陽性抗原：フコシル化AFP（AFP-L3／組換え体）
陰性抗原1：非フコシル化AFP（ヒト血清由来AFP（LEE biosolutions）のLCAレクチン非吸着画分）
陰性抗原2：フコシル化ALP（オリエンタル酵母/ 47787055）
・一次抗体
スクリーニング抗体：一次スクリーニング陽性の培養上清
ネガティブコントロール：ハイブリドーマ培地
・二次抗体
anti mouse-IgG(Fc) Ab -HRP（BET ／A90-131P）
anti mouse-IgM Ab -HRP（SBA／1020-05）
・ブロッキング剤：PVDF Blocking Reagent for Can Get Signal（TOYOBO／NYPBR01）
・洗浄液：TBST
・希釈液：1%BSA-TBST
・PVDFメンブレン（iBlot（登録商標） 2 Transfer Stacks, PVDF, mini／IB24002）
・ブロッティング装置（iBlot2）
・発光検出試薬ECL prime Western Blotting Detection Reagent(GEヘルスケア/RPN2232)
<Material>
-Screening antigen Positive antigen: Fucosylated AFP (AFP-L3 / recombinant)
Negative antigen 1: Non-fucosylated AFP (LCA lectin non-adsorbed fraction of human serum-derived AFP (LEE biosolutions))
Negative antigen 2: Fucosylated ALP (Oriental yeast / 47787055)
-Primary antibody Screening antibody: Culture supernatant positive for primary screening Negative control: Hybridoma medium-Secondary antibody
anti mouse-IgG (Fc) Ab -HRP (BET / A90-131P)
anti mouse-IgM Ab -HRP (SBA / 1020-05)
・ Blocking agent: PVDF Blocking Reagent for Can Get Signal (TOYOBO / NYPBR01)
・ Cleaning liquid: TBST
・ Diluted solution: 1% BSA-TBST
・ PVDF membrane (iBlot (registered trademark) 2 Transfer Stacks, PVDF, mini / IB24002)
・ Blotting device (iBlot2)
・ Emission detection reagent ECL prime Western Blotting Detection Reagent (GE Healthcare / RPN2232)
＜手法＞
(1)各スクリーニング抗原にNuPAGE LDS Sample Buffer (4X) (Thermo/NP0008)を1/4量、
NuPAGE Sample Reducing Agent (10X) (Thermo/NP0009)を1/10量添加し混和する。
(2)分子量マーカーおよび(1)の各スクリーニング抗原を下記抗原量で電気泳動（SDS-PAGE）する。
Lane０：分子量マーカー（MagicMark（商標） XP Western Protein Standard／LC5602）
Lane１：陽性抗原：フコシル化AFP（AFP-L3） 50ng
Lane２：陰性抗原１：非フコシル化AFP 50ng
Lane３：陰性抗原２：フコシル化ALP 50ng
(3)PVDFメンブレンにブロッティングする。
(4)PVDF Blocking Reagent for Can Get Signalに室温で1時間浸しブロッキングする。
(5)TBSTで3回洗浄する。
(6)一次抗体を1%BSA-TBSTで下記の希釈倍率に希釈し、4℃、Overnight反応させる。
スクリーニング抗体：1stスクリーニング陽性の培養上清 10倍希釈
ネガティブコントロール：ハイブリドーマ培地 10倍希釈
(7)TBSTで3回洗浄する。
(8)二次抗体（2種混合）を1%BSA-TBSTで下記の希釈倍率に希釈し、室温で1時間反応させる。
anti mouse-IgG(Fc) Ab -HRP 20,000倍希釈
anti mouse-IgM Ab -HRP 5,000倍希釈
(9)TBSTで3回洗浄する。
(10)化学発光にて検出
(11)陽性抗原にのみバンドが検出された5ウェルを陽性と判定し、クローニングに進めた。
<Method>
(1) Add 1/4 amount of NuPAGE LDS Sample Buffer (4X) (Thermo / NP0008) to each screening antigen.
Add 1/10 amount of NuPAGE Sample Reducing Agent (10X) (Thermo / NP0009) and mix.
(2) Molecular weight markers and each screening antigen of (1) are electrophoresed (SDS-PAGE) with the following antigen amounts.
Lane0: Molecular Weight Marker (MagicMark ™ XP Western Protein Standard / LC5602)
Lane1: Positive antigen: Fucosylated AFP (AFP-L3) 50ng
Lane2: Negative antigen 1: Non-fucosylated AFP 50ng
Lane 3: Negative antigen 2: Fucosylated ALP 50ng
(3) Blotting on PVDF membrane.
(4) Immerse in PVDF Blocking Reagent for Can Get Signal at room temperature for 1 hour to block.
(5) Wash with
(6) The primary antibody is diluted with 1% BSA-TBST to the following dilution ratio and reacted at 4 ° C. overnight.
Screening antibody: 1st screening positive culture supernatant 10-fold dilution Negative control: Hybridoma medium 10-fold dilution
(7) Wash with
(8) Dilute the secondary antibody (mixture of 2 types) with 1% BSA-TBST to the following dilution ratio and react at room temperature for 1 hour.
anti mouse-IgG (Fc) Ab -HRP 20,000 times diluted
anti mouse-IgM Ab -HRP 5,000 times diluted
(9) Wash with
(10) Detected by chemiluminescence
(11) The 5 wells in which the band was detected only in the positive antigen were judged to be positive, and cloning proceeded.
（４）クローニング
上記(３)で選定した5ウェルの細胞を限界希釈法により播種した。その培養上清を（２）の一次スクリーニングの要領で再度スクリーニングを行い、2クローンを得た。ELISAのデータを表4に示した。
(4) Cloning The 5-well cells selected in (3) above were seeded by the limiting dilution method. The culture supernatant was screened again in the same manner as in the primary screening of (2) to obtain 2 clones. The Elisa data are shown in Table 4.
上記2クローンの培養上清を（３）の二次スクリーニングと同様にしてウエスタンブロットを実施し、AFP-L3に特異的に反応することを確認した。ウエスタンブロットの結果を図1に示した。図1において、各レーンの抗原は次の通りである。
レーン1：フコシル化AFP（AFP-L3／組換え体）（陽性抗原）
レーン2：非フコシル化AFP（ヒト血清由来AFP（LEE biosolutions）のLCAレクチン非吸着画分）（陰性抗原）
レーン3：フコシル化ALP（オリエンタル酵母/ 47787055）（陰性抗原）
Western blotting was performed on the culture supernatants of the above two clones in the same manner as in the secondary screening of (3), and it was confirmed that they specifically react with AFP-L3. The results of Western blotting are shown in Fig. 1. In FIG. 1, the antigens in each lane are as follows.
Lane 1: Fucosylated AFP (AFP-L3 / recombinant) (positive antigen)
Lane 2: Non-fucosylated AFP (LCA lectin non-adsorbed fraction of human serum-derived AFP (LEE biosolutions)) (negative antigen)
Lane 3: Fucosylated ALP (Oriental yeast / 47787055) (negative antigen)
（５）エピトープの確認
これらのクローンが生産する抗体のエピトープの確認を行うため、免疫原に使用した糖ペプチドAの配列よりアミノ酸残基が少ない表5に示す糖ペプチドB（配列番号21）および非フコシル化糖ペプチドB（配列番号22）を認識するかを上記(２)と同様の手順で確認した。その結果を表6に示した。ここで陽性コントロール（PC）には糖ペプチドBに対してはフコースを認識するAALレクチン、非フコシル化糖ペプチドBに対してはGlcNAcを認識するWGAレクチンを用いた。陰性コントロール（NC）には緩衝液を用いた。
(5) Confirmation of epitopes In order to confirm the epitopes of the antibodies produced by these clones, glycopeptide B (SEQ ID NO: 21) and glycopeptide B (SEQ ID NO: 21) shown in Table 5 having fewer amino acid residues than the sequence of glycopeptide A used as an immunogen. It was confirmed by the same procedure as in (2) above whether the non-fucosylated glycopeptide B (SEQ ID NO: 22) was recognized. The results are shown in Table 6. Here, as a positive control (PC), an AAL lectin that recognizes fucose for glycopeptide B and a WGA lectin that recognizes GlcNAc for non-fucosylated glycopeptide B were used. A buffer solution was used for negative control (NC).
糖ペプチドのN末は、BSA-PEG4であり、C末はアミド化されている。
The N-terminal of the glycopeptide is BSA-PEG4, and the C-terminal is amidated.
全ての抗体が、抗原に用いた糖ペプチドAには反応し、糖ペプチドBには反応しなかった。この結果から、取得できた抗体のエピトープ配列は、少なくとも、表7に示す配列のどちらかを含む領域であることが分かった。 All antibodies reacted with glycopeptide A used as the antigen and not with glycopeptide B. From this result, it was found that the obtained epitope sequence of the antibody was a region containing at least one of the sequences shown in Table 7.
（６）CDR配列の確認
上記（４）で得た5クローンについて、抗体のCDR解析を行った。その結果、CDR配列は以下の2パターンの配列であった。1F8-A4の抗体のCDR配列（配列番号1〜6）を表8に示した。また、2F11-2A9の抗体のCDR配列（配列番号7〜12）を表9に示した。
(6) Confirmation of CDR sequence The CDR analysis of the antibody was performed on the 5 clones obtained in (4) above. As a result, the CDR sequence was an array with the following two patterns. The CDR sequences of the 1F8-A4 antibody (SEQ ID NOs: 1 to 6) are shown in Table 8. The CDR sequences of the 2F11-2A9 antibody (SEQ ID NOs: 7 to 12) are shown in Table 9.
上記で得られたクローンのうち、1F8-A4をI2-1F8と名付けて国際寄託(NITE BP-02264)した。また、2F11-2A9をI2-2F11と名付けて国際寄託(NITE BP-02263)した。 Of the clones obtained above, 1F8-A4 was named I2-1F8 and deposited internationally (NITE BP-02264). In addition, 2F11-2A9 was named I2-2F11 and made an international deposit (NITE BP-02263).
実施例２
サンドイッチELISAの構築およびSDSによる反応性への影響：
実施例１で得られたI2-1F8またはI2-2F11を用い、以下のようにしてサンドイッチELISAによる検出を行った。また、SDSによる反応性への影響を検討した。
＜材料＞
・抗体感作プレート（I2-1F8, I2-2F11／2.5μg/mL 100μL/well）
・リコンビナントAFP-L3抗原
・抗AFP抗体：Polyclonal Antibody to Alpha-Fetoprotein（WLS/# PAA153Hu01）
・標識抗体：Goat anti rabbit immunoglobulin-HRP
・Buffer A: 150mM NaCl + 1% BSA / 10mMリン酸緩衝液 (pH7)
・Buffer B: 150mM NaCl +0.05% Tween20 / 10mMリン酸緩衝液 (pH7)
Example 2
Construction of sandwich ELISA and impact of SDS on reactivity:
Using I2-1F8 or I2-2F11 obtained in Example 1, detection by sandwich ELISA was performed as follows. We also examined the effect of SDS on reactivity.
<Material>
・ Antibody sensitization plate (I2-1F8, I2-2F11 / 2.5 μg / mL 100 μL / well)
・ Recombinant AFP-L3 antigen ・ Anti-AFP antibody: Polyclonal Antibody to Alpha-Fetoprotein (WLS / # PAA153Hu01)
・ Labeled antibody: Goat anti rabbit immunoglobulin-HRP
・ Buffer A: 150 mM NaCl + 1% BSA / 10 mM phosphate buffer (pH 7)
・ Buffer B: 150 mM NaCl + 0.05% Tween20 / 10 mM phosphate buffer (pH 7)
＜手法＞
(1) （抗原前処理）20μg/mLのAFP-L3抗原溶液に2%、1%、0.5%、0.25%、0.13%、0.06%のSDS溶液を等量加え、混合後3分以上静置する。（前処理時SDS濃度：0.03 - 1%）
(2) 抗原濃度が1μg/mL, 0.5μg/mL, 0.25μg/mLとなるようにBuffer Aで希釈する。（最終SDS濃度：0.00075% - 0.1%）
(3) 抗体感作プレートに抗原溶液を100μL/well添加し、室温で60分反応させる。
(4) Buffer Bで各ウェルを洗浄後、400倍希釈した抗AFP抗体を100μL/well添加し、室温で60分反応させる。
(5) Buffer Bで各ウェルを洗浄後、4000倍希釈した標識抗体を100μL/well添加し、室温で40分反応させる。
(6) Buffer Bで各ウェルを洗浄後、HRP発色基質を100μl/well添加し、20分発色させる。
(7) 停止液を100μl/well添加して発色を停止させ、OD450を測定する。
<Method>
(1) (Antigen pretreatment) Add equal amounts of 2%, 1%, 0.5%, 0.25%, 0.13%, 0.06% SDS solution to 20 μg / mL AFP-L3 antigen solution, and allow to stand for 3 minutes or more after mixing. do. (SDS concentration during pretreatment: 0.03-1%)
(2) Dilute with Buffer A so that the antigen concentration becomes 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL. (Final SDS concentration: 0.00075% --0.1%)
(3) Add 100 μL / well of the antigen solution to the antibody sensitized plate and react at room temperature for 60 minutes.
(4) After washing each well with Buffer B, add 100 μL / well of anti-AFP antibody diluted 400-fold, and react at room temperature for 60 minutes.
(5) After washing each well with Buffer B, add 100 μL / well of the labeled antibody diluted 4000 times, and react at room temperature for 40 minutes.
(6) After washing each well with Buffer B, add 100 μl / well of HRP coloring substrate and let the color develop for 20 minutes.
(7) Add 100 μl / well of stop solution to stop color development, and measure OD450.
＜結果＞
I2-1F8を用いた場合の各種抗原濃度、最終SDS濃度、前処理SDS濃度におけるOD450の測定値を表10に示した。また、I2-2F11を用いた場合の各種抗原濃度、最終SDS濃度、前処理SDS濃度におけるOD450測定値を表11に示した。
<Result>
Table 10 shows the measured values of OD450 at various antigen concentrations, final SDS concentration, and pretreated SDS concentration when I2-1F8 was used. Table 11 shows the OD450 measured values at various antigen concentrations, final SDS concentration, and pretreated SDS concentration when I2-2F11 was used.
I2-1F8では最終SDS濃度が0.013%以下で、I2-2F11では最終SDS濃度が0.025%以下で抗原濃度依存的にシグナルが上昇した。 In I2-1F8, the final SDS concentration was 0.013% or less, and in I2-2F11, the final SDS concentration was 0.025% or less, and the signal increased in an antigen concentration-dependent manner.
実施例３
サンドイッチELISAの特異性確認：
実施例２で構築されたサンドイッチELISAについて以下のようにして特異性を確かめた。
＜材料＞
・抗体感作プレート（I2-1F8, I2-2F11／2.5μg/mL 100μL/well）
・陽性抗原：リコンビナントAFP-L3抗原
・陰性抗原１：非フコシル化AFP（ヒト血清由来AFP（LEE biosolutions）のLCAレク・チン非吸着画分）
・陰性抗原２：AFP以外のフコシル化タンパク質ALP（オリエンタル酵母）
・ビオチン化抗AFP抗体：anti AFP, Human (mouse)（ABV/ H00000174-M01）
・検出試薬：HRP-Conjugated Streptavidin（Thermo / N100）
・Buffer A: 150mM NaCl + 1% BSA / 10mMリン酸緩衝液 (pH7)
・Buffer B: 150mM NaCl +0.05% Tween20 / 10mMリン酸緩衝液 (pH7)
Example 3
Confirmation of sandwich ELISA specificity:
The specificity of the sandwich ELISA constructed in Example 2 was confirmed as follows.
<Material>
・ Antibody sensitization plate (I2-1F8, I2-2F11 / 2.5 μg / mL 100 μL / well)
-Positive antigen: Recombinant AFP-L3 antigen-Negative antigen 1: Non-fucosylated AFP (LCA lek-tin non-adsorbed fraction of human serum-derived AFP (LEE biosolutions))
-Negative antigen 2: Fucosylated protein ALP (Oriental yeast) other than AFP
-Biotinylated anti-AFP antibody: anti AFP, Human (mouse) (ABV / H00000174-M01)
-Detection reagent: HRP-Conjugated Streptavidin (Thermo / N100)
・ Buffer A: 150 mM NaCl + 1% BSA / 10 mM phosphate buffer (pH 7)
・ Buffer B: 150 mM NaCl + 0.05% Tween20 / 10 mM phosphate buffer (pH 7)
＜手法＞
(1) 20μg/mLの各抗原溶液に0.06%のSDS溶液を等量加え（変性）、あるいは加えずに（非変性）、混合後3分以上静置する。（前処理時SDS濃度：0.03%）
(2) 抗原濃度が1000ng/mL, 500ng/mL, 250ng/mL, 125ng/mL, 63ng/mL, 31ng/mLとなるようにBuffer Aで希釈する。
(3) 抗体感作プレートに抗原溶液を100μL/well添加し、室温で60分反応させる。
(4) Buffer Bで各ウェルを洗浄後、480倍希釈したビオチン化抗AFP抗体を100μL/well添加し、室温で60分反応させる。
(5) Buffer Bで各ウェルを洗浄後、10000倍希釈した検出試薬を100μL/well添加し、室温で60分反応させる。
(6) Buffer Bで各ウェルを洗浄後、HRP発色基質を100μl/well添加し、10分発色させる。
(7) 停止液を100μl/well添加して発色を停止させ、OD450を測定する。
<Method>
(1) Add an equal amount of 0.06% SDS solution to each 20 μg / mL antigen solution (denaturation) or no addition (non-denaturation), and let stand for 3 minutes or more after mixing. (SDS concentration during pretreatment: 0.03%)
(2) Dilute with Buffer A so that the antigen concentration is 1000 ng / mL, 500 ng / mL, 250 ng / mL, 125 ng / mL, 63 ng / mL, 31 ng / mL.
(3) Add 100 μL / well of the antigen solution to the antibody sensitized plate and react at room temperature for 60 minutes.
(4) After washing each well with Buffer B, add 100 μL / well of biotinylated anti-AFP antibody diluted 480 times, and react at room temperature for 60 minutes.
(5) After washing each well with Buffer B, add 100 μL / well of the detection reagent diluted 10000 times, and react at room temperature for 60 minutes.
(6) After washing each well with Buffer B, add 100 μl / well of HRP coloring substrate and let the color develop for 10 minutes.
(7) Add 100 μl / well of stop solution to stop color development, and measure OD450.
＜結果＞
I2-1F8を用いた場合の各種抗原濃度におけるOD450の測定値を表12および図２に示した。また、I2-2F11を用いた場合の各種抗原濃度におけるOD450測定値を表13および図３に示した。
<Result>
Table 12 and FIG. 2 show the measured values of OD450 at various antigen concentrations when I2-1F8 was used. In addition, Table 13 and FIG. 3 show OD450 measured values at various antigen concentrations when I2-2F11 was used.
I2-1F8およびI2-2F11は、変性状態、非変性状態のいずれにおいても、非フコシル化AFP、及びALPには反応を示さなかった。一方、I2-1F8およびI2-2F11はAFP-L3に対しては抗原濃度依存的にシグナルの上昇が見られた。特に、I2-1F8およびI2-2F11は変性状態ではAFP-L3に対する反応性が顕著に向上していた。これらのことから、I2-1F8およびI2-2F11はAFP-L3のフコースとペプチド部分を同時に認識することによりサンドイッチELISAでAFP-L3と特異的に反応していることが示された。また、I2-1F8およびI2-2F11は、抗原を0.03%SDSで前処理することにより強く反応することが示された。 I2-1F8 and I2-2F11 did not respond to non-fucosylated AFP and ALP in either the denatured or non-modified state. On the other hand, I2-1F8 and I2-2F11 showed an increase in signal for AFP-L3 in an antigen concentration-dependent manner. In particular, I2-1F8 and I2-2F11 had significantly improved reactivity to AFP-L3 in the denatured state. These results indicate that I2-1F8 and I2-2F11 specifically react with AFP-L3 in sandwich ELISA by simultaneously recognizing the fucose and peptide moieties of AFP-L3. It was also shown that I2-1F8 and I2-2F11 react strongly by pretreating the antigen with 0.03% SDS.
実施例４
天然ヒトAFP-L3との反応性：
I2-1F8が天然ヒトAFP-L3と反応するかどうかを以下のようにしてウェスタンブロットで確かめた。
＜材料＞
一次抗体：I2-1F8（10倍希釈ハイブリドーマ培養上清） 4℃ O/N
二次抗体：anti mouse-IgG(Fc) Ab -HRP（BET／#A90-131P）（20,000倍希釈）RT 1hr
Example 4
Reactivity with natural human AFP-L3:
Whether or not I2-1F8 reacts with natural human AFP-L3 was confirmed by Western blotting as follows.
<Material>
Primary antibody: I2-1F8 (10-fold diluted hybridoma culture supernatant) 4 ° C O / N
Secondary antibody: anti mouse-IgG (Fc) Ab-HRP (BET / # A90-131P) (20,000-fold dilution) RT 1hr
＜手法＞
一次抗体および二次抗体を以下のものに代える以外は実施例１（３）と同様にしてウエスタンブロットを行った。
<Method>
Western blotting was performed in the same manner as in Example 1 (3) except that the primary antibody and the secondary antibody were replaced with the following.
＜結果＞
ウエスタンブロットの結果を図4に示した。I2-1F8は天然ヒトAFP-L3にも反応を示した。図4において、各レーンの抗原は次の通りである。
レーン1: リコンビナントAFP-L3
レーン2: 非フコシル化AFP
レーン3: 天然ヒトAFP（ミュータスワコー AFP-L3用キャリブレータ1）
レーン4: 天然ヒトAFP-L3 (ミュータスワコー AFP-L3用キャリブレータ2)
<Result>
The results of Western blotting are shown in FIG. I2-1F8 also responded to natural human AFP-L3. In FIG. 4, the antigens in each lane are as follows.
Lane 1: Recombinant AFP-L3
Lane 2: Non-fucosilized AFP
Lane 3: Natural human AFP (
Lane 4: Natural Human AFP-L3 (Mutaswako AFP-L3 Calibrator 2)
実施例５
SDSによる反応性への影響：
実施例２で構築したELISAを用いて、SDSによる反応性への影響を検討した。
＜材料＞
抗体としてI2-1F8を用いる以外は実施例２と同じ材料を用いた。
Example 5
Impact of SDS on reactivity:
The effect of SDS on reactivity was examined using the Elisa constructed in Example 2.
<Material>
The same material as in Example 2 was used except that I2-1F8 was used as the antibody.
＜手法＞
(1)（抗原前処理）20μg/mLのAFP-L3抗原溶液に0.031%、0.016%、0.008%、0.004%のSDS溶液を等量加え、混合後3分以上静置する。
(2) 抗原濃度が0.4μg/mL, 0.2μg/mL, 0.1μg/mLとなるようにBuffer Aで希釈する。（最終SDS濃度：0.00032% - 0.00001%）
(3)以降の操作は実施例２と同様に行った。
<Method>
(1) (Antigen pretreatment) Add equal amounts of 0.031%, 0.016%, 0.008%, 0.004% SDS solution to 20 μg / mL AFP-L3 antigen solution, and allow to stand for 3 minutes or more after mixing.
(2) Dilute with Buffer A so that the antigen concentration becomes 0.4 μg / mL, 0.2 μg / mL, 0.1 μg / mL. (Final SDS concentration: 0.00032% --0.00001%)
The following operations were performed in the same manner as in Example 2.
＜結果＞
I2-1F8を用いた場合の各種抗原濃度、最終SDS濃度、前処理SDS濃度におけるOD450の測定値を表14に示した。
<Result>
Table 14 shows the measured values of OD450 at various antigen concentrations, final SDS concentration, and pretreated SDS concentration when I2-1F8 was used.
前処理SDS濃度が0.016%以下でも濃度依存的なシグナルを検出することができた。 A concentration-dependent signal could be detected even when the pretreated SDS concentration was 0.016% or less.
Claims (17)
軽鎖のＣＤＲが、配列番号４で示されるアミノ酸配列、配列番号５で示されるアミノ酸配列および配列番号６で示されるアミノ酸配列を含む、
請求項１〜６の何れかに記載の抗体。 The heavy chain CDR comprises the amino acid sequence set forth in SEQ ID NO: 1, the amino acid sequence set forth in SEQ ID NO: 2, and the amino acid sequence set forth in SEQ ID NO: 3.
The CDR of the light chain comprises the amino acid sequence set forth in SEQ ID NO: 4, the amino acid sequence set forth in SEQ ID NO: 5, and the amino acid sequence set forth in SEQ ID NO: 6.
The antibody according to any one of claims 1 to 6.
軽鎖のＣＤＲが、配列番号１０で示されるアミノ酸配列、配列番号１１で示されるアミノ酸配列および配列番号１２で示されるアミノ酸配列を含む、
請求項１〜６の何れかに記載の抗体。 The heavy chain CDR comprises the amino acid sequence set forth in SEQ ID NO: 7, the amino acid sequence set forth in SEQ ID NO: 8 and the amino acid sequence set forth in SEQ ID NO: 9.
The CDR of the light chain comprises the amino acid sequence set forth in SEQ ID NO: 10, the amino acid sequence set forth in SEQ ID NO: 11, and the amino acid sequence set forth in SEQ ID NO: 12.
The antibody according to any one of claims 1 to 6.
以下の構造を含む糖ペプチド抗原を非ヒト動物に免疫する工程を含む、
前記方法。 A method for producing an antibody according to any one of claims 1 to 6.
Including the step of immunizing a non-human animal with a glycopeptide antigen containing the following structure,
The method.
以下の構造を含む糖ペプチド抗原を非ヒト動物に免疫する工程を含む、
前記方法。 A method for producing a hybridoma that produces the antibody according to any one of claims 1 to 6.
Including the step of immunizing a non-human animal with a glycopeptide antigen containing the following structure,
The method.
前記捕捉用抗体または前記検出用抗体が請求項１〜８のいずれかに記載の抗体である、
フコシル化ＡＦＰ検出用キット。 Includes an antibody for capturing fucosylated AFP, an antibody for detecting fucosylated AFP, and a solid phase.
The capture antibody or the detection antibody is the antibody according to any one of claims 1 to 8.
Fucosilized AFP detection kit.
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