JP5487393B2 - Standardization method of antigen-specific IgG antibody titer measurement in human serum using ELISA method - Google Patents
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Description
本発明は、生体試料に含まれる抗原特異的なＩｇＧ抗体力価を標準化して測定する方法に関する。 The present invention relates to a method for standardizing and measuring antigen-specific IgG antibody titers contained in a biological sample.
以前より担がん患者の血清中には多くのがん抗原（ｐ５３、ＣＥＡ、ＭＵＣ１など）に対する自家抗体（auto-antibody）の存在を示唆する報告がこれまでになされている（非特許文献１〜３）。特にＰ５３に対する自家抗体の検出は、さまざまな固型がんにおいて報告されておりがん患者での血清マーカーとしての可能性も示唆されている（非特許文献４〜６）。また最近肺がんや前立腺がんにおいて複数のがん抗原に対する自家抗体を組み合わせることにより、がんの血清診断やファージペプチドディスプレイを用いた自家抗体アレイなどの開発も行われており、科学的にも注目されている（非特許文献７、８）。 Previously, reports suggesting the existence of auto-antibodies against many cancer antigens (p53, CEA, MUC1, etc.) in the serum of cancer-bearing patients have been made (Non-patent Document 1). ~ 3). In particular, detection of autoantibodies against P53 has been reported in various solid cancers, and its possibility as a serum marker in cancer patients has also been suggested (Non-Patent Documents 4 to 6). Recently, autologous antibody arrays using serodiagnosis of cancer and phage peptide display have been developed by combining autoantibodies against multiple cancer antigens in lung cancer and prostate cancer. (Non-Patent Documents 7 and 8).
現在、一般的に用いられている標準化方法では抗原固相化の代わりに抗ヒトＩｇＧ抗体をプレートに固相化し、測定する血清中抗原特異的ＩｇＧの代わりに標準化ヒトＩｇＧを段階希釈したものを用い測定し標準化に用いている。しかし、この方法ではプレートに固相化した捕獲抗体と検出側の抗体との交差反応により生じるバックグラウンドのため、標準化可能な抗体濃度の範囲に制限がかかるとともに、マニュアル操作では同一プレート上で測定するＩｇＧ濃度の高い血清サンプルからのＩｇＧの混入による影響を受けやすく、測定結果の再現性、信頼性が低いものであった。どの抗体価の測定系においても定性的な評価の粋を出ず、具体的な抗体価の定量・標準化する技術が必要とされていた。 Currently, the standardized method that is generally used is a method in which an anti-human IgG antibody is immobilized on a plate instead of antigen-immobilization, and standardized human IgG is serially diluted instead of antigen-specific IgG in serum to be measured. Used for measurement and standardization. However, this method limits the range of antibody concentrations that can be standardized due to the background generated by the cross-reaction between the capture antibody immobilized on the plate and the antibody on the detection side. It was easily affected by the contamination of IgG from a serum sample with a high IgG concentration, and the reproducibility and reliability of the measurement results were low. In any antibody titer measurement system, a technique for quantifying and standardizing a specific antibody titer was required without producing a qualitative evaluation.
血清中の抗体価測定にダイレクトＥＬＩＳＡの系を用いる際、プレート間の差を補正するために対照となる標準化された反応系（検量線等）を同一プレート上に置く必要がある。目的の特定抗原に対し反応するモノクローナルヒト抗体を入手できれば対照としての検量線の作成が可能だが、現状では入手不可能であるため、反応系を模倣した代用の対照標準化反応系が必要になる。本発明の課題は、様々ながん患者を含むヒト血清中に存在するがん抗原、又は感染症を惹起しうる病原体由来の抗原に特異的なＩｇＧ抗体価を定量し、標準化するための測定方法を提供することにある。 When using a direct ELISA system for measuring antibody titers in serum, it is necessary to place a standardized reaction system (such as a calibration curve) as a control on the same plate in order to correct for differences between the plates. If a monoclonal human antibody that reacts with a specific antigen of interest can be obtained, a calibration curve can be prepared as a control. However, since it is not currently available, a substitute control reaction system that mimics the reaction system is required. An object of the present invention is to measure and standardize IgG antibody titers specific for cancer antigens present in human serum including various cancer patients or antigens derived from pathogens that can cause infectious diseases. It is to provide a method.
本標準化方法は検体のＩｇＧを段階希釈するのではなく、捕獲側抗体を段階希釈し、高濃度（飽和量の）ヒトＩｇＧを検体添加の段階で用いることにより捕獲抗体をヒトＩｇＧで飽和させ、検出側抗体との交差反応性、およびＩｇＧ混入による影響を無視できるようにしたものである。より具体的には、まず固相化抗原と血清中抗体の複合体を模倣するために、抗原の代わりに固相化された抗ヒト抗体と精製ヒトＩｇＧの反応を用いる。（抗原、抗体の関係性は逆向きになる。）この際、固相化された抗ヒト抗体に精製ヒトＩｇＧの濃度系列物を添加するのではなく、抗ヒト抗体の濃度系列を作製し固相化したものに過剰量の精製ヒトＩｇＧを添加し飽和させた複合体を生成させる。この操作は、後で用いる標識抗ヒト抗体と固相化抗ヒト抗体の交差反応によるバックグラウンドの上昇を避けるためである。プレートウェルへの血清もしくは精製ヒトＩｇＧの添加後は酵素標識抗ヒト抗体、発色基質を測定ウェル、対照ウェルともに同様に加えて反応させ、吸光度を測定する。血清中の抗原特異的抗体が十分に希釈され、Ｏ．Ｄ．が１を超えない条件下ではサンプル、対照ともに濃度とＯ．Ｄ．が一次式の反応を示すことを実験により確認しており、以下の式で抗体価を相対化することが出来る。
血清サンプルＯ．Ｄ．の指数化
(指数値)＝標準線の傾き(固相化時の抗体の濃度(μg/ml)/Ｏ．Ｄ．)×(血清 Ｏ．Ｄ．)×(血清希釈倍率)
This standardization method does not serially dilute the IgG of the specimen, but serially dilutes the capture side antibody and saturates the capture antibody with human IgG by using a high concentration (saturated amount) of human IgG at the stage of specimen addition. The cross-reactivity with the detection side antibody and the influence of IgG contamination can be ignored. More specifically, first, in order to mimic the complex of the immobilized antigen and the antibody in serum, a reaction between the immobilized anti-human antibody and purified human IgG is used instead of the antigen. (Relationship between antigen and antibody is reversed.) At this time, the concentration series of the anti-human antibody is not prepared by adding the purified human IgG concentration series to the immobilized anti-human antibody. An excess of purified human IgG is added to the phased phase to produce a saturated complex. This operation is for avoiding an increase in background due to a cross-reaction between the labeled anti-human antibody and the immobilized anti-human antibody used later. After addition of serum or purified human IgG to the plate well, enzyme-labeled anti-human antibody and chromogenic substrate are added and reacted in the same manner in both the measurement well and the control well, and the absorbance is measured. Antigen-specific antibodies in serum are sufficiently diluted and D. Under the condition that the sample does not exceed 1, the concentration and O.D. D. Have shown by the experiment that the antibody titer can be relativized by the following formula.
Serum sample O.D. D. Indexing
(Index value) = Slope of standard line (concentration of antibody at solid phase (μg / ml) / OD) × (serum OD) × (serum dilution rate)
すなわち本発明は（１）（１ａ）抗原の代わりに使用する抗ヒト抗体を段階希釈し、抗ヒト抗体の濃度系列を作製して、各々固相化した複数の捕獲側抗体プレートを調製する工程；（１ｂ）抗原タンパク質を固相化した複数の抗原固定プレートを調製する工程；（２ａ）過剰量（飽和量）のヒトＩｇＧを用いることにより、工程（１ａ）で調製した捕獲側抗体プレート上の捕獲側抗体をヒトＩｇＧで飽和させ、洗浄してヒトＩｇＧ−捕獲側抗体複合体を調製する工程；（２ｂ）段階希釈したサンプルを、工程（１ｂ）で調製した抗原固定プレートに接触させ、サンプル中の標的抗体を抗原タンパク質と反応させ、洗浄して標的抗体−抗原複合体を調製する工程；（３ａ）工程（１ａ）で使用した抗ヒト抗体とエピトープを異にした抗ヒト抗体を標識した酵素標識抗ヒト抗体を、工程（２ａ）で調製したヒトＩｇＧ−捕獲側抗体複合体と反応させ、洗浄して酵素標識抗ヒト抗体−ヒトＩｇＧ−捕獲側抗体複合体を調製する工程；（３ｂ）工程（３ａ）で使用した酵素標識抗ヒト抗体を、工程（２ｂ）で調製した標的抗体−抗原複合体と反応させ、洗浄して酵素標識抗ヒト抗体−標的抗体−抗原複合体を調製する工程；（４ａ）工程（３ａ）で調製した酵素標識抗ヒト抗体−ヒトＩｇＧ−捕獲側抗体複合体に基質を添加して反応後、吸光度を測定し、捕獲側抗体と吸光度との標準曲線を作成する工程；（４ｂ）工程（３ｂ）で調製した酵素標識抗ヒト抗体−標的抗体−抗原複合体に基質を添加して反応後、吸光度を測定し、吸光度の測定結果と工程（４ａ）で作成した標準曲線から、サンプル中の標的抗体の抗体価を定量する工程；を備えた抗原特異的な抗体価の標準化された測定方法や、（２）サンプル中の標的抗体が、血清中に存在するがん抗原又は感染症を惹起しうる病原体由来の抗原に特異的なＩｇＧ抗体であることを特徴とする前記（１）記載の測定方法に関する。 That is, the present invention includes (1) (1a) a step of serially diluting an anti-human antibody used in place of an antigen to prepare a concentration series of anti-human antibody, and preparing a plurality of capture antibody plates each immobilized on a solid phase. (1b) a step of preparing a plurality of antigen-immobilized plates on which an antigen protein is immobilized; (2a) a capture-side antibody plate prepared in step (1 a ) by using an excess amount (saturation amount) of human IgG; Saturating the upper capture antibody with human IgG and washing to prepare a human IgG-capture antibody complex; (2b) contacting the serially diluted sample with the antigen-fixed plate prepared in step (1 b ) is, the target antibody in a sample is reacted with an antigen protein, washed and target antibody - preparing a antigen complex; (3a) step antihuman was different from an anti-human antibody and an epitope used in (1 a) The enzyme-labeled anti-human antibody labeled with the body is reacted with the human IgG-capture antibody complex prepared in step (2a) and washed to prepare an enzyme-labeled anti-human antibody-human IgG-capture antibody complex. Step: (3b) The enzyme-labeled anti-human antibody used in step (3a) is reacted with the target antibody-antigen complex prepared in step (2b), washed and enzyme-labeled anti-human antibody-target antibody-antigen complex (4a) A substrate is added to the enzyme-labeled anti-human antibody-human IgG-capture antibody complex prepared in step (3a), and after reaction, the absorbance is measured, and the capture antibody and absorbance are determined. (4b) Step of preparing a standard curve; (4b) After adding the substrate to the enzyme-labeled anti-human antibody-target antibody-antigen complex prepared in step (3b) and reacting, the absorbance is measured, and the absorbance measurement result and step From the standard curve created in (4a) Quantifying the antibody titer of the target antibody in the sample; and (2) a cancer antigen or infection in which the target antibody in the sample is present in the serum. The measurement method according to (1) above, which is an IgG antibody specific for an antigen derived from a pathogen capable of causing a disease.
本発明の抗体価の測定・標準化法により、適切な抗原タンパクが取得できればすべてのがん抗原や病原体由来の抗原に対する血清抗体価（患者または健常人）を測定評価することが可能となる。これにより抗体価の上昇している症例を選択し、その抗体陽性のＢ細胞を分離することによりヒト抗体の遺伝子配列の取得が極めて効率的に施行できる可能性がある。またがんや感染症のワクチン療法（ペプチド、樹状細胞その他）における治療効果を判定しうる診断技術（測定キット）として汎用化することも可能となる。以上の結果を検討すると本抗体価測定技術は、バイオサイエンスの発展に寄与しうる優れたツールとなりうる。 If an appropriate antigen protein can be obtained by the antibody titer measurement / standardization method of the present invention, serum antibody titers (patients or healthy individuals) against all cancer antigens and pathogen-derived antigens can be measured and evaluated. Thus, there is a possibility that acquisition of a human antibody gene sequence can be performed very efficiently by selecting a case in which the antibody titer is increased and isolating the antibody-positive B cell. It can also be used as a diagnostic technique (measurement kit) that can determine the therapeutic effect in vaccine therapy (peptides, dendritic cells, etc.) for cancer and infectious diseases. Considering the above results, this antibody titer measurement technique can be an excellent tool that can contribute to the development of bioscience.
本発明の抗原特異的な抗体価の標準化された測定方法としては、
（１ａ）抗原の代わりに使用する抗ヒト抗体を段階希釈し、抗ヒト抗体の濃度系列を作製して、各々固相化した複数の捕獲側抗体プレートを調製する工程；
（１ｂ）抗原タンパク質を固相化した複数の抗原固定プレートを調製する工程；
（２ａ）過剰量（飽和量）のヒトＩｇＧ標準品を用いることにより、工程（１ａ）で調製した捕獲側抗体プレート上の捕獲側抗体をヒトＩｇＧで飽和させ、洗浄してヒトＩｇＧ標準品−捕獲側抗体複合体を調製する工程；
（２ｂ）段階希釈したサンプルを、工程（１ｂ）で調製した抗原固定プレートに接触させ、サンプル中の標的抗体を抗原タンパク質と反応させ、洗浄して標的抗体−抗原複合体を調製する工程；
（３ａ）工程（１ａ）で使用した抗ヒト抗体とエピトープを異にした抗ヒト抗体を標識した酵素標識抗ヒト抗体を、工程（２ａ）で調製したヒトＩｇＧ−捕獲側抗体複合体と反応させ、洗浄して酵素標識抗ヒト抗体−ヒトＩｇＧ−捕獲側抗体複合体を調製する工程；
（３ｂ）工程（３ａ）で使用した酵素標識抗ヒト抗体を、工程（２ｂ）で調製した標的抗体−抗原複合体と反応させ、洗浄して酵素標識抗ヒト抗体−標的抗体−抗原複合体を調製する工程；
（４ａ）工程（３ａ）で調製した酵素標識抗ヒト抗体−ヒトＩｇＧ−捕獲側抗体複合体に基質を添加して反応後、吸光度を測定し、捕獲側抗体と吸光度との標準曲線を作成する工程；
（４ｂ）工程（３ｂ）で調製した酵素標識抗ヒト抗体−標的抗体−抗原複合体に基質を添加して反応後、吸光度を測定し、吸光度の測定結果と工程（４ａ）で作成した標準曲線から、サンプル中の標的抗体の抗体価を定量する工程；
の工程（１ａ）〜（４ｂ）を備えた、ＥＬＩＳＡ（Enzyme Linked Immuno-Sorbent Assay）法であれば特に制限されるものではなく、（１ａ）及び（１ｂ）の後に、さらにブロッキング工程を含むものであってもよい。本発明に用いるサンプルとしては、特に制限されるものではないが、例えば、被検者から採取した血液（血清又は血漿）、唾液、組織又は細胞抽出液などを具体的に挙げることができるが、なかでも、血清を用いることが好ましい。
As a standardized method for measuring the antigen-specific antibody titer of the present invention,
(1a) A step of serially diluting an anti-human antibody to be used in place of an antigen to prepare a concentration series of anti-human antibodies, and preparing a plurality of capture-side antibody plates each immobilized on a solid phase;
(1b) preparing a plurality of antigen-immobilized plates on which an antigen protein is immobilized;
(2a) By using an excess amount (saturation amount) of a human IgG standard product, the capture antibody on the capture antibody plate prepared in step (1a) is saturated with human IgG, washed and washed with a human IgG standard product— Preparing a capture antibody complex;
(2b) A step of bringing the serially diluted sample into contact with the antigen-immobilized plate prepared in step (1b), causing the target antibody in the sample to react with the antigen protein, and washing to prepare a target antibody-antigen complex;
(3a) An enzyme-labeled anti-human antibody labeled with an anti-human antibody having an epitope different from that of the anti-human antibody used in step (1a) is reacted with the human IgG-capture side antibody complex prepared in step (2a). Washing and preparing an enzyme-labeled anti-human antibody-human IgG-capture antibody complex;
(3b) The enzyme-labeled anti-human antibody used in step (3a) is reacted with the target antibody-antigen complex prepared in step (2b), washed, and the enzyme-labeled anti-human antibody-target antibody-antigen complex is washed. Preparing step;
(4a) After adding the substrate to the enzyme-labeled anti-human antibody-human IgG-capture side antibody complex prepared in step (3a), the absorbance is measured, and a standard curve of the capture side antibody and the absorbance is prepared. Process;
(4b) After adding the substrate to the enzyme-labeled anti-human antibody-target antibody-antigen complex prepared in step (3b) and reacting, the absorbance is measured, and the absorbance measurement result and the standard curve created in step (4a) Quantifying the antibody titer of the target antibody in the sample from;
The method is not particularly limited as long as it is an ELISA (Enzyme Linked Immuno-Sorbent Assay) method comprising the steps (1a) to (4b), and further includes a blocking step after (1a) and (1b). It may be. The sample used in the present invention is not particularly limited, and specific examples include blood (serum or plasma) collected from a subject, saliva, tissue or cell extract, Of these, serum is preferably used.
上記抗原としては特に制限されるものではないが、がん抗原ポリペプチド又はがん抗原タンパク質であることが好ましく、例えば、ＭＡＧＥ１、ＭＡＧＥ２、ＭＡＧＥ３等を好適に挙げることができ、上記抗ヒトＩｇＧ抗体としては特に制限されるものではなく、ポリクローナル抗体であってもモノクローナル抗体であってもよいが、異なるエピトープに対する２種類の抗ヒトＩｇＧ抗体を、それぞれ捕獲側抗体（固相化抗体）、検出用抗体として用いる必要がある。また、上記ブロッキング工程に用いるブロッキング剤としては、ＥＬＩＳＡ法において通常用いられるものであれば特に制限されるものではなく、例えば、ウシ血清アルブミン、カゼイン、スキムミルク、ゼラチン等を挙げることができ、０．１〜１０％ウシ血清アルブミン溶液、０．１〜１％カゼイン溶液として使用することができる。 Although it does not restrict | limit especially as said antigen, It is preferable that it is a cancer antigen polypeptide or cancer antigen protein, for example, MAGE1, MAGE2, MAGE3 etc. can be mentioned suitably, The said anti-human IgG antibody There is no particular limitation, and it may be a polyclonal antibody or a monoclonal antibody. Two types of anti-human IgG antibodies against different epitopes are used as a capture antibody (solid-phase antibody) and for detection, respectively. It needs to be used as an antibody. Further, the blocking agent used in the blocking step is not particularly limited as long as it is usually used in the ELISA method. Examples thereof include bovine serum albumin, casein, skim milk, gelatin and the like. It can be used as 1-10% bovine serum albumin solution, 0.1-1% casein solution.
上記酵素標識ヒトＩｇＧ抗体としては、特に制限されるものではないが、例えば、ビオチン標識抗ヒトＩｇＧ抗体や、アルカリフォスファターゼ標識ヒトＩｇＧ抗体、ホースラディッシュペルオキシダーゼ（ＨＲＰ）標識ヒトＩｇＧ抗体などを具体的に挙げることができるが、なかでも、ＨＲＰ標識ヒトＩｇＧ抗体であることが好ましい。酵素の基質としては、アルカリフォスファターゼに対する基質としてｐ−ニトロフェニルリン酸を、ＨＲＰに対する基質としてトリメチルベンジジン又はルトフェニレンジアミンを使用することができる。 The enzyme-labeled human IgG antibody is not particularly limited, but specific examples include biotin-labeled anti-human IgG antibody, alkaline phosphatase-labeled human IgG antibody, horseradish peroxidase (HRP) -labeled human IgG antibody, and the like. Among them, an HRP-labeled human IgG antibody is preferable. As the enzyme substrate, p-nitrophenyl phosphate can be used as a substrate for alkaline phosphatase, and trimethylbenzidine or ltophenylenediamine can be used as a substrate for HRP.
以下、実施例により本発明をより具体的に説明するが、本発明の技術的範囲はこれらの例示に限定されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.
＜抗原特異的ＩｇＧ抗体力価測定の標準化法の確立＞
がん抗原の一種であるＣＭＶ−ｐｐ６５タンパク質に対するＩｇＧの抗体価を、以下のＥＬＩＳＡ法により測定し標準化した。
１．固相化プレートの作製
抗原タンパク質（ＣＭＶ−ｐｐ６５タンパク質）を、ＰＢＳ又は塩化ナトリウムを１Ｍ含むＰＢＳで１００ｎｇ／ｍｌとなるように希釈し、９６穴アッセイプレート（ＩＷＡＫＩ社製）の３〜１２列に１００μｌ／ウェルずつ分注した。また、上記プレートの１及び２列目には、標準曲線作成のために、ＰＢＳで２００ｎｇ／ｍｌから逐次的２倍希釈した抗ヒトＩｇＧウサギ抗体（Dako cytomation社製）を、１００μｌ／ウェル（９６穴プレートの）ずつ分注した。３７℃で１時間インキュベートした後、さらに、４度で一晩インキュベートすることによりそれぞれのタンパク質を固相化した。固相化後のプレートを洗浄バッファー（０．０５％Ｔｗｅｅｎ−２０を含むＰＢＳ）で３回洗浄した後、２００μｌ／ウェルの飽和バッファー（３％ＢＳＡを含むＰＢＳ）を分注し、室温で２時間インキュベートすることによりブロッキングした。なお、上記の標準曲線用のヒトＩｇＧ添加量については、固相化したウサギ抗ヒトＩｇＧ抗体に対して飽和量となる量をあらかじめ検討し、図２Ａに示すように１００μｇ／ｍｌで吸光度が飽和することを確認している。
<Establishment of standardization method for antigen-specific IgG antibody titer measurement>
The antibody titer of IgG against CMV-pp65 protein, which is a kind of cancer antigen, was measured and standardized by the following ELISA method.
1. Preparation of solid-phase plate Antigen protein (CMV-pp65 protein) was diluted to 100 ng / ml with PBS or PBS containing 1 M of sodium chloride, and placed in 3 to 12 rows of 96-well assay plate (IWAKI). 100 μl / well was dispensed. In the first and second rows of the plate, an anti-human IgG rabbit antibody (Dako cytomation) serially diluted 2-fold from 200 ng / ml with PBS was prepared at 100 μl / well (96 Dispensed one at a time). After incubating at 37 ° C. for 1 hour, each protein was immobilized by further incubating overnight at 4 degrees. The plate after solid-phase immobilization was washed 3 times with a washing buffer (PBS containing 0.05% Tween-20), and then 200 μl / well of a saturated buffer (PBS containing 3% BSA) was dispensed, followed by 2 at room temperature. Blocked by incubating for a period of time. As for the amount of human IgG added for the above standard curve, the saturation amount with respect to the immobilized rabbit anti-human IgG antibody was examined in advance, and the absorbance was saturated at 100 μg / ml as shown in FIG. 2A. Make sure you do.
２．サンプルの測定
測定検体を、飽和バッファーで１／１０から逐次的１０倍に希釈し、固相化プレートの３〜１２列目（ＣＭＶ−ｐｐ６５タンパク質を固相化したウェル）に１００μｌ／ウェルずつ分注した。一方、固相化プレートの１、２列目（抗ヒトＩｇＧウサギ抗体を固相化したウェル）には、１００μｇ／ｍｌに希釈したヒトＩｇＧ標準品（ＳＩＧＭＡ社製）を１００μｌ／ウェルずつ分注した。プレートを室温で１時間インキュベートした後、洗浄バッファーで５回洗浄した。続いて、飽和バッファーで１０００倍に希釈したホースラディッシュペルオキシダーゼ（ＨＲＰ）標識抗ヒトＩｇＧヒツジ抗体（ＧＥヘルスケアバイオサイエンス社製）を、全てのウェルに１００μｌ／ウェルずつ加え、室温で１時間インキュベートした。その後、洗浄バッファーで７回洗浄し、基質液（TMB substrate reagent set；BD Biosciences社製）を、全てのウェルに１００μｌ／ウェルずつ加え、３７℃で３０分間インキュベートした。２Ｍ硫酸水溶液を５０μｌ／ウェルずつ添加して発色反応を停止させ、各ウェルの吸光度をプレートリーダー（ImmunoMini NJ-2300；バイオテック社製）を用いて４５０ｎｍと６２０ｎｍで測定した。
３．データの解析
図２Ｂに示すように、固相化した抗ヒトＩｇＧウサギ抗体の濃度と、得られた吸光度の間には正の相関が認められた。標準化用に固相化した抗ヒトＩｇＧウサギ抗体濃度希釈系列（≒固定化されたヒトＩｇＧ抗体量）と吸光度より一次の近似式を作成し、測定検体の吸光度を式に代入し抗原特異的ＩｇＧの抗体価を定量的に算出し、指数化したものをデータとして使用した。
2. Sample measurement The measurement sample was diluted 1/10 to 10-fold sequentially with a saturated buffer, and 100 μl / well was dispensed into the 3rd to 12th rows of the solid-phased plate (wells on which CMV-pp65 protein was solid-phased). Noted. On the other hand, in the first and second rows of the solid-phased plate (well in which the anti-human IgG rabbit antibody was solid-phased), a human IgG standard product (manufactured by SIGMA) diluted to 100 μg / ml was dispensed at 100 μl / well. did. The plate was incubated for 1 hour at room temperature and then washed 5 times with wash buffer. Subsequently, horseradish peroxidase (HRP) -labeled anti-human IgG sheep antibody (manufactured by GE Healthcare Biosciences) diluted 1000-fold with a saturated buffer was added to all wells at 100 μl / well and incubated at room temperature for 1 hour. . Thereafter, the plate was washed 7 times with a washing buffer, and a substrate solution (TMB substrate reagent set; manufactured by BD Biosciences) was added to all wells at 100 μl / well and incubated at 37 ° C. for 30 minutes. The color development reaction was stopped by adding 50 μl / well of 2M sulfuric acid aqueous solution, and the absorbance of each well was measured at 450 nm and 620 nm using a plate reader (ImmunoMini NJ-2300; manufactured by Biotech).
3. Analysis of Data As shown in FIG. 2B, a positive correlation was observed between the concentration of the immobilized anti-human IgG rabbit antibody and the obtained absorbance. Create a first-order approximate expression from the anti-human IgG rabbit antibody concentration dilution series (≒ immobilized human IgG antibody amount) immobilized for standardization and the absorbance, and substitute the absorbance of the sample to be measured into the antigen-specific IgG The antibody titer was quantitatively calculated and indexed to be used as data.
＜ヒト血清中の特定抗原に対するＩｇＧの抗体価の測定・標準化＞
１．ヒト血清の採取
メラノーマ患者の臨床検体を用いた研究は、国立がんセンター及び静岡がんセンターの倫理審査委員会において承認済みである。また、採血は、全てのメラノーマ患者及び健常人からインフォームドコンセントを得た上で行った。
<Measurement and standardization of IgG antibody titer against specific antigen in human serum>
1. Collection of human serum Studies using clinical specimens from melanoma patients have been approved by the Ethical Review Boards of the National Cancer Center and Shizuoka Cancer Center. In addition, blood collection was performed after obtaining informed consent from all melanoma patients and healthy individuals.
２．ＣＭＶ−ｐｐ６５抗原に対するＩｇＧ抗体価測定
まず、患者血清中にＣＭＶ−ｐｐ６５に対するＩｇＧが含まれるかどうかを検討する目的で、患者由来血清を１次抗体に使用したWestern blotting解析を行った。バキュロウイルスにて発現させたＣＭＶ−ｐｐ６５タンパク質、又は、ＣＭＶ−ｐｐ６５タンパク質を発現させたＨｉｇｈＦｉｖｅ昆虫細胞の細胞抽出液を電気泳動により分画してブロットした後、９例の患者由来血清（Ｍ１、Ｍ２、Ｍ３、Ｍ５、Ｍ６、Ｍ７、Ｍ９、Ｍ１８、ＭＳ７）を１次抗体として反応させた。その結果、図３に示すように、４症例（Ｍ５、Ｍ６、Ｍ１８、ＭＳ７）由来の血清により強い反応シグナルが検出されたことから、これらの血清中にＣＭＶ−ｐｐ６５に特異的な抗体が含まれていることが示された。
2. Measurement of IgG antibody titer against CMV-pp65 antigen First, Western blotting analysis using patient-derived serum as a primary antibody was performed for the purpose of examining whether IgG against CMV-pp65 is contained in patient serum. After fractionating and blotting a cell extract of CMV-pp65 protein expressed by baculovirus or HighFive insect cells expressing CMV-pp65 protein by electrophoresis, 9 patient-derived sera (M1, M2, M3, M5, M6, M7, M9, M18, MS7) were reacted as primary antibodies. As a result, as shown in FIG. 3, a strong reaction signal was detected in the sera from 4 cases (M5, M6, M18, MS7), and thus these sera contained antibodies specific for CMV-pp65. It was shown that.
次に、実施例１で確立したＥＬＩＳＡ法に用いる血清の最適希釈濃度を検討する目的で、ＣＭＶ−ｐｐ６５タンパク質を固相化したアッセイプレートに、一次抗体として上記の２症例（Ｍ１８、ＭＳ７）由来の血清を、二次抗体としてＨＲＰ標識抗ヒトＩｇＧヒツジ抗体を反応させ、吸光度を測定した。図４に結果を示すように、患者血清の希釈倍率は、５０〜１００００倍で測定が可能であることが明らかとなった。 Next, for the purpose of examining the optimal dilution concentration of serum used in the ELISA method established in Example 1, the above-mentioned two cases (M18, MS7) were derived as primary antibodies on the assay plate on which CMV-pp65 protein was immobilized. Was reacted with an HRP-labeled anti-human IgG sheep antibody as a secondary antibody, and the absorbance was measured. As shown in FIG. 4, it was revealed that the patient serum can be measured at a dilution rate of 50 to 10,000 times.
実施例１で確立した方法を用いて、メラノーマ患者３１例と健常人１０例の血清中のＣＭＶ−ｐｐ６５抗原に対するＩｇＧ抗体価を測定・標準化した。その結果、図５に示すように、カットオフ値を１とするとメラノーマ患者３１例中２３例で抗体価の上昇を認めた。一方、健常人では、明らかな陽性は１０例中４例のみであった。 Using the method established in Example 1, IgG antibody titers against CMV-pp65 antigen in the serum of 31 melanoma patients and 10 healthy subjects were measured and standardized. As a result, as shown in FIG. 5, when the cut-off value was 1, 23 of 31 melanoma patients showed an increase in antibody titer. On the other hand, in healthy individuals, only 4 out of 10 cases were clearly positive.
実施例１で確立した方法を用いて、メラノーマ患者３１例と健常人１１例の血清中のメラノーマ特異抗原（ＭＡＧＥ１、ＭＡＧＥ２、ＭＡＧＥ３）に対するＩｇＧ抗体価を測定・標準化した。各メラノーマ抗原１００ｎｇ／ｍｌ、陰性コントロールとしてＧＳＴタンパク質を使用した。一番上のデータは抗原を固相化しないバックグラウンドの数値を示す。ＭＡＧＥ１及びＭＡＧＥ２は全長を、ＭＡＧＥ３は部分配列（４４−１１４ａａ）を有する組換えタンパク質を固相化抗原として使用した。その結果、図６に示すように、カットオフ値を１とすると３１例中１５例が抗体陽性であった。ＭＡＧＥ２に対する抗体価は、同様に１０例、ＭＡＧＥ３に対する抗体価は１例のみが陽性であった。一方で健常人でもこれらの抗原に対する抗体陽性例が数例に認められた。 Using the method established in Example 1, IgG antibody titers against melanoma specific antigens (MAGE1, MAGE2, MAGE3) in the serum of 31 melanoma patients and 11 healthy subjects were measured and standardized. Each melanoma antigen was 100 ng / ml, and GST protein was used as a negative control. The top data shows the numerical value of the background which does not solidify an antigen. MAGE1 and MAGE2 used full length, and MAGE3 used the recombinant protein which has a partial sequence (44-114aa) as a solid-phased antigen. As a result, as shown in FIG. 6, when the cut-off value was 1, 15 out of 31 cases were antibody positive. Similarly, the antibody titer against MAGE2 was positive in 10 cases, and the antibody titer against MAGE3 was positive in only 1 case. On the other hand, in healthy individuals, several cases of antibody positive against these antigens were observed.
実施例１で確立した方法を用いて、メラノーマ患者２７例における樹状細胞ワクチン前後でのメラノーマ抗原に対するＩｇＧ抗体価の推移を測定・標準化した。静岡がんセンター倫理審査委員会にて承認された「悪性黒色腫に対する樹状細胞を用いた腫瘍特異的免疫療法」の臨床試験において、樹状細胞ワクチンの投与をうけたメラノーマ２７症例においてワクチン投与前（Ｐｒｅ）、ワクチン４回投与後（Ｐｏｓｔ）、６−８回投与後（Ｐｏｓｔ１）、１０回投与後（Ｐｏｓｔ２）の各時期に患者より血清を採取し、実験に用いた。なお、上記樹状細胞ワクチンは、樹状細胞をＭＡＧＥ１、ＭＡＧＥ２、ＭＡＧＥ３、Ｔｙｒｏｓｉｎａｓｅ等のメラノーマ抗原ペプチドに感作させたものを使用した。図７に示すように、樹状細胞ワクチン投与を受けた転移性メラノーマ患者における、ワクチン投与の前後でのメラノーマ抗原に対する血清中のＩｇＧ抗体価を測定した結果、ワクチン投与後にＩｇＧ抗体価が２倍以上の増加を認めた症例は、ＭＡＧＥ１で４例、ＭＡＧＥ２で４例、ＭＡＧＥ３で５例、ｔｙｒｏｓｉｎａｓｅで７例であった。また、臨床効果（腫瘍の縮小）を認めた症例（ＭＥＬ００１、ＭＥＬ０１８）では、すべての抗体価がワクチン後に上昇が認められた。 Using the method established in Example 1, the transition of IgG antibody titer against melanoma antigen before and after dendritic cell vaccine in 27 melanoma patients was measured and standardized. Vaccine administration in 27 cases of melanoma receiving dendritic cell vaccine in clinical trial of "Tumor specific immunotherapy using dendritic cells for malignant melanoma" approved by Shizuoka Cancer Center Ethics Review Committee Serum was collected from the patient at each stage before (Pre), after 4 doses of vaccine (Post), after 6-8 doses (Post1), and after 10 doses (Post2), and used for experiments. In addition, the said dendritic cell vaccine used what sensitized the dendritic cell to melanoma antigen peptides, such as MAGE1, MAGE2, MAGE3, and Tyrosinase. As shown in FIG. 7, as a result of measuring the IgG antibody titer in the serum against the melanoma antigen in the metastatic melanoma patient who received the dendritic cell vaccine administration, the IgG antibody titer doubled after the vaccine administration. There were 4 cases with MAGE1, 4 cases with MAGE2, 5 cases with MAGE3, and 7 cases with tyrosinase. Moreover, in the cases (MEL001, MEL018) in which clinical effects (tumor shrinkage) were observed, all antibody titers increased after the vaccine.
Claims (2)
（１ａ）抗原の代わりに使用する抗ヒト抗体を段階希釈し、抗ヒト抗体の濃度系列を作製して、各々固相化した複数の捕獲側抗体プレートを調製する工程；
（１ｂ）抗原タンパク質を固相化した複数の抗原固定プレートを調製する工程；
（２ａ）過剰量（飽和量）のヒトＩｇＧを用いることにより、工程（１ａ）で調製した捕獲側抗体プレート上の捕獲側抗体をヒトＩｇＧで飽和させ、洗浄してヒトＩｇＧ−捕獲側抗体複合体を調製する工程；
（２ｂ）段階希釈したサンプルを、工程（１ｂ）で調製した抗原固定プレートに接触させ、サンプル中の標的抗体を抗原タンパク質と反応させ、洗浄して標的抗体−抗原複合体を調製する工程；
（３ａ）工程（１ａ）で使用した抗ヒト抗体とエピトープを異にした抗ヒト抗体を標識した酵素標識抗ヒト抗体を、工程（２ａ）で調製したヒトＩｇＧ−捕獲側抗体複合体と反応させ、洗浄して酵素標識抗ヒト抗体−ヒトＩｇＧ−捕獲側抗体複合体を調製する工程；
（３ｂ）工程（３ａ）で使用した酵素標識抗ヒト抗体を、工程（２ｂ）で調製した標的抗体−抗原複合体と反応させ、洗浄して酵素標識抗ヒト抗体−標的抗体−抗原複合体を調製する工程；
（４ａ）工程（３ａ）で調製した酵素標識抗ヒト抗体−ヒトＩｇＧ−捕獲側抗体複合体に基質を添加して反応後、吸光度を測定し、捕獲側抗体と吸光度との標準曲線を作成する工程；
（４ｂ）工程（３ｂ）で調製した酵素標識抗ヒト抗体−標的抗体−抗原複合体に基質を添加して反応後、吸光度を測定し、吸光度の測定結果と工程（４ａ）で作成した標準曲線から、サンプル中の標的抗体の抗体価を定量する工程； A standardized method for measuring an antigen-specific antibody titer comprising the following steps.
(1a) A step of serially diluting an anti-human antibody to be used in place of an antigen to prepare a concentration series of anti-human antibodies, and preparing a plurality of capture-side antibody plates each immobilized on a solid phase;
(1b) preparing a plurality of antigen-immobilized plates on which an antigen protein is immobilized;
(2a) By using an excess amount (saturation amount) of human IgG, the capture side antibody on the capture side antibody plate prepared in step (1 a ) is saturated with human IgG, washed and human IgG-capture side antibody Preparing the complex;
(2b) a step of bringing the serially diluted sample into contact with the antigen-immobilized plate prepared in step (1 b ), reacting the target antibody in the sample with the antigen protein, and washing to prepare a target antibody-antigen complex;
(3a) The enzyme-labeled anti-human antibody labeled with an anti-human antibody having an epitope different from that of the anti-human antibody used in step (1 a ) is reacted with the human IgG-capture side antibody complex prepared in step (2a). And washing to prepare an enzyme-labeled anti-human antibody-human IgG-capture antibody complex;
(3b) The enzyme-labeled anti-human antibody used in step (3a) is reacted with the target antibody-antigen complex prepared in step (2b), washed, and the enzyme-labeled anti-human antibody-target antibody-antigen complex is washed. Preparing step;
(4a) After adding the substrate to the enzyme-labeled anti-human antibody-human IgG-capture side antibody complex prepared in step (3a), the absorbance is measured, and a standard curve of the capture side antibody and the absorbance is prepared. Process;
(4b) After adding the substrate to the enzyme-labeled anti-human antibody-target antibody-antigen complex prepared in step (3b) and reacting, the absorbance is measured, and the absorbance measurement result and the standard curve created in step (4a) Quantifying the antibody titer of the target antibody in the sample from;
The measurement method according to claim 1, wherein the target antibody in the sample is an IgG antibody specific for a cancer antigen present in serum or an antigen derived from a pathogen capable of causing an infection.
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