JP2011163992A - Immunoassay method of complex of ftcd and autoantibody thereof, kit used for the same, and cancer determination method using the same - Google Patents
Immunoassay method of complex of ftcd and autoantibody thereof, kit used for the same, and cancer determination method using the same Download PDFInfo
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Abstract
Description
本発明は、ホルミニノトランスフェラーゼ シクロデアミナーゼ（formininotransferase cyclodeaminase；ＦＴＣＤ）とその自己抗体との複合体を測定するための免疫測定方法及び免疫測定用キットに関するものである。ＦＴＣＤとその自己抗体との複合体は、特に担癌患者の血中に高値に出現するものであり、したがって、本発明は、ＦＴＣＤとその自己抗体との複合体を単に測定する方法を提供するだけでなく、癌の判定にも利用することができる。 The present invention relates to an immunoassay method and an immunoassay kit for measuring a complex of formininotransferase cyclodeaminase (FTCD) and an autoantibody thereof. The complex of FTCD and its autoantibodies is particularly high in the blood of cancer-bearing patients, and therefore the present invention provides a method for simply measuring the complex of FTCD and its autoantibodies. Not only can it be used to determine cancer.
ＦＴＣＤは、葉酸代謝及びグルタミン酸代謝の２種類の機能を持つ肝臓特異的に発現する酵素であることが知られている（非特許文献１）。
他方、アガロース２次元電気泳動に２Ｄ−ＤＩＧＥ法（two-dimensional fluorescence difference gel electrophrosis）を適用した改良アガロース２次元電気泳動法（非特許文献２）により、原発性肝細胞癌の癌部および周辺の非癌部組織の蛋白発現量の比較をプロテオーム解析を行ない、ＦＴＣＤが非癌部に多く発現されることが明らかになっている（非特許文献３）
FTCD is known to be a liver-specific enzyme that has two functions of folate metabolism and glutamate metabolism (Non-patent Document 1).
On the other hand, by the improved agarose two-dimensional electrophoresis method (Non-patent Document 2) applying 2D-DIGE method (two-dimensional fluorescence difference gel electrophrosis) to agarose two-dimensional electrophoresis, the cancerous part of the primary hepatocellular carcinoma and the surrounding area Proteome analysis is performed to compare the protein expression levels of non-cancerous tissues, and it has been revealed that FTCD is highly expressed in non-cancerous parts (Non-patent Document 3).
本発明者らは、癌が疑われる患者または癌患者由来の組織検体中に存在する、ＦＴＣＤの発現レベルを測定して、組織中の癌部は非癌部に比べて弱く発現することにより、それらの組織の癌部と非癌部の判別することができることを見出し、これに対し特許出願した（特願２００８年２６４７９４号）。本発明者らは、さらに血液検体中のＦＴＣＤの存在について研究を続けたところ、驚くべきことに、血液検体中にＦＴＣＤとその自己抗体との複合体が存在し、癌患者の場合、組織部とは逆に、その複合体の量が多いことを見出した。したがって、本発明の目的は、癌判定に応用することのできる、ＦＴＣＤとその自己抗体との複合体の測定方法を提供することである。 The present inventors measured the expression level of FTCD present in a tissue sample derived from a patient suspected of cancer or a cancer patient, and the cancerous part in the tissue was weakly expressed as compared to the non-cancerous part. It was found that cancerous and non-cancerous parts of these tissues could be distinguished, and a patent application was filed for this (Japanese Patent Application No. 2008-264794). The inventors further continued research on the presence of FTCD in a blood sample. Surprisingly, a complex of FTCD and its autoantibodies is present in the blood sample. On the contrary, we found that the amount of the complex was large. Therefore, an object of the present invention is to provide a method for measuring a complex of FTCD and its autoantibody that can be applied to cancer determination.
本発明者らは、検体中のＦＴＣＤとその自己抗体との複合体を免疫測定することにより、該複合体を測定することができ、それによって癌判定が可能になることを見出し、本発明を完成させた。
従って、本発明は、検体中のＦＴＣＤとその自己抗体との複合体を免疫測定することを特徴とする、検体中のＦＴＣＤとその自己抗体との複合体の免疫測定方法に関する。
更に、本発明は、ＦＴＣＤに対する試薬抗体およびその自己抗体に対する結合可能物質を含む、ＦＴＣＤとその自己抗体との複合体の免疫測定用キットに関する。
更に、本発明は、ＦＴＣＤとその自己抗体との複合体を測定することにより、癌であることを判定する、癌判定方法に関する。
The present inventors have found that the complex can be measured by immunoassay of a complex of FTCD and its autoantibody in a specimen, thereby enabling cancer determination. Completed.
Therefore, the present invention relates to a method for immunoassay of a complex of FTCD in a sample and its autoantibody, which comprises immunoassay of a complex of FTCD in the sample and its autoantibody.
Furthermore, the present invention relates to a kit for immunoassay of a complex of FTCD and its autoantibody comprising a reagent antibody against FTCD and a substance capable of binding to the autoantibody.
Furthermore, this invention relates to the cancer determination method which determines that it is cancer by measuring the composite_body | complex of FTCD and its autoantibody.
本発明においては、検体中、特に、血液由来検体中に存在するＦＴＣＤとその自己抗体との複合体を簡単に測定でき、原発性肝細胞癌などの癌患者の判定に有効である。 In the present invention, a complex of FTCD and its autoantibody present in a specimen, particularly in a blood-derived specimen, can be easily measured, and is effective in determining cancer patients such as primary hepatocellular carcinoma.
本発明のＦＴＣＤとその自己抗体との複合体の免疫測定方法は、一般的な蛋白質とその抗体との免疫複合体の免疫測定方法で知られている公知の方法をそのまま、応用できる。
本発明の免疫測定方法においては、例えば、検体中のＦＴＣＤとその自己抗体との複合体に、ＦＴＣＤに対する試薬抗体およびその自己抗体に対する結合可能物質を作用させ、得られる複合体と試薬抗体と結合可能物質との免疫複合物を測定することによりその複合体を測定することができる。
As a method for immunoassay of a complex of FTCD of the present invention and its autoantibody, a known method known as a method for immunoassay of an immune complex of a general protein and its antibody can be applied as it is.
In the immunoassay method of the present invention, for example, a reagent antibody against FTCD and a substance capable of binding to the autoantibody are allowed to act on a complex of FTCD and its autoantibody in a specimen, and the resulting complex is bound to the reagent antibody. The complex can be measured by measuring the immune complex with the potential substance.
本発明において、検体とは、生体由来の試料が好適で、特に、血液由来検体が好適であり、血液検体としては、全血、血漿、血清を例示できる。 In the present invention, the sample is preferably a biological sample, particularly preferably a blood-derived sample, and examples of the blood sample include whole blood, plasma, and serum.
本発明の免疫測定方法の測定対象は、ＦＴＣＤとその自己抗体との複合体である。本発明において、ＦＴＣＤは、正式名がホルムイミノトランスフェラーゼシクロデアミナーゼ（formiminotransferase cyclodeaminase）であり、Swiss-Prot entry O95954でコードされる分子量５９ｋＤａ、５４１アミノ酸で構成される蛋白質である。 The measurement target of the immunoassay method of the present invention is a complex of FTCD and its autoantibody. In the present invention, FTCD is a protein having a formal name of formiminotransferase cyclodeaminase and a molecular weight of 59 kDa and 541 amino acids encoded by Swiss-Prot entry O95954.
本発明において、ＦＴＣＤとその自己抗体との複合体とは、ＦＴＣＤとＦＴＣＤに対する自己抗体との免疫複合体を意味し、本明細書では単に複合体と記載することもある。
本発明において、自己抗体とは、自己の身体に存在する物質に対して自己の身体で産生される抗体であって、自己の身体に存在する物質がＦＴＣＤであり、そのＦＴＣＤに対する抗体をいう。
本発明において、ＦＴＣＤに対する試薬抗体とは、試薬として用いるＦＴＣＤと特異的に結合する抗体をいい、本明細書では単に試薬抗体と記載することもある。その試薬抗体は、その産生動物種としてヒト、マウス、ラット、ウサギ、ヤギ、ウマ等があり、それぞれに所定範囲の免疫グロブリンがある。その試薬抗体は、ＩｇＧ、ＩｇＭ、ＩｇＡ、ＩｇＥ、ＩｇＤのいずれでもよい。また、試薬抗体は、モノクローナル抗体、ポリクローナル抗体、及びこれらの断片（抗原と結合能を有するもので例えば、Ｈ鎖、Ｌ鎖、Ｆａｂ、Ｆ（ａｂ’）２等）のいずれでもよい。このような試薬抗体は、ＦＴＣＤ全長蛋白質またはその断片ペプチドを抗原として、上記した産生動物種に免疫して、その免疫動物から抗血清として得ることができ、また、免疫動物からの脾細胞とミエローマ細胞とを融合して、融合細胞から、ＦＴＣＤに対する抗体を産生する融合細胞をスクリーニングして、得られるハイブリドーマからモノクローナル抗体として得ることもできる。また、ＦＴＣＤに対する試薬抗体は、抗ＦＴＣＤ抗体として市販されており、それらの市販品を使用することもできる。
In the present invention, the complex of FTCD and its autoantibody means an immune complex of FTCD and an autoantibody against FTCD, and may be simply described as a complex in the present specification.
In the present invention, the autoantibody is an antibody produced in the own body against a substance present in the own body, and the substance present in the own body is FTCD, and refers to an antibody against the FTCD.
In the present invention, the reagent antibody against FTCD refers to an antibody that specifically binds to FTCD used as a reagent, and may be simply referred to as a reagent antibody in the present specification. The reagent antibodies include humans, mice, rats, rabbits, goats, horses, and the like as production animal species, and each has a predetermined range of immunoglobulins. The reagent antibody may be any of IgG, IgM, IgA, IgE, and IgD. The reagent antibody may be any of a monoclonal antibody, a polyclonal antibody, and a fragment thereof (having an antigen-binding ability, such as H chain, L chain, Fab, F (ab ′) 2, etc.). Such a reagent antibody can be obtained as an antiserum from the immunized animal by immunizing the above-mentioned animal species using the full-length protein of FTCD or a fragment peptide thereof as an antigen, and spleen cells and myeloma from the immunized animal. It is also possible to obtain a monoclonal antibody from the hybridoma obtained by screening a fused cell that produces an antibody against FTCD from the fused cell by fusing the cell. Moreover, the reagent antibody with respect to FTCD is marketed as an anti-FTCD antibody, and those commercial products can also be used.
本発明において、その自己抗体に対する結合可能物質とは、ＦＴＣＤに対する自己抗体と結合可能な物質であれば特に限定しないが、本明細書では単に結合可能物質と記載することがある。この様な結合可能物質としては、抗ＩｇＧ抗体、プロテインＡ、プロテインＧ、試薬としてのＦＴＣＤ抗原を用いることができ、そのうち、抗ＩｇＧ抗体が好ましい。
試薬としてのＦＴＣＤ抗原を用いる場合、ＦＴＣＤに対する自己抗体と抗原抗体反応しうる抗原であれば特に限定しないが、ＦＴＣＤ全長蛋白質、ＦＴＣＤ全長蛋白質の変異体であって該蛋白質と同様のＦＴＣＤに対する自己抗体と抗原抗体反応しうる機能を有し且つ該アミノ酸配列と９０％以上の相同性を有する蛋白質もしくはＦＴＣＤ全長蛋白質のアミノ酸配列において１個から数個のアミノ酸残基が欠失、置換もしくは付加したアミノ酸配列を有する蛋白質である変異体、ＦＴＣＤの断片ペプチドであってＦＴＣＤの自己抗体と抗原抗体反応しうるペプチドを例示できる。ＦＴＣＤ全長蛋白質は、Ａｂｎｏｖａ社より入手可能であるが、全アミノ酸配列が既知であるので、ＦＴＣＤ全長蛋白質やその変異体は、遺伝子組換え技術によっても合成できる。本発明においてＦＴＣＤの断片ペプチドを用いるときは、ＦＴＣＤ全長蛋白質を酵素分解等によって各種のペプチド断片に切断して作成してもよいし、市販の自動ペプチド合成装置を用いても容易に作成することができる。また、標的のＦＴＣＤの断片ペプチドを遺伝子組み換え技術によっても作成することができる。
そのようにして得られたＦＴＣＤ全長蛋白質の変異体や断片ペプチドを、ＦＴＣＤに対する自己抗体と反応させ抗原抗体反応をするものを選択して試薬としてのＦＴＣＤ抗原として用いることができる。本発明においては、上記した各ペプチド断片の全体のほか、その一部も使用できるし、それらの混合物も使用でき、これらも試薬としてのＦＴＣＤ抗原に包含される。
In the present invention, the substance capable of binding to the autoantibody is not particularly limited as long as it is a substance capable of binding to the autoantibody against FTCD, but may be simply described as a substance capable of binding in the present specification. As such a substance capable of binding, anti-IgG antibody, protein A, protein G, and FTCD antigen as a reagent can be used, and of these, anti-IgG antibody is preferable.
When the FTCD antigen is used as a reagent, it is not particularly limited as long as it is an antigen antibody-antibody reaction with an autoantibody against FTCD, but it is a FTCD full-length protein, a mutant of FTCD full-length protein, and the same autoantibody against FTCD as the protein An amino acid having a function capable of reacting with an antigen and an antibody and having 90% or more homology with the amino acid sequence or an amino acid sequence of a full-length FTCD protein, wherein one to several amino acid residues are deleted, substituted or added Examples thereof include a mutant that is a protein having a sequence, and a peptide peptide that is a fragment peptide of FTCD and can react with an autoantibody of FTCD in an antigen-antibody reaction. The full-length FTCD protein is available from Abnova, but since the entire amino acid sequence is known, the full-length FTCD protein and its variants can also be synthesized by gene recombination techniques. When using a fragment peptide of FTCD in the present invention, it may be prepared by cleaving the full-length FTCD protein into various peptide fragments by enzymatic degradation or the like, or easily using a commercially available automatic peptide synthesizer. Can do. Moreover, a fragment peptide of the target FTCD can also be prepared by a gene recombination technique.
A mutant or fragment peptide of the FTCD full-length protein thus obtained is allowed to react with an autoantibody against FTCD to undergo an antigen-antibody reaction, and can be used as an FTCD antigen as a reagent. In the present invention, in addition to the whole peptide fragments described above, a part of them can be used, or a mixture thereof can be used, and these are also included in the FTCD antigen as a reagent.
本発明においては、検体中のＦＴＣＤとその自己抗体との複合体に、ＦＴＣＤに対する試薬抗体およびその自己抗体に対する結合可能物質を作用させると、複合体と試薬抗体と結合可能物質との免疫複合物が生成する。その免疫複合物を測定するには、ＦＴＣＤに対する試薬抗体（試薬抗体）およびその自己抗体に対する結合可能物質（結合可能物質）のいずれかを標識成分で標識し、生成する免疫複合物中の標識成分を測定することによって、免疫複合物を測定するのが好ましい。
標識成分としては、酵素、放射性物質、蛍光物質、化学発光物質等常用される標識成分を使用することができるが、酵素や放射性物質が好ましい。
標識するための酵素としては、西洋ワサビペルオキシダーゼ(ＨＲＰ)、ウシ小腸アルカリフォスファターゼ、β−ガラクトシダーゼ、ウレアーゼ、グルコースオキシダーゼ等の酵素免疫分析法（ＥＩＡ）に常用される酵素が適宜使用され、これらの酵素に適合しＥＩＡで常用される発色基質が適宜使用される。発色基質としては、例えばＨＲＰの場合は、３，３′，５，５′−テトラメチルベンジジン（ＴＭＢＺ）、ＴＭＢＺ・ＨＣｌ、ＴＭＢＺ・ＰＳ、ＡＢＴＳ、ｏ−フェニレンジアミン、ｐ−ヒドロキシフェニル酢酸等が使用され、アルカリフォスファターゼの場合は、ｐ−ニトロフェニルフォスフェート、４−メチルウンベリフェリルフォスフェート等が使用され、β−ガラクトシダーゼの場合は、ｏ−ニトロフェニル−β−Ｄ−ガラクトピラノシド、４−メチルウンベリフェリルβ−Ｄ−ガラクトピラノシド等が使用される。
標識するための放射性物質としては放射性ヨウ素原子等を、蛍光物質としてはＦＩＴＣやローダミン等を、化学発光物質としてはルミノール等を例示することができる。
In the present invention, when a reagent antibody against FTCD and a substance capable of binding to the autoantibody are allowed to act on a complex of FTCD and the autoantibody in a sample, an immune complex of the complex, the reagent antibody and the substance capable of binding. Produces. In order to measure the immune complex, either a reagent antibody against FTCD (reagent antibody) or a substance capable of binding to the autoantibody (binding substance) is labeled with a labeling component, and the labeling component in the resulting immune complex It is preferred to measure immune complexes by measuring.
As the labeling component, commonly used labeling components such as enzymes, radioactive substances, fluorescent substances, and chemiluminescent substances can be used, but enzymes and radioactive substances are preferred.
As the enzyme for labeling, enzymes commonly used in enzyme immunoassay (EIA) such as horseradish peroxidase (HRP), bovine small intestine alkaline phosphatase, β-galactosidase, urease, glucose oxidase and the like are appropriately used. A chromogenic substrate that is suitable for EIA and commonly used in EIA is appropriately used. Examples of chromogenic substrates include 3,3 ', 5,5'-tetramethylbenzidine (TMBZ), TMBZ.HCl, TMBZ.PS, ABTS, o-phenylenediamine, p-hydroxyphenylacetic acid and the like in the case of HRP. In the case of alkaline phosphatase, p-nitrophenyl phosphate, 4-methylumbelliferyl phosphate and the like are used, and in the case of β-galactosidase, o-nitrophenyl-β-D-galactopyranoside, 4-Methylumbelliferyl β-D-galactopyranoside and the like are used.
Examples of radioactive materials for labeling include radioactive iodine atoms, examples of fluorescent materials include FITC and rhodamine, and examples of chemiluminescent materials include luminol.
本発明において標識成分を用いる場合、例えば、検体中のＦＴＣＤとその自己抗体との複合体に、水不溶性担体に結合しているＦＴＣＤに対する試薬抗体を作用させ、次いで、標識成分で標識されたその自己抗体に対する結合可能物質を作用させ、複合体と試薬抗体と結合可能物質との免疫複合物を生成させ、その免疫複合物に結合している標識成分を測定することによりその複合体を測定することが好ましい。また、検体中のＦＴＣＤとその自己抗体との複合体に、その自己抗体に対する結合可能物質が結合した水不溶性担体を作用させ、次いで、標識成分で標識されたＦＴＣＤに対する試薬抗体を作用させ、複合体と結合可能物質と試薬抗体との免疫複合物を生成させ、その免疫複合物に結合している標識成分を測定することによりその複合体を測定することもできる。 When a labeling component is used in the present invention, for example, a reagent antibody against FTCD bound to a water-insoluble carrier is allowed to act on a complex of FTCD and its autoantibody in a sample, and then labeled with the labeling component. Measures the complex by reacting a binding substance to the autoantibody to generate an immune complex of the complex, the reagent antibody, and the binding substance, and measuring the label component bound to the immune complex. It is preferable. Further, a complex of FTCD and its autoantibody in a sample is allowed to act on a water-insoluble carrier to which a substance capable of binding to the autoantibody is bound, and then a reagent antibody against FTCD labeled with a labeling component is allowed to act on the complex. It is also possible to measure the complex by generating an immune complex of the body, a substance capable of binding, and a reagent antibody, and measuring the label component bound to the immune complex.
水不溶化担体の調製は、蛋白質を固相面に結合する既知の方法を用いて容易に行うことができる。例えば、固相化担体としては、通常、ビーズ、マイクロプレート、チューブ等が用いられる。これらの固相面にＦＴＣＤに対する試薬抗体を結合する方法としては、物理吸着、化学結合等既知の固定化技術が適宜利用できる。
このようにして固相化したＦＴＣＤに対する試薬抗体に、ＦＴＣＤとその自己抗体との複合体とを含む検体とを接触させると、複合体中のＦＴＣＤ部分と試薬抗体とが結合する。さらに、その結合物に対し、標識成分で標識されたその自己抗体に対する結合可能物質（例えば標識抗ＩｇＧ抗体）を作用させると複合体と試薬抗体と結合可能物質との免疫複合物が生成する。その結果、生成する免疫複合物中の標識成分を測定することにより、検体中のＦＴＣＤとその自己抗体との複合体を測定することができる。
上記と同様にして、固相化担体にＦＴＣＤの自己抗体に対する結合可能物質を結合させて、固相化した結合可能物質に、ＦＴＣＤとその自己抗体との複合体とを含む検体とを接触させて、複合体中の自己抗体部分と結合可能物質とを結合させ、さらに、その結合物に対し、標識成分で標識されたＦＴＣＤに対する試薬抗体（例えば、抗ＦＴＣＤ抗体）を作用させて、複合体と結合可能物質と試薬抗体との免疫複合物を生成させて、同様にして、検体中のＦＴＣＤとその自己抗体との複合体を測定することもできる。
The water-insolubilized carrier can be easily prepared using a known method for binding a protein to a solid phase surface. For example, as the solid-phase support, beads, microplates, tubes and the like are usually used. As a method for binding a reagent antibody against FTCD to these solid phase surfaces, known immobilization techniques such as physical adsorption and chemical bonding can be used as appropriate.
When the reagent antibody against FTCD thus immobilized is brought into contact with a specimen containing a complex of FTCD and its autoantibody, the FTCD portion in the complex and the reagent antibody are bound. Further, when a substance capable of binding to the autoantibody labeled with a labeling component (for example, a labeled anti-IgG antibody) is allowed to act on the conjugate, an immunocomplex of the complex, the reagent antibody and the substance capable of binding is produced. As a result, the complex of FTCD and its autoantibodies in the specimen can be measured by measuring the label component in the generated immune complex.
In the same manner as described above, a substance capable of binding to the autoantibody of FTCD is bound to the immobilized carrier, and a specimen containing the complex of FTCD and the autoantibody is brought into contact with the immobilized substance capable of binding. Then, the autoantibody part in the complex is bound to the bindable substance, and a reagent antibody (for example, anti-FTCD antibody) against FTCD labeled with the labeling component is allowed to act on the complex, In the same manner, a complex of FTCD and its autoantibody in a sample can be measured by generating an immunocomplex of a binding substance and a reagent antibody.
本発明の免疫測定方法の典型的な例を以下に示す。
プレートに抗ＦＴＣＤ抗体を加え、低温例えば４℃で静置して感作し、その後、ＰＢＳ等の洗浄液で洗浄する。ついで、そのプレートをＢＳＡでコーティングし、抗ＦＴＣＤ抗体ＥＬＩＳＡプレートを作成する。希釈した検体を抗ＦＴＣＤ抗体ＥＬＩＳＡプレートに加え、加温例えば３７℃で静置し、次いでＰＢＳ等の洗浄液で洗浄をする。得られるプレートのウェルにＨＲＰ標識された抗ヒトＩｇＧ抗体を加え、加温例えば３７℃で静置する。ついで、ウェルをＰＢＳ等の洗浄液で洗浄した後、ＴＭＢＺを加え、例えば室温で静置した後、反応停止剤として１Ｎ硫酸を加える。吸光度はマイクロプレートリーダー（ＢｉｏＲａｄ社製）を用いて、波長４５０ｎｍにて吸光度を測定する。吸光度の値とあらかじめ作成しておいた検量線から、ＦＴＣＤとその自己抗体との複合体の値を求める。
A typical example of the immunoassay method of the present invention is shown below.
Anti-FTCD antibody is added to the plate, left to stand at a low temperature, for example, 4 ° C. for sensitization, and then washed with a washing solution such as PBS. The plate is then coated with BSA to make an anti-FTCD antibody ELISA plate. The diluted specimen is added to the anti-FTCD antibody ELISA plate, allowed to stand at a warm temperature, for example, 37 ° C., and then washed with a washing solution such as PBS. An HRP-labeled anti-human IgG antibody is added to the well of the resulting plate, and the plate is allowed to stand at a warm temperature, for example, 37 ° C. Next, the well is washed with a washing solution such as PBS, and then TMBZ is added. For example, after standing at room temperature, 1N sulfuric acid is added as a reaction terminator. Absorbance is measured at a wavelength of 450 nm using a microplate reader (BioRad). From the absorbance value and the calibration curve prepared in advance, the value of the complex of FTCD and its autoantibody is determined.
本発明の測定方法は、ＦＴＣＤに対する試薬抗体およびその自己抗体に対する結合可能物質を含む、ＦＴＣＤとその自己抗体との複合体の免疫測定用キットにより実施することができる。そのためのＦＴＣＤに対する試薬抗体、その自己抗体に対する結合可能物質は、本発明の測定方法で説明したとおりである。すなわち、例えば、ＦＴＣＤに対する試薬抗体およびその自己抗体に対する結合可能物質のいずれかを水不溶性担体に結合させた形態で、他のいずれかを標識成分で標識した形態で、キットの試薬成分とすることができる。その他の試薬成分として、界面活性剤、緩衝剤等の免疫測定法で常用されるものを適宜、加えてもよい。 The measurement method of the present invention can be carried out by a kit for immunoassay of a complex of FTCD and its autoantibody, which contains a reagent antibody against FTCD and a substance capable of binding to the autoantibody. Therefore, the reagent antibody against FTCD and the substance capable of binding to the autoantibody are as described in the measurement method of the present invention. That is, for example, either a reagent antibody against FTCD or a substance capable of binding to the autoantibody is bound to a water-insoluble carrier, and any of the other is labeled with a labeling component and used as a reagent component of the kit. Can do. As other reagent components, those commonly used in immunoassay methods such as surfactants and buffers may be added as appropriate.
本発明においては、ＦＴＣＤとその自己抗体との複合体を測定することにより、癌判定をすることができる。
また、一般にＦＴＣＤとその自己抗体との複合体の量が多いと、原発性肝細胞癌（原発性肝細胞癌、原発性胆管細胞癌など）、転移性肝癌等の肝癌、膀胱癌、乳癌、肺癌、卵巣癌、前立腺癌、甲状腺癌、皮膚癌などの癌が疑われ、本発明によりＦＴＣＤに対する自己抗体を測定することは、患者の癌疾患の判別に有効である。本発明においては、原発性肝細胞癌の判別に有効であり、例えば、健常人と、初発原発性肝細胞癌患者や再発原発性肝細胞癌患者との判別に有効である。
In the present invention, cancer can be determined by measuring a complex of FTCD and its autoantibody.
In general, when the amount of the complex of FTCD and its autoantibody is large, primary hepatocellular carcinoma (primary hepatocellular carcinoma, primary cholangiocellular carcinoma etc.), liver cancer such as metastatic liver cancer, bladder cancer, breast cancer, Cancers such as lung cancer, ovarian cancer, prostate cancer, thyroid cancer, and skin cancer are suspected, and measurement of autoantibodies against FTCD according to the present invention is effective in determining cancer diseases in patients. In the present invention, it is effective for discriminating primary hepatocellular carcinoma. For example, it is effective for discriminating between healthy individuals and patients with primary primary hepatocellular carcinoma or patients with recurrent primary hepatocellular carcinoma.
以下、本発明を実施例により更に詳細に説明するが、本発明は、これら実施例に何ら制限されるものではない。
実施例１
ＦＴＣＤとその自己抗体との複合体の測定
健常人、初発原発性肝細胞癌患者、及び再発原発性肝細胞癌患者から採取した血清検体について、ＦＴＣＤとその自己抗体との複合体の測定を、以下に具体的に説明するようにして行なった。
EXAMPLES Hereinafter, although an Example demonstrates this invention still in detail, this invention is not restrict | limited to these Examples at all.
Example 1
Measurement of the complex of FTCD and its autoantibody For serum samples collected from healthy individuals, patients with primary primary hepatocellular carcinoma, and patients with recurrent primary hepatocellular carcinoma, measurement of the complex of FTCD and its autoantibody This was carried out as specifically described below.
１．方法
（１）抗ＦＴＣＤ抗体ＥＬＩＳＡプレートの作成
ＥＬＩＳＡプレート（Ｎｕｎｃ社製，Ｍａｘｉｓｏｒｐ）に抗ＦＴＣＤ抗体（Ａｂｎｏｖａ社製，５μｇ／ｍＬ，１００μＬ／ｗｅｌｌ）を１晩４℃静置して感作し、その後、０．０５％Ｔｗｅｅｎ２０を含むＰＢＳ（２００μＬ／ｗｅｌｌ）で３回洗浄を行った。ついで、１．５％ＢＳＡ、１０％サッカロースを含むＰＢＳ（２００μＬ／ｗｅｌｌ）で１晩コーティングし、抗ＦＴＣＤ抗体ＥＬＩＳＡプレートを作成した。
1. Method (1) Preparation of anti-FTCD antibody ELISA plate Anti-FTCD antibody (Abnova, 5 μg / mL, 100 μL / well) is left on a ELISA plate (Nunc, Maxisorp) at 4 ° C. overnight to sensitize, Then, it wash | cleaned 3 times by PBS (200 microliters / well) containing 0.05% Tween20. Subsequently, it was coated overnight with PBS (200 μL / well) containing 1.5% BSA and 10% saccharose to prepare an anti-FTCD antibody ELISA plate.
（２）ＦＴＣＤとその自己抗体との複合体の測定
検出抗体としてＨＲＰ標識された抗ヒトＩｇＧ抗体（Ｚｙｍｅｄ社製）を、０．０５％ Ｔｗｅｅｎ２０を含むＰＢＳにて４０００倍に希釈したものを用いた。サンプル血清はＰＢＳにて１００倍に希釈した。その希釈したサンプルを抗ＦＴＣＤ抗体ＥＬＩＳＡプレートに１００μＬ／ｗｅｌｌずつ加え、１時間３７℃で静置し、その後、０．０５％ Ｔｗｅｅｎ２０を含むＰＢＳ（２００μＬ／ｗｅｌｌ）で３回洗浄を行った。得られるプレートのウェルに希釈したＨＲＰ標識された抗ヒトＩｇＧ抗体を１００μＬ／ｗｅｌｌずつ加え、３０分間３７℃で静置した。ついで、０．０５％Ｔｗｅｅｎ２０を含むＰＢＳ（２００μＬ／ｗｅｌｌ）で３回洗浄した後、ＴＭＢＺを１００μＬ／ｗｅｌｌずつ加え、１０分間室温で静置の後、反応停止剤として１００μＬ／ｗｅｌｌの１Ｎ硫酸を加えた。吸光度はマイクロプレートリーダー（ＢｉｏＲａｄ社製）を用いて、波長４５０ｎｍにて測定を行った。
なお、検体は健常人１６例、初発原発性肝細胞癌患者検体１６症例、再発原発性肝細胞癌患者１６例を用いた。
(2) Measurement of a complex of FTCD and its autoantibody As a detection antibody, an HRP-labeled anti-human IgG antibody (manufactured by Zymed) diluted 4000 times with PBS containing 0.05% Tween 20 is used. It was. Sample serum was diluted 100 times with PBS. The diluted sample was added to the anti-FTCD antibody ELISA plate at 100 μL / well, allowed to stand at 37 ° C. for 1 hour, and then washed three times with PBS containing 0.05% Tween 20 (200 μL / well). 100 μL / well of diluted HRP-labeled anti-human IgG antibody was added to each well of the resulting plate, and allowed to stand at 37 ° C. for 30 minutes. Next, after washing three times with PBS containing 0.05% Tween 20 (200 μL / well), 100 μL / well of TMBZ was added and left at room temperature for 10 minutes, and then 100 μL / well of 1N sulfuric acid was added as a reaction stopper. added. Absorbance was measured at a wavelength of 450 nm using a microplate reader (BioRad).
The specimens used were 16 healthy subjects, 16 primary hepatocellular carcinoma patient specimens, and 16 recurrent primary hepatocellular carcinoma patients.
２．結果
ＦＴＣＤとその自己抗体との複合体を測定した結果を用いた結果を、図１に示す。有意差検定はＫａｌｅｉｄａＧｒａｐｈ４．０を用い、Ｗｉｌｃｏｘｏｎの２標本検定にて統計処理した。
図１に示すように、健常人群と比較し、特に再発原発性肝細胞癌患者検体群のＦＴＣＤとその自己抗体との複合体は、大きな有意差を認めた。
2. Results The results of using the results of measuring the complex of FTCD and its autoantibody are shown in FIG. For the significant difference test, KaleidaGraph 4.0 was used, and statistical processing was performed by Wilcoxon two-sample test.
As shown in FIG. 1, compared with the healthy subject group, the complex of FTCD and its autoantibody particularly in the recurrent primary hepatocellular carcinoma patient sample group showed a significant difference.
以上に詳細に説明したように、血液由来検体などの検体中のＦＴＣＤとその自己抗体との複合体に、ＦＴＣＤに対する試薬抗体およびその自己抗体に対する結合可能物質を作用させ、得られる複合体と試薬抗体と結合可能物質との免疫複合物を測定することによりその複合体を測定することができ、それによって、原発性肝細胞癌などの癌判定が可能である。 As described in detail above, the complex and reagent obtained by allowing a reagent antibody against FTCD and a substance capable of binding to the autoantibody to act on a complex of FTCD and its autoantibody in a specimen such as a blood-derived specimen. The complex can be measured by measuring an immune complex of an antibody and a bindable substance, whereby cancer such as primary hepatocellular carcinoma can be determined.
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