JPH07260786A - Immunoassay using antibody having human originated constant region - Google Patents
Immunoassay using antibody having human originated constant regionInfo
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- JPH07260786A JPH07260786A JP4844294A JP4844294A JPH07260786A JP H07260786 A JPH07260786 A JP H07260786A JP 4844294 A JP4844294 A JP 4844294A JP 4844294 A JP4844294 A JP 4844294A JP H07260786 A JPH07260786 A JP H07260786A
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- JP
- Japan
- Prior art keywords
- antibody
- human
- antigenic substance
- derived
- immunoassay
- Prior art date
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Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、少なくとも一定領域が
ヒト由来である抗体を使用する、試料中の抗原性物質の
免疫測定方法に関するものである。詳しくは、少なくと
も一定領域がヒト由来である抗体を、ラジオイムノアッ
セイ（以下ＲＩＡ）、エンザイムイムノアッセイ（以下
ＥＩＡ）等に使用する、試料中の抗原性物質の測定方法
に関するものである。TECHNICAL FIELD The present invention relates to an immunoassay method for an antigenic substance in a sample, which uses an antibody in which at least a certain region is of human origin. More specifically, the present invention relates to a method for measuring an antigenic substance in a sample, which uses an antibody in which at least a certain region is derived from human, for radioimmunoassay (hereinafter RIA), enzyme immunoassay (hereinafter EIA) and the like.
【０００２】[0002]
【従来の技術】抗体は、生体内で抗体産生細胞により産
生され、生体防御機構において重要な役割を果たす蛋白
質で、特定の物質（抗原性物質）に特異的に結合する性
質があり、従来からこの性質を利用して特定の蛋白質や
糖鎖などの免疫測定用試薬として利用されてきた。抗体
を免疫測定用試薬として利用した測定方法として、例え
ばＲＩＡ、ＥＩＡなどが知られている。ＲＩＡやＥＩＡ
では、抗体として、抗原性物質に対する抗血清、ポリク
ロ−ナル抗体、モノクロ−ナル抗体が用いられている
が、近年では、品質の均一性という点が好まれ、でモノ
クロ−ナル抗体の利用が盛んである。2. Description of the Related Art Antibodies are proteins produced in vivo by antibody-producing cells and play an important role in the defense mechanism of the body. They have the property of specifically binding to a specific substance (antigenic substance). Utilizing this property, it has been used as a reagent for immunoassay of a specific protein or sugar chain. For example, RIA, EIA, etc. are known as measurement methods using an antibody as a reagent for immunoassay. RIA and EIA
Antibodies against antigenic substances, polyclonal antibodies, and monoclonal antibodies have been used as antibodies in recent years, but in recent years, homogeneity of quality has been favored, and thus monoclonal antibodies are widely used. Is.
【０００３】[0003]
【発明が解決しようとする課題】モノクロ−ナル抗体の
作製は、自体公知であるハイブリド−マ法（ケ−ラ−と
ミルシュタイン、Ｎａｔｕｒｅ、２５６、４９５、１９
７５年）を用いて行なうことができる。即ち、抗原性物
質で免疫された動物のＢ細胞と骨髄腫細胞を細胞融合し
てハイブリド−マを作製し、得られたハイブリド−マの
中から抗原性物質に結合する抗体を産生するハイブリド
−マを単離し、該ハイブリド−マを用いて作製する。免
疫する動物としてはマウスを用いるのが便利であり多用
されているが、ラット、ニワトリ、ウサギなども用いら
れる。The production of a monoclonal antibody is carried out by a hybridoma method known in itself (Keller and Milstein, Nature, 256 , 495, 19).
75 years). That is, B cells of an animal immunized with an antigenic substance and myeloma cells are fused to prepare a hybridoma, and a hybridoma producing an antibody that binds to the antigenic substance from the obtained hybridomas. Are isolated and made using the hybridomas. As an animal to be immunized, it is convenient to use a mouse and it is widely used, but a rat, a chicken, a rabbit and the like are also used.
【０００４】ハイブリド−マ法によりモノクロ−ナル抗
体を作製する際に、免疫する動物としてマウスを用いた
場合、その結果作製されるモノクロ−ナル抗体はマウス
抗体である。このようなマウス抗体を用いて、ＲＩＡや
ＥＩＡでヒト血清のようなヒト抗体を夾雑物として含有
する試料中の抗原性物質を測定する場合、いわゆるＨＡ
ＭＡ（ｈｕｍａｎ ａｎｔｉ−ｍｏｕｓｅ ａｎｔｉｂ
ｏｄｙ）がマウス抗体と抗原性物質との反応に干渉し、
測定値に影響が及ぶことがある。When a mouse is used as an immunizing animal when producing a monoclonal antibody by the hybridoma method, the resulting monoclonal antibody is a mouse antibody. When such a mouse antibody is used to measure an antigenic substance in a sample containing a human antibody such as human serum as a contaminant by RIA or EIA, a so-called HA is used.
MA (human anti-mouse antib)
ody) interferes with the reaction between the mouse antibody and the antigenic substance,
The measured value may be affected.
【０００５】ＨＡＭＡはマウス抗体を認識するヒト抗体
で、主にマウス抗体の一定領域を認識することが知られ
ている。具体的に測定値への影響が問題となる例として
は、例えば後述の実施例に示すように、固相に固定化し
た第１の抗体および標識した第２の抗体の両方にマウス
抗体を用いたワンステップサンドイッチＥＩＡでヒト血
清中の抗原性物質を測定した場合、実際の抗原性物質濃
度よりも高い値が検出されることが挙げられる。これ
は、第２の抗体が抗原性物質を介して第１の抗体と結合
する以外に、第２の抗体がＨＡＭＡを介して第１の抗体
に結合したり、抗原性物質又はＨＡＭＡを介して第１の
抗体に結合した第２の抗体がさらにＨＡＭＡを介して別
の第２の抗体に結合することによると考えられる。HAMA is a human antibody that recognizes mouse antibodies, and is known to mainly recognize a certain region of mouse antibodies. Specifically, as an example in which the influence on the measured value is a problem, for example, as shown in the Examples below, a mouse antibody is used for both the first antibody immobilized on a solid phase and the labeled second antibody. When an antigenic substance in human serum is measured by the one-step sandwich EIA, a higher value than the actual concentration of the antigenic substance is detected. This means that, in addition to the second antibody binding to the first antibody via the antigenic substance, the second antibody binds to the first antibody via HAMA, or via the antigenic substance or HAMA. It is considered that the second antibody bound to the first antibody is further bound to another second antibody via HAMA.
【０００６】ＨＡＭＡによる測定値への影響を低減する
方法として、測定の際、該抗原性物質の測定自体には影
響を及ぼさないマウス抗体を添加すること等が提案され
ている（例えば特開昭６１−６５１６２号公報等）。[0006] As a method for reducing the influence of HAMA on the measured value, it has been proposed to add a mouse antibody which does not affect the measurement itself of the antigenic substance during the measurement (for example, Japanese Unexamined Patent Publication No. 2006-242242) 61-65162, etc.).
【０００７】しかし、添加するマウス抗体が抗原性物質
とある程度以上の結合能を有する場合、測定値が影響さ
れる可能性がある、またＨＡＭＡには多様性があるため
に、添加したマウス抗体があらゆるＨＡＭＡに対して効
果を有するとは限らないという課題がある。実際、マウ
スＩｇＧを認識するＨＡＭＡに対して効果のあるマウス
抗体が、マウスＩｇＭを認識するＨＡＭＡに対しては効
果がないという場合がある。このように、マウス抗体を
添加してＨＡＭＡの影響を低減する場合、添加するマウ
ス抗体が抗原性物質の測定に影響を及ぼさず、かつＨＡ
ＭＡによる測定値への影響を低減し得ることを、実際に
使用する測定系、即ち、抗原性物質、固相に固定化した
第１の抗体、標識した第２の抗体等との組み合わせごと
に予め確認する必要がある。また当然のことながら、免
疫反応の際の反応液にマウス抗体を添加するという操作
が必要となる。However, when the added mouse antibody has a binding ability with the antigenic substance to a certain degree or more, the measured value may be affected, and since HAMA has a variety, the added mouse antibody may be There is a problem that it is not always effective for all HAMAs. In fact, a mouse antibody that is effective against HAMA that recognizes mouse IgG may be ineffective against HAMA that recognizes mouse IgM. Thus, when mouse antibodies are added to reduce the effect of HAMA, the added mouse antibodies do not affect the measurement of antigenic substances, and HA
The fact that the influence of MA on the measured value can be reduced is determined for each combination with the actually used measuring system, that is, the antigenic substance, the first antibody immobilized on the solid phase, the labeled second antibody, etc. It is necessary to confirm in advance. In addition, as a matter of course, the operation of adding the mouse antibody to the reaction solution during the immune reaction is required.
【０００８】一方、上述のハイブリド−マ法で免疫する
動物としてマウスを用いるかわりにマウス以外の動物
（ヒトを除く）を用いた場合にも、上述のＨＡＭＡの問
題と同様の問題が起こり得る。すなわち、免疫する動物
（ヒトを除く）由来の抗体を認識するヒト血清中のヒト
抗体が測定値に及ぼす影響が問題となる。ヒト以外の動
物由来の抗体はヒトにとっては異物であるため、ヒト血
清中にヒト以外の動物由来の抗体を認識する抗体（例え
ばＨＡＭＡはその一種である）が存在する可能性は常に
ある。On the other hand, when an animal other than a mouse (excluding human) is used instead of a mouse as an animal to be immunized by the hybridoma method, the same problem as that of HAMA described above may occur. That is, there is a problem of the influence of human antibodies in human serum that recognize antibodies derived from animals (excluding humans) to be immunized on the measured values. Since antibodies derived from animals other than humans are foreign to humans, there is always a possibility that antibodies that recognize antibodies derived from animals other than humans (for example, HAMA is one of them) are present in human serum.
【０００９】以上のように、ヒト抗体を夾雑物として含
有するヒト血清等の試料中の抗原性物質を抗体を用いる
免疫測定方法で測定する場合、ヒト以外の動物由来の抗
体を使用する限り、該抗体を認識するヒト血清中のヒト
抗体によって受ける測定値への影響を常に考慮しなけれ
ばならないという課題があった。[0009] As described above, when an antigenic substance in a sample such as human serum containing a human antibody as a contaminant is measured by an immunoassay method using an antibody, as long as an antibody derived from a non-human animal is used, There has been a problem that the influence on the measurement value, which is affected by the human antibody in human serum that recognizes the antibody, must always be considered.
【００１０】[0010]
【課題を解決するための手段】本発明者らは、上述の課
題について検討した結果、少なくとも一定領域がヒト由
来である抗体を使用することでＨＡＭＡによる問題を解
決し得ることを見出し本発明を完成させた。即ち本発明
は、少なくとも一定領域がヒト由来である抗体を使用す
る、ヒト由来の抗体を夾雑物として含有する試料中の抗
原性物質の免疫測定方法である。以下、本発明を詳細に
説明する。As a result of examining the above-mentioned problems, the present inventors have found that the problem of HAMA can be solved by using an antibody having at least a certain region of human origin. Completed That is, the present invention is an immunoassay method for an antigenic substance in a sample containing a human-derived antibody as a contaminant, which uses an antibody in which at least a certain region is human-derived. Hereinafter, the present invention will be described in detail.
【００１１】少なくとも一定領域がヒト由来である抗体
とは、ヒト以外の動物由来の抗体の可変領域がヒト由来
の抗体の一定領域と融合したヒト型キメラ抗体又は可変
領域及び一定領域が共にヒト由来であるモノクロ−ナル
抗体、即ちヒトモノクロ−ナル抗体そのものである。ヒ
ト型キメラ抗体は、例えば、抗原性物質に特異的に結合
するモノクロ−ナル抗体産生ハイブリド−マを作製し、
該ハイブリド−マから該モノクロ−ナル抗体のＨ鎖Ｖ領
域をコ−ドする遺伝子及びＬ鎖Ｖ領域をコ−ドする遺伝
子を単離し、該遺伝子をヒト型キメラ抗体発現ベクタ−
に組込んで組換え体分子を作製し、該組換え体分子を適
当な宿主細胞に導入して発現させることにより作製する
ことができる。An antibody in which at least a certain region is derived from human means a human chimeric antibody in which the variable region of an antibody derived from a non-human animal is fused with a certain region of an antibody derived from human, or both the variable region and the certain region are derived from human. Is a human monoclonal antibody itself, that is, a human monoclonal antibody itself. The human chimeric antibody is prepared by, for example, producing a monoclonal antibody-producing hybridoma that specifically binds to an antigenic substance,
A gene encoding the H chain V region of the monoclonal antibody and a gene encoding the L chain V region of the monoclonal antibody were isolated from the hybridoma, and the gene was expressed as a human chimeric antibody expression vector.
It can be prepared by incorporating the recombinant molecule into a suitable host cell and expressing it.
【００１２】抗原性物質に特異的に結合するモノクロー
ナル抗体を産生するハイブリドーマは、公知の方法（ケ
−ラ−とミルシュタイン、２５６、４９５、１９７５
年）に準じて作製することができ、例えば次の如くすれ
ば良い。（１）抗原性物質を用いて免疫した動物から抗
体産生細胞を調製し、（２）これとは別に骨髄腫細胞を
調製し、（３）これらの細胞を融合させ、（４）得られ
た融合細胞のうちハイブリドーマのみを選択的に増殖さ
せ、（５）得られたハイブリドーマから該抗原性物質に
特異的に結合する抗体を産生するハイブリドーマを検索
し、（６）該抗原性物質に特異的に結合する抗体を産生
するハイブリドーマをクローニングする。Hybridomas producing a monoclonal antibody that specifically binds to an antigenic substance can be obtained by a known method (Keller and Milstein, 256 , 495, 1975).
Year), and may be manufactured as follows, for example. (1) Antibody-producing cells were prepared from an animal immunized with an antigenic substance, (2) Myeloma cells were prepared separately, (3) These cells were fused, and (4) obtained. Among the fused cells, only the hybridoma is selectively grown, and (5) the obtained hybridoma is searched for a hybridoma producing an antibody that specifically binds to the antigenic substance, and (6) the hybridoma is specific to the antigenic substance. A hybridoma producing an antibody that binds to is cloned.
【００１３】モノクロ−ナル抗体産生ハイブリド−マか
らの該モノクロ−ナル抗体のＨ鎖Ｖ領域をコ−ドする遺
伝子及びＬ鎖Ｖ領域をコ−ドする遺伝子は、公知の方法
（Ｋａｎｅｋｏら、Ｊ．Ｂｉｏｃｈｅｍ．１１３、１１
４−１１７、１９９３年）に準じて単離することがで
き、例えば次の如くすれば良い。（１）モノクロ−ナル
抗体産生ハイブリド−マから染色体ＤＮＡを抽出し、
（２）該染色体ＤＮＡを適当な制限酵素で切断してＤＮ
Ａ断片の混合物を調製し（３）該ＤＮＡ断片の混合物を
ラムダファ−ジ等のクロ−ニングベクタ−に組込んでジ
ェノミックライブラリ−を作製し、（４）該ジェノミッ
クライブラリ−から、適当なＤＮＡプロ−ブ（例えば抗
体のＪ領域を含むＤＮＡプロ−ブ）とハイブリダイズす
るクロ−ンを単離し、（５）該クロ−ンの中から該モノ
クロ−ナル抗体のＨ鎖Ｖ領域をコ−ドする遺伝子ＤＮＡ
及びＬ鎖Ｖ領域をコ−ドする遺伝子ＤＮＡを含有するク
ロ−ンを選択する。The gene encoding the H chain V region and the gene encoding the L chain V region of the monoclonal antibody from the monoclonal antibody producing hybridoma can be obtained by known methods (Kaneko et al., J. Biochem.113, 11
4-117, 1993) and can be isolated, for example, as follows. (1) Extracting chromosomal DNA from a monoclonal antibody-producing hybridoma,
(2) DN by cutting the chromosomal DNA with an appropriate restriction enzyme
A mixture of A fragments is prepared (3) The mixture of DNA fragments is incorporated into a cloning vector such as lambda phage to prepare a genomic library, and (4) an appropriate DNA is prepared from the genomic library. A clone that hybridizes with a probe (for example, a DNA probe containing the J region of the antibody) is isolated, and (5) the H chain V region of the monoclonal antibody is cloned from the clone. Gene DNA
And a clone containing the gene DNA coding for the L chain V region is selected.
【００１４】なお、（１）で染色体ＤＮＡを抽出する代
わりにＲＮＡを抽出してもよく、この場合にはジェノミ
ックライブラリ−の代わりにｃＤＮＡライブラリ−を作
製した後、該モノクロ−ナル抗体のＨ鎖及びＬ鎖のＶ領
域をコ−ドする遺伝子ＤＮＡを単離すれば良い。また、
適当なＤＮＡプロ−ブとハイブリダイズするクロ−ンの
中から該モノクロ−ナル抗体のＨ鎖Ｖ領域をコ−ドする
遺伝子ＤＮＡ及びＬ鎖Ｖ領域をコ−ドする遺伝子ＤＮＡ
を含有するクロ−ンを選択するには、例えば、単離した
クロ−ンの塩基配列を決定して発現可能なＨ鎖Ｖ領域を
コ−ドする遺伝子ＤＮＡ及びＬ鎖Ｖ領域をコ−ドする遺
伝子ＤＮＡを含むことを確認し、更に該遺伝子を適当な
発現ベクタ−に組込んで組換え体分子を作製し、該組換
え体分子を適当な宿主細胞に導入して抗体分子を発現さ
せ、該抗体分子が抗原性物質を認識することを確認すれ
ばよい。RNA may be extracted instead of extracting the chromosomal DNA in (1). In this case, after preparing a cDNA library instead of the genomic library, H of the monoclonal antibody is prepared. The gene DNA encoding the V regions of the chain and the L chain may be isolated. Also,
A gene DNA encoding the H chain V region of the monoclonal antibody and a gene DNA encoding the L chain V region of clones that hybridize with an appropriate DNA probe.
In order to select clones containing, for example, the nucleotide sequence of the isolated clones is determined, and the gene DNA encoding the expressible H chain V region and the L chain V region are coded. It is confirmed that the gene contains the gene DNA, and the gene is further incorporated into an appropriate expression vector to prepare a recombinant molecule, and the recombinant molecule is introduced into an appropriate host cell to express the antibody molecule. It may be confirmed that the antibody molecule recognizes an antigenic substance.
【００１５】上記発現ベクタ−として、ヒト型キメラ抗
体発現ベクタ−を用いれば、該遺伝子ＤＮＡが該モノク
ロ−ナル抗体のＨ鎖Ｖ領域をコ−ドする遺伝子ＤＮＡ及
びＬ鎖Ｖ領域をコ−ドする遺伝子ＤＮＡであるかどうか
を確認できると同時に、ヒト型キメラ抗体も作製できる
が、単離した遺伝子ＤＮＡが該モノクロ−ナル抗体のＨ
鎖Ｖ領域をコ−ドする遺伝子ＤＮＡ及びＬ鎖Ｖ領域をコ
−ドする遺伝子ＤＮＡであるかどうかを確認する目的の
ためにはいかなる発現ベクタ−を用いても構わない。ま
た、用いる宿主細胞も用いる発現ベクタ−が発現できる
ものであればいかなる細胞であっても構わない。If a human chimeric antibody expression vector is used as the expression vector, the gene DNA codes the H chain V region of the monoclonal antibody and the L chain V region of the monoclonal antibody. It is possible to confirm whether or not it is the gene DNA to be produced, and at the same time, a human chimeric antibody can be prepared.
Any expression vector may be used for the purpose of confirming whether it is the gene DNA coding for the chain V region or the gene DNA coding for the L chain V region. Any host cell may be used as long as it can express the expression vector used.
【００１６】単離した遺伝子ＤＮＡが該モノクロ−ナル
抗体のＨ鎖Ｖ領域をコ−ドする遺伝子ＤＮＡ及びＬ鎖Ｖ
領域をコ−ドする遺伝子ＤＮＡであることを確認できれ
ば、該遺伝子をヒト型キメラ抗体発現ベクタ−に組込ん
で組換え体ＤＮＡ分子を作製し、該組換え体ＤＮＡ分子
を適当な宿主細胞に導入し、ヒト型キメラ抗体を発現さ
せればよい。The isolated gene DNA codes for the H chain V region of the monoclonal antibody and the L chain V
If it can be confirmed that it is a gene DNA coding for the region, the gene is incorporated into a human chimeric antibody expression vector to prepare a recombinant DNA molecule, and the recombinant DNA molecule is put into an appropriate host cell. It may be introduced to express a human chimeric antibody.
【００１７】抗原性物質に特異的に結合するヒトモノク
ロ−ナル抗体を産生するハイブリド−マは、公知の方法
（ケ−ラ−とミルシュタイン、Ｎａｔｕｒｅ、２５６、
４９５、１９７５年）に準じて作製することができる。
その際、細胞融合に用いるＢ細胞としては、抗原性物質
で免疫したヒトから採取したＢ細胞、偶然抗原性物質で
免疫されたヒトから採取したＢ細胞、ヒトからＢ細胞を
採取し、ｉｎ ｖｉｔｒｏで抗原性物質により免疫した
Ｂ細胞等を用いればよい。Hybridomas that produce human monoclonal antibodies that specifically bind to antigenic substances can be prepared by known methods (Keller and Milstein, Nature, 256 ,
495, 1975).
At that time, as B cells used for cell fusion, B cells collected from a human immunized with an antigenic substance, B cells collected from a human accidentally immunized with an antigenic substance, B cells collected from a human, and in vitro B cells and the like immunized with the antigenic substance may be used.
【００１８】ヒト以外の動物由来の抗体を認識するヒト
血清中のヒト抗体は、主にヒト以外の動物由来の抗体の
一定領域を認識する。従って、上述の如くして作製した
少なくとも一定領域がヒト由来である抗体にはほとんど
結合せず、それ故少なくとも一定領域がヒト由来である
抗体を免疫測定に用いれば、ＨＡＭＡ等に代表されるヒ
ト抗体による免疫測定の結果への影響を低減できる。Human antibodies in human serum that recognize non-human animal-derived antibodies mainly recognize a certain region of non-human animal-derived antibodies. Therefore, an antibody having at least a certain region derived from human, which is produced as described above, hardly binds, and therefore, an antibody having at least a certain region derived from human is used for immunoassay, a human represented by HAMA or the like. The influence of the antibody on the result of the immunoassay can be reduced.
【００１９】本発明における免疫測定方法としては、例
えばＲＩＡ（標識物質としてラジオアイソト−プを使用
する方法）やＥＩＡ（標識物質として、検出可能な信号
を発生し得る物質を発生する酵素を使用する方法）は勿
論、標識物質としてそれ自体が検出可能な信号を発生す
る蛍光物質や発光物質を用いる免疫測定方法を採用する
ことができる。取扱いの容易さの面でＥＩＡが好適であ
る。また、それぞれの免疫測定方法には、標識物質と結
合した既知量の抗原性物質を使用する、いわゆる競合法
と、固相化した第１の抗体及び標識物質と結合した第２
の抗体を使用するいわゆるサンドイッチ法があるが、測
定感度の良さ及び交叉反応による測定誤差の少なさとい
う面でサンドイッチＥＩＡが好ましい。なおサンドイッ
チ法には２ステップ法と１ステップ法があるが、いずれ
の場合も本発明は効果的である。特にサンドイッチ法に
おいては、ＨＡＭＡ等による測定値への影響を低減する
ために、２ステップ法においては第１の抗体と第２の抗
体の両方又はいずれか一方に一定領域がヒト由来である
抗体を使用し、１ステップ法においては少なくとも一定
領域がヒト由来である抗体を標識物質と結合させる第２
の抗体に使用すれば良い。As the immunoassay method in the present invention, for example, RIA (method using radioisotope as a labeling substance) or EIA (labeling substance, an enzyme generating a substance capable of generating a detectable signal) is used. In addition to the method), an immunoassay method using a fluorescent substance or a luminescent substance which itself generates a detectable signal can be adopted as the labeling substance. EIA is preferable in terms of easy handling. Further, in each of the immunoassay methods, a so-called competitive method, in which a known amount of an antigenic substance bound to a labeling substance is used, and a second antibody bound to a immobilized first antibody and a labeling substance are used.
There is a so-called sandwich method using the antibody of 1., but the sandwich EIA is preferable in terms of good measurement sensitivity and small measurement error due to cross-reaction. The sandwich method includes a two-step method and a one-step method, but the present invention is effective in any case. In particular, in the sandwich method, in order to reduce the influence of HAMA etc. on the measured value, in the two-step method, an antibody in which a certain region is derived from human is used in both or one of the first antibody and the second antibody. A second step of binding an antibody, of which at least a certain region is of human origin, to a labeling substance in the one-step method
Can be used for the antibody.
【００２０】本発明の免疫測定において対象とされる抗
原性物質は、それ自体は抗原性を有さないが、他の適当
なキャリア蛋白と結合した状態で抗原性を示す物質をも
包含する。より具体的に本発明の方法は、例えばＦＳ
Ｈ、ＬＨ、ＨＣＧ、成長ホルモン、ＰＲＬ、ＡＦＰ、Ｃ
ＥＡ、ＴＳＨ、ＦＥＲ、インシュリン、クレアチンキナ
−ゼ等の各種ホルモン又は生理物質はもとより、例えば
肝炎ウイルス、免疫不全ウイルス等、その測定が診断に
有用な抗原性物質を免疫測定方法により測定する際に使
用できる。The antigenic substance to be used in the immunoassay of the present invention includes a substance which does not have antigenicity per se but exhibits antigenicity when bound to another suitable carrier protein. More specifically, the method of the present invention is, for example, FS
H, LH, HCG, growth hormone, PRL, AFP, C
For measuring various hormones or physiological substances such as EA, TSH, FER, insulin, and creatine kinase, as well as, for example, hepatitis virus, immunodeficiency virus, and other antigenic substances whose measurement is useful for diagnosis by immunoassay. Can be used.
【００２１】[0021]
【発明の効果】以上の説明から明らかなように、本発明
によれば、少なくとも一定領域がヒト由来である抗体を
使用することにより、ヒト抗体を夾雑物として含有する
ヒト血清等の試料中の抗原性物質を測定する場合、該ヒ
ト抗体の免疫測定結果への影響を低減することが可能で
ある。従来、ヒト以外の動物由来の抗体を免疫測定に使
用した場合、ヒト以外の動物由来の抗体を認識するヒト
抗体により測定値が影響を受け、測定結果を用いた診断
等が困難になったり、免疫測定の結果とそれ以外の種々
の臨床検査の結果を併用する必要があったが、本発明の
ように少なくとも一定領域がヒト由来である抗体を使用
することでヒト以外の動物由来の抗体を認識するヒト血
清中のヒト抗体による測定値への影響を低減できるか
ら、結果として免疫測定結果を用いた診断が行い易くな
る。As is apparent from the above description, according to the present invention, by using an antibody in which at least a certain region is of human origin, a sample such as human serum containing human antibody as a contaminant can be used. When measuring an antigenic substance, it is possible to reduce the influence of the human antibody on the immunoassay result. Conventionally, when an antibody derived from a non-human animal is used in an immunoassay, the measured value is affected by a human antibody that recognizes an antibody derived from a non-human animal, and diagnosis using the measurement result becomes difficult, or the like. It was necessary to combine the results of immunoassays and the results of various other clinical tests, but by using an antibody in which at least a certain region is of human origin as in the present invention, an antibody of non-human animal origin can be obtained. Since the influence of the human antibody in the recognized human serum on the measurement value can be reduced, as a result, the diagnosis using the immunoassay result can be easily performed.
【００２２】[0022]
【実施例】以下、実施例を挙げて本発明を更に詳細に説
明するが、本発明はこれら実施例に限定されるものでは
ない。The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.
【００２３】実施例１ ヒト甲状腺刺激ホルモン（以下ＴＳＨ）に対するマウス
モノクロ−ナル抗体産生ハイブリド−マをハイブリド−
マ法（ケ−ラ−とミルシュタイン、Ｎａｔｕｒｅ、２５
６、４９５、１９７５年）に準じて作製し、２種類のハ
イブリド−マ（ともにＩｇＧクラスの抗体を産生）を確
立し、それぞれのハイブリド−マが産生する抗体をＴＳ
Ｍ１．３及びＴＳＮ２．９と命名した。ＴＳＭ１．３及
びＴＳＮ２．９を精製し、ＴＳＮ２．９を固相に固定化
して第１の抗体と、一方ＴＳＭ１．３を標識物質である
アルカリ性フォスファタ−ゼ（以下ＡＬＰ）と結合させ
て第２の抗体として、既知濃度のＴＳＨを含むヒト血清
試料について１ステップサンドイッチＥＩＡを行った。Example 1 A hybridoma producing a mouse monoclonal antibody against human thyroid stimulating hormone (TSH) was hybridized.
Ma method (Keller and Milstein, Nature, 25)
6 , 495, 1975) and established two types of hybridomas (both produce IgG class antibodies), and each hybridoma produces an antibody TS
It was named M1.3 and TSN2.9. TSM1.3 and TSN2.9 were purified, and TSN2.9 was immobilized on a solid phase to bind to the first antibody, while TSM1.3 was bound to alkaline phosphatase (hereinafter referred to as ALP) as a labeling substance to bind to the second antibody. As an antibody of 1., a one-step sandwich EIA was performed on a human serum sample containing a known concentration of TSH.
【００２４】その結果、ＴＳＨ濃度に相関した信号（Ａ
ＬＰによるｐ−ニトロフェニルフォスフェイトの分解に
より生ずるｐ−ニトロフェノ−ルの発色）が測定され
た。As a result, the signal (A
The color of p-nitrophenol produced by the decomposition of p-nitrophenyl phosphate by LP) was measured.
【００２５】実施例２ ＴＳＮ２．９産生ハイブリド−マを用いてＴＳＮ２．９
を産生させ、ペプシン消化によってＦ（ａｂ´）2 化を
行い、Ｆ（ａｂ´）2 化したＴＳＮ２．９を精製し、直
径１．２ｍｍの球状固相に固定化した。Example 2 Using TSN2.9-producing hybridoma, TSN2.9
Was produced and subjected to F (ab ′) 2 conversion by pepsin digestion, F (ab ′) 2 converted TSN2.9 was purified and immobilized on a spherical solid phase having a diameter of 1.2 mm.
【００２６】一方、ＴＳＭ１．３産生ハイブリド−マを
用いてＴＳＭ１．３を産生させ、ＴＳＭ１．３を精製し
た。また、ＴＳＭ１．３のキメラ化を行い（特願平５−
１５６７０７号参照）、キメラＴＳＭ１．３産生細胞を
用いてキメラＴＳＭ１．３を産生させ、キメラＴＳＭ
１．３を精製した。精製したＴＳＭ１．３及びキメラＴ
ＳＭ１．３について、ＴＳＨとの結合性を競合法により
調べた結果、ＴＳＭ１．３及びキメラＴＳＭ１．３はＴ
ＳＨに対して同等の結合性を有していることが確認され
た。次に、精製したＴＳＭ１．３あるいはキメラＴＳＭ
１．３をそれぞれウシ小腸アルカリホスファタ−ゼ（ザ
イモサイト社製）と結合した。On the other hand, TSM1.3 was purified using TSM1.3-producing hybridoma. In addition, we performed chimerization of TSM1.3 (Japanese Patent Application No. 5-
156707), chimeric TSM1.3 is produced using the chimeric TSM1.3 producing cell, and chimeric TSM is produced.
Purified 1.3. Purified TSM 1.3 and chimera T
As a result of investigating the binding property of SM1.3 with TSH by a competitive method, TSM1.3 and chimeric TSM1.3 showed T
It was confirmed that they have equivalent binding properties to SH. Next, purified TSM1.3 or chimeric TSM
Each of 1.3 was bound to bovine small intestine alkaline phosphatase (Zymosite).
【００２７】これらウシ小腸アルカリホスファタ−ゼ標
識ＴＳＭ１．３あるいはウシ小腸アルカリホスファタ−
ゼ標識キメラＴＳＭ１．３と、Ｆ（ａｂ´）2 化ＴＳＮ
２．９を固定化した固相を使用して、ＴＳＨ濃度の既知
な標準検体について、市販の免疫測定装置（ＡＩＡ−１
２００、東ソ−（株）製）を用いてＴＳＨ値の測定を行
った結果、標識ＴＳＭ１．３を用いた場合と標識キメラ
ＴＳＭ１．３を用いた場合でほぼ同一の測定値が得られ
た。These bovine small intestine alkaline phosphatase labeled TSM1.3 or bovine small intestine alkaline phosphatase
ZE-labeled chimeric TSM1.3 and F (ab ') 2 -modified TSN
Using a solid phase on which 2.9 is immobilized, a commercially available immunoassay device (AIA-1) is used for a standard sample with a known TSH concentration.
200, manufactured by Toso Co., Ltd., the TSH value was measured, and as a result, almost the same measurement values were obtained when the labeled TSM1.3 was used and when the labeled chimera TSM1.3 was used. .
【００２８】実施例３ 実施例２と同一の測定系を使用して、血清中のＨＡＭＡ
の値が高い４試料（Ａ、Ｂ、Ｃ、Ｄ）について、前記同
様にＴＳＨ値を測定した。Example 3 Using the same assay system as in Example 2, HAMA in serum was tested.
The TSH value was measured in the same manner as above for the four samples (A, B, C, D) having a high value of.
【００２９】なお測定に際し、ＨＡＭＡの影響を低減す
るために予め十分量のマウス抗体を検体に添加した場合
（ｍＹ＋）と添加しない場合（ｍＹ−）についてそれぞ
れ行った。なおマウス抗体の添加に先立って、添加回収
試験においてマウス抗体を共存させてＴＳＨ値の測定を
行い、該マウス抗体が測定系自体に影響を及ぼさないこ
と、及び、十分量のマウス抗体を添加することによりＨ
ＡＭＡによる測定値への影響が低減されることを予め確
認した。In the measurement, it was carried out when a sufficient amount of mouse antibody was previously added to the sample (mY +) and when it was not added (mY-) in order to reduce the influence of HAMA. Prior to the addition of the mouse antibody, the TSH value is measured in the addition recovery test in the presence of the mouse antibody, and the mouse antibody does not affect the measurement system itself, and a sufficient amount of the mouse antibody is added. By H
It was previously confirmed that the influence of AMA on the measured values was reduced.
【００３０】結果を表１に示す。表１は、実施例３で示
した方法で測定した、血清中ＴＳＨ値である。表１から
明らかなように、キメラＴＳＭ１．３を用いた場合に
は、ｍＹ非存在下でもＨＡＭＡによる測定値への影響が
十分に低減していることがわかる。The results are shown in Table 1. Table 1 shows serum TSH values measured by the method described in Example 3. As is clear from Table 1, when chimeric TSM1.3 was used, the influence of HAMA on the measured values was sufficiently reduced even in the absence of mY.
【００３１】[0031]
【表１】 [Table 1]
Claims (6)
体を使用する、ヒト由来の抗体を夾雑物として含有する
試料中の抗原性物質の免疫測定方法。1. A method for immunoassaying an antigenic substance in a sample containing a human-derived antibody as a contaminant, which uses an antibody in which at least a certain region is human-derived.
第１の抗体と測定可能な信号を発生し又は測定可能な信
号を発生し得る物質を発生する標識物質と直接又は間接
的に結合した第２の抗体を用いて、第１の抗体、抗原性
物質及び第２の抗体からなる免疫複合体を出現させる操
作を含む免疫測定方法において、前記第１又は第２の抗
体の両方又は一方に少なくとも一定領域がヒト由来であ
る抗体を使用することを特徴とする請求項１の免疫測定
方法。2. A first antibody bound directly or indirectly to an insoluble solid phase and directly or indirectly bound to a labeling substance which produces a measurable signal or a substance capable of producing a measurable signal. In the immunoassay method including the operation of causing an immunocomplex composed of the first antibody, the antigenic substance and the second antibody to appear using the second antibody described above, both or one of the first or second antibody The immunoassay method according to claim 1, wherein an antibody in which at least a certain region is derived from human is used for.
変領域とヒト由来の抗体の一定領域とが融合したヒト型
キメラ抗体であることを特徴とする請求項１又は２項の
免疫測定方法。3. The immunoassay according to claim 1 or 2, wherein the antibody is a human chimeric antibody in which a variable region of an antibody derived from a non-human animal is fused with a constant region of a human-derived antibody. Method.
徴とする請求項１又は２項の免疫測定方法。4. The immunoassay method according to claim 1 or 2, wherein the antibody is a human-derived antibody.
特徴とする請求項１〜４項いずれかに記載の免疫測定方
法。5. The immunoassay method according to any one of claims 1 to 4, wherein the antibody is a monoclonal antibody.
とを特徴とする請求項１又は２項の免疫測定方法。6. The immunoassay method according to claim 1 or 2, wherein the sample is a solution containing human serum.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4844294A JPH07260786A (en) | 1994-03-18 | 1994-03-18 | Immunoassay using antibody having human originated constant region |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4844294A JPH07260786A (en) | 1994-03-18 | 1994-03-18 | Immunoassay using antibody having human originated constant region |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH07260786A true JPH07260786A (en) | 1995-10-13 |
Family
ID=12803472
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP4844294A Pending JPH07260786A (en) | 1994-03-18 | 1994-03-18 | Immunoassay using antibody having human originated constant region |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH07260786A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002243740A (en) * | 2001-02-21 | 2002-08-28 | Morinaga & Co Ltd | Immunoassay reagent |
| JP2003530543A (en) * | 1999-12-06 | 2003-10-14 | バイオサイト インコーポレイテッド | Human antibodies as detection reagents |
| JPWO2009084369A1 (en) * | 2007-12-28 | 2011-05-19 | シスメックス株式会社 | Reagent for detection of HIV-1 antigen and detection method |
| WO2023008404A1 (en) * | 2021-07-26 | 2023-02-02 | デンカ株式会社 | Reagent containing humanized antibody |
-
1994
- 1994-03-18 JP JP4844294A patent/JPH07260786A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003530543A (en) * | 1999-12-06 | 2003-10-14 | バイオサイト インコーポレイテッド | Human antibodies as detection reagents |
| JP2002243740A (en) * | 2001-02-21 | 2002-08-28 | Morinaga & Co Ltd | Immunoassay reagent |
| JP4590117B2 (en) * | 2001-02-21 | 2010-12-01 | 森永製菓株式会社 | Immunoassay reagent |
| JPWO2009084369A1 (en) * | 2007-12-28 | 2011-05-19 | シスメックス株式会社 | Reagent for detection of HIV-1 antigen and detection method |
| WO2023008404A1 (en) * | 2021-07-26 | 2023-02-02 | デンカ株式会社 | Reagent containing humanized antibody |
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