JPS592346B2 - Two-component simultaneous quantitative enzyme immunoassay - Google Patents

Description

【発明の詳細な説明】 本発明は酵素免疫測定法のうち、被検液中に含有されて
いる２成分（２種類の抗原）を同時に定量しうる酵素免
疫測定法に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an enzyme immunoassay method that can simultaneously quantify two components (two types of antigens) contained in a test liquid.

酵素免疫測定法とは、生体由来の微量成分の定量法とし
て最近広く利用されるようになつた方法であり、そのｌ
例を説明すると次のようである。Enzyme immunoassay is a method that has recently become widely used as a method for quantifying trace components derived from living organisms.
An example is as follows.

一定量の抗体を、適当な担体（固相）例えばガラス製試
験管の内壁あるいはプラスチックビーズの表面に付着せ
しめて不溶化（固相化）し、これに対して一定量の酵素
標識抗原と測定すべき抗原を加えると、酵素標識抗原と
測定すべき抗原は、抗原−抗体反応によつて、上記固相
化した抗体に対して競合的に結合し、その時、抗体と結
合する酵素標識抗原の量は、測定すべき抗原の量に応じ
て変化するのス抗体に結合した酵素標識抗原の酵素の活
性を測定するか、逆に抗体に結合しなかつた酵素の活性
を測定することによつて測定すべき抗原の量を求めるこ
とができる。上記のような酵素免疫測定法によつて、微
量成分の測定をする場合、例えば新生児のマススクリー
ニングなどにおいては、その採取しうる試料は極めて微
量であり、かつ多くの成分について定量することすなわ
ち多項目の測定が要求される。A fixed amount of antibody is attached to a suitable carrier (solid phase) such as the inner wall of a glass test tube or the surface of plastic beads to make it insoluble (solid phase), and a fixed amount of enzyme-labeled antigen is measured against this. When the target antigen is added, the enzyme-labeled antigen and the antigen to be measured competitively bind to the immobilized antibody through an antigen-antibody reaction, and at this time, the amount of the enzyme-labeled antigen that binds to the antibody increases. can be measured by measuring the enzyme activity of the enzyme-labeled antigen bound to the antibody, which changes depending on the amount of antigen to be measured, or conversely by measuring the activity of the enzyme not bound to the antibody. The amount of antigen required can be determined. When measuring trace components using the enzyme immunoassay method described above, for example in mass screening of newborns, the sample that can be collected is extremely small, and it is difficult to quantify many components. Measurement of the item is required.

従つて２成分を同時に定量しつることは試料の使用量が
少なくですみ、かつ測定時間を短縮しうるという利点が
ある。本発明はこの要求を満たすために開発されたもの
であつて、以下本発明について説明する。被検液中に含
有されている２つの成分例えば抗原ＡおよびＢを同時に
定量する方法について述べる。Therefore, simultaneous quantitative determination of two components has the advantage of requiring less sample and shortening measurement time. The present invention was developed to meet this need, and will be described below. A method for simultaneously quantifying two components, such as antigens A and B, contained in a test liquid will be described.

第１図に示すように、抗Ａ抗体（抗体Ａ）を試験管１の
内壁に固相化し、一方、抗Ｂ抗体（抗体Ｂ）をビーズ２
の表面に固相化し、このビーズを試験管に入れた後、被
検液３を加え、次いで酵素標識抗原Ｂを加えると、試験
管内壁表面の抗体Ａには被検液中の抗原Ａが結合し、ピ
ース表面の抗体Ｂには、被検液中の抗原Ｂと、酵素標識
抗原Ｂが競合的に結合する。この場合、抗体Ｂに結合す
る酵素標識抗原Ｂの量は、測定すべき抗原Ｂの量に応じ
て定まる。上記結合が終了した後、ビーズを試験管から
取り出し、ビーズを洗浄し、ビーズ上において抗体Ｂに
結合している酵素の活性を測定すれｆ）抗原Ｂの量を知
ることができる。一方、ビーズを取り出した試験管を洗
浄し、これに酵素標識抗原Ａを加えて抗体Ａに結合せし
めた後、抗体Ａに結合している酵素の活性を測定すれば
、抗体Ａの量を求めることができる。下記に、チロキシ
ンＴ４と甲状腺刺激ホルモンＴＳＨを同時測定する方法
を例示し、本発明を説明する。As shown in Figure 1, anti-A antibody (antibody A) was immobilized on the inner wall of test tube 1, while anti-B antibody (antibody B) was immobilized on beads 2
After placing the beads in a test tube, test solution 3 is added, and then enzyme-labeled antigen B is added. Antigen A in the test solution is absorbed by antibody A on the inner wall of the test tube. Antigen B in the test liquid and enzyme-labeled antigen B competitively bind to antibody B on the surface of the piece. In this case, the amount of enzyme-labeled antigen B that binds to antibody B is determined depending on the amount of antigen B to be measured. After the above binding is completed, the beads are removed from the test tube, washed, and the activity of the enzyme bound to antibody B on the beads is measured. f) The amount of antigen B can be determined. On the other hand, the amount of antibody A can be determined by washing the test tube from which the beads were taken out, adding enzyme-labeled antigen A to it and binding it to antibody A, and then measuring the activity of the enzyme bound to antibody A. be able to. The present invention will be explained below by exemplifying a method for simultaneously measuring thyroxine T4 and thyroid stimulating hormone TSH.

。下記に示す試
薬を用いる。（１）緩衝液：（Ａ）０．０５Ｍリン酸緩
衝液 ＰＨ７．Ｏ（０．１（Ｆｌｌ牛血清アルブミンＢ
ＳＡｌＯ．９％塩化ナトリウム含有）（８） ０．０５
Ｍリン酸緩衝液 ＰＨ７．Ｏｌ〕（０．９（ｆｌ塩化ナ
トリウム含有）（１）抗Ｔ抗体固相化試験管：ベーリン
ガ一Ｔ４キツトの試験管（３）Ｔ ：マイルズ社製品 （４）Ｔ４標準液：Ｔ４Ｏ〜２００臂／ＴＩＩＪＩ，水
溶 １液（５）ペルオキシダーゼ標準Ｔ４：ベーリンガ
一社製Ｔ４キツトの標準Ｔ４（６）ＴＳＨ：カルピオケ
ム社製品 （７） ＴＳＨ標準液：ＴＳＨ卜１００μＵ／ｍノ ２
溶液（８）ペルオキシダーゼリシグマ社製品 ＨＯｒＳｅｒａｄｉＳｈｐｅｒＯｘｉｄａＳｅＴｙｐｅ
３２ＯＵ／３ｆ１（９）抗ＴＳＨ抗血清：ＬＴＢケミカ
ル社製品 ２（１１ペルオキシダーゼ標識ＴＳＨ：ペル
オキシダーゼをＮａＩＯを用いて酸化した後、塩基性水
浴液中でＴＨＳに結合せしめ、ＮａＢＨを用いて還元処
理した後、セフアデツクスＧ−１００を用いたゲルクロ
マト５グラフイ一によつて精製レ酵素活性訃よび免疫活
性の高い分画を使用する。. Use the reagents shown below. (1) Buffer: (A) 0.05M phosphate buffer PH7. O(0.1(Fll bovine serum albumin B
SAIO. Contains 9% sodium chloride) (8) 0.05
M phosphate buffer pH7. Ol] (0.9 (contains fl sodium chloride)) (1) Anti-T antibody immobilized test tube: Boehringer T4 kit test tube (3) T: Miles product (4) T4 standard solution: T4O ~ 200 mm /TIIJI, aqueous 1 solution (5) Peroxidase standard T4: Standard T4 of T4 kit manufactured by Boehringer (6) TSH: Product of Carpiochem (7) TSH standard solution: TSH 100μU/m2
Solution (8) Peroxidase Lysigma product HOrSeradiShperOxidaSeType
32OU/3f1 (9) anti-TSH antiserum: LTB Chemical Co. product 2 (11 peroxidase-labeled TSH: peroxidase was oxidized using NaIO, then bound to THS in a basic water bath solution, and reduced using NaBH. Thereafter, it was purified by gel chromatography using Sephadex G-100, and the fractions with high enzyme activity and immunoactivity were used.

（１１）抗家兎１ｇＧ抗体固相化ビーズ（第２抗体固相
化ビーズ）：アフイニテイークロマトグラフイ一によつ
（て精製した抗家兎１ｇ（／ｆ異抗体（山羊）の希釈液
（０Ｄ２８０ｎｍ０．１５〜０．２）にポリアセタール
ビーズを浸し、４℃で１日以上放置し、使用時に、緩衝
液（Ｂ）で洗い、ついで緩衝液（４）に１時間以上浸漬
する。(11) Anti-rabbit 1gG antibody-immobilized beads (second antibody-immobilized beads): Diluted solution of anti-rabbit 1g (/f different antibody (goat)) purified by affinity chromatography. (0D280nm 0.15-0.2) and left at 4° C. for 1 day or more. When used, wash with buffer (B) and then immerse in buffer (4) for 1 hour or more.

ｔ上記のようにして準備した試薬
を用い、下記に示す方法によつて測定を行なう。抗Ｔ４
抗体固相化試験管に、約１０万倍に希釈した抗ＴＳＨ血
清１００μムＴ標準液２０μムＴＳＨ標準液１００１ｔ
込ペルオキシダーゼ標準ＴＳＨｌＯＯｔＬノ緩衝液（Ａ
）５００μノ｝よび抗家兎１ｇＧ抗体固相化ビーズ１個
を入れ、４℃に１夜放置した後、ビーズを別の試験管に
移し、緩衝液Ｂで洗浄した後、緩衝液ＢＯ．６５Ｔｒｌ
Ｊ！／とｐ−ヒドロキシフエニルプａピオン酸５７１ｆ
／ｍノ水溶液５０Ｉｔノ｝よび０．０１％Ｈ２Ｏ２水溶
液５，０μノを加え、室温に訃いてｌ時間放置する。Using the reagents prepared as described above, measurements are performed by the method shown below. anti-T4
In an antibody-immobilized test tube, add 1001 tons of anti-TSH serum diluted approximately 100,000 times as 100 μm T standard solution and 20 μm TSH standard solution.
Contains peroxidase standard TSHlOOtL buffer (A
) and one anti-rabbit 1gG antibody-immobilized bead, and left at 4°C overnight.The beads were then transferred to another test tube, washed with buffer B, and then added with buffer BO. 65Trl
J! / and p-hydroxyphenylpionic acid 571f
50 μm of aqueous solution and 5.0 μm of a 0.01% H2O2 aqueous solution were added, and the mixture was allowed to stand at room temperature for 1 hour.

次いで１．２５％シアン化カリウム水溶液５０μノ寂よ
びＩＮ水酸化ナトリウム水溶液５０μノを加えて酵素反
応を止め、その螢光強度を、Ｅｘ３２ＯｎｒｒｋＥｍ４
Ｏ５ｎｍの光を用いて測定した。一方、上記ビーズを取
り去つた抗Ｔ４抗体固相化試験管にベルオキシダーゼ標
準Ｔ４溶液１ｍノを加え、室温で２時間放置した後、試
験管内の液を棄却し、緩衝液［Ｆ］）で試験管を洗浄し
た後、緩衝液（８）０．９ｍノ、ｐ−ヒドロキシフエニ
ルプロピオン酸５１Ｖｍノ水溶液５０μノ寂よび０．０
１％Ｈ２Ｏ２水溶液５０μノを加え、室温で１時間放置
する。Next, 50μ of a 1.25% potassium cyanide aqueous solution and 50μ of an IN sodium hydroxide aqueous solution were added to stop the enzyme reaction, and the fluorescence intensity was determined by Ex32OnrrkEm4.
Measurement was performed using O5 nm light. On the other hand, 1 m of peroxidase standard T4 solution was added to the anti-T4 antibody-immobilized test tube from which the beads had been removed, and after leaving it at room temperature for 2 hours, the solution in the test tube was discarded, and buffer solution [F]) was added. After washing the test tube, add 0.9 μm of buffer (8), 50 μm of p-hydroxyphenylpropionic acid 51 Vm aqueous solution, and 0.0 μm of p-hydroxyphenylpropionic acid.
Add 50μ of 1% H2O2 aqueous solution and leave at room temperature for 1 hour.

次いで１．２５％シアン化カリウム溶液５０μノ｝よび
ＩＮ水酸化ナトリウム水溶液５０μノを加えて酵素反応
を止め、その螢光強度を、上記と同様にＥｘ３２Ｏｎｍ
，．Ｅｍ４Ｏ５ｎｍの光を用いて測定した〇上記方法に
よつて、Ｔ４、ＴＳＨを測定する場合、Ｔ４として０ｎ
９／Ｔｎ）．２５ｎ９Δπム５０ｎ９／Ｍｊ．ｌＯＯｎ
ｇｒノ山一よび２００ｎ９Ｚ畷？水溶液を用い、ＴＳＨ
として０ＩｊＸＪ／ｉ又５μじＪｄ、１０μＶ／ＮＬｊ
！／Ｘ５ＯμＵへづよび１００μじ而Ｊの水溶液を用い
、下記に示す組合せによつてそれぞれ２０μノ Ｔ４｝
よび１００μＪＴＳＨを用へそれぞれの螢光強度を測定
して、第２図に示す検量線を得た。Next, 50μ of a 1.25% potassium cyanide solution and 50μ of an IN aqueous sodium hydroxide solution were added to stop the enzyme reaction, and the fluorescence intensity was adjusted to Ex32Onm in the same manner as above.
、． Measured using Em4O 5 nm light 〇 When measuring T4 and TSH by the above method, T4 is 0n.
9/Tn). 25n9Δπmu 50n9/Mj. lOOn
gr No Yamaichiyo and 200n9Z Nawate? Using an aqueous solution, TSH
As 0IjXJ/i and 5μJd, 10μV/NLj
! Using an aqueous solution of /
The fluorescence intensity was measured using 100 μJTSH and 100 μJTSH, and the calibration curve shown in FIG. 2 was obtained.

上記に示した方法に｝いて、Ｔ 標準液２０μノとＴＳ
Ｈ標準液１００μノを用いる代りに、被検液１２０μノ
を用いて上記の方法を繰返してそれぞれの螢光強度を測
定し、上記検量線から被検液中に含有されるＴ４｝よび
ＴＳＨを測定することができる。According to the method shown above, T standard solution 20μ and TS
Instead of using 100μ of the H standard solution, repeat the above method using 120μ of the test solution to measure the fluorescence intensity of each, and calculate the T4 and TSH contained in the test solution from the above calibration curve. can be measured.

さらにＴ４、ＴＳＨを下記に示す組合せによつて測定を
行い、第３図に示す結果を得た。Furthermore, T4 and TSH were measured using the combinations shown below, and the results shown in FIG. 3 were obtained.

この結果より、Ｔ４の測定に際して、第２抗体ビーズが
存在しても、その測定に何ら影響のないことがわかる。
上記本発明の方法によれば、微量の被検液しか得られな
い場合にも。This result shows that the presence of the second antibody beads has no effect on the measurement of T4.
According to the method of the present invention, even when only a small amount of test liquid can be obtained.

その試料を用いて２種類の成分を同時に測定することが
できる。Two types of components can be measured simultaneously using the sample.

【図面の簡単な説明】 第１図は本発明の方法を示す説明図、第２図訃よび第３
図は本発明の方法によつて得られた検量線を示す図であ
る。[Brief Description of the Drawings] Fig. 1 is an explanatory diagram showing the method of the present invention, Fig. 2 is an explanatory diagram showing the method of the present invention,
The figure shows a calibration curve obtained by the method of the present invention.

Claims (1)

【特許請求の範囲】[Claims] １ 被検液中に含有されている２種類の抗原ＡおよびＢ
を酵素免疫測定法によつて同時に定量する方法において
、抗Ａ抗体（抗体Ａ）をその内壁に固相化した試験管に
、抗Ｂ抗体（抗体Ｂ）をその表面に固相化したビーズ、
抗原ＡおよびＢを含有する被検液、および酵素標識抗原
Ｂを加え、抗原−抗体反応を行なわしめた後、ビーズを
取り出し、ビーズ上の抗体Ｂに結合している酵素の活性
を測定し、一方、ビーズを取り出した試験管に、酵素標
識抗原Ａを加え、抗原−抗体反応を行なわしめた後、試
験管内壁の抗体Ａに結合している酵素の活性を測定する
段階を含むことを特徴とする２成分同時定量酵素免疫測
定法。1 Two types of antigens A and B contained in the test liquid
In the method of simultaneously quantifying by enzyme immunoassay, a test tube with anti-A antibody (antibody A) immobilized on its inner wall, beads with anti-B antibody (antibody B) immobilized on the surface thereof,
After adding a test solution containing antigens A and B and enzyme-labeled antigen B to perform an antigen-antibody reaction, the beads are removed and the activity of the enzyme bound to antibody B on the beads is measured. On the other hand, the feature includes the step of adding enzyme-labeled antigen A to the test tube from which the beads have been taken out, causing an antigen-antibody reaction, and then measuring the activity of the enzyme bound to antibody A on the inner wall of the test tube. A two-component simultaneous quantitative enzyme immunoassay method.