JPS607363A - Measurement of antigen or antibody - Google Patents
Measurement of antigen or antibodyInfo
- Publication number
- JPS607363A JPS607363A JP11673683A JP11673683A JPS607363A JP S607363 A JPS607363 A JP S607363A JP 11673683 A JP11673683 A JP 11673683A JP 11673683 A JP11673683 A JP 11673683A JP S607363 A JPS607363 A JP S607363A
- Authority
- JP
- Japan
- Prior art keywords
- antigen
- antibody
- carrier
- labeled
- column
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
- G01N33/545—Synthetic resin
Abstract
Description
【発明の詳細な説明】
不発８Ａは免疫測定法による新規な抗原もしくは抗体の
測定方法に関する。DETAILED DESCRIPTION OF THE INVENTION Fusho 8A relates to a novel method for measuring antigens or antibodies by immunoassay.
免疫測定法とは、抗原（ハプテンを含む）で実験動物を
免疫して該抗原と特異的に結合する抗体を実験動物に作
らせ、この抗体と上記抗原との間の特異的な結合反応を
利用して、抗原または抗体を測定する方法である。Immunoassay is a method in which a laboratory animal is immunized with an antigen (including hapten), the animal is made to produce an antibody that specifically binds to the antigen, and a specific binding reaction between this antibody and the antigen is detected. This is a method for measuring antigens or antibodies.
この測定法では、測定対象物である抗原もしくは抗体と
、試薬として用いる抗原または抗体とを抗原抗体反応さ
せるに際し、後者の抗原または抗体を酵素、放射性同位
元素、蛍光物質、不対電子をもつ化合物等で標識してお
き、抗原抗体反応した標識抗原もしくａ標識抗体と、未
反応の標識抗原もしくは標識抗体の何れか一方の標識を
測定する。したがって、通常、抗原抗体反応した（Ｂｏ
ｕｎｄ） 標識抗原または標識抗体と、未反応の（Ｆｒ
ｅｅ）標識抗原または標識抗体とを、分離（Ｂ／Ｆ分離
）することが必要とされる。In this measurement method, when an antigen or antibody to be measured is subjected to an antigen-antibody reaction with an antigen or antibody used as a reagent, the latter antigen or antibody is mixed with an enzyme, a radioactive isotope, a fluorescent substance, or a compound with unpaired electrons. etc., and the label of either the labeled antigen or a-labeled antibody that has undergone antigen-antibody reaction, or the unreacted labeled antigen or labeled antibody is measured. Therefore, there is usually an antigen-antibody reaction (Bo
und) labeled antigen or labeled antibody and unreacted (Fr
ee) It is necessary to separate the labeled antigen or labeled antibody (B/F separation).
しかし、均一な溶液として抗原抗体反応を行なった場合
には、Ｂ／Ｆ９離が極めて繁雑となる。そこで、Ｂ／Ｆ
分離を容易にするため、抗原もしくは抗体を固体状の担
体に結合させ、こうして得られる同相化抗原もしくは固
相化抗体を用いることが知られている。例えば内径ｌχ
径程度毛細管自身を担体として毛細管内面に抗体を付着
せしめてＢ／Ｆ分離をしたり、スチレンビーズを担体と
して試験管内でＢ／Ｆ分離をしたりすることが行われて
いる。However, when the antigen-antibody reaction is performed in a homogeneous solution, B/F9 separation becomes extremely complicated. Therefore, B/F
In order to facilitate separation, it is known to bind an antigen or antibody to a solid carrier and use the resulting in-phase antigen or immobilized antibody. For example, the inner diameter lχ
B/F separation has been carried out by using a capillary tube itself as a carrier and attaching an antibody to the inner surface of the capillary tube, or by using styrene beads as a carrier to carry out B/F separation in a test tube.
しかしながら、１１１者は毛細管内面の表面積が小さく
固相化電が小さいので低ａ度域における測定対象物の定
量性が極めて悪いという欠点があり、後者は、試験管内
部の洗浄が不充分になＦＪ勝ちであシ又粒径の小さなビ
ーズを用いた場ある。However, the 111 tester has the disadvantage that the surface area of the inner surface of the capillary tube is small and the solid phase charge is small, so the quantitative performance of the measurement target in the low a degree region is extremely poor. There are cases where beads with small particle size are used in FJ Kachi.
本発明は、上記測定法の現状に鑑み、筒便忙して、精度
が高く、従来の機器を用いることにより容易に自動化が
可能な抗原もしくは抗体の測定方法を提供することを目
的とするもので、その要旨は、免疫測定法による抗原も
しくは抗体の測定方法において、測定対象物と同様の抗
原もしくは抗体又は測定対象物と反応する抗体もしくは
抗原が同相化された担体が充填されたカラムに、該カラ
ム中の担体に測定対象物と同様の抗原もしくは抗体が同
相化された場合は、測定試料と、測定対象物と反応する
既知量の標識抗体もしくは抗原とを導入し、又は、該カ
ラム中の担体に測定対象物と反応する抗体もしくは抗原
が同相化された場合は、測定試料と、測定対象物と同様
の既知量の標識抗原もしくは抗体、又は、試αｊ定対象
物と反応する既知量の標識抗原もしくは抗体とを導入し
、上記担体に結合した標識抗体もしくは標識抗原の量を
担体に結合した状想で測定することを特徴とする抗原も
しくは抗体の測定方法に存する。In view of the current state of the above measurement methods, the present invention aims to provide a method for measuring antigens or antibodies that is convenient, highly accurate, and easily automated using conventional equipment. , the gist of which is that in a method for measuring antigens or antibodies by immunoassay, a column filled with a carrier in which an antigen or antibody similar to the object to be measured, or an antibody or antigen that reacts with the object to be measured is made in phase, is used. If an antigen or antibody similar to the analyte is in phase with the carrier in the column, introduce the measurement sample and a known amount of labeled antibody or antigen that reacts with the analyte, or When antibodies or antigens that react with the analyte are in phase with the carrier, the measurement sample and a known amount of labeled antigen or antibody similar to the analyte, or a known amount of the labeled antigen or antibody that reacts with the analyte. The method of measuring an antigen or antibody is characterized in that a labeled antigen or antibody is introduced, and the amount of the labeled antibody or labeled antigen bound to the carrier is measured while the labeled antigen or antibody is bound to the carrier.
本発明において、測定対象物とは測定しようとする抗原
（ハプテンを含む）もしくけ抗体を意味し、測定対象物
と同様の抗原もしくは抗体とは、例えば人のインシュリ
ンを抗原として測定する場合の固相化抗体としては抗ヒ
トインシュリン抗体のみならずモルモット等に免疫して
得られた抗ヒトインシュリン抗体が使用可能なことから
明らかなように、測定対象物と同一の抗原もしくは抗体
又は測定対象物と類似の抗原性もしくは抗体活性を有す
る物質を意味する。In the present invention, the analyte refers to an antigen (including hapten) or a mechanism antibody to be measured, and an antigen or antibody similar to the analyte is, for example, a specific antibody when human insulin is measured as an antigen. As is clear from the fact that not only anti-human insulin antibodies but also anti-human insulin antibodies obtained by immunizing guinea pigs etc. can be used as the phasing antibody, it is possible to use anti-human insulin antibodies obtained by immunizing guinea pigs etc. Refers to substances with similar antigenicity or antibody activity.
抗原もしくは抗体を（ａ有化するのに用いる担体の材質
は、抗原もしくは抗体を安定な状態で結合し得る限り何
等限定されず、その具体例としてはポリスチレン、ポリ
オレフィン、セルロース、’Ｒ性ｙ カロース、ガラス
等が挙げられポリスチレンやセルロースＣ＊にアルミ基
が導入された熱可塑性セルロース）等が多用される。又
、担体は、洗浄液が残溜しない点で無孔性のものが好ま
しく、その寸法形状は、固相化率を高めることが出来、
カラムに供給する液体の流速を早くし得る点で粒径０．
１％〜ｏ、 ｓ ″ｇ程度のビーズや同程度の大きさの
フィルム状物が好適に用上記担体に抗原もしくは抗体を
同一化するには、抗原もしくは抗体を緩衝液中に添加、
溶解次いでとの担体を、例えば、Ｆ部にグラスクール等
が詰められた管状体に充填してカラムとし、更に下端を
粘土等でシールしく但し随時聞放可ｆＥ）、必要により
、緩衝液を添加する。場合によっては管状体に予め担体
を充填しておいてから抗原もしくけ抗体を結合させて同
相化してもよい。The material of the carrier used to prepare the antigen or antibody is not limited in any way as long as it can bind the antigen or antibody in a stable state, and specific examples include polystyrene, polyolefin, cellulose, , glass, etc., and polystyrene and thermoplastic cellulose (thermoplastic cellulose in which an aluminum group is introduced into cellulose C*) are often used. In addition, the carrier is preferably non-porous in that the cleaning solution does not remain, and its size and shape can increase the solid phase rate.
The particle size of 0.0.
Beads of approximately 1% to o.s''g or film-like materials of the same size are preferably used.To imitate the antigen or antibody to the above carrier, add the antigen or antibody to the buffer solution.
After dissolving the carrier, fill it into a column, for example, in a tubular body whose F part is filled with glass cooler, etc., and further seal the lower end with clay, etc. (However, it can be left open at any time fE). If necessary, add a buffer solution. Added. In some cases, the tubular body may be filled with a carrier in advance, and then the antigen or the antibody may be bound to the carrier to form the same phase.
上記管状体の容量は、小さ過ぎると担体充填量が少くな
り、従って試料の低濃度域の測定精度が悪くなるが、一
方多過ぎても特に測定精度が上るということはなくこの
種の試験の性質上試料は多量に採取されるものではない
ので、通゛縫は内径５〜２０％程度、高さｌＯ〜１００
％程度とされる。If the capacity of the above-mentioned tubular body is too small, the amount of carrier filled will be small, and therefore the measurement accuracy in the low concentration range of the sample will be deteriorated.On the other hand, if the capacity is too large, the measurement accuracy will not particularly increase, and this type of test Due to the nature of the sample, it is not necessary to collect a large amount of it, so the diameter of the thread is about 5 to 20%, and the height is lO to 100%.
It is said to be about %.
本発明における標識抗体もしくは標識抗原を調整する際
の標識としては、酵素、放射性同位元素（１１２５等）
、蛍光物質、不対電子をもつ化合物、色素等が使用され
、特に酵素や放射性同位元素が好適に用いられる。Labels used in the preparation of labeled antibodies or labeled antigens in the present invention include enzymes, radioactive isotopes (1125, etc.)
, fluorescent substances, compounds with unpaired electrons, dyes, etc. are used, and enzymes and radioactive isotopes are particularly preferably used.
上記担体が充填されたカラムに、通°帛は溶液状の測定
（、試料を添加、導入した後、標識抗体もしくは標識抗
原を添加、導入する。標識抗体もしくは標識抗原は予め
測定試料と混合しておいてもよい。Typically, a sample is added and introduced into a column filled with the above-mentioned carrier, and then a labeled antibody or labeled antigen is added and introduced.The labeled antibody or labeled antigen is mixed with the measurement sample in advance. You can leave it there.
本発明においては、カラムの下端に設けられた粘土等よ
りなるシールを除去して上記標識抗体もしく＃′ｉ標識
抗体をカラムに添加し、必要により更に緩衝液を添加す
ることにより容易にＢ／Ｆ分離が行われる。酵素標識を
用いた場合、上記担体に結合した標識抗体もしくは標識
抗原の量の測定は、更に基質を添加しカラム通過中に分
解された基質の量を、７０−セルによって、或いは一度
試験管等に採取してから通常の角型セルを用いることに
よって容易に行うことが出来、更に高速液体クロマトグ
ラフィーのポンプ、パルプ、オートナンプラーを適宜配
列結合すれば、測定が容易に自動化される。In the present invention, the labeled antibody or #'i labeled antibody is added to the column by removing the seal made of clay or the like provided at the bottom end of the column, and if necessary, by further adding a buffer solution, B. /F separation is performed. When enzyme labeling is used, the amount of labeled antibody or labeled antigen bound to the carrier can be measured by adding a substrate and measuring the amount of substrate decomposed during column passage using a 70-cell or once in a test tube. The measurement can be easily carried out by collecting the sample and using an ordinary rectangular cell, and furthermore, the measurement can be easily automated by appropriately arranging and connecting a high-performance liquid chromatography pump, pulp, and auto-numerator.
又放射性同位元素、不対電子をもつ化合物を標識として
用いた場合は、通常のカウンターを用いる仁とにより標
識抗体もしくは標識抗原を添加した後のカラムをそのま
ま測定対象とすることができる。更に、蛍光物質や色素
を標識とした場合は、通常の分光学的測定方法が採用さ
れる。Furthermore, when a radioactive isotope or a compound with unpaired electrons is used as a label, the column to which the labeled antibody or labeled antigen has been added can be directly used as a measurement target using a conventional counter. Furthermore, when fluorescent substances or dyes are used as labels, ordinary spectroscopic measurement methods are employed.
本発明における測定対象物の抗原の具体例としては、各
種ポリペプチド糸ホルモン、ステロイド糸ホルモン、ビ
ラ５２８１２１葉！、サイロキシン、トリヨードサイロ
ニン、補体、α−クエトプヮティン、カルジノエンブリ
オニック１ンチゲン、臓器および血液中の各種酵素およ
び蛋白質、ＨＢｓ抗原等の各種微生物抗原、植物ホルモ
ン、抗生物質、抗てんかん剤等の薬物等が挙げられ、そ
のうち特に重要なものは、甲状腺ホルモン、下垂体ホル
モン、ＨＢｓ抗原、インシュリン、免反グロブリンおよ
び肝酵素である。Specific examples of antigens to be measured in the present invention include various polypeptide thread hormones, steroid thread hormones, and Vira 528121 leaf! , thyroxine, triiodothyronine, complement, α-quetoputin, cardinoembryonic antigen, various enzymes and proteins in organs and blood, various microbial antigens such as HBs antigen, plant hormones, antibiotics, antiepileptic drugs, etc. Among these drugs, particularly important ones are thyroid hormone, pituitary hormone, HBs antigen, insulin, immunoglobulin, and liver enzymes.
本発明における抗体としては、上に述べた抗原に対応し
て動物体内で生産されるものが含まれる。Antibodies in the present invention include those produced within the animal body in response to the above-mentioned antigens.
又、本発明における測定対象物、同相化抗原もしくは抗
体、標識抗体もしくは抗原の王者の組合せに基づく本発
明方法の分〃（の例を以下に示す。Further, examples of the method of the present invention based on the combination of the object to be measured, the in-phase antigen or antibody, the labeled antibody, or the antigen in the present invention are shown below.
ｆｉｌ 測定対象物の抗原Ａと同様の抗原を固オ目イヒ
し、八と反応する標識抗体を用いる競合反応法０
（２） 測定対象物の抗体Ｂと同様の抗体を同相化し、
Ｂと反応する標識抗原を用いる焼全′反−ルー″７ｉ法
。fil Competitive reaction method 0 using a labeled antibody that reacts with an antigen similar to antigen A of the measurement target and reacting with 8 (2) An antibody similar to antibody B of the measurement target is made in phase,
7i method using a labeled antigen that reacts with B.
（３） 測定対象物の抗ｆ、（Ａと反応する抗体を固Ｉ
ｔｌ化し、Ａと同様の標識抗原を用いる競合反応法。(3) Anti-f of the measurement target, (I immobilize the antibody that reacts with A)
Competitive reaction method using the same labeled antigen as in A after tl conversion.
（４） 測定対象物の抗原Ａとｊ反応する抗体を同相化
し、Ａと反応する標識抗体を用いるサンドイツチ法。(4) Sand-Deutsch method in which an antibody that reacts with antigen A of the measurement target is brought into phase and a labeled antibody that reacts with A is used.
（５） 測定対象物の抗体Ｂと反応する第２抗体を同相
化し、Ｂと反応する標識抗原を用いるサンドイツチ法。(5) Sand-Deutsch method in which a second antibody that reacts with antibody B of the measurement target is made in phase and a labeled antigen that reacts with B is used.
（６） 測定対象物の抗体Ｂと反応する抗）帛を固４１
１化し、Ｂと反応する標識杭１鼠を用いるサンドイツチ
法。(6) Fix the anti-)cloth that reacts with antibody B of the measurement target 41
1 and reacts with B using the Sanderutsch method.
（７） 測定対象物の抗体Ｂと反１１′５する抗原を同
相化し、Ｂと反応する標識第２抗体を用いるサンドイツ
チ法。(7) Sanderch method, in which antibody B of the measurement target and an anti-11'5 antigen are brought into phase, and a labeled second antibody that reacts with antibody B is used.
尚、本発明においては、チオシアン酸ナトリラムを洗浄
することにより抗原もしくはｔ／Ｌ体が同相化きれた担
体に結合したｊｌ１１定対象物や標識抗体もしくは標識
抗原を担体から脱敲させて、同相化抗原もしくけ抗体を
繰返し使用することが可能である。In the present invention, by washing with sodium thiocyanate, the jl11 target substance, labeled antibody, or labeled antigen bound to the carrier in which the antigen or t/L form has been made in-phase is removed from the carrier, and the antigen or t/L form is made in-phase. It is possible to use antigens or antibodies repeatedly.
（以下余白）
本発明抗原もしくけ抗体の測定方法は、上述の通シの構
成罠なされ、抗原もしくは抗体が固相化された担体が、
Ｑ、ｊｉ充填されたカラムに、測定資料と、標識抗体も
しくは標識抗原を導入し、上記担体に結合した標識抗体
もしくは標識抗原の量を担体に結合した状態で測定する
ので、抗原もしくは抗体を毛細管内面に直接同相化する
方法や抗原もしくは抗体を同相化した担体を有底の試験
管に充填して測定する従来法と比較して、抗原もしくは
抗体の同相化量が多い為測定精度が高く、且つ比較的簡
便な操作でＢ／Ｆ分離（及び酵素標識を用いる場合は酵
素基質反応まで）をカラムで行う・ことができ、又、容
易に自動化が可能であるという利点を有する。(Left below) The method for measuring antigens or antibodies of the present invention has the above-mentioned general structure, in which the carrier on which the antigen or antibody is immobilized is
The measurement material and the labeled antibody or labeled antigen are introduced into the column filled with Q, ji, and the amount of the labeled antibody or labeled antigen bound to the carrier is measured while it is bound to the carrier, so the antigen or antibody is transferred to the capillary tube. Compared to the conventional method of in-phase formation directly on the inner surface or the conventional method of filling a bottomed test tube with a carrier with in-phase antigen or antibody, the measurement accuracy is high because the amount of in-phase antigen or antibody is large. In addition, it has the advantage that B/F separation (and up to the enzyme substrate reaction when using an enzyme label) can be performed using a column with a relatively simple operation, and it can be easily automated.
以下、実施例及び比較例により本発明の実施態様と効果
を詳細に説明する。単にチとあるのは重量％を意味する
。Hereinafter, embodiments and effects of the present invention will be explained in detail using Examples and Comparative Examples. The term ``chi'' simply means weight %.
実施例
サンドインチ法によるインシュリン抗原の測（Ａ）抗イ
ンツユリン抗体の固相化
モルモット産の抗ブタインシュリン抗血清を硫安沈澱法
によりＴ−グロブリン分画まで精製した。EXAMPLES Measurement of insulin antigen by Sand Inch method (A) Immobilization of anti-insulin antibody Anti-pig insulin antiserum from guinea pigs was purified to the T-globulin fraction by ammonium sulfate precipitation method.
このＴ−グロブリン分画をＰ Ｈ７，０の０．１Ｍリン
酸緩衝液（以下この液を０．１ ＭＰ、 Ｂ、（Ｐ Ｈ
７，０）と表わす）１００ｍｌ！に溶解して濃度１００
μｙ／−の浴液と、シ、粒径０．２〜０，６〜のポリス
ｆＶンピーズ担体５０ｙと混合し、３７℃で１時間イノ
キュベートした後、４°Ｃで１６時間放置した。This T-globulin fraction was dissolved in 0.1 M phosphate buffer with pH 7.0 (hereinafter referred to as 0.1 MP, B, (PH
7,0)) 100ml! Dissolved in to a concentration of 100
The bath solution of μy/− was mixed with 50y of polyfV beads carrier having a particle size of 0.2 to 0.6, and after incubating at 37° C. for 1 hour, it was left to stand at 4° C. for 16 hours.
次いで０．１ＭＰ、Ｂ、（ＰＨ７，０）で充分洗浄し、
０．１１ＶＩ Ｎａｐ ｌ 、１ ｍＭＭｇＣｅ２．
０．１ ％ＮａＮ３゜０．１％牛血悄アルブミンを含む
０．０２Ｍ２Ｍリン酸緩衝液Ｈ７，４，以下緩衝液Ａと
ｄう）２０ｄ中に移し、４℃で１６時間インキュベート
した。インキュベート後の担体を取シ出し、長さが約７
−１中央郡の内径が７％で先細にされたポリプロピレン
製管状体（エツペンドル７社製。Next, wash thoroughly with 0.1MP, B, (PH7,0),
0.11 VI Napl, 1 mM MgCe2.
The mixture was transferred to 0.02M 2M phosphate buffer H7.4 (hereinafter referred to as buffer A) containing 0.1% NaN3 and 0.1% bovine blood albumin for 20 days and incubated at 4°C for 16 hours. After incubation, the carrier was taken out and the length was about 7 mm.
-1 A tapered polypropylene tubular body with an inner diameter of 7% in the center (manufactured by Etsupendor 7).
１００〜１０００ ｐ ｌ用）で先端にグラスクールを
詰めたものに０．６ｆずつ分取してカラムを作成した。100 to 1000 pl), the tip of which was packed with a glass cooler, was fractionated in 0.6 f increments to prepare a column.
分注後、カラム先端（下端）を粘土（クレイアダムス社
製「クール・イーズ」）Ｋよシノールし、カラムを緩衝
液Ａで満たして上端をパラフィンフィルム（アメリカン
−カン・カンパニーの商品名）でノールし４℃に保持し
た。After dispensing, fill the tip (lower end) of the column with clay (Kool-Ease, manufactured by Clay Adams) K, fill the column with buffer A, and cover the upper end with paraffin film (trade name of American Can Company). The mixture was heated and kept at 4°C.
（Ｂ）酵素標識抗体の、、Ｉ４整 酵素としてβ−ガラクトシダーゼを用いた。(B) I4 alignment of enzyme-labeled antibody β-galactosidase was used as the enzyme.
（Ａ）で用いた抗イン／ニリン抗血清のＴ−グロブリン
分画のＳＨ基とβ−ガラクト７ダーゼのＳＨ基とをＮ−
（ｍ−マレイミドベンゾオキ／）サクシイミドによシ架
檎し、変性アガロースからなるカラム（サルマゾア社製
、セファロースＣＬ−６Ｂ）によシ梢製して酵素標識抗
体を得た。The SH group of the T-globulin fraction of the anti-in/nilin antiserum used in (A) and the SH group of β-galact7dase
The enzyme-labeled antibody was obtained by cross-linking with (m-maleimidobenzoyl/)succinimide and column-coating with a column made of denatured agarose (Sepharose CL-6B, manufactured by Sarmazoa).
（Ｃ）定量曲線の作成
精製フ゛タインシュリン （ノボ社製、アクトラビッド
ＭＣ）を緩衝液Ａで稀釈し濃度６４０゜３２０．１６０
＋ ８０．４０．２０ （μｕｎｉｔ／ｍｅ）の稀釈液
及び緩衝液［株］を用意し、（Ａ）で調整したカラムに
１００μｌずり添加した。添加の際、カラム下端の７−
ルをとυ、添加後再びノールをして３０℃で１時間イン
キュベートした。(C) Creation of quantitative curve Purified protein insulin (Actravid MC, manufactured by Novo) was diluted with buffer A to a concentration of 640°320.160.
+ 80.40.20 (μunit/me) dilution solution and buffer solution [stock] were prepared, and 100 μl was added to the column prepared in (A). During addition, the 7-
After adding the solution, the solution was added again and incubated at 30°C for 1 hour.
次いで上下端の７−ルを除去し２ ｍｅの緩衝液への添
加にエリカラムを洗浄し、５４０ｍｕｎｉｔの酵素を含
む３００μｌの酵素標識抗体を添加し、上下端を７−ル
し３７℃で２時間インキュベートした。Next, remove the 7-rings at the upper and lower ends, add 2 me to the buffer solution, wash the Elicolumn, add 300 μl of enzyme-labeled antibody containing 540 units of enzyme, remove the 7-rings from the upper and lower ends, and incubate at 37°C for 2 hours. Incubated.
その後、７−ルを取って４−の緩働ｉＡでカラムを洗浄
し、緩衝液Ａに基質として０−ニトロフェニル−β−Ｄ
ガラクトビラノンドを溶〃トした０、５チ溶液を０．５
ｄ添加後再び７−ルをして３７℃で１時間インキュベー
トした。次いで、酵素反応停止剤として０．１ Ｍの炭
酸ナトリウム２−を加えて酵素反応を停止させると共に
管状体を洗浄し、カラムを通過した基質分解生成物０−
ニトロフェノール液を採取し、波長４２０ｎｍの元の吸
光度を測定した。かかる定量を３回行い平均値によりブ
タインシュリン濃度〜吸光度曲線を作成し第１図のＥ線
で示した。After that, remove 7-L, wash the column with 4-Slow iA, and add 0-nitrophenyl-β-D as a substrate to Buffer A.
0.5% solution of galactobiranondo
After adding d, the mixture was heated again at 7° C. and incubated at 37° C. for 1 hour. Next, 0.1 M sodium carbonate 2- was added as an enzyme reaction terminator to stop the enzyme reaction, and the tubular body was washed, and the substrate decomposition products 0-
The nitrophenol solution was collected and its original absorbance at a wavelength of 420 nm was measured. Such quantification was carried out three times, and a porcine insulin concentration-absorbance curve was created using the average value and is shown as line E in FIG.
比較例１
実施例の囚で調整した濃度１００ ／ｌ ９ ／ ｒＪ
のＴ−グロブリン分画溶液を１００μｌ試験管にとり、
実施例で用いた緩衝液Ａを０．５ ｍｅ加え、実施例で
用いたポリスチレンピーズ０．５ｙを加え３７℃で１時
間インキュベートした。次いで実施例の（Ｃ）で調整し
たズタイン７ユリン稀釈液を加え、２ｍｌの緩衝液Ａで
吸引洗浄し実施例の（Ｂ）で調整した酵素標識抗体を３
００Ｊｌ／加えた後３７℃で２時間インキュベートした
。その後２ ｍｌの緩衝液で２回吸引洗浄し、実施例と
同様ＫＬ、−ｒＯ，５％０−ニトロフェニル−β−Ｄ
−カラクジトビ２フフド溶液を０．５ ｒｎｌ添加し、
３７℃で１時間インキュベートした。次いで２ ｒｎｅ
の０、１ Ｍ炭酸ナトリクムを加えて酵素反応を停止さ
せ、Ｏ−二トロフェノール溶液の波長４２０ｎｍの光の
吸光度を測定した。結果は第１図の■線の通）であった
。Comparative Example 1 Concentration adjusted with Example 100/l 9/rJ
Take 100 μl of the T-globulin fraction solution in a test tube,
0.5 me of buffer A used in the example was added, 0.5 y of the polystyrene beads used in the example were added, and the mixture was incubated at 37°C for 1 hour. Next, the Zustein 7 urin diluted solution prepared in Example (C) was added, and the enzyme-labeled antibody prepared in Example (B) was washed by suction with 2 ml of buffer A.
After adding 00 Jl/ml, it was incubated at 37°C for 2 hours. Thereafter, suction and washing was performed twice with 2 ml of buffer, and as in the example, KL, -rO, 5% 0-nitrophenyl-β-D
- Add 0.5 rnl of Karakjitobi 2 Fufudo solution,
Incubate for 1 hour at 37°C. Then 2 rne
The enzyme reaction was stopped by adding 0 or 1 M sodium carbonate, and the absorbance of the O-nitrophenol solution at a wavelength of 420 nm was measured. The results were as indicated by the ■ line in Figure 1).
比較例２
比較例１において濃度１００μｙ／−〇丁−グロブリン
分画溶液を５００μｌ試験管にとシ直径６．３％のポリ
スチレンピーズ１個を用いる以外は比較例１と同様の測
定を行った。結果は第１図の■線の通りであった。Comparative Example 2 Measurements were carried out in the same manner as in Comparative Example 1, except that a globulin fraction solution with a concentration of 100 μy/−〇 was placed in a 500 μl test tube and one polystyrene bead with a diameter of 6.3% was used. The results were as indicated by the ■ line in Figure 1.
燗寺堵４４４ジ祠シ致う４シ卓４禰嶋徳４過ユ・度＝−
１かした４ぼ〒第１図から比較例２では抗原の定量が殆
んど不可能でおることが明らかである。Kanji To 444 Ji Shrine 4 Shi Taku 4 Nejima Toku 4 Passage = -
It is clear from FIG. 1 that it is almost impossible to quantify the antigen in Comparative Example 2.
又、比較例１では抗原濃度Ｏｕｎｉｔ ／ ｄの値が異
常に高く、従って低ｉ度域における測定値の信頼性が乏
しく、又比較的高濃度の領域においても測定値のバラツ
キが太きい。これは繰返し行われた吸引洗浄の過程でピ
ーズが吸引によシ一部失われた為と思われる。Furthermore, in Comparative Example 1, the value of the antigen concentration Ounit/d is abnormally high, so the reliability of the measured values in the low i degree range is poor, and the measured values also vary widely even in the relatively high concentration range. This seems to be because some of the beads were lost by suction during the repeated suction cleaning process.
第１図は実施例及び比較例によシ作成されたイン／ニリ
ンの定量曲線でｂる。
Ｅ：実施例による定量曲線
■：比較例１による定量曲線
■：比較例２による定量曲線
特許出願人
積水化学工業株式会社
代表者 藤 沼 基 利FIG. 1 shows a quantitative curve of in/niline prepared in Examples and Comparative Examples. E: Quantitative curve according to Example ■: Quantitative curve according to Comparative Example 1 ■: Quantitative curve according to Comparative Example 2 Patent applicant Mototoshi Fujinuma, Representative of Sekisui Chemical Co., Ltd.
Claims (1)
いて、測定対象物と同様の抗原もしくは抗体又は測定対
象物と反応する抗体もしくは抗原が固相化された担体が
充填されたカラムに、ｆ漬方ラム中の担体に測定対象物
と同様の抗原もしくは抗体が固相化された場合は、測定
試料と、測定対象物と反応する既知量の標識抗体もしく
は抗原とを導入し、又は、該カラム中の担体に測定対象
物と反応する抗体もしくは抗原が同相化された場合は、
測定試料と、測定対象物と同様の既知量の標識抗原もし
くは抗体、又は、該測定対象物と反応する既知量の標識
抗原もしくは抗体とを導入し、上記担体に結合した標識
抗体もしくは標識抗原の量を担体に結合した状０で測定
することを特徴とする抗原もしくは抗体の測定方法。 ２ 担体が無孔性である第１．１１Ｊ記載の測定方法。 λ 担体が粒径０．１〜５％のビーズである第１項又は
第２項記載の測定方法。 克 担体がポリスチレン製である第１項〜第３゛何れか
１項に記載の測定方法。 飄 担体がアミ７基の導入された熱可塑性セルロース製
である第１項〜第３゛項何れか１項に記載の測定方法。[Scope of Claims] L In a method for measuring an antigen or antibody by immunoassay, a column filled with a carrier immobilized with an antigen or antibody similar to the object to be measured, or an antibody or antigen that reacts with the object to be measured. If an antigen or antibody similar to the object to be measured is immobilized on the carrier in the f-soaked lamb, introduce the measurement sample and a known amount of labeled antibody or antigen that reacts with the object to be measured, Or, if the antibody or antigen that reacts with the analyte is in phase with the carrier in the column,
A measurement sample and a known amount of labeled antigen or antibody similar to the measurement target, or a known amount of labeled antigen or antibody that reacts with the measurement target are introduced, and the labeled antibody or labeled antigen bound to the carrier is 1. A method for measuring an antigen or antibody, which comprises measuring the amount of an antigen or antibody bound to a carrier. 2. The measuring method according to Section 1.11J, wherein the carrier is non-porous. The measuring method according to item 1 or 2, wherein the λ carrier is beads having a particle size of 0.1 to 5%. The measuring method according to any one of items 1 to 3, wherein the carrier is made of polystyrene. The measuring method according to any one of items 1 to 3, wherein the carrier is made of thermoplastic cellulose into which 7 amino groups have been introduced.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11673683A JPS607363A (en) | 1983-06-27 | 1983-06-27 | Measurement of antigen or antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11673683A JPS607363A (en) | 1983-06-27 | 1983-06-27 | Measurement of antigen or antibody |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS607363A true JPS607363A (en) | 1985-01-16 |
Family
ID=14694512
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP11673683A Pending JPS607363A (en) | 1983-06-27 | 1983-06-27 | Measurement of antigen or antibody |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS607363A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6418216A (en) * | 1987-07-13 | 1989-01-23 | Tdk Corp | Ceramic capacitor and composite component |
| JPS6421912A (en) * | 1987-07-16 | 1989-01-25 | Tdk Corp | Manufacture of ceramic chip capacitor |
| JP2009513403A (en) * | 2003-06-26 | 2009-04-02 | コンチネンタル・テベス・アーゲー・ウント・コンパニー・オーハーゲー | Piston type accumulator |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS49125517A (en) * | 1973-03-19 | 1974-12-02 | ||
| JPS51123819A (en) * | 1975-04-07 | 1976-10-28 | Summa Corp | Fixed immune adsorbing agent |
| JPS57103649A (en) * | 1980-12-18 | 1982-06-28 | Asahi Chemical Ind | Sterilized gamma-globulin fixing column |
| JPS57150433A (en) * | 1981-03-12 | 1982-09-17 | Kuraray Co Ltd | Carrier for immobilizing physiologically active material and selective adsorbent, selective electrode and analytical column using said carrier |
-
1983
- 1983-06-27 JP JP11673683A patent/JPS607363A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS49125517A (en) * | 1973-03-19 | 1974-12-02 | ||
| JPS51123819A (en) * | 1975-04-07 | 1976-10-28 | Summa Corp | Fixed immune adsorbing agent |
| JPS57103649A (en) * | 1980-12-18 | 1982-06-28 | Asahi Chemical Ind | Sterilized gamma-globulin fixing column |
| JPS57150433A (en) * | 1981-03-12 | 1982-09-17 | Kuraray Co Ltd | Carrier for immobilizing physiologically active material and selective adsorbent, selective electrode and analytical column using said carrier |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6418216A (en) * | 1987-07-13 | 1989-01-23 | Tdk Corp | Ceramic capacitor and composite component |
| JPS6421912A (en) * | 1987-07-16 | 1989-01-25 | Tdk Corp | Manufacture of ceramic chip capacitor |
| JPH0513528B2 (en) * | 1987-07-16 | 1993-02-22 | Tdk Electronics Co Ltd | |
| JP2009513403A (en) * | 2003-06-26 | 2009-04-02 | コンチネンタル・テベス・アーゲー・ウント・コンパニー・オーハーゲー | Piston type accumulator |
| JP4723488B2 (en) * | 2003-06-26 | 2011-07-13 | コンチネンタル・テベス・アーゲー・ウント・コンパニー・オーハーゲー | Piston type accumulator |
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