JPS5960260A - Enzyme immunological measurement - Google Patents

Abstract

PURPOSE:To enable an accurate measurement of the density of an antigen by arranging the action of an insoluble antibody in the presence of serum protein and polyalkylene glycol when the insoluble antibody specifically to be bonded to a measuring material (anitgen or the like) and an antibody labelled by an enzyme are allowed to react upon the measuring substance. CONSTITUTION:An antibody (b) supported on an insoluble carrier and an isolated antibody (d) labelled by an enzyme is made to react upon a measuring substance (a) (antigen, hapten or the like) to measure the activity of the enzyme in a bond type labelled antibody d1 by separating the antibody d1 in the insoluble antibody-the measuring substance-antibody from an isolated antibody d2 through competition reaction between the antibodies (b) and (d), serum protein obtained from a bovine, horse, goat or the like and a compound with an average molecular weight of 1,000-2,000 such as polyethylene glycol and polypropylene glycol are added to the reaction system. This permits the absorbance of coloring and fluorescence to be presented as shown by the curve A in stead of in the inverted-V pattern as shown by the curve B as generated in the reaction between the enzyme and the substrate when the density of the measuring substance is high thereby enabling an accurate measurement of the density.

Description

【発明の詳細な説明】 本発明は酵素免疫測定法に関するものであり、その目的
は画素免疫測定法の操作を簡略化し標準曲線の逆Ｖ字型
を回ＩＨｔすることにある。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an enzyme immunoassay, and its purpose is to simplify the operation of the pixel immunoassay and to repeat the inverted V-shape of the standard curve.

従来より生体内の微力１成分の測定に非常に特異的かつ
鋭敏な抗原抗体反応を利用する方法が数多く開発され、
臨床検査分野において非常に多く利用されている。その
中でも、放射免疫？Ｉｕｌ＋定法（以下ＲＩ　Ａと略す
）、酵素免疫測定法（以下ＥＩＡと略す）は、１ａｌｌ
＋定感度が非常に高く、定量性に優れているため広い応
用範囲を有しており、測定可能な物質も、ペグチド、ホ
ルモン、免疫グロブリン等の高分子物質からステロイド
、薬物等の低分子物質まで多種多様にわたっている。し
かじなからＲＩ　Ａはラジオアイソトープを使用するた
め、被爆、その取扱い管理、廃棄等、種々の問題をかか
えている。そのためＩＬ　Ｉ　Ａに代わる測足法として
、標識物質としてのラジオアイソトープを使用ぜず、酵
素を標識物質とするＥＩＡが近年脚光をあびてきた。Ｅ
　Ｉ　Ａの測定原理は競合法及びサンドイツチ法と呼ば
れる二種類の測定原理が広く用イラれている。以下サン
ドインチ法についてｎ９明する。Numerous methods have been developed that utilize highly specific and sensitive antigen-antibody reactions to measure single components in vivo.
It is widely used in the field of clinical testing. Among them, radioimmunity? Iul+ standard method (hereinafter abbreviated as RIA), enzyme immunoassay (hereinafter abbreviated as EIA), 1all
+ It has a very high sensitivity and excellent quantitative properties, so it has a wide range of applications, and the substances that can be measured range from high-molecular substances such as pegtides, hormones, and immunoglobulins to low-molecular substances such as steroids and drugs. There are many different types. However, since RIA uses radioisotopes, there are various problems such as exposure to radiation, handling and disposal, and so on. Therefore, EIA, which does not use a radioisotope as a labeling substance and uses an enzyme as a labeling substance, has been attracting attention in recent years as a foot measurement method to replace ILIA. E
Two types of IA measurement principles are widely used: the competition method and the Sand-Deutsch method. The sandwich method will be explained below.

１ず、サンドイツチ法の測定原理の概要を図面によって
説明する。第１図に示すように、測定物質（以下、抗原
と呼ぶ）ａとその抗原に対する抗体を不溶化した不溶化
抗体ｂ’に反応させる。両者ν」、抗＋＋５を抗体反応
を生じ、ネ゛す合体Ｃを形成する（第１反応）。この？
ニア合体ｃｆ反応混合液よシ分離し、測定抗原に対する
抗体にｒ）γ素全結合させた酵素標識抗体（１の−＞ｊ
憂１１と反応させる（第２反応）。ｊｉｊ：素イ４＋！
識抗体は杓合体Ｃに結合するが、複合体Ｃの結合ス１ヒ
を越えたｆｊｉの１：ン素標識抗体は遊離状態で存在す
る。次に前期複合体に結合した標識抗体ｄ１と遊１ｉ、
ＩＵ状態のｒｉｊＱ素（：“１ｖ識抗体ｄ２を分離し、
いずれかのｌ′ｉ、素活４ツＦを測定する。同時に既知
濃度の標準物質を用いて同様Ｋ（パｆ１作して４！１１
準曲Ｉｖｉ！を作成し、これによって未知の抗原量を求
める。First, an overview of the measurement principle of the Sand-Deutsch method will be explained using drawings. As shown in FIG. 1, a substance to be measured (hereinafter referred to as antigen) a and an insolubilized antibody b' prepared by insolubilizing an antibody against the antigen are reacted. Both ν' and anti++5 cause an antibody reaction and form a combined compound C (first reaction). this?
The enzyme-labeled antibody (1->j
Make it react with Ui 11 (second reaction). jij: Prime 4+!
The labeled antibody binds to complex C, but the fji 1:9-labeled antibody that has exceeded the bound limit of complex C exists in a free state. Next, labeled antibody d1 bound to the prophase complex and antibody 1i,
rijQ element in IU state (: "1v recognition antibody d2 is separated,
Measure either l'i and elemental activity 4F. At the same time, using a standard substance with a known concentration, similarly
Quasi-piece Ivi! and calculate the amount of unknown antigen.

上記ザンドイノチ法は測定しうる抗原量の変化が酵素標
識抗体の活性の変化箕に対応するので広い範囲にわたる
抗原の測定が可能である。しかしながら測定法は、第１
反応、第２反応、酵素反応という三段階のＪ・■作が必
要で非常に煩雑であるという欠点を有している。そこで
これらの欠点を改良したＥ　ｉ　Ａが４！ｆｌ；−；」
昭５３−４７５１８号公報、５３−４７５１９号公報に
おいて報告されている。それによると、第１反応終了後
、抗原抗体反応複合物を混合液よシ分離せず、標識抗体
を加えるという方法と、測定物質、同相化抗体、イ＝識
抗体を同時に作用させ、次いで固相上の酵素活性を測定
する方法とが記載されている。これらの方法は、分Ｓ操
作が１回で済み、操作が簡略になったＥ　Ｉ　Ａの改良
法である。In the above Zando Inochi method, a change in the amount of antigen that can be measured corresponds to a change in the activity of the enzyme-labeled antibody, so it is possible to measure a wide range of antigens. However, the measurement method
It has the disadvantage that it is very complicated because it requires three steps of reaction, second reaction, and enzyme reaction. Therefore, E i A that has improved these shortcomings is 4! fl;-;”
It is reported in Publications No. 53-47518 and No. 53-47519. According to this, after the first reaction, the antigen-antibody reaction complex is not separated from the mixed solution and a labeled antibody is added, and the measuring substance, in-phase antibody, and i=identifying antibody are reacted simultaneously, and then immobilized. A method for measuring enzyme activity on a phase is described. These methods are improved methods of EIA, requiring only one minute S operation and simplifying the operation.

しかしながら、この改良法においては、抗原１．ｔの増
加に従って標準曲線が逆Ｖ字型を示す事が報告されマい
る。この場合、抗原量が非常に多い場合には、見かけ上
実際の抗原量よりも非常に低い抗原量として検出してし
まい、擬隘性を示してしまうか、あるいは検体を希釈し
て再測定しなければならず、測定の二度手間になり操作
を簡便化するための改良法としては意味がない、特に測
定物質（抗原）、不溶化抗体、酢素神識抗体金同時に反
応させ、抗原抗体複合物に結合した酵素標識抗体または
、遊離状態の酵素標識抗体の酵素活性を測定するＥ　Ｉ
　Ａでは、一般には特開昭５３　４７５１９号公報にも
示されている様に抗原ｈ１が増加すると第２図１３に示
す如く、逆Ｖ字型の標準曲線が得られる。However, in this improved method, antigen 1. It has been reported that the standard curve exhibits an inverted V-shape as t increases. In this case, if the amount of antigen is extremely large, the amount of antigen detected will appear to be much lower than the actual amount, resulting in a false detection, or the sample may need to be diluted and re-measured. Therefore, it is meaningless as an improved method to simplify the operation because it requires two steps of measurement.In particular, it is necessary to react the substance to be measured (antigen), insolubilized antibody, and acetic acid antibody gold at the same time, and the antigen-antibody complex is E I to measure the enzyme activity of an enzyme-labeled antibody bound to a substance or a free enzyme-labeled antibody
In A, generally, as shown in JP-A-53-47519, when the antigen h1 increases, an inverted V-shaped standard curve is obtained as shown in FIG. 2, 13.

本発明者らはこのオ・口準曲綜の逆Ｖ字型を７９子決す
るため種々？ｉＪＦ究した結果、庶くべきことに血清蛋
白質全主体とする媒ａ中にポリアルキレングリコールの
有効ｉ１１を添加し、反応を行うと標準曲線の逆Ｖ字型
を回避できることを見い出し本発明に到Ｐ；？シた。The present inventors have various methods to determine the inverted V-shape of this O-mouth quasi-curved helix. As a result of our research on iJF, we discovered that it is possible to avoid the inverted V-shape of the standard curve by adding effective i11 of polyalkylene glycol to a medium mainly consisting of all serum proteins and carrying out the reaction, which led to the present invention. P;? Shita.

すなわち不発り−１は側５ピ物質に、この物質に特異的
に結合する抗体全不溶化した不溶化抗体および抗体に酵
素を結合させた酵素標識抗体を作用させ、側だ物質にｉ
’、！ｊ合した７、ｑ、％素（；ヤ識抗体または結合し
なかった？４チ素４：ｌ識抗体の酵素活性を測定するこ
とにより、１ｌｌｌ！定物質の′１１１を測定する酵素
免疫測定法において、血清３（ｙ白質およびポリアルキ
レングリコールの存在下に不溶化抗体全作用させること
を特徴とする酵素免１−′ヒ測定法である。In other words, in case of misfire-1, an insolubilized antibody that specifically binds to the substance is insolubilized, and an enzyme-labeled antibody in which an enzyme is bound to the antibody is applied to the substance on the side.
',! Enzyme immunoassay to measure '111 of 1lll! substance by measuring the enzyme activity of the combined 7, q, % element (;Ya antibody or the unbound ?4 element 4:l antibody) This method is an enzyme immunoassay method characterized by allowing the total effect of insolubilized antibodies in the presence of serum 3 (y white matter and polyalkylene glycol).

本発明ではＩ＝；　Ｉ　Ａ　Ｋおける操作の煩雑性及び
標準曲線逆Ｖ字型（抗原量が異常に高い場合には低値を
示してし甘う）という欠点を有しない。The present invention does not have the drawbacks of complicated operations and an inverted V-shaped standard curve (low values are easily shown when the amount of antigen is abnormally high).

不発ツＪの酵素免疫法は測定物７りに、この物質に特異
的に結合する抗体を不溶化した不溶化抗体および抗体に
酵素を結合させた酵素標識抗体を作用させ、測定物質に
結合した酵素標識抗体または結合しなかった酵素標識抗
体の酵素活性を測定することにより、測定物質の９’ｆ
　ｆ　１ｌｌｌ定する方法である。In the enzyme immunoassay method of Futsutsu J, an insoluble antibody, which is an insoluble antibody that specifically binds to the substance, and an enzyme-labeled antibody, which is an antibody bound to an enzyme, are applied to the substance to be measured, and the enzyme label bound to the substance to be measured is detected. By measuring the enzyme activity of the antibody or unbound enzyme-labeled antibody, the 9'f of the test substance can be determined.
This is a method to determine f 1llll.

反応形態としては下記のものがあげられる。Examples of reaction forms include the following.

（Ａ）　　測定物質に不溶化抗体を作用させ、次いで標
識抗体を作用させる。(A) Let an insolubilized antibody act on the substance to be measured, and then let a labeled antibody act on it.

０３）　　測定物質に標識抗体を作用させ、次いで不溶
化抗体を作用させる。03) A labeled antibody is allowed to act on the substance to be measured, and then an insolubilized antibody is made to act on the substance to be measured.

（０測定物質、不溶化抗体および標識抗体の三者を同時
に反応させる。(The three substances, the substance to be measured, the insolubilized antibody, and the labeled antibody, are reacted simultaneously.

物に三者を同時に反応させる（Ｑの方法が好せしい。Make all three react to an object at the same time (method Q is preferable).

本発明では（５）測定物質と不溶化抗体、（至）測定物
質−（・ツ識抗体結合体と不溶化４＋’ｉ体、（Ｑ測定
物質、不溶化抗体および標識抗体を血清蛋白質およびポ
リアルキレングリコール存在下で行なう。In the present invention, (5) a measuring substance and an insolubilized antibody, (to) a measuring substance-(・antibody conjugate and an insolubilized 4+'i form, Do it below.

本発明のポリアルキレングリコールとしてはボリエチレ
ングリコール、ポリプロピレングリコール、ポリ子トラ
メチレングリコールなどがある。Examples of the polyalkylene glycol of the present invention include polyethylene glycol, polypropylene glycol, and polytramethylene glycol.

木兄ψＩにおいて用いるポリアルキレングリコールの平
均分子（１１は１．、ＯＯＯ〜２０，０００、好寸しく
け４．０００〜６．０００のものである。添加量として
は、約１〜１０５ｎ　ｆｆ　対’：（：　ｆｉ４：　（
Ｗ／　Ｖ　）　％　、好ましくは３〜６　！Ｒ（ｉ：対
答ｔｉｔＣＷ／Ｖ　）％である。１重量対容量（Ｗ／〜
′）チ未満であると、本発明の目的が達成されない。１
０重量対対答Ｌｉ゛（Ｗ／Ｖ　’）％を越える添加は血
ｒ／ｉ　；）（白りｔの沈ン讃ヲ引き起こし、好ましい
ものではない。又、血清道イ白質としては、牛血清、牛
アルブ］ン、ヤギ血ｆｉ！、ウマ梅漬、家兎血清等多種
挙げられる。添加１１トとしては約１〜３０容量チ、好
ましくは５〜１０容量係である。１容斯チ未満であると
本発明の［」的が達成されない。まだ３０容−ｒａ：％
を越えると正確な測定ができない。The average molecule of the polyalkylene glycol used in Kinei ψI (11 is 1., OOO ~ 20,000, suitable size is 4.000 ~ 6.000. The amount added is about 1 ~ 105 n ff vs. ':(: fi4: (
W/V)%, preferably 3-6! R(i: response titCW/V)%. 1 weight to capacity (W/~
') If it is less than H, the object of the present invention will not be achieved. 1
Addition of more than 0% by weight vs. response Li゛(W/V') causes blood r/i;) (whitening) and is not desirable. Various types include serum, bovine albumen, goat blood fi!, horse plum pickle, rabbit serum, etc.The amount to be added is approximately 1 to 30 volume units, preferably 5 to 10 volume units.Less than 1 volume volume. If so, the objective of the present invention will not be achieved.It is still 30 volume-ra:%
Accurate measurements cannot be made if the value exceeds this value.

本発明全実施例により具体的に説明する。The present invention will be specifically explained using all embodiments.

実施例１　ヒトイムノグロブリンＥの測定ａ）ヒトイノ
、ノブロブリンＩｉｉ　（以下■Ｌ？Ｅと略す）稗準溶
液の調整 Ｉ、Ｅ高値のヒト血清（ヘキスト社製）’１ＷＨＯのＩ
　ｙ　Ｅ　［準品７５１５ｏ２を用いて、−元放射状免
疫拡散法により濃度を検定し、家兎血清で、２５．５０
．１００．２００．４００．８００．１，６００．３．
２００．６、４００．９，６００　Ｕ／ｍｌのｆＡ度に
調製した。Example 1 Measurement of human immunoglobulin E a) Preparation of human immunoglobulin III (hereinafter abbreviated as ■L?E) subliminal solution I, human serum with high E value (manufactured by Hoechst) '1 WHO I
y E [The concentration was assayed by the radial immunodiffusion method using quasi-product 7515o2, and the concentration was determined to be 25.50 in rabbit serum.
．． 100.200.400.800.1,600.3.
It was prepared to fA degrees of 200.6, 400.9,600 U/ml.

ｂ）抗１，１弓抗体の製造 ヒトＩｙＥ’ｆｆｉフロイントの完全アジ該バンドとと
もに家兎に免疫した。得ら１．た抗血清ヲ３３％　’Ｌ
Ｗ酸ナトリウムで塩析しイムノグロブリン画分ｔ　４４
）た。このイムノグロブリン画分をヒトＩ。b) Production of anti-1,1 antibody A rabbit was immunized with the human IyE'ffi Freund's complete horsetail band. Obtained 1. Antiserum 33%'L
Salting out with sodium W acid, immunoglobulin fraction t44
)Ta. This immunoglobulin fraction was used as human I.

Ｇ、ＴＭ、Ｉ、Ａ、、　Ｉ、Ｄを結合させたセファロ−
？ ス４１３カラムに通し、素通りしたＩ、ｈｉＫ特異的な
Ｉ、（＃画分金得、抗ヒ）Ｉ、ＩＪ抗体とした。Cephalocarbon combined with G, TM, I, A,, I, D
? The mixture was passed through a 413 column to obtain I, hiK-specific I, (#fraction gold, anti-human) I, and IJ antibodies.

Ｃ）抗Ｉ、Ｅ抗体−酵素結合物の製造 ５■の西洋わさびペルオキシダーゼ（以下、ＨＲＰと略
す。東洋紡績株式会社製グレード１−Ｃ）　’ｆｆ１ｌ
−の０．３Ｍ炭酸水素ナトリウム溶液に溶解し、Ｑ、１
ｍ７！の１％２．４ジニトロフルオロベンゼンを加えて
室温で１８ｅｊ間攪拌した。この溶液ＫＯ，０６Ａ４過
ヨウ素酸ナトリウム溶液１ｍｅを加え３０分間室温で］
１゛ノ拌後、１．０ｒｎＩ！の０．１６　Ｍエチレング
リコール（′１才液を加え、室τ、情で１時間攪拌し体
ｋＯ，ｏＩ　Ｍ炭ｎ２！ｌＪｊＭ　（ｐｌ−１９，５）
　Ｉｃ　１０　Ｗ／ｍｌ　（Ｄ濃度に溶解しだ抗ＩｙＥ
抗体溶液０．５−を加え、室温で３時間（１〕拌後５■
の水素化ホウ素ナトリウムを加え、室温でネらＶｃ１時
間反応させた。反りス（′！Ｊ２（ＩＱで分画鞘製して
抗Ｉ、Ｅ抗体ＨＲＰ結合物（」ソ下標ｉｉｉ’＆抗体と
呼ぶ）を得た。C) Production of anti-I, E antibody-enzyme conjugate 5■ Horseradish peroxidase (hereinafter abbreviated as HRP. Grade 1-C manufactured by Toyobo Co., Ltd.) 'ff1l
- dissolved in 0.3M sodium bicarbonate solution, Q, 1
m7! of 1% 2.4 dinitrofluorobenzene was added and stirred at room temperature for 18ej. Add 1me of this solution KO, 06A4 sodium periodate solution and keep at room temperature for 30 minutes]
After 1 inch stirring, 1.0rnI! Add 0.16 M ethylene glycol (1 year old solution) and stir for 1 hour in a room temperature.
Ic 10 W/ml (anti-IyE dissolved at D concentration
Add 0.5-ml of antibody solution and stir at room temperature for 3 hours (1).
of sodium borohydride was added, and the mixture was allowed to react at room temperature for 1 hour. The anti-I, E antibody HRP conjugate (referred to as ``III'&antibody'') was obtained by fractionation using warp ('!J2) (IQ).

ｄ）　　抗１　ｙ　Ｅ　ｂ″［体ポリスチレンボール結
合物のｊＦＪ　造直径０．６３５ｃｌｎのポリスチレン
ポールを前記ｂ）で製造した抗Ｉ　、　Ｅ抗体全ＰＢＳ
Ｋｌグ／−の濃度に７６解した抗体溶液中に４°Ｃ２４
時間浸′ｍ後、抗体ｆｒＩ液を除去し、］、’　Ｉ−３
８で４回洗浄した。これに１容量ヴ牛アルブミンを含む
Ｐ　ＴＪ　Ｓ　金加え保存し、抗１ｙＪ’＝抗体ポリス
チレンボール結合物（以下抗体Ａｉ’ｆ合ボールと略す
）を得た。d) Anti-1 y E b'' [jFJ of polystyrene ball conjugate] Anti-I, E antibody prepared in b) above using a polystyrene pole with a diameter of 0.635 cln, whole PBS
4°C 24°C in an antibody solution diluted to a concentration of Klg/-.
After soaking for an hour, remove the antibody frI solution, ], ' I-3
8 and washed four times. To this was added 1 volume of P TJ S gold containing bovine albumin and stored to obtain an anti-1yJ'=antibody polystyrene ball conjugate (hereinafter abbreviated as antibody Ai'f conjugate ball).

ｅ）　　ｘｙＥの測定 前記ａ）で調２整した各険度のＩ　、　Ｅ溶液の５０ｐ
Ｌを試験管にとり抗体結合ボールを加え、さらに下記の
緩衡剤で適当な吸光度が得られるように希釈したＰ、ｑ
　Ｒ？＆抗体’ｆｆ’ｏ、５ｍｅ、加え３７”Ｃｌ２Ｏ
分反応させた。e) Measurement of xyE 50p of I and E solutions of each steepness adjusted in a) above.
Take L in a test tube, add antibody-bound balls, and then dilute P and q with the following buffer to obtain an appropriate absorbance.
R? & Antibody 'ff'o, 5me, plus 37" Cl2O
It was allowed to react for a minute.

Ａ：（本発明法）３重Ｊ肛対容量（Ｗ／Ｖ）係ポリエチ
レングリコール＃６，０ＯＯＣ半井什学社製）、５容お
：％ぼ兎血清含有Ｐ　ＨＳ　ｐＨ７，２１３：５容１．
（チ坂兎血清含有Ｐ　Ｂ　Ｓ　ｐＨ７，２Ｃ：３Ｍ引゛
対対答チ（Ｗ／Ｖ　）％ＰＥＧ＃６，０００含有１）　
ＲＳ　ｐｉ−＋　７．２ 反応終了りさ、反応混合物をアスピレータ−で吸引除去
し、蒸留水１　ｍｅで３回洗浄し、これに０−フェニレ
ンジアミン３００１ｑ／ｄｌと２．０％過酸化水素水１
　ｍｅ／ｄｌとを含む０．１Ｍクエン酸−０，２Ｍリン
酸２プトリウム緩衡液ｐＩ−（５，０の基タノｆ溶液０
．５ｍｌを加え、室温、ＩＩｌ′ｆ所で６０分間反応さ
せた。ＩＮ硫酸２　ｍｌで反応全停止させ４９２ｎｍで
の吸光度を測定した、このときの伸率曲線を第２図に示
した。A: (Method of the present invention) 3-fold J anal volume (W/V) polyethylene glycol #6,0OOC (manufactured by Hani Jiugaku Co., Ltd.), 5 volumes O: % rabbit serum-containing PHS pH 7,213: 5 volumes 1 ．．
(PBS containing Chisaka rabbit serum pH 7,2C: 3M vs. response (W/V)% PEG #6,000 content 1)
RS pi-+ 7.2 After the reaction is completed, the reaction mixture is removed by suction using an aspirator, washed three times with 1 ml of distilled water, and 3001 q/dl of 0-phenylenediamine and 1 ml of 2.0% hydrogen peroxide are added to this.
0.1M citric acid-0,2M diptrium phosphate buffer pI-(5,0 base tanof solution 0
．． 5 ml was added, and the reaction was allowed to proceed for 60 minutes at room temperature. The reaction was completely stopped with 2 ml of IN sulfuric acid, and the absorbance at 492 nm was measured. The elongation curve at this time is shown in FIG.

７’６　Ｎ’Ｊ剤Ｉ＋　−１）　ｆはｉ−ｉ＋；　２　
図ＶＣ示ｔ　如＜　通Ｖ　字Ｘ’２　ノイ・”′ζζ凹
曲線イ（ＪらＪｌ、た。本発明法のｌ）　Ｅ　Ｇを含む
緩１１１・■剤Ａでは緩衡剤ＩＪ−Ｄの場合のような逆
Ｖ字型の４へ゛（準曲腺はｊ！ＩらｆＬず、（１ミキ陰
性を示す場合もなくｔＶ＋作は非常Ｖｃ１７！１単であ
った５、次に本発明のＰ　Ｅ　Ｇ含有緩衡剤Ａ全用いて
作製した神準曲め！ｆ：オリ用した正常人およびアトピ
ー性アレルギー患者血清中のＩ、Ｅ測定例を述べる。測
定（テヘ体５０μｔ４・そｎ４’れ試験管にとり、これ
にｄ）で製造した抗体結合ビーズを加え、さらにＣ）で
製造した（・、＋ｉ識抗体を本発明法のＰ　ＩＪ　Ｇ含
有緩衡剤Ａで仲偲曲線をやＩるときと同じ割合で希釈し
たものを０．５ｍｌ加え３７°Ｃで１２０分間反応させ
た。以下標準面イＩ泉作製の場合と同様に操作し、４９
２ｎｍ［おける吸光度を測＞〆した。第２図Ａの標準曲
線を利用して各々の測定１６２体の示す吸光度よりＩ、
Ｅ量をｔ’Ｊ出した。＋（ｊ、果を第１表に示した。参
考のため、ｆｆｉ、ｉｔ　２図Ｈの４．１準曲線を利用
した場合も示す。7'6 N'J agent I+ -1) f is i-i+; 2
Figure VC shows t As shown in Figure VC. To the inverted V-shaped 4 (semi-curved glands are j! An example of measuring I and E in the serum of a normal person and an atopic allergy patient using PEG-containing buffer A! The antibody-conjugated beads produced in step d) were added to the test tube, and the antibody-bound antibody produced in step C) was added to the test tube. Add 0.5 ml of diluted material in the same proportion as when preparing the standard surface I and react at 37°C for 120 minutes.
The absorbance was measured at 2 nm. Based on the absorbance of each of the 162 samples measured using the standard curve in Figure 2A, I,
The amount of E was taken out as t'J. +(j, results are shown in Table 1. For reference, the case where ffi, it 4.1 quasi-curve of Fig. 2 H is used is also shown.

第１表 実施例２ 実施例１においてＰＥＧ−＃１０００　（牛丼化学社製
）について同様の実験全行なった。ＰＥＧ＃１０００は
１０重量対容量（Ｗ／Ｖ）％濃Ｕ！とじた。結果は第３
図に示した。ＰＥＧを含まない緩衡剤Ｂの場合は逆Ｖ字
型の４４：ｉ識曲線が得られたがＰＥ’Ｃ７ｉ含む本発
明性緩衝剤への場合は逆Ｖ字型イ翠準曲腺は得られず、
ｊヅ！作も簡単であった。Table 1 Example 2 All experiments similar to those in Example 1 were conducted using PEG-#1000 (manufactured by Gyudon Kagaku Co., Ltd.). PEG #1000 is 10% weight to volume (W/V) concentration U! Closed. The result is the third
Shown in the figure. In the case of buffer B which does not contain PEG, an inverted V-shaped 44:i curve was obtained, but in the case of the buffer of the present invention containing PE'C7i, an inverted V-shaped I green subcurved curve was obtained. Unable to do so.
jzu! It was also easy to make.

実施例３　インスリンの測定 ａ）インスリン伸率溶液の調整 ブタインスリン（シグマ社製）’ｅＷＩ−ＩＱの標準品
６６／／３０４ｆｔ用い、ラジオイムノアッセイ−で検
定し、■ｑＩ）牛アルブミンを含む生理食塩水で５０．
１００．２００．５００．１，０００．２，０００．５
，０００ｉｏ、ｏｏｏ、２０，０００”％ｇの濃度に調
整した、１））　　抗インスリン抗体の製造 ５りのブタインスリン（シグマ社製）、３７のＩｆ　ｐ
ｅｒｔｕ！１ｓｉｓ　ｖａｃｃｉｎｅ　（リリー社製）
にｐＨ２，，３の０．３％’Ｖ％フェノール７　ｍｌを
加えフロイントの完全アジ〜バンド１０ｍｅと混合し、
モルモツトに１週問おきに投与した。最終投与の１週間
後にに′１１動脈より探面してモルモット抗インスリン
血χ１１１　ｋ　イ４）だ。この抗血ｆ：（ｋ硫酸ナト
リウムで２回ＪＭ：ｉ析し、グロブリン分画全分取し、
抗インスリン抗体とした。Example 3 Measurement of insulin a) Adjustment of insulin elongation solution Porcine insulin (manufactured by Sigma) Tested by radioimmunoassay using standard product 66//304ft of eWI-IQ, ■qI) Physiological saline containing bovine albumin 50.00 with water.
100.200.500.1,000.2,000.5
,000io,ooo, adjusted to a concentration of 20,000''%g, 1)) Preparation of anti-insulin antibody 5 porcine insulin (manufactured by Sigma), 37 If p
Ertu! 1sis vaccine (manufactured by Lilly)
Add 7 ml of 0.3%'V% phenol at pH 2,3 and mix with 10 me of Freund's complete azide band.
It was administered to guinea pigs every other week. One week after the final administration, guinea pig anti-insulin blood was detected from the '11 artery (4). This anti-blood f:(k) was subjected to JM:i analysis twice with sodium sulfate, and the entire globulin fraction was collected.
It was used as an anti-insulin antibody.

Ｃ）抗インスリン抗体、酵素結合物の製造法抗インスリ
ン抗体を０．ＯＩＭ炭酸緩衡液（ｐｉｉ９．５　）　Ｋ
　１０　ｍｆ／ｌｄ　の濃度に調整した抗インスリン抗
体溶液０．５ｄｉ用い、実施例１−Ｃ）で示す方法でペ
ルオキシダーゼ抗インスリン抗体結合物を作製した。C) Method for producing anti-insulin antibodies and enzyme conjugates. OIM carbonic acid buffer (PII9.5) K
A peroxidase anti-insulin antibody conjugate was prepared by the method shown in Example 1-C) using 0.5 di of an anti-insulin antibody solution adjusted to a concentration of 10 mf/ld.

ｄ）　　抗インスリン抗体ポリスチレンボール結合物の
製造 前記ｂ）で製造した抗インスリン抗体金用い実施例１−
ｄ）で示す方法で抗インスリン抗体ポリスチレンボール
結合物を作製した。d) Production of anti-insulin antibody polystyrene ball conjugate Example 1 using anti-insulin antibody gold produced in b) above
An anti-insulin antibody polystyrene ball conjugate was prepared by the method shown in d).

ｅ）インスリンの測定 前記ａ）で調整した各濃度のインスリン標準陪液の１０
０１ノｔを試験管にとシ、抗体結合ボールを加えさらに
下記の緩衝剤で希釈したＷ識抗体を０５ゴ加え３７°Ｃ
ｌ２Ｏ分反応させた。e) Measurement of insulin 10% of the insulin standard solution of each concentration prepared in a) above.
Transfer 01 to a test tube, add an antibody binding ball, add 05 to 05 antibody diluted with the following buffer, and heat at 37°C.
The reaction was carried out for 120 minutes.

Ｅ：（本発明法）２チＰ　Ｅ　Ｇ＃　２０，０００　（
牛丼化学）含有０．０３　Ｍ　Ｐ　ＨＳ　ｐｉ−１７，
２Ｆ：（対照）　０．０３　Ｍ　Ｐ　Ｂ　Ｓ　ｐＨ７，
２以下実ノフイハ例１−ｅ）で示す方法で、（１′ｐ準
インスリ／ン・測定し１１り光度を得た。この時の標準
曲線を第４図に示した。E: (method of the present invention) 2chi P E G# 20,000 (
Beef bowl chemical) containing 0.03 M P HS pi-17,
2F: (control) 0.03 M PBS pH7,
By the method shown in Example 1-e), (1'p quasi-insulin) was measured to obtain a luminous intensity of 11. The standard curve at this time is shown in FIG.

緩衡剤１・〕では第４回に示す如く逆Ｖ字型の標準曲線
が得らｊｔだが、ｌ）　Ｅ　Ｇを含む本発明法の緩衝剤
Ｆでは逆Ｖ字型の（４：ｌ（準曲線は得られなかった。Buffer 1.] gives an inverted V-shaped standard curve as shown in Part 4, but buffer F of the present invention, which contains G No quasi-curve was obtained.

【図面の簡単な説明】[Brief explanation of the drawing]

第１図は−リンドイソチ法の測定原理を示す。図中、ａ
　ｉ、ｉ　）ノ”：、原、Ｉ）　Ｉｔよ不溶化抗体、Ｃ
は抗原抗体複合物、（ｊｌは結合型標識抗体、ｄ２は遊
離型酵素標識抗体を示す 第２図はＩ　、　Ｊｒ、の（・１ζ準曲線を示し、Ａは
本発明法で得らオ］、たＩ、］うの標準曲線（ＰＥＧ＃
２，０００）、ＢはＰＥＧなしの」１ツ合、Ｃは家兎血
清なしの場合、ＤはＰ　Ｅ　Ｇおよび家兎血清なしの場
合の標準曲線を示す。 第３図は［、Ｅの標準曲線を示し、Ａは不発肌性でイ（
ＩられたＴ、ｌ弓の標準曲線（ＰＥＧ＃１，０００　）
、Ｂは対照のＸｙＥ悸準曲線を示す。 第４図はインスリンの標準曲線を示し、Ｅは本発明法で
得られたインスリンの標準曲線（ＰＥＧ＃２０．０００
）、）゛は対照のインスリン（傳準曲線を示す。 特許出航人　東洋紡績株式会社 手　　続　　補　　正　　書 昭和５７年ＬＱ月２・を日 １４　　事件の表示 昭和５７年特許願第１７２１８３号 ２　発明の名利、 酵素免疫４１１１定法 ８、　補正をする者 事件との関係　　特許出願人 大阪市北区堂島浜二丁目２番８号 明細書の発明の詳細な説明の欄 「前期」を「前記」に訂正する。 （２）同第９百第３．５．９行目 第１Ｏ頁第４，１７行ｌ」 第１１頁第１，２〜３，３，６．１１行目第１３頁第１
，３行目 第１４頁第３，１６６行 目１５頁第４，５行目 「緩衝」を１級衝」に訂正する。 （３）同第１３頁第２行目 「標識」を「標ｒい」に訂正する。 （４）同第１３頁第８行目 「ラジオイムノアッセイ−」を「ラジオイムノアッセイ
」に訂正する。FIG. 1 shows the measurement principle of the Lindoisothi method. In the figure, a
i, i)ノ”:, Hara, I) Ityo insolubilized antibody, C
is the antigen-antibody complex, (jl is the bound labeled antibody, and d2 is the free enzyme-labeled antibody. Figure 2 shows the (・1ζ quasi-curve of I, Jr, and A is the o obtained by the method of the present invention). , taI,] standard curve (PEG#
2,000), B shows the standard curve without PEG, C shows the standard curve without rabbit serum, and D shows the standard curve without PEG and rabbit serum. Figure 3 shows the standard curve of [, E, A is non-inflammatory skin and A (
Standard curve of I-shaped T, l-bow (PEG #1,000)
, B shows the XyE normal curve of the control. FIG. 4 shows the standard curve of insulin, E is the standard curve of insulin obtained by the method of the present invention (PEG #20.000
),)'' indicates the control insulin (standard curve) Patent originator: Toyobo Co., Ltd. Procedures Amendment: LQ, 1982, July 2nd, 2014, 14th, 1981 Case description: 1982 Patent Application No. 172183 2 Invention Relation to the case of the person making the amendment, Enzyme Immunology 4111 Statutory Law 8, Patent applicant 2-2-8 Dojimahama, Kita-ku, Osaka City, Detailed description of the invention column is corrected to ``earlier term'' to ``aforesaid''. (2) No. 900, line 3, 5, 9, page 10, lines 4, 17 l, page 11, lines 1, 2 to 3, 3, 6.11, page 13, 1
, line 3, page 14, line 3,166, page 15, lines 4 and 5, ``buffer'' is corrected to ``1st grade shock''. (3) On page 13, line 2, "marker" is corrected to "marker". (4) On page 13, line 8, "Radioimmunoassay" is corrected to "Radioimmunoassay."

Claims (1)

【特許請求の範囲】[Claims] 測定物質に、この物質に特異的に結合する抗体を不溶化
した不溶化抗体および抗体に酵素を結合させたｔｌ）’
　２標ａ１に抗体を作用させ、測定物質に結合しだｒＦ
１素標識抗体または結合しなかった酵素標識抗体の酵素
活性を測定することによシ、測定物質の−Ｉ１１を測定
する「１デ素免疫測定法において、血清蛋白質およびポ
リアルキレングリコールの存在下に不溶化抗体全作用さ
せることを特徴とする酵素免疫測定法。An insolubilized antibody in which an antibody that specifically binds to the substance to be measured is insolubilized, and an enzyme bonded to the antibody tl)'
Antibodies are applied to 2-label a1, and rF binds to the substance to be measured.
In the ``1 element immunoassay method, which measures -I11 of the analyte by measuring the enzyme activity of the 1 element labeled antibody or the unbound enzyme-labeled antibody, in the presence of serum protein and polyalkylene glycol. An enzyme immunoassay method characterized by allowing the entire insoluble antibody to act.