JP2000329764A - Immunoassay and reagent - Google Patents
Immunoassay and reagentInfo
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- JP2000329764A JP2000329764A JP11143249A JP14324999A JP2000329764A JP 2000329764 A JP2000329764 A JP 2000329764A JP 11143249 A JP11143249 A JP 11143249A JP 14324999 A JP14324999 A JP 14324999A JP 2000329764 A JP2000329764 A JP 2000329764A
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- reagent
- antigen
- substance
- measured
- immunoassay
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Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は免疫学的測定法及び
測定試薬に関する。より詳細には、臨床検査などの分野
で利用される免疫学的測定法において、反応系の夾雑物
質による非特異的反応を抑制し得る免疫学的測定法及び
それに使用される試薬に関する。[0001] The present invention relates to an immunoassay and an assay reagent. More specifically, the present invention relates to an immunological assay used in the field of clinical testing and the like, which relates to an immunological assay capable of suppressing a non-specific reaction due to a contaminant in a reaction system and a reagent used therefor.
【０００２】[0002]
【従来の技術】臨床検査などの分野では試料中の測定対
象物質の測定法として、従来から，測定対象物質と当該
対象物質と抗原抗体反応可能な物質との抗原抗体反応を
使用した免疫学的測定法が汎用されている。係る測定法
としては、例えば、免疫拡散法（ＳＲＩＤ法）、免疫比
濁法、赤血球凝集法、ラテックス等の担体を用いた方
法、ＲＩＡ法、ＥＩＡ法が挙げられる。このような免疫
学的測定法では、目的とする抗原抗体反応のほかに試料
中の補体、免疫複合体、抗ヒトマウス抗体、脂質等の夾
雑物質との非特異的反応による凝集や吸着が生じ易く、
このことが原因で測定精度及び信頼性に欠けるという問
題があった。従来、係る非特異的反応を抑える方法とし
て、デキストラン硫酸、ヘパリン、ポリスチレンスルホ
ン酸、コンドロイチン硫酸等のポリアニオンを反応系に
添加する方法が知られている。2. Description of the Related Art In the field of clinical testing, etc., as a method for measuring a substance to be measured in a sample, an immunological reaction using an antigen-antibody reaction between the substance to be measured and a substance capable of reacting with the substance and an antigen-antibody has been conventionally used. Measurement methods are widely used. Examples of such a measuring method include an immunodiffusion method (SRID method), an immunoturbidimetric method, a hemagglutination method, a method using a carrier such as latex, an RIA method, and an EIA method. In such an immunoassay, in addition to the desired antigen-antibody reaction, aggregation or adsorption due to non-specific reactions with complements, immune complexes, anti-human mouse antibodies, lipids, and other contaminants in the sample occurs. Easy,
For this reason, there has been a problem that measurement accuracy and reliability are lacking. Conventionally, as a method for suppressing such non-specific reactions, there is known a method of adding a polyanion such as dextran sulfate, heparin, polystyrene sulfonic acid, or chondroitin sulfate to a reaction system.
【０００３】[0003]
【発明が解決しようとする課題】しかし、上記のポリア
ニオンを使用する方法では、非特異的反応を抑制するに
はポリアニオンの濃度を高める必要があるので反応液の
粘度が高くなり、そのため反応性が高くなりすぎ、バッ
クグランド値が上がるなどの欠点を有していた。本発明
は、このような従来技術の欠点を解消するためになされ
たもので、本発明者らが鋭意研究した結果、従来の免疫
学的測定法の問題点を改良して非特異的反応を除去でき
る方法を見出して完成したものである。即ち、本発明は
測定精度に優れ、臨床検査の分野などで有用に利用でき
る免疫学的測定法を提供することを目的とする。However, in the above-mentioned method using a polyanion, the viscosity of the reaction solution increases because the concentration of the polyanion must be increased in order to suppress the non-specific reaction. It had drawbacks such as being too high and increasing the background value. The present invention has been made in order to solve such disadvantages of the prior art, and as a result of diligent research conducted by the present inventors, it has been found that the problems of the conventional immunological assay method can be improved to improve the non-specific reaction. We have found a method that can be removed and completed it. That is, an object of the present invention is to provide an immunological measurement method which is excellent in measurement accuracy and can be usefully used in the field of clinical tests and the like.
【０００４】[0004]
【課題を解決するための手段】上記課題を解決すべくな
された本発明の免疫学的測定法は、反応系に多価フェノ
ール、特にカリクサレン類を添加することを特徴とする
ものである。また、本発明の免疫学的測定法用試薬は、
試料中の測定対象物質と当該測定対象物質と抗原抗体反
応可能な物質との抗原抗体反応を使用した免疫学的測定
法に使用される試薬であって、当該試薬が多価フェノー
ルを含有することを特徴とするものである。すなわち、
多価フェノール（好ましくはカリクサレン類）を反応系
に添加することにより、補体などの夾雑物質の非特異的
反応が抑えられ、測定精度が改善されるものである。The immunological assay of the present invention which has been made to solve the above-mentioned problem is characterized by adding a polyhydric phenol, particularly a calixarene, to a reaction system. Further, the reagent for immunological assay of the present invention,
A reagent used for an immunological assay using an antigen-antibody reaction between a substance to be measured in a sample and a substance capable of reacting with the substance to be measured and an antigen-antibody reaction, wherein the reagent contains a polyhydric phenol. It is characterized by the following. That is,
By adding polyhydric phenol (preferably calixarene) to the reaction system, non-specific reaction of contaminants such as complement is suppressed, and measurement accuracy is improved.
【０００５】[0005]
【発明の実施の形態】本発明において、免疫学的測定法
としては、測定対象物質と当該対象物質と抗原抗体反応
可能な物質との抗原抗体反応を使用した免疫学的測定法
であれば特に限定されないが、例えば、免疫拡散法（Ｓ
ＲＩＤ法）、免疫比濁法、赤血球凝集法、ラテックス等
の担体を用いた方法、ＲＩＡ法、ＥＩＡ法が挙げられ
る。BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, an immunoassay is particularly preferably an immunoassay using an antigen-antibody reaction between a substance to be measured and a substance capable of reacting with the substance and an antigen and antibody. Without limitation, for example, immunodiffusion (S
RID method), immunoturbidimetry, hemagglutination method, method using a carrier such as latex, RIA method, and EIA method.
【０００６】本発明の方法における測定対象物として
は、免疫学測定法で測定される物質であれば特に限定さ
れず種々の物質（例えば，抗原、ハプテン、抗体、ホル
モン、薬剤等）が挙げられる。より具体的には、例え
ば、ＣＲＰ、フィブリン及びフィブリノーゲン分解産
物、ＩｇＧ、ＩｇＡ、ＩｇＭ、ＩｇＥ、ＩｇＤ、抗スト
レプリジンＯ、リウマチ因子、トランスフェリン、ハプ
トグロビン、α１−アンチトリプシン、α１−アシドグ
リコプロテイン、α２−マクログロブリン、ヘモぺキシ
ン、アンチトロンビン−ＩＩＩ、α−フェトプロテイ
ン、ＣＥＡ（カルシノエンブリオニツク抗原）、フェリ
チン、ＨＢｓ−Ａｇ（Ｂ型肝炎外被抗原）、Ａｎｔｉ−
ＨＢｓ（抗Ｂ型肝炎外被）、ＨＢｅ−Ａｇ（Ｂ型肝炎ｅ
抗原）、Ａｎｔｉ−ＨＢｅ（抗Ｂ型肝炎ｅ）、Ａｎｔｉ
−ＨＢｃ（抗Ｂ型肝炎コア）などを挙げることができ
る。これらの物質を含む試料（検体）としては、例え
ば、血清、血漿、尿、髄液、リンパ液などを挙げること
ができる。The subject to be measured in the method of the present invention is not particularly limited as long as it is a substance to be measured by an immunological assay, and various substances (eg, antigens, haptens, antibodies, hormones, drugs, etc.) can be mentioned. . More specifically, for example, CRP, fibrin and fibrinogen degradation products, IgG, IgA, IgM, IgE, IgD, anti-strepridin O, rheumatoid factor, transferrin, haptoglobin, α1-antitrypsin, α1-acid glycoprotein, α2 -Macroglobulin, hemodoxin, antithrombin-III, α-fetoprotein, CEA (carcinoembryonic antigen), ferritin, HBs-Ag (hepatitis B envelope antigen), Anti-
HBs (anti-hepatitis B envelope), HBe-Ag (hepatitis B e
Antigen), Anti-HBe (anti-hepatitis B e), Anti
-HBc (anti-hepatitis B core) and the like. Samples (specimens) containing these substances include, for example, serum, plasma, urine, cerebrospinal fluid, and lymph.
【０００７】上記測定対象物質と抗原抗体反応しうる物
質（以下、免疫反応物質という）としては、測定対象物
が抗原、ハプテン等の場合はその抗体が、測定対象物が
抗体の場合にはその抗原が用いられる。免疫反応物質と
しての抗原及び抗体は慣用の方法にて調製することがで
きる。また、抗体は、ポリクローナル抗体、モノクロー
ナル抗体のいずれでもよく、更に、Ｆ（ａｂ）２画分、
Ｆ（ａｂ’）２画分などであってもよい。The substance (hereinafter referred to as an immunoreactive substance) capable of antigen-antibody reaction with the above-mentioned substance to be measured is an antibody when the substance to be measured is an antigen, a hapten or the like, and an antibody when the substance to be measured is an antibody. An antigen is used. Antigens and antibodies as immunoreactive substances can be prepared by conventional methods. The antibody may be either a polyclonal antibody or a monoclonal antibody. Further, the F (ab) 2 fraction,
The F (ab ') 2 fraction may be used.
【０００８】本発明で使用される多価フェノールは、例
えば、カテキン、ケルセチン、イソフラボン、タンニ
ン、カテコール、レゾルシノール、オイゲノール、カリ
クサレン類などが挙げられ、これらの多価フェノールは
２種以上を併用してもよい。上記の多価フェノールにお
いて、カリクサレン類が好適に使用され、カリクサレン
類はフェノールを基本骨格とし、フェノールの４〜８分
子をメチレン基で環状に重合させた環状オリゴマーであ
る。カリクサレン類としては特に制限されず各種のもの
を使用することができ、例えば、カリクス（４）アレン
［Ｃａｌｉｘ（４）ａｒｅｎｅ］、カリクス（６）アレ
ン、カリクス（８）アレン、硫酸カリクス（４）アレ
ン、硫酸カリクス（６）アレン、硫酸カリクス（８）ア
レン、酢酸カリクス（４）アレン、酢酸カリクス（６）
アレン、酢酸カリクス（８）アレン、カルボキシカリク
ス（４）アレン、カルボキシカリクス（６）アレン、カ
ルボキシカリクス（８）アレン、カリクス（４）アレン
アミン、カリクス（６）アレンアミン、カリクス（８）
アレンアミンなどが例示され、硫酸カリクス（６）アレ
ン、硫酸カリクス（８）アレンが好ましい。これらのカ
リクサレン類は２種以上を併用してもよい。反応系にお
ける多価フェノールの濃度は、０．０５〜５０ｍＭ程
度、好ましくは、０．１〜２０ｍＭ程度となるように調
整されて使用される。更に、反応系には、必要に応じ
て、ポリエチレングリコール、デキストラン、ゼラチ
ン、塩化ナトリウム、ＥＤＴＡ等の慣用の添加剤を加え
てもよい。The polyhydric phenol used in the present invention includes, for example, catechin, quercetin, isoflavone, tannin, catechol, resorcinol, eugenol, calixarene, and the like. These polyhydric phenols may be used in combination of two or more. Is also good. In the above polyhydric phenols, calixarenees are preferably used, and the calixarenees are cyclic oligomers having phenol as a basic skeleton and 4 to 8 molecules of phenol polymerized cyclically with methylene groups. The calixarenes are not particularly limited, and various types can be used. For example, calix (4) arene [Calix (4) arene], calix (6) arene, calix (8) arene, calix sulfate (4) Allene, calix sulfate (6) Allene, calix sulfate (8) Allene, calix acetate (4) Allene, calix acetate (6)
Allene, calix acetate (8) allene, carboxy calix (4) allene, carboxy calix (6) allene, carboxy calix (8) allene, calix (4) allenamine, calix (6) allenamine, calix (8) )
Allenamine and the like are exemplified, and calix sulfate (6) arene and calix sulfate (8) arene are preferable. Two or more of these calixarenees may be used in combination. The concentration of the polyhydric phenol in the reaction system is adjusted to about 0.05 to 50 mM, preferably about 0.1 to 20 mM before use. Further, conventional additives such as polyethylene glycol, dextran, gelatin, sodium chloride, and EDTA may be added to the reaction system as needed.
【０００９】本発明の方法の一例を免疫比濁法にて試料
中のＣＲＰを測定する例をもって説明すると、まず、適
当な緩衝液（例えば、ＨＥＰＥＳ緩衝液、ｐＨ７．５程
度）に多価フェノール（好ましくはカリクサレン類）を
終濃度として０．０５〜５０ｍＭとなるように添加し、
更に必要に応じてポリエチレングリコール、ＥＤＴＡ、
塩化ナトリウムなどの添加剤を適宜添加した第１試薬を
調製し、一方、適当な緩衝液に（例えば、ＨＥＰＥＳ緩
衝液、ｐＨ７．５程度）に抗ＣＲＰ抗体（例えば、抗ヒ
トＣＲＰヤギ血清等）を添加し、更に必要に応じてポリ
エチレングリコール、塩化ナトリウムなどの添加剤を適
宜添加した第２試薬を調製しておく。そして、ＣＲＰを
含有する試料（例えば、血清等）を第１試薬で適宜希釈
した後、２０〜４０℃程度（通常、３７℃程度）で２〜
１０分間程度（通常、５分間程度）加温する。次いで、
適当量の第２試薬を添加し、２０〜４０℃程度（通常、
３７℃程度）で２〜１０分間程度（通常、５分間程度）
加温した後、波長３４０ｎｍでの吸光度を測定し、予め
作成した検量線に基づきＣＲＰ濃度を求めることができ
る。吸光度の測定は、エンドポイント法、レートアッセ
イ法のいずれで行ってもよい。なお、本発明の方法は上
記の方法に限定されるものではなく、使用する免疫学的
測定法に応じて適宜変更して実施することとができ、使
用する緩衝液、測定条件なども適宜変更すればよい。An example of the method of the present invention will be described with reference to an example in which CRP in a sample is measured by an immunoturbidimetric method. First, a polyhydric phenol is added to an appropriate buffer (for example, HEPES buffer, about pH 7.5). (Preferably calixarene) so that the final concentration is 0.05 to 50 mM,
If necessary, polyethylene glycol, EDTA,
A first reagent to which an additive such as sodium chloride is appropriately added is prepared. On the other hand, an anti-CRP antibody (for example, anti-human CRP goat serum or the like) is added to an appropriate buffer (for example, HEPES buffer, about pH 7.5). And a second reagent to which additives such as polyethylene glycol and sodium chloride are appropriately added as needed is prepared. After appropriately diluting a sample containing CRP (for example, serum or the like) with the first reagent, the sample is cooled to about 20 to 40 ° C. (generally, about 37 ° C.).
Heat for about 10 minutes (usually about 5 minutes). Then
Add an appropriate amount of the second reagent, and add about 20 to 40 ° C (usually,
(About 37 ° C) for about 2 to 10 minutes (usually about 5 minutes)
After heating, the absorbance at a wavelength of 340 nm is measured, and the CRP concentration can be determined based on a previously prepared calibration curve. The measurement of the absorbance may be performed by any of the end point method and the rate assay method. Note that the method of the present invention is not limited to the above method, and can be carried out by appropriately changing the method according to the immunological assay method to be used. do it.
【００１０】本発明の試薬は、上記の方法に使用される
試薬であって、当該試薬が多価フェノールを含有するこ
とからなる。上記の試薬は、免疫学的測定法の種類に応
じて適宜設定することができ、また複数の構成試薬から
なるキットであってもよく、この場合には構成試薬の少
なくとも１種が多価フェノールを含有すればよい。[0010] The reagent of the present invention is a reagent used in the above method, which reagent contains a polyhydric phenol. The above reagents can be appropriately set according to the type of the immunological assay, and may be a kit comprising a plurality of constituent reagents. In this case, at least one of the constituent reagents is a polyhydric phenol. May be contained.
【００１１】[0011]
【発明の効果】本発明の方法によれば、試料中の補体子
等の夾雑物質による非特異的反応が多価フェノールによ
り抑制され、更に多価フェノールは反応液の粘度を上昇
させることがないので反応性が高くなりすぎることがな
い。従って、本発明の方法によれば、免疫学的測定法の
測定精度を著しく向上させることができるという効果を
奏する。また、本発明の試薬は多価フェノールを含有す
ることからなり、本発明の試薬によれば、上記の効果を
発現し得る試薬を得ることができる。According to the method of the present invention, nonspecific reactions due to contaminants such as complements in a sample are suppressed by polyhydric phenol, and polyhydric phenol can increase the viscosity of the reaction solution. The reactivity does not become too high. Therefore, according to the method of the present invention, there is an effect that the measurement accuracy of the immunological assay can be significantly improved. Further, the reagent of the present invention contains a polyhydric phenol. According to the reagent of the present invention, a reagent capable of exhibiting the above effects can be obtained.
【００１２】[0012]
【実施例】以下、実施例に基づいて本発明をより詳細に
説明するが、本発明はこれらの実施例に限定されるもの
ではない。 実施例１ ヒト血清中のＣＲＰを測定するために、試料としてヒト
血清１０例を使用した。そして、ヒト血清１５μＬに下
記の第１試薬２５０μＬを加え、３７℃で５分間加温
後、更に下記の第２試薬を５０μＬを加え、３７℃で５
分間加温した後、波長３４０ｎｍでの吸光度を測定し、
ＣＲＰ濃度既知の標準試料を用いて同様な操作により予
め作成した検量線からＣＲＰの値に換算した（測定Ａと
いう）。一方、第１試薬に硫酸カリクス（６）アレン５
ｍＭを加えたものを第３試薬として調製する。この第３
試薬を第１試薬の代りに使用する以外は、測定Ａと同様
に操作し、ＣＲＰの値に換算した（測定Ｂという）。更
に、測定Ａ及びＢで用いたヒト血清試料と同じ試料を予
め５６℃で３０分間加温して非動化し、補体成分を不活
性化したものについて測定Ａと同様に測定し、ＣＲＰ値
に換算した（測定Ｃという）。これらＡ〜Ｃの測定法に
より得られた結果を第１表に示す。EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples. Example 1 In order to measure CRP in human serum, 10 human sera were used as samples. Then, 250 μL of the following first reagent was added to 15 μL of human serum, and the mixture was heated at 37 ° C. for 5 minutes, and then 50 μL of the following second reagent was added.
After heating for minutes, the absorbance at a wavelength of 340 nm was measured,
A standard curve with a known CRP concentration was converted into a CRP value from a calibration curve prepared in advance by the same operation (referred to as measurement A). On the other hand, calix sulfate (6) allene 5 was used as the first reagent.
A solution to which mM is added is prepared as a third reagent. This third
Except that the reagent was used instead of the first reagent, the operation was performed in the same manner as in Measurement A, and converted into a CRP value (referred to as Measurement B). Furthermore, the same sample as the human serum sample used in Measurements A and B was heated in advance at 56 ° C. for 30 minutes to immobilize it, and the complement component was inactivated. (Referred to as measurement C). Table 1 shows the results obtained by the measurement methods A to C.
【００１３】第１試薬 ５０ｍＭ ＨＥＰＥＳ緩衝液 ｐＨ７．５ ４％ ポリエチレングリコール６０００ １％ ＮａＣｌ １ｍＭ ＥＤＴＡ 第２試薬 ５０ｍＭ ＨＥＰＥＳ緩衝液 ｐＨ７．５ １％ ＮａＣｌ １５％ 抗ヒトＣＲＰウサギ血清（長瀬産業社製）First reagent 50 mM HEPES buffer pH 7.5 4% Polyethylene glycol 6000 1% NaCl 1 mM EDTA Second reagent 50 mM HEPES buffer pH 7.5 1% NaCl 15% Anti-human CRP rabbit serum (manufactured by Nagase & Co., Ltd.)
【００１４】[0014]
【表１】 [Table 1]
【００１５】表１に示されるように、測定Ｂの測定値は
測定Ａよりも低く、また測定Ｂ及びＣの測定値はほぼ一
致していることから、反応系にカリクサレン類を添加す
る本発明の方法（測定Ｂ）は非特異的反応を抑制できる
ことが明かになった。As shown in Table 1, the measured value of the measurement B is lower than that of the measurement A, and the measured values of the measurements B and C are almost the same. Therefore, in the present invention, a calixarene is added to the reaction system. It was found that the method (measurement B) can suppress non-specific reactions.
Claims (3)
質と抗原抗体反応可能な物質との抗原抗体反応を使用し
た免疫学的測定法において、多価フェノールの存在下に
反応を行うことを特徴とする免疫学的測定法。In an immunoassay using an antigen-antibody reaction between a substance to be measured in a sample and a substance capable of reacting with the substance to be measured and an antigen-antibody reaction, the reaction is performed in the presence of a polyhydric phenol. Characteristic immunoassay.
請求項１記載の免疫学的測定法。2. The immunoassay according to claim 1, wherein the polyhydric phenol is a calixarene.
質と抗原抗体反応可能な物質との抗原抗体反応を使用し
た免疫学的測定法に使用される試薬であって、当該試薬
が多価フェノールを含有することを特徴とする免疫学的
測定法用試薬。3. A reagent for use in an immunological assay using an antigen-antibody reaction between a substance to be measured in a sample and a substance capable of reacting with the substance to be measured and an antigen-antibody reaction, wherein the reagent is a polyvalent reagent. A reagent for an immunoassay, comprising phenol.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11143249A JP2000329764A (en) | 1999-05-24 | 1999-05-24 | Immunoassay and reagent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11143249A JP2000329764A (en) | 1999-05-24 | 1999-05-24 | Immunoassay and reagent |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JP2000329764A true JP2000329764A (en) | 2000-11-30 |
Family
ID=15334363
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP11143249A Pending JP2000329764A (en) | 1999-05-24 | 1999-05-24 | Immunoassay and reagent |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2000329764A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008249603A (en) * | 2007-03-30 | 2008-10-16 | Sysmex Corp | Specimen pretreatment liquid for immunoassay, reagent kit for immunoassay, and immunoassay method |
| WO2010001619A1 (en) | 2008-07-04 | 2010-01-07 | 積水メディカル株式会社 | Method for enhancing sensitivity or method for avoiding influence of hemoglobin in immunological measurement |
-
1999
- 1999-05-24 JP JP11143249A patent/JP2000329764A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008249603A (en) * | 2007-03-30 | 2008-10-16 | Sysmex Corp | Specimen pretreatment liquid for immunoassay, reagent kit for immunoassay, and immunoassay method |
| WO2010001619A1 (en) | 2008-07-04 | 2010-01-07 | 積水メディカル株式会社 | Method for enhancing sensitivity or method for avoiding influence of hemoglobin in immunological measurement |
| EP2295969A1 (en) * | 2008-07-04 | 2011-03-16 | Sekisui Medical Co., Ltd. | Method for enhancing sensitivity or method for avoiding influence of hemoglobin in immunological measurement |
| EP2295969A4 (en) * | 2008-07-04 | 2012-04-04 | Sekisui Medical Co Ltd | Method for enhancing sensitivity or method for avoiding influence of hemoglobin in immunological measurement |
| US8722342B2 (en) | 2008-07-04 | 2014-05-13 | Sekisui Medical Co., Ltd. | Method for enhancing sensitivity or method for avoiding influence of hemoglobin in immunological measurement |
| EP3062104A1 (en) | 2008-07-04 | 2016-08-31 | Sekisui Medical Co., Ltd. | Method for enhancing sensitivity or method for avoiding influence of hemoglobin in immunological measurement |
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