JP2002340896A - Immunoassay reagent - Google Patents
Immunoassay reagentInfo
- Publication number
- JP2002340896A JP2002340896A JP2001152701A JP2001152701A JP2002340896A JP 2002340896 A JP2002340896 A JP 2002340896A JP 2001152701 A JP2001152701 A JP 2001152701A JP 2001152701 A JP2001152701 A JP 2001152701A JP 2002340896 A JP2002340896 A JP 2002340896A
- Authority
- JP
- Japan
- Prior art keywords
- antibody
- antigen
- immunoassay reagent
- measured
- reaction
- 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.)
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Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、不溶性担体を使用
した免疫学的な凝集反応に基づいて被測定物質を測定す
る免疫測定試薬に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunoassay reagent for measuring an analyte based on an immunological agglutination reaction using an insoluble carrier.
【０００２】[0002]
【従来の技術】各種生体成分から特定の被測定物質を検
出もしくは定量するため、被測定物質である抗原（又は
抗体）に対応する抗体（又は抗原）との免疫学的な凝集
反応を光学的に観察するか、又は、目視（肉眼）で観察
する方法が行われている。2. Description of the Related Art In order to detect or quantify a specific substance to be measured from various biological components, an immunological agglutination reaction with an antibody (or antigen) corresponding to an antigen (or antibody) as the substance to be measured is optically measured. Or a method of observing with the naked eye.
【０００３】又、検出感度をより向上させるために、被
測定物質である抗原（又は抗体）に対応する抗体（又は
抗原）を不溶性担体に感作させ、被測定物質との抗原抗
体反応により生じた不溶性担体の凝集の度合いを検出す
ることにより、被測定物質を測定する方法が近年広く行
われている。このような測定法としては、例えば、ラテ
ックス凝集法や赤血球凝集法等が挙げられる。In order to further improve the detection sensitivity, an antibody (or antigen) corresponding to the antigen (or antibody) to be measured is sensitized to an insoluble carrier, and the antibody is generated by an antigen-antibody reaction with the substance to be measured. In recent years, a method of measuring a substance to be measured by detecting the degree of aggregation of an insoluble carrier has been widely used. Examples of such a measuring method include a latex agglutination method and a red blood cell agglutination method.
【０００４】このような測定法に用いられる免疫測定試
薬では、抗原抗体反応を促進し検出感度を向上させるた
めに、各種の水溶性添加物を含有させることが行われて
おり、例えば、特開昭５８−４７２５６号公報には、抗
原又は抗体を担持した不溶性微粒子と、抗体及び／又は
抗原とを水溶液中で反応させ、反応混合物に光を照射し
て反応の進行度を光学的に測定する抗原抗体反応の測定
法において、水溶液中にポリエチレングリコールを共存
させる方法が開示されている。[0004] The immunoassay reagent used in such an assay contains various water-soluble additives in order to promote the antigen-antibody reaction and improve the detection sensitivity. JP-A-58-47256 discloses that an insoluble fine particle carrying an antigen or an antibody is reacted with an antibody and / or an antigen in an aqueous solution, and the reaction mixture is irradiated with light to optically measure the progress of the reaction. A method for coexisting polyethylene glycol in an aqueous solution in a method for measuring an antigen-antibody reaction is disclosed.
【０００５】又、例えば、特開平５−１８０８３８号公
報や特開平６−１６７４９３号公報等には、水溶性添加
物のなかでも特にプルランに代表されるマルトトリオー
ス類のような水溶性糖重合体を用いることにより、抗原
抗体反応以外の非特異的凝集反応の抑制と特異的凝集反
応の増大とによる高感度化を図る測定法が開示されてい
る。Further, for example, JP-A-5-180838 and JP-A-6-167493 disclose a water-soluble sugar such as maltotriose represented by pullulan among water-soluble additives. There is disclosed a measurement method for increasing the sensitivity by suppressing the nonspecific agglutination other than the antigen-antibody reaction and increasing the specific agglutination by using the union.
【０００６】しかし近年、臨床現場からは、早期診断に
よる早期治療を実現する目的から、従来にも増してより
微量の被測定物質を検出するために、試薬の反応性を増
大することが強く要望されている。However, in recent years, there has been a strong demand from clinical sites to increase the reactivity of reagents in order to detect a smaller amount of a substance to be measured than ever before, in order to realize early treatment by early diagnosis. Have been.
【０００７】[0007]
【発明が解決しようとする課題】本発明の目的は、上記
要望に鑑み、高感度で非特異反応が少なく、微量の被測
定物質を的確に検出することのできる免疫測定試薬を提
供することにある。SUMMARY OF THE INVENTION An object of the present invention is to provide an immunoassay reagent which is highly sensitive, has little nonspecific reaction, and can accurately detect a trace amount of a substance to be measured. is there.
【０００８】[0008]
【課題を解決するための手段】本発明者は、従来法の問
題を解決するため鋭意検討を重ねた結果、被測定物質で
ある抗原（又は抗体）と対応する抗体（又は抗原）を感
作した不溶性担体の免疫学的な凝集反応の度合いを検出
することにより被測定物質を測定する免疫測定試薬にお
いて、上記免疫測定試薬にエルシナンを含有させること
により、従来のマルトトリオース類よりも飛躍的に高い
特異的反応の増大効果が得られることを見出し、本発明
を完成するに至った。Means for Solving the Problems The inventors of the present invention have conducted intensive studies in order to solve the problems of the conventional method, and as a result, have sensitized the antigen (or antibody) which is the substance to be measured with the corresponding antibody (or antigen). In the immunoassay reagent for measuring the substance to be measured by detecting the degree of immunological agglutination of the insoluble carrier, the erucinate is added to the immunoassay reagent, so that it is far more dramatic than conventional maltotrioses. It was found that a high specific reaction increasing effect was obtained, and the present invention was completed.
【０００９】即ち、請求項１に記載の発明による免疫測
定試薬は、被測定物質である抗原（又は抗体）と対応す
る抗体（又は抗原）を感作した不溶性担体及びエルシナ
ンが含有されてなることを特徴とする。That is, the immunoassay reagent according to the first aspect of the present invention comprises an insoluble carrier sensitized with an antigen (or antibody) as a substance to be measured and an antibody (or antigen) corresponding thereto, and ercinan. It is characterized by.
【００１０】本発明で用いられるエルシナンは、例えば
特公昭５５−２７５６１号公報に開示されているよう
な、エルシノエ（Ｅｌｓｉｎｏｅ）属に属する微生物を
栄養培養して得られる、主な繰り返し単位が〔３）−Ｇ
ｌｃ−（１→４）−Ｇｌｃ（１→４）−Ｇｌｃ−（１
→〕（但し、Ｇｌｃはα−Ｄ−グルコピラノース残基を
示す）の構造を有しているグルカンである。又、例えば
エルシナンのエステル化物やエルシナンのエーテル化物
などのエルシナンの各種誘導体も本発明で用いられるエ
ルシナンに包含される。これらのエルシナンは、単独で
用いられても良いし、２種類以上が併用されても良い。The main repeating unit of elsinan used in the present invention is [3] obtained by vegetatively culturing a microorganism belonging to the genus Elsinoe as disclosed in, for example, Japanese Patent Publication No. 55-27561. ) -G
lc- (1 → 4) -Glc (1 → 4) -Glc- (1
→] (where Glc represents an α-D-glucopyranose residue). In addition, various derivatives of ercinan such as esterified ercinan and etherified ercinan are also included in ercinan used in the present invention. These ercinans may be used alone or in combination of two or more.
【００１１】上記エルシナンは、特に限定されるもので
はないが、その分子量が約１万から１０００万以上のも
のが好適に用いられるが、なかでもゲル濾過等により分
子量を５万から５０万としたものがより好適に用いられ
る。The above-mentioned elsinan is not particularly limited, but those having a molecular weight of about 10,000 to 10,000,000 or more are preferably used. Among them, the molecular weight is adjusted to 50,000 to 500,000 by gel filtration or the like. Are more preferably used.
【００１２】本発明の免疫測定試薬は、被測定物質であ
る抗原（又は抗体）と、これに対応する抗体（又は抗
原）を感作した不溶性担体とを凝集反応させる際に、こ
の凝集反応系（以下、単に「反応系」と略記する）にエ
ルシナンが含有されているように構成される。The reagent for immunoassay of the present invention is used for agglutination reaction between an antigen (or antibody) to be measured and an insoluble carrier sensitized with a corresponding antibody (or antigen). (Hereinafter simply referred to as “reaction system”) contains ercinan.
【００１３】反応系にエルシナンを含有させる方法とし
ては、被測定物質である抗原（又は抗体）と、これに対
応する抗体（又は抗原）を感作した不溶性担体との反応
系にエルシナンを含有させ得る方法であれば如何なる方
法であっても良く、特に限定されるものではないが、例
えば、不溶性担体の懸濁液中にエルシナンを含有させる
方法、凝集反応時に使用する緩衝液中にエルシナンを含
有させる方法、不溶性担体の懸濁液中及び緩衝液中の双
方にエルシナンを含有させる方法、或いは、不溶性担体
の懸濁液及び緩衝液以外の構成試薬を更に１種類もしく
は２種類以上設け、この構成試薬の少なくとも１種類中
にエルシナンを含有させる方法等が挙げられ、いずれの
方法が用いられても良い。又、これらの方法は、単独で
用いられても良いし、２種類以上が併用されても良い。
更に、エルシナンは、予め水溶液状として用いられても
良いし、固体状のままで用いられても良い。As a method of including ercinnan in a reaction system, ercinan is added to a reaction system between an antigen (or antibody) to be measured and an insoluble carrier sensitized with the corresponding antibody (or antigen). Any method may be used as long as it is a method for obtaining, and is not particularly limited.For example, a method in which elsinan is contained in a suspension of an insoluble carrier, a method in which elsinan is contained in a buffer used during an agglutination reaction Or a method in which ercinan is contained in both the suspension of the insoluble carrier and the buffer, or one or more components other than the suspension of the insoluble carrier and the buffer are further provided. A method in which ercinane is contained in at least one of the reagents is mentioned, and any method may be used. These methods may be used alone or in combination of two or more.
Furthermore, elsinan may be used in advance as an aqueous solution or may be used as it is in a solid state.
【００１４】本発明の免疫測定試薬において、エルシナ
ンの含有量は、最終的な試薬性能を考慮して適宜設定さ
れれば良く、特に限定されるものではないが、被測定物
質である抗原（又は抗体）と、これに対応する抗体（又
は抗原）を感作した不溶性担体との反応系の０．０１〜
１０重量％であることが好ましく、より好ましくは０．
１〜５重量％である。In the immunoassay reagent of the present invention, the content of elsinan may be appropriately set in consideration of the final reagent performance, and is not particularly limited. Antibody) and an insoluble carrier sensitized with the corresponding antibody (or antigen).
It is preferably 10% by weight, more preferably 0.1% by weight.
1 to 5% by weight.
【００１５】本発明で用いられる不溶性担体としては、
特に限定されるものではないが、例えば、ポリスチレ
ン、スチレン−スチレンスルホン酸塩共重合体、メタク
リル酸重合体、アクリル酸重合体、アクリロニトリル−
ブタジエン−スチレン共重合体、塩化ビニル−アクリル
酸エステル共重合体、酢酸ビニル−アクリル酸エステル
共重合体などのラテックス粒子；不溶性アガロース、セ
ルロース、不溶性デキストランなどの有機高分子粒子；
シリカ、アルミナなどの無機粒子；金、チタン、鉄、ニ
ッケルなどの金属粉末；赤血球などの生体由来粒子等が
挙げられ、好適に用いられるが、なかでもラテックス粒
子が特に好適に用いられる。これらの不溶性担体は、単
独で用いられても良いし、２種類以上が併用されても良
い。The insoluble carrier used in the present invention includes:
Although not particularly limited, for example, polystyrene, styrene-styrene sulfonate copolymer, methacrylic acid polymer, acrylic acid polymer, acrylonitrile-
Latex particles such as butadiene-styrene copolymer, vinyl chloride-acrylate copolymer, vinyl acetate-acrylate copolymer; organic polymer particles such as insoluble agarose, cellulose, and insoluble dextran;
Inorganic particles such as silica and alumina; metal powders such as gold, titanium, iron and nickel; living body-derived particles such as erythrocytes; and the like are preferably used. Among them, latex particles are particularly preferably used. These insoluble carriers may be used alone or in combination of two or more.
【００１６】上記不溶性担体の平均粒子径は、検出濃度
や測定機器によっても異なり、特に限定されるものでは
ないが、一般的には０．０５〜５．０μｍの範囲で適宜
選択されることが好ましい。The average particle size of the insoluble carrier varies depending on the concentration to be detected and the measuring instrument, and is not particularly limited, but is generally appropriately selected in the range of 0.05 to 5.0 μm. preferable.
【００１７】本発明の免疫測定試薬において、不溶性担
体に感作する抗体（又は抗原）としては、生体由来のも
の、遺伝子組み換え技術等を用いて得られたもの、或い
は、それらの処理物等が用いられ、その具体例として
は、特に限定されるものではないが、例えば、変性γ−
グロブリン、リウマチ因子、ストレプトリジンＯ（ＳＬ
Ｏ）、抗ＳＬＯ抗体、カルジオライピン、抗カルジオラ
イピン抗体、インスリン、抗インスリン抗体、Ｃ反応性
蛋白（ＣＲＰ）、抗ＣＲＰ抗体、α−フェトプロテイン
（ＡＦＰ）、抗ＡＦＰ抗体、癌胎児性抗原（ＣＥＡ）、
抗ＣＥＡ抗体、ＨＢｓ抗原、抗ＨＢｓ抗体、抗トレポネ
ーマ抗体に対する抗原、ＨＣＶ抗原等の公知の抗体（又
は抗原）が挙げられ、好適に用いられる。これらの不溶
性担体に感作する抗体（又は抗原）は、単独で用いられ
ても良いし、２種類以上が併用されても良い。In the immunoassay reagent of the present invention, the antibody (or antigen) sensitizing the insoluble carrier may be one derived from a living organism, one obtained by using a genetic recombination technique, or a processed product thereof. It is used, as a specific example thereof is not particularly limited, for example, modified γ-
Globulin, rheumatoid factor, streptolysin O (SL
O), anti-SLO antibody, cardiolipin, anti-cardiolipin antibody, insulin, anti-insulin antibody, C-reactive protein (CRP), anti-CRP antibody, α-fetoprotein (AFP), anti-AFP antibody, carcinoembryonic antigen (CEA),
Known antibodies (or antigens) such as an anti-CEA antibody, an HBs antigen, an anti-HBs antibody, an antigen against an anti-treponemal antibody, and an HCV antigen can be used, and are preferably used. These antibodies (or antigens) sensitizing these insoluble carriers may be used alone or in combination of two or more.
【００１８】本発明の免疫測定試薬においては、非特異
反応を抑制するためや免疫測定試薬自体の安定性を高め
るために、特に限定されるものではないが、例えば、ア
ルブミン、カゼイン、ゼラチンなどの蛋白質、これら蛋
白質の分解物、アミノ酸、界面活性剤等の１種類もしく
は２種類以上を前記不溶性担体に更に担持させても良
い。The immunoassay reagent of the present invention is not particularly limited in order to suppress non-specific reactions and to enhance the stability of the immunoassay reagent itself. Examples of the immunoassay reagent include albumin, casein, gelatin and the like. One or more of proteins, degradation products of these proteins, amino acids, surfactants and the like may be further carried on the insoluble carrier.
【００１９】抗体（又は抗原）を感作した不溶性担体の
本発明の免疫測定試薬による凝集反応の度合いは、光学
的に観察するか、又は、目視（肉眼）で観察することに
より、検出される。The degree of agglutination of the insoluble carrier sensitized with the antibody (or antigen) by the immunoassay reagent of the present invention can be detected by optical observation or visual observation (visual observation). .
【００２０】抗体（又は抗原）を感作した不溶性担体の
凝集の度合いを光学的に観察して、検出する方法として
は、特に限定されるものではないが、例えば、散乱光強
度、吸光度又は透過光強度の増加もしくは減少を測定す
る方法や、これらの方法を併用する方法等が挙げられ
る。The method of optically observing and detecting the degree of aggregation of the insoluble carrier sensitized with the antibody (or antigen) is not particularly limited, and examples thereof include, for example, scattered light intensity, absorbance and transmission. Examples include a method of measuring an increase or decrease in light intensity, and a method of using these methods in combination.
【００２１】又、抗体（又は抗原）を感作した不溶性担
体の凝集の度合いを目視（肉眼）で観察して、検出する
方法としては、特に限定されるものではないが、例え
ば、被測定物質と上記不溶性担体とを含む水溶液を例え
ばガラス板のような判定板上で混合し、１〜５分間揺り
動かした後、凝集の有無や程度を判定する方法等が挙げ
られる。上記凝集の有無や程度の判定は、単なる目視
（肉眼）による判定であっても良いし、像を撮影し、画
像処理を施すことによる判定であっても良い。The method of visually observing (visually) the degree of aggregation of the insoluble carrier sensitized with the antibody (or antigen) is not particularly limited. And an aqueous solution containing the insoluble carrier and the above-mentioned insoluble carrier are mixed on a determination plate such as a glass plate, shaken for 1 to 5 minutes, and then the presence or absence and degree of aggregation are determined. The determination of the presence or absence and the degree of the aggregation may be made by simple visual inspection (the naked eye) or may be made by photographing an image and performing image processing.
【００２２】本発明の免疫測定試薬を用いる抗原抗体反
応の条件は、通常の条件と同様で良く、反応媒体として
は、被測定物質である抗原（又は抗体）の種類に応じた
各種緩衝液が用いられる。この緩衝液は、被測定物質を
失活させることがなく、且つ、抗原抗体反応を阻害しな
いようなイオン濃度やｐＨを有するものであれば良く、
その具体例としては、特に限定されるものではないが、
例えば、リン酸緩衝液、グリシン緩衝液、トリス緩衝
液、グッド緩衝液等の公知の各種緩衝液が挙げられ、好
適に用いられる。これらの緩衝液は、単独で用いられて
も良いし、２種類以上が併用されても良い。The conditions for the antigen-antibody reaction using the immunoassay reagent of the present invention may be the same as those under ordinary conditions. As the reaction medium, various buffers depending on the type of the antigen (or antibody) to be measured are used. Used. This buffer does not deactivate the substance to be measured, and may have an ion concentration or pH that does not inhibit the antigen-antibody reaction.
Specific examples thereof are not particularly limited,
For example, various known buffers, such as a phosphate buffer, a glycine buffer, a Tris buffer, and a Good buffer, may be used, and are preferably used. These buffers may be used alone or in combination of two or more.
【００２３】上記抗原抗体反応時には、本発明の課題達
成を阻害しない範囲で必要に応じて、例えば感度をより
高めるために、特に限定されるものではないが、例え
ば、ポリエチレングリコ−ル、カルボキシルメチルセル
ロ−ス、デキストラン、ポリビニルピロリドン、ポリグ
リコシルエチルメタクリレート、プルラン等の各種水溶
性高分子が添加されても良い。これらの水溶性高分子
は、単独で用いられても良いし、２種類以上が併用され
ても良い。At the time of the antigen-antibody reaction, if necessary, for example, in order to further increase the sensitivity, there is no particular limitation as long as the achievement of the object of the present invention is not hindered. For example, polyethylene glycol, carboxymethyl Various water-soluble polymers such as cellulose, dextran, polyvinylpyrrolidone, polyglycosylethyl methacrylate, and pullulan may be added. These water-soluble polymers may be used alone or in combination of two or more.
【００２４】又、上記抗原抗体反応時には、本発明の課
題達成を阻害しない範囲で必要に応じて、例えば特異反
応性をより高めたり、免疫測定試薬自体の安定性をより
向上させるために、特に限定されるものではないが、例
えば、アルブミン、カゼイン、ゼラチンなどの蛋白質や
その分解物、塩化コリンなどの第４級アンモニウム塩、
ＥＤＴＡ、ポリアニオン、カオトロピックイオン（例え
ば、Ｃｌ- 、Ｉ- 、ＳＣＮ- 等）、アミノ酸、界面活性
剤等の各種水溶性添加剤が添加されても良い。これらの
水溶性添加剤は、単独で用いられても良いし、２種類以
上が併用されても良い。In the above-mentioned antigen-antibody reaction, if necessary, for example, in order to further increase the specific reactivity or to further improve the stability of the immunoassay reagent itself as long as the object of the present invention is not hindered, Although not limited, for example, proteins such as albumin, casein, and gelatin and their degradation products, quaternary ammonium salts such as choline chloride,
Various water-soluble additives such as EDTA, polyanions, chaotropic ions (eg, Cl − , I − , SCN −, etc.), amino acids, and surfactants may be added. These water-soluble additives may be used alone or in combination of two or more.
【００２５】上記抗原抗体反応時の反応系のｐＨは、特
に限定されるものではないが、５〜１０であることが好
ましく、より好ましくは６〜８である。又、反応温度
は、特に限定されるものではないが、０〜５０℃である
ことが好ましく、より好ましくは２０〜４０℃である。
尚、反応時間は、適宜設定されれば良い。The pH of the reaction system at the time of the antigen-antibody reaction is not particularly limited, but is preferably from 5 to 10, and more preferably from 6 to 8. The reaction temperature is not particularly limited, but is preferably from 0 to 50 ° C, more preferably from 20 to 40 ° C.
The reaction time may be set as appropriate.
【００２６】[0026]
【作用】本発明の免疫測定試薬は、被測定物質である抗
原（又は抗体）と対応する抗体（又は抗原）を感作した
不溶性担体及びエルシナンが含有されてなるので、上記
エルシナンの働きにより、抗原抗体反応による特異的な
凝集反応が促進され、微量の被測定物質でも高感度で検
出することができる。The immunoassay reagent of the present invention comprises an insoluble carrier sensitized with an antigen (or an antibody) as a substance to be measured and an antibody (or an antigen) corresponding thereto, and ercinan. A specific agglutination reaction due to an antigen-antibody reaction is promoted, and a very small amount of the analyte can be detected with high sensitivity.
【００２７】[0027]
【発明の実施の形態】本発明をさらに詳しく説明するた
め以下に実施例を挙げるが、本発明はこれら実施例のみ
に限定されるものではない。尚、以下の実施例及び比較
例においては、抗トレポネーマ抗体測定用の免疫測定試
薬を作製し、評価した。BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples. In the following Examples and Comparative Examples, immunoassay reagents for measuring anti-treponema antibody were prepared and evaluated.
【００２８】（実施例１） （１）検体希釈用緩衝液の調製 牛血清アルブミンを１重量％含有するリン酸緩衝液に、
特公昭５５−２７５６１号公報に記載されている方法で
製造したエルシナン１重量％を添加し、溶解して、検体
希釈用緩衝液を調製した。Example 1 (1) Preparation of Sample Dilution Buffer A phosphate buffer containing 1% by weight of bovine serum albumin was added to
1% by weight of ercinan produced by the method described in JP-B-55-27561 was added and dissolved to prepare a sample dilution buffer.
【００２９】（２）トレポネーマ・パリダム由来抗原感
作ラテックス液の調製 トレポネーマ・パリダム由来抗原を３０μｇ／ｍｌの濃
度で０．０１Ｍリン酸緩衝液（ｐＨ：７．４）に溶解し
た溶液０．４ｍｌに、平均粒子径が０．４０μｍのポリ
スチレンラテックス（固形分：１０重量％、積水化学工
業社製）０．１ｍｌを添加し、４℃で６０分間撹拌混合
した。次に、この溶液に、牛血清アルブミンを１重量％
含有する０．１Ｍリン酸緩衝液（ｐＨ：７．４）を０．
５ｍｌ添加し、４℃で９０分間撹拌混合した後、回転数
１５０００ｒｐｍで１５分間遠心分離した。次いで、得
られた沈澱物に、牛血清アルブミンを１重量％含有する
０．１Ｍリン酸緩衝液（ｐＨ：７．４）４ｍｌを添加
し、ラテックスを再懸濁させて、トレポネーマ・パリダ
ム由来抗原感作ラテックス液を調製した。(2) Preparation of Treponema pallidum-derived antigen-sensitized latex solution 0.4 ml of a solution of Treponema pallidum-derived antigen in a concentration of 30 μg / ml in 0.01 M phosphate buffer (pH: 7.4) To this was added 0.1 ml of a polystyrene latex having an average particle diameter of 0.40 μm (solid content: 10% by weight, manufactured by Sekisui Chemical Co., Ltd.), followed by stirring and mixing at 4 ° C. for 60 minutes. Next, 1% by weight of bovine serum albumin was added to this solution.
0.1M phosphate buffer (pH: 7.4) containing 0.1M phosphate buffer.
After adding 5 ml and stirring and mixing at 4 ° C. for 90 minutes, the mixture was centrifuged at 15,000 rpm for 15 minutes. Next, 4 ml of a 0.1 M phosphate buffer (pH: 7.4) containing 1% by weight of bovine serum albumin was added to the obtained precipitate, and the latex was resuspended to obtain an antigen derived from Treponema pallidum. A sensitized latex solution was prepared.
【００３０】（３）抗トレポネーマ抗体測定用の免疫測
定試薬の作製 上記（１）の検体希釈用緩衝液からなる第１試薬と上記
（２）のトレポネーマ・パリダム由来抗原感作ラテック
ス液からなる第２試薬とから構成される２液型の抗トレ
ポネーマ抗体測定用の免疫測定試薬を作製した。(3) Preparation of Immunoassay Reagent for Anti-Treponemal Antibody Measurement The first reagent consisting of the buffer for diluting the specimen of (1) above and the antigen sensitizing latex derived from Treponema pallidum of (2) above. An immunoassay reagent for measuring a two-pack anti-treponema antibody composed of two reagents was prepared.
【００３１】（４）抗トレポネーマ抗体標準液の調製 梅毒陽性標準血清（５濃度、積水化学工業社製）を各１
ｍｌの精製水で溶解して、抗トレポネーマ抗体標準液を
調製した。抗体価は、０、３６、１２０、２４２、４５
６Ｔ．Ｕ．であった。尚、上記Ｔ．Ｕ．は、抗トレポネ
ーマ抗体測定試薬（商品名「メディエースＴＰＬＡ」、
積水化学工業社製）で血清を測定した場合の抗トレポネ
ーマ抗体の抗体価を表す単位である。上記トレポネーマ
抗体測定試薬「メディエースＴＰＬＡ」では、上記抗体
価が１０Ｔ．Ｕ．以上の場合、陽性と判定される。(4) Preparation of Anti-Treponemal Antibody Standard Solution Syphilis-positive standard serum (5 concentrations, manufactured by Sekisui Chemical Co., Ltd.) was used for each one.
The resultant was dissolved in ml of purified water to prepare an anti-treponema antibody standard solution. The antibody titers are 0, 36, 120, 242, 45
6T. U. Met. Note that the above T.P. U. Is an anti-treponemal antibody measurement reagent (trade name “MEDIACE TPLA”,
It is a unit that indicates the antibody titer of the anti-treponema antibody when the serum is measured using Sekisui Chemical Co., Ltd.). In the above-mentioned treponemal antibody measurement reagent “MEDIACE TPLA”, the above antibody titer is 10T. U. In the above cases, it is determined to be positive.
【００３２】（５）測定用検体の準備 健常人の血清１０例及び梅毒患者の血清５例をそれぞれ
測定用検体として準備した。(5) Preparation of Samples for Measurement Ten sera of healthy individuals and five sera of syphilis patients were prepared as measurement samples.
【００３３】（６）抗トレポネーマ抗体標準液の測定及
び抗トレポネーマ抗体検量線の作成 抗トレポネーマ抗体標準液２０μｌを秤取し、これに上
記（１）の検体希釈用緩衝液（第１試薬）２１０μｌを
混合し、３７℃で一定時間保持した後、上記（２）のト
レポネーマ・パリダム由来抗原感作ラテックス液（第２
試薬）３０μｌを添加し、攪拌混合した。次いで、約８
０秒間から３００秒間の波長７００ｎｍでの吸光度の変
化量を測定し、吸光度変化量（ΔＡｂｓ）とした。尚、
上記吸光度の変化量の測定は、日立自動分析装置（型
式：「７１５０型」、日立製作所社製）を用いて行っ
た。得られた吸光度変化量（△Ａｂｓ）と抗トレポネー
マ抗体標準液の抗体価（Ｔ．Ｕ．）から、抗トレポネー
マ抗体検量線を作成した。(6) Measurement of Anti-Treponemal Antibody Standard Solution and Preparation of Anti-Treponemal Antibody Standard Curve 20 μl of the anti-treponema antibody standard solution was weighed, and 210 μl of the sample dilution buffer (first reagent) described in (1) above was weighed. And kept at 37 ° C. for a certain period of time. Then, the antigen-sensitized latex solution derived from Treponema pallidum (2)
(Reagent) was added and mixed by stirring. Then, about 8
The amount of change in absorbance at a wavelength of 700 nm from 0 seconds to 300 seconds was measured and defined as the amount of change in absorbance (ΔAbs). still,
The change in the absorbance was measured using a Hitachi automatic analyzer (model: “7150”, manufactured by Hitachi, Ltd.). An anti-treponema antibody calibration curve was prepared from the obtained absorbance change (ΔAbs) and the antibody titer (TU) of the anti-treponema antibody standard solution.
【００３４】（７）測定用検体の抗トレポネーマ抗体価
の測定 上記（５）で準備した測定用検体を用い、それぞれ上記
（６）の場合と同様の操作を行って、吸光度変化量を求
めた。次いで、上記（６）で作成した抗トレポネーマ抗
体検量線を用いて、それぞれの測定用検体の吸光度変化
量からそれぞれの測定用検体の抗トレポネーマ抗体価
（Ｔ．Ｕ．）を求めた。(7) Measurement of Anti-Treponemal Antibody Titer of Sample for Measurement Using the sample for measurement prepared in the above (5), the same operation as in the above (6) was performed to determine the amount of change in absorbance. . Next, using the anti-treponema antibody calibration curve prepared in (6) above, the anti-treponema antibody titer (TU) of each measurement sample was determined from the change in absorbance of each measurement sample.
【００３５】（比較例１）検体希釈用緩衝液の調製にお
いて、エルシナン１重量％の代わりに、プルラン１重量
％を添加したこと以外は実施例１の場合と同様にして、
上記（５）で準備した測定用検体の抗トレポネーマ抗体
価（Ｔ．Ｕ．）を求めた。(Comparative Example 1) In the preparation of the sample dilution buffer, the same procedure as in Example 1 was carried out except that 1% by weight of pullulan was added instead of 1% by weight of elsinan.
The anti-treponemal antibody titer (TU) of the measurement sample prepared in the above (5) was determined.
【００３６】（比較例２）検体希釈用緩衝液の調製にお
いて、エルシナン１重量％の代わりに、ポリエチレング
リコール（分子量：２０万）１重量％を添加したこと以
外は実施例１の場合と同様にして、上記（５）で準備し
た測定用検体の抗トレポネーマ抗体価（Ｔ．Ｕ．）を求
めた。Comparative Example 2 The procedure of Example 1 was repeated, except that 1% by weight of polyethylene glycol (molecular weight: 200,000) was added instead of 1% by weight of elsinan in the preparation of the sample dilution buffer. The anti-treponema antibody titer (TU) of the measurement sample prepared in the above (5) was determined.
【００３７】実施例１、及び、比較例１及び比較例２の
結果は表１に示すとおりであった。The results of Example 1 and Comparative Examples 1 and 2 are as shown in Table 1.
【００３８】[0038]
【表１】 [Table 1]
【００３９】表１から明らかなように、本発明による実
施例１の免疫測定試薬は、反応系にプルランを含有させ
た比較例１の免疫測定試薬及び反応系にポリエチレング
リコールを含有させた比較例２の免疫測定試薬に比較し
て、反応性（吸光度変化量）が大幅に向上していた。
又、本発明による実施例１の免疫測定試薬は、反応性
（吸光度変化量）が大幅に向上したにもかかわらず、健
常人の血清は陰性、梅毒患者の血清は陽性と的確に判定
されており、優れた特異反応性を示した。As is clear from Table 1, the immunoassay reagent of Example 1 according to the present invention is the immunoassay reagent of Comparative Example 1 containing pullulan in the reaction system and the comparative example containing polyethylene glycol in the reaction system. As compared with the immunoassay reagent No. 2, the reactivity (absorbance change amount) was significantly improved.
In addition, the immunoassay reagent of Example 1 according to the present invention was able to accurately judge that the serum of a healthy person was negative and the serum of a syphilis patient was positive even though the reactivity (absorbance change) was significantly improved. And showed excellent specific reactivity.
【００４０】[0040]
【発明の効果】以上述べたように、本発明の免疫測定試
薬は、被測定物質である抗原（又は抗体）と対応する抗
体（又は抗原）を感作した不溶性担体及びエルシナンが
含有されてなるので、上記エルシナンの働きにより、抗
原抗体反応による特異的な凝集反応性を大幅に向上させ
ることができる。即ち、本発明の免疫測定試薬は、高感
度で非特異反応が少なく、微量の被測定物質を的確に検
出することができるものである。As described above, the immunoassay reagent of the present invention comprises an insoluble carrier sensitized with an antigen (or antibody) as a substance to be measured and an antibody (or antigen) corresponding thereto, and ercinan. Therefore, the specific agglutinating reactivity due to the antigen-antibody reaction can be significantly improved by the action of ercinan. That is, the immunoassay reagent of the present invention is highly sensitive, has little nonspecific reaction, and can accurately detect a trace amount of the substance to be measured.
Claims (1)
応する抗体（又は抗原）を感作した不溶性担体及びエル
シナンが含有されてなることを特徴とする免疫測定試
薬。1. An immunoassay reagent comprising an insoluble carrier sensitized with an antibody (or antigen) corresponding to an antigen (or antibody) to be measured and ercinan.
Priority Applications (1)
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|---|---|---|---|
| JP2001152701A JP2002340896A (en) | 2001-05-22 | 2001-05-22 | Immunoassay reagent |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2001152701A JP2002340896A (en) | 2001-05-22 | 2001-05-22 | Immunoassay reagent |
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| Publication Number | Publication Date |
|---|---|
| JP2002340896A true JP2002340896A (en) | 2002-11-27 |
Family
ID=18997330
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|---|---|---|---|
| JP2001152701A Pending JP2002340896A (en) | 2001-05-22 | 2001-05-22 | Immunoassay reagent |
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| Country | Link |
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| JP (1) | JP2002340896A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60258125A (en) * | 1984-06-06 | 1985-12-20 | Hayashibara Biochem Lab Inc | Water-soluble dried material containing proteinic physiologically active substance |
| JPH06167493A (en) * | 1992-11-30 | 1994-06-14 | Kyowa Medex Co Ltd | Measuring method for immunity |
| JPH07179494A (en) * | 1993-12-21 | 1995-07-18 | Hayashibara Biochem Lab Inc | Polypeptide, its production and use |
-
2001
- 2001-05-22 JP JP2001152701A patent/JP2002340896A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60258125A (en) * | 1984-06-06 | 1985-12-20 | Hayashibara Biochem Lab Inc | Water-soluble dried material containing proteinic physiologically active substance |
| JPH06167493A (en) * | 1992-11-30 | 1994-06-14 | Kyowa Medex Co Ltd | Measuring method for immunity |
| JPH07179494A (en) * | 1993-12-21 | 1995-07-18 | Hayashibara Biochem Lab Inc | Polypeptide, its production and use |
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