JPH08193999A - Immune measuring method - Google Patents
Immune measuring methodInfo
- Publication number
- JPH08193999A JPH08193999A JP395995A JP395995A JPH08193999A JP H08193999 A JPH08193999 A JP H08193999A JP 395995 A JP395995 A JP 395995A JP 395995 A JP395995 A JP 395995A JP H08193999 A JPH08193999 A JP H08193999A
- Authority
- JP
- Japan
- Prior art keywords
- antibody
- antigen
- reaction
- immune complex
- solution
- 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
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Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、臨床検査の分野で用い
られ、測定対象物質に対する抗原または抗体の凝集反応
を利用した免疫測定法に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunoassay method used in the field of clinical examination and utilizing the agglutination reaction of an antigen or an antibody against a substance to be measured.
【０００２】[0002]
【従来の技術】各種生体成分から特定の物質を検出もし
くは定量するために、抗原抗体反応が利用され、一般的
には測定対象となる抗原または抗体に対応する抗体また
は抗原を反応させる方法が用いられる。測定方法として
は、不溶性の担体を使用する方法と担体を使用せず直接
凝集を検出する方法があり、前者の方法としては、測定
する抗原に対する抗体をラテックス等を担体として担持
させ、その免疫反応により生じた凝集の度合いを測定す
ることにより、試料中の抗原を検出あるいは定量するも
のである。2. Description of the Related Art An antigen-antibody reaction is used to detect or quantify a specific substance from various biological components. Generally, an antigen to be measured or an antibody corresponding to the antibody or a method of reacting the antigen is used. To be As the measuring method, there are a method of using an insoluble carrier and a method of directly detecting agglutination without using a carrier.The former method carries an antibody against the antigen to be measured as a carrier such as latex, and its immune reaction. The antigen in the sample is detected or quantified by measuring the degree of aggregation generated by.
【０００３】凝集の度合いを検出する方法としては、凝
集の有無を肉眼で判定する方法、反応液に光を照射して
散乱光または透過光を測定する方法があり、肉眼判定は
定性法または半定量法として用いられ、光学的な測定法
は抗原の定量法として用いられている。As a method for detecting the degree of agglutination, there are a method of visually judging the presence or absence of agglutination, and a method of irradiating the reaction solution with light to measure scattered light or transmitted light. It is used as a quantitative method, and the optical measurement method is used as a quantitative method of antigens.
【０００４】上記凝集法以外に、抗原抗体反応の検出を
酵素反応を利用して発色としてとらえる酵素免疫測定法
（ＥＩＡ）、放射免疫測定法（ＲＩＡ）、補体結合反応
を利用する方法等がある。In addition to the above-mentioned agglutination method, there are an enzyme immunoassay method (EIA), a radioimmunoassay method (RIA), a method using a complement fixing reaction, etc., which detects the detection of an antigen-antibody reaction as a color development utilizing an enzyme reaction. is there.
【０００５】上記補体結合反応は、抗原抗体反応におい
て免疫複合体が生じると補体を結合し、活性化する反応
であり、補体の反応量を測定することによって抗原また
は抗体を定量する。補体結合反応を利用する方法におい
て、検出感度を改善する方法として、コングルチニン、
免疫コングルチニン、リウマチ因子等を免疫複合体の補
体と結合させることが開示されている（特表昭５７−５
０１６９１号公報）。The above-mentioned complement binding reaction is a reaction that binds and activates complement when an immune complex is generated in the antigen-antibody reaction, and the antigen or antibody is quantified by measuring the reaction amount of complement. In a method utilizing a complement fixation reaction, as a method for improving the detection sensitivity, conglutinin,
It has been disclosed that immune conglutinin, rheumatoid factor, etc. are bound to the complement of the immune complex (Table 57-5).
No. 01691).
【０００６】しかしながら、補体結合反応においては補
体系そのものが複雑な系であり、免疫グロブリンＧ（Ｉ
ｇＧ）のクラスによっては補体を結合せず、また血球の
調製や管理に手間がかかり、さらに補体の調製は氷冷下
で行わなければならず、保存も困難である。キット化さ
れたものでも安定性は２か月程度であるといった種々の
問題点がある。However, in the complement fixation reaction, the complement system itself is a complicated system, and immunoglobulin G (I
gG) does not bind complement depending on the class of gG), and it takes time and effort to prepare and manage blood cells. Furthermore, preparation of complement must be carried out under ice cooling, and storage is also difficult. There are various problems that the kit is stable for about 2 months.
【０００７】[0007]
【発明が解決しようとする課題】本発明は上記問題点を
解決するものであり、その目的とするところは、血球調
製の手間をなくし、簡便で安定かつ特異的に免疫複合体
を測定し、抗原または抗体を測定する免疫測定法を提供
することである。DISCLOSURE OF THE INVENTION The present invention is intended to solve the above-mentioned problems, and an object of the present invention is to eliminate the trouble of blood cell preparation, to measure an immune complex simply, stably and specifically, It is to provide an immunoassay for measuring an antigen or an antibody.
【０００８】[0008]
【課題を解決するための手段】本発明の免疫測定法は、
検体中の対象物質の抗原と、該抗原に対する抗体Ａの抗
原抗体反応により生じる免疫複合体を、免疫複合体に対
する抗体Ｂを用いて凝集反応により測定することを特徴
とする。上記抗体Ｂは、本発明１においては上記抗原と
抗体Ａの免疫複合体Ｉに対する抗体であり、本発明２に
おいては抗原抗体反応により生じる免疫複合体IIに対す
る抗体である。The immunoassay method of the present invention comprises:
It is characterized in that an antigen of a target substance in a sample and an immune complex formed by an antigen-antibody reaction of antibody A against the antigen are measured by agglutination reaction using antibody B against the immune complex. The antibody B is an antibody against the immune complex I of the antigen and the antibody A in the present invention 1, and an antibody against the immune complex II generated by an antigen-antibody reaction in the present invention 2.
【０００９】本発明の免疫測定法は、検体中の対象物質
の抗体Ａ’と、該抗体Ａ’に対応する抗原の抗原抗体反
応により生じる免疫複合体を、免疫複合体に対する抗体
Ｂを用いて凝集反応により測定することを特徴とする。
上記抗体Ｂは、本発明３においては上記抗原と抗体Ａの
免疫複合体Ｉに対する抗体であり、本発明４においては
抗原抗体反応により生じる免疫複合体IIに対する抗体で
ある。In the immunoassay method of the present invention, an antibody A'of a target substance in a sample, an immune complex formed by an antigen-antibody reaction of an antigen corresponding to the antibody A ', and an antibody B against the immune complex are used. It is characterized by being measured by an agglutination reaction.
The above-mentioned antibody B is an antibody against the immune complex I of the above-mentioned antigen and the antibody A in the present invention 3, and an antibody against the immune complex II generated by an antigen-antibody reaction in the present invention 4.
【００１０】上記免疫複合体は免疫反応による抗原抗体
複合物であり、さらに補体反応により補体が結合された
ものも含まれる。上記抗体Ｂは、免疫複合体のみと反応
し、上記抗原、抗体Ａ及び抗体Ａ’とは反応しないもの
である。The above-mentioned immune complex is an antigen-antibody complex by an immune reaction, and further includes one in which complement is bound by a complement reaction. The antibody B reacts only with the immune complex and does not react with the antigen, the antibody A and the antibody A ′.
【００１１】上記抗体Ａは、例えば、対応する抗原で適
当な動物を免疫する等の公知の方法で得られる。上記抗
体Ｂは、同様に、抗原抗体反応によって生じる免疫複合
体で適当な動物を免疫する等の公知の方法で得られる。
この時得られる抗血清に、上記免疫複合体と交差反応す
る可能性のある抗原、抗体等の免疫原を、そのまま又は
セファデックス、セファロース等の粒子に結合させて混
合する等、公知の方法により吸収を行うことで、免疫複
合体にのみ反応する抗体Ｂが得られる。The above-mentioned antibody A can be obtained by a known method such as immunizing a suitable animal with a corresponding antigen. Similarly, the antibody B can be obtained by a known method such as immunizing a suitable animal with an immune complex generated by an antigen-antibody reaction.
The antiserum obtained at this time is mixed with an immunogen such as an antigen or an antibody that may cross-react with the above-mentioned immune complex, as it is or after being bound to particles such as Sephadex or Sepharose and mixed, by a known method. Upon absorption, antibody B that reacts only with the immune complex is obtained.
【００１２】本発明により測定される検体は、抗原、抗
体等の免疫学的に活性な被測定物質を含有する試料、特
に生体試料である。生体試料としては、例えば、血液、
胸水、腹水、リンパ液等の体液、尿、便、汗等の***
物、及び組織の抽出物などが挙げられる。The sample measured by the present invention is a sample, especially a biological sample, containing an immunologically active substance to be measured such as an antigen and an antibody. As the biological sample, for example, blood,
Pleural fluid, ascites, body fluids such as lymph, excrements such as urine, feces and sweat, and extracts of tissues can be mentioned.
【００１３】上記被測定物質としては、抗原抗体反応し
うる抗原、抗体等の免疫学的に活性なあらゆる物質、す
なわち従来測定されていた物質のいずれもが包含され、
例えばタンパク質、ポリペプチド、多糖類、脂質、ステ
ロイド等が挙げられる。上記タンパク質としては、例え
ば、アルファフェトプロテイン（ＡＦＰ）、フィブリノ
ーゲン分解産物（ＦＤＰ）、Ｃ反応性タンパク（ＣＲ
Ｐ）、ヒト絨毛性ゴナドトロピン（ＨＣＧ）等の血液中
タンパク質、Ｂ型肝炎ウイルス（ＨＢ）、Ｃ型肝炎ウイ
ルス（ＨＣ）、ヒト免疫不全ウイルス（ＨＩＶ）等の感
染症に対する抗原及びその抗体、レセプター、酵素など
が挙げられる。The above-mentioned substances to be measured include all immunologically active substances such as antigens capable of antigen-antibody reaction, antibodies, etc., that is, any substances that have been conventionally measured,
Examples thereof include proteins, polypeptides, polysaccharides, lipids and steroids. Examples of the protein include alpha-fetoprotein (AFP), fibrinogen degradation product (FDP), C-reactive protein (CR).
P), blood proteins such as human chorionic gonadotropin (HCG), antigens against infectious diseases such as hepatitis B virus (HB), hepatitis C virus (HC), human immunodeficiency virus (HIV), and their antibodies, receptors , Enzymes, etc.
【００１４】本発明の測定法においては、測定系中に対
象物質の抗原又は抗体Ａ’、それらに対応する抗体Ａ又
は抗原、及び免疫複合体に対する抗体Ｂが含有されてい
ればよく、上記抗体Ｂは、不溶性担体に担持されていて
もよい。さらに、上記測定系中には必要に応じて反応促
進物質を添加してもよい。反応促進物質としては、例え
ば、ポリエチレングリコール、ポリグリコシルメタクリ
レート、ポリビニルピロリドン、カルボキシメチルセル
ロース、デキストラン、プルラン等が挙げられる。In the assay method of the present invention, it is sufficient that the assay system contains the antigen or antibody A ′ of the target substance, the antibody A or antigen corresponding thereto, and the antibody B against the immune complex. B may be supported on an insoluble carrier. Furthermore, a reaction accelerating substance may be added to the above measurement system, if necessary. Examples of the reaction accelerating substance include polyethylene glycol, polyglycosyl methacrylate, polyvinylpyrrolidone, carboxymethyl cellulose, dextran, pullulan and the like.
【００１５】上記不溶性担体としては、例えば、有機高
分子粉末、無機物質粉末、微生物、血球及び細胞膜片、
プラスチック製マイクロタイタープレート等が挙げられ
る。上記有機高分子粉末としては、例えば、不溶性アガ
ロース、セルロース、不溶性デキストラン等の天然高分
子粉末、メタクリル酸重合体、アクリル酸重合体、ポリ
スチレン、スチレン−スチレンスルホン酸塩共重合体、
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等の合成高分子粉末などが
挙げられ、特に合成高分子粉末を均一に懸濁させたラテ
ックスが好ましい。Examples of the insoluble carrier include organic polymer powder, inorganic powder, microorganisms, blood cells and cell membrane pieces,
Examples include plastic microtiter plates. As the organic polymer powder, for example, insoluble agarose, cellulose, natural polymer powder such as insoluble dextran, methacrylic acid polymer, acrylic acid polymer, polystyrene, styrene-styrene sulfonate copolymer,
Acrylonitrile-butadiene-styrene copolymer, vinyl chloride-acrylic acid ester copolymer, vinyl acetate-
Examples thereof include synthetic polymer powders such as acrylic acid ester copolymers, and a latex in which the synthetic polymer powders are uniformly suspended is preferable.
【００１６】上記無機物質粉末としては、例えば、金、
チタン、鉄、ニッケル等の金属片、シリカ、アルミナ、
炭素末などが挙げられる。上記不溶性担体の平均粒径
は、測定方法、測定機器によって異なるが、通常０．０
５〜１．０μｍ、好ましくは０．０５〜０．５μｍのも
のが用いられる。使用する不溶性担体の濃度は、光学的
観察に用いる場合には通常０．０５〜３．０ｍｇ／ｍ
ｌ、好ましくは０．２〜１．０ｍｇ／ｍｌであり、目視
観察に用いる場合には通常０．５〜２０ｍｇ／ｍｌ、好
ましくは１．０〜５．０ｍｇ／ｍｌである。上記不溶性
担体に免疫複合体に対する抗体Ｂを担持させる方法とし
ては、例えば、化学的または物理的結合により感作させ
る方法が挙げられる。As the above-mentioned inorganic substance powder, for example, gold,
Metal pieces such as titanium, iron, nickel, silica, alumina,
Carbon powder etc. are mentioned. The average particle size of the insoluble carrier varies depending on the measuring method and measuring equipment, but is usually 0.0
The thickness is 5 to 1.0 μm, preferably 0.05 to 0.5 μm. The concentration of the insoluble carrier used is usually 0.05 to 3.0 mg / m when used for optical observation.
1, preferably 0.2 to 1.0 mg / ml, and usually 0.5 to 20 mg / ml, preferably 1.0 to 5.0 mg / ml when used for visual observation. Examples of the method of allowing the insoluble carrier to carry the antibody B against the immune complex include a method of sensitizing by chemical or physical binding.
【００１７】本発明の免疫測定法において、上記抗原抗
体反応は通常の条件で行われ、反応におけるｐＨは、通
常５〜１０、好ましくは６〜８である。使用される緩衝
液としては、例えば、リン酸緩衝液、トリス緩衝液、グ
リシン緩衝液、クエン酸緩衝液等が挙げられる。反応温
度は、通常０〜５０℃、好ましくは２０〜４０℃であ
る。反応時間は通常２０秒〜３０分、好ましくは１〜１
５分である。In the immunoassay method of the present invention, the above-mentioned antigen-antibody reaction is carried out under ordinary conditions, and the pH in the reaction is usually 5-10, preferably 6-8. Examples of the buffer solution used include phosphate buffer solution, Tris buffer solution, glycine buffer solution, citrate buffer solution and the like. The reaction temperature is generally 0 to 50 ° C, preferably 20 to 40 ° C. The reaction time is usually 20 seconds to 30 minutes, preferably 1 to 1
5 minutes.
【００１８】抗原抗体反応を測定する際の測定系として
は、例えば、免疫比濁法、ラテックス凝集法、血球凝集
法等が利用される。上記反応により生じた凝集の度合い
を測定する方法としては、凝集の程度を光学的に観察す
る方法あるいは目視により観察する方法等が挙げられ
る。As a measuring system for measuring the antigen-antibody reaction, for example, an immunoturbidimetric method, a latex agglutination method, a hemagglutination method and the like are used. Examples of the method for measuring the degree of aggregation generated by the above reaction include a method of optically observing the degree of aggregation and a method of visually observing the degree.
【００１９】光学的に観察する方法においては、上記反
応後の混合液の散乱光強度、吸光度、または透過光強度
を検出する。測定方法は公知の方法に従い、用いる不溶
性担体の粒径、濃度、反応時間によって散乱光強度、吸
光度、または透過光強度の増加もしくは減少を測定す
る。またこれらの方法は単独で用いられてもよいし、併
用されてもよい。目視により観察する方法においては、
凝集の有無を判定する際に単に肉眼で判定する以外に、
ビデオカメラで撮影し画像処理を施すことによって判定
することもできる。In the optical observation method, the scattered light intensity, the absorbance, or the transmitted light intensity of the mixed solution after the above reaction is detected. According to a known method, the increase or decrease in scattered light intensity, absorbance, or transmitted light intensity is measured according to the particle size, concentration, and reaction time of the insoluble carrier used. In addition, these methods may be used alone or in combination. In the visual observation method,
In addition to the naked eye when determining the presence or absence of aggregation,
It can also be determined by shooting with a video camera and performing image processing.
【００２０】[0020]
【実施例】本発明を実施例につき説明する。実施例にお
いては以下の試薬を用いた。 ・リン酸緩衝液（ＰＢＳ）：リン酸１ナトリウム（２水
和物）、リン酸２ナトリウム（２水和物）及び塩化ナト
リウムを、リン酸の最終濃度が０．０２Ｍ、塩化ナトリ
ウムの最終濃度が０．１５Ｍ、ｐＨが７．２となるよう
に、精製水に溶解した溶液 ・１％ＢＳＡ−ＰＢＳ：ＰＢＳ１００ｍｌに牛血清アル
ブミン（ＢＳＡ、試薬特級）１ｇを溶解した溶液 ・５％ＢＳＡ−ＰＢＳ：ＰＢＳ１００ｍｌに牛血清アル
ブミン（ＢＳＡ、試薬特級）５ｇを溶解した溶液 ・カップリングバッファー：炭酸水素ナトリウム、塩化
ナトリウムの最終濃度がそれぞれ０．１Ｍ、０．５Ｍ、
ｐＨが８．３となるように精製水に溶解した溶液EXAMPLES The present invention will be described with reference to examples. The following reagents were used in the examples. -Phosphate buffer (PBS): monosodium phosphate (dihydrate), disodium phosphate (dihydrate) and sodium chloride, the final concentration of phosphoric acid is 0.02M, the final concentration of sodium chloride Of 0.15 M and pH of 7.2 dissolved in purified water-1% BSA-PBS: 1 ml of bovine serum albumin (BSA, reagent grade) dissolved in 100 ml of PBS-5% BSA-PBS : Solution in which 5 g of bovine serum albumin (BSA, reagent grade) is dissolved in 100 ml of PBS-Coupling buffer: final concentrations of sodium hydrogen carbonate and sodium chloride are 0.1 M and 0.5 M, respectively.
A solution dissolved in purified water to a pH of 8.3
【００２１】〔免疫複合体に対する抗体の作成〕 １）免疫複合体の作成 ヤギ抗ヒトＣＲＰ抗体（コスモバイオ社製）を１０ｍｇ
／ｍｌとなるようにＰＢＳで調製した溶液２ｍｌに、ヒ
トＣＲＰ陽性血清（ＣＲＰ１０ｍｇ／ｄｌ相当、ＣＤＰ
社製）を２ｍｌ添加し、３７℃で２時間インキュベート
した。その後１５０００回転で２時間遠心し、得られた
沈殿を２ｍｌのＰＢＳに分散させ、抗原抗体反応による
免疫複合体を得た。この試料をＳＤＳ−ポリアクリルア
ミドゲル電気泳動法で確認したところ、分子量１５００
００以上に位置しており、上記試料は抗体ではなく免疫
複合体であることが確認された。[Preparation of Antibody Against Immune Complex] 1) Preparation of Immune Complex 10 mg of goat anti-human CRP antibody (manufactured by Cosmo Bio Inc.)
2 ml of a solution prepared with PBS so that the amount of the CRP is 10 ml / ml, human CRP positive serum (CRP 10 mg / dl equivalent, CDP
2 ml) was added, and the mixture was incubated at 37 ° C for 2 hours. Then, it was centrifuged at 15,000 rpm for 2 hours, and the obtained precipitate was dispersed in 2 ml of PBS to obtain an immune complex by an antigen-antibody reaction. When this sample was confirmed by SDS-polyacrylamide gel electrophoresis, the molecular weight was 1500.
It was confirmed that the above sample was an immune complex, not an antibody.
【００２２】２）免疫複合体抗血清の作成 上記で得られた免疫複合体溶液０．３ｍｌをウサギの耳
静脈に注射し、通常の方法により免疫を行った（「続生
化学実験講座５ 免疫生化学研究法」日本生化学会編・
東京化学同人社発行(1986)参照) 。その後２週間おきに
２回、さらにその１か月後に１回、免疫複合体溶液０．
３ｍｌを注射した。その後採血を行い、４０ｍｌの血液
を得、血清として約２０ｍｌの免疫複合体抗血清を得
た。2) Preparation of Immune Complex Antiserum 0.3 ml of the immune complex solution obtained above was injected into the ear vein of a rabbit, and immunization was carried out by a usual method (see "Seikagaku Jikken Koza 5 Biochemistry Research ”edited by The Biochemical Society of Japan
Published by Tokyo Kagaku Dojinsha (1986)). After that, twice every two weeks, and once a month later, once again, the immune complex solution 0.
3 ml was injected. After that, blood was collected to obtain 40 ml of blood and about 20 ml of immune complex antiserum as serum.
【００２３】３）免疫原に対する吸収 ヒトＣＲＰ陽性カップリングゲルの作成 あらかじめカップリングバッファーに透析したヒトＣＲ
Ｐ陽性血清（ＣＲＰ２０ｍｇ／ｄｌ相当、ＣＤＰ社製）
２０ｍｌと、膨潤・洗浄した臭化シアン活性化セファロ
ース４Ｂ（ファルマシアバイオテク社製）１０ｍｌを混
合し、４℃で一晩静置した。その後この混合液を２００
０回転で５分間遠心分離し、得られたゲルを０．２Ｍグ
リシン−塩酸溶液（ｐＨ２．３）２０ｍｌで２回洗浄し
た後、０．２Ｍグリシン−塩酸溶液（ｐＨ２．３）５０
ｍｌ中で一晩静置した。これをカップリングバッファー
及び０．１Ｍ酢酸緩衝液（ｐＨ４．０）各２０ｍｌで交
互に２回ずつ洗浄してヒトＣＲＰ陽性カップリングゲル
を得、カップリングバッファー１０ｍｌ中に保存した。3) Absorption against immunogen Preparation of human CRP positive coupling gel Human CR dialyzed against a coupling buffer in advance
P-positive serum (CRP 20 mg / dl equivalent, manufactured by CDP)
20 ml and 10 ml of swollen and washed cyanogen bromide activated Sepharose 4B (Pharmacia Biotech) were mixed and left at 4 ° C. overnight. Then add this mixture to 200
After centrifuging at 0 rotation for 5 minutes, the obtained gel was washed twice with 20 ml of 0.2 M glycine-hydrochloric acid solution (pH 2.3), and then 0.2 M glycine-hydrochloric acid solution (pH 2.3) 50.
Let stand overnight in ml. This was alternately washed twice with 20 ml each of coupling buffer and 0.1 M acetate buffer (pH 4.0) to obtain a human CRP-positive coupling gel, which was stored in 10 ml of coupling buffer.
【００２４】ヤギＩｇＧカップリングゲルの作成 ヤギＩｇＧ（生化学工業社製）を２０ｍｇ／ｍｌとなる
ようにカップリングバッファーに溶解した。この液１ｍ
ｌと、膨潤・洗浄した臭化シアン活性化セファロース４
Ｂ（ファルマシアバイオテク社製）１０ｍｌを混合し、
４℃で一晩静置した。その後この混合液を２０００回転
で５分間遠心分離し、得られたゲルを０．２Ｍグリシン
−塩酸溶液（ｐＨ２．３）２０ｍｌで２回洗浄した後、
０．２Ｍグリシン−塩酸溶液（ｐＨ２．３）５０ｍｌ中
で一晩静置した。これをカップリングバッファー及び
０．１Ｍ酢酸緩衝液（ｐＨ４．０）各２０ｍｌで交互に
２回ずつ洗浄してヤギＩｇＧカップリングゲルを得、カ
ップリングバッファー１０ｍｌ中に保存した。Preparation of Goat IgG Coupling Gel Goat IgG (manufactured by Seikagaku Corporation) was dissolved in a coupling buffer at 20 mg / ml. 1m of this liquid
1 and swollen / washed cyanogen bromide activated sepharose 4
Mix 10 ml of B (Pharmacia Biotech),
Let stand overnight at 4 ° C. Thereafter, this mixed solution was centrifuged at 2000 rpm for 5 minutes, and the obtained gel was washed twice with 20 ml of a 0.2 M glycine-hydrochloric acid solution (pH 2.3).
The mixture was allowed to stand overnight in 50 ml of 0.2 M glycine-hydrochloric acid solution (pH 2.3). This was alternately washed twice with 20 ml each of coupling buffer and 0.1 M acetate buffer (pH 4.0) to obtain a goat IgG coupling gel, which was stored in 10 ml of coupling buffer.
【００２５】免疫複合体抗血清のヒトＣＲＰ陽性カッ
プリングゲル及びヤギＩｇＧカップリングゲルによる吸
収 上記２）で得られた免疫複合体抗血清１０ｍｌを、上記
ヒトＣＲＰ陽性カップリングゲル５ｍｌと混合し、４
℃で一晩静置した。その後この混合液を２０００回転で
１０分間遠心分離し、上清を回収した。次いでこの１０
ｍｌを上記ヤギＩｇＧカップリングゲル５ｍｌと混合
し、４℃で一晩静置した。その後この混合液を２０００
回転で１０分間遠心分離し、その上清を回収し、免疫複
合体抗血清中の免疫原の吸収を行った。Absorption of Immune Complex Antiserum by Human CRP Positive Coupling Gel and Goat IgG Coupling Gel 10 ml of the immune complex antiserum obtained in 2) above was mixed with 5 ml of the above human CRP positive coupling gel, Four
Let stand overnight at 0 ° C. Thereafter, this mixed solution was centrifuged at 2000 rpm for 10 minutes, and the supernatant was recovered. Then this 10
ml was mixed with 5 ml of the above goat IgG coupling gel, and the mixture was allowed to stand overnight at 4 ° C. Then add this mixture to 2000
Centrifugation was performed for 10 minutes by rotation, the supernatant was collected, and the immunogen in the immune complex antiserum was absorbed.
【００２６】４）免疫複合体抗血清からの抗体の精製 上記３）で得られた免疫複合体抗血清を通常の塩析法
により精製を行った（「続生化学実験講座５ 免疫生化
学研究法」日本生化学会編・東京化学同人社発行(1986)
参照) 。免疫複合体抗血清５ｍｌを攪拌しながら、飽和
硫安２．５ｍｌを滴下し、攪拌しながら室温で６０分放
置した。その後１００００回転で１０分間遠心分離し、
得られた沈渣にＰＢＳ５ｍｌを添加した。これに飽和硫
安２．５ｍｌを再度滴下し、攪拌しながら室温で６０分
放置した後、１００００回転で１０分間遠心分離し、沈
渣を得た。これを５ｍｌのＰＢＳで溶解し、５００ｍｌ
のＰＢＳで５時間透析を行った。この操作を３回繰り返
し、免疫複合体抗血清からの抗体を精製した（以下「複
合体抗体」とする）。4) Purification of Antibody from Immune Complex Antiserum The immune complex antiserum obtained in 3) above was purified by the usual salting-out method ("Zokusei Chemistry Laboratory 5 Immunobiochemistry Research"). Law "edited by The Biochemical Society of Japan, published by Tokyo Kagaku Dojinsha (1986)
See). 2.5 ml of saturated ammonium sulfate was added dropwise while stirring 5 ml of immune complex antiserum, and the mixture was left at room temperature for 60 minutes while stirring. After that, centrifuge at 10,000 rpm for 10 minutes,
5 ml of PBS was added to the obtained precipitate. To this, 2.5 ml of saturated ammonium sulfate was added again, and the mixture was left for 60 minutes at room temperature with stirring and then centrifuged at 10,000 rpm for 10 minutes to obtain a precipitate. Dissolve this with 5 ml of PBS, 500 ml
It dialyzed with PBS of 5 hours. This operation was repeated 3 times to purify the antibody from the immune complex antiserum (hereinafter referred to as “complex antibody”).
【００２７】（実施例１） 〔複合体抗体結合ラテックス液の調製〕上記で得られた
複合体抗体を１ｍｇ／ｍｌとなるようにＰＢＳに分散さ
せた。この溶液９００μｌと粒径０．２μｍのポリスチ
レンラテックス（積水化学社製）１００μｌを混合し、
４℃で一晩静置した。次いでこの液を遠心分離し、上清
を除去して得られる沈殿に１％ＢＳＡ−ＰＢＳを１ｍｌ
添加し、３７℃で２時間ブロッキングした。その後生理
食塩水で３回洗浄し、１％ＢＳＡ−ＰＢＳを２ｍｌ添加
し、複合体抗体結合ラテックス液を得た。(Example 1) [Preparation of complex antibody-bound latex solution] The complex antibody obtained above was dispersed in PBS so as to have a concentration of 1 mg / ml. 900 μl of this solution and 100 μl of polystyrene latex (manufactured by Sekisui Chemical Co., Ltd.) having a particle size of 0.2 μm are mixed,
Let stand overnight at 4 ° C. Then, this solution is centrifuged, and the precipitate obtained by removing the supernatant is supplemented with 1 ml of 1% BSA-PBS.
It was added and blocked at 37 ° C. for 2 hours. Then, it was washed three times with physiological saline, and 2 ml of 1% BSA-PBS was added to obtain a complex antibody-bound latex solution.
【００２８】〔ヒトＡＦＰの測定〕ヒツジ抗ヒトＡＦＰ
抗体（生化学工業社製）を１ｍｇ／ｍｌとなるようにＰ
ＢＳに希釈し、この１ｍｌを上記複合体抗体結合ラテッ
クス液２ｍｌに添加し、室温で１時間攪拌してラテック
ス測定液を調製した。２５０ｎｇ／ｍｌのＡＦＰ標準品
（デンカ生研社製、ラテックス試薬シリーズ標準液）
を、５％ＢＳＡ−ＰＢＳで順次２倍に希釈し、１２５ｎ
ｇ／ｍｌ、６２．５ｎｇ／ｍｌの標準品を調製した。[Measurement of human AFP] Sheep anti-human AFP
Antibody (Seikagaku Corporation) P to 1mg / ml
It was diluted with BS, 1 ml of this was added to 2 ml of the above complex antibody-bound latex solution, and the mixture was stirred at room temperature for 1 hour to prepare a latex measurement solution. 250 ng / ml AFP standard product (Denka Seiken Co., Ltd., latex reagent series standard solution)
Is serially diluted 2-fold with 5% BSA-PBS to obtain 125n
Standards of g / ml and 62.5 ng / ml were prepared.
【００２９】上記２５０、１２５、６２．５ｎｇ／ｍｌ
のＡＦＰ標準品、及び５％ＢＳＡ−ＰＢＳ（０ｎｇ／ｍ
ｌＡＦＰ標準品）各１００μｌを１ｍｌの１％ＢＳＡ−
ＰＢＳに添加し、混合した。次いでこれに上記ラテック
ス測定液２００μｌを添加し、生化学自動分析装置（日
立Ｕ−３２００型、日立製作所社製）により、光路長１
ｃｍのセルを用い、測定波長５７０ｎｍで５分間の吸光
度変化を測定した。また上記ラテックス測定液を４℃で
５か月間保存した後、同様に標準品を添加して測定を行
った。以上の結果を表１に示す。The above 250, 125, 62.5 ng / ml
AFP standard product and 5% BSA-PBS (0 ng / m
lAFP standard) 100 μl of each 1 ml of 1% BSA-
Added to PBS and mixed. Next, 200 μl of the above latex measurement solution was added to this, and an optical path length of 1 was measured with a biochemical automatic analyzer (Hitachi U-3200, manufactured by Hitachi, Ltd.).
The change in absorbance for 5 minutes was measured at a measurement wavelength of 570 nm using a cm cell. The latex measurement solution was stored at 4 ° C. for 5 months, and then a standard product was added in the same manner for measurement. Table 1 shows the above results.
【００３０】[0030]
【表１】 [Table 1]
【００３１】（実施例２）ストレプトリジンＯ（還元
型、４０ＩＵ、ＲＢＩ社製）を、ＢＣＡ蛋白測定キット
（Ｐｉｅｃｅ社製）で蛋白濃度が１ｍｇ／ｍｌとなるよ
うにＰＢＳに分散させた。また上記４）で得られた複合
体抗体を１ｍｇ／ｍｌとなるようにＰＢＳに分散させ、
この１ｍｌと上記ストレプトリジンＯ液１ｍｌを混合
し、測定液を調製した。５００ＩＵ／ｍｌの抗ストレプ
トリジンＯ（ＡＳＬＯ）標準品（デンカ生研社製、ラテ
ックス試薬シリーズ標準液）を、５％ＢＳＡ−ＰＢＳで
順次２倍に希釈し、２５０ＩＵ／ｍｌ、１２５ＩＵ／ｍ
ｌの標準品を調製した。Example 2 Streptolysin O (reduced type, 40 IU, manufactured by RBI) was dispersed in PBS using a BCA protein measurement kit (manufactured by Piece) so that the protein concentration was 1 mg / ml. Further, the complex antibody obtained in 4) above is dispersed in PBS so as to be 1 mg / ml,
This 1 ml was mixed with 1 ml of the above Streptolidine O liquid to prepare a measurement liquid. 500 IU / ml anti-streptolidine O (ASLO) standard product (Denka Seiken Co., Ltd., latex reagent series standard solution) was serially diluted 2-fold with 5% BSA-PBS, 250 IU / ml, 125 IU / m.
1 standard was prepared.
【００３２】上記５００、２５０、１２５ＩＵ／ｍｌの
ＡＳＬＯ標準品、及び５％ＢＳＡ−ＰＢＳ（０ＩＵ／ｍ
ｌＡＳＬＯ標準品）各１００μｌを、５％ポリエチレン
グリコール６０００（和光純薬社製）を含む１％ＢＳＡ
−ＰＢＳ１ｍｌに添加し、混合した。次いでこれに上記
測定液２００μｌを添加し、生化学自動分析装置（日立
Ｕ−３２００型、日立製作所社製）により、光路長１ｃ
ｍのセルを用い、測定波長３４０ｎｍで５分間の吸光度
変化を測定した。また上記測定液を４℃で３か月間保存
した後、同様に標準品を添加して測定を行った。以上の
結果を表２に示す。The above 500, 250 and 125 IU / ml ASLO standard products, and 5% BSA-PBS (0 IU / m
100 μl each of 1% BSA containing 5% polyethylene glycol 6000 (manufactured by Wako Pure Chemical Industries, Ltd.)
-Added to 1 ml of PBS and mixed. Next, 200 μl of the above-mentioned measurement solution was added to this, and an optical path length of 1c was obtained by using a biochemical automatic analyzer (Hitachi U-3200, manufactured by Hitachi, Ltd.).
The change in absorbance for 5 minutes was measured at a measurement wavelength of 340 nm using a m cell. Further, the above-mentioned measurement solution was stored at 4 ° C. for 3 months, and then a standard product was added in the same manner for measurement. Table 2 shows the above results.
【００３３】[0033]
【表２】 [Table 2]
【００３４】[0034]
【発明の効果】本発明の免疫測定法は上述のとおりであ
り、免疫複合体の抗体を用いることにより、検体中の抗
原または抗体を特異的にかつ簡便に測定でき、さらに安
定性に優れる。EFFECT OF THE INVENTION The immunoassay method of the present invention is as described above, and by using the antibody of the immune complex, the antigen or antibody in the sample can be specifically and easily measured, and the stability is excellent.
Claims (4)
Ａ、及び抗原抗体反応によって生じる免疫複合体Ｉに対
する抗体Ｂを混合し、上記抗原と抗体Ａの抗原抗体反応
によって生じる免疫複合体Ｉと、上記抗体Ｂとの抗原抗
体反応によって生じる凝集の度合いを測定する方法であ
って、上記抗体Ｂが上記抗原及び抗体Ａと反応しないこ
とを特徴とする免疫測定法。1. An immune complex I generated by an antigen-antibody reaction of the above-mentioned antigen and antibody A by mixing a specimen containing an antigen, an antibody A against the antigen, and an antibody B against an immune complex I generated by the antigen-antibody reaction. A method for measuring the degree of aggregation caused by an antigen-antibody reaction with the antibody B, wherein the antibody B does not react with the antigen and the antibody A.
Ａ、及び抗原抗体反応によって生じる免疫複合体Ｉに対
する抗体Ｂを混合し、上記抗原と抗体Ａの抗原抗体反応
によって生じる免疫複合体IIと、上記抗体Ｂとの抗原抗
体反応によって生じる凝集の度合いを測定する方法であ
って、上記抗体Ｂが上記抗原及び抗体Ａと反応しないこ
とを特徴とする免疫測定法。2. An immune complex II formed by an antigen-antibody reaction of the above-mentioned antigen and antibody A by mixing a specimen containing an antigen, an antibody A against the antigen, and an antibody B against an immune complex I generated by the antigen-antibody reaction. A method for measuring the degree of aggregation caused by an antigen-antibody reaction with the antibody B, wherein the antibody B does not react with the antigen and the antibody A.
応する抗原、及び抗原抗体反応によって生じる免疫複合
体Ｉに対する抗体Ｂを混合し、上記抗体Ａ’と抗原の抗
原抗体反応によって生じる免疫複合体Ｉと、上記抗体Ｂ
との抗原抗体反応によって生じる凝集の度合いを測定す
る方法であって、上記抗体Ｂが上記抗体Ａ’及び抗原と
反応しないことを特徴とする免疫測定法。3. A sample containing an antibody A ′, an antigen corresponding to the antibody A ′, and an antibody B against an immune complex I generated by an antigen-antibody reaction are mixed, and the antibody A ′ and the antigen are reacted by an antigen-antibody reaction. The resulting immune complex I and the above antibody B
A method for measuring the degree of agglutination caused by an antigen-antibody reaction with the antibody, wherein the antibody B does not react with the antibody A ′ and the antigen.
応する抗原、及び抗原抗体反応によって生じる免疫複合
体Ｉに対する抗体Ｂを混合し、上記抗体Ａ’と抗原の抗
原抗体反応によって生じる免疫複合体IIと、上記抗体Ｂ
との抗原抗体反応によって生じる凝集の度合いを測定す
る方法であって、上記抗体Ｂが上記抗体Ａ’及び抗原と
反応しないことを特徴とする免疫測定法。4. A sample containing an antibody A ′, an antigen corresponding to the antibody A ′, and an antibody B against an immune complex I generated by an antigen-antibody reaction are mixed, and the antibody A ′ and the antigen-antibody reaction of the antigen are mixed. The resulting immune complex II and the above antibody B
A method for measuring the degree of agglutination caused by an antigen-antibody reaction with the antibody, wherein the antibody B does not react with the antibody A ′ and the antigen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP395995A JPH08193999A (en) | 1995-01-13 | 1995-01-13 | Immune measuring method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP395995A JPH08193999A (en) | 1995-01-13 | 1995-01-13 | Immune measuring method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH08193999A true JPH08193999A (en) | 1996-07-30 |
Family
ID=11571642
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP395995A Pending JPH08193999A (en) | 1995-01-13 | 1995-01-13 | Immune measuring method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH08193999A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002010760A1 (en) * | 2000-07-27 | 2002-02-07 | Kyowa Medex Co., Ltd | Method of immunity examination with insoluble carrier particle and reagent therefor |
| WO2005114186A1 (en) | 2004-05-20 | 2005-12-01 | Wako Pure Chemical Industries, Ltd. | Method of assaying hyaluronic acid by using hyaluronic acid-binding protein |
| CN109991405A (en) * | 2017-12-29 | 2019-07-09 | 博阳生物科技（上海）有限公司 | A kind of immunity detection reagent and its application |
| CN109991411A (en) * | 2017-12-29 | 2019-07-09 | 博阳生物科技（上海）有限公司 | The method of immunity of target antibody and its application in a kind of detection sample to be tested |
-
1995
- 1995-01-13 JP JP395995A patent/JPH08193999A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002010760A1 (en) * | 2000-07-27 | 2002-02-07 | Kyowa Medex Co., Ltd | Method of immunity examination with insoluble carrier particle and reagent therefor |
| JP4950406B2 (en) * | 2000-07-27 | 2012-06-13 | 協和メデックス株式会社 | Immunoassay method using insoluble carrier particles and reagent thereof |
| US8431415B2 (en) | 2000-07-27 | 2013-04-30 | Tfb, Inc. | Immunoassay using insoluble carrier particles and reagent therefor |
| WO2005114186A1 (en) | 2004-05-20 | 2005-12-01 | Wako Pure Chemical Industries, Ltd. | Method of assaying hyaluronic acid by using hyaluronic acid-binding protein |
| JPWO2005114186A1 (en) * | 2004-05-20 | 2008-03-27 | 和光純薬工業株式会社 | Method for measuring hyaluronic acid using hyaluronic acid binding protein |
| JP2011090004A (en) * | 2004-05-20 | 2011-05-06 | Wako Pure Chem Ind Ltd | Hyaluronic acid measuring kit for immune-aggregation method |
| JP4730304B2 (en) * | 2004-05-20 | 2011-07-20 | 和光純薬工業株式会社 | Method for measuring hyaluronic acid using hyaluronic acid binding protein |
| CN109991405A (en) * | 2017-12-29 | 2019-07-09 | 博阳生物科技（上海）有限公司 | A kind of immunity detection reagent and its application |
| CN109991411A (en) * | 2017-12-29 | 2019-07-09 | 博阳生物科技（上海）有限公司 | The method of immunity of target antibody and its application in a kind of detection sample to be tested |
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