JPS638561A - Composition and method for immuno-analysis - Google Patents
Composition and method for immuno-analysisInfo
- Publication number
- JPS638561A JPS638561A JP15165386A JP15165386A JPS638561A JP S638561 A JPS638561 A JP S638561A JP 15165386 A JP15165386 A JP 15165386A JP 15165386 A JP15165386 A JP 15165386A JP S638561 A JPS638561 A JP S638561A
- Authority
- JP
- Japan
- Prior art keywords
- marker
- antigen
- antibody
- substance
- immunoassay
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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Abstract
Description
【発明の詳細な説明】
［発明の目的］
（産業上の利用分野）
本発明は試料中の特定の抗原又は抗体を定量分析するた
めの免疫分析用組成物及びこの組成物を用いた免疫分析
法に関するものである。Detailed Description of the Invention [Object of the Invention] (Industrial Application Field) The present invention relates to an immunoassay composition for quantitatively analyzing a specific antigen or antibody in a sample, and an immunoassay using this composition. It is about law.
（従来の技術）
従来の免疫分析方法として、例えば、ラジオアイソトー
プで標識した抗体（又は抗原）と生体より採取した試料
中の抗原（又は抗体）との抗原抗体反応を利用して試料
中の特定の抗原（又は抗体）を定損分析するラジオイム
ノアッセイ法や、酵素で、標識した抗体く又は抗原〉と
生体より採取した試料中の抗原（又は抗体）との抗原抗
体反応により抗原抗体結合物を得、その抗原抗体結合物
に標識した酵素による酵素反応を利用して試料中の特定
の抗原（又は抗体）を定量分析するエンザイムイムノア
ツセイ払等がある。(Prior art) As a conventional immunoassay method, for example, identification in a sample is performed using an antigen-antibody reaction between an antibody (or antigen) labeled with a radioisotope and an antigen (or antibody) in a sample collected from a living body. Radioimmunoassay method, which analyzes the loss of antigen (or antibody) in a living body, or an antigen-antibody reaction between an enzyme-labeled antibody or antigen and an antigen (or antibody) in a sample collected from a living body, is used to detect antigen-antibody conjugates. There are enzyme immunoassays that quantitatively analyze a specific antigen (or antibody) in a sample using an enzyme reaction with a labeled enzyme on the antigen-antibody conjugate.
しかしながら、前記ラジオイムノアッセイ法は、放射性
物質を利用するので設備が大がかりになるという欠点が
あり、また、ラジオイムノアッセイ法およびエンザイム
イムノアツセイ法のいずれにおいても、十分な検出感度
に達するまでには数時間から数十時間を要して分析に長
時間を要するという欠点がある。However, the radioimmunoassay method has the disadvantage that it requires large-scale equipment because it uses radioactive substances, and in both the radioimmunoassay method and the enzyme immunoassay method, it takes several times to reach sufficient detection sensitivity. The drawback is that analysis takes a long time, ranging from hours to several tens of hours.
上記問題点を解決した新規免疫分析法として、本発明者
らは先に赤血球、リポソーム等の組成物を利用した方法
を提案したく特願昭５７−１３５４８７号、同昭５７−
１３９４５４号）。As a new immunoassay method that solves the above-mentioned problems, the present inventors first proposed a method using compositions such as red blood cells and liposomes.
No. 139454).
ところで、このような組成物として用いられるリポソー
ムとしてはその脂質膜組成および形状から種々のものが
知られている（ＬｉｌｌＯＳＯｌｌｌｅ Ｔｅｃｈｎｏ
ｌＯ（ＩＶ ｖｏｌ、 １〜３ 、 Ｅｄ、ｂｙＧｒｅ
ｇｏｒｙ Ｇｒｅ（］０ｒｉａｄｉＳ。By the way, various types of liposomes used in such compositions are known depending on their lipid membrane compositions and shapes (LillOSOlle Techno.
lO(IV vol, 1-3, Ed, byGre
gory Gre(]0riadiS.
ＣＲＣＰＲＥＳＳ、 ＩＮｃ 、 Ｆｌｏｕｄａ、 １
９８４ ）。CRCPRESS, INc, Flouda, 1
984).
そのうち、リポソームを形状的に分類すると、その製造
方法により多層膜タイプ（ＭＬＶ）及び単＠膜タイプ（
ｔＪＬＶ）に分けられる。Among them, liposomes can be classified according to their shape into multilayer type (MLV) and single membrane type (MLV) depending on the manufacturing method.
tJLV).
ところで、免疫分析用組成物の条件としては、（１）均
一な粒度、
（２）マーカー含有率が高い（＝増［１〕率大）、（３
）安定性、
の３点が重要なポイントとなると思われる。By the way, the conditions for the composition for immunoassay are (1) uniform particle size, (2) high marker content (=high increase [1] rate), (3)
) Stability is considered to be the three important points.
しかしながら、現状のリポソームは上記条件に照らして
みると必ずしも満足のゆくものではない。However, current liposomes are not necessarily satisfactory in light of the above conditions.
すなわら、ＭＬＶの問題点としては、
（ａ）粒度が不均一：そのシＪ法からいって均一な粒度
が得がたく通常０．１〜１０μｍ（φ）の分布を有して
いる、
（ｂ）多層膜の間にマーカー液がサンドイッチされてい
る：このため膜破壊された時必ずしも１００％のマーカ
ーが溶出しない、
（Ｃ）バックグラウンドが高い、
等の問題がある。In other words, the problems with MLV are: (a) Nonuniform particle size: It is difficult to obtain a uniform particle size using the J method, and the particle size usually has a distribution of 0.1 to 10 μm (φ); (b) The marker liquid is sandwiched between the multilayer membranes: Therefore, there are problems such as not necessarily 100% of the marker eluting when the membrane is destroyed; and (C) high background.
また、ＵＬＶの問題点としては、
（ａ＞脂質膜が一層のみのため胞を大きくすると安定性
に欠ける、
（ｂ）安定な大きざとしては６００Ａ以下であり、この
ためマーカー含有量が低く増幅効果を期待出来ない、
等がおる。In addition, the problems with ULV are: (a) Since the lipid membrane is only one layer, it lacks stability if the cell is enlarged. (b) The stable size is less than 600A, so the marker content is low and amplification is poor. There are cases where you cannot expect the results to be effective.
（発明が解決しようとする問題点）
本発明は上述した従来の組成物の欠点をなくし、免疫分
析法に現想的な組成物を求めるべく検討した結果完成さ
れたものである。(Problems to be Solved by the Invention) The present invention was completed as a result of studies aimed at eliminating the drawbacks of the conventional compositions mentioned above and seeking a modern composition for immunoassay.
即ち、均一な粒度を有し、マーカー含有１が高く、バッ
クグラウンドが低く、しかも安定性に冨む組成物及びこ
の組成物を用いた免疫分析方法を提供することを目的と
するものである。That is, the object is to provide a composition having uniform particle size, high marker content 1, low background, and high stability, and an immunoassay method using this composition.
［発明の構成コ
（問題点を解決するための手段）
本発明の免疫分析用組成物は、均一粒度でかつマーカー
を含有した核物質とこの核物質を被覆する単層膜とを有
して構成される。[Configuration of the Invention (Means for Solving the Problems) The composition for immunoassay of the present invention comprises a nuclear material having a uniform particle size and containing a marker, and a monolayer film covering this nuclear material. configured.
また、本発明の免疫分析方法は、均一粒度でかつマーカ
ーを含有した核物質とこの核物質を被覆する単層膜とを
有する免疫分析用組成物における前記単層膜に抗原又は
抗体を固定化するものである。Further, the immunoassay method of the present invention provides an immunoassay composition comprising a nuclear material having a uniform particle size and containing a marker and a monolayer film covering the nuclear material, in which an antigen or an antibody is immobilized on the monolayer film. It is something to do.
（作用）
上記構成の免疫分析用組成物は、均一粒度でかつマーカ
ーを含有した核物質の外周を単層膜で被覆したものであ
るため、この組成物自体の粒度が均一化するとともに、
予めマーカーを各物質に含有しこれを単層膜で被覆して
いるので、外部からのマーカー吸着によるバックグラウ
ンドの高騰が防止され安定性が向上する。(Function) The composition for immunoassay having the above structure has a uniform particle size and coats the outer periphery of the marker-containing nuclear material with a single layer film, so that the particle size of the composition itself becomes uniform, and
Since markers are contained in each substance in advance and coated with a single layer film, background increases due to adsorption of markers from the outside are prevented and stability is improved.
また、本発明方法によれば、上記組成物に抗原又は抗体
を固定化するものであるから、短時間の分析で十分な検
出感度を１ｑられる。Furthermore, according to the method of the present invention, since the antigen or antibody is immobilized on the above-mentioned composition, a sufficient detection sensitivity of 1q can be achieved with a short analysis time.
（実施例）
以下に本発明の実施例を第１図及び第２図を参照して詳
細に説明する。(Example) Examples of the present invention will be described in detail below with reference to FIGS. 1 and 2.
均一な粒度を有し、マーカー含有量が多く、安定性を有
する核物質（リポソーム）１として、均−な親水性多孔
質又は籠状物質を用い、その核物質１にマーカー２とな
るべき酵素又は色素体を担持せしめ当該核物質１の外周
を脂質単層膜３で被覆する。A homogeneous hydrophilic porous or cage-like material is used as the core material (liposome) 1, which has uniform particle size, high marker content, and stability, and the enzyme to be the marker 2 is added to the core material 1. Alternatively, the outer periphery of the nuclear material 1 is coated with a lipid monolayer film 3 with a plastid supported thereon.
前記核物質１としては、粒度の均一な親水性ゲル状物質
例えばセファデックスＴＨ，セファロースＴＨ，キトン
サンビーズ、シリカゲルビーズ、ポーラスガラスピーズ
及びそれらのイオン交換体が応用可能である。As the core substance 1, hydrophilic gel-like substances with uniform particle size, such as Sephadex TH, Sepharose TH, chiton sun beads, silica gel beads, porous glass beads, and ion exchangers thereof can be used.
これらの物質を核物質１とし、これに脂質単層膜３を被
覆せしめれば自ずから完成したリポソームの粒度は均一
となり、本来不安定であった脂質単層ｆｆ１Ｊ３も核物
質１という骨組みの上に構築せしめる事により、膜自体
が安定化される。If these substances are used as the core material 1 and coated with the lipid monolayer membrane 3, the particle size of the completed liposome will become uniform, and the lipid monolayer ff1J3, which was originally unstable, will also be formed on the framework of the nuclear material 1. The membrane itself is stabilized by this construction.
上記組成物を免疫分析に供するためには、内部にマーカ
ー２を含有させ、更に外部に抗原５又は抗体（第１抗体
４又は第２抗体６）を感作する必要がある。In order to subject the composition to immunoassay, it is necessary to contain the marker 2 inside and to sensitize the outside with antigen 5 or antibody (first antibody 4 or second antibody 6).
このようなマーカー２としては、アルカリホスファター
ゼ（ＡＬＰ＞、β−ガラクトトシダーゼ。Such marker 2 includes alkaline phosphatase (ALP>, β-galactosidase).
パーオキシダーゼ等酵素又は各ｒＢ素に対応する基質、
更には蛍光色素をはじめとする各種色素体を用いること
ができる。An enzyme such as peroxidase or a substrate corresponding to each rB element,
Furthermore, various color bodies including fluorescent dyes can be used.
酵素としては上記記載のものに限定される訳ではないが
、安定かつ比活性の高いものが望ましい。Although the enzyme is not limited to those described above, it is desirable to use one that is stable and has high specific activity.
また、色素体としては、リポソーム自体のバックグラウ
ンドを低下させる面からも例えばカルボキシフルオレセ
イン又はカルセイン等の自己消光型蛍光物質が望ましい
。Further, as the pigment, a self-quenching fluorescent substance such as carboxyfluorescein or calcein is preferable from the viewpoint of reducing the background of the liposome itself.
前記核物質１に前記マーカー２を担持せしめる方法とし
ては吸着又は固定化が考えられる。すなわち、一般に酵
素に関して固定化が、その他の基質又は色素体に関して
は吸着が望ましい。酵素の固定化については幸い前記ゲ
ル状物質が各種官能基を有しているため、常法に従って
結合させる事が可能である。Possible methods for carrying the marker 2 on the nuclear material 1 include adsorption or immobilization. That is, in general, immobilization is desirable for enzymes, and adsorption is desirable for other substrates or pigments. Fortunately, the gel-like substance has various functional groups, so it is possible to immobilize the enzyme using conventional methods.
一方、吸着については核物質１をイオン交換体とする事
により、上述した吸着を実現可能とすることができｉｏ
このようにマーカー２を担持させた核物質１に脂質単層
膜３を被覆せしめるには、従来用いられて来たリポソー
ム及び一般マイクロカプセル調整法が適用可能である、
即ち、（ロータリーエバポレータ）＋（ソニック）法、
相分離法、液中乾燥法、スプレードライ法等である。On the other hand, regarding adsorption, the above-mentioned adsorption can be realized by using the nuclear material 1 as an ion exchanger. In this way, the nuclear material 1 carrying the marker 2 is coated with the lipid monolayer membrane 3. Conventionally used liposome and general microcapsule preparation methods can be applied to
That is, (rotary evaporator) + (sonic) method,
These methods include phase separation method, submerged drying method, and spray drying method.
尚、この場合の脂質単層膜３の組成は従来のリポソーム
に用いられて来た組成がそのまま適用出来る。また、被
覆後、必要に応じて原料脂質の相転移温度以上に加温し
たり、超音波処理を行なう等の処理をしてもよい。In this case, the composition of the lipid monolayer membrane 3 can be the same as that used for conventional liposomes. Further, after coating, treatments such as heating to a temperature higher than the phase transition temperature of the raw material lipid or ultrasonic treatment may be performed as necessary.
本実施例方法では、抗原５又は抗体（４又は６）を脂質
単層膜３の外周に感作させる。In the method of this example, the outer periphery of the lipid monolayer membrane 3 is sensitized with the antigen 5 or antibody (4 or 6).
感作方法としては従来知られている脂質成分（フォスフ
ァヂジルエタノールアミン又は糖脂質）を利用する方法
の他、前記核物質に疎水性物質よりなるスペーサ１１を
結合せしめ、そのスペーサ１１の先端に抗原５又は抗体
（４又は６）を感作させることも可能である。As a sensitization method, in addition to a method using a conventionally known lipid component (phosphadidylethanolamine or glycolipid), a spacer 11 made of a hydrophobic substance is bonded to the core substance, and the tip of the spacer 11 is It is also possible to sensitize the antigen 5 or antibody (4 or 6) to the antigen 5 or antibody (4 or 6).
スペーサ１１は疎水性物質であることから、脂゛ 質単
層膜３を貫通可能でその先端をこの膜の外表に突出させ
ることが出来る。Since the spacer 11 is a hydrophobic substance, it can penetrate the lipid monolayer membrane 3 and its tip can protrude to the outer surface of this membrane.
この様にして調整した核物質１としてのリポソームを抗
原抗体反応に供せしめると、当該リポソーム表面で抗原
５と抗体（／１又は６）が結合して抗原抗体結合物にな
る。When the liposome as the nuclear material 1 prepared in this manner is subjected to an antigen-antibody reaction, the antigen 5 and the antibody (/1 or 6) are combined on the surface of the liposome to form an antigen-antibody conjugate.
この抗原抗体結合物に対して補体７を添加すると前記抗
原抗体結合物により補体７が活性化され、通常のルート
に゛より膜障害が発生し、脂質単層膜３に孔８が必く。When complement 7 is added to this antigen-antibody conjugate, complement 7 is activated by the antigen-antibody conjugate, membrane damage occurs due to the normal route, and pores 8 are required in the lipid monolayer membrane 3. Ku.
従って、これまで脂質単層膜３により隔絶されていたリ
ポソーム内外は孔８によりつながり、例えばマーカー２
が基質９又は色素体の場合には、内部より外部への流出
が生じ、酵素の場合は外液中の基質９が流入して短時間
に酵素反応を誘起せしめる。Therefore, the inside and outside of the liposome, which were previously separated by the lipid monolayer membrane 3, are connected through the pore 8, and, for example, the marker 2
When it is a substrate 9 or a plastid, it flows out from the inside to the outside, and when it is an enzyme, the substrate 9 in the external solution flows in and induces an enzyme reaction in a short time.
その結果の色素体又は生成物１０を第３図に示す検量線
を用いて定母する事により、［組成物（リポソーム）の
破壊恒１＝［検液中の抗原又は抗体量］として締出する
事が可能となる。By standardizing the resulting plastids or products 10 using the calibration curve shown in Figure 3, it can be determined that the destruction constant of the composition (liposome) 1 = [the amount of antigen or antibody in the test solution]. It becomes possible to do so.
尚、水沫によるとマーカー２は予め核物質１にしっかり
と担持せしめた後、核物質１の外周を脂質単層膜３で被
覆するため、従来悩まされていた様な脂質膜に吸着した
マーカーによるバックグラウンドの高騰は皆無となる。According to Mizuyoshi, the marker 2 is firmly supported on the nuclear material 1 in advance, and then the outer periphery of the nuclear material 1 is covered with a lipid monolayer film 3. There will be no background spikes.
以上述べたように本実施例の組成物は核物質１の粒度を
揃える事により目的に応じた均一の粒度の組成物を調整
でき、測定結果の再現性向上を図ることができる。As described above, in the composition of this example, by making the particle size of the nuclear material 1 uniform, a composition with a uniform particle size can be adjusted according to the purpose, and the reproducibility of measurement results can be improved.
また、脂質単層膜３が単層のため補体活性化による膜障
害の効果が直接的である利点があるまた、単層の脂質膜
ではあるが内部に核物質１を有しているために安定であ
るとともに、従来のリポソーム調整法に比ベスケールア
ップが簡単となる。In addition, since the lipid monolayer membrane 3 is a single layer, it has the advantage that the effect of membrane damage due to complement activation is direct.Also, although it is a monolayer lipid membrane, it has nuclear substance 1 inside. It is stable and easy to scale up compared to conventional liposome preparation methods.
さらに、スペーサ１１を用いると膜脂質組成に関係なく
抗原又は抗体を感作可能となり、より安定な脂質組成を
選択することができる。Furthermore, by using the spacer 11, it is possible to sensitize the membrane with an antigen or antibody regardless of the membrane lipid composition, and a more stable lipid composition can be selected.
本発明は上述した実施例に限定されるものではなくその
要旨の範囲内で種々の変形が可能であることはいうまで
もない。It goes without saying that the present invention is not limited to the embodiments described above, and that various modifications can be made within the scope of the invention.
（実験例１）；各物質への酵素固定化
１ｍｌのキトサンビーズ〈キトパール１Ｍ）（バック）
と０．５ｍｌのアルカリ性ホスファターゼ／リン酸バッ
ファ（ＰＢ）（ｐＨ７，０＞（１，０００１Ｕ／ｍ１）
）と、ｏ、ｇｍ＋、６％のグルタルアルデヒド／リン酸
バッファ（ｐＨ７，０＞とを容器に入れ、温度条件５℃
で６０分間ゆっくり撹拌しつつ反応させた後、５ｍｌの
ＰＢＳ （ＰＢに０゜８５％量の塩化ナトリウムを添加
）を添加し、ざらに、５°Ｃで容器を３０００ｒｌ）ｍ
の回転速度で１０分間回転攪拌させた。そして、沈澱物
を収集しこれを５ｍｌのＰＢで３回洗浄してアルカリ性
ホスファターゼ固定化キトサンビーズ（ＡＬＰ／ＣトＩ
ＩＯ＞を１ｑた。このアルカリ性ホスファターゼ固定化
キトサンビーズのＡＬＰ活性は４００ＩＵ／ｍＮ回収率
８０％）であった。(Experiment Example 1) ; Enzyme immobilization on each substance 1 ml of chitosan beads <Chito Pearl 1M) (back)
and 0.5 ml alkaline phosphatase/phosphate buffer (PB) (pH 7.0>(1,0001 U/ml)
) and o, gm+, 6% glutaraldehyde/phosphate buffer (pH 7,0>) were placed in a container, and the temperature condition was 5°C.
After reacting with slow stirring for 60 minutes, 5 ml of PBS (added 85% sodium chloride to PB) was added, and the container was heated to 3000 ml at 5° C.
The mixture was rotated and stirred for 10 minutes at a rotational speed of . Then, the precipitate was collected and washed three times with 5 ml of PB, and the precipitate was washed with alkaline phosphatase-immobilized chitosan beads (ALP/C to I).
1q of IO>. The ALP activity of the alkaline phosphatase-immobilized chitosan beads was 400 IU/mN (recovery rate 80%).
実験例２：脂質膜コーティング
５００μｍのＡＬＰ／ＣＨＩＴＯの付層水分を除去し、
１ｍｌの脂質溶液（クロロホルム中にコレストロール１
０「ｎμｍ、ジパルミトイルホスファチジルコリン１０
ｍμ１．ジチオピリジル・ジパルミトイルホスファチジ
ルエタノールアミン５ｍμｍを含有したもの）に添加し
攪拌した後、混液を激しく攪拌させている１００ｍ１の
ＰＢＳ中に添加し、陰性にて溶媒（クロロホルム）を除
去し 。Experimental example 2: removing the moisture from the lipid membrane coating of 500 μm ALP/CHITO,
1 ml of lipid solution (1 ml of cholesterol in chloroform)
0"nμm, dipalmitoylphosphatidylcholine 10
mμ1. After stirring, the mixture was added to 100 ml of PBS with vigorous stirring, and the solvent (chloroform) was removed under negative conditions.
た。Ta.
イして、この混液を４５°Ｃに加温しつつ１０分間に拝
した。次に５分間超音波処理を実施し、ざらに、５°Ｃ
で容器を３００Ｏｒｐｍの回転速度で１Ｑ分間回転攪拌
させた。そして、沈澱物を収集しこれを５ｍｌのＰＢＳ
で３回洗浄して有核リポソームを得た。ＡＬＰ活性は３
２０ＩＵ／ｍｌ （回収率８０％）であった。The mixture was heated to 45°C for 10 minutes. Next, perform ultrasonication for 5 minutes, and heat at 5°C.
The container was rotated and stirred for 1Q minutes at a rotational speed of 300 rpm. Then, collect the precipitate and add it to 5ml of PBS.
was washed three times to obtain nucleated liposomes. ALP activity is 3
It was 20 IU/ml (recovery rate 80%).
（実験例３）：有核リポソームへの抗体感作５００μｍ
の有核リポソームを５００μｍの５ＰＤＰ （Ｎ−サク
シイミジル−３−（２−ピリジルチオノプロピオネート
）化抗ヒトＡ Ｆ Ｐ抗体（ヤギ由来、蛋白質５００μ
ＣＩ／ｍ ｌ ＞に添１叫し、２０’Ｃで一夜ゆっくり
攪拌した。次に５°Ｃで容器を３００Ｏｒｐｍの回転速
度で１０分間回転攪拌させた。そして、沈澱物を収集し
これを５ｍｌのＰＢＳで３回洗浄して高ヒトＡＦＰ抗体
監査リポソーム（監査リポソーム）を得た。蛋白質感作
率は２４％であった。(Experiment Example 3): Antibody sensitization to nucleated liposomes 500 μm
A nucleated liposome of 500 μm was prepared using 5PDP (N-succinimidyl-3-(2-pyridylthionopropionate))-conjugated anti-human AFP antibody (goat-derived, protein 500 μm).
CI/ml> was added and slowly stirred at 20'C overnight. Next, the container was rotated and stirred at 300 rpm for 10 minutes at 5°C. Then, the precipitate was collected and washed three times with 5 ml of PBS to obtain a high human AFP antibody audit liposome (audit liposome). The protein production rate was 24%.
（実験例４）：ＡＦＰの測定
ＰＢＳ中の１０μｍのヒトＡＦＰ標準液［（０，１，１
０，２０，５０，１００，５００゜１０００ｕＱ／＋１
１＞／？イクロタイタウエル１をＰＢＳ中の１０μｍ、
３％の感作リポソームに添加し、攪拌した後、３７°Ｃ
で５分間反応させた。(Experimental Example 4): Measurement of AFP 10 μm human AFP standard solution in PBS [(0,1,1
0,20,50,100,500゜1000uQ/+1
1>/? 10 μm of Icrotita Well 1 in PBS;
Add to 3% sensitized liposomes, stir, and then heat to 37°C.
The mixture was allowed to react for 5 minutes.
そして、さらにこの混液をＰＢＳ中の１０μｍの高ヒト
ＡＦＰ抗体くウサギ由来）に添加し、３７℃で５分間反
応させた。次にこの混液と１５０μｍの補体（４ＣＨ５
０／ｍｌ）、１０ｍｕＩの４−メチルウンベリフェリル
リン酸含有ＧＶＢ″＋（こ３７°Ｃ，３０分反応させた
。Then, this mixed solution was further added to a 10 μm high human AFP antibody (derived from rabbit) in PBS, and reacted at 37° C. for 5 minutes. Next, this mixture and 150μm complement (4CH5
0/ml), 10 muI of 4-methylumbelliferyl phosphate-containing GVB''+ (this was reacted at 37°C for 30 minutes.
蛍光測定時の励起波長（ＥＸＣｉ ｔａｔ ｉ ｏｎ）
λＥＸは４９０ｎｍ、蛍光放射波長（Ｅｍｉｓｓｉｏｎ
）λ［ｍは５３０ｎｍであった。Excitation wavelength during fluorescence measurement (EXCi tation)
λEX is 490 nm, fluorescence emission wavelength (Emission
)λ[m was 530 nm.
し発明の効果１
以上詳述した本発明によれば、目的に応じた均一の粒度
を有し、マーカー含有母が多くてバックグラウンドも低
く、しかも安定性に優れた免疫分析用の組成物を調整用
きる。Effects of the Invention 1 According to the present invention detailed above, a composition for immunoassay that has a uniform particle size according to the purpose, contains a large amount of markers, has a low background, and has excellent stability. Can be adjusted.
また、本発明方法によれば、短時間の分析で充分な検出
感度が得られ免疫分析方法を提供することができる。Further, according to the method of the present invention, sufficient detection sensitivity can be obtained with short-time analysis, and an immunoassay method can be provided.
第１図、第２図はそれぞれ有核リポソームによる免疫反
応の概略説明図、第３図はＡＦＰと相対蛍光強度との関
係を示す検酊線を示づグラフである。
１・・・・・・核物質、２・・・・・・マーカー、３・
・・・・・肥質単層膜、
４・・・・・・第１抗体、
５・・・・・・抗原、
６・・・・・・第２抗体。
代理人 弁理士 則 近 憲 佑
同 大胡典夫
第１図
第２図FIGS. 1 and 2 are schematic explanatory diagrams of the immune reaction caused by nucleated liposomes, and FIG. 3 is a graph showing a test line showing the relationship between AFP and relative fluorescence intensity. 1...Nuclear material, 2...Marker, 3.
... Hypertrophic monolayer, 4 ... First antibody, 5 ... Antigen, 6 ... Second antibody. Agent Patent Attorney Norio Chika Yudo Norio Ogo Figure 1 Figure 2
Claims (8)
核物質を被覆する単層膜とを有することを特徴とする免
疫分析用組成物。(1) A composition for immunoassay comprising a nuclear material having a uniform particle size and containing a marker and a monolayer film covering the nuclear material.
溶解作用を受ける脂質膜である特許請求の範囲第１項記
載の免疫分析用組成物。(2) The composition for immunoassay according to claim 1, wherein the membrane covering the outer periphery of the nuclear substance is a lipid membrane that undergoes a dissolving action due to complement activity.
特許請求の範囲第１項若しくは第２項記載の免疫分析用
組成物。(3) The composition for immunoassay according to claim 1 or 2, wherein the core substance is a porous or cage-like hydrophilic substance.
核物質を被覆する単層膜とを有する免疫分析用組成物に
おける前記単層膜に抗原又は抗体を固定化することを特
徴とする免疫分析方法。(4) An immunoassay composition comprising a nuclear material having a uniform particle size and containing a marker and a monolayer film covering the nuclear material, characterized in that an antigen or antibody is immobilized on the monolayer film. Analysis method.
介して抗原又は抗体を固定化するものである特許請求の
範囲第４項記載の免疫分析方法。(5) The immunoassay method according to claim 4, wherein an antigen or antibody is immobilized on the nuclear material via a spacer that penetrates a peripheral monolayer membrane.
求の範囲第４項記載の免疫分析方法。(6) The immunoassay method according to claim 4, wherein an enzyme is immobilized on the nuclear material.
請求の範囲第４項記載の免疫分析方法。(7) The immunoassay method according to claim 4, wherein a substrate is adsorbed to each of the substances.
許請求の範囲第４項記載の免疫分析方法。(8) The immunoassay method according to claim 4, wherein a plastid is adsorbed to the nuclear substance.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP15165386A JPS638561A (en) | 1986-06-30 | 1986-06-30 | Composition and method for immuno-analysis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP15165386A JPS638561A (en) | 1986-06-30 | 1986-06-30 | Composition and method for immuno-analysis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS638561A true JPS638561A (en) | 1988-01-14 |
Family
ID=15523277
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP15165386A Pending JPS638561A (en) | 1986-06-30 | 1986-06-30 | Composition and method for immuno-analysis |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS638561A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6165798A (en) * | 1996-10-10 | 2000-12-26 | University Of British Columbia | Optical quantification of analytes in membranes |
| JP2008122236A (en) * | 2006-11-13 | 2008-05-29 | Ulvac Japan Ltd | Fixing method of lipid to solid phase surface of sensor, measuring method by sensor, and lipid film fixing sensor |
-
1986
- 1986-06-30 JP JP15165386A patent/JPS638561A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6165798A (en) * | 1996-10-10 | 2000-12-26 | University Of British Columbia | Optical quantification of analytes in membranes |
| JP2008122236A (en) * | 2006-11-13 | 2008-05-29 | Ulvac Japan Ltd | Fixing method of lipid to solid phase surface of sensor, measuring method by sensor, and lipid film fixing sensor |
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