JPH11174057A - Immunity measuring base material - Google Patents
Immunity measuring base materialInfo
- Publication number
- JPH11174057A JPH11174057A JP34559897A JP34559897A JPH11174057A JP H11174057 A JPH11174057 A JP H11174057A JP 34559897 A JP34559897 A JP 34559897A JP 34559897 A JP34559897 A JP 34559897A JP H11174057 A JPH11174057 A JP H11174057A
- Authority
- JP
- Japan
- Prior art keywords
- molecule
- molecules
- substrate
- immobilized
- base material
- 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
- 239000000463 material Substances 0.000 title abstract description 18
- 230000036039 immunity Effects 0.000 title abstract 3
- 238000001179 sorption measurement Methods 0.000 claims abstract description 36
- 125000000524 functional group Chemical group 0.000 claims abstract description 18
- 238000005259 measurement Methods 0.000 claims abstract description 14
- 239000000758 substrate Substances 0.000 claims description 30
- 238000003018 immunoassay Methods 0.000 claims description 22
- 125000003277 amino group Chemical group 0.000 claims description 7
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 7
- 230000035945 sensitivity Effects 0.000 abstract description 10
- 239000007790 solid phase Substances 0.000 abstract description 8
- 230000000903 blocking effect Effects 0.000 description 10
- 238000005406 washing Methods 0.000 description 10
- 102000009027 Albumins Human genes 0.000 description 9
- 108010088751 Albumins Proteins 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 238000000034 method Methods 0.000 description 7
- 235000005956 Cosmos caudatus Nutrition 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 5
- 239000008363 phosphate buffer Substances 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 239000004793 Polystyrene Substances 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 239000002585 base Substances 0.000 description 4
- 239000002981 blocking agent Substances 0.000 description 4
- 108040007629 peroxidase activity proteins Proteins 0.000 description 4
- 102000013415 peroxidase activity proteins Human genes 0.000 description 4
- 229920002223 polystyrene Polymers 0.000 description 4
- 238000004381 surface treatment Methods 0.000 description 4
- 229920001342 Bakelite® Polymers 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 239000004637 bakelite Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 2
- 101000930457 Rattus norvegicus Albumin Proteins 0.000 description 2
- 150000001718 carbodiimides Chemical class 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 230000008105 immune reaction Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000008055 phosphate buffer solution Substances 0.000 description 2
- 229920003229 poly(methyl methacrylate) Polymers 0.000 description 2
- 239000004926 polymethyl methacrylate Substances 0.000 description 2
- 235000020183 skimmed milk Nutrition 0.000 description 2
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 230000000274 adsorptive effect Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 229910052786 argon Inorganic materials 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- XUCNUKMRBVNAPB-UHFFFAOYSA-N fluoroethene Chemical group FC=C XUCNUKMRBVNAPB-UHFFFAOYSA-N 0.000 description 1
- 239000001307 helium Substances 0.000 description 1
- 229910052734 helium Inorganic materials 0.000 description 1
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 239000011261 inert gas Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- -1 polypropylene Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、抗原抗体反応を利
用して抗体又は抗原を検出する免疫測定で、抗原又は抗
体を基材に固相化して測定する固相化法に用いる基材の
材質及び表面処理に関するものである。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunoassay for detecting an antibody or an antigen by utilizing an antigen-antibody reaction. It relates to material and surface treatment.
【０００２】[0002]
【従来の技術】従来の固相化法による免疫測定において
は、ポリスチレン等のプラスチック基材に非特異的吸着
又は共有結合を利用して抗体又は抗原等の分子を固相化
し、固相化した分子に対して免疫反応を利用してサンプ
ル溶液中の目的の分子を補足させ、最終的にラジオアイ
ソトープ、酵素、蛍光物質等で標識した分子を目的の分
子に特異的に補足させて定量する方法が一般的に行われ
ている。免疫測定は、研究開発の分野だけではなく、臨
床検査といった医療の分野でも多く用いられており、測
定結果が事実と誤った値であった場合、人命に係わる重
篤な事態を引き起こす恐れもあるため、測定感度、安定
性（再現性）については特に重要視すべきである。2. Description of the Related Art In a conventional immunoassay using a solid phase immobilization method, a molecule such as an antibody or an antigen is immobilized on a plastic substrate such as polystyrene using nonspecific adsorption or covalent bonding. A method in which a target molecule in a sample solution is captured using an immune reaction to the molecule, and finally, a molecule labeled with a radioisotope, an enzyme, a fluorescent substance, or the like is specifically captured and quantified. Is commonly done. Immunoassays are used not only in research and development but also in medical fields such as clinical tests, and if the measurement result is incorrect, it may cause a serious situation that can be life-threatening. Therefore, measurement sensitivity and stability (reproducibility) should be particularly emphasized.
【０００３】本来、抗原抗体反応は非常に特異的で安定
な反応であるため、その反応を利用した免疫測定法は理
論上非常に特異的、かつ高感度に目的分子を検出出来る
はずである。しかし、従来の方法においては、固相化分
子の固相化量を上げるために高吸着の材料又は表面処理
を行った材料を使用した基材を用いているため、固相化
分子以外の物質が非特異的吸着によって測定系に残留
し、その結果、感度、安定性が低下してしまう。[0003] Since an antigen-antibody reaction is originally a very specific and stable reaction, an immunoassay utilizing this reaction should theoretically be able to detect a target molecule with very specific and high sensitivity. However, in the conventional method, since a substrate using a material having high adsorption or a material subjected to a surface treatment is used to increase the amount of the immobilized molecule, a substance other than the immobilized molecule is used. Remains in the measurement system due to non-specific adsorption, resulting in reduced sensitivity and stability.
【０００４】尚、従来の免疫測定法においては前述のよ
うな固相化分子以外の非特異的吸着を防ぐために固相化
分子を固相化した後に、スキムミルクやアルブミン等の
一般的にブロッキング剤と呼ばれる吸着性の高い蛋白で
基材表面を覆い、他の分子の非特異的吸着を阻害するブ
ロッキング操作を行っていたが、ブロッキング操作に
は、ブロッキング剤の種類、溶媒の種類、ｐＨ、温度、
反応時間等のブロッキングの条件を間違うと全く効果が
得られず、条件の検討に時間を要すること、又最適な条
件を選択してもブロッキングによって完全に基材への吸
着を防ぐことは不可能であること、例え完全にブロッキ
ングが達成されたとしても、ブロッキング剤上への非特
異的吸着が避けられないこと、更に、固相化分子が低分
子量であった場合、分子量の比較的大きなブロッキング
剤の下に隠れてしまうといった問題点、及びブロッキン
グ効果及び非特異的吸着は測定時の湿度にも影響を受け
るために従来の免疫測定において測定毎の再現性が得ら
れない場合が多いといった問題点もあり、ブロッキング
によって完全に非特異的吸着を防ぐことは出来ず、結果
的にばらつきを増大させる原因になっていた。[0004] In the conventional immunoassay, after a solid-phased molecule is immobilized in order to prevent non-specific adsorption other than the above-mentioned solid-phased molecule, a blocking agent such as skim milk or albumin is generally used. The surface of the substrate was covered with a highly adsorptive protein, called blocking, and a blocking operation was performed to prevent nonspecific adsorption of other molecules.However, the blocking operation includes the type of blocking agent, type of solvent, pH, and temperature. ,
If the blocking conditions such as reaction time are incorrect, no effect can be obtained at all, and it takes time to study the conditions. Even if the optimal conditions are selected, it is impossible to completely prevent adsorption to the substrate by blocking That, even if the blocking is completely achieved, non-specific adsorption on the blocking agent is unavoidable, furthermore, if the solid-phased molecule has a low molecular weight, a relatively large blocking of the molecular weight The problem of hiding under the agent, and the problem that the blocking effect and non-specific adsorption are often affected by the humidity at the time of measurement, so that reproducibility for each measurement cannot be obtained in conventional immunoassays in many cases. Due to this point, non-specific adsorption could not be completely prevented by the blocking, and as a result, the variation was increased.
【０００５】[0005]
【発明が解決しようとする課題】本発明は、上述のよう
な従来の問題点を解決すべく鋭意検討の結果なされたも
ので、非特異的吸着を無くすことで目的以外の分子が測
定系へ残留することを無くし、その結果従来の免疫測定
より高感度で安定性（再現性）に優れた免疫測定用基材
の提供を目的とするものである。DISCLOSURE OF THE INVENTION The present invention has been made as a result of intensive studies in order to solve the above-mentioned conventional problems. By eliminating non-specific adsorption, molecules other than the target molecules can be transferred to a measurement system. An object of the present invention is to provide a substrate for immunoassay which eliminates the residue and as a result has higher sensitivity and better stability (reproducibility) than conventional immunoassays.
【０００６】[0006]
【課題を解決するための手段】即ち本発明は、測定に使
用する分子の非特異的吸着が２５℃において５×１０-4
ｎｍｏｌ／ｃｍ2以下である基材表面に、固相化に用い
る分子との結合が可能な官能基を有する免疫測定用基材
である。That is, the present invention provides a method for measuring the non-specific adsorption of a molecule used for measurement at 25 ° C. at 5 × 10 -4.
This is an immunoassay substrate having a functional group capable of binding to a molecule used for immobilization on the surface of the substrate having a thickness of nmol / cm 2 or less.
【０００７】[0007]
【発明の実施の形態】本発明においては、従来の高吸着
な基材とは逆に、基材自体に分子の非特異的な吸着が５
×１０-4ｎｍｏｌ／ｃｍ2以下である材料又は表面処理
を施したものを使用し、最初に固相化する分子は基材表
面に存在または生成させた官能基を利用し、イオン結合
もしくは共有結合によって固定化させることとした。そ
れによってばらつきの原因の一つであるブロッキング操
作は不要となり、目的以外の分子は系に残留することな
く、高感度で安定な測定が可能になる。更に、固相化分
子の固相化量を表面官能基だけで制御することで高感度
以外のメリットも生まれる。DESCRIPTION OF THE PREFERRED EMBODIMENTS In the present invention, contrary to the conventional high-adsorption base material, non-specific adsorption of molecules to the base material itself is 5%.
A material having a surface treatment of × 10 −4 nmol / cm 2 or less is used, and molecules to be first immobilized are ionic-bonded or shared by utilizing functional groups present or generated on the substrate surface. It was immobilized by bonding. This eliminates the need for a blocking operation, which is one of the causes of variation, and enables high-sensitivity and stable measurement without leaving molecules other than the target in the system. Further, by controlling the amount of immobilized molecules by only the surface functional groups, advantages other than high sensitivity can be obtained.
【０００８】即ち、従来の免疫測定用基材においては、
多くの非特異的吸着が存在したため、固相化分子の固相
化量は溶液の濃度もしくは固相化時間で調節するしかな
かった。しかし、安定した固相化を行うには固相化分子
を過剰量加えて過剰時間吸着させることが必須であり、
基材が有する吸着能力以下の量を固相化する事は、固相
化量が安定せず非常に困難であった。That is, in a conventional substrate for immunoassay,
Due to the presence of many non-specific adsorptions, the amount of immobilized molecules could only be adjusted by the concentration of the solution or the time of immobilization. However, in order to perform stable solid-phase immobilization, it is essential to add an excessive amount of solid-phased molecules and perform adsorption for an excessive time,
It was very difficult to solidify an amount less than the adsorption capacity of the substrate because the amount of solidification was not stable.
【０００９】本発明の免疫測定用基材においては、固相
化分子の固相化量は、基材表面の官能基の数だけで決定
されるため、官能基密度を制御する事で固相化量を制御
することが可能である。例えば低濃度の固相化分子を使
用する場合は、基材表面の官能基密度を下げる、つまり
固相化分子濃度が官能基密度に対して過剰量になる程度
に官能基密度を下げることで、安定した固相化を行うこ
とが出来る。一般的に使用される抗原等の固相化分子は
特に高価なものが多く、低濃度で安定した固相化が出来
れば、試薬節約の意味でも非常に有意義である。In the immunoassay substrate of the present invention, the amount of immobilized molecules is determined only by the number of functional groups on the surface of the substrate. It is possible to control the amount of conversion. For example, when using low-concentration immobilized molecules, the functional group density on the substrate surface is reduced, that is, the functional group density is reduced to such an extent that the immobilized molecule concentration becomes excessive with respect to the functional group density. And a stable solid phase can be obtained. Many commonly used immobilized molecules such as antigens are particularly expensive, and if stable immobilization can be performed at a low concentration, it is very significant in terms of saving reagents.
【００１０】また、臨床検査用として用いる場合、検査
時に固相化から行うのではなく、固相化分子を基材に固
相した状態のものを一度に大量に作成し、保存してお
き、検査時にはすでに固相化している基材を取り出して
使用されるのが一般的であるが、非特異的吸着が存在す
ると、非特異的吸着により固相化された分子は不安定で
あるため保存状況によっては基材表面から脱離してしま
い、経時的にばらつきを発生する事が多い。しかし、本
発明の免疫測定用基材においては非特異的吸着は存在せ
ず、安定な共有結合、イオン結合によって固相化されて
いるため、固相化後の保存にも安定である。In addition, when used for a clinical test, a large number of solid-phased molecules are prepared and stored at once, instead of being immobilized at the time of the test. During inspection, it is common to remove the substrate that has already been immobilized and use it.However, if non-specific adsorption is present, the molecules immobilized due to non-specific adsorption are unstable and should be stored. Depending on the situation, they are detached from the surface of the base material, and often vary with time. However, the non-specific adsorption does not exist in the immunoassay substrate of the present invention, and the substrate is immobilized by stable covalent and ionic bonds.
【００１１】本発明の免疫測定用基材は、分子の非特異
的吸着が５×１０-4ｎｍｏｌ／ｃｍ2以下といった非特
異的吸着を起こしにくい材料表面に固相化分子との結合
可能な官能基を生成させている点が特徴であり、最初に
固相化分子を該官能基と結合させた後は、分子が基材表
面に非特異的に吸着することは殆どなく目的の分子のみ
が特異的な免疫反応により基材表面に補足され、結果と
して高感度で安定した結果を得ることが出来る。前述の
通り、感度、安定性に最も優れているのは、目的以外の
分子の非特異的吸着が全く起こらない表面であるが、実
際、固相化法による免疫測定を行う場合、固相化分子の
固相化量は、５×１０-3程度であるため、分子の非特異
的な吸着は５×１０-4ｎｍｏｌ／ｃｍ2以下で有れば問
題はない。The substrate for immunoassay according to the present invention is capable of binding to immobilized molecules on the surface of a material which is less likely to cause nonspecific adsorption, such as 5 × 10 −4 nmol / cm 2 or less. The feature is that a functional group is generated.After the solid-phased molecule is first bound to the functional group, the molecule hardly non-specifically adsorbs to the substrate surface, and only the target molecule is formed. Is captured on the substrate surface by a specific immune reaction, and as a result, a highly sensitive and stable result can be obtained. As described above, the surface with the highest sensitivity and stability has no non-specific adsorption of molecules other than the target, but in fact, when performing immunoassay by the solid-phase method, Since the amount of immobilized molecules is about 5 × 10 −3 , there is no problem if the nonspecific adsorption of molecules is 5 × 10 −4 nmol / cm 2 or less.
【００１２】分子の非特異的な吸着が５×１０-4ｎｍｏ
ｌ／ｃｍ2以下である表面を得るために使用する材料と
しては、分子の非特異的な吸着が５×１０-4ｎｍｏｌ／
ｃｍ2以下である材料、若しくは分子の非特異的な吸着
が５×１０-4ｎｍｏｌ／ｃｍ2以下である表面処理を施
した材料で有れば特に限定するものではないが、ポリプ
ロピレン、ポリテトラフルオロエチレン（ＰＴＦＥ）等
の高分子が挙げられる又、ポリスチレンの様に非特異的
吸着の起こりやすい材料を使用する場合には、プラズマ
暴露によるカルボキシル基及び／または水酸基の導入、
ポリメチルメタクリレートを使用する場合であればアル
カリによる表面部分加水分解でカルボキシル基を導入す
る等の表面改質により非特異的な吸着が５×１０-4ｎｍ
ｏｌ／ｃｍ2以下である表面を実現することが出来る。
また分子の非特異的な吸着が５×１０-4ｎｍｏｌ／ｃｍ
2以下である材料をコーティングしても良い。Non-specific adsorption of molecules is 5 × 10 -4 nmo
As a material used to obtain a surface of 1 / cm 2 or less, nonspecific adsorption of molecules is 5 × 10 −4 nmol /
cm 2 or less is material, or is not particularly limited as long in a non-specific adsorption was subjected to a 5 × 10 -4 nmol / cm 2 or less is a surface treatment material molecules, polypropylene, polytetra Polymers such as fluoroethylene (PTFE) may be used. In addition, when a material such as polystyrene which is apt to cause non-specific adsorption is used, introduction of carboxyl groups and / or hydroxyl groups by plasma exposure,
In the case of using polymethyl methacrylate, nonspecific adsorption is 5 × 10 −4 nm by surface modification such as introduction of a carboxyl group by partial surface hydrolysis with alkali.
ol / cm 2 or less can be realized.
In addition, non-specific adsorption of molecules is 5 × 10 −4 nmol / cm.
A material having 2 or less may be coated.
【００１３】基材表面に有する分子との結合が可能な官
能基は特に限定するものではないが、基材表面に有する
官能基がアミノ基である場合は、分子内にアミノ基を有
する固相化分子であればグルタルアルデヒドを介して固
相化する事が出来、又、分子内にカルボキシル基を有す
る固相化分子であればカルボジイミドを利用して固相化
する事が出来る。基材表面に有する官能基がカルボキシ
ル基である場合は、分子内にアミノ基を有する固相化分
子であれば、カルボジイミドを利用して固相化する事が
出来る。The functional group capable of binding to the molecule on the substrate surface is not particularly limited. However, when the functional group on the substrate surface is an amino group, a solid phase having an amino group in the molecule is used. The immobilized molecule can be immobilized via glutaraldehyde, and the immobilized molecule having a carboxyl group in the molecule can be immobilized using carbodiimide. When the functional group on the substrate surface is a carboxyl group, any solid-phased molecule having an amino group in the molecule can be solid-phased using carbodiimide.
【００１４】基材表面に官能基を付与する方法として
は、特に限定するものではないが、一般的に行われてい
るプラズマ暴露による官能基付与の方法を用いることが
出来る。例えば、アンモニアプラズマ又は窒素と酸素の
混合プラズマによってアミノ基を付与することが出来、
アルゴン、ヘリウム等の不活性ガスのプラズマ又は、酸
素プラズマによってカルボキシル基を導入することが出
来る。The method for imparting a functional group to the substrate surface is not particularly limited, but a generally used method for imparting a functional group by plasma exposure can be used. For example, an amino group can be provided by ammonia plasma or a mixed plasma of nitrogen and oxygen,
A carboxyl group can be introduced by plasma of an inert gas such as argon or helium or oxygen plasma.
【００１５】[0015]
【実施例】以下、実施例によって本発明を更に具体的に
説明する。 （実施例１）市販のポリスチレン製９６穴ＥＬＩＳＡ用
プレート（住友ベークライト製 ＭＳ−８４９６Ｆ）を
窒素と酸素の混合プラズマで１５分処理を行い、基材表
面に水酸基とアミノ基を生成させたものを実施例１とし
た。 （実施例２）９６穴マイクロプレートをポリメチルメタ
クリレートで成形、成形後ウェルに２Ｎの水酸化ナトリ
ウムを分注、５０℃で１０時間処理して表面部分加水分
解を行い、表面にカルボキシル基と水酸基を生成させた
ものを実施例２とした。 （比較例）市販のポリスチレン製９６穴ＥＬＩＳＡ用プ
レート（住友ベークライト製 ＭＳ−８４９６Ｆ）を比
較例として用いた。The present invention will be described more specifically with reference to the following examples. (Example 1) A 96-well commercially available polystyrene ELISA plate (MS-8496F, manufactured by Sumitomo Bakelite) was treated with a mixed plasma of nitrogen and oxygen for 15 minutes to produce a hydroxyl group and an amino group on the substrate surface. Example 1 was used. (Example 2) A 96-well microplate was molded with polymethyl methacrylate. After molding, 2N sodium hydroxide was dispensed into the wells, and the mixture was treated at 50 ° C for 10 hours to partially hydrolyze the surface. Example 2 was produced. Comparative Example A commercially available polystyrene 96-well ELISA plate (MS-8496F, manufactured by Sumitomo Bakelite) was used as a comparative example.
【００１６】（非特異的吸着性の比較）非特異的吸着性
を比較するため、酵素で標識した抗ヒトＩｇＧ抗体（コ
スモバイオ 製）を１００ｎｇ／ｍｌ、１μｇ／ｍｌ、
１０μｇ／ｍｌ、１００μｇ／ｍｌの濃度系列で、各濃
度を２４ウェルづつ分注し、３時間吸着させた。尚、実
施例１及び実施例２のプレートについては、予めアミノ
基又はカルボキシル基を失活させてから検討に用いた。
標識酵素は、ペルオキシターゼを用いて、吸光度から吸
着量を求め、ウェルと溶液の接触面積から密度として算
出した。結果は、図１の通りで、実施例１、実施例２の
プレートともに比較例に比べ非特異的吸着性が低下して
いることを確認した。(Comparison of non-specific adsorptivity) In order to compare the non-specific adsorptivity, an anti-human IgG antibody (manufactured by Cosmo Bio) labeled with an enzyme was added at 100 ng / ml, 1 μg / ml,
In a concentration series of 10 μg / ml and 100 μg / ml, each concentration was dispensed in 24 wells and adsorbed for 3 hours. In addition, about the plate of Example 1 and Example 2, after deactivating amino group or carboxyl group beforehand, it was used for examination.
For the labeled enzyme, the amount of adsorption was determined from the absorbance using peroxidase, and calculated as the density from the contact area between the well and the solution. The results are as shown in FIG. 1, and it was confirmed that the non-specific adsorptivity of both the plates of Example 1 and Example 2 was lower than that of the comparative example.
【００１７】（免疫測定感度の比較）免疫測定感度の比
較として、固相化抗体として抗ラットアルブミン抗体、
測定抗原としてラットアルブミン、標識抗体としてペル
オキシターゼ標識抗ラットアルブミン抗体を用いて免疫
測定を行った。各プレートの測定法は下記の通り。(Comparison of immunoassay sensitivity) As a comparison of immunoassay sensitivity, an anti-rat albumin antibody was used as an immobilized antibody.
Immunoassay was performed using rat albumin as a measurement antigen and peroxidase-labeled anti-rat albumin antibody as a labeled antibody. The measurement method for each plate is as follows.
【００１８】（実施例１）プレートの各ウェルに、ｐＨ
７．４のリン酸緩衝液で２％に希釈したグルタルアルデ
ヒド（電子顕微鏡用２０％ 和光純薬製）を２００μＬ
／ウェルで分注し、３７℃で２時間静置した。次に、洗
浄液（０．０５％Ｔｗｅｅｎ２０入りｐＨ７．４のリン
酸緩衝液）３００μＬ／ウェルで３回洗浄した後に、ｐ
Ｈ７．４のリン酸緩衝液で５μｇ／ｍｌに希釈した抗ラ
ットアルブミン抗体（コスモバイオ製）を２００μＬ／
ウェルで分注し３７℃で２時間静置した。(Example 1) pH of each well of a plate
200 μL of glutaraldehyde (20% manufactured by Wako Pure Chemical Industries, Ltd. for electron microscope) diluted to 2% with a phosphate buffer of 7.4
Per well and allowed to stand at 37 ° C. for 2 hours. Next, after washing three times with 300 μL / well of a washing solution (phosphate buffer solution of pH 7.4 containing 0.05% Tween 20), p
An anti-rat albumin antibody (manufactured by Cosmo Bio) diluted to 5 μg / ml with a phosphate buffer of H7.4 was added at 200 μL /
The mixture was dispensed into wells and allowed to stand at 37 ° C. for 2 hours.
【００１９】次に、洗浄液３００μＬ／ウェルで３回洗
浄した後に、ラットアルブミン（コスモバイオ製）を1
μｇ／ｍｌ、０．５μｇ／ｍｌ、０．２５μｇ／ｍｌ、
０．１２５μｇ／ｍｌの濃度系列で各濃度２４ウェルづ
つ１００μＬ／ウェルで分注し、室温で１時間静置し
た。次に洗浄液３００μＬ／ウェルで３回洗浄した後
に、ｐＨ７．４のリン酸緩衝液で２μｇ／ｍｌに希釈し
たペルオキシターゼ標識抗ラットアルブミン抗体（コス
モバイオ製）を２００μＬ／ウェルで分注し、室温で１
時間静置した。次に洗浄液３００μＬ／ウェルで３回洗
浄した後に、発色キット（ペルオキシターゼ用発色キッ
トＴ 住友ベークライト製）を使用して発色、プレート
リーダーで４５０ｎｍの波長の吸光度を測定した。Next, after washing three times with 300 μL / well of a washing solution, 1 ml of rat albumin (manufactured by Cosmo Bio) was added.
μg / ml, 0.5 μg / ml, 0.25 μg / ml,
The solution was dispensed at a concentration series of 0.125 μg / ml at a concentration of 24 wells of 100 μL / well for each 24 wells and allowed to stand at room temperature for 1 hour. Next, after washing three times with 300 μL / well of a washing solution, a peroxidase-labeled anti-rat albumin antibody (manufactured by Cosmo Bio) diluted to 2 μg / ml with a phosphate buffer of pH 7.4 was dispensed at 200 μL / well, and the mixture was added at room temperature. 1
Let stand for hours. Next, after washing three times with 300 μL / well of a washing solution, color was developed using a color developing kit (color developing kit T for peroxidase, manufactured by Sumitomo Bakelite), and absorbance at a wavelength of 450 nm was measured with a plate reader.
【００２０】（実施例２）プレートの各ウェルに、ｐＨ
５．８のリン酸緩衝液で１０％に調製した水溶性カルボ
ジイミド（和光純薬製）を２００μＬ／ウェルで分注
し、３７℃で１時間静置した。以降、抗体分注から発色
まで前記実施例１と同条件にて実施。(Example 2) pH of each well of a plate was
A water-soluble carbodiimide (manufactured by Wako Pure Chemical Industries, Ltd.) adjusted to 10% with a phosphate buffer solution of 5.8 was dispensed at 200 μL / well and allowed to stand at 37 ° C. for 1 hour. Thereafter, the procedure from antibody dispensing to color development was carried out under the same conditions as in Example 1 above.
【００２１】（比較例）ｐＨ７．４のリン酸緩衝液で５
μｇ／ｍｌに希釈した抗ラットアルブミン抗体（コスモ
バイオ製）を２００μＬ／ウェルで分注し、４℃で２０
時間静置した。次に、洗浄液３００μＬ／ウェルで３回
洗浄した後に、ブロッキング剤（スキムミルクをｐＨ
７．４のリン酸緩衝液で５％に調製）２００μＬ／ウェ
ルで分注し、室温で２時間静置した。以降アルブミン分
注から発色まで前記実施例１と同条件にて実施。結果は
図２の通りで、実施例１、２共に比較例と較べてアルブ
ミン濃度に対する吸光度値に直線性が得られ、各濃度に
おける２４ウェルのデータの変動係数（ＣＶ％）も低
く、安定していた。(Comparative Example) A phosphate buffer having a pH of 7.4
An anti-rat albumin antibody (manufactured by Cosmo Bio) diluted to μg / ml was dispensed at 200 μL / well and 20 μl at 4 ° C.
Let stand for hours. Next, after washing three times with 300 μL / well of a washing solution, a blocking agent (skim milk was adjusted to pH
(Prepared to 5% with 7.4 phosphate buffer)) Dispensed at 200 μL / well and allowed to stand at room temperature for 2 hours. Thereafter, the procedure from albumin dispensing to color development was performed under the same conditions as in Example 1. The results are as shown in FIG. 2. In both Examples 1 and 2, linearity was obtained in the absorbance value with respect to the albumin concentration as compared with the comparative example, and the coefficient of variation (CV%) of the 24-well data at each concentration was low and stable. I was
【００２２】[0022]
【発明の効果】本発明の免疫測定用基材は、測定に使用
する分子の非特異的吸着が２５℃において５×１０-4ｎ
ｍｏｌ／ｃｍ2以下である基材表面に、固相化に使用す
る分子の結合が可能な官能基を有することで、目的以外
の分子が測定系に残留して結果に影響を及ぼすことな
く、高感度、安定な免疫測定が可能になる。また、固相
化分子の固相化量は、官能器量によって制御することが
可能であるため、測定する物質の溶液濃度に応じて固相
化分子の固相化量を調節することが出来る。また、不安
定な非特異的吸着が存在しないため、固相化分子固相化
後も安定であり、分子を固相化した状態で保存した基材
を用いても優れた再現性が得られる。According to the immunoassay substrate of the present invention, the nonspecific adsorption of the molecules used for the measurement is 5 × 10 −4 n at 25 ° C.
By having a functional group capable of binding molecules used for solid-phase immobilization on the surface of the base material of not more than mol / cm 2 , molecules other than the target remain in the measurement system without affecting the results, Highly sensitive and stable immunoassay is possible. In addition, since the amount of the immobilized molecule can be controlled by the amount of the functional device, the amount of the immobilized molecule can be adjusted according to the solution concentration of the substance to be measured. In addition, since there is no unstable non-specific adsorption, the solid phase is stable even after immobilizing the molecule, and excellent reproducibility can be obtained even when using a substrate stored with the molecule immobilized. .
【図１】非特異的吸着性の比較として、蛋白溶液濃度と
吸着量の関係を示す。FIG. 1 shows the relationship between the concentration of a protein solution and the amount of adsorption as a comparison of nonspecific adsorption.
【図２】免疫測定感度の比較として、アルブミン濃度と
吸光度の関係を示す。FIG. 2 shows the relationship between albumin concentration and absorbance as a comparison of immunoassay sensitivity.
Claims (2)
５℃において５×１０-4ｎｍｏｌ／ｃｍ2以下である基
材表面に、固相化分子との結合が可能な官能基を有する
ことを特徴とする免疫測定用基材。1. Non-specific adsorption of a molecule used for measurement is 2
An immunoassay substrate comprising, on a substrate surface having a molecular weight of 5 × 10 −4 nmol / cm 2 or less at 5 ° C., a functional group capable of binding to an immobilized molecule.
官能基が、アミノ基および／またはカルボキシル基であ
る請求項１記載の免疫測定用基材。2. The substrate for immunoassay according to claim 1, wherein the functional group capable of binding to a molecule on the surface of the substrate is an amino group and / or a carboxyl group.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP34559897A JPH11174057A (en) | 1997-12-15 | 1997-12-15 | Immunity measuring base material |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP34559897A JPH11174057A (en) | 1997-12-15 | 1997-12-15 | Immunity measuring base material |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH11174057A true JPH11174057A (en) | 1999-07-02 |
Family
ID=18377681
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP34559897A Pending JPH11174057A (en) | 1997-12-15 | 1997-12-15 | Immunity measuring base material |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH11174057A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007288133A (en) * | 2006-03-24 | 2007-11-01 | Jsr Corp | Magnetic particles and method for producing the same |
| WO2008038774A1 (en) | 2006-09-28 | 2008-04-03 | Fujifilm Corporation | Instrument for biochemical use having surface under the inhibition of nonspecific adsorption |
| US8703289B2 (en) | 2005-11-01 | 2014-04-22 | Jsr Corporation | Organic polymer particles and process for producing the same, magnetic particles for diagnostics, carboxyl group-containing particles and process for producing the same, and probe-bound particles and process for producing the same |
-
1997
- 1997-12-15 JP JP34559897A patent/JPH11174057A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8703289B2 (en) | 2005-11-01 | 2014-04-22 | Jsr Corporation | Organic polymer particles and process for producing the same, magnetic particles for diagnostics, carboxyl group-containing particles and process for producing the same, and probe-bound particles and process for producing the same |
| JP2007288133A (en) * | 2006-03-24 | 2007-11-01 | Jsr Corp | Magnetic particles and method for producing the same |
| US7713627B2 (en) | 2006-03-24 | 2010-05-11 | Jsr Corporation | Magnetic particles comprising an organic polymer layer and method for producing the same |
| WO2008038774A1 (en) | 2006-09-28 | 2008-04-03 | Fujifilm Corporation | Instrument for biochemical use having surface under the inhibition of nonspecific adsorption |
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