JP3284896B2 - Enzyme immunoassay device and method - Google Patents
Enzyme immunoassay device and methodInfo
- Publication number
- JP3284896B2 JP3284896B2 JP26030196A JP26030196A JP3284896B2 JP 3284896 B2 JP3284896 B2 JP 3284896B2 JP 26030196 A JP26030196 A JP 26030196A JP 26030196 A JP26030196 A JP 26030196A JP 3284896 B2 JP3284896 B2 JP 3284896B2
- Authority
- JP
- Japan
- Prior art keywords
- label
- antibody
- antigen
- developing solution
- membrane
- 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.)
- Expired - Lifetime
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Description
【０００１】[0001]
【発明の属する技術分野】本発明は、検体中の抗原又は
抗体を検出するために用いられる酵素免疫アッセイ装置
及びそれを用いた酵素免疫アッセイ方法に関する。The present invention relates to an enzyme immunoassay apparatus used for detecting an antigen or an antibody in a specimen and an enzyme immunoassay method using the same.
【０００２】[0002]
【従来の技術】検体中の抗原又は抗体を簡便に検出する
装置として、酵素免疫アッセイ装置が用いられている。
従来の酵素免疫アッセイ装置を図２に模式的に示す。従
来の酵素免疫アッセイ装置は、ニトロセルロース等の材
質から成るメンブレン部２０と、その両端にそれぞれ設
けられた、吸水性の材質から成る展開液パッド部２２及
び吸収パッド部２４を有する。メンブレン部２０には、
標識体を点着した標識体点着部２６が設けられている。
標識体点着部２６には、タンパク質の非特異的吸着を防
止するためにメンブレンをブロッキングした後、検出す
べき抗原又は抗体と反応する抗体又は抗原が酵素標識さ
れた標識体が点着されている。さらに、標識体点着部２
６と吸収パッド部２４との間には、検出ライン２８が設
けられている。検出ライン２８には、検出すべき抗原又
は抗体と反応する抗体又は抗原が固定化されている。2. Description of the Related Art An enzyme immunoassay device is used as a device for easily detecting an antigen or an antibody in a sample.
FIG. 2 schematically shows a conventional enzyme immunoassay apparatus. The conventional enzyme immunoassay device has a membrane portion 20 made of a material such as nitrocellulose, and a developing solution pad portion 22 and an absorption pad portion 24 made of a water-absorbing material provided at both ends thereof. In the membrane part 20,
A marker spot application section 26 on which a marker is spotted is provided.
In the labeled body spotting portion 26, after blocking the membrane in order to prevent non-specific adsorption of protein, a labeled body in which an antibody or antigen that reacts with the antigen or antibody to be detected is enzyme-labeled is spotted. I have. Further, the marker body spotting part 2
A detection line 28 is provided between 6 and the absorption pad section 24. An antibody or antigen that reacts with the antigen or antibody to be detected is immobilized on the detection line 28.
【０００３】使用時には、検体液を標識体点着部２６に
加えると共に展開液パッド部に展開液を加える。展開液
は、標識体の標識酵素の基質であって、酵素反応により
発色する基質を含む。標識体点着部２６に点着されてい
る標識体は検体液中の抗原又は抗体と反応し、展開液パ
ッド部２２から流れてくる展開液により流されると共
に、該展開液中の基質と反応する。これらが展開液によ
り流されて検出ライン２８に到達すると、検体中の抗原
又は抗体が検出ライン２８に固定化されている抗体又は
抗原にトラップされ、そこに留まる。この場合、検体中
の抗原又は抗体は、標識体と結合しているから標識体も
検出ラインに留まる。従って、検体液中に検出すべき抗
原又は抗体が含まれている場合には検出ライン２８上で
発色が観察される。なお、展開液及びトラップされなか
った他の成分は吸収パッド部２４に吸収される。一方、
検体液中に検出すべき抗原又は抗体が含まれない場合に
は、標識体は検出ライン２８にトラップされないので、
そのまま吸収パッド部２４に吸収されてしまい、検出ラ
イン２８上に発色は観察されない。従って、検出ライン
が発色するか否かにより検体中に検出すべき抗原又は抗
体が含まれるか否かを知ることができる。[0003] In use, a sample solution is added to the marker spotting portion 26 and a developing solution is added to the developing solution pad portion. The developing solution contains a substrate for the labeling enzyme of the label, which develops a color by an enzyme reaction. The label spotted on the label spotting section 26 reacts with the antigen or antibody in the sample solution, is washed by the developing solution flowing from the developing solution pad section 22, and reacts with the substrate in the developing solution. I do. When these are flowed by the developing solution and reach the detection line 28, the antigen or antibody in the sample is trapped by the antibody or antigen immobilized on the detection line 28 and stays there. In this case, since the antigen or antibody in the sample is bound to the label, the label remains on the detection line. Therefore, when an antigen or an antibody to be detected is contained in the sample liquid, color development is observed on the detection line 28. The developing liquid and other components not trapped are absorbed by the absorption pad section 24. on the other hand,
When the antigen or antibody to be detected is not contained in the sample liquid, the label is not trapped in the detection line 28,
The light is absorbed by the absorption pad portion 24 as it is, and no color is observed on the detection line 28. Therefore, whether or not the sample contains an antigen or antibody to be detected can be determined based on whether or not the detection line is colored.
【０００４】また、カラーラテックスを用いた免疫アッ
セイ装置も知られている。この装置では、検出すべき抗
原又は抗体と反応する抗体又は抗原をカラーラテックス
粒子に結合したものが標識体として用いられる。この装
置を模式的に図３に示す。メンブレン３０の一端に標識
体点着部３２が設けられ、ここに、前記標識体が含有さ
れている。一方、検出ライン３６には検出すべき抗原又
は抗体と反応する抗体又は抗原が固定化されている。検
体液は、標識体点着部３２の中にある試料点着部位３４
に加えられる。検体液及び検体液により溶かされた標識
体が反応しながら流れ、検体液中に検出すべき抗原又は
抗体が含まれる場合には標識体が、上記と同様、検出ラ
イン３６にトラップされる。標識体はカラーラテックス
を含むので、検出ライン３６が着色する。一方、検体液
中に検出すべき抗原又は抗体が含まれない場合には、標
識体が検出ライン３６にトラップされないので検出ライ
ン３６は着色しない。従って、検出ライン３６が着色さ
れるか否かにより、検体液中に検出すべき抗原又は抗体
が含まれるか否かを知ることができる。[0004] Immunoassay devices using color latex are also known. In this device, an antibody or antigen that reacts with the antigen or antibody to be detected is bound to color latex particles as a label. This device is schematically shown in FIG. A marker spot application portion 32 is provided at one end of the membrane 30 and contains the marker. On the other hand, an antibody or antigen that reacts with the antigen or antibody to be detected is immobilized on the detection line 36. The sample liquid is applied to the sample spotting portion 34 in the label spotting portion 32.
Is added to The sample liquid and the label dissolved by the sample liquid flow while reacting, and when the antigen or antibody to be detected is contained in the sample liquid, the label is trapped in the detection line 36 as described above. Since the label contains a color latex, the detection line 36 is colored. On the other hand, when the antigen or antibody to be detected is not contained in the sample liquid, the detection line 36 is not colored because the label is not trapped in the detection line 36. Therefore, whether or not the antigen or antibody to be detected is contained in the sample liquid can be known from whether or not the detection line 36 is colored.
【０００５】[0005]
【発明が解決しようとする課題】しかしながら、従来の
免疫アッセイ装置では、判定時に陰性であった結果が経
時的に陽性に変化することがあるという問題があった。
従って、複数の検体を同一条件で判定する場合には、一
定の判定時間で判定するか又は一定時間経過後に反応停
止液を加えて反応を停止させてから判定する必要があっ
た。しかしながら、臨床検査のように、多数の検体を並
行して検査する場合には、全ての検体について同一の判
定時間で判定を行うことや同一の時間経過後に反応停止
液を加えることは困難である。また、反応停止液を加え
ると工程が増し、煩雑になる。However, the conventional immunoassay apparatus has a problem that a negative result at the time of determination may change to a positive one over time.
Therefore, when a plurality of specimens are determined under the same conditions, it is necessary to make a determination in a fixed determination time or to stop the reaction by adding a reaction stop solution after a lapse of a certain time. However, when a large number of samples are tested in parallel as in a clinical test, it is difficult to make a determination for all the samples at the same determination time or to add a reaction stop solution after the same time has elapsed. . In addition, the addition of a reaction stop solution increases the number of steps and complicates the process.
【０００６】従って、本発明の目的は、判定時に陰性で
あった判定結果が経時的に陽性に変換することがない酵
素免疫アッセイ装置及び該装置を用いた酵素免疫アッセ
イ方法を提供することである。Accordingly, it is an object of the present invention to provide an enzyme immunoassay apparatus in which a negative judgment result at the time of judgment is not converted to a positive result with time, and an enzyme immunoassay method using the apparatus. .
【０００７】[0007]
【課題を解決するための手段】本願発明者らは、鋭意研
究の結果、酵素の基質を従来のように展開液中に含める
のではなく、メンブレンに乾燥状態で含有させることに
より上記目的が達成できることを見出し本発明を完成し
た。Means for Solving the Problems As a result of intensive studies, the inventors of the present invention have achieved the above object by allowing the enzyme substrate to be contained in a dry state in the membrane, instead of being included in the developing solution as in the prior art. We have found that we can do this and completed the present invention.
【０００８】すなわち、本発明は、メンブレン部と、該
メンブレン部の両端にそれぞれ設けられた展開液パッド
部と吸収パッド部とを具備し、前記メンブレン部に検出
すべき抗原又は抗体と反応する抗体又は抗原が標識酵素
により標識された標識体が含有された標識体含有部が設
けられ、該標識体含有部と前記吸収パッドの間には、検
出すべき抗原又は抗体と反応する抗体又は抗原が前記メ
ンブレン部に固定化された検出部が設けられており、か
つ、前記標識体含有部と前記展開液パッド部との間のメ
ンブレン部に、酵素反応により発色する、前記標識酵素
の基質が乾燥状態で含有されている基質含有部が設けら
れている、酵素免疫アッセイ装置を提供する。さらに本
発明は、上記本発明の装置を用い、検体を前記標識体含
有部又は該標識体含有部と前記展開液パッドに挟まれる
メンブレン部の領域上のいずれかの部位に点着し、前記
展開液パッドから展開液を前記メンブレン部に流し、前
記検出部における発色を調べることを含む、酵素免疫ア
ッセイ方法を提供する。[0008] That is, the present invention comprises a membrane portion, a developing solution pad portion and an absorption pad portion provided at both ends of the membrane portion, respectively, and the antibody reacting with the antigen or antibody to be detected in the membrane portion. Alternatively, a labeled body-containing portion containing a labeled body in which an antigen is labeled with a labeling enzyme is provided, and an antibody or antigen that reacts with the antigen or antibody to be detected is provided between the labeled body-containing portion and the absorption pad. A detection part fixed to the membrane part is provided, and a substrate of the labeling enzyme, which is colored by an enzyme reaction on the membrane part between the label containing part and the developing solution pad part, is dried. An enzyme immunoassay device provided with a substrate-containing portion contained in a state. Further, the present invention, using the apparatus of the present invention, spotted a sample on the label-containing portion or any site on the region of the membrane portion sandwiched between the label-containing portion and the developing solution pad, An enzyme immunoassay method is provided, comprising flowing a developing solution from a developing solution pad to the membrane section and examining color development at the detection section.
【０００９】[0009]
【発明の実施の形態】本発明の酵素免疫アッセイ装置の
好ましい態様を図１に模式的に示す。基本的な構成は図
２に示す上記した従来のアッセイ装置と同じである。す
なわち、本発明のアッセイ装置は、メンブレン部１０を
含む。メンブレン部は、従来と同様、通常、ニトロセル
ロースメンブレンのような材質から成り、通常、長方形
状である。メンブレン部１０の両端には、展開液パッド
部１２と吸収パッド部１４とがそれぞれ設けられてい
る。これらは、従来と同様、吸水性の材質、例えば、ス
ポンジ、吸水性の不織布、ろ紙等からなる。展開液パッ
ド部１２及び吸収パッド部１４の厚さは、多量の液を含
浸できるように、メンブレン部１０の厚さよりも通常厚
く形成されているが、このことは必須的ではない。メン
ブレン部１０上に設けられる標識体含有部１６（後述）
と吸収パッド１４の間には検出部１８が設けられてい
る。検出部１８には、従来と同様、検出すべき抗原又は
抗体と反応する抗体又は抗原が固定化されている。検出
部１８は、従来と同様線状に形成されていることが好ま
しいが、必ずしも線状である必要はなく、円形等の他の
形状であってもよい。また、従来と同様、メンブレン部
１０は、タンパク質の非特異的吸着を防止するために、
ＢＳＡ等でブロッキングされている。DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 1 schematically shows a preferred embodiment of the enzyme immunoassay device of the present invention. The basic configuration is the same as the above-mentioned conventional assay device shown in FIG. That is, the assay device of the present invention includes the membrane unit 10. The membrane portion is usually made of a material such as a nitrocellulose membrane, as in the related art, and usually has a rectangular shape. A developing liquid pad section 12 and an absorption pad section 14 are provided at both ends of the membrane section 10, respectively. These are made of a water-absorbing material, for example, sponge, water-absorbing nonwoven fabric, filter paper, and the like, as in the conventional case. The thickness of the developing liquid pad portion 12 and the absorption pad portion 14 is usually formed to be thicker than the thickness of the membrane portion 10 so that a large amount of liquid can be impregnated. However, this is not essential. Marker-containing portion 16 provided on membrane portion 10 (described later)
A detection unit 18 is provided between the and the absorption pad 14. An antibody or antigen that reacts with the antigen or antibody to be detected is immobilized on the detection unit 18 as in the related art. The detection unit 18 is preferably formed in a linear shape as in the related art, but is not necessarily required to be linear, and may be another shape such as a circle. Further, as in the prior art, the membrane portion 10 is used to prevent non-specific adsorption of proteins.
Blocked by BSA or the like.
【００１０】標識体含有部１６には、検出すべき抗原又
は抗体と反応する抗体又は抗原を酵素標識した標識体が
乾燥状態で含有されている。標識酵素としては、従来と
同様、酵素免疫分析で多用されている、アルカリフォス
ファターゼ、ペルオキシダーゼ、β−ガラクトシダーゼ
等を用いることができる。なお、標識体は、メンブレン
に直接点着してもよいが、メンブレン上に吸水性の材
質、例えば、スポンジ、吸水性不織布、ろ紙等からなる
標識体パッドを設け、これに標識体を含有させることも
できる。この場合には、含有させる標識体の量を多くす
ることができ、標識体が展開液により流れやすく、ま
た、多量の検体液を用いることができるので、検出感度
を高くすることができる。The label containing section 16 contains a label in which the antibody or antigen that reacts with the antigen or antibody to be detected is enzyme-labeled in a dry state. As a labeling enzyme, alkaline phosphatase, peroxidase, β-galactosidase and the like, which are frequently used in enzyme immunoassays, can be used as in the past. The marker may be spotted directly on the membrane, but a marker pad made of a water-absorbing material, for example, sponge, water-absorbent nonwoven fabric, filter paper, etc., is provided on the membrane, and the marker is contained in this. You can also. In this case, the amount of the label to be contained can be increased, and the label can easily flow into the developing solution, and a large amount of the sample solution can be used, so that the detection sensitivity can be increased.
【００１１】本発明の酵素免疫アッセイ装置では、従来
の装置と異なり、標識酵素の基質を乾燥状態で含む基質
含有部１９がメンブレン１０に設けられている。基質含
有部１９は、標識体含有部１６と展開液パッド部１２の
間に設けられる。基質含有部１９に含有される基質の量
は、特に限定されず、各検査によっても異なるが、通
常、２０〜２００μｇ程度である。なお、基質は、従来
と同様、酵素反応により発色する、標識酵素の基質であ
る。In the enzyme immunoassay device of the present invention, unlike the conventional device, a substrate containing portion 19 containing a substrate of a labeled enzyme in a dry state is provided on the membrane 10. The substrate containing section 19 is provided between the label containing section 16 and the developing solution pad section 12. The amount of the substrate contained in the substrate-containing portion 19 is not particularly limited and varies depending on each test, but is usually about 20 to 200 μg. In addition, the substrate is a substrate of the labeling enzyme that develops a color by an enzyme reaction as in the related art.
【００１２】使用時には、検体液を標識体含有部１６又
は該標識体含有部１６と前記展開液パッド１２に挟まれ
るメンブレン部の領域上のいずれかの部位に加えると共
に展開液パッド部１２に展開液を加える。なお、検体液
と標識体との反応を十分に行うために検体液は標識体含
有部１６に加えることが好ましい。従来と異なり、展開
液中には、標識体の標識酵素の基質が含まれない。展開
液パッド部１２に加えられた展開液は、基質含有部１９
中の基質を溶かして流れ、標識体含有部１６に達する。
標識体含有部１６に含まれている標識体は検体液中の抗
原又は抗体と反応し、展開液パッド部１２から流れてく
る展開液により流されると共に、該展開液中の基質と反
応する。これらが展開液により流されて検出部１８に到
達すると、検体中の抗原又は抗体が検出部１８に固定化
されている抗体又は抗原にトラップされ、そこに留ま
る。この場合、検体中の抗原又は抗体は、標識体と結合
しているから標識体も検出ラインに留まる。従って、検
体液中に検出すべき抗原又は抗体が含まれている場合に
は検出部１８上で発色が観察される。なお、展開液及び
トラップされなかった他の成分は吸収パッド部１４に吸
収される。一方、検体液中に検出すべき抗原又は抗体が
含まれない場合には、標識体は検出部１８にトラップさ
れないので、そのまま吸収パッド部１４に吸収されてし
まい、検出部１８上に発色は観察されない。従って、検
出部が発色するか否かにより検体中に検出すべき抗原又
は抗体が含まれるか否かを知ることができる。なお、展
開液及びトラップされなかった他の成分は吸収パッド部
１４に吸収される。一方、検体液中に検出すべき抗原又
は抗体が含まれない場合には、標識体は検出部１８にト
ラップされないので、そのまま吸収パッド部１４に吸収
されてしまい、検出部１８上に発色は観察されない。従
って、検出部が発色するか否かにより検体中に検出すべ
き抗原又は抗体が含まれるか否かを知ることができる。
なお、前記標識体と前記基質の一部は展開液が前記検出
部に到達する前に反応して発色しつつ移動しても構わな
いが、検出部では展開液中に免疫アッセイを行うために
十分な基質量を含有している。この場合、形成された発
色体のうち、前記検出部にトラップされなかったものは
前記吸収パッド部に吸収される。At the time of use, the sample liquid is added to the label-containing part 16 or any part of the region of the membrane part sandwiched between the label-containing part 16 and the developing liquid pad 12 and spread on the developing liquid pad part 12. Add the liquid. It is preferable that the sample liquid is added to the label-containing section 16 in order to sufficiently perform the reaction between the sample liquid and the label. Unlike the conventional method, the developing solution does not contain a substrate for the labeling enzyme of the label. The developing solution added to the developing solution pad section 12 is supplied to the substrate containing section 19.
The substrate inside is dissolved and flows, and reaches the labeled body-containing portion 16.
The label contained in the label containing section 16 reacts with the antigen or antibody in the sample liquid, flows with the developing liquid flowing from the developing liquid pad section 12, and reacts with the substrate in the developing liquid. When these are flowed by the developing solution and reach the detection unit 18, the antigen or antibody in the sample is trapped by the antibody or antigen immobilized on the detection unit 18 and stays there. In this case, since the antigen or antibody in the sample is bound to the label, the label remains on the detection line. Therefore, when an antigen or an antibody to be detected is contained in the sample liquid, color development is observed on the detection unit 18. The developing liquid and other components not trapped are absorbed by the absorption pad section 14. On the other hand, if the antigen or antibody to be detected is not contained in the sample liquid, the label is not trapped by the detection section 18 and is absorbed by the absorption pad section 14 as it is, and color development is observed on the detection section 18. Not done. Therefore, whether or not the antigen or antibody to be detected is contained in the sample can be known based on whether or not the detection section develops color. The developing liquid and other components not trapped are absorbed by the absorption pad section 14. On the other hand, if the antigen or antibody to be detected is not contained in the sample solution, the label is not trapped by the detection section 18 and is absorbed by the absorption pad section 14 as it is, and color development is observed on the detection section 18. Not done. Therefore, whether or not the antigen or antibody to be detected is contained in the sample can be known based on whether or not the detection section develops color.
The label and a part of the substrate may move while reacting and developing a color before the developing solution reaches the detection unit.However, the detection unit performs an immunoassay in the developing solution. Contains sufficient base mass. In this case, of the formed color bodies, those which are not trapped by the detection section are absorbed by the absorption pad section.
【００１３】なお、上記説明では、酵素標識する物質及
び検出部に固定化する物質を抗体又は抗原としたが、本
願明細書で言う「抗体又は抗原」とは、抗原抗体反応が
可能な物質であれば、例えば抗体断片（Fab フラグメン
ト、F(ab')2 フラグメント等）やハプテン等も包含する
意味で用いている。In the above description, the substance to be labeled with an enzyme and the substance to be immobilized on the detection section are referred to as an antibody or an antigen. However, the term “antibody or antigen” as used herein refers to a substance capable of an antigen-antibody reaction. If present, the term is used to include, for example, antibody fragments (Fab fragments, F (ab ') 2 fragments, etc.) and haptens.
【００１４】従来の装置では、基質が展開液中に含ま
れ、展開液は多量に用いられるから、発色反応がいつま
でも続く。これに対し、本発明のアッセイ装置では、基
質含有部１９に含まれる基質の全量が展開液で流されて
しまった後には、基質が存在しなくなるので、発色反応
はそれ以上進まない。従って、判定時に陰性と判定され
たものが経時的に陽性に変化することはない。また、本
発明のアッセイ装置では、基質が乾燥状態で維持される
ので、従来のように展開液に溶けた状態で液体で維持さ
れるのに比較して保存安定性が高い。In the conventional apparatus, since the substrate is contained in the developing solution and the developing solution is used in a large amount, the coloring reaction continues forever. On the other hand, in the assay device of the present invention, after the entire amount of the substrate contained in the substrate-containing portion 19 has been washed away with the developing solution, the substrate is not present, and the color reaction does not proceed further. Therefore, what is determined to be negative at the time of determination does not change to positive over time. Further, in the assay device of the present invention, since the substrate is maintained in a dry state, storage stability is higher than that in the conventional case where the substrate is maintained in a liquid state while being dissolved in a developing solution.
【００１５】[0015]
【実施例】以下、実施例に基づき本発明をより具体的に
説明する。もっとも、本発明は下記実施例に限定される
ものではない。Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.
【００１６】実施例１、比較例１ ５ｍｍｘ５０ｍｍのニトロセルロースメンブレンからな
るメンブレン部の両端に、吸水性の不織布から成る展開
液パッド部及び吸水パッド部を設けた。メンブレン部の
吸水パッド部よりの部分に、抗ヘモグロビンポリクロー
ナル抗体を点着し、乾燥し、検出ラインとした。次いで
メンブレンをブロッキング後、５ｍｍ四方、厚さ１ｍｍ
の吸水性不織布からなる標識体パッドをメンブレン上に
設けた。該標識体パッドには、１５μｇ／ｍｌの濃度で
アルカリフォスファターゼ標識抗ヘモグロビンポリクロ
ーナル抗体を含む溶液１０μｌを点着し、乾燥させた。
さらに、実施例１の装置では、標識体パッドと展開液パ
ッド部との間のメンブレン部に、基質含有部を設け、ア
ッセイ装置とした。基質含有部は、２０ｍｇ／ｍｌの５
−ブロモ−４−クロロ−３−インドリル−リン酸ナトリ
ウム（ＢＣＩＰ，Ｎａ）溶液５μｌを点着し、乾燥させ
る操作を３回繰り返すことにより形成した。一方、比較
例１の装置では、基質含有部を設けなかった。 Example 1 and Comparative Example 1 A developing liquid pad and a water absorbing pad made of a water-absorbing nonwoven fabric were provided at both ends of a nitrocellulose membrane of 5 mm × 50 mm. An anti-hemoglobin polyclonal antibody was spotted on a portion of the membrane portion from the water-absorbing pad portion, and dried to form a detection line. Then, after blocking the membrane, 5 mm square, 1 mm thick
Was provided on the membrane. 10 μl of a solution containing an alkaline phosphatase-labeled anti-hemoglobin polyclonal antibody at a concentration of 15 μg / ml was spotted on the label pad and dried.
Furthermore, in the device of Example 1, a substrate-containing portion was provided in the membrane portion between the label pad and the developing solution pad portion, and used as an assay device. The substrate-containing part was 5 mg of 20 mg / ml.
5 μl of a solution of -bromo-4-chloro-3-indolyl-sodium phosphate (BCIP, Na) was spotted and dried three times. On the other hand, in the device of Comparative Example 1, the substrate-containing portion was not provided.
【００１７】５、１０又は２０ｎｇ／ｍｌのヘモグロビ
ンを含む試料２５μｌを標識体パッドに点着した後、展
開パッドに展開液２００μｌを添加して測定開始とし
た。展開液は、実施例１では０．１Ｍ ２，２’−イミ
ノジエタノール−塩酸緩衝液ｐＨ１０、１ｍＭ ＭｇＣ
ｌ2 、０．０５％Ｔｗｅｅｎ２０（商品名）を用いた。
比較例１では、実施例１で用いた展開液に０．３ ｍｇ
／ｍｌの濃度でＢＣＩＰ，Ｎａを加えたものを展開液と
して用いた。展開液添加後８分を判定時間とし、さらに
開始後１０分及び３０分後に判定を行った。なお、各試
験は２回ずつ行った。結果を表１及び表２に示す。After 25 μl of a sample containing 5, 10, or 20 ng / ml hemoglobin was spotted on the pad for the label, 200 μl of the developing solution was added to the developing pad to start the measurement. In Example 1, the developing solution was 0.1 M 2,2'-iminodiethanol-hydrochloric acid buffer pH 10, 1 mM MgC
l 2 , 0.05% Tween 20 (trade name) was used.
In Comparative Example 1, 0.3 mg was added to the developing solution used in Example 1.
A solution to which BCIP and Na were added at a concentration of / ml was used as a developing solution. The determination time was 8 minutes after the addition of the developing solution, and the determination was made 10 minutes and 30 minutes after the start. Each test was performed twice. The results are shown in Tables 1 and 2.
【００１８】[0018]
【表１】 [Table 1]
【００１９】[0019]
【表２】 [Table 2]
【００２０】表１と表２の比較から、比較例１の装置を
用いた場合には、全ての場合において経時的に判定結果
が陰性から陽性に変わっているのに対し、本発明の装置
を用いた場合には、陰性から陽性への変化はほぼないこ
とがわかる。From the comparison between Tables 1 and 2, when the apparatus of Comparative Example 1 was used, the judgment result changed from negative to positive over time in all cases, whereas the apparatus of the present invention was used. It can be seen that there was almost no change from negative to positive when used.
【００２１】比較例２ 図３に示すような、カラーラテックスを用いる従来のア
ッセイ装置を作製した。検出ラインは実施例１と同様に
作製した。実施例１と同様な検体液１００μｌを試料点
着部位に添加して測定開始とした。試料添加後８分を判
定時間とし、さらに開始後１０分及び３０分後に判定を
行った。結果を下記表３に示す。 Comparative Example 2 A conventional assay device using a color latex as shown in FIG. 3 was prepared. The detection line was produced in the same manner as in Example 1. The measurement was started by adding 100 μl of the same sample liquid as in Example 1 to the sample spotting site. The judgment time was 8 minutes after the sample addition, and the judgment was made 10 minutes and 30 minutes after the start. The results are shown in Table 3 below.
【００２２】[0022]
【表３】 [Table 3]
【００２３】表３から、従来のカラーラテックスを用い
た方法でも判定結果が陰性から陽性に変化することがわ
かる。From Table 3, it can be seen that the judgment result changes from negative to positive even in the method using the conventional color latex.
【００２４】[0024]
【発明の効果】本発明により、判定時の判定結果が経時
的に陰性から陽性に変化しない酵素免疫アッセイ装置及
びそれを用いた酵素免疫アッセイ方法が提供された。According to the present invention, there is provided an enzyme immunoassay apparatus and an enzyme immunoassay method using the same, wherein the judgment result at the time of judgment does not change from negative to positive with time.
【図１】本願発明の酵素免疫アッセイ装置の好ましい態
様の一例を模式的に示す図である。FIG. 1 is a diagram schematically showing an example of a preferred embodiment of an enzyme immunoassay device of the present invention.
【図２】従来の酵素免疫アッセイ装置を模式的に示す図
である。FIG. 2 is a diagram schematically showing a conventional enzyme immunoassay device.
【図３】カラーラテックスを用いる従来のアッセイ装置
を模式的に示す図である。FIG. 3 is a diagram schematically showing a conventional assay device using color latex.
１０ メンブレン部 １２ 展開液パッド部 １４ 吸収パッド部 １６ 標識体含有部 １８ 検出部 １９ 基質含有部 ２０ メンブレン部 ２２ 展開液パッド部 ２４ 吸収パッド部 ２６ 標識体点着部 ２８ 検出ライン ３０ メンブレン部 ３２ 標識体点着部 ３４ 試料点着部位 ３６ 検出ライン DESCRIPTION OF SYMBOLS 10 Membrane part 12 Developing liquid pad part 14 Absorbing pad part 16 Labeling substance containing part 18 Detecting part 19 Substrate containing part 20 Membrane part 22 Developing liquid pad part 24 Absorbing pad part 26 Label spotting part 28 Detection line 30 Membrane part 32 Label Body spotting part 34 Sample spotting part 36 Detection line
───────────────────────────────────────────────────── フロントページの続き (72)発明者 芦原 義弘 東京都新宿区西新宿２丁目７番１号 富 士レビオ株式会社内 (56)参考文献 特開 平５−52836（ＪＰ，Ａ) 特開 平６−180320（ＪＰ，Ａ) 特開 平６−341989（ＪＰ，Ａ) 特開 平６−167497（ＪＰ，Ａ) 特開 昭64−63865（ＪＰ，Ａ) (58)調査した分野(Int.Cl.7，ＤＢ名) G01N 33/543 521 ──────────────────────────────────────────────────続 き Continuation of front page (72) Inventor Yoshihiro Ashihara 2-7-1, Nishishinjuku, Shinjuku-ku, Tokyo Inside Fuji Rebio Co., Ltd. (56) References JP-A-5-52836 (JP, A) JP-A-6-180320 (JP, A) JP-A-6-341989 (JP, A) JP-A-6-167497 (JP, A) JP-A-64-63865 (JP, A) (58) Fields investigated (Int .Cl. 7 , DB name) G01N 33/543 521
Claims (4)
にそれぞれ設けられた展開液パッド部と吸収パッド部と
を具備し、前記メンブレン部に検出すべき抗原又は抗体
と反応する抗体又は抗原が標識酵素により標識された標
識体が含有された標識体含有部が設けられ、該標識体含
有部と前記吸収パッドの間には、検出すべき抗原又は抗
体と反応する抗体又は抗原が前記メンブレン部に固定化
された検出部が設けられており、かつ、前記標識体含有
部と前記展開液パッド部との間のメンブレン部に、酵素
反応により発色する、前記標識酵素の基質が乾燥状態で
含有されている基質含有部が設けられている、酵素免疫
アッセイ装置。1. A membrane comprising a developing solution pad portion and an absorption pad portion provided on both ends of the membrane portion, respectively, wherein the antibody or antigen reacting with the antigen or antibody to be detected is labeled on the membrane portion. A label-containing portion containing a label labeled with an enzyme is provided, and between the label-containing portion and the absorbent pad, an antibody or antigen that reacts with the antigen or antibody to be detected is provided on the membrane portion. An immobilized detection unit is provided, and a substrate of the labeling enzyme, which is colored by an enzyme reaction, is contained in a dry state in a membrane portion between the labeling body-containing portion and the developing solution pad portion. An enzyme immunoassay device provided with a substrate-containing portion.
吸水性の材料から形成された標識体パッドである請求項
１記載の装置。2. The apparatus according to claim 1, wherein the marker-containing portion is a marker pad formed of a water-absorbing material containing the marker.
を前記標識体含有部又は該標識体含有部と前記展開液パ
ッドに挟まれるメンブレン部の領域上のいずれかの部位
に点着し、前記展開液パッドから展開液を前記メンブレ
ン部に流し、前記検出部における発色を調べることを含
む、酵素免疫アッセイ方法。3. The method according to claim 1, wherein a sample is spotted on the label-containing part or on any part of an area of a membrane part sandwiched between the label-containing part and the developing solution pad. An enzyme immunoassay method, comprising flowing a developing solution from the developing solution pad to the membrane section and examining color development at the detection section.
前記検出部に到達する前に反応して発色しつつ移動し、
検出部では免疫アッセイを行うために十分な基質が展開
液中に含まれる請求項３記載の方法。4. The label and a part of the substrate react with each other before the developing solution reaches the detection unit and move while developing color,
4. The method according to claim 3, wherein a sufficient amount of a substrate for performing an immunoassay is contained in the developing solution in the detection unit.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP26030196A JP3284896B2 (en) | 1995-09-08 | 1996-09-09 | Enzyme immunoassay device and method |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP25675795 | 1995-09-08 | ||
| JP7-256757 | 1995-09-08 | ||
| JP26030196A JP3284896B2 (en) | 1995-09-08 | 1996-09-09 | Enzyme immunoassay device and method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH09133681A JPH09133681A (en) | 1997-05-20 |
| JP3284896B2 true JP3284896B2 (en) | 2002-05-20 |
Family
ID=26542885
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP26030196A Expired - Lifetime JP3284896B2 (en) | 1995-09-08 | 1996-09-09 | Enzyme immunoassay device and method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3284896B2 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005331463A (en) * | 2004-05-21 | 2005-12-02 | Denka Seiken Co Ltd | Device and detection method |
| JP2009258136A (en) * | 2009-08-10 | 2009-11-05 | Denka Seiken Co Ltd | Device and detection method |
| US8119420B2 (en) | 2006-03-13 | 2012-02-21 | Fujirebio Inc. | Absorption pad for immunoassay, strip for immunoassay, and immunoassay apparatus |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2326712B (en) * | 1997-06-25 | 2001-09-26 | Fujirebio Kk | Colour devoloping method,enzyme immunoassay using the colour developing method,and immunochromatography incorporating the enzyme immunoassay |
| CA2846247A1 (en) * | 2011-09-16 | 2013-03-21 | Credo Biomedical Pte Ltd | Molecular diagnostic assay device and method of use |
-
1996
- 1996-09-09 JP JP26030196A patent/JP3284896B2/en not_active Expired - Lifetime
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005331463A (en) * | 2004-05-21 | 2005-12-02 | Denka Seiken Co Ltd | Device and detection method |
| JP4544907B2 (en) * | 2004-05-21 | 2010-09-15 | デンカ生研株式会社 | Device and detection method |
| US8119420B2 (en) | 2006-03-13 | 2012-02-21 | Fujirebio Inc. | Absorption pad for immunoassay, strip for immunoassay, and immunoassay apparatus |
| JP2009258136A (en) * | 2009-08-10 | 2009-11-05 | Denka Seiken Co Ltd | Device and detection method |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH09133681A (en) | 1997-05-20 |
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