JPH0453579Y2 - - Google Patents
Info
- Publication number
- JPH0453579Y2 JPH0453579Y2 JP13630986U JP13630986U JPH0453579Y2 JP H0453579 Y2 JPH0453579 Y2 JP H0453579Y2 JP 13630986 U JP13630986 U JP 13630986U JP 13630986 U JP13630986 U JP 13630986U JP H0453579 Y2 JPH0453579 Y2 JP H0453579Y2
- Authority
- JP
- Japan
- Prior art keywords
- substance
- paper
- kit
- enzyme
- container
- 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
Links
- 239000000126 substance Substances 0.000 claims description 62
- 108090000790 Enzymes Proteins 0.000 claims description 29
- 102000004190 Enzymes Human genes 0.000 claims description 29
- 238000006243 chemical reaction Methods 0.000 claims description 26
- 239000003153 chemical reaction reagent Substances 0.000 claims description 13
- 238000004040 coloring Methods 0.000 claims description 13
- 238000009739 binding Methods 0.000 claims description 11
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 6
- 230000027455 binding Effects 0.000 claims description 6
- 238000002372 labelling Methods 0.000 claims description 6
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 5
- 239000006172 buffering agent Substances 0.000 claims 1
- 239000000123 paper Substances 0.000 description 39
- 239000000243 solution Substances 0.000 description 21
- 238000012360 testing method Methods 0.000 description 18
- 238000000034 method Methods 0.000 description 12
- 239000004033 plastic Substances 0.000 description 11
- 229920003023 plastic Polymers 0.000 description 11
- 239000007864 aqueous solution Substances 0.000 description 10
- 210000002700 urine Anatomy 0.000 description 8
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 6
- 238000003018 immunoassay Methods 0.000 description 6
- 239000000463 material Substances 0.000 description 5
- 239000011087 paperboard Substances 0.000 description 5
- 230000009870 specific binding Effects 0.000 description 5
- 239000004698 Polyethylene Substances 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
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 4
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000002953 phosphate buffered saline Substances 0.000 description 4
- -1 polyethylene Polymers 0.000 description 4
- 229920000573 polyethylene Polymers 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000000853 adhesive Substances 0.000 description 3
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 3
- 239000012491 analyte Substances 0.000 description 3
- 229940088597 hormone Drugs 0.000 description 3
- 239000005556 hormone Substances 0.000 description 3
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 3
- 235000017557 sodium bicarbonate Nutrition 0.000 description 3
- 239000007790 solid phase Substances 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 230000005856 abnormality Effects 0.000 description 2
- 230000001070 adhesive effect Effects 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000000967 suction filtration Methods 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 239000002313 adhesive film Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004880 lymph fluid Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 239000005871 repellent Substances 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 229920002379 silicone rubber Polymers 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
Landscapes
- Spectrometry And Color Measurement (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Description
【考案の詳細な説明】
［産業上の利用分野］
本考案は生体由来物質を定性的ないしは半定量
的に、且つ極めて簡易に測定するためのキツト、
特に抗体を固定して成るロ紙上に於ける呈色反応
を利用して前記測定を行なうためのキツトに係わ
る。[Detailed description of the invention] [Field of industrial application] The present invention is a kit for qualitatively or semi-quantitatively and extremely easily measuring biological substances.
In particular, the present invention relates to a kit for carrying out the above-mentioned measurement using a color reaction on a sheet of paper on which antibodies are immobilized.
［従来の技術］
病態を正しく把握するためには、血清、尿又は
リンパ液等の体液試料中の特定成分（例えばホル
モンなど）値の変動の具合を知ることが必要であ
る。[Prior Art] In order to accurately understand the pathological condition, it is necessary to know how the values of specific components (eg, hormones, etc.) in body fluid samples such as serum, urine, or lymph fluid vary.
更に、病気が大事に至らないうちに治療する為
には、健康状態に於いて前記成分を定期的に検出
して変動を常時観察し、その異常を早期発見する
ことが重要である。 Furthermore, in order to treat diseases before they become serious, it is important to periodically detect the above-mentioned components in a healthy state, constantly observe fluctuations, and detect abnormalities at an early stage.
近年、このような特定成分を測定し得る方法と
して、酵素標識物質を結合した物質と他の生体由
来物質との間の特異的結合反応を利用するものが
開発されている。 In recent years, methods that utilize specific binding reactions between substances bound to enzyme labeling substances and other biological substances have been developed as methods capable of measuring such specific components.
一般に酵素免疫測定法と呼ばれるものは上記測
定方法の代表的な例であり、放射性同位元素を標
識物質として用いた以前の方法と較べて特に安全
性の点で非常に優れたものである。 What is generally called enzyme immunoassay is a typical example of the above measurement method, and is extremely superior in terms of safety, especially compared to previous methods that use radioactive isotopes as labeling substances.
上記の酵素免疫測定法の中でもヘテロジーナス
法と呼ばれる一般的な方法に於いては、被検物質
と特異的に反応する物質（例えば、抗原又は抗体
等）を固相と呼ばれる物質に固定し、各反応段階
後の洗浄操作によつて、いわゆるＢ（bound）／
Ｆ（free）分離を行なつている。この固相として
従来用いられている物質には、ポリスチレンビー
ズ、ガラスビーズ、デキストラン、シリコンデイ
スク、シリコンラバー、アガロース及びロ紙等が
ある。 Among the enzyme immunoassay methods mentioned above, in a common method called the heterogeneous method, a substance that specifically reacts with the test substance (e.g., an antigen or an antibody) is immobilized on a substance called a solid phase. By washing operations after each reaction step, the so-called B (bound)
F (free) separation is performed. Materials conventionally used as this solid phase include polystyrene beads, glass beads, dextran, silicon disks, silicon rubber, agarose, and paper.
［考案が解決しようとする問題点］
しかしながら、従来の上記酵素免疫測定を行な
う為の装置は、主に、設備等の充実している実験
室や検査室等で用いられることを前提として作ら
れているものである。従つて、これらの装置は比
較的高価であり、且つ、前記の洗浄という繁雑な
操作が要求されるものである。[Problems to be solved by the invention] However, the conventional enzyme immunoassays mentioned above were mainly designed to be used in well-equipped laboratories and examination rooms. It is something that Therefore, these devices are relatively expensive and require complicated cleaning operations.
そこで、病気を早期に発見する為に、一般の家
庭で誰でも手軽に扱え、前記の特定成分の検出を
日常的に簡単にし得る為の方法及びその為の安価
で簡易な装置が強く望まれているものである。 Therefore, in order to detect diseases at an early stage, there is a strong desire for a method and an inexpensive and simple device that can be easily used by anyone at home and that can easily detect the above-mentioned specific components on a daily basis. It is something that
本考案者は以上の課題を解決する為に、物質間
の特異的結合反応及び標識物質である酵素を触媒
とする呈色反応を利用した、家庭等に於いて一般
人が誰でも極めて簡単に生体由来物質を定性的又
は半定量的に測定することのできる安価で簡易な
キツトを開発し、本考案に至つたものである。 In order to solve the above-mentioned problems, the inventor of the present invention has developed a method that can be used by any ordinary person at home, etc., to easily develop a biological system using a specific binding reaction between substances and a coloring reaction catalyzed by an enzyme, which is a labeling substance. The present invention was achieved by developing an inexpensive and simple kit that can qualitatively or semi-quantitatively measure derived substances.
［問題点を解決するための手段］
即ち、本考案は、生体由来物質を測定するため
のキツトであり、折りたたみ容器と、該生体由来
物質と特異的に結合し得る物質をロ紙上に固定し
て成る領域、該生体由来物質と特異的に結合し得
る物質であつて酵素で標識されて成る該物質を含
有する溶液が封入されている領域及び前記標識酵
素と反応し得る発色試薬を含有する溶液が封入さ
れている領域を有するフイルム状支持体とからな
り、前記フイルム状支持体が前記容器の底部に接
合されていることを特徴とする前記キツトに係わ
るものである。[Means for Solving the Problems] That is, the present invention is a kit for measuring biological substances, which includes a foldable container and a substance that can specifically bind to the biological substances fixed on a piece of paper. a region containing a solution containing a substance capable of specifically binding to the biological substance and labeled with an enzyme, and a coloring reagent capable of reacting with the labeled enzyme. The present invention relates to the kit, characterized in that the kit comprises a film-like support having a region in which a solution is enclosed, and the film-like support is joined to the bottom of the container.
本考案のキツトで用いる折りたたみ容器は、反
応に必要な量の尿、血液等の検体を反応に必要な
時間に亘つて収容・保持することができるだけの
容量及び強度を備えたものであるならばどのよう
な大きさ、形状及び材質を有するものでも構わな
いが、経済性、輸送性及び在庫性等の観点から、
例えば、実用新案出願公告昭51−52545号公報に
開示の、非熱接着性基材である板紙製の折りたた
みコツプが好適なものである。 The collapsible container used in the kit of the present invention must have sufficient capacity and strength to contain and hold the amount of urine, blood, or other specimen required for the reaction for the time required for the reaction. It does not matter what size, shape, and material it has, but from the viewpoint of economy, transportability, stockability, etc.
For example, a folding cup made of paperboard, which is a non-thermal adhesive base material, disclosed in Utility Model Application Publication No. 52545/1980 is suitable.
該折りたたみ容器の内表面は例えばポリエチレ
ンのような撥水性及び熱接着性のフイルムで被覆
されていると好ましい。 The inner surface of the collapsible container is preferably coated with a water-repellent and heat-adhesive film such as polyethylene.
本考案のフイルム状支持体は、例えばプラスチ
ツク又は紙等のような、前記検体（尿、血液等）
中で不活性な材質からできており、薄いスリツプ
状の形態のものが好適である。 The film-like support of the present invention, such as plastic or paper, can be used to support the specimen (urine, blood, etc.).
It is preferably made of an inert material and is in the form of a thin slip.
好ましくは、生体由来物質と特異的に結合し得
る物質が固定されるロ紙が適当な手段により該フ
イルム状支持体上に接合されている。又、このロ
紙がそのまま本考案のフイルム状支持体として機
能することも可能である。 Preferably, a paper on which a substance capable of specifically binding to a biological substance is immobilized is bonded onto the film-like support by an appropriate means. It is also possible for this paper to function as the film-like support of the present invention as it is.
前記の溶液が封入されている領域は、好ましく
は、袋状に形成されており、その袋が前記フイル
ム状支持体上に適当な手段により接合されてい
る。 The region in which the solution is enclosed is preferably formed in the form of a bag, and the bag is bonded onto the film-like support by suitable means.
所望の被検物質の種類に対応して、複数個の前
記ロ紙がフイルム状支持体上に接合されていても
良い。 A plurality of the above-mentioned strips of paper may be bonded onto a film-like support depending on the type of desired analyte.
この袋は、通常の中性領域の水溶液を安定に保
持し得、板紙の角、例えば本考案のキツトに於け
る折りたたみ容器の角にあたる部分でひつかくこ
とにより容易に破れる性質のものであればどのよ
うな材質のものでも良いが、特にビニール製の袋
が好適である。 This bag can stably hold a normal aqueous solution in the neutral range, and can be easily torn by being hit by the corners of the paperboard, for example, the corners of the folding container in the kit of the present invention. The bag may be made of any material, but a plastic bag is particularly suitable.
化学の分野に於いて使用される通常のロ紙であ
れば本考案のロ紙として好適に用いることができ
るが、東洋ロ紙のNo.２からNo.８までのものが本考
案のキツトに用いるロ紙としては特に好ましい。
ロ紙は呈色反応が視覚的に確認し得る程度の任意
の大きさであれば良い。 Any ordinary paper used in the field of chemistry can be suitably used as the paper in the present invention, but Toyo paper from No. 2 to No. 8 can be used in the kit of the present invention. It is particularly preferable for the paper to be used.
The paper may have any size as long as the color reaction can be visually confirmed.
被検物質としては、例えば、HCG，FSH，
LH，T3，T4，ETR及びTSH等のホルモン、
CEA及びAFP等の腫瘍マーカー並びにHBs抗原、
更にそれら各物質に対する各種抗体又は受容体の
ような生体由来物質が挙げられる。 Examples of test substances include HCG, FSH,
Hormones such as LH, T 3 , T 4 , ETR and TSH,
Tumor markers such as CEA and AFP and HBs antigen,
Further examples include biologically derived substances such as various antibodies or receptors for each of these substances.
従つて、本考案のキツトのロ紙に固定させる物
質としては、上記生体由来物質と特異的に結合し
得る物質、即ち、例えば被検物質がHCGの場合
にはロ紙に固定させる物質としては抗HCG抗体
が好ましい。更に、被検物質と特異的に結合し得
る物質が抗体である場合には、該抗体はモノクロ
ーナル抗体であることが好ましい。 Therefore, the substance to be immobilized on the paper of the kit of the present invention is a substance that can specifically bind to the above-mentioned biological substance, that is, for example, when the test substance is HCG, the substance to be immobilized on the paper is Anti-HCG antibodies are preferred. Furthermore, when the substance that can specifically bind to the test substance is an antibody, the antibody is preferably a monoclonal antibody.
被検物質とロ紙に固定された物質との特異的結
合反応としては前記のような抗原−抗体反応の他
に、例えば、リガンド（ホルモン）−レセプタ−
間の反応が挙げられる。 In addition to the above-mentioned antigen-antibody reaction, the specific binding reaction between the test substance and the substance immobilized on the paper includes, for example, a ligand (hormone)-receptor reaction.
Examples include reactions between.
所望の物質（例えば被検物質に対する抗体）
は、例えば、Porath，J.の方法により、BrCNで
ロ紙を活性化した後に該物質をロ紙に固定化させ
る（Porath，J.，Aspberg，K.，Drevin，H.，
＆ Axen，R.，Prepeation of cyanogen
bromide activated agarose gels.J.
Chromatography，86：53，1973参照）。 Desired substance (e.g. antibody against test substance)
have, for example, activated Ro paper with BrCN and then immobilized the substance on Ro paper by the method of Porath, J. (Porath, J., Aspberg, K., Drevin, H.,
& Axen, R., Prepetion of cyanogen
bromide activated agarose gels.J.
Chromatography, 86: 53 , 1973).
本考案のフイルム状支持体は折りたたみ容器の
底部に、例えば、両面テープのような接着剤で直
接固定されているか、又は他の適当な手段により
底部に接合されている。 The film-like support of the present invention is fixed directly to the bottom of the collapsible container, for example with an adhesive such as double-sided tape, or bonded to the bottom by other suitable means.
標識物質として用いることのできる酵素として
は、酵素免疫測定法の分野で通常使用されるもの
であればどれでもよいが、西洋ワサビペルオキシ
ダーゼ（HRP）が好適なものである。 As the enzyme that can be used as a labeling substance, any enzyme commonly used in the field of enzyme immunoassay may be used, but horseradish peroxidase (HRP) is preferred.
該酵素は例えば、グルタルアルデヒド架橋法
（AVRAMEAS，S.，Immunochemistry，６：
43−52，1969参照）及び過ヨウ素酸架橋法
（NAKANE，Ｐ，K.，＆ KAWAOI，A.，J.
Histochm，Cytochem.，22：1084−1091，1974
参照）などの前記酵素免疫測定法の分野に於いて
通常行われる方法で目的物質（例えば、前記抗
HCG抗体）と結合させることができる。酵素で
標識された該物質は好ましくは、例えば、リン酸
緩衝食塩水などの緩衝溶液中に溶解されている。 The enzyme can be crosslinked, for example, by the glutaraldehyde crosslinking method (AVRAMEAS, S., Immunochemistry, 6:
43-52, 1969) and periodate cross-linking method (NAKANE, P., K., & KAWAOI, A., J.
Histochm. Cytochem. 22: 1084-1091, 1974
The target substance (e.g., the above-mentioned antibody) is detected by a method commonly used in the field of the above-mentioned enzyme immunoassay.
The enzyme-labeled substance is preferably dissolved in a buffer solution, such as, for example, phosphate-buffered saline.
更にまた、酵素免疫測定法に於いて通常用いら
れる方法によつて、ビオチン及びアビジンにより
上記反応の測定感度を高めることもできる
（Heggeness and Ash（1977）参照）。 Furthermore, the measurement sensitivity of the above reaction can be increased by using biotin and avidin by a method commonly used in enzyme immunoassay (see Heggeness and Ash (1977)).
発色試薬としては、該標識酵素を触媒として呈
色反応を生起し得るようなもの、例えば酵素が前
記HRPの場合には、２，２−アジノビス（３−
エチルベンゾチアゾリン−６スルホン酸）
（ABPC）又はOPT、及びH2O2であり、この場
合ABPC又はOPTはH2O2により酸化されてそれ
ぞれ青色又は赤色の呈色反応を示す。 The coloring reagent is one that can cause a coloring reaction using the labeled enzyme as a catalyst, for example, when the enzyme is HRP, 2,2-azinobis(3-
ethylbenzothiazoline-6 sulfonic acid)
(ABPC) or OPT, and H 2 O 2 . In this case, ABPC or OPT is oxidized by H 2 O 2 and exhibits a blue or red color reaction, respectively.
これらの発色試薬は、例えば、リン酸緩衝食塩
水又は水に溶解された溶液状となつており、H2
O2とABPC、又はH2O2とOPTはそれぞれ別々の
袋に封入されていることが好ましい。 These coloring reagents are, for example, in the form of a solution dissolved in phosphate buffered saline or water, and H 2
Preferably, O 2 and ABPC or H 2 O 2 and OPT are each sealed in separate bags.
本考案のキツトは更に、呈色反応の標準として
用いる色調表及び操作マニユアル等を含むことが
できる。 The kit of the present invention can further include a color table used as a standard for color reaction, an operation manual, and the like.
［作用・効果］
本考案のキツトに於いては、まず、折りたたみ
容器を組み立て、尿、血液等の検体を十分量この
容器に受け取り、該容器の底部に接合されている
ロ紙と接触させ、このロ紙に固定されている物質
と上記検体中に含まれている被検物質との間で、
一定時間特異的結合反応を生起せしめ、次いで該
検体を容器から捨て去り、その後に、酵素標識さ
れた物質の溶液が封入されている袋を破き、その
溶液を該容器の底部に接合されているロ紙と一定
時間接触させ、このロ紙に前記反応により特異的
に結合された被検物質と該酵素標識された物質と
の間で、一定時間特異的結合反応を生起せしめ、
次いで発色試薬を含む溶液が封入された袋を破
き、その溶液を前と同様にロ紙と一定時間接触さ
せ、ロ紙上に前記結合被検物質を介して固定化さ
れた該標識酵素を触媒として呈色反応をロ紙上で
生起せしめ、その呈色反応により、目的とする被
検物質を定性的又は半定量的に測定するものであ
る。[Operation/Effect] In the kit of the present invention, first, a collapsible container is assembled, a sufficient amount of a sample such as urine or blood is received in this container, and the sample is brought into contact with the paper bonded to the bottom of the container. Between the substance fixed on this paper and the test substance contained in the sample,
A specific binding reaction is allowed to occur for a certain period of time, and then the sample is discarded from the container. After that, the bag containing the solution of the enzyme-labeled substance is torn, and the solution is poured into the bottom of the container. contacting with a paper for a certain period of time to cause a specific binding reaction for a certain period of time between the test substance specifically bound to the paper by the reaction and the enzyme-labeled substance;
Next, the bag in which the solution containing the coloring reagent was sealed is torn, and the solution is brought into contact with the paper for a certain period of time in the same manner as before, and the labeled enzyme immobilized on the paper via the bound analyte is catalyzed. A color reaction is caused on the paper, and the target test substance is measured qualitatively or semi-quantitatively based on the color reaction.
尚、この操作の際に、上記の袋を同時に破いて
封入されている各溶液を同時にロ紙と接触させる
こともできる。 Incidentally, during this operation, it is also possible to tear the bag at the same time and bring the enclosed solutions into contact with the paper at the same time.
尿、血液等の検体中に目的とする被検物質が存
在しているときには、その被検物質がロ紙上に特
異的に結合し、更にその被検物質に酵素標識され
た物質が特異的に結合して該標識酵素の触媒作用
によりロ紙上で呈色反応が生起する訳である。 When the target test substance is present in a sample such as urine or blood, the test substance specifically binds to the paper, and furthermore, the enzyme-labeled substance to the test substance specifically binds to the test substance. Upon binding, a coloring reaction occurs on the paper due to the catalytic action of the labeled enzyme.
このキツトの特徴は、上記反応に於いて固相で
あるロ紙が容器の底部に接合されていることによ
り、従来この種の測定装置で必要とされていた洗
浄操作というわずらわしい段階が不必要になつた
ことである。即ち、各反応終了後に、単に容器を
傾けるか又は各反応液を容器外に単に捨て去るこ
とにより、ロ紙に未結合の被検物質及び呈色反応
溶液がロ紙上から排除される結果、各反応成分の
いわゆる（bound）／Ｆ（free）分離が、従来は
必要とされていた洗浄というわずらわしい操作な
しに、極めて容易に行なえるようになつたもので
ある。 The feature of this kit is that the solid phase in the above reaction is bonded to the bottom of the container, eliminating the need for the troublesome cleaning step that was previously required with this type of measuring device. It's summer. That is, after each reaction is completed, by simply tilting the container or simply discarding each reaction solution out of the container, the analyte unbound to the paper and the colored reaction solution are removed from the paper, and as a result, each reaction is completed. The so-called (bound)/F (free) separation of components can now be performed extremely easily without the troublesome operation of washing that was previously required.
この結果、本発明のキツトを使用することによ
つて、生体由来物質を家庭等で極めて簡便に且つ
経済的に測定することができるようになり、この
キツトを用いてこのような測定を日常的、定期的
に行なうことにより生体の異常を早期発見するこ
とが可能になたものである。 As a result, by using the kit of the present invention, biological substances can be measured extremely easily and economically at home, etc., and this kit can be used to carry out such measurements on a daily basis. By performing this on a regular basis, it has become possible to detect abnormalities in the body at an early stage.
以下、添附図面に基づき本考案を説明する。添
附図面で示される本考案のキツトは本考案を具体
化したものであつて本考案の内容はこれらに限定
されるものではない。 The present invention will be explained below based on the accompanying drawings. The kit of the present invention shown in the accompanying drawings embodies the present invention, and the content of the present invention is not limited thereto.
第１図は本考案キツトのフイルム状支持体であ
るプラスチツク片１の拡大平面図である。プラス
イツク片の中央部上には被検物質と特異的に結合
し得る物質を固定して成るロ紙２が接合されてい
る。 FIG. 1 is an enlarged plan view of a plastic piece 1 which is the film-like support of the kit of the present invention. A piece of paper 2 on which a substance that can specifically bind to the test substance is immobilized is bonded to the center of the plastic piece.
被検物質と特異的に結合し得る物質であつて酵
素で標識されて成る該物質を含有する溶液が封入
されている袋３及び前記標識酵素と反応し得る発
色試薬を含有する溶液が封入されている袋４，
４′は、前記ロ紙２の片側にこれらも前記ロ紙２
と同様にプラスチツク片１上に接合されている。 A bag 3 encloses a solution containing a substance capable of specifically binding to a test substance and labeled with an enzyme, and a solution containing a coloring reagent capable of reacting with the labeled enzyme. bag 4,
4' are also attached to one side of the above paper 2.
It is similarly bonded onto the plastic piece 1.
第２図は、本考案の生体由来物質測定キツトの
折りたたみ容器５の外観展開図である。一部に切
欠穴６を設けた板紙７の内表面一面にポリエチレ
ンフイルム８が貼合されており、板紙７の四辺の
縁に沿つて一定巾の熱接着部９が設けられてい
る。中央部１０と、それに平行した折り込み部１
１，１２に沿つた１３及び１４にミシン目が形成
されている。これによつてこの折りたたみ容器は
Ｗ状に折りたたみ込まれ、熱接着部９によつて密
閉され、偏平な袋状形態をとるものである。 FIG. 2 is a developed view of the foldable container 5 of the kit for measuring biological substances of the present invention. A polyethylene film 8 is laminated to the entire inner surface of a paperboard 7 having a cutout hole 6 in a part thereof, and heat-bonded parts 9 of a constant width are provided along the edges of the four sides of the paperboard 7. A central part 10 and a folded part 1 parallel to it
Perforations are formed at 13 and 14 along lines 1 and 12. As a result, the foldable container is folded into a W-shape and sealed by the heat bonding part 9, so that it takes the form of a flat bag.
折りたたみ容器５を組み立てたときにその底部
となる折り込み部１１，１２の内面に点線で示さ
れるように前記プラスチツク片１が接合されてい
る。 The plastic piece 1 is bonded to the inner surfaces of the folded portions 11 and 12, which form the bottom of the foldable container 5 when assembled, as shown by dotted lines.
折りたたみ容器５の上部に形成されたミシン目
１５に沿つてこの上部を切り取つてこの容器を開
封する。次いで、熱接着部９を各々指でつまみ、
左右から中央部に向けて軽く押すことによつて折
り込み部１１，１２は開き、更に折りたたみ容器
に対して縦に形成されているミシン目１６及び１
７によつて折りたたみ容器が全体として折り込み
部１１及び１２を底部とする四角い升状のコツプ
状に組み立てられる。 The upper part of the foldable container 5 is cut off along the perforations 15 formed in the upper part to open the container. Next, pinch the heat-adhesive parts 9 with your fingers,
By pressing lightly from the left and right toward the center, the folded parts 11 and 12 open, and the perforations 16 and 1 formed vertically with respect to the folded container are opened.
7, the folding container is assembled as a whole into a rectangular box-shaped pot having folded portions 11 and 12 as the bottom.
このコツプの内表面はポリエチレンフイルム８
で一様に覆われている為に、コツプ内に入れた液
体検体は漏洩することなく保持される。 The inner surface of this tip is made of polyethylene film 8.
Because it is uniformly covered with water, the liquid sample placed inside the cup is retained without leaking.
以下、本考案のキツトの作製及びそれに用いた
測定の一実施例を示す。 An example of the production of the kit of the present invention and the measurements used therein will be shown below.
［実施例］
Ｉ ロ紙の前処理
東洋ロ紙（No.６）100gを室温で20〜60分間蒸
留水７mlで浸潤させ、ロ紙に対して３〜10重量％
の蒸留水を含有させた。次に、これに３〜10重量
％のBrCN水溶液７mlを撹拌しながら加えた。こ
の後直ちに、1MのNaOH水溶液を加えて前記
BrCN溶液をPH10〜10.5に調節した。２〜５分後
に、４℃に冷した0.005MのNaHCO3水溶液を140
ml添加して撹拌し、最後にこの溶液をブフナー漏
斗を通してロ過し、前記反応溶液を除去した。
NaHCO3による洗浄操作を３〜４回繰返した。[Example] I Pretreatment of Ro paper 100 g of Toyo Ro paper (No. 6) was infiltrated with 7 ml of distilled water for 20 to 60 minutes at room temperature, and a concentration of 3 to 10% by weight was added to the Ro paper.
of distilled water. Next, 7 ml of a 3-10% by weight BrCN aqueous solution was added to this with stirring. Immediately after this, add 1M NaOH aqueous solution and
The BrCN solution was adjusted to pH 10-10.5. After 2-5 minutes, add 0.005M NaHCO 3 aqueous solution cooled to 4℃ to 140℃.
ml was added and stirred, and finally the solution was filtered through a Buchner funnel to remove the reaction solution.
The washing operation with NaHCO3 was repeated 3-4 times.
次にこのブフナー漏斗上のロ紙を、それぞれ約
50mlの30重量％、70重量％、100重量％及び100重
量％の冷アセトン水溶液（４℃）で順次洗浄し
た。洗浄した前処理済のロ紙を冷蔵庫にて１〜２
時間かけて乾燥させた。このロ紙はその後−30℃
で保存すれば約２年間は活性が保持された状態で
安定である。 Next, place each piece of paper on this Buchner funnel, about approx.
It was washed sequentially with 50 ml of cold acetone aqueous solutions (4° C.) of 30%, 70%, 100% and 100% by weight. Place the washed and pre-treated paper in the refrigerator for 1-2 minutes.
It took a while to dry. This paper is then heated to -30℃.
It is stable and retains its activity for about 2 years if stored at room temperature.
抗体の固定
目的とするHCG抗原量（800IU／ｌ〜
1000IU／ｌ以上）を検出し得るだけの抗HCG抗
体（UCB（ベルギー）製）を含む水溶液20〜30ml
中に前記前処理済のロ紙100gを入れ４℃で36時
間〜72時間マグネチツクスターラーを用いて撹拌
させながら反応させた。その後、過剰の該抗体溶
液を除去し、重曹水（0.1M）を70〜100ml加えて
混和後、吸引ロ過で重曹水を除いた。次にβ−エ
タノールアミン（0.05M）の0.1M NaHCO3水溶
液を70〜100ml加え室温で２時間半〜３時間半マ
グネチツクスターラーにて撹拌した。その後、吸
引ロ過で前記β−エタノールアミン溶液を除去
し、次にロ紙を0.1MのNaHCO3水溶液約100mlで
洗浄し、次に0.1Mアセテート緩衝液（PH3.5〜
4.5）約100mlで３〜４回繰り返し洗浄した。 Immobilization of antibody Target amount of HCG antigen (800IU/l ~
20-30 ml of an aqueous solution containing enough anti-HCG antibody (manufactured by UCB (Belgium)) to detect 1000 IU/l or more
100 g of the pretreated paper was placed inside and reacted at 4° C. for 36 to 72 hours with stirring using a magnetic stirrer. Thereafter, excess of the antibody solution was removed, 70 to 100 ml of aqueous sodium bicarbonate (0.1M) was added and mixed, and the aqueous sodium bicarbonate was removed by suction filtration. Next, 70 to 100 ml of a 0.1M NaHCO 3 aqueous solution of β-ethanolamine (0.05M) was added and stirred with a magnetic stirrer for 2.5 to 3.5 hours at room temperature. Thereafter, the β-ethanolamine solution was removed by suction filtration, and the filter paper was then washed with about 100 ml of 0.1 M NaHCO 3 aqueous solution, followed by 0.1 M acetate buffer (pH 3.5 ~
4.5) Repeated washing 3 to 4 times with approximately 100 ml.
その後、ロ紙を0.5〜0.2重量％のBSAを含むリ
ン酸緩衝食塩水（0.05〜0.1重量％のTween20，
PH7.4〜PH7.6）約100mlにて２〜３回洗浄し、冷
蔵庫に保存した（乾燥状態または、リン酸緩衝食
塩水中でも保存が可能）。 After that, the paper was soaked in phosphate buffered saline containing 0.5-0.2 wt% BSA (0.05-0.1 wt% Tween20,
PH7.4-PH7.6) Washed 2-3 times with about 100 ml and stored in the refrigerator (can be stored in dry state or in phosphate buffered saline).
抗体と標識酵素との複合体の作製
目的とするHCG抗原量（800IU／ｌ〜
1000IU／ｌ以上）を検出し得るだけの抗HCG抗
体（前記と同じもの）水溶液50mlを約等量の
HRP水溶液（２〜５重量％）マグネチツクスタ
ーラを使用して、４℃にて２〜３時間インキユベ
ーシヨンした。 Preparation of a complex between antibody and labeled enzyme Target amount of HCG antigen (800 IU/l ~
Approximately equal volume of 50 ml of anti-HCG antibody (same as above) aqueous solution capable of detecting 1000 IU/l or more)
HRP aqueous solution (2-5% by weight) was incubated at 4° C. for 2-3 hours using a magnetic stirrer.
発色試薬の作製
20〜30mg／mlの２，２−アジノビス（３−エチ
ルベンゾチアゾリン−６−スルホン酸）及び５〜
10重量％のH2O2を含む水溶性の発色試薬0.5〜１
mlをビニール袋に封入し折りたたみ容器内に収容
した。 Preparation of coloring reagent 20-30 mg/ml of 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and 5-30 mg/ml
Water-soluble color reagent containing 10% by weight H2O2 0.5-1
ml was sealed in a plastic bag and placed in a foldable container.
以上の各反応試薬から成る本発明のキツトを用
いて次のように尿検体中のHCGの量を測定した。 The amount of HCG in a urine specimen was measured as follows using the kit of the present invention comprising the above-mentioned reaction reagents.
まず初めに、前述したように折りたたみ容器を
組み立て、各尿検体約５mlをこの容器にとつた。
数十秒後にこの尿を捨てた。 First, a collapsible container was assembled as described above, and approximately 5 ml of each urine sample was placed in this container.
The urine was discarded after several tens of seconds.
次に、前記HRP−抗HCG抗体複合体溶液が封
入されているビニール袋及び発色試薬が封入され
ているビニール袋を同時に破いてこれらの溶液と
ロ紙を接触させた。 Next, the plastic bag containing the HRP-anti-HCG antibody complex solution and the plastic bag containing the coloring reagent were simultaneously torn to bring these solutions into contact with the paper.
約２分後に容器を傾けて、反応液を容器のすみ
に集め、ロ紙と該反応後とを分離した。 After about 2 minutes, the container was tilted, the reaction solution was collected in the corner of the container, and the filter paper and the reaction product were separated.
陽性の場合はロ紙が青色を呈した。 If the test was positive, the paper turned blue.
以上の検査結果を市販の検査装置を使用して同
一検体について得られた結果と比較した。 The above test results were compared with results obtained for the same specimen using a commercially available testing device.
市販品との相関
検体No. 本発明のキツト 持田ラテツクス
試薬を用いる装置
１〜10 陽 性 陽 性
11〜20 陰 性 陰 性
ｎ＝20例のうち、陽性10例、陰性10例について
いずれも両者の間に100％の相関がみられた。Correlation with commercial products Specimen No. Kit of the present invention Mochida Latex Device using reagent 1 to 10 Positive Positive 11 to 20 Negative Negative Out of n = 20 cases, 10 positive cases and 10 negative cases were both tested. There was a 100% correlation between the two.
第１図は本考案のプラスチツク片１の拡大平面
図である。第２図は本考案の折りたたみ容器５の
外観展開図である。
１……プラスチツク片、２……ロ紙、３，４，
４′……袋、５……折りたたみ容器、６……切欠
穴、７……板紙、８……ポリエチレンフイルム、
９……熱接着部、１０……中央部、１１，１２…
…折り込み部、１３，１４，１５，１６，１７…
…ミシン目。
FIG. 1 is an enlarged plan view of a plastic piece 1 of the present invention. FIG. 2 is an external development view of the foldable container 5 of the present invention. 1... Plastic piece, 2... Paper, 3, 4,
4'...Bag, 5...Folding container, 6...Notch hole, 7...Paperboard, 8...Polyethylene film,
9...Thermal bonding part, 10...Central part, 11, 12...
...folding part, 13, 14, 15, 16, 17...
…Perforation.
Claims (1)
り、折りたたみ容器と、該生体由来物質と特異
的に結合し得る物質をロ紙上に固定して成る領
域、該生体由来物質と特異的に結合し得る物質
であつて酵素で標識されて成る該物質を含有す
る溶液が封入されている領域及び前記標識酵素
と反応し得る発色試薬を含有する溶液が封入さ
れている領域を有するフイルム状支持体とから
なり、前記フイルム状支持体が前記容器の底部
に接合されていることを特徴とする前記キツ
ト。 (2) 生体由来物質と特異的に結合し得る物質が抗
体であることを特徴とする実用新案登録請求の
範囲第１項に記載のキツト。 (3) 抗体が抗HCG抗体であることを特徴とする
実用新案登録請求の範囲第２項に記載のキツ
ト。 (4) 標識酵素が西洋ワサビペルオキシダーゼであ
り、発色試薬が２，２−アジノビス（３−エチ
ルベンゾチアゾリン−６−スルホン酸）及び過
酸化水素であることを特徴とする実用新案登録
請求の範囲第２項又は第３項に記載のキツト。 (5) 標識酵素が西洋ワサビペルオキシダーゼであ
り、発色試薬がOPT及び過酸化水素であるこ
とを特徴とする実用新案登録請求の範囲第２項
又は第３項に記載のキツト。 (6) 呈色反応の標準として色調表を含む実用新案
登録請求の範囲第１項ないし第５項のいずれか
に記載のキツト。 (7) 生体由来物質と特異的に結合し得る物質であ
つて酵素で標識されて成る該物質を含有する溶
液が更に緩衝剤を含むことを特徴とする実用新
案登録請求の範囲第１項ないし第６項のいずれ
かに記載のキツト。[Claims for Utility Model Registration] (1) A kit for measuring biological substances, comprising a foldable container, an area on which a substance capable of specifically binding to the biological substances is immobilized on a piece of paper, and a kit for measuring biological substances. A region in which a solution containing a substance that can specifically bind to a biological substance and is labeled with an enzyme is enclosed, and a solution containing a coloring reagent that can react with the labeled enzyme is enclosed. and a film-like support having a region in which the kit is formed, the film-like support being joined to the bottom of the container. (2) The kit according to claim 1, wherein the substance that can specifically bind to a biological substance is an antibody. (3) The kit according to claim 2, wherein the antibody is an anti-HCG antibody. (4) Utility model registration claim No. 1 characterized in that the labeling enzyme is horseradish peroxidase and the coloring reagents are 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and hydrogen peroxide. The kit according to item 2 or 3. (5) The kit according to claim 2 or 3, wherein the labeling enzyme is horseradish peroxidase, and the coloring reagents are OPT and hydrogen peroxide. (6) The kit according to any one of claims 1 to 5 for utility model registration, which includes a color tone table as a standard for color reaction. (7) Utility model registration claims 1 to 5, characterized in that the solution containing the substance, which is labeled with an enzyme and is capable of specifically binding to a biological substance, further contains a buffering agent. The kit according to any of paragraph 6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP13630986U JPH0453579Y2 (en) | 1986-09-05 | 1986-09-05 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP13630986U JPH0453579Y2 (en) | 1986-09-05 | 1986-09-05 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6344162U JPS6344162U (en) | 1988-03-24 |
| JPH0453579Y2 true JPH0453579Y2 (en) | 1992-12-16 |
Family
ID=31039297
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP13630986U Expired JPH0453579Y2 (en) | 1986-09-05 | 1986-09-05 |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0453579Y2 (en) |
-
1986
- 1986-09-05 JP JP13630986U patent/JPH0453579Y2/ja not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6344162U (en) | 1988-03-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US5571667A (en) | Elongated membrane flow-through diagnostic device and method | |
| US6140136A (en) | Analytical test device and method of use | |
| US7344893B2 (en) | Immuno-gold lateral flow assay | |
| US4948726A (en) | Enzyme immunoassay based on membrane separation of antigen-antibody complexes | |
| EP0310406B1 (en) | Directed flow diagnostic device and method | |
| JP3358737B2 (en) | Assays with improved dose response curves | |
| US20010021536A1 (en) | Even fluid front for liquid sample on test strip device | |
| US6689317B1 (en) | Immunoassay apparatus for diagnosis | |
| JPS61247965A (en) | Enzyme immunological measurement method | |
| JPH0755808A (en) | Diagnostic method | |
| JPH06317595A (en) | Complementary visual sense signal immunoassay | |
| US5958339A (en) | Format for immunoassay in thin film | |
| JP2002507725A (en) | Immunoassay device and method | |
| JPH11153600A (en) | Test piece for immunoassay and measuring method using the same | |
| JPH10177028A (en) | Method for measuring specific substance in papillary secretion | |
| CA2008100A1 (en) | Method for the determination of immunologically detectable substance | |
| EP0402023B1 (en) | Elongated membrane flow-through diagnostic device and method | |
| EP0603958A1 (en) | Improvement of the dynamic range in specific binding assays | |
| WO1999060401A1 (en) | Immunoassay reagents and immunoassay method | |
| JPH0453579Y2 (en) | ||
| JP2001059845A (en) | Method and element for dry analysis | |
| US5296356A (en) | Enzyme-immunoassay method for the determination of an analyte | |
| EP0333801B1 (en) | Test strip for diagnosis by multi-immunoassay system | |
| JPH05209879A (en) | Immunological method for detecting hemoglobin | |
| JPH04324358A (en) | Measurement method of human hemoglobin in dejection |