JPS63289453A - Method and reagent kit for measuring human hemoglobin in biological component - Google Patents
Method and reagent kit for measuring human hemoglobin in biological componentInfo
- Publication number
- JPS63289453A JPS63289453A JP12253987A JP12253987A JPS63289453A JP S63289453 A JPS63289453 A JP S63289453A JP 12253987 A JP12253987 A JP 12253987A JP 12253987 A JP12253987 A JP 12253987A JP S63289453 A JPS63289453 A JP S63289453A
- Authority
- JP
- Japan
- Prior art keywords
- human
- measured
- human hemoglobin
- polyclonal antibody
- antigen
- 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.)
- Granted
Links
- 102000001554 Hemoglobins Human genes 0.000 title claims abstract description 23
- 108010054147 Hemoglobins Proteins 0.000 title claims abstract description 23
- 238000000034 method Methods 0.000 title claims description 38
- 239000003153 chemical reaction reagent Substances 0.000 title claims description 5
- 238000006243 chemical reaction Methods 0.000 claims abstract description 24
- 230000004520 agglutination Effects 0.000 claims abstract description 22
- 210000004369 blood Anatomy 0.000 claims abstract description 11
- 239000008280 blood Substances 0.000 claims abstract description 11
- 210000002700 urine Anatomy 0.000 claims abstract description 7
- 239000004816 latex Substances 0.000 claims description 21
- 229920000126 latex Polymers 0.000 claims description 21
- 230000002776 aggregation Effects 0.000 claims description 9
- 238000004220 aggregation Methods 0.000 claims description 8
- 230000002550 fecal effect Effects 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 2
- 239000000427 antigen Substances 0.000 abstract description 22
- 102000036639 antigens Human genes 0.000 abstract description 22
- 108091007433 antigens Proteins 0.000 abstract description 22
- 238000005259 measurement Methods 0.000 abstract description 12
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 230000003287 optical effect Effects 0.000 abstract description 3
- 239000012472 biological sample Substances 0.000 abstract description 2
- 208000001118 melena Diseases 0.000 abstract description 2
- 238000007689 inspection Methods 0.000 abstract 1
- 230000000007 visual effect Effects 0.000 abstract 1
- 239000002245 particle Substances 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- 238000007796 conventional method Methods 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 239000004793 Polystyrene Substances 0.000 description 3
- 238000000691 measurement method Methods 0.000 description 3
- 229920002223 polystyrene Polymers 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000007822 coupling agent Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 210000003608 fece Anatomy 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000001235 sensitizing effect Effects 0.000 description 2
- 229920003051 synthetic elastomer Polymers 0.000 description 2
- 239000005061 synthetic rubber Substances 0.000 description 2
- 230000002485 urinary effect Effects 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 101100501250 Caenorhabditis elegans elo-3 gene Proteins 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 229910021555 Chromium Chloride Inorganic materials 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 238000005054 agglomeration Methods 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- QSWDMMVNRMROPK-UHFFFAOYSA-K chromium(3+) trichloride Chemical compound [Cl-].[Cl-].[Cl-].[Cr+3] QSWDMMVNRMROPK-UHFFFAOYSA-K 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 208000035861 hematochezia Diseases 0.000 description 1
- 208000006750 hematuria Diseases 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- -1 polyethylene Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- DVKJHBMWWAPEIU-UHFFFAOYSA-N toluene 2,4-diisocyanate Chemical compound CC1=CC=C(N=C=O)C=C1N=C=O DVKJHBMWWAPEIU-UHFFFAOYSA-N 0.000 description 1
Abstract
Description
【発明の詳細な説明】
〈産業上の利用分野〉
この発明は、抗ヒト・ヘモグロビンポリクローナル抗体
を用いた生体成分中のヒト・ヘモグロビン（以下ヒト・
Ｈｂと略称する）の測定方法及び測定用試薬キットに圓
するものである。[Detailed Description of the Invention] <Industrial Application Field> The present invention is directed to the detection of human hemoglobin (hereinafter referred to as human hemoglobin) in biological components using an anti-human hemoglobin polyclonal antibody.
The present invention relates to a method for measuring Hb (abbreviated as Hb) and a reagent kit for the measurement.
〈従来の技術〉
抗原抗体反応を利用した生体の微量成分を分析する方法
として、ポリクローナル抗体感作担体とフリーな抗原を
液体中で混合して生ずる凝集反応を、スライド凝集板上
で目視的に、または光学的に測定する方法が知られてい
る。<Prior art> As a method for analyzing trace components of living organisms using antigen-antibody reactions, a polyclonal antibody-sensitized carrier and a free antigen are mixed in a liquid, and the agglutination reaction that occurs is visually observed on a slide agglutination plate. , or optical measurement methods are known.
この方法は、多種類の生体成分を含む試料の中で目的と
する生体成分を高選択的、かつ高感度的に測定可能なた
め、一般に広く使用されており、その中には、例えば抗
ヒト・Ｈｂポリクローナル抗体をラテックス粒子に結合
させた抗体感作ラテックス粒子と、糞便中のヒト・Ｈｂ
とを反応させ、糞便中のヒト・Ｈｂを測定する方法（い
わゆるラテックス凝集法）がある（特開昭５９−１２５
０６４号公報参照）。This method is generally widely used because it can measure target biological components with high selectivity and sensitivity in samples containing many types of biological components.・Antibody-sensitized latex particles with Hb polyclonal antibodies bound to latex particles and human Hb in feces.
There is a method of measuring human Hb in feces (so-called latex agglutination method) by reacting with
(See Publication No. 064).
〈発明が解決しようとする問題点〉
しかしながら、前記した従来の分析方法（ラテックス凝
集法）においては、免疫学的反応の特有の性質として、
一定量のポリクローナル抗体感作担体に対し、測定対象
である抗原濃度を増加さ、 せていく場合に、ある濃度
までは抗原抗体反応による凝集反応が増加していくが、
一定量の抗原濃、 度を越えると、逆に凝集反応が抑制
されるいわゆる地帯現象がみられる。このため高濃度の
抗原領域においで、抗原濃度を異常に低濃度に測定して
しまうという、測定方法としては重大な欠陥をもってい
る方法である。<Problems to be Solved by the Invention> However, in the conventional analysis method described above (latex agglutination method), as a characteristic characteristic of the immunological reaction,
When increasing the concentration of the antigen to be measured against a fixed amount of polyclonal antibody-sensitized carrier, the agglutination reaction due to the antigen-antibody reaction increases up to a certain concentration;
When the antigen concentration exceeds a certain level, a so-called zone phenomenon occurs in which the agglutination reaction is suppressed. For this reason, this method has a serious flaw as a measurement method in that it measures an abnormally low antigen concentration in a region of high antigen concentration.
そこで、本発明者等は抗ヒト・Ｈｂポリクローナル抗体
を使用して、凝集反応によって、ヒト・Ｈｂｊ＆測定す
る方法について種々検討した結果、別に測定対象である
ヒト・Ｈｂを使用してヒト・Ｈｂ感作担体を製作し、こ
れを抗ヒト・）−１ｂポリクロ一ナル抗体（溶液、また
は感作担体）とし、前記反応系に添加（導入）してフリ
ーのヒト・Ｈｂを測定する新たな方法を開発して、本発
明を完成した。Therefore, the present inventors have investigated various methods for measuring human Hbj & Hb by agglutination reaction using anti-human Hb polyclonal antibodies. A new method for measuring free human Hb was created by preparing a carrier, using it as an anti-human )-1b polyclonal antibody (solution or sensitized carrier), and adding (introducing) it to the reaction system. and completed the present invention.
〈問題を解決するための手段〉
すなわち、この発明は測定対象であるヒト・Ｈｂと特異
的に反応するポリクローナル抗体（溶液または感作担体
）と測定対象であるヒト・Ｈｂ感作担体とが反応して生
ずる凝集反応を、測定対象であるフリーのヒト・Ｈｂが
阻止する程度（度合）を目視的または光学的に測定する
ことによって、フリーのヒト・Ｈｂを免疫学的に高特異
的、かつ高感度に測定することができるようにすると共
に、フリーのヒト・Ｈｂ（抗原）が高濃度（こ存在する
場合においても、ラテックス凝集法における地帯現象を
生じさせることなく、精確に測定することができるよう
にした生体成分中のヒト・ヘモグロビンの測定方法及び
測定用試薬キットを提供することを目的として開発した
ものである。<Means for solving the problem> In other words, the present invention provides a method for reacting a polyclonal antibody (solution or sensitized carrier) that specifically reacts with human Hb, which is the measurement target, with a human/Hb sensitized carrier, which is the measurement target. By visually or optically measuring the degree to which free human Hb, the measurement target, inhibits the agglutination reaction that occurs, free human Hb can be detected with high immunological specificity and In addition to enabling highly sensitive measurement, even when free human Hb (antigen) is present at high concentrations, it is possible to accurately measure without causing the zone phenomenon in the latex agglutination method. This invention was developed for the purpose of providing a method for measuring human hemoglobin in biological components and a reagent kit for the measurement.
本発明に係る生体成分中のヒト・Ｈｂの測定方法は、ヒ
ト・Ｈｂと特異的に反応するポリクローナル抗体（溶液
または感作担体）と測定対象であるフリーのヒト・Ｈｂ
とが反応しても凝集しにくく、目視、または光学的に測
定困難な場合に特に有効である。The method for measuring human Hb in biological components according to the present invention uses a polyclonal antibody (solution or sensitized carrier) that specifically reacts with human Hb and free human Hb to be measured.
It is particularly effective in cases where it is difficult to agglomerate even if the two react with each other, and it is difficult to measure visually or optically.
また、ヒト便中、または尿中に血液が高濃度に存在する
場合（例タール便または血尿）に低濃度と間違えること
なく測定できるという点においてもラテックス凝集法に
比べて優れでいる。It is also superior to the latex agglutination method in that when blood is present at a high concentration in human stool or urine (eg, tarry stool or hematuria), it can be measured without mistaking it as a low concentration.
次に、本願発明に係る抗ヒト・Ｈｂポリクローナル抗体
を用いた生体成分中のヒト・Ｈｂの分析方法の原理をよ
り詳細に記載する。Next, the principle of the method for analyzing human Hb in biological components using the anti-human Hb polyclonal antibody according to the present invention will be described in more detail.
従来使用されていたポリクローナル抗体感作担体は、こ
れをフリーの抗原と混合すると、ポリクローナル抗体が
、抗原分子上の多種の抗原決定基と次々と反応し凝集塊
となる。しかしこの凝集塊が目視的、または光学的に測
定可能な程度に生成するには長時間を要する場合が多く
、精確かつ迅速性を至上命令とする分析方法としては問
題があった。When a conventionally used polyclonal antibody sensitized carrier is mixed with a free antigen, the polyclonal antibody reacts with various antigenic determinants on the antigen molecule one after another, forming an aggregate. However, it often takes a long time to form such agglomerates to the extent that they can be measured visually or optically, which is a problem for analytical methods that require precision and speed as paramount requirements.
そこで、本願発明においでは測定対象と同一の抗原（ヒ
ト・・Ｈｂ）感作担体を別に製作し、このヒト・Ｈｂ感
作担体を前記反応系内に添加して抗ヒト・Ｈｂポリクロ
ーナル抗体（溶液または感作担体）と凝集反応を確実に
生じさせ、測定対象であるフリーのヒト・Ｈｂ（抗原）
は、前記凝集反応を阻止する要因（成分）として機能さ
せることとした。これにより、前記反応において主する
凝集の程度を目視的、または光学的に測定する（測定対
象であるフリーのヒト・Ｈｂを添加する場合と、添加し
ない場合についで）こと１こより測定対象であるフリー
のヒト・１−１ｂを、高特異的、かつ高感度に測定する
ことを可能とすると共に、前記ヒト・Ｈｂが高濃度（こ
存在する場合においてもラテックス凝集法におけるよう
な地帯現象の発生する余地をなくして、ヒト・Ｈｂ濃度
を異常に低濃度に測定することを防止したものである。Therefore, in the present invention, a carrier sensitized with the same antigen (human...Hb) as that to be measured is separately manufactured, and this carrier sensitized with human Hb is added to the reaction system to form an anti-human/Hb polyclonal antibody (solution). or sensitized carrier) to ensure an agglutination reaction, and free human Hb (antigen) to be measured.
was made to function as a factor (component) for inhibiting the aggregation reaction. As a result, the main degree of aggregation in the reaction can be visually or optically measured (with and without addition of free human Hb, which is the measurement target). It is possible to measure free human Hb with high specificity and sensitivity, and even when the human Hb is present at a high concentration, a zone phenomenon like that in the latex agglutination method occurs. This prevents abnormally low human Hb concentrations from being measured.
次に本発明の具体例として、ポリクローナル抗体に抗ヒ
ト・Ｈｂポリ、クローナル抗体を、抗原にはヒト・Ｈｌ
）７＆用い、これらから各々、抗ヒト・Ｈｂポリクロー
ナル抗体溶液または感作担体、及びヒト・Ｈｂ感作担体
を製造し、これを使用して便潜血と尿潜血を測定する方
法についで述べる。Next, as a specific example of the present invention, an anti-human Hb polyclonal antibody is used as a polyclonal antibody, and a human Hb polyclonal antibody is used as an antigen.
) 7&, respectively, to produce an anti-human Hb polyclonal antibody solution or sensitized carrier, and a human Hb sensitized carrier from these, and to use these to measure fecal occult blood and urinary occult blood.
本発明の抗ヒト・ヘモグロどンポリクローナル抗体の作
製法は、通常の文献に記載されでいる方法で充分であり
、特別な方法を必要としない。The anti-human hemoglodon polyclonal antibody of the present invention can be produced by methods described in ordinary literature, and no special methods are required.
本発明に使用する担体粒子は、間接凝集反応用のものを
使用すればよい。すなわち、ポリエチレンラテックス、
各種ラテックス、動物の赤血球、カオリシ、炭素粒子等
を使用することができる。The carrier particles used in the present invention may be those for indirect aggregation reactions. i.e. polyethylene latex,
Various types of latex, animal red blood cells, Kaorishi, carbon particles, etc. can be used.
本発明の抗ヒト・ヘモグロビンポリクローナル抗体やヒ
ト・ヘモグロビン抗原を担体に感作する方法も一般の抗
体または抗原を感作する公知の方法によればよい。例え
ば、物理的に吸着させる方法は、最も一般的である。ま
た、カルボキシル化ポリスチレンラテックスによる共有
結合法、ゲルタールアルデヒド、トリレンジイソシアナ
ート、カルボジイミド類、及び塩化クロム等のいわゆる
カップリング剤を使用する方法もある。The method for sensitizing a carrier with the anti-human hemoglobin polyclonal antibody or human hemoglobin antigen of the present invention may be any known method for sensitizing a general antibody or antigen. For example, physical adsorption is the most common method. There are also covalent bonding methods using carboxylated polystyrene latex, and methods using so-called coupling agents such as geltaraldehyde, tolylene diisocyanate, carbodiimides, and chromium chloride.
次に具体的手法について更に詳細に述べる。Next, the specific method will be described in more detail.
（ａ）抗ヒト・Ｈｂポリクロナール抗体感作担体（ラテ
ックス）の調製法
抗ヒト・Ｈｂポリクローナル抗体と担体（ラテックス粒
子）との結合は、物理的結合、あるいは一般の化学的結
合により行なうことができる。(a) Preparation method of anti-human Hb polyclonal antibody sensitized carrier (latex) The binding between the anti-human Hb polyclonal antibody and the carrier (latex particles) can be carried out by physical bonding or general chemical bonding. .
次にその結合方法の具体例を示す。Next, a specific example of the coupling method will be shown.
（イ）物理的方法
ポリスチレンラッテクス粒子（日本合成ゴム■製、直径
４６７１ｍ）の懸濁液を、０．１５Ｍ塩化ナトリウムを
含む〇二 １Ｍトリス塩酸緩衝液（ｐＨ８，０）で１０
倍に稀釈し、この緩衝液で３度遠心洗浄する。この沈澱
を前記緩衝液で１％に調製した懸濁液とする。これに抗
ヒト・Ｈｂポリクロナール抗体（０，６ｍ９／ｍｌ２）
を添加し、室温で１時間攪拌した後、ラテックス（こ結
合しない余剰の抗体を遠心洗浄する。これに牛血清アル
ブミン０．０１％を含有する０、１５Ｍ塩化ナトリウム
含有トリス−塩酸緩衝液（ｐＨ８゜Ｏ）を添加し、′１
％ラテックス懸濁液に調製する。(b) Physical method A suspension of polystyrene latex particles (manufactured by Nippon Synthetic Rubber ■, diameter 4671 m) was mixed with 1 M Tris-HCl buffer (pH 8.0) containing 0.15 M sodium chloride for 10 min.
Dilute it twice and centrifuge and wash it three times with this buffer. This precipitate is made into a 1% suspension with the above buffer. In addition to this, anti-human Hb polyclonal antibody (0.6m9/ml2)
After stirring at room temperature for 1 hour, the latex (excess antibody that does not bind to the latex) is washed by centrifugation.゜O) and '1
% latex suspension.
（ロ）化学的方法
カルボキシル基を導入したポリスチレンラッテクス粒子
（日本合成ゴム■製、直径０．４９７ ｕｍ）の１０％
懸濁液を０．１Ｍトリス塩酸緩衝液（ｐＨ８，０）ｒｌ
Ｏ倍に稀釈する。この稀釈液に、抗ヒト・Ｈｂポリクロ
ナール抗体（０，６ｍ９／ｍβ）とカップリング剤を順
に添加し、よく攪拌して共有結合させる。ラテックスに
結合しない余剰の抗体を遠心洗浄する。これに牛血清ア
ルブミン０．０１％を含有する０、１５Ｍ塩化ナトリウ
ム含有トリス−塩酸緩衝液（ｐＨ８，０）を添加し、１
％ラテックス懸濁液に調製する。。(b) Chemical method: 10% of polystyrene latex particles (manufactured by Japan Synthetic Rubber ■, diameter 0.497 um) into which carboxyl groups have been introduced.
The suspension was added to 0.1M Tris-HCl buffer (pH 8,0) rl.
Dilute O times. To this diluted solution, anti-human Hb polyclonal antibody (0.6m9/mβ) and a coupling agent are sequentially added and stirred well to cause covalent bonding. Excess antibody that does not bind to the latex is washed away by centrifugation. To this was added 0.15 M sodium chloride-containing Tris-HCl buffer (pH 8.0) containing 0.01% bovine serum albumin, and
% latex suspension. .
（ｂ）ヒト・Ｈｂ感作担体（ラテックス粒子）の調製法 抗体に準する。(b) Preparation method of human Hb sensitized carrier (latex particles) Similar to antibodies.
〈実施例１〉
スライド凝集板法による便中潜血の検出健康者の便１９
を０．１５Ｍ−ＮａＣ１含有。<Example 1> Detection of occult blood in stool by slide agglutination plate method Stool 19 of a healthy person
Contains 0.15M-NaCl.
ｏ、ＩＭ−トリス−塩酸緩衝液１００ｍｊｌ！に懸濁し
、遠心分離機にかけ上清液を採取する。次にヒト・Ｈｂ
を前記上清液に添加し、１００００μ９／ｍｌｌ、２０
００ｕ９／ｍ１２，４００ｕｑ／、ｍ１！、８０ｕ９／
ｒｒ１．１．６ｕｑ／ｒｒｌ、３．２ｕｑ／ｍ１１．０
．６４ｕ９／ｍｕ、０．１２８Ｌ１９／ｍ１２，０．０
２５６ｕ９／ｍＣＯｕｑ／ｒｒｌ濃度のサンプルを各々
調製する。これらの各、濃度のサンプルの４．Ｏｕ Ｉ
Ｉを、各々スライド凝集板上に滴下した後、前記（ａ）
の（イ）で調製した抗ヒト・Ｈｂポリクロナール抗体溶
液、または抗体感作担体、及び前記（ｂ）で調製したヒ
ト・Ｈｂ抗原感作担体２０μβを追加して滴下し、２分
周ゆるやかに攪拌しながら観察する。同時に従来法（抗
ヒト・Ｈｌ）ポリクロナール抗体感作担体２０ｕｌｌ、
上記サンプル ４０μβ）も行なった。o, 100 mjl of IM-Tris-HCl buffer! Suspend in a centrifuge and collect the supernatant. Next, human Hb
was added to the supernatant, 10,000μ9/ml, 20
00u9/m12,400uq/, m1! ,80u9/
rr1.1.6uq/rrl, 3.2uq/m11.0
．． 64u9/mu, 0.128L19/m12, 0.0
Each sample is prepared at a concentration of 256u9/mCOuq/rrl. 4 of each of these samples at a concentration. Ou I
After dropping I onto each slide aggregation plate, the above (a)
Add dropwise the anti-human Hb polyclonal antibody solution prepared in (a) or the antibody sensitized carrier and 20 μβ of the human Hb antigen-sensitized carrier prepared in (b) above, and stir gently for 2 minutes. Observe while doing so. At the same time, 20 ul of conventional method (anti-human/Hl) polyclonal antibody sensitized carrier,
The above sample (40μβ) was also tested.
その結果を次表に示す。The results are shown in the table below.
註 ＋は凝集を、また−は非凝集を表わす。Note: + indicates aggregation, and - indicates non-aggregation.
この表の結果によれば、本願発明に係る方法は、測定対
象であるフリーのヒト・ＨＭＩ（０，６４ＬＩ９／ｍβ
以下低濃度であれば、抗ヒト・Ｈｂポリクローナル抗体
（溶液、または感作担体）とヒト・Ｈｂ感作担体との凝
集反応を阻止しないのに対し、３．２ｕｑ／ｍＪ以上１
００００ｕｑ／ｍｌｌの範囲（約３０００倍）で凝集反
応を阻止している。このことは、抗原であるヒト・Ｈｂ
が大過剰存在しても、低濃度と区別できることを示して
いる。これに対し従来法における抗ヒト・Ｈｉ）ポリク
ローナル抗体感作担体と抗原であるヒト・Ｈｂとの凝集
反応が生ずる範囲は、０．６４ｕｑ／ｍＩｌ〜１６Ｌ１
９／ｍｌの範囲（約２５倍）と非常に狭い、従って、抗
原であるヒト・Ｈｂ８０ｕｑ／ｍＡ以上２０００ｕ９／
ｍＩ２の範囲では、０．１２８ｕ９／ｍβ以下の濃度と
同一視され、非常に高濃度のヒト・Ｈｂを異常に低濃度
に測定してしまうのである。According to the results of this table, the method according to the present invention can be used to measure free human HMI (0,64LI9/mβ
If the concentration is lower than 3.2 uq/mJ, the agglutination reaction between the anti-human Hb polyclonal antibody (solution or sensitized carrier) and the human Hb sensitized carrier will not be inhibited.
The agglutination reaction is inhibited within the range of 0,000 uq/ml (approximately 3,000 times). This means that the antigen human Hb
This shows that even if there is a large excess, it can be distinguished from a low concentration. On the other hand, in the conventional method, the range in which the agglutination reaction between the anti-human Hi) polyclonal antibody sensitized carrier and the antigen human Hb occurs is 0.64 uq/ml to 16 L1.
The range of 9/ml (approximately 25 times) is very narrow, therefore, the antigen human Hb 80 uq/mA or more
The mI2 range is equated with a concentration of 0.128u9/mβ or less, and an extremely high concentration of human Hb is measured at an abnormally low concentration.
すなわち、本願発明に係るヒト・Ｈｂ感作担体を使用す
る方法は、抗ヒト・Ｈｂポリクローナル抗体が溶液であ
っても、また感作担体であっても、フリーのヒト・Ｈｂ
が少なくとも３．２μ９／ ｍ １１以上あれば、ヒト
・Ｈｂ感作担体との凝集反応を阻止できるので測定可能
である。これに対し従来法は、フリーのヒト・Ｈｂの濃
度が０．６４−１６ｕ９／ｍｉ７存在する場合に限られ
、８０ｕｑ／ｍ４２以上の高濃度のフリーのヒト・Ｈｂ
を、０．１２８ｕ９／ｍｕ以下の低濃度の場合と同様、
測定することができない。That is, the method of using the human/Hb sensitized carrier according to the present invention is applicable to free human/Hb polyclonal antibodies, whether they are in solution or in the sensitized carrier.
If it is at least 3.2μ9/m11 or more, it can be measured because the agglutination reaction with the human Hb-sensitized carrier can be inhibited. In contrast, the conventional method is limited to cases where the concentration of free human Hb is 0.64-16u9/mi7, and is limited to cases where the concentration of free human Hb is 0.64-16u9/mi7.
As in the case of low concentration below 0.128u9/mu,
cannot be measured.
く寅施例２〉
スライド凝集法による尿中潜血の検出
健康者の尿を採取し、遠心分離機にかけ上清液を採取す
る。その上清液を０．３Ｍ−ＮａＣＬ含有の０．２Ｍ１
−リス塩酸緩衝液と等量混合した後ヒト−Ｈｂを添加し
、１００００ｕｑ／ｍβ、２０００ｕｑ／ｍＩ２，４０
０ｕ９／ｍＩ２．８０Ｌ１９／ｍ１１．１６ｕｑ／ｍｆ
、３．２μ９／ｍｕ。Example 2 Detection of urinary occult blood by slide agglutination method Urine from a healthy person is collected and centrifuged to collect the supernatant. The supernatant was 0.2M1 containing 0.3M-NaCL.
- After mixing an equal amount with Lis-HCl buffer, add human-Hb, 10000uq/mβ, 2000uq/mI2,40
0u9/mI2.80L19/m11.16uq/mf
, 3.2μ9/mu.
０．６４ｕ９／ｍｕ、０．１２８ｕｑ／ｍｎ。0.64u9/mu, 0.128uq/mn.
０．０２５６ｕ９／ｍＣＯｕｑ／ｍＩｌ濃度のサンプル
を各々調製する。これらの各濃度のサンプルの４０ｕＡ
を、各々スライド凝集板上に滴下した後、前記（ａ）の
（イ）で調製した抗ヒト・Ｈｂポリクロナール抗体溶液
、または抗体感作担体、及び前記（ｂ）で調製したヒト
・Ｈｂ抗原感作担体２０ｕＡＮ追加して滴下し、２分間
ゆるやかに攪拌しながら観察する。Each sample is prepared at a concentration of 0.0256u9/mCOuq/ml. 40uA of samples at each of these concentrations
were dropped onto the slide agglutination plate, and then the anti-human Hb polyclonal antibody solution prepared in (a) (b) or the antibody sensitized carrier and the human Hb antigen sensitized carrier prepared in (b) above were added. Add 20 μAN of the working carrier and drop it, and observe while stirring gently for 2 minutes.
その結果を次表に示す。The results are shown in the table below.
この表あ結果によれば、本願発明に係る方法は、測定対
象であるフリーのヒト・Ｈｂが便中であれ、尿中であれ
、それぞれに特有に共存する物質に影響されることなく
、測定対象であるヒト・Ｈｂを高選択的、かつ高感度に
測定可能であることを示している。According to the results shown in this table, the method according to the present invention can measure free human Hb, which is the measurement target, regardless of whether it is in stool or urine, without being affected by substances that coexist in each case. This shows that the target human Hb can be measured with high selectivity and sensitivity.
〈実施例３〉
分光光度計を用いた便中潜血の検出（反応）実施例１で
使用したサシプル１００ｍＩｌに抗体０．０２ｍｑ、ま
たは抗ヒト・Ｈｂポリクローナル感作担体０．０１％を
含む０．１５トリス−塩酸緩衝液２５００ｕｆと、前記
（Ｂ）で調製したヒトーＨｂ感作担体（０，１％）４０
０ｕＩ２１８加え、手速く混合した後、光路長１０ｍｍ
のセルに入れ、分光光度計（日立３２０型）を用い、波
長７００ｎｍにおける、反応開始０．５分〜５．５分間
の吸光度を測定した。<Example 3> Detection (reaction) of fecal occult blood using a spectrophotometer 0.02 mq of antibody or 0.01% of anti-human Hb polyclonal sensitized carrier was added to 100 ml of Sacipul used in Example 1. 15 2500 uf of Tris-HCl buffer and the human Hb sensitized carrier (0.1%) prepared in (B) above 40
Add 0 uI218 and mix quickly, then optical path length 10 mm
The absorbance was measured at a wavelength of 700 nm from 0.5 minutes to 5.5 minutes from the start of the reaction using a spectrophotometer (Hitachi Model 320).
その結果は第１図の通りであった。The results were as shown in Figure 1.
第１図に示される標準曲線から、便中ヒト・Ｈｂ濃度を
実施例１のスライド凝集法より、更に高感度かつ高濃度
に測定することができることがわかる。The standard curve shown in FIG. 1 shows that the human Hb concentration in stool can be measured with higher sensitivity and higher concentration than the slide agglutination method of Example 1.
また、第１図から従来法の欠点である［高濃度抗原域に
おいて、抗原濃度を異常に低濃度に測定してしまう」と
いう欠陥を回避できることは明らかである。Furthermore, from FIG. 1, it is clear that the drawback of the conventional method, which is that the antigen concentration is measured at an abnormally low concentration in a high antigen concentration area, can be avoided.
〈発明の効果〉
以上のようにこの発明ｆこ係る生体成分中のヒト・Ｈｂ
の測定方法によれば、多くの他種の物質を含む生体試料
（得にヒト便中、または尿中）の目的とする微量の潜血
を簡便に、高選択的かつ高感度で測定できると共に、高
濃度のヒト・Ｈｂを低濃度と間違えることなく測定でき
るという効果を有する。<Effects of the Invention> As described above, this invention
According to this measurement method, a trace amount of occult blood can be easily measured with high selectivity and sensitivity in a biological sample (particularly human stool or urine) containing many other types of substances, and It has the effect of being able to measure high concentrations of human Hb without mistaking them for low concentrations.
第１図は、フリーのヒト・Ｈｂ濃度と吸光度の関係を示
す標準曲線である。
手続補正書（自発）
昭和６３年０７月２７日
特許庁長官 吉 １）文 毅 殿
ｌ事件の表示
昭和６２年特許願第１２２５３９号 ゛２発明の名
称
生体成分中のヒト・ヘモグロビンの測定方法及び測定用
試薬キット
３補正をする者
事件との関係 特許出願人
堤第−ビル４階１’ＥＬＯ３（５９１）０７８５■「明
細書の発明の詳細な説明の欄」
６補正の内容
（１）明細書の第・１１ページを別紙のようの訂正する
。
に滴下した後、前記（ａ）の（イ）で調製した抗ヒト・
Ｈｂポリクロナール抗体溶液、または抗体感作担体、及
び前記（ｂ）で調製したヒト・Ｈｂ抗原感作担体２０色
文を追加して滴下し、２分間ゆるやかに攪拌しながら観
察する。同時に従来法（抗ヒト＠Ｈｂポリクロナール抗
体感作担体２゜ＩＬ文、上記サンプル ４０ ＪＬａｎ
も行なった。
その結果を次表に示す。
註 十は凝集を、またーは非凝集を表わす。FIG. 1 is a standard curve showing the relationship between free human Hb concentration and absorbance. Procedural amendment (spontaneous) July 27, 1988 Director General of the Japan Patent Office Yoshi 1) Indication of the Moon Takeshi l case 1988 Patent Application No. 122539 ゛2 Name of the invention Method for measuring human hemoglobin in biological components and Measurement reagent kit 3 Relationship with the case of the person making the amendment Patent applicant Tsutsumi Building 4th floor 1' ELO 3 (591) 0785 ■ "Column for detailed explanation of the invention in the specification" 6 Contents of the amendment (1) Details Make the corrections on page 11 of the book as shown in the attached sheet. After dropping the anti-human drug prepared in (a) (b) above,
The Hb polyclonal antibody solution or the antibody sensitized carrier and the human Hb antigen sensitized carrier prepared in (b) above are added dropwise and observed for 2 minutes while stirring gently. At the same time, the conventional method (anti-human @Hb polyclonal antibody sensitized carrier 2°IL, the above sample 40 JLan
I also did The results are shown in the table below. Note: Ten represents agglomeration or non-aggregation.
Claims (6)
を混合して生ずる凝集反応と、前記（Ａ）、及び（Ｂ）
成分に、更に次に示す（Ｃ）成分を含有する溶液を添加
混合して生ずる凝集反応の両者の程度を比較測定するこ
とを特徴とする生体成分中のヒト・ヘモグロビンの測定
方法。 （Ａ）測定対象であるヒト・ヘモグロビンと特異的に反
応するポリクローナル抗体 （Ｂ）測定対象であるヒト・ヘモグロビン感作担体 （Ｃ）測定対象であるヒト・ヘモグロビン(1) Aggregation reaction that occurs by mixing solutions containing the following components (A) and (B), and the above-mentioned (A) and (B).
A method for measuring human hemoglobin in a biological component, which comprises adding and mixing a solution containing the following component (C) to the component, and comparing and measuring the degree of agglutination reaction between the two components. (A) Polyclonal antibody that specifically reacts with the human hemoglobin to be measured (B) Human hemoglobin sensitized carrier to be measured (C) Human hemoglobin to be measured
存在する場合の特許請求の範囲第１項記載の生体成分中
のヒト・ヘモグロビンの測定方法。(2) The method for measuring human hemoglobin in a biological component according to claim 1, in which the human hemoglobin to be measured is present in fecal occult blood.
存在する場合の特許請求の範囲第１項記載の生体成分中
のヒト・ヘモグロビンの測定方法。(3) The method for measuring human hemoglobin in a biological component according to claim 1, in which the human hemoglobin to be measured is present in urine occult blood.
の範囲第１項記載の生体成分中のヒト・ヘモグロビンの
測定方法。(4) A method for measuring human hemoglobin in a biological component according to claim 1, which uses a polyclonal antibody as a solution.
請求の範囲第１項記載の生体成分中のヒト・ヘモグロビ
ンの測定方法。(5) A method for measuring human hemoglobin in a biological component according to claim 1, which uses a polyclonal antibody sensitized to a carrier.
と抗ヒト・ヘモグロビン抗体（溶液または感作担体）の
少なくとも二者からなるか、更には測定対象であるヒト
・ヘモグロビンの標準品を添付した三者からなることを
特徴とする生体成分中のヒト・ヘモグロビンの測定用試
薬キット。(6) The main composition consists of at least two components: a human hemoglobin latex solution and an anti-human hemoglobin antibody (solution or sensitized carrier), or moreover, three components with a standard product of human hemoglobin to be measured. A reagent kit for measuring human hemoglobin in biological components, characterized by comprising:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62122539A JP2571383B2 (en) | 1987-05-21 | 1987-05-21 | Method for measuring human hemoglobin in biological components and reagent kit for measurement |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62122539A JP2571383B2 (en) | 1987-05-21 | 1987-05-21 | Method for measuring human hemoglobin in biological components and reagent kit for measurement |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS63289453A true JPS63289453A (en) | 1988-11-25 |
| JP2571383B2 JP2571383B2 (en) | 1997-01-16 |
Family
ID=14838367
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62122539A Expired - Fee Related JP2571383B2 (en) | 1987-05-21 | 1987-05-21 | Method for measuring human hemoglobin in biological components and reagent kit for measurement |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2571383B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE4037724C2 (en) * | 1989-12-18 | 2003-04-10 | Princeton Biomeditech Corp | Devices for immunoassays and their materials |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5748658A (en) * | 1980-09-08 | 1982-03-20 | Teikoku Hormone Mfg Co Ltd | Immunologic measuring method and reagent for measurement |
| JPS61228351A (en) * | 1985-04-02 | 1986-10-11 | Kyoto Ikagaku Kenkyusho:Kk | Method for detecting hemoglobin in excretion |
-
1987
- 1987-05-21 JP JP62122539A patent/JP2571383B2/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5748658A (en) * | 1980-09-08 | 1982-03-20 | Teikoku Hormone Mfg Co Ltd | Immunologic measuring method and reagent for measurement |
| JPS61228351A (en) * | 1985-04-02 | 1986-10-11 | Kyoto Ikagaku Kenkyusho:Kk | Method for detecting hemoglobin in excretion |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE4037724C2 (en) * | 1989-12-18 | 2003-04-10 | Princeton Biomeditech Corp | Devices for immunoassays and their materials |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2571383B2 (en) | 1997-01-16 |
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