JPH0334589B2 - - Google Patents
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- Publication number
- JPH0334589B2 JPH0334589B2 JP58147118A JP14711883A JPH0334589B2 JP H0334589 B2 JPH0334589 B2 JP H0334589B2 JP 58147118 A JP58147118 A JP 58147118A JP 14711883 A JP14711883 A JP 14711883A JP H0334589 B2 JPH0334589 B2 JP H0334589B2
- Authority
- JP
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- Prior art keywords
- red blood
- antigen
- type
- blood cells
- hemagglutination
- Prior art date
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
- G01N33/555—Red blood cell
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Cell Biology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Microbiology (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Mycology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Description
技術分野
本発明は被検液中の２種以上の抗原または抗体
を一度の測定で高感度に検出しうる赤血球凝集反
応試薬に関する。
技術水準
現在、赤血球凝集（HA）反応試薬としてはＢ
型肝炎の特定試薬、肝ガンのアルフアフエトプロ
テインの判定試薬等、数々の試薬が作られてい
る。これらの試薬は放射能あるいは酵素を用いた
高感度試薬と同様に、簡単でしかも安価に利用で
きる利点がある。
ところで従来は試薬の性質上、単品の抗原また
は抗体物質について高感度で判定することに重点
が置かれており、赤血球に単品の抗原または抗体
物質を感作させた試薬が用いられていた。
しかし、一度の測定結果で症状の進行を判断で
きるように、数種の抗原物質または抗体物質を検
出できる試薬が求められている。また、自然には
混合する機会の非常に少ない抗原物質または抗体
物質を併せて検出できるような試薬を用いて、統
一的な判断を正しく得たい場合もある。
後者の例としては、先述の肝炎の判定の際に、
免疫学的に差を生じないサブタイプを判定できる
試薬が該当する。
即ち、例えば、日本・韓国等の東南アジアで
は、adタイプのＢ型肝炎の表面抗原が多いが、
欧米ではayタイプが多いと言われている。そこ
でad，ayの両タイプの抗原物質または抗体物質
を併せて検出できるような試薬があれば便宜的で
ある。
従つて、本発明は一度に複数の抗原物質また
は、抗体物質を高感度に検出しうる試薬を提供す
ることを目的とするものであり、その要旨は２種
以上の抗原または２種以上の抗体を任意の一種ず
つ順次赤血球に感作させて成る赤血球凝集反応試
薬にある。
本発明において赤血球に感作される２種以上の
抗原物質としては、同種抗原中の異種サブタイプ
抗原群（例えば、Ｂ型肝炎Ｓ抗原のadタイプと
ayタイプ）、あるいは同一の生理活性を有する蛋
白群（例えば、人尿性ウロキナーゼと組織プラス
ミノーゲンアクチベーター）等が用いられ、また
２種以上の抗体物質も上記抗体と同様の関連性を
有するものが用いられることは容易に理解される
であろう。
この複数の抗原物質を感作させる赤血球は、従
来赤血球凝集反応用として用いられているものが
使用でき、特に動物を選ぶ必要はないが、安定で
しかも感度の高い試薬を作るためには、ヒツジ、
ニワトリ、ヒトのＯ型赤血球等が望ましい。赤血
球は、通常生理食塩水で十分洗浄した後、グルタ
ールアルデヒド、ホルマリン等で処理して安定化
される。補助的にタンニン酸を用いることもでき
る。赤血球は５−15μm程度のものが好ましい。
かかる赤血球の抗原または抗体による感作は公
知の方法、例えば（医学のあゆみ、78759（1970））
に記載の方法等によつて実施することができる。
赤血球への抗原または抗体の感作は緩衝液中で
行うのが良く、一般には赤血球浮遊液と抗原また
は抗体含有液を混合することにより行う。
ところで、数種の抗原物質または数種の抗体物
質を同時に赤血球に感作させた赤血球の血球凝集
反応の感度は、各々の抗原物質または抗体物質を
単独で赤血球に感作させた場合に比べて劣つてい
ることが多い。
ところが、まず、任意の一種の抗原物質または
抗体物質を赤血球に感作した後、緩衝液で十分に
洗浄し、続いて次の別種の抗原物質または抗体物
質を感作するという操作を繰り返して得た試薬は
すぐれた感度を有することを、本発明者らは見い
出した。従つて、本発明においては、任意の一種
の抗原または抗体を赤血球に感作させた後、次の
別種のを抗原または抗体を感作するという操作を
繰り返すことが好ましい。本操作はPH５〜8.5、
感作させる抗原物質または抗体物質濃度（即ち、
蛋白濃度）が、280nmの波長における吸光度が、
0.05〜0.1となる濃度程度で行う。
感作赤血球は凍結乾燥して、例えばバイアル中
に、好ましくはナトリウムアジト等の保存剤を添
加して、封入しておくことが望ましい。
本発明の赤血球凝集反応試薬は、単一の赤血球
に生理活性においてなんらかの関連性を有する複
数の抗体物質または抗原物質を感作させているの
で、一度に複数の抗原物質または抗体物質を検出
しうるという効果を有する。
以下に、本発明を実施例によつて更に具体的に
説明する。
実施例 １
２％グルタール・アルデヒドおよびタンニン酸
10mg／dlで処理したヒンジ固定赤血球を充分に洗
浄し、終濃度10％ヘマトクリツト値になるよう
に、高度精製したＢ型肝炎のＳ抗原（比活性：mg
当たりRPHA値で１：30000）のadタイプ−波長
280nmで吸光度0.05の値にした等量−をPH5.5で混
合し、室温で12時間撹拌して感作する。終了後、
0.1Mリン酸緩衝液（PH6.5）で数回、充分に洗浄
し、続いてayタイプの同様Ｓ抗原（比活性：mg
当たりRPHA値で１：20000）を上記と全く同様
の方法で感作する。感作後充分に全量の10倍程度
の液量の上記リン酸緩衝液で６回程度洗浄する。
こうして得られた感作赤血球の性能を調べるた
めにayタイプ単独で感作した赤血球と、赤血球
凝集反応の比較を行つた。試料は、サブタイプが
判明している血漿を用いた。結果を後記第１表に
示す。第１表に示したように、実施例１の感作赤
血球は両サブタイプについて、単独感作型とほぼ
同程度の感度を示た。
実施例 ２
２％グルタールアルデヒドおよびタンニン酸12
mg／dlで処理したヒツジ固定化赤血球を充分に洗
浄し、終濃度10％ヘマトクリツト値になるよう
に、高度精製した人尿性ウロキナーゼ（比活性：
70000単位／E280nm以上）−波長280nmで吸光度
0.05の値にした等量−をPH6.5で混合し、室温で
12時間撹拌して感作する。感作終了後、0.1Mリ
ン酸緩衝液（PH6.5）で数回、充分に洗浄し、続
いて組織より抽出した後に高度精製した組織プラ
スミノーゲンアクチベーター（比活性：７万単
位／E280nm以上）を上記と全く同様の方法で感
作する。感作後、充分に全量の10倍程度の液量の
上記リン酸緩衝液で６回程度洗浄する。
かくして得られた試薬の感度の判定は、人尿性
と組織性の各々のプラスミノーゲンを賦活化する
活性物質の力価を測定することにより行つた。そ
の結果、実施例２で作製した試薬は、単品の抗原
を感作させた試薬と同程度の感度を示した。
Technical Field The present invention relates to a hemagglutination reagent that can detect two or more types of antigens or antibodies in a test liquid with high sensitivity in a single measurement. Technical level Currently, B as a hemagglutination (HA) reaction reagent
A number of reagents have been produced, including a specific reagent for type hepatitis and a reagent for determining alpha fetoprotein for liver cancer. These reagents have the advantage of being simple and inexpensive to use, similar to highly sensitive reagents using radioactivity or enzymes. Conventionally, due to the nature of reagents, emphasis has been placed on highly sensitive determination of single antigens or antibody substances, and reagents have been used in which red blood cells are sensitized with single antigens or antibody substances. However, there is a need for reagents that can detect several types of antigenic substances or antibody substances so that the progression of symptoms can be determined from a single measurement result. In addition, there are cases in which it is desired to obtain a uniform and correct judgment by using a reagent that can detect both antigenic substances and antibody substances, which have very few chances of being mixed together in nature. As an example of the latter, when determining hepatitis mentioned earlier,
Reagents that can determine subtypes that do not cause immunological differences are applicable. That is, for example, in Southeast Asia such as Japan and South Korea, there are many AD type hepatitis B surface antigens,
It is said that the AY type is more common in Europe and America. Therefore, it would be convenient to have a reagent that can detect both ad and ay types of antigenic substances or antibody substances. Therefore, an object of the present invention is to provide a reagent that can detect multiple antigenic substances or antibody substances at once with high sensitivity. The red blood cell agglutination reaction reagent consists of sequentially sensitizing red blood cells with any one of the following. In the present invention, the two or more antigenic substances that sensitize red blood cells include a heterogeneous subtype antigen group among homogeneous antigens (for example, hepatitis B S antigen ad type and
ay type) or a group of proteins with the same physiological activity (e.g., human urinary urokinase and tissue plasminogen activator), etc., and two or more types of antibody substances also have the same relationship as the above antibodies. It will be easy to understand what is used. The red blood cells that are used to sensitize these multiple antigenic substances can be those conventionally used for hemagglutination reactions, and there is no need to choose a particular animal, but in order to make a stable and highly sensitive reagent, sheep ,
Chicken, human type O red blood cells, etc. are preferable. Red blood cells are usually stabilized by being thoroughly washed with physiological saline and then treated with glutaraldehyde, formalin, or the like. Tannic acid can also be used as a supplement. Preferably, the red blood cells have a diameter of about 5-15 μm. Sensitization of such red blood cells with antigens or antibodies can be carried out using known methods, for example (Igaku no Ayumi, 78759 (1970)).
This can be carried out by the method described in . Sensitization of red blood cells with an antigen or antibody is preferably carried out in a buffer, and is generally carried out by mixing a red blood cell suspension with a solution containing the antigen or antibody. By the way, the sensitivity of the hemagglutination reaction of red blood cells obtained by simultaneously sensitizing red blood cells with several types of antigenic substances or several types of antibody substances is higher than that when red blood cells are sensitized with each antigenic substance or antibody substance alone. Often inferior. However, red blood cells are first sensitized with any type of antigenic substance or antibody substance, washed thoroughly with a buffer solution, and then sensitized with the next different type of antigenic substance or antibody substance. The inventors have found that the reagent has excellent sensitivity. Therefore, in the present invention, it is preferable to repeat the procedure of sensitizing red blood cells with any type of antigen or antibody and then sensitizing them with another type of antigen or antibody. This operation is performed at a pH of 5 to 8.5.
The sensitizing antigenic substance or antibody substance concentration (i.e.
The absorbance at a wavelength of 280 nm is
Do this at a concentration of 0.05 to 0.1. It is desirable that the sensitized red blood cells be lyophilized and sealed, for example, in a vial, preferably with the addition of a preservative such as sodium azide. The hemagglutination reagent of the present invention sensitizes a single red blood cell to a plurality of antibody substances or antigen substances that have some relationship in physiological activity, and therefore can detect a plurality of antigen substances or antibody substances at the same time. It has this effect. EXAMPLES Below, the present invention will be explained in more detail with reference to Examples. Example 1 2% glutaraldehyde and tannic acid
Hinge-fixed red blood cells treated with 10 mg/dl were thoroughly washed, and highly purified hepatitis B S antigen (specific activity: mg) was added to a final concentration of 10% hematocrit.
per RPHA value of 1:30000) ad type-wavelength
Sensitize by mixing equal amounts of 0.05 absorbance at 280 nm at pH 5.5 and stirring at room temperature for 12 hours. After the end,
After thorough washing several times with 0.1M phosphate buffer (PH6.5), the same S antigen of the ay type (specific activity: mg
(RPHA value 1:20000) was sensitized in exactly the same manner as above. After sensitization, wash thoroughly about 6 times with the above phosphate buffer solution in an amount about 10 times the total volume. In order to investigate the performance of the sensitized red blood cells thus obtained, we compared the red blood cell agglutination reaction with red blood cells sensitized with ay type alone. The sample used was plasma of known subtype. The results are shown in Table 1 below. As shown in Table 1, the sensitized red blood cells of Example 1 showed almost the same sensitivity as the single sensitized type for both subtypes. Example 2 2% glutaraldehyde and tannic acid 12
After thoroughly washing the fixed sheep red blood cells treated with mg/dl, highly purified human urinary urokinase (specific activity:
70000 units/E280nm or more) - Absorbance at wavelength 280nm
Equal amounts of - to a value of 0.05 were mixed at pH 6.5 and heated at room temperature.
Stir for 12 hours to sensitize. After sensitization, the tissue was thoroughly washed several times with 0.1M phosphate buffer (PH6.5), and then highly purified tissue plasminogen activator (specific activity: 70,000 units/E280nm) was extracted from the tissue. (above)) is sensitized in exactly the same manner as above. After sensitization, wash thoroughly about 6 times with the above phosphate buffer solution in an amount about 10 times the total amount. The sensitivity of the thus obtained reagent was determined by measuring the titer of the active substance that activates human urinary and tissue plasminogen. As a result, the reagent prepared in Example 2 exhibited sensitivity comparable to that of the reagent sensitized with a single antigen.
【表】【table】
Claims (1)
の一種ずつ順次赤血球に感作させて成る赤血球凝
集反応試薬。 ２ 抗原がＢ型肝炎Ｓ抗原のadタイプおよびＢ
型肝炎Ｓ抗原のayタイプの２種である特許請求
の範囲第１項記載の赤血球凝集反応試薬。 ３ 抗体がＢ型肝炎Ｓ抗原のadタイプに対する
抗体およびＢ型肝炎Ｓ抗原のayタイプに対する
抗体の２種である特許請求の範囲第１項記載の赤
血球凝集反応試薬。 ４ 抗原がヒト尿性ウロキナーゼおよび組織プラ
スミノゲンアクチベーターの２種である特許請求
の範囲第１項記載の赤血球凝集反応試薬。 ５ 抗体がヒト尿性ウロキナーゼの抗体および組
織プラスミノゲンアクチベーターの抗体の２種で
ある特許請求の範囲第１記載の赤血球凝集反応試
薬。 ６ 感作条件が、PH５〜8.5、蛋白濃度が280nm
の波長における吸光度が0.05〜0.1となる濃度で
ある特許請求の範囲第１〜５のいずれかの項記載
の赤血球凝集反応試薬。 ７ 赤血球がヒツジ、ニワトリまたはヒトＯ型赤
血球である特許請求の範囲第１〜６のいずれかの
項記載の赤血球凝集反応試薬。[Scope of Claims] 1. A red blood cell agglutination reaction reagent obtained by sequentially sensitizing red blood cells with any one of two or more antigens or two or more antibodies. 2 Antigen is hepatitis B S antigen ad type and B
The hemagglutination reagent according to claim 1, which is two types of ay type hepatitis S antigen. 3. The hemagglutination reaction reagent according to claim 1, wherein the antibodies are two types: an antibody against the ad type of hepatitis B S antigen and an antibody against the ay type of hepatitis B S antigen. 4. The hemagglutination reagent according to claim 1, wherein the antigens are two types: human urinary urokinase and tissue plasminogen activator. 5. The hemagglutination reagent according to claim 1, wherein the antibodies are two types: an antibody against human urinary urokinase and an antibody against tissue plasminogen activator. 6 Sensitization conditions are PH5-8.5, protein concentration 280nm
The hemagglutination reaction reagent according to any one of claims 1 to 5, which has a concentration such that the absorbance at a wavelength of 0.05 to 0.1. 7. The hemagglutination reagent according to any one of claims 1 to 6, wherein the red blood cells are sheep, chicken, or human type O red blood cells.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP14711883A JPS6038656A (en) | 1983-08-10 | 1983-08-10 | Red corpuscle agglutination reaction reagent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP14711883A JPS6038656A (en) | 1983-08-10 | 1983-08-10 | Red corpuscle agglutination reaction reagent |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6038656A JPS6038656A (en) | 1985-02-28 |
| JPH0334589B2 true JPH0334589B2 (en) | 1991-05-23 |
Family
ID=15422934
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP14711883A Granted JPS6038656A (en) | 1983-08-10 | 1983-08-10 | Red corpuscle agglutination reaction reagent |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS6038656A (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5079173A (en) * | 1987-08-19 | 1992-01-07 | Shionogi & Co., Ltd. | Methods, hybridomas, monoclonal antibodies and sensitized cells for measuring hbs antigen |
| JPH032565A (en) * | 1989-05-30 | 1991-01-08 | Olympus Optical Co Ltd | Immunoassay |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5821565A (en) * | 1981-07-31 | 1983-02-08 | Fuji Photo Film Co Ltd | Microcapsule for detecting variety of antibody and detection method thereby |
-
1983
- 1983-08-10 JP JP14711883A patent/JPS6038656A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6038656A (en) | 1985-02-28 |
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