JPH0648999B2 - Method for detecting alkaline phosphatase - Google Patents
Method for detecting alkaline phosphataseInfo
- Publication number
- JPH0648999B2 JPH0648999B2 JP20329287A JP20329287A JPH0648999B2 JP H0648999 B2 JPH0648999 B2 JP H0648999B2 JP 20329287 A JP20329287 A JP 20329287A JP 20329287 A JP20329287 A JP 20329287A JP H0648999 B2 JPH0648999 B2 JP H0648999B2
- Authority
- JP
- Japan
- Prior art keywords
- alkaline phosphatase
- detecting
- detecting alkaline
- detection sensitivity
- labeling
- 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
Links
Description
本発明は、生物研究分野、医学研究分野、特に臨床検査
に用いられるアルカリホスファターゼの検出方法に関す
るものである。The present invention relates to a method for detecting alkaline phosphatase used in the fields of biological research and medical research, particularly in clinical tests.
従来、アルカリホスファターゼをその存在部位（in sit
u)で検出する方法としては、ナフトールＡＳリン酸を用
いる方法、ナフトールＡＳ−ＭＸリン酸を用いる方法、
インドキシルリン酸を用いる方法、インドキシルリン酸
とテトラゾリウム系化合物を用いる方法等、多くの方法
が知られている。 しかしながら、現在では、検出感度や検出した試料の保
存安定性の点から、インドキシルリン酸とテトラゾリウ
ム系化合物を用いる方法が最もよく用いられている。Conventionally, alkaline phosphatase has a site (in sit
As the method for detecting in u), a method using naphthol AS phosphoric acid, a method using naphthol AS-MX phosphoric acid,
Many methods are known, such as a method using indoxylphosphoric acid and a method using indoxylphosphoric acid and a tetrazolium compound. However, at present, the method using indoxylphosphoric acid and a tetrazolium compound is most often used from the viewpoint of detection sensitivity and storage stability of the detected sample.
ところで、ビオチンとアビジンの親和力、そして、抗原
とその特異抗体の親和力は極めて高いことが知られてい
る。医学、生物学の研究分野や臨床検査では、この性質
を利用して、これらの組の何れかを標織し、それをマー
カーとして、それぞれと親和性の高い物質（標的物質）
の存在部位を検出するのに用いられている。その標識に
は、放射性同位元素、蛍光色素、さらに、最近では酵素
がよく用いられている。 標識酵素として、当初はペルオキシダーゼが用いられて
いたが、現在はその検出感度、および酵素の反応産物の
安定性が優っているアルカリホスファターゼがこの標識
に多用されている。酵素による標的物質の検出感度は、
放射性同位元素による標識に較べてまだ低いが、放射性
同位元素による被爆の危険性、放射性同位元素の減衰に
よる標識物質の使用可能期間の限定等を考慮すると、酵
素による標識の利用頻度はこれからも増していくと考え
られる。 そこで、アルカリホスファターゼの検出感度を高め、ア
ルカリホスファターゼに関わる試験研究、臨床検査をさ
らに発展させるために、新たな処置を講じようとするも
のである。By the way, it is known that the affinity between biotin and avidin, and the affinity between antigen and its specific antibody are extremely high. In the fields of medical and biological research and clinical examinations, this property is used to weave one of these sets, and use it as a marker for substances with high affinity for each (target substance).
It is used to detect the existence site of. Radiolabels, fluorescent dyes, and recently, enzymes are often used for the labeling. Peroxidase was initially used as a labeling enzyme, but nowadays, alkaline phosphatase, which is superior in its detection sensitivity and stability of the reaction product of the enzyme, is widely used for this labeling. The detection sensitivity of the target substance by the enzyme is
Although it is still lower than labeling with radioisotopes, the frequency of use of labels by enzymes will continue to increase in view of the danger of exposure to radioisotopes and the limitation of the usable period of labeled substances due to decay of radioisotopes. It is thought to go. Therefore, in order to improve the detection sensitivity of alkaline phosphatase and further develop the test research and clinical tests related to alkaline phosphatase, it is intended to take new measures.
本発明は、上記の問題点を解決するために、アルカリホ
スファターゼを検出する反応において、インドキシルリ
ン酸とテトラゾリウム系化合物を含む反応液に強塩基酸
を側鎖に持つ高分子物質を添加することを特徴とするも
のである。The present invention, in order to solve the above problems, in the reaction for detecting alkaline phosphatase, to add a polymer substance having a strong basic acid in the side chain to the reaction solution containing indoxyl phosphate and a tetrazolium compound It is characterized by.
上記の方法によって本発明の検出方法は、アルカリホス
ファターゼの検出感度を飛躍的に高めることができる。By the above method, the detection method of the present invention can dramatically increase the detection sensitivity of alkaline phosphatase.
以下、本発明の実施例について説明する。まず、 10mM Tris−HCl(pH9.0) 5mM ＭｇＣｌ２ 0.1％ ２−アクリルアミド−２メチルプロパンスルホ
ン酸（ＡＭＰＳ）のホモポリマー0.7％アガロース溶液 以上でアビジン−アルカリホスファターゼ複合体を固定
化したニトロセルロース膜に薄い層を作り、アガロース
を固形化させた。 次に、このアガロースの上に従来の反応液と同量の５−
bromo−４−chloroindoxyl phosphate（ＢＣＩＰ）とni
tro blue tetrazolium（ＮＢＴ）を含む 10mＭ Tris−HCl(pH9.0) 5mM ＭｇＣｌ２ 0.01%ＡＭＰＳのホモポリマー 0.7％アガロース溶液 を重層し、これをアルカリホスファターゼの検出に用い
た。 その結果、従来の方法に比べて、約３倍の検出感度が得
られた。Examples of the present invention will be described below. First, 10 mM Tris-HCl (pH 9.0) 5 mM MgCl 2 0.1% 2-acrylamido-2methylpropanesulfonic acid (AMPS) homopolymer 0.7% agarose solution A nitrocellulose membrane with avidin-alkaline phosphatase complex immobilized thereon A thin layer was made to solidify the agarose. Next, on this agarose, the same amount of 5-
Bromo-4-chloroindoxyl phosphate (BCIP) and ni
A 10 mM Tris-HCl (pH 9.0) 5 mM MgCl 2 0.01% AMPS homopolymer 0.7% agarose solution containing tro blue tetrazolium (NBT) was overlaid and used for the detection of alkaline phosphatase. As a result, detection sensitivity about three times higher than that of the conventional method was obtained.
以上説明したように、本発明のアルカリホスファターゼ
の検出方法によれば、インドキシルリン酸とテトラゾリ
ウム系化合物によるアルカリホスファターゼの検出に際
し、強塩基酸を側鎖に持つ高分子物質を添加したので、
試験研究、臨床検査において、試料の微量化および解析
の高精度化をもたらす。 また、アルカリホスファターゼ標識による標的物質の検
出感度は、放射性同位元素による標識に比べてまだ低い
のが現状であるが、本発明による検出方法の導入によっ
て、検出感度がかなり改善されるため、放射性同位元素
による標識から、安全で、かつ作業効率のよいアルカリ
ホスファターゼによる標識への移行がさらに促進され
る。As described above, according to the method for detecting alkaline phosphatase of the present invention, in detecting alkaline phosphatase with indoxyl phosphate and a tetrazolium compound, a polymer substance having a strong basic acid as a side chain is added,
In test studies and clinical examinations, it will lead to miniaturization of samples and higher accuracy of analysis. Further, the detection sensitivity of the target substance by the alkaline phosphatase label is still lower than that by the labeling with the radioisotope, but the introduction of the detection method according to the present invention significantly improves the detection sensitivity. The transition from elemental labeling to alkaline phosphatase labeling, which is safe and efficient, is further promoted.
Claims (1)
合物によるアルカリホスファターゼの検出に際し、強塩
基酸を側鎖に持つ高分子物質を添加することを特徴とす
るアルカリホスファターゼの検出方法。1. A method for detecting alkaline phosphatase, which comprises adding a polymer having a strongly basic acid as a side chain when detecting alkaline phosphatase with indoxyl phosphate and a tetrazolium compound.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP20329287A JPH0648999B2 (en) | 1987-08-14 | 1987-08-14 | Method for detecting alkaline phosphatase |
| US07/895,172 US5443958A (en) | 1987-08-14 | 1992-06-05 | 2-acrylamine-2-methyl-1-propanesulfonic acid enhancement of alkaline phosphatase label detection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP20329287A JPH0648999B2 (en) | 1987-08-14 | 1987-08-14 | Method for detecting alkaline phosphatase |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6447399A JPS6447399A (en) | 1989-02-21 |
| JPH0648999B2 true JPH0648999B2 (en) | 1994-06-29 |
Family
ID=16471625
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP20329287A Expired - Lifetime JPH0648999B2 (en) | 1987-08-14 | 1987-08-14 | Method for detecting alkaline phosphatase |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0648999B2 (en) |
-
1987
- 1987-08-14 JP JP20329287A patent/JPH0648999B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6447399A (en) | 1989-02-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0227795B2 (en) | Method for performing nucleic acid hybridization assays | |
| US4994373A (en) | Method and structures employing chemically-labelled polynucleotide probes | |
| RU2107730C1 (en) | Molecular probe | |
| CA1148859A (en) | Simultaneous assay of two hepatitis viruses using a solid phase | |
| EP0440647A1 (en) | Labeled nucleic acid probe | |
| JPS59104555A (en) | Method of measuring terminal deoxynucleotidyltransferase | |
| EP0097341B1 (en) | Sensitive tests for malignancies based on dna detection | |
| CN110196334A (en) | Thrombomodulin chemiluminescence immunoassay immue quantitative detection reagent box and preparation method thereof | |
| JP2001512691A (en) | An electrochemical reporter system for examining immunoassay analysis and molecular biological processes | |
| Ritter et al. | Improved radioimmunoassay for creatine kinase isoenzymes in plasma. | |
| JP2540482B2 (en) | In vivo labeling of polynucleotide sequences | |
| JPS6112547B2 (en) | ||
| US4372941A (en) | Novel radioassay procedure | |
| AU647431B2 (en) | An agent for improving the recovery of annexins | |
| Sauvaigo et al. | Fast solid support detection of PCR amplified viral DNA sequences using radioiodinated or hapten labelled primers | |
| CN103597351A (en) | Method for inhibiting non-specific reaction in PIVKA-II measurement reagent | |
| Headings et al. | A radioimmunoassay for quantifying carbonic anhydrase isozymes in crude lysates | |
| JPH0648999B2 (en) | Method for detecting alkaline phosphatase | |
| JPS63282659A (en) | Method of measuring nucleic acid and measuring agent | |
| Hawley et al. | Biogenesis of mitochondrial membranes in Neurospora crassa: Mitochondrial protein synthesis during conidial germination | |
| Vanderheiden | Micro assay of ITP pyrophosphohydrolase by liquid scintillation | |
| JPH07509058A (en) | Chemiluminescent assay method for dsDNA antibodies | |
| US20090311719A1 (en) | In vitro method for diagnosing neurodegenerative diseases | |
| Cagen | Advances in prostaglandin and thromboxane research: Vol. 5. Edited by JC Fröhlich, Raven Press, New York, 1978, 250 pp. $24.00 | |
| Arakawa | Development of highly sensitive analytical methods for biologically relevant materials and their pharmaceutical applications |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EXPY | Cancellation because of completion of term |