CN1595150A - DAB coloration liquid as a novel stabilized instant substrate - Google Patents
DAB coloration liquid as a novel stabilized instant substrate Download PDFInfo
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- CN1595150A CN1595150A CN 03150896 CN03150896A CN1595150A CN 1595150 A CN1595150 A CN 1595150A CN 03150896 CN03150896 CN 03150896 CN 03150896 A CN03150896 A CN 03150896A CN 1595150 A CN1595150 A CN 1595150A
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- Prior art keywords
- electron donor
- substrate
- dab
- colour developing
- color
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Abstract
This invention belongs to immune organization chemical field and adopts new bubatrate, DAB color reagent comparing to the current color reagent, which is easy to be polluted, harm to human. This color reagent comprises buffer liquid, auricome bubatrate, color organic electron donor and carbohydrate electron donor as stabilizer and a polymer. Wherein, the polymer is used to improve the dissolution of the color electron donor and reduce its oxidation reaction. This invention can be used in immune group analysis and nucleic acid cross analysis.
Description
Technical field
The invention belongs to the immunohistochemistry field, major product is stable instant substrate-DAB liquid that develops the color.This substrate-DAB solution comprises damping fluid (first-elected imidazole buffer), hydrogen peroxide substrate, the organic electronic donor of colour developing and as the carbohydrates electron donor of stabilizing agent.Substrate-DAB solution has superoxide activity completely.
Background technology
The detection of superoxide activity has been widely used in analytical chemistry, biological chemistry and the clinical chemistry.In majority detects, analyte response in the electron donor of superoxide and colour developing and the sample, this analyte may be catalyzer (as the heme in forensic samples or ight soil, urine, blood plasma, the serum, hemochrome etc.) or the material (enzyme mark peroxidase) relevant with catalyzer.If contain this analyte in the sample, then peroxidation can take place, and in several seconds to several hours visible chromogenic reaction take place.The active detection of peroxidating also is applicable to the positioning analysis that is fixed on analyte on filter membrane, cell or tissue section, running gel, the hybridization spot.
Enzyme labeling commonly used in immunology or the nucleic acid detection system is horseradish peroxidase (HRP), at hydrogen peroxide (H
2O
2) exist under the situation, HRP catalysis of phenol, naphthols, amine, diamines or other can be created in the compound of visible chromogenic substrate under optics and electron microscope and colourimetry or the naked eyes.HRP can resolve into the oxygen G﹠W with 2 hydrogen peroxide molecule, in this reaction, at first HRP supplies with hydrogen peroxide molecule with a pair of electronics, then HRP obtains electronics from suitable organic donor, and the organic donor complex of HRP-can form precipitation solubility or soluble at reactive site when reaction was finished.
Summary of the invention
(1) goal of the invention
The weak point of conventional method of analysis at present is the oxidation reaction of robotization, be that detected superoxide activity may be that superoxide and organic electronic donor just directly reacted " background " that causes before adding catalyzing enzyme, is limited in several hours the effective time of indicator; This kinds of oxidation reaction also may be the pollution that dyeing liquor and oxide in trace quantities since agent such as transiting state metal cause.Therefore purpose of the present invention just provides stable substrate-colour developing liquid compound, visible to be suitable for, hot metering, spectrophotometric, reflectometer analysis; And provide the using method of these stable substrates-colour developing liquid compound.
(2) invention advantage
The colour developing liquid of conventional method separates with substrate to be deposited, and substrate-colour developing liquid will be pre-mixed during test.By comparison, the present invention is stable substrate-colour developing liquid compound, has both saved the storage area, shortens detection time again, improves sensitivity and the reliability that detects simultaneously; And DAB is carcinogen, and product of the present invention has avoided operating personnel to contact with the direct of DAB, has reduced harmfulness.
(3) summary of the invention describes in detail
The present invention includes: buffer system (pH3-8), colour developing electron donor and as carbohydrates form electron donor and a polymkeric substance of stabilizing agent.
The carbohydrates electron donor can be selected from following material: monose, disaccharides, polysaccharide, their can suppress to develop the color autoxidation activity of electron donor.
The pH of substrate buffer solution adopts imidazoles or contains other buffer solution mixture of imidazoles between 3-8; Although different superoxide analyses can be carried out in the pH of broad scope, the optimal pH that most of HRP analyzes is between 4-6.When polymkeric substance existed, imidazoles-HCl was owing to the dissolving power of colour developing electron donor strong surge capability strong and between pH5.0-7.8 is become desirable damping fluid; And imidazoles can improve the oxidability of DAB, and its final concentration is best results when 1-500mM especially 50mM.
Polymkeric substance is the high molecular weight molecules that a class is formed by the simple molecules that repeats, and polymer molecule forms ethylene linkage between carbon carbon in the addition polymerization reaction mostly.Adding polymkeric substance in substrate solution can improve the dissolubility of colour developing electron donor and reduce its spontaneous oxidation reaction.First-elected polymkeric substance is polyethylene glycol PEG or propyleneglycoles PG, and final concentration is between 1-1000mM..
Because strong dissolving power and avirulence, the present invention advises adopting hydrogen peroxide and perhydrit as superoxide, final concentration between 0.1-10mM, they highly sensitive in alkyl peroxide.
Embodiment
(1) substrate-DAB composite formulations:
Working concentration: the 50mM imidazoles, pH6.0 contains 7.5mM PEG (molecular weight 3350), 2.94mM H
2O
2With 2.77mMDAB-4HCl and 10mM wood sugar.
General preparation storing solution (20 *, the pH7.5 of 100mL, 0.5M Tris-HCl solution):
6.8g imidazoles, final concentration 1M
2.51g PEG (molecular weight 3350), final concentration 7.5mM
2g DAB-4HCl, final concentration 55.4mM
The 150mg wood sugar, final concentration 10mM
H
2O
2(20 *) storing solution: 0.45%H
2O
2Solution
(2) SABC detects
When SABC detected, after the HRP reagent of finishing sample was hatched and washed sample, promptly available product of the present invention developed the color:
1.H
2O
2With substrate-DAB compound equal proportion mixing.This solution keeps in Dark Place, and is effective in 30 minutes.
2. the solution of first step preparation covers sample, hatches 4-10 minute, and microscopically is observed coloration result, is pale brown look.
(3) result
Immunohistochemical analysis result shows, and is simpler and more direct, quick than traditional DAB development process based on the development process of substrate-DAB compound, have higher detection sensitivity and a reliability.
Claims (1)
1. the present invention relates to the chromogenic reaction of immunohistochemical analysis, can be used for immunohistochemical analysis and Western hybridization analysis.This substrate-DAB solution comprise damping fluid (first-elected imidazole buffer, pH3-8), the hydrogen peroxide substrate, the organic electronic donor of colour developing is as carbohydrates electron donor and polymkeric substance of stabilizing agent.Its characteristic is the adding of carbohydrates electron donor and polymkeric substance, has reduced the autoxidation activity of colour developing electron donor, improves the dissolubility of colour developing electron donor.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03150896 CN1595150A (en) | 2003-09-10 | 2003-09-10 | DAB coloration liquid as a novel stabilized instant substrate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03150896 CN1595150A (en) | 2003-09-10 | 2003-09-10 | DAB coloration liquid as a novel stabilized instant substrate |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1595150A true CN1595150A (en) | 2005-03-16 |
Family
ID=34659781
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 03150896 Pending CN1595150A (en) | 2003-09-10 | 2003-09-10 | DAB coloration liquid as a novel stabilized instant substrate |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1595150A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104155167A (en) * | 2013-05-27 | 2014-11-19 | 厦门菲尔科生物技术有限公司 | Low-background P16-INK4a staining kit and application method thereof |
| CN104714007A (en) * | 2009-10-20 | 2015-06-17 | 丹麦达科有限公司 | Immunochemical detection of single target entities |
| CN110361245A (en) * | 2019-07-30 | 2019-10-22 | 河南赛诺特生物技术有限公司 | Colour reagent box and its application is immunized in a kind of DAB |
| CN110907255A (en) * | 2019-12-10 | 2020-03-24 | 武汉赛维尔生物科技有限公司 | Ready-to-use immunohistochemical staining solution and preparation method and application thereof |
| CN111426826A (en) * | 2018-12-24 | 2020-07-17 | 武汉赛维尔生物科技有限公司 | Immunohistochemical staining solution and application thereof |
-
2003
- 2003-09-10 CN CN 03150896 patent/CN1595150A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104714007A (en) * | 2009-10-20 | 2015-06-17 | 丹麦达科有限公司 | Immunochemical detection of single target entities |
| CN104155167A (en) * | 2013-05-27 | 2014-11-19 | 厦门菲尔科生物技术有限公司 | Low-background P16-INK4a staining kit and application method thereof |
| CN111426826A (en) * | 2018-12-24 | 2020-07-17 | 武汉赛维尔生物科技有限公司 | Immunohistochemical staining solution and application thereof |
| CN110361245A (en) * | 2019-07-30 | 2019-10-22 | 河南赛诺特生物技术有限公司 | Colour reagent box and its application is immunized in a kind of DAB |
| CN110361245B (en) * | 2019-07-30 | 2021-08-03 | 河南赛诺特生物技术有限公司 | DAB (digital audio broadcasting) immunochromatography kit and application thereof |
| CN110907255A (en) * | 2019-12-10 | 2020-03-24 | 武汉赛维尔生物科技有限公司 | Ready-to-use immunohistochemical staining solution and preparation method and application thereof |
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