CN101169367A - Fucosidase diagnosis reagent kit and fucosidase activity concentration determination method - Google Patents
Fucosidase diagnosis reagent kit and fucosidase activity concentration determination method Download PDFInfo
- Publication number
- CN101169367A CN101169367A CNA2006100972155A CN200610097215A CN101169367A CN 101169367 A CN101169367 A CN 101169367A CN A2006100972155 A CNA2006100972155 A CN A2006100972155A CN 200610097215 A CN200610097215 A CN 200610097215A CN 101169367 A CN101169367 A CN 101169367A
- Authority
- CN
- China
- Prior art keywords
- reagent
- fucosidase
- cupferron
- stabilizing agent
- coenzyme
- 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.)
- Pending
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a diagnostic reagent box of fucosidase, meanwhile, the invention also relates to a method for detecting the activity of the fucosidase, and the compositions and the component of reagent, and belongs to the technical field of medical examination and determination. The reagent box contains buffer solution, fucose dehydrogenase, fucose glucoside, coenzyme, bluestone, new cupferron and stabilizer. The sample is mixed with the reagent at certain volume ratio to impel the series of enzymatic and chemical reaction between the sample and the reagent, and the reactant is then put under the analyzer of visible light to detect the raising speed of absorbency at the 455nm of the main wavelength, thus, the activity degree of the fucosidase can be calculated. The invention which can completely obtain the needed test result with the help of the analyzer of visible light is easy to be popularized.
Description
Technical field
The present invention relates to a kind of fucosidase diagnostic kit, the invention still further relates to the method for measuring fucoidan glycosidase activity simultaneously, belong to medical test determination techniques field.
Background technology
French scholar Deugnier in 1980 etc. discover that Patients with Primary serum fucosidase raises, and are confirmed by numerous research institutes.So have the people to propose the tumor markers that fucosidase can be used as primary carcinoma of liver, improved diagnosis and treatment value greatly with the alpha-fetoprotein associating or with reference to using.Recent study finds that fucosidase is all significant to generation, the development of some disease.
Serum fucoside enzymatic determination mainly contains fluorescence method and colourimetry.Colourimetry is the substrate colour developing with 4-nitrobenzene α-L-rock algae pyranoside or 4-nitrobenzene alpha-L-fucosidase, and this law is easy, and equipment requirements is not high, extensively adopts both at home and abroad.
Summary of the invention
The technical problem to be solved in the present invention is: propose a kind of enzymatic reaction (EnzymaticCatalytic Reaction) technology and coupling chemical reaction developing technology utilized, the variation of continuous monitoring 455nm wavelength place absorbance, measured the method for fucoidan glycosidase activity, simultaneously, the present invention also will provide in order to realize the fucosidase diagnostic kit of this method, adopt this kit not only can be ultraviolet analyser or half, carrying out fucoidan glycosidase activity on the automatic clinical chemistry analyzer measures, and finding speed is fast, the accuracy height, thereby can obtain practical applying.
Fucoidan glycosidase activity assay method principle of the present invention is as follows:
Fucoside
FucosidaseFucose+corresponding alcohols
Fucose+coenzyme
FDRock algae ketose+reduced coenzyme
Reduced coenzyme+Cu
2+---→ coenzyme+Cu
+
Cu
++ new cupferron---→ Cu
+-Xin cupferron compound
Rock algae ketose: Fuculose
New cupferron: Neocuproine
This method is used fucosidase enzymatic reaction continuous monitoring method, fucosidase enzymolysis fucoside discharges fucose, effect by FD produces reduced coenzyme, in order to increase reaction sensitivity, reduced coenzyme reduction bivalent cupric ion generates univalent copper ion, and univalent copper ion and new cupferron (Neocuproine) form yellow compound.The end reaction thing is placed under visible light analysis instrument or half, the automatic clinical chemistry analyzer, detect Cu
+-Xin cupferron compound calculates the active size measurement result of fucosidase in the speed that predominant wavelength 455nm absorbance rises.
Experiment shows, takes all factors into consideration from the accuracy of measurement result and economy two aspects of preparation cost, and no matter be single agent or two agent, the fucosidase diagnostic kit of the present invention of following composition relation is comparatively desirable:
PH of buffer 8.5 100mmol/L
Stabilizing agent 20mmol/L
FD 200U/L
Fucoside 5mmol/L
Coenzyme (oxidized form) 4mmol/L
Copper sulphate 50mmol/L
New cupferron 100mmol/L
Fucosidase diagnostic kit of the present invention can be single agent, comprising:
Damping fluid, stabilizing agent, FD, fucoside, coenzyme, copper sulphate, new cupferron.Kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
Also above-mentioned single agent reagent can be made into following pair of agent reagent:
Reagent 1
Damping fluid, stabilizing agent, coenzyme, copper sulphate, new cupferron.
Reagent 2
Damping fluid, stabilizing agent, FD, fucoside.
FD, fucoside, coenzyme, copper sulphate, the new position of cupferron in reagent 1 or reagent 2 can not limit.Kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
Also above-mentioned single agent reagent can be made into following three doses of reagent:
Reagent 1
Damping fluid, stabilizing agent, coenzyme.
Reagent 2
Damping fluid, stabilizing agent, copper sulphate, new cupferron.
Reagent 3
Damping fluid, stabilizing agent, FD, fucoside.
FD, fucoside, coenzyme, copper sulphate, the new position of cupferron in reagent 1, reagent 2 or reagent 3 can not limit.Kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
No matter be single agent, two agent or three doses, the present invention measures the method for fucoidan glycosidase activity, and its oxidized coenzyme can be NADP
+, NAD
+Or thio-NAD
+In a kind of.
Embodiment
The present invention is further illustrated below in conjunction with examples of implementation.
Embodiment one
The fucosidase diagnostic reagent of present embodiment is a single reagent, comprising:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
FD 200U/L
Fucoside 5mmol/L
Coenzyme (oxidized form) 4mmol/L
Copper sulphate 50mmol/L
New cupferron 100mmol/L
Reagent divides the bottle of packing into after all dissolving and preparing, and carries out freeze drying, makes powdered reagent; Before the use, add pure water, use after redissolving.
On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance≤0.1, test predominant wavelength 455nm, test commplementary wave length 660nm, the volume ratio of tested fucosidase sample and reagent is 1/25, the Direction of Reaction is positive reaction (reaction of rising), about about 1 minute of time delay is about 2 minutes detection times.
After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects the speed that predominant wavelength 455nm absorbance rises, thereby calculates the active size of fucosidase.
Embodiment two
The fucosidase diagnostic reagent of present embodiment is a double reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme (oxidized form) 4mmol/L
Copper sulphate 50mmol/L
New cupferron 100mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
FD 200U/L
Fucoside 5mmol/L
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid double reagent, can directly use.
On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance≤0.1, test predominant wavelength 455nm, test commplementary wave length 660nm, the volume ratio of tested fucosidase sample and reagent 1, reagent 2 is 2/20/5, the Direction of Reaction is positive reaction (reaction of rising), about about 1 minute of time delay is about 2 minutes detection times.
After adding sample and reagent, make them mixed and have reaction, reactant places biochemical analysis the most at last
Under the instrument, detect the speed that predominant wavelength 455nm absorbance rises, thereby calculate fucosidase
Active size.
Embodiment three
The fucosidase diagnostic reagent of present embodiment is three reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme (oxidized form) 3mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Copper sulphate 50mmol/L
New cupferron 100mmol/L
Reagent 3
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
FD 200U/L
Fucoside 5mmol/L
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid three reagent, can directly use.
When measuring fucoidan glycosidase activity, on automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance≤0.1, test predominant wavelength 455nm, test commplementary wave length 660nm, the volume ratio of tested fucosidase sample and reagent 1, reagent 2, reagent 3 is 4/40/5/5, the Direction of Reaction is positive reaction (reaction of rising), and about about 1 minute of time delay is about 2 minutes detection times.
After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects the speed that predominant wavelength 455nm absorbance rises, thereby calculates the active size of fucosidase.
In a word, experiment showed, and adopt assay method of the present invention can draw required measurement result by general biochemical analyzer fully, and highly sensitive, degree of accuracy good, be not subjected in the pollution of allogenic material, easy to utilize.
Claims (6)
1. a fucosidase (EC 3.2.1.51) activity determination method, its method principle is as follows:
Rock algae ketose: Fuculose
New cupferron: Neocuproine
The end reaction thing is placed under visible light analysis instrument or half, the automatic clinical chemistry analyzer, detect Cu
+The speed that-Xin cupferron compound predominant wavelength 455nm absorbance rises calculates the active size measurement result of fucosidase.
2. fucosidase diagnostic kit, principal ingredient comprises:
Damping fluid 20---500mmol/L
Stabilizing agent 1---50mmol/L
FD 50---10000U/L
Fucoside 2---30mmol/L
Coenzyme (oxidized form) 0.5---9.0mmol/L
Copper sulphate 10---100mmol/L
New cupferron 20---200mmol/L
It is characterized in that: kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
3. according to the described fucosidase diagnostic kit of claim 2, it is characterized in that:
By damping fluid, stabilizing agent, FD (L-fucose dehydrogenase EC 1.1.1.122, EC 1.1.1.116), fucoside, coenzyme, copper sulphate (CuSO
4), new cupferron (2,9-dimethyl-1,10-phenanthroline; Neocuproine) form single agent reagent.
4. according to the described fucosidase diagnostic kit of claim 2, it is characterized in that:
By damping fluid, stabilizing agent, FD (L-fucose dehydrogenase EC 1.1.1.122, EC 1.1.1.116), fucoside, coenzyme, copper sulphate (CuSO
4), new cupferron (2,9-dimethyl-1,10-phenanthroline; Neocuproine) form two agent reagent; Reagent 1 is made up of damping fluid, coenzyme, copper sulphate, new cupferron; Reagent 2 is made up of damping fluid, stabilizing agent, FD, fucoside.
5. according to the described fucosidase diagnostic kit of claim 2, it is characterized in that:
By damping fluid, stabilizing agent, FD (L-fucose dehydrogenase EC 1.1.1.122, EC 1.1.1.116), fucoside, coenzyme, copper sulphate (CuSO
4), new cupferron (2,9-dimethyl-1,10-phenanthroline Neocuproine) forms multi-agent reagent; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme; Reagent 2 is made up of damping fluid, stabilizing agent, copper sulphate, new cupferron; Reagent 3 is made up of damping fluid, stabilizing agent, FD, fucoside.FD, fucoside, coenzyme, copper sulphate, the new position of cupferron in reagent 1, reagent 2 or reagent 3 can not limit.
6. according to the described fucosidase diagnostic kit of claim 2, it is characterized in that: also comprise stabilizing agent 1-4000mmol/L or 0.1%-100% volume ratio.Described stabilizing agent is: ammonium sulfate (AmmoniaSulfate), glycerine (Glycerol), propylene glycol (Propylene Glycol), ethylene glycol (Ethylene glycol) and at least one of the preservatives.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2006100972155A CN101169367A (en) | 2006-10-24 | 2006-10-24 | Fucosidase diagnosis reagent kit and fucosidase activity concentration determination method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2006100972155A CN101169367A (en) | 2006-10-24 | 2006-10-24 | Fucosidase diagnosis reagent kit and fucosidase activity concentration determination method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101169367A true CN101169367A (en) | 2008-04-30 |
Family
ID=39390063
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2006100972155A Pending CN101169367A (en) | 2006-10-24 | 2006-10-24 | Fucosidase diagnosis reagent kit and fucosidase activity concentration determination method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101169367A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111154833A (en) * | 2020-01-03 | 2020-05-15 | 浙江夸克生物科技有限公司 | α -L-fucosidase determination kit |
-
2006
- 2006-10-24 CN CNA2006100972155A patent/CN101169367A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111154833A (en) * | 2020-01-03 | 2020-05-15 | 浙江夸克生物科技有限公司 | α -L-fucosidase determination kit |
| CN111154833B (en) * | 2020-01-03 | 2022-08-23 | 浙江夸克生物科技有限公司 | alpha-L-fucosidase determination kit |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101329345A (en) | Glutathion diagnosis / determination reagent kit and method for determining glutathion concentration | |
| CN101169366A (en) | Fucosidase diagnosis reagent kit and fucosidase activity determination method | |
| CN101169367A (en) | Fucosidase diagnosis reagent kit and fucosidase activity concentration determination method | |
| CN101169368A (en) | Fucosidase diagnosis reagent kit and fucosidase activity concentration determination method | |
| CN101173901A (en) | N-acet-beta-amino glucosidase diagnostic reagent kit and method for measuring active concentration of N-acet-beta-amino glucosidase | |
| CN101464332A (en) | Selenium diagnosis/measuring reagent kit and method for measuring selenium concentration | |
| CN101173902A (en) | N-acet-beta-amino glucosidase diagnostic reagent kit and method for measuring active concentration of N-acet-beta-amino glucosidase | |
| CN101173931A (en) | Zinc diagnosis/measuring reagent kit and method for measuring zinc concentration | |
| CN101329264A (en) | Sucrase diagnosis / determination reagent kit and method for determining sucrase active concentration | |
| CN101750354A (en) | Adenosine deaminase diagnostic reagent (kit) and adenosine deaminase activity concentration measuring method | |
| CN101173903A (en) | N-acet-beta-amino glucosidase diagnostic reagent kit and method for measuring active concentration of N-acet-beta-amino glucosidase | |
| CN101750355A (en) | Adenosine deaminase diagnostic reagent (kit) and adenosine deaminase activity concentration measuring method | |
| CN101173898A (en) | Monoamine oxidase diagnostic reagent kit and method for measuring active concentration of monoamine oxidase | |
| CN101169447A (en) | Ferrous ion diagnosis/determination reagent kit and ferrous ion concentration determination method | |
| CN101464328A (en) | Selenium diagnosis/measuring reagent kit and method for measuring selenium concentration | |
| CN101464329A (en) | Selenium diagnosis/measuring reagent kit and method for measuring selenium concentration | |
| CN101750382A (en) | Diagnostic reagent (kit) of adenosine deaminase and method for measuring activity concentration of adenosine deaminase | |
| CN101329257A (en) | Nitrous acid determination reagent kit and method for determining nitric acid concentration | |
| CN101620167A (en) | Adenosine deaminase diagnosis kit and method for determining adenosine deaminase activity concentration | |
| CN101329329A (en) | Oxalic acid diagnosis / determination reagent kit and method for measuring oxalic acid concentration | |
| CN101750381A (en) | Adenosine deaminase diagnostic reagent (kit) and adenosine deaminase activity concentration measuring method | |
| CN101329332A (en) | Oxalic acid diagnosis / determination reagent kit and method for measuring oxalic acid concentration | |
| CN101620066A (en) | 5'nucleotidase diagnosis kit and method for determining 5'nucleotidase activity concentration | |
| CN101464339A (en) | Selenium diagnosis/measuring reagent kit and method for measuring selenium concentration | |
| CN101464334A (en) | Selenium diagnosis/measuring reagent kit and method for measuring selenium concentration |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Open date: 20080430 |