CN1975383A - Method for measuring diethyl p-nitrophenyl phosphate esterase active concentration and diagnostic kit - Google Patents
Method for measuring diethyl p-nitrophenyl phosphate esterase active concentration and diagnostic kit Download PDFInfo
- Publication number
- CN1975383A CN1975383A CN 200610161226 CN200610161226A CN1975383A CN 1975383 A CN1975383 A CN 1975383A CN 200610161226 CN200610161226 CN 200610161226 CN 200610161226 A CN200610161226 A CN 200610161226A CN 1975383 A CN1975383 A CN 1975383A
- Authority
- CN
- China
- Prior art keywords
- paraoxonase
- reagent
- diagnostic kit
- active concentration
- phenylacetate
- 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
This invention disclosed a determination method for paraoxonase activity and the reagent box. The procedures are as follows: react with phenylacetate to produce acetic acid, oxidate the deoxidize style coenzyme to coenzyme, measure the coenzyme at 340nm absorbency, calculate the activity and concentration. The reagent box contains buffer, deoxidize style coenzyme, phenylacetate and stabilizer. The result obtained by this method was precise and accurate.
Description
Technical field
The present invention relates in the fields such as medicine, food, environment detection to the paraoxonase active concentration, relate in particular to and utilize enzymic colorimetric and enzyme (even) united method to measure the method for paraoxonase active concentration, and the paraoxonase diagnostic kit that makes thus, belong to paraoxonase active concentration technical field of analysis and detection.
Background technology
Paraoxonase in the serum, mainly at liver expression, its effect is a phosphate in the hydrolysis tissue.Paraoxonase in the serum is relevant with the high and low density lipoprotein metabolism in the human body.Paraoxonase can be protected low-density lipoprotein, avoids oxidative modification, so, can think that paraoxonase is a kind of enzyme with antiatherosclerosis and antiinflammatory action.The increase of blood plasma paraoxonase activity, make the autoantibody level of anti-oxidant modification type low-density lipoprotein in the blood plasma reduce by 18.8%, and the increase of paraoxonase activity on the high-density lipoprotein (HDL) makes hdl level raise 9.1%.
The active concentration of the paraoxonase in smoker's body is low than non-smoker or ES's active concentration, thereby antioxidation also decreases, cause producing in the blood vessel more cholesterol deposition, and unnecessary cholesterol can cause blood vessel blockage, finally causes heart disease or apoplexy to take place.Except that smoking, diabetes also reduce relevant with the paraoxonase level with aging.
Summary of the invention
The objective of the invention is: a kind of method of measuring the paraoxonase active concentration is provided, and uses the formulated paraoxonase diagnostic kit of this method.
For realizing purpose of the present invention, a kind of method of utilizing enzymic colorimetric (Enzymatic ColorimetricMethod) and enzyme (even) united method (Couple Reaction) to measure the paraoxonase active concentration, adopt following steps to carry out:
At first,, make it to take place following reaction with sample of measuring and the reagent mixing that contains phenylacetate, aldehyde dehydrogenase and reduced coenzyme,
Then, the end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect the speed that predominant wavelength 340nm absorbance descends, calculate the active concentration size of paraoxonase.
Above-mentioned utilization utilizes enzymic colorimetric and enzyme (even) united method to measure in the method for paraoxonase active concentration, and described reduced coenzyme is a kind of among NADH, NADPH, thio-NADH or the thio-NADPH.
Realize that paraoxonase diagnostic kit of the present invention can be single agent, is grouped into by following one-tenth:
Damping fluid 20～500mmol/L,
Stabilizing agent 5～100g/ml,
Reduced coenzyme 0.1～0.35mmol/L,
Aldehyde dehydrogenase 1000～80000U/L,
Phenylacetate 1～50mmol/L.
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 the various compositions in above single agent can be carried out formulated in combination and become two agent, such as:
| Agent I | Stabilizing agent | 0.1～3mol/L |
| Reduced coenzyme | 0.1～0.35mmol/L | |
| Phenylacetate | 1～50mmol/L | |
| Reagent II | Damping fluid | 20～500mmol/L |
| Stabilizing agent | 5～50％v/v | |
| Aldehyde dehydrogenase | 1000～80000U/L |
The prescription of two agent is not limited only in the above-mentioned table listed, wherein the composition of reagent I: reduced coenzyme, phenylacetate can be placed on reagent II; Aldehyde dehydrogenase among the reagent II also can be put into reagent I, so can form multiple formulations, enumerates no longer one by one.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.
Reagent can also be made into following three reagent:
Similar with two agent, three doses prescription also is not limited only to above-mentioned prescription, wherein the reduced coenzyme among the reagent I can be placed among reagent II or the reagent III, phenylacetate among the reagent II can be placed among reagent I or the reagent III, aldehyde dehydrogenase among the reagent III also can be put among reagent I or the reagent II, so can form multiple formulations, enumerate no longer one by one.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.
In addition, for reducing the cross influence between each reagent composition, the stability of maintenance reagent, so that standing storage, usually add stabilizing agent in the middle of the reagent I/ reagent II of above single agent, two agent, three doses the reagent I/ reagent II/ reagent III, active concentration is at 0.1～3mol/L, or 5～100g/ml, or within 5～50%v/v scope.
Material with used as stabilizers can be: at least a in ammonium sulfate (Ammonia Sulfate), glycerine (Glycerol), propylene glycol (Propylene Glycol), the ethylene glycol (Ethylene glycol).
No matter studies show that, take all factors into consideration from the accuracy of measurement result and economy two aspects of preparation cost, be single agent, two agent or three doses, and the diagnosis/detection kit of following formula components relation is comparatively desirable, also is preferred version of the present invention:
Damping fluid 100mmol/L,
Stabilizing agent 2mol/L,
Reduced coenzyme 0.25mmol/L,
Aldehyde dehydrogenase 8000U/L,
Phenylacetate 16mmol/L.
The present invention utilizes enzymic colorimetric (Enzymatic Colorimetric Method) and enzyme (even) united method (Couple Reaction) technology, continuous monitoring reduced form nicotinamide coenzyme (reduced coenzyme) is in the variation of 340nm wavelength place absorbance, measured the method for paraoxonase active concentration, simultaneously, the present invention gives in order to realize the paraoxonase diagnostic kit of this method, adopt this reagent not only can be ultraviolet analyser or half, carry out the paraoxonase active concentration determination on the automatic clinical chemistry analyzer, and finding speed is fast, the accuracy height, thereby can obtain practical applying.
The outstanding substantive distinguishing features and the obvious improvement of technical solution of the present invention mainly shows:
(1) the present invention utilizes enzymic colorimetric and enzyme (even) united method to measure the paraoxonase active concentration fully, and test result is accurate;
(2) composition of participation reaction all adds, and is not subjected to the pollution of inside and outside source material, test process degree of accuracy height;
(3) this method is easy, easy to operate, can obtain testing result fast, and the reaction be under buffer conditions, to carry out, do not pollute the environment;
(4) but this method just fast detecting on general ultraviolet/visible light analysis instrument or semi-automatic/automatic clinical chemistry analyzer does not need special or additional instruments, testing cost is cheap, is convenient to apply in the industry;
(5) use the reagent that assay method provided by the invention can be made various ways such as liquid reagent, powdered reagent, be used for measuring the size of various sample paraoxonase active concentrations;
(6) liquid paraoxonase diagnosis/detection kit provided by the invention, good stability has guaranteed the application testing effect well.Be made into after two agent or three doses, can further reduce the cross influence between the various compositions, testing result is more credible, and reagent is more stable, can store for a long time.
Embodiment
The assay method of a kind of paraoxonase active concentration of the present invention and paraoxonase diagnostic kit utilize paraoxonase (Paraoxonase; EC 3.1.1.2) enzyme (idol) connection aldehyde dehydrogenase (aldehyde dehydrogenase; EC 1.2.1.3; EC 1.2.1.4; EC 1.2.1.5) enzymatic reaction continuous monitoring method.The reaction of paraoxonase enzymolysis phenylacetate produces acetate, the effect of uniting aldehyde dehydrogenase again by (idol), reduced coenzyme (absorption peak being arranged at the 340nm place) is oxidized into coenzyme (not having absorption peak at the 340nm place) the most at last, thereby measured the speed that reduced coenzyme descends in 340nm place absorbance, by measuring the speed that 340nm place absorbance descends, can calculate the active concentration size of paraoxonase.
Below in conjunction with specific embodiment technical solution of the present invention is described further.These examples only are some exemplary applications, can not be interpreted as a kind of restriction to claim protection domain of the present invention.
Embodiment one (single agent)
Prepare the paraoxonase diagnostic kit by following composition and consumption:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L,
Ammonium sulfate 80g/ml,
NADH 0.25mmol/L，
Aldehyde dehydrogenase 8000U/L,
Phenylacetate 16mmol/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 1.8 ± 0.7, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested paraoxonase sample and reagent is 1: 25, the Direction of Reaction is negative reaction (reaction descends), about about 1 minute of time delay, about 2 minutes detection times, theoretical k value-4180.
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 340nm absorbance descends, thereby calculates the active concentration size of paraoxonase.
Embodiment two (two agent)
Prepare the paraoxonase diagnostic kit by following composition and consumption:
Reagent I---
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L,
Glycerine 2mol/L,
thio-NADH 0.25mmol/L，
Phenylacetate 12mmol/L;
Reagent II---
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L,
Propylene glycol 50%v/v,
Aldehyde dehydrogenase 12000U/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 1.8 ± 0.7, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested paraoxonase sample and reagent I, reagent II is 2: 20: 5, the Direction of Reaction is negative reaction (reaction descends), about about 1 minute of time delay, about 2 minutes detection times, theoretical k value-2170.
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 340nm absorbance descends, thereby calculates the active concentration size of paraoxonase.
Embodiment three (three doses)
Prepare the paraoxonase diagnostic kit by following composition and consumption:
Reagent I---
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L,
Ammonium sulfate 80g/ml,
NADPH 0.25mmol/L；
Reagent II---
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L,
Ethylene glycol 2mol/L,
Phenylacetate 20mmol/L;
Reagent III---
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L,
Glycerine 50%v/v,
Aldehyde dehydrogenase 6000U/L.
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid three reagent, can directly use.
When measuring the paraoxonase active concentration, on automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance 1.8 ± 0.7, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested paraoxonase sample and reagent I, reagent II, reagent III is 4: 40: 5: 5, and the Direction of Reaction is negative reaction (reaction descends), about about 1 minute of time delay, about 2 minutes detection times, theoretical k value-2170.
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 340nm absorbance descends, thereby calculates the active concentration size of paraoxonase.
Through experimental verification, adopt other various reduced form chromogens combinations of putting down in writing in the above summary of the invention all can reach purpose of the present invention, in view of situation such as determination step and above embodiment roughly the same, do not separately enumerate.
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, and is easy to utilize.
Claims (6)
1. the assay method of paraoxonase active concentration may further comprise the steps:
1. with sample of measuring and the reagent mixing that contains phenylacetate, aldehyde dehydrogenase and reduced coenzyme, make it to take place following reaction,
2. the end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect the speed that predominant wavelength 340nm absorbance descends, calculate the active concentration size of paraoxonase.
2. paraoxonase diagnostic kit, reagent is grouped into by following one-tenth in the box:
Damping fluid 20～500mmol/L,
Stabilizing agent 0.1～3mol/L, or
5～100g/ml, or
5～50％v/v，
Reduced coenzyme 0.1～0.35mmol/L,
Aldehyde dehydrogenase 1000～80000U/L,
Phenylacetate 1～50mmol/L.
3. paraoxonase diagnostic kit according to claim 2 is characterized in that: described reduced coenzyme is a kind of among NADH, NADPH, thio-NADH or the thio-NADPH.
4. paraoxonase diagnostic kit according to claim 2 is characterized in that: described stabilizing agent is at least a in ammonium sulfate, glycerine, propylene glycol, the ethylene glycol.
5. any one paraoxonase diagnostic kit in the claim 2～4 is characterized in that: described reagent is made into single agent, two agent or three doses.
6. any one paraoxonase diagnostic kit in the claim 2～4, it is characterized in that: described kit is powdered reagent box or liquid reagent box.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610161226 CN1975383A (en) | 2006-12-15 | 2006-12-15 | Method for measuring diethyl p-nitrophenyl phosphate esterase active concentration and diagnostic kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610161226 CN1975383A (en) | 2006-12-15 | 2006-12-15 | Method for measuring diethyl p-nitrophenyl phosphate esterase active concentration and diagnostic kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1975383A true CN1975383A (en) | 2007-06-06 |
Family
ID=38125606
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200610161226 Pending CN1975383A (en) | 2006-12-15 | 2006-12-15 | Method for measuring diethyl p-nitrophenyl phosphate esterase active concentration and diagnostic kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1975383A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101329257B (en) * | 2007-06-21 | 2013-06-19 | 苏州艾杰生物科技有限公司 | Nitrous acid determination reagent kit and method for determining nitric acid concentration |
| CN104762309A (en) * | 2015-04-07 | 2015-07-08 | 安徽省农业科学院水稻研究所 | Phosphate mannose isomerase (PMI) gene OsPMI2 originating from oryza sativa and application thereof |
| CN105510308A (en) * | 2015-12-25 | 2016-04-20 | 江苏迈源生物科技有限公司 | Activity testing method for paraoxonase and kit of paraoxonase |
-
2006
- 2006-12-15 CN CN 200610161226 patent/CN1975383A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101329257B (en) * | 2007-06-21 | 2013-06-19 | 苏州艾杰生物科技有限公司 | Nitrous acid determination reagent kit and method for determining nitric acid concentration |
| CN104762309A (en) * | 2015-04-07 | 2015-07-08 | 安徽省农业科学院水稻研究所 | Phosphate mannose isomerase (PMI) gene OsPMI2 originating from oryza sativa and application thereof |
| CN105510308A (en) * | 2015-12-25 | 2016-04-20 | 江苏迈源生物科技有限公司 | Activity testing method for paraoxonase and kit of paraoxonase |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1975383A (en) | Method for measuring diethyl p-nitrophenyl phosphate esterase active concentration and diagnostic kit | |
| CN1995975A (en) | Method for detecting N-acetyl-beta-amino glucosaccharase activity and diagnosis kit therefor | |
| CN101173901A (en) | N-acet-beta-amino glucosidase diagnostic reagent kit and method for measuring active concentration of N-acet-beta-amino glucosidase | |
| CN101169366A (en) | Fucosidase diagnosis reagent kit and fucosidase activity determination method | |
| CN101082575A (en) | Method for measuring citric acid concentration and citric acid diagnose reagent kit | |
| CN101169372A (en) | Glutamate dehydrogenase diagnosis reagent kit and glutamate dehydrogenase activity and concentration determination method | |
| CN101082568A (en) | Method for measuring acetic acid concentration and acetic acid diagnose reagent kit | |
| CN1920532A (en) | Method for NH3 concentration determination and reagent kit for NH3 diagnosis | |
| CN101173895A (en) | Paraoxonase diagnostic reagent kit and method for measuring active concentration of paraoxonase | |
| CN1975431A (en) | Method for assaying ion calcium concentration and ion calcium diagnostic kit | |
| CN101082572A (en) | Method for measuring aminoglutaric acid concentration and aminoglutaric acid diagnose reagent kit | |
| CN1987472A (en) | Method for detecting manganese comcentration and manganese diagnostic kit | |
| CN1786692A (en) | Process for determining content of potassium ion by enzyme method and kit for diagnosing potassium ion thereof | |
| CN1786694A (en) | Process for determining content of carbon dioxide and kit for diagnosing carbon dioxide therefor | |
| CN1975430A (en) | Method for assaying carbon dioxide concentration and carbon dioxide diagnosing reagent kit | |
| CN1975384A (en) | Fucosidase activity assaying method and fucosidase diagnostic kit | |
| CN101082569A (en) | Method for measuring amino acid concentration and amino acid diagnose reagent kit | |
| CN1778955A (en) | Determination of inorganic phosphorus and its diagnostic reagent kit | |
| CN101082571A (en) | Method for measuring malic acid concentration and malic acid diagnose reagent kit | |
| CN1570645A (en) | Quantitative detection reagent for activity of semen lactate dehydrogenase X isozyme | |
| CN101082570A (en) | Method for measuring ethyl hydrate concentration ethyl hydrate diagnose reagent kit | |
| CN1778941A (en) | Determination of inorganic phosphorus and its diagnostic reagent kit | |
| CN1789427A (en) | Monoamine oxidase activity determination method and monoamine oxidase diagnostic kit | |
| CN101169373A (en) | Adenosine deaminase diagnosis reagent kit and adenosine deaminase activity determination method | |
| CN101169374A (en) | Adenosine deaminase diagnosis reagent kit and adenosine deaminase activity and concentration determination method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |