CN101089593A - Homotype semicystionl diagnostic kit and investigating method for homotype semicystinol concentration - Google Patents
Homotype semicystionl diagnostic kit and investigating method for homotype semicystinol concentration Download PDFInfo
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- CN101089593A CN101089593A CN 200610085364 CN200610085364A CN101089593A CN 101089593 A CN101089593 A CN 101089593A CN 200610085364 CN200610085364 CN 200610085364 CN 200610085364 A CN200610085364 A CN 200610085364A CN 101089593 A CN101089593 A CN 101089593A
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- nitrobenzoic acid
- dithiobis
- homocysteine
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- stabilizing agent
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Abstract
A homocysteine diagnosis kit consists of compositions as buffer solution; 5,5' dithiobis-( 2-nitrobenzoicacid ); cystathionine upsilon-synthase EC 2.5.1.48 and stabilizer. Its method for determining concentration of homocysteine is also disclosed.
Description
Technical field
The present invention relates to a kind of homotype semicystionl diagnostic kit, the invention still further relates to the assay method of measuring homocysteine concentration simultaneously, belong to medical test determination techniques field.
Background technology
The method of measuring homocysteine in blood plasma has a variety of, comprise high performance liquid chromatography (HPLC), amino-acid analyzer determination method, the chromatography of ions, enzyme-linked immunosorbent assay (EIA), capillary gas chromatography-mass spectrum, fluorescence polarization immunoassay (FPIA), electrochemical process and enzyme process or the like.
1. high performance liquid chromatography (HPLC): be classical reference method, but it has the operation more complicated, test consuming time longly, the shortcoming that the test figure variance ratio is bigger is replaced by enzyme-linked immunosorbent assay, fluorescence polarization assay method and enzyme process aspect clinical practice gradually.High performance liquid chromatography is: use earlier reductive agent to make that the homocysteine of form of ownership is transformed into the reduction form in the blood sample, with the fluorescer generation homocysteine-fluorescent material compound of deriving, the sample after will deriving carries out stratographic analysis then.Absorption affinity difference because of the relative different material of chromatographic stationary, therefore moving phase is also different with its order that elutes, according to this principle separately with the different material in the sample, detect the fluorescence intensity of homocysteine-fluorescent material compound under the wash-out with fluorescence detector, itself and standard items/interior target ratio are compared and calculate, just can measure the level of blood total homocysteine.
2. enzyme-linked immunosorbent assay (EIA): be the common method of measuring homocysteine level clinically, be characterized in operating more convenient, quick, good reproducibility, method is reliable and stable.Shortcoming is most of operation or manual operations, and is more consuming time etc.Its fundamental analysis principle of enzyme-linked immunosorbent assay: use enzyme that the homocysteine of form of ownership in the blood sample is transformed into S-adenosine-L-homocysteine earlier, the monoclonal or the polyclonal antibody that add the anti-S-adenosine of enzyme target-L-homocysteine then, adopt the principle of competition combination, the standard items of varying level are combined with the enzyme labelled antibody competition, with chromogenic reagent with stop reagent and develop the color respectively and stop, produce the typical curve of homocysteine concentration and coloring intensity then.Sample is also handled by same steps as, just can find the concentration of its homocysteine on typical curve.
3. the chromatography of ions: be mainly used in experimental study, less clinically use.Its fundamental analysis principle is a kind of of stratographic analysis, mainly is that its stationary phase adopts ion exchange material, homocysteine can be separated with other material according to its absorption affinity difference to different ions and measure.
4. electrocapillary phoresis method: be mainly used in experimental study, the report in clinical use is arranged.Its characteristics are similar to efficient liquid-phase chromatography method, as it operation more complicated, the test long and bigger shortcoming of test figure variance ratio consuming time arranged.The fundamental analysis principle: use earlier reductive agent to make that the homocysteine of form of ownership is transformed into the reduction form in the blood sample, with the fluorescent reagent generation homocysteine-fluorescent material compound of deriving, the sample after will deriving carries out the electrocapillary phoresis analysis then.Sample is positioned in the high-voltage electric field about 10 kilovolts, because the electrically charged difference of various materials, its isoelectric point is also inequality, therefore its migration velocity in electric field is also just different, homocysteine and other material in the sample can be separated according to this principle, detect the fluorescence intensity of homocysteine-fluorescent material compound again with fluorescence detector, itself and standard items/interior target ratio are compared and calculate, just can measure the level of total homocysteine.
5. fluorescence polarization method: be the reasonable method of measuring homocysteine level clinically, this method full automation instrumental analysis, have fast, accurately, advantage easily.Its fundamental analysis principle: the fluorescence polarization intensity of the homocysteine that free homocysteine and anti-monoclonal antibody combine in the sample is different, sample, standard items are combined with the labeling antibody competition, adopt the principle of competing combination to produce the typical curve of homocysteine concentration and fluorescence polarization intensity, on typical curve, just can find the concentration of its homocysteine.
6. enzyme process: be method of testing of coming out newly developed in response to the market demand, at present domestic have multinomial patented claim: CN98807531.8 to utilize homocysteine to come homocysteine content in the measuring samples; CN200410016789.6 utilizes the cyclic amplification effect of HcyMetase (E.C.2.1.1.10) and S-Adenosylhomocysteine synthase (E.C.3.3.1.1), add adenosine deaminase, purine nucleoside phosphorylase, xanthine oxidase, peroxidase etc., or chromogenic reactions such as adenosine deaminase, glutamte dehydrogenase are measured homocysteine content; CN200510053210.8 utilizes the L-methionine Gamma-catenase to act on and produces fluorescence with fluorescent Compound D MPD2HCl effect again after homocysteine makes it to produce sulfuretted hydrogen, this method is used the test of full-automatic fluorescence analyser, have fast, accurately, advantage easily.Its fundamental analysis principle: with the genetic recombination enzyme homocysteine is decomposed, the sulfuretted hydrogen product that is formed forms the fluorescent chemicals that can measure with fluorescent color-developing agent generation chemical reaction again.
Summary of the invention
The technical problem to be solved in the present invention is: propose a kind of enzyme reaction coupling chemical reaction technology of utilizing, the sulfuretted hydrogen and 5 that metering is produced by cystathionine Gamma synthase (cystathionine γ-synthase EC 2.5.1.48) enzymolysis homocysteine, 5-dithiobis-(2-nitrobenzoic acid) (5,5 '-dithiobis-(2-nitrobenzoic acid), DTNB) chemical reaction taking place generates 5-sulfenyl-2-nitrobenzoic acid, and in the variation of 412nm wavelength place absorbance, measured the method for homocysteine concentration, simultaneously, the present invention also will provide in order to realize the homotype semicystionl diagnostic kit of this method, adopt this kit not only can be visible light analysis instrument or half, carry out the mensuration of homocysteine concentration on the automatic clinical chemistry analyzer, and finding speed is fast, the accuracy height, thereby can obtain practical applying.
Homocysteine method for measurement of concentration principle of the present invention is as follows:
Homocysteine+succinic acid
Cystathionine Gamma synthaseSulfuretted hydrogen+
Amber acidic group-homoserine
Sulfuretted hydrogen+5,5-dithiobis-(2-nitrobenzoic acid) → 5-sulfenyl-2-nitrobenzoic acid
+ sulphur
This method is used cystathionine Gamma synthase (cystathionine γ-synthase EC 2.5.1.48) coupling chemical reaction terminal colorimetric analysis.Cystathionine Gamma synthase (cystathionine γ-synthaseEC 2.5.1.48) enzymolysis homocysteine produces sulfuretted hydrogen, again by coupling 5, the chemical reaction of 5-dithiobis-(2-nitrobenzoic acid), produce 5-sulfenyl-2-nitrobenzoic acid (absorption peak being arranged) at the 412nm place, thereby measured the degree that 412nm place absorbance rises, can be calculated the size of homocysteine concentration.
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 homotype semicystionl diagnostic kit of the present invention of following composition relation is comparatively desirable:
Damping fluid 100mmol/L
Stabilizing agent 50mmol/L
Cystathionine Gamma synthase 5000U/L
5,5-dithiobis-(2-nitrobenzoic acid) 0.8mmol/L
Homotype semicystionl diagnostic kit of the present invention can be single agent, comprising:
Damping fluid, stabilizing agent, 5,5-dithiobis-(2-nitrobenzoic acid), cystathionine Gamma synthase.
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, cystathionine Gamma synthase.
Reagent 2
5,5-dithiobis-(2-nitrobenzoic acid).
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.
Embodiment
The present invention is further illustrated below in conjunction with examples of implementation.
Embodiment one
The homocysteine diagnosis reagent of present embodiment is single reagent, comprising:
Damping fluid 100mmol/L
Stabilizing agent 30mmol/L
Cystathionine Gamma synthase 4000U/L
5,5-dithiobis-(2-nitrobenzoic acid) 0.4mmol/L
On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance≤0.1, test predominant wavelength 412nm, test commplementary wave length 660nm, the volume ratio of tested homocysteine sample and reagent is 1/25, the Direction of Reaction is positive reaction (reaction of rising), detection method is an end-point method, about 5 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 degree that predominant wavelength 412nm absorbance rises, thereby calculates the size of homocysteine concentration.
Embodiment two
The homocysteine diagnosis reagent of present embodiment is double reagent, comprising:
Reagent 1
PH of buffer 8.2 100mmol/L
Stabilizing agent 40mmol/L
Cystathionine Gamma synthase 3000U/L
Reagent 2
5,5-dithiobis-(2-nitrobenzoic acid) 0.6mmol/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 412nm, test commplementary wave length 660nm, the volume ratio of tested homocysteine sample and reagent 1, reagent 2 is 2/20/5, the Direction of Reaction is positive reaction (reaction of rising), detection method is an end-point method, about 5 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 degree that predominant wavelength 412nm absorbance rises, thereby calculates the concentration of homocysteine.
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 (5)
1. homocysteine method for measurement of concentration, its method principle is as follows:
Homocysteine+succinic acid
Cystathionine Gamma synthaseSulfuretted hydrogen+amber acidic group-homoserine
Sulfuretted hydrogen+5,5-dithiobis-(2-nitrobenzoic acid) → 5-sulfenyl-2-nitrobenzoic acid+sulphur
End reaction thing 5-sulfenyl-2-nitrobenzoic acid (5-thio-2-nitrobenzoate) is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect the big or small degree that predominant wavelength 412nm absorbance rises, calculate homocysteine concentration measurement result.
2. homotype semicystionl diagnostic kit, principal ingredient comprises:
Damping fluid 20-500mmol/L
Stabilizing agent 1-50mmol/L
Cystathionine Gamma synthase 500-20000U/L
5,5-dithiobis-(2-nitrobenzoic acid) 0.2-5mmol/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 homotype semicystionl diagnostic kit of claim 2, it is characterized in that:
By damping fluid, stabilizing agent, 5, and 5-dithiobis-(2-nitrobenzoic acid) (5,5 '-dithiobis-(2-nitrobenzoic acid), DTNB), cystathionine Gamma synthase (cystathionine γ-synthaseEC 2.5.1.48) forms single agent reagent.
4. according to the described homotype semicystionl diagnostic kit of claim 2, it is characterized in that:
By damping fluid, stabilizing agent, 5, and 5-dithiobis-(2-nitrobenzoic acid) (5,5 '-dithiobis-(2-nitrobenzoic acid), DTNB), cystathionine Gamma synthase (cystathionine γ-synthase EC 2.5.1.48) forms two agent reagent; Reagent 1 is made up of damping fluid, stabilizing agent, cystathionine Gamma synthase; Reagent 2, by 5,5-dithiobis-(2-nitrobenzoic acid) formed.
5. according to the described homotype semicystionl 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 (Ammonia Sulfate), glycerine (Glycerol), propylene glycol (PropyleneGlycol), ethylene glycol (Ethylene glycol) and at least one of the preservatives.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103575730A (en) * | 2013-10-21 | 2014-02-12 | 华南农业大学 | Kit for detecting modification capacity of protein alkylation modifier PEG (polyethylene glycol) and using method thereof |
| CN105486666A (en) * | 2014-09-19 | 2016-04-13 | 苏州贝和医疗科技有限公司 | Homocysteine detection method and equipment thereof |
-
2006
- 2006-06-12 CN CN 200610085364 patent/CN101089593A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103575730A (en) * | 2013-10-21 | 2014-02-12 | 华南农业大学 | Kit for detecting modification capacity of protein alkylation modifier PEG (polyethylene glycol) and using method thereof |
| CN103575730B (en) * | 2013-10-21 | 2016-03-30 | 华南农业大学 | A kind of kit and using method thereof detecting protein alkylation dressing agent PEG Modifying Capability |
| CN105486666A (en) * | 2014-09-19 | 2016-04-13 | 苏州贝和医疗科技有限公司 | Homocysteine detection method and equipment thereof |
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Application publication date: 20071219 |