CN101762509A - Homocysteine diagnosis/determination reagent (kit) and homocysteine concentration determination method - Google Patents
Homocysteine diagnosis/determination reagent (kit) and homocysteine concentration determination method Download PDFInfo
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- CN101762509A CN101762509A CN200810244210A CN200810244210A CN101762509A CN 101762509 A CN101762509 A CN 101762509A CN 200810244210 A CN200810244210 A CN 200810244210A CN 200810244210 A CN200810244210 A CN 200810244210A CN 101762509 A CN101762509 A CN 101762509A
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Abstract
The invention relates to a homocysteine diagnosis/determination reagent (kit) using enzyme-colorimetry and enzyme-linked method technology. Meanwhile, the invention also relates to a homocysteine concentration determination method, reagent composition and component, which belongs to the technical filed of medical test and determination. The reagent (kit) of the invention mainly includes: buffer solution, reduced coenzyme, succinic acid-yl-homoserine, homocysteine desulfhydrase, cystathionine Gamma synthase, succinate semialdehyde dehydrogenase and stabilizing agent; samples and reagent are mixed according to a certain volume ratio to generate a series of enzymatic reactions; then the reactants are arranged under an ultraviolet/visible light analyzer to detect the decreasing degree of absorbance at the 340nm position of a dominant wave so as to measure and calculate the concentration of the homocysteine.
Description
Technical field
The present invention relates to a kind of homocysteine diagnosis/mensuration reagent (box), the invention still further relates to the method for 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 cycle amplification method (EnzymaticRecycling Method) of utilizing, enzymic colorimetric (Enzymatic Colorimetric Method) and enzyme (even) united method (Couple Reaction) technology, monitoring reduced form nicotinamide coenzyme (reduced coenzyme) is in the variation of 340nm wavelength place absorbance, measured the method for homocysteine concentration, simultaneously, the present invention also will provide in order to the homocysteine diagnosis of realizing this method/mensuration reagent (box), adopt this reagent not only can be ultraviolet analyser or half, carry out the homocysteine concentration determination 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 of the present invention is as follows:
Homocysteine+water
The homocysteine desulfhydraseSulfuretted hydrogen+ammonia+alpha-oxo-butyric acid sulfuretted hydrogen+amber acidic group-homoserine
Cystathionine Gamma synthaseSuccinic acid+homocysteine succinic acid+reduced form is auxilliary
Succinic semialdehyde dehydrogenaseSuccinic acid semialdehyde+coenzyme+water
This method is used homocysteine desulfhydrase (homocysteine desulfhydrase; EC4.4.1.2) enzyme (idol) connection cystathionine Gamma synthase (cystathionine γ-synthase; EC 2.5.1.48), succinic semialdehyde dehydrogenase (succinate semialdehyde dehydrogenase; EC 1.2.1.16; EC 1.2.1.24) enzymatic reaction continuous monitoring speed ratio color method.Homocysteine desulfhydrase is as the effect enzyme, and function is for to separate generation sulfuretted hydrogen with homocysteine.Cystathionine Gamma synthase is as cyclophorase: become sulfuretted hydrogen again homocysteine once more, it is recycling that homocysteine constantly is repeated.Succinic semialdehyde dehydrogenase is as the colour developing enzyme, reduced coenzyme (absorption peak being arranged at the 340nm place) is oxidized into coenzyme (not having absorption peak at the 340nm place), thereby measured the speed that reduced coenzyme descends in 340nm place absorbance, by measuring the speed that 340nm place absorbance descends, can calculate the concentration of homocysteine.
Experiment shows, takes all factors into consideration from the accuracy of measurement result and economy two aspects of preparation cost, no matter is single agent, two agent or three doses, and homocysteine diagnosis of the present invention/the mensurations reagent (box) of following composition relation is ideal comparatively:
Damping fluid 100mmol/L
Stabilizing agent 500mmol/L
Reduced coenzyme 0.25mmol/L
Homocysteine desulfhydrase 10000U/L
Cystathionine Gamma synthase 12000U/L
Succinic semialdehyde dehydrogenase 10000U/L
Amber acidic group-homoserine 8mmol/L
Homocysteine diagnosis of the present invention/mensuration reagent (box) can be single agent, comprising:
Damping fluid, stabilizing agent, reduced coenzyme, homocysteine desulfhydrase, cystathionine Gamma synthase, succinic semialdehyde dehydrogenase, amber acidic group-homoserine.
Reagent (box) can be 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, reduced coenzyme, amber acidic group-homoserine.
Reagent 2
Damping fluid, stabilizing agent, homocysteine desulfhydrase, cystathionine Gamma synthase, succinic semialdehyde dehydrogenase.
Reduced coenzyme, homocysteine desulfhydrase, cystathionine Gamma synthase, succinic semialdehyde dehydrogenase, the position of amber acidic group-homoserine in reagent 1 or reagent 2 can not limit.Reagent (box) can be dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
Above-mentioned single agent reagent can also be made into following three doses of reagent:
Reagent 1
Damping fluid, stabilizing agent, reduced coenzyme, amber acidic group-homoserine.
Reagent 2
Damping fluid, stabilizing agent, cystathionine Gamma synthase, succinic semialdehyde dehydrogenase.
Reagent 3
Damping fluid, stabilizing agent, homocysteine desulfhydrase.
Reduced coenzyme, homocysteine desulfhydrase, cystathionine Gamma synthase, succinic semialdehyde dehydrogenase, the position of amber acidic group-homoserine in reagent 1, reagent 2 or reagent 3 can not limit.Reagent (box) can be 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 homocysteine concentration, and its reduced coenzyme can be a kind of among NADPH, NADH or the thio-NADH.
Embodiment
The present invention is further illustrated below in conjunction with examples of implementation.
Embodiment one
The homocysteine diagnosis of present embodiment/mensuration reagent is single reagent, comprising:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Reduced coenzyme 0.25mmol/L
Homocysteine desulfhydrase 10000U/L
Cystathionine Gamma synthase 12000U/L
Succinic semialdehyde dehydrogenase 10000U/L
Amber acidic group-homoserine 8mmol/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 homocysteine sample and reagent is 1/25, the Direction of Reaction is negative reaction (reaction descends), about about 0 minute of time delay is 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 speed that predominant wavelength 340nm absorbance descends, thereby calculates the concentration of homocysteine.
Embodiment two
The homocysteine diagnosis of present embodiment/mensuration reagent is double reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Reduced coenzyme 0.25mmol/L
Amber acidic group-homoserine 8mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Homocysteine desulfhydrase 10000U/L
Cystathionine Gamma synthase 12000U/L
Succinic semialdehyde dehydrogenase 10000U/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 homocysteine sample and reagent 1, reagent 2 is 2/20/5, the Direction of Reaction is negative reaction (reaction descends), about about 0 minute of time delay is 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 speed that predominant wavelength 340nm absorbance descends, thereby calculates the concentration of homocysteine.
Embodiment three
The homocysteine diagnosis of present embodiment/mensuration reagent is three reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Reduced coenzyme 0.25mmol/L
Amber acidic group-homoserine 8mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Cystathionine Gamma synthase 12000U/L
Succinic semialdehyde dehydrogenase 10000U/L
Reagent 3
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Homocysteine desulfhydrase 10000U/L
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid three reagent, can directly use.
When measuring homocysteine 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 homocysteine sample and reagent 1, reagent 2, reagent 3 is 4/40/5/5, the Direction of Reaction is negative reaction (reaction descends), and about about 0 minute of time delay is 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 speed that predominant wavelength 340nm absorbance descends, thereby calculates the concentration of homocysteine.
The applicant adopts other assay methods of putting down in writing in the above summary of the invention all can reach purpose of the present invention through experimental verification, in view of situation such as determination step and above embodiment roughly the same, do not separately enumerate.
In a word, experimental results show that: adopt assay method of the present invention can draw required measurement result by general biochemical analyzer fully---the blank reagent absorbance changes (Δ A/min)≤0.001; Absorbance time response curve should be decline curve until terminal point; Reagent can be surveyed effectively, and (R 〉=0.99) linear range can reach 600 μ mol/L; The inaccuracy of reagent test, its relative deviation be no more than ± and 4%; The coefficient of variation (CV)≤2% of the precision of reagent test (repeatability); The sensitivity of reagent can reach 0.008 ± 0.004 Δ A/ μ mol/L; Reagent is preserved down at 2-8 ℃, and activity can be stablized 1 year;---the present invention is highly sensitive, degree of accuracy good, the linear range broadness, and stationary phase is long, is enough to easy to utilize.
Claims (6)
1. homocysteine method for measurement of concentration that utilizes enzyme cycle amplification method, enzymic colorimetric and enzyme-linked method technology, its method is as follows:
Homocysteine+water
The homocysteine desulfhydraseSulfuretted hydrogen+ammonia+alpha-oxo-butyric acid
Sulfuretted hydrogen+amber acidic group-homoserine
Cystathionine Gamma synthaseSuccinic acid+homocysteine
Succinic acid+reduced form is auxilliary
Succinic semialdehyde dehydrogenaseSuccinic acid semialdehyde+coenzyme+water
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 concentration measurement result of homocysteine.
2. homocysteine diagnosis/mensuration reagent (box), principal ingredient comprises:
Damping fluid 20---500mmol/L
Stabilizing agent 1---4000mmol/L
Reduced coenzyme 0.1---0.35mmol/L
Homocysteine desulfhydrase 1000---80000U/L
Cystathionine Gamma synthase 1000---80000U/L
Succinic semialdehyde dehydrogenase 1000---80000U/L
Amber acidic group-homoserine 1---50mmol/L
The concentration of reagent composition not necessarily is only limited to above-mentioned scope; Effect is better in this scope, and outside this scope, reagent still can reagentia.
It is characterized in that: reagent (box) can be 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 homocysteine diagnosis of claim 2/mensuration reagent (box), it is characterized in that:
Form single agent reagent by damping fluid, stabilizing agent, reduced coenzyme, homocysteine desulfhydrase, cystathionine Gamma synthase, succinic semialdehyde dehydrogenase, amber acidic group-homoserine.
4. according to the described homocysteine diagnosis of claim 2/mensuration reagent (box), it is characterized in that:
Form two agent reagent by damping fluid, stabilizing agent, reduced coenzyme, homocysteine desulfhydrase, cystathionine Gamma synthase, succinic semialdehyde dehydrogenase, amber acidic group-homoserine; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, amber acidic group-homoserine; Reagent 2 is made up of damping fluid, stabilizing agent, homocysteine desulfhydrase, cystathionine Gamma synthase, succinic semialdehyde dehydrogenase.Reduced coenzyme, homocysteine desulfhydrase, cystathionine Gamma synthase, succinic semialdehyde dehydrogenase, the position of amber acidic group-homoserine in reagent 1 or reagent 2 can not limit.
5. according to the described homocysteine diagnosis of claim 2/mensuration reagent (box), it is characterized in that:
Form multi-agent reagent by damping fluid, stabilizing agent, reduced coenzyme, homocysteine desulfhydrase, cystathionine Gamma synthase, succinic semialdehyde dehydrogenase, amber acidic group-homoserine; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, amber acidic group-homoserine; Reagent 2 is made up of damping fluid, stabilizing agent, cystathionine Gamma synthase, succinic semialdehyde dehydrogenase; Reagent 3 is made up of damping fluid, stabilizing agent, homocysteine desulfhydrase.Reduced coenzyme, homocysteine desulfhydrase, cystathionine Gamma synthase, succinic semialdehyde dehydrogenase, the position of amber acidic group-homoserine in reagent 1, reagent 2 or reagent 3 can not limit.
6. according to the described homocysteine diagnosis of claim 2/mensuration reagent (box), 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 (Propylene Glycol), ethylene glycol (Ethylene glycol) and at least one of the preservatives.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102539692A (en) * | 2010-12-13 | 2012-07-04 | 苏州艾杰生物科技有限公司 | Determination method of ammonia (ammonium ion) and diagnosis/determination reagent kit of ammonia (ammonium ion) |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102539692A (en) * | 2010-12-13 | 2012-07-04 | 苏州艾杰生物科技有限公司 | Determination method of ammonia (ammonium ion) and diagnosis/determination reagent kit of ammonia (ammonium ion) |
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Application publication date: 20100630 |